In Vitro And Comparison Of Biological Characteristics Of Rabbit's Osteoblasts From Cortical Bone And Other Different Sources

Objective:By comparing the in vitro culture and parts of osteogenic functions of rabbit's osteoblasts from cortical bone and other different sources,identify and explore the osteogenic capacity of osteoblasts from different sources,and through the continuous explant culture of cortical bone and related testing,explore the feasibility of continuous explant culture.Methods:Cortical bone of upper limb,ilium,periosteum,and bone marrow obtained by operation from 3-month-old New Zealand white rabbits(no limit of male or female), were dealt with explant culture and enzyme digestion respectively to obtain osteoblasts. Bone marrow stem cells were extracted by density gradient centrifugation separation and induced into osteoblasts when detected.We observated the morphology,adhesion and growth of osteoblasts with inverted phase contrast microscope daily,pured cells using the different adherent speed of cells.Cells were divided into cortical bone group, periosteum group,cancellous bone group,stem cells induced group and stem cells' routine group which weren't induced.We got P3 cells from different sources to carry out the following experiments.Firstly,we stained the cells with crystal violet to observe the morphology of cells,determined the cell proliferation curve by MTT,then used nitrophenol method to determine the alkaline phosphatase(ALP) activity and ELISA for the osteocalcin(BGP) expression and so on.We collected the cortical bone after primary culture to carry out the second culture,then got P4 cells to carry out the following experiments,compared the cell growth and ALP,BGPexpression of the two explants.Experimental data would be analyzed by Statistical software,indicated as mean±standard deviation((?)±s),P＜0.05 was of statistical significance. Results:1,Morphological observation:No morphological difference was found among the various sources.In primary culture,cancellous bone group and cortical bone group were like long spindle,ranged radially around the bone chips.Periosteal bone group were like spindle and triangular.Some cancellous bone group were slightly flat with longer protuberance,cortical bone group with slightly shorter protuberance,and the protuberance of periosteal bone group was shorter.Stem cells were of irregular shape, appeared shuttle-like,and polygon shape,with shortest and more protuberance.After being subcultured,cells from various sources appeared the same.Stem cells looked more regular after being induced,just like other cells,to be long shuttle like,angular shape and irregular shape and so on.There was also no morphological difference between the two explants.2,Proliferation:The cell growth curve is like "S".For the cell double time, periosteal group and stem cells' routine group was about five days,stem cells (induced)was 7 days,cortical bone and cancellous bone groups were both between the two,about 6 days.At the proliferation rate,the first day there was no difference in each group(P＞0.05);on the third day the stem cells which have been induced began to drop behind other groups(P＜0.05).On the fifth day and seventh day,no difference was found within the cortical and cancellous bone group.And they were second to the pefiosteum group and stem cells'routine group,while only the difference of cancellous bone group growing slower than periosteal group and stem cells'routine group was of statistical value(P＜0.05).There was also no difference between periosteal group and stem cells'routine group(P＞0.05).The proliferation ability of cells from the two continuous explants was of no significant difference(P＞0.05).3,Osteogenic capacity:(1) The alkaline phosphatase(ALP) activity:Absorbance of various cells increased with the cultured time,that is,ALP activity increased with the time.It increased gradually in the early and middle cultured time,but the rate of increase slowed down in the later time.While stem cells'routine group didn't be found obvious ALP activity.ALP activity comparation of the cortical bone group,periosteal group,cancellous bone group and stem cells induced group:ALP activity of periosteal group began to be higher than the other groups excluding the cortical bone group on the 7^th day(P＜0.05),no difference was found between periosteal and cortical bone group(P＞0.05).On the 12^th day,the ALP activity order(strong to weak) was the periosteal group,then the cortical bone group,following the cancellous bone group, and stem cells induced group(P＜0.05).The periosteal group and the cortical bone group which two groups were of no difference between were stronger than the other two groups-cancellous bone group and stem cells induced group on the 20^th day.And the cancellous bone group express stronger ALP activity than stem cells induced group. On the 28^th day,the same result appeared again as it on the 12^th day.Cells from the two continuous explants expressed the same ALP activity(P＞0.05).(2)Osteocalcin(BGP) expression:Osteocalcin activity of the cells from different sources except the stem cells'routine group increased with the culture time.The stem cells'routine group didn't express obvious osteocalcin activity.On the 12^th and 20^th day,the same result was firstly the periosteal group,then the cortical bone group,following the cancellous bone group,and stem cells induced group(P＜0.05).On the 28^th day,it was almost the same with the result of the 20^th day,but the difference between the periosteal and cortical bone groups,the cortical and cancellous bones was unconspicuous(P＞0.05).Cells from the two continuous explants expressed the same BGP activity(P＞0.05).Conclusion:1,Osteoblasts could be successfully cultured from cortical bone,periosteum, cancellous bone and bone marrow stem cells.There was no significant morphological difference within the cells of different sources,most of them were like long shuttle, triangle and irregular shape.2,Proliferation ability of cells from different sources was as follow:Periosteal group and stem cells'routine group were the first,then the stem cells induced group were the last one,and the cortical and cancellous bone group were placed in the middle center without any difference found between them.The stem cells(not induced) owned no obvious osteogenic capacity.Osteogenic capacity of osteoblasts from different sources was as follow:It was firstly the periosteal group,then the cortical bone group, following the cancellous bone group,and stern cells induced group.3,The osteoblasts from cortical bone was comparatively pure.No significant difference of biological characteristics was discovered within cells from the two cortical explants culture.We can make the best of bone chips by the continuous explant culture,to save a lot of money and time.