The Research Of Pre-lining Modified Demineralized Bone Tissue

Partâ… The study about enhancing demineralized bone cell adhesion by Fn-lining modifiedObjectiveTo explore Surface Pre-coating method of decalcified bone decellularized,to increase MCSs cells' biocompatibility,attachment and growth on the tissue Engineering material.MethodsPick out suitable bovine bone as the tissue Engineering material.Acellular tissue matrix is made through a gentle multi-process decellularization protocols and cross-linked by dye-mediated photooxidation.Apply Fibronectin as Pre-coating groups,PBS liquid as the compared group.Six different compositions were deposed onto surface of 18 tissue sheets with a dose of 30μl,and each sheet,which was cut out at 1 cm₂,was implanted with mesenchymal stem cells 5×10⁵ on surface.Then culture the sheet under static condition for 7 days.Took the sample at 1^st,4^th,7^thday,depart cells from sheet surface with enzyme and count numbers.Took samples for HE stain and took photos on microscope.The 7^thday samples of the compared and the Pre-coating groups had scanning and transmission electron microscope checks.By comparing each groups' effect on increasing cell's biocompatibility,growth and disposition,explore the best surface pre-coating method of decellularized tissue sheets,so as to get the best endothelialization of tissue sheet.Results(1)Cell number:In 7 days of culture course,Pre-coating groups' surface cells,were more than compared group's(p＜0.005).(2)All samples had a section after paraffin imbedding,then had a HE stain and took photos on microscope.At 1st day,each groups' surface had a cell layer,spread closely,cellular nucleus could see an accumulation.At 4th day,the compared group's surface cells had a decoherence,while the pre-coating group gained a cell layer well.At 7th day,the compared were scarce of cells,fibre below was partly uncoated.The pre-coating group's surface cells had formed a integrity layer,linked with fibre below.(3)Took the 7^thsample for a scanning electron microscope.On the compared group's surface,the cells were scarce and no conjunction linked between cells or cell and sheet.The Pre-coating group had more cells.Cellular skeleton was formed and linked with tissue sheet fibre.Among cells, there was fusiform-shaped axon as linkage,which was a tight connection of cells and fibre.(4)Transmission electron microscope could see vacant brand among surface cells and fibre below sheet.ConclusionDecellularized sheets are capable of growing MCSs cells.Under Fn modification,the sheet gain an augment of cells' biocompatibility, attachment and growth.Pre-coating is better than compared on increasing cells' attachment and growth.Partâ…¡Histological evaluation of bovine decalcified bone treated with decellularization and photooxidation technique in vivoObjectiveThe purpose of this study was to investigate the histological change of bovine decalcified bone treated with decellularization and photooxidation technique in vivo and evaluate the possibility of which as a tissue engineering scaffold in clinical application.MethodsValved bovine bone treated with decelluarization and photooxidation method were implanted in young dogs long leg bone.The tissues were explanted and analyzed by gross examination,light microscopy,and electron microscopy in 1 month,6 months and 1 year after being implanted.Tissue contents of collagen,elastin and glycosaminoglycans were assayed respectively to the explanted tissues.Neutral salt soluble fractions,acetic acid soluble fractions and pepsin-digested fractions of collagen were extracted and assayed for these samples.Amino acid contents of different implantation samples were assayed.ResultsOf 7 dogs subjected implantation,1 died of in operation and the other 6 ones survived.2 dogs sacrifaced in 1 month after operation, another 2 dogs sacrifaced in 6 month and the others sacrifaced in 1 year.All the luminal,wall were still soft and smooth without calcification, thrombosis and hemangioma.No graft stenosis was observed in all animals.The valves all functioned well and the thicknesses of which were not changed.Endothelium-like cells had migrated in the perianastomotic regions.Macrophages appeared in 1 month but disappeared in 6-month and 1-year dogs.Fibroblasts and myofibroblasts infiltrated and grew from outer layer to the middle layer and the regeneration of novel collagen.Degradation of elastin fibers took place in the repcellular regions and only a few new elastin fibers were found near the outer layer to the 1-year implanted conduits.Calcification of 1-year implantation sample was found only in the needling area in the anastomotic regions. Collagen content decreased in 1 month and 3 month,and increased in 6 month;elastin increased in 1 month,but not changed in 3 month and 6 month.Amino acid analysis showed that the amino acids decreased from decellularizaton and photooxidation procedure increased in implantation samples.ConclusionsThe preliminary results in vivo showed decellularizaton and photooxidation treated tissue having growth potentiality,which surpport the bone as a tissue engineering scaffold.