Multidimensional biomimetic strategy integrating structural, functional and biological cues for enhanced tendon-to-bone interface regeneration

Abstract

Failed tendon-bone interface (TBI) healing post-reconstruction is primarily attributed to persistent pathological microenvironments and insufficient fibrocartilage differentiation. However, effective tissue engineering strategies that concurrently regulate pathological microenvironments and promote fibrocartilage regeneration remain lacking. To address this, we developed a biomimetic strategy integrating fibrocartilage stem cells (FCSCs) with a novel dual-crosslinked BGBT hydrogel. This hydrogel, composed of bacterial cellulose, gelatin methacryloyl, borax, and tannic acid, mimics the native fibrocartilage extracellular matrix architecture, provides mechanical support, and regulates the microenvironment to promote fibrocartilage regeneration. In vitro, BGBT hydrogel significantly enhanced FCSCs proliferation and chondrogenic differentiation while modulating Sharpey fiber formation and inflammatory responses. In vivo, the BGBT@FCSC group exhibited superior new bone formation, biomechanical properties, and histological restoration of the tendon-bone interface compared to controls. RNA sequencing revealed that BGBT activated the PI3K pathway, driving FCSCs toward functional fibrocartilage lineages. This study presents a multifaceted biomimetic approach that integrates structural, functional, and biological cues to enhance TBI regeneration, offering a promising solution for clinical applications.

Graphical abstract

Schematic illustration of BGBT hydrogel fabrication and the BGBT@FCSC therapeutic strategy. After encapsulating fibrocartilage-derived stem cells (FCSCs), the hydrogel drives functional tendon-to-bone interface repair in rats by: (i) enhancing fibrocartilage matrix regeneration, (ii) suppressing Sharpey-fiber formation, (iii) modulating the inflammatory microenvironment, and (iv) inhibiting bacterial growth.

1. Introduction

Ligament tears, ruptures and avulsion injuries are common clinical challenges in orthopedics, often requiring surgical repair [1]. Although autologous ligament grafts remain the “gold standard” for reconstruction, the healing quality of the tendon-to-bone interface (TBI) postoperatively remains suboptimal [2,3]. The native TBI features a four-layer gradient structure-comprising bone, mineralized fibrocartilage, non-mineralized fibrocartilage, and tendon-that enables stress buffering and efficient load transfer via stiffness gradation. Specifically, the mineralized fibrocartilage layer provides a critical transition zone that facilitates the gradual change in stiffness from bone to tendon, while the non-mineralized fibrocartilage acts as a lubricating and shock-absorbing layer. Conversely, postoperative interfaces are typically dominated by Sharpey fibers, a scar-like tissue rich in type I collagen (COL1) but lacking structural hierarchy, predisposing the interface to stress concentration, loosening and rerupture [4]. Reconstructing functional fibrocartilaginous gradients to restore biomechanical adaptation thus represents a critical unmet need in orthopedic regenerative medicine.

The failure of TBI healing stems from dual challenges: insufficient regenerative cells and dysregulated microenvironments [5,6]. During early healing, the injured interface is dominated by M1 macrophages, which create an inflammatory milieu characterized by the sustained release of proinflammatory cytokines, such as Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-6 (IL-6). These cytokines not only suppress the recruitment of endogenous stem/progenitor cells but also drive fibroblast hyperactivation, leading to aberrant deposition of COL1 and the formation of Sharpey fibers [7,8]. This pathological cascade hampers fibrocartilage regeneration and osseointegration, trapping the interface in a cycle of scar formation, mechanical failure and reinjury. Consequently, early-stage replenishment of regenerative cells, coupled with microenvironmental remodeling, is pivotal to breaking this impasse.

Mesenchymal stem cells (MSCs) have been regarded as an alternative therapeutic option for bone tissue regeneration due to their multipotency. However, their clinical translation is hampered by low survival rates, unpredictable differentiation and microenvironmental dependency [[9], [10], [11], [12]]. The selection of MSC sources is particularly critical for achieving controlled and stable fibrocartilage regeneration in TBI repair. Recently identified fibrocartilage stem cells (FCSCs) exhibit unique advantages: originating from fibrocartilaginous tissues, they demonstrated intrinsic affinity for TBI microenvironments and differentiated into chondrogenic and osteogenic lineages in vivo, positioning them as ideal candidates for interface regeneration [13,14]. Utilizing an appropriate hydrogel biomaterial as a cell delivery vehicle can create a conducive microenvironment that not only prolongs cell viability but also provides essential factors to guide MSCs differentiation into the desired lineages [15,16]. Nevertheless, technical hurdles persist in maintaining FCSC viability, guiding lineage-specific differentiation and modulating host microenvironments via carrier systems [17]. Current hydrogel carriers, while mimicking the three-dimensional (3D) architecture of the extracellular matrix (ECM), often suffer from inadequate mechanical strength, limited bioactivity and poor integration with host tissues [18]. For instance, conventional gelatin methacryloyl (GelMA) hydrogels lack biomechanical adaptability to meet TBI’s dynamic stress demands, while single-drug delivery systems fail to concurrently achieve anti-inflammatory, antibacterial and pro-regenerative functions [19,20]. These limitations underscore the urgent need for multifunctional smart carriers.

To address these challenges, we developed a novel dual-crosslinked network hydrogel (BGBT) based on bacterial cellulose (BC), GelMA and tannic acid (TA) through a biomimetic design. First, BGBT achieves micro- and nanostructural biomimicry. BC forms a nanofibrous network (20–100 nm) that topologically resembles the collagen fibers (type I/II collagen, 50–150 nm) in native fibrocartilage matrices [21]. Such nanoscale biomimetic architectures can regulate FCSC alignment via contact guidance, promoting the ordered secretion of fibrocartilage-specific ECM components [e.g., COL2, aggrecan (AGG)] [22,23]. Simultaneously, GelMA’s photocurable properties enable in situ formability, while its 3D microporous structure (50–200 μm pores) facilitates cell infiltration and nutrient exchange [24]. Second, BGBT enables multifunctional microenvironmental regulation. TA acts as a crosslinker, forming hydrogen bonds with BC and GelMA via phenolic hydroxyl, carboxyl and amino groups, while π-π stacking interactions further enhance compressive modulus [25,26]. Moreover, TA’s anti-inflammatory and antioxidative properties suppress M1 macrophage polarization and activate SRY-related high-mobility group box 9 (SOX9) signaling to drive chondrogenic differentiation of MSCs [27]. Notably, TA exhibits broad-spectrum antibacterial activity by disrupting microbial membrane integrity through its pyrogallol and catechol groups [28]. This “mechanical-biochemical-antibacterial” multifunctionality overcomes the limitations of conventional single-activity hydrogels.

This study proposes a “structural-functional-biological triple-bioinspired” strategy by integrating FCSCs with the BGBT hydrogel delivery system. Structural biomimicry is achieved through BC’s nanofibrous topology, which mimics native fibrocartilage matrices to guide FCSC-driven ECM deposition. Functional biomimicry harnesses TA’s microenvironmental regulation and GelMA’s cell-adhesive properties to sustain FCSC viability and activity. Biological biomimicry capitalizes on FCSCs’ innate capacity to recapitulate the cellular composition and regenerative behavior of native TBI. The hydrogel exhibits dynamic crosslinking via chemical borate ester bonds, endowing it with exceptional cell adhesion and shape adaptability. By synergizing FCSCs’ regenerative potential with BGBT’s multidimensional regulatory functions, this study aims to achieve integrated therapeutic goals: “sharpey fiber regulation, microenvironment remodeling and regeneration guidance.” This approach offers a breakthrough solution for functional TBI healing and mechanistic exploration (see Fig. 1).

Fig. 1.

Fabrication and application of BGBT dual-crosslinked network hydrogel loaded with FCSCs for treatment of TBI in rats. Created with BioRender.com. (FCSCs, fibrocartilage stem cells; TBI, tendon-to-bone interface.)

2. Results

2.1. Isolation and characterization of FCSCs

FCSCs were isolated from rat temporomandibular joint cartilage using established protocols. Flow cytometric analysis confirmed successful FCSC isolation, with cells exhibiting strong positivity for MSC markers (CD29, CD90 and CD44) and robust negativity for hematopoietic lineage markers (CD34 and CD45) (Fig. 2B) [29]. Bone marrow-derived mesenchymal stem cells (BMSCs) were isolated according to prior studies [30].

Fig. 2.

Isolation and characterization of FCSCs. (A) Schematic diagram illustrating the extraction, identification and subsequent experimental procedures for FCSCs. (B) Flow cytometric analysis of surface antigens on FCSCs. (C, D) Relative gene expression of COLI and COL2 in FCSCs and BMSCs following 14 days of chondrogenic induction, scale bar, 200 μm. (E) Chondrogenic pellet assays showing the aggregation of FCSCs and BMSCs under chondrogenic induction medium (CID). (F) Alcian Blue staining of FCSCs and BMSCs after 14 days of chondrogenic induction, scale bar, 200 μm. Data are presented as mean ± SD. ∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Two-tailed unpaired Student’s t-test for double comparison.

To evaluate the fibrocartilaginous differentiation potential of FCSCs, we compared fibrocartilage-related gene expression profiles between FCSCs and BMSCs following chondrogenic induction. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed that FCSCs cultured in chondrogenic induction medium (CIM) exhibited significant upregulation of COL1 and COL2 mRNA levels, whereas BMSCs showed no notable change in COL1 expression (Fig. 2C and D, Table S3).

To further validate these findings, Alcian blue staining demonstrated more intense ECM staining in FCSCs compared to BMSCs after chondrogenic induction, indicating enhanced glycosaminoglycan deposition (Fig. 2E). In chondrogenic pellet assays, FCSCs aggregated into macroscopically visible spheroids within 2 days of induction, whereas BMSCs failed to form cohesive aggregates. After 28 days of induction, FCSC-derived spheroids remained intact following PBS washing and medium replacement, while BMSC aggregates disintegrated and were removed during washing (Fig. 2F).

Collectively, these results demonstrated the superior fibrocartilaginous differentiation potential of FCSCs over BMSCs.

2.2. Fabrication and characterization of BGBT hydrogel

Three hydrogel formulations-GelMA, BGB (BC-GelMA pre-hydrogel) and BGBT (TA-incorporated BGB)-were fabricated (Fig. 3A). The BGB hydrogel exhibited UV-triggered crosslinking due to the photocurable properties of GelMA (Fig. 3B).

Fig. 3.

Fabrication and characterization of the BGBT hydrogel. (A) Schematic diagram of the BGBT hydrogel fabrication process. (B) Photocuring of the BGB hydrogel. (C) Photographs and SEM images of the photocured hydrogels. Scale bars, 300 μm and 200 μm, respectively. (D) Fourier-transform infrared spectroscopy (FTIR) analysis of the hydrogels. (E) Rheological properties of the hydrogels at room temperature (RT). (F) Weight and time relationship of the hydrogels soaked in simulated body fluid at room temperature (RT). (H) Swelling properties of the hydrogels. (G) Degradation properties of the hydrogels.

SEM imaging revealed that GelMA, BGB and BGBT hydrogels possessed three-dimensional porous networks with interconnected macropores (Fig. 3C). Notably, BGBT displayed heterogeneous pore sizes (20–200 μm), providing adequate spatial architecture for FCSC migration and survival.

FTIR spectroscopy was employed to analyze the chemical structures of Borax(B), BC, GelMA, TA, BGB and BGBT (Fig. 3D). Compared to B, BC and GelMA, BGB exhibited two intensified C-H stretching vibration peaks (3000-2800 cm⁻¹) and disappearance of the O-H bending vibration peak at 1108 cm⁻¹, confirming hydrogen bond formation between BC and GelMA. The attenuation of B-O symmetric stretching vibration peaks (995, 941, 821 and 705 cm⁻¹) in B indicated molecular crosslinking between borate ions and the BC-GelMA network. Finally, the main absorption peaks in the BGBT spectrum resemble those of BGB, indicating minimal structural differences between the two hydrogels. In BGBT, reduced O-H (3286 and 1062 cm⁻¹) and N-H (1633 and 1515 cm⁻¹) bending vibrations suggested interactions between TA and amino groups in BGB.

Rheological analysis demonstrated that BGBT exhibited higher storage modulus (G′) than BGB and GelMA, along with reduced loss modulus (G″) compared to BGB (Fig. 3E), indicating enhanced elasticity and deformation resistance. These improvements were attributed to the dual-network entanglement (BC/GelMA) and TA-mediated crosslinking, ensuring structural stability under mechanical loading.

Swelling tests revealed that BGBT achieved a maximum water absorption capacity 3.14-fold higher than GelMA, with a swelling profile similar to BGB (Fig. 3F and H). This enhancement likely originated from the hydroxyl-rich BC network, which facilitated interstitial fluid absorption to fill tendon-bone gaps.

Degradation kinetics showed complete hydrogel dissolution within 12 days (Fig. 3G), aligning with the inflammatory phase peaking at ∼2 weeks post-surgery. This temporal matching ensures sustained mechanical support and bioactive factor release during critical healing stages.

Adhesion testing via pull-off and overlap shear assays demonstrated superior bone-binding strength of BGBT over GelMA (Pull-off: 0.613 ± 0.214 kPa vs. 0.180 ± 0.092 kPa; Overlap shear: 0.440 ± 0.105 kPa vs. 0.075 ± 0.020 kPa) (Fig. S1). This enhancement arose from synergistic non-covalent interactions (BC hydroxyls and TA catechol groups), preventing hydrogel dislodgement during tendon graft passage through bone tunnels.

2.3. Cytocompatibility of BGBT hydrogel

To evaluate the effects of BGBT hydrogel on FCSC proliferation, we first performed Cell Counting Kit-8 assays after a 3-day incubation with varying concentrations (Fig. 4A). The results indicated that BGBT concentrations ranging from 100 to 500 μg/ml significantly enhanced FCSC proliferation. Specifically, a concentration of 100 μg/ml was found to be the most effective. Consequently, this concentration was selected for subsequent functional experiments. The cytocompatibility of BGBT and its precursors was further assessed using live/dead assays with FCSCs (Fig. 4B), fibroblasts (NIH3T3) (Fig. 4C) and macrophages (RAW264,7) (Fig. 4E). All cell types maintained high viability when cultured with BGBT hydrogel extracts, confirming its non-cytotoxicity (Fig. 4D and G). To evaluate FCSC growth within hydrogels, 3D fluorescence imaging of live/dead-stained constructs revealed that most FCSCs remained viable when encapsulated in BGBT hydrogel, exhibiting higher proliferative activity compared to those in GelMA and BGB hydrogels (Fig. 4F and H). Besides, the migratory capacity of FCSCs in BGBT hydrogel was investigated via scratch (Fig. 4J and L) and Transwell assays (Fig. 4I and K). FCSCs in BGBT group exhibited significantly enhanced migration potential relative to control and BGB groups, indicating that BGBT provides a favorable microenvironment for cellular recruitment and tissue integration.

Fig. 4.

Cell compatibility assessment of the BGBT hydrogel. (A) Proliferation activity of FCSCs treated with varying concentrations of BGBT hydrogel extracts (n = 5). (B) Live/dead staining of FCSCs co-cultured with different hydrogel extracts. Scale bars, 1000 μm. (C, D) Live/dead staining and semi-quantitative analysis of NIH3T3 cells co-cultured with different hydrogel extracts. Scale bars, 500 μm (n = 3). (E, G) Live/dead staining and semi-quantitative analysis of RAW264.7 cells co-cultured with BGBT hydrogel extracts. Scale bars, 500 μm (n = 3). (F, H) Three-dimensional scanning images and semi-quantitative analysis of live/dead staining for FCSCs within different hydrogel. (I, K) Scratch assay and semi-quantitative analysis of FCSCs co-cultured with BGBT hydrogel extracts. Scale bars, 200 μm (n = 3). (J, L) Transwell assay and semi-quantitative analysis of FCSCs co-cultured with BGBT hydrogel extracts. Scale bars, 200 μm (n = 6). Data are presented as mean ± SD. ns = not significant, ∗P < 0.01, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Two-tailed unpaired Student’s t-test for double comparison; Two-sided ANOVA with a Tukey’s post hoc test for multiple comparisons.

2.4. In vitro chondrogenic induction of FCSCs by BGBT hydrogel

RT-qPCR analysis revealed that FCSCs cultured with BGBT hydrogel extracts exhibited significant upregulation of chondrogenic markers (SOX9 and AGG) compared to controls, demonstrating BGBT’s pro-chondrogenic differentiation potential (Fig. 5D and E). Immunofluorescence staining further confirmed enhanced secretion of fibrocartilaginous ECM components [SOX9, AGG and collagen type II alpha 1 chain (COL2A1)] by FCSCs in BGBT group, indicating a microenvironment conducive to differentiation and ECM synthesis (Fig. 5A–C and F-H). Comparative analysis of GelMA, BGB and BGBT hydrogel extracts on FCSCs over 10 days of co-culture showed that BGB and BGBT groups induced higher cellular aggregation and glycosaminoglycan deposition, as evidenced by toluidine blue (Fig. 5I) and Alcian blue staining (Fig. 5J). Notably, the BGBT group exhibited the highest degree of cellular aggregation, suggesting that BC contributes to chondrogenic priming, while TA acts as a critical bioactive factor driving FCSC differentiation. Collectively, these results demonstrated that BGBT hydrogel promotes FCSC fibrocartilaginous differentiation, highlighting its therapeutic potential for tendon-bone interface regeneration.

Fig. 5.

Impact of BGBT hydrogel on the fibrocartilage differentiation of FCSCs. (A–C) Immunofluorescence staining of FCSCs co-cultured with BGBT hydrogel extracts. Scale bars, 250 μm. (D, E) Relative gene expression of SOX9 and Aggrecan in FCSCs co-cultured with different hydrogel extracts (n = 3). (F–H) Semi-quantitative analysis of SOX9, Aggrecan and COL2A1 immunofluorescence (n = 10). (I, J) Toluidine blue staining (I) and Alcian blue staining (J) of FCSCs co-cultured with different hydrogel extracts. Scale bars, 500 μm and 200 μm, respectively. Data are presented as mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Two-tailed unpaired Student’s t-test for double comparison; Two-sided ANOVA with a Tukey’s post hoc test for multiple comparisons.

2.5. Multifunctional bioactivities of BGBT hydrogel: in vitro anti-inflammatory, antibacterial and anti-fibrotic effects

Using NIH3T3 fibroblasts as an in vitro model, we investigated the effects of BGBT hydrogel on Sharpey fiber formation. RT-qPCR and immunofluorescence analyses revealed that BGBT treatment significantly downregulated collagen type I (COL1) and heat shock protein 47 (HSP47) expression while upregulating collagen type III (COL3) and laminin levels compared to controls (Fig. 6A–D). Immunofluorescence further confirmed reduced collagen type I alpha 1 chain (COL1A1) and elevated collagen type III alpha 1 chain (COL3A1) protein expression in BGBT-treated cells (Fig. 6E–G). These findings indicate that BGBT hydrogel selectively regulates collagen synthesis: suppressing fibroblast-driven COL1 (associated with Sharpey fiber maturation and scarring) while promoting COL3 (linked to healthy tissue regeneration) (Fig. 6H). Notably, COL1 in native tendon-bone interfaces primarily originates from osteoblasts and fibrochondrocytes, whereas fibroblast-derived COL1 contributes to pathological fibrosis. BGBT exhibits cell-specific modulation of COL1: it enhances COL1 synthesis in FCSCs while inhibiting this process in fibroblasts. This differential regulation of COL1 expression across cell types provides a basis to infer reduced Sharpey fiber formation-given that excessive COL1 deposition by fibroblasts is a key driver of such scar-like tissue-and further suggests a favorable microenvironment for fibrocartilage regeneration. On the other hand, in LPS-stimulated RAW264.7 macrophages, BGBT hydrogel significantly attenuated M1 polarization, as evidenced by reduced mRNA levels of pro-inflammatory markers (iNOS, TNFα, IL-1β) and restored expression of anti-inflammatory markers (CD206, Arg1) (Fig. 6K–M, P-R). Immunofluorescence corroborated these results, showing decreased iNOS and increased CD206 protein expression in BGBT-treated cells (Fig. 6I–J, N-O). This dual regulation at transcriptional and protein levels highlights BGBT’s capacity to rebalance macrophage phenotypes, favoring a pro-regenerative M2 state. Furthermore, BGBT hydrogel exhibited broad-spectrum antibacterial activity against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in antibacterial assays, forming distinct inhibition zones absent in GelMA and BGB controls (Fig. 6S and T). This antimicrobial property was attributed to TA, which disrupts microbial membrane integrity via its polyphenolic groups. Collectively, BGBT hydrogel synergistically modulates macrophage polarization, regulates pathological Sharpey fiber formation and inhibits bacterial colonization, creating a regenerative microenvironment conducive to tendon-bone interface healing. These multifunctional properties could address the tripartite challenges of inflammation, fibrosis and infection in interfacial repair.

Fig. 6.

Effects of BGBT hydrogel on the extracellular microenvironment. (A–D) Gene expression of laminin, col3A1, COL1A1 and hsp47 in NIH3T3 cells (n = 3). (E–G) Immunofluorescence images and semi-quantitative analysis of COL1A1 and COL3A1 protein expression in NIH3T3 cells (n = 8). Scale bars, 80 μm. (H) Schematic illustration of BGBT inhibition of Sharpey’s fibers. (I, J) Immunofluorescence images and semi-quantitative analysis of iNOS protein expression in RAW264.7 cells (n = 8). Scale bars, 250 μm. (K–M) Pro-inflammatory gene expression (iNOS, TNFα, IL-1β) in RAW264.7 cells (n = 3). (N, O) Immunofluorescence images of CD206 protein expression in RAW264.7 cells. Scale bars, 250 μm. (P, R) Anti-inflammatory gene expression (CD206, ARG1) in RAW264.7 cells (n = 3). (Q) Schematic illustration of macrophage RAW264.7 polarization. (S, T) Inhibition zone assay and quantitative analysis of S. aureus, E. coli and P. aeruginosa by the hydrogel (n = 3). Scale bars, 30 mm. (U) Schematic illustration of bacterial inhibition by hydrogel BGBT. Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Two-tailed unpaired Student’s t-test for double comparison; Two-sided ANOVA with a Tukey’s post hoc test for multiple comparisons. S. aureus: Staphylococcus aureus; E. coli: Escherichia coli; P. aeruginosa: Pseudomonas aeruginosa.

2.6. Biosafety and therapeutic efficacy of FCSCs-laden BGBT hydrogel for tendon-bone interface healing in vivo

At 2- and 4-weeks post-surgery, no wound dehiscence was observed in any group. Histopathological evaluation via Hematoxylin and Eosin (H&E) staining of heart, liver, spleen, lung and kidney tissues from the negative control (NC), BGBT hydrogel alone (BGBT), free FCSCs (FCSC) and FCSCs-laden BGBT hydrogel (BGBT@FCSC) groups at 4 weeks revealed no significant pathological alterations compared to controls (Fig. S2), confirming the biosafety of the BGBT@FCSC strategy.

New bone formation at the tendon - bone interface is an inevitable consequence of fibrocartilage calcification and serves as a key indicator for assessing the repair of this interface. In this study, Micro - CT scanning was used to evaluate new bone formation in the rat tibial tendon - bone reconstruction model across four groups: NC, BGBT, FCSC and BGBT@FCSC. Three-dimensional reconstructions demonstrated bone tunnel varying degrees of contraction with irregular surfaces due to neo bone growth in other groups (Fig. 7C). The BGBT@FCSC group exhibited greater tunnel contraction than NC and FCSC groups at both 2 and 4 weeks (Fig. 7D and H, Table S2). Quantitative analysis indicated that, except for the NC and FCSC groups, the other two groups exhibited increasing trends in bone volume/tissue volume (BV/TV) and bone surface area/tissue volume (BS/TV) from 2 to 4 weeks post - operation (Fig. 7E, F, I, J). Specifically, the BGBT group’s BS/TV rose significantly from 0.54 % ± 0.098 % at 2 weeks to 1.13 % ± 0.193 % at 4 weeks. In contrast, the BGBT@FCSC group demonstrated notable increases in BV/TV, BS/TV and tissue mineral density (TMD) during this period (Fig. 7G–K). At 2 weeks post - operation, this group had significantly higher BS/TV than the others and higher BV/TV than the NC and FCSC groups. Although not always statistically significant, the BGBT@FCSC group’s data trends were consistent and higher than those of the other groups. These findings collectively suggest enhanced neo bone formation in the BGBT@FCSC group, indicating its superior potential for tendon-bone interface regeneration.

Fig. 7.

Impact of FCSCs-laden BGBT hydrogel on functional recovery of tendon-bone interface in rats. (A) Schematic and anatomical illustration of the preparation process for the rat tibial tendon-bone interface reconstruction model. (B) 3D reconstruction of the tibial bone tunnel from micro-CT scans. Scale bars, 2 mm and 5 mm, respectively. (C–K) Quantitative assessment of micro-CT parameters for new bone at 2- and 4-weeks post-surgery (n = 3): bone tunnel diameter; Bone Volume ratio to Total Volume (BV/TV); Bone Surface ratio to Total Volume (BS/TV); Tissue Mineral Density (TMD). (L) Photograph of the tendon-tibia complex undergoing tensile testing. (M–R) Quantitative evaluation of tensile test parameters at 2- and 4-weeks post-surgery (n = 3): maximum load; tension-distance curve; stiffness. Data are presented as mean ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Two-sided ANOVA with a Tukey’s post hoc test for multiple comparisons.

In addition, pull-out testing was performed to evaluate the biomechanical properties of the tendon-bone interface in the NC, BGBT hydrogel, FCSC and BGBT@FCSC groups. At 2- and 4-weeks post-surgery, the BGBT@FCSC group demonstrated significantly higher pull-out force compared to the NC and FCSC groups (Fig. 7M and P, Table S2), indicating superior interfacial fixation strength. To quantify interface stiffness, the load-displacement curves were analyzed using piecewise function modeling (Fig. 7N and Q). The linear segment of the curve was selected, with its slope serving as a measure of stiffness. The BGBT@FCSC group exhibited greater stiffness than other groups at both 2 and 4 weeks (Fig. 7O and R), further confirming its enhanced efficacy in stabilizing the tendon-bone interface.

Histological analysis via H&E staining and Masson’s trichrome staining revealed distinct differences in TBI healing among groups at 2 and 4 weeks postoperatively (Fig. 8A–D). At 2 weeks postoperatively, NC and FCSC groups exhibited extensive inflammatory cell infiltration, disorganized fibrovascular tissue and irregularly arranged collagen fibers. By contrast, the BGBT and BGBT@FCSC groups showed attenuated inflammatory responses, improved collagen alignment, increased matrix deposition and blurred TBI boundaries. At 4 weeks postoperatively, no obvious new bone trabeculae were observed in the NC or FCSC groups; Sharpey fibers served as the primary structure connecting the tendon and bone at the transition interface, with slightly indistinct interface boundaries. In contrast, the BGBT@FCSC group formed a continuous transitional zone at the TBI, consisting of new bone trabeculae - calcified fibrocartilage - non-calcified fibrocartilage - tendon and the collagen fiber alignment was significantly more regular than that in the other groups.

Fig. 8.

Impact of FCSCs-laden BGBT hydrogel on histological structure restoration. (A, B) Representative H&E staining images of different groups. Scale bar, 200 μm and 100 μm, respectively. (C, D) Representative Masson staining images of different groups. Scale bar, 200 μm and 100 μm, respectively. (E–H) Representative immunofluorescence staining images for COL1A1 and COL2A1 of different groups. Scale bar, 100 μm. (I, J) Quantitative evaluation of immunofluorescence staining at 2- and 4-weeks post-surgery (n = 3): COL1A1 and COL2A1. (K) Histological score for TBI healing (n = 6). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Two-sided ANOVA with a Tukey’s post hoc test for multiple comparisons. Tendon-bone boundary: red dashed line; tidemark: black dashed line. B: old bone; NB: new bone; T: tendon; FG: fibrocartilage; CFC: calcified fibrocartilage; NFC: non-calcified fibrocartilage.

Immunohistochemical staining was performed to evaluate the expression of COL1A1 and COL2A1 (Fig. 8E–J). At 2 weeks postoperatively, the NC group [integrated optical density (IOD): 0.278 ± 0.004] and FCSC group (IOD: 0.273 ± 0.009) maintained significantly high COL1A1 expression, indicating excessive activation of fibroblasts and accumulation of Sharpey fibers. The BGBT group showed a marked reduction in COL1A1 expression (IOD: 0.171 ± 0.007, P < 0.0001), which was consistent with in vitro findings, confirming its role in regulating collagen expression in Sharpey fibers. Notably, the BGBT@FCSC group maintained the highest COL2A1 expression at both 2 weeks (IOD: 0.261 ± 0.008, P < 0.0001) and 4 weeks (IOD: 0.272 ± 0.009, P < 0.0001) postoperatively. Furthermore, at the 4-week timepoint, COL1A1 levels in the BGBT@FCSC group (IOD: 0.236 ± 0.005) were significantly elevated relative to the BGBT-only group (IOD: 0.193 ± 0.006, P < 0.001). These findings suggest that under the regulatory influence of BGBT hydrogel, FCSCs actively contribute to the synthesis of fibrocartilaginous matrix components.

Histological scoring was conducted based on three criteria: cellular morphology of interface tissue, extent of fibrocartilage tissue and interface tissue transition from bone to tendon (Fig. 8K). The BGBT@FCSC group achieved significantly higher scores than the other groups at both 2 and 4 weeks postoperatively, confirming its efficacy in promoting TBI healing.

Taken together, BGBT@FCSC promotes structural repair of the TBI by suppressing inflammatory responses, reducing COL1 accumulation in Sharpey fibers and facilitating fibrocartilage regeneration.

2.7. Underlying mechanisms of FCSCs-laden BGBT hydrogel in tendon-bone interface regeneration

To elucidate the biological mechanisms underlying BGBT-mediated tendon-bone healing, RNA sequencing (RNA-Seq) was performed on samples from the NC and BGBT groups at 2 weeks post-surgery using the MGI DNBSEQ-T7 platform. Principal component analysis (PCA) and violin plots revealed distinct transcriptomic profiles between the two groups (Fig. 9B and C). Volcano plot analysis identified 440 downregulated and 946 upregulated differentially expressed genes (DEGs) (|log2(FC)| ≥1, P ≤ 0.05) (Fig. 9D). Gene Ontology (GO) enrichment analysis demonstrated that these DEGs were predominantly associated with inflammatory response and ECM organization in biological processes, signaling receptor binding in molecular functions and integral plasma membrane components in cellular components (Fig. 9F). These findings align with BGBT’s observed anti-inflammatory and ECM-modulatory effects in vitro.

Fig. 9.

Therapeutic mechanisms of BGBT hydrogel on tendon-bone interface healing in rats. (A) Gross specimen photos for sequencing. (B, C) Principal component analysis and violin plots illustrating the relationships among replicate samples between the control and BGBT groups (n = 3). (D, E) Volcano plots and heatmaps based on differentially expressed genes (DEGs) between the control and BGBT groups (∣log2 (FC)∣≥ 1, P ≤ 0.05). (F) GO analysis displaying the enrichment of DEGs from BP, CC and MF categories in the control and BGBT groups. (G) Bubble plot for KEGG pathway enrichment analysis. (H, I) GSEA confirms the upregulation of the TGF-β and PI3K pathways in the BGBT group. (J–L) RT-qPCR analysis of gene expression in FCSCs with different interventions for 7 days (n = 3). (M − P) Western blot and semi-quantitative evaluation of protein expression in FCSCs with different interventions for 7 days. Scale bars, 200 μm (n = 3). Data are presented as mean ± SD. ns = not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001. Two-sided ANOVA with a Tukey’s post hoc test for multiple comparisons.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis further highlighted DEG enrichment in inflammation-related pathways (TNF, NF-κB and Jak-STAT signaling) and osteochondral development pathways (TGF-β, HIF-1 and PI3K-Akt signaling) (Fig. 9G). Moreover, Gene Set Enrichment Analysis (GSEA) results corroborated the KEGG findings, showing that the Transforming Growth Factor-beta (TGF-β) and PI3K pathways were upregulated in the BGBT group (Fig. 9H and I). Based on statistical significance (P-value), enrichment factor and gene count, the PI3K-Akt pathway was prioritized as a key regulator. Western blot analysis confirmed that BGBT hydrogel extracts significantly upregulated PI3K, COL2A1 and COL1A1 expression in FCSCs after 7 days of co-culture (Fig. 9M), indicating that PI3K pathway activation drives fibrocartilage differentiation. Conversely, PI3K inhibition with BKM120 suppressed COL2A1 and COL1A1 expression at both transcriptional and translational levels (Fig. 9J–L, N-P). Uncropped Western blot images are provided in Fig. S4. Overall, these findings demonstrated that BGBT hydrogel orchestrates tendon-bone interface regeneration by activating the PI3K-Akt pathway to direct FCSC differentiation toward functional fibrocartilage lineages.

3. Discussion

This study provides robust evidence that the integration of FCSCs with the double-network BGBT hydrogel enhances TBI healing by regulating inflammation, modulating fibrosis and promoting fibrocartilage regeneration-with the PI3K signaling axis primarily mediating the pro-fibrocartilage regeneration effect.

Early tendon-bone interface healing often results in mechanically inferior fibrous connections, which delay rehabilitation and compromise joint functionality [31,32]. Fibrocartilage regeneration is critical for restoring biomechanical competence during this phase [5,33]. This study proposes a tripartite biomimetic strategy: (1) cellular biomimicry via FCSC transplantation to replenish fibrocartilage precursors; (2) structural biomimicry using BGBT hydrogel to replicate native fibrocartilage ECM architecture; and (3) functional biomimicry through microenvironmental modulation-promoting FCSC-driven matrix synthesis while regulating fibroblast-mediated Sharpey fiber formation and suppressing M1 macrophage polarization.

Tendon-bone interface (TBI) healing proceeds in three phases: inflammation, proliferation and remodeling [34]. In rat models, the inflammatory phase (postoperative weeks 1–2) features rapid macrophage recruitment, M1 polarization and upregulated pro-inflammatory cytokines (TNF-α, IL-1β and iNOS)-a phase critical for clearing tissue debris and foreign materials. However, excessive inflammation induces aberrant COL1 deposition in Sharpey fiber regions, suppresses Sox9 expression and fibrocartilage cell function and ultimately impairs gradient architecture integrity. The proliferative phase (weeks 3–4) focuses on stem cell recruitment, proliferation and lineage-specific differentiation, whereas the remodeling phase (weeks 4–6) involves neofibrocartilage maturation and mineralization. The proposed BGBT@FCSC strategy targets early TBI healing intervention. BGBT hydrogel shortens inflammation by modulating macrophage polarization and reducing pro-inflammatory cytokines, while reprogramming Sharpey-fiber collagen deposition and curbing excessive COL1 accumulation and thus accelerates transition to the proliferative-remodeling phases. Additionally, scaffold-loaded FCSCs serve as an in situ seed cell reservoir, continuously providing progenitor cells for fibrocartilage regeneration and directly participating in functional reconstruction of TBI gradient fibrocartilaginous architecture.

COL1 serves as a defining component of fibrocartilage, distinguishing it from other cartilage types [35]. Moreover, COL1 exhibits spatially regulated expression in the native TBI [36]. In the osseous region, COL1 is predominantly secreted by osteoblasts, forming a highly mineralized and mechanically robust bone matrix. Within the calcified fibrocartilage layer, fibrochondrocytes orchestrate COL1 synthesis, blending it with COL2 to create a transitional matrix. In contrast, the non-calcified fibrocartilage layer is COL2-dominant, with reduced COL1 contribution. Reconstituting this COL1-enriched architecture is pivotal for functional tendon-bone interface regeneration, underscoring the necessity of targeted fibrocartilage induction-a central focus of this study. Embree et al. [29] first directly confirmed the existence of FCSCs on the temporomandibular joint condyle through stem cell lineage tracing, revealing their exceptional chondrogenic potential. S. Zhu et al. [37] further demonstrated FCSCs’ superior chondrogenic differentiation capacity compared to human orofacial MSCs. In our study, BMSCs served as controls. FCSCs not only exhibited significantly higher expression of AGG and COL2 but also outperformed BMSCs in synthesizing COL1, the hallmark component of fibrocartilage (Fig. 2C–E). This dual advantage in matrix synthesis and spheroid formation (Fig. 2F) validates FCSCs’ suitability for biomimetic fibrocartilage regeneration strategies.

The BGBT hydrogel-a dual-network system integrating GelMA, BC, borax and TA-addresses critical limitations of existing carriers. Macroscopically, its robust viscoelasticity and phenolic hydroxyl-mediated adhesion stabilize the tendon-bone gap. Microstructurally, the nanofibrous BC network (20–100 nm) mimics native collagen topology, guiding FCSC alignment and matrix deposition. Degradation kinetics (complete within 2 weeks) temporally align with inflammatory resolution, shielding FCSCs during early healing. Functionally, TA’s polyphenolic moieties confer anti-inflammatory, antibacterial and anti-fibrotic activities, synergizing with FCSCs to suppress Sharpey fiber formation while promoting fibrocartilage regeneration. Our in vitro findings demonstrate the remarkable immunomodulatory capacity of BGBT hydrogel in regulating macrophage polarization. The hydrogel effectively suppresses M1 macrophage differentiation while promoting polarization toward the M2 phenotype. This phenotypic shift holds critical therapeutic implications, as pro-inflammatory M1 macrophages exacerbate inflammatory responses through cytokine secretion, whereas M2 macrophages exhibit anti-inflammatory properties and facilitate tissue repair processes [8]. Consistent with cellular observations, transcriptomic profiling revealed significant enrichment of inflammation-related pathways during early tendon-bone interface reconstruction in BGBT-treated specimens. Mounting evidence associates imbalanced M1/M2 macrophage ratios with delayed tissue regeneration and suboptimal healing outcomes at tendon-bone junctions [[38], [39], [40]]. Our data suggest that BGBT hydrogel accelerates transition to the proliferative healing phase through timely modulation of macrophage phenotypic switching and inflammatory control during critical early stages.

Notably, inflammation constitutes a double-edged sword in tissue repair-while excessive inflammation impedes regeneration, appropriate inflammatory responses remain essential for pathogen clearance and initiating repair cascades. Excessive anti-inflammatory approaches risk oversuppressing this physiological process, potentially compromising wound defense mechanisms. The BGBT hydrogel addresses this therapeutic paradox through innovative incorporation of tannic acid, a polyphenolic compound conferring dual protective effects. Beyond its established anti-inflammatory capacity in regulating cytokine release and macrophage polarization, the hydrogel simultaneously exhibits potent antimicrobial activity. This synergistic design ensures adequate infection control while maintaining appropriate inflammatory balance, thereby creating an optimal microenvironment for accelerated tendon-bone interface healing.

Pro-inflammatory macrophages are critically implicated in fibroblast-mediated fibrotic cascades. Yaozeng Xu et al. demonstrated in a murine tendon-bone healing model that heightened M1 macrophage infiltration correlates with peritendinous fibroblast activation and fibrosis, while inflammatory suppression attenuates fibrotic progression [7]. Although acute inflammation mediated by M1 macrophages plays a role in pathogen clearance and debris removal during early tendon-bone interface healing, their prolonged retention aberrantly stimulates fibroblasts to overproduce ECM components, ultimately driving the formation of biomechanically inferior Sharpey fibers.

Fibroblast-derived ECM primarily comprises collagen isoforms COL1A1 and COL3A1, which exhibit distinct roles in tissue repair. COL1A1 dominates hypertrophic scar tissue and mature Sharpey fibers [41], whereas COL3A1, highly expressed during embryonic scarless healing [42], facilitates provisional tissue stabilization. Early Sharpey-like fibers, characterized by COL3-rich loose fibrous networks, play pivotal roles in initial tendon-bone interface repair, bone growth and dental anchorage [43,44]. However, persistent inflammatory stimuli drive a pathological transition toward disorganized, densely packed COL1-dominated Sharpey fibers [43], highlighting the temporal and compositional specificity of collagen isoforms in healing outcomes. Our data demonstrate that BGBT hydrogel suppresses COL1A1 while upregulating COL3A1 expression in NIH3T3 fibroblasts. This regulatory shift mimics embryonic scarless healing by establishing COL3-based provisional scaffolds that enable cell migration and matrix remodeling, while preventing rigid scar formation from COL1 over-deposition. By preserving spatial flexibility and biochemical compatibility, BGBT creates a permissive niche for fibrocartilage regeneration. We propose that BGBT orchestrates tendon-bone interface restoration through dual mechanisms: upstream regulation, which attenuates M1 macrophage-derived pro-fibrotic signals to disrupt COL1-driven Sharpey fiber overproduction and direct matrix reprogramming, which redirects fibroblast collagen synthesis toward regenerative COL3, thereby recapitulating embryonic-like matrix dynamics.

Excessive inflammation disrupts fibrocartilage regeneration through dual mechanisms: pro-inflammatory cytokines directly inhibit chondrocyte differentiation and matrix synthesis, while matrix metalloproteinase activation accelerates degradation of COL2 and proteoglycans within fibrocartilaginous matrices, critically impairing tissue restoration [45]. Furthermore, sustained inflammatory stimuli induce fibroblast hyperactivation, driving disordered COL1 secretion that spatially competes with and functionally inhibits the production of fibrocartilage-specific matrix components, ultimately forming scar tissue with poor mechanical properties [7]. Early suppression of inflammatory responses is therefore critical to prevent aberrant COL1 deposition in Sharpey fibers and establish a favorable microenvironment for functional fibrocartilage regeneration. The proposed BGBT@FCSC strategy targets early TBI healing intervention. BGBT hydrogel curtails inflammation by modulating macrophage polarization and reducing pro-inflammatory cytokines. Simultaneously, it reprograms Sharpey-fiber collagen deposition and curbs excessive COL1 accumulation, accelerating the transition to the proliferative-remodeling phase. Additionally, scaffold-loaded FCSCs serve as an in situ seed cell reservoir, providing progenitor cells for fibrocartilage regeneration and participating in functional reconstruction of TBI gradient fibrocartilaginous architecture.

Macrophage polarization (M1/M2) serves as a pivotal regulator of this balance. M1 macrophages exacerbate pathological microenvironments by secreting TNF-α and IL-1β, which activate NF-κB signaling to suppress chondrogenic genes (e.g., Sox9, AGG) while recruiting neutrophils and pro-fibrotic cells, amplifying localized inflammation and fibrosis [46,47]. In contrast, M2 macrophages resolve inflammation, protect chondroprogenitor cells and activate Smad/MAPK pathways to drive MSC chondrogenesis, thereby promoting COL2 and proteoglycan synthesis [48]. During later healing phases, M2 dominance facilitates MSC recruitment to support interface proliferation and repair [49]. The BGBT hydrogel synergistically accelerates this transition by dual mechanisms: (1) promoting M2 polarization to expedite resolution of the inflammatory phase, and (2) delivering exogenous FCSCs to directly participate in in situ matrix synthesis, including glycosaminoglycans, COL2 and spatially organized COL1. This integrated strategy-combining anti-inflammatory, antimicrobial, Sharpey fiber-modulating and FCSC-mediated regenerative functions-creates a mutually reinforcing microenvironment that bypasses fibrotic scarring while enabling functional fibrocartilage restoration.

To comprehensively assess therapeutic outcomes at the tendon-bone interface, we performed radiological, biomechanical and histological analyses. Micro-CT imaging revealed significantly enhanced neo-bone formation in both the BGBT and BGBT@FCSC groups compared to controls, indicating that BGBT not only remodels the fibrocartilage microenvironment but also promotes peripheral bone ingrowth. Previous studies have established that neo-ossification at the bony end of the tendon-bone interface strengthens tendon attachment and improves pull-out resistance [50]. The observed bone in growth in our study likely enhances fibrocartilage mineralization, facilitating the establishment of a graded transition zone encompassing bone, calcified fibrocartilage and non-calcified fibrocartilage, thereby accelerating graft integration within the bone tunnel. Biomechanical testing demonstrated that the BGBT@FCSC group achieved the highest maximum pull-out load, attributable to its multi-biomimetic therapeutic strategy that integrates FCSC-derived fibrocartilage regeneration with microenvironmental structural and functional support. In contrast, the FCSC-alone group exhibited mechanical performance comparable to the NC group, likely due to rapid cell loss or macrophage-mediated clearance of unprotected FCSCs during the inflammatory phase. Histological findings corroborated these results: both NC and FCSC groups displayed predominant loose fibrovascular tissue and disorganized Sharpey fibers, explaining their inferior mechanical properties. Notably, compared to the NC control, the BGBT group demonstrated reduced inflammatory infiltrates, more aligned collagen architecture and downregulated expression of COL1A1 (a marker of fibrotic scarring), consistent with the in vitro observation that BGBT suppressed COL1 expression in NIH3T3 cells, thus validating its dual anti-inflammatory and collagen remodeling capabilities in vivo. Critically, at 4 weeks post-surgery, the BGBT@FCSC cohort established an early-stage fibrocartilaginous transition zone characterized by a gradient of calcified and non-calcified fibrocartilage, accompanied by significantly upregulated expression of fibrocartilage-specific markers COL1A1 and COL2A1. This outcome underscores FCSCs compensate for the limited regenerative capacity of resident cells during initial healing, ultimately accelerating reconstruction of the TBI’s gradient architecture.

GO enrichment analysis of DEGs between BGBT-treated and NC groups demonstrated significant associations with biological processes, including inflammatory response and ECM organization. Molecular functions were enriched in signaling receptor binding, while cellular components highlighted integral plasma membrane constituents and extracellular space, collectively implicating BGBT’s dual roles in modulating inflammatory pathways and ECM dynamics-consistent with our in vitro cellular findings. KEGG pathway analysis further revealed DEG enrichment in environmental information processing and signal transduction pathways. Key inflammatory pathways (e.g., TNF-α, NF-κB and Jak-STAT signaling pathway) and osteochondral developmental pathways (e.g., TGF-β, HIF-1 and PI3K-Akt signaling pathway) were prominently represented. Based on combined metrics of p-value, enrichment factor and gene count, the PI3K-Akt signaling pathway emerged as a priority candidate regulatory pathway of tendon-bone interface regeneration.

The critical role of the PI3K signaling pathway in the regenerative healing of the tendon-bone interface has been emphasized. On one hand, the PI3K-AKT signaling pathway is associated with immune regulation. Wang et al. [51] discovered that magnesium alloy materials activate the PI3K/AKT pathway, inducing macrophages to polarize towards the M2 phenotype (characterized by upregulated expression of Arg1), which in turn secretes anti-inflammatory factors (such as IL-10) and inhibits pro-inflammatory factors (such as TNF-α). This mechanism significantly promotes tendon-bone interface healing and reduces local inflammatory responses in rotator cuff injury repair. On the other hand, numerous studies have confirmed that the PI3K/Akt signaling axis and its downstream key molecules (e.g., GSK3β, mTOR) play a central regulatory role in chondrogenic differentiation [52]. TGF-β acts upstream of this axis: by activating the PI3K/Akt pathway, it not only upregulates the expression of SOX9 and COL2A1 but also inhibits the production of matrix metalloproteinase 13 (MMP13). These effects collectively drive chondrocyte differentiation, enhance the anabolism of ECM and alleviate cartilage damage. Therefore, it is hypothesized that the PI3K-Akt signaling pathway serves as the pivotal pathway through which BGBT hydrogel induces gradient fibrocartilage regeneration at the TBI. Given the centrality of fibrocartilage regeneration in tendon-bone healing, we focused on FCSCs to validate BGBT’s PI3K-Akt-mediated effects. The expression results of genes and proteins confirmed that BGBT activates PI3K signaling, subsequently upregulating AGG, COL2A1 and COL1A1 expression-key drivers of fibrocartilage matrix synthesis. This links PI3K pathway activation to FCSC-driven ECM remodeling, promoting fibrocartilage regeneration.

This study has several limitations that merit consideration. First, to exclude mechanical variables, a tibia–tendon model with a load profile distinct from clinical ACL or rotator-cuff injuries was employed [53]. Second, animal experiments only included tissue harvests up to 4 weeks postoperatively-a timeframe insufficient to evaluate long-term fibrocartilage maturation and mechanical durability. These outcomes are critical for clinical translation. Third, the work primarily focused on fibrocartilage formation as the core therapeutic target, with limited attention to other cell types critical for the TBI’s “tendon-fibrocartilage-bone” gradient transition (e.g., osteoblasts mediating bone-fibrocartilage integration, tenocytes maintaining tendon structural integrity). This narrow focus restricts understanding of how BGBT@FCSC regulates the full TBI enthesis, as successful repair depends on coordinated crosstalk between these cell populations. These limitations underscore the need for long-term and high-load animal studies to assess fibrocartilage maturation and mechanical durability, alongside investigations into multi-cell type interactions, to fully validate BGBT@FCSC’s translational potential.

4. Conclusion

In summary, this study presents a novel biomimetic strategy for TBI repair by integrating a BGBT hydrogel scaffold with FCSCs. Unlike conventional hydrogel or stem cell therapies, this scaffold not only provides structural support but also activates the PI3K signaling pathway to drive FCSC differentiation, while simultaneously modulating fibroblast-mediated Sharpey-like fiber deposition and macrophage polarization. These synergistic actions ameliorate the pathological microenvironment and, together with FCSCs, achieve functional TBI regeneration, offering promising potential for enhancing graft integration in ligament reconstruction or rotator-cuff repair. By transcending conventional single-level biomimicry, this work establishes a “structural adaptation-functional activation-biological self-organization” continuum, offering a holistic solution that bridges mechanical reinforcement, biochemical signaling and cellular self-organization. This paradigm shift advances TBI regeneration from isolated structural repair to functionally coordinated tissue restoration.

CRediT authorship contribution statement

Zhaoquan Liang: Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Qiang Xiao: Methodology, Investigation, Conceptualization. Yuelin Wu: Software, Methodology, Data curation. Da Song: Methodology, Formal analysis, Data curation. Yucong Li: Methodology. Jingle Chen: Validation. Qili Sun: Investigation. Zhenyu Yang: Methodology. Tao Peng: Methodology. Yeyang Wang: Methodology, Conceptualization. Chao Xie: Writing – review & editing, Supervision, Funding acquisition, Formal analysis, Conceptualization. Li Zhang: Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used CHATGPT3.5 in order to check grammar and polish the English language quality. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

Declaration of competing interest

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

Appendix A. Supplementary data

The following is the supplementary data to this article:

Data availability

Data will be made available on request.