In the field of vascular tissue engineering,current technological advancements are focused on the proli-feration and secretion of collagen-rich extracellular matrix(ECM)by vascular smooth muscle cells(VSMCs)in vitro,coupled with specific mechanical stimuli to reconstruct biomaterials with desired mechanical properties.The opti-mal oxygen concentration is crucial for three-dimensional cell culture in vitro.However,there is a lack of sufficient research regarding its application in tissue-engineered vascular graft(TEVG).To this end,this study designed vary-ing oxygen concentration environments to determine the optimal value for VSMCs culture by assessing cell prolifera-tion activity.Furthermore,collagen gene expression and protein secretion were measured under normoxic and hy-poxic conditions,along with quantification of total collagen content deposited in the cell layer using hydroxyproline assay.Subsequently,VSMCs were seeded on polyglycolic acid(PGA)scaffolds for three-dimensional culturing to form TEVG,and the effects of hypoxic conditions on TEVG cultivation were observed through histological staining and total collagen quantification.The results indicate that VSMC cell activity increased most rapidly at 7%oxygen concentration;under hypoxic conditions,an upregulation of type I(Col I)and type Ⅲ(Col Ⅲ)collagen gene expres-sion was observed,with an increased secretion of collagen into the culture medium between days 5 and 9,particu-larly Col Ⅲ.Furthermore,the total collagen content on the 11th day demonstrates a 3.1-fold increase relative to the normoxic group.The 7%oxygen concentration facilitated collagen deposition during the three-dimensional cul-ture of VSMCs on PGA scaffolds,resulting in a 2.09-fold increase in total collagen content compared to the nor-moxic group.Additionally,the formation of more dense collagen fibrils was observed.These findings indicate that the utilization of a hypoxic environment can enhance the collagen content in TEVG,thereby providing a foundation for the further optimization of in vitro culture conditions for TEVG.
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