| Objective:To identify the rabbit ulna cortical bone -derived cells that obtained through enzy-me digestion, researches its ability to grow and multiply in vitro, and discuss the most approp-riate cell algebra as the seed cells. To exam rabbit cortical bone -derived osteoblasts's growth situation on the self-made calf deproteinised cancellous bone,freeze-dried cancellous bone (XKC-FDB series of bone) and absorbability gelatin sponge, appraise the biological compatib-ility of three kind of support materials and rabbit cerebral cortex bone osteoblast; otherwise we used different cell density raising with three kind of support materials, to discuss the most appropriate cell density.For bone tissue engineering's cell source,carrier materials and further animal testing provided further experimental evidence.Methods and Result:Ulna cortical bone obtained by operation from 3-month-old New Zealand white rabbits (no limit of male or female), were dealt with enzyme digestion to obtain cells. With Microsco-pe observed cellular form and multiplication characteristic everyday; assay cell growth and proliferation with MTT,drawing the growth curve. Detection of the second,third,6th,7th and 8th generation cells's ALP.Appraisal cells with the method of staining cells with ALP,I collagen and Bone calcium element. Results:The isolated and cultured primary cells morphol-ogy were similar to fibroblasts, with good proliferation in vitro, cells's doubling time was about 7 days, and all test results were positived. Cell proliferation rate, the first 1-2 days, each generation of cell did not have the difference (P>0.05), from the third day the 7th,8th generat-ion cells significantly lagged behind the second,third and 6 th generation, continued to the first 10 days (P<0.05), while second,third and 6th generation,7th and 8th generation had no significant difference (P> 0.05). ALP:the second,third and 6th generation of osteoblast's ALP examination were higher than the 7th,8th generation of cells's ALP (P<0.05).Osteoblast were inoculated on three kind of scaffold materials,culturing in vitro,using mic-roscope and scanning electron microscopic observed of cells's growth and proliferation on th-e scaffold materials, then conducting cell adhesion rate,MTT, ALP and bone gla protein detec-tion.Result: microscope showed that three kind of scaffold materials were porous, pores con -nected between each other. And with the culture time extended, cells grew and proliferated rapidly. Scanning electron microscope showed that the third day, there were some cells adher-ed and spreaded on the scaffold materials; the 6th day, cells were fully extended, showing long spindle; the 10th day, cells secreted lots of extracellular matrix, overlaped grew and connected into the network, particularly in two kinds of bone materials. MTT tests showed that three kinds of scaffold materials did not affect the osteoblast's growth and proliferation in the early time,later (9th and 12th day) they would promote osteoblast's proliferation and secre-tion,and compared with the control group there were significant difference (P<0.05), while the three kind of scaffold materials had no significant difference between each groups(P> 0.05). cell adhesion rate:when the cell density was 1×106/ml, the absorbability gelatin sponge had the highest cell adhesion rate:45.52±3.15%.ALP:when the cell density was 1×105/ml, there were no significance difference of each group (P>0.05), The cell density was 1×106/ml, three kind of support material group were higher than the control group (P<0.05),but the three kind of scaffold materials had no significant difference between group, when the cell density was 1×106/ml and 1×107/ml, absorbability gelatin sponge group> self-made calf deproteinise-d cancellous bone> freeze-dried cancell -ous bone> control group. The 12d, each group of support material ALP when the cell density was 6×106/ml> each group of support material ALP when the cell density is 1×107/ml.BGP:The 14th day, self-made calf depro -teinised cancellous bone and freeze-dried cancellous bone group was obviously higher than the control group and absorbability gelatin sponge group (P<0.05); The 21 st day and the 28th day, self-made calf deproteinised cancellous bone> freeze-dried cancellous bone >absorbabil -ity gelatin sponge>control group(P<0.05).Conclusion:1,Rabbit ulna cortical bone can cultivate osteoblasts; Osteoblasts have the function of proliferation and secretory in vitro.The sceond,third and 6th generation cells have stronger capacity of proliferation and secretory than the 7th and 8th generation cells. New Zealand big white rabbit cortical bone -derived osteoblasts's ideal cell algebra is 2nd-6th generation as seed cells.2,The ideal cells seeded density of three kind of support materials and rabbit cortical bone-derived osteoblast are 1×106/ml-6×106/ml. With the cell density increased, the cell adhesion rate is declining. When the cell density is 1×106/ml, the absorbability gelatin sponge has the highest cell adhesion rate :45.52±3.15%.3,In the ideal inoculation density, ALP:absorbability gelatin sponge group> self-made calf deproteinised cancellous bone>freeze-dried cancellous bone>control group. BGP:self-made calf deproteinised cancellous bone>freeze-dried cancellous bone>absorbability gelatin sponge>control group.4,The self-made calf deproteinised cancellous bone that this experiment made with the rabbit ulna cortical bone -derived osteoblasts has good biological compatibility and bone inductivity, moreover it prepared conveniently, has the low price, so it is the very good bone substitution material. The self-made calf deproteinised cancellous bone and the rabbit ulna cortical bone-derived osteoblasts conformed to construct organization project bone, and carry on further experiment is feasible.
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