Research On Guniea Pig MSCs Induced By BMP2 To Osteoblast And Preparation Of Tissue Engineering Artificial Auditory Ossicle

Objective:To observe the compounded culture degree of guinea bone marrow stromal cells (MSCs) and nano-hydroxyapatite (nHA), analysis the feasibility of tissue-engineering artificial auditory ossicle. Methods:(1) Using the different growth characteristics of MSCs compared with other cells, culture of MSCs was isolated from the bone marrow of guinea pig by adhesion separation. The shape and growth condition of MSCs was observed by phase-contrast inverted fluorescent microscope. The surface molecule expressions of cells were examined CD29, CD44, CD45 by flow cytometric analyses. Bone morphogenetic protein 2 was uesd for cell amplification and differetiation. Induced cells were stained immunohistochemically with Collage I and were detected with alkaline phosphatas (ALP), alizarin red. MSCs was induced to osteoblast in order to detect the differentiated capability. (2) To culture bone marrow stromal cells and nano-hydroxyapatite analyze their adhesion situation by scanning electron microscope at Id,3d,5d,7d, lOd. Through energy spectrum analysis, to observe the ability of bone-formation in body. (3) Supported material with bone marrow stromal cells and nano-hydroxyapatite after 5d were implanted in successful otocyst model of guniea pig, in 2 weeks,4weeks,8weeks, l0weeks,12weeks, there were obsevered the ability of osteogenetic by pathology, scanning electrion microscope and energy spectrum analysis on tissue-engineering artificial otosteo. Results:(1)â‘ MSCs detection:The morphology of MSCs is uniform, monolayer and nemaline grow. Their common marker CD29 is 95.7%, CD44 is 70%, CD45 is 0.7%.â‘¡Osteoblast detection:The inducted MSCs had the characteristics of osteoblast morphological by inverted microscopic; Immunohistochemi-cal collagen type I can be observed in cells had yellow-brown solids precipitation, alkaline phosphatase saw cell cytoplasm within a purple grain, alizarin red dyed visible induction osteoblast calcium nodules form. (2) After three days cultivation, many of MSCs attached the material surface with stable form, proliferation activity and terrific extensibility; Composite material had all covered MSCs at 5 days, cell surface had a large number of secretory granules, soma amplify obviously, edge of cells was complete and fibroid extension; 7 days later cells gradually fall from the material surface, still attached to the material surface cells appear "star" or" pseudopods"; 10 days after cells flake off the material surface. (3) The tissue-engineering artificial otosteo were packed with abundance of fibrillar connective tissue in implanted 10 weeks, combining with the surrounding tissue; 3 months after back to implant, there are bone lacuna and almost the same microelement detected by scanning electron microscope and radioactivity spectrum analyzer. Conclusion:It can be explained that nano-hydroxyapatite retain a good biocompatibility which is beneficial MSCs to adhesion, proliferation and combination and be used to construct the tissue-engineering artificial otosteon.