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A review of biomimetic scaffolds for bone regeneration: Toward a cell‐free strategy

Abstract In clinical terms, bone grafting currently involves the application of autogenous, allogeneic, or xenogeneic bone grafts, as well as natural or artificially synthesized materials, such as polymers, bioceramics, and other composites. Many of these are associated with limitations. The ideal scaffold for bone tissue engineering should provide mechanical support while promoting osteogenesis, osteoconduction, and even osteoinduction. There are various structural complications and engineering difficulties to be considered. Here, we describe the biomimetic possibilities of the modification of natural or synthetic materials through physical and chemical design to facilitate bone tissue repair. This review summarizes recent progresses in the strategies for constructing biomimetic scaffolds, including ion‐functionalized scaffolds, decellularized extracellular matrix scaffolds, and micro‐ and nano‐scale biomimetic scaffold structures, as well as reactive scaffolds induced by physical factors, and other acellular scaffolds. The fabrication techniques for these scaffolds, along with current strategies in clinical bone repair, are described. The developments in each category are discussed in terms of the connection between the scaffold materials and tissue repair, as well as the interactions with endogenous cells. As the advances in bone tissue engineering move toward application in the clinical setting, the demonstration of the therapeutic efficacy of these novel scaffold designs is critical.

vide mechanical support while promoting osteogenesis, osteoconduction, and even osteoinduction. There are various structural complications and engineering difficulties to be considered. Here, we describe the biomimetic possibilities of the modification of natural or synthetic materials through physical and chemical design to facilitate bone tissue repair. This review summarizes recent progresses in the strategies for constructing biomimetic scaffolds, including ion-functionalized scaffolds, decellularized extracellular matrix scaffolds, and micro-and nano-scale biomimetic scaffold structures, as well as reactive scaffolds induced by physical factors, and other acellular scaffolds. The fabrication techniques for these scaffolds, along with current strategies in clinical bone repair, are described. The developments in each category are discussed in terms of the connection between the scaffold materials and tissue repair, as well as the interactions with endogenous cells. As the advances in bone tissue engineering move toward application in the clinical setting, the demonstration of the therapeutic efficacy of these novel scaffold designs is critical. time-consuming therapies, supervisory issues, and high costs, need to be optimized. [1][2][3] In view of this, the field of acellular biomaterials is progressing and is becoming a practical alternative to cell-based therapies.
Previously, acellular materials only were regarded as fillers for the tissue defects, 4 but now are able to be engineered into scaffolds that can interact with surrounding cells and tissues to alter the traditional recovery processes from disease or trauma. 3 In this review, we address an acellular approach utilizing cell-free biomaterials which can be modified through physical and chemical strategies and takes advantage of the capacity for tissue regeneration via interaction with local stem cells and surrounding tissues and which promises to avoid the scientific and regulatory disputes of cellbased materials.
Mesenchymal stem cells (MSCs) have great potential in cell-based treatments for tissue repair and regeneration and are used extensively, because of their proliferation, multilineage potential, immune regulatory, and anti-inflammatory effects. However, as exogenous cells, there is insufficient understanding of the interplay between the cells and the implanted materials. There are also risks of supraphysiological dosages required to produce the necessary efficacy, potential sideeffects, and the challenges of achieving the ideal release kinetics to stimulate the surrounding cells. These factors indicate the necessity of using cell-free materials. For acellular materials, it is important to focus on the characteristics of the designed scaffolds, as well as their biodegradability, porosity, biocompatibility, and, with reference to bone regeneration, their osteoconduction.
Natural bone is composed of complicated hierarchical architectures from nanoscale to macroscale, combining distinctive biological properties and high mechanical strength. The native bone matrix is composed of inorganic components (hydroxyapatite) and organic components (collagen-I), which have been widely used in simulated biomimetics due to their outstanding osteoconductivity and biocompatibility. 5,6 Foreign ions (such as Zn 2+ , Sr 2+ , Si 4+ ) could be doped in hydroxyapatite (HA) or another natural or polymeric material, even bioceramic material, that would effectively mimic the mineralization process of natural bone and hence promote osteoinduction and osteointegration. [7][8][9][10] Another biomimicry target is the extracellular matrix (ECM), a complex network of polysaccharides and proteins secreted and regulated by cells that provides biochemical signals for the modulation of cell activities and also as a bridge for connecting cells and materials. 11,12 Decellularized extracellular matrix (dECM) deposited on a biphasic calcium phosphate (BCP) scaffold was prepared through two different methods and was shown to be effective in promoting the bioactivity of scaffolds and providing an appropriate microenvironment for tissue regeneration, especially for osteogenesis. 13 In addition, we consider literature describing the mimicry of native bone ECM for bone tissue engineering. 14 This review focuses on the recreation of the chemical and physical cues within native ECM in relation to different aspects, aiming to apply this knowledge to the development of acellular materials for bone regeneration.
Additionally, the defined control of topological features of scaffold materials is dependent on ordered and elaborated preparation methods such as advanced three-dimensional (3D) printing technology. Variations in surface roughness and fiber alignment, especially interconnected pore structures, could be prepared by 3D printing technology that is able to produce sophisticated architectures with 3D features. 15,16 By mimicking such micro-/nanostructural characteristics of bone tissues, cell actions such as migration, adhesion, proliferation, as well as differentiation could be regulated, further promoting bone regeneration. 17,18 Meanwhile, in addition to biochemical signals, ambient physical stimuli such as electrical and magnetic factors, can also influence cells and are able to further prompt bone regeneration. [19][20][21] Based on previous reports, bone tissue, which possesses piezoelectric properties, can generate charges or potentials in response to mechanical stimuli and have the capacity of enhancing bone growth. 22 The application of magnetoelectric scaffolds and restoration of the physiological electric microenvironment in bone tissue regeneration can further regulate cell fate and optimize biomaterial design. 19,23 Evolving strategies that combine external environmental physical cues with the intrinsic features of materials and modulated scaffold systems can thus be utilized to synergistically drive bone regeneration. Moreover, mechanical parameters of materials or cells that can be controlled are important for regulating cellular fate. [24][25][26] For example, 3D scaffolds coated with Ti surfaces can provide similar rigidity to cartilage (0.5-3 MPa), allowing cell growth. 27 Hydrogels which possess flexible and tunable stress relaxation could guide cell behavior and fate. It has been found that cells cultured in gels with faster relaxation, spreading of MSCs was faster, as well as boosting both the proliferation and osteogenic differentiation of the cells. 25 Innate growth factors can be stimulated by cellular adhesive forces, a feature that has been used by flexible aptamer technology to produce mimics of the transforming growth factor-beta large latent complex.
Traction forces can thus act as triggers activating specific biological responses and thus have potential applications in both biological research and regenerative medicine. 26 Lastly, energy-driven, photothermally modified, thermodynamically controlled, and photoluminescent biodegradable materials have been prepared by researchers. [28][29][30] Tissue regeneration is dependent upon cellular bioenergetics (CBE) which, within bioenergetic-active material (BAM) scaffolds, promote mitochondrial membrane potential (ΔΨm) to provide elevated bioenergetic levels and further accelerate bone repair. 28 For the simultaneous treatment of osteosarcoma and tissue regeneration in clinical terms, an innovative multifunctional scaffold with temperature-controlled characteristics has been reported which can efficiently eliminate human osteosarcoma cells at 48 C, while enhancing osteogenesis of BMSCs at a temperature of 42 ± 0.5 C using 808-nm near-infrared (NIR) light irradiation. 30 This review aims to describe the manufacture of biomimetic bone materials, including the different methods used, their structures, and scales from microscale to macroscale, to promote the physical and chemical modification of structural surface features to regulate bone growth. We describe the latest progress in biomimetic strategies, including ion doping, functionalization of the dECM, and ambient physical stimulation, from micro-/nanoscale to macroscale, as well as the advantages of other functional scaffold materials ( Figure 1). Cellular responses to these scaffolds in vitro, as well as the in vivo process of new bone formation produced by these strategies will be highlighted. This summary of recent advances in these fields identifies important issues and future directions for the design of biomimetic scaffold materials, specifically in terms of promoting cellular behavioral changes toward substrates in the process of bone tissue regeneration. 31

| BIOMIMETIC ION-FUNCTIONALIZED SCAFFOLDS
Several trace inorganic ions have been discovered that are conducive to bone tissue regeneration. 32 This has inspired researchers to explore various bioactive glass dissolution products and doping strategies, as well as synthetic HA, bioactive glass, and other materials involving natural/polymer materials. In comparison with other cues of promoting osteogenesis, the superiority of applying inorganic ions to facilitate bone trauma repair is multifaceted, including cost-efficiency, improved stability and simplicity, and outstanding efficacy at low concentrations. [33][34][35] In this section, we will pay attention to ion-doped and dissoluble scaffold materials with enhanced biological activities involving osteogenesis, angiogenesis and antibacterial properties that are involved in the application of these ion-relevant materials. Figure 2 shows the distinctive therapeutic effects of these ions toward bone regeneration released from a biomaterial scaffold.

| Ion-doped materials
Neščáková et al. fabricated mesoporous bioactive glass nanoparticles (MBGNs) using the SiO 2 -CaO system with Zn 2+ ions being doped in MBGNs. The Zn-MBGNs were able to gradually release zinc ions to the medium and also showed an enhanced ability to adsorb proteins. 10 Among the different bioactive metal ions tested, strontium has been extensively investigated in the context of bone repair materials due to its structural and physicochemical similarity to calcium, promoting bone regeneration and inhibiting bone resorption. 10,[36][37][38] Lei et al. developed a SrHAP/chitosan (CS) nanohybrid scaffold by freeze-drying technology. The SrHAP nanocrystals can uniformly disperse into the scaffolds and, with the release of Sr 2+ ions, cell proliferation, and osteogenic differentiation can be improved. Additionally, the presence of strontium in the scaffolds promoted ECM mineralization, alkaline phosphatase (ALP) activity, and expression of the osteogenic genes ALP and COL-1. 39 Since the 1970s, the possibility of using silicon, usually acting as the silicate ion (Si 4− ), for bone formation has emerged. 40 Si has a critical role in the metabolism of bone formation and is utilized to induce hydroxyapatite precipitation into the matrix by elevating its concentration at the early stages of bone calcification. 33 Mao et al. created bioactive bone regeneration particles (BRPs) using amorphous calcium phosphate and 58S bioglass, composed of β-tricalcium phosphate (β-TCP) and calcium silicate, that could enhance bone regeneration. The BRPs also showed outstanding osteoinduction and osteoconduction for alveolar bone repair. 41 Magnesium accounts for approximately half the mineral complement of bone tissue. 42 It is also essential for many metabolic reactions. 43 Among many effects of magnesium ions, its direct influence on osteogenesis is significant. Yoshiwaza et al. found that Mg 2+ improved ECM mineralization in human bone marrow stromal cells (BMSCs), enhancing the expression of collagen-X and vascular endothelial growth factor (VEGF). 44 Hung et al. demonstrated that Mg 2+ initially induces an osteogenic effect in the marrow cavity before motivating BMSC differentiation into osteoblasts through activation of the canonical Wnt signaling pathway. They further demonstrated the effective application of Mg-based devices in therapy, especially in the bone regeneration field. 45 Minardi et al. constructed a bio-inspired biomimetic osteogenic niche with osteoinductive potential, composed of magnesium-doped hydroxyapatite/type-I collagen, which represents a critical advance in acellular off-the-shelf substitutes for bone regeneration applications. 46 F I G U R E 1 Schematic diagram of construction strategies for biomimetic bone scaffolds for cell guidance in bone regeneration Calcium is the most abundant mineral in the body and is stored mainly in the skeleton. 47 During the process of bone remodeling, the extracellular calcium ion concentration can be elevated to some extent through bone-resorbing osteoclasts. 48 This resorption can be inhibited, as can be the proliferation and differentiation of MSCs [49][50][51] and the osteoblasts can be stimulated. 52,53 During the 1980s, researchers found that an extracellular G-protein-coupled receptor, namely the calcium sensor receptor (CaSR), 54 can be activated, resulting in increased levels of calcium, which are then able to promote proliferation, chemotaxis, and osteogenic differentiation of BMMSCs in a dose-dependent manner. 55 In view of the composition of natural bone and the significant role of calcium in cellular activities, diverse materials composed of calcium phosphate have emerged as bone substitute treatments. [56][57][58] However, although the deposition of calcium phosphate on the surfaces of these bone replacement materials may benefit osseointegration, the calcium deficiency remains a problem in bone regeneration. 59 It is known that ionic dissolution products have beneficial effects on cellular activities, suggesting that dissociated calcium and phosphate ions may promote the osteogenic differentiation of osteoblasts. 60 Because of the beneficial antimicrobial properties of the silver ion (Ag + ) in tissue regeneration applications, incorporation of Ag + into tissue engineering scaffolds could be useful to inhibit infections with minimal adverse effects. 32 Qing T. et al. found that silver-based nanoparticles were able to facilitate the proliferation and differentiation of MC3T3-E1 cells, further contributing to the upregulation of bone formation and regulation markers. 61 3D scaffolds incorporating AgNP-loaded nHA@RGO have been investigated. These composite scaffolds have been shown to effectively eliminate infection and inhibit the formation of biofilm, further facilitating bone repair. 62 Iron is indispensable for a wide variety of cellular processes in the human organism, [63][64][65] including the synthesis of DNA, RNA, and proteins, as well as electron transport processes, cellular proliferation, and differentiation. 66,67 In bone regeneration, in vitro experiments have shown inhibition of osteogenic lineage differentiation in human osteoblasts concomitant with decreased calcification caused by iron overload. [68][69][70] Furthermore, in vivo studies in zebrafish larvae have shown reduced osteoblast function and mineralization caused by iron overload resulting in the augmented generation of reactive oxygen species. 59 Deferoxamine, an iron chelator able to remove iron throughout the body, is able to counteract this effect in osteoblasts progenitors and has been applied extensively in osteogenesis. 70 72 They found that osteoblastic differentiation was inhibited as the increase of iron concentration in a concentration-dependent manner while a mild iron deficiency caused an elevation in cellular activity. However, osteoblastic differentiation may be restricted at critically low iron levels. Consequently, the potential advantages of iron in tissue regeneration need further exploration.

| THE POTENTIAL OF DECELLULARIZED EXTRACELLULAR MATRIX SCAFFOLDS
The extracellular matrix (ECM) is a complex network of structural and functional molecules secreted by cells. 73 All tissues and organs are thus largely composed of cells and ECMs. The main components of the ECM are (i) proteoglycans and glycosaminoglycans (GAGs), (ii) filamentous proteins such as collagen and elastin, (iii) adhesive proteins such as laminin, vitronectin, and fibronectin. Bone ECM has both inorganic and organic constituents. The inorganic part, consisting of calcium phosphate, mainly in the form of hydroxyapatite (HA), is the source of bone strength, 74 while the organic part, composed mostly of type I collagen, provides the tissue and cell with flexibility and F I G U R E 2 Schematic of inorganic ions released from a biomaterial scaffold and their associated therapeutic effects toward bone regeneration. MSC, mesenchymal stem cell adhesion, respectively. Decellularized bone is frequently used as a special scaffold material in bone tissue engineering, due to its ability to eliminate cellular components and antigenicity and its osteogenic and biomechanical properties as well as its physiological similarity to the bone matrix.
Boram et al. demonstrated that the potential of a biphasic calcium phosphate (BCP) scaffold with attached dECM in bone tissue engineering. Rat BMSCs were cultured on porous BCP scaffolds for 3 weeks, after which the decellularized ECM-deposited scaffolds (dECM-BCP) were further utilized for study in vitro ( Figure 3). The results indicated that the BCP scaffold with ECM was enhanced the bioactivity of the materials, as well as offering a stable microenvironment for osteogenesis. 13 Wang et al. showed that adipose-derived ECM (A-ECM) could be combined with chitosan/gelatin conducive to the attachment and growth of BMSCs. Thus, for ECM scaffolds with poor mechanical properties, the association of chitosan/gelatin with the ECM can promote not only the strength of the ECM scaffolds but also the bioactivity of composite scaffolds, while simultaneously enhancing the osteogenic ability of chitosan. 12 In contrast to decellularized ECM scaffolds, Platelet-rich fibrin (PRF), which functions as a growth factor vector, has been widely used in the field of soft and hard tissue regeneration. [75][76][77] However, the bioactive stability of decellularized PRF (DPRF) is unknown. Chi et al.
investigated whether the incorporation of DPRF into the chitosan/gelatin scaffold could synergistically improve both the bioactivity of the C/G scaffold and the strength of PRF, due to the suitable biocompatible and mechanical properties of C/G scaffolds, but found a lack of bioactivity. Ultimately, the merging of DPRF can not only promote BMSC adhesion, proliferation, and osteogenic differentiation with a suitable microenvironment in vitro but also expedite bone repair in vivo. 78

| MICRO/NANO-STRUCTURAL FEATURES OF THE BIOMIMETIC SCAFFOLD
Tissue engineers need to mimic the micro/nano-architecture of natural bone to investigate the means of stimulating effective tissue growth. To understand the intrinsic osteoinduction of materials, probing the tunable structural properties of scaffolds is necessary. Of these features, primary concern are gross features such as the surface roughness and morphologies of materials on which cells proliferate and attach, in addition to substrate modulus and pore size conducive to osteogenic differentiation, and distinctive structural components such as fibrils of particular sizes and interconnectivity. 79

| Nanostructured surfaces and interfaces
The design of materials to direct cell behaviors and thus to promote tissue repair is of great concern in tissue engineering and is essential F I G U R E 3 Schematic diagram of the procedure to generate cell-derived extracellular matrix deposited on biphasic calcium phosphate (BCP) scaffolds. The rat-derived bone marrow mesenchymal stem cells were seeded on BCP scaffolds, cultured for 1 week in growth medium, and incubated for 3 weeks with osteogenic medium followed by decellularization using freezing and thawing and sodium dodecyl sulfate treatments. The decellularized extracellular matrix-biphasic calcium phosphate (dECM-BCP) scaffolds were evaluated in vitro for the improvement of bioactive materials. [80][81][82] MSC differentiation may be triggered by surface topography or by the release of growth factors, calcium, and phosphate by inflammatory cells such as macrophages, monocytes, and osteoclasts. 83  It was found that these surfaces were able to promote cell attachment and spreading as well as stimulating proliferation and osteogenic differentiation in rBMSCs. These effects were enhanced by the incorporation of Si, with the best effects produced using a Si-substituted HAp bioceramic with a nanorod surface. 89

| Micro/nano-porous structures of the scaffolds
Bone tissue engineering scaffolds require interconnected 3D pore structures that permit cell infiltration as well as allowing nutrient access and waste removal. 90 Most studies describe surfaces that only permit one-way guidance, resulting in only transverse or longitudinal migration of cells and asymmetric repair of the tissue defect. 91,92 Thus, a necessity for symmetrical regeneration is facilitated migration of the cells into the center of scaffolds. It has been found that scaffolds with oriented porous structures are effective in this regard 93,94 as oriented pores are able to promote cell infiltration allowing improved tissue regeneration both in vivo and in vitro. 95 Dai et al. reported an O-HA-MA/PLGA scaffold with radial pores prepared by directed cooling, freeze-drying, and PLGA infiltration ( Figure 5). It was found that this type of scaffold allowed bone marrow stem cell (BMSC) aggregation characterized by spherical cell morphology, while the cell-free hybrid scaffold facilitated regeneration by the recruitment of surrounding BMSCs and chondrocytes rather than preseeding any type of cells. 96  challenge. 98 Although there are a variety of products, including bone void filters and temporary scaffolds as well as reports of new biomaterials, many of these have surgical, technical, and manufacturing shortcomings. 99,100 Evidence indicates that inadequate contact with host bone tissue adversely affects osseointegration. 101 Ceramics and cements, both natural and synthetic, are frequently used for the clinical treatment of bone defects; 99 however, rigid ceramics are difficult to machine, indicating that ceramic constructs cannot be simply shaped and tuned in clinical treatment to accommodate the defect site. 102 In addition, the fragile and brittle nature of ceramics has restricted their application. Recently, a novel material allowing shape recovery has appeared to be a groundbreaking application in regeneration medicine. 103 The shape recovery feature guarantees scaffold implantation in a compressed form with minimally invasive surgery, avoiding the technical, surgical, and manufacturing limitations and allowing the scaffold to fit into the defect site. Moreover, as native bone ECM has a nanofibrous physical structure with 65-70 wt% inorganic composition, 104 107,108 Furthermore, chitosan undergoes a glass transition on hydration, endowing the chitosan scaffolds with shape recovery properties. 109 The prepared SiO 2 NF-CS scaffolds show shape recovery on hydration as well as good elasticity and resistance to fatigue. They have been shown to be effective in promoting hMSC differentiation in vitro as well as self-fitting to bone defect sites and promoting bone regeneration in vivo 110 ( Figure 6).

| PHYSICAL FACTORS AND INDUCEMENT-REACTIVE SCAFFOLDS
The strategies of constructing acellular scaffolds involve not only the optimization of internal structures, surface modification, and growth factor delivery of the 3D biomaterials, but also a consideration of external physical cues, including electrical, mechanical, and magnetic stimuli, as well as photothermal drive can influence biological processes, including bone regeneration. 21,112,113 Thus, synergistic regulation of bone repair has been employed, combining external physical stimuli with the internal structures of scaffolds, in particular, those features that are responsive to stimuli ( Figure 8).

| Photothermally-controlled bone scaffolds
Recently, photothermal therapy (PTT) has been utilized for eliminating tumors and stimulating tissue regeneration. [114][115][116][117][118] PTT and radiotherapy (RT) are extensively employed in clinical treatment where, in combination with nanomedicine, they have proved successful. 119,120 Various biomaterials with photothermal effects have been reported past a few years involving gold nanoparticles 121-123 and carbon nanomaterials. [124][125][126] Among these, graphene oxide (GO) has shown great promise due to its ability to absorb near-infrared (NIR) light, its high photothermal transforming efficiency, and biocompatibility. 127  The selective mineralization process in the collagenous gap areas was modulated by a high energy level of PAA-Ca media through an energetically downhill process. Among the three groups, it was found that HIMC, with structural and functional features similar to native bone tissue, provided a beneficial microenvironment for cell homing and multidifferentiation, while recruiting native stem cells for bone defect repair. 29

| Electrically and magnetically guided bone scaffolds
Since the discovery of the bioelectrical properties of bone in the 1950s, electrical stimuli have been recognized in clinical therapy as a supplement to speed fracture healing and promote spinal fusion. 20,133,134 Because of the presence of electric current and potential in native bone and periosteum, it has been suggested that they play an essential role in sustaining bone quality and volume. 135,136 Several capacitive biomaterials capable of storing electrical charge on their surfaces have been discovered. These biomaterials have shown promise in the bone regeneration field. Recent findings have demonstrated that electrical stimuli can drive bone cells to migrate, proliferate, and differentiate at particular sites in vitro. [137][138][139][140] Clinical results also indicated that electrical stimuli could boost bone healing via interactions between bioelectrics and charged biomolecules. 135 Histological and SEM results showed accelerated healing and interfacial bonding between the implant and the bone tissue. 141 Since electric microenvironment-stimulated wound repair has been proposed as a cue for bone regeneration, recovery of a damaged physiological potential microenvironment appears to be an effective strategy for bone regeneration. 142 Figure 11). Using the 2D membranes, a 40% increase in cell proliferation was observed compared to controls, while use of the 3D scaffolds resulted in a 134% increase in proliferation. 19 Besides the application of electromagnetic effects for bone regeneration, pure has emerged in clinical therapy for bone healing for many years, though with limitations. 147 in vitro studies have determined that static magnetic fields (SMF) and EMF could both promote osteoblast differentiation, 148,149 while in vivo findings also demonstrated that SMF and EMF can improve bone healing and enhance bone integration with grafts. 150 It has been assumed that the underlying biological mechanism might be cell membrane deformation and cytoskeletal restructuring induced by magnetic stimuli, owing to the presence of water acting as a diamagnetic fluid, triggering mechanically stimulated signaling pathways to promote osteoblast differentiation. 151 Magnetic tissue engineering scaffolds, especially magnetic bone scaffolds that have exhibited prospects for enhancing bone regeneration, have been constructed based on these natural biological reactions through embedding magnetic nanoparticles (MNPs) into diverse matrices. [152][153][154] For instance, the embedding of MNPs into PCL nanofiber scaffolds indicated that the composite scaffolds not only enhanced the osteogenic differentiation of rat MSCs in vitro but also promoted vascularization and bone regeneration in vivo. 155 However, the underlying mechanisms of interactive response between the magnetic scaffolds and cells or tissues remain obscure. One hypothesis is that the blended MNPs ameliorate physical properties, such as mechanical features, hydrophilicity, and degradation rate, thus enhancing cell adhesion and bone regeneration. Another possible reason may be the generation of an internal magnetic field induced by the incorporation of MNPs, thereby affecting cell behavior.
F I G U R E 1 1 Construction of magnetoelectric (ME) inverse opal scaffolds and structural characterization of ME NPs and the ME scaffold. (a) Scheme showing the construction steps starting with the assembly of gelatin spheres, followed by (i) their infiltration with a solution of PLLA and ME nanoparticles and (ii) the removal of gelatin spheres to obtain 3D and porous ME scaffolds. SEM images of (b) assembled gelatin template and the inset show its magnified image. (c) SEM image showing the top view of a 3D ME scaffold and the inset shows its magnified image demonstrating a uniform porous structure. (d) SEM image presenting a cross-sectional view of a uniform and well-connected ME scaffold. (e) Scheme showing the ME effect induced enhanced cell proliferation on 3D scaffolds under the influence of AC magnetic fields Source: Adapted with permission (Mushtaq et al. 19 ) Besides the use of MNPs-embedded magnetic scaffolds, the stimulation of external magnetic fields can be utilized to promote scaffold fixation 156 and drive cells toward angiogenesis and osteogenesis. 157 For instance, an external SMF applied on magnetic PCL/MNP scaffolds for osteoblast differentiation and bone formation has been studied showing that the external SMF stimuli not only facilitated osteoblast differentiation in vitro, but significantly promoted new bone regeneration in mouse skull defects, in comparison to magnetic scaffolds alone. 158 In addition，an external magnetic field not only can stimulate cells toward bone regeneration but can produce a direct effect on angiogenesis. Sapir et al. demonstrated that an external alternating magnetic field prompted the formation of vessels in endothelial cells in magnetically reaction alginate scaffolds. 159 However, the underlying mechanisms of how external magnetic fields motivate osteogenesis and angiogenesis are still unknown and it is assumed that microdeformation induced by magnetic scaffolds may produce bending and stretching forces that could mechanically stimulate cells. 157,160

| Mechanically sensitive bone scaffolds
At the molecular level, cell behavior, for instance in MSCs, may well be regulated by the mechanical properties of materials as the capacity of a material substrate to either store or dispel cellular forces could contribute to a strong cue to cells interacting with it. [161][162][163] Regarding bone which is composed of diverse cells and ECM and is a mechanically sensitive tissue, the effect of mechanical forces on the remodeling and structural improvement of bone has been long known. 164 In particular, the effect of mechanical cues on cell behavior is important for sustaining bone tissue homeostasis. 151 External mechanical forces have been suggested to be a potential of modulating MSC differentiation in vitro. As an example, a materials approach with a tunable rate of stress relaxation of hydrogels for the 3D culture of cells has been reported. The study aimed to investigate the effects of hydrogel substrate viscoelasticity and stress relaxation on cell proliferation, spread, and MSC differentiation under 3D conditions. It was found that with the initial elastic modulus of 17 KPa in fast-relaxing hydrogels, MSCs could develop a mineralized, collagen-1-rich matrix resembling that of bone. 25 Thus, the characteristic of cell mechanosensitivity to substrate through stress relaxation is a promising design parameter for biomaterials for bone repair. Another study demonstrated that mechanical stimuli in vitro could produce highly mineralized bone formation. Steinmetz et al. found that human MSC differentiation could be regulated by dynamic mechanical stimuli causing expression of collagen-I as well as the formation of mineral deposits in the bone layer of an osteochondral hydrogel. 165 Moreover, external mechanical stimulation can be combined with the natural physical characteristics of a scaffold for cell regulation. For instance, the patterns of aligned and unaligned nanofibers affecting MSC differentiation under tensile pressure have been used with good effect. 166 Besides external mechanical factors, research has focused on the important role of intrinsic forces of the scaffold materials. 112 Scaffolds that show beneficial mechanical signals via physical cues, such as stiffness and other mechanical properties, can generate internal mechanical forces to facilitate cell differentiation 167 (Figure 12). For instance, on 2D membranes with 0.1-1 kPa stem cells developed into neurons.
When grown on stiffer substrates (20-80 kPa), the cells had a greater probability of turning into bone cells. 168,169 It appears that the stiffness of the structure is more significant with a 3D substrate stiffness ranging from 0.7 to 3 MPa resembling that of cartilage being optimal for osteoblast functionality. In another study, Maggi et al. aimed to investigate the efficacy of stiffness of 3D nanoarchitected scaffolds on stress and mineralization in osteoblast-like cells. The fabricated 3D scaffolds with tetrakaidecahedral geometry, referred to as nanolattices, had a stiffness range of 0.7-100 MPa with each type of nanolattice under mechanical assays. It was found that 3D scaffolds with Ti-coated surfaces under optimal microenvironmental conditions may facilitate cell growth, as the stiffness resembles that of cartilage (~0.5-3 MPa). 27

scaffolds. Both in vitro experiments with cells and in vivo experiments
with subcutaneous implantation in rats indicated that the low scaffolds could promote osteogenesis and bone regeneration. 170 Although it is widely known that MSCs and osteoblasts respond to both substrate topography and stiffness, 171 the underlying mechanism still being investigated.

| OTHER ACELLULAR BONE SCAFFOLD MATERIALS
Apart from the strategies of constructing cell-free scaffolds for bone regeneration described above, other methods will also be discussed,  Figure 14). The multifunctional composite scaffolds provide barriers, osteoconduction, and the release of bioactive substances, promoting mineralization and bone regeneration in vitro and in vivo, and also provide a novel strategy for clinical bone repair. 182 The ECM acts as the mechanical support for cells in vivo while matrix-cell interactions are fundamental in modulating cell activities. 183 To advance cell viability and function within materials, immobilizing bioactive ligands on 3D scaffolds is essential for interaction with stem cells. 184,185 Recently, a surface ligand with a specific amino acid sequence, namely the tripeptide arginine-glycine-aspartic acid (RGD) motif, has been integrated with transmembrane cell adhesion proteins for improving cellular adhesion to the ECM. 186 The RGD motif is a cell-recognizable sequence discov- can be constructed. Results indicated that the use of the RGDCfunctionalized scaffolds produced increased ALP activity and upregulated osteocalcin expressed compared to controls. 187 Furthermore, a cell-free and growth factor-free hydrogel that can induce angiogenesis, osteogenesis, and innervation has been created. The formation of new blood vessels is critical for the recovery from tissue damage, especially prior to new bone formation, due to the nutrition and oxygen requirements of growing tissues. However, the role of nervous tissue is poorly described compared to angiogenesis although innervation is necessary for the regeneration and repair of many tissues. 188  following tissue injury, a synthetic biomaterial showed the sequestration of innate adenosine and the acceleration of bone repair in the fracture site of a murine model. 189 Graphene, a new allotrope of carbon with a 2D honeycomb lattice, has received great attention in the fields of materials science [190][191][192] together with its derivatives. With the ability to accelerate differentiation in MSCs and osteoblasts, graphene is attractive for bone tissue engineering. 193 Furthermore, due to its superior mechanical properties and biocompatibility, graphene has the potential for incorporation with other scaffold materials to intensify their mechanical features and promote bioactivity. Additionally, reduced graphene oxide (rGO), also known as chemically modified graphene, has numerous carboxyl groups on its surface 194 which can be modified to endow bioactive functionality to recognize specific targets. 195 For instance, singlestranded oligonucleotide aptamers that can bind to target molecules with high specificity and affinity. 196 In this respect, a hierarchically macro-mesoporous bioactive glass (MBG) with an osteoblast-specific aptamer and rGO surface coating has been prepared as a novel 3D bioactive porous scaffold (Figure 15a,b). With the combination of scaffolds to promote bone regeneration. In this report, three types of silica precursors were acted for surface biosilicification in situ on collagen scaffolds derived from porcine DCB as a template. Due to the surface functionalization, these nSC scaffolds provided the topographical and chemical cues to produce an osteoinductive microenvironment that could facilitate native MSC recruitment and osteogenesis with a probable underlying mechanism being the abundance of negatively charged silanol groups (Si-OH) of the nanosilica on the scaffolds that interact with surrounding mineral ions. 199 In view of both in vitro and in vivo results, the nanosilica-functionalization scaffolds promoted in situ bone regeneration and, moreover, indicated great potential for the treatment of clinical bone defects. 200 In addition to immobilization of the above-mentioned bioactive agents for promoting osteogenesis, combining these with anti-inflammatory and antibiotic drugs also assists bone repair. 201 Tetracycline (TC) has been applied widely as an antibacterial agent 202 due to its well-known antimicrobial activities. Besides its affinity for Ca 2+ , intermolecular interactions, including Van der Waals forces and hydrogen bonding, between the hydroxyl groups of TC and the apatite, facilitate delivery to the bone. 203,204 Choosing an appropriate material as carrier is significant not only to prolong the drug release but also to produce additional therapeutic effects. Polyurethane, as a synthetic polymer material, has been shown to enhance new bone formation.
The use of polyurethane as an injectable biodegradation scaffold has been found to significantly increase antibiotic release compared to poly(methyl methacrylate) (PMMA) beads, together with facilitating long-term drug release. 205 Another report showed that a slower release could be realized through the combination of the porous polyurethane scaffold and decreased water solubility of vancomycin. Due to the reduction of water solubility, the burst release was reduced by the precipitation of the hydrophilic vancomycin hydrochloride. 206  In summary, biological functionalization of scaffold material surfaces not only involving immobilization of proteins, bioactive peptides, and specific aptamers, but also modification of nanosilica, is a broadly investigated field with significant potential for translating into clinical application. Due to cell/GF-free, one-step surgery, the functionalization of scaffold surfaces can simultaneously recruit and provide adhesive surfaces for cells to augment their biological responses, further enhancing tissue regeneration.
Instead of the traditional invasive materials for bone fracture repair, these adhesives may revolutionize bone-implanted surgeries. [211][212][213] Many adhesives can satisfy biological criteria, such as biocompatibility and cell attachment while the dense layer provided by the adhesives does not cause bone cell ingrowth because of its nondegradability and chemical integrity. 214 Thus, it is imperative to improve on the advan- Although the MSC-secreted EVs have significant prospects in the tissue regeneration field, many challenges need to be resolved before clinical application, including the choice of optimal MSC source and route of administration, as well as a complete understanding of the bioactive constituents and mechanisms of action. 231

| PRODUCTION TECHNIQUES FOR CELL-FREE BIOMIMETIC SCAFFOLDS
The manufacture of acellular biomimetic scaffolds for bone regeneration, including traditional and free-form technologies, have been investigated in novel regenerative tissue engineering. We have previously probed the characteristics and applications of cell-free scaffolds; here, we investigate the manufacture techniques of both conventional and novel bone scaffolds.

| The fabrication of ion-functionalized scaffolds
Inorganic ions with the capability of rapid dispersion through the cellular membrane and regulation of different cellular activities have F I G U R E 1 7 (a) Schematic of the chemical structures and proposed in vitro or in vivo degradation mechanism of bioenergetic-active material (BAMs). (b) Potential mechanism of degradation fragments mediated bioenergetic effects for enhanced bone regeneration. α-KG, α-ketoglutarate; NADH, reduced form of NAD + Source: Adapted with permission (Tang et al. 28 ) been extensively studied. 35,232 Because of the different properties of the various ions, distinct encapsulation techniques allowing specific spatial and temporal release to the damaged sites have been investigated. 233 The inherently stable nature of these ions allows them to be used in various ways. Freeze-drying, as a traditional production technique, is commonly used. 7,39,41,46 This can lead to the formation of solvent ice crystals surrounded by polymer aggregates. When the solvent undergoes direct sublimation from the solid phase into gas, an interconnected porous structure emerges within the dry polymer scaffold. Apart from lyophilization, solid-state sintering 9 and the microemulsion-assisted sol-gel approach 10 have also been used in the manufacture of biomimetic ion-functionalized scaffolds. Therefore, compared to their ionic counterparts, zwitterionic detergents cause less protein denaturation and less removal of cellular components. 247,248 To add a beautiful thing to a contrasting beautiful thing, enzymatic decellularization is often applied after chemical decellularization, leading to further cellular degradation and the elimination of and ASCs, as well as related mechanisms, were systematically investigated. 85,86 The combination of nanostructures and silicon-substitution on hydroxyapatite for bone regeneration has also been demonstrated.
The calcium silicate used as raw material was produced by sintering chemically precipitated CaS powders, and, furthermore, Si-substituted HAp scaffolds were also produced through hydrothermal reactions into nanosheet and nanorod surfaces. 89 96 As another radially-aligned scaffold, fibrous material scaffolds coated with polydopamine have been created by electrospinning to guide directional migration of MSCs. 97 It is relatively complicated to repair the bone defect with irregular shapes and hierarchical structures that represent a combination of soft and hard tissues. A hierarchically structured scaffold for repair of tendon-to-bone has been designed and manufactured. Three regions of the scaffold were arranged as follows: collagen fibers are deposited on the tendon side, composed of an array of channels and alignments, the middle region is regarded as a region of stress transfer and has a mineral gradient, while the bone side is a mineralized inverse opal that promotes the integration of the scaffold with the bone. In terms of the manufacturing process, first, the HAp gradient was created by layer-by-layer coating, then the HAp/PLGA-coated scaffolds were machined through a CO 2 laser, and, finally, the composite scaffolds were completed by removing the opaline lattice which acted as a sacrifice template. 98 Due to the shape memory effect, chitosan can be utilized to construct various porous scaffolds for repairing irregular bone defects. 109 114 Other bifunctional scaffolds have also been reported, including the fabrication of a 3D-printed bioceramic scaffold with a uniformly self-assembled Ca-P/polydopamine nanolayer surface, which is both biocompatible and biodegradable, as well as incorporating the superior photothermal effects of polydopamine. 132 The production of MoS 2 nanosheets and AKT bioceramic scaffolds via a 3D printing method and a hydrothermal approach has also been reported. 115  Hummer's method, following which the GO/nHA was dispersed in deionized water mixed with the CS scaffolds and subsequently lyophilized. 30 Utilizing the highly efficient NIR photothermal effect, the BPs-PLGA scaffolds with complete biodegradation could facilitate osteogenesis in vitro and in vivo. In brief, the BPs were prepared via solvent exfoliation of bulk BP crystals, after which the BPs-PLGA were made by mixing the BPs and PLGA, followed by slow evaporation of the solvent. 116 27 In another study, 3D demineralized bone scaffolds with different mechanical properties were constructed through decalcification using EDTA-2Na for different lengths of time.
These scaffolds with distinctive compressive modules were then investigated in cell experiments and animal studies. 170

| The manufacture of other acellular bone scaffolds
Considering the specific cell-free bone scaffolds for bone regeneration, we will first discuss the immobilization of growth factors. Fibronectin (FN) domains have been used to bind growth factors.
Specifically, a recombinant fragment of FN containing three fibrinbinding sequences, was first produced, followed by the binding of three growth factors, VEGF-A165, PDGF-BB, and BMP-2, to the domains for accelerating skin repair and bone repair. 217  Recently, the use of adhesives has led to the realization of these goals.
A novel class of chemical compounds has been derived from dental resin composites and self-etch primers with the adhesives being methodically designed and manufactured using visible light thiol-ene coupling. Moreover, the precision of the adhesive strength was completed through fiber-reinforced adhesive patch methods. 211 Xu et al. reported a bone adhesive with a pore structure that was superior to commercial bone adhesives which may not adequately support cell infiltration. First, PEG and PEG/PSC porogens were prepared by heatmelting, grinding, and sieving. Then, the initial PEG/CA pore-forming adhesives were prepared through the incorporation of the PEG porogens into the mixture containing CA monomers and the PTSA stabilizer. Furthermore, the bioactive PSC/PEG/CA pore-forming adhesives were constructed by the incorporation of PSC/PEG composite porogens with prewrapped PSC bioactive glass. 215 Table 1  The iliac crest, as main source of autografts, is the preferred harvesting site. The cancellous bone can be collected intraoperatively and used for preparing bone blocks or bone chips for the filling of bone defects. 258 To overcome the problem of vascularization, a vascularization cortical autograft has been produced to reconstruct large bone defects. 259 The applications of a free fibula flap in mandibular and maxillary reconstruction have showed a superior graft survival ratio. 260,261 Bone allografts, as a substitute for autografts, are commonly harvested from living donors or cadavers, followed by processing and, finally, transplantation into another patient 262 ; these are useful due to their easy availability in different sizes and shapes. 263 Besides the application of allografts alone, their combination with autologous concentrated bone marrow cells also has been reported. [264][265][266] Xenografts harvested from different species have been reported in clinical treatment; these are advantageous due to availability and their compatible porosity for bone tissue growth and similar mechanical properties to native bone. Karalashvili et al.
reported a case utilizing decelluarized bovine bone to repair a large bone defect which showed long-term retention of the graft shape without resorption and bone integration. 267 The bovine cancellous bone also played a significant role in the management of tibial fractures in elderly patients, showing good healing results. 268 Nevertheless, the use of xenografts has been hampered by issues such as graft rejection and failure of tissue integration. [269][270][271][272] Apart from autologous, allogeneic, and xenogeneic grafts, synthetic scaffolds have been extensively used. HAp scaffolds with loaded MSCs showed notable osteogenic ability with no adverse responses after tumor curettage. 273 Interconnected porous calcium hydroxyapatite loaded with bone marrow mononuclear cells was effective in the repair of osteonecrosis and avoided collapse. 274 Comparing the efficacy and safety between HAp/collagen and β-TCP, the former showed superior bone repair capability but with a higher incidence of untoward effects. 275 Combining the β-TCP scaffolds with MSCs also has been reported; these showed more trabecular remodeling in clinical femoral defects with the addition of MSCs. 276  shown. Calcium phosphate cement produced at ambient temperatures from hydrolysis is distinct from CaP ceramics. These cements are commonly used as fillers via injection or as scaffolds by 3D printing 279,280 ; however, due to slow degradation, delayed bone repair occurred. 281 The use of 3D printing, especially 3D printed ceramic scaffolds [282][283][284] is able to mimic the microarchitecture and sophisticated anatomical structures of the patient's anatomy. 285 However, the potential challenges may hamper the translation of 3D printing bioceramics to clinical treatment. The information related to clinical trials and results were added in Table 2.
Although a great deal of research has been published on bone tissue regeneration, a few factors still hinder the translation from basic research to the clinic. These include the following scientific and technological challenges: (a) The most appropriate cell type for use in bone regenerative therapy is still unclear. Although mesenchymal stem cells have been applied clinically and experimentally, risks remain. 286,287 Embryonic (ESCs) and induced-pluripotent stem cells are indispensable in adult tissue repair and in addition, still require further laboratory manipulation, (b) Sufficient vascularization is required for the survival of the cells and the subsequent bone repair. However, infiltration of neovessels often lacks depth of penetration, limiting the size of viable bone constructs that can be implanted, 288 F I G U R E 1 8 A diagrammatic representation of the initial stages of implant osseointegration for nonfunctionalized (current orthopedic implants) and biomolecule-functionalized implants. After implantation, the implant surface interacts with the biological environment. The surfaces of nonfunctionalized implants become coated in proteins from the environment forming a variable protein layer (as described by the Vroman effect). The cellular response to the variable protein layer changes with the composition leading to unfavorable immune response, infection, or failure to integrate. Biomolecule-functionalized surfaces produce a defined layer of biomolecules for a more controlled biological response leading to improved osseointegration Source: Adapted with permission (Stewart et al. 173 ) brittleness, unsuitability for load-bearing clinical treatment, and potential harmful effects of toxic solvents and high temperatures on cell viability. [291][292][293] In addition, the current 3D bioprinters lack the capacity of describing internal pore architecture, requiring further optimization for clinical translation, 294  As for the future outlook for ion-functionalized materials, compared to single ion applications for bone regeneration, multielemental composites have been considered to be able to further enhance bone regeneration, or even synergistically improve bone repair; for example, silver is used for its antimicrobial activity and other ions for the promotion of osteogenesis and angiogenesis. Another example is the demonstrated synergistic effects of Co 2+ and Mg 2+ with HA on osteogenesis and angiogenesis. 300 In the future, more attention should be paid to these multielement applications including the use of graded materials that can release ions sequentially. Another major concern is the precise control of the kinetics of ion release. With further research, ion-functionalized materials will show great potential for bone tissue regeneration. 31 The future prospects in decellularized tissue engineering should be considered. If biomaterials can perfectly mimic the structural and functional properties of the native tissue ECM, including encapsulation of the cell, stimulation of cell growth and ECM production, they may represent ideal scaffolds for tissue repair in the field of tissue engineering. 301 Although synthetic materials have their benefits, such as tunable physical and chemical properties, they are unable to fully replicate the functions and structures of the native tissue, even with modifications or the addition of bioactive factors. 302 Hence, the use of dECM in tissue engineering will create an environment that is able to mimic the characteristics of native tissue and to repair the injury site. However, limitations of the application of dECM in standard clinical therapies still exist. Various methods for decellularization have been reported, making it difficult to make decisions on which method is best for a specific application. Keeping the balance between eliminating fully cellular components so as not to trigger an immune reaction and trying to preserve the ECM composition to maintain biological activity is a great challenge that needs further research. 303 Additionally, improving the methods to enhance the physical and chemical properties of the dECM while utilizing its inherent regenerative capacities will be the key to offer feasible treatment schemes for bone tissue regeneration.
The promise of tunable morphological and structural properties of certain materials has great potential. As described above, the complex micro-and nano-scale architectures present in native bone tissue have been utilized many times in tissue engineering. Future work is required to address the intricacies of these features in 3D spatial modeling. It appears that the nanostructured surfaces (nanosheet and nanorod), rather than the release of Si ions, contribute most to the early cellular response while Si ions are mainly responsible for the promotion of BMSC differentiation. 87  has been investigated, based on BMP-2 cues and different degrees of substrate stiffness. Hence, the combination of mechanobiological and biochemical phenomena will be the novel direction in the field of future bone tissue regeneration. 304 As interdiscipline of materials and medicine, the materdicine should draw more our attention to the underlying mechanisms of the interactions between the grafted scaffold materials and the microenvironment of the bone defect areas. 305 The biological functionalization of material surfaces, due to its superiority on the improvement of biofunction, has been widely investigated. For example, the protein functionalization which requires the specific active sequence of the biomolecules in the microenvironment can attract resident endogenous cells and allow them to adhere gradually to the surfaces, 173 indicating the critical importance of incorporating these biomolecules into the design of biomaterials and scaffolds for bone repair ( Figure 18).Furthermore, sequestration of innate proteins such as growth factors by biomaterial-based approaches is conducive to avoid the requirement of exogenous administration and has more potential in regenerative medicine. 306 In comparison to other publications resembling this review article, 14 Conceptualization; data curation; formal analysis; funding acquisition; resources; writing-original draft; writing-review and editing.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

DATA AVAILABILITY STATEMENT
The data supporting this systematic review are from previously reported studies and datasets, which have been cited. Data sharing is not applicable to this article as no new data were created or analyzed in this study.