Bone marrow mesenchymal stem cells overexpressing FGF-2 loaded onto a decellularized extracellular matrix hydrogel for the treatment of osteoarthritis

Abstract

Osteoarthritis, as one of the major disabling diseases in the elderly, has a long-term impact on patients’ quality of life and brings heavy medical and social burden. The pathogenesis of osteoarthritis is still unclear, and the main pathological changes include chondrocyte death and osteochondral damage. Therefore, how to solve the cartilage damage caused by osteoarthritis has become the key and difficult point in the clinical treatment of osteoarthritis. Bone marrow mesenchymal stem cells (MSCs) have the potential for self-renewal and multidirectional differentiation, and their engineering has been a hot research topic for the treatment of cartilage damage in recent years. In this study, an injectable hydrogel with stable and continuous release of growth factors was successfully prepared by modifying bone marrow mesenchymal stem cells to overexpress fibroblast growth factor-2 (FGF-2) and piggybacking on a decellularized extracellular matrix (dECM) hydrogel for the repair of cartilage injury in osteoarthritis. This hydrogel demonstrated excellent biocompatibility both in vitro and in vivo. In 3D cell culture, BMSCs in the dECM hydrogel survived, proliferated, and produced abundant cartilage-specific extracellular matrix and growth factors, promoting BMSC differentiation into hyaline chondrocytes. In vitro and in vivo experiments, along with RNA-seq analysis, showed that engineered BMSCs loaded onto the dECM hydrogel could inhibit chondrocyte apoptosis and boost BMSC differentiation. In summary, dECM hydrogels carrying FGF-2 overexpressing bone marrow mesenchymal stem cells have great prospects in accelerating osteochondral defect repair and delaying the progression of osteoarthritis.

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1. Introduction

Osteoarthritis (OA), a prevalent musculoskeletal disorder, has a significant correlation with the aging process. The cartilage of the knee is the most frequently damaged area, and the pathology is characterized by degeneration, sclerosis, neovascularization and synovitis of the articular cartilage. Disease progression causes joint stiffness, pain, and even loss of motor function—ranking OA as the top disabling musculoskeletal disorder. Long-term impacts include reduced patients’ quality of life, which not only increases personal burden but also imposes heavy pressure on society.^1,2 Articular cartilage lacks blood vessels, nerves, and lymphatics, resulting in very limited self-healing and regeneration after damage.³ Currently, most treatments for such injuries produce fibrocartilage (instead of functional hyaline cartilage) at the defect site. Although these treatments provide pain relief and spawn cartilage-like tissue, fibrocartilage is far inferior to normal hyaline cartilage in terms of mechanical properties and durability, and the long-term therapeutic effects remain unsatisfactory.⁴ Therefore, how to solve the cartilage damage caused by OA has become the key and difficult point in treating osteoarthritis in the clinic.

In the past few years, the use of stem cell-based therapeutic approaches has garnered substantial interest and has been increasingly implemented in the area of OA injury regeneration.⁵ Mesenchymal stem cells (MSCs) represent a class of stem cells endowed with the capacities of self-renewal and multidirectional differentiation. These cells can be obtained from diverse tissue sources and possess stable biological characteristics. It has a wide range of sources, is easy to isolate and culture, and possesses functions such as multipotent differentiation, tissue repair, anti-inflammatory activity, and immunomodulatory effects. Recent studies have shown that MSCs regulate the local microenvironment of damaged cartilage via inherent anti-inflammatory and immunomodulatory functions and secretion of diverse bioactive factors, thus protecting cartilage from further damage.⁶ Among these, bone marrow mesenchymal stem cells (BMSCs) are the most widely used types of MSCs. Extracted by bone marrow aspiration, BMSCs have little cellular damage, and they are fast-proliferating and genetically stable and can easily expand in vitro. Several clinical trials have demonstrated their effectiveness in promoting articular cartilage regeneration.^7–9 BMSCs serve as optimal seed cells for OA cell therapy. This is attributed to their characteristics of multidirectional differentiation potential, low immunogenicity, and high portability.

Despite the great potential of BMSCs as seed cells for the treatment of cartilage damage in osteoarthritis, direct cell injection often faces challenges such as insufficient cell viability and low survival rates. Ideal biomaterials should have low cytotoxicity, suitable biodegradability, and outstanding biocompatibility in order to create a favorable milieu for stem cells.¹⁰ Extracellular matrix (ECM) decellularized bioscaffolds, as ideal carriers, not only have good biocompatibility and low cytotoxicity but also have been widely used in the treatment of osteoarthritis, lumbar disc degeneration and other diseases.^11–13 Moreover, decellularization of the extracellular matrix not only reduces its immunogenicity but also preserves its bioactive components, effectively avoiding the induction of immune responses in the host.^14,15 The decellularized extracellular matrix (dECM) represents a highly promising cellular scaffold. Dwikora et al. demonstrated that decellularized bovine cartilage scaffold sponges could enhance the adhesion, proliferation, and chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (BMSCs) even in the absence of any external inducing stimuli.¹⁶ Furthermore, it has been demonstrated that scaffolds made from the decellularized porcine cartilage matrix, when loaded with autologous chondrocytes, are capable of triggering the generation of new cartilage.¹⁷ Although dECM is therapeutically effective, it is usually made in membrane or powder form, which largely limits its application in vitro and in vivo. Recent studies have shown that the biological activity of the extracellular matrix of dECM materials is not affected when they are made into a hydrogel state.¹⁵ In addition, dECM hydrogels can be injected as a gel-adhesive liquid and can fit perfectly in the area of cartilage defects. Based on these advantages, we finally selected dECM hydrogel as a biomaterial for cartilage implantation.

Cell growth factors play a wide range of roles in bone injury repair and are hot spots in cartilage tissue engineering research. During the induction of cartilage-directed differentiation, appropriate stimulatory factors are necessary. Several cytokines, including insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor 2 (FGF-2), transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF), have been shown to either trigger or facilitate chondrocyte differentiation. FGF-2 is a polypeptide consisting of 155 amino acids. It exhibits extensive mitogenic activities and participates in a diverse array of biological processes, such as embryonic growth and development, cell division and differentiation, tissue repair, and tumor growth and invasion.^18,19 Moreover, FGF-2 is ubiquitously distributed within the body and possesses the ability to promote cell proliferation. It plays a crucial role in tendon-to-bone healing, cartilage repair, bone repair, and nerve regeneration.²⁰ It has been shown that FGF-2 induces up-regulation of bone morphogenetic protein-3 (BMP-3) in a rabbit joint model of cartilage injury, which in turn leads to favorable cartilage repair.²¹ Not only that, but the mechanism by which FGF-2 stimulates chondrocyte proliferation has a strong correlation with Sox9, which promotes the production of glycosaminoglycans (GAGs) as a way to protect cartilage and promote cartilage repair.²² Meanwhile, FGF-2 also promotes the proliferation of bone marrow MSCs and induces their differentiation towards cartilage by activating pathways such as FGF/FGFR2, P13K-Akt, and ERK l/2.²³ However, the therapeutic efficacy of genetically engineered modified BMSCs overexpressing FGF-2 coupled with injectable dECM hydrogels for OA treatment has not been reported.

In this study, we successfully loaded engineered BMSCs onto dECM hydrogels to give BMSCs overexpressing FGF-2 (F-BMSCs) a suitable microenvironment for value-addition and differentiation, while FGF-2 in turn promotes the value-addition of bone marrow mesenchymal stem cells and their differentiation into cartilage to enhance the therapeutic effect on OA. Rheometry and scanning electron microscopy (SEM) were used to analyze the hydrogels’ mechanical characteristics and microstructure. Next, in vitro two- and three-dimensional cell cultures were performed to evaluate the cytocompatibility of the hydrogels. BMSCs were transfected to overexpress FGF-2 protein using a lentivirus encoding FGF-2. The pro-chondrogenic differentiation and anti-apoptotic abilities of F-BMSCs were evaluated in a co-culture system. Finally, the therapeutic efficacy of the developed combination therapy was investigated in vivo by micro-computed tomography (micro-CT) and histological analysis. In conclusion, this study confirmed the positive phase effect of dECM hydrogel-coupled F-BMSCs on cartilage repair in OA at four levels—tissue engineering, animal tissue specimens, in vitro cellular studies, and animal model experiments—and further elucidated the mechanism of delaying OA progression, emphasizing the good injectability of dECM hydrogel-coupled F-BMSCs and their great potential to promote cartilage repair. It provides new ideas for the clinical treatment of OA.

2. Materials and methods

2.1 Porcine cartilage decellularization treatment and histological and biochemical evaluation

2.1.1 Porcine cartilage decellularization. Fresh knee cartilage from 6–7-month-old pigs was cut into 5–10 mm³ pieces, alternately rinsed with PBS and 75% ethanol, and subjected to repeated freeze–thaw cycles (brief liquid nitrogen freezing followed by 37 °C water bath reheating). The treated cartilage was placed in sterile ultrapure water in a sealed container and shaken to dislodge cleaved chondrocytes. After discarding the water, 1% Triton X-100 solution was added for shaking. Following thorough rinsing, the cartilage was incubated in DMEM high glucose medium (10% FBS, 1% penicillin–streptomycin) at 37 °C with 5% CO₂. The medium was discarded, and the tissue was shaken in PBS. The remaining chondrocytes were removed via further freeze-thawing. The dECM was lyophilized, ground into powder, and stored in a sealed container at −80 °C.^24,25

2.1.2 Histological and biochemical evaluations. HE staining and Masson staining were used to observe the decellularization of cartilage tissues. The DNA content of the decellularized cartilage tissues was detected using a Quant-iT™ PicoGreen® dsDNA kit (Invitrogen, P7589, China) to characterize the degree of decellularization. A Hydroxyproline Assay Kit (Sigma, MAK008, China) was used to detect the collagen content. A Dimethylmethylene Blue (DMMB) Colorimetric Assay Kit (HL, HL19239.2, Shanghai) was used to detect the glycosaminoglycan (GAG) content of decellularized tissues and assess the extent of cartilage tissue damage to the extracellular matrix (ECM) during the decellularization process.

2.2 dECM hydrogel preparation and characterization

2.2.1 Synthesis of dECM hydrogels. First, pepsin was dissolved with 0.1 M hydrochloric acid, which was used to dissolve dECM after adjusting its concentration to 1 mg mL⁻¹, mixed thoroughly and placed on a shaker for 48 hours at room temperature for digestion. After complete digestion, the solution was manipulated on ice by adding 1 M NaOH to adjust the solution to neutral pH (7.0), followed by placing it at 37 °C for 1 h to obtain the gel, which was then diluted to 0.3 mg mL⁻¹, 3 mg mL⁻¹ and 10 mg mL⁻¹ concentrations of the dECM hydrogel and set aside.

2.2.2 Characterization of the dECM hydrogels. A scanning electron microscope (SEM; EHT = 3 kV; aperture size = 30 μm, Carl Zeiss AG, Germany) was used to view the scaffolds. A rheometer (Haake Mars 40, Germany) was used to assess the dECM hydrogels’ rheological characteristics. All experiments employed a plate–plate geometry with a 40 mm diameter and a 1 mm plate–plate spacing. For one hour, the dECM droplets were left on the plates at room temperature. Using amplitude testing at a fixed frequency, frequency testing at a fixed strain, and time scanning at a fixed strain and frequency, the energy storage modulus (G′) and loss modulus (G′′) were determined as functions of amplitude, frequency, and time, respectively.

2.3 Isolation and culture of BMSCs and chondrocytes

Bone marrow MSCs were isolated from the bone marrow of suckling SD rats for culture as previously described.²⁶ Briefly, the femoral stem and tibia of suckling SD rats were aseptically separated, with surface tissues stripped clean; the bone ends were cut to expose the marrow cavity, which was then repeatedly rinsed with PBS until grayish white. The rinsate was centrifuged, and the precipitate was resuspended in complete medium (MEM-alpha, Gibco; 10% FBS, Gibco; 1% penicillin–streptomycin) and seeded into cell culture flasks. Cells were cultured at 37 °C with 5% CO₂, with the medium changed every 2–3 days, and used for subsequent experiments after reaching the second passage. Chondrocytes were isolated and cultured as previously described.²⁷

2.4 BMSCs’ fine remodeling with overexpression of FGF-2

The pHBLV-EF1-FGF-2-CMV-ZsGreen-T2A-Puro vector (HANBIO Shanghai) was constructed by inserting FGF-2 and enhanced green fluorescent protein genes into the pHBLV-EF1-MCS-CMV-ZsGreen-T2A-Puro plasmid. This lentiviral shuttle plasmid, along with the psPAX2 and pMD2.G vectors (triple plasmid system) and Lipofectamine 3000 (Thermo Fisher), was used to transfect 293T cells; after viral transcription, translation, and assembly, the overexpression lentivirus (LV-op-FGF-2) was generated, and its titer was measured via dilution counting. Second-generation BMSCs in good condition were seeded into 6-well plates (30%–50% confluence) and transfected with LV-op-FGF-2 at 37 °C with 5% CO₂ overnight. The next day, half the volume of fresh medium plus the virus (per instructions) was added; the remaining medium was supplemented 4 hours later, and again after another 4 hours. After 24 hours of infection, the virus-containing medium was replaced, and cells were cultured for another 24 hours. The transfection efficiency was checked via fluorescence microscopy, and FGF-2 gene/protein expression in BMSCs was detected by western blotting (WB) and quantitative PCR (qPCR). The cytocompatibility of dECM hydrogels with BMSCs was assessed via co-culture (0.3 mg ml⁻¹ and 3 mg ml⁻¹ hydrogels) using live/dead staining (Yeasen, 40747ES76) and CCK-8 (Beyotime, C0038). BMSCs were seeded into 6-well plates (6 × 10⁴ per well) into three groups (control, 0.3 mg ml⁻¹ dECM, 3 mg ml⁻¹ dECM). After cell adherence, 100 μl of hydrogel (per concentration) was added, and live/dead staining was imaged via fluorescence microscopy (Zeiss) on days 1, 3, and 7. For CCK-8, BMSCs were seeded into 96-well plates (5 × 10³ per well), with viability tested at predetermined time points per kit instructions.

2.5 Identification of the migration ability of BMSCs induced by dECM hydrogels

We established three experimental groups: the control group, the LPS-treated group, and the LPS + dECM hydrogel group. A Transwell apparatus (BIOFIL, TCS003024), which was a 24-well plate featuring 8 μm pores, was employed for this experiment. In the control group, solely 500 μl of MEM-α complete medium was placed into the lower compartments of the Transwell. In contrast, for the LPS-treated group, 500 μl of MEM-α complete medium blended with an LPS inducer was added to the lower wells. As for the LPS + dECM hydrogel group, around 50 μL of 3 mg ml⁻¹ dECM hydrogel was incorporated into the lower wells along with the medium. Following this, 100 μL of a cell suspension containing 1 × 10⁴ BMSCs was introduced into the upper wells of the Transwell. The plates were then incubated at 37 °C in an environment with 5% CO₂ enrichment. After incubation, the leftover medium was carefully collected at 24, 48, and 72 hours and then washed three times with 1× PBS. Following a 15-minute fixation period with 4% paraformaldehyde, the cells were stained with a 1% crystal violet solution for an additional 15 minutes at room temperature. After three additional washes with PBS and air-drying, the Transwell inserts were flipped over onto glass slides. Photographs were taken using an Olympus microscope (Japan) to count and record the number of BMSCs passing through the membrane.

2.6 Identification of the chondrogenic differentiation capacity of BMSCs overexpressing FGF-2

2.6.1 Toluidine blue staining. Group interventions were performed, with group 1 receiving BMSCs-only (control), group 2 F-BMSCs, and group 3 F-BMSCs + dECM. Toluidine blue staining was performed to identify cartilage in combination with cell morphology (cells appeared as purplish-red heterostained granules with small, rounded nuclei in blue color). Cells were aspirated from the medium in 6-well plates, washed twice using 1× PBS for 1 min each time, and then stained by adding toluidine blue staining solution (Solarbio, G3660, China) 500 μl per well for 5 min. To homogenize the staining solution, an equal amount of distilled water was added, gently shaken, and allowed to stand for fifteen minutes, and then washed finally twice using distilled water for 30 s each time. An appropriate amount of distilled water was added to completely submerge and subsequently observed using a microscope (Zeiss, Germany).

2.6.2 Safranin-O staining. Safranin-O staining was used to detect chondrocyte GAG production. Chondrocytes were inoculated in six-well plates (n = 6 × 10⁴) and divided into the blank group, OA control group, 3 mg ml⁻¹ dECM group and dECM + F-BMSC group. After overnight incubation of the cells, the appropriate drugs were added to each group for intervention. Safranin-O staining was performed after 24 and 72 h of co-culture. After discarding the medium, the cells were washed 3 times with 1× PBS in six-well plates, fixed by adding 4% paraformaldehyde (Biosharp, China) for 15 min, discarding the paraformaldehyde, washed 3 times using 1× PBS, and then stained for 8 min with Safranin-O staining workup, discarded the staining solution, rinsed three times with 1× PBS, and finally examined under a microscope (Zeiss, Germany).

2.6.3 QRT-PCR assay. The cartilage-related genes Col2a1, Col1a1, Aggrecan and Sox9 were measured for mRNA expression levels using QRT-PCR based on the previously indicated groupings. Total cellular RNA was isolated using an RNAeasy™ Plus Animal RNA Isolation Kit (Spin Column, Beyotime, R0027, China). One microgram of the extracted RNA was then reverse-transcribed into complementary DNA (cDNA) using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, China). The amplification reaction system's total volume was set at 10 μL. This reaction combination included 2.5 μL of cDNA, 1.7 μL of nuclease-free water, 0.4 μL of primers, and 5 μL of SYBR Premix Ex Taq Mix (Thermo Fisher Scientific, USA). By utilizing the 2^−ΔΔCt method to normalize the control group and then comparing it with the other experimental groups, the expression levels of the target mRNA were examined (the experiment was repeated three times).

2.6.4 Western blotting. The expression levels of a series of cartilage characteristic indicator proteins, such as Sox-9, Aggrecan, Collagen-I, Collagen-II, etc., were detected by western blotting. Cell culture was performed using the previously mentioned groupings. RIPA lysis buffer (Beyotime, China) was used to extract cellular proteins on ice. A BCA Protein Assay Kit was used to measure the protein content of each sample. Subsequently, SDS-PAGE gels were prepared for electrophoresis using a PAGE Gel Rapid Preparation Kit (EpiZyme, PG111). After the electrophoresis was finished, the proteins were transferred onto nitrocellulose membranes (Beyotime, FFN08, China) with a pore size of 0.22 nm. A protein-free quick blocking buffer (EpiZyme, PS108P) was used to block the nitrocellulose blotting membranes for half an hour at room temperature. Then, the membranes were washed three times with 1× TBS/Tween buffer (TBST, EpiZyme, PS103S). Next, primary antibodies diluted with a universal antibody diluent (NCM, HYcezmbio, HYC00833) were added. The main antibodies were Aggrecan (SAB, 45068, diluted at 1 : 1000), Collagen-II (Proteintech, 28459-1-AP, diluted at 1 : 1000), Collagen-I (Proteintech, 14695-1-AP, diluted at 1 : 1000), and Sox9 (Abcam, ab185966, diluted at 1 : 1000). After that, the membranes were incubated at 4 °C for the entire night. The next day, the secondary antibody (Thermo Fisher Scientific) was incubated for 1 h. Finally, the membranes were analyzed by enhanced chemiluminescence detection (the experiment was repeated three times).

2.6.5 Immunofluorescence (IF) staining. The expression levels of Collagen-I and Collagen-II were detected by immunofluorescence staining. Cell culture was carried out on 24-well plates according to the above groups, fixed for 15 minutes with 4% paraformaldehyde, washed three times with 1× PBS for 5 minutes each, and permeabilized for 3 minutes using a prepared 0.2% Triton X-100 solution. After blocking with 3% bovine serum albumin for 2 h at room temperature, 300 μl per well of diluted primary antibodies, Collagen II (1 : 600, Proteintech, China) and Collagen I (1 : 30 00, Proteintech, China) were used for an overnight incubation at 4 °C. The next day, the cells were washed three times with 1× PBS, treated for two hours in a dark environment with fluorescent secondary antibodies (1 : 20 00, Abcam, ab150077), stained for ten minutes with DAPI, and examined for cell crawling behavior using a research-grade fluorescence microscope (Zeiss, Germany).

2.7 Effect of cartilage dECM hydrogels loaded with F-BMSCs on chondrocyte proliferation in SD rats

Second-generation SD mammary rat chondrocytes were inoculated into 24-well plates at a cell density of 1 × 10⁴ per well. The chondrocytes were then divided into four groups: group 1 was the chondrocyte-only control group; group 2 was the LPS inflammation model group; group 3 was the LPS + dECM hydrogel group; and group 4 was the LPS + dECM hydrogel loaded with F-BMSC group. The groups were then given the interventions, and an EdU cell proliferation assay kit (Beyotime, C0078S, China) was used to assess the impact of chondrocyte proliferation on in SD mammary rats as directed. 24-well plates were incubated for 48 hours. Each well received 10 μM EdU marker, and the wells were co-cultured for three hours at 37 °C with 5% CO₂. After that, cells were permeabilized for 10 minutes with 0.3% Triton X-100 in PBS, washed three times with 1× PBS, and fixed for 15 minutes at room temperature with 4% paraformaldehyde. Afterwards, the cells were co-incubated with an anti-EdU working solution for 15 minutes. Then, at room temperature and protected from light, the cell nuclei were stained with a 1× Hoechst 33342 solution for 10 minutes. A fluorescence microscope (Zeiss, Germany) was utilized to observe and record the quantity of positive cells. The growth and apoptosis of chondrocytes were determined through live/dead staining and safranin staining.

2.8 Assessment of the effect of cartilage dECM hydrogels loaded with F-BMSCs on chondrocyte apoptosis

According to the grouping: group 1 was the pure chondrocyte control group; group 2 was the LPS inflammation model group; and group 3 was the LPS + dECM hydrogel + F-BMSC group. After the grouping intervention of chondrocytes in suckling SD mice, chondrocyte apoptosis was detected using flow cytometry, and to assess the impact of the hydrogels on chondrocyte apoptosis, the same procedures were carried out to measure the protein and mRNA expression levels of apoptosis-related indexes (Bcl-2, Bax, Caspase-3).

2.9 mRNA sequencing probes the mechanism of dECM hydrogels piggybacking F-BMSCs to promote OA cartilage damage repair

The chondrocytes were divided into three groups for intervention, in which group 1 was the normal chondrocyte group, group 2 was the OA group, and group 3 was the dECM + F-BMSC treatment group. RNA sequencing was performed on the intervened chondrocytes to understand the mRNA expression profiles of each experimental group, so as to further explore the potential mechanism of the composite hydrogel to promote cartilage regeneration in OA. Following the normal extraction procedure, RNA was extracted from the cells using an RNAeasy™ Plus Animal RNA Isolation Kit with a Spin Column (Beyotime, R0027, China). An Agilent 2100 Bioanalyzer (Agilent 2100, Germany) was then used to strictly control the quality of the RNA samples. After the quality of the library was assessed, RNA sequencing was performed. Finally, the sequence information about the fragments to be sequenced was obtained (Fig. S1). After sequencing, RStudio (R 4.3.1) was used to analyze the sequencing results and identify differentially expressed genes (DEGs). The R software package (clusterProfiler) was employed for Gene Ontology (GO) biological function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA). The comprehensive analysis was designed to unravel the latent biological functions and regulatory pathways related to the genes. This will contribute to a deeper understanding of the therapeutic mechanisms of complex hydrogels.

2.10 In vivo animal experiments to evaluate the ability of dECM hydrogels loaded with F-BMSCs for cartilage damage repair in osteoarthritis

The Guangxi Medical University Animal Care and Use Committee approved the procedures for animal care and experiments (Ethical Approval No. 2024-E646-01). Twenty-four male Sprague–Dawley (SD) rats weighing between 220 and 250 g and 7 to 8 weeks of age were chosen and obtained from Guangxi Medical University's Animal Experiment Center.

Twenty-four rats were randomly assigned to 4 groups (n = 6 per group) via double-blinded randomization: blank (sham surgery), OA control, OA + dECM hydrogel, and OA + dECM + F-BMSC groups. After 6 weeks of OA modeling, the OA control, OA + dECM hydrogel, and OA + dECM + F-BMSC groups received weekly knee injections of PBS, dECM hydrogel, and dECM hydrogel + F-BMSCs, respectively, for 12 weeks. Animal experiments were performed by other members of our team to ensure blinding. We performed OA modeling in SD rats according to the method described in the literature,¹⁴ and after anesthesia, the right hind limb was prepared and sterilized, and a longitudinal incision of approximately 3 cm was made from the partial medial aspect of the knee joint. The infrapatellar plica attached to the intercondylar fossa was cut to visualize the ACL beneath it. The ACL was exposed and the ACL was cut with microscopic scissors close to the femur. The knee drawer test was performed to confirm the complete dissociation of the ACL, the wound was sterilized, and the wound was closed layer by layer. Following eight weeks of therapy, the rats were euthanized by intraperitoneal injection under overdose anesthesia (150 mg kg^–1 sodium pentobarbital), and each group's knee joints were taken. The Osteoarthritis Research Society International (OARSI) score, H&E (Solarbio, China) staining, and Safranin-O solid green (Solarbio, China) staining were used to evaluate the degree of osteoarthritis and the healing of cartilage injury in the rats in each group. Immunohistochemistry was used to detect the expression of cartilage-related proteins and apoptosis indicators (Col-2, Aggrecan, Bax, Casp3) in each group. TUNEL staining was used to further assess the apoptosis in each group. In addition, micro-CT was used to macroscopically evaluate the osteochondral formation and repair of rat knee joints.

2.11 Statistical analysis

The mean ± standard deviation is used to present all of the experiment data. We used SPSS 64.0 and GraphPad Prism 8 for statistical analysis. A P-value below 0.05 was considered statistically significant. ANOVA, or one-way analysis of variance, was used to find significant differences across several groups. To compare two sets of data, a t-test was carried out. Regarding the RNA sequencing data, RStudio was used to conduct differential gene expression (DGE) analysis, GO biological function analysis, KEGG pathway enrichment analysis, and GSEA.

3. Results

3.1 Preparation and characterization of dECM hydrogels

The steps of the present study were unfolded in accordance with Fig. 1, and we successfully prepared dECM hydrogels by the method described above. HE and Masson staining showed that dECM had fewer nuclei/cells but relatively intact ECM structure compared to normal cartilage (Fig. 2a and b). This reduction in cellular components was confirmed by DNA quantification: dECM had a drastically lower DNA content (10.91 ng mg⁻¹) than normal cartilage (60.86 ng mg⁻¹), nearly reaching undetectable levels (Fig. 2c). There was a substantial difference (P < 0.05) when compared with normal cartilage. To ensure that the extracellular collagen matrix of the cartilage was not harmed by the decellularization process, we measured the amounts of collagen and GAG in the cartilage tissues. The results indicated that there was not a significant difference in the collagen and GAG contents between the decellularized cartilage and normal cartilage tissues (Fig. 2d and e). This suggests that the cartilage ECM structure was not seriously harmed during the decellularization process. The extracted dECM was lyophilized using a freeze-dryer. It was then ground into a powder using a grinder and stored in a −80 °C refrigerator for further studies.

Fig. 1 Schematic diagram of this study.

Fig. 2 Preparation and physicochemical characterization of dECM hydrogels. (a) HE staining (scale bar: 100 μm/50 μm). (b) Masson staining (scale bar: 100 μm/50 μm). Quantification of (c) DNA content (n = 3), (d) GAG content (n = 3), and (e) collagen content (n = 3). (f) The macroscopic morphologies of dECM hydrogels at different concentrations: ①, ② and ③> represent 0.3 mg ml⁻¹, 3 mg ml⁻¹ and 10 mg ml⁻¹ dECM hydrogels respectively. (g) Internal microstructures of the dECM hydrogels at three concentrations under scanning electron microscopy (n = 3). (h) Rheological test: frequency test of fixed strain (stress). (i) Rheological testing: time scanning with fixed strain and frequency. (j) Rheological test: amplitude test at fixed frequency. Data are expressed as mean ± SD, #: P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05, ns: no significance.

After determining the success of decellularization, we prepared dECM at three concentrations, 0.3 mg ml⁻¹, 3 mg ml⁻¹, and 10 mg ml⁻¹, respectively (Fig. 2f). SEM showed the internal microstructures of the dECM hydrogels with different concentrations, and the dECM hydrogel with a concentration of 3 mg ml⁻¹ exhibited more porous structures, which was believed to be favorable for cell infiltration and proliferation (Fig. 2g). Subsequently, we selected 3 mg ml⁻¹ of dECM hydrogel for rheological testing. As can be seen in Fig. 2h, there is a small difference between the energy storage modulus (G′) and loss modulus (G′′), which indicates that the dECM hydrogel has better dynamic properties such as injectability and self-healing. In the time–stress curves, we applied the same deformation to the dECM hydrogel with a jig, under which the G′ and G′′ of the hydrogel were characterized. As can be seen in Fig. 2i, the energy storage modulus and loss modulus of the 3 mg ml⁻¹ dECM hydrogel are relatively stable under a certain pressure, and do not change with the extension of time, which indicates that the internal structure of the hydrogel is relatively homogeneous and stable. Subsequently, we characterized the stress–strain behavior of the dECM hydrogels. The rheometer will apply a horizontal shear force to the hydrogel between the fixtures, and then produce a certain deformation of the hydrogel itself, and with the extension of time, the deformation produced by the rheometer gradually increases. In this process, the rheometer evaluates the hydrogel's resistance to horizontal shear by measuring its storage modulus and loss modulus. The horizontal deformation produced by the rheometer in the initial state is small, and the energy storage modulus and loss modulus of the 3 mg ml⁻¹ dECM hydrogel are similar to those of the hydrogels in the corresponding groups of the time–stress curves, which indicates that the hydrogel can still maintain the stability of its internal structure under lower horizontal strains (Fig. 2j). The energy storage modulus of the hydrogel shows a decreasing trend with increasing stress, and the loss modulus also increases with increasing horizontal deformation. This indicates that the internal cross-linking structure of the dECM hydrogel breaks during the gradual increase of horizontal deformation, and when the energy storage modulus intersects with the loss modulus curve, the structure of the hydrogel itself may have been damaged, and then fracture occurs. From Fig. 2j, we can still see that although the relative hydrogel structure is damaged at large horizontal deformations, the cartilage tissue is less exposed to horizontal shear forces from the contralateral cartilage tissue and more exposed to direct or indirect vertical forces from the contralateral joints, so that the hydrogel tissue can still maintain a relatively stable internal structure at low horizontal deformations, which is also in line with the need for the experimental investigation of the mechanical environment suitable for the regeneration of cartilage.

3.2 Cytocompatibility assay of dECM hydrogels

Second- or third-generation BMSCs were used for two-dimensional cell co-culture with dECM hydrogels, and the cytocompatibility of the dECM hydrogels was assessed by live/dead staining, CCK-8 assay, and inflammatory index assay. The cell survival of BMSCs within the hydrogels was assessed by fluorescence microscopy on days 1, 3 and 7 of culture (Fig. 3a). Live/dead staining indicated that the number of consistently stocked cells exceeded 95% in all groups during cell culture, with the group at a concentration of 3 mg mL⁻¹ showing the most favorable results (Fig. 3b). This demonstrated the significant pro-proliferative effect of the dECM hydrogel co-culture.

Fig. 3 Study on the biocompatibility of dECM hydrogels. (a) Live/dead staining of 0.3 mg ml⁻¹ and 3 mg ml⁻¹ dECM hydrogels co-cultured with chondrocytes for 1 day, 3 days, and 7 days: green for live chondrocytes and red for dead chondrocytes (scale bar = 100 μm). (b) Determination of cell viability after live/dead staining. (c) The cell viability of chondrocytes co-cultured with dECM hydrogels at various concentrations was measured by CCK-8. (d–f) The mRNA expression of matrix metalloproteinase 13 (MMP13) and inflammatory factors (IL-6, IL-1β) after co-culture of the dECM hydrogel with chondrocytes was detected by RT-PCR, and the involvement of the dECM hydrogels in inducing inflammatory responses of chondrocytes was analyzed (n = 3).

The influence of dECM hydrogels on the proliferation of BMSCs under two-dimensional (2D) culture conditions was further investigated using the CCK-8 assay. Cells were grouped into the blank group, the 0.3 mg ml⁻¹ dECM group, and the 3 mg ml⁻¹ dECM group. CCK-8 assays were carried out on days 1, 3, and 7 of culture. The outcomes indicated that when BMSCs were cultured in 2D with the 3 mg ml⁻¹ dECM hydrogel for 7 days, the cells proliferated remarkably and exhibited the highest survival rate. We further measured the vitality of BMSCs based on the results of the CCK-8 assay. It was demonstrated that when the cells were cultured for up to 7 days, the cell viability in the 3 mg ml⁻¹ hydrogel group significantly exceeded 100% (Fig. 3c). This once again confirmed that the dECM hydrogel possessed excellent biocompatibility. Therefore, we ultimately selected the 3 mg ml⁻¹ dECM hydrogel for subsequent experimental investigations.

Subsequently, to determine whether the dECM hydrogel had an inflammation-inducing effect on chondrocytes, we employed QRT-PCR to measure the levels of inflammatory mediators (MMP 13, IL-1β, IL-6). We established three groups: a control group, an LPS-induced inflammatory model group, and a dECM hydrogel group. The results showed that the LPS group had the highest mRNA expression of inflammatory mediators and the most significant inflammatory response. On the other hand, the dECM hydrogel group's performance was comparable to that of the control group. Compared to the LPS group, the control and dECM hydrogel groups displayed noticeably decreased expression levels (Fig. 3d–f). All of these results indicated that the dECM hydrogel did not cause an inflammatory reaction in chondrocytes and could be utilized safely in future investigations.

3.3 Bone marrow mesenchymal stem cell transformation by overexpressing FGF-2

In order to engineer BMSCs overexpressing FGF-2, we transfected second-generation BMSCs with a lentivirus overexpressing FGF-2, observed the transfection efficiency under a fluorescence microscope, and detected the expression of the FGF-2 gene and protein in BMSCs by quantitative PCR and WB technology. The transfection rate peaked at 72 h post-transfection with the strongest fluorescence intensity (Fig. S2a). We divided the cells into three groups according to the value of the multiplicity of infection (MOI = 10, MOI = 30 and MOI = 50, respectively) for the determination of the optimal MOI value. The results showed that the lentivirus infection was most efficient when the MOI value was 50, so we finally chose the MOI value of 50 for subsequent experiments. In order to test whether the transfected BMSCs could be enough to persistently express FGF-2, we performed quantitative PCR and WB to detect the mRNA and protein expression of FGF-2 in BMSCs 72 hours after transfection, and the results suggested that the expression of FGF-2 in the transfected stem cells was significantly higher than that in the normal group (Fig. S2b–d). In summary, we constructed F-BMSCs that could persistently express FGF-2.

3.4 Identification of the chondrogenic differentiation ability of F-BMSCs

The ability of BMSCs to differentiate chondrogenically is essential for the repair of damaged cartilage. In order to determine the chondrogenic induction properties of F-BMSCs loaded onto dECM hydrogels, grouping interventions were performed: in group 1 BMSCs-only (control), in group 2 F-BMSCs, and in group 3 dECM + F-BMSCs were incubated at 37 °C in chondrogenic-inducing medium for 7 days. Then immunofluorescence staining, toluidine blue staining, quantitative PCR and WB were performed to detect cartilage-related indexes. As shown in Fig. 4a, the immunofluorescence staining revealed the high expression of Collagen-I and Collagen-II proteins in the dECM + F-BMSC group, and the difference between the dECM + F-BMSC-treated group and the blank group was significant (Fig. 4b), suggesting that the dECM + F-BMSC combination helped to promote cartilage repair. And the toluidine blue staining further confirmed that F-BMSCs equipped with dECM hydrogels could promote the differentiation of stem cells into chondrocytes, and chondrocytes could be identified by the appearance of purplish-red heterostained particles with small and rounded nuclei in blue color in the cells (Fig. 4c). To verify the chondrogenic differentiation of BMSCs within dECM hydrogels, WB and QRT-PCR assays were carried out. These assays aimed to measure the protein and mRNA expression of genes associated with chondrogenesis. Compared to the blank group, the dECM + F-BMSC group showed significantly higher expression of chondrogenic markers (Col-I, Col-II, SOX9, Aggrecan) at both gene and protein levels (Fig. 4d–l), indicating that dECM hydrogels enhance the chondrogenic potential of F-BMSCs.

Fig. 4 Identification of the cartilage differentiation ability of F-BMSCs. (a) Cellular immunofluorescence detection of Collagen-II and Collagen-I protein expression: green fluorescence of the target protein and blue fluorescence of the nucleus (scale bar = 200 μm). (b) Quantification of the immunofluorescence intensity of Collagen-I and Collagen-II proteins in each group. (c) The ability of BMSCs to differentiate into chondrocytes was detected by toluidine blue staining: chondrocytes were purplish red and bone marrow mesenchymal stem cells were blue. (d–g) The expression levels of chondrogenic genes (COL-I, COL-II, SOX9, Aggrecan) were detected by QRT-PCR (n = 3). (h–l) Western blotting was used to detect the expression and quantitative analysis of cartilage characteristic proteins (n = 3).

3.5 Characterization of the dECM hydrogel-induced directional migration capacity of BMSCs

We evaluated the migration ability of BMSCs at 24 hours, 48 hours, and 72 hours using a Transwell assay to verify that the dECM hydrogel could cause the directional migration of F-BMSCs (Fig. 5a). Microscopic examination revealed that the number of migrating BMSCs was notably decreased under the influence of LPS-induced inflammation. In contrast, the dECM hydrogel group was capable of inducing the directional migration of BMSCs. The number of migrating BMSCs in this group was substantially greater than that in the normal group and the LPS group following the addition of the dECM hydrogel (Fig. 5b–e). These findings demonstrated that even in the inflammatory environment of OA, the dECM hydrogel could effectively draw in and cause BMSCs to migrate to the site of cartilage destruction, assisting in the repair of damaged cartilage tissue.

Fig. 5 Identification of the directed migration ability of BMSCs induced by the dECM hydrogel. (a) Transwell assay to determine the ability of the dECM hydrogel to induce the directed migration of bone marrow mesenchymal stem cells. (b) Crystal violet staining of different experimental groups after 24, 48 and 72 h of culture: positive cells were purplish red. (c–e) Quantitative analysis of crystal violet staining positive cells in different experimental groups after 24, 48 and 72 h culture.

3.6 Effect of dECM hydrogels loaded with F-BMSCs on chondrocyte proliferation and apoptosis in SD rats

The proliferation and apoptosis of chondrocytes are crucial for the regeneration of damaged cartilage tissue.²⁸ Osteoarthritic tissues have significantly lower proliferative activity and higher rates of chondrocyte apoptosis.^29,30 We evaluated the effect of dECM hydrogels loaded with F-BMSCs on chondrocyte proliferation using EdU assay. Following LPS intervention, the number of proliferating cells significantly decreased, and chondrocytes displayed a remarkable growth-inhibited state. In contrast, even in the LPS-induced inflammatory state, the group treated with dECM combined with F-BMSCs was still able to promote chondrocyte proliferation (Fig. 6a and b). To further confirm this conclusion, we performed senna staining and live/dead staining to further view the glycosaminoglycan deposition and value-added of the chondrocytes, as shown in Fig. 6c and d. Chondrocytes in the inflammation group had the least amount of inflammation-stimulated GAG content, whereas the dECM + F-BMSC group could promote the deposition of chondrocyte GAG. In addition, the live/dead staining led to a similar conclusion: the chondrocyte mortality was significantly higher in the inflammation group compared to the normal and dECM + F-BMSC groups (Fig. 6e and f). Flow cytometry showed significantly higher LPS-induced chondrocyte apoptosis in the OA group, whereas F-BMSCs loaded onto the dECM markedly reduced chondrocyte apoptosis (Fig. 7a and b). Apoptosis-related genes (Bax, Bcl-2, Casp3) were further detected via immunofluorescence staining, western blotting (WB), and qPCR; the results confirmed that dECM + F-BMSCs downregulated Bax and Casp3 and upregulated Bcl-2 (Fig. 7c–k). According to the aforementioned findings, the dECM hydrogel backed by F-BMSCs may prevent chondrocyte apoptosis and undo the effects of LPS-induced inflammation on chondrocyte growth.

Fig. 6 Effect of F-BMSCs supported by dECM hydrogels on chondrocyte proliferation in SD rats. (a and b) EdU assay was used to evaluate the effect of dECM hydrogels loaded with F-BMSCs on chondrocyte proliferation and quantitative analysis was performed: proliferated cells were labeled with red fluorescence and nuclei were labeled with blue fluorescence (scale bar = 50 μm). (c and d) F-BMSC + dECM hydrogel was used to characterize glycosaminoglycan deposition in chondrocytes after 1 day and 3 days of intervention and quantitative analysis: red labeled chondrocytes (scale bar = 100 μm). (e and f) Live/dead staining further assessed chondrocyte proliferation between groups on days 1, 3, and 5: live cells were labeled with green fluorescence and dead cells with red fluorescence (scale bar = 100 μm).

Fig. 7 Effect of F-BMSCs loaded onto the dECM hydrogel on apoptosis of SD rat chondrocytes. (a and b) Flow cytometry was used to detect the effect of F-BMSCs supported by the dECM hydrogel on chondrocyte apoptosis and its quantitative analysis. (c and d) Immunofluorescence staining was used to detect the expression levels of apoptosis-related proteins (Bax, Bcl-2, Casp3): green fluorescence was labeled as the target protein, and blue fluorescence was labeled as the nucleus (scale bar = 20 μm). (e–h) The expression of apoptosis-related proteins was detected and quantified by western blotting (n = 3). (i–k) QRT-PCR was used to detect the expression of apoptosis-related genes (n = 3).

3.7 Investigation of the therapeutic impact of dECM hydrogels piggybacking F-BMSCs by mRNA sequencing

Firstly, we conducted quality control on the sequenced data (Fig. S3 and S4). The differences in transcriptional profiles and possible biological functions between the normal group, the group stimulated by OA inflammation, and the group treated with the F-BMSC + dECM composite hydrogel were then examined using subsequent differential expression analysis, GO functional enrichment analysis, and KEGG pathway enrichment analysis. Fig. 8a–c shows the volcano plots that show the DEGs between the various groups. We visualized the DEGs between the group stimulated by OA inflammation and the group treated with the dECM composite hydrogel (Fig. 8d). In this comparison, 310 genes were down-regulated and 232 genes were up-regulated. The key biological roles of the DEGs in the OA group and the composite hydrogel group were next investigated using GO enrichment analysis from the perspectives of molecular function (MF), biological processes (BP) and cellular components (CC). The results showed that the DEGs in the OA and hydrogel treatment groups were predominantly associated with biological processes such as extracellular matrix, oxidoreductase activity, and pyrophosphatase activity (Fig. 8e). To delve deeper into the signaling pathways associated with these DEGs, we carried out KEGG analysis. The outcomes revealed that these DEGs were predominantly enriched in pathways such as ferroptosis, ECM–receptor interaction, and oxidative phosphorylation (Fig. 8f). Moreover, to further explore the biological functions of chondrocytes following treatment with the F-BMSC + dECM hydrogel, we conducted a GSEA. From the analysis, it can be seen that the biological functions related to DNA replication and repair showed an up-regulation trend after the F-BMSC + dECM hydrogel treatment (Fig. 8g and h), whereas the biological functions related to nitrogen compound transport were down-regulated (Fig. 8i). This phenomenon suggests that the F-BMSC + dECM hydrogel treatment may induce the inflammation-stimulated chondrocytes to enhance the DNA replication and repair ability, which in turn promotes the repair process of chondrocytes; on the other hand, it reduces the apoptosis and senescence of chondrocytes triggered by reactive oxygen species and nitrogen compounds. Furthermore, we noticed that the hydrogel-treated group had a down-regulated mTOR pathway, whereas the OA group had an up-regulated one (Fig. 8j). This suggests that the healing impact of the dECM hydrogel piggybacking F-BMSCs on OA chondrocytes can be induced by controlling the mTOR signaling pathway and limiting the onset of chondrocyte apoptosis and senescence, thereby protecting the OA cartilage from further damage.

Fig. 8 mRNA sequencing analysis of the therapeutic effects of the dECM hydrogel equipped with F-BMSCs. (a–c) Volcanic maps of DEGs between different experimental groups. (d) Heat maps showing DEGs between the dECM hydrogel treatment group and the OA group: red represents high expression and blue represents low expression. (e) GO functional enrichment analysis of DEGs between the dECM hydrogel treatment group and the OA group. (f) KEGG enrichment analysis of DEGs between the dECM hydrogel treatment group and the OA group. (g–j) GSEA plots.

3.8 Therapeutic effects of dECM hydrogels loaded with F-BMSCs on cartilage damage in rat osteoarthritis

To assess the therapeutic efficacy of dECM hydrogels loaded with F-BMSCs on osteochondral injury in OA in vivo, an OA model was developed using SD rats, and intervention treatments were implemented. Twenty-four SD rats were randomly assigned to four groups. In the blank group and the OA group, PBS was administered. In the dECM hydrogel group, the dECM hydrogel was injected into the knee joint. In the experimental group, the knee joint was injected with a combination of the dECM hydrogel and F-BMSCs. The rats were humanely put down twelve weeks after the hydrogel was administered, and samples were taken for pertinent studies to assess the hydrogel's ability to repair osteochondral damage caused by OA. First, based on macroscopic observation, when compared with the other groups, the group treated with the dECM hydrogel loaded with BMSCs exhibited newly regenerated hyaline-cartilage-like tissue 12 weeks after the treatment. The untreated group, on the other hand, only had a rough surface, noticeable osteophytes, little new tissue growth, and less than ideal cartilage regeneration (Fig. 9a). Each group's cartilage damage was assessed using the International Cartilage Repair Society (ICRS) score in order to quantify it. According to the results, the dECM hydrogel + F-BMSC treatment had a favorable effect on cartilage regeneration, and their score was comparable to that of the normal group (Fig. 9b). A microscopic reconstruction of the rat knee joint was performed using micro-CT in order to examine the growth of osteophytes. The micro-CT showed rough, uneven cartilage surfaces on the femoral condyle, femoral trochlea, and tibial plateau, with extensive osteophytes on the bone surface in the OA group (Fig. 9c). After 12 weeks of dECM hydrogel + F-BMSC treatment, the femoral condyle and tibial plateau cartilage surfaces were smoother than those in the OA group, and the osteophyte formation was significantly reduced (Fig. 9c). To more accurately quantify the above results, we quantitatively analyzed the total volume fraction of osteophytes (osteophyte bone volume/total volume, BV/TV), trabecular bone number (Tb.N), and trabecular bone separation (Tb.Sp). The total volume fraction of osteophytes can indirectly reflect the change in the total number of osteophytes. The total volume fraction of osteophytes in the OA group was significantly greater than those of the normal group and the group treated with the dECM hydrogel in combination with F-BMSCs, as shown in Fig. 9d. This finding suggested that the total volume of osteophytes in the OA group was significantly greater compared to the normal group and the dECM hydrogel + F-BMSC treatment group, thereby highlighting the therapeutic efficacy of the dECM hydrogel + F-BMSC treatment. The trabecular bone number in the OA group was substantially lower than that in the normal group, according to the quantitative analysis of trabecular bone numbers. Although the trabecular bone number was higher in the dECM + F-BMSC group than that in the OA group, the difference was not statistically significant (Fig. 9e). In the analysis of trabecular bone separation, the normal group and the dECM + F-BMSC treatment group had considerably larger trabecular bone separation than the OA group (Fig. 9f). These results indicate that treatment with dECM hydrogel + F-BMSCs promoted the formation of subchondral bone and trabecular bone in OA rats, reduced the generation of osteophytes in OA, restored the smooth state of the joint surface, and had a significant repair effect on cartilage injury caused by OA. To further observe the situation of cartilage regeneration at the histological level, hematoxylin and eosin (H&E) staining and Safranin-O staining were performed. The results showed that the cartilage defect in the OA group showed a rough surface and poor fibrocartilage regeneration at 12 weeks. The cartilage surface was not smooth, the continuity was interrupted, and osteophytes were formed. Although some new soft tissue and bone tissue were also formed in the dECM hydrogel group at 12 weeks, the dECM hydrogel treatment group loaded with F-BMSCs showed a smoother and continuous surface, and the formation of many cartilage-like tissues and chondrocytes (Fig. 10a). According to the Osteoarthritis Research Society International (OARSI) scoring system, the OA group's cartilage injury score was significantly higher than the normal group's. However, after the intervention with the dECM hydrogel combined with F-BMSCs, the score decreased significantly (Fig. 10b and c). In addition, immunohistochemistry (IHC) was used to detect Collagen-II, ACAN proteins, and apoptosis-related proteins (Bax and Casp3) in cartilage tissue. The results showed that the expression of Collagen type II and ACAN proteins in the OA group was significantly decreased. However, 12 weeks after treatment with dECM hydrogel + F-BMSCs, the expression of Collagen type II and ACAN proteins increased (Fig. 10d–f). Moreover, the expression of apoptotic proteins in the dECM hydrogel + F-BMSC treatment group decreased significantly (Fig. 10g–i). Consistent results were derived from TUNEL staining (Fig. 10j and k). The data indicated that the treatment group with dECM hydrogel + F-BMSCs could remarkably decrease the apoptotic level in OA. These findings further corroborate that the dECM hydrogel loaded with F-BMSCs exerts a reparative effect on OA-induced osteochondral injury. The underlying mechanism involves the inhibition of apoptosis and the promotion of in vivo cartilage regeneration, thereby contributing to the restoration of damaged osteochondral tissues in the context of OA.

Fig. 9 The therapeutic effects of dECM hydrogel-modified F-BMSCs on the SD rat OA model were observed through macroscopic and micro-CT observation. (a and b) Quantitative analysis of macroscopic images and ICRS scores in each group after 12 weeks of treatment (n = 6). (c) Microscopic CT scan images of each group (n = 6). (d–f) Total volume fraction of osteophytes (BV/TV), quantitative analysis of the trabecular bone number (Tb.N), and quantitative analysis of trabecular bone separation (Tb.Sp) (n = 6).

Fig. 10 Histological staining and immunohistochemical evaluation of the therapeutic effects of F-BMSCs loaded onto the dECM hydrogel in the SD rat OA model. (a–c) H&E and Safranin-O/green staining to evaluate the repair effect of the dECM hydrogel on OA damaged osteochondral tissues after 12 weeks of treatment (n = 6). (d–f) Immunohistochemical analysis was performed to evaluate the expression and quantification of cartilage characteristic proteins (Aggrecan, Collagen II) in each group after 12 weeks of treatment (n = 3). (g–i) Immunohistochemical analysis was performed to evaluate the expression and quantification of apoptotic proteins (Bax, Casp3) in each group after 12 weeks of treatment (n = 3). (j–k) TUNEL staining and quantitative analysis (n = 3).

4. Discussion

BMSCs are recognized as ideal seed cells for osteoarthritis (OA)-related cartilage repair due to their multidirectional differentiation potential and immunomodulatory properties. However, direct BMSC injection in clinical practice is plagued by poor cell viability and limited functional retention.^31,32 Concurrently, traditional dECM scaffolds are usually made into membranes or powders, which cannot meet the requirements of minimally invasive intra-articular drug delivery and limit their translational application. To address these key challenges, this study developed a composite therapeutic system that combines FGF-2-overexpressing bone marrow mesenchymal stem cells (F-BMSCs) with injectable dECM hydrogels and discussed the main findings and their implications for materials science and OA treatment here.

Injectable dECM hydrogels represent a pivotal innovation in biomaterial design for cartilage repair, with their performance validated through material characterization and functional data. Firstly, the physicochemical properties of the hydrogel were optimized for clinical applicability: the 3 mg ml⁻¹ dECM hydrogel exhibits a porous internal microstructure (Fig. 2g), which facilitates cell infiltration and nutrient exchange. Rheological analyses confirmed its injectability and structural stability (Fig. 2h and i). These characteristics enable the material to undergo in situ gelation at cartilage defects, conforming to the irregular defect morphology (Fig. 2f), and to withstand mechanical stimuli during joint movement—an essential feature for maintaining a supportive microenvironment for BMSCs. Secondly, we verified the retention of biological components in the dECM: the contents of collagen and GAGs preserved after decellularization (Fig. 2d and e) are comparable to those of native cartilage, while the DNA residue is minimized (<10.91 ng mg⁻¹, Fig. 2c) to reduce immunogenicity. The retained ECM components mimic the native cartilage niche, which serves as a critical driver for subsequent F-BMSC functions. Finally, live/dead staining and CCK-8 assays confirmed that the material does not induce inflammation. Collectively, these data demonstrate that the 3 mg ml⁻¹ dECM hydrogel overcomes the limitations of traditional dECM scaffolds (e.g., poor injectability and insufficient cell support) and provides a standardized biocompatible carrier for F-BMSCs, meeting the core requirement of structure–function synergy.

Chondrocyte apoptosis plays a critical role in the pathogenesis of OA. Studies have shown that in human OA tissue samples, the severity of cartilage destruction exhibits a positive correlation with matrix depletion and chondrocyte apoptosis.³³ Inhibiting chondrocyte apoptosis is therefore a critical step in preventing and managing OA. BMSCs are a common source of multipotent cells for regenerating and repairing damaged tissues, with adipogenic, osteogenic, and chondrogenic properties. They can also secrete various factors to exert anti-inflammatory, immunomodulatory, and anti-fibrotic effects. BMSCs have been shown to inhibit OA-induced chondrocyte senescence and apoptosis.³⁴ Liu et al. demonstrated that exosomes derived from MSCs promote chondrocyte proliferation and inhibit apoptosis via the miR-206/GIT1 axis, thereby facilitating cartilage regeneration.³⁵ Additionally, Wen et al. suggested that MSC-derived exosomes (MSC-exo) may inhibit autophagy and apoptosis of OA chondrocytes through the PI3K/Akt/mTOR signaling pathway.³⁶ These studies fully confirm the anti-apoptotic and pro-proliferative effects of MSCs. We used dECM to create a specialized microenvironment suitable for F-BMSC proliferation and chondrogenic differentiation. Intriguingly, this composite system reversed lipopolysaccharide (LPS)-induced chondrocyte apoptosis: flow cytometry showed a reduced apoptosis rate (Fig. 7a and b), while the anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic proteins Bax/Casp3 were downregulated (Fig. 7c–k). The dECM hydrogel promotes F-BMSC survival and differentiation, and F-BMSCs secrete FGF-2 to protect chondrocytes. This synergy highlights the ability of the composite hydrogel system to address OA pathological processes, such as chondrocyte loss, chondrocyte apoptosis, matrix degradation, and inflammation.

Accumulating evidence indicates that OA is a degenerative disease associated with metabolic disturbances³⁷—specifically, glycolysis–oxidative phosphorylation imbalance may accelerate OA progression.^38,39 Excessive nitric oxide (NO) production induces MAPK phosphorylation, which further promotes p53 phosphorylation; this triggers cleavage of cysteinyl aspartate-3 (caspase-3) and PARP, ultimately leading to chondrocyte apoptosis.^40–42 In addition, MAPK phosphorylation also activates mTOR, a process that promotes the abnormal proliferation and hypertrophic differentiation of chondrocytes, as well as increases the expression and activity of extracellular matrix-degrading enzymes (e.g., matrix metalloproteinases, MMPs), which leads to a decrease in cartilage elasticity and quality and accelerates the pathological process of OA. Combined with mRNA sequencing results, we propose that F-BMSCs loaded onto the dECM restore chondrocyte metabolic balance (glycolysis vs. oxidative phosphorylation) by regulating the MAPK/NF-κB/PI3K-AKT-mTOR pathway. This regulation further reduces nitrogen compound and catabolic enzyme (e.g., MMP) production, thereby inhibiting chondrocyte apoptosis, alleviating cartilage matrix degradation, and maintaining cartilage homeostasis to promote repair.

Compared with existing OA treatment strategies, the core advantage of the dECM/F-BMSC system constructed in this study lies in the synergistic effect of the “injectable scaffold–mesenchymal stem cell–growth factor” complex. Scaffold-free BMSC injection, while minimally invasive, suffers from a high cell loss rate; pure dECM hydrogel, despite its low immunogenicity, has limited chondrogenic efficiency. In contrast, the dECM/F-BMSC system exhibits superior therapeutic efficacy due to the dual advantages of 3D microenvironment construction and sustained growth factor release. The clinical value of this composite system is directly linked to addressing unmet needs in OA treatment, as validated by in vivo experiments in SD rats. Firstly, the injectability of the hydrogel enables minimally invasive administration via intra-articular injection, avoiding the trauma of open surgery and aligning with clinical preferences for minimally invasive procedures. Secondly, sustained therapeutic effects were observed at 12 weeks: macroscopic analysis revealed regenerated hyaline cartilage-like tissue (Fig. 9a) and the improvement of ICRS score (Fig. 9b). Micro-CT showed reduced osteophyte formation (BV/TV, Fig. 9d) and a smoother cartilage surface (Fig. 9c). Histologically, HE staining and Safranin-O staining confirmed the restoration of cartilage surface continuity and a decreased OARSI score (Fig. 10a–c); immunohistochemistry showed increased Col II/Aggrecan expression and decreased Bax/Casp3 expression (Fig. 10d–i). TUNEL staining further confirmed reduced chondrocyte apoptosis (Fig. 10j and k). These results indicate that the composite hydrogel not only alleviates symptoms but also delays OA progression, addressing the limitations of current small-molecule therapies that only target inflammation. Finally, the low immunogenicity and high biocompatibility of the dECM hydrogel reduce the risk of xenograft rejection, laying a foundation for the clinical safety evaluation of this OA treatment strategy.

Despite confirming the composite hydrogel's efficacy in hyaline cartilage regeneration, this study has limitations. First, the OA model was only established via anterior cruciate ligament transection (ACLT) in SD rats. Rats differ greatly from humans in knee cartilage thickness and weight-bearing, and no validation was performed in large animals (e.g., rabbits, miniature pigs), limiting the rodent model's translational value. Second, only 12 weeks’ therapeutic effects were assessed, with no evaluation of long-term repaired tissue stability (e.g., hyaline cartilage degradation, osteophyte recurrence, and chronic foreign body reactions from residual dECM). Furthermore, although dECM's low immunogenicity was confirmed via DNA residue testing, we did not measure anti-porcine dECM antibody levels in rats or long-term intra-articular macrophage polarization changes, leaving delayed xenogeneic immune rejection not ruled out. Additionally, after lentivirus-mediated stable FGF-2 expression in F-BMSCs, we did not evaluate F-BMSCs’ in vivo proliferation controllability or whether FGF-2's aberrant peri-articular tissue stimulation activates tumor-related pathways (e.g., PI3K-AKT-mTOR), lacking critical safety validation. FGF9, FGF18, FGF21, TNF-β, TGF-β and BMP2 were not used as direct detection indicators, nor were their independent contributions clarified via blocking experiments. This prevented full elucidation of their interactions with FGF2—a key area to supplement in subsequent research. Lastly, mechanistic studies on the composite hydrogel are limited to mRNA sequencing, requiring additional fundamental research to verify these mechanisms.

5. Conclusion

In summary, we engineered BMSCs to overexpress FGF-2 and loaded them onto the dECM hydrogel, successfully preparing a hydrogel material that can significantly inhibit cell apoptosis and inflammatory responses and effectively promote the proliferation and chondrogenic differentiation of BMSCs. The underlying mechanism may be achieved by regulating the MAPK/NF-κB/PI3K-AKT-mTOR signaling pathway, thereby restoring the metabolic balance of chondrocytes and delaying the progression of OA. This achievement provides a new direction for the treatment of osteoarthritis and the repair of osteochondral injuries.

Author contributions

Yue Qiu: writing—original draft, methodology, and conceptualization. Bo Yu: software and methodology. Cancai Jiang: formal analysis. Huangyi Yin: data curation. Jinzhi Meng: visualization. Hongtao Wang: resources and conceptualization. Lingyun Chen: conceptualization. Yang Cai: visualization. Tianyu Ren: investigation. Qingfa Qin: resources. Jia Li: writing—review & editing. Jun Yao: writing—review & editing, resources, and funding acquisition.

Conflicts of interest

No conflicts of interest exist in the submission of this manuscript, and the manuscript is approved by all authors for publication.

Ethics approval statement

This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Ethical Approval No. 2024-E646-01) and all experiments were performed in strict accordance with the guidelines.

Data availability

Data will be made available upon request.

Supporting information Fig. S1 Illumina sequencing diagram. Fig. S2 Overexpression of FGF-2 in BMSCs remodeling. (a) Observation of transfection efficiency of BMSCs transfected with lentivirus overexpressing FGF-2: BMSCs successfully transfected were marked with green fluorescence, with a scale bar of 100 μm. (b–d) Western blot and QRT-PCR were used to detect the protein and gene expression levels of FGF-2 in BMSCs. Data are expressed as mean ± standard deviation, **** : P < 0.0001, ***: P < 0.001, **: P < 0.01, *: P < 0.05, ns: no significance (n = 3). Fig. S3 Distribution of RNA sequencing data error rates. (a–i) Represent the distribution of sequencing data error rate of NC group, OA group and F-BMSCs + dECM hydrogel treatment group respectively: the abscissa is the base position of reads, and the ordinate is the single base error rate. Fig. S4 A/T/G/C content distribution inspection. (a–i) represents the A/T/G/C content distribution of NC group, OA group and F-BMSCs + dECM hydrogel group respectively: the abscissa is the base position of reads, and the ordinate is the percentage of the five base types of ATGCN. Fig. S5. Filtering of raw sequencing data. (a–i) Remove reads with adapters; Remove reads containing N (where N represents uncertain base information); Remove low-quality reads (reads with Qphred <=5 alkaline bases accounting for more than 50% of the entire read length). Fig. S6. Venous blood detection of SD rats. (a) Detection of ALT in venous blood of SD rats. (b) Detection of AST in venous blood of SD rats. (c) Detection of CREA in venous blood of SD rats (n = 4). See DOI: https://doi.org/10.1039/d5bm00920k.

Acknowledgements

This study was financially supported by the Guangxi Science and Technology Project (Grant Number: GuikeAD19254003), The First Affiliated Hospital of Guangxi Medical University Innovation Team Cultivation Program (Grant Number: YYZS2023004) and the Nanning Qingxiu District Science and Technology Plan Project (Grant Number: 2020018).

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Footnote

† These authors contributed equally to this work.

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