Research status of biomaterials based on physical signals for bone injury repair

Abstract

Bone defects repair continues to be a significant challenge facing the world. Biological scaffolds, bioactive molecules, and cells are the three major elements of bone tissue engineering, which have been widely used in bone regeneration therapy, especially with the rise of bioactive molecules in recent years. According to their physical properties, they can be divided into force, magnetic field (MF), electric field (EF), ultrasonic wave, light, heat, etc. However, the transmission of bioactive molecules has obvious shortcomings that hinder the development of the tissue-rearing process. This paper reviews the mechanism of physical signal induction in bone tissue engineering in recent years. It summarizes the application strategies of physical signal in bone tissue engineering, including biomaterial designs, physical signal loading strategies and related pathways. Finally, the ongoing challenges and prospects for the future are discussed.

1. Introduction

Bone defects are often caused by trauma, bone tumor resection, arthritis, etc., and their regeneration and repair are challenging tasks in clinical applications. There are several partially overlapping phases of bone regeneration. During the anabolic phase, cells gather at the defect site to form a cartilaginous callus and ensure a blood supply. After chondrocyte apoptosis, cartilage resorption, and secondary osteogenesis are carried out in the metabolic phase with the activity of osteoblasts and osteoclasts. Finally, remodeling of the defective portion leads to bone regeneration. The complexity of bone injury increases the risk of delayed healing and non-healing, and improper treatment may cause a variety of complications, such as inadequate angiogenesis and an insufficient number of progenitor cells required for new bone formation, ultimately resulting in the inability of the natural healing process to succeed [1]. Insufficient bone union leads to bone nonunion or segmental defect in 10 % of all cases of bone injury [2,3]. The present benchmark for addressing bone injuries is bone grafting, encompassing both autografts and allografts. However, an autograft is limited by the bone obtained volume, possible complications (lesions at the site of acquisition, local hematoma), and remodeling of implanted bone, etc. [4,5], which indicates autograft cannot fully meet clinical requirements. The poor integration and vascularization of host bone tissue also limit the clinical application of allograft [6,7]. Autologous bone transplantation is a common method to repair bone nonunion at present. The procedure involves removing bone from the iliac crest of the patient and grafting it onto the site of the fracture, which has good histocompatibility and non-immune immunogenicity [8]. Autografts contain growth factors (GF), osteoprogenitor cells, and a three-dimension matrix, which are important components of osteoinduction, bone conduction, and osseointegration. However, disadvantages of autologous bone transplantation such as hemorrhaging, swelling, infectious complications, persistent discomfort, potential harm to the donor site, hypersensitivity reactions, and scarring have been reported recently [9]. In addition, the source of autologous bone grafts is limited and is not able to deal with large bone defects [10]. Allogeneic bone transplantation is another common treatment option for bone nonunion. Still, there are also concerns about a lack of donors, the spread of viral illnesses, bacterial contamination, or rejection by the immune system [11]. Furthermore, distraction osteogenesis is a relatively mature treatment technique, but this treatment takes a long time, brings a strong sense of pain and may lead to poor healing and refracture due to the non-standard operating procedure of treatment [12]. Artificial bone material such as calcium phosphate shows low mechanical strength. Acrylic cement has poor biocompatibility, lacks bone conductivity, and the released heat during polymerization causes osteonecrosis. Bioceramics, such as hydroxyapatite, tricalcium phosphate, and PEEK, have poor osteoinductive properties, high prices, and limited clinical applications [13]. Therefore, a more effective clinical treatment is urgently needed.

The swift advancement of biological tissue engineering technology has introduced fresh perspectives and approaches to investigating bone biomaterials science 1995 [14]. In this modern era, with the continuous advancement of cell biology and material science, bone tissue engineering with seed cells, growth factors, and bone biomaterials as the main elements have been developed, which provides more possibilities for the clinical treatment of bone defect repair [15].

Bone tissue engineering does not require the transplantation of other parts of the patient's body to regenerate the damaged tissues. Moreover, bone tissue engineering can better mimic bone-cartilage tissue in structure and function, which provides a more promising method for bone defect treatment. However, currently, no scaffold perfectly mimics the structural characteristics of bone in terms of angiogenesis, absorbability, and bone regeneration. The majority of current studies have focused on incorporating active biomolecules and cells into scaffolds to mimic the signaling cascade that takes place during natural tissue regeneration to address the above issues [16,17]. However, some challenges are hindering this kind of treatment, the optimal dosage and delivery route of bioactive molecules still need to be further studied.

Over the past few decades, physical cues, including mechanical, optical, thermal, electrical, acoustic, and magnetic signals have emerged as promising options for directing stem cell differentiation as their enhanced safety, cost-effectiveness, and adjustable local characteristics compared to biological and chemical cues [18,19]. At the same time, the rapid development of biomaterial science has brought bright prospects for bone tissue engineering. Due to their special modifications of biomaterials, the physical properties of biomaterials have been greatly enhanced and the application strategies of bone tissue engineering have been expanded when compared with traditional materials. Not to mention, by adjusting the composition and structure of the material, biomaterials can respond to a variety of physical signals, combining the synergistic effects of multiple physical signals, to better repair bone defects. This suggests that biomaterials based on physical signals are a promising strategy for bone tissue engineering. At present, some therapeutic methods based on physical signals have been applied in clinical practice, such as exercise training, electrical stimulation, magnetotherapy, and ultrasound in rehabilitation therapy (see Fig. 1). Therefore, biomaterials based on physical signals have great research value and application prospects in the field of bone repair.

Fig. 1.

Physical stimulation signals for bone defect repair, including magnetic, light, heat, electrical, acoustic, and mechanical signals.

2. Biomaterials based on physical signals for bone defect repair

2.1. Magnetic signal

Magnetic stimulation has been found to play a crucial role in the biological processes of mesenchymal stem cell attachment, movement, growth, and specialization, thereby facilitating bone regeneration [20]. Bone repair can be divided into several stages, including the early stage: inflammatory stage; The middle stage: cartilaginous osteogenesis, teleost osteogenesis and the late stage: bone remodeling. Magnetic fields (MFs) can activate a variety of cell signaling pathways, regulate the degree of bone mineralization, and stimulate the early healing of patients with femoral shaft fracture [21].

The effect of magnetic signals on cells may affect cell function by Ca²⁺ or by using magnetic materials to affect cell biochemical signals. Intracellular Ca²⁺ is generally considered to be the main research object in the magnetic field mechanism as Ca²⁺ is one of the main factors in converting magnetic signals into biological signals. By influencing ion channels, MFs are able to promote Ca²⁺ transfer from the membrane side to the cytoplasm and subsequently increase intracellular Ca²⁺ concentration [22]. On top of that, the electrodynamic effects of the magnetic field are not limited to ion channels on the plasma membrane, but also moving charged particles in and out of the cytoplasm. Ca²⁺ channels are activated by static MFs, and Ca²⁺ inflow further activates p38 mitogen-activated protein kinase signaling channels, thereby affecting the recombination of cell bone shelves and enhancing cell anisotropy [23]. Studies have shown that MFs can not only promote Ca²⁺ concentration in the nucleus, but also increase extracellular Ca²⁺ concentration and subsequently promote Ca²⁺ deposition significantly, thus affecting the biological effects of cells. In addition, magnetic signals affect the permeability of cell membranes, inhibit the release of inflammatory factors, activate macrophages, and regulate the function of immune cells, thereby reducing inflammation, removing necrotic cells and debris at the injured site, and further promoting bone healing by activating immune effects [[24], [25], [26]].

It is worth mentioning that, the effect of the magnetic field on bone repair is affected by the intensity of the magnetic field. MFs can be classified into four levels based on their intensity: Weak MFs: <1 mT (millitesla); Moderate MFs: 1 mT ∼ 1 T (tesla); Strong MFs: 1 T ∼ 5 T; Ultra-strong MFs: >5 T [27]. Strong MFs can significantly change the membrane potential of cells, which has a significant impact on the properties and biological functions of cells [28]. Currently, moderate-intensity MFs are the most widely used. Recent research has found that moderate-strength MFs can promote cell adhesion, proliferation, migration, and differentiation, and can enhance the reparative effects on rabbit cartilage injury [29,30]. Furthermore, immune cells are only slightly affected by low-frequency alternating MFs but are strongly influenced by moderate-intensity MFs and high-gradient MFs (HGMFs). Larger immune cells, such as macrophages, are more sensitive to HGMF, which polarizes the cells towards an anti-inflammatory M2 phenotype [26].

Due to the effect of magnetic signals on osteogenesis, many biomaterials based on magnetic signals have been studied for bone defect treatments. Magnetic nanoparticles (MNPs) are capable of responding to external MFs, giving them the ability to respond to magnetic stimulation by incorporating them into the scaffold [31]. Recent research has implanted a gelatine sponge scaffold loaded with superparamagnetic ferric oxide nanoparticles into the extraction fossae of rat mandibular incisor teeth, and the results showed significantly enhanced bone regeneration after implantation. The protein cap on the MNP composite scaffold is able to promote cell proliferation through the MAPK signaling pathway [32]. What's more, scaffolded adsorption of proliferation-related proteins, particularly the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways and ion channel-related proteins, were enhanced. The enhanced cell proliferation induced by the MNP composite scaffold might be attributed to the activation of the MAPK/ERK signaling pathway via the adsorption of proteins involved in the upstream cascade of receptors. It has been found that magnetic hydroxyapatite scaffolds can stimulate osteoblast growth and multiplication in osteoporosis models by changing the “cargo” of exosomes from osteoclasts and reducing the efficiency of exosome uptake by osteoblasts [33]. Cai et al. combined a 100 mT moderate-intensity stationary magnetic field with polylactic acid/ferric oxide MNP scaffolds and found that the magnetic field could further regulate the proliferation and osteogenic differentiation of MC3T3-E1 cells induced by MNP. The cell adhesion rate on the scaffold, calcium salt deposition, and alkaline phosphatase (ALP) expression was significantly increased under the continuous stimulation of a constant 100 mT magnetic field [34]. Wang et al. examined the ability of the MNP composite scaffold to promote bone formation in vivo under an external magnetic field by establishing a rabbit femoral condyle defect model. Results showed that a magnetic field of 15 Hz/1 mT could promote osteogenic differentiation, and bone regeneration in the defect area and subsequently improve the fusion of graft and host bone [35]. Bin et al. incorporated superparamagnetic Fe₃O₄ nanoparticles into Poly (L-lactic acid) (PLLA) bone scaffolds to produce small-scale MFs, thereby continuously and stably stimulating cells. The results showed that the magnetic scaffolds stimulated the adhesion, expansion, and communication of MG63 cells, enhancing ALP activity and subsequently improving cell growth and specialization (see Fig. 2) [36].

Fig. 2.

The micromagnetic field generated by the Fe₃O₄-containing nanoparticles scaffold promotes bone defect repair [36]. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

In summary, these studies have demonstrated the great potential of magnetic stimulation in bone tissue engineering . Different MFs have different effects. Selecting the appropriate magnetic field to activate multiple biological effects can play an important role in orthopedic treatment, and it is a safe and effective biophysical stimulation method. Despite intensive preclinical exploration in magnetic responsivity-based biomaterials, the clinical applications are very limited. Due to safety issues reported in clinical trials, most materials are still not allowed for clinical applications, and there is still much room for the development of biomaterials based on magnetic response in the future [37,38].

2.2. Light signal

In modern medicine, light is widely used to treat skin, muscle, and eye diseases [39,40]. In contrast to magnetic energy, light isis an easily controlled and widely used physical signal as an external force to control stimulus-responsive biomaterials. Light is regarded as an appropriate physical signal for bone tissue engineering due to its range of advantages such as noninvasive, non-contact, precise, controllable, and good tissue penetration. Light stimulation can affect cell signaling pathways, promote angiogenesis and osteogenic differentiation, and play a role in the early and middle stages of bone repair [41].

The mechanism of direct biological regulation by light is mainly through the absorption of photons by cytochrome C oxidase to improve mitochondrial function and promote ATP generation while affecting related enzymes in cell pathways and promoting the expression of various genes [42], promote bone tissue regeneration. Pyo's team studied the effects of 808 nm laser irradiation on human osteoblasts in hypoxia environment, and results showed that photobiological regulation could promote the expressions of bone morphogenetic protein 2, osteocalcin (OCN), and transforming growth factor β1 genes in hypoxia environment [43]. Michael irradiated New Zealand white rabbits with 820 nm gallium-aluminum-As laser in mandibular distraction osteogenesis. Results showed that photobiological regulation is able to encourage the regeneration of bone tissue during distraction osteogenesis and combined with weak laser for distraction osteogenesis, thus shortening the treatment cycle [43,44]. Khadra examined the mechanism of low-intensity laser therapy that influenced the multiplication and specialization of human osteoblast cells grown on titanium implant surfaces. Results revealed that photobiological regulation was able to stimulate cell growth and specialization in the vicinity of the implant, thus promoting the early healing of the implant and surrounding bone tissue [45]. Moreover, light signals can stimulate cells to produce molecules such as reactive oxygen species and nitric oxide, which can exert immune effects, regulate inflammatory responses, and help the body remove dead tissue at the site of injury [46,47]. Light stimulation can affect a variety of immune cells during fracture healing, such as macrophages, T cells, and B cells, regulate their activity and distribution, and contribute to the immune response and tissue repair at the wound site [48,49]. It can accelerate the fracture healing process by regulating inflammatory response and immune cell function.

The wavelength of light affects cell function. Ultraviolet (UV) (200–400 nm), visible (vis) (400–700 nm), and near-infrared (NIR) (700–1300 nm) light are the three main light subclasses commonly used in phototherapy. As the wavelengths of these light types differ, they also have distinct effects on stem cell differentiation [[50], [51], [52]]. Vis light has the potential to promote osteogenic differentiation. Exposure to red light (630 nm) at 15 mW cm⁻² and 4 J cm⁻² was found to significantly increase the expression of ALP and OCN in rat bone marrow-derived mesenchymal stem cells (BMSCs). Exposing amniotic fluid-derived stem cells (AFSCs) to green light (525 nm) and blue light (470 nm) of Vis light, rather than red light, was found to more effectively enhance their osteogenic differentiation potential [52]. Additionally, NIR spectroscopy is emerging as a non-invasive method for evaluating cartilage pathology. Studies have shown that in the 4000–5100 cm⁻¹ range, the penetration depth can reach approximately 1–2 mm, in the 5100–7000 cm⁻¹ range it can reach around 3 mm, and in the 7000–9000 cm⁻¹ frequency range, the penetration depth can be up to approximately 5 mm. The 4000–7000 cm⁻¹ frequency range within the NIR spectrum has been identified as the most suitable for non-invasively assessing the influence of subchondral bone on overlying cartilage tissue [53].

However, compared to NIR, Vis light is limited in clinical applications due to its insufficient penetration depth and potential tissue damage. Therefore, there is a pressing need to find methods that can in situ convert NIR to Vis light, providing localized stimulation to stem cells. The emergence of upconversion nanoparticles (UCNPs) provides new possibilities [54]. UCNPs are a class of nanomaterials composed of lanthanide-ion species, with tremendous preclinical application potential. They are capable of converting long-wavelength (low-energy) excitation light in the UV, Vis, and NIR spectral regions into shorter-wavelength (higher-energy) emission. Rare earth-doped UCNPs exhibit excellent photostability and minimal autofluorescence, which can be leveraged to convert NIR light into UV or Vis light, thereby enhancing the penetration depth.

The cellular response to light exposure demonstrates the great potential of improving the treatment of bone defects. Light signals are normally used as an initiator for drug-responsive release or in conjunction with other physical signals such as photothermal therapy, and photoelectric therapy, for indirect repair of bone injury repair. Zhang et al. synthesized photoresponsive microgels that can be loaded with dexamethasone (DEX) and released in a photo-triggered manner under UV light. The DEX released can initiate the transformation of hMSC into osteoblasts, which is a crucial step towards bone defect repair [55]. Lijun Kuang et al. prepared an injectable with multiple functions in situ-generated calcium phosphate nanoparticle (ICPN) coordination poly (dimethyl aminoethyl methacrylate-Co-2-hydroxyethyl methacrylate) (DHCP) hydrogel loaded with PTH for NIR stimulated release, to achieve bone regeneration in the ovariectomy (OVX) model [56]. Besides, strontium (Sr) serves as an alternative bone-inducing element that stimulates bone formation. By combining strontium chloride (SrCl₂) and BP nanosheets into polylactic acid-glycolic acid (PLGA) microspheres, a NIR light-triggered Sr²⁺ delivery system was devised for the regeneration of bone tissue [57]. Wan et al. successfully fabricated a programmed drug delivery system by incorporating the NIR photoresponsive polydopamine-coated hydroxyapatite nanoparticles (nHA@PDA) into thermally responsive hydroxybutyl chitosan (see Fig. 3). This intelligent hydrogel composite is capable of initiating a swift release of SIM, enabling the recruitment of endogenous stem cells at the early stages of bone repair. At the same time, the increased PGL release triggered by flexible and NIR light is able to promote osteogenic differentiation of migrating cells via a safe and reliable epigenetic regulation mechanism. The stable release of these two drugs on demand is an efficient method for bone defect repair [58].

Fig. 3.

(A) A programmed delivery system is achieved by integrating a thermo-responsive hydrogel with NIR light-responsive PDA-coated nHA nanoparticles, enabling the precise and on-demand release of simvastatin and pargyline. (B) A transwell chemotaxis assay is conducted with mBM-MSCs. Cells migrated to the lower chamber were subsequently stained with crystal violet, and the quantity of migrated cells was counted. (C) Micro-CT images of the calvarial bone defect at 12 weeks after operation [58]. © 2022 The Authors. Published by Elsevier B.V.

These studies demonstrated that photobiostimulation, in which light of a specific wavelength can activate cell signaling pathways, promote the proliferation and differentiation of osteocytes, and accelerate the healing of bone injury. It can also play a better therapeutic effect by combining with other materials. The different application modes of optical signals in bone repair provide a new biophysical treatment method for orthopedic clinical practice.

2.3. Heat signal

Hyperthermia has been used since ancient times to stimulate the healing of physical diseases and repair of bone defects. The scientific principle of thermal osteogenesis has been validated through in previous vitro studies [[59], [60], [61], [62]]. Thermal stimulation can promote osteogenesis and mineralization, upregulate heat shock proteins (HSPs) in mesenchymal stem cells (MSCs), enhance blood flow, and facilitate nutrient exchange [63], mainly affecting the early and middle stages of bone repair.

Thermal stimulation can promote osteogenesis by promoting blood circulation in the bone defect area [64]. Studies on relevant mechanisms and pathways have shown that heat stimulation can up-regulate the expression of HSP induced by heat shock, and further activate the regulatory protein kinase (ERK) pathway to promote osteogenic differentiation, thereby promoting fracture healing [65,66]. The metabolic evolution of bone structure and morphology occurs throughout human life, and heat plays an important role in this process. Early studies using 915 MHz microwave therapy on rabbit femurs found that thermal therapy enhanced the formation of new bone trabeculae, increased cortical bone density, and accelerated bone remodeling [67]. Kristi for Sunderic et al. tested the effect of thermal stimulation on osteogenic differentiation by periodically applying mild heating to BMSCs. Results showed that thermal stimulation alleviated the cytokine-induced cell growth inhibition and promoted maximal proliferation of hMSCs. ALP and calcium precipitation levels increased significantly after thermal stimulation [68]. Moreover, Thermal stimulation can enhance the phagocytosis of macrophages and remove cell debris and necrotic tissue from the injured site. Heat treatment can also activate T cells and B cells and improve the body's immune response [[69], [70], [71]]. Modulating the local inflammatory response and the function of immune cells creates a favorable microenvironment for bone healing [72]. This suggests that thermal stimulation can promote bone regeneration from BMSCs.

Previous studies have shown that elevated temperatures by 2–4 °C above normal body temperature significantly boosts osteogenic activity, manifesting in the upregulation of ALP expression and improved mineralization capacity of MSCs [[73], [74], [75]]. When the temperature is maintained between 40–43 °C (104–109.4 °F), it is beneficial for the healing of bone defects [76]. The optimal temperature for maximizing osteogenic activity is around 42 °C (107.6 °F). Not to mention, the heating frequency also affects the differentiation efficiency. Applying thermal therapy twice a day shows the best results [73]. The above research suggests thermal stimulation can effectively repair bone defects.

However, due to the external tissue coverage of bone, the external heat source is not highly conductive to the human body. Therefore, the derived photothermal therapy, microwave heating and magnetic heating therapy have shown significant effects in the field of bone defects. Indirect photothermal activation of biomaterials sensitive to external fields, such as hydrogels or nanoparticles, can also stimulate subsequent cellular functions. Liping Tong et al. prepared a bone substitute with black phosphorus nanosheets and polylactic acid-glycolic acid copolymer (BPs@PLGA), and used NIR light-mediated mild heat to effectively up-regulate the expression of HSPs and promote bone formation in vitro and in vivo. This BPs@PLGA bone replacement provides a new direction for the development of remote controlled bone replacements via photothermal therapy [63]. Currently, the reported photothermal conversion agents primarily suffer from limited tissue penetration depth, which restricts their widespread application. As well as, most of these agents consist of non-biologically active materials that may induce undesirable systemic side effects at the time of implantation. Chen Yang et al. fabricated a 3D printed scaffold with the NIR-II photothermal conversion agent (Wesselsite [SrCuSi₄O₁₀] nanosheets (SC NSs). The designed 3D composite scaffolds triggered osteosarcoma ablation by extensive thermotherapy generated by NIR-II light (see Fig. 4). Moreover, it also stimulated cell proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) as well as human umbilical vein endothelial cells (HUVECs) in vitro, and ultimately enhanced vascularized bone regeneration in vivo due to controlled and sustained release of bioactive ions (Sr, Cu, and Si) [77]. Wang et al. synthesized RGD-coated magnetic iron oxide nanoparticles (MION; CoFe₂O₄@MnFe₂O₄) embedded in agarose, using the magnetothermal effect of the material system to repair bone defects. Mild thermal stimulation induced by magnetic field promotes osteogenic differentiation and biomineralization via PI3K/Akt activated by HSP. At the same time, cobalt collaborates with thermal stimulation to promote the formation of blood vessels, demonstrating the great potential of magnetothermal therapy in the treatment of bone defects [78].

Fig. 4.

(A) 3D printed scaffolds combined with NIR-II photothermal therapy of osteosarcoma to improve vascularized bone regeneration. (B) Thermal imaging of the scaffold under 1064 nm laser irradiation. (C) In vivo vascularized bone regeneration [77]. Copyright © 2021 John Wiley &amp; Sons, Inc. All rights reserved.

According to the above studies, heat has multiple functions in bone injury repair, such as promoting blood circulation, reducing inflammatory symptoms, stimulating osteocyte activity, and promoting bone repair. Hyperthermia at the appropriate temperature can play a multifaceted physiological regulatory role and play an important role in the conservative treatment and surgical repair of bone injuries. As a safe and effective biophysical stimulation method, hyperthermia offers new possibilities for orthopedic treatment.

2.4. Electrical signal

Bioelectricity is an important physiological activity in the human body. Many physiological functions of the human body are realized through endogenous electric fields, such as membrane potential, nerve potential, etc [79]. Based on the research of endogenous bioelectricity, exogenous electrical stimulation therapy has been extensively employed as a promising medical intervention for the treatment of nerves, muscles, and bones [[80], [81], [82]]. Electric field (EF) stimulation can elicit various responses in stem cells, affecting key signaling pathways and cellular reactions, including the involvement of reactive oxygen species and HSPs, the regulation of intracellular calcium concentration, and ATP production, as well as processes related to cell surface receptor clustering and cytoskeleton remodeling [83], Electrical stimulation can promote vascularization, proliferation and differentiation of osteoblasts, mainly in the middle and late stages of bone repair [84,85].

The effects of EF on cells are multifaceted. The changing EF can change the charge distribution on the plasma membrane surface, induce the release of intracellular Ca²⁺, and cause downstream cascade reactions [86]. Pulsed electromagnetic fields can activate the FAK/Rho GTPases signaling pathway by increasing intracellular Ca²⁺ concentration, thus promoting cell migration and differentiation and reconstructing cytoskeleton [87]. In addition, electrical stimulation plays an important role in regulating osteoblast differentiation [88]. In vitro, electrical stimulation can increase the proliferation and activity of osteoblasts and promote the expression of osteocyte and OCN in osteoblasts, both of which are key proteins that increase the mineralized parts of the bone. Electrical stimulation has been demonstrated to enhance mRNA expression of the type I collagen gene in osteoblasts, leading to elevated collagen-I protein levels [89,90], electrical stimulation creates an environment conducive to bone growth. For example, when stimulated by a direct current, the cathode undergoes a chemical reaction to form hydroxide ions. The increase in the ion concentration reduces the oxygen concentration and increases the local pH value, thus promoting osteoblast osteogenesis and reducing osteoclast activity [91]. Moreover, the electric field can also play an immunomodulatory role by affecting the cell membrane potential, thereby regulating the expression of inflammatory factors such as cytokines and chemokines, and affecting the activation, migration, and differentiation of immune cells [[92], [93], [94]].

The effect of the electric field on the osteoblast is affected by the voltage. In the study by Ercan et al., the optimal voltage window to maximize osteoblast density on nanotubular titanium was found to be 1 V–15 V and 20 Hz (IonOptix) [95]. In the study by Aaron et al., it was determined that EF amplitudes in the range of 10–1000 V/m were sufficient to achieve an effect without cell damage [96], electrical stimulation enhances bone recombination and healing by helping to support the implantation of cells into new bone and remodeling old bone (Osteoblasts), as well as by optimizing the function and activity of bone cells themselves by altering ion channel activity in cell function.

In the study of cells regulated by electrical stimulation, biomaterials are often used in combination with electrical stimulation. Exogenous electrical stimulation provides a physiologically suitable environment to replace endogenous EFs, more accurately load damaged tissues, and promote tissue regeneration and repair [97]. In recent years, increasing attention has been directed towards nanometer conductive materials and electroresponsive materials. Liudmila Leppik et al. exposed adipose tissue-derived mesenchymal stem cells (AT-MSC) seeded on a β-tricalciumphosphate (β-TCP) scaffold to ES and found that ES promotes bone differentiation. Then, in vivo, experiments demonstrated that ES could elicit elevated expression of osteogenic marker genes, increase new bone mass and mechanical properties, reduce fibrous tissue, and promote vascularization [98]. Electrical stimulation can promote cell proliferation. Cui et al. integrated the hBMP-4 gene fragment into a non-viral artificial recombinant plasmid vector (pSTAR) to construct a doxycycline-inducible expression vector (phBMP-4), controlled the release of phBMP-4 by electroactive scaffold, and controlled gene release under electrical stimulation with improved cell proliferation ability. In order to enhance in vitro osteogenic differentiation and effective bone healing ability [99]. This kind of electroactive and controlled-release scaffold provides a new idea for bone tissue engineering. Liu et al. developed batiO3-coated 3D printed porous Ti₆Al₄V scaffoldings (pTi/BaTiO₃) with appropriate mechanical properties, providing space for bone and blood vessel growth, as well as a physiological electrical microenvironment for bone regeneration. The scaffold is capable of generating an endogenous EF through mechanical deformation, thus promoting the formation of PDGF-BB/PDGFR-β and its downstream molecules via an autocrine pathway, thus promoting the vascularization of bone tissue and providing a better basis for bone growth. The piezoelectric effect can effectively promote the increase of intracellular calcium ion concentration, and eventually lead to elevated intracellular P38 phosphorylation levels, which leads to the upregulation of osterix expression, and ultimately realizes the osteogenic differentiation of MSCs [100]. Li et al. developed a graphene-cellulose (G-C) -based bone scaffold (see Fig. 5), a device with electrically active G-C “paper” in a polystyrene chamber, for simultaneous cell culture and ES to stimulate osteogenic differentiation of adipose-derived stem cells (ADSCs). This device may provide a new therapeutic strategy for bone injury repair [101].

Fig. 5.

Electrical stimulation-induced osteogenesis of human adipose-derived stem cells using a conductive graphene-cellulose scaffold [101]. © 2019 Elsevier B.V. All rights reserved.

In summary, weak electrical stimulation can activate osteocytes, improve the activity and mineralization level of osteoblasts, accelerate the healing process of fracture, regulate the differentiation of stem cells, and is applied in the field of bone regeneration combined with biomaterials. Electrical stimulation therapy is safe, reliable and adjustable, and has a broad prospect in orthopedic clinical applications in the future.

2.5. Acoustic signal

Acoustic signals have been utilized extensively for the diagnosis and treatment of various diseases for a considerable period of time. Sound waves carry momentum and energy, which will also be transferred to scatterers in the medium through scattering and refraction, resulting in acoustic radiation forces. Acoustic radiation force can be used to manipulate particles and microbubbles, which has broad application prospects in biomedicine [102]. Ultrasound can play a role in all stages of bone repair, which can promote angiogenesis, chondrogenesis, proliferation and differentiation of osteoblasts. Long-term ultrasound stimulation in the late stage of fracture can help to increase bone mineral density and promote fracture remodeling and reconstruction at the fracture end. Ultrasound provides suitable biophysical stimulation for bone tissue engineering and accelerates the repair of bone defects [[103], [104], [105]].

Low-intensity pulsed ultrasound (LIPUS) is a non-invasive physical therapy modality that has gained widespread acceptance in clinical orthopedics [106]. Ultrasound can develop the disease clinically and has satisfied effects on kidney stones, liver, bile, pancreas, and spleen. It is a non-invasive, low-cost diagnostic method. Compared with radioactivity examination, ultrasound has incomparable safety advantages. On the other hand, low-intensity ultrasound can promote osteogenic differentiation, and many clinical studies on the use of low-intensity ultrasound to treat bone disunion and promote bone healing. In 2000, LIPUS was approved by the US Food and Drug Administration (FDA) for the clinical treatment of fractures and nonunions. The influence of ultrasound in bone injury repair is multifaceted. Firstly, LIPUS can alter cell signaling, stimulate proteoglycan synthesis, increase intracellular calcium ion concentration, and accelerate fracture healing [[107], [108], [109]]. Secondly, LIPUS increases levels of vascular endothelial growth factor A (VEGF-A), which accelerates vascularization. Finally, LIPUS has been shown to enhance the maturity of new bone tissue and facilitate the occurrence of endochondral osteogenesis [[110], [111], [112]]. What's more, it is worth mentioning that acoustic stimulation can increase local blood circulation and promote the clearance of inflammatory substances, inhibiting the expression of inflammatory factors and reducing inflammatory response. It regulates the function of immune cells, promotes the clearance of damaged tissues, and further treats bone injury by exerting immune effects [[113], [114], [115], [116]].

With the development of bone tissue engineering, ultrasound has been used to deliver growth factors and genes, as well as scaffold design and assembly. Yanni He et al. designed an acoustic response scaffold (ARS) and embedded it into a growth factor-loaded hydrogel to obtain a biomimetic hydrogel scaffold. Endogenous BMSCs were recruited through ultrasonic (p-US) responsive release of growth factors. The sound scaffold greatly promotes cell adhesion and growth and can be used for bone defect regeneration [117]. This study offers a novel and efficient approach for repairing orthotopic bone defects by ultrasound signal stimulation. Maxim Bez et al. implanted collagen scaffolds into the critical bone defects of pigs and recruited endogenous stem cells into the defects. Two weeks later, bone morphogenetic protein-6 (BMP-6) plasmid DNA and microvesicles were injected into the nonunion site, and the microvesicles were sonicated to help the recruited osteoprogenitor cells take up BMP-6. Ultrasound can promote BMP-6 secretion and fracture healing [118].

Furthermore, ultrasonic stimulation can be combined with other physical stimulation for bone tissue engineering, such as sonoelectric stimulation and sonothermal stimulation. Jie Chen et al. proposed a treatment strategy that combined piezoelectric batio3-coated Ti6Al4V scaffolds with LIPUS. The piezoelectric effect of ultrasound and piezoelectric coating can activate intracellular mitochondria by promoting cell proliferation and reducing cell apoptosis. The coordinated stimulation of ultrasonic and piezoelectric scaffolds can up-regulate the expression of osteogenic genes and promote bone healing [119]. Sandra Camarero-Espinosa et al. developed a Janus scaffold formed spontaneously by phase separation of polycaprolactone (PCL) and polylactide (PLA), which can be activated by ultrasound to generate vibrations that are delivered to cells (see Fig. 6). PLA and PCL represent active and supporting materials respectively. Remote stimulation of the Janus scaffold promoted cell proliferation, matrix deposition and osteogenic differentiation of human bone marrow-derived stromal cells (hBMSC) through the formation and activation of voltage-gated calcium channels [120]. In addition, ultrasound-targeted microbubble destruction technology, as the latest targeted gene delivery system with physical properties, has proven to be a promising new strategy for non-invasive, targeted drug and gene delivery [[121], [122], [123]]. The principle of UTMD is the targeted release of drug-loaded or gene-loaded microbubbles under the action of ultrasound and the targeted destruction of microbubbles to cause local regional drug aggregation, which can achieve an increase in local regional drug concentration. The principle of UTMD combined with microbubbles to deliver drugs or genes is achieved by the cavitation effect. The cavitation effect refers to the expansion and contraction of microbubbles caused by the change of periodic sound pressure during ultrasound irradiation, which breaks the shell of microbubbles and releases drugs or genes [124,125]. Gong et al. developed VEGF-loaded PLGA microbubbles (MBs) combined with ultrasound-targeted microbubble disruption (UTMD) to improve angiogenesis and osteogenesis in bone defect repair. The release curve indicated that UTMD promoted the burst release of VEGF from PLGA MBs. Results showed that in combination with UTMD, VEGF MBs significantly up-regulated the expression of osteogenesis-related genes and promoted calcium deposition, which effectively promoted the formation of new bone and the development of blood vessels at the injury site [126].

Fig. 6.

(A) Schematic representation of the process to create phase-segregated scaffolds. The materials were first blended to obtain filaments, which were then cut into particles and fed to a 3D printer (left), where phase segregation occurred as the material was deposited (middle) to produce the Janus scaffolds (right). (B) Collagen I, collagen X, RunX2, and OCN gene expression and cell number (C) were also analyzed. (D) Alizarin red staining (left) showing increased calcium deposition on Janus and PCL scaffolds stimulated with ultrasound compared to static cultures [120]. Copyright © 2021 Springer Nature. All rights reserved.

Therefore, as a safe and effective biophysical stimulation method, ultrasound can play an important role in all stages of fracture healing and is widely used in orthopedic clinical treatment. Furthermore, ultrasound also plays a key role in bone defect repair and postoperative rehabilitation, providing patients with a full range of non-invasive physical therapy. Due to its safety, adjustability, and multiple physiological regulation effects, ultrasound therapy has been widely used in the field of orthopedics and will continue to play an important role in the conservative treatment and surgical repair of bone injuries.

2.6. Mechanical signal

The human body can be affected by various mechanical effects during motion. These stresses include compressive stress, tensile stress, bending stress, torsional stress, and shear stress. It has been found that bone homeostasis and remodeling during bone injury repair are regulated by external mechanical stimuli [127]. Mechanical signals can promote the proliferation and differentiation of osteocytes, angiogenesis and activate cell signaling pathways. It mainly acts on the initial stage of bone repair [128,129].

Mechanical stress can stimulate osteoblasts to produce a variety of biological effects, such as promoting the proliferation and differentiation of osteoblasts, changing the morphology of osteoblasts, affecting the function of osteoblasts, regulating the metabolism of osteoblasts and the levels of cytokines [130,131]. Mechanical stimulation signals regulate the phenotypic differentiation of stem cells through different signaling pathways and biochemical signals. ALP, type I collagen (COLI), osteopontin and osteopontin were promoted via appropriate mechanical stimulation. In addition, mechanical signals can regulate the release of inflammatory factors [132], such as interleukins and prostaglandins. Appropriate mechanical loading is beneficial to promote the blood circulation of damaged tissues and the clearance of inflammatory products [133]. It can also affect the cytotoxicity of NK cells and enhance the innate immune response [134]. At present, the specific mechanism of intracellular mechanical signal transduction is not clear, and the studies mainly focus on the BMPs/Smad pathway, TGF-6 signaling pathway, MAPK pathway, Runx2/osterix signaling pathway, Notch pathway and Wnt pathway [[135], [136], [137], [138]]. It is of great significance for the development of biomedical research and the diagnosis and treatment of clinical diseases to be familiar with various loading methods of mechanical stress stimulation and to study the mechanism of stress on osteoblasts at the cellular and molecular levels.

Additionally, the magnitude of mechanical force is closely related to the osteogenic effect. Professor Frost pointed out the relationship between mechanics and bone formation in his “Theory of Mechanical Stability”. Phoonsuk Limraksasin et al. exposed cells to shear loads of various magnitudes (0.15, 0.5, and 1.5 Pa) over a period of two days. Among the tested magnitudes, a shear stress of 0.5 Pa elicited the most robust osteogenic gene expression and the largest mineral deposition. This response correlated with increased expression of connexin 43 (Cx43) and phosphorylated Erk1/2, indicating that shear stress of 0.5 Pa effectively enhanced osteogenic differentiation of induced pluripotent stem cells (iPSCs). In part, it is achieved through Cx43 and Erk1/2 signaling pathways [139]. The results reveal the potential application of shear stress in regenerative bone therapy.

Bone tissue engineering is achieved through the interaction of scaffold materials with cells and tissues [140]. Among commonly used “smart” materials, mechanoresponsive biomaterials can translate mechanical stimulation into interfacial regulation of scaffolds, osteoblasts, and mesenchymal stem cell (MSC) precursors [141], the combination of regenerative medicine and rehabilitation therapy can be used to repair the tissue structure of damaged bone by using bond formation/breakage, structural change, or the responsiveness of nanoparticles. In order to achieve the recovery of physiological function, Wei et al. established a dynamic mechanical potential model based on alginate hydrogels, which can easily achieve dynamic mechanical conversion by introducing or removing Ca²⁺. Results showed that hardened hydrogels (from soft to hard) inhibit the osteogenic differentiation of human mesenchymal stem cells (hMSCs) at the early stage, but eventually promote the osteogenic differentiation of hMSCs over a long period of time [142]. In contrast, softened hydrogels (hard to soft) still retained strong osteogenic differentiation at the early stage, although they eventually showed lower levels of osteogenic differentiation than hard hydrogels. Ângela Semitela et al. fabricated novel smart scaffolds for bone tissue engineering using graphene oxide (GO) derivatives with 3D Marine sponge protein (MS) (see Fig. 7). MS was coated layer-by-layer with polyethylene imine (PEI) and graphene oxide (GO). GOPEI@MS the composite exhibits shape memory capability in compression tests under wet conditions. Dynamic tests showed that the GOPEI@MS material was able to be applied as a mechanical stimulation-responsive scaffold to promote osteogenesis by strongly triggering the mineralization of the bone matrix [143]. Due to the piezoelectric properties of bone, the electric potential will be generated during mechanical deformation, and respond to electrical stimulation with the generation of mechanical stress. The use of piezoelectric materials in combination with mechanical and electrical stimulation for the treatment of bone defects has been widely reported. Zheng et al. combined the novel piezoelectric Ca/Mn co-doped BaTiO₃ (CMBT) nanofibers with PLLA as CMBT nanofibers to form a network in the PLLA matrix. PLLA/CMBT composites significantly improved the osteogenic activity in vitro and in vivo. Furthermore, the piezoelectric PLLA/CMBT composite has strong antibacterial and anti-inflammatory activities, which is more conducive to the repair of bone defects [144]. Shen et al. constructed a PLGA scaffold with high pore connectivity and modified it with VEGF. rBMSCs and HUVECs were co-cultured on scaffolds to promote vascularization and bone tissue regeneration. Hydrostatic pressure stimulation was applied to the scaffolds to investigate whether loading stimulation could promote osteogenesis and angiogenesis. Results showed that under hydrostatic pressure culture conditions, the cells showed higher CD31 expression, and the formation of new capillaries in the cell-scaffold structure was conducive to calcium deposition in vivo and promoted bone regeneration [145].

Fig. 7.

Graphene/sponge biomimetic scaffolds that promote osteogenesis through mechanical stimulation [143].

In conclusion, mechanical stimulation plays an important role in bone repair. The mechanical environment is a key factor in regulating the activity of osteocytes and inducing bone formation. Appropriate mechanical stimulation can accelerate fracture healing and bone regeneration. In clinical practice, external mechanical stimulation therapy, such as low-intensity shock waves and vibration, has been used to treat delayed fracture union and osteoporosis. In the future, combined with 3D printing, biomaterials, and other technologies, mechanical stimulation can further optimize bone tissue engineering and improve the repair effect. In conclusion, the application of mechanical stimulation in bone repair is promising and is expected to become an emerging strategy for orthopedic treatment.

3. Conclusion and prospective

In the study of tissue engineering, providing cells with appropriate growth information such as growth factors, biochemical molecules, and physical signals, is the decisive factor to guide the fate of stem cells. Regulation of stem cell differentiation by growth factors is highly specific and effective. However, due to the easy diffusion and poor localization of growth factors in the body environment, it may cause side effects and dysfunction of other tissues in the body. Similar limitations exist in inducing the differentiation of biochemical molecules into stem cells. In recent years, physical stimulation has shown promising potential as the fourth cornerstone of bone tissue engineering [146]. The regulation of stem cell differentiation by physical factors has attracted increasing attention. The development of appropriate physical stimuli to enhance cell recruitment, osteogenic differentiation, angiogenesis, and immune modulation is imperative. Several physical signals, including sound waves, light, electrical stimulation, and external MFs, have been shown to affect stem cell differentiation [18,19].

This article reviews the research status of biomaterials based on physical signals for bone injury repair (see Table 1). It can be divided into mechanical force, MFs, EF, ultrasound, light, and heat according to their physical properties. It is worth mentioning that bone tissue engineering based on physical signals shows unique advantages of long-term stability in vivo, controlled drug release, and high safety. However, most of these systems have only been studied in animal models or even in vitro, and further studies are required before clinical application. In addition, science the specific mechanisms by which physical signals affect the life activities of cells are still not clear, there is a lack of in vivo detection methods. In this context, the exploration of biological mechanisms based on physical signals is a key issue for designing the next generation of physical signal-based biomaterials. With the increasing research of biomaterials based on physical signals, researchers need to understand more about how these physical signals interact with each other and their mechanism of action, as well as quantify the impact of physical signal strength and action time on tissue regeneration. Although there are technical differences in biomaterials with various physical signals, the synergistic use of multiple physical stimuli in biomaterials to promote the repair of bone defects is the future direction of bone tissue engineering development. For example, light can be precisely localized, but has poor tissue penetration depth. Ultrasound has good tissue penetration and is easy to control. How to combine ultrasonic localization, magnetic navigation and appropriate stimulation to achieve precise localization and release of drugs. Moreover, biomaterials based on physical signals have improved the accuracy of tissue engineering, but there is still room for further improvement. Recently designed micromotors can control directional motion by ultrasound or magnetic drive, enabling local drug delivery at a predetermined location rather than relying on local blood circulation. A better understanding of these physical signals facilitates the development of more effective combinations of physical signals to address bone tissue engineering problems. In conclusion, the use of physical signals to design biomaterials for bone tissue engineering has achieved increasing research results in both preclinical and clinical practice stages. This suggests that biomaterials based on physical signals hold great promise in bone injury repair.

Table 1.

Physical signals for bone injury repair: advantages, limitations.

Physical signal	Advantages	Limitations
Magnetic	➢High control accuracy ➢High spatio-temporal controllability ➢Deep tissue penetration	➢The adverse effects of magnetic field stimulation have not been overcome and still need to be further studied
Light	➢Equipment price is low and convenient to use ➢Photosensitive materials can be used to diagnose and treat bone defects	➢The tissue penetration depth was shallow ➢Some photosensitizers have physiological toxicity ➢Some light intensities and wavelengths can cause tissue damage
Heat	➢Ease of use ➢Often used in conjunction with other physical stimuli	➢The heating range is difficult to control accurately ➢Strict thermal control is required ➢Lack of real-time thermal feedback from the tissue
Electrical fields	➢Easy to operate ➢Tissue electrophysiological activity can be monitored	➢Some implant designs are invasiveMay cause tissue damage
Acoustic fields	➢High spatio-temporal controllability ➢Deep tissue penetration	➢High power ultrasound may cause tissue necrosis
Mechanical fields	➢Consistent with the mechanical properties of bone tissue ➢Often used in conjunction with other physical stimuli	➢Some implant designs are invasive ➢May cause damage to nerves, joints and other tissues

Author contributions

Qi Sun: Writing – original draft, Funding acquisition. Chaohua Li: Data curation. Qishun Liu: Resources. Yuanbin Zhang: Formal analysis. Baisong Hu: Validation. Qi Feng: Visualization. Yong Lang: Writing – review & editing, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.