Study Of Silk Artificial Ligament Compound With Marrow Mesenchymal Stem Cells

ObjectiveModified the surface texture of the silk artificial ligaments made by ourselves inseveral different methods, choose the best methods through an observation of the seedcells’ growth and adherence situation, and provide a good way to compound the seed cellsto silk artificial ligament in tissue engineering ligaments.MethodsDegum the silk artificial ligaments with pancreatic enzymes and sodium carbonateand test the degumming loss rate. Cross link the ligaments with1-3-(3ethyl-two methylpropyl ammonia (EDC) and N-hydroxyl amber imide (NHS) and then test the degree ofcross linking with enzyme standard instrument law. Observe the surface changes of theligaments before and after the cross link with fluorescence microscope and scanningelectron microscope.The ligaments were divided into experimental group, reference group and controlgroup. The ligaments of the reference group only compound with collagen withoutcross-linking; but with cross-linking in the experimental group; and with no specialtreatment of the control group. We chose the rabbit’s Marrow mesenchymal stem cells,MSCs as our seed cells. The cells were cultured and generated to the eighth generation, wetest them with CD34, CD44, CD50immunohistochemical and flow cytometry to ensurethey were MSCs indeed, adjust the cell destiny to1×106/L and planted into ligaments ofdifferent groups. Observe and compare the cell growth situation with enzyme standardinstrument law, fluorescence microscope and scanning electron microscope.ResultThe degumming loss rate was around29%using pancreatic enzymes and sodiumcarbonate. The max degree of cross linking happened when the EDC concentration was3g/L. Under the observation of fluorescence microscope, the surface of untreated silk stayintegrity; the degummed silk with a fluffy texture and smooth surface, with silk fiber exposed. Under the observation of SEM, the surface of untreated silk were unsmooth, withsericin around the silk fiber, the degummed silk with a smooth surface, we can see the fiberseparately.BSCs cultured with whole blood explants–attached method, the CD34antibody werenegative and the CD44, CD50, STRO-1antibodies were positive. CD34+cells were10%and CD44+cells were42%in a flow cytometry test, which performance characteristics forstem cells. Under the observation of fluorescence microscope, there were fewer cells in thenone cross-linked group and the cells were not extended. Cells in the cross-linked groupextended greatly and with a high adhesive rate. The surface of the silk were unsmooth andthere were smaller spaces between the silk fibers. Under the observation of SEM, cellswere adherence with collagen tightly and extended greatly with more tentacles. Which wassignificantly better than the other groups through an one-way ANOVA analyst (P<0.05).Conclusion1. The marrow mesenchymal stem cells can promote the differentiation of a variety ofdifferent cells, and shows a good proliferation ability in our experiment, is the ideal choiceof the seed cells.2. Collagen protein has a good biocompatibility, is a good silk surface modificationagent. And EDC has low toxicity, could be used for a good crosslinking agent.3. Silk artificial ligament compound with collagen can adhere more cells after beencrosslinked with EDC/NHS.