Effects Of Hypoxic Porous Tantalum-osteoblast Compound On Osteoblast Proliferation, Synthesis And Expression Of COL-â… , OC MRNA In Vitro

Objective To explore the effect of hypoxia porous tantalum-osteoblast compound on osteoblast(OB) proliferation, synthesis and expression of COL- Ⅰ, OC m RNA in vitro,so as to provide the experimental basis for clinical application in vivo.Methods 1 MG63 osteoblasts were isolated. The type Ⅰ collagen and Alkaline phosphatase(ALP) of OB were detected by immunocytochemistry and ALP staining. The morphological properties of the porous tantalum were observed by SEM. The 3rd generation of normal OB were seeded on the porous tantalum into concentration of 1×106/L-1 cells and cultured. 2% p O2 and 20% p O2 were set as hypoxia and normoxic environment model respectively. Hypoxia group(hypoxia osteoblasts pure cultured) and hypoxia tantalum group(hypoxia osteoblasts and porous tantalum co-cultured) were set as the experimental group, with normoxic control group(normoxic osteoblasts cultured alone) for comparison. 2 The morphology and growth of OB were observed under inverted phuse contrast microscope. The osteoblasts were co-cultured with porous tantalum in vitro, the morphology, proliferation was observed under SEM and TEM. 3 Each groups of OB growth and proliferation were detected by CCK-8 assay. 4 Each groups of osteoblast COL-Ⅰ、OC secretory were detected by ELISA assay. 5 Immunohistochemitry were used to determine the protein expression of each groups of osteoblasts COL-Ⅰ 、OC. 6. Osteoblasts COL-Ⅰ、OC gene expression were detected by RT-QPCR assay.Results 1 Porous tantalum scaffolds surface and cross section were evenly distributed needlepoint cellular porosity. The observed result of canning electron microscope showed that the pore sizes of porous tantalum ranged from 400~600μm, which had a similar threedimensional connected hole morphology to cancellous bone. 2 Normoxic osteoblasts grew well. Short-term hypoxia osteoblasts morphology and quality did not change significantly. The forth day hypoxia osteoblast were less than the normoxic osteoblasts. The seventh day hypoxia osteoblasts were not fully covered the bottom, part of them were drop off. Osteoblasts adhered, grew and secreted extracellular matrix in the microporous of porous tantalum scaffolds under SEM. A large number of organelles and secretory vesicles, large nucleolus, rich chromatin could be seen in normoxic porous tantalum osteoblasts compound cell cytoplasm under TEM. A large number of villous membrane protrusions, karyopyknosis, chromatin condensation, and swelling mitochondria could be seen in hypoxia porous tantalum osteoblasts compound cell cytoplasm under TEM. 3 The result of CCK-8 assay showed that the proliferation of hypoxia osteoblast was lower than that of the normoxic control group(P<0.05). The proliferation of normoxic tantalum group osteoblasts was higher than that of the normoxic control group. The proliferation ofhypoxia group and hypoxia tantalum group osteoblasts was lower than that of the normoxic tantalum group. The more severe the degree of hypoxia was, the cell proliferation was weaker(P<0.05). 4 The result of ELISA assay showed that the COLI、OC secretory of hypoxia group and hypoxia tantalum group osteoblasts was lower than normoxic tantalum group(P<0.05). The COL-Ⅰ secretory of hypoxia tantalum group osteoblasts was higher than that of the hypoxia group at the seventh day. The OC secretory of hypoxia tantalum group osteoblasts was higher than that of the hypoxia group at the fifth and the seventh day. 5 The result of immunocytochemistry assay showed that the COL-Ⅰ、OC protein expression of hypoxia group and hypoxia tantalum group osteoblasts was lower than normoxic tantalum group(P<0.05). 6 RT-QPCR analysis shows that the expression of COL- Ⅰ, OC m RNA of hypoxia group and hypoxia tantalum group osteoblasts was lower than normoxic tantalum group(P<0.05).Conclusion 1. Porous tantalum- osteoblast complexes cultured in different hypoxic(3%PO2、2%PO2、1%PO2), with time extending hypoxia decreased osteoblast of groups proliferation. Furthermore, the hypoxia groups of obvious effect were 2%PO2 and 1%PO2. 2. Severe hypoxia lead to porous tantalum-osteoblast complexes cell ultrastructure lesion such as mitochondrial swelling, mitochondria decreased or disappeared, rough endoplasmic reticulum degranulated and so on. 3. Hypoxia decreased that osteoblast secrete extracelluar matrix COL-Ⅰ、OC. However, the three-dimensional structure of porous tantalum could ease the reduction of COL-Ⅰ、OC secretion of osteoblast when tantalum and osteoblast co-cultured. 4. Hypoxia decreased expression of COL-Ⅰ, OC m RNA of tantalum- osteoblast complexes. Constructing bone tissue engineering scaffold material, especially when applied to repair of bone defect, the PO2 of injury site should be considered due to suitable PO2 is beneficial to the growth of osteoblasts.