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29Three-dimensional (3D)-printed bioactive scaffolds that can be produced rapidly could offer an individualized approach for treating full-thickness skin defects. Decellularized extracellular matrix (dECM) and mesenchymal stem cells have been proven to support wound healing. Adipose tissues obtained by liposuction are rich in adipose-derived dECM (adECM) and adipose-derived stem cells (ADSCs) and thus represent a natural source of bioactive materials for 3D bioprinting. Herein, ADSC-laden 3D-printed bioactive scaffolds consisting of gelatin methacryloyl (GelMA), hyaluronic acid methacryloyl (HAMA), and adECM were fabricated with dual properties of photocrosslinking in vitro and thermosensitive crosslinking in vivo . adECM was prepared by decellularization of human lipoaspirate and mixed as a bioactive material with GelMA and HAMA to form a bioink. Compared with the GelMA-HAMA bioink, the adECM-GelMA-HAMA bioink had better wettability, degradability, and cytocompatibility. Full-thickness skin defect healing in a nude mouse model showed that ADSC-laden adECM-GelMA-HAMA scaffolds accelerated wound healing by promoting faster neovascularization, collagen secretion, and remodeling. ADSCs and adECM collectively conferred bioactivity on the prepared bioink. This study represents a novel approach to enhancing the biological activity of 3D-bioprinted skin substitutes by adding adECM and ADSCs derived from human lipoaspirate and may provide a promising therapeutic option for full-thickness skin defects., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2023, Zhang D, Fu Q, Fu H, et al.)

Objective: To explore the feasibility of three-dimensional (3D) bioprinted adipose-derived stem cells (ADSCs) combined with gelatin methacryloyl (GelMA) to construct tissue engineered cartilage., Methods: Adipose tissue voluntarily donated by liposuction patients was collected to isolate and culture human ADSCs (hADSCs). The third generation cells were mixed with GelMA hydrogel and photoinitiator to make biological ink. The hADSCs-GelMA composite scaffold was prepared by 3D bioprinting technology, and it was observed in general, and observed by scanning electron microscope after cultured for 1 day and chondrogenic induction culture for 14 days. After cultured for 1, 4, and 7 days, the composite scaffolds were taken for live/dead cell staining to observe cell survival rate; and cell counting kit 8 (CCK-8) method was used to detect cell proliferation. The composite scaffold samples cultured in cartilage induction for 14 days were taken as the experimental group, and the composite scaffolds cultured in complete medium for 14 days were used as the control group. Real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect cartilage formation. The relative expression levels of the mRNA of cartilage matrix gene [(aggrecan, ACAN)], chondrogenic regulatory factor (SOX9), cartilage-specific gene [collagen type Ⅱ A1 (COLⅡA1)], and cartilage hypertrophy marker gene [collagen type ⅩA1 (COLⅩA1)] were detected. The 3D bioprinted hADSCs-GelMA composite scaffold (experimental group) and the blank GelMA hydrogel scaffold without cells (control group) cultured for 14 days of chondrogenesis were implanted into the subcutaneous pockets of the back of nude mice respectively, and the materials were taken after 4 weeks, and gross observation, Safranin O staining, Alcian blue staining, and collagen type Ⅱ immunohistochemical staining were performed to observe the cartilage formation in the composite scaffold., Results: Macroscope and scanning electron microscope observations showed that the hADSCs-GelMA composite scaffolds had a stable and regular structure. The cell viability could be maintained at 80%-90% at 1, 4, and 7 days after printing, and the differences between different time points were significant ( P <0.05). The results of CCK-8 experiment showed that the cells in the scaffold showed continuous proliferation after printing. After 14 days of chondrogenic induction and culture on the composite scaffold, the expressions of ACAN, SOX9, and COLⅡA1 were significantly up-regulated ( P <0.05), the expression of COLⅩA1 was significantly down-regulated ( P <0.05). The scaffold was taken out at 4 weeks after implantation. The structure of the scaffold was complete and clear. Histological and immunohistochemical results showed that cartilage matrix and collagen type Ⅱ were deposited, and there was cartilage lacuna formation, which confirmed the formation of cartilage tissue., Conclusion: The 3D bioprinted hADSCs-GelMA composite scaffold has a stable 3D structure and high cell viability, and can be induced differentiation into cartilage tissue, which can be used to construct tissue engineered cartilage in vivo and in vitro .