Repair of Osteochondral Defect with Acellular Cartilage Matrix and Thermosensitive Hydrogel Scaffold

Abstract

In the present study, acellular cartilage matrix (ACM) was modified with poly-l-lysine/hyaluronic acid (PLL/HA) multilayers via detergent-enzyme chemical digestion and layer-by-layer self-assembly technology. This modified ACM was then loaded with Transforming Growth Factor Beta 3 (TGF-β3) and incorporated into a thermosensitive hydrogel (TH) to create a HA/PLL-ACM/TH composite scaffold with sustained-release function. This study aimed to evaluate the efficacy of this novel composite scaffold in promoting chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and facilitating osteochondral defect repair. In vitro, isolated, and cultured rat BMSCs were inoculated in equal amounts into TH, ACM/TH, and HA/PLL-ACM/TH groups, with or without TGF-β3 supplementation, for 21 days. Western blot (WB) analysis and immunofluorescence staining were employed to assess the expression levels of collagen II, aggrecan, and SOX-9. In vivo, osteochondral defect was created in the Sprague–Dawley rat trochlea using microdrilling. TH, ACM/TH, and HA/PLL-ACM/TH scaffolds, with or without TGF-β3, were implanted into the defect. After 6 weeks, the repairs were evaluated macroscopically, using Micro computed tomography (micro-CT), histological analysis, and immunohistochemistry. The results demonstrated that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 significantly upregulated the expression of collagen II, aggrecan, and SOX-9 compared with the control and other experimental groups. Furthermore, at 6 weeks postsurgery, the HA/PLL-ACM/TH group loaded with TGF-β3 exhibited superior tissue formation on the joint surface, as confirmed by micro-CT and histological evidence, indicating improved osteochondral repair. These findings suggest that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 holds promise as a therapeutic strategy for osteochondral defect and offers a novel approach for utilizing acellular cartilage microfilaments.

Impact Statement

The cartilage matrix was modified by decellularized and layer-by-layer self-assembly technology and still retained glycosaminoglycans and collagen II to a certain extent. The hyaluronic acid/poly-l-lysine–acellular cartilage matrix/thermosensitive hydrogel (HA/PLL-ACM/TH) composite scaffold exhibited thermoresponsive properties. HA/PLL-ACM/TH loaded with TGF-β3 demonstrated the most effective and prolonged sustained release. In this study, HA/PLL-ACM/TH scaffolds loaded with TGF-β3 were most effective in facilitating the repair of osteochondral defect.

Introduction

Articular cartilage injuries are a frequent occurrence in clinical practice, leading to joint pain, dysfunction, and other clinical symptoms that significantly impact patient quality of life.¹ Recent years have witnessed a sharp rise in the incidence of cartilage injuries due to factors such as the increasing economic standard, the growing aging population, and a rise in sports injuries, traffic accidents, and arthritis.^2,3 Currently, established treatment options for articular cartilage injury include subchondral grinding microfracture, subchondral bone drilling, autologous chondrocyte implantation, and autologous cartilage transplantation.^3–5 However, these conventional methods have limitations and often fail to achieve optimal therapeutic outcomes.^3,6,7 The continual development and refinement of tissue engineering technology offer promising new avenues for repairing cartilage injuries.^4,8

Scaffold materials play a crucial role in tissue engineering. Acellular tissue matrix scaffolds are particularly advantageous for cartilage engineering due to their resemblance to the complex natural structure and biological characteristics of native tissues.⁴ Acellular cartilage matrix (ACM) has emerged as a promising material in this field.^9–11 Advancements in decellularization techniques have paved the way for the utilization of xenogeneic chondrocyte extracellular matrix (ECM). Currently, various decellularized scaffolds, including porcine decellularized peritoneum, porcine decellularized heart valve, and decellularized dermal matrix, are being explored for clinical applications.^12–15 Notably, Liu et al. made a significant contribution by developing injectable cell carriers using self-assembled nanofibrous hollow microspheres fabricated from star-shaped biodegradable polymers.¹⁶

Layer-by-layer (LbL) assembly technology, a widely used technique in biomaterials science,^17,18 offers a versatile coating method for improving material performance and biocompatibility. This approach relies on electrostatic interactions to construct multilayer films through the alternating adsorption of oppositely charged polyelectrolyte cations and anions onto a material surface. Hyaluronic acid/poly-l-lysine (HA/PLL) polyelectrolyte multilayers, for instance, are nanoscale materials with good biocompatibility, making them suitable for surface modification of various biological materials.^19,20

The development of injectable scaffold materials offers a two-pronged benefit: reduced surgical invasiveness and faster postoperative recovery times. Upon delivery to the defect site, these materials readily conform to the defect margins and solidify into gel scaffolds possessing sufficient mechanical strength.²¹ For instance, injectable thermoreversible poly(DL-lactic-coglycolic acid)-poly(ethylene glycol)-poly(DL-lactic-coglycolic acid) (PDLLA-PEG-PDLLA) copolymeric thermogels undergo spontaneous transformation into hydrogels within just 2 min at physiological temperatures, both in vitro and in vivo.²² Notably, injectable PDLLA-PEG-PDLLA has already been combined with multilayered, modified platelet lysates for osteochondral defect repair.²³ These findings collectively suggest that PDLLA-PEG-PDLLA holds promise as a future injectable thermosensitive hydrogel (TH).

In the present study, ACM was isolated from rabbit costal cartilage through physical and chemical treatments. To enable sustained release of TGF-β3, the ACM surface was then modified with a HA/PLL multilayer film, allowing for TGF-β3 loading. Finally, the modified ACM was incorporated into a TH (PDLLA-PEG-PDLLA) to create the final HA/PLL-ACM/TH composite scaffold with sustained TGF-β3 release capability. This study was designed to evaluate the potential of the HA/PLL-ACM/TH composite scaffold in two aspects: (1) its ability to induce chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) in vitro; (2) its efficacy in promoting osteochondral defect repair in a rat model.

Materials and Methods

Reagents

The following materials were used in this study: injectable TH PDLLA-PEG-PDLLA (State Key Laboratory of Biotherapy, Sichuan University, China), TGF-β3 (PeproTech, USA), primary antibodies against aggrecan, collagen II (Col-II), SOX-9, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, UK), goat antirabbit (Boster, China), Cell Counting Kit-8 (CCK-8) (Dojindo, Japan), and Alexa Fluor® 488-labeled and Alexa Fluor® 594-labeled goat antirabbit immunoglobulin G (H + L) second antibody (Jackson ImmunoResearch, USA).

Construction of the HA/PLL-ACM/TH composite scaffold

Preparation of ACM

Rabbit costal cartilage slices were obtained under aseptic conditions within 6 h postmortem and subsequently crushed and decellularized. Briefly, the cartilage slices were washed and crushed in phosphate-buffered saline (PBS) containing 3.5% (w/v) phenylmethylsulfonylfluoride (Merck, Germany) and 0.1% (w/v) ethylenediaminetetraacetic acid (EDTA) (Sigma, UK) to inhibit protease activity. This protease inhibition was maintained throughout all subsequent treatments unless otherwise specified. The resulting cartilage fragment suspension was rotated at 2000 rpm in a Beckman (USA) X-22R centrifuge. The supernatant was discarded, and the pellet was further centrifuged at 7000 rpm for 5 min. This differential centrifugation process yielded cartilage microfilaments with a diameter of ∼500 nm to 5 μm. The microfilaments were then gently stirred in 1% TritonX-100 hypotonic Tris-HCl at 4°C for 12 h followed by immersion in 10 mM Tris-HCl (pH 7.5) containing 50 U/mL deoxyribonuclease I and 1 U/mL ribonuclease A (both Sigma) at 37°C for 12 h. Subsequently, the microfilaments were washed with PBS without protease inhibition. The resulting ACM microfilaments were further washed with sterile PBS and prepared as a 30 mg/mL suspension. For subsequent multilayer assembly experiments, a separate 100 mg/mL ACM suspension was prepared, with 1 mL aliquots being transferred to Eppendorf (EP) tubes for centrifugation and precipitation.

The assembly of HA/PLL multilayer films

A 5 mg/mL solution of polyethyleneimine (PEI) was added to the centrifuged EP tube and evenly distributed across the sample by gentle agitation. The mixture was incubated for 30 min to allow for the adsorption of a positively charged PEI layer onto the surface of the ACM. Subsequently, the EP tube was centrifuged to remove the excess PEI solution. The remaining material was then washed with Double Distilled Water (ddH₂O) by gentle agitation for 10 min, followed by centrifugation to remove any unbound PEI. This process of alternating immersion in HA (3 mg/mL) and PLL (1 mg/mL) solutions for 10 min followed by a 15-s wash with ddH₂O and centrifugation was repeated to create 15 bilayers of HA/PLL on the ACM surface. To cross-link the assembled multilayers, a solution containing N-hydroxysulfosuccinimide (11 mg/mL) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (30 mg/mL) was added to the layered ACM. The mixture was then gently agitated and incubated overnight. The following day, after cross-linking was complete, the supernatant was removed by centrifugation. The remaining material was washed three times with PBS and lyophilized for 4 h to obtain the final HA/PLL-ACM composite scaffold. Finally, the scaffolds were sterilized using ⁶⁰Co γ-irradiation.

The construction of HA/PLL-ACM/TH composite scaffolds containing TGF-β3

One hundred milligrams of HA/PLL-ACM was placed in an EP tube containing 100 µL of TGF-β3 solution (4 μg/mL) and incubated overnight at 4°C. After sterilization, 1 mL of TH was added to the EP tube and gently stirred to distribute the ACM evenly. This resulted in the formation of a HA/PLL-ACM/TH composite scaffold containing TGF-β3. The HA/PLL-ACM/TH composite scaffold exhibited a sol-to-gel transition, remaining liquid at 4°C and 25°C and transforming into a gel at 37°C, demonstrating good temperature sensitivity.

Characterization of the HA/PLL-ACM/TH composite scaffold

Scanning electron microscope assessment

The samples of ACM and HA/PLL-ACM were sputter-coated with gold–palladium and then observed using a scanning electron microscope (SEM; Hitachi S-4000, Japan). SEM imaging enabled an objective assessment of the size of ACM and the aggregation state of HA/PLL-ACM.

The sustained-release level of TGF-β3 was detected by HA/PLL-ACM/TH composite scaffolds

To evaluate the sustained-release properties of the novel HA/PLL-ACM/TH composite scaffold, it was compared with TH and ACM/TH scaffolds containing an identical volume (4 μg/mL) of TGF-β3. All groups exhibited a gel state at 37°C. For the in vitro sustained-release experiment, 1 mL of preheated PBS (37°C) was added to the surface of each gel. At designated time points (1, 2, 5, 8, 12, 18, and 25 days), the PBS was collected and replaced with 1 mL of fresh preheated PBS. The collected PBS samples were stored at −20°C. The TGF-β3 release profile was determined using a TGF-β3 enzyme-linked immunosorbent assay kit (Dongge Boye, China). The sustained-release experiment was performed five times for each of the three groups.

Cell proliferation assay

Cytotoxicity of the composite scaffold extracts was evaluated using cultured rat BMSCs. HA/PLL-ACM/TH in its gel state was incubated in Dulbecco’s modified Eagle’s medium (DMEM) at 37°C for 24 h to generate the extract. The CCK-8 assay was employed, following the manufacturer’s instructions, to assess the impact of various HA/PLL-ACM/TH extract concentrations on cell proliferation. Briefly, BMSCs in suspension were seeded into a 96-well plate at a density of 3 × 10³ cells/well and incubated overnight at 37°C. The culture medium was then replaced with fresh media containing different concentrations of the HA/PLL-ACM/TH extracts (0%, 25%, 50%, and 100%). Cell viability was measured using the CCK-8 assay after 1, 2, 3, 4, 5, and 6 days of culture. The culture medium was replaced every other day. To perform the CCK-8 assay, 100 μL of medium containing 10% CCK-8 solution was added to each well, followed by incubation for 1 h at 37°C and 5% carbon dioxide (CO₂). Subsequently, a microplate spectrophotometer (Leica Microsystems, Germany) was used to measure the absorbance of each well at a wavelength of 450 nm. All experiments were performed in quintuplicate.

In vitro cell experiment

Isolation and culture of BMSCs

One-week-old Sprague–Dawley rats were obtained from the Animal Experimental Center of Wenzhou Medical University (China). BMSCs were isolated and cultured from the femurs of these rats using the whole bone marrow adherent method.²⁴ The isolated cells were inoculated onto 25 cm² petri dishes and cultured in an incubator containing 5% CO₂ at 37°C. After 24 h, the culture medium was replaced to remove nonadherent cells. The remaining adherent cell colonies were cultured for 14 days until they reached 80% confluence. Then, they were digested with 0.25% trypsin for 3 min before subculture. Only the second-passage cells were used in subsequent experiment.

The seeding of BMSCs

Sterilized scaffold materials from each group were placed in a 24-well plate and allowed to solidify into gels at 37°C. Subsequently, BMSCs were seeded onto the scaffolds at a density of 5 × 10⁴ cells per well. Complete DMEM was replaced every 3 days throughout the 21-day culture period. The experiment consisted of the following groups: (1) blank control, (2) TH (no TGF-β3), (3) TH (with TGF-β3), (4) ACM/TH (no TGF-β3), (5) ACM/TH (with TGF-β3), (6) HA/PLL-ACM/TH (no TGF-β3), and (7) HA/PLL-ACM/TH (with TGF-β3).

Protein extraction and WB analysis

Whole-cell protein lysates were prepared from chondrocytes using a mixture of Radio Immunoprecipitation Assay (RIPA) lysis buffer and phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluorid (PMSF) in a 100:1 ratio. The extracts were incubated on ice for 10 min and then centrifuged at 12,000 rpm for 15 min at 4°C. Protein concentration for each sample group was determined using the Bicinchoninic Acid Assay (BCA) protein assay kit (Beyotime, China). A total of 40 μg of protein from each sample was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad, USA). The membrane was then blocked with 5% nonfat milk for 2 h. Following three washes with TBST [Tris Buffered Saline (TBS) containing 0.1% Tween-20], the membranes were incubated with primary antibodies against Col-II (1:1000 dilution), aggrecan (1:500 dilution), SOX-9 (1:100 dilution), and GAPDH (1:5000 dilution) overnight at 4°C. Subsequently, the membranes were incubated with the corresponding secondary antibodies for 2 h at room temperature. Finally, the membranes were washed three times with TBST, and protein band signals were quantified using Image Lab 3.0 software (Bio-Rad Laboratories Inc.).

Immunofluorescence assay

For Col-II staining, harvested BMSCs from each group were seeded onto coverslips placed in a 6-well plate. The cells were then washed with PBS three times for 5 min each wash. Subsequently, fixation was performed using 4% paraformaldehyde solution for 15 min, followed by three additional PBS washes (5 min each). The cells were then permeabilized with 0.5% Triton X-100 for 15 min. After rinsing with PBS, the cells were blocked with 5% goat serum for 1 h at 37°C. Following additional PBS washes, the cells were incubated with a primary antibody against Col-II (dilution 1:200) overnight at 4°C. The next day, the coverslips were washed three times and incubated with Alexa Fluor® 594 and Alexa Fluor® 488 conjugated secondary antibodies (dilution 1:200) for 45 min at room temperature. Finally, the cells were stained with 4',6-Diamidino-2-phenylindole (DAPI) for 10 min. Fluorescence microscopy (Olympus Inc., Japan) was used to capture images of the cells. ImageJ software version 2.1 (USA) was then employed to quantify the fluorescence intensity.

In vivo studies

Establishment of animal models

Thirty-five male Sprague–Dawley rats, 8 weeks old and weighing 250 ± 15 g (n = 5 per group), were obtained from the Animal Center of the Chinese Academy of Sciences (China). The Animal Care and Use Committee of Wenzhou Medical University approved the experiment, and all procedures adhered to established guidelines. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (2.5%, 30 mg/kg). The left leg was shaved and disinfected with povidone–iodine swabs. A medial parapatellar incision was made on the left knee, and the patella was dislocated laterally to expose the trochlear surface. A cylindrical osteochondral defect measuring 1.8 mm in diameter and 1 mm in depth was drilled in the center of the trochlea using a miniature electric drill. Seven groups (n = 5/group) were randomly assigned from the total population: (1) Control group: the defect was not treated; (2) TH group: the defect was treated with TH (without TGF-β3); (3) TH group (containing TGF-β3); (4) ACM/TH group: the defect was treated with ACM/TH (without TGF-β3); (5) ACM/TH group (containing TGF-β3); (6) HA/PLL-ACM/TH group: the defect was treated with HA/PLL-ACM/TH (without TGF-β3); and (7) HA/PLL-ACM/TH group (containing TGF-β3). All scaffold materials were sterilized (using ⁶⁰Co γ-irradiation) before implantation. Following surgery, rats were housed in cages, allowed free movement, and provided unrestricted access to food and water. Intramuscular injections of penicillin were administered for 3 days postsurgery. After 6 weeks, the rats were euthanized with pentobarbital sodium administered intravenously, and their knees were harvested for evaluation of gross appearance and histological analysis.

The macroscopic evaluation and micro-CT analysis

The gross appearance of the trochlear groove was evaluated and photographed. The International Cartilage Repair Society (ICRS) scoring system²⁵ was employed to assess the effectiveness of osteochondral defect repair. Micro-CT (Micro-CT M100, SCANCO Medical, Switzerland) was used to quantify new bone formation within the femoral condyle defect. The entire defect area was defined as the region of interest for the sample to evaluate the extent of bone regeneration. Mimics software (Materialise, Belgium) was used to perform 2D reconstruction and quantitative analysis. The final quantitative results showed the bone volume to total volume (BV/TV) ratio for each implant group. A higher BV/TV ratio indicated greater bone ingrowth into the defect.

Histological and immunohistochemical analysis

Six weeks following the initial surgery, knee specimens were collected and fixed in 4% paraformaldehyde for 24 h. Decalcification was then performed using 10% EDTA for 8 weeks. Subsequently, the specimens were dehydrated through a graded alcohol series, embedded in paraffin wax, and sectioned at a thickness of 5 μm. Following dehydration, the sections underwent staining with Harris’ hematoxylin and eosin Y (Sigma-Aldrich, USA) and toluidine blue (TB; Sigma-Aldrich) according to established protocols. Additionally, immunohistochemical localization of Col-II was performed using an anti-Col-II antibody (ARG20787; Arigo Biolaboratories, Taiwan) to assess positive Col-II staining within the regenerated tissue.

Statistical analysis

All experiments were independently replicated five times. The results were presented as mean ± standard deviation. Statistical analysis of all experimental data was performed using SPSS 20.0 statistical software. One-way analysis of variance followed by Tukey’s post hoc test was used to compare between groups. A p-value <0.05 was statistically significant.

Results

Characterization of the HA/PLL-ACM/TH scaffold

Histological analysis was performed to evaluate the acellular integrity and retention of ECM within the ACM. Fluorescence staining with DAPI revealed the absence of cells or cell fragments in the ACM compared with normal rabbit costal cartilage (Fig. 1A, B). This suggests successful decellularization. Furthermore, positive staining with Safranin O indicated the presence of glycosaminoglycans (GAGs) within the ACM to a certain extent (Fig. 1C), suggesting partial preservation of the ECM. Interestingly, immunofluorescence analysis also demonstrated the retention of Col-II within the ACM to some extent (Fig. 1D).

FIG. 1.

(A) DAPI staining of normal cartilage. (B) DAPI staining of ACM. (C) Safranin O staining of ACM, showing positive results. (D) Immunofluorescence examination of ACM, demonstrating the presence of collagen II components. ACM, acellular cartilage matrix.

SEM revealed a clear distinction between ACM and HA/PLL-ACM. The unmodified ACM exhibited a loose and disorganized structure. In contrast, the HA/PLL multilayer films applied to the ACM resulted in a closely packed and layered organization, creating a 3D architecture (Fig. 2).

FIG. 2.

Comparison of ACM before and after modification by HA/PLL multilayer films. (A) ACM. (B) HA/PLL-ACM; ×400. HA/PLL, hyaluronic acid/poly-l-lysine.

The HA/PLL-ACM/TH composite scaffold exhibited thermoresponsive properties, remaining liquid at 4°C and persisting in a liquid state upon increasing the temperature to 25°C. Only at 37°C does HA/PLL-ACM/TH solidify into a gel (Fig. 3A), demonstrating its favorable temperature sensitivity. The CCK-8 assay revealed no significant difference in absorbance between the extracts at various concentrations, indicating that the HA/PLL-ACM/TH scaffold exhibits no cytotoxic effect on BMSCs (Fig. 3B). As shown in Figure 3C, HA/PLL-ACM/TH loaded with TGF-β3 demonstrated the most effective and prolonged sustained release compared with TH and ACM/TH scaffolds, with sustained release persisting for ∼18 days.

FIG. 3.

(A) Thermoresponsive properties of HA/PLL-ACM/TH. The physical state of HA/PLL-ACM/TH at 4°C, 25°C, and 37°C is shown. Scale bar = 1 cm. (B) Cytotoxicity of HA/PLL-ACM/TH scaffolds. Cell viability of BMSCs cultured with HA/PLL-ACM/TH extracts at various concentrations is presented. Data are represented as mean ± standard deviation (SD) (n = 5). No significant differences were observed between groups (p > 0.05). (C) Sustained release of TGF-β3 from different scaffolds. The cumulative release of TGF-β3 from TH, ACM/TH, and HA/PLL-ACM/TH scaffolds over 18 days is depicted. Data are represented as mean ± SD (n = 5). Significant differences are indicated: *p < 0.01 compared with TH; ##p < 0.01 compared with ACM/TH. BMSC, bone marrow mesenchymal stem cell; TH, thermosensitive hydrogel.

Coll-II, aggrecan, and Sox9 expression levels

WB analysis was employed to investigate the expression levels of Sox9, a crucial protein for BMSC differentiation into chondrocytes,²⁶ and Col-II and aggrecan, the main components of the chondrocyte ECM. WB results revealed significantly higher expression of Col-II, aggrecan, and Sox9 in the ACM/TH+TGF-β3 and HA/PLL-ACM/TH+TGF-β3 groups compared with the control group (Fig. 4A). Conversely, no significant differences were observed in the expression levels of these proteins in the TH, TH+TGF-β3, ACM/TH, and HA/PLL-ACM/TH groups relative to the control. Furthermore, the HA/PLL-ACM/TH+TGF-β3 group exhibited significantly higher expression of Col-II, aggrecan, and Sox9 compared with the ACM/TH+TGF-β3 group. Consistent with the WB findings, Col-II immunofluorescence staining demonstrated significantly upregulated Col-II expression in the HA/PLL-ACM/TH+TGF-β3 and ACM/TH+TGF-β3 groups, with the former group displaying a notably higher expression level (Fig. 4B). In conclusion, these data suggest that while the ACM/TH+TGF-β3 composite scaffolds can induce BMSC chondrogenic differentiation to some extent, the HA/PLL-ACM/TH+TGF-β3 scaffolds with the multilayer modification exhibit the most pronounced effect on BMSC chondrogenic differentiation. No significant changes were observed in the other groups compared with the control.

FIG. 4.

(A) The expression levels of collagen II, aggrecan, and Sox9 in each group were quantified via western blot analysis. (B) The expression levels of collagen II were evaluated via immunofluorescence and DAPI staining for nuclei. The data shown represent the average value and SD for each group. Statistical significance is indicated by asterisks: ** denotes p < 0.01 compared with the control group; ## denotes p < 0.01 for the difference between HA/PLL-ACM/TH (containing TGF-β3) and ACM/TH (containing TGF-β3). Five samples were analyzed per group (n = 5).

Osteochondral defect model

To evaluate the efficacy of various scaffolds in repairing osteochondral defect in vivo, a cylindrical osteochondral defect (diameter: 1.8 mm, depth: 1 mm) was created in the femoral condyle of Sprague–Dawley rats using a microdrill. The different scaffolds were then implanted into the defect. Notably, the presence of the TH component within the scaffolds facilitated a good fit between the scaffold and the defect margins.

Macroscopic evaluation of osteochondral repair and micro-CT evaluation

No signs of infection or immune rejection were observed, and all rats survived until the designated euthanasia time point. Six weeks postsurgery, all rats were euthanized, and tissue samples were collected. Macroscopic evaluation revealed the presence of thickened translucent tissue at the osteochondral defect sites in the ACM/TH and HA/PLL-ACM/TH groups containing TGF-β3, suggesting osteochondral regeneration and repair. In contrast, the defect sites in the control group and other treatment groups exhibited a sunken and rough appearance (Fig. 5A). The ICRS scoring system was employed to assess the gross appearance and repair status of osteochondral defect in the rats.²⁵ Both the HA/PLL-ACM/TH and ACM/TH groups received higher scores compared with the control and TH groups, while the HA/PLL-ACM/TH group containing TGF-β3 exhibited significantly better cartilage repair scores than the ACM/TH group containing TGF-β3 (Fig. 5C).

FIG. 5.

Macroscopic evaluation and micro-CT evaluation. (A) Macroscopic views of treated osteochondral defects at 6 weeks postsurgery. (B) Micro-CT imaging of treated osteochondral defects at 6 weeks postsurgery. (C) ICRS gross evaluation score of osteochondral defects at 6 weeks postsurgery. (D) The ratio of new bone tissue volume to total volume in different scaffold groups (BV/TV). The figure shows the mean ± SD of the experimental data for each group. **p < 0.01 (versus the control group); the significant difference between HA/PLL-ACM/TH (containing TGF-β3) and ACM/TH (containing TGF-β3) is expressed as^##p < 0.01; n = 5. ICRS, International Cartilage Repair Society.

Micro-CT was employed to evaluate the efficacy of various scaffolds in repairing subchondral bone after 6 weeks of implantation. Representative coronal images obtained following 2D reconstruction are presented in Figure 5B. Quantitative analysis of BT/VT revealed that the HA/PLL-ACM/TH and ACM/TH groups exhibited significantly greater subchondral bone repair compared with the control and TH groups. Furthermore, the HA/PLL-ACM/TH scaffold loaded with TGF-β3 demonstrated significantly improved subchondral bone repair at the defect site compared with the ACM/TH scaffold with TGF-β3 (Fig. 5D).

Histological and immunohistochemical assessment

TB staining was used to visualize GAGs within the cartilage tissue. The defect sites in the control and TH groups exhibited minimal positive GAG staining, indicating poor tissue growth. Compared with the control group, the ACM/TH and HA/PLL-ACM/TH groups (without TGF-β3) displayed significantly improved tissue growth. Additionally, the ACM/TH group containing TGF-β3 showed some positive GAG staining. Notably, the defect surface of the HA/PLL-ACM/TH group containing TGF-β3 exhibited a layer of GAG-positive chondroid tissue, and this group demonstrated the most significant improvement in tissue growth at the defect site compared with all other groups (Fig. 6A).

FIG. 6.

Microscopic evaluation of osteochondral defect repair. (A) Toluidine blue stains of osteochondral defect repair. (B) Collagen II immunostaining of osteochondral defect repair.

Immunohistochemical staining revealed Col-II formation on the defect surface. Minimal to no positive staining for Col-II was observed in the defect areas of the control and TH groups. The ACM/TH and HA/PLL-ACM/TH groups without TGF-β3 exhibited negligible Col-II deposition on the surface. The defect surface of the ACM/TH group containing TGF-β3 displayed some Col-II formation, but the distribution was uneven. Interestingly, the HA/PLL-ACM/TH group containing TGF-β3 demonstrated robust Col-II staining on the articular surface (Fig. 6B).

Histological analysis of TB and Col-II immunostaining revealed that HA/PLL-ACM/TH scaffolds loaded with TGF-β3 exhibited superior potential for repairing osteochondral defect in rats compared with other groups.

Discussion

The success of tissue-engineered scaffolds hinges on the selection of biomaterials and their fabrication methods.²⁵ Chondrocyte ECM is a natural biomaterial that offers a microenvironment conducive to cell proliferation and differentiation. Consequently, it has emerged as a promising candidate material for articular cartilage repair. However, sourcing allogeneic articular cartilage for scaffold production presents limitations in both availability and cost in clinical settings.²⁷ Therefore, the utilization of ECM materials derived from heterologous tissues for treating osteochondral defect holds significant research potential and offers broad applicability. In this study, we employed a natural, acellular, ECM-based biomaterial as the core component of our scaffold. This ACM underwent a decellularization process, resulting in the removal of double-stranded DNA while preserving GAGs and Col-II.

LbL self-assembly technology, a development based on L-B films introduced in 1991, offers a novel approach for fabricating multilayer films. This technique has gained widespread application in the field of tissue engineering. Park et al. successfully employed LbL self-assembly to transform poly(lactic-co-glycolic acid) microspheres into drug delivery vehicles capable of sustained TGF-β3 release with a prolonged duration of action.²⁸ Additionally, studies have demonstrated that LbL-modified platelet lysates with HA/PLL multilayers exhibit sustained release of growth factors, thereby accelerating skin defect healing in rats.²³ Inspired by these findings, the present study aimed to modify ACM with HA/PLL multilayers to achieve enhanced sustained release of TGF-β3.

The limited space within the joint cavity has long posed a significant challenge for conventional surgical procedures, driving the development of minimally invasive techniques. These minimally invasive approaches necessitate novel biomaterials with specific properties. Thermoresponsive hydrogels, capable of transitioning between liquid and gel states in response to external temperature changes, hold great promise for various applications. Prior research has explored the combination of nanoparticle-coated platelet solubilizer with injectable PDLLA-PEG-PDLLA for cartilage tissue engineering.²³ Demonstrating minimal cytotoxicity and hemolysis, PDLLA-PEG-PDLLA THs have emerged as promising injectable biomaterials for medical applications.²² In this study, we utilized injectable PDLLA-PEG-PDLLA as a carrier material and incorporated it with HA/PLL-multilayer-modified ACM loaded with TGF-β3 to create the HA/PLL-ACM/TH+TGF-β3 composite scaffold. Our cytotoxicity testing confirmed the biocompatible nature of the composite scaffold components.

Sox9, Col-II, and aggrecan are well-established markers of chondrogenic differentiation in BMSCs. Sox9 protein plays a critical role in directing BMSC differentiation toward the chondrocyte lineage, while Col-II and aggrecan are the major ECM components of articular cartilage. In vivo experiments demonstrated that the ACM/TH+TGF-β3 composite scaffold could induce chondrogenic differentiation of BMSCs to some extent. However, the HA/PLL-ACM/TH+TGF-β3 group exhibited significantly higher expression levels of Sox9, Col-II, and aggrecan, indicating a superior ability to promote chondrogenic differentiation. Notably, in vivo experiments revealed that HA/PLL-ACM/TH composite scaffolds with sustained release of TGF-β3 demonstrated the most effective repair of the osteochondral defect model in rats.

This study successfully generated an ACM from rabbit costal cartilage using a series of physical, chemical, and enzymatic treatments. Fluorescence and histological staining confirmed the absence of DNA within the ACM, while GAG and Col-II components were demonstrably preserved. The ACM was then modified with HA/PLL multilayers and subsequently cross-linked to create a 3D scaffold structure. Experimental results revealed that the HA/PLL-ACM/TH composite scaffold exhibited a favorable sustained-release effect. Whether the composite scaffold could further prolong the time for sustained release of growth factors is worthy of further exploration. Because of its thermosensitive characteristics, it can form a gel at 37°C, the hydrogel can be filled in any shape to fill the osteochondral defect in the animal body, and it is more suitable for the defect site. Notably, in vitro cell experiments demonstrated that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 (HA/PLL-ACM/TH+TGF-β3) displayed superior efficacy in promoting chondrogenic differentiation of BMSCs. Furthermore, in vivo animal experiments revealed that the HA/PLL-ACM/TH+TGF-β3 composite scaffold group achieved the most effective repair of osteochondral defect in rats. These findings collectively suggest that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 holds promise as a therapeutic strategy for osteochondral defect and offers a novel approach for utilizing acellular cartilage microfilaments.

This study also has limitations. In this study, the ECM was obtained from rabbit costal cartilages. The xenogeneic ECM might produce rejection when implanted in rats. However, the resources of allogeneic ECM are limited while there are much more abundant resources to obtain heterogeneous ECM. Therefore, the decellularization technology needs to be further verified by in vivo experiment. TGF-β3 plays an important role in chondrogenesis of cartilage mesenchymal stem cells.^29,30 The loading of BMSCs in hydrogels for tissue engineering applications remains controversial. BMSCs loading can achieve better tissue regeneration, but the time-consuming and high cost of this procedure limits its application. In addition, the viability of the transplanted cells was low due to insufficient nutrients infiltrating into the hydrogel after implantation. Therefore, cell-free hydrogels have attracted great attention, and it is important to recruit stem cells from the host for successful regeneration.³¹ Our TH composite scaffold takes advantage of this property. The experimental results show that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 demonstrated significantly improved subchondral bone repair at the defect site. However, whether there is a scaffold with better biocompatibility and less rejection still needs further exploration.

Footnotes

Authors’ Contributions

S.Z.: Conceptualization, methodology, validation, writing—original draft, and writing—review and editing. G.X.: Conceptualization, investigation, formal analysis, data curation, and writing—review and editing. Z.Z.: Validation, data curation, and software. T.C.: Validation, investigation, and software. Y.H.: Funding acquisition, resources, and supervision.

Disclosure Statement

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 article.

Funding Information

This research was supported by Zhejiang Provincial Medical and Health Technology Foundation of China (No. 2019KY465) and Wenzhou Science and Technology Bureau (No. Y20210048, Y20240102).

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