## 4 Discussion  
The findings of this study highlight the potential of pcECM-DM as a promising alternative to periosteal patches in ACI. The successful adhesion and uniform distribution of rabbit chondrocyte cells on the pcECM-DM, as demonstrated by DAPI-PKH26 staining, indicate that this novel biomaterial provides a conducive environment for cell attachment and growth. Post-cell-seeding, the cells successfully attached to the pcECM-DM, indicating that the membrane provides a conducive environment for cell adhesion. The observed increase in cell density at 48 h compared to 24 h suggests that the pcECM-DM supports initial cell attachment and promotes cell proliferation over time. This is a critical factor for effective cartilage regeneration, as higher cell density is essential for forming a robust ECM. [PERSON] et al. discussed cell adhesion as a dynamic process resulting from specific interactions between cell surface molecules and their appropriate ligands, emphasizing the role of ECM in supporting cell adhesion and proliferation [27]. [PERSON] et al. showed that the cell-derived ECM scaffold might provide a favorable environment functionally and structurally for chondrocytes [19]. The ability of the membrane to support cell adhesion and proliferation underscores its suitability for cartilage repair applications. The live/dead assay results demonstrated a high proportion of viable cells at 24 and 48 h. This indicates that the pcECM-DM provides a supportive environment for cell survival and growth, which is crucial for successful cartilage regeneration. Additionally, the results demonstrated that the pcECM-DM can induce the proliferation of rabbit chondrocytes at both 24 and 48 h. The observed increase in cell proliferation over time highlights the ability of the pcECM-DM to support cell growth and viability. Similar studies have discussed the enhanced proliferation of chondrocytes, emphasizing the importance of biocompatibility and cellular proliferation for tissue engineering applications [28]. The results of this study demonstrate the successful chondrogenic differentiation of rabbit chondrocytes and bone marrow-derived mesenchymal stem cells (BMSCs) on the  
Figure 7: Proteomics analyzed the function diagram of pcECM-DM. pcECM-DM. Safranin-O (SO) staining confirmed the presence of ECM production and typical chondrocyte morphology, indicating that the pcECM-DM provides a conducive environment for chondrogenesis. Chondrogenic differentiation was observed for chondrocytes, with and without TGF\(\beta\) treatment and with and without pcECM-DM. This suggests that the pcECM-DM alone is sufficient to support chondrogenic differentiation, and the addition of TGF\(\beta\) further enhances this process. For BMSCs, chondrogenic differentiation was observed in pcECM-DM treatment, both with and without TGF\(\beta\), and in TGF\(\beta\) treatment without pcECM-DM. However, chondrogenic differentiation could not be expressed without TGF\(\beta\) and pcECM-DM treatments, highlighting the synergistic effect of these two factors in promoting chondrogenesis. The ability of pcECM-DM to support chondrogenic differentiation and extracellular matrix production, both independently and in combination with TGF\(\beta\), suggests its suitability for clinical applications in cartilage repair. The results of this study provide valuable insights into the molecular functions of the pcECM-DM, as illustrated in the function diagram (Figure 7). The identified molecular functions include structural molecule activity, catalytic activity, and various binding activities such as anion binding, protein binding, glycosaminoglycan binding, and growth factor binding. These functions are crucial for maintaining the structural integrity and biochemical interactions of ECM. The significant enrichment of genes associated with ECM organization, cell adhesion, and cartilage development further underscores the potential of pcECM-DM in supporting tissue regeneration.