Advances in polymer-based hydrogel systems for adipose-derived mesenchymal stem cells toward bone regeneration

Abstract

Bone regeneration for non-load-bearing defects remains a significant clinical challenge requiring advanced biomaterials and cellular strategies. Adipose-derived mesenchymal stem cells (AD-MSCs) have garnered significant interest in bone tissue engineering (BTE) because of their abundant availability, minimally invasive harvesting procedures, and robust differentiation potential into osteogenic lineages. Unlike bone marrow-derived mesenchymal stem cells, AD-MSCs can be easily obtained in large quantities, making them appealing alternatives for therapeutic applications. This review explores hydrogels containing polymers, such as chitosan, collagen, gelatin, and hyaluronic acid, and their composites, tailored for BTE, and emphasizes the importance of these hydrogels as scaffolds for the delivery of AD-MSCs. Various hydrogel fabrication techniques and biocompatibility assessments are discussed, along with innovative modifications to enhance osteogenesis. This review also briefly outlines AD-MSC isolation methods and advanced embedding techniques for precise cell placement, such as direct encapsulation and three-dimensional bioprinting. We discuss the mechanisms of bone regeneration in the AD-MSC-laden hydrogels, including osteoinduction, vascularization, and extracellular matrix remodeling. We also review the preclinical and clinical applications of AD-MSC-hydrogel systems, emphasizing their success and limitations. In this review, we provide a comprehensive overview of AD-MSC-based hydrogel systems to guide the development of effective therapies for bone regeneration.

Key Words: Mesenchymal stem cells; Adipose-derived mesenchymal stem cells; Bone tissue engineering; Hydrogels; Bone regeneration; Polymers

Core Tip: This review highlights the critical need for advanced biomaterials in bone regeneration, emphasizing hydrogels as optimized scaffolds for adipose-derived mesenchymal stem cell delivery. The innovative aspect of this study lies in its comprehensive analysis of polymer-based hydrogels synthesis, advanced embedding techniques such as three-dimensional bioprinting, and osteoinductive modifications to enhance bone regeneration. Advancements in biomaterial engineering and stem cell technology will be essential for developing effective and clinically applicable bone tissue engineering strategies. Future research should focus on addressing scalability, regulatory challenges, and long-term safety to facilitate clinical translation.

INTRODUCTION

Reconstruction of critical-sized bone defects, defined as osseous voids that fail to heal spontaneously, remains an unresolved clinical challenge[1], despite the intrinsic regenerative potential of bone tissue[2]. Current standard-of-care strategies, primarily autologous bone grafting, offer a triad of osteogenic, osteoinductive, and osteoconductive cues[3]. However, these are constrained by donor site morbidity, limited graft volume, and extended operative time[4]. Allografts and xenografts bypass the tissue availability issue, but have immunogenicity, pathogen transmission, and inconsistent resorption kinetics risks[5], often culminating in poor graft-host integration and compromised biomechanical performance[6,7]. These limitations have led to the evolution of bone tissue engineering (BTE) as a transdisciplinary strategy that integrates materials science, stem cell biology, and orthopedic surgery[8] to biofabricate constructs capable of promoting de novo bone formation[9]. Central to this paradigm is the development of scaffolds that recapitulate the structural, mechanical, and biochemical characteristics of the native extracellular matrix (ECM), thereby facilitating cell adhesion, migration, proliferation, and osteogenic differentiation[10]. Among the various biomaterial platforms that have been investigated, hydrogels have attracted considerable interest[11]. Hydrogels are three-dimensional (3D), hydrophilic polymer networks capable of absorbing and retaining substantial water content. They mimic the viscoelasticity and porosity of native soft tissues[12]. This renders them suitable for minimally invasive delivery, as well as dynamic crosstalk with embedded or infiltrating cells[13].

Hydrogels uniquely enable the spatiotemporal modulation of the cellular microenvironment[14]. Their tunable mechanical properties, degradation profiles, and biochemical functionalization allow for precise control over the stiffness, matrix elasticity, and ligand presentation, each of which is known to govern mesenchymal stem cell (MSC) fate via integrin-mediated signaling and mechanotransduction pathways[15]. Moreover, hydrogels can serve as reservoirs for osteoinductive factors, such as bone morphogenetic proteins (BMPs), delivering them in a sustained or stimuli-responsive manner to enhance osteogenesis[16]. To fully harness the regenerative potential of these scaffolds, they must be integrated into biologically responsive cell populations capable of executing complex reparative programs[17].

MSCs are pivotal cellular agents in BTE owing to their multilineage differentiation capacity, immunomodulatory properties, and secretion of trophic factors that orchestrate tissue regeneration[18]. Although bone marrow-derived MSCs (BM-MSCs) have historically been the prototypical choice, their clinical utility is increasingly constrained by the need for invasive harvesting, reduced proliferative capacity due to donor age, and low cell yields[19]. In contrast, adipose-derived MSCs (AD-MSCs) are more accessible, abundant, and phenotypically stable[20]. AD-MSCs harvested using minimally invasive liposuction techniques exhibited high proliferative indices, robust immunosuppressive capabilities, and comparable, if not superior, osteogenic potential under appropriate osteoinductive priming[21].

Furthermore, AD-MSCs possess a highly active secretome rich in cytokines, growth factors, and extracellular vesicles, including exosomes, which modulate the bone-healing milieu by enhancing angiogenesis, recruiting endogenous progenitor cells, and attenuating inflammatory cascades[22]. These properties render AD-MSCs particularly well suited for incorporation into hydrogel-based scaffolds, forming a biointeractive interface where cellular viability, spatial organization, and osteoinductive signaling can be precisely modulated[23]. Advanced encapsulation techniques, including direct cell loading and 3D bioprinting, now permit spatially controlled cell placement within structurally defined hydrogel architectures[24], enabling the fabrication of "living scaffolds" that dynamically remodel and integrate with host tissue[25].

Despite the growing interest in hydrogel–stem cell hybrids, there remains a conspicuous gap in the literature at the intersection of AD-MSC biology and hydrogel engineering for bone regeneration[26]. Existing studies often examine these components in isolation and lack integrative frameworks to evaluate their synergistic potential across the preclinical and translational domains. This review addresses this critical gap by offering a comprehensive, mechanistic, and application-focused analysis of AD-MSC-laden hydrogel systems for bone regeneration. We begin with a technical overview of hydrogel synthesis, characterization, and functionalization, followed by an in-depth discussion of AD-MSC isolation, culture, and embedding strategies. We then explore the mechanistic underpinnings of AD-MSC-mediated osteogenesis within hydrogels, including immunomodulation and angiogenic coupling. Finally, we discuss the preclinical and clinical landscapes, highlighting emerging opportunities and translational challenges. To the best of our knowledge, this is the first review to specifically delineate the role of AD-MSCs embedded within hydrogel scaffolds in BTE, providing a roadmap for future biomaterial–cell hybrid strategies aimed at clinically relevant skeletal regeneration.

BTE

BTE fundamentally relies on the development of biomaterial scaffolds that recapitulate the native ECM to facilitate cellular adhesion, proliferation, and differentiation, thereby promoting functional bone regeneration[27]. Traditional scaffold materials include a broad spectrum of polymers and ceramics, each with its own distinct advantages and limitations. Polymers are classified into natural and synthetic polymers, and have been used in various biomedical applications, including BTE[28]. Natural polymers, including collagen (COL), gelatin (GEL), chitosan (CS), and silk fibroin, are inherently biocompatible and bioactive, promoting cell-matrix interactions; however, they often have poor mechanical robustness and batch-to-batch variability[29]. Synthetic polymers, such as polylactic acid, polyglycolic acid, and their copolymers, poly (lactic-co-glycolic acid) (PLGA), offer tunable degradation rates and mechanical properties; however, their hydrophobicity and acidic degradation byproducts can induce local inflammation and impair cellular viability[30]. Ceramic-based materials, such as hydroxyapatite (HAp), β-tricalcium phosphate, and bioactive glasses provide excellent osteoconductivity and mechanical strength closely resembling the mineral phase of native bone[31]. However, their brittleness, lack of intrinsic bioactivity, and difficulty in molding complex architectures restrict their applications, particularly in load-bearing or irregular defect sites[32]. Thus, composite materials integrating polymers and ceramics aim to synergize osteoconductivity with mechanical flexibility, but frequently involve complex fabrication processes and pose challenges in reproducibility and scale-up[33].

Hydrogels, defined as 3D hydrophilic polymer networks capable of retaining high water content, are an exceptional class of biomaterials in BTE because of their biomimetic physicochemical properties and versatile functionalization potential[34]. Their intrinsic softness and viscoelasticity closely resembles those of the native bone marrow microenvironment, providing a conducive niche for cell survival and lineage specification[35]. Hydrogels enable the fine-tuning of mechanical stiffness, degradation kinetics, and porosity, which are parameters that critically influence MSC mechanotransduction and osteogenic differentiation via integrin-mediated pathways and cytoskeletal remodeling[36]. Furthermore, hydrogels provide minimally invasive delivery modalities, including injectable formulations that conform to irregular defect geometries, ensuring intimate host-tissue integration[37]. Chemical modification strategies, such as the incorporation of arginyl-glycyl-aspartic acid peptides, enzymatically degradable crosslinks, and growth factor tethering enhance bioactivity and promote sustained osteoinductive signaling[38,39]. Recent advances in stimuli-responsive and self-healing hydrogel systems have provided dynamic microenvironments that can adapt to the evolving regenerative milieu, facilitating coordinated matrix remodeling and vascular ingrowth, which are essential for robust bone regeneration[40]. Collectively, hydrogels provide an unparalleled platform for next-generation BTE scaffolds by combining structural mimicry with customizable biochemical and biomechanical cues critical for successful bone tissue formation.

SYNTHESIS AND CHARACTERIZATION OF HYDROGELS IN BTE

Hydrogels serve as crucial biomimetic scaffolds in the BTE, with fabrication strategies tailored to balance mechanical robustness, biocompatibility, and degradation. By mimicking the ECM, they create a favorable microenvironment for cell adhesion, proliferation, and lineage commitment[41]. As shown in Figure 1, hydrogels can be derived from natural, synthetic, or hybrid polymers. Natural polymers, such as GEL, CS, alginate (ALG), and hyaluronic acid (HA) provide intrinsic bioactivity and osteoconductivity but have poor mechanical properties and exhibit rapid degradation.

Figure 1 Classification and advantages of polymer-based hydrogels used in bone tissue engineering. BTE: Bone tissue engineering.

To address the mechanical and structural limitations of natural polymer-based hydrogels, synthetic polymer-based hydrogels, such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), pluronic, and polyacrylamide, are used the mechanical properties, degradation rates, and structural integrity of these hydrogels can be precisely controlled. However, the lack of intrinsic bioactivity necessitates additional modifications or the incorporation of bioactive molecules to support cellular responses. To harness the benefits of both classes, hybrid hydrogels combine the biological functionality of natural polymers with the tunable mechanical properties of synthetic polymers. This integrated approach provides controlled mechanical strength, enhances osteoconductivity, facilitates cell infiltration, and promotes vascularization, making the hybrid hydrogels particularly effective for complex bone tissue regeneration strategies. To ensure that these engineered hydrogels meet biological requirements, they are typically characterized by their structural, mechanical, and functional properties. Techniques, such as spectroscopy (for chemical composition), rheology and compression testing (for viscoelasticity and stiffness), and microscopy (for morphology, porosity, and cell–material interactions) are commonly used. These evaluations establish a direct link between the fabrication strategy and the ability of hydrogels to support osteogenesis and angiogenesis.

In addition to their polymeric sources, hydrogels are further categorized based on their molecular crosslinking mechanisms and functional responses, as summarized in Figure 2. Physical crosslinking methods (hydrogen bonding, ionic interactions, and hydrophobic forces) create reversible polymer networks that are conducive to cell encapsulation, but generally exhibit limited mechanical strength, making them unsuitable for load-bearing bone defects[42,43]. In contrast, chemical crosslinking strategies (click chemistry, Schiff base reactions, Michael addition, and photoinitiated polymerization) establish stable covalent bonds, resulting in hydrogels with enhanced stiffness and structural integrity, which are necessary for physiological loading[43,44]. Importantly, these crosslinking approaches not only determine material stability, but also influence cellular viability, proliferation, and signaling events that drive osteogenesis and angiogenesis.

Figure 2 Classification of hydrogels in bone tissue engineering based on molecular bonding and function. Physically crosslinked hydrogels rely on non-covalent interactions, while chemically crosslinked hydrogels form stable covalent bonds via Schiff base formation, click chemistry, or Michael’s addition. Functional categorization includes cell-encapsulated hydrogels, which provide biomimetic microenvironments for bone regeneration, and stimuli-responsive hydrogels, which dynamically adapt to external physical, chemical, and biochemical cues, enhancing regenerative capabilities.

From a functional perspective, hydrogels can be classified as cell-encapsulated or stimuli-responsive systems. Cell-encapsulating hydrogels provide biomimetic 3D microenvironments that preserve cell viability and support osteogenic differentiation. Stimuli-responsive hydrogels can be engineered with pH-, temperature-, or enzyme-sensitive linkers that adapt to physical, chemical, or biochemical cues. Such adaptability regulates scaffold degradation and controls the release of bioactive factors, thereby fine-tuning the temporal signaling for osteogenesis[45-49]. Advanced fabrication technologies, such as 3D bioprinting and microfluidics allow high-resolution spatial control of hydrogel composition and cell distribution, replicating the anisotropic microarchitecture of bones[50,51]. The integration of these fabrication platforms with bioactive chemistries allows the precise spatiotemporal regulation of hydrogel properties, thereby enhancing osteoinductive signaling and improving regenerative outcomes[45,46].

Miao et al[52] developed a DNA-based nanocomposite hydrogel functionalized with dexamethasone via hydrogen bonding, which demonstrated significant osteoinductive potential and effective in vivo bone regeneration in non-load-bearing models. Xiang et al[53] fabricated PVA/HAp/tannic acid/COL hydrogels stabilized by hydrogen bonding, which enhanced mouse preosteoblastic cells (MC3T3-E1) proliferation and osteogenic differentiation of mouse preosteoblastic cells and promoted bone regeneration in rat femoral defects. Wang et al[54] engineered a PDLLA-PEG-PDLLA hydrogel embedded with metformin-loaded mesoporous silica nanoparticles and stromal cell-derived factor-1, leveraging hydrophilic-hydrophobic interactions to restore osteogenic capacity and promote periodontal bone repair under diabetic conditions. Similarly, Guo et al[55] reported that PLGA-PEG-PLGA hydrogels incorporating calcium silicate phases formed via hydrophobic interactions facilitate new bone formation in rabbit femoral condylar defects.

Biocompatibility and biodegradability are essential considerations in hydrogel scaffold design, with degradation rates tuned to parallel new bone matrix deposition to avoid adverse inflammatory and fibrotic responses[56]. Natural polymers, such as CS and HA have intrinsic bioactivity and enzymatic degradability but require chemical modification or composite formation to enhance their mechanical stability and achieve controlled degradation profiles[30,57]. Synthetic hydrogels, particularly PEG-based networks, provide reproducible hydrolytic and enzymatic degradation kinetics, allowing precise scaffold resorption timing[58,59]. Chen et al[60] demonstrated that 3D-printed PEG-co-poly (glycerol sebacate) acrylate (PEGSA)/HA gyroid scaffolds, combined with MSC-laden hydrogels, exhibited excellent biocompatibility and osteoinductive properties, significantly improving bone regeneration in vivo. Noory et al[61] designed 3D-printed hybrid scaffolds with dexamethasone-loaded polycaprolactone microparticles, enabling sustained osteogenic drug delivery and favorable biocompatibility, markedly enhancing osteogenic marker expression and alkaline phosphatase activity. Vu et al[62] fabricated N,O-carboxymethyl CS, aldehyde HA, and HAp into poly(ε-caprolactone) (PCL) hybrid scaffolds with mechanical properties analogous to those of cancellous bone, supporting hydrogel retention and interconnectivity; these scaffolds demonstrated biodegradability and facilitated bone regeneration in rabbit tibial defects, illustrating clinical potential. Advances in hydrogel synthesis and rigorous characterization have established hydrogels as adaptable, biomimetic scaffolds that deliver critical structural and biochemical signals for bone regeneration. Table 1[63-70] summarizes the key hydrogel systems and their efficacies in guiding future scaffold designs for BTE.

Table 1 Hydrogel systems and their efficacy, guiding future scaffold design in bone tissue engineering.

Scaffold compositions	Fabrication methods	In vitro models	In vivo models	Inferences	Ref.
Chondroitin-6-sulfate (CHA)/glycol CS (GCS)/maleic anhydride-modified polyethylene glycol (DF-PEG) (CHA/SF/GCS/DF-PEG) hydrogels	Freeze drying	BM-MSCs, human embryonic kidney cells (HEK293), mouse osteoblastic progenitor cells, mouse macrophages (RAW264.7)	Adult mouse fracture model (BALB/c)	Hydrogel with BM-MSC-exo regulated macrophage polarization and angiogenesis to enhance bone regeneration	[63]
Methacrylate-silk fibroin/nano-HAp hydrogels	Photo-crosslinking	RAW 264.7 cells, BM-MSCs	Rat femoral defect	Elevated osteochondral repair by macrophage M2 polarization has been reported	[64]
Ultrasound-triggered ultrashort peptide SESSE nanofiber hydrogels	Ionic gelation method	BM-MSCs	C57BL/6J mouse bone defect model	Increased M2 polarization led to secretion of BMP-2 and IGF-I, and enhanced bone regeneration	[65]
Alpha-tricalcium phosphate/COL bioceramic ink/sanitizer-based hydrogels	3D-bioceramic printing	MC3T3 cells	-	Improved mechanical properties, increased cellular growth and osteogenic activity have been reported	[66]
Nano-HAp/GEL methacrylate/oxidized sodium ALG hydrogels loaded with BMP7	Photo-crosslinking	Rat BM-MSCs	Rat distal femur defect model	Enhanced osteogenic differentiation and bone formation have been reported	[67]
GEL-Bletilla striata polysaccharide-mesoporous bioactive glass hydrogels	Schiff base reaction and hydrogen bonding	Bone marrow-derived macrophages	Rat cranial defect model	Enhanced immunomodulation and bone regeneration were reported	[68]
Poly (L-glutamic acid) (PLG) grafted with tyramine (PLG-g-TA) polymer, VEGF, and strontium-doped borosilicate bioactive glass nanoparticles	Enzyme cross-linking	Rat BM-MSCs, human umbilical vein endothelial cells, and Raw264.7 cells	Rat cranial bone defects	Enhanced vascularization, osteogenesis, and angiogenesis have been reported	[69]
Amino-functionalized barium titanate nanoparticle hydrogels	Schiff base reaction and hydrogen bonding	BM-MSCs	Rat calvarial defect model	Enhanced ALP, BMP2, OPN, OCN, and COL-I expression and new bone formation have been reported	[70]

ALG: Alginate; BM-MSC: Bone marrow-derived mesenchymal stem cell; CS: Chitosan; GEL: Gelatin; Hap: Hydroxyapatite; VEGF: Vascular endothelial growth factor; 3D: Three-dimensional.

AD-MSC-LADEN HYDROGELS FOR BONE REGENERATION

AD-MSCs are a highly accessible and potent cell source for BTE. When incorporated into hydrogels as 3D and hydrated polymer networks, AD-MSCs benefit from a biomimetic microenvironment that supports their survival, proliferation, and osteogenic differentiation (Figure 3). Hydrogels address the intrinsic challenges of MSC-based therapies, such as poor retention and limited engraftment, and provide tunable physicochemical properties that emulate the native bone niche. The convergence of refined AD-MSC isolation protocols with advanced hydrogel encapsulation techniques, including direct cell encapsulation and 3D bioprinting, has markedly enhanced the regenerative potential of cell-laden constructs.

Figure 3 Schematic representation of adipose-derived mesenchymal stem cells-laden hydrogels for bone regeneration. The hydrogel matrix incorporates extracellular matrix components, such as collagen, fibrin, and hyaluronic acid, along with bioactive molecules, such as ascorbic acid, dexamethasone, and β-glycerophosphate to enhance osteogenic differentiation. Various sources of adipose-derived mesenchymal stem cells, including subcutaneous adipose tissue, bone marrow adipose tissue, infrapatellar fat pad, and visceral adipose tissue, contribute to the regenerative potential. The combined effect of these components facilitates bone tissue regeneration by promoting osteogenic differentiation and extracellular matrix deposition. AD-MSC: Adipose-derived mesenchymal stem cells.

Tissue sources and isolation of AD-MSCs

AD-MSCs can be isolated from diverse adipose depots, including subcutaneous, visceral, infrapatellar fat pads, and bone marrow-associated adipose tissue, offering a unique cellular profile relevant for bone regeneration. Enzymatic digestion, predominantly using collagenase, followed by systematic enzyme inactivation and differential centrifugation, remains the cornerstone for high-yield, high-purity AD-MSC isolation. For instance, Labedz-Maslowska et al[71] described isolation of human AD-MSCs from adipose tissue through collagenase digestion and centrifugation at 370 × g for 10 minutes at 16 °C, yielding stromal vascular fractions enriched for mesenchymal progenitors. Ghorbani et al[72] optimized MSC isolation from liposuction aspirates using collagenase digestion, followed by phosphate-buffered saline addition and centrifugation at 2000 rpm for 5 minutes to maximize cell viability and yield. Tareen et al[73] compared multiple isolation strategies across subcutaneous, omental, and perirenal fat depots, confirming that enzymatic digestion with collagenase, enzyme inactivation via Dulbecco’s modified Eagle’s medium, and centrifugation at 548 × g for 10 minutes effectively isolated MSCs with preserved multipotency. Extending these methods to bovine adipose tissue, Lee et al[74] demonstrated that collagenase digestion combined with fetal bovine serum quenching and centrifugation at 415 × g for 5 minutes effectively isolated primary cells from intermuscular adipose tissue. In subsequent experiments, these cells were cultured in AD-MSC proliferation medium to support MSC growth. Thus, MSCs were isolated without compromising their proliferative capacity or doubling time. Collectively, these studies emphasize the necessity of stringent enzymatic digestion parameters to obtain AD-MSC populations suitable for downstream applications.

Purification and culture of AD-MSCs

Post-isolation purification is critical to obtain homogenous AD-MSC populations with consistent regenerative capacities. Ficoll-Paque density gradient centrifugation is widely used to enrich MSCs fractions, as demonstrated by Laloze et al[75], who verified cell identity using flow cytometry and expanded cells under standardized culture conditions. Ishii et al[76] further refined purification by sequential filtration through 100- and 40-μm strainers prior to culturing in DMEM/F-12 supplemented with 10% fetal bovine serum under controlled CO₂ and temperature conditions. Absari et al[77] emphasized repeated passaging and homogenization combined with flow cytometric characterization to maintain the cell phenotype and multipotency. Rigorous purification and culture protocols are required to ensure that functional AD-MSCs are capable of robust osteogenic differentiation upon encapsulation.

Embedding AD-MSCs in hydrogels for bone regeneration

Embedding AD-MSCs within hydrogels offers a 3D, cell-friendly milieu that recapitulates critical ECM cues, thereby sustaining cellular functions essential for bone repair.

Direct encapsulation: Direct encapsulation involves homogeneously suspending AD-MSCs in hydrogel precursors prior to polymerization, which supports uniform cell distribution and cell-matrix interactions critical for differentiation. Lee et al[78] engineered an injectable nano-HAp-based hydrogel encapsulating human AD-MSCs, which significantly enhanced osteogenic differentiation in vitro and accelerated bone regeneration in a rat calvarial defect model. Haddad-mashadrizeh et al[79] encapsulated human AD-MSCs in CS-β-glycerol phosphate-hydroxyethyl cellulose-based hydrogels, demonstrating robust cell viability and osteogenic capacity. In a complementary strategy, Guo et al[80] embedded AD-MSC-derived extracellular vesicles within thermosensitive pluronic F127 hydrogels, which potentiated osteochondral regeneration in rat models via enhanced osteogenic signaling. These studies underscore the efficacy of direct encapsulation in maintaining AD-MSC function and enhancing bone regeneration through cell-scaffold synergy.

3D bioprinting for spatial precision: 3D bioprinting has revolutionized scaffold fabrication by enabling precise spatial placement of AD-MSCs within architecturally complex hydrogels, thereby mimicking the hierarchical structure and heterogeneity of native bone tissue. Li et al[81] demonstrated a bilayer hydrogel scaffold combining decellularized ECM (dECM) and AD-MSC-derived exosomes fabricated via 3D bioprinting, achieving region-specific osteochondral regeneration in vivo by guiding endogenous bone marrow stromal cells. Zhou et al[82] reported that 3D-bioprinted self-assembled nanopeptide hydrogels encapsulating AD-MSCs preserved high viability and supported multilineage differentiation along the osteogenic, adipogenic, and endothelial lineages. Koivunotko et al[83] highlighted the proangiogenic advantage of 3D-bioprinted nano-fibrillated cellulose hydrogels supporting AD-MSC viability and pericyte-like differentiation, which enhanced endothelial tubular formation in vitro, indicating an improved vascularization potential that is critical for bone healing. These advances confirmed that 3D bioprinting provides structural and functional fidelity for recreating the bone microenvironment.

Mechanisms of bone regeneration in AD-MSC-laden hydrogels

Bone regeneration is a spatially and temporally orchestrated biological process involving osteogenic cells, ECM components, and bioactive factors that restore skeletal architecture and mechanical function[84]. Hydrogels are powerful platforms for BTE owing to their ECM-mimicking properties, injectability, and tunable degradation kinetics[85]. Owing to their abundance, immunomodulatory properties, and osteogenic potential, AD-MSCs are frequently encapsulated in these matrices to facilitate localized bone regeneration. The crosstalk between AD-MSCs and hydrogel scaffolds enhances osteogenic lineage commitment via paracrine signaling and cell–matrix interactions, resulting in spatially controlled bone formation[26].

In vivo investigations demonstrated the therapeutic potential of AD-MSC-laden hydrogels in regenerating critical-size defects and osteochondral lesions. For example, thiol-modified poly(N-isopropylacrylamide)-grafted CS (TNC) hydrogels loaded with human AD-MSCs (hAD-MSCs) were evaluated in a monosodium iodoacetate-induced osteoarthritis (OA) rabbit model. After 4 weeks, micro-computed tomography (micro-CT) revealed an increase in bone volume fraction (BV/TV) from 21.79% in the empty control to 33.29% in the TNC + hAD-MSCs group and 38.17% when co-delivered with etanercept (TNF-α inhibitor). By 12 weeks, the BV/TV further increased to 48.79% and 50.05%, respectively, comparable to normal bone values (approximately 55%), and trabecular thickness also showed significant improvement[86]. Similarly, Tu et al[87] used microbial transglutaminase-cross-linked GEL hydrogels containing osteogenically preconditioned AD-MSC spheroids, which significantly enhanced mineral deposition and bone formation in a diabetic rat model by day 28. Preconditioned cells showed higher alizarin red staining in vitro, and in vivo data confirmed superior trabecular number, bone volume, and expression of bone sialoproteins compared with those in controls.

To further optimize the osteoinductive microenvironment, bilayered hydrogel systems have been engineered using both structural and chemical cues. A representative example is a bilayer scaffold composed of COL-carbonyl dihydrazide, oxidized chondroitin sulfate, and PEG-diacrylate (that incorporates a sublayer of zinc-doped HAp. This biomimetic architecture promoted the robust osteogenic differentiation of AD-MSCs, as evidenced by increased mineralized matrix deposition and improved bone morphometric indices on micro-CT and histology[88]. Similarly, freeze-dried carboxyl dextran was grafted PCL hydrogels provided a highly porous, hydrophilic environment for efficient adipose-derived stem cell (AD-SC) infiltration and proliferation. Scanning electron microscopy and fluorescence imaging confirmed uniform cell distribution, whereas DNA quantification demonstrated the proliferation of AD-SCs in the hydrogel over time[89].

Beyond the biochemical composition, physical and photonic modulation of hydrogel-encapsulated AD-MSCs enhance regeneration outcomes. Methacrylated GEL hydrogels loaded with AD-MSCs exhibited significantly improved mineralized tissue formation upon photobiomodulation. After 20 weeks, these constructs displayed reduced microporosity and improved matrix organization, with histological evidence of bone remodeling and AD-MSC integration at the defect site[90]. In the context of tendon-bone interface repair, tendon-derived hydrogels (tHG) facilitates AD-MSC homing to the injury site, accompanied by increased cellularity and immune cell infiltration. These findings suggest that tHG promotes regeneration via stem-cell recruitment and immune crosstalk[91].

Beyond structural and regenerative outcomes, recent studies have begun to elucidate the signaling mechanisms underlying AD-MSC–hydrogel interactions. For instance, Cao et al[92] reported that AD-MSC-derived exosomes enriched in miR-21-5p promote angiogenesis in endothelial progenitor cells and accelerate bone repair by activating the NOTCH1/DLL4/VEGFA pathway. In their study, rats in the HA group were treated with HA gel alone, while the HA+ADSCs group received a mixture of HA gel and ADSCs. The HA + Exo-L and HA + Exo-H groups were administered HA gel containing low (5 μg/mL) and high (20 μg/mL) concentrations of ADSC-derived exosomes, respectively. Micro-CT analysis showed that the levels of bone regeneration parameters were markedly upregulated in the HA + ADSCs, HA + Exo-L, and HA + Exo-H groups, indicating that ADSC-derived exosomes effectively accelerate cranial defect repair (Figure 4). These findings highlight that hydrogel-based delivery platforms can support osteogenesis and vascularization, which are indispensable for sustained bone regeneration. While most studies emphasize macroscopic repair outcomes, emerging evidence indicates that paracrine signaling, integrin-matrix interactions, and Wnt/β-catenin signaling may also contribute to the coupling of angiogenesis and bone formation. Nevertheless, the systematic characterization of these molecular pathways in AD-MSC-laden hydrogels remains limited, representing an important direction for future research.

Figure 4 In vivo evaluation of ADSC-derived exosomes in cranial defect repair. A: Effects of ADSC-derived exosomes on cranial defect regeneration. Effects of ADSC-derived exosomes on repairment of cranial defects and micro-computed tomography analysis results. The images of cranial defects in each group before collecting skull tissues; B: The three-dimensional reconstruction images of cranial defects in each group from the frontal and lateral positions[92]. Citation: Cao L, Sun K, Zeng R, Yang H. Adipose-derived stem cell exosomal miR-21-5p enhances angiogenesis in endothelial progenitor cells to promote bone repair via the NOTCH1/DLL4/VEGFA signaling pathway. J Transl Med 2024; 22: 1009. Copyright ©The Author(s) 2024. Published by Springer Nature.

Expanding upon cell-intrinsic strategies, genetically engineered or secretome-optimized AD-MSCs delivered within ECM-mimetic hydrogels offer advanced therapeutic efficacy. Yu et al[93] demonstrated that intra-articular injection of transforming growth factor beta-1-overexpressing AD-MSCs using injectable hydrogels mitigated OA progression, promoted anabolic responses in chondrocytes, and reduced cartilage degeneration and subchondral bone loss. In another study, the stable integration of TiO₂ nanoparticles and curcumin enhanced the physicochemical characteristics of a hydrogel, particularly its swelling capacity, which supported nutrient diffusion and cell viability. The composite hydrogel exhibited excellent cytocompatibility, promoting increased viability of AD-MSCs. Importantly, the co-loaded hydrogel significantly stimulated osteogenic differentiation, as evidenced by the upregulation of Runx-2 and osteocalcin (OCN) expression, along with elevated ALP activity and calcium deposition. These findings highlight the osteoinductive potential of this scaffold, making it a promising biomaterial for advancing BTE and regenerative therapies[94]. A 3D-osteogel system, incorporating various MSC types, including AD-MSCs, BM-MSCs, and hair follicle-derived autologous MSCs derived from the hair follicle outer root sheath (MSCORS), supported sustained osteogenic differentiation, with MSCORS exhibiting the most potent mineral deposition and expression of OCN and BMP2[95]. Furthermore, thermosensitive injectable exosome hydrogels (dECM^@exo) derived from the nucleus pulposus ECM and loaded with AD-MSC exosomes provide a cell-free yet bioactive scaffold that modulates inflammation, preserves disc homeostasis, and suppresses pyroptosis in intervertebral disc degeneration models[96]. Collectively, these findings reinforced the ability of AD-MSC-laden hydrogels, whether cell-based or cell-free, to orchestrate multifactorial bone regeneration in diverse injury models. Table 2[97-104] summarizes key preclinical investigations using AD-MSCs in hydrogel-based scaffolds for BTE applications. Collectively, these findings demonstrated that AD-MSC-laden hydrogels incorporated with bioactive nanoparticles, growth factors, or ECM-mimetic polymeric matrices effectively enhanced osteogenic differentiation and bone regeneration. Among these, ECM-mimetic hydrogel designs are particularly promising as they provide a supportive microenvironment for AD-MSCs while enabling the sustained release of bioactive factors.

Table 2 Preclinical investigations employing adipose-derived mesenchymal stem cells in hydrogel-based scaffolds for bone tissue engineering applications.

Cell sources	Hydrogel compositions	Fabrication methods	In vitro models	In vivo models	Inferences	Ref.
Immortalized adipose-derived stem cells (AD-SCs)	Immortalized AD-SCs were embedded within the dextran hydrogel for photobiomodulation (PBM) treatment	Physical gelation of dextran-based hydrogel (commercially prepared)	AD-SCs	-	Optimized PBM combined with dextran hydrogels enhanced osteogenic differentiation of AD-SCs by upregulating osteogenic markers (RUNX2, BGLAP, BGN, and SOST)	[97]
SIRT1 + adipose-derived mesenchymal stromal stem cells (ASCs)	SIRT1 + ASCs in ALG hydrogels	Polymerization	ASCs	5-year-old mare with subchondral bone cyst	Pretreatment of ASCs with resveratrol and 5-azacitidine, followed by encapsulation in 3D-ALG hydrogel, promoted the replacement of subchondral bone cysts with normal bone tissue and facilitated complete recovery	[98]
Human adipose-derived stem cells (hAD-SCs)	hAD-SCs seeded with COL-GEL composite scaffolds containing fluorapatite (FA) nanoparticles	Blending of COL and GEL with FA nanoparticles followed by crosslinking to form a composite hydrogel scaffold	hAD-SCs	-	Increased ALP activity and calcium deposition as well as the expression of osteogenic genes, including Runx2, Col-I, ALP, and OCN, and the synthesis of proteins, such as OCN have been reported	[99]
hAD-SCs	VEGF-ADSCs were seeded on whitlockite (WH-C)	Cryogelation	hAD-SCs	Mouse calvarial bone defect model	VEGF-transduced AD-MSCs seeded on WH-C-reinforced GEL/heparin cryogels enhanced osteogenesis and angiogenesis, leading to significantly improved bone regeneration in a mouse calvarial defect model	[100]
hAD-SCs	O-carboxymethyl CS (O-CMC)/acetylsalicylic acid/hAD-SCs hydrogel	Chemically cross-linked hydrogel formation using EDC/NHS chemistry followed by freeze-drying to fabricate a porous 3D-scaffold	-	Wistar rat skull defect model	The ASA-crosslinked O-CMC hydrogel seeded with hAD-SCs significantly enhanced cranial bone regeneration in rats, as evidenced by CT imaging and histological analyses	[101]
Rabbit AD-SCs from inguinal adipose tissue	HA-grafted-CS-grafted-poly(N-isopropylacrylamide) (HA-g-CS-g-PNIPAM) hydrogel with platelet-rich plasma and biphasic calcium phosphate	HA-g-CS-g-PNIPAM hydrogel synthesized via EDC/NHS coupling and free radical polymerization, exhibiting thermo-gelling sol-to-gel transition at approximately 37 °C	Rabbit AD-SCs	Rabbit calvarial bone defect model	The injectable HA-g-CS-g-poly (N-isopropylacrylamide) (HA-CPN) hydrogel scaffold incorporated with platelet-rich plasma and biphasic calcium phosphate significantly enhanced proliferation, osteogenic differentiation, and mineralization of adipose-derived stem cells in vitro and promoted robust bone regeneration in vivo	[102]
hAD-SCs	CS and β-glycerol phosphate	In situ thermosensitive gelation	hAD-SCs cultured in 2D monolayer and 3D-CS/β-GP hydrogel	-	The quantum dots-βcyclodextrin-histidine labeled hAD-SCs laden CS hydrogel (QD-βCD-His@Dex) nanocarrier enhanced osteogenic differentiation compared to that with free Dex, sustained ALP activity and calcium deposition in a 3D-hydrogel, and upregulated RUNX2 and OPN gene expression	[103]
Immortalized AD-MSCs	Fast-dextran hydrogel disc	Direct encapsulation	Immortalized AD-MSCs	-	Elevated ATP levels, improved cell viability, and preserved membrane integrity have been reported	[104]

ALG: Alginate; BTE: Bone tissue engineering; COL: Collagen; CS: Chitosan; CT: Computed tomography; GEL: Gelatin, HA: Hyaluronic acid; AD-MSC: Adipose-derived mesenchymal stem cell; 3D: Three-dimensional; VEGF: Vascular endothelial growth factor.

AD-MSCs promote bone regeneration predominantly via exosome-mediated immunomodulatory mechanisms These exosomes are enriched in microRNA-451a (miR-451a), which plays a pivotal role in regulating macrophage polarization. Specifically, AD-MSC-Exos facilitated the conversion of pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages, creating a favorable microenvironment for osteogenesis. Mechanistically, miR-451a targets and suppresses the macrophage migration inhibitory factor (MIF), a key modulator of inflammation. Downregulation of MIF led to reduced expression of M1 markers (iNOS, CD86, and TNF-α) and enhanced expression of M2 markers (CD206 and IL-10). This shift in the macrophage phenotype contributes to reduced inflammation and enhanced bone healing. Furthermore, when ADMSC-Exos were delivered via GEL nanoparticle hydrogels, sustained release, preserved bioactivity, and significantly enhanced bone regeneration in vivo. Histological and micro-CT analyses in rat skull defect models demonstrated that GEL nanoparticle-Exos promoted superior bone formation compared with that in controls, highlighting the therapeutic potential of ADMSC-Exos in BTE applications[105].

Sadeghian-Nodoushan et al[106] demonstrated that a novel, cell-free BTE scaffold composed of an ALG hydrogel, cobalt ferrite nanoparticles, and exosomes derived from hADMSC significantly enhanced osteogenic differentiation. The incorporation of magnetic nanoparticles and exosomes into a hydrogel scaffold led to increased cell proliferation, elevated ALP activity, and greater ECM mineralization, particularly when exposed to a static magnetic field. Flow cytometry confirmed the expression of CD105, a surface marker associated with both mesenchymal identity and angiogenic activity, suggesting its potential role in vascularization. These findings highlight the synergistic effects of magnetic stimulation and bioactive exosomal components within a hydrogel matrix, establishing a promising strategy for enhancing bone regeneration and supporting angiogenic processes.

TRANSLATIONAL PROGRESS AND CHALLENGES OF AD-MSC-LADEN FROM PRECLINICAL TO CLINICAL BONE REGENERATION

The transition of AD-MSC-laden hydrogels from the bench to bedside has substantial potential for regenerative orthopedics, particularly in bone repair. These hydrogel systems provide biomimetic environments that support cell viability, osteogenic differentiation, and matrix deposition. However, translating this technology into clinical applications requires a rigorous evaluation of its scalability, reproducibility, regulatory compatibility, and long-term safety. Several preclinical studies have advanced the translational landscape by offering important insights and simultaneously illuminating persistent challenges. Bhattacharjee et al[107] demonstrated one such advancement by developing an amniotic membrane-based injectable hydrogel system delivering AD-MSCs for OA therapy. This combinatorial platform facilitated the intra-articular retention of stem cells, closely mimicked the native synovial microenvironment, and promoted cartilage repair while exerting anti-inflammatory effects in a collagenase-induced OA rat model. The synergy between the amniotic membrane and AD-MSCs resulted in superior chondroprotective outcomes compared with that of monotherapies, establishing the translational viability of these biohybrid systems for degenerative joint disorders. Despite these promising findings, the study emphasized unresolved barriers, including the necessity for validation in large animal models, mechanistic elucidation of cellular interactions, and consistency in therapeutic efficacy and biosafety, including critical factors for regulatory clearance and clinical acceptance.

Hosseinzadeh et al[101] explored a novel O-CMC hydrogel crosslinked with acetylsalicylic acid (ASA) and incorporated it into human AD-MSCs for bone tissue regeneration. In addition to providing mechanical stability, ASA functioned as a bioactive crosslinker, enhancing the osteoinductive capacity of the composite scaffold. In a rat calvarial defect model, this system significantly improved cellular recruitment, proliferation, and new bone formation, as shown by using micro-CT and histological evaluation (H&E and Masson’s trichrome staining). The use of ASA as a functional therapeutic crosslinker is a strategic innovation in hydrogel design. Nevertheless, this study highlights key translational requirements, such as the need for extended biocompatibility profiling, immunological risk assessment, biodegradation kinetics, and scalable manufacturing processes. Furthermore, progression toward human applications will require efficacy validation in large animal models and alignment with regulatory frameworks governing advanced therapeutic medicinal products. Despite these advances, several controversies in the field remain unresolved. Reported outcomes vary depending on the animal model used, with small animal models frequently demonstrating robust osteogenic or chondrogenic repair. However, these effects are not always reproducible in larger or more clinically relevant models, raising questions about the true translatability of the current findings. Similarly, AD-MSCs are subject to donor-to-donor variability influenced by age, health status, and isolation protocols, which complicates the reproducibility and generalization of the findings. Hydrogel scaffold fabrication also introduces inconsistencies, as small changes in crosslinking chemistry or bioactive incorporation can markedly alter the degradation, mechanical strength, and cellular responses. Consequently, while preclinical evidence consistently supports the hydrogel-mediated enhancement of AD-MSC survival, retention, and paracrine activity, other proposed benefits, particularly those related to long-term integration, stable immunomodulatory function, and superiority over alternative MSC sources, remain unclear. In summary, although AD-MSC-laden hydrogels demonstrated encouraging regenerative potential in preclinical models, their clinical translation is constrained by multifaceted challenges. Thus, despite the increasing research interest, preclinical outcomes should be interpreted cautiously until reproducibility across different animal models, donor cell sources, and scaffold fabrication methods is consistently demonstrated.

CONCLUSION

AD-MSC-laden hydrogels are a sophisticated strategy for bone regeneration that integrates cellular osteogenic capacity with customizable biomaterial scaffolds that provide mechanical support and biochemical cues. Recent advancements in hydrogel engineering with polymeric composite materials have enabled the precise modulation of physicochemical properties, enhancing AD-MSC viability, proliferation, and osteogenic differentiation in preclinical models. Despite these promising outcomes, translation into clinical practice is hindered by critical challenges, including scalable production, reproducible quality control, immunogenicity, regulatory compliance, and long-term safety assessment. Comprehensive mechanistic studies are required to elucidate cell–matrix interactions and host immune responses to optimize scaffold design. Future research must prioritize standardized manufacturing protocols and robust clinical evaluations to establish their efficacy and safety. Interdisciplinary collaboration spanning biomaterial science, stem cell biology, and clinical research are essential to establish the full therapeutic potential of AD-MSC-laden hydrogels as advanced personalized bone-regenerative therapies.