| On the median and late stage of osteoarthritis, joint cartilage is destructed extensivly,and synovial membrane is severely destroyed .Because synovial membrane is main source of nutritious materials of joint cartilage,single treatment for joint cartilage is not very effective.This research constructs three dimensional cell culture in vitro with the scaffold of gelatin sponge as a beneficial attempt to cure osteoarthritis by the method of artificial synovium transplantation.Materials and Methods: Complex culture of cell and scaffold: The synovium is separated from joint in surgical patient of condylar hyperplasia .Superior layer synovium that inner layer is cut away is flushed,and then is minced by Imm3 .The blocks are collected and dispersed before cell culture at 37癈 in the condition of 5% CO2. The scaffolds are tailored to cylinder by 1cm of the diameter,2mm of the thickness.and then sterilized and saturated for 30 min.The cells of three passages are used as seeding cell and put into the scaffold in the ultimate high density by 1×105 cell/ml .And the volume of cell admixture can just cover the scaffold.Observation complex scaffold with light microscope and AO fluorescence.and with scanning and transmission electron microscope: After the cells are seeded into the scaffold for 2 weeks,the scaffold are taken out and fixed with 10% polyformalin.l%AO staining for lmin,CaCl2 washing for 2min,PBS lavaging for some time, and Fluorescent stimulating.And observed by Von Kossa's and oil red O staining.The scaffold was fixed by 2% Glutaraldehyde after complex culture 2 weeks, the parts of samples are dehydrated with glycolonitrile in the serials of concentration, and observed by the SEM after spouted by the gold.Others of samples are fixed by 2% osmium tetroxide, and then embedded in the resin and sectioned before observation.Results and Conlusion: In the condition of monolayer cell culture,the cells on the wall arrange like the reticulum and most of cells are scattered while are rarely directed and radiated in the order,Although the cells can exhibit polygon-like in some area where the amount of cell increase rapidly epthelial-like shape, most are short spheroid shape after several passages.Observations of complex scaffold with light microscope and AO fluorescence staining show that, three passage cells may fill up with the whole space of scaffold after 2 w with the cells proliferation.Microhole of the honeycomb-like scaffold is made of the gelatin fibrils in the full dimensions. the cells seeded in the scaffold attach to the fibrils and exhibit the little prustusion,which can be observed in all focus of microscope.HE staining shows that,the endoplasm of type B cells in the scaffold exhibit slightly blue staining ,and nuclear deep blue staining is short spheroid in the center of the cell, nucleolus areseen clearly .A lot of secretory materials lie at the surrounding of clusters of cells. Consquence of Von Kossa's and oil red O staining is negative.Observation of SEM shows, a large amount of synovial cell is short spheroid shape, squamous contour and stretched on the surface of fibrils in the scaffold.Ramifing intercellular crosslines exist so that cells can adhesion with each other.A lot of microvilli cover the surface of the cell. From observation of TEM, the chromatin of nuclear is very loose and scattered around the cell nuclear.there are abound of developed rough endoplasmic reticula, mitochondria, Golgi complex, filaments and lysosome in the endoplasma,and large amount of secretory granules in the long and thick protiberances and on the fringe of the cell. Content of secretory granules is reticulum materials.There are microvilli-like fingers on the surface of cells,and atype cellular connection between the cellular membrane.Discussion: The cells in the scaffold are short spheroid shape and their endoplasma are clear,which demonstrates the type B cell is some kind of fibroblast. Observation through SEM shows that, structure of villi around the cells, play a critical role on adhesion of synovium and funct... |
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