添加i-PRF的不同静电纺丝方式对肌腱干细胞分化的影响

Effects of Different Electrospinning Methods with i-PRF on the Differentiation of Tendon Stem Cells

摘要: 目的：本实验主要为了研究可注射型富血小板纤维蛋白通过普通静电纺丝与同轴静电纺丝两种方式添加到纳米纤维中后，对于肌腱干细胞分化的影响。方法：已知富血小板纤维蛋白有着促进肌腱–骨愈合的作用。本研究通过两种不同静电纺丝方式，制备了两种纳米纤维支架。实验①组：普通静电纺丝：以体积比等于3:1的乙酸甲酸为溶剂，将i-PRF和PCL (聚酯纤维)分别溶解后进行静电纺丝。实验②组：同轴静电纺丝：以体积比为等于3:1的乙酸甲酸为溶剂，将PCL (聚酯纤维)溶解作为壳层而i-PRF作为芯层，进行同轴静电纺丝。之后我们在材料上进行肌腱干细胞的种植，并在第3、7天行CCK-8检测；另外在第7天我们进行了RunX2和Tenascin C的荧光检测，通过两个实验验证两种材料对肌腱干细胞分化的影响。结果：CCK-8实验结果显示肌腱干细胞在两种材料上均可增殖，3天时，对照组及两实验组无明显差异，7天时，对照组OD值高于两实验组，两实验组无明显差异，我们考虑是材料诱导肌腱干细胞进行了分化，故增殖减少。7天时，免疫荧光结果显示，同轴静电纺丝组的RunX2和Tenascin C荧光强度更高，与普通静电纺丝组有着明显差异(p < 0.05, p < 0.01)，即该组对肌腱干细胞分化有着更明显的促进作用。结论：同轴静电纺丝相比普通静电纺丝能更好地保留i-PRF的生物活性，能更好地诱导肌腱干细胞的成骨和成肌腱分化，对于负载生物活性物质的纳米纤维支架制备提供一种新的思路。

Abstract: Objective: the main purpose of this study was to study the effect of injectable platelet-rich fibrin on the differentiation of tendon stem cells after being added to nanofibers by ordinary electrospinning and coaxial electrospinning. Methods: it is known that platelet-rich fibrin can promote tendon-bone healing. In this study, two kinds of nanofiber scaffolds were prepared by two different electrospinning methods. Experimental group 1: ordinary electrospinning: i-PRF and PCL (polyester fiber) were dissolved in acetic acid formic acid equal to 3:1 by volume and then electrospun. Experimental group 2: coaxial electrospinning was carried out with acetic acid formic acid (volume ratio = 3:1) as solvent, PCL (polyester fiber) as shell and i-PRF as core. After that, we implanted tendon stem cells on the material, and detected CCK-8 on the 3^rd and 7^th day. In addition, on the 7^th day, we carried out the fluorescence detection of RunX2 and Tenascin C, and verified the effect of the two materials on the differentiation of tendon stem cells through two experiments. Results: the results of CCK-8 experiment showed that tendon stem cells could proliferate on both materials. On the 3^rd day, there was no significant difference between the control group and the two experimental groups. On the 7^th day, the OD value of the control group was higher than that of the two experimental groups, and there was no significant difference between the two experimental groups. We considered that the material induced the differentiation of tendon stem cells, so the proliferation decreased. On the 7^th day, the immunofluorescence results showed that the fluorescence intensity of RunX2 and Tenascin C in the coaxial electrospinning group was significantly higher than that in the ordinary electrospinning group (p < 0.05, p < 0.01), that is, this group could promote the differentiation of tendon stem cells more obviously. Conclusion: compared with conventional electrospinning, coaxial electrospinning can better retain the biological activity of i-PRF and better induce osteogenesis and tendon differentiation of tendon stem cells. This provides a new idea for the preparation of nanofiber scaffolds loaded with bioactive substances.