| Objective:The study, using the basic fibroblast growth factor and compression force to stimulate bone marrow-derived mesenchymal stem cells(BM-MSCs) and using histology, immunohistochemistry and enzyme linked immunosorbent assay (ELISA) to examine the typeâ… collagen and glycosmioglycan(GAG) of extracellular matrix produced by BM-MSCs after the stimulation of biochemistry and mechanics, evaluates the feasibility of BM-MSCs being the seeding cells of temporomandibular joint disc tissue engineering. It will also be an important step towards the further study of TMJ disc tissue engineering.Methods:1. In bacteria-free environment,3-5ml bone marrow was obtained from healthy goat aged about one to three months old by the No.18 bone marrow puncture needle and used percoll lymphocyte separating medium to get the mononuclear cell layer by density gradient centrifugation. We observed the cells'shape by inverted phase contrast microscope every day and got their growth curves.2. The passage 3 and 4 BM-MSCs were induced to chondrocyte-like cells or fibroblast-like cells respectively with inducing medium containing 5ng/ml and 10ng/ml bFGF. The typeâ… collagen of supernatant medium was examined by using ELISA at 7,14,21 day. After 1,2,3 weeks' induction, each group was tested by toluidine blue staining, safranin O staining and picrosirus red staining for typeâ… collagen and GAG.Transmission electron microscope (TEM) was used to observe the ultrastructure of the induced cells.3. The passage 3 and 4 BM-MSCs were induced to fibroblast-like respectively with inducing medium containing bFGF at different concentrations for two weeks. The induced cells/agarose gel constructs were cultured for 6 days and then undertaken dynamic compression 0.2MPa,0.1 Hz 2hrs/day for a week.14 micrometer thickness sections were used to examine the collagen typeâ… and GAG distribution by toluidine blue staining, safranin O staining and picrosirus red staining.Results:1. The primary passage goat BM-MSCs were cultured for 24hrs, from which we can observe cells. Most of them are round shaped, some are triangle, spindle shaped. After 72hrs, half-exchanging medium, most cells are triangle, spindle shaped and some also suspend in the medium. Cultured for about 7-10 days, cells reached 90% fusion, connecting to each other, paved bottom of the bottle. After passaged 12hrs, attachment efficiency can reach 90%, and most of cells are triangle shaped, spreading all bottom of the bottle in 6-7 days.2. Induced BMSCs by induction medium containing bFGF reduced proliferation rate, most of them are triangle, spindle shaped like chondrocytes. Histology, immunohistochemical staining and ELISA all exhibited positive results.TEM denoted that induced BMSCs are round shaped, and there are plenty of well-developed mitochondria, Golgi complex, rough endoplasmic reticulum and also lots of lysosomes and glycogen granules.3. BMSCs induced by combination bFGF and dynamic compression increased expression of synthesized ECM and this was confirmed by the result of histology, immunohistochemical staining and ELISA.Conclusion: BMSCs could be isolated and purified with density gradient centrifuge. Sufficient amount of BMSCs could be obtained through in vitro amplification and meet requirements for the TMJ disc tissue engineering. After several passages, cultured cells still sustain their original shape, they have basic characteristics of seeding cells biologically and morphologically. By a specific induced medium, BMSCs can be differentiated to chondrocyte-like cells or fibroblast-like cells, and express ECM component of the native TMJ disc tissue. So BMSCs are expected to be ideal seeding cells for TMJ disc tissue engineering in future study. |
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