Study Of The Bone Mesenchymal Stem Cells Tenogenic Differentiation Induced By The Union Of Uniaxial Stretch And Aligned Nanofiber

Objective:(1)To evaluate the electrostatic spinning technique was used to prepare an orientation arrangement of polylactic acid polylactic acid(PLA)nanofiber scaffolds as tissue engineering ligament scaffold feasibility and advantages;(2)To observe the effects of nanofiber arrangement and uniaxial stretch on cytoskeleton and differentiation of bone mesenchymal stem cells(BMSCs);(3)To discuss and analyze the roles of rho-associated protein kinase(ROCK)and focal adhesion kinase(FAK)in the differentiation process of BMSCs into ligament cells induced by nanofiber arrangement and uniaxial stretch.Method:(1)Rabbit BMSCs were obtained by whole-bone marrow adherent method,and their osteogenic,lipogenic differentiation potential and surface antigen expression were detected.(2)Electrostatic spinning technology was used to prepare oriented and randomly arranged nanofiber scaffolds and to detect their mechanical properties.(3)The skeletal changes of BMSCs were observed by F-actin fluorescence staining under static and tensile conditions after 7 days of culture on aligned and random nanofiber scaffolds.After administration of ROCK inhibitor(Y-27632),BMSCs were cultured on nanofiber scaffolds for 7 days under static and tensile conditions,respectively,and cytoskeleton were observed.(4)After BMSCs were cultured on aligned and random nanofiber scaffolds under static and tensile conditions for 7 days,RT-PCR was used to detect the mRNA expression of ligament differentiation-related markers(COL1A2,COL3A1,Tenomodulin,Tenascin C),ROCK and FAK of BMSCs on aligned and random nanofiber scaffolds.After administration of Y-27632,the mRNA expression of the above gene was detected.(5)BMSCs were cultured on aligned and random nanofiber scaffolds under static and tensile conditions for 7 days respectively.Western blot was used to detect the expression of BMSCs ligament associated protein(Collagen ?,Collagen ?,Tenomodulin,Tenascin C),FAK,ROCK and osteogenic associated protein RUNX2 on aligned and random nanofiber scaffolds.After administration of Y-27632,the expression level of these proteins was detected.Results:(1)The rabbit BMSCs obtained by the whole bone marrow adherence method had good proliferation ability,and could differentiate into bone and lipid in a stable manner,and the expression of surface antigen was consistent with the characteristics of BMSCs.(2)The PLA aligned nanofiber scaffolds prepared by electrostatic spinning technology have good biocompatibility and mechanical properties close to human ligaments.(3)Under static conditions,the cytoskeleton on the aligned scaffold extended parallel to the direction of fiber arrangement,and the cells were fusiform,while the cytoskeleton on the random scaffold extended at will,and the cells were polygonal.Y-27632 inhibited the cytoskeleton on the aligned scaffold extended parallel to the direction of fiber arrangement.Under the tensile condition,the cytoskeleton was extended in parallel along the tensile direction,and Y-27632 significantly inhibited the cytoskeleton extension along the tensile direction.(4)Under static condition,compared with the random nanofiber scaffolds,the mRNA expression of ligament differentiation-related marker(COL3A1,Tenomodulin,Tenascin C)and the mRNA expression of ROCK and FAK increased significantly(P < 0.01).Under uniaxial stretch,compared with the static fiber scaffold,the mRNA expression of ligament differentiation-related marker(COL1A2,COL3A1,Tenomodulin)in BMSCs increased(P < 0.01),and the mRNA expression of ROCK and FAK also increased(P < 0.05).After administration of Y-27632,these mRNA levels decreased.(5)Under static condition,compared with the random nanofiber scaffolds,the expression of ligament related protein(Collagen ?,Collagen ?,Tenascin C)in BMSCs on the aligned nanofiber scaffolds increased,the expression of ROCK increased significantly(P < 0.05),and the expression of osteogenic related protein RUNX2 decreased.Under uniaxial tension,compared with the static fiber scaffold,the expression of(Collagen I,Collagen III,Tenascin C),ROCK and FAK increased,while the expression of RUNX2 decreased significantly(P < 0.01).After Y-27632 administration,the expression level of these proteins decreasedConclusion:(1)The aligned nanofiber scaffolds prepared from PLA by electrospinning has good mechanical properties,and BMSCs can achieve similar cell arrangement in ligament tissues,and have the potential to differentiate into ligament like cells,which can be used as ideal scaffolds for tissue-engineered ligaments(2)During the differentiation of BMSCs induced by uniaxial stretch combined with aligned nanofiber,the change of cytoskeleton plays an important role,which may be one of the early intracellular structural signals regulating the differentiation of BMSCs into ligament like cells.(3)In vitro small amplitude and low frequency cyclic uniaxial tensile stimulation combined with oriented nano scaffolds can promote the differentiation of BMSCs into ligament like cells.FAK and ROCK may be involved in the process of nanofiber arrangement and mechanical stimulation induced differentiation of BMSCs into ligament like cells,but the specific mechanism needs further study.