In Vitro Research On Bone Mesenchymal Stem Cells Inducing To Ligament Fibroblasts By Mechanical Stretching And Indirect Co-culture

BackgroundLigaments play a critical important role in stabilization of joint motion and joint stability. The incidence and chronic fatigue are the most common reasons of ligament disruptions. With lack of blood supplying, ligament has usually a poor healing capacity which leads to severe dysfunction of ligament. Many outstanding athletes have to give up their career forever. Four options have been utilized for the repair or replacement of damaged ligaments using xenograft, autograft, allograft and synthetic materials in surgical therapy. But all these methods are pressed for a long term promise. A Research on potential tissue engineered ligament has been going on for some time, with the hope of overcoming the present problem. Thus, it may provide an available solution in arthrosis treatment option for ligament reconstruction.Cells are very important in enforcing ligament properties, activating genes, and self-renewing of ligament in an artificial ligament. How to choose an ideal seeding cell? Which microenvironment condition is favorable for the seeding cells? These questions are worth to be researched. The objective of our present study is trying to induce BMSCs differentiating into ligament fibroblasts. We firstly, indirect cocultured BMSCs with ligament fibroblasts; secondly, applied uniaxial stretching on BMSCs, then changes of the ligament specific proteins in BMSCs were evaluated in order to test the feasibility to induce BMSCs differentiating into ligament.Methods1. Rat and human mesenchymal stem cells (BMSCs) were isolated and separated by Percoll using intensity gradient centrifugation and selected during the passages. BMSCs were identified through morphologic (TEM, SEM) and by phenotype molecule (CD44, CD90, CD45, CD34 for rBMSCs and CD44, CD45, CD34 for hBMSCs) assessments. And they have been induced to differentiate into osteoblasts and adipocytes.2. The mRNA expressions of collagen typeⅠ,Ⅲand tenascin-C by rBMSCs from passage 1 to passage 6 were analysed by real time RT-PCR.3. After non-contact co-culture with ligament fibroblasts during 3, 6 and 12 days, the transcriptional activities of collagen typeⅠ,Ⅲand tenascin-C mRNA were assessed by real time RT-PCR. At similar time-points, the protein levels of collagenⅠand collagenⅢin the BMSCs were detected by radioimmunoassay and immunocytochemical analysis.4. After treated with a cyclic 10％uniaxial stretching at 1 Hz, the length, width and orientation of the BMSCs were determined; mRNA level of typesⅠandⅢcollagen and tenascin-C were quantitated by real time RT-PCR; and the protein expressions of them were assessed by radioimmunoassay and immunocytochemistry; Confocal fluroscent microscopy was used to observe cytoskeleton changes.Results1. The morphologic feature showed that the rBMSCs and hBMSCs had the general appearance of stem cells, i.e., larger nuclei and minor plasm. By means of FCM, rBMSCs had the phenotype of positive expression of CD44,CD90 and negative expression of CD45, CD34; hBMSCs had the phenotype of positive expression of CD44 and negative expression of CD45 and CD34; Moreover, BMSCs obtained in our experiment could be differentiated into osteoblasts and adipocytes in the presence of conditional culture medium.2. The mRNA of collagen typeⅠ,Ⅲand tenascin-C were expressed stably by rBMSCs from passage 1 to passage 6.3. Compared to control cell groups, BMSCs co-cultured with ligament fibroblasts showed an enhancement of mRNA expression for collagen typeⅠ, typeⅢwith a 2-fold increase in the ratio of collagenⅢ/Ⅰmeasured at 6th day (p＜0.05, vs. their control groups) and for tenascin-C at 12th day. Levels of collagens typeⅠand typeⅢproteins were significantly documented at 12th day, as well as for collagenⅢ/Ⅰ, by immunoassay. Microscopic investigation showed that co-cultured BMSCs didn't display significant changes in their morphology.4. It was shown that the uniaxial stretching lengthened and rearranged the orientation of cells. The mRNA level of collagen typesⅠandⅢwere up-regulated after a 12-hour stretching compared with the control group (p＜0.05, vs. their control groups), while significant increase of protein was observed only after a 24-hour stretching. Whereas, the up-regulation of tenascin-C mRNA expression occurred after a 24-hour stretching. Cytoskeleton has also been changed by stretching stimulations.ConclusionsThese data suggest:1. The cells we obtained from rat and human bone marrow are exactly the BMSCs.2. Rat BMSCs of P1 to P6 express stably mRNA of collagen typeⅠ, typeⅢand tenascin-C.3. Indirect co-culture with ligament fibroblast may promote mesenchymal stem cells transforming into ligament fibroblasts.4. The cyclic stretching promotes the synthesis of collagen typesⅠandⅢand tenascin-C by the rat BMSCs.