Engineered self-assembling hydrogel systems for advanced guided bone regeneration: structural optimization and biofunctional modulation

Abstract

Self-assembling hydrogels have emerged as a promising class of biomaterials for bone regeneration due to their highly tunable properties, stimulus responsiveness, and excellent biocompatibility. While current research predominantly focuses on their applications as structural scaffolds or conventional drug delivery systems, recent advancements highlight their potential for spatiotemporal regulation of bone repair through advanced morphological engineering and multifunctional module integration. This review synthesizes recent progress in self-assembling hydrogel-based strategies aimed at enhancing bone regeneration, emphasizing their dual roles as bioresponsive carriers and architectural regulators. Particular emphasis is placed on their capacity for controlled release of bioactive agents and dynamic adaptation to microenvironmental stimuli, particularly when implementing synergistic design principles combining structural optimization with functional multiplexing. The interplay between these modalities within three-dimensional spatial contexts is systematically analyzed, revealing critical synergies that potentiate osteogenic outcomes. Finally, unresolved challenges related to degradation kinetics, vascularization induction, and precise spatiotemporal control are critically evaluated, along with proposed solutions to advance clinical translation. These collective insights underscore the transformative potential of self-assembling hydrogels in addressing the complexities of critical-sized bone defect regeneration while identifying key directions for future investigative efforts.

Graphical Abstract

Introduction

Bone regeneration following bone defects resulting from diverse etiologies constitutes a multifaceted, meticulously orchestrated regenerative process. The utilisation of materials to fill bone defects, with the aim of promoting bone regeneration, represents a persistent domain of research [1]. Traditional bone defect repair materials such as bone cement are being replaced by a number of new individually customized grafts such as composite ceramics and laponite [2–4]. Despite the fact that new bone fillers are more adaptable and have superior bone-forming properties, they still have disadvantages such as non-degradability, inability to cope with complex changes in the physiological environment and lack of immunomodulatory properties [5–8]. To overcome these limitations, self-assembling hydrogels are receiving increasing attention as novel injectable fillers to promote bone regeneration, particularly in targeted bone repair and regeneration in osteoporotic, locally infected and tumour settings [9–12]. 

Recently, the use of self-assembling hydrogels as drug-carrying platforms for delivering osteogenesis-related drugs and stimulus-responsive strategies for modulating drug release has opened new novel directions for bone regeneration (Table 1). Compared with the drawbacks of traditional bone defect repair materials, such as limited availability, immune rejection, poor osteoinduction and osteoconductivity, and poor biocompatibility and degradation properties, self-assembling hydrogels resemble the environment of a cytoplasmic matrix that promotes cellular adhesion, migration, proliferation, and division, and their degradation products are naturally occurring harmless proteins, which make them good scaffolding materials for regenerative medicine [13–15]. Self-assembling hydrogels are very flexible materials that can respond to complex environmental changes and exhibit stable performance with appropriate chemical modifications. Currently, the unique structure and tunable physicochemical properties of self-assembling hydrogels have received extensive attention [16, 17].

Table 1.

Growth factors involved in the healing of bone defects [25–27]

Growth factor	Mechanism of action	Limitations
BMP-2 (Bone morphogenic protein)	Induces osteoblasts proliferation and mesenchymal cells (MSCs) differentiation Induces VEGF-A secretion therefore has a role in angiogenesis	Needs to be delivered in a controlled manner Variable outcomes have been seen in humans Limited capacity to initiate vascular proliferation
CGF(Concentrated Growth Factor)	Induces osteogenic differentiation of BMSCs.
VEGF (Vascular endothelial growth factor)	Induces endothelial cells mitogenesis Attracts MSCs and induces their differentiation	Delivered alone they lead to the inability to produce organized bone regeneration
PDGF (Platelet derived growth factor)	Attracts cells that stabilize growing vasculature Recruits MSCs Upregulates VEGF production
FGF (Fibrobast growth factor)	Involved in the formation of new capillaries
IGF (insulin like growth factor)	Involved in adult neo angiogenesis
TGF-β(Transcription growth factor β)	TGF-β1 promotes the expression of cell adhesion molecules and cell condensation. TGF-β3 majorly promotes cell proliferation.

At present, most academic research has focused on the use of “ordinary hydrogels” as carriers for drug delivery but is limited by the instability of the hydrogel itself when subjected to external stimuli, especially in the face of changes in the surrounding physiological environment, such as an increase in pH and temperature, making it difficult to regulate the rate of drug release, poor targeting and limited drug loading. Poor targeting and limited drug loading are also important factors limiting its therapeutic effect. Therefore, the therapeutic effect of a drug release system using an “ordinary hydrogel” as the carrier is often unsatisfactory, or its application has great limitations.

To date, only a few hydrogels have been approved for clinical therapeutic and tissue engineering applications, e.g., hyaluronic acid hydrogels and some special liposomal hydrogels [18]. To address these issues, self-assembling hydrogels have been designed to change their morphology spontaneously in response to changes in the physicochemical properties of the internal environment or to indirectly control drug release on the basis of external artificial modulation. The changes are often reversible and can adjust the rate of drug release according to the actual healing situation of the desired part of the body, effectively avoiding excessive drug release and improving the therapeutic index, such as multiresponsive self-assembling hydrogels for diabetic wound healing and thermoresponsive artificially regulated smart self-assembling hydrogels for promoting vascular regeneration [19, 20].

The unique microstructure and highly editable functionality of self-assembling hydrogels can perfectly compensate for the shortcomings of traditional bone-enhancing materials. Self-assembling hydrogels are small organic molecules that can form immobile colloidal structures in water by forming water-encapsulated supramolecular networks through the process of molecular self-assembly at very low concentrations (the mass fraction can be less than 1%). These small organic molecules that can form hydrogels form ordered aggregates mainly through intermolecular noncovalent interactions such as hydrogen bonding, π‒π interactions, electrostatic forces, van der Waals forces, etc., and further self-assemble to form multilayer structures. Individual noncovalent interactions are usually weak and reversible; however, when they act together, they form robust self-assembling structures [21]. This unique structure allows the self-assembling hydrogel to adapt to the changing physicochemical environment during bone regeneration at the bone defect site while ensuring sufficient drug-carrying space, maintaining the stability of the encapsulation, and achieving the therapeutic effect of long-lasting and stable induced bone regeneration. Moreover, the unique stimulus responsiveness of the self-assembling hydrogel allows it to support the spatial and temporal adjustment of drug release and structural changes in vivo and in vitro, perfectly adapting to the physiological state of bone regeneration at various stages, and fully improving the utilization rate of the drug.

Traditional hydrogel systems face significant challenges in practical applications, particularly for the repair of large-sized and load-bearing bone defects, substantially limiting their clinical utility. A fundamental limitation is their inherently inadequate mechanical properties. These materials typically exhibit low elastic modulus, insufficient compressive and tensile strength, and poor fracture toughness. This mechanical mismatch with native bone tissue renders them susceptible to failure under physiological loading conditions. Consequently, this critical shortfall impedes the widespread clinical adoption of hydrogels for functional bone restoration.

The limitations of traditional hydrogels underscore the critical need and unique potential of self-assembling hydrogels (Fig. 1). The key advantage of self-assembly resides in the precise control over nano- and micro-scale architectures achieved through the programmable interactions of designed molecular building blocks. This inherent microstructural tunability provides a robust foundation for engineering enhanced mechanical performance. By strategically selecting self-assembling components (e.g., peptides, polymers, supramolecular units) and optimizing assembly parameters, hydrogel networks with significantly improved stiffness, strength, and toughness can be fabricated – properties essential for withstanding the mechanical demands inherent to bone regeneration sites. Furthermore, the inherent structural programmability of self-assembling hydrogels, coupled with their highly tailorable functionality, facilitates the precise integration of biochemical cues (e.g., growth factors) and enables the design of customized degradation profiles within mechanically robust matrices (Fig. 2).

Fig. 1.

Abstract Schematic of self-assembling hydrogels in bone regeneration. This schematic summarizes the assembly mechanisms of the self-assembling hydrogel system, its application as a drug delivery platform, and the guidance of stimuli-responsive behavior for bone regeneration

Fig. 2.

Schematic illustration of bone regeneration and fracture repair accomplished with the assistance of self-assembling hydrogels. The schematic includes instructions for whole-body multiple-outgrowth bone regeneration repair assisted by self-assembling hydrogels, such as cranial defect repair and femoral fracture healing and regeneration, and illustrates the various forms of self-assembling hydrogels that can be used to act on the human body, including bioscaffolds, direct injections, microneedles, and small-molecule drug-carrying systems

The capacity of self-assembling hydrogels to address the mechanical deficiencies of traditional systems via microstructural engineering provides a compelling rationale for investigating drug delivery strategies within these platforms. The potential to combine necessary mechanical support with sophisticated localized therapeutic release capabilities positions self-assembling hydrogels as promising candidate materials for comprehensive bone regeneration.

This article provides a comprehensive review of self-assembling hydrogels with a focus on their application in bone tissue regeneration. We systematically examine the fundamental principles and distinctive microarchitectures underlying these materials, while critically evaluating their current clinical implementations for osteogenic induction. The discussion encompasses both structural merits and functional limitations inherent in hydrogel design strategies, followed by a categorization framework for polymeric substrates employed in self-assembling hydrogel fabrication. Particular emphasis is placed on innovative drug delivery paradigms and stimulus-responsive systems engineered for bone regeneration applications. Through detailed analysis of preparation methodologies and application-specific optimization strategies, we highlight recent advancements in programmable hydrogel platforms that respond to physiological or external stimuli. The review concludes with an assessment of persistent challenges in clinical translation, proposing essential physicochemical criteria for ideal hydrogel scaffolds and outlining future research directions. We further explore the potential integration of emerging smart materials with self-assembly technologies to develop next-generation regenerative solutions.

Morphological design and characteristics of self-assembling hydrogels

Classification

Self-assembling hydrogels can be classified by many factors, such as the source; preparation method; lattice structure, including nonpermanent, i.e., physically cross-linked, such as hydrogen-bonded hydrogels, and permanent, i.e., chemically cross-linked, irreversible; size and morphology, such as spherical hydrogels, membrane hydrogels, fibrous hydrogels at the macroscopic level, and microscopic micro/nanomicrosphere hydrogels; environmental sensitivity, such as physical, chemical and biochemical stimuli; charge of the polymer network, including nonionic, ionic, zwitterionic and amphoteric; and degradability, including biodegradable and nonbiodegradable hydrogels (Fig. 3). The different structural designs of self-assembling hydrogels, such as micelles, hydrogels, and vesicles, are also important factors in their classification, and different building structures can confer different properties and applications to self-assembling hydrogels [22].

Fig. 3.

Schematic of two typical synthesis and application schemes for hydrogels. Process 1: By completing in vitro premixing and self-assembly with exogenous drugs at a specific pH, asepticization and strength alteration under UV light irradiation, hydrogel scaffolds are generated for the healing and support of bone defects. Process 2: The self-assembling hydrogel is cocultured with autologously derived osteogenic factors (e.g., MSCs), and the hydrogel loaded with osteogenic factors is directly injected into the bone defect site to complete targeted release

The morphological design of self-assembling hydrogels is based on their assembly principles. Coiled coils and β-sheets are well-known methods of secondary peptide self-assembly, and scholars have investigated the purpose of changing the secondary structure of self-assembling hydrogels by switching the peptide’s secondary structure and connecting linear synthetic molecules to existing self-assembling peptides to change their secondary structure [23].

Self-assembling hydrogels are constructed by the self-assembly of monomer components into polymer-like protofibrils through noncovalent interactions and the subsequent entanglement of these protofibrils into an extended network that causes aqueous solvent gelation. The essence of self-assembling hydrogels is that small organic molecules form a supramolecular network structure that encapsulates water through the process of molecular self-assembly under very low concentration conditions (the mass fraction can be less than 1%), thus forming a stable colloidal structure in water. The chemical bonding through which small organic molecules combine to form self-assembling hydrogels is also an important classification factor for self-assembling hydrogels. For example, ordered aggregates can be formed through intermolecular noncovalent interactions such as hydrogen bonding, π‒π interactions, electrostatic forces, van der Waals forces, etc., and further self-assembly to form multilayer structures [21].

Self-assembling hydrogels are constructed by the self-assembly of monomer components into polymer-like protofibrils through noncovalent interactions, and the subsequent entanglement of these protofibrils into an extended network leads to aqueous solvent gelation. Self-assembling hydrogels have been shown to have advantages over covalently bonded hydrogels in biomedical applications, including tissue engineering and drug delivery. Another important classification factor for self-assembling hydrogels is the biological or biomimetic molecular components that make.

Self-assembling hydrogels, including proteins, peptides, amino acids, carbohydrates, therapeutic agents, and oligonucleotides, are also responsible for their biocompatibility [14, 24].

Characteristics

Self-assembling hydrogels have many properties, and these properties in turn determine the way they are not used in the fields of biomedical and tissue engineering (Fig. 4). The hydrogel material based on deacetylated chitosan has the characteristics of strong water absorption, excellent compatibility, degradability and inexpensive construction and is primarily used as a wound dressing [28, 29]. The hydrogel materials with the addition of collagen and gelatin have good thermal and chemical stability, high mechanical strength, biodegradability, nontoxicity, convenient cross-linking with other substances and high biocompatibility properties and are currently mostly used for bone reconstruction and drug delivery [30, 31]. Hydrogel materials with added heparin can be combined with various growth factors to promote angiogenesis and the growth of cells [31, 32]. Excellent biocompatibility and biodegradability [33], injectability, outstanding absorbability [34], good mechanical stability [35], adjustable stiffness [36], easily adjustable hydrophilicity/hydrophobicity [37], high sensitivity to stimuli [38], and good tissue viscoelasticity [39] are the main properties of self-assembling hydrogels that have been widely noted. Currently, self-assembling hydrogels have been proposed for several biological applications and diagnostic studies, including drug delivery and tissue engineering [40, 41] (Fig. 5). For the application of self-assembling hydrogels in the biomedical field, three significant properties are highlighted below.

Fig. 4.

Comparison of conventional hydrogels and self-assembling peptide hydrogels for biomedical applications. (A) Scheme for enhancing the mechanical properties of self-assembling hydrogels by adding polymer colloids [42]. (B) Self-assembly process for self-assembling peptide hydrogels and biomedical applications [38]. (C) Properties of hydrogels for biosensing applications [33]

Fig. 5.

Schematic representation of self-assembling modes of several DNA hydrogels [39]. (A) Shows the initial DNA template used as a starting structure for assembly. (B) Illustrates rolling circle amplification (RCA), producing a long single-stranded DNA (ssDNA) used as a building block. (C) Represents a patterning step using ssDNA. (D) Continues the assembly process with a different single-stranded DNA, suggesting modular construction of the hydrogelf

Mechanical properties

Hydrogels are hydrophilic macromolecular polymer networks that naturally result in good water absorption, as well as flexibility, versatility, stimulated responsiveness and soft structure [43]. In particular, self-assembling hydrogels also exhibit unique viscoelasticity when the material changes, a property that results in both viscous and elastic properties due to conformational changes within the polymer structure [44] (Table 2). The viscoelasticity of self-assembling hydrogels is an important factor in their ability to mimic the natural ECM microenvironment in the human body [45].

Table 2.

Comparative mechanical properties of traditional hydrogels vs. Self-Assembled hydrogels for bone regeneration

Property	Traditional hydrogels	Self-assembled hydrogels	Ref
Elastic modulus	Typically low (0.1–20 kPa); limited adjustability \	Highly tunable (5–500 kPa); can be tailored to match bone mechanical microenvironment	[55, 56]
Compressive Strength	Generally weak (< 50 kPa); prone to structural collapse	Enhanced (0.1–5 MPa); improved structural integrity under load	[57, 58]
Elasticity Recovery	Often exhibits incomplete recovery; significant hysteresis	High resilience; minimal permanent deformation due to dynamic crosslinking	[59, 60]
Fatigue life	Low cyclic durability; rapid crack propagation under repeated loading	Superior fatigue life; stable performance under prolonged physiological loading conditions	[61, 62]
Applicability in bone regeneration	Restricted to non-load-bearing applications	Suitable for minimally load-bearing and critical-size bone defect regeneration	[63–65]

Self-assembling hydrogels are not mechanically strong by nature, but their mechanical properties can be altered by incorporating different materials into the hydrogel. Chemical cross-linking is an effective mechanism for enhancing the mechanical properties of hydrogels, but additional synthetic steps that may require changes in the formation and processing of the target hydrogel are needed [46]. Adding and changing the polymer species in the polymer network of self-assembling hydrogels, such as polyethylene glycol (PEG) [47], hyaluronic acid [48], alginates [49] and proteins [42], which can be incorporated into hydrogels to tune their mechanical properties and change their elastic modulus while also introducing new functions, is another approach to improve their mechanical properties. For example, reinforcing mechanically weak hydrogels with fibres is a promising way to obtain strong materials for biomedical applications while preserving a favourable cellular environment [50]. Fibre-reinforced hydrogels also spontaneously produce layered structures similar to those found in natural tissues, such as articular cartilage, which facilitates cell growth and drug absorption [51].

Another advantage of self-assembling hydrogel materials is that they maintain high tensile modulus after moulding while retaining injectability and can be customised to achieve the desired implant or injection geometry. Specifically, the optimised self-assembling hydrogel exhibits suitable injection time, with tensile strength enhanced to 321.1 kPa and fracture energy reaching 29.0 kJ/m² [52]. Edited injectable self-assembling hydrogel can achieve a compressive modulus of 1 MPa at merely 5.0 wt% polymer concentration while incorporating ultra-low-molecular-weight components (MW < 300 Da)—a feat rarely attainable with conventional hydrogels at equivalent concentrations [53]. Additionally, by adjusting the cross-linking method and degree of cross-linking, the degradation rate, porosity, or release profile can be easily modulated [54]. The translation of hydrogel-based scaffolds into bone regeneration therapies is critically dependent on their mechanical properties, which must closely mimic the native bone microenvironment to provide adequate structural support and biointegration. Conventional hydrogels often fall short in meeting the stringent mechanical demands of load-bearing bone defects due to their inherent limitations in stiffness, strength, and fatigue resistance. In contrast, self-assembled hydrogels exhibit highly tunable and superior mechanical behavior, owing to their nanoscale architectural control and dynamic crosslinking mechanisms. The following table provides a comparative overview of key mechanical properties between traditional and self-assembled hydrogels, highlighting the latter’s enhanced suitability for functional bone tissue engineering.

Biodegradability

Another important advantage of self-assembling hydrogels proposed for drug delivery, tissue engineering, wound healing, and bone regeneration applications is their biodegradability [40]. The major degradation approaches of polymer hydrogels can be categorized as hydrolysis, controlled degradation via enzymes and stimulation of the corresponding degradation.

Hydrogels themselves are highly water-containing polymer networks similar to the ECM environment of biological cells and exhibit natural biocompatibility and degradability. The degradability and degradation rate of these materials are accomplished mainly by tuning the degree of polymer degradation. The degradation of peptide-based self-assembling hydrogels can be achieved via the use of proteases (Fig. 6). For example, endothelial cell-derived matrix metalloproteinases (MMPs) attack collagen by changing the peptide sequence to achieve controlled degradation of peptide hydrogels [66, 67].

Fig. 6.

Biodegradable properties and applications of self-assembling hydrogels. (A) Environmentally triggered self-assembly and degradation of hydrogels [66]. (B) Realization of spatiotemporally controlled release of metalloproteinase-9 (MMP-9)-responsive self-assembling hydrogel structures [67]. (C) Schematic representation of self-assembling hydrogels for tissue engineering and bone regeneration applications [40]

In addition to cleavable sequences, the secondary structure of the peptide hydrogel also affects MMP-based degradation [68]. A starch hydrogel was synthesized via a thiol‒alkene click reaction between thiol starch (St-SH) and allyl starch (AS). The biodegradation of this hydrogel proceeded rapidly in the presence of α-amylase. The rate of biodegradation can be controlled by the thiol-ene molar ratio or α-amylase concentration [69].

The biodegradation of self-assembling hydrogels can be modulated and influenced through multiple approaches, including the use of hydrophilic peptide sequences, peptide concentration, molecular design strategies for self-assembly, and crosslinking density modulation. Specifically, the incorporation of hydrophilic short peptides as self-assembly building blocks for hydrogel construction enables the conferral of injectability, reversibility, and excellent biodegradability to the hydrogel system while preserving the inherent functionality of the peptides [70]. Conversely, incorporating specific amino acid residues into hydrophilic self-assembling peptide hydrogels can significantly reduce the degradation rate through enhanced proteolytic stability, thereby endowing the self-assembling hydrogel system with superior drug delivery capabilities [71].

When hydrogel systems are formed through the self-assembly of two or more peptides or other components, both peptide concentration and specific spatial conformations influence the system’s biodegradation. For dual-component supramolecular self-assembling peptide hydrogels, increasing peptide concentration while maintaining a fixed stoichiometric ratio enhances the physical properties of the hydrogel, concomitantly reducing its degradation rate [72]. Deng et al. designed hydrogel system loaded with biotin-modified exosomes via self-assembly to achieve sustained drug release for up to 7 days in vitro. Following subcutaneous implantation, stable retention of osteogenic components within the hydrogel was observed for as long as 28 days. This system demonstrates excellent compatibility with the regeneration of complex periodontal tissues, including acellular cementum, periodontal ligaments (PDLs), and alveolar bone [73]. Distinctive spatial conformations, such as the DNA-like architecture characteristic of self-assembling nucleotide-peptide hydrogel systems, can impart tunable biodegradation profiles to the hydrogel system tailored for specific therapeutic requirements [74].

Adjustments in molecular design and crosslinking strategies for self-assembling hydrogels also critically impact the biodegradation profile of the entire system. For instance, low-molecular-weight hydrogels formed through non-covalent self-assembly demonstrate more desirable degradation kinetics than conventional hydrogel carriers for biomedical applications [75]. Strategic molecular design of the constituent peptides enables precise modulation of multiple properties in self-assembling hydrogels, including biodegradation kinetics. For instance, a binary hydrogel system composed of two oppositely charged dipeptides can undergo in situ gelation via electrostatic interactions between the peptides. This system exhibits tunable mechanical properties, with an elastic modulus ranging from 4 to 62 kPa, alongside controllable biodegradability spanning from several days to weeks. Such features make it an ideal platform for mimicking the natural microenvironment of various cell types [76].

Notably, in addition to the factors mentioned above that affect the degradation rate of self-assembling hydrogels, the biodegradation rate of hydrogels is also related to their water absorption rate and functional base sequence [40, 66]. Stimulus-responsive hydrogels can spontaneously accomplish degradation by responding to stimuli generated by changes in environmental temperature, pH, light, electromagnetism, etc [77, 78].

Stimuli-responsive properties

A complex physicochemical environment often exists at the site of a bone defect, which hinders autologous bone repair and bone regeneration to promote the transport and efficacy of drugs and bioactive components. Conventional drug delivery systems and bone graft materials are often unable to cope with the complex biological environment at the injury site. Self-assembling hydrogels are naturally reversible and dynamic through spontaneous ordered bonding in the presence of noncovalent bonds, which endows the molecular structure of hydrogels with unique stimulus-responsive and self-repairing capabilities [19]. Owing to this unique advantage, self-assembling hydrogels can be perfectly adapted to complex biological environments, responding to external or internal stimuli on the basis of mimicking the microenvironment of natural tissues to easily achieve a single-stimulus response, double-stimulus response or even multiple-stimulus response, triggering and modulating drug release [79].

The stimulus responsiveness of self-assembling hydrogels to the environment, which can be artificially edited and regulated, is also an important property for their application in medicine. The microenvironment of a diseased part of the human body is very different from that of a normal part, e.g., elevated temperature, elevated pH and increased secretion of specific enzymes. After editing, the self-assembling hydrogel can respond to changes in the ambient temperature, pH, electric charge, magnetic field, light, glucose, or enzyme environment, adjusting the timing and conditions of its own self-assembly and thus regulating the precise delivery and release of local drugs [78, 80].

There are many viable strategies for designing self-assembling hydrogels for stimulus responsiveness, and they share the same principle of combining the ability to recognize a target stimulus with the ability to induce structural changes in the self-assembling hydrogel network [80–84]. For example, target recognition events are combined with the response behavior of pH- or temperature-responsive polymers [78, 81]. Strategically designing the response of self-assembling hydrogels for use in drug delivery systems by determining the type of stimuli that may be present at the lesion site is currently the main option for the application of self-assembling responsive hydrogels [77].

Self-assembling hydrogels for guiding bone regeneration in drug delivery systems

The ideal drug delivery system releases the drug continuously in a controlled manner, maintaining the appropriate drug concentration in the defect area without affecting healthy bone tissue [85]. The transportation of osteogenic actives and biomaterials in specific carriers to the site of bone defects for limited and sustained administration is an emerging strategy to promote bone regeneration [86]. The secondary structure of self-assembling hydrogels, such as peptides, is very easy to break and reorganize in response to environmental changes, which is an important basis for self-assembling hydrogels to be used as drug carriers to achieve precise drug delivery and controlled release, which is fundamentally different from traditional drug delivery systems [87].

The different binding modes of self-assembling hydrogels also determine their form of action. For example, self-assembling hydrogels are bound by dynamic reversible covalent bonds, and when the surrounding environment changes, the reversible covalent bonds are destroyed, and the wrapped drug is released, thus realizing the controlled release of the drug. Self-assembling hydrogels bound by stable chemical bonds, on the other hand, can be used as carriers to release drugs slowly to achieve the sustained release of drugs [88, 89]. The controlled solid‒liquid conversion of self-assembling hydrogels allows them to be used as drug-carrying substrates, which can be directly injected or used as coating materials for bone defect surfaces. Moreover, the unique three-dimensional network structure, high hydrophilicity, biocompatibility, and biodegradability of self-assembling hydrogels determine their excellent drug-carrying capacity and minimize adverse biological reactions, which are difficult to compare with those of traditional drug delivery systems. As summarized in Table 3, seven major bioactive cargo types delivered via self-assembling hydrogels were systematically categorized based on their loading mechanisms and release behaviors. Accumulating evidence confirms that self-assembling hydrogels can serve as effective drug carriers in bone regeneration applications, improving drug stability while demonstrating excellent spatiotemporal control release characteristics and significantly enhancing therapeutic efficacy [90–94].

Table 3.

Therapeutic agents delivered by self-assembling hydrogels

Therapeutic agent	Examples	Molecular mechanisms	Delivery method	Advantages	Limitations	Potential solutions	Ref
Small-molecule drugs	Adriamycin, paclitaxel, antibiotics	Hydrophobic interaction, hydrogen bonding	Physical embedding/chemical bonding in self-assembling hydrogel networks	Slow/controlled release; Reduce systemic toxicity; Increase local concentration; Extend duration of action	Limited drug loading capacity due to poor solubility; risk of burst release	Use of cyclodextrins or liposomes for solubilization; development of dual-crosslinked or nanocomposite hydrogels for more controlled release; pro-drug strategies	[95, 96]
miRNA/mRNAs	miR-21 inhibitor, miR-34a mimetic	Electrostatic complexation (polyplexes/nanoplexes), covalent conjugation to carriers	Electrostatic adsorption/covalent coupling/encapsulation in nanocomposite hydrogels	Protect nucleic acid degradation; Targeted delivery; Improve transfection efficiency; Local regulation of gene expression	Low cellular uptake and endosomal escape efficiency; off-target effects; instability during scale-up	Chemical modification of nucleic acids; peptide-based endosomolytic agents; lipid-assisted nanoparticles; freeze-drying for improved storage stability	[97, 98]
Exosomes	Stem cell-derived exosome, tumour exosomes	Membrane fusion, endocytosis, ligand-receptor interactions	Physical embedding/in situ loading of hydrogel networks	Maintains exosome activity; Synergises with hydrogel to promote tissue repair; Slow release prolongs action time; Low immunogenicity	Difficult isolation and purification; limited loading capacity; poorly controlled release kinetics	Microfluidic isolation techniques; engineering exosomes with targeting peptides; hybrid systems with injectable microspheres; stimulus-responsive hydrogels for triggered release	[99, 100]
Bioactive factors	VEGF, BMP-2, EGF, nerve growth factors	Affinity binding, covalent immobilization, electrostatic encapsulation	Covalent fixation/affinity binding/physical encapsulation	Spatial positioning release; Protective factor activity; Reduction of enzymatic inactivation; Simulation of ECM microenvironment	High cost; loss of bioactivity during encapsulation; insufficient local concentration due to rapid diffusion	Fusion with stabilizing protein domains; site-specific conjugation; use of heparan sulfate mimetic peptides; co-delivery with protease inhibitors	[101, 102]
Metal ions		Ionic coordination, cross-linking via dynamic bonds, redox activity	Ion coordination integration/as a cross-linking agent for self-assembling hydrogels	Ion-controlled release synergistic therapy (antibacterial/osteogenic); Responsive degradation (e.g., pH/enzyme-triggered); Enhanced hydrogel mechanical properties	Concentration-dependent cytotoxicity (e.g., Ag⁺); potential local accumulation; disruption of ionic homeostasis	Chelator-controlled release systems; biodegradable ion-doped nanoparticles; combination with antioxidant agents to mitigate oxidative stress	[52, 103,103,104]
Cells	Human/animal-derived MSCs	Cell-matrix adhesion (integrin-mediated), paracrine signaling	3D encapsulation in hydrogel networks/microsphere composites/growth factor co-delivery	Promotes in situ regeneration of tissues; Paracrine effects enhance repair; Hydrogels mimic the extracellular matrix (ECM) microenvironment	Low cell viability post-encapsulation; poor control over differentiation; immune rejection; limited long-term functionality	Genetic modification to enhance survival/differentiation; use of immunomodulatory hydrogels; oxygen-releasing or nutrient-enriched scaffolds; bioprinting for spatial organization	[105, 106]
Plant-extracted components	Baicalin, curcumin, leucoanthocyanidin	Hydrophobic encapsulation, π–π stacking, integration into amphiphilic networks	Hydrophobic encapsulation/self-assembling hydrogel network cross-linking	Improve water solubility/bioavailability; Reduce irritation; Multi-component synergistic drug delivery	Poor standardization; chemical instability; low loading efficiency; lack of pharmacokinetic studies	Nanoemulsion-based encapsulation; solid dispersion techniques; chemical stabilization; pharmacokinetic optimization via PEGylation or liposomal formulation	[107–109]

Small-molecule drugs

Current clinical approaches to bone regeneration remain predominantly dependent on protein-based growth factor therapies, as exemplified by INFUSE^® – the commercially available BMP-2 delivery system approved by the FDA [110]. While these biologics have demonstrated therapeutic potential, their clinical efficacy is substantially compromised by intrinsic challenges in controlled drug delivery and pharmacological maintenance [111].

Self-assembling hydrogel systems have emerged as promising alternatives to address critical limitations of conventional small-molecule therapeutics, particularly concerning aqueous solubility and structural stability. Through their unique molecular organization and tunable rheological properties, these materials demonstrate enhanced pharmacokinetic profiles when serving as versatile delivery platforms, exhibiting multifunctional therapeutic capabilities in localized anti-inflammatory interventions, wound management, and antimicrobial/antitumor applications.

Injectable hydrogel systems containing bioactive molecules, such as doxorubicin (DOX), are a novel treatment option for localised bone tumours. To address the issue of osteogenesis inhibition caused by excessive local DOX concentrations, a new injectable self-assembling hydrogel system that combines photodynamic and chemotherapy has been developed. Fu J et al. demonstrated that a self-assembling hydrogel system incorporating the small molecule drugs osteoprotegerin Icariin (ICA) and DOX exhibits excellent drug loading capacity and sustained drug release properties. The bioactive small molecule ICA incorporated into the self-assembling hydrogel system exhibits significant bone induction and regeneration potential [95]. This research is expected to become a new paradigm for osteosarcoma treatment, achieving a new balance between bone tumour suppression and osteogenic protection through the combination of self-assembling hydrogel systems and bioactive small molecules.

Self-assembling hydrogels have been widely used as therapeutic cell carrier materials to deliver drugs for articular cartilage repair [112]. Self-assembling hydrogels for the delivery of small-molecule drugs can be prepared in a variety of ways (Fig. 7). Zhang, KY et al. prepared a bioactive nanocomposite hydrogel based on hyaluronic acid and self-assembling magnesium pamidronate nanoparticles, which could adapt to irregularly shaped bone defects in terms of mechanical properties and, over time, promoted the osteogenic differentiation of encapsulated human mesenchymal stem cells (hMSCs) and the activation of alkaline phosphatase (ALP) by magnesium ions released from the hydrogel in a positive feedback-regulated manner. The release of Dex, a small-molecule drug encapsulated in a hydrogel, is accelerated in the form of positive feedback regulation, contributing to the promotion of bone regeneration [113].

Fig. 7.

Self-assembling hydrogels loaded with drugs promote bone regeneration. (A) Schematic representation of a “smart” hydrogel that mediates cofactor-assisted activation of biomarker-responsive drug delivery via a positive feedback loop (highlighted in red) to increase osteogenesis in encapsulated hMSCs [113]. (B) Schematic diagram of the injection site of the HA-Pam-Mg nanocomposite hydrogel. (C) Therapeutic options and drug delivery strategies for regeneration during fracture healing [111]

Compared with free drugs, small-molecule drugs that directly self-assemble into supramolecular hydrogels through intermolecular π‒π interactions and hydrogen bonding have better drug potency in addition to excellent biostability, sustained drug release, and reversible stimulus response properties [114]. Self-assembling hydrogels loaded with anti-inflammatory small-molecule drugs have been shown to self-assemble in water and spontaneously form hydrogels consisting of three-dimensional networks of nanofibers under acidic conditions for targeted drug delivery and sustained release [115] (Fig. 8).

Fig. 8.

Self-assembling hydrogels for drug delivery. (A) Schematic representation of the miRNA nanodelivery system [116]. (B) Schematic diagram of self-assembling hydrogel-encapsulated exosomes for bone regeneration [91]. (C) Schematic of in situ hydrogel formation via the thiol–acrylate reaction and encapsulation of RNA-PEI nanocomplexes and hMSCs [117]. (D) Schematic illustration of the self-assembly of HDMC for targeted drug delivery in cancer cells [118]. (E) Schematic diagram of a self-assembling glycocontaining compound hydrogel for the treatment of mucosal wounds [115]. (F) Schematic representation of a self-assembling hydrogel based on a precursor drug [89]

Self-assembling natural small molecule drug hydrogels can spontaneously form without structural modification and can even serve as natural carriers. An herbal natural product, rhubarbic acid, which directly self-assembles into a hydrogel through noncovalent interactions, has been reported in the literature. This hydrogel showed excellent stability, sustained release and a reversible stimulatory response. Compared with the in vitro equivalent of free drug, the rhubarbic acid hydrogel reduces neuroinflammation with long-lasting effects and minimal cytotoxicity [114]. As technology advances and drug development becomes increasingly predictive, personalized and regenerative, precise structural and functional control of nanomaterials at the molecular level is needed to create customized nanostructures. Self-assembly can fulfil these needs by allowing the synthesis of functional nanomaterials from precisely designed building blocks. Thus, self-assembling hydrogels possess unique advantages over traditional drug carriers as drug carriers [71].

Despite offering well-defined pharmacokinetics that facilitate basic osteogenic induction, small-molecule drugs are fundamentally limited by their inability to orchestrate the complex genetic programs necessary for functional bone regeneration. This gap motivates the adoption of miRNAs/mRNAs, which enable epigenetic reprogramming of MSCs through RNA interference or gene activation, thereby fundamentally promoting bone regeneration.

miRNAs/mRNAs

The regulation of gene expression levels via the delivery of synthetic microRNAs (miRNAs) is a well-established gene therapy approach. MicroRNAs serve as effective molecular managers that can regulate multiple endogenous signalling pathways simultaneously [119]. MicroRNA delivery provides a way to mimic natural RNA to the maximum extent possible to simulate the bone regeneration microenvironment [116]. Hypertrophic cartilage formation is a major setback for mesenchymal stem cell (MSC)-mediated cartilage repair, and overcoming it requires optimal differentiation [120]. Previous studies have shown that miRNAs act on bone marrow mesenchymal stem cells (BMSCs) in cartilage differentiation and promote bone tissue regeneration and repair by suppressing the expression of specific genes, with significant success in promoting articular cartilage growth and treating massive bone defects [121]. However, direct delivery of miRNAs is very challenging, resulting in inactivation, low transfection efficiency and a short half-life [122]. Self-assembling hydrogels can provide a stable microenvironment for miRNA delivery and can precisely modulate miRNA release [123] (Fig. 9).

Fig. 9.

Mechanisms of RNA vaccine delivery. (A) Schematic mechanism of vaccine antigens with nonamplifying mRNAs (namRNAs) versus self-amplifying mRNAs (samRNAs). The choice of the appropriate route of administration may influence the efficacy of RNA drugs [129]. (B) Mechanisms of action of mRNA vaccines and strategies for RNA delivery [132]. (C) Schematic showing the various osteogenic proteins for which mRNAs could be encoded and the ways in which the synthesized mRNAs could be formulated for application [133]

One type of hydrogel formed from polyethylene glycol (PEG) and cross-linked by a click reaction is loaded with cholesterol-modified miR-26a, which is covalently bound to the fibres of the hydrogel by UV-cleavable linkers, thus preventing the accidental release of miRNA. This hydrogel allows for light-controlled release of miRNA in vitro and in vivo, resulting in bone regeneration [124].

RNA interference (RNAi) may be an effective and valuable tool to promote functional tissue growth, as short interfering RNAs (siRNAs) and miRNAs can block the expression of genes that negatively affect tissue regeneration [117]. The delivery of RNAi molecules may be a valuable strategy to guide cellular behavior in tissue engineering applications. A PEG hydrogel formed in situ by a Michael-type reaction was proposed that allows the encapsulation of hMSCs and the accompanying siNoggin and/or miRNA-20a. Researchers have selected these RNAs to inhibit the BMP-2 antagonists noggin and/or PPAR-y (BMP-2-mediated negative regulator of osteogenesis), thereby promoting hMSCs and subsequent osteogenic differentiation and repair of critical-sized rat cranial defects. Compared with the controls, the in situ-formed polyethylene glycol hydrogel system provided an exciting platform for critical size bone repair while enabling local controlled delivery of RNAi molecules to the encapsulated hMSCs and surrounding cells, resulting in greatly enhanced bone regeneration and repair at the defect area [117].

Wang, WC et al. developed a self-assembling RNA hydrogel consisting of a tumor suppressor miRNA (miRNA-205) and an antimetastatic miRNA (miRNA-182). This self-assembling miRNA hydrogel efficiently delivered DNA CpG and short hairpin RNA (shRNA) cofactors, the MnO2-loaded photodynamic drug chlorine e6 (MnO2@Ce6), and the chemotherapeutic drug doxorubicin (DOX) into MDA-MB-231 cells, alleviating tumor hypoxia while precisely targeting drug delivery. This strategy can be used to design any other miRNA as a vector to treat cancer in combination with other therapeutic approaches, thus overcoming the limitations of current cancer therapies [118].

Currently, one of the major advances in the field of bone regeneration therapeutics is the development of mRNAs. mRNA is already widely used in vaccines and can be used as a potential inducer of bone regeneration [125]. Owing to the innate nature of mRNAs, such as their relatively large size compared with those of microRNAs or oligonucleotides, the rapid degradation of mRNAs even before they reach the cellular compartment and preservation of translation efficiency are great challenges for the stable delivery of mRNA therapeutics [126]. One of the proven methods to improve the delivery efficiency of mRNA particles is their encapsulation via various chemicals, including but not limited to polymers, lipids, and dendrimers [127, 128]. The use of cationically charged lipids and lipid-like materials as encapsulation and delivery media for mRNAs has been one of the oldest and most well-studied approaches to encapsulate mRNAs in the form of liposomes [129, 130]. Self-assembling hydrogels are currently being promoted as popular materials for RNA vaccine delivery systems to promote bone regeneration [131, 132]. Self-assembling hydrogels based on fibronectin, calcium phosphate, PLGA, collagen, and similar biodegradable materials can serve as excellent matrices for bone regeneration. Self-assembling hydrogels have several distinct advantages as drug carriers for delivering mRNA vaccines. First, hydrogels are usually delivered to specific sites, which is compatible with the requirement of local delivery of the intended mRNA particles [133]. Second, the use of bone regeneration-assisted hydrogel matrices to carry mRNAs that promote bone differentiation could theoretically lead to further enhancement of bone regeneration [134]. In addition, the hydrogel can also be tuned for specific spatiotemporal release of mRNA particles [135].

Hydrogels offer a unique strategy for the localized delivery of RNA, overcoming some of the difficulties associated with systemic RNA delivery. They enable localized, controlled and sustained delivery of high payloads while maintaining the biological activity of the RNA. As a result, off-target effects and the need for multiple payload administrations in systemic delivery can be avoided [135].

A nanofiber trivalent peptide hydrogel vaccine has been constructed via spontaneous supramolecular coassembly of three antigenic epitope-coupled peptides, which can mimic the fibrous structure and biological functions of the extracellular matrix and naturally occurring protein assemblies; accurately and flexibly load each antigenic peptide at a defined ratio to promote antigen presentation in dendritic cells; and significantly improve the CD8 + T-cell response and secretion of interferon-gamma (IFN-γ), which provides a simple, customizable, and multifunctional approach for the development of tumor vaccines with significant therapeutic effects [136].

Peptide hydrogel vaccines with self-adjuvant properties were successfully prepared via peptide supramolecular assembly to increase the immunogenicity of antigens, and a strategy to improve the efficiency of the antigen-specific T-cell response was also demonstrated in the studies of Wei Wei and Wang et al. [137]. The presence of bone tumors tends to produce an osteolytic response that prevents bone regeneration. This self-assembling hydrogel vaccine can elicit a better immune response than the bare RNA vaccine but potentially does not cause a localized inflammatory response, an advantage not available with other vectors (Fig. 10).

Fig. 10.

(A) A supramolecular self-assembling hydrogel vaccine with a triple antigenic epitope-conjugated peptide improved the CD8 + T-cell immune response [136]. (B)(C) Formation of a coassembled, self-adjuvant peptide hydrogel vaccine with nanofiber structures and vaccination-mediated E. G7-OVA lymphoma immunotherapy [137]. (D) Schematic diagram of the UMSC-EV-loaded self-healing hydrogel tested in rats with induced femoral condylar defects [138]

Although nucleic acids provide precise genetic control, their vulnerability to enzymatic degradation and poor cellular internalization hinders clinical translation. Natural exosomes emerge as evolutionarily optimized nanocarriers that not only protect therapeutic RNAs but also deliver endogenous bioactive cargoes (e.g., signalling proteins, lipids), leveraging inherent homing capabilities for targeted bone defect repair.

Exosomes

Exosomes are highly specialized and functional bilayer membrane particles. They are thought to be carriers for the transport and delivery of large amounts of proteins, lipids and nucleic acids (genes, noncoding) [139]. Exosomes are released by a variety of cells, including mesenchymal stem cells. They are considered to be an efficient means of communication between cells for growth and functional purposes [140]. Exosomes are also actively involved in tissue damage repair. Exosomes affect target cells involved in tissue repair by facilitating the exchange of mRNAs, peptides and proteins [141]. The major contribution of exosomes to bone regeneration is to promote scaffold functionalization by integrating exosomes from BMP2-stimulated macrophages to avoid heterotopic bone formation and reduce adverse effects. The scaffold also promotes osteogenesis [142]. Exosomes produced by specialized cells, such as umbilical cord MSC-derived exosomes, encapsulated in hydrogels can accelerate bone repair by enhancing angiogenesis at the target site [143].

Purified, unformulated exosomes are taken up by the reticuloendothelial system and are thus rapidly cleared from the body [141]. However, the exosomes in protective hydrogels protect them from degradation and are stored in the hydrogel to provide sustained therapeutic effects [17]. Exosomes formulated in biocompatible and biodegradable hydrogels can be introduced near or at the target tissue site and facilitate the local delivery of highly concentrated therapeutic molecules encapsulated in exosomes [144]. Owing to the structural and physicochemical properties of hydrogels, the release of exosomes can be controlled, and their functional properties can be adjusted by regulating the degradation rate of the hydrogel matrix [145, 146]. In recent years, a new type of cell-free tissue engineering (TE), namely, the use of exosome-loaded hydrogels to promote bone regeneration, has been investigated and developed [138, 147, 148].

A study revealed that synovial-derived MSCs encapsulated in chitosan overexpressed miRNA-126-3p in the presence of hydroxyapatite (HAp) nanoparticles. miRNA-126-3p overexpression delayed the release of MSCs from exosomes (from 2 to 6 days). The integration of BMP2-activated macrophage-derived exosome carrier hydrogels into titanium nanotubes has been shown to be important for enhanced osteogenesis [142]. A self-healing CHA/SF/GCS/DF-PEG hydrogel containing hucMSC-derived exosomes was successfully prepared by Li Wang et al. This hydrogel has a high self-healing ability, perfect surface morphology, precipitated CHA crystals with a small size and low crystallinity of bone minerals, similar to natural crystals, and is expected to be a novel bone grafting material. hucMSC-derived exosomes can persist for a long time at the target site after addition and effectively promote bone repair and regeneration [138].

Exosomes can diffuse through blood vessels and activate stem cells to participate in bone repair; however, existing exosome (Exo) delivery strategies are unable to reach the angiogenic stage after bone injury. The prevailing strategy is to develop intelligently engineered and naturally derived nanovesicles to target specific tissues and cells, in which liposomes stand out among different types of self-assembling nanovesicles owing to their amphiphilicity and nontoxicity [149]. Compared with liposome-based self-assembling nanovesicles, self-assembling hydrogel carriers, although slightly lacking in amphiphilicity, their excellent biocompatibility and highly editable stimulus responsiveness may be able to replace liposomes as more desirable carriers for the targeted transport of exosomes in future studies.

To address the targeted delivery of exosomes, a research team utilized the principle of coassembly to design an enzyme-sensitive Exo delivery material that responds to new blood vessel formation during the angiogenic stage. They used an injectable MMP1-sensitive hydrogel microsphere (KGE) that was made via a microfluidic chip prepared with a mixture of self-assembling peptides (KLDL-MMP1), GelMA and BMSC-Exos. In vivo and in vitro experiments demonstrated that self-assembling hydrogel microspheres carried exosomes and responded to neovascularization temporally and spatially, thus maximizing the ability of exosomes to recruit stem cells [150].

Notably, the use of protein hydrogels for diffusion-based exosome release leads to burst release of exosomes, and a cellulose-based self-assembling cryogenic sponge has been developed to provide controlled exosome release by altering exosome structure in response to temperature changes [151]. The transport of exosomes by traditional hydrogel protein carriers suffers from difficulty in controlling slow release; however, self-assembly may provide a solution to this dilemma. Currently, there have been successful cases of self-assembling forms of other substances, such as liposomes and cellulose chains; however, self-assembling hydrogels are still lacking in this direction, and their unique stimulus responsiveness may be a breakthrough in research.

Beyond exosomal mediation, direct delivery of biological factors remains indispensable for robust osteoinduction. Growth factors like BMP-2/VEGF exemplify high-potency osteogenic agonists that activate conserved signaling pathways, yet require advanced stabilization strategies to overcome their short half-lives in hostile defect microenvironments.

Biological factors

Self-assembling hydrogels are being actively developed for biologic drug delivery, and current clinical translation is limited primarily by low drug loading capacity, insufficient mechanical rigidity, poor storage stability, and poor drug release, which often results in shorter release durations and burst release [152].

The secretion of bioactive substances by live probiotics can accelerate wound healing, but the clinical application of antibiotics inhibits the survival of probiotics. A study by Jiangfan, Yu et al. successfully designed and prepared a Gel/L@FeTA hydrogel. Lactobacillus Royale layer-by-layer self-assembling with Schiff base gels were complexed with precursors of OHA and CCS through layered self-assembly. These biocompatible hydrogels had high water absorption, degradation, and mechanical properties, which improved the survival of Lactobacillus Royceae even in the presence of antibiotics. Thus, the loading of hydrogels containing encapsulated probiotics promotes immunomodulation, angiogenesis and other beneficial processes for in vitro wound healing [153]. This study presents a novel approach to the development of biomaterials for clinical wound management based on self-assembling hydrogels. Pan et al. also proposed a cell sheet capture and release platform based on self-assembling hydrogels, a biological platform that can respond to changes in the sugar content and temperature of the environment to change the self-assembling structure to release inactivated cell sheets with bioactivity [154], which may be a new direction in the investigation of the use of self-assembling hydrogels for the transportation of biologically factorized drugs. The stimuli-responsive, nontoxic and self-healing properties of self-assembling hydrogels allow them to be perfectly loaded with biological factors and adapted to the complex biological environment of the bone defect site, accomplishing the controlled release of biological factors, and there is a great deal of research potential in the targeted transportation of biological factors to promote bone regeneration via self-assembling hydrogel platforms.

Whereas biological factors dominate clinical bone regeneration, the critical role of inorganic components in bone metabolism often be neglected. Metal ions (e.g., Sr²⁺, Mg²⁺, Cu²⁺) serve as ionic osteogenic modulators that enhance angiogenesis-collagen mineralization coupling, offering cost-effective alternatives with superior thermal/chemical stability.

Metal ions

Among the numerous metal ions that maintain the ecological balance of the body, calcium cations (Ca²⁺), magnesium cations (Mg²⁺), and zinc cations (Zn²⁺) promote bone defect repair and bone regeneration in various ways [155–158]. How to deliver these metal ions stably and with local sustained release and on-demand administration are the main clinical limitations [159]. Owing to their biocompatibility and low immunogenicity, hydrogel scaffolds are promising and widely applicable platforms for the use of various therapeutic agents to promote bone tissue regeneration.

Magnesium cations (Mg²⁺) are emerging therapeutic agents for inducing vascularized bone regeneration. Mg²⁺ not only modulates cellular behavior but also stimulates local bone formation and healing [158]. However, given that a continuous supply of Mg²⁺ may prevent bone regeneration and biomineral deposition during late maturation, the controlled delivery of Mg²⁺ via dual or multiple ion systems, especially layered therapeutic ion release systems, often results in better results in clinical applications [160]. A nanocomposite hydrogel based on hyaluronic acid and self-assembling magnesium bisphosphonate (BP-Mg) nanoparticles was developed in 2017 to enhance the hydrogel network structure, promote the mineralization of the hydrogel and mediate the sustained release of Mg2+ [161]. The released Mg²⁺ ions promote stem cell adhesion and spreading on the hydrogel substrate and promote the osteogenesis of seeded hMSCs in vitro. This cell-free hydrogel can support in situ bone regeneration without the use of exogenous cells or inducers, thus greatly simplifying the approach to bone regeneration therapy [161]. Sustained release of Mg²⁺ mediated by an injectable self-healing adhesive hydrogel can also effectively promote regeneration at the fibrocartilage interface, with experimental results superior to those of magnesium-based alloy orthopedic implants, which are limited in rotator cuff repair [162].

Filipinin (SF) hydrogels have significant limitations in current clinical applications because of the lack of reversible cross-linking, the use of nonphysiological conditions, and the difficulty in controlling the gelation time. The above problems can be solved via metal ion coordination self-assembly. Liyang, Shi et al. achieved in situ assembly of SF hydrogels with shear-thinning and autonomous self-healing properties through calcium ion dynamic metal ligand coordination chemistry, which enabled in vitro stem cell proliferation and accelerated bone regeneration of critical size defects of the cranial bone without the need to provide any additional morphogenetic factors. This type of hydrogel has great potential for clinical applications in bone regeneration [163].

Another idea of using metal ions and hydrogels to promote bone regeneration is the direct preparation of functional bone regeneration hydrogels. A self-assembling chelating peptide hydrogel loaded with clusters of osteogenic metal ions extracted from processed pyrite broth, including Fe²⁺, Cu²⁺, Zn²⁺, Mn²⁺, Mg²⁺, and Ca²⁺ ions, was named processed pyrite amine hydrogel (PPH) [164]. Xu, L et al. suggested that, as a reservoir of beneficial metal ion clusters in bone regeneration, the PPH regenerates through the regulation of a variety of genes. These genes are involved mainly in extracellular matrix synthesis, cell adhesion and migration, cytokine expression, antibacterial activity and inflammation. Thus, the PPH accelerated the progression of various bone healing stages and shortened the bone healing cycle by 4 weeks [164]. Their experiments demonstrated that engineered metal ion cluster hydrogels are novel, simple and commercially available bone regeneration hydrogels with potential clinical use (Fig. 11).

Fig. 11.

Metal ion-loaded hydrogel/self-assembling hydrogel for bone regeneration. (A) Schematic representation of the magnesium 2+-thiol coordination of hydrogels prepared by mixing 4-arm-PEG-SH with MgCl2. Ki67 expression in BMSCs cultured in vitro [159]. (B) Schematic representation of the ability of a magnesium ion-containing self-healing hydrogel to enhance regeneration at the fibrocartilage interface in a rotator cuff tear model [162]. (C) Schematic diagram of processed pyritum self-assembling peptide chelating hydrogel (PPH) construction and promotion of bone defect regeneration [164]

Research on the combination of self-assembling hydrogels and metal ions to promote bone regeneration is still limited, and the research direction is focused mostly on the osteogenic effect of specific metal ions and lacks in-depth exploration of the hydrogel as a carrier; thus, the material properties of self-assembling hydrogels are not fully understood, and scholars can attempt to combine the reversible assembly of self-assembling hydrogels and the loading of metal ions with the local sustained delivery of the drug to investigate a more efficient platform for the delivery of the hydrogel.

Despite the bioinorganic utility, ionic cues alone cannot replicate the dynamic cellular interactions of native osteogenesis. Cell-based self-assembling hydrogel delivery systems—particularly stem cells genetically engineered to overexpress osteogenic factors—provide living microfactories for sustained trophic factor secretion and direct matrix deposition at defect sites.

Cells

In vitro cell culture has been an important tool for biomedical research. Since hydrogels contain a large amount of water, which is close to the composition of the extracellular matrix, hydrogel materials have been widely used in in vitro three-dimensional cell culture. DNA, as a kind of polymer compound, can self-assemble to form hydrogels through complementary base pairing. Compared with traditional cell culture hydrogel materials, such as sodium alginate and peptides, the application of self-assembling DNA hydrogels to three-dimensional cell culture will enable the preparation of highly intelligent and tunable in vitro cell culture scaffolds. The self-assembling DNA hydrogels used as cell culture platforms can be gel-forming at nanomolar DNA concentrations and have adjustable mechanical properties and thixotropic rheology, with the ability to undergo repeated gel formation and cell recycling [165].

Mesenchymal stem cells (MSCs) are promising candidates for various tissue cell therapies because of their self-renewal ability, multispectral differentiation potential, immunomodulatory properties and very promising strategy for nonscaffolded bone regeneration [166–168]. The mechanism by which mesenchymal stem cells (MSCs) promote bone regeneration and repair is mainly by being recruited to the site of injury and differentiating into chondrocytes or osteoblast precursors after the inflammatory phase in the bone repair and regeneration phase, leading to bone repair and regeneration [169, 170]. Direct application of MSCs and their secreted ECM to the bone defect area can result in osteogenic effects [171]. However, direct injection often leads to uncontrolled cell proliferation and differentiation, reduced cell viability and poor grafting [172]. In addition, although MSCs have multidirectional differentiation potential, they do not have the ability to differentiate into osteoblasts by themselves; they need to be stimulated by various growth and differentiation factors for further transformation into osteoblasts and chondrocytes [173]. Many publications have shown that the extraction, expansion and implantation methods of MSCs can significantly affect bone formation outcomes [173].

Hydrogels have been widely used as drug carriers for bone regeneration and osteochondral repair [174]. The advantages of self-assembling hydrogels as carriers of MSCs for promoting bone regeneration are numerous. First, self-assembling hydrogel gels have been designed to specifically adapt to the size of the bone defect, while the internal space can support the morphology of the cells [172]. Self-assembling hydrogel materials inherently possess a 3D mesh structure that allows for cell encapsulation and storage. The encapsulation of cells in microscale hydrogels can simulate a three-dimensional (3D) microenvironment to support cell viability and function and protect cells from environmental stresses, which has been widely used in tissue regeneration and cell therapy [175]. Second, hydrogel materials are not toxic to MSCs [176]. In addition, hydrogel materials are highly biocompatible and have low immunogenicity, which can create a microenvironment conducive to the osteogenic differentiation of MSCs, including recruited endogenous cells [177–179]. The ability of hydrogel materials to achieve targeted and precise delivery and sustained release of drugs and therapeutic cells is also one of the advantages of their use as MSC carriers for bone regeneration and bone repair [180] (Fig. 12).

Fig. 12.

Strategies for hydrogels to contribute to bone by inoculating or targeting transported cells. (A) Schematic diagram of the BMSC-loaded liquid hydrogel for bone defect regeneration [174]. (B) Design of clay composite hydrogels for controlled drug delivery and drug loading and the mechanical properties and effects of composite hydrogels [152]. (C) Preparation of cell-permeable and injectable (Ci-I) gelatin hydrogels and the use of Ci-I hydrogels as therapeutic carriers (cells and drugs) to promote bone defect regeneration in a rat model [182]. (D) Schematic illustration of nanogel (NG) assembly and peptide grafting. The spatiotemporal release profiles of BMP-2 and VEGF were determined via a patterned hydrogel-based sustained release system [186]

The high water content, tunable mechanical properties and diverse synthesis strategies make hydrogels ideal cell carriers and reservoirs for controlled drug release [94]. Biopolymer hydrogels have been widely used as carriers of therapeutic cells and drugs for biomedical applications [181]. One key to the preparation of hydrogel carriers for the transport of MSCs to promote bone regeneration and bone repair is to make them highly dynamic networks, thus allowing the encapsulation of cells while being injectable [182, 183]. Another key factor is that hydrogel materials used as MSC transport carriers are also able to mediate the continuous delivery of small-molecule drugs (e.g., chymotrypsin) to promote stem cell differentiation [184]. It is also important for supporting the infiltration of external cells and thus participates in the healing process of endogenous cells [182]. The synthesis of biochemical components that promote stem cell differentiation directly onto injectable hydrogel materials for the purpose of supporting MSC proliferation and pleiotropic differentiation is also a viable strategy [185].

Engineered cells demonstrate high therapeutic potential but face substantial obstacles in clinical implementation due to regulatory and scalability hurdles. Plant-extract components (e.g., icariin, salvianolic acid B) present a translational bridge of promoting bone regeneration through multi-targeted pharmacological actions that simultaneously suppress inflammation, stimulate angiogenesis, and promote mineralization.

Plant-extracted components

Plant-extracted components consist of a complex and diverse set of active components, including alkaloids, organic acids, flavonoids, terpenoids, polysaccharides and proteins. Different components can self-assemble to form nanoparticles through different intermolecular noncovalent bonding forces (hydrogen bonding, electrostatic forces, van der Waals forces, π‒π interactions, hydrophobic interactions and coordination interactions) [187]. Plant-extracted components can be self-assembling at various stages, including freshness, preparation, decoction, molding and storage of the preparation, and final administration to the body. Taking traditional Chinese medicine as an example, the nanostructure generated by the self-assembly of multiple components during the decoction process of the Chinese medicinal compound ‘Astragali Yin San Liang San’ is the material basis for the therapeutic effect of the medicine [188].

However, the self-assembly of traditional plant-extracted components is based primarily on noncovalent bonding, which often results in poor physicochemical stability. Carrier-free nanopreparations may lack specific targeting in disease treatment. The transport of self-assembling nanomicelles from plant extract ingredients via stable carriers with immunological properties, such as exosomes, can effectively solve this problem [189] (Fig. 13). There have been various results of herbal self-assembly combined with hydrogels for promoting bone regeneration and wound healing. For example, the combination of ACSCs and AHI-loaded mesoporous silica nanoparticles into chitosan-based hydrogels with dehydrated epimedium (AHI), a flavonoid compound, enables sustained drug delivery to promote cartilage regeneration [190]. As another example, researchers have recently discovered that a natural isoquinoline alkaloid (BBR) with photoinducible properties ubiquitously present in herbs such as cypress and Rhizoma Coptidis is capable of generating large amounts of reactive oxygen species (ROS) in the presence of light, which can be used to kill bacteria. Therefore, researchers have designed hybridized hydrogels with AIE properties self-assembling from N-(9-fluorenylmethoxycarbonyl)-L-phenylalanine (Fmoc-F) and BBR. The hydrogel was shown to exhibit potent antimicrobial activity against Escherichia coli and Staphylococcus aureus and was able to penetrate biocoated membranes and eradicate intramembrane bacteria [191]. Puerarin (PUE), a traditional Chinese medicine ingredient extracted from Pueraria lobata, is able to prepare self-assembling nanofiber hydrogels containing herbal medicine ingredients through effective collision and self-assembly between the molecules of Pueraria lobata and chitosan (CS) achieved by mechanical force, and such self-assembling hydrogels containing herbal medicine ingredients have been proven to have good antimicrobial and anti-inflammatory effects, which can effectively promote the healing of wounds [192].

Fig. 13.

Self-assembly of plant-extracted components. (A) The main type of supramolecular interaction that drives the self-assembly process of natural herbal molecules [193]. (B) Illustration of the fabrication and cartilage regeneration application of the injectable GOAS-ACSC-engineered nanocomposite [190]. (C) Self-assembly of Fmoc-F and berberine chloride (BBR) into a novel hybrid hydrogel for efficient sterilization and wound healing [191]. (D) Self-assembly of puerarin (PUE) and chitosan (CS) by mechanical force collision to prepare nanofiber hydrogels containing traditional Chinese medicine components [192]. (E) Self-assembling drug carriers with pharmacological activities in plant-extracted components [187]

Luo W et al. reported a small-molecule self-assembling hydrogel capable of directly loading bioactive compounds derived from traditional Chinese medicine (e.g., glycyrrhizic acid, berberine, and anthraquinones) under physiological conditions, eliminating the need for additional drug-loading procedures or inert carrier components. This hydrogel enables effective drug delivery and sustained release without the need for polymer carriers or exogenous inactive substances as media, thereby significantly enhancing biosafety and clinical translation potential [108]. Studies by Zhao W et al. further demonstrated that integrating plant extracts into self-assembling supramolecular hydrogels significantly enhances the stability of labile phytochemicals and enables targeted delivery, leading to enhanced therapeutic efficacy [107].

Future integration of self-assembling hydrogels with natural plant extracts would leverage the inherent capacity of natural plant molecules to form multicomponent co-assemblies. This strategy, when combined with synergistic therapeutic approaches targeting specific pathologies, enables multi-target intervention through coordinated modulation of disease-relevant pathways. Capitalizing on the exceptional biocompatibility of these hydrogels significantly enhances the physicochemical stability and functional persistence of self-assembling systems incorporating natural plant extracts, thereby accelerating clinical translation.

Stimulus-responsive self-assembling hydrogels guide bone regeneration

The process of spontaneous organization of hydrogel supramolecules into ordered structures under specific conditions through noncovalent bonding interactions (e.g., hydrogen bonding, van der Waals forces, electrostatic interactions, π‒π stacking, etc.) is the basic principle of their self-assembly. Different self-assembly processes determine the different morphologies of self-assembling hydrogels, such as poly(vinyl alcohol) (PVA) hydrogels, in which hydrogen bonds can form between hydroxyl groups, which leads to the self-assembly of PVA molecular chains in water to form a fibrous chain structure and, ultimately, a three-dimensional network structure. Hydrophobic groups spontaneously aggregate in a water environment; thus, the self-assembly of hydrogel molecules can lead to the formation of a spherical hydrogel with hydrophobic microregions, and the self-assembly of hydrogel molecules results in the formation of spherical aggregates with good dispersion and stability in water. In addition to these two common forms, edited self-assembling hydrogels can also form special structures, such as sheets, tubes, and vesicles, which can be used for capturing, storing and transporting the active ingredients of some specific drugs in living cells. These self-assembly processes are reversible and dynamic, resulting in hydrogel supramolecular structures with unique stimulus responsiveness and self-repair capabilities.

Since the networks in self-assembling hydrogel structures are fragile and reversible networks formed by noncovalent forces, these gels can respond to external stimuli, such as temperature, pH, or ions, and can be endowed with unique mechanical properties, such as reversible healing and solution‒solid alteration [194]. This is the theoretical basis for self-assembling hydrogels based on responsive strategies. 32. In recent decades, self-assembling hydrogels based on responsive strategies have attracted increasing attention in the fields of environmental remediation, biosensors, drug delivery, optical switches and self-healing materials. Hydrogels are responsive to external stimuli such as light, temperature, pH, mechanical forces (e.g., ultrasound), magnetic fields, and molecular analytes, and they can be used for functions such as on-demand release of drugs or therapeutic molecules [195].

Self-assembling hydrogel motifs allow the precise regulation of noncovalent interactions and the modulation of peptide assembly and properties via a variety of physiological parameters, such as pH, temperature, ions, and enzymes. Properly designed peptide-based self-assembling hydrogels can undergo a sol‒gel transition in response to physiological stimuli, facilitating rapid hemostasis and wound filling during injection [33, 40]. It is particularly beneficial for the appositional filling and hemostasis of irregular and deep wounds. In addition, controlled release of therapeutic drugs for wounds can also be achieved via the use of stimulus-responsive materials [196].

Since Wichterle first introduced hydrogels into the biomedical field in 1960 [197], hydrogels have evolved from simple inert gels to complex, variable and controllable “smart” gels with sensitive or responsive properties and are increasingly being used in bone regeneration and bone repair [198]. Stimulus-responsive self-assembling hydrogels have a high water content, high sensitivity, and tunable structural and physicochemical properties that allow them to respond rapidly to external environmental stimuli in the form of swelling, contraction, or sol‒gel phase transitions [199]. Depending on the response conditions, self-assembling hydrogels can be divided into physical responses, chemical responses and biochemical responses according to the source of the response to stimuli [200]. In a physical response, a hydrogel will respond to changes in the physical environment, such as a thermal response, a light response, a magnetic response and a mechanical response. The chemical response refers to the response of a hydrogel to changes in the chemical environment, such as changes in pH, oxidation reactions and ionic strength. Biochemically reactive hydrogels, on the other hand, respond to changes in the biochemical environment, such as glucose and enzymes. Numerous studies have shown that injuries to bone or cartilage tissue, including those caused by fractures or inflammation, can cause changes in the microenvironment, such as changes in enzymes [201–204], pH [205], temperature [206] and redox reactions [207] at the damaged site. These characteristic changes can directly stimulate the responsive hydrogel, triggering a change in its properties and leading to drug release [91]. In addition to these internal stimuli, responsive changes in hydrogels can also be triggered by external stimuli such as near-infrared (NIR) light [208] for bone and cartilage tissue engineering. The application of stimulus-responsive hydrogels facilitates on-demand release, ensuring that growth factors or cells reach specific defective tissues, providing mechanical and biological support for bone and cartilage defects [209]. Responsiveness to external or internal stimuli further enhances the material’s tunability, enabling greater design flexibility. When integrated with established osteogenic materials, stimulus-responsive systems more readily demonstrate their therapeutic potential for bone regeneration [210].

Stimuli-responsive self-assembling hydrogels dynamically modulate physicochemical properties in response to microenvironmental or external signals, enabling spatiotemporally precise therapeutic strategies for bone regeneration. Table 4 systematically synthesizes the operating mechanisms, applications in bone regeneration, and current technological limitations of 12 stimulus-responsive modalities, providing critical design references and selection guidelines for developing advanced intelligent materials for bone regeneration.

Table 4.

Stimuli-responsive self-assembling hydrogels in bone regeneration: mechanisms, applications, and limitations

Responsiveness	Mechanism	Bone regeneration applications	Primary limitations	Ref
Photo	Photothermal/photochemical reactions (e.g., NIR light activation, UV crosslinking)	Spatiotemporal delivery of growth factors (BMP-2/VEGF); minimally invasive in situ gelation	Limited tissue penetration depth (< 5 cm); potential long-term phototoxicity	[103, 211, 212]
Thermo	LCST/UCST phase transition behavior (e.g., thermosensitive aggregation of PNIPAM)	Injectable hydrogels for irregular defects; thermal-triggered stem cell differentiation	Narrow physiological temperature fluctuation (± 2 °C); suboptimal sensitivity; local inflammation risk	[213, 214]
Mechanical	Stress-induced conformational changes/bond cleavage (e.g., disulfide rearrangement)	Load-bearing bone repair (mimicking piezoelectricity); mechanotransduction-driven osteogenesis	Complex in vivo mechanical environment; poor controllability; fatigue failure risks	[53, 215]
Magnetic/electric	Magnetic field-driven nanoparticle heating/displacement; electric field-modulated ion channels/protein conformation	Targeted BMP-2 delivery via magnetic particles; electrostimulated calcium deposition	Strong-field interference with medical devices; biocompatibility concerns of implants	[216, 217]
pH	Protonation/deprotonation-induced swelling/collapse (e.g., polyacrylic acids)	Smart antibacterial release in infected defects (pH >7.4 inhibition); osteoclast-targeted drug delivery	Compromised efficiency due to physiological pH buffering	[106, 218]
ROS	Oxidative cleavage of sensitive bonds (e.g., thioketal, selenide)	ROS scavenging during inflammation; controlled antioxidant release for osteoblast protection	Rapid material depletion under chronic inflammation	[219, 220]
Ion	Ionic crosslinking/coordination (e.g., Ca²⁺-alginate, Zn²⁺-histidine)	Biomimetic mineralization for osseointegration; ion-activated osteogenic signaling (e.g., CaSR)	Narrow triggering window due to homeostatic ion concentrations	[164, 221]
Glucose	Glucose oxidase-mediated acidification/boronate ester dissociation	Combinatorial therapy for diabetic defects: glucose-triggered insulin/bone-healing factor release	Enzyme deactivation by protein fouling; slow response kinetics	[222, 223]
ATP	ATP aptamer conformational change/ATP hydrolysis energy utilization	Acute trauma energy metabolism regulation; BMP-2 release at high cellular-activity sites	Low delivery efficiency (intracellular [ATP] > >extracellular [ATP])	[224, 225]
Enzyme	Specific enzymatic cleavage (e.g., MMP, ALP-sensitive sequences)	Targeted anti-resorptive drug delivery; enzyme-cascaded osteogenic signal amplification	Inter-patient enzyme variability; off-target cleavage risks	[150, 226]
Ultrasound	Cavitation effects/mechanical wave-induced microbubble collapse/polymer degradation	Non-invasive activation for deep defects; sonoporation-enhanced stem cell penetration	Energy attenuation in tissue; limited spatial precision	[227, 228]
Dual/multi	Synergistic mechanisms (e.g., photothermal + pH, pH/glucose dual-responsive)	Microenvironment-adaptive therapy: anti-infection + vascularization + mineralization	Synthetic complexity; circuit logic design challenges; scalability issues	[212, 229, 230]

Photo response

Hydrogels are networks of polymers dispersed in aqueous solvents. The volume of hydrogels with photoreactive molecules bonded to the polymer network can change in response to light. Light triggers chemical reactions of photoreactive molecules, such as photoisomerization of spirochromes, and changes the interaction parameters between the polymer network and the solvent molecules, thus driving the diffusion of the solvent molecules and inducing volume changes in the hydrogel. The photo-responsive kinetics are governed by simultaneous reactions and diffusion [231]. Hydrogels with photo-responsive mechanical properties have found broad biomedical applications, including the delivery of bioactive molecules or drugs [232, 233], cell culture [234, 235], biosensing [236], and tissue engineering [235, 237, 238]. Specifically, in the field of bone regeneration, photo-responsive self-assembling hydrogels can be used for controlled drug release and fracture restoration [91].

The versatile covalent combinations of self-assembling hydrogels allow them to mimic some of the properties of natural materials and have excellent self-healing qualities. An example of the design of dynamic colloidal PC hydrogels with self-healing and injectable properties through a reversible boronic acid covalent binding strategy is presented here. Pan et al. ingeniously constructed dynamic colloidal PC hydrogels through reversible cross-linking of adjacent particles in self-assembling hydrogel colloidal crystals, which exhibited perfect self-healing of physical damage and rapid recovery of structural color after injection [239]. This example not only demonstrates the excellent editable nature of self-assembling hydrogels and their wide range of material applications but also provides a good idea for directing self-assembling hydrogels for bone regeneration promotion.

Dynamic polymer‒nanoparticle interactions can be used to engineer hydrogels in situ [240]. A range of self-assembling in situ-forming hydrogels have been developed on the basis of the inherent hydrophobic or electrostatic properties of polymers and nanoparticles. Hydrophobic modifications can improve the adsorption of hydrophobic nanoparticles by polymer chains. Inspired by these designs, the hydrophobicity of polymers and nanoparticles can be controlled by external stimuli such as light. This in situ gelation approach can lead to the formation of crosslinked networks for prolonged local retention or on-demand release of drugs for the regeneration of drugs and drug delivery [241]. On the basis of the above studies, Akhilesh K. Gaharwar’s team proposed in situ gelation by combining NIR-responsive molybdenum disulfide (MoS2) with temperature-responsive poly(n-isopropylacrylamide) (PNIPAm) networks, which was reported for the first time for initiating the gelation of a self-assembling hydrogel by using NIR light without the use of other reagents [242]. The molybdenum disulfide nanoparticles embedded in the hydrogel network can be used as drug carriers, which endows light-responsive self-assembling hydrogels with great potential for regenerative medicine and drug delivery.

Photo responsiveness enables precise tunability and control over the structural morphology of self-assembling carrier systems (Fig. 14). This capability offers a highly promising strategy for reconstructing complex bone defects while facilitating spatiotemporally controlled delivery of osteogenic agents. Yun J et al. developed an in situ photo-crosslinking bioadhesive hydrogel (iPBAH) as a multifunctional bone graft adhesive. By incorporating mussel-derived adhesive protein (MAP), the system achieves rapid light-triggered crosslinking in situ, exhibiting superior mechanical properties and biocompatibility [243]. This study underscores the significant potential of light-responsive hydrogels for enhancing bone regeneration in anatomically complex defect sites. Kim M et al. recently developed a near-infrared(NIR) light-activated angiogenesis nanoparticle (NM) capable of on-demand release of bone-inducing growth factors. Specifically, they combined the angiogenic component (VEGF mimetic peptide) and the osteogenic factor (bone morphogenetic protein 2 (BMP-2)) with elastin-like peptide (ELP) to form NM through self-assembly, and utilised gold nanoparticles (AuNP) to create a near-infrared responsive system. This system facilitates the sequential release of VEGF mimetic peptides and BMP-2 under NIR light activation, providing spatiotemporal control over drug delivery aligned with distinct stages of bone healing [211]. This NIR-triggered strategy offers a promising approach for remote-controllable therapeutic release in bone regeneration.

Fig. 14.

Photo-responsive self-assembling hydrogels for biomolecule delivery and biosensing. (A) Schematic representation of the fabrication of injectable photostretchable hydrogels by metal-directed assembly of His6-labelled proteins [237]. (B) Preparation and characterization of light-responsive injectable self-healing hydrogels formed on the basis of the covalent cross-linking of phenylboronic acid lipid bonds. Synthesis of PBA-microgels. Photocrosslinking of PBA microgels induced by photoinitiated polymerization of a cis-diol-containing glycopolymer [239]

The unparalleled spatiotemporal precision of photo-responsive systems is tempered by the fundamental limitation of tissue penetration, which constrains their clinical utility for treating deep-seated defects. This inherent challenge motivates the development of thermo-responsive hydrogels, which leverage endogenous physiological temperature gradients to achieve non-invasive activation across diverse anatomical contexts.

Thermos response

Thermos responsive hydrogels undergo a sol‒gel transition at ambient temperature and induce heat. As this transition occurs, the volume, mechanical properties, and internal structure of the thermos responsive hydrogel change [244]. The temperature threshold that triggers the response mechanism on the basis of the ambient temperature is called the critical solution temperature (CST). Hydrogels with LCSTs, such as poly-N-isopropylacrylamide (pNIPAM), are subject to entropy-driven shrinkage at temperatures above the critical temperature and expand back to their normal state at low temperatures. In contrast, hydrogels with UCSTs, such as poly(acrylic-coacrylamide), expand at temperatures above the critical temperature and contract at low temperatures [245].

For the purposes of easy injection, drug delivery, and providing rigid support for the cell growth microenvironment at the site of bone defects, the most commonly used materials in bone repair and tissue engineering are LCST-type thermos responsive hydrogels, the main components of which are poly-N-isopropylacrylamide (PNIPAAm) [246, 247] and its derivatives, which can increase the lowest critical solution reaction temperature by means of chemical cross-linking [248]. This thermos responsive hydrogel is kept in solution at room temperature for mixing with drugs and other therapeutic components, and after injecting it into the corresponding bone defect site, the temperature of the external environment increases to the LCST, triggering the hydrogel thermal response mechanism to undergo a sol‒gel transition, changing its internal structure and hydrophilic shape to release drugs and growth factors for the purpose of promoting bone restoration and bone regeneration [249] (Fig. 15). Natural PNIPAM hydrogels are very fragile and are rarely used for practical purposes. Strategies to enhance the mechanical properties of PNIPAM hydrogels by modifying their polymer networks or adding nanomaterials have been widely investigated [250]. Polyethylene glycol is also a common raw material for the preparation of mechanically tough and thermos responsive self-assembling hydrogels [251].

Fig. 15.

Thermoresponsive hydrogels for bone regeneration. (A) Schematic diagram of the use of thermal hydrogels for bone defect modelling [254]. (B) Sol-to-gel phase transition at different temperatures [257]. (C) Schematic synthesis of dual supramolecular self-assembling hydrogels with significant thermoresponsive properties used to control the slow release of encapsulated drugs at elevated ambient temperatures [252]. (D) A novel thermosensitive in situ mold hydrogel based on methyl cellulose (MC) and Persian gum (PG) for bone regeneration studies: Schematic diagram of thermosensitive hydrogel preparation and regeneration in rat femoral defects [253]

Composite hydrogels have been widely used as drug carriers in the biomedical field, and the design of stimuli-responsive hydrogels has received extensive attention for improving the in vivo delivery of drugs. By utilizing the thermoresponsiveness of the polymer LCST, the slow release of drugs in vivo can be better achieved. Jun, Li’s team developed a hydrogel based on dual supramolecular self-assembly. The addition of the thermos responsive unit PNIPAAM enables it to have variable strength, which can be enhanced in situ to realize slow release in vivo. After degradation of the gel, the drug is further released as micelles, which improve drug uptake and help overcome resistance to multiple drugs in tumors [252].

In recent years, the strategy of preparing novel thermally responsive hydrogels by blending or covalently cross-linking with some osteogenic materials or high-mechanical-strength copolymers to enhance the mechanical and biological properties of hydrogels in bone tissue engineering has been increasingly adopted by scholars. The new thermosensitive hydrogel provides rapid gelation at physiological temperatures, high mechanical strength, injectability and improved bone regeneration effects [253, 254].

Thus, thermos responsive hydrogels with high elasticity and fast response are urgently needed. This has been achieved via the use of ABA triblock copolymer flower-like nanogels with hydrophilic polymer rings, and it has been identified as a new and effective method for constructing tough and elastic hydrogel networks [255]. Thermoresponsive hydrogels containing allogeneic bone graft material (BGM) exhibit a sol‒gel transition at physiological temperatures. In animal model studies of cranial defects, hydrogels containing BGM significantly improved bone regeneration compared with that in the untreated and hydrogel-only groups [256].

The capacity of thermoresponsive hydrogels to sense and respond to deep-tissue temperature gradients is counterbalanced by their inherent absence of dynamic mechanoadaptive properties, which are critical for bone repair. Consequently, mechanical responsiveness has emerged as a pivotal design paradigm, where adequate tensile modulus facilitates the reconstruction of geometrically complex bone defects through mechanotransduction-driven osteogenesis.

Mechanical response

Due to their high similarity to the natural extracellular matrix and suitability for minimally invasive procedures, injectable hydrogels have been highly attractive biomaterials in the fields of cartilage and bone tissue engineering. However, for conventional hydrogel materials, injectability and tensile modulus are often inversely correlated. The current study shows that the mechanical response of hydrogels varies systematically with the polymer concentration, which in turn may be correlated with the concentration dependence of the crystallinity and mechanical response [258]. Well-designed self-assembly systems offer an effective solution to address this challenge.

Dr. Wang Z and his team constructed a multiple hydrogen bonding system and developed a supramolecular hydrogel with a compressive modulus of 2.6 MPa. This system resolves the trade-off between strength and injectability through a dynamically reorganizable hydrogen-bonding mechanism. This self-assembling hydrogel system achieves a storage modulus of 1 MPa at an ultralow concentration (5.0 wt%), representing the strongest supramolecular hydrogel reported to date composed of ultra-low molecular weight (MW < 300) gelators. Furthermore, it exhibits excellent shear-thinning injectability and can complete its sol-to-gel transition within seconds after injection [53].

The therapeutic effect on chronic infected wounds has been unsatisfactory. Liu X’s team, inspired by the grinding treatment of traditional Chinese medicines, introduced mechanical force to promote effective molecular collision and self-assembly of chitosan (CS) and puerarin (PUE) to prepare herbal hydrogels. The dynamic self-assembly and alignment of CS and PUE formed special C@P nanofibrous structures, which endowed the C@P hydrogels with special physicochemical and biological properties. Moreover, the C@P hydrogel showed excellent antimicrobial ability, which could promote the healing of noninfected wounds by regulating the immune response, as well as good biocompatibility and antimicrobial and anti-inflammatory abilities, which showed great advantages in the treatment of chronically infected wounds, and it is expected to be a dressing with synergistic antimicrobial and immune-modulating effects for the treatment of infected wounds [259].

Biocomposite nanomaterials are very suitable for cell attachment and proliferation. The addition of composite bionanomaterials to mechanically responsive hydrogels not only leads to a significant increase in their mechanical stiffness and physiological stability but also allows the hydrogels to have a stronger mechanical response [260]. This finding is also supported by the study of Kumar, A et al., who enhanced the mechanical, biomineralization and cellular responses of nanocomposite hydrogels via the use of bioactive glass (BG) and elosite nanotubes (HNTs) to promote bone tissue regeneration [261].

The mechanical behavior of cartilage tissue plays a crucial role in the physiological mechanical transduction process of chondrocytes and in pathological changes such as osteoarthritis [262]. Cartilage tissue equivalents formed from hydrogels containing chondrocytes can provide a solution for replacing damaged cartilage. Previous approaches have typically utilized elastic hydrogels [263]. Owing to their structural similarity to natural cartilage, double-layered hydrogels have been suggested as candidates for cartilage replacement. The bilayer hydrogels loaded with TiO2 nanoparticles (NPs) possess a high elastic modulus and stiffness and exhibit good mechanical response [264]. A study by Lee, HP et al. revealed that elastic stress may limit the formation of the cartilage matrix and alter the phenotype of chondrocytes. A faster mechanical relaxation of the gel can promote chondrocyte formation and a significant increase in the volume of the interconnected cartilage matrix [265]. These findings suggest that the mechanical response of hydrogels, particularly mechanical relaxation, is key to designing articular cartilage replacement materials and promoting cartilage restoration.

Since articular cartilage is susceptible to mechanical overload, the sensitivity of the mechanical response affects the efficacy of the therapeutic mechanical response of hydrogels for bone regeneration. BMMCs in 3D chondroitin sulfate-tyramine/gelatin (CS-Tyr/gel) hydrogels may have an advantage over chondrocytes in cartilage regeneration applications because of their lower sensitivity to mechanical loading [266].

Beyond passive mechanoadaptation and basic stress-response behaviors, active modulation of physical properties enables targeted adaptation to dynamic bone defect microenvironments. Magnetic/electric field responsiveness—as a frontier technology discussed in the subsequent section—facilitates remote spatiotemporal control over self-assembling hydrogel systems. This approach provides capabilities for modulation of mechanical properties and therapeutic release kinetics.

Magnetic/electric field response

Different small molecules can self-assemble to form different basic structures, such as nanofibers, nanotubes, and nanosheets, which determine the basic physicochemical properties of the hydrogels they form and, in turn, their applications. For example, owing to their unique mesh structure, nanonetwork structures form self-assembling hydrogels that are naturally tensile resistant and allow for high-speed movement and exchange of substances within their network structure. Inspired by these findings, some scholars have developed new hydrogel materials based on self-assembling nanonetworks. Compared with conventional conductive materials, self-assembling hydrogel materials have an order of magnitude greater conductivity because of their easy preparation and high strength [267] (Fig. 16).

Fig. 16.

A method for fabricating conductive hydrogels on the basis of hybrid assemblies of polymer nanofiber networks. (A)(C) Schematic of nanofiber template-guided synthesis of percolating polypyrrole (PPy) networks and SEM images of PPy particles and the associated polymer networks. CNHs in soft bioelectronics. Electrochemical impedance spectroscopy and cyclic voltammetry measurements. Photographs of patterned CNHs and electrodes and EMG recordings of hands [267]. (B) Composite nanohydrogels based on starch and codoped zinc ferrite nanoparticles (Co 0.50 Zn 0.50 Fe2O4, which undergo changes in drug release) can respond to external magnetic fields [268]. (D) Multifunctional magnetic field-responsive hydrogels based on methacrylated chondroitin sulfate (MA-CS) and iron-based superparamagnetic nanoparticles (MA-CS MNPs) [269]

The use of electrically/magnetically responsive hydrogels as scaffolds can effectively promote cell proliferation after receiving remote magnetic stimulation, suggesting that hydrogel matrices with remotely stimulated magnetostrictive biomaterials can be used for bone tissue engineering to promote bone regeneration [270].

Magnetic fields are triggers with the advantages of temporal control, noninvasiveness and safety without contact. Magnetic field-responsive hydrogels have many advantages, including rapid response, precise control, noninvasiveness, tissue permeability and wide range [271]. Like NIR, magnetic fields can enhance osteogenesis through photothermal effects and the immune microenvironment. Studies have shown that magnetic fields can induce osteogenic and chondrogenic differentiation in BMSCs, thereby promoting bone healing [272]. Magnetic fields can also modulate cell behavior and improve cell-biomaterial interactions, and magnetic field-responsive hydrogels have been shown to be a viable strategy to enhance bone repair by modulating the immune microenvironment [273].

Magnetically stimulated responsive hydrogels are mainly used to modulate the release of drugs, growth factors and therapeutic cells in a controlled manner on demand [274]. For example, photocrosslinked magnetically responsive hydrogels made of chondroitin methacrylate sulfate (MA-CS) enriched with a tunable characteristic platelet lysate (PL) can modulate lysis, degradation, and release of PL-derived growth factors under external magnetic field (EMF) stimulation to affect cell morphology and the expression and synthesis of tendons and bone-like matrices [275]. Magnetically responsive hydrogels prepared via novel magnetic fabrication techniques can exhibit a controlled layered structure inside and can be morphologically altered by external stimuli, suggesting the possibility of precise transport and layered delivery of targeted drugs [276]. An electric field-responsive hydrogel can induce its own swelling response in response to changes in the external electric field, enabling the release of the encapsulated drug from the matrix. Once electrical stimulation is stopped, the gel is able to return to its original size. By controlling the applied voltage and duration, the drug release rate and dosage can be precisely controlled [199, 269].

Coblending or covalent polymerization of iron-containing components with hydrogels is a common way to prepare magnetically responsive hydrogels. The deformation of the iron gel depends on the polymer concentration, calcium concentration, iron oxide concentration and magnetic field strength [268].

Novel magnetically responsive hydrogels doped with magnetism by in situ precipitation using protoelastin are also available. In vitro studies have shown that these materials exhibit rapid magnetic responsiveness and support cell viability [277]. This new magnetoresponsive composite hydrogel is expected to be used as a soft tissue regeneration platform for articular cartilage regeneration and repair.

DNA hydrogels, with a variety of designable stimulus-responsive units, are biocompatible and degradable, making them ideal carriers for drug release and substance detection, and the process of drug release can be modulated by changing the structure of the DNA monomers used in the preparation of the hydrogels. Prof. Dongsheng Liu’s team constructed a DNA-MNP hydrogel that can be remotely controlled by integrating DNA-modified magnetic nanoparticles into the body of the DNA hydrogel. By applying various triggers, such as temperature, enzymes and magnetic fields, the DNA-MNP hydrogel can specifically undergo a gel‒sol transition, which can be remotely regulated to move to a specific location and release drugs for drug delivery and cancer therapy. This is the first study in which DNA hydrogels were shown to be endowed with magnetic responsiveness [278].

The complexation of xanthan gum and chitosan is a new exploration in which the two can undergo self-assembly in the presence of magnetic nanoparticles (Fe3O4-mNPs) to form ion-complexed magnetically responsive polyelectrolyte hydrogels in the presence of glucuronic acid. The data showed that the proliferation and adhesion of fibroblasts on the gels were significantly enhanced under the influence of an applied magnetic field. The addition of magnetic nanoparticles also significantly increased the mechanical strength of the gel and improved its rheological properties (elevated energy storage modulus) [200]. Therefore, magnetically responsive hydrogels are expected to have potential applications in tissue engineering.

Notably, the solvation traits of magnetically responsive hydrogels can be very complex, and preparing and simulating their behavior under stimulation is important for the use of smart materials in biomedical applications. Artificial neural networks (ANNs) in MATLAB were used to simulate these behaviors. ANN models with correlated input variables are able to accurately predict the complex swelling and heating behavior of magnetic field-responsive poly(NIPAAm-co-VSA)/(FeO4)-O-3 IPN hydrogel composites [279].

Electrical stimulation can affect transmembrane potential and growth factor signalling to regulate osteogenesis, whereas electrical stimulation can induce cell proliferation, adhesion and mineralization [280]. On the basis of this mechanism, several electrically responsive hydrogels combined with conductive nanoparticles have been developed to promote bone repair [281]. Under exogenous ES, conductive hydrogels enhance osteogenesis, increase M2 macrophage polarization, and promote angiogenesis and bone regeneration [282]. Thus, electrically responsive hydrogels provide a unique and effective strategy for enhancing direct osteogenesis, modulating the immune microenvironment and facilitating neovascularization for bone healing. The treatment of diabetic wounds remains a great clinical challenge, and the regulation of the regenerative microenvironment is crucial for diabetic wound treatment. Zheng Longpo et al. designed and prepared a novel charge-driven self-assembling microsphere hydrogel scaffold (SMHS) based on charge interactions, combining positively charged chitosan methacryloyl (CS) microspheres containing black phosphorus (BP) with negatively charged hyaluronic acid (HA) microspheres containing basic fibroblast growth factor (bFGF). Owing to the presence of BP, near-infrared (NIR) irradiation had a significant effect on the degradation and drug release behavior of the SMHS. SMHS + NIR significantly accelerated the wound healing process by inhibiting the inflammatory response and promoting angiogenesis and tissue remodelling [283]. This self-assembling dose‒responsive hydrogel achieves both long-term treatment with drug delivery and short-term treatment with photothermal therapy, which holds great promise for diabetic wound treatment and other tissue regeneration applications.

Externally Magnetic/electric field response enables precise spatiotemporal manipulation, however, its clinical translation is hindered by complex physiological environments. This limitation necessitates microenvironment-responsive systems such as pH-triggered self-assembling hydrogels, which initiate intelligent therapeutic release in response to pathological acidosis within infectious defects or osteoclast-mediated resorption regions.

pH response

Different acidities and alkalinities exist in living organisms, and pH has become an important reactive component in the preparation of responsive biomaterials. The construction of chemical bonds with responses under different pH conditions in hydrogel systems can endow traditional hydrogels with pH responsiveness, thus improving their applicability in living organisms and exerting better therapeutic effects.

Compared with conventional drug delivery systems or free drugs for therapeutic purposes, self-assembling pH-responsive hydrogels tend to achieve better cellular uptake and less cytotoxicity when used as drug delivery systems [284] (Fig. 17). Self-assembling hydrogels that produce a sol‒gel phase transition in response to the acidic microenvironment in vivo, which in turn releases the drug, are often used for localized oncology chemotherapy, with the advantages of simplicity of preparation, injectability, ease of identification of the site of mutation, long retention of the oncology drug at the target site, and almost negligible side effects [285]. This pH-responsive drug delivery strategy is mostly realized via self-assembling peptide hydrogels [286].

Fig. 17.

(A) Schematic representation of the pH-responsive release of Cur from the AGC16/NTS hydrogel and the uptake of Cur by cancer cells [284]. (B) Demonstration of the pH-triggered self-assembly behavior of CMCh-ACP hybrid nanoparticles and the injectability of the formed gels [106]. (C) Hydrogel functional properties for controlled RNA delivery [135]. (D) Schematic representation of widely studied self-assembly systems and four major peptide self-assembly systems with representative examples [196]

In addition to being used as drug delivery systems, pH-responsive self-assembling hydrogels have potential applications in the field of bone tissue engineering. Effective bone tissue engineering can restore bone and bone function damaged by trauma and/or certain medical conditions. This technique centers on providing an ideal scaffolding material for the mesenchymal stem cells (MSCs) mentioned above [112]. The material should support MSC proliferation and cell adhesion in addition to possessing mechanical strength. Self-assembling hydrogels perfectly fulfil these conditions and can be combined with the current widely used 3D printing technology. Chen Zhao et al. subsequently developed a pH-triggered, self-assembling and bioprintable composite nanoparticle hydrogel scaffold based on the combination of carboxymethyl chitosan (CMCh) and amorphous calcium phosphate, which has excellent biocompatibility, effectively supports MSC proliferation and cell adhesion, and enhances BMP9-induced osteogenic differentiation, whereas the hybrid hydrogel itself is osteoinductive, which can be triggered by the pH of the controlled assembly method for easy preparation [106].

A response mechanism based on the acidic microenvironment in vivo enables the controlled self-assembly of hydrogels in vivo. A new strategy to construct functional hydrogels through the transformation of pH-responsive nanoparticles is proposed. Under acidic conditions, pH-sensitive nanoparticles can be hydrolysed to release hydrophilic host molecules, which self-assemble with guests to form hydrogels through enhanced host‒guest interactions. The nanoparticles that can be converted into hydrogels showed significant protective effects against ethanol- or drug-induced gastric injury in mice. In addition, proton-driven hydrogel self-assembly of pH-responsive nanoparticles can be triggered by the inflammatory microenvironment. Thus, transformable nanoparticles may also serve as functional nanocarriers for different therapeutic agents for the treatment of inflammatory diseases [287]. This study accomplished self-assembly of hydrogels in vivo on the basis of a pH-responsive mechanism with satisfactory therapeutic efficacy and demonstrated the great potential of pH-responsive self-assembling hydrogels in the treatment of inflammatory diseases.

The pH-triggered release mechanism, while effective against infection, inadequately addresses oxidative stress, which is a hallmark of inflammatory bone loss. ROS-responsive designs thus constitute a logical progression, integrating oxidative bond cleavage to scavenge free radicals while concurrently delivering cytoprotective agents.

ROS response

A modest increase in ROS leads to enhanced cell proliferation, survival and benign immune responses, whereas ROS stress destroys the antioxidant capacity of cells, thereby damaging nucleic acids, proteins and lipids and leading to oncogenic mutations and cell death [288]. The scavenging of excess ROS from the body eliminates oxidative stress and accelerates local regenerative processes. Highly biocompatible hydrogels are promising therapeutic drug platforms developed for ROS scavenging and have synergistic effects with other drugs or therapeutic molecules [289] (Fig. 18).

Fig. 18.

(A) Mechanisms of ROS scavenging by therapeutic hydrogels [289]. Schematic illustration of the ROS-responsive hydrogel for the sustained codelivery of photosensitizers and ICB antibodies [291]. (B) Ion-triggered hydrogels self-assembling from statistical copypeptides: structure of the statistical copolypeptide poly(l-lysine-stat-l-tyrosine), image of the hydrogel in PBS buffer on a spatula and examples of hydrogels dyed with rhodamine and illustration of the gelation process [295]. (C) Sugar and temperature dual-responsive hydrogels based on PNIPAAm copolymers containing phenylboronic acid (PBA) moieties were prepared for harvesting cell slices [154]

The active release of drugs and ROS scavenging in response to high levels of ROS in the in vivo microenvironment constitute highly effective strategies for reducing the inflammatory response and accelerating wound healing. Lei, H et al. prepared the first TA-siRNA nanogel on the basis of the self-assembly of tannic acid (TA) and short interfering RNA (siRNA). This highly efficient and biodegradable nanogel was crosslinked with poly(vinyl alcohol) (PVA), human-like collagen (HLC), TA, and borax to prepare adaptive conductive PHTB (TA-siRNA) hydrogels. In the presence of high levels of reactive oxygen species (ROS), the ROS-responsive borate bonds in the hydrogel were oxidized and broken, and the TA-siRNA nanogel was released into the cells to reduce MMP-9 expression [290]. The development of this ROS-responsive self-assembling hydrogel provides a new approach to accelerate diabetic wound healing, fracture repair and bone regeneration.

Although ROS-responsive hydrogels are now widely used as ROS-scavenging platforms [289] for drug delivery and controlled release [291] and to promote articular cartilage repair [292], self-assembling ROS-responsive hydrogels still lack practical clinical applications. Compared with traditional hydrogels, self-assembling hydrogels can be synthesized more easily and are more editable, and self-assembly in vivo can achieve better therapeutic effects. ROS-responsive self-assembling hydrogels have great potential for alleviating inflammatory responses, modulating local ROS levels and drug release.

Effective bone regeneration represents a dual challenge: mitigating inflammatory damage via ROS scavenging represents only one facet, as success depends equally on the pro-regenerative participation of various ions in the local microenvironment. Ion-responsive hydrogels bridge this functional gap through programmable ion exchange kinetics (e.g., Ca²⁺/PO₄³⁻ release), reproducing the hierarchical mineralisation process of natural bone matrix.

Ion response

Self-assembly is a popular means of designing hydrogels today. Compared with traditional ways of designing hydrogels, which include physical cross-linking and chemical cross-linking, it is easier and more efficient to obtain hydrogel complexes and diverse functions by controlling the self-assembly pathway. Owing to the high sensitivity of self-assembling peptide hydrogels to environmental conditions, such as light, temperature, and ionic concentration, stimuli-responsive peptide hydrogels are increasingly being studied. Controlling and directing the supramolecular structure of low-molecular-weight peptide hydrogel agents through ion-mediated methods is an innovative bottom-up approach for designing materials.

The synergistic effect of divalent metal ions in promoting the hydrogelation of short collagen self-assembling peptides was first reported by Vijay Kumar Pal et al. Their study revealed that the introduction of biologically relevant metal ions (Ca2+/Mg2+) into short peptide self-assembling hydrogels could enable them to overcome the limitation of self-assembly into multiscale structures at physiological pH and highlighted that in the presence of metal ions, the gelation equilibrium point of negatively charged peptides changed significantly and showed a transition from sol to gel [293]. This finding offers the possibility of fabricating novel self-assembling hydrogel materials that control cellular behavior in complex physiological environments. In addition, Vijay Kumar Pal et al. reported that, compared with peptide gelling agents, ionically coordinated peptide hydrogels have tunable mechanical strength and better cellular response [293].

Pooja Sharma et al. aimed to understand the role of different metal ions and anions in regulating the self-assembly of peptide amphiphiles. They reported that judicious incorporation of histidine in peptide amphiphiles confers ion-responsive behavior to the hydrogels and that the self-assembly pathway of such histidine-based dipeptide amphiphiles is largely influenced by various metal salts [294]. Histidine-based dipeptide amphiphiles undergo a gel-to-sol transition at physiological pH under the influence of Cu²⁺, Ni²⁺, and Co²⁺, which shifts gelation to pH 3.0. However, in the presence of Fe²⁺ and Mn²⁺, weak histidine–metal ion interactions still maintain the gels at physiological pH [294].

In addition to single peptide self-assembling hydrogels that can be modulated and designed in an ion-responsive manner, research on ion-responsive copolymerized self-assembling peptide hydrogels has also seen breakthroughs in recent years. Poly(amino acid) hydrogels are known to be prepared via two main methods: intermolecular cross-linking and self-assembly of block copolymers. Although the assembly of block polyamino acids into gels on the basis of secondary structure effects has been widely reported, there are no cases of direct gel formation from random copolymers.

Bing Wu et al. reported for the first time that copolymers of lysine and tyrosine are able to form hydrogels directly in an ionic environment and that their gel-forming mechanism relies on ion-triggered rearrangement of the polypeptide structure, which leads to a shift from an intramolecular secondary structure to an intermolecular secondary structure. The diffusion and mechanical properties of these hydrogels can be tuned by adjusting the peptide chain length and ionic strength [295]. Bing Wu et al. reported that the gelation of this polymerized peptide is due to the presence of salt ions, which induce the formation of a stable intermolecular β-sheet secondary structure as well as the aggregation of α-helices among the copolymerized peptides, a behavior unique to statistical lysine/tyrosine copolymer peptides [295] (Fig. 18). Although this behavior is not observed in any other amino acid combination or arrangement, this simple but unique gelation mechanism provides ideas to inform the design of future copolymer peptide hydrogels. Moreover, the fact that these porous hydrogels were formed in physiological buffer solution offers the possibility of realizing the in vivo self-assembly of copolypeptide hydrogels with promising biological applications.

Whereas ionic cues facilitate mineralization processes, they fail to address metabolic dysregulation in comorbid pathologies such as diabetic bone defect healing. Consequently, glucose-responsive self-assembling hydrogel systems provide a combinatorial therapeutic strategy that dynamically modulates local glycemic levels and orchestrates the release of osteogenic factors in response to hyperglycemic fluctuations. This closed-loop regulation corrects impaired bone regeneration by rescuing compromised osteogenesis through metabolic normalization.

Glucose response

Polymers with phenylboronic acid as the glucose-sensitive moiety are common glucose-sensitive materials. The phenylboronic acid moiety generally exists in a hydrophobic, undissociated form, and when glucose is present, the binding of glucose to phenylboronic acid increases the ionization of phenylboronic acid, leading to increased hydrophilicity of the phenylboronic acid-containing polymers. Using this glucose-sensitizing mechanism, glucose-responsive hydrogels can be designed for bioreactive delivery and therapeutic use.

Phenylboronic acid (PBA)-based polymeric hydrogels are widely recognized as among the most promising insulin delivery systems to replace frequent insulin injections. However, most of these hydrogels are based on a variety of PBA-containing acrylamide monomers and are far from being used in clinical practice [296, 297].

The hyperglycemic microenvironment in diabetic patients exacerbates the local inflammatory response and severely affects the rate of wound healing, including surface skin wounds and deep bone regeneration, increasing the risk of fragility fractures [298]. Cyclic glucose-responsive insulin delivery can effectively control blood glucose and, on this basis, accelerate wound healing and improve the quality of life and health of diabetic patients. Therefore, research on insulin-bearing delivery systems is a top priority [299] (Fig. 19). Owing to their excellent biocompatibility, degradability, efficient drug-bearing ability, protective properties, and controlled release, self-assembling hydrogels are compatible with closed-loop glucose-responsive insulin delivery.

Fig. 19.

(A) Glucose-responsive pH-sensitive self-assembling peptide hydrogels loaded with glucose oxidase, catalase and insulin [299]. (B) Schematic illustration of insulin release from a pH-sensitive injectable self-assembling peptide hydrogel loaded with insulin- and glucose-specific enzymes [300]. (C) Schematic of an adenosine triphosphate (ATP)-responsive self-assembling DNA nanohydrogel for fluorescence imaging and chemotherapy for specific activation of cancer cells [302]. (D) Hybridized nanocomposite hydrogels with shear-thinning and self-healing properties based on self-assembling thiored hyaluronic acid (HA-SH) and bioactive silver lignin nanoparticles (Ag@Lig NPs) [310]. (E) Self-assembling injectable hydrogels based on 3,4-dihydroxyphenylalanine (DOPA) and peptides release inflammatory memory peptides and angiogenic peptides in response to mycobacterial enzymes for anti-inflammatory and proangiogenic purposes [311]

One possible idea is the use of pH-sensitive peptide hydrogel glucose-responsive insulin delivery systems loaded with insulin and glucose-specific enzymes. Insulin and enzymes are loaded via a self-assembly process under physiological conditions. When hyperglycemia occurs, the enzymatic conversion of glucose to gluconic acid leads to a local decrease in pH, and mutual repulsion between the peptide molecules causes the hydrogel to be broken down, which triggers the release of insulin [300].

Beyond glucose, abnormal adenosine triphosphate (ATP) gradients serve as biochemical markers of cellular stress in trauma sites. ATP-responsive self-assembling hydrogel platforms capitalize on this biomarker, utilizing aptamer conformational switches to deliver therapeutics preferentially to high-metabolic-activity zones thereby promoting bone regeneration.

ATP response

Tumor marker-responsive materials show great potential for biosensing and controlled drug delivery in tumor therapeutics [301]. However, despite their smart controllability and selectivity, their limited drug loading capacity and susceptibility to unstable drug release limit the practical clinical utility of many responsive systems. Therefore, the development of a novel drug delivery strategy with precise controlled release and high efficacy is imperative. In this context, DNA hydrogels have unique advantages as stimuli-responsive drug delivery platforms. Hydrogels are composed mainly of highly biocompatible polymer molecules with low biotoxicity and high water content, which naturally possess a porous network structure and are considered ideal drug carriers. In addition, the programmability of functional nucleic acids allows DNA hydrogels to respond to different environments as needed. Thus, DNA hydrogels with tumor marker-responsive properties can be designed to improve the specificity of tumor treatment strategies and the efficiency of drug therapy.

Owing to the differences in ATP levels between cancer cells and normal cells, ATP-responsive DNA hydrogels can serve as representative tumor marker-responsive DNA hydrogel drug delivery systems. Chunhua Lu et al. developed a self-assembling ATP-responsive AAL-nanogel for specific fluorescence imaging and chemotherapy of cancer cells. The purpose of this self-assembling DNA hydrogel is to construct an ATP-responsive drug delivery system by assembling two polyacrylamide-DNA (PAA-DNA) copolymers, ATP aptamer linker substances, and the chemotherapeutic drug DOX in specific ratios. The AAL-nanogel responds to high levels of ATP through the formation of an ATP/aptamer complex, which leads to AAL-nano gel decomposition. The AAL-nanogel-loaded chemotherapeutic drug DOX was then released, enabling specific activation of fluorescence imaging and selective chemotherapy for cancer cells [302] (Fig. 19). This therapeutic strategy is expected to enable qualitative analysis of intracellular ATP levels and demonstrate self-assembly, a novel method for constructing tumor marker-responsive drug carriers.

ATP-responsive DNA hydrogels for tumor treatment have shown promising results in both fluorescence imaging and drug delivery in recent years. Jingyi Yang et al. developed a novel DNA hybridization hydrogel for fluorescence-guided surgical resection (Apt-HEX/Cp-BHQ1 gel or AHB gel) that showed strong fluorescence at high ATP concentrations (100–500 μm), corresponding to the TME, but little fluorescence at lower ATP concentrations (10–100 nm), such as those in normal tissue. The AHB gel shows strong fluorescence at high ATP concentrations (100–500 μm) (corresponding to the TME) but shows little fluorescence at low ATP concentrations (10–100 nm) (such as those found in normal tissues), thus creating a clear boundary between the high- and low-ATP regions and enabling precise imaging of tumor tissues [303]. Zhuang Liu et al. constructed an in situ ATP-responsive CpG oligonucleotide-releasing smart hydrogel using sodium alginate and an ATP aptamer, which can form an in situ ALGA-Aapt/CpG smart hydrogel with long-term residence at the tumor site. Once ICD-induced chemotherapy or radiation therapy is applied, the smart hydrogel can release CpG stimulated by ATP leakage from dead tumor cells, achieving the simultaneous release of immune adjuvants and low-dose chemotherapy/radiotherapy drugs, which provides a remarkable synergistic response in clearing the already tumor and improving the efficacy of radiotherapy [304]. Considering the unique top-down editable nature of self-assembling hydrogels, building on existing research on ATP-responsive hydrogel platforms for tumor therapy would be promising if they could be better combined with the programmability of functional nucleic acids.

In accordance with these findings, self-assembling hydrogels for cellular fluorescence imaging have been studied for a long time. Wang Hu et al. constructed a novel bioresponsive supramolecular fluorescent hydrogel through electrostatic interactions between sodium poly(p-styrenesulfonate) and tetraphenylene (TPE) derivatives containing two quaternary ammonium cations. This self-assembly process led to the aggregation of TPE, a typical chromophore with aggregation-induced emission (AIE) properties, thus conferring AIE fluorescence properties to this hydrogel. This supramolecular fluorescent hydrogel undergoes a gel-to-sol transition induced by high ATP, and the gel state and fluorescence intensity can be restored upon the addition of ATPase [305]. This self-assembling hydrogel can cross the cell membrane into the cytoplasm on the basis of its fluorescence properties and ATP responsiveness and thus can be used for cellular imaging.

While ATP responsiveness enables spatially targeted delivery of osteogenic agents, it exhibits inherent limitations in substrate specificity for bone remodeling pathways. Enzyme-responsive self-assembling hydrogel systems address this constraint through protease-cleavable linkers (e.g., MMP-sensitive peptide sequences), achieving pathology-dependent therapeutic liberation synchronized with osteoclast/osteoblast activity dynamics.

Enzyme response

Self-assembling supramolecular hydrogels exhibit tunable molecular recognition and have a wide range of applications in wound healing, anti-inflammatory and antimicrobial activities, and drug release. Synergistic Coupling of Enzymatic and Gel Reactions Enables the Construction of Unique Stimulus-Responsive Self-assembling Hydrogel Materials for Sensing Various Disease-Related Biomarkers [306]. In addition to enzymes acting as switches to trigger small-molecule self-assembly, enzyme-responsive self-assembling hydrogels also exhibit excellent performance in antimicrobial, hemostatic and anti-inflammatory responses [307]. Sunnapu, O et al. developed an anti-inflammatory agent, gingerone (the active ingredient in ginger), encapsulated in a modified injectable enzyme-responsive self-assembling hydrogel, which releases gingerone in the presence of inflammation-associated protein hydrolysing enzymes, and the released gingerone retains its anti-inflammatory properties for localized anti-inflammatory effects [308].

The traditional approaches to overcome impediments to healing, such as chronic wound infection, uncontrolled inflammation, impaired cellular function, poor angiogenesis and enhanced protease activity, are mainly surgical debridement, exudate absorption and the use of conventional drug-carrying hydrogel dressings, which have a low overall healing capacity [309]. Perez-Rafael, S et al. reported a simple and versatile route for the preparation of biocompatible multifunctional self-assembling hydrogels via polymer‒nanoparticle interactions. The core idea of this study is to prevent wound chronicity and promote wound healing by utilizing the in situ self-assembly of thiolated hyaluronic acid and hybridized silver-lignin nanoparticles (Ag@Lig NPs) to produce nanocomposite hydrogels capable of simultaneously inhibiting the overexpression of severe protein hydrolases and oxidative enzymes, as well as a high bacterial load [310] (Fig. 19).

A self-assembling nanofibrous supramolecular hydrogel was developed by Chawla, V et al. This hydrogel couples the peptide chain of the peptide lauric acid peptide with positively charged lysine side chains, which is highly stable for proteolytic hydrolase degradation and exhibits efficient antimicrobial activity against Escherichia coli and Staphylococcus aureus. This hydrogel also shows excellent antioxidant activity and pro-cellular proliferative capacity in in vitro experiments, and its loaded nanoparticles are highly effective in the treatment of disease because they upregulate pro-angiogenic genes, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF2), and epidermal growth factor (EGFR) to promote angiogenesis, which can provide a favourable wound healing environment by restoring damaged cellular functions [309].

Diabetic foot wound therapy needs to address the major problem of refractory ulcers due to immunodeficiency, ischemia and a high-glucose microenvironment [311]. Immune mediator derived from melanoma 2 (AIM2) deficiency can effectively improve the immunodeficient microenvironment of inflammatory memory peptides. Cheng, PK et al. coupled inflammatory memory peptides along with angiogenic peptides to a peptide backbone and then formed an anisotropic hydrogel via self-assembly of β-folded peptides on the basis of hydrophobicity and DOPA oxidation, and Mycobacterium avium subsp. mucilaginosum was inoculated into the hydrogel to confer enzyme responsiveness. Peptide hydrogels transferred to diabetic ischemic ulcers via subcutaneous injection via the release of inflammatory memory peptides from the mucinophilic trehalase response effectively increase local environmental immunity and act together with proangiogenic peptides to promote the healing of diabetic ischemic ulcers [311].

Enzyme-responsive self-assembling hydrogels enable localized drug delivery to match disease activity, which is of great value in the treatment of a number of chronic, dynamically changing diseases, such as rheumatoid arthritis. Vemula, P.K. et al. reported an injectable self-assembling nanofiber hydrogel that encapsulates and releases reagents in response to specific enzymes that are significantly upregulated in diseased states, including matrix metalloproteinases (MMP-2 and MMP-9) and esterases. They also demonstrated that this self-assembling nanofiber gel can withstand the shear forces that may be experienced in dynamic environments such as joints and can remain stable after being injected into healthy joints in mice [312]. This report has implications for therapeutic strategies for chronic bone-related diseases, and future research could refine a highly biocompatible, enzyme-responsive drug delivery system that releases drugs only during deterioration and self-titrates according to the level of inflammation.

The combination of short peptide self-assembly and enzyme catalysis can also be applied for rapid hemostasis of wounds. Chen, CX et al. designed an amphiphilic short peptide, I(3)QGK, which self-assembles into long nanoribbons in aqueous solution. Upon addition of transglutaminase (TGase), the peptide solution undergoes a pronounced sol‒gel transition, rapidly forming a rigid hydrogel with shear-thinning and immediate recovery properties. Chen, CX et al. reported that, since factor XIII in blood is converted to active TGase (factor XIIIa) by thrombin during bleeding, the peptide solution, unlike other hemostatic methods or materials, can be used as an anticoagulant. showed more rapid and effective hemostasis by gelling blood and promoting self-assembly of platelet adhesion [313].

Tumor cell resistance and damage to normal cells caused by conventional tumor treatments, such as radiotherapy, are important obstacles to the effectiveness of cancer treatment. The team of Tanaka developed an enzyme-responsive self-assembling hydrogel that uses the enzyme MMP-7, which is highly expressed in tumor cells, to cut the precursor of the hydrogel, and the main part of the cut hydrogel can self-assemble into nanofibers, which can enter tumor cells through cytophagy. After being cut, the main part of the hydrogel can self-assemble into nanofibers, which can enter the tumor cells through cytotoxicity and generate a certain viscosity in the tumor cells, thus interfering with the normal physiological activities of the tumor cells and achieving the purpose of apoptosis of the tumor cells, and the normal cells are not affected [314]. This method can specifically target tumor cells without damaging normal cells and is expected to be a new means to kill tumor cells and overcome the drug resistance of tumor cells in the clinic.

Despite the biochemical precision, enzyme-triggered systems require molecular recognition events that may be delayed in hypovascular defects. Ultrasound responsiveness provides complementary spatiotemporal control, utilizing non-invasive acoustic energy to penetrate deep tissues and induce on-demand cargo release through cavitation effects.

Ultrasound response

Hydrogels composed of an extracellular matrix (ECM) are used as a matrix for tissue culture and have been used in many preclinical and clinical applications to facilitate the repair and reconstruction of various tissues. However, these ECM hydrogel materials are prepared via lengthy methods that focus on the enzymatic dissolution of the ECM with acidic proteases in acidic solutions, which may affect the structure and function of the natural protein. Stephen F. Badylak’s team reported a method for preparing ECM bioscaffold hydrogels via ultrasonic cavitation. By adjusting the temperature, the dissolved ECM can be induced to rapidly self-assemble into a gel, and the material properties of the gel can be tailored by adjusting the ECM concentration and sonication parameters [315]. Compared with conventional enzymatic digestion, this method enables the rapid formation of ECM hydrogels without the need for acidic solutions or protease digestion, which significantly improves the rheological properties and processing time and serves as a platform for the large-scale production of ECM hydrogels [315] (Fig. 20). This method provides great convenience for in vitro studies of osteogenesis-related cells.

Fig. 20.

(A) Method for preparing hydrogels from ECM bioscaffolds via ultrasonic cavitation. Dissolved ECM can be induced to rapidly self-assemble into gels by adjusting the temperature [315]. (B) Multistimulus-responsive biointerfaces based on silicon modified with copolymers containing a thermoresponsive component (poly(N-isopropylacrylamide)) and a component phenylboronic acid [19]

The preceding sections establish that single-stimulus systems exhibit inherent trade-offs between precision, penetration depth, and biological relevance. Dual/multi-responsive hydrogels transcend these compromises by integrating orthogonal triggers (e.g., pH-glucose, enzyme-pH), thereby enabling context-aware therapeutic actions across the dynamic bone healing continuum.

Dual/multi-response

The microenvironment at the site where bone regeneration needs to be promoted is often characterized by increased acidity, inflammation, and complex levels of ROS when the body is suffering from other diseases or severe injuries, where stimuli-responsive hydrogels alone are insufficient or ineffective at achieving the goals of bone regeneration in complex microenvironments [316]. Therefore, dual- and multistimulus-responsive hydrogels have been developed to promote bone regeneration under complex microenvironmental changes. High levels of inflammation and oxidative stress in the microenvironment due to glucose fluctuations induce metabolic disturbances and mitochondrial dysfunction in diabetic patients, leading to slow healing of diabetic bone defects and difficult tissue regeneration.

Diabetic patients with peripheral vasculopathy of the lower extremities and insufficient blood supply to foot wounds, as well as higher glucose levels in foot wounds, are more likely to experience increased bacterial growth and stimulated inflammation than wounds in the general population. The microenvironment of the diabetic foot is characterized by a low pH and high glucose levels, and the development of pH/glucose dual-responsive drug-carrying systems specific to the diabetic foot is challenging. Prof. Pauline Guo’s team designed a pH/glucose dual-responsive release of the diabetes therapeutic drug metformin in a multifunctional hydrogel (PC/GO/Met), which achieves healing of diabetic foot wounds by selectively releasing the drug through the two dynamic covalent bonds of Schiff base and phenylboronic acid and is able to reduce the immune response and enhance coagulation, as well as possessing excellent antimicrobial and antioxidative stress properties [20]. This unique delivery system is highly effective in reducing the local inflammatory response caused by diabetic feet and accelerating wound healing.

Some components with cellular activity, such as hBMSCs, tend to promote bone tissue regeneration at the site of bone defects; however, in the context of the complex intraorganism environment, how to rationally achieve targeted transport and improve the limit of transport has been a key constraint to its clinical application. Under such a premise, self-assembling hydrogel carriers capable of realizing dual correspondence between sugars and temperature provide a new direction to solve this challenge. Pan et al. developed a sugar and temperature dual-responsive hydrogel based on a phenylboronic acid (PBA) moiety and PNIPAAm copolymer for capturing cell sheets, which is characterized by the effective release of cell sheets at lower temperatures and elevated sugar concentrations [154].

Artificially designed biointerfaces based on stimulus response can serve as a dynamic tool to modulate the biological internal environment and improve the effectiveness of related therapies. Considering the complexity of biological environments, biointerfaces with multistimulus response properties have been widely developed in the biomedical field. Herein, a dynamic self-assembling hydrogel biointerface with both a thermoresponsive component (poly(N-isopropylacrylamide)), a pH-responsive molecule (diol), and a sugar-responsive component (dynamic borate bonding) is presented, which responds to ambient temperature, pH, and sugar concentration to regulate the adsorption and release of proteins and the attachment and detachment of bacteria and cells on the surface [19]. Compared with single-stimulus-responsive biomaterials, this biointerface is more adaptable to the complex biological environment of diabetic bone defects (high sugar content, fluctuating pH, and higher temperature than other skin surfaces) and is expected to promote bone regeneration in diabetic bone defects, shorten the therapeutic process, and improve treatment efficacy through infection prevention, the modulation of inflammatory responses, and specific drug delivery.

Conclusions and future perspectives

This comprehensive analysis of self-assembling hydrogels in bone-related biomedical applications over the past two decades reveals their dual functional superiority as drug carriers and stimuli-responsive platforms, characterized by three critical merits: (1) exceptional biocompatibility enabling site-specific therapeutic delivery with minimized systemic drug loss, (2) programmable responsiveness permitting spatiotemporal control of bioactive release, and (3) structural editability facilitating customized mechanical and biochemical microenvironments. Current implementations predominantly exploit their passive drug-loading capacity and single-stimulus responsiveness, achieving baseline requirements for localized osteogenic factor delivery. However, fundamental limitations persist in optimizing carrier-drug interaction dynamics and systematically correlating structural parameters with release kinetics – gaps exacerbated by the prevalent focus on empirical material combinations rather than mechanistic investigations of intrinsic carrier properties.

While self-assembling hydrogels exhibit considerable promise for bone regeneration, their clinical translation remains impeded by significant challenges. Beyond promising proof-of-concept studies, concerted efforts are required to address critical barriers, particularly in load-bearing scenarios. Key among these is ensuring precise spatiotemporal matching between the evolving mechanical and biological demands of bone defects and the scaffold’s properties, while simultaneously maintaining robust bone-implant integration stability. Furthermore, as drug delivery platforms, these hydrogels must demonstrate responsive behavior to dynamic physiological cues (e.g., pH, temperature) and ensure the preservation and controlled, tailorable release kinetics of encapsulated bioactive factors to sustain therapeutic efficacy.

To bridge this translational gap, future research may consider prioritising the development of hydrogels meeting essential physicochemical criteria for clinical bone repair scaffolds. An ideal construct should possess: tunable and dynamically adaptive tensile modulus approximating native bone at the implantation site; drug release kinetics meticulously synchronised with new bone formation rates; spatiotemporally controllable delivery of osteogenic/angiogenic signals; highly interconnected porous architectures facilitating rapid vascularisation and osseointegration; excellent biocompatibility with minimal chronic inflammation; injectability enabling minimally invasive deployment; and robust manufacturability ensuring batch-to-batch reproducibility under stringent quality control.

Future research should focus on addressing the key challenges in the clinical translation of self-assembling hydrogel systems for bone repair. Priority areas include the development of “smart” hydrogels that respond to bone-specific microenvironmental cues—such as pH shifts, enzymatic activity, and mechanical loading—to enable on-demand modulation of their properties; the design of advanced delivery systems based on endogenous triggers, cascade release mechanisms, and more stable and cost-effective bioactive agents, such as biomimetic peptides, small molecules, and extracellular vesicles; and elucidating the mechanisms underlying material-host interactions, particularly immune responses, to actively harness these biological processes for enhanced bone regeneration. Comprehensive long-term evaluations using clinically relevant endpoints in standardized load-bearing bone defect models in large animals (e.g., sheep, minipigs) are essential. Furthermore, computational modeling and artificial intelligence should be integrated to facilitate the rational design and optimization of self-assembling systems. Ultimately, a multidisciplinary, consortium-based approach is imperative to translate the fundamental potential of self-assembling hydrogels into viable clinical solutions for bone defect repair.

Author contributions

All the authors have read and approved the final review manuscript. Y.Wang and Y.Yang conceived the article. Y.Wang wrote the first version of the manuscript.W, Geng created the outline and formatted the figures. Y.Li, Y.Qian and R.Fan formatted the tables. Z.Sun and J.Guan checked the English. Y.Qiao D.Geng oversaw and directed the entire work. Y.Wang, Y.Yang and Y.Hong contributed equally to this work.

Funding 

This work was supported by the National Natural Science Foundation of China [Grant No. 82572432].

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.