关键词: 静电纺丝, 脱细胞软骨基质, 光交联改性, 软骨修复, 组织工程, 软骨再生

Abstract: BACKGROUND: Tissue engineering strategies, particularly those based on decellularized cartilage matrix and biomimetic scaffolds, offer new therapeutic directions for cartilage regeneration. 
OBJECTIVE: To develop a photocrosslinkable nanofiber scaffold loaded with decellularized cartilage matrix and evaluate its potential for cartilage tissue engineering.
METHODS: (1) Decellularized cartilage matrix was prepared by enzymatic digestion and a combined chemical-ultrasonic method. A photocrosslinkable polyvinyl alcohol-glycidyl methacrylate polymer was synthesized. 8% polymer solution and 3% decellularized cartilage matrix solution were mixed at volume ratios of 8:1, 4:1, and 2:1 to prepare electrospinning precursor solutions. Nanofiber scaffolds were fabricated using electrospinning technology, designated as PVA-GMA-dECM(8:1), PVA-GMA-dECM(4:1), and PVA-GMA-dECM(2:1), respectively. A polyvinyl alcohol-glycidyl methacrylate nanofiber scaffold (PVA-GMA) was also prepared. The microstructure of each scaffold was characterized. Chondrocytes were co-cultured non-contact with the four types of ultraviolet-irradiated crosslinked scaffolds using Transwell chambers, and cell proliferation was detected by the CCK-8 assay. Based on morphological observations and CCK-8 results, a suitable scaffold was selected for subsequent experiments. (2) The surface water contact angle and maximum tensile stress of PVA-GMA, PVA-GMA-dECM(8:1), and PVA-GMA-dECM(4:1) were measured. Based on these results, a suitable scaffold was selected for further experiments. Chondrocytes were co-cultured non-contact with ultraviolet-irradiated crosslinked PVA-GMA and PVA-GMA-dECM(4:1) using Transwell chambers. EdU staining and live/dead staining were used to assess cell proliferation and viability, and scratch assay and invasion assay were used to evaluate cell migration ability. (3) Using a Transwell chamber, chondrocytes were co-cultured non-contact with ultraviolet-irradiated cross-linked PVA-GMA and PVA-GMA-dECM (4:1). After chondrogenic induction culture, the chondrogenic differentiation efficiency was evaluated by Alcian blue staining, RT-qPCR (detecting type II collagen and Sox9 mRNA expression), immunofluorescence staining (detecting type II collagen and aggrecan expression), and western blot assay (detecting type II collagen and Sox9 protein expression).
RESULTS AND CONCLUSION: (1) Scanning electron microscopy showed that a high proportion of decellularized cartilage matrix (2:1) affected the fiber morphology, while a 4:1 ratio yielded well-structured nanofibers. CCK-8 assay showed that PVA-GMA-dECM promoted ATDC5 chondrocyte proliferation compared with PVA-GMA. Based on the results of these two experiments, PVA-GMA-dECM (2:1) was excluded from subsequent experiments. The water contact angle of PVA-GMA-dECM (4:1) was lower than that of PVA-GMA and PVA-GMA-dECM (8:1), and the maximum tensile strength was greater than that of PVA-GMA and PVA-GMA-dECM (8:1). Therefore, PVA-GMA-dECM (8:1) was excluded from subsequent experiments. Live/dead staining and EdU staining showed that both PVA-GMA and PVA-GMA-dECM (4:1) maintained cell viability, and PVA-GMA-dECM (4:1) had a better cell proliferation effect than PVA-GMA. PVA-GMA-dECM (4:1) had a stronger cell migration ability than PVA-GMA. PVA-GMA-dECM (4:1) had a stronger chondrogenic differentiation efficiency than PVA-GMA. (2) The results show that PVA-GMA-dECM (4:1) exhibits excellent bioactivity and can effectively promote chondrocyte proliferation, migration, and chondrogenic differentiation, making it a promising scaffold material for cartilage tissue engineering.

Key words: electrospinning, decellularized cartilage matrix, photocrosslinking modification, cartilage repair, tissue engineering, cartilage regeneration