Nanofiber scaffold for bone tissue engineering: Mechanism, challenge and future prospect

Abstract

Nanofiber scaffold has built a bionic microenvironment for bone marrow mesenchymal stem cells by highly simulating the topological structure of natural extracellular matrix. Its ordered fiber network effectively guides the directional migration and spatial arrangement of cells through the mechanical signal transduction mediated by integrin. Surface functionalization can synergistically activate the osteogenic transcription network and significantly enhance the osteogenic differentiation potential of cells. The precise design of scaffold stiffness affects the cell fate choice by regulating the nuclear translocation of mechanical sensitive factors. This triple cooperative strategy of “physical topology-biochemical signal-mechanical microenvironment” effectively overcomes the biological inertia of traditional scaffolds and provides a dynamic and adjustable platform for bone defect repair. Looking forward to the future, breaking through the bottleneck of clinical transformation such as long-term intelligent slow release of functional factors and in situ efficient construction of vascular network is the key to promoting nanofiber scaffolds from basic research to precise bone regeneration treatment.

Key Words: Nanofibrous scaffolds; Triple synergistic regulation; Bone regeneration; Mechanobionics; Precision bone tissue engineering

Core Tip: Nanofiber scaffold accurately mimics the topological structure of natural bone extracellular matrix, and regulates the behavior of bone marrow mesenchymal stem cells through physical topological guidance, biochemical signal activation and mechanical microenvironment programming, providing a dynamic adjustable platform for bone defect repair. However, clinical transformation is restricted by bottlenecks such as functional factor delivery, vascularization and mechanical adaptation. This review analyzes the triple coordinated regulation network and dynamic interaction mechanism, evaluates the transformation prospects of intelligent materials and bionic design, and provides theoretical support for the research and development of precise bone regeneration strategies.

INTRODUCTION

Bone defects caused by trauma, tumors, infection, and degenerative diseases often exceed the self-healing threshold, constituting a major clinical challenge in orthopedics[1,2]. Worldwide, there are more than 2 million bone graft procedures annually, with an annual growth rate of 13%[3]. Although autologous grafts offer osteoconductivity, osteoinductivity, and osteogenic activity, they are associated with donor site morbidity (up to 20%) and limited bone availability[4,5]. Allografts and metal implants carry risks of immune rejection, infection, and stress shielding effects, often requiring secondary revision surgery[6]. Bone tissue engineering aims to create bioactive bone substitutes through an integrated strategy of “scaffolds, cells, and biological factors”. The core concept is that the scaffold should serve as a dynamic extracellular matrix (ECM) simulator to actively regulate cell adhesion, migration, proliferation, and differentiation[7,8]. However, traditional ceramic/polymer scaffolds often fail to fully replicate native bone ECM functions due to their structural inertness and lack of bio-signaling, leading to inefficient new bone integration[9,10]. In contrast, natural bone ECM is composed of 50-500 nm collagen nanofibers, and its topological characteristics directly instruct bone marrow mesenchymal stem cells (BMSCs) to undergo osteogenic differentiation[11,12].

Nano-fiber scaffolds prepared by electrospinning and self-assembly technology, with high specific surface area, adjustable porosity/orientation, accurately bionic ECM nano-topology, and build intelligent cell microenvironment through surface chemistry and mechanical programmability. This research paradigm marks the in-depth application of “mechano-bionics” in bone tissue engineering: That is, it is no longer limited to morphological bionics, but deeply simulates the mechanical signal transmission and transduction logic of natural bone ECM, and actively writes a “mechanical program” to regulate the fate of cells through material design. This supports the biomedical and clinical application of nanofiber scaffolds (Figure 1)[13]. The core advantage of the scaffold stems from its triple coordinated regulation of BMSCs, which is the concrete embodiment of the concept of mechanical bionics: Physical topological guidance: Ordered fibers are guided by mediated by integrins contact, which activates Ras homolog gene (Rho)/a Rho-associated coiled coil-forming protein kinase pathway to drive cell directional migration[14,15]. Biochemical signal activation: Immobilization of bone morphogenetic protein (BMP) 2/arginine-glycine-aspartic acid (RGD) on the surface activates BMP/Smad and Wnt/β-catenin axes in equal time and space, and up-regulates the expression of runt-related transcription factor 2 (Runx2)/Osterix (Osx)[16,17]. Mechanical microenvironment programming: Scaffold stiffness regulates fibrochondrogenesis through Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding domain (TAZ) nuclear translocation[18]. Although nanofiber scaffolds show significant advantages in basic research, their clinical transformation is still limited by bottlenecks such as uncontrollable delivery of functional factors, inefficient construction of vascular network and lack of dynamic mechanical adaptation[19,20]. Therefore, this review focuses on the dynamic interaction mechanism of this strategy in bone defect repair by systematically analyzing the triple cooperative regulation network of “physical topology-biochemical signal-mechanical microenvironment” in nanofiber scaffold. The application prospect of intelligent response materials, bionic vascularization design and mechanical adaptive scaffold is evaluated, aiming at providing a theoretical framework for developing accurate bone regeneration strategies based on mechanical bionics.

Figure 1 Biomedical and clinical application of nanofiber scaffold. With three-dimensional printing, electrospinning and other technologies as the core preparation process, nanofiber scaffolds radiate in many fields: Constructing bionic microenvironment in tissue engineering and regenerative medicine to repair defects. As a drug delivery system, accurate drug delivery; empower medical devices, prostheses and orthotics to achieve functional adaptation; power-assisted surgical instruments and guide plates to improve operating accuracy; used for radiotherapy equipment and phantom to optimize radiotherapy scheme; training models and simulators can also be developed to assist medical teaching and training, showing its multiple values in biomedical clinical scenes in an all-round way and providing material support for cross-disciplinary medical innovation.

NANOFIBER SCAFFOLD IN BONE TISSUE ENGINEERING

Material science basis and selection strategy of nanofiber scaffold

The triple coordinated regulation of physical, biochemical, and mechanical signals is fundamentally determined by the material properties of the nanofiber scaffold itself. Materials from different sources (natural, synthetic, and composite) possess inherent differences in their physicochemical properties, which directly determine the scaffold’s degradation behavior, mechanical strength, and biological activity. These factors profoundly affect the scaffold’s synergistic regulatory efficiency and spatiotemporal dynamics (Table 1). Therefore, to achieve precise bone regeneration, material selection must align with the intended regulatory goals. The triple coordinated regulation of physical, biochemical, and mechanical signals is fundamentally determined by the material properties of the nanofiber scaffold itself. Materials from different sources (natural, synthetic, and composite) possess inherent differences in their physicochemical properties, which directly determine the scaffold’s degradation behavior, mechanical strength, and biological activity. These factors profoundly affect the scaffold’s synergistic regulatory efficiency and spatiotemporal dynamics (Table 1). Therefore, to achieve precise bone regeneration, material selection must align with the intended regulatory goals.

Table 1 Comparison of characteristics of nanofiber scaffolds with different material types.

Types	Representative materials	Advantage	Disadvantage	Influence on “triple coordinated regulation”
Natural polymer	Collagen, gelatin, silk fibroin, chitosan	Inherent cell recognition site, excellent cell compatibility and biodegradability	The mechanical properties are poor, the degradation rate is fast and uncontrollable, and the difference between batches is large	Born with biological activity, it is easy for cells to adhere and recognize. However, rapid degradation leads to premature loss of topological structure and collapse of mechanical support, which not maintain long-term mechanical microenvironment regulation. Rapid degradation will lead to the sudden release of encapsulated growth factors, and it is difficult to realize long-term intelligent slow release
Synthetic polymer	PLGA, PCL, PLA, PLLA	Excellent and adjustable mechanical properties, controllable degradation rate and stable structure	The surface is usually biologically inert, hydrophobic and lacks cell-specific recognition sites	It can provide long-term and stable topological guidance and mechanical support. However, it must be endowed with biological activity through surface functionalization, otherwise it will be difficult for cells to use effectively
composite material	PCL/collagen, PLGA/bioactive glass	Combining the biological activity of natural materials and the mechanical/degradation controllability of synthetic materials	The preparation process is complex, and the interface bonding between the two materials is the key	Provide stable physical and mechanical signals; natural polymer components or bioactive ceramics provide biochemical signals and improve hydrophilicity

PLGA: Poly (lactic-co-glycolic acid); PCL: Polycaprolactone; PLA: Poly (lactic acid); PLLA: Poly-L-lactic acid.

The modification strategies and effects of different materials are different. For scaffolds based on natural materials (such as collagen), which are rich in functional groups, BMP2 can be immobilized by mild chemical crosslinking, but its rapid degradation makes the factor release cycle short and explosive, which is difficult to match the osteogenesis process for several weeks[21]. On the contrary, synthetic polymer scaffolds [such as polycaprolactone (PCL)] need plasma treatment or copolymerization to introduce active functional groups, but their slow and controllable degradation behavior can be combined with microsphere encapsulation technology to realize long-term and steady-state release of vascular endothelial growth factor and other factors, which better simulates the expression curve of physiological factors[22]. Synthetic polymers [such as poly-L-lactic acid PLLA, poly (lactic-co-glycolic acid)] are ideal materials to study the effect of stiffness (such as by adjusting fiber density and diameter) on cell differentiation because of their high modulus and slow degradation, and can provide continuous and stable mechanical signals[23]. However, scaffolds made of pure natural materials often have low modulus, and their stiffness will drop sharply with time due to hydrolysis during the culture process, which leads to unstable mechanical signals and it is difficult to draw exact conclusions[24]. Therefore, in the research that needs to accurately control the mechanical microenvironment, synthetic polymers or composite natural materials are often used to enhance the mechanical properties. Most synthetic polymers (such as PCL) show more obvious elastic behavior and slow stress relaxation. However, natural hydrogel materials (such as gelatin) have more obvious viscous behavior and faster stress relaxation rate[25]. By compounding gelatin with PCL, the stress relaxation rate of the composite scaffold can be accurately controlled, which makes it closer to the dynamic mechanical environment of natural bone, thus guiding cell behavior more effectively[26].

Therefore, material selection is not a simple distinction between “advantages and disadvantages”, but a strategic decision based on the spatiotemporal requirements of bone defect repair. Synthetic polymers and their composites are superior substrates for providing long-term mechanical support in large bone defects, whereas natural materials are more advantageous in contexts that prioritize rapid cell recruitment and initial regeneration. All subsequent “coordinated control” strategies must be built upon a deep understanding of this materials science foundation.

Regulation of physical topological structure

The physical topology of nanofiber scaffold can actively guide the spatial behavior of BMSCs through the nano-scale characteristics of bionic natural bone ECM[14]. The scale effect of fiber diameter directly affects the cell adhesion mode and activates the transcription program of osteogenic differentiation[27]. Fiber orientation simulates the anisotropy of collagen arrangement in bone tissue, induces cells to extend and migrate along the main axis polarity, and forms bionic tissue structure[28]. The optimization of pore structure needs to give consideration to nutrient transport and mechanical support, and multi-stage pore design can coordinate cell infiltration efficiency and scaffold stability[29]. The core of this regulation lies in the mediated by integrins mechanical signal transduction: After the topological features are recognized by the integrin receptor on the cell membrane surface, the signal cascade of focal adhesion kinase and Rho guanosine triphosphate enzyme is triggered, and the RhoA/Rho-associated coiled coil-forming protein kinase signal cascade drives actin to contract, forming stress fiber bundles, and then driving actin cytoskeleton reorganization[30,31]. This process enables BMSCs to perceive the fiber direction and adjust the migration direction, and finally realize the spatial orderly arrangement[32]. At present, the topology is accurately constructed by electrospinning and three-dimensions printing. The former can prepare parallel/radial oriented fiber network by electric field regulation, which simulates the collagen arrangement pattern of periosteum[33]. The latter designed a three-dimensional gradient pore scaffold with the help of melt-electrowriting and other technologies to reproduce the bionic structure transition from cancellous bone to cortical bone[34]. These strategies provide key technical support for bionic design of physical microenvironment

Optimization of fiber diameter, orientation and porosity: Simulating nanoscale topological characteristics of natural ECM: Physical topology is the core of nanofiber scaffold to regulate cell behavior, and its design needs to accurately mimic the nano-characteristics of natural ECM, and build a functional microenvironment through the collaborative optimization of fiber diameter, orientation and porosity. Among them, the nanoscale regulation of fiber diameter affects the cell recognition mechanism. When the diameter matches the natural ECM collagen fiber, it can increase the contact between the cell receptor and the fiber, promote integrin clustering and adhesion spot assembly, and then regulate the cell morphology and differentiation[35]. Studies have shown that submicron fibers are easy to induce BMSCs to differentiate into osteoblasts, while micron coarse fibers tend to support cell proliferation[36]. Fiber orientation determines cell arrangement and functional directionality. The oriented fiber structure of bionic natural bone ECM can induce cells to extend along the long axis of fiber polarity through contact guidance effect, which not only drives cytoskeleton recombination, but also regulates the spatial specific expression of osteogenesis-related genes. Anisotropic fiber network simulates the layered structure of bone cortex, and isotropic disordered fibers are suitable for bionic cancellous bone reticular structure[37,38]. Porosity and connectivity are the core of ensuring cell infiltration and nutrient exchange, and the scaffold should have 50%-90% porosity and 100-500 μm pore size distribution, which not only provides channels for cell migration, but also maintains mechanical stability[39]. Highly connected pores can promote the uniform distribution of cells, avoid the apoptosis of cells in the central area due to insufficient nutrition, and provide space for new blood vessels, so as to realize the spatial-temporal coordination of vascularization and osteogenesis[40].

Mediated by integrins mechanical signal transduction: Fiber network guiding the directional migration and arrangement of BMSCs: The physical topological characteristics of fiber network can accurately regulate the directional migration and arrangement of BMSCs through the mechanical signal transduction pathway mediated by integrin[41]. When BMSCs come into contact with the surface of nanofibers, integrin receptors on the cell surface (such as α5β1 and αvβ3) will recognize the topological sites on the fiber surface and activate conformationally, and then connect with cytoskeleton (microfilament and microtubule system) through adapter molecules such as ankylin and neurin, forming a functional “cell-scaffold” mechanical coupling interface[42]. Fiber orientation transmits directional mechanical signals through this interface, inducing cytoskeleton to recombine along the fiber axis, making actin filaments aggregate into stress fibers parallel to the fiber long axis, and driving the directional migration of cells. This migration depends on the cascade activation of Rho family guanosine triphosphate enzymes, and realizes directional movement by regulating the periodic formation and retraction of pseudopods[43,44]. Three-dimensional PCL nanofiber scaffolds can guide cell arrangement, and radially arranged scaffolds show stronger ability to promote cell proliferation, can guide tissue arrangement and remodeling, and support the regeneration speed of bone tissue significantly[45]. At the same time, the stiffness gradient of fiber network affects the force sensing process mediated by integrin: The high stiffness region activates YAP/TAZ and other factors by enhancing the tension of adhesive spots, and promotes osteogenic differentiation[46]. Low stiffness region may induce fat differentiation tendency[47]. In addition, by changing the local mechanical microenvironment, the branching structure and crossing nodes of fibers guide BMSCs to selectively aggregate in specific areas, forming a cell nest structure similar to natural bone tissue. This structural guidance cooperates with biochemical signals to maintain stem cell dryness or start the differentiation process, and provide a spatial coordinate system for bone tissue regeneration[48,49].

Synergistic activation of biochemical signals

Surface functionalization strategy: Modification of growth factors, peptides and bioactive ions: Surface functionalization is the core strategy of nanofiber scaffold to simulate natural bone microenvironment. By integrating growth factors, bionic peptides and bioactive ions, a multi-dimensional signal network is constructed to provide synergistic regulatory signals for bone repair[50,51]. Among them, the precise integration of growth factors is the key link: BMP2 is immobilized on the surface of scaffold through covalent coupling, which can stably bind to cell receptors and start osteogenic signal pathway, providing the core driving force for osteogenic differentiation[52]. Vascular endothelial growth factor is loaded by biodegradable polymer microspheres, which can be released slowly with the degradation of the carrier, continuously promote angiogenesis, provide sufficient nutrition supply for the bone repair area, and ensure the metabolic demand in the repair process. Biomimetic polypeptide modification focuses on optimizing the cell-scaffold interaction[53]. Among them, RGD sequence can not only significantly enhance cell adhesion, but also activate intracellular survival signals, reduce the occurrence of apoptosis, and lay a solid foundation for cell colonization and subsequent differentiation[54]. Bioactive ion doping further enriches the signal level: Sr²⁺ can regulate calcium signal pathway and realize two-way regulation of osteogenesis and osteoclast activity[55]. SiO participates in the process of cell metabolism, which effectively promotes collagen synthesis and mineralization. Both of them participate in nuclear transcription regulation through slow-release mechanism, and jointly fine regulate the expression of osteogenesis-related genes to improve the efficiency of bone repair[56].

Activation of osteogenic transcription network: Expression regulation of key factors such as Runx2 and Osx: The high specific surface area of nanofiber scaffold makes it possible to realize the stable fixation and sequential release of osteogenic signal molecules (such as BMP2-2 and RGD polypeptide) through the dual strategy of “covalent surface coupling + fiber internal embedding” different from the non-specific adsorption of traditional blocky or porous scaffolds, nano-grooves on the surface of nanofiber can maintain the exposure of active sites of signal molecules through spatial conformation constraints. At the same time, the interwoven structure of fibers can construct a “signal molecular gradient distribution”, guide BMSCs to differentiate directionally along the signal gradient, and then realize the orderly activation of osteogenic transcription network (Runx2/Osx)[57,58]. Bioactive ions (such as Sr, SiO) can stabilize the conformation of transcription factor complex, prolong the expression time of osteogenic related genes, and ensure the continuous and efficient osteogenic differentiation process[59]. This synergistic effect of “topological anchoring-signal slow release” is the core advantage of nanofiber scaffolds different from general scaffolds, which can significantly improve the temporal and spatial accuracy of osteogenic gene expression.

The oriented nanofibers of biomimetic natural bone ECM (such as parallel arrangement and radial radiation) can make BMSCs extend along the fiber long axis polarity through “contact guidance” mediated by integrin, and this cell morphological change will activate actin contraction related signals, thus enhancing the nuclear binding efficiency of YAP and Runx2, forming a synergistic amplification loop of “topological signal-transcription factor”, and finally promoting the efficient expression of osteogenesis related genes[60,61]. This fiber orientation-dependent transcription regulation model is difficult to be reproduced by traditional non-nano scaffolds (such as porous ceramics and macro-polymer scaffolds) because it cannot provide accurate nano-scale spatial guidance signals.

Dynamic adjustment of mechanical microenvironment

Effect of stent stiffness on the fate of BMSCs: From YAP/TAZ nuclear translocation to the balance of osteogenic-adipogenic differentiation: As the core parameter of mechanical microenvironment, scaffold stiffness deeply affects the osteogenic-adipogenic differentiation balance by regulating the nuclear translocation process of YAP/TAZ in BMSCs[62]. When the stiffness of the scaffold matches the natural bone tissue, the cell senses the mechanical signal through the adhesion spot mediated by integrin, which promotes the reorganization of the cytoskeleton and produces tension. This mechanical stimulus can be transmitted to the cell to release anchor of YAP/TAZ from the cytoskeleton, and to make it break away from the phosphorylation degradation pathway and enter the cell nucleus, and activate osteogenesis-related genes (such as collagen type I alpha 1 chain and alkaline phosphatase) in cooperation with Runx2 and other osteogenic transcription factors, thus promoting BMSCs to differentiate into osteoblasts[63]. However, when the stiffness of the scaffold is reduced to the level of adipose tissue, the tension of the adhesion spot is weakened, which leads to YAP/TAZ staying in the cytoplasm and being ubiquitinated and degraded, losing the activation of osteogenic genes. Meanwhile, adipogenic transcription factors such as peroxisome proliferator-activated receptor γ take the regulatory advantage and induce the cells to differentiate into adipocytes[62]. In a word, the gradient change of scaffold stiffness can also be localized by YAP/TAZ gradient, forming a regional distribution of osteogenic-adipogenic differentiation in space. This stiffness-dependent fate regulation mechanism provides a key target for accurately guiding the differentiation direction of BMSCs.

Stress relaxation and viscoelasticity: Dynamic response characteristics of simulating physiological and mechanical environment: Stress relaxation and viscoelasticity are the core dynamic response characteristics of the scaffold to simulate the physiological and mechanical environment. Under physiological conditions, natural ECM such as bone tissue is not a rigid structure, but has a stress relaxation behavior that gradually decreases with time. This viscoelastic characteristic enables cells to experience continuous mechanical stimulation instead of static load when stressed[64]. When the scaffold has proper viscoelasticity, its deformation will develop slowly with time under the action of external force, and dynamic mechanical signals will be transmitted to cells through the integrin-cytoskeleton pathway, which will promote the expansion and contraction of pseudopodia and cell migration[65]. At the same time, the dynamic tension change during stress relaxation can adjust the nuclear shuttle frequency of YAP/TAZ, and avoid the imbalance of differentiation caused by persistent nuclear retention in static high stiffness environment[66]. The research shows that the scaffold simulating the viscoelastic characteristics of bone tissue can activate the osteogenic transcription network more effectively, and its regulation effect is better than that of the simple rigid or completely elastic scaffold[67]. This dynamic mechanical response characteristic is an important symbol for the scaffold to realize bionic physiological microenvironment.

The innovation of triple synergy strategy

The advantages of the triple synergy strategy not only stem from the independent effects of physical, biochemical and mechanical signals, but also from the close dynamic crosstalk among the three, which together constitute a perception response adaptation intelligent regulatory network to accurately guide the osteogenic fate of BMSCs[68]. Its core crosstalk mechanism is reflected in the following aspects: (1) Physical topology enhances biochemical signal sensitivity: The orientation and topology of nanofibers are not passive “tracks”, which can actively regulate cell morphology and membrane tension, thereby affecting the aggregation and activation efficiency of growth factor receptors[69]. For example, BMSCs stretching along directional fibers produce polar tension in their cytoskeleton, which can promote the coupling of BMP type II receptors and type I receptors and the formation of lipid rafts, thereby significantly enhancing the sensitivity to BMP2 signals[70]. This means that under the same BMP2 concentration, the directional topology can realize the effect of “physical signal amplifying biochemical signal”; (2) Mechanical microenvironment regulates the transcription efficiency of biochemical signals: Scaffold stiffness directly regulates the activity and synergy of osteogenic transcription factors through YAP/TAZ pathway[71]. On the rigid scaffold, YAP/TAZ not only acts as a co-activator, but also forms a transcription enhancement complex with Smad complex and β-catenin, which are combined in the promoter regions of Runx2 and Osx, greatly improving the transcription efficiency of osteogenic genes[72,73]. On the contrary, even if BMP2 exists on the soft scaffold, YAP/TAZ will retain the cytoplasm, which will greatly reduce the transcription output of biochemical signals[71]. This explains why “stiffness matching” is a necessary condition for biochemical signals to play their roles; and (3) Biochemical signal feedback regulates mechanical perception: Biochemical signals can also reversely regulate cells’ perception of physical and mechanical signals. For example, the combination of RGD polypeptide and integrin can not only provide adhesion sites, but also up-regulate the expression of integrin subunits (such as αv, β1) and activate RhoA, thus enhancing the contractility of actin, thus enhancing the cell’s perception ability and response amplitude to scaffold stiffness (that is, the cell’s own “mechanical phenotype” is reshaped by biochemical signals)[74]. This enables the biochemically functionalized scaffold to guide cell differentiation more effectively through mechanical signals.

Therefore, the essence of triple synergy can be summarized as follows: A physical topology provides spatial coordinates and initial mechanical instructions for cells, thereby enhancing their capacity to capture and interpret biochemical signals; biochemical signals, in turn, optimize cellular perception and response to physical and mechanical cues by regulating the cell’s mechanical phenotype; ultimately, the final cell fate decision (such as YAP/TAZ nuclear translocation) is determined by the integrated cooperation of the mechanical microenvironment and biochemical signal-driven transcriptional complexes. This dynamic, bidirectional crosstalk network transcends a simple superposition of independent signals and achieves precise spatiotemporal regulation of the bone regeneration process.

CURRENT CHALLENGES AND TECHNICAL BOTTLENECKS

Achieving the long-term and intelligent sustained release of functional factors remains a key challenge in the field of bone repair. The process of bone healing is complex, with different stages having distinct requirements for functional factors such as growth factors and bioactive ions[75]. Currently, although numerous carriers for factor delivery exist, including nanofiber scaffolds and hydrogels, precise control over the release rate and duration of these factors remains difficult to achieve[76]. Taking BMP2 as an example, its rapid diffusion and inactivation in vivo often necessitate the use of high doses, which not only increases costs but also elevates the risk of side effects such as heterotopic ossification[77]. Traditional sustained-release systems are often incapable of dynamically responding to changes in the bone repair microenvironment and cannot release factors on demand[78]. Therefore, there is an urgent need to develop intelligent carriers that can sense in vivo microenvironmental cues and regulate factor release accordingly.

Vascularization is the core element of bone tissue engineering to successfully repair large-scale bone defects, but the efficiency of in situ construction of vascular network is seriously limited at present[79]. The slow revascularization of bone defect leads to the death of cells due to ischemia and hypoxia, which affects bone regeneration[80]. It has been reported that short nanofibers assembled with mesoporous silica nanoparticles loaded with dimethyloxalylglycine and three-dimensional printed scaffolds containing strontium hydroxyapatite/PCL can easily realize adjustable porous structure by changing the density of nanofibers, and at the same time, due to the framework effect of strontium hydroxyapatite/PCL strong compressive strength will be obtained. Due to the different degradation performance between electrospun nanofibers and three-dimensional printed microfibers, the sequential release of dimethyloxalylglycine and Sr ions is realized, which will significantly promote angiogenesis and osteogenesis by stimulating endothelial cells and osteoblasts, and effectively accelerate tissue ingrowth and vascularized bone regeneration by activating hypoxia inducible factor-1α pathway and immunomodulation[81]. Introducing fish collagen to modify the poly (lactic-co-glycolic acid) shell of coaxial fiber and loading baicalin into PCL-baicalin scaffold can regulate inflammation and osteoclast differentiation, which is beneficial to neovascularization and bone formation[82]. In addition, in the complex microenvironment in vivo, neovascularization is easily disturbed by inflammation and immune response, and its stability is poor[83]. It is urgent to develop a material system that can simulate the natural angiogenesis microenvironment, and combine efficient cell recruitment and factor delivery technology to improve the efficiency of in situ construction of vascular network and realize the efficient synergy between vascularization and osteogenesis in bone repair.

From laboratory research to clinical application, bone tissue engineering products are facing the challenges of scale and standardization. In terms of large-scale production, the current preparation process is complex and requires high equipment and operation, resulting in low yield and high cost, which is difficult to meet a large number of clinical needs[84,85]. Taking three-dimensional printed bone scaffold as an example, the printing speed is slow and the material waste is serious, which limits its large-scale promotion[86]. It is the key to accelerate the clinical transformation of bone tissue engineering products to establish standardized preparation process, quality control system and clinical evaluation standard and improve the large-scale production capacity of products.

CONCLUSION

Nanofiber scaffold has constructed a bionic microenvironment suitable for BMSCs by highly simulating the topological structure of natural ECM, and its core regulation mechanism is embodied in triple synergy: Ordered fiber network guides the directional migration and spatial arrangement of cells through mechanical signal transduction mediated by integrin. Surface functionalization synergistically activates the osteogenic transcription network and enhances the osteogenic differentiation potential of cells. The precise design of scaffold stiffness affects the choice of cell fate by adjusting the nuclear translocation of the mechanical sensitive factors. This strategy breaks through the biological inertia of traditional scaffolds and provides a dynamic adjustable platform for bone defect repair (Figure 2). However, the limitations are obvious, and the long-term intelligent slow release of functional factors is difficult, which is easily affected by microenvironment and leads to unstable activity. In situ construction of vascular network is inefficient, and it is difficult to support the nutritional needs of large-scale bone defect repair, which limits its clinical application effect.

Figure 2 Triple synergistic regulation of nanofiber scaffold for bone tissue engineering. Nanofiber scaffolds precisely regulate bone regeneration through three synergistic mechanisms: Scaffolds construct bionic microenvironment from physical topological structure (fiber network and other characteristics), biochemical signals (bearing functional factors) and mechanical microenvironment (adaptive mechanical properties), and finally achieve bone defect repair, osteoporosis reversal and functional bone tissue reconstruction by inhibiting cell death, promoting migration and proliferation and optimizing extracellular matrix/cytokine synthesis. ECM: Extracellular matrix.

Future research needs to focus on the core challenges. First, overcome the problem of long-term intelligent slow-release of functional factors, develop a dynamic delivery system that responds to changes in microenvironment, and ensure that factor activity and release time match the bone regeneration process. The second is to improve the efficiency of in situ construction of vascular network, and promote the rapid formation and maturity of functional blood vessels by optimizing the cooperative strategy of scaffold topology and biochemical signals. The third is to promote clinical transformation, solve the problems of large-scale production and standardization, and establish a unified evaluation system covering material properties and biocompatibility, so as to clear the obstacles from basic research to clinical application. In the future, it is necessary to rely on three-dimensional bio-printing and artificial intelligence aided design to realize personalized matching between the scaffold and the characteristics of bone defect and stem cells of patients. Finally, the nano-fiber scaffold will be transformed from laboratory research to precise bone regeneration treatment, and the clinical application pattern of bone tissue engineering will be reshaped, providing efficient and safe solutions for the repair of various bone defects.