Abstract
Black phosphorus (BP) has attracted considerable attention as a biodegradable, stimuli-responsive 2D nanomaterial, emerging as a powerful theragenerative platform that integrates disease modulation with tissue regeneration. While earlier studies focused mainly on its anticancer properties, this review provides the first comprehensive analysis of BP as a theragenerative agent, unifying its disease-modulating capacity with its ability to stimulate tissue regeneration across multiple organs. BP exhibits several shared advantages: its degradation releases bioactive phosphate ions that support tissue repair; its highly reactive surface promotes cell interactions and enables efficient drug loading and delivery; its responsiveness to external stimuli, such as Near-infrared (NIR) light, ultrasound, and electrical signals, allows precise, on-demand therapeutic activation; and its ability to modulate reactive oxygen species (ROS) and immune modulation helps balance inflammation and regeneration. These properties collectively enhance osteogenesis and implant integration in bone, accelerate wound healing in skin, promote neural repair and redox homeostasis, protect cardiac tissue, and support recovery in kidney and liver injuries. By highlighting these mechanisms, this review emphasizes BP's versatility as a multifunctional nanomaterial capable of addressing pathological conditions while simultaneously stimulating endogenous regenerative pathways, thereby laying the foundation for its translation into next-generation theragenerative platforms.
Keywords: Black phosphorus, 2D nanomaterials, Stimuli-responsive, Disease therapy, Tissue regeneration
Graphical abstract
BP-based platforms for disease therapy and tissue regeneration. Created in BioRender. Bigham, A. (2025) https://BioRender.com/svxjxcz.
Highlights
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BP uniquely integrates disease therapy with tissue regeneration.
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BP degrades into bioactive phosphate ions and responds to NIR, ultrasound, and electrical cues.
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BP supports bone repair, accelerates wound healing, promotes neural recovery, and protects major organs.
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BP is a potent theragenerative platform for next-generation regenerative medicine.
1. Introduction
Various conditions, including trauma, degenerative diseases, accidents, cancer surgeries, etc., can cause tissue defects that require intervention. Besides how a medical practitioner should address the issue, these defects are prone to other potential dangers for patients—infection, chronic wounds, residual cancerous cells, etc., which can further complicate the condition [1,2]. Typically, grafts taken from either the patient or a donor are used with risks of morbidity. Tissue engineering, on the other hand, proposes two approaches for tissue regeneration—ex vivo and in situ. The former approach adopts a scaffold that is involved with related biomolecules and cells outside of the body, followed by being implanted in the defect's site, while the latter approach manipulates the inner regenerative potential [3,4]. Using ex vivo approaches, a biologically relevant scaffold/construct is fabricated to be substituted into the defect and to recapitulate the tissue's function. Since this approach has some limitations, more attention has been given to in situ tissue regeneration through those scaffolds. In this way, bioactive cues loaded onto/into these biomaterials can guide functional regeneration at the site of action and eliminate problems related to cell loading and maintenance. Nonetheless, other complications, as mentioned before, like cancerous cells, infection, etc., may intervene in the regeneration process, necessitating the design and development of effective advanced therapies [[5], [6], [7]].
Different organs and tissues face unique challenges in therapy and regeneration. In bone, defects may result from trauma, degenerative conditions, and tumor removal, and the main goal is to restore the damaged area and promote new bone growth using suitable biomaterials [[8], [9], [10]]. In the skin, chronic wounds caused by diabetes are a major clinical problem. High blood sugar can lead to poor blood flow, oxidative stress, prolonged inflammation, and delayed healing, often requiring advanced biomaterials that can accelerate repair, prevent infection, and support tissue regeneration [[11], [12], [13], [14], [15], [16], [17]]. Neurodegenerative diseases, such as Alzheimer's (AD), Parkinson's (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), involve the gradual loss of neurons, protein aggregation, oxidative stress, and neuroinflammation [18,19]. Current treatments are largely limited to symptom relief, highlighting the need for innovative strategies, where nanomaterials could offer targeted delivery, antioxidation, and neural repair [20]. Heart injuries, including myocardial infarction, cause reduced blood flow, excessive ROS production, inflammation, and cell death, with limited treatment options. In this case, the potential therapeutic agent's ability to scavenge ROS and capture metal ions could help protect and repair cardiac tissue [21,22]. Similarly, acute kidney and liver injuries involve oxidative stress and inflammation, underscoring the importance of biocompatible, ROS-scavenging materials for functional recovery [23,24].
Various 2D nanomaterials have attracted research interest for biomedical applications due to their unique physicochemical properties—such as their easy-to-functionalize nature, high surface area, etc. Due to their photocatalytic properties, they can produce heat upon irradiation with external light, a process known as photothermal therapy (PTT), as well as reactive oxygen species (ROS), which form the basis of photodynamic therapy (PDT) [[25], [26], [27]]. Nonetheless, their high surface area enables loading various drug molecules onto the 2D nanomaterials, thereby providing control over the release rate. However, careful evaluation of these 2D materials' safety revealed that, for instance, graphene at a concentration of 10 mg kg−1 can cause lung lesions in mice, leading to pulmonary edema, and molybdenum sulfide, a well-known 2D nanomaterial, induces cell toxicity at the concentration of 25 μg mL−1 in the exposure of human lung epithelial cells in vitro [[28], [29], [30]]. Black phosphorus (BP) is a 2D nanomaterial with a tunable bandgap and significant inherent advantages over other 2D materials. BP is a mono-elemental compound composed of phosphorus, which is distributed abundantly all over the Earth's crust. Since phosphorus is considered an essential element in the human body, BP exhibits excellent cytocompatibility and biocompatibility. It is known that once BP is exposed to biological media, it undergoes degradation, releasing phosphate groups [31,32]. Previous studies have reported the positive effects of those phosphate groups on bioactivity and on triggering osteogenic cell responses [[32], [33], [34]]. Nonetheless, there is a unique feature of BP that sets it apart in the family of 2D nanomaterials. It has been shown that BP can selectively suppress the growth and proliferation of cancer cells, without negatively affecting healthy tissues and cells, through different mechanisms of action, mainly related to differences in BP degradation rates in cancer and healthy cells [35]. The degradation rate of BP in a tumor's environment is higher than in normal tissues due to the higher metabolism of cancerous cells, which accelerates BP degradation and ROS formation [35,36]. BP responds to external stimuli, such as light and ultrasound, in addition to its inherent anticancer activity, making it a multifunctional biomaterial for various therapeutic approaches [[37], [38], [39]]. On the downside, BP is highly prone to oxidation, so optical-related features will be negatively affected as oxidation progresses. Therefore, to control the degradation rate of BP in biological environments, as desired, different strategies have been proposed [40,41].
Different strategies have been employed to reduce the oxidation rate of BP and improve its functionality over time for biological applications. One of the early approaches was encapsulating BP inside polymer matrices [42]. An important strategy to prevent oxidation during encapsulation was the use of water-free systems, such as incorporating BP inside poly (lactic-co-glycolic acid) (PLGA). There are other strategies as well, such as developing injectable hydrogels, electrospun fibers, 3D scaffolds, hybrid materials, etc., based on BP for various biomedical applications, specifically tissue engineering and regenerative medicine [32,33,[43], [44], [45], [46], [47], [48]]. The primary objective is to decrease the degradation rate, where necessary. However, in some cases, early degradation can be advantageous for tissue regeneration, as the released phosphate groups can promote cell proliferation and differentiation. The degradation of BP is accompanied by ROS generation because of the high reactivity rate of this material with oxygen due to the lone pair of electrons in each phosphorus atom [49,50]. These ROS are double-edged swords, good when it comes to cancer therapy, risky to the healthy cells if the concentration goes beyond their tolerance, and so more and more attention has been given to passivating the surface of BP as much as possible [31,51]. Modification of BP's surface with metal ions is another effective approach by which not only does the ROS generation decrease, but also the metal ion itself can add new functionality to BP—regeneration ability, reinforced photocatalytic activity, and antibacterial activity [20].
This review presents the first comprehensive, organ-spanning evaluation of BP as a theragenerative nanomaterial, integrating its disease-modulating capabilities with its ability to promote functional tissue regeneration (Scheme 1). While BP's anticancer properties and drug delivery potential across different organs have been extensively reviewed in our prior work [31], this review shifts the focus to its underexplored yet rapidly growing role in supporting tissue repair and regeneration across various pathological conditions. We systematically categorize and discuss BP-based strategies for enhancing regeneration across six major tissue systems—bone, skin, neural, cardiac, hepatic, and renal tissues —particularly in contexts involving trauma, ischemia, infection, inflammation, and degenerative damage. By organizing the literature into targeted subcategories for each organ system, we provide a clear and in-depth understanding of BP's biological interactions, microenvironment-dependent therapeutic mechanisms, and translational potential. Nonetheless, the BP nanomaterials' physicochemical properties, including the surface modification strategies adopted to either passivate or functionalize BP, have been thoroughly covered. Moreover, a comparison with other well-known biomaterials is provided to highlight how BP's unique combination of multifunctionality and biocompatibility may facilitate its future clinical translation. This review, therefore, not only fills a critical gap in the current literature but also establishes a unified framework for future studies exploring BP as a versatile, bioactive platform in regenerative medicine.
Scheme 1.
BP-based platforms for disease therapy and tissue regeneration across different organs. Created in BioRender. Bigham, A. (2025) https://BioRender.com/94gn9vb.
2. BP's physicochemical properties
BP is a layered two-dimensional allotrope composed of puckered sheets of sp3-hybridized phosphorus atoms, held together by van der Waals forces. This corrugated lattice produces a pronounced in-plane anisotropy, distinguishing armchair and zigzag directions with different mechanical, thermal, and electronic transport properties [52,53]. This anisotropy manifests as higher thermal conductivity along the zigzag direction and higher electrical conductivity along the armchair direction, allowing directional control over heat dissipation and charge flow in engineered systems [54,55]. Unlike graphene—which is structurally planar, chemically inert, and lacks a bandgap—BP possesses a thickness-dependent direct bandgap ranging from ∼0.3 eV in bulk to ∼2.0 eV in monolayer form, known as phosphorene. This feature enables strong optical absorption and highly efficient photothermal and photodynamic conversion in the Near-infrared (NIR) region, which are central to many of its therapeutic applications [56,57]. Combined with high carrier mobility and strong surface reactivity, these intrinsic physicochemical properties explain why BP has rapidly gained attention across biomedicine, electronics, sensing, catalysis, and energy conversion technologies [[58], [59], [60], [61], [62]]. However, the same lone-pair electron configuration that contributes to BP's semiconducting and optical properties also makes it susceptible to oxidation [63]. In aqueous, oxygen-rich environments, BP gradually forms PxOy intermediates, which then degrade to phosphate ions. While this biodegradability ensures physiological compatibility, uncontrolled oxidation can lead to a loss of photothermal efficiency, diminished electronic properties, and a limited functional lifetime in biomedical settings [31]. For this reason, stability engineering is a foundational requirement in the design of BP-based therapeutic platforms, and nearly every application discussed in this review employs a purposeful stabilization or encapsulation strategy.
A variety of surface modification and matrix-encapsulation approaches have been used to regulate BP stability and function (Scheme 2 and Table 1) [41,64]. Polymer passivation is a widely adopted strategy: PEG coatings provide steric shielding and improve solubility, circulation time, and colloidal stability, enabling the use of BP in renal, hepatic, and vascular injury without premature degradation [17,[65], [66], [67]]. Polydopamine (PDA) coating, obtained via simple alkaline self-polymerization, forms a tightly adherent protective layer that scavenges oxygen species while simultaneously enhancing photothermal conversion, as PDA strongly absorbs in the NIR region [[68], [69], [70]]. PLGA encapsulation was among the earliest approaches to protect BP against oxidation, as it is encapsulated within a hydrophobic matrix that prevents oxidation during processing and provides sustained-release capability in drug delivery applications [42,71,72]. Silk fibroin (SF) passivation, introduced during exfoliation, establishes hydrophobic-hydrophilic interactions that slow oxidation in aqueous environments and prevent aggregation, preserving optical performance for extended periods, where uncoated BP degrades rapidly [73,74]. Beyond polymers, coordination chemistry provides another robust stabilization route. Metal ions such as calcium, copper, and lanthanides can occupy reactive phosphorus sites, thereby reducing their susceptibility to oxidation [[75], [76], [77], [78], [79]]. Notably, lanthanide coordination also enables imaging, enabling multimodal platforms that couple NIR therapy with fluorescence or MRI [77]. Likewise, mineral-based encapsulation, such as the bioactive glass-black phosphorus quantum dots (BPQDs) nanocomposite fabricated in our study, demonstrates how confining BPQDs within amphiphilic liquid-crystal domains substantially slows oxidation while permitting integration into mineralizing microenvironments—particularly beneficial for bone-related applications [32]. Integration into tissue engineering architecture further influences BP's physicochemical behavior. In 3D scaffolds, BP interacts with mineral phases and extracellular matrix surrogates, where hydration and ion exchange modulate degradation rates and phosphate release kinetics [33,80]. In hydrogels, BP's NIR absorption provides on-demand thermal control over gel swelling, stiffness, and drug release profiles [[81], [82], [83]]. In gene and molecular delivery systems, cationic surface coatings such as polyethyleneimine enable efficient nucleic acid loading and intracellular trafficking while maintaining BP's photothermal responsiveness [84,85]. Cell membrane coating has also been employed as a biomimetic strategy to stabilize and target BP nanomaterials. By cloaking BPQDs or nanosheets with different cell membranes, the resulting core–shell structures inherit surface proteins that regulate immune recognition, circulation time, and homotypic targeting toward diseased tissues [[86], [87], [88]]. This coating not only shields BP from oxidative degradation but also preserves photothermal performance and improves site-specific accumulation without requiring synthetic ligand modification [85,87,89]. Each platform imposes a distinct microenvironmental context—hydration, ionic concentration, mechanical stress, and host-protein adsorption—that determines how rapidly BP oxidizes and how long its photothermal and electrical properties are maintained. Collectively, BP's layered structure, anisotropic conductivity, tunable semiconducting bandgap, and high photothermal efficiency support its remarkable versatility. Rather than representing a limitation, BP's chemical instability serves as a tunable design parameter; it can be leveraged to achieve short-lived antimicrobial and tumoricidal activity or mitigated through stabilization strategies to enable long-term functionality in tissue repair and controlled drug delivery. By modulating particle size, surface passivation chemistry, coordination interactions, encapsulation architectures, and integration into scaffold or hydrogel platforms, BP can be precisely engineered to achieve application-specific stability and functional behavior.
Scheme 2.
Main approaches towards surface modification or incorporation of BP nanomaterials for biomedical applications. Created in BioRender. Bigham, A. (2025) https://BioRender.com/jnm6oye.
Table 1.
Physicochemical properties of BP nanomaterials, either surface-modified or incorporated into various platforms, for biomedical applications.
| Composition | BP synthesis, size, and thickness | Particle size (nm) Zeta potential (mV) Photothermal-related parameters | Surface modification | Remarks | Ref. |
|---|---|---|---|---|---|
| BPQDs-poly lactic-co-glycolic acid (PLGA) | BPQDs:
|
BPQDs-PLGA: Particle size: 102.8 ± 35.7 nm
|
Encapsulation of BPQDs inside PLGA nanospheres |
|
[42] |
| BP-poly ethylene glycol (PEG) | BP nanosheets:
|
BP-PEG: Particle size: ∼100 nm
|
Surface functionalization of BP nanosheets with PEG-NH2 |
|
[90] |
| BP-silk fibroin (SF) | BP nanosheets:
|
BP-SF: Particle size: 131.9 ± 15.00 μm
|
SF nanofibers were self-assembled around the BP nanosheets and formed microspheres |
|
[73] |
| BP-poly dopamine (PDA) | BPQDs:
|
BP-PDA: Particle size: ∼150 nm
|
PDA was formed around the BPQDs in an alkaline medium in situ |
|
[70] |
| Cu2+-modified BP | BP nanosheets:
- Average thickness: 2 nm - Zeta potential: −53.70 ± 2.48 mV |
BP@Cu2+: Particle size:
|
Cu2+ ions made the π–π stacking interactions with the BP nanosheets |
|
[78] |
| Calcium phosphate-mineralized BP | BP nanosheets:
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BP@Cu2+: Particle size: 181.5 nm
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A layer of calcium phosphate mineral was formed on the surface of BP nanosheets |
|
[75] |
| BP@Mesoporous silica-PEG | BP nanosheets:
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BP@mSiO2-PEG-cRGD: Particle size: 511.90 nm
|
Sequential surface modification of BP nanosheets with mesoporous silica and PEG-cRGD |
|
[67] |
| Adipose-derived stem cell exosomes (ADSC-Exos) coated BPQDs |
BPQDs:
|
ADSC-Exos coated BPQDs: Particle size: 150 nm
|
BPQDs were encapsulated within exosome membranes |
|
[88] |
3. BP degradation, redox duality, and microenvironment-dependent therapeutic outcomes across organs
BP undergoes a characteristic oxidative degradation process in physiological environments, wherein its surface progressively converts to PxOy intermediates and ultimately to biocompatible phosphate ions [91]. While the chemical pathway is consistent, this transformation leads to different biological outcomes across tissues. The same degradation event can produce either pro-oxidant or antioxidant effects, depending on the surrounding microenvironment [50,92]. This dynamic redox duality is central to BP's ability to unify disease modulation with tissue regeneration across multiple organ systems (Scheme 3). For instance, wound and tumour micro-environments are typically highly metabolically active because rapidly proliferating cells demand large amounts of energy and biosynthetic precursors. This elevated metabolic flux, combined with hypoxia and dysfunctional vasculature, drives a shift toward glycolysis and lactate production—leading to acidification of cytoplasm and extracellular milieu [93]. Concurrently, tumors generate elevated ROS through mitochondrial dysfunction, inflammatory infiltration, and hypoxia-induced signaling [94], which further attacks BP's reactive surface. The combined effects of acidity, oxidative stress, and hypoxia create a biochemical niche that destabilizes BP more rapidly than in healthy tissue, thereby enhancing its degradation kinetics and amplifying its ROS-generating therapeutic activity. A similar microenvironment-driven shift in BP's redox behavior is also observed in wounded or infected tissues, where elevated ROS levels accelerate BP oxidation and trigger a transient surge in ROS production. In these conditions, BP acts as a pro-oxidant agent, amplifying oxidative stress and damaging microbial membranes, or inducing cancer cell apoptosis [35,95]. For example, BP-based wound dressings leverage this mechanism to eradicate multidrug-resistant bacteria, where photothermal activation under NIR irradiation produces localized hyperthermia and oxidative membrane disruption while avoiding systemic oxidative damage. This pro-oxidant behavior is intentionally localized and often externally controlled, ensuring on-demand antibacterial action while preserving adjacent healthy tissue [[96], [97], [98]]. In contrast to these pathological settings, when BP encounters a physiologically normal tissue environment, its redox behaviour shifts markedly. Indeed, in healthy tissues, BP acts as a ROS scavenger rather than a ROS generator. This is because healthy tissues maintain relatively low basal ROS levels and a physiologically neutral pH, ensuring slow, controlled BP degradation. Indeed, this slow oxidation of BP consumes ROS species as the phosphorus reacts to form PxOy intermediates, thereby acting as a net ROS sink [91]. The resulting antioxidant effect aids tissue homeostasis and repair, underscoring how BP's redox behaviour is highly dependent on the local microenvironment. This mechanism becomes critical in the case of inflammatory tissue injury, in which moderate ROS production and persistent pro-inflammatory cytokine release create a microenvironment that impairs healing and promotes tissue damage [99].
Scheme 3.
Schematic illustration of microenvironment-adaptive redox regulation ability of BP. Created in BioRender. Bigham, A. (2025) https://BioRender.com/uwhlpeq.
In recent studies, BP—especially in the form of ultrasmall quantum dots or surface-modified nanosheets (e.g., PDA, tannic acid (TA), and PEG)—has been shown to exhibit strong ROS-scavenging and metal-ion-chelating effects [22,24,100,101]. A representative example is a PEGylated BP nanoplatform co-delivering the Nrf2-activating metabolite 4-octyl itaconate (4-OI), which demonstrated robust antioxidative regulation in acute kidney injury by promoting nuclear translocation of Nrf2 and upregulating downstream antioxidant effectors while simultaneously suppressing NF-κB–mediated pro-inflammatory signaling [92]. Similarly, BPQDs have been shown to alleviate acute liver injury by quenching excessive intracellular ROS and stabilizing mitochondrial function, thereby preventing hepatocyte apoptosis and restoring metabolic homeostasis [102]. In these cases, slow, controlled BP degradation is essential to maintaining antioxidant activity without reintroducing oxidative imbalance, while shifting the tissue environment from a destructive, high-ROS, inflammatory state toward a regenerative, low-ROS, pro-repair state. The situation in bone presents a distinct but integrative case in which both redox regulation and ion release are needed to coordinate immunomodulation, osteogenesis, and matrix mineralization. Controlled degradation of BP embedded in scaffolds provides phosphate ions that directly support hydroxyapatite (HA) nucleation and bone matrix maturation, thereby enhancing mechanical stability and integration with the host bone tissue during the treatment period [33,72]. Moreover, BP influences early-stage immune responses by modulating macrophage polarization. BP-based scaffolds have been shown to activate the IL-33 axis, promoting the transition from pro-inflammatory M1 macrophages to pro-regenerative M2 phenotypes, thereby establishing a cytokine environment favorable to osteoblast differentiation and angiogenesis [80,103,104]. In this tissue, BP does not serve purely as a ROS generator or scavenger; instead, it balances oxidative signaling—supporting initial inflammatory clearance while later suppressing NF-κB–mediated chronic inflammation and promoting reparative signaling pathways such as PI3K/Akt, Wnt/β-catenin, and BMP/SMAD [105,106]. Thus, bone regeneration requires moderate, phase-appropriate BP degradation kinetics, distinguishing it from both antibacterial/anticancer and ischemic injury contexts.
Thus, BP's theragenerative capacity is not merely a sum of its material properties, but a function of how degradation rate, formulation, and tissue biology intersect. By tuning particle size, surface stabilization, composite embedding, and external stimuli, BP can be tailored to operate as a pro-oxidant, antioxidant, or immune-instructive agent in a highly organ-specific manner [31]. This adaptive versatility enables BP to unify disease therapy and tissue regeneration across multiple systems, as discussed in detail in the following sections.
4. Bone-related applications of BP-based systems
Bone is a rigid organ that serves multiple critical functions, including providing structural support for the body, producing white and red blood cells, protecting organs, and storing essential minerals, among others [107,108]. A wide variety of factors can result in bone defects that cannot be healed spontaneously and naturally, requiring medical intervention. Such defects may arise from fractures due to aging and the increased prevalence of trauma [109]. Currently, the gold standard for bone tissue regeneration is autogenous bone transplantation; however, this approach has several shortcomings that limit its widespread application. Additionally, there are other concerns, such as bacterial infection and cancer recurrence, which have led the scientific community to seek alternatives [110,111]. BP has been among the potential biomaterials investigated in recent years, with multifunctionality for bone tissue engineering [112]. Since the by-product of BP is phosphate, it not only impacts and stimulates the bone regeneration rate directly, but also has a high affinity towards Ca2+ ions to form carbonated HA, followed by being adhered to the host bone [113]. Nonetheless, the stimulus-responsivity of BP opens new doors to other issues like infection, cancer cells, osteoarthritis, etc. [35]. Some BP-based platforms used in bone tissue engineering are listed in Table 2. This section covers and discusses the most recent advances in bone tissue engineering using BP.
Table 2.
Physicochemical and biological properties of BP-containing platforms for bone therapy and regeneration.
| Composition | Synthesis | Physicochemical properties | Application | Animal model and cycle | In vitro and in vivo | Remarks | Ref. |
|---|---|---|---|---|---|---|---|
| BP-coated polycaprolactone (PCL) 3D-printed scaffolds | 3D printing of PCL scaffolds, alkaline treatment for surface functionalization, and deposition of BP nanosheets | Alkaline-treated BP nanosheets: Zeta potential: −34.16 ± 0.52 mV Particle size: 175.73 ± 4.23 nm PTT in vitro: 808 nm NIR at 1.0 W cm−2 for 2 min. PTT in vivo: 808 nm NIR at 1.0 W cm−2 for 2 min daily up to 8 weeks. |
Bone regeneration with the aid of mild hyperthermia |
|
In vitro: A substantial increase in osteogenic-related proteins' expression level, particularly for NIR-irradiated BP-coated scaffolds. In vivo: Only the BP-coated scaffolds, under NIR irradiation, almost completely filled the bone defect. |
|
[114] |
| BP-coated PCL 3D-printed scaffolds | 3D printing of PCL scaffolds, alkaline treatment for surface functionalization, and deposition of BP nanosheets | – | Bone immunomodulation and regeneration |
|
In vitro: Scaffolds with three BP doses were tested. -Only the highest dose was cytotoxic to bone marrow stem cells. BP below 16 μg mL−1 was safe for macrophages and increased interleukin-33, promoting immunomodulation and bone healing. In vivo: PCL and BP-coated scaffolds were implanted at three doses. The medium dose showed the best bone ingrowth, while the high dose and control did not fully fill the defect. |
|
[115] |
| BP-coated 3D-printed scaffolds | 3D-printing of scaffolds composed of PCL and poly (caprolactone fumarate) (PCLF) followed by deposition of BP and graphene oxide (GO) | – | Bone immunomodulation and regeneration |
|
In vitro: BP-GO scaffolds recruited the most macrophages, efficiently loaded interleukin-4 to induce M2 polarization, and promoted osteogenic marker expression in bone marrow stem cells after 14 days. In vivo: Implanted in rat calvarial defects for 4 weeks, BP-GO scaffolds largely filled the defect with new bone. |
|
[106] |
| BP-poly lactic-co-glycolic acid (PLGA)-SrCl2 | Oil-in-water emulsion solvent evaporation method | Particle size: 45.4 ± 12.9 μm Zeta potential: PTT in vitro: 808 nm NIR at 1.0 W cm−2 for 10 min. PTT in vivo: 808 nm NIR at 1.0 W cm−2 for 5 min at day 0 and repeated at 1, 2, 3, 4, and 8 weeks postoperatively. |
Ion delivery and bone regeneration |
|
In vitro: The samples were exposed to bone marrow-derived mesenchymal stem cells; over 95 % of the cells remained alive in all groups. In vivo: Compared to PLGA, the PLGA-SrCl2 could form more new bone tissue in the defect, thanks to the strontium release.
|
|
[71] |
| BP-polydopamine (PDA)-ZnO-coated titanium implants | The surface coatings were applied through immersion | – | Antibacterial activity and bone regeneration |
|
In vitro: In the absence of NIR, the antibacterial efficiency was lower than 40 %, while with NIR it reached up to 70 %.
|
|
[116] |
| A multifunctional hydrogel-coated implant | The hydrogel made of gelatin methacrylate/dopamine methacrylate-PDA-BP nanosheets was coated on the implant via spin coating | Particle size: BP nanosheets: 450 nm PDA-BP: 567.2 nm Zeta potential: BP nanosheets: −25 mV PDA-BP: −48 mV PTT in vitro: 808 nm NIR at 1.0 W cm−2, 10 min/day for 1, 3, and 7 days. PTT in vivo: 808 nm NIR at 1.0 W cm−2 for 10 min on days 0, 2, 4, 8, 16, and 20. |
Anticancer, antibacterial, and bone regeneration |
|
In vitro: After 14 days, the expression of collagen-1 and runt-related transcription factor 2 (Runx2) was significantly improved in the cells exposed to the coated implants.
|
|
[117] |
4.1. BP for bone cancer therapy and regeneration
Primary bone cancer is among the rare cancer types; however, secondary bone malignancies caused by metastasis, particularly from breast and prostate cancers, are more common and often lead to the formation of bone tumors. Surgical removal of these tumors typically results in bone defects, which require the use of biomaterials capable of stimulating the regeneration rate [118]. One of the early studies targeting bone cancer therapy and regeneration through BP was published in 2018 [33]. Taking advantage of stimulus-responsivity, BP-coated on the 3D bioactive glass scaffolds could produce heat in the exposure to NIR and lead to tumor growth suppression. The regenerative potential of BP-coated scaffolds was tested in vivo on Sprague–Dawley rats; cranial defect models were established, followed by implantation of the 3D scaffolds. At 8 weeks post-implantation, it was observed that newly formed osseous tissues were formed in the defects treated with the scaffolds, and they were degraded and absorbed. The micro-CT images revealed that the BP-bioactive glass scaffolds showed competitively better reconstructive outcomes than the scaffolds without BP [33]. Our group later discovered the in vitro inherent anticancer activity and regeneration potential of BP against osteosarcoma and osteoblast cells, respectively, with and without NIR irradiation. The BP could suppress the cancer cells' growth at specific concentrations, while the same concentrations did not have any negative effect on the healthy counterpart [35]. Hydrogels are of particular interest in tissue engineering because of their similarity to natural tissue, and they are excellent candidates for drug delivery applications due to their high loading capacity along with a sustained release over an intended time [119]. A bifunctional injectable hydrogel based on chitosan was designed in which BP and doxorubicin had been encapsulated for bone cancer therapy and regeneration. To turn the chitosan hydrogel into thermosensitive, β-Glycerol phosphate disodium salt was added to the chitosan solution, and then BP (50 μg mL−1) and doxorubicin were added one by one. The in vitro anticancer effect showed that the combination of PTT and the drug release could kill all the cancer cells, whereas neither PTT alone nor chemotherapy led to sufficient anticancer activity. The regenerative capability of hydrogel was assessed in vitro through Alkaline phosphatase activity (ALP) and alizarin red staining (ARS) against MC3T3-E1 cells. Compared to the control represented by chitosan, the BP-incorporated hydrogel could induce Alkaline phosphatase nodules at a higher density than the control; this marker is considered an early indicator of osteogenic differentiation in osteoblasts. Mineralization in the extracellular matrix was also evaluated through ARS, as it is the most significant late-stage osteogenic differentiation marker. The calcium nodules formed after 14 days were competitively higher in BP samples, suggesting the higher mineralization rate of BP [120]. Via the freeze-drying technique, a multifunctional scaffold was developed, consisting of HA, BP, chitosan, and hydroxypropyltrimethyl ammonium chloride-chitosan, for PTT-assisted bone cancer therapy and regeneration. Interestingly, this study has effectively employed mild hyperthermia (∼42 °C) to stimulate osteogenesis through the upregulation of heat shock proteins. The overall results of osteogenic studies showed that the expression of ALP, collagen type I (COL I), and osteocalcin (OCN) in the cells treated with the mild hyperthermia of multifunctional scaffold was 1.64, 1.31, and 1.27 times higher than that of the scaffold not being exposed to the laser irradiation in vivo [121]. Our group has recently reported the development of a theragenerative nanoplatform for bone cancer therapy and tissue regeneration. The platform was composed of Pluronic F127, bioactive glass, and BPQDs, which were synthesized through a two-step synthesis strategy in which the BP nanosheets were introduced into the sol-gel medium along with the bioactive glass precursors. The mixture underwent microwave irradiation to transform the nanosheets into quantum dots in situ, which were then distributed throughout the hybrid material. The anticancer activity of the hybrid was assessed with and without NIR irradiation; the hybrid was found to induce anticancer suppressive effects on osteosarcoma cells without NIR, and in the presence of the laser, the anticancer potency was strengthened thanks to PTT. The regenerative potential of the hybrid platform was also assessed against osteoblast and mesenchymal stem cells; the hybrid material stimulated the proliferation of osteoblast cells thanks to the release of osteogenic ions (e.g., calcium, silicon, and phosphorus) and also led to the differentiation of stem cells towards osteoblast cells [32]. Using a biomineralization-inspired strategy, BP nanosheets were surface-modified with fructose-incorporated calcium phosphate for a combination of cancer therapy and bone tissue regeneration. BP with an abundant phosphorus source is utilized as an excellent growth substrate to form calcium phosphate in situ, thereby increasing the degradation rate in the tumor microenvironment while enhancing BP's stability within normal cells. Since tumor cells exhibit higher expression of glucose transporter proteins compared to healthy cells, the surface-modified BP showed a pH-responsive behavior and underwent faster degradation in 143B osteosarcoma cells, which altered the cellular ATP level and led to apoptosis. On the other side, the release of calcium and phosphorus ions from the platform was found to induce positive effects on the osteogenic differentiation and mineralization of pro-osteoblastic cells; the liberated calcium ions would bind to calmodulin, and this phenomenon activates the calcium/calmodulin-dependent signaling cascades leading to the improvement in the osteoblast differentiation and mineralization [122].
4.2. Stimuli-responsive BP systems for bone repair
PTT is a well-known method in cancer therapy by which the cancerous cells get ablated and killed through the generation of localized high-energy thermal effects by nanomaterials upon NIR irradiation [123]. Besides the anticancer activity of PTT, it has been shown that mild hyperthermia has a remarkable ability to stimulate and activate the expression of crucial factors in cells, such as ALP, mineralization, heat shock protein, etc., contributing to bone regeneration [124,125]. Porous AuPd alloy was used as a hyperthermia agent to assess whether mild hyperthermia can accelerate bone regeneration. Under NIR irradiation, the cell proliferation and bone formation were greatly accelerated in vitro. The in vivo studies that were performed on cranial defects (8 mm in diameter) exhibited almost 97 % coverage of the defect after 6 weeks by newly formed bone tissue, and it turned out that the mild hyperthermia induced the Wnt signaling pathway [126]. Another study has reported on the development of microspheres composed of BP, strontium chloride, and PLGA for bone regeneration and indicated that the NIR-activated microspheres facilitated the bone regeneration rate in rats’ femoral defects [71]. One of the main challenges in PTT bone repair is the formation of non-degradable or toxic by-products liberated from the PTT agents. These agents include MXenes, metal-organic frameworks, and gold nanoparticles, among others [124,127]. However, degradation of BP is synchronized with the release of phosphate anions, which are naturally present in the body and are essential for various biological processes [31].
4.2.1. PTT-enabled BP for bone regeneration
A multifunctional photothermal scaffold composed of PLGA and BP was fabricated to promote bone regeneration. The fabrication strategy involves the synthesis of PLGA microspheres loaded with bone morphogenetic protein-2 (BMP-2) on one side, and the development of a BP-incorporated PLGA scaffold through the freeze-drying technique on the other side. Then the microspheres were coated on the scaffolds to yield a multifunctional scaffold. A series of in vitro studies assessed the effects of BMP-2 release and hyperthermia on cell viability and osteogenic differentiation. The proliferative activity of periosteal-derived stem cells revealed that protein release and effects of hyperthermia significantly improved the viability. Moreover, the same trend was repeated for the mRNA expression of ALP, COL I, osteopontin (OPN), and runt-related transcription factor 2 (Runx2) genes. Using a Western blot, the mild hyperthermia effect was further analyzed to determine if the heat had a significant effect on heat shock proteins; it turned out that the protein expression of HSP70 and HSP27 in the BP-incorporated samples exposed to NIR was significantly higher than that of those without NIR exposure [128]. Heat shock proteins are responsible for folding newly synthesized or misfolded proteins, which in turn affects the cell's proliferation rate and differentiation. HSP70 and HSP27 are highly expressed in osteoblast cells in newly formed bone regions. Interestingly, the upregulation of these proteins is associated with the stimulation of several osteogenic-related genes [129,130]. A fish-derived scaffold synthesized from the decellularized fish scale and fish gelatin was developed to stimulate the bone regeneration rate. BP nanosheets were incorporated into a gelatin methacrylate hydrogel network synthesized from fish gelatin to endow it with PTT activity upon NIR exposure. In vitro assays showed that BP-incorporated scaffolds could promote osteogenic differentiation without NIR, and the relative mRNA levels of Runx2 and bone morphogenetic protein-2 were elevated. Under NIR irradiation, the BP-coated scaffolds exhibited a much higher transcription level of osteogenic markers. The biomaterials were also applied in vivo on a mouse skull, and an NIR laser was applied to the defect twice a week. During each cycle, the temperature was raised to 42C for 10 min; better recovery of bone defects was observed in defects treated with BP-incorporated materials exposed to NIR [131]. Injectable BP/SF microspheres were developed as bioinspired, porous, nanofibrous scaffolds to address some challenges in bone regeneration—poor cell viability after injection and lack of directional differentiation (Fig. 1(I)). SF was self-assembled into nanofibers to form highly porous microspheres that mimic the extracellular matrix, providing mechanical stability, enhancing cell adhesion, and protecting loaded cells during injection for efficient delivery. Exfoliated BP nanosheets were incorporated to endow the microspheres with tunable photothermal properties and sustained PO43− ion release. Under NIR irradiation, the BP/SF microspheres acted as a “micro-hotbed,” promoting osteogenic differentiation by regulating key osteogenesis-related genes and signaling pathways. In vitro, BP/SF enhanced the proliferation, adhesion, and osteogenesis of bone marrow mesenchymal stem cells. In vivo, the system significantly accelerated bone defect repair, demonstrating improved bone regeneration through effective cell delivery and synergistic photothermal and phosphate-mediated osteogenic stimulation (Fig. 1(II)) [73]. A recent study employed BP as bone-mimicking seeds, drawing a parallel between bone regeneration and the natural growth of plant seeds (Fig. 1(III)). Their main aim was to address the deficiency of calcium ion enrichment and mineralization in conventional 3D-printed scaffolds. In this regard, they took advantage of amine functionalization on the PCL scaffold's surface to capture BP nanosheets via electrostatic interactions between phosphate and amine groups; the high affinity of phosphate groups on oxidized BP for calcium ions accelerates the endogenous regeneration of bone defects. In vitro osteogenic performance of PCL and PCL-BP scaffolds in the presence of bone marrow-derived mesenchymal stem cells with and without NIR irradiation was evaluated. Cell viability and proliferation showed no statistically significant differences between the groups and the control, indicating scaffold compatibility with cells. However, the difference was evident in the assessment of the scaffolds' osteogenic performance, including ALP, ARS, Runx2, OPN, and osterix. By day 14, the levels of ALP and ARS in the scaffolds exposed to NIR were 2.6 and 9.3 times higher than those of non-irradiated scaffolds, respectively, indicating the impact of mild hyperthermia on bone regeneration. A substantial increase in the upregulation of OPN, Runx2, and osterix proteins was observed in the cells treated with BP-coated scaffolds exposed to NIR compared to the control group. In the case of in vivo experiments (Fig. 1(IV)), although all the bone defects treated with different scaffolds experienced new bone formation from the edge of the defect towards the center, the NIR-irradiated scaffold achieved the best level of skull defect repair, with almost complete bone healing after 8 weeks. The histological analysis reveals that the bone defect in the control group was mainly colonized by soft tissue with negligible formation of new bone tissue. In contrast, for the BP-coated scaffolds, specifically those exposed to NIR, there is noticeable new bone growth [114].
Fig. 1.
BP nanosheets accelerate bone regeneration under NIR stimulation. (I) Schematic illustration of the fabrication of BP/SF microspheres and their application as injectable “micro-hotbeds” for efficient cell delivery and enhanced bone regeneration. (II)In vivo evaluation of bone regeneration using BP/SF with NIR irradiation: (A) Schematic representation of BP/SF-assisted bone regeneration under NIR. (B) Micro-CT reconstruction images showing superficial and cross-sectional views at 4 and 8 weeks post-implantation. Quantitative micro-CT analyses: (C) Bone volume fraction (BV/TV), (D) trabecular thickness (Tb.Th), and (E) trabecular separation (Tb.Sp). Statistical significance: *P < 0.05, **P < 0.01 vs. blank control; #P < 0.05, ##P < 0.01 vs. NIR-BP/SF. Reprinted from Ref. [73] with permission from Elsevier. (III) (A) The natural process of seed growth, which is resembled by this study as a way to induce bone growth by BP-coated scaffolds. (B) Schematic illustration of BP nanosheets coating on the surface of alkaline-treated PCL scaffolds for bone regeneration under the NIR stimulation. (IV) In vivo performance of the scaffolds in skull bone defects. (A–D) Animal model, scaffold implantation, the apparatus for NIR irradiation, and the digital photographs of the scaffold-filled defects after 4/8 weeks. (E) The defect area related to each group is indicated through X-ray and micro-CT images after implantation. (F) The quantitative results from the micro-CT. (G) Histological analysis to assess the newly formed bone in the defects. Reproduced under the terms of the Creative Commons Attribution License [114]. Copyright 2024, Wiley.
4.2.2. Electrical stimulation-enabled BP for bone regeneration
Electricity has been shown to improve bone regeneration by modulating key cellular behaviors (e.g., proliferation, differentiation, and migration). Both external and endogenous electrical stimuli can accelerate the bone healing process by promoting osteogenesis and enhancing collagen synthesis [132,133]. To harness these features, electrically responsive biomaterials have been designed to generate endogenous electrical fields through the incorporation of conductive elements, such as graphene, polypyrrole, and others, thereby facilitating osteoblast differentiation [134]. In addition, these materials can also be engineered to release bioactive agents in a controlled manner, promoting osteogenesis in the electrically active scaffolds/materials, providing mechanical support and electrical cues that boost bone regeneration [135].
The periosteum is vital for bone healing, and based on previous studies, it thickens within 24 h after the occurrence of a bone injury. Moreover, it is known that the periosteum contributes to the initiation of endochondral osteogenesis [136]. This tissue is rich in nerve, and the sensory nerves throughout the periosteum can promote osteogenesis through the Calcitonin gene-related peptide secretion, the release of phosphorus, etc. [137]. A recent interesting study has developed an electrically activated platform to promote neurogenic bone regeneration using BP nanosheets. A fibrous core-shell structure composed of poly-caprolactone (core) and decellularized nerve matrix biomimetic periosteum (shell) was fabricated using a coaxial electrospinning technique. Next, the BP nanosheets were deposited on the surface of the shell through electrostatic interactions. The shell layer provided the proper extracellular matrix, and the nanosheets could induce an endogenous electric field that facilitates bone regeneration through the sensory nerve (Fig. 2(I)). Periosteal mesenchymal stem cells were exposed to the samples to detect their osteogenic transformation upon being stimulated by the electricity in vitro; the bone marrow stem cells were cultured with sensory neurons’ extracellular fluid. The ALP of BP-loaded samples was 5.29 times higher than that of non-loaded ones, and also the osteogenic-related mRNAs were assessed using RT-qPCR, which revealed that the expression of OPN, OCN, Col1, and RUNX2 was significantly higher for the BP-loaded samples. The efficacy of scaffolds to repair skull defects was determined in vivo (Fig. 2(II)). Using microcomputed tomography, the extent of tissue repair was assessed, and in order to destroy the sensory nerves, capsaicin was applied. The results indicated a better healing efficacy of the BP-loaded samples compared to the non-loaded ones; the defect size reduced by 93.36 % after 8 weeks, whereas the PCL alone could reach 22.19 %. The scaffolds were then applied in vivo in the skulls of mice to determine the neurogenic and osteogenic effects through tissue sectioning and immunofluorescence staining. It was found that in regions with greater nerve aggregation, the degree of osteogenesis was elevated, and the expression of NF200 and OCN was remarkably higher in the BP-loaded scaffolds (Fig. 2(III)) [138]. Although applying external electrical stimulation to electrically responsive biomaterials has been shown to accelerate bone regeneration effectively, the size and weight of the device limit the procedure, underscoring the need for alternative approaches [132,139]. A recent study has developed a self-powered electric pulse stimulator that does not require batteries and circuits to address this issue (Fig. 3). This system consisted of two parts: a flexible piezoelectric nanogenerator and an injectable hydrogel consisting of PDA-coated BP nanosheets and alginate methacryloyl. The addition of PDA-BP nanocomposites increased the viscosity and mechanical properties of the injectable hydrogel while also imparting it with good electrical conductivity. In vitro studies on bone marrow stem cells showed that the addition of BP nanocomposite did not prevent cell growth and was highly biocompatible. Upon electrical stimulation, the conductive hydrogel was found to promote cell proliferation. Using Transwell plates, the migration of stem cells was examined with and without electrical stimulation. Exposing the plates to a homemade electrical stimulation system for 1 h indicated an improvement in cell migration in the stimulated hydrogel. The performance of the platform in promoting bone regeneration was tested in vivo, and the surgical images show how the platform was implanted in rats. Using platinum wires incorporated into the hydrogel, the nanogenerator placed in front of the knee joint was connected to the femoral defect. After two weeks, the microcomputed tomography images confirmed that the platform was positioned stably without displacement. The platform could efficiently generate biphasic electric pulse signals from knee joint movements. Both the quantitative results and the microcomputed tomography images demonstrated significantly better bone regeneration performance (higher bone density, bone volume-to-total volume ratio, trabecular number, and thickness) in the platform with electrical stimulation compared to the other groups. The histological analysis revealed more newly formed bone and a more even distribution of collagen in the defects treated with the platform and electrical stimulation [133].
Fig. 2.
Neurogenic bone regeneration through a core-shell fibrous structure coated with BP nanosheets. (I) A schematic illustration showing the potential of a surface-modified fibrous scaffold composed of poly-caprolactone (core), decellularized nerve matrix biomimetic periosteum (shell), and BP nanosheets for neurogenic bone regeneration. (II) Implantation of scaffolds in vivo. (A,B) Micro-computed tomography related to periosteal bone defects, 8 weeks of treatment with four samples after denervation. (C,D) Micro-computed tomography of the cross-section of the treated bone defects after denervation. (E,F) The 3D images taken from microcomputed tomography were used to reconstruct defects in different samples. (G,H) Proportion of defect area plus the BV/TV analysis. (III) Immunofluorescence analysis of the defect area treated with poly-caprolactone and the BP-loaded scaffold. Abbreviations: Poly-caprolactone: PCL, poly-caprolactone-decellularized nerve matrix biomimetic periosteum: PD, and black phosphorus: BP. Reprinted from Ref. [138] with permission from Wiley.
Fig. 3.
Design and fabrication of an electric-stimuli platform consisting of a nanogenerator and a conductive hydrogel made of PDA-coated BP nanosheets for the purpose of bone tissue regeneration. (On the left side) (A,B) The process of preparing a conductive hydrogel that contains PDA-coated BP nanosheets. (C) SEM image of the UV-cured hydrogel displaying a porous design. (D) Evaluation of the electrical conductivity of the hydrogels. (E) The stimulation of bone marrow stem cells using the electric-stimuli platform. (F) Live/dead staining results of the cells treated with various samples (scale bar: 300 μm). (G) The viability of stem cells grown on platforms with and without electrical stimulation. (H) Evaluation of cell migration using a Transwell assay (scale bar: 200 μm). (I) The quantitative findings from the migration assay. (J) Immunofluorescence staining results for cells, showcasing nuclei (blue), vinculin focal points (green), and cellular F-actin (red) (scale bar: 200 and 40 μm). (K) The count of focal adhesions. (On the right side) (A) Surgical photos depicting the implantation of the platform. (B) 3D microcomputed tomography images confirming the stable placement of the HTP-NG after a two-week period. (C) Analysis of the distribution of electrical potential within the platform using finite element modeling. (D) Measurement of the voltage output from the implanted platform when the rat walked at a speed of 1 km per hour, two weeks post-surgery. (E) Visualization of bone regeneration following treatment with various samples through 3D, sagittal, and transverse images. (F–I) Quantitative assessment of the images concerning bone tissue regeneration. (J) Histological examination related to the femurs of rats treated with distinct samples (scale bar: 500 μm). Statistical significance indicated as: ***P < 0.001, **P < 0.01, and *P < 0.05. Abbreviations: Black phosphorus: BP, polydopamine: PDA, cross-linked extracellular matrix-alginate methacrylate-black phosphorus-polydopamine: EABP, and electrical stimulation: ES. Reproduced under the terms of the Creative Commons Attribution License [133]. Copyright 2025, Nature.
4.3. BP-mediated immunomodulation in bone regeneration
Upon implantation of a biomaterial into a bone defect, immune cells actively migrate to the site of implantation, initiating a localized inflammatory response. In response to injury, infection, or introduction of a foreign substance to the body, inflammation takes place to eliminate harmful agents, clear damaged tissue, and initiate the healing process [140]. If the inflammatory response is mild, the macrophages secrete numerous cytokines, leading to an anti-inflammatory response and the activation of somatic cells to promote wound healing. However, if the inflammatory response becomes excessive or chronic, it leads to complications such as impaired healing, decreased efficacy of the biomaterial, and eventually implant failure [141]. It has been observed that adequate polarization of macrophages towards the M2 phenotype leads to the creation of an osteoimmune-friendly environment in which the stimulation of pro-osteogenic growth factors is promoted. In this regard, a wide variety of techniques have been adopted to achieve such an environment for bone implants, among which alteration in the surface topography and wettability, using immune-regulative ions (e.g., Cu2+, Ca2+, Sr2+, etc.), cytokines, and growth factors can be enumerated [[142], [143], [144], [145], [146], [147]].
One of the earliest reports on the intrinsic immunomodulation effects of BP was published in 2023 [115], where a 3D-printed scaffold composed of polycaprolactone was designed and BP nanosheets were loaded in the scaffold. The incorporation of BP in the scaffold was found to significantly accelerate the bone regeneration rate compared to the unloaded scaffold. Deeper investigations revealed that the BP-loaded scaffold created an osteogenic immunological environment in which the expression of pro-inflammatory factors was promoted, and the expression of anti-inflammatory factors such as interleukin-10 (IL-10) and insulin-like growth factor-1 (IGF-1) was enhanced in the subsequent stages. Looking at the BP's potential to manipulate the immunomodulatory behavior of scaffolds revealed that BP nanosheets stimulate the expression of interleukin-33 (IL-33) through macrophages, which amplifies the inflammatory response at early stages while suppressing inflammation later. The expression of IL-33 directly affects the bone marrow stem cells and promotes their osteogenic differentiation, facilitating bone healing. In vitro experiments, macrophages were exposed to BP at various concentrations for 24 h. BP concentrations up to 32 μg mL−1 were found to inhibit macrophage activity, whereas concentrations below 16 μg mL−1 were safe. BP at 5 μg mL−1 significantly stimulated the expression of IL-33, whereas higher concentrations had a negative effect on its expression. Besides its immunomodulating effect, the in vitro bone regeneration potential of IL-33 was evaluated in comparison to bone marrow stem cells. Exposing the cells to IL-33 at a concentration of 50 ng mL−1 resulted in an improvement in ALP, BMP-2, COL I, and OPN after 7 days. This study has further assessed this effect in vivo by experimenting on IL-33 knockout mice and their wild-type littermates. Micro-computed tomography images showed that the lack of this type of interleukin resulted in thinning of the bone cortex and sparse bone trabeculae, whereas its presence stimulated the formation of bone mass and differentiation of stem cells towards osteogenesis [104]. A BP-loaded PLGA scaffold was fabricated through 3D printing to assess its osteo-immunomodulation and osteogenesis. As macrophages play a decisive role in the inflammatory response and tissue remodeling, they were exposed to the scaffolds in vitro to investigate the potential for manipulating the polarization of macrophages. The RAW264.7 cells were cultured with the BP-loaded scaffolds, and after 24 h, it was found that the expression of CD86, a surface marker that is strongly expressed in M1-type macrophages, was reduced significantly in comparison with other groups. On the other hand, the expression of CD163, a surface marker that is strongly expressed in M2-type macrophages, was increased significantly. The upregulation of M2-type macrophage gene markers in the presence of BP-loaded scaffolds was assessed on IL-4-treated RAW264.7 cells after 12 and 24 h; it turned out that the mRNA levels of those markers, including CD206, TGF-β, IL-10, and Arg-1, were elevated. The obtained results indicated the capability of BP-loaded scaffolds to recruit macrophages and alleviate inflammation via altering their phenotype towards M2 to promote bone regeneration. To support the in vitro studies related to the immunomodulatory effects of the BP-loaded scaffolds, the scaffolds' immunomodulatory effects were tested on the SAON rat model. The macrophages isolated from rat femoral slices were immunostained with CD86 and CD163 antibodies to identify M1 and M2 macrophage phenotypes, respectively. The unloaded and BP-loaded PLGA scaffolds were observed to recruit M2 macrophages, and the expression levels of CD86 were lower for the BP-loaded scaffold than for the control and the PLGA scaffold. The immunofluorescence results confirmed that the BP-loaded PLGA scaffold could alter the macrophage phenotype from M1 to M2 [103]. Elsewhere, BP nanosheets were first surface-modified with Mg2+-TA, then incorporated into an SF hydrogel for immunomodulation, angiogenesis, and osteogenesis. The in vitro immunomodulatory ability of the hydrogels was evaluated using RAW264.7 cells. The levels of inflammatory cytokines were significantly higher in the control and unloaded hydrogels. On the contrary, the anti-inflammatory cytokines were secreted more by the BP-incorporated hydrogels, showing BP's anti-inflammation effect on macrophage phenotypic switching. Critical-sized defects were induced on the SD rats' calvarial bone, and the hydrogels were injected into the defects, followed by irradiation to form a gel. IL-10, a marker of M2 macrophages, increased significantly after application of the hydrogel. The expression of M1 and M2 macrophage markers, including iNOS and CD206, was assessed, respectively, and the BP-incorporated hydrogel altered the M1 phenotype into M2 [148]. BPQDs-modified adipose-derived mesenchymal stem cells were developed to address an inflammatory environment in the alveolar bone defect and promote regeneration. Using the paracrine pathway, the platform was found to manipulate macrophage polarization towards an M2 phenotype in vivo during periodontitis. Further studies on the osteogenic potential of the platform revealed that the BPQDs promoted and accelerated the osteogenic rate through different signaling pathways—Wnt/β-catenin and bone morphogenic protein-2/SMAD5/Runx [105]. Another strategy to apply immunomodulatory effects on bone tissue is to adopt immunomodulatory cytokines. A recent study took advantage of this approach and developed a 3D-printed scaffold composed of PCL and poly (caprolactone fumarate). Followed by the fabrication of scaffolds, they underwent surface modifications with graphene oxide nanosheets, BP nanosheets, and a combination of both to achieve a hetero-nanostructure. Next, IL-4 cytokines were loaded onto the surface-modified scaffolds to modulate the polarization of macrophages towards the M2 phenotype and establish a healthy microenvironment for bone regeneration. The idea behind applying graphene oxide and BP nanosheets was to enhance cell attachment and provide a continuous release of phosphate ions, thereby promoting cell proliferation and osteogenesis. Besides those properties, the nanosheets helped to increase the loading efficiency of cytokines on the 3D-printed scaffolds (Fig. 4(I)). For the in vitro immunomodulation assessment, four groups of scaffolds were prepared, including unmodified, BP-coated, graphene oxide-coated, and BP-graphene oxide-coated samples. All samples were soaked in rat interleukin-4 solution to allow cytokine adsorption. Compared to unmodified and BP-modified scaffolds, the loading efficiency of cytokines on the heterostructure (BP and graphene oxide) and graphene-oxide-coated scaffolds was higher. Speaking of the release study, it was evident that the same samples that could load more cytokines also exhibited a more controlled release rate, whereas the unmodified and BP-coated ones showed a burst release in the first two days, followed by a plateau up to 10 days. The adhesion of bone marrow-derived macrophages on different scaffolds was tested, and all the samples exhibited higher attachment of macrophages on the surface than on the unmodified scaffolds. Notably, the heterostructure modified with BP and graphene oxide showed the highest attraction of macrophages, indicating its potential for macrophage adhesion and proliferation. The change in macrophage phenotype was assessed in vitro upon exposure to different scaffolds; after culturing with macrophages, the scaffolds were immunolabeled with CD206 and CD68, markers of the M2 phenotype. Analysis of all immune-modified scaffolds revealed CD206+ macrophages; however, the heterostructured scaffold outperformed in terms of the CD206+ macrophage ratio and achieved the most potent macrophage phenotype polarization among all scaffolds (Fig. 4(II)). Various scaffolds were implanted into a rat calvarial bone defect for up to 4 weeks to determine if they can support bone repair. Micro-computed tomography images showed that the 3D-printed scaffolds implanted induced notable new bone formation, whereas the empty control exhibited minimal new bone formation. The scaffolds modified with both BP and graphene oxide, plus loaded with IL-4, could significantly promote bone regeneration, and the defect was largely filled with new bone (Fig. 4(III)) [106].
Fig. 4.
Osteo-immune-modulation using a surface-modified 3D-printed scaffold loaded with interleukin-4. (I) (a,b) Schematic illustration of immune modulation using 3D-printed surface-modified scaffolds with BP, graphene oxide, and the combination of both on which interleukin-4 was loaded. (II) (a) A scheme related to the loading of cytokines on the scaffolds. (b,c) Loading efficiency and release kinetics of cytokines. (d) Flow cytometry analysis of the phenotype of macrophages after being stained with CD68 and CD206. (e) iNOS as the marker of M1 phenotype. (f) CD206 is the marker of the M2 phenotype. (g,h) Quantitative analysis related to iNOS+ and the CD206+ (*: P < 0.05). (III) (a) Micro-computed tomography images of bone defects treated with different samples for up to 4 weeks. (b–d) New bone area, BV/TV ratio, and bone mineral density in the bone defects (*: P < 0.05). Reproduced under the terms of the Creative Commons Attribution License [106]. Copyright 2024, KeAi Communications Co.
4.4. BP as a multifunctional delivery platform for osteogenic bioagents
The delivery of various bioactive agents (e.g., drugs, ions, genes) to bone through different systems is of great importance as it induces therapeutic outcomes and promotes healing and regeneration. These delivery platforms can load or encapsulate bioactive moieties in high quantities and enable precise control over the release rate to stimulate osteogenesis, promote vascularization, and modulate inflammation [149]. BP nanosheets have a high surface area, making them suitable candidates for delivery applications. Moreover, upon BP degradation, phosphate groups are released, thereby improving osteogenesis [31]. Such strategies are able to overcome the limitations of traditional approaches through mimicking natural healing processes, which offer enhanced outcomes in bone regeneration, especially in complex and critical-sized defects [150].
4.4.1. Ion delivery
Ions such as Mg2+, Sr2+, Ca2+, etc. can directly influence cellular processes involved in bone healing. These bioactive ions increase biomineralization, stimulate osteoblast proliferation, modulate osteoclast activity, and promote vascularization [151]. The controlled release of these ions from biomaterials and/or scaffolds provides localized therapeutic effects, stimulating targeted regeneration and addressing the challenges associated with delayed healing [152]. Strontium is a trace element with a strong potential for inducing osteoblast differentiation and bone formation while reducing the activity of osteoclast cells. One commercially available strontium-related product is strontium ranelate (PROTELOS®), approved in Europe to treat osteoporosis-related diseases [[153], [154], [155]]. Strontium has been incorporated into different substrates to achieve a long-term steady release over time for bone regeneration, but the regulation of strontium release from those compounds remains a challenge [156,157]. Through the incorporation of strontium chloride and BP nanosheets into PLGA microspheres, a stimuli-responsive carrier for bone tissue regeneration was designed. To assess if strontium release can be triggered by laser light irradiation, the samples were exposed to NIR (808 nm, 1 W cm−1) for different periods of time. Since the glass transition temperature of PLGA is 45 °C, laser irradiation stimulated the encapsulated BP nanosheets, leading to an increase in temperature beyond the threshold and resulting in the collapse and formation of cracks in the PLGA microspheres, as well as faster strontium release. Using an inductively coupled plasma, the concentration of released strontium from the microspheres after NIR was determined, and the concentration was found in the range of 10.72–18.94 mg mL−1 up to 28 days. The release rate of strontium without NIR irradiation and BP nanosheets was also assessed; interestingly, even without NIR, the incorporation of BP significantly increased the ion's release rate, which was attributed to the BP's degradation into PO43−, accelerating the degradation of PLGA. The samples were tested in terms of cell viability against bone marrow-derived stem cells, and all of them showed excellent cell viability. The samples were then implanted into rat femoral defects for 8 weeks to assess the bone regeneration capability. Thanks to the strontium release, the new bone formation was significantly higher for the PLGA-SrCl2 than for the PLGA. However, compared to all groups, the PLGA-SrCl2-BP stimulated by NIR could form greater new bone formation [71]. In order to achieve simultaneous angiogenesis and neurogenesis in bone repair, a periosteum-stimulating bilayer hydrogel was designed and developed. In this regard, BP nanosheets were modified with magnesium ions to not only stabilize the nanosheets but also provide a sustained release of magnesium over time. The bilayer hydrogel was composed of an upper and a bottom structure; magnesium-modified BP was incorporated into gelatin methacryloyl hydrogel to form the upper side, and the bottom side was comprised of a double-network hydrogel system consisting of gelatin methacryloyl, polyethylene glycol diacrylate, and β-tricalcium phosphate. The upper hydrogel served as a bionic periosteal structure, stimulating angiogenesis through its compounds, which induced and facilitated endothelial cell migration and upregulated the expression of nerve-related proteins in neural stem cells. On the other side, the bottom side promoted bone marrow mesenchymal stem cells and induced osteogenic differentiation. Different hydrogels were applied in vivo in rat skull defects for up to 12 weeks. Those hydrogels were the unmodified bilayer hydrogel, β-tricalcium phosphate-, pristine BP-, and magnesium-modified BP-incorporated bilayer hydrogels. The results demonstrated that the BP-incorporated ones had a greater capacity to form new bone tissue. However, the magnesium-modified one could almost fill the whole defect with newly formed bone due to early vascularization and neurogenesis, whereas the rest failed to do so [20].
4.4.2. Gene/growth factor delivery
Thanks to the high surface area and functional chemical groups of BP nanosheets, they can potentially bind macromolecular DNA strands to deliver them effectively, followed by controlling the release rate [158,159]. A dynamic DNA hydrogel was engineered with vascular endothelial growth factor (VEGF)-decorated BP nanosheets, followed by integration into a 3D-printed scaffold for improved vascularized bone regeneration (Fig. 5(A)). VEGF is known to degrade in physiological media and lose its bioactivity easily. Adopting biomaterials as carriers for these growth factors can preserve their activity and provide a sustained release in the targeted tissue. The growth factor-decorated BP nanosheets were embedded in the physical network of DNA and then incorporated into PCL scaffolds to form a gel-scaffold structure. Different samples were produced with and without BP to better understand the effect of BP on the functionality of growth factors. The release of growth factors was monitored for 6 days; a burst release was visible for the scaffold without BP and this sample almost released over 95 % of the growth factors on the 6th day, whereas the one with BP had a much slower release and achieved 60 % at the same time interval demonstrating the effect of BP on the long-term retention of growth factors. To determine whether the encapsulated growth factors have retained their bioactivity in the scaffolds, they were exposed to human umbilical vein endothelial cells to assess their effect on migration. The wounded area was found to be almost healed after 12 h of exposure to growth factor-decorated BP scaffolds, whereas the rest required more time to fill the scratch. Moreover, the tube-forming ability of cells on different samples was tested, and, again, the growth factor-decorated BP scaffolds significantly promoted in vitro tube formation. The scaffolds were then implanted in vivo up to 8 weeks. Generally speaking, the bone tissue started to grow from the edge to the center of the defect along the direction of the scaffolds’ filaments, and the highest bone formation percentage belonged to the growth factor-decorated BP scaffolds [80]. Another double-network hydrogel was fabricated via an air-in-water emulsion template. This hydrogel consisted of a double physical/chemical crosslinking network (gelatin methacrylate and DNA) with an interconnected microporous structure to provide and facilitate cell infiltration and tissue ingrowth (Fig. 5(B)). The Apt19S nucleic acid aptamer was anchored to the hydrogel network, which has a high affinity towards stem cells and can recruit them. BP nanosheets were also incorporated into the hydrogel to regulate osteogenesis and enhance mineralization, owing to their high affinity for calcium ions, thereby promoting the formation of calcium phosphate. Since the recruitment of mesenchymal stem cells to the bone defect area is of great importance, the efficiency of an anchored aptamer in attracting stem cells was tested in vitro. Exposing hydrogels to different cell lines, including L929, bone marrow mesenchymal stem cells, and RAW264.7, showed that the aptamers selectively bound to the stem cells. The aptamer-functionalized hydrogel was applied in vivo in a cranial defect of Sprague-Dawley rats. Specific antigens, such as CD44, CD45, and CD29, were identified using flow cytometry to determine the percentage of bone marrow mesenchymal stem cells in the bone defect. It was found that the cells expressing both CD44 and CD29 were nearly 6.6 % at the site of action treated with aptamer-functionalized BP hydrogel, and this was significantly higher compared to other study groups. Moreover, the percentage of recruited stem cells with negative expression of CD45 was approximately 45 % demonstrating the high capability of aptamer-functionalized BP hydrogel to recruit more stem cells than other samples in the bone defect [160].
Fig. 5.
Bioactive agents-modified BP-based platforms for bone tissue regeneration. (A) The integration of VEGF-decorated BP nanosheets into a DNA pre-gel, followed by being incorporated into a 3D-printed PCL scaffold, to induce vascularization and bone regeneration. Reprinted in accordance with the Creative Commons Attribution License [161]. Copyright 2024, KeAi Communications Co. (B) Schematic illustration of step-by-step (I) preparation of a double hydrogel network composed of DNA and gelatin methacrylate, followed by (II) imparting BP nanosheets and an aptamer to recruit and differentiate mesenchymal stem cells for accelerated bone regeneration (III). Reproduced under the terms of the Creative Commons Attribution License [160]. Copyright 2024, Wiley.
4.5. Antibacterial and infection-preventive BP platforms
As society continues to age, a growing number of elderly individuals are prone to degenerative conditions such as bone fractures, osteoporosis, etc. This has led to a significant rise in the use of biomedical implants for orthopedic procedures. Millions of these implants are inserted into patients worldwide annually, placing a considerable economic and psychological burden on healthcare systems [162]. Although these implantable devices are beneficial, there is a significant risk of infection during and after surgery. Bacteria tend to accumulate on implant surfaces, forming biofilms within a matrix of extracellular polymeric substances. Elimination of these biofilms is a difficult task to achieve due to both the protective nature of the matrix and the developing resistance of bacteria to different antibiotics [163]. Failure to treat these bacterial infections may result in chronic infection necessitating implant replacement, which exacerbates patient suffering and healthcare costs [164]. Therefore, the development of innovative and effective antibacterial/antimicrobial strategies to impede implant-related infections becomes critically important in the field.
4.5.1. BP-mediated PTT and PDT antibacterial activity in bone
Different strategies have been adopted to prevent bacterial infection of bone implants, including the application of antibacterial surface modifications, antibiotic loading, and hyperthermia [[165], [166], [167], [168], [169], [170], [171]]. The first method is among the most widely studied approaches, involving the application of a wide variety of antibacterial nanomaterials, including zinc, silver, and copper, in different forms to the implant's surface via various techniques. This method has shown promising results; however, it remains a passive approach, often lacking sufficient potency against different pathogens and carrying a high risk of cytotoxicity due to the release of metal ions at the defect site [172,173]. In contrast, exploiting stimuli-responsive coatings via NIR laser irradiation to generate ROS and heat has shown great potential. This approach is active and can be repeatedly applied until the bacteria are completely eradicated [174,175]. Thanks to its excellent optoelectronic and photosensitive properties, BP can serve as an efficient photosensitizer, producing ROS and heat upon NIR laser irradiation [176]. A multifunctional bone scaffold was developed to achieve sequential PTT, enabling antibacterial activity at early stages (<50 °C) followed by bone regeneration at mild hyperthermic conditions (40–42 °C). BP nanosheets were coordinated with a zinc sulfonate ligand, which was subsequently integrated onto the surface of an HA scaffold. In this system, BP nanosheets act as efficient photothermal agents, while the zinc sulfonate ligand enhances the thermal sensitivity of peri-implant bacteria by inducing envelope stress, thereby improving antibacterial efficacy at mild temperatures. The moderate photothermal environment ensures biosafety while the sustained release of Zn2+ and PO43− ions from the scaffold further promotes osteogenesis during the later stages of bone healing [177]. Another study reported an interesting surface modification of metallic implants with a nanocomposite consisting of PDA-coated BP nanosheets and zinc oxide nanowires. Both the PTT and the release of zinc ions exerted a more effective bactericidal effect against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) biofilms. The step-by-step coating process and the surface topography at each stage are indicated in Fig. 6(I (a,b)). After PDA deposition, the titanium surface remained smooth, and the polymer significantly improved the wear resistance. The coating has two advantages: improved adhesion at the interface of the implant and the host tissue, and it also provides functional groups, such as amine and catechol, which facilitate the deposition of BP and zinc oxide nanowires on the titanium. The deposition of inorganic nanomaterials adjacent to the PDA resulted in numerous granular and sheet-like structures attributed to BP nanosheets. The light-responsivity behavior of different coatings was assessed through 5 min exposure to NIR (808 nm, 0.5 W cm−2) (Fig. 6(II(a-d)); the bare titanium implant was found to increase the temperature up to 35.5C, whereas the PDA- and PDA-zinc oxide-coated ones raised the temperature to nearly 45C, thanks to the photothermal conversion of PDA. The BP-deposited sample outperformed the rest and reached nearly 50C, stemming from the double photothermal conversion effect (BP and PDA). Notably, the addition of zinc oxide did not have a significant effect on the PTT of the implants [116]. Since zinc is known to have antibacterial properties, the release of zinc ions from the surface-modified implants was measured after soaking in phosphate-buffered saline. The implant was observed to release zinc ions in a sustained manner up to 21 days, and the concentration reached nearly 5 ppm. It has been reported that 0.65 ppm is the minimum concentration at which zinc induces a bactericidal effect [178]. Interestingly, the release of zinc accelerated when NIR laser irradiation was applied. The antibacterial capability of different samples was assessed in vitro, both with and without NIR, against S. aureus and E. coli. In the absence of NIR, the antibacterial efficiency was found to be lower than 40 %. However, when the NIR laser was applied, a significant increase in the antibacterial activity of PDA-coated implants was observed, reaching 70 %. Normally, antibacterial efficiency below 90 % is insufficient, as the bacteria can quickly grow back. The combination of BP and PDA-coated implants can achieve more than 98 % efficiency against both pathogens in the presence of the NIR laser, as shown in Fig. 6(III) [116]. Importantly, these BP-coated implants were also assessed for cytotoxicity. In vitro studies consistently showed that osteoblasts and fibroblasts remained viable, and that controlled photothermal conditions did not impair cell proliferation or the expression of osteogenic markers. Likewise, the ZnO nanostructures exhibited low cytotoxicity, and the PDA/BP/ZnO coatings showed excellent cytocompatibility under NIR irradiation, likely due to the mild photothermal effect, the intrinsic biocompatibility of BP, and the hydrophilic PDA layer enriched with polar functional groups that promote cell attachment and spreading. In vivo studies further corroborated these findings. Rats in all treatment groups exhibited steady weight gain throughout a 28-day study period, and histological examination of major organs revealed no signs of tissue damage or pathological abnormalities [116].
Fig. 6.
Applying a nanocomposite consisting of BP nanosheets on titanium against bacteria-associated infections in bone tissue engineering. (I) (a) Step-by-step surface modification of the implants with PDA, PDA-BP, and PDA-BP-ZnO. (b) SEM micrographs of surface-modified titanium implants. (II) Results related to the PTT potential of different surface-modified implants. (a,b) Photothermal images and curves related to the samples. (c) Photothermal curves were obtained after applying the laser with different power densities. (d) Temperature alteration related to the PDA-BP-ZnO-coated implants for 30 min. (e,f) The release curves of zinc ions from the implants with and without NIR irradiation. (III) The bactericidal activity of the surface-modified implants against different pathogens. (a–c) The bactericidal efficiency of different samples with and without NIR. (d,e) Protein leakage from the bacteria treated with the surface-modified implants. (f) Schematic illustration of how the heat generated from the photosensitizers leads to membrane damage and protein leakage to the bacteria. Reprinted from Ref. [116] with permission from Elsevier.
4.5.2. BP-mediated SDT antibacterial activity in bone
Sonodynamic therapy (SDT) is an innovative, noninvasive approach that combines sonosensitizers and low-intensity ultrasound to damage and eradicate tumors or bacteria by inducing sensitized reactions that produce ROS. Compared to light-based therapies such as PTT and PDT, SDT offers significant benefits, including deeper tissue penetration and fewer side effects [179]. Extensive research has highlighted its effectiveness in treating multidrug-resistant bacterial infections, atherosclerotic peripheral artery disease, and cancer [[180], [181], [182]]. Studies have shown that ultrasound can penetrate biological tissues up to 40 mm, nearly five times the 8.5 mm penetration depth of NIR light. Given that orthopedic implants are often encased in both soft and hard tissues, SDT's ability to reach deeper tissue layers makes it a promising strategy for managing implant-related infections [[183], [184], [185]]. A double-layer surface coating of BP-PDA was formed on a titanium implant to facilitate the elimination of bacteria and bone integration. This study has taken advantage of both light- and ultrasound-responsivity of BP against infection. To prepare the coatings, the implant was first homogeneously coated with PDA, followed by immersion in the BP-PDA nanocomposite for the second surface modification. The SEM micrographs of the surface-modified implants confirmed the deposition of a large quantity of BP nanosheets and PD nanoparticles, which were distributed uniformly all over the titanium surface. The photothermal conversion of the samples was determined under an 808 nm NIR laser with various power densities (0.5–1.5 W cm−1). The changes in temperature were monitored up to 10 min; the titanium, PDA-coated, and PDA-BP-coated titanium implants reached 36.5 °C, 42.9 °C, and 48.4 °C, respectively, indicating the effects of PDA and BP on photothermal conversion rate. To assess the SDT potential of samples, electron spin resonance spectroscopy was adopted, which can detect ROS via dimethyl-1-pyrroline N-oxide and 2,2,6,6-tetramethylpiperidine. Upon exposure to ultrasound, a large amount of hydroxyl radicals was detected. Among the samples, only the BP-included coating induced the formation of hydroxyl radicals, while the other two groups failed to do so. S. aureus was employed to test the bactericidal capability of BP-PDA-coated implants with and without NIR and ultrasound irradiation. The live-dead assay, bacterial colony images, and the micrographs obtained by SEM and TEM of bacteria on the implant surface indicated that only the combination of PTT and SDT was strong enough to completely eradicate the bacteria. Notably, the biocompatibility of coated implants was also assessed in vitro, showing that rat bone marrow mesenchymal stem cells remained vital on the coated surfaces and that the application of PTT and SDT had a negligible impact on cell growth. Regarding the bone regeneration potential of the surface-modified implants, a series of biological tests was conducted. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells with and without PTT and SDT was assessed. The samples demonstrated excellent cell compatibility, with late-stage osteogenic proteins, such as osteocalcin and RUNX2, being expressed, along with ALP and mineralization, indicating the high potential of surface-modified samples in vitro. Notably, neither PTT nor SDT weakened the in vitro cell viability and osteogenic differentiation. The in vivo studies were conducted using a rat model with an S. aureus-infected tibia defect. The surface-modified implant, triggered by both PTT and SDT, exhibited an antibacterial efficiency of 96.8 %, comparable to the value of the vancomycin antibiotic powder (98.0 %), which was introduced directly into the defect. The Von Gieson staining revealed that the bone-implant contact ratio of the BP-PDA-coated implants was significantly higher than that of the other groups [34].
4.5.3. BP-enhanced implant/scaffold coatings for antibacterial activity
One of the well-known effective strategies to enhance the efficiency of surface modification on titanium implants is to increase surface roughness and change surface functional groups. There are various pre-coating strategies, spanning from sandblasting to alkalization and electro-plasma oxidation, also known as micro-arc oxidation [169,170,186]. For instance, titanium implants were surface-modified through immersion in NaOH to facilitate the deposition of a nanocomposite of BP, HA, and chitosan, thereby enhancing antibacterial activity and improving osteointegration. The first surface modification in NaOH has resulted in a micro- and nanoporous structure, along with titanium hydroxyl functional groups, both of which improve the efficiency of the second surface coating's deposition. Electrophoretic deposition was employed to deposit the nanocomposite. In this regard, a slurry consisting of HA (10 mg mL−1), chitosan (2 mg mL−1), and BP (0.5 mg mL−1) in deionized water was prepared to carry out the surface coating on the implants. The photothermal conversion was assessed, and the BP-included coating could raise the temperature up to 58.6C, while the rest could reach even 40C. With and without NIR irradiation, the antibacterial activity showed that chitosan indicated a significant increase in antibacterial activity; however, the most effective treatment belonged to the BP coating in the presence of NIR, which nearly eradicated both pathogens. The in vitro cell viability and expression levels of osteogenesis-related proteins indicated that the ternary nanocomposite not only preserved osteoblast viability and functionality, demonstrating that antibacterial performance can be achieved without sacrificing cytocompatibility, but also outperformed the other groups promoting osteoblast differentiation, thanks to the presence of BP and HA [187]. Besides metallic implants for bone repair, polyetheretherketone (PEEK) is widely used as a bone implant material due to several advantages, including excellent biocompatibility, intrinsic radiolucency, and chemical stability. Moreover, PEEK's elastic modulus is similar to that of human cortical bone; however, this polymer is biologically inert, incapable of facilitating osseointegration with the host bone, and is easily colonized by bacteria due to its inert nature [[188], [189], [190]]. To address the bio-inertness of PEEK, a double-surface coating strategy was applied to achieve effective osteogenicity and antibacterial activity. The implants were first sulfonated in sulfuric acid for 5 min and then transferred to an autoclave for hydrothermal treatment at 120C for 6 h to remove residual sulfuric acid. The sulfonated implants were then soaked in a PDA-BP solution for 12 h to form the second surface layer. Eventually, the PDA-BP-coated PEEK was immersed in a solution containing the bioactive short peptide E7 for 24 h to graft the peptide onto the surface. This peptide has the potential to recruit stem cells, thereby improving osseointegration and osteogenesis. A significant improvement in cell attachment, proliferation, and early- and late-stage osteogenic gene expression was observed in cells treated with PDA-BP-E7-coated implants in in vitro studies. The multifunctional coating exhibited excellent photothermal conversion and strong antibacterial activity, thanks to the PTT. The PTT potential of implants was also tested in vivo in a rat femoral infection model, and under NIR irradiation, the multifunctional implant could effectively eliminate bacteria and promote osseointegration [191]. Although PEEK is a high-performance polymer alternative to metal biomaterials, it suffers from disadvantages, such as processing difficulties and low solubility, both of which lead to higher costs. Therefore, there were efforts to find alternatives [[192], [193], [194]]. A recent study has focused on poly (phthalazinone ether nitrile ketone) instead of PEEK, and this material exhibits mechanical properties similar to those of human bone while also being easy to process. To turn the implant into a bioactive substrate, a hydrogel composed of gelatin methacrylate/dopamine methacrylate containing PDA-BP was coated on the surface. Approximately 85 % (E. coli) and more than 70 % (S. aureus) of bacteria were found dead as a result of 10 min exposure to the laser light, and the ROS produced by BP was the dominating reason for the antibacterial activity. Relevantly, no cytotoxicity was observed in vitro in pre-osteoblasts, even at a high concentration (200 μg mL−1), highlighting the biosafety of BP and its potential for antimicrobial activity within a safe therapeutic window. The surface-modified implant was also tested for in vitro and in vivo osteogenicity; histological analysis revealed that the coated implants were more tightly integrated with the surrounding bone tissue, showing the formation of denser, more mature bone, further highlighting its good biocompatibility and bone regenerative ability. Indeed, the PDA was found to consume the excessive ROS and play an antioxidant role, while the BP contributed to the new bone formation by degrading into PO43− [117].
By providing a supportive microenvironment that mimics the extracellular matrix, hydrogels enhance cell adhesion, proliferation, and differentiation in bone tissue regeneration. Moreover, they can deliver bioactive molecules, such as growth factors and osteoinductive factors, that accelerate healing and promote tissue repair/regeneration [195,196]. Building on these properties, an interesting study designed a photosensitive conductive hydrogel for the treatment of infected bone defects. BP nanosheets were modified with magnesium and then incorporated into gelatin methacrylate. The excellent photo-responsivity, including PDT and PTT of the hydrogel, thanks to BP, led to the prevention of continuous deterioration of the bone environment due to bacterial infection (Fig. 7). Using the spread plate method, the antibacterial efficacy of hydrogels was tested in vitro against E. coli and S. aureus. In the absence of NIR irradiation, the hydrogels showed negligible antibacterial activity. However, applying the NIR laser to the hydrogels was synchronized with an antibacterial efficiency of more than 90 % against both pathogens. The promotion of osteogenic differentiation was examined through the expression of different markers (e.g., ALP, mineralization, COL I, RUNX2, and OPN), and the magnesium-modified BP-incorporated hydrogel showed excellent osteogenic activity in vitro. In addition to in vitro investigations, this study has evaluated the antibacterial activity and osteogenic potential of hydrogels in a Sprague-Dawley rat model with an infected cranial defect. Upon applying NIR to the bone defect for 5 min, the local temperature rose to 50C, which was strong enough to eradicate the bacteria. Speaking of bone regeneration, the bone-to-tissue volume ratio in the hydrogels incorporating magnesium-modified BP nanosheets was significantly higher than in other groups after 8 weeks post-implantation [197].
Fig. 7.
Treatment of infected bone using a conductive and photosensitive hydrogel incorporating magnesium-modified BP nanosheets. (I) Schematic illustration of designing the hydrogel and how using PTT gets rid of bacterial infection, and through the release of magnesium and phosphate, promotes bone regeneration. (II) (A–E) photothermal conversion of the hydrogels and antibacterial activity in vitro. (III) (A–C) In vivo experiments including the bone healing potential of the hydrogels in the cranial defect Sprague-Dawley rat model for up to 8 weeks and the related histological analysis (*P < 0.05). Abbreviations: Gelatin methacrylate: GelMa, GelMA-BP: GB, and GelMA-magnesium-BP: GPM. Reprinted from Ref. [197] with permission from Wiley.
5. BP-based platforms for skin repair and regeneration
The skin is regarded as the largest organ of the human body, and it has a crucial role as a protective layer against pathogens, moisture loss, and environmental insult. However, the integrity of the skin is severely compromised through injuries such as infection, diabetic wounds, burns, etc., leading to chronic wounds with prolonged and delayed healing and a high risk of complications [166,180]. For example, diabetic wounds are characterized by excessive oxidative stress, impaired angiogenesis, and chronic inflammation, while burn injuries result from extensive tissue damage, both of which are highly susceptible to infection [3]. Traditional approaches fail to address all the challenges, highlighting the importance of advanced biomaterials capable of promoting rapid healing, immunomodulation, antimicrobial defense, and tissue regeneration [[198], [199], [200]]. BP with unique physicochemical properties such as stimuli-responsivity, high biocompatibility, and degradation into bioactive phosphate ions has shown promising potential for skin tissue engineering. It has been indicated that BP-incorporated biomaterials could accelerate wound closure through modulation of inflammation, stimulating angiogenesis, and improving cell proliferation (Table 3), making these platforms a potential game-changer for treating complex wounds [[201], [202], [203]].
Table 3.
Physicochemical and biological properties of the BP-containing platforms applied in skin therapy and tissue regeneration.
| Composition | Synthesis | Physicochemical properties | Application | Animal model and cycle | In vitro and in vivo | Remarks | Ref. |
|---|---|---|---|---|---|---|---|
| Silk fibroin (SF)-modified BP nanosheets |
|
Particle size: 220 ± 142 nm PTT in vitro: 808 nm NIR at 0.5 W cm−2 for 10 min. PTT in vivo: 808 nm NIR at 1.0 W cm−2 for 5 min. |
Infected wound healing | Wound healing: Full-thickness 5 mm2 infected wound in Kunming mice (E. coli, 1 × 105 CFU mL−1); 5 days. |
|
|
[204] |
| BP-loaded hybrid inverse opal heparin-hyaluronic acid hydrogel microneedles |
|
– | Chronic wound healing | Diabetic wound infection: Full-thickness 1 cm2 dorsal wounds in streptozotocin-induced diabetic Sprague-Dawley rats; 12 days. Scar inhibition: 8 mm full-thickness ear wounds in New Zealand white rabbits; 35 days |
|
|
[83] |
| BP nanosheets-incorporated catechol-modified chitosan and oxidized dextran |
|
– |
Burn wound healing | Wound healing: Full-thickness burn wound infection in ICR mice (E. coli, 1 × 105 CFU mL−1); 21 days |
|
|
[205] |
| 4-octyl itaconate (4-OI)-modified BP injectable hydrogels |
|
BP-PEG: −4.88 mV PTT in vitro: 808 nm NIR at 1 W cm−2 for 5 min. – |
Diabetic skin wound healing | Wound healing: Streptozotocin-induced diabetic full-thickness skin wound model in Sprague-Dawley rats (1.5 cm diameter); 6 weeks |
|
|
[206] |
5.1. BP for chronic wound therapy and tissue regeneration
Diabetes is considered a major medical problem with detrimental effects, often leading to dysfunction of other organs and/or tissues. In recent years, the incidence of diabetes has risen significantly, becoming a substantial burden on society. Among its common complications, diabetic foot ulcers are particularly costly and complicated to treat. It is estimated that nearly 34 % of patients diagnosed with diabetes developed foot ulcers [207]. With limited therapeutic options, around 25 % of these patients face the risk of amputation and long-term disability. Hyperglycemia leads to tissue ischemia, impaired nutrient and oxygen delivery, and delayed wound healing by damaging microvascular endothelial cells and compromising vascular function. This environment is characterized by excessive oxidative stress, prolonged inflammation, and an increased risk of infection, all of which require more functional platforms to address them effectively [3,208]. In this regard, BP, with antibacterial properties, strong photothermal conversion, angiogenic promotion, and immunomodulatory effects, has been extensively adopted for accelerated wound healing [206,209]. 4-OI is a derivative of itaconic acid and has attracted considerable attention as a naturally occurring metabolite capable of inducing antioxidant and anti-inflammatory properties [210]. The anti-inflammatory potential of 4-OI has been widely assessed in various inflammatory diseases, including hepatic injury, acute lung injury, and systemic sclerosis [[211], [212], [213]]. A recent study utilized 4-OI in diabetic wound healing by anchoring it on BP nanosheets and then incorporating it into gelatin methacrylamide hydrogel. Upon applying the NIR laser to the 4-OI-BP-entrapped hydrogel, it underwent rapid gelation and formation of a membrane on the wound. The strong photothermal conversion of BP led to potent PDT and PTT against bacterial infection. In the absence of laser irradiation, BP nanosheets played a carrier role in the hydrogel and took control over the release rate of 4-OI; the release of 4-OI was found to synergistically improve antioxidant activity, followed by alleviation of excessive oxidation damage to endothelial cells, stimulating vascularization, and accelerating diabetic wound closure [206]. Hypoxia is a major challenge in delayed wound healing in diabetes. Chronic hypoxia typically occurs in diabetic wounds, particularly foot ulcers, resulting from poor blood circulation, which increases bacterial infection and slows the healing rate [214]. The main reasons why hypoxia occurs in diabetic wounds are as follows: weak blood flow due to damage to blood vessels by high blood sugar levels, impaired angiogenesis, dysfunctional immune response, and excessive inflammation [[215], [216], [217]]. To address the hypoxic environment of diabetic wounds, a multifunctional wound dressing was fabricated using electrospinning technique. Hemoglobin, as an oxygen carrier, was attached to BP nanosheets, followed by self-assembly with quaternized chitosan onto an electrospun poly-l-lactide nanofiber layer-by-layer. Upon conversion of NIR light irradiation to heat by BP nanosheets, hemoglobin would be stimulated to liberate oxygen in situ in a dynamic on/off mode. Across a broad range of dosages, the degraded by-products released from BP nanosheets upon NIR irradiation exhibited good cytocompatibility. Besides the PTT effect on oxygen release, the generated heat by BP nanosheets increased bacterial susceptibility to quaternized chitosan, resulting in effective antibacterial properties. In diabetes, persistent hypoxia combined with hyperglycemia disrupts the stability and function of HIF-1α. The multifunctional wound dressing effectively reduced HIF-1α expression and improved endothelial cell tube formation under conditions of 1 % O2 and high glucose, simulating the diabetic microenvironment. Furthermore, in vivo studies using a streptozotocin-induced diabetic model demonstrated that, in the presence of NIR, the platform accelerated the wound-healing process [218]. Another strategy to address the complex environment of diabetic wounds is to deliver bioactive moieties that improve tissue regeneration, reduce inflammation, and combat bacterial infections. There are different platforms to load drug molecules/growth factors, among which nanoparticles, hydrogels, and scaffolds can be enumerated; these platforms can provide sustained release of growth factors, antimicrobial agents, and antioxidants over time to reduce inflammation, prevent infection, stimulate angiogenesis, and collagen synthesis [219]. Using self-assembled microspheres, an injectable hydrogel was developed to deliver a growth factor for the treatment of diabetic wounds. The hydrogel was composed of two different microspheres: one containing BP-chitosan methacryloyl and the other containing basic fibroblast growth factor-hyaluronic acid methacryloyl. Since the chitosan-coated microspheres were positively-charged and the counterpart was negatively-charged, there was an electrostatic interaction between the microspheres, forming an annealed particle hydrogel with a hierarchical microporous structure suitable for cell infiltration and being injected in irregular defects (Fig. 8(A)). BP at various concentrations, including 10, 100, and 200 ppm, was incorporated into the hydrogel and exposed to NIR at 808 nm, 1 W cm−2 for 10 min to determine the photothermal conversion. A concentration-dependent behavior was observed, and from the lowest to the highest BP concentration, the temperature increase was 8.2, 23.4, and 37.3 °C, respectively. Notably, the strong photothermal conversion of BP led to an increase in the degradation rate of the hydrogel and faster release of the growth factor. The in vitro cell viability assays revealed that the hydrogel + NIR had suitable viability and stimulated the proliferation of NIH/3T3 and human umbilical vein endothelial cells. The immunomodulatory potential of the hydrogel was tested against RAW 264.7 cells, and the flow cytometry result exhibited that the hydrogel promoted macrophage polarization. The in vivo studies on the control and the hydrogel with and without NIR irradiation up to 14 days demonstrated that the combination of hydrogel with PTT had an average healing area of ∼78 % and ∼93 % after 7 and 14 days, respectively, which was competitively higher than other groups (Fig. 8(B)) [209].
Fig. 8.
Treatment of diabetic wounds using a charge-driven hydrogel. (A) The multifunctional hydrogel is capable of promoting angiogenesis, accelerating wound healing, and modulating inflammation. (B)In vivo experiments in diabetic models with full-thickness skin defects. (a) The treated wounds with different samples, along with a schematic diagram of wounds being healed by the platforms up to 14 days. (c) Histological analysis of the wound tissue sections treated with the platforms after 7 and 14 days (Scale bar = 200 μm); the blue arrows, red arrows, and yellow stars represent the granulation tissue gap, newly formed blood vessels, and hair follicles, respectively. (d–f) Quantitative data related to the relative wound area, granulation tissue gap, and epidermis thickness. *P < 0.05, **P < 0.01. Reprinted from Ref. [209] with permission from Wiley.
5.2. BP systems for infection control and healing acceleration
It is known that over 65 % of chronic wound infections are caused by S. aureus, and the resistant type of this bacterium, known as multidrug-resistant S. aureus, has become a significant clinical challenge with a high risk of mortality for the patients [220]. BP nanoflakes have been assessed in terms of their antimicrobial properties, revealing a direct effect on the bacteria's viability through direct physical contact-driven cell membrane damage and the generation of ROS. Besides these two mechanisms of action, upon exposure to an external NIR laser, the effect was intensified due to PTT and PDT [221]. Few-layered BP nanosheets were directly placed in contact with drug-resistant pathogenic fungi, Cryptococcus neoformans and methicillin-resistant S. aureus, and after 2 h, about 97.9 % and 99.3 % of the pathogens became non-viable, respectively. Notably, the experiment was performed under dark conditions without using NIR [222]. Alongside antimicrobial testing, Shaw et al. also performed cytotoxicity and biocompatibility assessments to ensure that antibacterial potency does not come at the expense of cell safety. Indeed, in this study, BP's effect on human and murine fibroblasts was investigated, demonstrating acceptable biocompatibility at therapeutically relevant concentrations [222]. A recent study has investigated the antibacterial and regenerative properties of bare BP nanoflakes through in vitro studies and infected murine wound models in vivo without the stimulation of NIR irradiation. The BP nanoflakes' size was nearly 850 nm, observed through TEM and SEM, and the average thickness and lateral size were 41 ± 33 nm and 259 ± 179 nm, respectively. To ensure therapeutic relevance, their cytotoxicity was assessed in HaCaT keratinocytes and HFF fibroblasts across concentrations ranging from 312.5 to 5000 μg mL−1. This evaluation revealed a clear safety window, with low and moderate doses showing good cytocompatibility in both cell lines; toxicity was observed only at the highest concentrations, and HaCaTs were more sensitive than HFFs, exhibiting significantly reduced viability above 2500 μg mL−1 [223]. In addition, the in vitro antibacterial activity was assessed against S. aureus (Xen 29 and MRSA) and S. epidermidis, P. aeruginosa, and E. coli; all of them were exposed to the BP nanoflakes at concentrations of 750–1500 μg mL−1 to determine the minimum inhibitory concentration. This concentration was found to be about 750 μg mL−1 for E. coli and S. epidermidis, whereas for S. aureus and P. aeruginosa, this concentration was twofold. The Live-Dead assay showed that S. aureus treated with the BP nanoflakes experienced a 62 % loss of viability within the first 2 h, which increased to 80 % after 6 h, and finally reached approximately 99 % after 24 h. Finally, the efficacy of BP nanoflakes was assessed in a murine S. aureus acute wound infection model at a concentration of 1500 μg mL−1 (a concentration deemed safe in in vitro evaluation) for up to 7 days. Here, topical application of BP nanoflakes resulted in a ∼3-log reduction in bacterial load and accelerated wound closure, re-epithelialization, and reduced inflammation compared to controls [223]. Notably, histological analysis revealed no signs of systemic toxicity, inflammation, necrosis, or BP accumulation in major organs, including the heart, kidney, liver, lung, and spleen, further highlighting their promise as safe and efficient candidates for next-generation wound care and anti-infective therapies.
Although BP has demonstrated suitable antibacterial activity with and without NIR irradiation, some studies have focused on further improving this effect by utilizing well-known inorganic antibacterial agents [[224], [225], [226]]. A research study has developed a multifunctional hydrogel based on BP nanosheets with synergistic antioxidant and antibacterial properties for wound healing applications. To prevent BP's oxidation and endow it with improved antibacterial properties, they chose to deposit metal ions on the nanosheets' surface via metal-ion coordination. Zn2+ ions were surface-coated on the BP, and then both of them were covered by TA based on mussel-inspired chemistry. The BP-Zn2+-TA nanocomposite was then incorporated into a hyaluronic acid-based hydrogel through physical blending to develop a multifunctional hydrogel with antioxidative, antibacterial, photothermal, tissue adhesive, and regenerative properties. The in vitro antibacterial activity was assessed against E. coli and S. aureus on solid agar media; the bare hydrogel showed no obvious inhibition zone after 18 h of co-incubation with the bacteria, while the other groups, specifically the one containing BP-Zn2+-TA nanocomposite, exhibited significant antibacterial capacity. The antibacterial activity was also assessed in the presence of NIR irradiation, where all bacteria treated with BP-incorporated samples were killed after 15 min of laser irradiation, whereas the samples without BP did not exhibit this effect. Using diabetic mice, a bacterial-infected full-thickness skin wound model has been established to assess the antimicrobial and wound-healing potential of samples in vivo. After 3 days of treatment, the BP-incorporated and BP-Zn2+-TA nanocomposite hydrogels showed better results in terms of wound closure, and on day 7, the BP-Zn2+-TA nanocomposite hydrogel could achieve a wound closure ratio of 70 %, whereas the commercial wound dressing (Tegaderm™) could reach 37.6 % [224]. Silver has long been a well-known antimicrobial agent, and its ions can penetrate the bacterial cytoplasm and cytoplasmic membrane, resulting in bacterial death [227]. An injectable hyaluronic acid hydrogel containing BP and silver ions was developed, in which the silver ions formed complexes with thiolate hyaluronic acid and phosphate groups on the surface of BP nanosheets. The antibacterial activity of samples in the presence and absence of the NIR laser was assessed using the disk-diffusion method. Under applied NIR laser (808 nm), the BP-incorporated hydrogel increased the temperature to nearly 45C after 120 s, followed by a plateau up to 400 s, but the temperature was sufficiently high to kill the bacteria (2.6396 ± 0.069 cm2). Without using NIR, the hydrogel containing silver ions formed an inhibition area about 2.2236 ± 0.033 cm2, whereas the BP-included hydrogel reached 2.2766 ± 0.024 cm2, which was significantly higher than the hydrogel without BP, which could be possibly related to the sharp edge of BP, inducing a bactericidal effect. The efficacy of hydrogels was tested in vivo by applying them to a full-thickness infected wound defect with a diameter of 1 cm up to 14 days. Among the samples, only the BP-incorporated hydrogel exposed to NIR could almost recover the infected wound by day 10 (94.17 % ± 60.89 %), while the rest failed to do so even by day 14 [226]. A series of nanofibrous wound dressings consisting of PCL, silver nanoparticles, and BP were electrospun for the treatment of infected wound healing. The idea behind designing such a platform was to capitalize on the release of silver nanoparticles/ions against bacterial biofilms and take advantage of the strong photothermal conversion rate of BP to induce additional antibacterial activity through PTT, thereby facilitating the further delivery of silver from PCL fibers. Besides the anti-infection potential of the platform, the synergistic effects of silver and BP were found to reduce inflammation and promote granulation tissue formation, angiogenesis, and collagen deposition, all of which lead to accelerated wound healing (Fig. 9(I)). The in vitro antibacterial properties and bacterial biofilm ablation of the platforms were assessed against multidrug-resistant S. aureus. The samples used were as follows: PCL (PL-0), PCL/Ag (PL-1), and PCL/Ag/BP (PL-2) fibrous wound dressings. As indicated in Fig. 9(II), in the absence of NIR, the samples inhibited the proliferation bacteria effectively due to the release of silver ions; the sample named PL-3 was the PCL/Ag/BP, which was exposed to NIR irradiation, and it almost eradicated both the multidrug-resistant S. aureus and E. coli, showing the potency of faster silver release and PTT. This sample also eradicated bacterial biofilms, resulting in a significant decrease in the counts of live bacteria. The nanofibrous wound dressings were applied to infected wounds up to 15 days in vivo to check on their antibacterial activity and healing capacity (Fig. 9(III)). The PL-3 could significantly accelerate wound healing; by day 15, the PL-3-treated wound was completely healed. Using LB plates, bacterial counts from wounds treated with each sample were quantified. The highest bacterial disinfection was achieved by PL-3, thanks to the combination of silver release and PTT [225].
Fig. 9.
A bi-functional fibrous wound dressing for infected wound healing. (I) (a,b) Step-by-step preparation and functionality of PCL/Ag/BP electrospun dressing for infection wound healing applications. (II) Antibacterial properties of nanofibrous platforms. (a) The antibacterial mechanism of action of different samples with and without NIR irradiation. (b) The MRSA proliferation rate was treated with different samples (B.C: PBS, P.C: antibiotic, and NIR: laser irradiation). (c) LB dishes showing the survival of bacteria treated with the samples and (d) the inhibition rate. (e) Live/Dead assay of bacteria and (f) the SEM micrographs related to each sample exposed to the bacteria. (g) 3D CSLM images of the bacterial biofilms treated with PL-0, PL-1, PL-2, PL-3, from left to right, respectively. (h) The quantitative analysis of the biofilm treatment. (i,j) Alteration of ROS levels, (k) protein leakage, and (l) cellular ATP of bacteria treated with different samples. *P < 0.05, **P < 0.01, and ***P < 0.001. Reprinted in accordance with the Creative Commons Attribution License [225]. Copyright 2023, Elsevier.
5.3. Externally-triggered BP platforms for wound healing and skin repair
Stimuli-responsive platforms incorporating BP have shown significant potential in enhancing wound healing and skin tissue regeneration through external triggers such as NIR irradiation [228], electrical stimulation [229,230], thermo-sensitive [231], and sonodynamic therapy [232]. One of the critical issues in the wound healing process is the frequent need for dressing changes or removal of hydrogels, which can lead to injury and pain [233]. In this regard, a study has designed a stimulus-responsive platform to address this issue. A new sprayable BP-containing hydrogel was developed with antibacterial, antioxidation, and angiogenesis properties for wound healing. The hydrogel was prepared by mixing BP-incorporated functionalized dextran and oxidized hyaluronic acid, which had been functionalized with dopamine. The hydrogel was found to have suitable adhesiveness due to the aldehyde groups and dopamine in the hydrogel, and also, the catechol groups could alleviate oxidative stress in the wound resulting from long-term inflammation. Applying NIR irradiation in this study had two purposes; once it came to on-demand removal of the hydrogel, the NIR laser could elevate the temperature due to the BP, turning the gel into a sol. The existence of thermally reversible C=C double bonds in the hydrogel's structure enabled such a phenomenon. Another point of NIR irradiation was to take advantage of the PTT potential of incorporated BP for antibacterial activity applications. Nonetheless, vascularization and tissue remodeling were facilitated through encapsulation of VEGF in the hydrogel (Fig. 10(A)). The normalized G′ changes were measured at various temperatures. Histidine solutions were prepared, followed by immersing the hydrogel. Then, the temperature was increased from 50 to 70C, and the G′ decreased. It was observed that this effect accelerated the degradation of the hydrogel till it completely dissolved. Speaking of PTT potential of the BP-incorporated hydrogel, different amounts of BP were added to the hydrogel, spanning from 0.25 to 2 mg mL−1; increasing the BP content was synchronized with the sharp temperature elevation from 46 to 61.3C. To determine the antibacterial activity of the platform using PTT, the hydrogels were exposed to E. coli and S. aureus in the presence of NIR irradiation (5 min, 1 W cm−2). Data revealed that the viability of each bacterium was as follows: E. coli (3.8 %) and S. aureus (3.01 %). However, for the hydrogel without BP and the BP-incorporated hydrogel without NIR laser, the survival rates of both pathogens were above 90 %. To confirm the facile removal of the hydrogel from the wound bed, it was sprayed onto an open wound in vivo, and after 3 min, it turned into a gel. Next, the wound was dressed with gauze immersed in histidine, and in the meantime, the NIR laser was applied. The temperature during this process was preserved below 50C, and after 20 min, they could remove the hydrogel from the wound with a small residue left behind, exhibiting the efficacy of the NIR in the wound healing process [228]. A NIR-responsive composite patch integrating BP nanosheets was designed and fabricated for wet skin adhesion with multifunctional features—hemostasis, anticancer activity, and sensing (Fig. 11(I)). The platform was composed of methacrylate anhydride-modified hyaluronic acid, gelatin, and PVA, in which the BP nanosheets were dispersed. The addition of BP was found to improve the water absorption capacity of the platform. Due to the triple molecular network of the patch and the homogeneously distributed BP nanosheets along the patch, it showed excellent flexibility and strength, and even after water uptake, it could maintain the physicochemical properties. The adhesive capability of the BP-integrated patch was tested on wet porcine skin and wet nude mouse skin, yielding nearly 171 and 252 kPa, respectively. In both wet and dry states, BP-induced photothermal conversion occurred in the patches in response to NIR irradiation. Speaking of anticancer therapy, skin cancer was induced in a nude mouse model, and once the tumors reached a size of nearly 200 mm3, they were removed, and instead, the patches were adhered to the surgical site. Exposure of the adhered patches to 808 nm, 1 W cm−2 laser irradiation for 5 min was synchronized with a temperature increase of up to ∼55 °C. Interestingly, the control and the NIR applied (without the patch) groups experienced tumor recurrence, showing the inefficient effect of NIR alone on the tumor growth, whereas the wounds treated with the patch exposed to the NIR did not experience tumor reemergence within 4 weeks (Fig. 11(II)) [234].
Fig. 10.
Stimuli-responsive BP-containing platforms for skin wound treatment and regeneration. (A) A multifunctional NIR-responsive sprayable hydrogel for wound healing. Reprinted from Ref. [228] with permission from Wiley. (B) Hyaluronic acid-dopamine hydrogel containing BP nanosheets, responsive to external electrical stimulus for antibacterial activity and accelerated wound healing. Reprinted from Ref. [229] with permission from Wiley. (C) Preparing ultrasound-responsive electrospun nanocomposite wound dressing for infected wound healing. (a) Step-step formation of 2D-2D nanostructure of V2C nanoflakes deposited onto BP nanosheets, followed by being incorporated into PLGA nanofibers. (b) The electrospun wound dressing's potential upon being activated by ultrasound irradiation. Reprinted from Ref. [232] with permission from Wiley.
Fig. 11.
A multifunctional nanocomposite patch integrating BP nanosheets for hemostasis, sensing, cancer therapy, and tissue regeneration. (I) The multifunctional nanocomposite patch with strong adherence to the wet tissue indicates that incorporating BP nanosheets improved water absorption, followed by adherence to the skin. (II)In vivo studies related to the anticancer activity of the patches. (a) Treatment schedule up to 28 days. (b) Thermal images and curves related to the patch on the mouse skin exposed to the NIR irradiation for 300 s. (c) Changes in the mice's body weight throughout the experiment, treated with various samples. (d) Changes in the tumor volume after resection treated with different samples. (e) Images obtained before and after surgery related to the mice treated with the samples. (f) Histological analysis of different organs of mice treated with the samples. Abbreviations: S: control, CP: composite patch, CPB: composite patch containing BP nanosheets. Reprinted in accordance with the Creative Commons Attribution License [234]. Copyright 2024, Nature.
Beyond their role in photothermal cancer therapy, such functional biomaterials, especially when engineered to be electroactive, are attracting increasing scientific interest. Recent advances in electroactive biomaterials have opened up promising avenues for not only regulating cellular electrical activity and stimulating tissue regeneration, but also for monitoring cell behavior through bioelectrical signals. Given the high vascularization and innervation of skin tissue, electroactive biomaterials are being actively explored for their potential in promoting skin tissue regeneration [235,236]. Due to the BP's appropriate electrical properties, it was adopted as a conductive agent in a hydrogel that consists of dopamine and hyaluronic acid. The hydrogel was found to have pH-responsivity resulting from the coordination of ferrite ions and catechol groups; transformation in the hydrogel's phase took place upon changing the pH, leading to the smart release of BP in a mild acidic medium of the wound. Upon the release of BP nanosheets, the applied electrical stimulus led to significant antibacterial properties and enhanced wound healing (Fig. 10(B)). The electrical activity of the hydrogels was evaluated in terms of antibacterial activity against E. coli, a model bacterium. Both the control hydrogel without BP and the BP-incorporated hydrogel without electrical stimulation showed weak antibacterial activity after 120 min of co-incubation with bacteria. Interestingly, the electrical stimulation was applied to the bacteria without using any material, and as the stimulation time increased, the antibacterial activity rose from 20 % after 30 min to 50 % after 120 min. The strongest effect was observed with the BP hydrogel, achieving a bactericidal effect of over 90 % after 1 h and nearly 100 % after 2 h. The reason for the stronger effect in this study was attributed to the better electrical conductivity of BP, as well as the ROS produced by BP under the action of electrical stimulation. The potential of this electrically active hydrogel was tested in vivo. The wound that received the BP hydrogel and was exposed to the electrical current showed a significant reduction in size and scab, and the recovery was much faster than the other treated wounds, indicating the dual effects of BP release, which eradicated the bacteria and prevented the infection, and also the electrical activity of BP, promoting the wound healing [229].
Sono-piezodynamic therapy is a non-invasive approach that has recently attracted attention. It leverages ultrasound to activate piezoelectric nanomaterials, leading to the generation of ROS. In cancer therapy, oxidative stress and apoptosis in tumor cells take place as a result of ROS generation. This therapeutic approach offers an oxygen-independent treatment, particularly effective against hypoxic tumors. In antibacterial applications, the formation of ROS disrupts bacterial membranes and inhibits biofilm formation, making sono-piezodynamic therapy a promising alternative to antibiotics and an effective strategy against drug-resistant bacteria [237,238]. To combat drug-resistant infections and stimulate cutaneous regeneration, an electrospun nanocomposite wound dressing comprised of PLGA, BP nanosheets, and V2C was fabricated, which was responsive to ultrasound for sono-piezodynamic therapy. One of the innovative aspects of this study was the formation of a 2D-2D structure through hydrothermal treatment (Fig. 10(C)). The reason for preparing such a structure relates to the improvement in both chemodynamic and sono-piezodynamic capabilities. The 2D V2C nanoflakes were covalently bonded to the surface of BP nanosheets and formed the 2D-2D structure, and then this 2D-2D structure was combined with PLGA through electrospinning for wound healing; there are numerous advantages relating to the integration of this 2D-2D nanocomposite into the scaffold like improved structural stability, sustained release of nanocomposite, biocompatibility, and above them endowing catalytic generation of ROS in response to ultrasound stimulation. Upon applying ultrasound to the platform, the electron transfer improves from the ultrasound-stimulated BP nanosheets to the V2C nanoflakes, facilitating the formation of positively charged holes and negatively charged electrons. Therefore, the generation of ROS is accelerated, leading to effective bacterial sterilization. Once the ultrasound irradiation is removed, the platform begins to scavenge excessive ROS and bacteria, playing an antioxidant role on the wound bed, which decreases the inflammatory response and promotes skin repair. The anti-infection potential of wound dressings was assessed in vitro against S. aureus, E. coli, and drug-resistant E. coli with and without ultrasound irradiation. In the absence of ultrasound, the PLGA fibrous dressing demonstrated nearly zero antibacterial efficacy, and the 2D-2D-incorporated PLGA scaffold exhibited a minimal effect, ranging from 10 % to 17 % against different pathogens. However, following ultrasound irradiation, the antibacterial efficacy was significantly improved, with an outstanding efficiency of nearly 100 %, thanks to sono-piezodynamic therapy. The nanofibrous wound dressings were also applied to a full-thickness infected wound model in vivo for 7 days. The interstitial fluid secreted from the infected wound was collected after ultrasound irradiation and cultured to assess antibacterial efficacy. It was found that the 2D-2D-incorporated PLGA and Amoxicillin-treated groups were the only ones that reduced microbial growth, whereas the rest failed to do so. In terms of tissue regeneration, from day 3, the 2D-2D-incorporated PLGA sample accelerated wound healing, and the wound areas treated with this sample were significantly smaller than the ones treated with other groups [232].
6. BP for neurodegenerative disease therapy and neuro-regeneration
Neurodegenerative disorders represent a diverse and currently untreatable collection of diseases characterized by the gradual deterioration of nerve cells in both the central and peripheral nervous systems. Key examples include PD, AD, ALS, and HD. These conditions are generally linked to the progressive loss of neurons and synaptic connections, ultimately leading to impairments in cognitive function, motor skills, and behavior. Although the precise causes may differ, many neurodegenerative diseases display common pathological characteristics such as protein misfolding and aggregation (for instance, amyloid-β in AD and α-synuclein in PD), mitochondrial dysfunction, oxidative stress, neuroinflammation, and synaptic toxicity [239,240]. Current therapeutic options are predominantly aimed at alleviating symptoms and do not succeed in halting or reversing the progression of the disease, highlighting the critical need for novel treatment strategies that can tackle these intricate and multifaceted disorders at both molecular and systemic levels. In this section, the multifaceted potential of BP nanomaterials in overcoming current barriers to neurodegenerative disease treatment will be explored, with subsequent sections delving into recent advancements and experimental validations of BP-enabled strategies for brain-targeted therapy and nerve regeneration.
6.1. BP nanomaterials as smart delivery systems for efficient BBB penetration
The blood-brain barrier (BBB) acts as a robust, selective permeability barrier, hindering the penetration of harmful substances into the central nervous system (CNS) while facilitating the transport of vital nutrients and ions [241]. This selectivity, however, creates considerable obstacles to delivering therapeutic agents to the brain, especially when addressing neurodegenerative diseases. Within this framework, BP has recently attracted notable interest due to its distinctive physicochemical characteristics and low toxicity [242]. Multiple studies have shown that BP-based nanomaterials can penetrate the BBB in vitro, attributed to intrinsic properties such as size and surface charge [243,244]. A research study by Xie et al. explored the pathways of BP nanosheet absorption in an in vitro BBB model utilizing various endocytosis inhibitors. The results indicated that cerebrovascular endothelial cells primarily internalize BP nanosheets via caveolae- and clathrin-mediated pathways (Fig. 12(I)) [243]. Despite their intrinsic ability to cross the BBB, surface functionalization approaches have been used to facilitate BP-based nanomaterials' cellular uptake and transcytosis across the endothelial cells of the BBB. For instance, BP-based nanocarriers functionalized with lactoferrin [245], a brain-targeting ligand capable of crossing the BBB via receptor-mediated transcytosis in brain capillary endothelial cells [246], showed increased BBB penetration (Fig. 12(II)) [245]. Furthermore, lactoferrin receptor overexpression is often observed in PD patients, making lactoferrin an especially promising targeting moiety for anti-Parkinsonian therapies [245]. BP nanosheets and BPQDs microbubbles were obtained via focused ultrasound, an approach that ensures a high degree of temporal and spatial delivery specificity [247]. However, BBB penetration by BP-based nanomaterials has been more often ensured by NIR irradiation and PTT. Indeed, BP possesses a tunable bandgap that ranges from 0.3 eV (bulk) to 2.0 eV, allowing absorption across the ultraviolet to infrared spectrum [248]. When irradiated with NIR light (typically 700–1000 nm), BP exhibits strong photothermal performance, with great absorption and efficient heat generation due to its broadband optical properties. If such localized heating has been extensively used for several biomedical purposes, including tumor ablation and on-demand drug release as previously discussed, it can also be used to transiently modulate endothelial tight junctions, thus enhancing BBB permeability [249]. Other studies have also demonstrated that NIR treatment may also promote caveolae- and clathrin-mediated endocytosis and uptake, further enhancing BPNs penetration across the BBB [243]. The better permeability of BP nanosheets with the aid of PTT through the BBB was demonstrated both in an in vitro model of the BBB barrier [243,244] and in several in vivo studies [244,245,250]. In vitro, NIR irradiation was employed to enhance BPNs crossing through a bEnd.3 cell monolayer seeded in trans-wells emulating the BBB barrier [243], indicating improved BBB permeability by the photothermal effect of NIR-irradiated BP nanosheets without any negative effect on bEnd.3 viability [244].
Fig. 12.
NIR-induced BP BBB crossing and neuromodulation. (I) Schematic representation showing that BP is able to penetrate the brain parenchyma through intercellular and intracellular pathways. Reprinted from Ref. [243] with permission from Elsevier. (II) An illustration showing BP particles functionalized with lactoferrin successfully traversing the BBB upon NIR irradiation, providing site-specific treatment for Parkinson's disease. Reprinted from Ref. [244] with permission from Wiley. (III) Above, a schematic illustration of BP-flake–mediated neuromodulation for attenuating aberrant electrical activity in epilepsy. Below are the electrophysiological traces of hippocampal neuronal spikes showing reduced epileptic activity following transcranial NIR irradiation. Reprinted from Ref. [251] with permission from the American Chemical Society.
In in vivo studies, to leverage the BP photothermal effect, mice were administered BP nanosheets intravenously, and their heads were then exposed to an 808 nm laser. The PTT potential of BP exposed to laser has been shown using a thermal image camera, whereas Evans blue co-injection was used to demonstrate BBB penetration by BP nanosheets [244,245]. Importantly, no cell or tissue damage was found as assessed with hematoxylin and eosin histological analysis after NIR irradiation [244,245], confirming the safety of mild localized hyperthermia [252]. Evans blue staining also revealed minimal dye accumulation in the brain 48 h post-treatment, indicating that NIR irradiation did not compromise BBB permeability [245]. Relevantly, a recent study has demonstrated BP-enabled NIR neuromodulation, owing to NIR light's deep tissue penetration and precision. In particular, millisecond-scale NIR illumination of neurons induced intercellular calcium signaling with high spatial and temporal resolution, triggered action potentials in hippocampal brain slices, and suppressed epileptic signals in a mouse model of epilepsy [251]. Additionally, by adjusting NIR irradiation parameters, it was possible to achieve frequency-dependent modulation of neural activity, highlighting BP responsiveness to NIR irradiation as a promising and innovative therapeutic avenue for treating hippocampal epilepsy (Fig. 12(III)) [251]. While NIR light enables non-invasive localized heating to enhance BBB permeability of BP-based nanomaterials, excessive or prolonged irradiation could damage vascular structures or trigger inflammatory responses, potentially increasing the risk of cerebral thrombosis [249]. However, the temperature elevation induced by BP-mediated NIR photothermal therapy can be monitored and precisely controlled by adjusting irradiation parameters, making it a versatile, minimally invasive tool for targeted treatment. Furthermore, studies have shown no evidence of thrombosis on MRI scans of mice treated with injected BP nanosheets and NIR irradiation, suggesting a low risk of cerebral thrombosis during treatment [244]. These findings underscore the potential of BP and NIR irradiation as a synergistic approach to transiently modulate the BBB, enabling targeted delivery of therapeutic agents to the brain and holding promise for advancing the treatment of neurodegenerative disorders.
6.2. Targeting proteinopathies with BP to treat neurodegenerative disorders
Neurodegenerative diseases, including AD, PD, HD, ALS, and various prion disorders, represent a growing global health challenge due to their increasing prevalence and lack of curative treatments [253]. A common pathological hallmark among these disorders is the abnormal accumulation and aggregation of misfolded proteins, a phenomenon broadly referred to as proteinopathy. These misfolded proteins—such as amyloid-β (Aβ) and tau in AD, α-synuclein in PD, huntingtin in HD, and TDP-43 in ALS—undergo conformational changes that promote their self-assembly into toxic oligomers and insoluble fibrillar aggregates [239,240,254]. The formation of such aggregates disrupts cellular homeostasis through multiple mechanisms, including impairment of proteostasis, mitochondrial dysfunction, oxidative stress, synaptic toxicity, and neuroinflammation [254]. Several therapeutic strategies have been developed to target proteinopathies in neurodegenerative diseases, aiming to prevent or reverse the aggregation of misfolded proteins and mitigate their toxic effects. These approaches include small molecules that inhibit protein aggregation or promote disaggregation, monoclonal antibodies designed to enhance clearance of pathological proteins via the immune system, and gene-silencing techniques [255]. Additionally, molecular chaperones and proteostasis regulators have been explored to stabilize protein folding and enhance degradation pathways [256]. Despite promising preclinical results, the clinical translation of these therapies has faced significant challenges, often due to poor BBB permeability, off-target effects, or insufficient engagement with pathological aggregates. In this context, recent studies have shown that BP-based nanomaterials have great potential for neuropathies treatment, offering a novel platform for multimodal therapeutic strategies that can address the diverse mechanisms underlying neurodegenerative disease progression. For instance, Liam et al., reported the development of BP nanomaterials—BP nanosheets and quantum dots modified with titanium sulfonate ligand (TiL4) to improve their stability, demonstrating that both nanomaterials were able to inhibit the fibrillization of Aβ40, the major component of the extracellular plaque found in AD, by adsorbing Aβ40 monomers [257] and shedding light on the employment of BPs as novel therapeutic avenues in the prevention or treatment of neurodegenerative diseases. Another study has demonstrated that BP nanosheets can also directly interact with α-syn fibrils, promoting their disaggregation as a potential treatment for PD (Fig. 13(I)) [250]. Specifically, BP nanosheets demonstrate a selective affinity for α-syn through van der Waals interactions and have been shown to activate autophagy. This activity helps regulate α-syn levels, restore mitochondrial function, decrease ROS production, and prevent neuronal death and synaptic loss in PC12 cells. Importantly, BP nanosheets were able to protect dopaminergic neurons in vivo and to alleviate motor impairments in both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mouse models and hA53T α-syn transgenic mice [250], highlighting the potential of BP nanomaterials as multifunctional nanomedicines capable of simultaneously targeting α-syn and ROS species, offering therapeutic benefits for PD.
Fig. 13.
(I) A conceptual overview is presented to illustrate how BP nanosheets exert neuroprotective actions in PD models in both C. elegans and mice. (A) The first panel depicts how BP nanosheets can cross the BBB and reach neural tissues. (B) The second panel outlines their protective influence in a C. elegans model of PD induced by 6-hydroxydopamine (6-OHDA). (C) Under pathological conditions, misfolded α-synuclein aggregates compromise mitochondrial performance and disturb redox balance, creating a cycle that promotes further α-synuclein aggregation and PD progression. Following BP nanosheets administration, these nanosheets help diminish α-synuclein accumulation through two complementary pathways: direct binding and breakdown of aggregated α-synuclein, and activation of autophagy to clear remaining aggregates. By reducing the burden of toxic α-synuclein species, BPNSs help preserve mitochondrial function and maintain oxidative stability, ultimately reducing ongoing α-synuclein misfolding. Reprinted from Ref. [250] with permission from Wiley. (II) The fabrication process and the multifunctional anti-PD actions of the “Swiss Army Knife” nanoplatform based on BP. The photothermal properties of BP enable a temporary, controllable increase in BBB permeability, thereby facilitating greater accumulation of the BP–matrine (MT) system at PD-affected sites. Once delivered to the lesion area, the BP-MT complex provides a coordinated therapeutic response. BP and MT together act as potent ROS scavengers, leading to a cascade of beneficial cellular effects, including reducing mitochondrial oxidative damage, suppressing neuroinflammatory responses, and reducing α-synuclein buildup. These improvements collectively support healthier dopamine metabolism and help restore the pool of functional synaptic vesicles, ultimately contributing to a stronger antiparkinsonian outcome. Reprinted from Ref. [260] with permission from Elsevier. (III) The development and therapeutic potential of β-carotene–modified BPQDs for promoting nerve regeneration in peripheral nerve injury. (A) The first section illustrates how BPQDs are functionalized with β-carotene and subsequently incorporated into a GelMA/PEGDA hydrogel scaffold, creating a composite system (BPQD@β-carotene) suitable for nerve repair applications. (B) The second part showcases the biological benefits of this hybrid material, demonstrating its ability to support Schwann cell–mediated neural regeneration, stimulate the formation of new blood vessels, and modulate inflammatory responses in favor of tissue healing. (C) Implantation of the BPQD@β-carotene–loaded scaffold improves functional recovery in rat and beagle dog peripheral nerve injury models by enhancing axonal remyelination, promoting axonal regrowth, and facilitating robust intraneural angiogenesis. Reprinted from Ref. [261] with permission from the American Chemical Society.
6.3. Drug-loaded BP-based nanomaterials for neurodegenerative disease therapy and nerve repair
In contrast to other 2D materials such as graphene and MoS2, BP features a distinctive puckered lattice structure that provides a much higher surface-to-volume ratio, thereby improving its drug-loading capability [258]. Despite this advantage, the application of BP nanosheets as a drug delivery system for neurodegenerative disease therapy has only recently been explored. Relevantly, BP nanosheets loaded with paeoniflorin (Pae), a well-characterized natural compound with established neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced dopaminergic neurotoxicity in dopaminergic neurons [259] have been recently developed to treat PD [245]. Relevantly, Pae-loading significantly reduced BP oxidation, mitigating the oxidation-induced reductions in absorption values [245], suggesting enhanced stability compared to BP. Notably, these Pae-loaded nanocarriers also displayed excellent photothermal performance upon NIR irradiation and were able to efficiently cross the BBB, improving motor coordination and activity in a MPTP-induced mouse model of PD as assessed using the rotarod, pole, and open-field test. In addition, these BP-loaded nanomaterials were shown to significantly protect from MPTP-induced DA neuronal loss in the substantia nigra, to mitigate MPTP-induced reduction of dopamine and its metabolites, and to promote neuronal antioxidant activity through activation of superoxide dismutase and glutathione, further highlighting the potential of BP-based nanocarriers loaded with Pae to treat PD [245].
Similarly, matrine (MT) loaded BP nanosheets have also been synthesized for PD treatment [260]. MT loading did not interfere with BP photothermal conversion efficiency, as MT-loaded BP nanosheets were able to induce a sufficient thermal effect to ensure BBB crossing. Moreover, the combined antioxidant effects of BP and MT were used to restore redox balance, alleviate neuroinflammation, reduce α-syn protein aggregation, and improve dopamine metabolism and synaptic vesicle availability. MT-loaded BP nanosheets also protected dopaminergic neurons from MPTP-induced toxicity (Fig. 13(II)) and improved motor deficits in a mouse model of PD, indicating its great therapeutic potential [260]. Loaded BP nanosheets have also been developed for the treatment of AD. For instance, BP nanosheets loaded with methylene blue (MB) have been developed to treat AD owing to the ability of MB to inhibit apoptosis and prevent Tau hyperphosphorylation [243]. The unique structure of MB allows it to adsorb onto BP nanosheets via a simple self-assembly process in water, where the positively charged MB interacts electrostatically with the abundant lone pair electrons present on the BP surface. MB loading slightly decreased photothermal conversion efficiency, probably due to partial oxidation during the drug-loading process. Nevertheless, MB-loaded BP nanosheets maintained effective photothermal conversion capabilities and demonstrated potential for photothermal transport across the BBB. Relevantly, these MB-loaded BP nanosheets were able to improve learning and memory deficits in a mouse model of AD, as assessed using the Morris water maze, the novel object recognition, and the paired-associate learning tests [243].
Li et al. have developed BP nanosheets conjugated with the thioflavin-T derivative, 4-(6-methyl-1,3-benzothiazol-2-yl) phenylamine (BTA), which shows a high affinity to Aβ monomers. Modification with BTA resulted in enhanced stability against rapid degradation within hours and endowed the nanoplatform with the ability to oxidize Aβ upon NIR-irradiation through the generation of a high quantum yield of singlet oxygen (1O2), thereby inhibiting Aβ aggregation and cytotoxicity [262]. BP nanosheets have also been used as a drug-delivery system for the antidepressant drug fluoxetine [263]. Here, fluoxetine loading was performed through a self-assembly process as previously, and fluoxetine binding to BP nanosheets was mediated via electrostatic interactions, as confirmed by an increase in zeta potential. Such modification also improved dispersion in water. Furthermore, a slight redshift in absorption was found in fluoxetine-loaded BP nanosheets compared to free fluoxetine, suggesting an interaction between Flu and the BP nanosheets and highlighting the stability of the nanocomposites. Such nanocarriers showed excellent photothermal behavior, resulting in the release of nearly 90 % of the encapsulated drug within 30 min. Relevantly for their future clinical applications, treatment with fluoxetine-loaded BP nanosheets significantly ameliorated depressive-like behaviors in a mouse model of depression as measured by the sucrose preference, the tail suspension, the forced swim tests, and the coat score assay [263]. Furthermore, unlike conventional fluoxetine therapy, drug-loaded BP nanosheets significantly enhanced depressive-like behaviors in a mouse model of depression after only two weeks of treatment by inducing an increase in brain-derived neurotrophic factor production in the hippocampus and reducing neuronal excitability of pyramidal neurons in the amygdala, two important brain regions involved in the pathophysiology of depression. This dual action, by significantly shortening the duration of treatment, may offer a rapid-acting antidepressant approach [263].
Modified BP has also been employed for nerve injury repair applications. For instance, BP nanosheets loaded with stromal cell-derived factor 1 (SDF1-α) have been successfully employed to treat cavernous nerve injury in rats [264]. Similarly, BPQDs functionalized with the antioxidant β-carotene (BPQD@β-carotene) have been employed for peripheral nerve injury repair. Such functionalization, which enhances the stability of BP, promotes neurogenesis in vitro by activating the PI3K/Akt and Ras/ERK1/2 signaling pathways in Schwann cells at the genetic, protein, and metabolite levels. Relevantly, when BPQD@β-carotene was incorporated into a GelMA/PEGDA scaffold and applied in both a rat sciatic nerve defect model and a beagle dog nerve crush model, the resulting scaffolds were able to promote nerve regeneration and improve myelination. Indeed, immunofluorescence labelling of regenerated nerves revealed increased expression of both neuronal (β3tubulin, growth-associated protein 43-GAP-43) and myelin markers (myelin basic protein–MBP) (Fig. 13(III)), demonstrating strong therapeutic potential for peripheral nerve repair and may serve as a promising candidate for clinical translation [261]. Similarly, BPQDs modified with epigallocatechin-3-gallate, the most abundant polyphenol in green tea with great anti-inflammatory capabilities, were synthesized and incorporated into hydrogels (E@BP). These hydrogels effectively reduced inflammation in primary neurons and supported neuronal regeneration in vitro. In vivo, E@BP promoted the repair of spinal cord tracts’ structural and functional integrity by increasing phosphorylation of key proteins in the AKT signaling pathway, thereby aiding the recovery of motor neuron function after transplantation in a rat model of spinal cord injury [265].
Treating large gaps in peripheral nerves has also been achieved through the development of multifunctional BP-based materials that combine BP's regenerative potential with biologically relevant proteins and growth factors involved in nerve regeneration. For instance, the combinatorial effect of incorporating BP and neuregulin 1, a protein involved in nerve regeneration, has been used to enhance neuronal outgrowth in PC12 cells and to stimulate Schwann cell proliferation. The therapeutic efficacy of the conduits was further validated in vivo in a rat model of nerve injury [266].
6.4. BP-mediated electrical modulation to promote neural repair
Conductive materials offer promising benefits in electroactive tissues (e.g., cardiac muscle and nerves) and have recently been explored for peripheral nerve repair [267,268]. BP possesses highly anisotropic physical characteristics—including its electron mass and pore architecture—due to its unique band dispersion, which is crucial to its high electrical conductivity of up to 300 S m−1 [62]. Given that BP demonstrates superior conductivity compared to other two-dimensional materials, it holds potential to restore impaired electrical function in damaged neurons [269]. This capability has been employed to develop BP-based materials able to support neural regeneration.
Exposure to BP nanosheets enhances both the viability and neural differentiation of neural progenitor cells through activation of the Nrf2 pathway. In a spinal cord injury mouse model, BP nanosheets exhibited a strong neuroprotective effect, reducing glial scar formation and promoting axonal regeneration, thereby enhancing nerve repair outcomes [270]. In another study, Qian et al. developed BP/PCL nanoscaffolds utilizing a concentrically integrative layer-by-layer bioassembly technique. In this approach, using dichloromethane, BP nanoplates were dispersed in a PCL solution, and the resulting suspension was sprayed onto a rotating mold to fabricate the nanoscaffold. The BP-based conduits offered long-term mechanical stability, preventing collapse and nerve-end entrapment, while also allowing sustained BP release. Moreover, incorporation up to 0.5 % BP into the scaffold resulted in an electrically conductive structure that remained stable for up to four months post-implantation in vivo, a time necessary to ensure that BP nanoscaffolds could effectively bridge 20 mm peripheral nerve gaps and facilitate in situ neurogenesis [271]. The addition of BP was used to enhance the conductivity of interpenetrating polymer networks composed of glycyrrhizic acid (GA), a natural compound extracted from the roots of licorice plants, and photo-crosslinked methacrylated SF (Fig. 14(A)) [74]. BP incorporation promoted neuronal differentiation and axonal regeneration while suppressing astrocyte differentiation. Concurrently, the sustained gradual release of GA mitigated inflammation by modulating the polarization of macrophages toward the M2 phenotype and reducing M1 pro-inflammatory activity (Fig. 14(B)). Additionally, the SF/BP/GA composite hydrogel significantly enhanced spinal cord regeneration and improved motor function recovery, highlighting the potential of the synergistic action of BP and GA in spinal cord injury repair [74].
Fig. 14.
(A) Schematic representation of the synthesis procedure of SF/BP/GA hydrogels and the mechanism underlying their ability to facilitate spinal cord neuron regeneration. (B) Immunofluorescence micrographs showing inducible nitric oxide synthase (iNOS)-positive M1 RAW264.7 macrophages and arginase-1 (Arg-1)-positive M2 RAW264.7 macrophages treated with SF-based hydrogels. Reprinted from Ref. [74] with permission from Wiley. (C) Immunofluorescence micrographs showing labeling for Tuj1 and Nestin (green) and GFAP and MAP2 (red). Reprinted from Ref. [272] with permission from Wiley.
Electrical conductivity enables BP-based materials to be used for delivering electrical stimulation. Indeed, electrical stimulation has emerged as a promising therapeutic strategy in the field of neural repair, offering new avenues for restoring function following nervous system injuries or neurodegenerative diseases [273,274]. Indeed, conductive gelatin methacryloyl hydrogel containing BP nanosheets has been developed and used for electrical stimulation delivery [272]. Here, BP nanosheets' interaction with the polymeric matrix was strengthened through a PDA coating via in situ oxidative polymerization, which also enhanced BP nanosheets' stability and boosted the hydrogels’ electrical conductivity. These composite scaffolds demonstrated excellent electrical conductivity, biocompatibility, and biodegradability. Notably, the incorporation of BP@PDA significantly enhanced mesenchymal stem cell differentiation into neural-like cells under synergistic electrical stimulation (Fig. 14(C)) [272]. Wireless electrical stimulation was also obtained using rotating magnetic fields [275]. Here, the incorporation of BP nanoplates in a dual-crosslinked hydrogel composed of chitosan and SF was also used to create a conductive hydrogel able to generate stable electrical signals under the influence of rotating magnetic fields [275]. Indeed, wireless electrical stimulation facilitated by the hydrogel promoted the differentiation of neural stem cells into neurons, a process linked to activation of the PI3K/AKT signaling pathway. In vivo, the BP-loaded hydrogels significantly improved behavioral performance and electrophysiological neuronal properties in a complete spinal cord injury transection model by mitigating inflammation and promoting the differentiation of endogenous neural stem cells into functional neurons under rotating magnetic field stimulation [275].
6.5. Antioxidant and ion scavenging potential of BP
ROS are chemically reactive molecules formed as inherent byproducts of normal cellular metabolic processes [276]. While physiologically relevant at low concentrations, excessive ROS accumulation leads to oxidative stress, which plays a central role in the pathogenesis of numerous diseases, including neurodegenerative disorders [276].
BP nanosheets have been widely used for their antioxidant capacity, playing a major role in the development of effective ROS scavengers for the treatment of neurodegenerative diseases. Structurally, a single phosphorus atom within BP nanosheets is covalently bonded to three neighboring atoms, with the lone pair electrons exposed on the surface. These electrons are readily oxidized by oxygen molecules, leading to BP nanosheets oxidation and initiating its degradation into phosphoric acid upon subsequent reaction with water, an outcome that exerts no toxic effect on the host organism [277].
This redox-responsive behavior makes BP nanosheets suitable for treating oxidative stress-induced cell damage and death. For instance, BP nanosheets have been shown to contribute to the elimination of ROS species [243,263] and preserve mitochondrial membrane potential, thus inhibiting cell apoptosis [263]. In vivo, treatment with BP nanosheets promoted neuronal antioxidant activity through activation of glutathione and superoxide dismutase [245]. Two key players in the reaction that catalyzes the dismutation of superoxide anion radical to molecular oxygen and harmless hydrogen peroxide, which is then neutralized, therefore amplifying the BP antioxidant effect.
The ROS scavenging activity of BP may also play a pivotal role in protecting transplanted stem cells from oxidative stress-induced cell damage, enhancing their resilience [278]. While stem cell transplantation holds great promise for tissue repair, particularly in neurological injury recovery, its efficacy in promoting effective neural regeneration remains often limited [279,280]. A major obstacle is the poor survival rate of transplanted stem cells, particularly in the hostile, inflammatory microenvironments that follow injury or surgery, where oxidative stress and inflammation contribute to extensive cell damage and death. This significantly hampers the ability of transplanted cells to differentiate into mature and functional neurons and thus participate in neural circuit reconstruction [281].
Recent advances have focused on enhancing stem cell resilience through the use of nanomaterial-based pretreatment. Various nanomaterials and nanocomposites have shown promise in improving stem cell survival and function. These materials often possess multi-antioxidant properties and can modulate cellular behavior by interacting with specific signaling pathways involved in stem cell homing and differentiation, as well as promoting neurotrophic factors secretion [282]. If pretreatment with these nanomaterials has shown promise to enhance stem cell transplantation, their clinical translation and effective application are significantly hampered by their complex synthesis, poor biodegradability, and potential long-term toxicity, highlighting the urgent need for the development of simple, bioactive, and biocompatible nanomaterials. Notably, a recent study has leveraged the redox-responsive behavior of BP nanosheets to demonstrate their broad applicability in enhancing the survival of stem cells in the presence of oxidative stress, further elucidating the underlying mechanisms of their biological effects on stem cells. Various stem cell types were pretreated with BP nanosheets in vitro to assess their resilience to oxidative damage. BP nanosheets-treated stem cells exhibited significantly reduced ROS levels upon stimulation with H2O2 [278]. Treatment with BP nanosheets also improved stem cell viability and proliferation after H2O2 exposure, as well as increasing the activation of Nrf2-dependent antioxidant pathways in stem cells, further demonstrating the ability of BP nanosheets to enhance the antioxidant defense mechanisms of stem cells in vitro [278]. Remarkably, in vivo transplantation experiments in a rat model of stroke induced by middle cerebral artery occlusion confirmed that BP nanosheets-treated neural progenitor cells exhibited higher survival rates and were more effective at reducing lipid peroxidation, inflammation, and neuronal apoptosis, resulting in enhanced neurological recovery [278].
The presence of lone-pair electrons on phosphorus atoms endows BP with multifunctional reactivity, allowing it to serve not only as an effective scavenger of ROS, but also as a selective binder of biologically relevant metal ions. This property is particularly relevant in pathological conditions characterized by metal ion dysregulation, such as the elevated levels of Cu2+ observed in neurodegenerative diseases [283,284]. Cu2+ has been shown to bind to amyloid-β peptides, contributing to their anomalous interaction and plaque formation [284]. In addition, Cu2+ dysregulation has been implicated in AD-associated oxidative stress [285,286]. Cu2+ can induce oxidative damage of critical cellular components, such as DNA, lipids, and proteins, and the disruption of signaling pathways, leading to neurodegeneration [254,284,286]. In this context, chelation therapy has emerged as a promising neuroprotective strategy, and BP, with its metal-ion and ROS-scavenging activity, is the ideal candidate to capture excess redox-active metal ions in the brain, forming non-toxic metal complexes and preventing ROS-mediated toxic effects. BP nanosheets have indeed been shown to efficiently and selectively bind Cu2+, thereby protecting and increasing the viability of neuronal cells from Cu2+-induced neurotoxicity [244]. Furthermore, BP nanosheets, by decreasing intracellular ROS content in neuronal cells, also contributed to maintaining the integrity and functionality of the mitochondrial membrane, inhibiting cell apoptosis [244]. Further pinpointing the role of BP nanosheets as promising candidates for neuroprotective nanomedicine in the treatment of neurodegenerative diseases.
Stroke is a major neurological disorder and the second leading cause of death and disability, with ischemic stroke accounting for approximately 80–85 % of cases. It is worth mentioning that ischemic stroke arises from transient or permanent cerebral artery occlusion that induces ischemia–reperfusion injury and neurological dysfunction [287]. Existing therapeutic strategies, including surgical recanalization and thrombolytic agents, are limited by invasive risks, short therapeutic windows, and suboptimal pharmacokinetics, leaving many patients without effective treatment [288]. The pathological progression of ischemic stroke is primarily driven by oxidative stress and inflammation; reperfusion generates excessive ROS (H2O2, O2−, •OH), leading to redox imbalance, mitochondrial damage, and neuronal apoptosis, while activation of the NF-κB pathway promotes microglial polarization toward the pro-inflammatory M1 phenotype and enhances cytokine release [289]. ROS further amplifies these inflammatory responses by stimulating microglial activation and leukocyte infiltration, thereby exacerbating tissue damage. These interconnected mechanisms highlight redox regulation and immunomodulation as critical therapeutic targets for ischemic stroke [290].
BP has recently been employed for ischemic stroke therapy due to its ROS scavenging ability and great potential for drug delivery [[291], [292], [293]]. A targeted drug-delivery platform was designed by integrating Angiopep-2, BP nanosheets, and resveratrol to efficiently transport therapeutics across the BBB. The resulting system showed uniform particle size, high stability, and strong responsiveness to NIR light, enabling controlled drug release in acidic environments typical of damaged brain tissue. The nanoparticles effectively accumulated in the brain and demonstrated notable protective effects, including reductions in lactate dehydrogenase and malondialdehyde levels, suppression of caspase-3 and tumor necrosis factor-α expression, and clear improvements in neurological behavior in animal models. It also decreased brain edema and infarct volume while maintaining excellent biocompatibility [292]. An interesting study has leveraged the negative surface charge and high surface area of BP nanosheets to load positively charged urokinase plasminogen activator to address oxidative stress generated during thrombolysis (Fig. 15(A)). Different samples, including pristine BP nanosheets, urokinase plasminogen activator, PBS, and urokinase plasminogen activator-loaded BP, were exposed to blood clots in vitro, and the drug-loaded BP dissolved more than 50 mg of thrombi after 2 h, while the others had lower dissolution rates. The ROS-scavenging ability of nanoplatforms was tested; BP nanosheets effectively scavenged H2O2, resulting in rapid concentration decreases due to redox-driven degradation of the nanosheets. They also efficiently removed •OH, with increasing BP dosage. In contrast, their scavenging of O2•- was limited. Importantly, BP nanosheets remaining after the drug release retained their structural characteristics and continued to eliminate ROS. In cell studies, BP nanosheets significantly protected SH-SY5Y neuronal cells from H2O2-induced damage, increasing cell viability up to 92 % at higher concentrations. Regarding BBB penetration, BP under NIR irradiation generated heat, temporarily enhancing permeability, allowing BP nanosheets to enter the brain. Evans blue and fluorescence imaging confirmed that only BP combined with NIR effectively crossed the BBB, supporting their use for targeted neuroprotection. In a mouse model of middle cerebral artery occlusion, intravenous injection of drug-loaded BP combined with 808 nm laser irradiation markedly reduced brain infarct size from ∼37 % to ∼9 % and improved neurological scores, whereas laser or BP nanosheets alone had little effect. These results indicate that the residual BP nanosheets after the drug release provide strong neuroprotection in ischemic stroke [291]. A multifunctional nanotherapeutic was developed by loading BP nanosheets with Mg2+ ions and PDA to treat ischemic stroke (Fig. 15(B)). BP nanosheets efficiently scavenged excessive ROS and prevented neuronal apoptosis. Mg2+ exerted anti-inflammatory effects by promoting microglial polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype, and PDA enhanced the stability of BP nanosheets. In a rat model of ischemic stroke, the platform improved the ischemic microenvironment, reduced infarct volume, protected neurons, and promoted neurofunctional recovery, including neural and vascular regeneration. The nanosystem was found to be biodegradable and biocompatible, minimizing long-term risks. The results of this study showed that this platform provides a synergistic, multi-target approach combining antioxidant, anti-apoptotic, anti-inflammatory, and neurorepair effects, representing a promising therapeutic strategy for ischemia-reperfusion injury [293].
Fig. 15.
BP nanomaterials for ischemic stroke and neuron protection. (A) Schematic illustration of the BP–urokinase plasminogen activator (uPA) synthesis and therapeutic process in a mouse model of middle cerebral artery occlusion (MCAO): (i) uPA is initially released to achieve thrombolysis; (ii) residual BP nanosheets cross the BBB upon 808 nm laser irradiation; (iii) the BP nanosheets that enter the brain function as ROS scavengers, providing neuroprotection. Reprinted from Ref. [291] with permission from the Royal Society of Chemistry. (B) An illustration showing the synthesis of BP-loaded with PDA-Mg2+, its ability to relieve oxidative stress, and its targeting of inflammatory microglia in ischemic stroke. Reprinted from Ref. [293] with permission from the Royal Society of Chemistry.
7. Other organs
While BP applications in bone, skin, and neural tissue have been widely explored, recent studies have expanded its potential to other vital organs such as the heart, liver, and kidneys. In these contexts, BP-based platforms have been investigated for their ability to modulate inflammation, promote tissue repair, and enhance the delivery of drugs. This section highlights recent advances in applying BP for therapeutic and regenerative purposes in cardiovascular, hepatic, and renal systems.
7.1. Heart
7.1.1. Myocardial infarction
As a serious threat to human health, myocardial infarction is a critical disease with an annual incidence rate of nearly 1 million worldwide; two main important factors causing myocardial infarction are thrombosis and atherosclerosis in the coronary arteries, which cause a reduction of myocardial blood flow [294]. It is known that following myocardial infarction, there is excessive ROS generation and overexpression of hypoxia-inducible factor-1α in the affected area, leading to the apoptosis of cardiomyocytes, inflammation, and other detrimental effects, all of which deteriorate cardiac function. The primary treatment remains the rapid restoration of blood flow to the ischemic tissue [21,295]. Some previous studies have reported the ROS scavenging effect of BP and its ability to capture transition metal ions, such as copper [100,258], both of which can alleviate the myocardial infarction condition. To improve cardiac function, decrease cardiomyocyte inflammation, and promote cardiac repair, an injectable hydrogel composed of alginate and hyaluronic acid incorporating a BP-PDA nanocomposite was designed and prepared (Fig. 16(A)). The hydrogel was found to have suitable biocompatibility, biodegradability, and electrical conductivity, which facilitated the release of phosphate in the surrounding medium; the phosphorus could react with ROS in the myocardium to form nontoxic phosphate. Alleviation of inflammation was achieved by the hydrogel through the inhibition of the NF-κB signaling pathway and the effective scavenging of ROS. The ROS scavenging ability of BP-PDA incorporated hydrogels was tested in vitro, and the nanocomposite was added to the hydrogel in various concentrations—10, 20, 50, and 100 μg mL−1. The highest concentration had nearly 90 % scavenging efficiency in clearing OH− groups, and thus it was chosen for animal studies. Speaking of in vitro cell viability, the cytotoxicity of hydrogels was assessed against H9C2 cells over a 24-h period, and no significant difference was observed compared to the control group. The hydrogel was then tested in vivo on a rat model of myocardial infarction, and the hydrogel demonstrated a strong protective effect on the rats. Compared to the myocardial infarction control group, the rats treated with this hydrogel had a survival rate of more than 80 %. To assess cardiac function after treatment, echocardiography was used to evaluate the rats at 24 h, 7 days, 14 days, and 28 days post-treatment. The left ventricle diameter after 28 days of treatment with the hydrogel increased for the myocardial infarction rats, whereas the left ventricle systolic function decreased compared to the control group. Moreover, improvement in the left ventricular ejection fraction and the left ventricular shortening fraction was observed for the hydrogel-treated animals. It is known that myocardial infarction causes collagen deposition and ventricular wall thinning, leading to myocardial fibrosis. For cases treated with the hydrogel, H&E staining revealed that the ventricular wall was significantly thicker than in the control group [296]. Another study reported on the loading and sustained release of BP-PDA nanocomposite in the myocardium (infarct region), but in a different hydrogel—gelatin and chitosan (Fig. 16(B)). It was proposed that the combination of BP-PDA would alleviate oxidative stress and influence macrophage phenotype in the injured region, resulting in effective heart tissue repair. The BP-PDA integrated hydrogel was evaluated for its mechanical characteristics, swelling behavior, injectability, adhesion properties, and phosphorus release. It was observed that the incorporation of the BP-PDA nanocomposite enhanced the mechanical properties to levels comparable to those of natural heart tissue, and the hydrogel exhibited suitable swelling characteristics, allowing it to easily pass through a 1 mL syringe. The phosphorus release was examined. During the initial soaking phase, the release rate was rapid, which then decreased over time, with detectable phosphorus remaining even at day 21, indicating the hydrogel's ability to sustain release. The biological outcomes demonstrated that the continuous release of BP-PDA eliminated excess ROS in the area affected by myocardial infarction, and the hydrogel also suppressed the HIF-1α/NF-κB inflammatory signaling pathway. The polarization of M1 macrophages towards M2 occurred as a result, achieving an anti-inflammatory response, which prevents the progression of adverse infarction [22].
Fig. 16.
Different platforms prepared using BP for addressing myocardial infarction and improving cardiac function. (A) Step-by-step preparation of alginate and hyaluronic acid hydrogel incorporating BP-PDA nanocomposite to alleviate inflammation in myocardial infarction conditions and repair the damaged tissue. Abbreviations: ALGHA: alginate-hyaluronic acid hydrogel, DA: polydopamine. Reprinted in accordance with the Creative Commons Attribution License [296]. Copyright 2023, the American Chemical Society. (B) The development of gelatin and chitosan hydrogel incorporating BP-PDA nanocomposite to eliminate excessive ROS and change the macrophage phenotype, reaching an anti-inflammatory response in the infarcted region. The continuous release of BP-PDA from the hydrogel inhibited the progression of adverse infarction and led to the enhancement of cardiac tissue function. Reproduced under the terms of the Creative Commons Attribution License [22]. Copyright 2024, the American Association for the Advancement of Science. (C) A multifunctional conductive hydrogel composed of hyaluronic acid and dopamine, co-encapsulating exosomes and BP-PDA nanocomposite for myocardial infarction. Reprinted from Ref. [47] with permission from the American Chemical Society. (D) Magnesium-modified BP nanosheets-incorporated PVA-based hydrogel for myocardial infarction therapy and cardiac tissue repair. Reprinted in accordance with the Creative Commons Attribution License [298]. Copyright 2024, Springer.
The mentioned studies took advantage of the antioxidant effects of BP and PDA nanocomposite to alleviate inflammation in the infarcted region and elevate the tissue regeneration, while other studies capitalized on the electrical conductivity of BP for cardiac repair [47,297]. Using aldehyde-modified hyaluronic acid and photo-responsive gelatin methacryloyl, a hydrogel was synthesized, and 3,3′-dithiobis (propionic hydrazide) and BP nanosheets were added to this hydrogel as a cross-linker and the conductive component. The in-situ gelatin and mechanical properties of the hydrogel were manipulated to be suitable for myocardium after being injected into the infarcted region. 3,3′-Dithiobis (propionic hydrazide), a strong antioxidant, helped modulate the oxidative stress microenvironment in the damaged area. In this way, cellular oxidative stress was reduced, and the polarization of macrophages towards an M2 phenotype created an environment conducive to tissue regeneration. Another interesting aspect of using this cross-linker was its ability to decrease the oxidation rate of BP nanosheets, thanks to its ROS scavenging property, thereby preserving the nanosheets' conductivity while matching it with that of myocardial tissue under infarcted conditions. The in vitro results showed that the hydrogel's electrochemical properties stimulated cardiomyocytes maturation by upregulating the expression of Cx43 protein and also compensated for the impaired electrical pathways that occurred during the maturation phase. Maintaining these electrical pathways enhances cardiac signal transmission and maintains the cardiac contraction function. Speaking of biosafety, this study revealed that the hydrogel underwent gradual degradation, and in particular, BP nanosheets decomposed into non-toxic phosphate and phosphonate groups in vivo, followed by their absorption by the body without any problem [297]. A new exosome-loaded hydrogel made of hyaluronic acid and dopamine was designed, and BP-PDA had been co-encapsulated along with the exosomes to be applied in an animal myocardial infarction model (Fig. 16(C)). After performing various in vitro studies, including cell compatibility and cell migration, the hydrogels were applied in vivo to a rat model of myocardial infarction. This study hypothesized that the multifunctional hydrogel could provide mechanical support in the injected region, induce tissue repair with the aid of exosomes, and exhibit electrical conductivity due to the BP nanosheets. Different groups were tested, such as the hydrogel-exosome, the hydrogel-BP-PDA, and the hydrogel-exosome-BP-PDA. In terms of restorative capability, the hydrogel with exosomes or BP-PDA showed virtually similar effects, but when combined, they exhibited a cumulative effect. Masson staining revealed the formation of dense fibrous connective tissue for all the treated groups, but the least fibrotic area belonged to the animals treated with the combination of BP and exosomes (15.7 ± 1.2 %). This sample could also induce the greatest amount of neovascularization in the tested groups. Moreover, the lowest neutrophil infiltration and an increase in M2 macrophages were found for the hydrogel containing both BP and exosomes, indicating the inhibition of the inflammatory response. The expression of connexin 43, as an intercellular junction protein, was significantly improved in the damaged area for the animals treated with the nanocomposite hydrogel compared to the other samples. A series of arrhythmia-inducing experiments was performed to understand the electrical activity of myocardial tissues. For the nanocomposite hydrogel, the number of arrhythmia events was reduced compared to the hydrogel alone, and the effective refractory period was prolonged, indicating recovery of myocardial tissue electrical activity [47]. A PVA-based ROS-responsive hydrogel was fabricated for the inhibition of post-myocardial infarction. BP nanosheets were also included in the platform, but first surface-modified with Mg2+ ions to increase the nanosheets' chemical stability and take advantage of the Mg2+ sustained release to promote angiogenesis through improving vascular endothelial cell proliferation (Fig. 16(D)). The Mg2+-modified BP was then dispersed into the dopamine solution at pH = 8.5 to yield the Mg2+-modified BP-PDA nanocomposite. The composite hydrogel was tested for ROS removal in the presence of H2O2 against rat H9C2 myocardial cells. Different concentrations of the hydrogel were applied, and 100 μg mL−1 was found to be the optimized concentration, showing excellent anti-oxidative stress effects. Cell viability was assessed up to 48 h, and the cell density increased for all samples, showing the good cell compatibility of the hydrogels. The in vivo results showed that BP nanosheets decreased the oxidative stress-inflammation reaction chain by down-regulating certain proteins related to the NF-κB signaling pathway and stimulated vascular regeneration in the damaged area by releasing Mg2+ ions. Regarding angiogenesis, the release of Mg2+ ions was found to activate the PI3K-Akt pathway [298].
7.1.2. Atherosclerosis
Atherosclerosis is another cardiovascular disease with high potential for human mortality, originating from vascular endothelial dysfunction, and is accompanied by a chronic inflammatory response. Upon endothelial damage, low-density lipoprotein forms under the damaged area, leading to the excessive formation of ROS, endothelial dysfunction, and the oxidative modification of lipoproteins. As a result of this oxidative modification, macrophages are stimulated to generate more ROS, express more pro-inflammatory factors, and subsequently, chronic inflammation develops in the plaque [299]. The primary treatments for atherosclerosis are currently surgery and medication. Medication, as the standard strategy, is based on lipid-lowering therapies; however, the challenges are related to the systemic administration of these drugs—poor accumulation, rapid clearance from the body, etc. On the other hand, surgery treatments such as stenting and vascular bypass are only applied to patients with severe atherosclerosis, and restenosis is considered a common side-effect of this approach, lowering the long-term therapeutic outcomes [300,301]. Since BP has been shown to scavenge excessive ROS and alleviate acute kidney injury [100], and regulate macrophage phenotypes, it is expected to be a promising candidate for treating atherosclerosis.
A recent study utilized BP nanosheets, which have great potential for drug delivery in atherosclerosis treatment (Fig. 17(I)). Resolvin D1 was loaded onto the nanosheets, and to endow the drug-loaded BP with suitable colloidal stability and targeting ability towards macrophages, it underwent a surface coating with S2P-PEG-NH2 through an electrostatic interaction. Notably, S2P is a peptide capable of targeting stabilin-2, which is overexpressed on macrophages in the plaque area related to atherosclerosis. Since the progression of atherosclerosis is related to the ROS, the assessment of the antioxidation potential of the platform against H2O2, •OH, and O2•− was of great importance; the platform was found to act as a potent antioxidant agent, scavenging a broad spectrum of ROS. An in vitro model (lipopolysaccharide-treated RAW264.7 cells) was developed to assess the ROS-scavenging and anti-inflammatory potential of the platform. The platform was found to attenuate the overproduction of ROS in lipopolysaccharide-treated cells and also suppressed the pro-inflammatory cytokines secreted by the cells. Since the platform had a targeting agent on the surface, the biodistribution and pharmacokinetics were assessed in vivo. Using 64Cu positron emission tomography, the platforms were tracked, and the S2P-modified nanosheets were found in atherosclerotic Apoe−/− mice in higher quantities than the non-modified ones. Additionally, the ROS-responsive release of Resolvin D1 was observed, demonstrating anti-inflammatory efficacy. The anti-atherosclerotic efficacy of the platform was then assessed in plaque-bearing Apoe−/− mice. Checking on the arterial walls through Oil Red O staining showed the reduction of atherosclerotic plaque area [302]. Somewhere else, BP nanosheets were coated with platelet membranes to target macrophages in atherosclerotic plaques. Small interfering RNA was loaded onto the BP nanosheets before being encapsulated into the cell membrane, with the intention of silencing Ca2+/calmodulin-dependent protein kinase γ in macrophages. The step-by-step synthesis of the platform and its targeting of atherosclerotic plaques is illustrated in Fig. 17(II). The platform was found to scavenge excess ROS in macrophages, restore efferocytosis in macrophages through the inhibition of Ca2+/calmodulin-dependent protein kinase γ, while stimulating the expression of the c-Mer proto-oncogene tyrosine kinase. The potential of the platform was tested in vivo in ApoE–/– mice after a high-fat diet, and it was revealed to largely inhibit the progression of atherosclerosis in the animal model [87]. Interestingly, BPQDs were applied directly after PEGylation in an atherosclerotic mouse model for 12 weeks as a preventive drug. In parallel, simvastatin—a well-established drug for atherosclerosis—was used as a reference to evaluate the therapeutic potential of BPQDs. Mice were fed either a normal or a high-fat diet, with the latter group developing significantly larger plaque areas. After 12 weeks of treatment with BPQDs or simvastatin, both groups showed reduced plaque formation; however, the BPQDs-treated mice exhibited less aortic plaque than those receiving simvastatin. To investigate the underlying mechanism, macrophages were exposed to oxidized low-density lipoprotein to induce foam cell formation. The results demonstrated that BPQDs promoted autophagy, thereby modulating intracellular lipid metabolism (Fig. 17(III)) [303]. A complementary study was performed by the same group on the BPQDs’ efficacy in treating atherosclerosis. Similar to their previous study, the BPQDs were found to decrease atherosclerotic plaques and also partially restore vascular elasticity. More comparative analyses were performed between the BPQDs and simvastatin; the BPQDs outperformed the drug in terms of safe and rapid removal of the plaque (Fig. 17(IV)) [304].
Fig. 17.
Anti-atherosclerotic efficacy of BP-based platforms. (I) Step-by-step preparation of the platform; the BP nanosheets were modified with PEG-S2P/R to improve the colloidal stability and targeting ability of the platform, and then RvD1 was loaded onto the nanosheets. The platform was found to alleviate the excessive oxidative stress and reduce atherosclerotic plaque area in vitro and in vivo. Reprinted in accordance with the Creative Commons Attribution License [302]. Copyright 2024, Nature. (II) Surface modification and loading of siRNA onto the BP nanosheets, followed by being encapsulated in the platelet membrane for targeted delivery to macrophages in atherosclerotic plaques; the platform was found to decrease inflammation and excessive ROS, while promoting efferocytosis, leading to the inhibition of atherosclerosis progression. Reprinted from Ref. [87] with permission from Elsevier. (III) Applying PEGylated-BPQDs to prevent atherosclerosis in a high-fat diet animal model up to 12 weeks. Reprinted in accordance with the Creative Commons Attribution License [303]. Copyright 2024, Impact Journals LLC. (IV) Surface-modified BPQDs for prevention of atherosclerosis. Reprinted in accordance with the Creative Commons Attribution License [304]. Copyright 2024, Impact Journals LLC.
7.2. Kidney and liver
Acute kidney and liver injuries are among the acute organ injuries with a high morbidity rate and mortality in patients. These injuries are known to be associated with excessive ROS generation, which can interact with proteins, nucleic acids, and lipids, stimulating oxidative stress and inflammatory responses. Moreover, renal infiltration of those ROS may trigger kidney damage and cause severe injury to the kidneys [23,305]. Furthermore, acute liver injury is associated with oxidative stress due to ROS overproduction. [102,306]. Since current clinical approaches to address these injuries are limited, and the lack of potent drug-based treatments is particularly notable [307], designing and developing biocompatible biomaterials with excellent ROS scavenging ability for antioxidation therapy is of great importance.
The first report on the potential of BP nanomaterials for treating kidney injury was published in 2020, revealing BP as a nanodrug without any payload for acute kidney injury. To test the ROS scavenging capability of BP nanosheets, they were exposed to H2O2, •OH, and O2•−, which are regarded as the main sources of cellular ROS. Using electron spin resonance, the signals related to •OH, and O2•− were found to decrease significantly upon the addition of BP nanosheets to the solutions. The H2O2 scavenging was assessed through Raman spectroscopy, due to the strong Raman signal of H2O2 around 900 cm−1. At a concentration of 100 μg mL−1, the BP could scavenge H2O2 with 90 % efficiency within 5 min, which is considered fast and efficient among the antioxidation agents used in biomedicine. The protective effect of BP nanosheets was further assessed in vitro in the exposure of Hek293 cells, which had been treated with H2O2 half an hour before the BP addition. Without the addition of BP, half of the cells died due to the excessive H2O2, whereas in the presence of BP (5 μg mL−1), about 82 % of the cells remained alive. Notably, the cytocompatibility of BP in the absence of H2O2 was also assessed in the range of 10–400 μg mL−1, with negligible cytotoxicity at the highest concentration. To indicate BP nanosheets' transportation, they were modified with Cy5; fluorescence imaging showed that the BP nanosheets, after being injected intravenously, accumulated in the kidney, and the highest signal intensity was yielded after 0.5 h. Up to 12 h, the fluorescent intensity decreased gradually in the glomerulus, indicating the degradation of BP nanosheets. Then, the BP nanosheets were tested in vivo in mice with acute kidney injury; the injury was induced by intramuscular injection of 50 % glycerol into dehydrated healthy mice. As a result of the glycerol injection, excessive ROS damaged the renal tubules. Similar to the biodistribution study on healthy mice, fluorescence imaging showed that the BP nanosheets accumulated in the kidneys 5 min post-injection and reached a maximum after 1 h, and eventually disappeared after 12 h. The kidneys were harvested after 12 h, and as expected, the BP nanosheets were passively accumulated mainly in the kidneys. On the other hand, the low fluorescence intensity of Cy5 was observed in other organs, such as the heart, liver, spleen, and lung. To determine the efficacy of nanosheets in treating acute kidney injury, blood test analyses were performed on treated and non-treated mice. Blood urea nitrogen and serum creatinine were related to renal dysfunction, and these two had reduced levels in the BP-treated group, indicating the alleviation of ROS content. Interestingly, as a positive control, two well-known antioxidant agents used for kidney injury were applied—amifostine and N-acetylcysteine at a concentration of 1.2 mg mL−1. However, at this concentration, neither of them yielded a remarkable treatment effect, while the BP nanosheets suppressed both creatinine and urea indices at the same dose, [100]. A year later, another paper was published on the BP's potential in acute kidney and liver injuries, but this time, BPQDs. The authors leveraged the strong photothermal conversion rate of BPQDs for NIR real-time imaging, and fluorescence emission was observed in the second NIR bio-window, which could be tuned by adjusting the size of BPQDs. The nanoparticles could be tracked in the organs using real-time NIR-II fluorescence, and the BPQDs could effectively eliminate excessive ROS in acute kidney and liver injury models [102]. Elsewhere, ultra-small BPQDs with a diameter of nearly 2 nm were synthesized and explored for photoacoustic imaging of acute kidney injury. Upon NIR irradiation, the PEGylated BPQDs generated strong photoacoustic signals, by which kidney dysfunctions were successfully detected in the mouse model, followed by easy clearance through the renal system due to their size [308]. A recent study has designed PEGylated BP, followed by another surface modification, to convert the passive BP nanosheets into an active targeting platform for acute kidney injury. LTH peptide and 4-OI were anchored onto the nanosheets to specifically target Kim-1 protein in the renal tubular epithelial cells and compensate for excessive ROS. Moreover, upon the degradation of nanosheets in the damaged area, the release of 4-OI was facilitated, which further improved the antioxidation ability and enhanced the Nrf2/Keap1 pathway. It is worth mentioning that this study revealed that efferocytosis was improved due to ROS removal, which subsequently inhibited the progression of inflammation [92]. BPQDs were obtained in a recent study via ultrasonic crushing with a size of 3–5 nm as a free-radical scavenger for acute kidney and liver injuries (Fig. 18(I)). The scavenging potential of QDs was assessed in vitro using the DPPH method, and at a concentration of 0.625 μg mL−1, 80 % of DPPH was decomposed. Through complementary analysis, it was revealed that the BPQDs at 2.5 μg mL−1 achieved a good clearance effect against DPPH, ABTS·, OH·, and O2−·. The cytocompatibility and cytoprotective effect of BPQDs were evaluated against HEK293 with and without H2O2. The MTT assay showed no cytotoxic effects up to 25 μg mL−1. In the presence of H2O2, the BPQDs at a concentration of 0.1 μg mL−1 exhibited a good cytoprotective effect, preventing cell death caused by excessive ROS. The BPQDs were tested both in vivo and in a glycerin-induced acute kidney injury animal model. In the experiment, there were three groups: BPQDs, NAC (a clinical drug), and a control, which did not receive any treatment and died after 5 days. The BPQDs were injected at a concentration of 5 mg kg−1, and the NAC at 160 mg kg−1; the mice treated with both groups were found to survive the entire therapeutic period (Fig. 18(II)). It indicates that the BPQDs yielded the same results as the clinical drug, but at a much lower concentration [307].
Fig. 18.
Ultra-small BPQDS for acute kidney and liver injuries. (I) Schematic illustration of BPQDs preparation and their ROS scavenging potential for ROS-induced acute kidney and liver injuries. (II) Testing the efficacy of BPQDs in vivo in glycerin-injected mice. (a) Schematic of establishing the in vivo model and the treatment schedule. (b,c) Survival rate after two weeks, and weight changes of the treated mice after 24 h. (d,e) The CRE and BUN indices obtained after 24 h from blood analysis (*, **, and *** denote P < 0.05, P < 0.01, and P < 0.001 compared to the control group). (f) Histological analysis of kidney tissues from animal models treated with different samples. Abbreviations: Acute kidney injury: AKI, creatinine: CRE, blood urea nitrogen: BUN. Reprinted from Ref. [307] with permission from Wiley.
8. Comparative perspective: positioning BP among conventional regenerative biomaterials and other 2D nanomaterials
Conventional regenerative biomaterials such as HA, bioactive glass, and calcium phosphate-based ceramics have long been regarded as the foundation of bone tissue reconstruction and are also used in soft tissue regeneration as well [309]. Their clinical success stems from their structural compatibility, osteoconductivity, and ion-release behavior, yet their therapeutic role remains essentially passive [310]. These materials lack inherent photothermal responsiveness or external stimulus sensitivity, limiting their application to static scaffold roles rather than multifunctional therapeutic platforms [179]. On the other hand, carbon-based 2D nanomaterials such as graphene and graphene oxide offer high mechanical strength, electrical conductivity, and large surface area, making them promising for neural interfaces, conductive scaffolds, and drug loading [311]. However, their major drawback is their extremely poor biodegradability, which often leads to long-term retention and chronic inflammatory responses unless extensively functionalized. Their biological activity is therefore not intrinsic but dependent on heavy surface modification, which introduces complexity, variability, and translational uncertainty. Furthermore, graphene materials typically lack the ability to release physiologically beneficial ions and do not exhibit microenvironment-adaptive oxidative behavior [312]. Transition metal dichalcogenides, such as MoS2 and WS2, have been widely explored for photothermal and photodynamic therapy, owing to their strong NIR absorbance and catalytic properties [313]. However, their degradation products include transition-metal ions, raising concerns about cytotoxicity, long-term accumulation, and metabolic clearance [314]. These materials perform well in short-term tumor ablation but face significant barriers to chronic or regenerative applications where biosafety and controlled breakdown are essential. Their therapeutic activity is mainly unidirectional—tumor-killing—without capacity to participate in immune homeostasis, endogenous repair signaling, or metabolic recovery (Table 4) [315,316].
Table 4.
Comparison of BP nanomaterials with conventional biomaterials and 2D nanomaterials for biomedical applications.
| Material | Biodegradability | Intrinsic Anticancer Activity | Biomineralization/Bone Regeneration | Stimuli Responsiveness (PTT/PDT/SDT) | Biocompatibility | Key Limitations | Ref. |
|---|---|---|---|---|---|---|---|
| BP | Degrades to physiological phosphate | Strong, intrinsic + stimuli-enhanced (ROS modulation, mitochondrial disruption, membrane damage) | Excellent (phosphate release leads to CaP nucleation, followed by enhanced osseointegration) | Excellent (NIR PTT, PDT, SDT responsive) | High when surface-stabilized (PEG, PDA, TA, lipids, coordination) | Oxidation instability; production standardization needed | [32,33,50] |
| Nano-Hydroxyapatite | Very slow but bioresorbable, depending on crystallinity | Moderate, mainly in bone tumors (induces mitochondrial apoptosis + immune activation) | Excellent (native osteoconductivity and osteointegration) | None | Excellent | Anticancer effects are context-specific and significantly weaker/selective compared with BP; they lack external controllability | [[319], [320], [321], [322]] |
| Bioactive glass/CaP Ceramics | Controlled degradation via ion release | No intrinsic anticancer activity | Excellent (bioactive ionic dissolution stimulates osteogenesis) | None | Good | Brittle; lacks adaptive therapeutic function | [[323], [324], [325]] |
| Graphene/Graphene Oxide | Non-degradable/persistent | Weak to moderate, often requires drug/photothermal loading | No inherent biomineralization, unless combined with CaP phases | Mild PTT, but dependent on defect density and doping | Variable; functionalization-dependent | Risk of long-term inflammation, accumulation | [326,327] |
| MoS2/WS2 | Slow degradation, potential ion release | Moderate, mostly stimuli-dependent PTT/PDT cytotoxicity | No inherent biomineralization | Strong PTT/PDT potential | Can be bioactive, but metal ion toxicity risk | Not ideal for regenerative implantation; unclear long-term clearance | [[328], [329], [330]] |
BP occupies a unique position among these materials due to its biodegradable, bioresorbable nature and its ability to degrade into phosphate ions, which are inherently involved in cellular metabolism, ATP formation, nucleic acid synthesis, and mineralized tissue formation [31]. Unlike HA or bioactive glass, BP is not merely structurally bioactive—it is dynamically biofunctional, responsive to external stimuli to induce PTT, PDT, and SDT, and capable of participating in oxidative signaling, immune modulation, and intracellular biochemical regulation [92,100,148]. Unlike graphene or MoS2, BP does not rely on extensive surface modification to become biocompatible [317,318]. BP nanomaterials inherently promote in situ biomineralization due to their surface chemistry. As BP undergoes gradual oxidation, it releases PO43− ions, which act as coordinating ligands to capture Ca2+ ions present in the surrounding medium. This local enrichment in Ca2+ and PO43− fosters the nucleation and deposition of new calcium phosphate nanocrystals, thereby supporting osseointegration [32,33]. Notably, the most important feature that distinguishes BP nanomaterials within the 2D nanomaterial family is their intrinsic anticancer activity, which enables them to selectively induce apoptosis in cancer cells without affecting healthy cells [32,35]. Importantly, BP possesses stimuli responsiveness that enables spatiotemporal control of therapeutic output, while stabilization strategies such as PEGylation, PDA coating, metal coordination, and micellar encapsulation have now demonstrated reliable modulation of oxidation rate without compromising functional performance. The competitive advantage of BP, therefore, lies not in replacing existing materials but in filling the functional gap between passive structural biomaterials and non-degradable nanotherapeutics (Scheme 4). BP provides structural ion release, controllable degradation, high photothermal conversion efficiency, electrical and redox activity, and immunoregulatory capacity, thereby enabling it to unify disease therapy and tissue regeneration within a single material platform (Table 4). Its remaining challenges—standardization of production, control of oxidation kinetics, and scale-up of reproducible stabilization strategies—are active areas of research rather than intrinsic limitations. As such, BP represents a next-generation, microenvironment-responsive theragenerative material positioned at the interface of nanomedicine, biomaterials science, and regenerative therapy.
Scheme 4.
Comparison of BP nanomaterials with conventional biomaterials and 2D nanomaterials for biomedical applications. Created in BioRender. Bigham, A. (2025) https://BioRender.com/cxtopja.
9. Clinical translation roadmap
While BP shows great promise in preclinical regenerative and therapeutic applications, moving from the lab to the clinic is complex and goes beyond just materials design and biological performance. This section highlights some important considerations that are often overlooked. A major challenge is that current animal models do not fully replicate human physiology. Small animals like mice, rats, and rabbits are useful for initial proof-of-concept studies, but large animals—such as pigs for heart applications or sheep for bone repair—are needed to better evaluate physiological responses, immune reactions, and mechanical performance before clinical translation. For organs like the brain and heart, large-animal studies are also essential to test effectiveness, delivery methods, dosing, and safety. Using imaging and functional outcome measures over time can help predict how treatments will work in humans. So far, the majority of studies have exploited the NIR light responsivity of BP systems (PTT and PDT) for various biomedical applications, which, although they offer valuable therapeutic effects, pose challenges for clinical application because precise energy delivery to deep tissues is required. Potential solutions include focusing more on the SDT potential of BP, as ultrasound has higher penetration depth than the NIR, and using fiber-optic or catheter-based NIR delivery, combined with imaging guidance (MRI or ultrasound, photoacoustic imaging) to improve the precise energy delivery to deep tissue, and the development of standardized protocols to ensure uniform treatment while avoiding damage to healthy tissue.
BP-incorporated platforms must be designed with specific organs in mind. In bone, they need to provide mechanical support, promote mineralization, and release phosphate in a controlled way to aid regeneration. In neural tissues, they should cross the BBB safely, avoid toxicity, and reduce oxidative stress to protect neurons. In heart and blood vessels, BP should conduct electrical signals, be flexible to match tissue movement, and regulate oxidative stress to protect against ischemic injury. Tumors or infected tissues benefit from BP's ability to generate localized oxidative stress and heat, enabling targeted destruction of cancer cells or bacteria. In the liver and kidney, BP's antioxidant and biocompatible properties help prevent damage during inflammation or ischemia. By carefully matching disease, tissue type, and BP properties—including particle size, surface coating, embedding, and external triggers—researchers can design BP-incorporated biomaterials optimized for each tissue. Using BP's directional electrical conductivity, tunable heat absorption, and controlled degradation, it is possible to fine-tune these materials for specific organs, combining therapy and regeneration across multiple systems.
In this context, a rational alignment between disease type, tissue biology, and material design is essential for developing BP-based composites that are truly tailored to the needs of each target organ. The required degradation profile, mechanical performance, and loading capacity for therapeutic agents will differ substantially between, for example, load-bearing bone defects, soft tissue regeneration, and cardiovascular applications. Systematic optimization of BP content, functionalization strategies, and composite architecture for each indication will therefore be critical to translate promising preclinical data into clinically relevant products.
A further challenge for translation is the current regulatory uncertainty. BP represents a borderline technology at the interface between medical devices and medicinal products, and its final classification will depend on the primary mechanism of action and intended use. BP-based systems designed mainly for structural support or localized release from an implantable scaffold are more likely to be evaluated under the medical device framework, whereas formulations exerting a predominantly pharmacological, immunological, or metabolic effect could fall under the medicinal product legislation. This ambiguity is compounded by the fact that, unlike conventional polymeric, ceramic, or metallic implants—where decades of regulatory experience and standardized testing frameworks exist—2D nanomaterials such as BP still occupy a regulatory “gray zone,” requiring dedicated degradation studies, nanomaterial-specific toxicological assessment, and harmonized biological assays.
Within the European context, several key legal instruments will have to be considered from the outset and systematically monitored to ensure compliance: Directive 2001/83/EC and Regulation (EC) No 726/2004 on medicinal products for human use, Regulation (EU) 2017/745 on medical devices, and Regulation (EU) 2017/746 on in vitro diagnostic medical devices. Early and proactive engagement with regulatory authorities (e.g., EMA and national competent authorities), as well as with notified bodies, together with the involvement of toxicologists, regulatory scientists, and clinicians, will be crucial to clarify the regulatory status of BP-based technologies and to de-risk their pathway towards clinical translation and market access. Translating BP into the clinic requires close collaboration across disciplines. Progress will depend not only on advanced material design but also on integrating biological knowledge, clinical needs, manufacturing constraints, and device engineering. The standardization of BP synthesis, degradation profiles, and biological testing protocols will be essential to improve reproducibility, enable cross-laboratory validation, and ultimately pave the way for first-in-human trials.
10. Conclusion and future perspectives
BP stands out within the 2D nanomaterial family due to its unique integration of physicochemical and biological functionalities. Beyond its tunable electronic structure and high surface area, BP exhibits intrinsic anticancer activity [35], excellent biocompatibility [32], and controllable biodegradability [31]. Moreover, its strong responsiveness to external stimuli such as light and ultrasound, along with its capacity to modulate ROS levels, endows BP with remarkable versatility for synergistic therapeutic and regenerative actions [22,67,90,92,318]. These distinct features collectively establish BP as a multifunctional platform that seamlessly bridges therapy and tissue regeneration, thereby defining a new paradigm in theragenerative technologies.
Over the past decade, remarkable technological advances have expanded BP's utility across diverse organs for disease therapy and tissue regeneration. In bone tissue engineering, BP-based scaffolds and coatings have demonstrated a unique dual function—simultaneously eradicating residual tumor cells through photothermal effects while promoting osteogenesis via phosphate release, immunomodulatory signaling, and mild hyperthermia (PTT) [34,81,104,114]. In wound healing, its capacity to generate ROS and localized hyperthermia has led to the development of multifunctional dressings that integrate antibacterial, anti-inflammatory, and angiogenic effects [44,96]. In neuroregeneration, functionalized BP nanosheets and quantum dots have shown the ability to cross the BBB, attenuate oxidative stress, and guide neuronal differentiation [20,101]. Similarly, in cardiac, hepatic, and renal models, BP-containing hydrogels and nanostructures have demonstrated powerful antioxidant, anti-inflammatory, and tissue-reparative properties, restoring organ function with minimal systemic toxicity [24,47,100]. Collectively, these technological breakthroughs highlight BP's distinct potential to unify therapeutic efficacy and regenerative stimulation within a single, adaptive nanoplatform. While these advances mark a new technological frontier, the clinical translation of BP-based systems remains constrained by several unresolved challenges. To transform BP from an experimental material into a clinically viable platform, these technological capabilities must be aligned with pressing clinical demands and standardized evaluation frameworks.
Although significant progress has been made in the design and preclinical evaluation of BP-based systems, several aspects still require careful consideration before clinical implementation. BP's chemical instability in oxygenated and aqueous environments, while increasingly mitigated by surface passivation and composite strategies, can still affect reproducibility, shelf-life, and safety if not rigorously controlled. In parallel, heterogeneity in synthesis routes continues to challenge batch-to-batch consistency and regulatory standardization [31]. From a clinical perspective, more comprehensive toxicological, pharmacokinetic, and long-term biocompatibility studies are needed to fully elucidate BP's degradation behaviour, metabolic fate, and potential accumulation. These issues do not negate the substantial technological advances achieved so far, but they highlight the key domains in which further work is required for reliable, safe, and scalable translation.
Future research should therefore focus on bridging these technological and clinical gaps through targeted innovation and a carefully translational strategy. (I) Materials optimization: Developing stable and scalable BP formulations through surface passivation, encapsulation, and hybridization with organic or inorganic frameworks will be essential to preserving functionality under physiological conditions. Advanced characterization standards and reproducible synthesis pipelines should be established to enable large-scale manufacturing and regulatory approval. (II) Functional integration: BP's inherent photothermal, piezoelectric, and electrical properties should be leveraged in smart biomedical systems, including flexible electronics, wearable sensors, and wireless stimulators, for real-time therapy and diagnostics. Coupling BP with bioactive molecules, aptamers, or therapeutic peptides may also enable disease-specific, on-demand therapeutic responses. (III) Safety and translational evaluation: Long-term toxicological and biodistribution studies in large-animal models, together with standardized data repositories, will be crucial for clinical validation. Coordinated efforts among material scientists, clinicians, and regulatory agencies are needed to establish clear guidelines for BP-based biomaterials. (IV) Emerging interdisciplinary directions: Integrating artificial intelligence and high-throughput screening could accelerate the optimization of BP compositions and predict in vivo behavior. Furthermore, BP's immunomodulatory and microbiome-interactive features open new frontiers in immunoengineering, cancer immunotherapy, and inflammation-associated disorders. Addressing these priorities will be key to transforming BP from a promising nanomaterial into a clinically adaptable, multifunctional platform that satisfies requirements for technological innovation, clinical efficacy, and translational feasibility. In this perspective, BP-based platforms have the potential to contribute to next-generation, patient-tailored therapies at the interface between regenerative medicine, immunotherapy, and bioelectronics, thereby addressing patients' needs and advancing the goals of precision and personalized medicine.
CRediT authorship contribution statement
Ashkan Bigham: Writing – original draft, Visualization, Validation, Software, Data curation, Conceptualization. Anna Mariano: Writing – original draft, Conceptualization. Aldo R. Boccaccini: Writing – review & editing, Visualization. Luigi Ambrosio: Writing – review & editing, Visualization, Supervision. Maria Grazia Raucci: Writing – review & editing, Supervision, Resources.
Ethics approval and consent to participate
The study does not include.
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Clinical study
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Experimentation on animals
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Human subjects
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Declaration of competing interest
Aldo R.Boccaccini is an associate editor for Bioactive Materials and was not involved in the editorial review or the decision to publish this article. The authors have declared that there is no conflict of interest.
Acknowledgement
This study was funded under the National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.1, Call for tender No. 104 published on 2.2.2022 by the Italian Ministry of University and Research (MUR), funded by the European Union – NextGenerationEU– Project SMART4SCLERO 2022W8SLM – CUP B53D23008940006 - Grant Assignment Decree No. 966 adopted on 30.06.2023 by the Italian Ministry of Ministry of University and Research (MUR) and ASTROTALK Project – AFOSR Grant Agreement nr. FA9550-23-1-0736 – CUP B53C23006960005.
Footnotes
Peer review under the responsibility of editorial board of Bioactive Materials.
Contributor Information
Ashkan Bigham, Email: ashkan.bigham@unina.it.
Maria Grazia Raucci, Email: mariagrazia.raucci@cnr.it.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.