Articular cartilage defects are challenging to repair due to the avascular, aneural, and lymphatic nature of the tissue and the complexities of regenerating its structure to meet mechanical demands. Current strategies, including auto/allografts and stem cell therapies, have limitations such as secondary trauma, low cell survival rates, and fibrocartilage formation. The incorporation of stem cells into biomaterials offers promise. While adipose-derived stem cells (ADSCs), bone marrow mesenchymal stem cells (BMSCs), and synovial membrane mesenchymal stem cells (SM-MSCs) have been explored, they have drawbacks like invasive acquisition and limited proliferation. Urine-derived stem cells (USCs) are a novel type of MSC with potential for robust proliferation and multipotent differentiation. Previous research has shown their chondrogenic potential *in vitro*, but *in vivo* studies are scarce. Biomaterials for cartilage tissue engineering include natural materials (chitosan, collagen, gelatin, fibrin) and synthetic materials (PEG, PCL, PLA). Synthetic materials may lack tissue integration, while natural materials can trigger immune responses. Decellularized extracellular matrix (dECM) offers a potential solution by removing immunogenic cells while retaining bioactive components. This study uses an injectable dECM hydrogel combined with USCs to address the challenges of cartilage regeneration, focusing on the immunomodulatory capacity of the material and the role of macrophages (M1 and M2 subtypes) in the repair process.

The introduction extensively reviews existing literature on cartilage defect repair strategies, highlighting the limitations of autologous chondrocyte implantation (ACI) and the use of various stem cell types (ADSCs, BMSCs, SM-MSCs). It discusses the challenges associated with current biomaterials, emphasizing the advantages of decellularized extracellular matrix (dECM) due to its biocompatibility and reduced immunogenicity. The literature review also underscores the importance of considering the immunomodulatory capacity of biomaterials, particularly the role of macrophages in influencing cartilage repair.

The study involved isolating and characterizing human USCs from urine samples, assessing their proliferation capacity using CCK-8 and CyQUANT assays, and confirming their MSC characteristics through flow cytometry analysis of surface markers. The multi-lineage differentiation potential of USCs was evaluated using osteogenic, adipogenic, and chondrogenic induction media, followed by staining and RT-qPCR analysis. An injectable dECM hydrogel was developed from pig articular cartilage through a decellularization process involving lyophilization and enzymatic digestion. The dECM hydrogels were characterized using various techniques, including SEM, FT-IR, and rheometry, to assess their structure, composition, and mechanical properties. The biocompatibility of the dECM hydrogels was assessed using CCK-8, Live/Dead staining, and SEM, evaluating USC proliferation and morphology within the hydrogel. The chondrogenic capacity of the USCs within the dECM hydrogel was determined *in vitro* using chondrogenic induction medium and assessing GAGs, collagen II production, and expression of chondrogenic genes (ACAN, SOX9, COL-II). The immunomodulatory effects of the dECM hydrogel were evaluated *in vitro* using RAW264.7 macrophages, analyzing macrophage polarization (M1 vs M2) through microscopy, immunofluorescence staining (iNOS, CD206), flow cytometry (CD86, CD206), and RT-qPCR (iNOS, TNF-α, ARG-1, CD206). *In vivo* studies used a rat model of full-thickness cartilage defect, comparing the regenerative capacity of USCs alone, dECM hydrogel alone, and the combination of both. Cartilage regeneration was assessed through gross observation, H&E staining, Safranin O/Fast Green staining, immunohistochemistry (Aggrecan, COL-I, COL-II), and ICRS macroscopic scoring.

USCs exhibited excellent proliferation and multipotency, expressing MSC markers and differentiating into osteoblasts, adipocytes, and chondrocytes. The dECM hydrogels displayed appropriate gelation properties and a porous structure. *In vitro*, the dECM hydrogels demonstrated good biocompatibility, supporting USC proliferation and chondrogenic differentiation, significantly increasing GAG and collagen II production and expression of chondrogenic genes in the presence of chondrogenic induction medium. The dECM hydrogels demonstrated immunomodulatory effects, promoting M2 macrophage polarization, reducing pro-inflammatory cytokine expression (iNOS, TNF-α), and increasing anti-inflammatory markers (CD206, ARG-1). *In vivo*, in a rat cartilage defect model, the USCs-laden dECM hydrogels showed superior cartilage regeneration compared to controls (USCs alone, dECM alone, or untreated defects). This was evidenced by gross observation, histological analysis (H&E, Safranin O staining), immunohistochemistry (Collagen I, II, Aggrecan), and ICRS scoring. The combination of USCs and dECM hydrogel resulted in significantly better cartilage repair and integration with the subchondral bone compared to either treatment alone.

The study successfully demonstrated the potential of an injectable dECM hydrogel encapsulating USCs for cartilage regeneration. The findings highlight the synergistic effects of the biomaterial and the stem cells, with the dECM hydrogel providing a suitable microenvironment for chondrogenesis and the USCs contributing to both tissue regeneration and immunomodulation. The immunomodulatory capacity of the dECM hydrogel, favoring M2 macrophage polarization, is a crucial aspect contributing to the success of cartilage repair. The *in vivo* results confirm the efficacy of the USCs-laden dECM hydrogel in promoting cartilage regeneration and structural restoration in a rat model. The results support the further development of this approach as a promising therapeutic strategy for cartilage defect repair.

This study successfully developed an injectable dECM hydrogel combined with USCs for cartilage regeneration. The combination demonstrated superior chondrogenic and immunomodulatory capacities *in vitro* and *in vivo*, leading to enhanced cartilage repair. Future studies should focus on optimizing the hydrogel composition, exploring clinical translation, and investigating long-term outcomes in larger animal models.

The study used a rat model, which may not fully recapitulate human cartilage regeneration. The long-term effects of the USCs-laden dECM hydrogel on cartilage regeneration remain to be fully investigated in larger animal models and ideally through clinical trials. The sample size for the *in vivo* study could be considered relatively small, warranting further investigation with larger cohorts.