Emerging Strategies for Bioactive Agent-Loaded Xenogeneic Bone Scaffolds in Regenerative Medicine: A Comprehensive Review

Introduction

The global population is aging, with a notable increase in the elderly demographic in Asia, where the number of older individuals is rising by 3% annually. This trend has led to a significant increase in the incidence of bone-related diseases worldwide.¹ A study indicates that in 2019 alone, approximately 178 million people worldwide experienced fractures, representing a nearly 34% increase over the past three decades.² Concurrently, around 2.2 million bone grafts were performed globally each year, incurring costs of approximately $2.5 billion, a figure that is gradually rising in response to the expanding aging population.³ Bone is a rigid tissue characterized by a highly vascularized, three-dimensional (3D) composite porous structure, composed of organic and inorganic materials in varying proportions (with organic matter constituting 30% and inorganic matter 70%).⁴ This tissue serves multiple functions, including protection, movement, mineral storage, accommodation of bone marrow, and maintenance of the body’s structural integrity.^5,6 Additionally, bones possess the ability to undergo automatic repair and regeneration in response to certain injuries during their development and continuous remodeling throughout adulthood.⁷ However, the capacity for self-repair and regeneration themselves is limited when faced with severe bone damage (defined as a bone defect size greater than 2 cm or loss of bone circumference exceeding 50%) resulting from severe trauma, bone diseases, congenital deformities, or cancer.⁸ Autologous bone grafting is regarded as the “gold standard” for optimal tissue acceptance and osteogenesis, however, it presents challenges such as the need for secondary surgery, donor site injuries, insufficient bone graft volume, limited availability, and potential complications.^9,10 Allografts serve as an alternative, yet they carry risk of immunogenic reactions and viral transmission.¹¹ Problems associated with the integration and compatibility of inert non-bioactive metal implants with surrounding tissues and natural bone, mismatch of biodegradable metal grafts with the rate of bone healing, and lack of long-term in vivo safety validation have hindered their widespread clinical application.¹² To mitigate these limitations, more effective strategies required, and bone tissue engineering (BTE) utilizing biomaterials, cells, and bioactive molecules has emerged as a promising alternative to autografts and allografts.¹³ BTE aims to replicate the three primary properties of bone: the osteoconductivity that facilitates new bone growth on the implant surface, the osteoinductiveness of mesenchymal stem cells (MSCs) that generates produce osteoblasts and chondroblasts in response to growth factors, and the osteogenic properties of bone grafting that support the formation and growth of new bone.^14–16 This approach allows for load shifting during weight-bearing activities and ensures biocompatibility with host tissues.^17–19 BTE is rapidly evolving as a solution for bone defects of varying sizes, centering on the fabrication of scaffolds that provide a biological and mechanical framework for cell adhesion, differentiation, and mineralized matrix deposition.^20,21 In recent years, the research frontier in BTE has shifted from inert structural supports to the development of “integrated bioactive scaffolds” capable of actively responding to the complex physiological environment. By incorporating bioactive components (such as metal ions, growth factors, or therapeutic drugs) into biomimetic porous structures, researchers have achieved precise regulation of the bone regeneration process. In-depth in vitro studies have elucidated their multi-faceted mechanisms of action: for instance, Yoda1-loaded polycaprolactone microfibrous scaffolds promote osteogenic differentiation by activating the Piezo1-F-actin signaling axis;²² non-linear metamaterial scaffolds induce tissue strain conducive to bone repair through their unique mechanical design;²³ manganese dioxide (MnO₂)-loaded scaffolds exhibit excellent reactive oxygen species (ROS) scavenging capacity, thereby protecting cells from oxidative stress-induced damage and promoting osteogenic differentiation;²⁴ furthermore, pH-responsive tea polyphenol-magnesium nanoscale scaffolds can intelligently release antibacterial/osteogenic components and polarize macrophages towards a pro-regenerative M2 phenotype.²⁵ The clarification of these in vitro mechanisms provides a solid foundation for in vivo applications. Animal model studies have confirmed that such functionalized scaffolds significantly enhance bone regeneration by ameliorating the immune microenvironment and promoting angiogenesis. For example, non-linear metamaterial scaffolds have been demonstrated to substantially increase the mineralized area in large segmental bone defects.²³ Notably, this strategy also shows great potential for post-resection bone defect repair in osteosarcoma: sonodynamically activated Ir-TiO₂/hydroxyapatite scaffolds can generate ROS within the acidic tumor microenvironment to ablate cancer cells, while simultaneously alleviating hypoxia in the neutral bone defect area to facilitate repair.²⁶ Moreover, reprogramming tumor-associated macrophages and other immunomodulatory mechanisms opens new avenues for achieving synergistic anti-tumor and osteogenic therapy.²⁷ Collectively, these systematic studies establish an “ in vitro mechanism exploration - in vivo functional validation” paradigm, highlighting the core value of bioactive scaffolds in guiding functional regeneration through multi-dimensional modulation of the bone microenvironment, thereby laying a robust foundation for advancing their clinical translation. Consequently, bone-engineered scaffolds infused with active ingredients represent a promising strategy for the repair of bone defects. This narrative review aims to synthesize the current landscape of the rapidly evolving field of bone tissue engineering, with a particular focus on the design and efficacy of scaffolds incorporating bioactive components. To this end, a systematic literature search was conducted to identify relevant articles published between 2015 and 2025, placing a strong emphasis on recent advances (2020–2025). The search was performed across PubMed, Web of Science Core Collection, and Scopus using customized query strings that combined keywords from the following categories: bone defects (eg, “bone defect” OR “bone regeneration”), scaffold technology (eg, “bone scaffold” OR “3/4D printed scaffold” OR “intelligent scaffold”), and bioactive components (eg, “bioactive” OR “growth factor” OR “exosome” OR “BMPs” OR “natural polymers” OR “traditional Chinese medicine”). This rigorous methodology ensures a comprehensive and contemporary overview, enabling this review to delineate the characteristics of ideal bone scaffolds, critically evaluate the advantages and limitations of current bioactive strategies, and highlight key challenges and future directions in the field.

Xenograft Bone Scaffolds

To fully comprehend the tools utilized in bone regeneration therapy, it is essential to understand mechanisms underlying bone repair and regeneration. Bone trauma triggers a cascade of events involving the simultaneous interaction of various growth factors, cells, and the extracellular matrix (ECM).²⁸ Bone repair and regeneration can be divided into three distinct phases: ① Inflammatory phase: Following a fracture, a fibrin-rich clot is formed, which releases cytokines that recruit inflammatory cells, including lymphocytes, macrophages, eosinophils, and neutrophils, to the injury site.²⁹ Concurrently, growth factors such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), produced by inflammatory cells, stimulate the proliferation and differentiation of MSCs to initiate the repair process and enhance fracture healing.³⁰ ②Fibrovascular phase: This phase is characterized by vascular remodeling and the recruitment of MSCs, which differentiate into chondrocytes and osteoblasts to facilitate bone regeneration.³¹ ③ Bone formation phase: following the fibrovascular phase, the MSCs present in the callus differentiate into osteoblasts or chondrocytes, thereby accelerating bone formation (Figure 1).²⁸ Osteoclast-mediated bone degradation produces bone isolating factors such as transforming growth factor-beta (TGF-β), along with other essential factors (complement 3a, Wnt10b, BMP-6, and SLIT3) that are critical for osteogenesis.³² The final stage of fracture repair involves the reconstruction of the bone callus into mature lamellar bone.³³ As one of the three major components of BTE, bone scaffolds play a crucial role in bone repair and regeneration. They are designed to promote the interaction between cells and the extracellular bone matrix,³⁴ providing a complex micro/nano environment that mimics the ECM function of natural bone tissue.^35–37 The ideal bone scaffold should possess the following characteristics: ① First, the scaffold material must be non-toxic, including its degradation products, which should be safely excreted from the body without significant impact on other organs.³⁸ ② It should feature customizable shapes and adequate mechanical properties to provide structural support for specific bone defects, while also exhibiting highly porous 3D structures that facilitate a suitable microenvironment for ECM formation, cellular activity, oxygen diffusion, nutrient delivery, and waste removal.^39,40 ③ The scaffold should be biodegradable, with a degradation rate that aligns with the rate of bone formation. Rapid degradation may lead to the loss of scaffold integrity before ECM formation, resulting in mechanical instability, while slow degradation could restrict cell infiltration and nutrient exchange, thereby delaying new bone growth.^41–43 ④ The scaffold should be osteoconductive, allowing primary bone and progenitor cells to adhere to and migrate across rough surfaces, ultimately proliferating and laying down new matrix.⁴⁴ ⑤ It should also be osteoinducible, capable of gradually releasing inducers that promote vascularization and osteogenesis, while recruiting progenitor cells through biomolecular signaling to stimulate new bone formation.^37,45 ⑥ It should be biocompatible to minimize the risk of adverse or inflammatory reactions.⁴⁶ ⑦ Furthermore, it should have the appropriate porosity of the scaffold, the level of porosity in the scaffold directly affects cell attachment, biodegradation, drug release rate, and angiogenesis, pore sizes exceeding 300 µm appear to promote the formation of new bone and capillaries, whereas pore sizes smaller than 100 µm may hinder mass transport and cell migration. Generally, smaller pore sizes (less than 75 µm) are conducive to the formation of fibrous tissues, while intermediate pore sizes (75–100 µm) and larger pore sizes (greater than 200 µm) are better suited for the development of unmineralized and fully mineralized bone tissue.^47–49 ⑧Finally, the scaffold should be radiographically permeable, enabling the differentiation between new bone and implanted bone scaffolds, which aids in the effective assessment of bone tissue regeneration.^36,46 To enhance the biological activity of bone scaffolds, drugs, growth factors, and other biological components can be incorporated into the material during scaffold preparation. This approach offers the potential for customizing scaffolds for specific patient applications in the future (Figure 2). For example, based on calcined bovine bone, a gene-activated xenograft scaffold was developed to deliver CKIP-1 siRNA, silencing a key negative regulator of osteogenesis. This action upregulates critical osteogenic markers (ALP, COL-1, OCN, BMP-2, Runx2), thereby enabling rapid bone defect repair and high-quality regeneration without compromising biosafety.⁵⁰ A systematic review have similarly shown that decellularized xenograft bone scaffolds, by significantly mitigating immunogenicity and enhancing bone regeneration coupled with their innate porous and osteoconductive nature, represent a highly promising, cost-effective, and accessible alternative to autologous bone grafts.⁵¹

Figure 1 Schematic representation of representative pathways influencing MSCs osteogenesis. (By FigDraw). Abbreviation: MSC, mesenchymal stem cells.

Figure 2 Schematic representation of repairing bone defect with scaffold as carrier and loading various active ingredients. (By FigDraw). Abbreviations: BMPs, bone morphogenetic proteins; MSC, mesenchymal stem cells; RPR, platelet-rich plasma; TCMs, Traditional Chinese Medicine ingredients.

Combined with Bone Morphogenetic Proteins

Since Urist’s discovery of bone morphogenetic protein (BMP) from demineralized bone matrix(DBM),⁵² there has been no single class of bone substitute that has been as extensively studied and published. BMPs are a group of highly conserved and structurally similar functional proteins that belong to the TGF-β family. These proteins exhibit a variety of biological activities, including the stimulation of DNA synthesis and cell replication, which subsequently promotes the differentiation of MSCs into chondroblasts and preosteoblasts, thereby inducing new bone formation.^53,54 Different types of BMPs have different roles in contributing to bone and angiogenesis (Table 1).^55–64

Table 1 The Role of Different Subtypes of BMPs in Promoting Angiogenesis and Osteogenesis

Additionally, BMPs facilitate the differentiation of osteoclasts from hematopoietic stem cells and enhance their activity both directly and indirectly through the RANK-RANKL signaling pathway.⁶⁵ BMPs play a morphogenic role in the development and maintenance of tissue structures throughout the body by stimulating the mineralization, differentiation, and survival of osteoblasts, which are crucial for maintaining healthy bone.^66,67 Among the BMP family members, BMP-2, BMP-4, BMP-6, and BMP-7 are significant coordinators of MSCs, bone marrow (BM) stromal cells, and osteoblast precursor cells, inducing their differentiation into osteoblasts and exerting the greatest influence on osteoblast differentiation,^68,69 Furthermore, BMP-2, 4 and 7 are also involved in the self-renewal and maintenance of hematopoietic stem cells (HSCs), as well as in progenitor cell expansion and hematopoietic differentiation, which are vital for hematopoiesis.⁷⁰ Recombinant BMP-2 (rhBMP-2) is the most widely used osteoinductive BMP in preclinical trials, promoting bone regeneration while stimulating angiogenesis in the defect area. It has been approved by the Food and Drug Administration (FDA) as an osteoinductive growth factor for clinical applications.⁷¹ Osteoinductive devices containing rhBMP-2 and bovine collagen carriers have been approved for anterior lumbar interbody fusion (ALIF), acute tibial fractures, and maxillofacial reconstruction.^72–75 Gonzaga et al⁷⁶ evaluated the repair of cranial bone defects associated with rhBMP-2 in rats, utilizing autografts, allograft bone, and xenograft bone. Their findings demonstrated that rhBMP-2 significantly improved bone repair when administered alone or in conjunction with various types of bone grafts. Similarly, Issa et al⁷⁷ conducted comparative studies that indicated different graft types enhanced bone formation, primarily attributed to rhBMP-2, thereby accelerating the repair process. Echoing these findings, Cohen et al⁷⁸ elucidated the dual role of different bone graft materials as rhBMP-2 carriers in modulating the bone regeneration microenvironment: on the one hand, the bone scaffolds (particularly alloplast) potently activate osteoblasts by sustaining the long-term release of rhBMP-2, directly promoting bone formation; on the other hand, scaffolds (such as xenograft) can also modulate the immune response of neutrophils via rhBMP-2, thereby fostering a more tolerant local environment for bone regeneration. Consequently, the capacity of different graft materials to enhance bone formation stems not only from their delivery of rhBMP-2 but is also closely linked to the immunomodulatory properties they confer. Pecin et al⁷⁹ implanted a mixture of autologous blood clots containing rhBMP-6 and ceramic particles into a pet dog with a multi-segment defect of approximately 3cm in the right front leg, resulting from a gunshot wound. A follow-up examination four months post-treatment revealed that the dog’s leg function was nearly fully restored. Concurrently, CT slice analysis of the affected leg indicated recovery, cortical re-bridging, and even renewal of the medullary canal. Su et al⁸⁰ employed a collagen-hydroxyapatite (CHA) nanocomposite scaffold to load BMP-2, which was then implanted into the femoral defect of rabbits. Micro-CT and histological staining results demonstrated that bone formation occurred more rapidly compared to controls lacking rhBMP-2, achieving a higher degree of mineralization. Moreover, rhBMP-2 not only accelerated and enhanced bone formation but also expedited the degradation of CHA. This further corroborates that BMPs are both necessary and sufficient for ectopic bone induction and serve as potent agents for the repair of large in situ bone defects.⁸¹ However, BMPs are soluble proteins, and therapeutic doses of BMP can easily diffuse into surrounding tissues before reaching the defect site. Increasing the dose to sustain bone production in the body may lead to significant side effects, such as hematoma, inflammatory reactions, and heterotopic ossification.^82,83 Consequently, BMPs require a carrier system capable of accurately delivering BMPs to the defect site, better maintaining BMP concentration, and facilitating prolonged BMPs release.^84,85 An increasing body of research has demonstrated that the combination of BMPs with various types of scaffolds in BTE can not only maximize BMPs affinity, but also optimize bone repair and regeneration to the greatest extent (Table 2).^86–95

Table 2 BMPs Combined with Various Types of Scaffolds in BTE are Reported in the Literature

Combined with Platelet-Rich Plasma

Platelet-rich plasma (PRP) is a platelet concentrate obtained through the centrifugation of peripheral blood from allogeneic or autologous sources.^96,97 It contains platelet concentrations that are 3 to 10 times higher than those found in blood (exceeding 1×10⁹ platelets/mL). This concentrate is rich in fibrin, platelets, and a variety of growth factors, including PDGF-AA, PDGF-BB, PDGF-AB, TGF-β₁ and TGF-β₃, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (BFGF) and insulin-like growth factor (IGF), which are released upon activation through several methods, such as calcium chloride, thrombin, and soluble type I collagen (COL1).^98–100 In related studies, PDGF has been shown to promote angiogenesis, macrophage activation, fibroblast proliferation, chemotaxis, collagen synthesis, and osteocyte proliferation. TGF is involved in the proliferation of fibroblasts, synthesis of COL1 and fibronectin, induction of bone matrix deposition, and inhibition of bone resorption. PDGF can stimulate epidermal regeneration, keratinocyte proliferation, and dermal fibroblast activity to enhance wound healing, as well as promote the production and action of other growth factors. VEGF stimulates vascular endothelial cell vascularization, while IGF exerts a chemotactic effect on fibroblasts, stimulates protein synthesis, and promotes bone formation.¹⁰¹ These growth factors collectively endow PRP with a wide range of regenerative functions, including stem cell proliferation and recruitment, cytokine activation, modulation of fibroblast pathways, regulation of local inflammatory and fibrotic responses, and stimulation of neovascularization.^102,103 Furthermore, PRP has demonstrated protective effects against chondrocyte apoptosis, inhibition of the inflammatory process, enhancement of cartilage repair, and stimulation of the migration and chondrogenic differentiation of human subchondral progenitor cells.^104,105 These properties have been reported in clinical studies across various medical fields, including orthopedics, plastic surgery, dermatology, and oral implantation.¹⁰⁶ Yu et al¹⁰⁷ investigated the potential of autologous PRP as a bone inductive factor to enhance bone induction and promote bone regeneration by combining bone marrow-MSCs withβ-tricalcium phosphate (β-TCP) ceramics. The results indicated that MSCs exhibited robust proliferation at all time points when exposed to autologous PRP. Furthermore, osteogenic ability tests revealed that the synthesis levels of alkaline phosphatase (ALP) activity and osteocalcin (OC) were significantly elevated after 7 and 14 days of culture. Several in vitro studies have also demonstrated that the inclusion of PRP enhances MSC proliferation, suggesting that PRP could serve as a valuable adjunct to MSC and fresh demineralized bone (FDB) grafts.¹⁰⁸ Variations in concentration lead to differences in the ability to stimulate chemotaxis (directed cell migration) and overall migration rates in MSCs. Holmes et al¹⁰⁹ examined the effects of varying concentrations of PRP and bone marrow concentrate (BMC) on MSCs chemotaxis, revealing that all biologics significantly increased the percentage of MSC migration, with no notable differences observed between BMC and high-concentration PRP when compared to low-concentration PRP. Although naturally prepared PRP is theoretically effective in promoting bone tissue regeneration, it is typically administered in the form of a liquid gel, which may not adequately meet the spatial size and mechanical strength requirements necessary for effective bone regeneration.¹¹⁰ Liu et al¹¹¹ developed a method incorporating PRP-gel, human umbilical cord mesenchymal stem cells (HUMSC), and nanohydroxyapatite, utilizing a novel tissue-engineered bone graft composed of polyamide 66 (nHA-PA66). This study aimed to investigate whether the composite scaffold could enhance angiogenesis and the bone repair capacity in rat femoral large bone defects (LBD). The results demonstrated that the composite scaffold significantly promoted both angiogenesis and bone regeneration in rat LBD, suggesting its potential for further application in the clinical treatment of LBD. A prospective clinical study confirms that the combination of platelet-rich fibrin (PRF) and deproteinized bovine bone mineral (DBBM) safely and effectively achieves horizontal maxillary ridge augmentation, leveraging PRF’s adhesive properties and healing-promotion capacity to enable significant bone gain without conventional barrier membranes and with zero complications.¹¹² Similarly, Hao et al¹¹³ created a personalized PCL/β-TCP/PRP active scaffold layer through 3D printing, utilizing bioink derived from the patient’s autologous PRP. This scaffold was applied to patients for the repair and reconstruction of bone defects following tibial tumor resection. The findings indicated that leveraging the combination of PRP and 3D printing technology could lead to significant clinical advancements compared to traditional bone implant materials. However, another study revealed that the combination of PRP and PuraMatrix did not effectively promote bone regeneration.¹¹⁴ The conflicting outcomes regarding the efficacy of PRP in enhancing osteogenic activity may be attributed to various factors, including differences in PRP preparation protocols, concentrations, animal models, the use of autologous versus allogeneic applications, and methods of administration.^115,116 The healing and regeneration of bone tissue is a prolonged process that typically requires 3 to 6 months to achieve sufficient strength. To ensure the sustained release of essential growth factors, which maximizes their regenerative potential, it is necessary to incorporate a scaffold with a controlled-release system for PRP.¹¹⁷ Additionally, the bioactive molecules present in PRP play a crucial role in BTE, and their synergistic combination with MSCs after encapsulation within the scaffold demonstrates a promising effect on osteogenesis.¹¹⁸ In conclusion, PRP holds significant potential for applications in BTE.

Combined with Natural Products

Natural products are polymers derived from plants, animals, or microorganisms, primarily including: chitosan, collagen, alginate, cellulose and hyaluronic acid. These materials provide natural cell adhesion sites, mimic the natural ECM of bone, and yield non-cytotoxic degradation products such as carbon dioxide and water.^119,120 Natural products are characterized by their convenient extraction, renewability, biodegradability, excellent plasticity, superior biocompatibility and minimal adverse immune effects. As such, they represent an emerging class of materials in the field of biomedicine, with widespread applications in the preparation of BTE scaffold materials. However, due to their large molecular weight, the hydrolysis of these polymers into non-toxic product can be time-consuming.¹²¹ For instance, chitosan, a well-known natural polymer sourced from the ocean, serves not only as a scaffold component but also as a therapeutic delivery platform to facilitate bone and cartilage repair both in vitro and in vivo.¹²² Chitosan can be chemically modified and functionalized with various bioactive materials, including organic molecules and inorganic nanoparticles, to achieve synergistic osteogenesis. Notably, chitosan-based organic matrices can function as templates to regulate the in situ growth of hydroxyapatite (HAP). Additionally, chitosan scaffolds can serve as controlled-release platforms to deliver potent osteoinductive molecules and functional bioactive ions, or can be combined with responsive materials to enhance bone regeneration through exogenous physical stimulation.¹²³ Soriente et al¹²⁴ prepared chitosan/hydroxyapatite (CS/HA) composite scaffolds featuring a three-dimensional porous structure derived from chitosan, utilizing a combination of the sol-gel method and freeze-drying technique. This approach elicited a favorable biological response in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) into the osteoblast phenotype. Collagen, which is abundantly present in the ECM of various natural tissues such as dentin, bone, skin, tendons, pancreas and cartilage, exhibits a high swelling index and low antigenicity.¹²⁵ However, its inadequate mechanical properties restrict its direct application as a bone substitute, leading to the development of various high-strength composite scaffolds that incorporate collagen.¹²¹ The collagen-based composite scaffolds have been shown to enhance the maintenance and function of cultured osteoblasts in vitro, promote matrix mineralization, and elevate the activity of alkaline phosphatase (ALP) osteoblastase.¹²⁶ Furthermore, scaffolds implanted in vivo demonstrate significant osteogenic properties in critical bone defects.¹²⁷ Tsai et al¹²⁸ prepared a COL-HANF composite scaffold utilizing collagen and Hap-CaO fibers (HANFs), and confirming the composite scaffold’s potential for in bone tissue regeneration through both in vivo and in vitro experiments. Alginate(Alg), a marine-derived polysaccharide, is a sodium and/or potassium salt of alginic acid extracted from brown algae, consisting of polymers with varying concentrations of d-manuronic acid (M) and glucuronic acid (G) residues.^129,130 Naruphontjirakul et al¹³¹ prepared a composite scaffold of zinc-containing and strontium sol-gel-derived bioactive glass nanoparticles (Zn-Sr-BGNP) incorporated into Alg. This study confirmed that the composite scaffold significantly promotes biological activity, increases osteocyte proliferation, enhances calcium deposition, and facilitates cell attachment, demonstrating great potential for bone tissue regeneration and repair. Complementing the approach of enhancing alginate scaffolds with bioactive inorganic ions, the study by Hamrun et al¹³² demonstrates that an optimal combination of chitosan, alginate, and fucoidan creates a fully organic scaffold with superior porosity (86.86% porosity), biocompatibility (0.43% hemolysis), and inherent osteogenic activity, driven by fucoidan-mediated upregulation of key bone markers like BMP-2. Cellulose, a linear polysaccharide widely found in various natural sources such as cotton, bast plants, wood, and bamboo, as well as in certain organisms like bacteria, fungi, and algae, is one of the most common polysaccharides utilized for creating bone substitutes.¹³³ Cellulose and its derivatives have been extensively employed to deliver growth factors and antibiotics directly to damaged bone tissue, thereby promoting tissue repair and presenting broad application prospects in bone tissue engineering.¹³⁴ Patel et al¹³⁵ developed biodegradable cellulose nanoparticle-reinforced chitosan/silk fibroin (CS/SF/CNPs) scaffolds through 3D printing and characterized them using various spectroscopic techniques. Enhanced bone regeneration was observed in a rat calvarial defect model, confirming this composite scaffold as a promising material for bone healing applications. In summary, leveraging the structural characteristics and performance advantages of natural products in combination with other biomaterials to achieve mutual complementarity and synergistic promotion will facilitate the design of more effective composite scaffolds for bone repair and regeneration (Table 3).^136–148

Table 3 Natural Products Combined with Scaffolds in BTE are Reported in the Literature

Combined with Traditional Chinese Medicine Ingredients

Traditional Chinese Medicine ingredients (TCMs) are widely sourced from herbs, animals, and minerals found in nature. Their unique chemical structures confer distinct pharmacological activities, and their ease of extraction and preparation often makes them more cost-effective. Furthermore, the active components of TCMs are generally considered safe alternatives to some synthetic drugs, potentially reducing long-term side effects, a claim supported by centuries of application within the Chinese population.^149,150 Pharmacological studies have fully demonstrated the significant potential of TCMs in bone tissue regeneration. Its role extends beyond merely preventing or alleviating osteoporotic symptoms to achieving systemic regulation of bone homeostasis through multi-component and multi-target synergistic mechanisms. On one hand, active constituents of TCMs directly act on bone cells by activating key osteogenic transcription factors such as Runx2 and upregulating osteogenic markers like osteocalcin, while simultaneously modulating the RANKL/RANK/OPG/Wnt/β-catenin signaling axis to suppress osteoclast activity, thereby precisely balancing bone metabolic processes (Figure 3). On the other hand, they improve the pathological bone microenvironment by inhibiting inflammatory responses, mitigating oxidative stress, and exerting estrogen-like effects. These estrogenic actions further enhance intestinal calcium absorption, reduce calcium excretion, and stimulate calcitonin synthesis, collectively contributing to increased bone mineral density.^151,152 One notable example is Icariin (ICA), a flavonoid that activates the ER-α-Wnt/β-catenin signaling pathway. This activation is specifically linked to osteoblast proliferation and differentiation, enhancing mineralization and the expression of bone-related genes and proteins. Additionally, ICA induces bone formation by inhibiting the transformation of osteoblasts into adipocytes and the formation and differentiation of osteoclasts.^153,154 In earlier related experiments, researchers soaked porous β-TCP in an ICA solution and subsequently implanted it deep into the dorsal muscles of rats. After three months, it was observed that this treatment promoted the proliferation and differentiation of rat Ros17/28 cells and induced new bone formation, while no significant osteogenesis was detected in the control group.¹⁵⁵ Additionally, ICA, due to its estrogen-like structure, binds to estrogen receptors and promotes angiogenesis by stimulating endothelial cell migration, proliferation, and tubular formation, thereby influencing bone metabolism.¹⁵⁶ One study demonstrated that ICA increased the expression of VEGF in rat adipose-derived stem cells (ASCs), with a concentration of 10–7 M ICA yielding the most pronounced effect in promoting VEGF secretion in ASCs. VEGF exerts a paracrine effect between ICA-induced ASCs and rat endothelial progenitor cells (EPCs), confirming that scaffolds incorporating ICA possess significant osteogenic and angiogenic potential.¹⁵⁷ Beyond this, Icariin also directly promotes osteogenic differentiation in Osteonecrosis of femoral head (ONFH) through a synergistic mechanism. Research indicates that it initiates this process by activating the PI3K/AKT pathway, which induces epigenetic reprogramming—specifically, the upregulation of UTX and downregulation of EZH-2. This reduces global H3K27me3 levels, ultimately promoting the expression of key osteogenic genes like RUN-2 and BMP-2.¹⁵⁸ However, ICA has low bioavailability and a short half-life (1–2 hours), making it prone to the loss of bioactive components in a fluid environment, and the molecule is easily eliminated from the body.¹⁵⁹ Given the duration required for bone repair and healing (3–6 months), it is crucial for BTE to consider combining ICA with appropriate carriers to achieve long-term administration and stable drug release concentrations that to act on surrounding tissues. To delay the release rate of ICA, researchers utilized ICA CS/nHA microspheres, which also provide a suitable microenvironment for the attachment and proliferation of osteoblasts.¹⁶⁰ In an experiment investigating a model of tibial plateau defects in adult rabbits, researchers developed a core scaffold containing ICA chitosan microspheres using freeze-drying technology. They conducted 3D CT reconstruction of the tibial plateau bone defect post-implantation at 4 to 12 weeks, which demonstrated osteogenesis in the area of composite scaffold implantation. Additionally, there was an observed increase in the expression of bone mineral density (BMD), connective density (Conn.Dn), ALP, COL1, osteopontin (OPN), and osteocalcin (OC), as assessed through histological and histochemical evaluations.¹⁶¹ However, to effectively implement TCM in the treatment of bone diseases, several issues should be addressed: ① lot-to-lot heterogeneity and reproducibility, ② determination of effective dosage, ③ purity and potential unknown toxicity, and ④ lack of uniform standards. Enhancements in previous methodologies will minimize variability between TCM batches by eliminating impurities and isolating the desired active compounds. Furthermore, TCM-based clinical and preclinical trials should focus on dose optimization, confirmation of toxicity ranges, and the expansion of knowledge regarding pharmacological mechanisms and interactions associated with TCM. Therefore, a comprehensive understanding of the relationship between implantable stents and TCM, along with process optimization and drug loading strategies, is essential (Table 4).^162–175

Figure 3 Schematic representation of the effects of TCMs on vascularization and osteogenesis, taking ICA as an example. (By FigDraw). Abbreviations: EC, Endothelial Cell; HIF-α, Hypoxia-inducible factor –α; TCMs, Traditional Chinese Medicine ingredients; VEGFR, vascular endothelial growth factor receptor.

Table 4 Representative TCMs Combined with Scaffolds in BTE are Reported in the Literature

Combined with Exosomes

MSCs play a crucial role in bone repair and regeneration. Their significance lies primarily in two aspects: their ability to migrate to and home in on the site of injury, and their capacity to differentiate into osteoblasts and chondroblasts, ultimately maturing into osteocytes and chondrocytes. This differentiation is essential for the reconstruction and repair of bone tissue defects.¹⁷⁶ Furthermore, MSCs possess unique immunomodulatory properties that enable them to evade recognition by the immune system, thereby avoiding attacks from the host’s immune response.¹⁷⁷ Additionally, MSCs may enhance bone regeneration by modulating the microenvironment, with the release of paracrine factors and specific microRNAs also contributing to their differentiation.¹⁷⁸ Despite the substantial number of clinical trials investigating MSC-based therapies, no single approach has yet received approval for the treatment of bone defects. The clinical application of MSCs for bone repair and regeneration encounters several significant challenges.¹⁷⁹ First, there is a need to improve the effectiveness of MSC implantation and their survival at bone defect sites.¹⁸⁰ Second, it is crucial to prevent to avoid unnecessary differentiation of MSCs during ex vivo expansion.¹⁸¹ Third, strategies should be developed to apply MSCs to larger bone defects without compromising their biological efficacy.¹⁸² Lastly, ethical concerns surrounding MSC-based therapies must be addressed, particularly regarding the potential risk of tumorigenesis.¹⁸³ As noted, there is increasing evidence that MSCs exert their effects primarily through paracrine mechanisms, with a growing focus on MSC-derived vesicles, various factors (including chemokines, growth factors, and cytokines), ECM, and metabolites that are instrumental in tissue repair and regeneration.¹⁸⁴ Extracellular vesicles (EVs) from diverse cellular sources orchestrate key processes in bone regeneration, including osteogenesis, angiogenesis, and immunomodulation. Specifically, EVs derived from mesenchymal stem cells promote osteogenic differentiation via miRNAs such as miR-21 and miR-26a, enhance angiogenesis through factors like HIF-1α and VEGF, and induce M2 macrophage polarization via miR-451a. Osteoclast-derived EVs facilitate bone formation via miR-324 and RANKL reverse signaling, while stimulating vessel formation through PDGF-BB, thereby mediating the “coupling” between bone resorption and formation. Moreover, osteocyte-derived EVs act as mechanical signal carriers to enhance osteogenic potential, and immune cell-derived EVs, such as those from M2 macrophages, activate the BMP2/Smad5 pathway via miR-5106, collectively supporting bone healing.¹⁸⁵ MSc-derived exosomes are nanoscale, lipid bilayer- encased structures capable of transporting functional biological macromolecules, including proteins, lipids, RNA, growth factors, and cytokines. These exosomes have garnered significant interest due to their ability to convey information to both adjacent cells and those located distantly from their origin, thereby influencing cellular functions.^186,187 Exosomes possess several advantages, such as the capacity to traverse biological barriers into deep tissues, promote regeneration, induce stem cell differentiation, and trigger specific immune responses (Figure 4). Additionally, they demonstrate a natural targeting ability, along with favorable properties including biodegradability, biocompatibility, and stability, which contribute to their safety concerning tumorigenicity and immunogenicity.^188–190 The beneficial effects of MSC-derived exosomes on bone repair and regeneration may arise from: ① the direct regulation of osteogenic differentiation in adjacent target cells, facilitated by the activation of MSC exosomes, through various signaling cascades (including BMP/Smad, Wnt/β-catenin, and PI3K/AKT), which induce the proliferation and differentiation of osteoblasts, as well as the recruitment of endogenous MSCs to the site of bone defects, thus promoting bone regeneration.^191–193 ② the stimulation of local angiogenesis and inhibition of bone resorption to repair bone defects, where the activation of the AKT/mTOR signaling pathway may enhance osteogenesis, optimizing the microenvironment and creating ideal conditions for bone regeneration.^194–196 However, exosomes may be are captured by the reticuloendothelial system or rapidly cleared by the mononuclear phagocyte system in vivo, leading to short retention time and insufficient effective concentration at the lesion site, thereby impairing their regeneration and repair capabilities.¹⁹⁷ Additionally, the inherent limited targeting capacity of exosomes makes it difficult to precisely deliver their therapeutic cargo to specific injury sites, which severely restricts their reparative efficacy. By employing engineering strategies such as genetic editing and surface modification to optimize their targeting ability and cargo, combined with advanced delivery systems like hydrogels and 3D-printed scaffolds to achieve localized controlled release, their therapeutic efficacy can be significantly enhanced.^198,199 Wan et al²⁰⁰ demonstrated that a composite scaffold composed of the natural polysaccharides gelatin and hyaluronic acid serves as an efficient delivery system for exosomes. By sustaining the release of TGF-β from the exosomes, the scaffold activates the PI3K/Akt signaling pathway, thereby synergistically enhancing osteogenic differentiation and angiogenesis, which ultimately accelerates bone regeneration and repair in a bone defect model. Similarly, Luo et al²⁰¹ developed a dual-biomimetic system integrating a 3D-printed trabecular-mimetic Ti-6Al-4V scaffold (BTPS) with hypoxia-induced exosome-loaded PEGDA/GelMA microspheres (PGHExo). The BTPS replicated native bone’s geometry and mechanical properties, effectively mitigating stress shielding. Sustained release of exosomes from the microspheres activated key pathways (including MAPK, mTOR, HIF-1, and VEGF), synergistically enhancing osteogenesis and angiogenesis. This combined structural and biochemical strategy significantly improved in vivo bone regeneration and neovascularization, highlighting the powerful synergy between 3D-printed porous scaffolds and exosome-based delivery in bone tissue engineering. The emergence of composite scaffold materials in BTE can further extend the characteristics of exosomes and optimize their application in bone repair and regeneration. However, several key challenges remain, such as the isolation and purification of exosomes, and there is a lack of a unified, efficient, and convenient method to ensure that the isolated exosomes possess standardized characterization and quality control.²⁰² Furthermore, unclear regulatory pathways and the absence of long-term safety evaluation systems collectively represent critical barriers to the broad clinical application of exosomes.¹⁹⁹ Additionally, although exosome-loaded scaffold composites have achieved a certain degree of slow release, the optimal release rate for bone regeneration has yet to be explored, Nonetheless, this area remains a promising field for bone repair and regeneration.²⁰³

Figure 4 Schematic representation of exosome formation and its effect on osteogenesis and cartilage. (By FigDraw).

Challenges and Prospects

As one of the three core elements of BTE, scaffolds play a crucial role in enhancing cell viability, increasing cell attachment area, facilitating homing, promoting osteogenic differentiation, ensuring host integration, and providing load-bearing support.³⁸ Consequently, a wide variety of scaffold composites have been developed. The combination of an active ingredient or drug with osteogenic potential and a scaffold significantly enhances the effectiveness of bone repair and regeneration. However, these bioactive material-loaded scaffold systems face challenges in the in vivo/vitro environment. The “bioactive” evaluation is reduced to in vitro ALP staining or ex vivo Micro-CT bone mass, and this “cell-molecule game” is severely disconnected from clinical function recovery. Key indicators such as nerve regeneration, mechanical adaptive remodeling, and metabolic homeostasis regulation are missing. It is necessary to establish a “full-dimensional biological activity” evaluation system, including structure (integration of bone-scaffold interface), function (recovery of ability to contrast lateral construction), metabolism (dynamic monitoring of calcium and phosphorus homeostasis), and system regulation (level of systemic inflammatory factors). In addition, traditional studies have focused too much on the direct regulation of MSCs and osteoblasts, while ignoring that macrophage polarization (M1/M2) is the “switch” of bone regeneration. Lack of immune microenvironment responsive loading scaffolds can lead to a vicious cycle of chronic inflammation, fiber encapsulation, and repair failure after implantation. Therefore, scaffold design needs to shift from “osteogenesis-centric theory” to “immune-osteogenic coupling theory”, and immunomodulators should be listed as the core active components in parallel with BMPs, and angiogenesis, stem cell homing and osseointegration repair cascades should be realized by reprogramming macrophage phenotypes.^204,205 The long-term treatment of orthopedic-related diseases necessitates the maintenance of good stability and compatibility between the loaded active ingredient and the scaffold, as well as enduring effectiveness to achieve a synergistic effect greater than the sum of its parts. The unknown mechanism of multifactor synergism in composite scaffolds is also another major challenge. For example, the combination of BMP-2 and VEGF can synchronously promote osteogenesis and vascularization, but the difference in the timing of the release of the two (VEGF is released earlier than that of BMP-2) significantly affects the repairing effect.²⁰⁶ And also for example, the multicomponent of TCMs (eg, tanshinone and panax notoginseng saponins) may exhibit synergistic effects of osteogenesis and anti-inflammation through multi-target regulation (eg, activation of the AKT pathway, inhibition of NF-κB inflammatory signaling), exhibiting synergistic effects of bone promotion.^207–210 Current research predominantly adheres to a rudimentary “load-and-release” paradigm, overlooking the stage-specific temporal demands of bone healing (eg, sequential prioritization of vascularization preceding osteogenesis) and spatial heterogeneity (the difference in the microenvironment between the center and the edge of the defect). This static delivery approach leads to insufficient bioavailability of the active ingredient, while burst release-induced off-target effects may even impede repair. Next-generation bioactive scaffolds necessitate evolution toward “dynamically programmable delivery systems” that enables the spatiotemporal-precise delivery of active ingredients through smart materials, mimicking endogenous healing signal chains (eg, sequential activation from VEGF to BMP-2).^211–213 Furthermore, the paradox of clinical translation, the “healing hallucination” of loaded scaffolds is mostly limited to small animal models, and the conclusion of “high cure rate” based on rodent non-weight-bearing bone defects (cranial/femoral condyle) may seriously mislead the transformation expectations, and such models cannot simulate the blood supply disorders, mechanical loads, and immunosenescence microenvironment of human weight-bearing bones. There is an urgent need to establish clinical predictive evaluation criteria, including the requirement of large animal weight-bearing models (such as sheep tibial segment defects), the inclusion of pathological microenvironment (such as diabetes/osteoporosis/infection), and the endpoint indicators focusing on functional reconstruction (such as biomechanical strength ≥ 80% of natural bone).^214,215

BTE integrates multiple disciplines, including immunology, cell biology, materials science, and manufacturing, fostering multidisciplinary collaboration and innovation in the field of bone repair and regeneration. In terms of cost control and regulatory approval, the utilization of waste biomass such as crustacean shells, fish bones, and eggshells as raw materials can significantly reduce the procurement cost of biomaterials.^216,217 Furthermore, the regulatory approval (such as CE mark, FDA) of natural material-based products like Bio-Oss (bovine bone-derived) and Algipore (algae-derived) paves a clear regulatory path for the development of more complex smart scaffold systems, thereby accelerating their clinical translation.²¹⁷ Additionally, the introduction of 4D printing technologies enables the fabrication of personalized scaffolds that integrate microscopic active ingredient loading with macroscopic morphological adaptation.²¹⁸ Studies have demonstrated that 4D-printed composite scaffolds loaded with osteogenic active components can enhance the bone defect healing rate by nearly 40–50% in animal models.^219–221 The convergence of these fields generates novel ideas that enhance the clinical application of bone defect repair and regeneration. Furthermore, the advent of 3D and 4D printing technologies enables the creation of microstructures tailored for loading active ingredients, as well as macrostructures suited for bone defects of varying shapes and sizes, thereby facilitating individualized treatment. These technologies have also improved the capability of composite scaffolds to deliver osteogenic substances through the integration of external fields, such as acoustic, electric, or magnetic fields, demonstrating significant potential in BTE. With the rapid advancement of artificial intelligence (AI) technology, it is possible to combine it with multi-omics technology (genomics, proteomics, metabolomics) to systematically analyze how multiple components in TCMs (such as flavonoids, saponins, etc.) work together on osteogenesis through a complex interaction network.²²² Looking ahead, the future development of composite scaffolds in bone repair and regeneration is anticipated to focus on innovations in biomaterials, intelligent design, personalized customization, bioprinting technology, the integration of tissue engineering and stem cell technologies, multifunctionality, comprehensive treatment approaches, and clinical translation. These advancements aim to establish more efficient, safe, and personalized treatment protocols for bone defect repair.

Conclusion

While the application of single active components demonstrates specific efficacy, their inherent limitations—such as side effects, poor in vivo stability, and single-target actions—often hinder their ability to mimic the complex physiological microenvironment and multi-signaling synergy inherent in bone repair. It is the clear recognition of these limitations that has driven the evolution of bone tissue engineering toward intelligent scaffold systems. The overarching goal of such intelligent scaffold design is not the crude stacking of functions, but rather the maximal activation of the body’s innate repair potential with minimal intervention. This necessitates the systematic integration of the unique advantages offered by different components: for instance, the potent osteoinductive capacity of BMPs, the safety and richness of growth factors in PRP, and the multi-target regulatory potential of natural products and TCMs. The “multimodal coordinated regulation” system built upon these elements marks a fundamental paradigm shift in bone repair strategy—from passive physical support to active biological regulation. Such a scaffold is no longer merely an implant, but a “bioactive platform” capable of precisely modulating the local microenvironment in both time and space to orchestrate the repair process. To facilitate the successful translation of this platform from concept to clinical practice, future research must concentrate on addressing several pivotal challenges: conducting robust efficacy validation in large animal models, establishing standardized characterization and quality control protocols for bioactive components (such as exosomes), and actively promoting the development of a forward-looking regulatory framework. Addressing these hurdles will accelerate the clinical adoption of this transformative technology and ultimately bring tangible hope to patients.