| ObjectiveIn bone tissue engineering, a great deal of biodegraded and ab-sorbable material has been researched widely. The scaffold provides the base of cell adhesion, growth and differentiation, also the scaffold is the structure that fixes growth factors in cell components and maintains three dimension spatial configuration to create new tissue. Although someone has narrated cell framework of acellular membrane, no ideal preparation of AECM or histomorphometry of bone lacunas have been reported in details. This experiment prepares ACEM by removing cell components and carries morphologic study to provide foundation for new natural supporting material of bone tissue engineering. Bone marrow stromal stem cell ( MSCs) can easily expand in vitro, and can give rise to a broad spectrumof fully differentiated connective tissues under appropriate experimental conditions. MSCs have a number of potential therapeutic applications. Their applications in the tissue engineering of bone are not enough. We used MSCs as seed cells and cultured the cells combined with AECM in vitro, and observed the integration of the donor cells. This provided the morphologic foundation for new natural supporting scaffold of tissue engineering of bone.Materials and Methods1. MaterialsKunMing mice (from animal center of china medical university) . Triton - X100, trypsin, Dulbecco 's Modified Eagle 's Medium (DMEM) (CIBCO, LTD) , calf serum (CS).2. AECM preparationAfter anesthetized by sodium pentobarbital, two sides of femurs of mice were taken down. Getting ride of the periosteum and being incised into patches, the bones were washed by PBS. Bone patches were put into jar containing 0.05M Tris - HCL( pH7.4) buffer and protein-ase inhibitors (0. 1ug/ml aprotinin,0. 5ug/ml leupeptin,0. 6ug/ml pepstatinA). The samples were then immersed in Tris - HC1 buffer containing 3%Triton x - 100 and proteinase inhibitors, distilled water washed bone patches after stirring at 4℃ constant temperature and the samples were digested by DNase and RNase at 371 for 12 hours. Then the samples were immersed into Tris - HC1 buffer containing 3% Triton x - 100 and washed by distilled water again. Finally the samples were reserved in Tris - HC1 buffer at 4℃. When decalcification bones were needed, AECM was put into Von Ebnel liquid for 30 days. Part of them was stained by HE staining.3. The culture and identification of MSCs in vitroThe primary culture of MSCs, which were collected from femoral marrow of mice about a month old by PBS, centrifugalized in the rate of 1000r/min, mixed with DMEM + 10% CS, cultured in the condition of 37℃, 5% CO2, and refreshed the DMEM every one day. Harvested them after they formed one layer, the cells were washed, trypsinized for 3 min, and suspended in Dmem + 10% CS. part ofthem was stained by alkaline phosphatase staining, alizarin S staining and Mallory staining.[3,4,5].4. MSCs cultured in AECMMSCs were collected in DMEM at the consistency of 5X104/ml and transplanted into AECM. MSGs were cultured in the condition of 37℃, 5%CO2 The DMEM were refreshed 8 hours later, Then refreshed every one - day.5. Histological examinationHie cells were observed by phase contrast microscope every day, after cultured for 7 days, the AECM was taken out; part of them was fixed by formaldehyde for 24 hours and paraffining, HE staining. Part of them was fixed by 2. 5 glutaraldhyde and observed by Scanning e-lectron microscope.Results1. Histology of AECMUnder light microscope, collagen fibers arranged uniformly. It' s blankness in ellipse bone lacunas and there were no osteocyte nucleus and other configuration.2. The conformation and growth of MSCsMSCs were diffusible in distribution, cell body was transparent 1-2 days after, the cells began adherent one week later, CFU - F wasobserved and single layer was formed in about 2 weeks. At this time,the active reaction of ectoblastic alkaline phosphatase marker could beseen. Alizarin S staining and Mallory staining was p... |
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