Evaluation of an AD-MSC Supernatant-Loaded Thermosensitive Hydrogel for Cartilage Protection in Osteoarthritis

Abstract

Knee osteoarthritis (KOA) is a degenerative joint disorder characterized by chronic inflammation and progressive cartilage degradation. Mesenchymal stem cell (MSC)-based therapies have demonstrated therapeutic potential; however, increasing evidence suggests that their efficacy primarily arises from paracrine factors, highlighting the potential of cell free approaches. In this study, we developed an injectable, thermosensitive composite hydrogel incorporating adipose-derived MSC (AD-MSC) supernatant within a Pluronic F-127 (PF-127)/sodium hyaluronate (HA) matrix. The hydrogel exhibited a solution state at a low temperature and rapidly transitioned into a stable gel at a physiological temperature without chemical crosslinkers. Microstructural analysis revealed a porous, interconnected three-dimensional network favorable for the sustained release of bioactive factors. In a rat model of KOA, intra-articular administration of the AD-MSC supernatant-loaded hydrogel significantly improved joint architecture and locomotor performance, alleviated synovial inflammation, and preserved cartilage integrity. Radiographic and histological assessments demonstrated reduced cartilage degeneration and subchondral bone alterations. Moreover, the treatment markedly decreased intra-articular levels of proinflammatory cytokines (IL-1β and TNF-α) and the cartilage degradation marker CTX-II in a time-dependent manner. These findings indicated that the sustained local delivery of AD-MSC-derived supernatant effectively modulated joint inflammation and attenuated cartilage degeneration, with the hydrogel serving primarily as a delivery vehicle for these bioactive factors. This cell-free injectable biomaterial platform could offer a promising therapeutic strategy for the treatment of knee osteoarthritis.

1. Introduction

Knee osteoarthritis (KOA) is a prevalent degenerative joint disease characterized by progressive cartilage destruction, synovial inflammation, and subchondral bone remodeling [1,2]. It represents a leading cause of pain, disability, and reduced quality of life worldwide. Current clinical treatments, including nonsteroidal anti-inflammatory drugs and intra-articular corticosteroids, primarily provide symptomatic relief and fail to halt disease progression [3,4,5,6,7,8]. Consequently, the development of disease-modifying strategies that target both inflammation and cartilage degeneration remains an unmet clinical need [9,10].

Mesenchymal stem cells (MSCs) have attracted considerable attention for osteoarthritis treatment due to their immune-modulatory and regenerative properties [11,12,13,14]. Among them, adipose-derived mesenchymal stem cells (AD-MSCs) are particularly appealing because of their abundance, ease of isolation, and robust secretory activity [15]. Increasing evidence suggests that the therapeutic benefits of MSCs are largely mediated by paracrine mechanisms rather than direct cell engraftment or differentiation [16,17]. MSC-derived secretome contain a broad spectrum of bioactive factors, including cytokines [18], interleukins [19], growth factors [20], and extracellular vesicles [21], which collectively modulate inflammation, inhibit cartilage matrix degradation, and promote tissue repair [22]. However, the direct administration of MSC secretome is limited by rapid diffusion, short residence time, and reduced bioavailability within the joint cavity.

Injectable hydrogels have emerged as promising delivery platforms for intra-articular therapies due to their ability to provide localized retention and controlled release with a minimally invasive administration [23]. Thermosensitive hydrogels, in particular, enable liquid injection at low temperatures followed by in situ gelation at physiological temperatures, making them well-suited for joint applications. Pluronic F-127 (PF-127), a triblock copolymer with excellent thermosensitive properties, has been widely explored for injectable hydrogel systems [24,25,26]. Sodium hyaluronate (HA), a natural component of synovial fluid and cartilage extracellular matrix, offers high biocompatibility, lubrication, and chondro protective effects [27,28,29,30]. Combining PF-127 and HA provides a synergistic scaffold with favorable physicochemical and biological characteristics for cartilage-related applications.

In this study, we have developed a cell-free, thermosensitive composite hydrogel by incorporating AD-MSC supernatant into a PF-127/HA matrix. The hydrogel is designed to achieve injectable delivery, rapid in situ gelation, and a sustained release of bioactive factors without the use of chemical crosslinkers. We have systematically characterized its physicochemical properties and microstructure and evaluated its therapeutic efficacy in a rat model of knee osteoarthritis. Radiological, behavioral, histological, and biochemical analyses have been performed to assess joint inflammation, cartilage degeneration, and functional recovery. By integrating AD-MSC-derived bioactivity with an injectable hydrogel platform, this work aimed to provide a translational strategy for the treatment of knee osteoarthritis.

2. Results

2.1. Isolation and Characterization of Adipose-Derived Mesenchymal Stem Cells (AD-MSCs)

Adipose-derived mesenchymal stem cells (AD-MSCs) were successfully isolated from subcutaneous abdominal adipose tissue of rabbits and were expanded in vitro under serum-free culture conditions. Primary cultures predominantly consisted of adherent cells with a short spindle-shaped morphology, accompanied by a small proportion of polygonal cells scattered throughout the culture. By the third passage, the cells exhibited stable adherence, good viability, and a homogeneous spindle or fusiform morphology, typically arranged in a whirlpool-like pattern (Figure 1A).

Flow cytometric analysis was performed to evaluate the expression of surface antigen markers characteristic of AD-MSCs. The results demonstrated high expression levels of CD73 and CD90 (81.4% and 86.0%, respectively), while the hematopoietic marker CD34 was minimally expressed (0.64%) (Figure 1B), consistent with the established immunophenotypic profile of AD-MSCs.

The multipotent differentiation capacity of AD-MSCs was further confirmed through adipogenic, osteogenic, and chondrogenic differentiation assays (Figure 1C). Following 10 days of adipogenic induction, numerous small and dispersed lipid droplets were observed under light microscopy, accompanied by a gradual transition of cell morphology from spindle-shaped to rounded. Oil Red O staining revealed lipid droplets by staining them red. After 21 days of osteogenic induction, cells transformed from spindle-shaped to a polygonal morphology, with increased cell size, enhanced cytoplasmic projections, and enlarged, rounded nuclei. Fine extracellular mineralized nodules formed island-like aggregates, which were stained orange-red with Alizarin Red S. During chondrogenic induction, noticeable morphological changes were observed as early as day 7, characterized by extensive vacuole formation. Alcian Blue staining revealed blue-stained cartilaginous nodules, indicating the presence of acidic polysaccharides and confirming chondrogenic differentiation.

Collectively, these results demonstrate that viable high-purity AD-MSCs with characteristic immunophenotypes and robust multilineage differentiation potential were successfully obtained.

2.2. Thermosensitive Behavior and Microstructural Features of the AD-MSC Supernatant-Loaded Composite Hydrogel

The AD-MSC supernatant was successfully incorporated with Pluronic F-127 (PF-127) and sodium hyaluronate (HA) to form a composite hydrogel without the use of chemical crosslinkers. The hydrogel exhibited pronounced thermosensitive behavior, remaining in a free-flowing liquid state at 4 °C and rapidly undergoing sol–gel transition at a physiological temperature (37 °C) to form a stable solid gel (Figure 2A). This temperature-dependent phase transition enabled injectable delivery at a low temperature and localized gelation during in vivo conditions.

Scanning electron microscopy of hydrogels gelled at 37 °C revealed a uniformly distributed porous microstructure (Figure 2B). The hydrogel displayed small, regularly shaped pores interconnected by continuous channels, forming a well-defined three-dimensional network. This porous architecture provided a high surface area and strong fluid absorption capacity, supporting the efficient loading and sustained release of bioactive factors from the AD-MSC supernatant. The interconnected network further facilitated nutrient diffusion and gas exchange, creating a favorable microenvironment for tissue repair.

Collectively, these results demonstrated that the AD-MSC supernatant-loaded composite hydrogel possessed injectable thermosensitive behavior and a highly porous internal structure, supporting its suitability for intra-articular delivery and localized retention of bioactive components.

2.3. Effects on Joint Behavior and Functional Recovery

To investigate the therapeutic efficacy of an AD-MSC supernatant hydrogel in knee osteoarthritis (KOA) and joint injury, a unilateral KOA model was established in the right-side knees of Sprague-Dawley rats and treated via intra-articular injection. Treatment outcomes were systematically evaluated using behavioral assessments, radiographic imaging, gross morphological observation, and histopathological analysis.

To assess the changes in knee joint function and locomotor recovery, behavioral scoring and gait analysis were performed at 2 week intervals (Figure 3A,B). Footprint analysis revealed regular, symmetrical gait patterns with uniform stride length in the sham group. In contrast, the rats in the KOA control group exhibited disorganized footprints and inconsistent stride lengths, indicative of impaired locomotor function. Mild lameness was evident at early stages and progressively worsened over time. By weeks 4 and 6, behavioral scores were significantly elevated, with some animals showing reluctance to bear weight on the affected limb. Marked differences in footprint-ink density further reflected pain-related avoidance and functional limitations. In the treatment group, behavioral scores began to decrease as early as 2 weeks post-injection, accompanied by an alleviation of lameness, increased range of motion, and improved weight-bearing capacity. By weeks 4 and 6, several animals exhibited near-normal gait patterns, with footprints largely symmetrical and comparable to those of the sham group. Quantitative analysis demonstrated a progressive increase in behavioral scores in the control group, whereas scores in the treatment group showed a sustained decline, with statistically significant differences between the groups at corresponding time points. These findings indicated that the AD-MSC-conditioned medium as a loaded hydrogel exerted a pronounced therapeutic effect on joint function recovery.

2.4. X-Ray Analysis and Histological Assessment

To further elucidate the structural basis underlying joint dysfunction, gross morphological and radiographic evaluations were conducted (Figure 4A,B). Gross observation revealed that the cartilage surfaces in the sham group were smooth, glossy, and semi-translucent, with well-defined margins and no evidence of osteophyte formation. In contrast, the KOA control group displayed progressively deteriorating joint morphology, characterized by dull and rough cartilage surfaces with yellowish or pale discoloration by week 2, pronounced cartilage defects and trabecular deformation by week 4, and visible joint effusion by week 6.

Rats in the treatment group exhibited a marked morphological improvement. The cartilage surfaces appeared smoother with a healthier luster, coloration approached normal levels, areas of cartilage degeneration were significantly reduced, and cartilage margins were more regular. Turbid joint fluid also notably decreased over time.

Radiographic analysis further corroborated these observations. In the sham group, knee joints displayed intact bone architecture, uniform bone density, clear joint spaces, and an absence of osteophytes. In the KOA control group, mild joint space narrowing was detectable as early as week 2, followed by progressive joint space loss, increased radiographic opacity, osteophyte formation, joint effusion, and subchondral bone sclerosis from weeks 4 to 6, reflecting ongoing cartilage degeneration and bone remodeling. In contrast, rats receiving the AD-MSC-conditioned medium as a loaded hydrogel showed substantial attenuation in these pathological changes. At 2 weeks, joint space and bone density were largely preserved. By weeks 4 and 6, although mild osteophyte formation was observed in some animals, joint spaces remained relatively clear, joint effusion was reduced, osteophyte development was limited, and subchondral sclerosis was significantly alleviated compared with the control group.

Histological examinations were performed to assess the microscopic alterations in cartilage structure and joint pathology (Figure 4C,D). In the sham group, hematoxylin and eosin (H&E) staining demonstrated smooth and intact cartilage surfaces, abundant chondrocytes with regular morphology, and an orderly cellular arrangement, with no evidence of fibrous hyperplasia or inflammatory cell infiltration. Toluidine blue (TB) staining showed uniformly intense blue-purple coloration, indicating a dense and homogeneous cartilage matrix with preserved biological function.

At week 2, the KOA control group exhibited inflammatory cell infiltration (black arrows) and the widespread disorganization of chondrocyte alignment (red arrows) through H&E staining. TB staining revealed a disrupted cartilage surface continuity (black arrows) and a reduced chondrocyte density (red arrows). In the treatment group, mild surface irregularities and decreased chondrocyte numbers were observed at this time point; however, TB staining showed largely continuous cartilage surfaces, suggesting the initiation of the reparative processes.

By week 4, the KOA control group displayed persistent inflammatory cell infiltration (black arrows) with fibrous tissue infiltration extending into the subchondral bone (yellow arrows). TB staining revealed exacerbated degenerative changes, including uneven cartilage surfaces, a loose matrix structure, and a severely disordered chondrocyte arrangement. In contrast, the treatment group showed a preserved subchondral bone architecture without significant fibrous proliferation. Chondrocytes gradually regained an organized arrangement, and TB staining demonstrated improved intensity and uniformity, indicating ongoing cartilage matrix restoration.

At week 6, severe cartilage destruction was evident in the KOA control group, with near-complete replacement of the cartilage by fibrous tissue (yellow arrows), extensive inflammatory cell infiltration (green arrows), and large areas of cartilage loss via TB staining. In contrast, the treatment group exhibited a smooth and intact cartilage surface, abundant chondrocytes with an orderly alignment, and extensive, uniformly deep blue-purple TB staining, indicative of a restored cartilage matrix composition.

Collectively, these results demonstrated that the AD-MSC-conditioned medium as a loaded hydrogel effectively ameliorated articular cartilage degeneration, promoted extracellular matrix repair, and exerted robust chondroprotective and regenerative regulatory effects in a rat model of knee joint injury.

2.5. Regulatory Effects of AD-MSC Supernatant-Loaded Hydrogel on Inflammation and Cartilage Degeneration

To evaluate the therapeutic effects of the AD-MSC supernatant-loaded hydrogel on intra-articular inflammatory responses and cartilage degeneration, we quantified the levels of inflammatory cytokines (IL-1β and TNF-α) and the cartilage metabolic marker CTX-II in rat synovial fluid using enzyme-linked immunosorbent assays (ELISA).

In the control group, IL-1β levels increased progressively over time (Figure 5A), with marked elevations at weeks 4 and 6, indicating an enhanced inflammatory response during KOA progression. In the treatment group, IL-1β levels were comparable to those of the control group at week 2 but were significantly reduced at week 4 and further decreased at week 6, approaching near-normal levels.

TNF-α expression in the control group showed a sustained time-dependent increase, reaching high levels at week 6, consistent with persistent immune activation and synovial inflammation (Figure 5B). In contrast, TNF-α levels in the treatment group did not differ at week 2 but were significantly suppressed from week 4 onward, with further reductions at week 6, demonstrating a robust anti-inflammatory effect.

CTX-II levels in the control group increased continuously over time (Figure 5C), reflecting ongoing cartilage matrix degradation and progressive joint degeneration. Treatment with the AD-MSC supernatant-loaded hydrogel significantly reduced CTX-II levels at week 4, with a more pronounced effect at week 6, indicating effective inhibition of cartilage matrix breakdown and the promotion of cartilage preservation.

Overall, the AD-MSC supernatant-loaded hydrogel markedly attenuated intra-articular inflammation and cartilage degeneration by suppressing proinflammatory cytokines and limiting cartilage matrix degradation. These findings provided strong evidence supporting its therapeutic potential for knee osteoarthritis.

3. Discussion

In this study, we developed an injectable thermosensitive composite hydrogel incorporating an adipose-derived MSC (AD-MSC) supernatant and demonstrated its therapeutic potential for knee osteoarthritis (KOA). By integrating bioactive factors derived from the AD-MSC supernatant with biocompatible scaffold materials, the hydrogel achieved localized retention, sustained bioactivity, and effective modulation of inflammation and cartilage degeneration in vivo. Collectively, our findings supported a cell-free, biomaterial-based strategy for KOA treatment.

A major challenge in stem-cell-based therapies is the limited survival, engraftment, and safety of transplanted cells. Increasing evidence has indicated that the therapeutic effects of MSCs are largely mediated by their secretome rather than direct cell replacement [31,32,33]. Published evidence further indicated that the use of MSC-derived secretome components—such as soluble proteins, growth factors, and extracellular vesicles/exosomes—exhibits substantially lower immunogenicity compared with direct cell transplantation [34]. On this basis, we adopted a serum-free culture system to obtain high-purity AD-MSC supernatant and used it as the primary therapeutic component. This approach avoided risks associated with live cell transplantation while preserving the broad anti-inflammatory and regenerative potential of MSC-derived bioactive factors.

Previous studies have reported that xenogeneic MSC-derived secretome did not induce significant immune rejection in rat models of osteoarthritis [35]. Consistent with these findings, in our study, the continuous monitoring of body weight, general health status, and local joint conditions revealed no evidence of weight loss, aggravated joint swelling, or other clinical signs suggestive of immune rejection throughout the experimental period, further supporting the low immunogenicity of MSC-derived products. AD-MSC from rabbits can be readily obtained through minimally invasive procedures and relatively straightforward isolation techniques. Moreover, rabbit AD-MSC demonstrate rapid in vitro proliferation and high culture stability, enabling the efficient generation of substantial quantities of bioactive secretome suitable for experimental and potential therapeutic applications [36,37,38].

Previous studies have reported that Pluronic F-127 (PF-127) hydrogel alone did not produce significant therapeutic effects, serving primarily as a biocompatible and widely used thermosensitive carrier for drug and bioactive factor delivery [39]. Although hyaluronic acid (HA) has demonstrated chondroprotective properties, its long-term efficacy, particularly at extended time points such as six months, has not consistently shown significant improvement [40]. In our study, the composite hydrogel constructed from PF-127 and HA exhibited pronounced thermosensitive behavior, enabling minimally invasive intra-articular injection followed by rapid gelation at physiological temperatures. This property is particularly advantageous for joint applications, where rapid clearance by synovial fluid can often limit the efficacy of soluble therapeutics. The sol–gel transition allowed the hydrogel to form in situ, promoting the localized retention of the AD-MSC supernatant at the lesion site and prolonging its biological activity.

Microstructural analysis revealed a uniformly porous and interconnected three-dimensional network, which is critical for both sustained release and biological function. The porous architecture facilitated efficient loading and gradual diffusion of bioactive factors while supporting nutrient transport and gas exchange. Such features have been known to enhance tissue compatibility and regenerative outcomes, and likely contributed to the observed reduction in inflammation and cartilage degeneration in the treated animals.

Therapeutically, the AD-MSC supernatant-loaded hydrogel significantly improved radiological features, joint morphology, and locomotor function in the KOA rat model. Histological analyses further demonstrated the preservation of cartilage structure as well as the reduction and attenuation of inflammatory infiltration. These structural improvements were accompanied by marked suppression of key inflammatory cytokines (IL-1β and TNF-α) and the cartilage degradation marker CTX-II. IL-1β and TNF-α are central mediators of synovial inflammation and cartilage catabolism, while CTX-II reflects ongoing collagen type II breakdown. Their coordinated downregulation indicated that the hydrogel not only mitigated inflammation but also directly interfered with the molecular processes driving cartilage degeneration [41,42].

Notably, the therapeutic effects of the hydrogel became more pronounced over time, suggesting a sustained and cumulative biological impact. This time-dependent efficacy was consistent with a controlled release from the hydrogel matrix and supported its role as a long-acting intra-articular therapeutic system. Compared with conventional viscosupplements or anti-inflammatory drugs, this strategy can offer the combined advantages of biological regulation, structural support, and prolonged residence time [43,44,45].

Despite the encouraging findings of this study, several limitations should be considered. Although toluidine blue staining provided a reliable semi-quantitative assessment of cartilage matrix alterations and enabled evaluation of cartilage degeneration and repair, additional approaches—such as Safranin O staining, OARSI scoring, and COL2 immunohistochemistry—could further enhance the comprehensiveness and standardization of histological assessments. Moreover, the present study primarily focused on the overall therapeutic effect at the level of the entire knee joint, offering a holistic perspective on joint repair. While we believe this approach appropriately reflected global disease modification, separate and more detailed analyses of the femoral and tibial cartilage may provide additional region-specific insights. Future studies will aim to incorporate more refined regional evaluations and to validate the therapeutic efficacy in large animal models, thereby strengthening the evidence base for potential clinical translation.

4. Materials and Methods

4.1. Isolation, Culture, and Identification of Adipose-Derived Mesenchymal Stem Cells (AD-MSCs)

New Zealand rabbits were anesthetized with ether prior to the collection of adipose tissue from the inguinal region. After removing blood vessels and connective tissues, the isolated adipose tissue was repeatedly rinsed, minced into small fragments, and then centrifuged at 1300 rpm for 5 min. An equal volume of adipose tissue digestive enzyme mixture was added, followed by oscillatory digestion at 37 °C for 30–60 min. The digestion reaction was terminated with serum-free complete medium. The resulting filtrate was passed through a 100-mesh cell strainer and centrifuged again at 1300 rpm for 5 min. After discarding the supernatant, the cell pellet was resuspended in a serum-free medium. The cell concentration was adjusted to 1 × 10⁵ cells/mL, and the cells were seeded into T25 culture flasks, which were then incubated in a humidified incubator at 37 °C with 5% CO₂. The first medium change was performed 48 h after seeding, and subsequent medium changes were carried out every 3 days. When the cell confluency reached 80–90%, the cells were passaged. For phenotypic identification, well-growing third-passage (P3) cells were adjusted to a density of 1 × 10⁵ cells/mL and seeded into 24-well plates. Flow cytometry was used to detect the expression of surface markers including CD73, CD90, and CD34. Additionally, the adipogenic (Oil Red O staining), osteogenic (Alizarin Red S staining), and chondrogenic (Alcian Blue staining) differentiation potentials of P3 cells were evaluated, and the adipogenic, osteogenic, and chondrogenic differentiation assays results were observed under a microscope.

4.2. Preparation, Thermosensitivity Detection, and Scanning Electron Microscopy (SEM) Observation of AD-MSC-Conditioned Medium Hydrogel

Adipose-derived mesenchymal stem cells were cultured until the third passage (80% cell confluency), after which the medium was replaced with a low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) for an additional 48 h of culture. The conditioned medium was then collected and temporarily stored at 4 °C. Subsequently, the conditioned medium was centrifuged at 4000 rpm for 30 min to remove cell debris, followed by sterile filtration through a 0.22 µm filter to obtain the AD-MSC conditioned medium. This conditioned medium was mixed with Pluronic F-127 (PF-127) and sodium hyaluronate (HA) at a ratio of 20% PF-127/0.3% HA.

An aliquot of 1 mL of the mixture was pipetted into a 2 mL vial and placed in a biochemical incubator at 37 °C to determine the hydrogel properties. The morphological transition process (sol–gel phase transition) of the hydrogel was dynamically observed by incubating the samples at 4 °C (low-temperature environment) and 37 °C (simulated in vivo physiological temperature environment) to systematically evaluate the thermosensitive characteristics of the hydrogel.

After solidification at 37 °C, the prepared hydrogel was fixed, subjected to gradient dehydration, and freeze-dried. Following gold sputtering, the internal microstructure of the hydrogel was observed and characterized using a scanning electron microscope (SEM).

4.3. Establishment and Grouping of Animal Models

A total of 35 healthy male Sprague-Dawley rats aged 8 weeks with a body weight of 220 g were randomly selected (Changsheng Bio, Shenyang, China). Five were assigned to the blank group, while the remaining 30 rats underwent unilateral modeling of the right-side knee joint. After anesthesia, the rats were fixed in a supine position, and a 3 cm longitudinal incision was made on the medial side of the right-side knee joint. The patellar ligament was exposed and subjected to blunt dissection; subsequently, the medial meniscal ligament, medial collateral ligament (MCL), medial meniscus, and anterior cruciate ligament (ACL) were transected. The wound was irrigated, repositioned, and sutured. Rats in the sham operation group only underwent exposure of the patellar ligament followed by direct suturing without further surgical intervention. Postoperatively, the rats were observed daily, and penicillin was intramuscularly injected once a day for three consecutive days to prevent infection. After the successful establishment of the model, the rats were randomly divided into the model group and the hydrogel treatment group (15 rats per group). Following the surgical induction of osteoarthritis, the rats were housed under standard conditions for two weeks, during which they gradually developed typical early KOA symptoms. Once successful modeling was confirmed, the corresponding injections were initiated. The model group received an intra-articular injection of normal saline once a week, whereas the hydrogel treatment group was administered 100 µL of hydrogel via intra-articular injection once a week for six consecutive weeks. The blank group received no treatment. Evaluations were performed at two, four, and six weeks after the initiation of treatment.

4.4. Multidimensional Evaluation Indicators for Knee Osteoarthritis (KOA)

Knee osteoarthritis (KOA) was evaluated using multiple indicators, including behavioral assessment, gross observation, imaging examination, and histopathological section analysis.

Behavioral Assessment: Tests were performed every two weeks during the administration period. The plantar surfaces of the rats’ hind paws were coated with ink, and the rats were placed on a runway (1 m in length × 0.21 m in width) to walk freely. Scoring was based on the color intensity of the ink footprints on the runway using the following criteria: 0 points: the ink color of the left and right footprints was consistent; 1 points: the color of the left footprint was less than 25% darker than that of the right footprint; 2 points: the color of the left footprint was 25–50% darker than that of the right footprint; 3 points: the color of the left footprint was 50–75% darker than that of the right footprint; 4 points: the color of the left footprint was 75% or more darker than that of the right footprint.

Scoring was based on the color intensity of the ink footprints on the runway using the following criteria:

Score	Scoring Criterion
0	The ink color of the left and right footprints was consistent
1	The color of the left footprint was less than 25% darker than that of the right footprint
2	The color of the left footprint was 25–50% darker than that of the right footprint
3	The color of the left footprint was 50–75% darker than that of the right footprint
4	The color of the left footprint was 75% or more darker than that of the right footprint

Gross Observation of Articular Cartilage: A preliminary assessment of the degree of cartilage repair was conducted. The pathological changes in the repair tissue in the injured area were carefully examined, including color, luster, irregularity, and the presence of depressions or protrusions.

Imaging Examination: Examinations were performed prior to treatment and then once every two weeks during the treatment period. Rats were anesthetized and fixed in an appropriate position to stabilize the affected thigh. A high-frequency X-ray machine was used to obtain anteroposterior (AP) view images of the knee joint for detecting intra-articular changes, such as osteophyte formation, synovial fluid secretion, and cartilage damage.

Histopathological Sections: The rats were euthanized, and the tissues of the right-side knee joint (cartilage samples) were harvested. After fixation in paraformaldehyde for 24 h, the tissues were decalcified in 10% EDTA (pH 7.4) for 21 days, embedded in paraffin, and cut into 5 μm thick sections. Serial sections were obtained from the medial and lateral compartments at 200 μm intervals. The selected sections were deparaffinized in xylene, rehydrated through a graded ethanol–water series, and subsequently stained with hematoxylin and eosin (H&E) and toluidine blue (TB) to observe the histopathological changes.

4.5. Detection of Inflammatory Factors and Chondrometabolic Markers

After the rats were anesthetized, synovial fluid was collected, and commercial sandwich enzyme-linked immunosorbent assay (ELISA) kits were used to determine the levels of interleukin-1β (IL-1β, ABclonal Biology Inc., Wuhan, China, Catalogue No.: RK00009), tumor necrosis factor-α (TNF-α, ABclonal Biology Inc., Wuhan, China, Catalogue No.: RK00029), and C-terminal telopeptide of type II collagen (CTX-II, Jianglai Biology Inc., Shanghai, China, Catalogue No.: JL52522).

4.6. Data Analysis

All data were analyzed using SPSS 24.0 and GraphPad Prism 8.0.1. Group differences were compared using t-tests (and nonparametric tests). A p < 0.05 was considered statistically significant, while a p < 0.01 was considered highly significant.

5. Conclusions

This study demonstrates that an AD-MSC supernatant-loaded thermosensitive composite hydrogel can effectively suppress intra-articular inflammation, inhibit cartilage matrix degradation, and promote cartilage repair in KOA. By combining the advantages of cell-free regenerative therapy with an injectable biomaterial platform, this approach represents a promising and translational strategy for osteoarthritis treatment.