Experiment Of Allogenic Deproteinized Bone Combined Fibrin Glue Constructing Bone Tissue Engineering Scaffold

Objective 1. To evaluate the difference of grow and differentiation of themarrow stem cells(BMSC) between allogenic deproteinized bone combined fibringlue and allogenic deproteinized bone alone as scaffold for bone tissue engineering invitro. 2. To investigate the difference between allogenic deproteinized bone combinedfibrin glue and allogenic deproteinized bone alone as scaffold for bone tissueengineering through Ectopic osteogenesis in vivo. 3. To make the model of bonedefect in New Zealand rabbits and investigate the difference between allogenicdeproteinized bone combined fibrin glue and allogenic deproteinized bone alone asscaffold for bone tissue engineering through comparing the effect of repaired bonedefect in vivo.To provid one more suitable compound scaffold for bone tissueengineering.Methods 1. Deproteinized bone was made first. Cancellous bone was obtainedfrom both sides iliac bone of the rabbit and then it was cut to different size.TheCancellous bone was processed by H₂O₂, chloroform-methanol,ethanediamine anddehydrated alcohol to be Deproteinized and then exposured under Co⁶⁰ after freezedrying. BMSC of rabbit was obtained from bone marrow aspiration and then culturedin vitro. About 5×10⁵ third generation BMSC was used in the experiment. Allogenicdeproteinized bone combined fibrin glue in experiment group, allogenic deproteinizedbone alone in control group.BMSC were co-cultured with two groups in vitro. The cell grow and differentiation state was detected through optical microscope observing,electron microscope scanning, DNA content assaying and calcium nodus staining ateach time point. 2.28 adult rabbits was divided to three groups randomly.12 inexperiment group,12 in control group and 4 in blank group. Allogenic deproteinizedbone+FG+BMSC in experiment group, allogenic deproteinized bone+BMSC incontrol group and allogenic deproteinized bone alone in blank group. Correlateddetection was carried on at 4, 8, 12weeks after operation in experiment group andcontrol group and 2, 4weeks in blank group. BMSC were co-cultured with two groupsin vitro,then were implanted into back muscle of the rabbits. The osteogenesis ofmaterials with cells were studied through gross observation, alkaline phosphataseactivity assay, biomechanics test, histological observation and quantitative analysis.Immune response was judged by lymphocyte quantity of the histology and analysis ofCD4⁺,CD8⁺ cell of body. 3. 12mm radius bone defect was made in both sides of 20adult rabbits.18 of 20 animals was divided to experiment group in the right andcontrol group in the left. The other 2 animals was in blank group. Allogenicdeproteinized bone+FG+BMSC in experiment group, allogenic deproteinizedbone+BMSC in control group and allogenic deproteinized bone alone in blank group.Correlated detection was carried on at 4, 8, 12weeks after operation in experimentgroup. BMSC were co-cultured with two groups in vitro,then were implanted torepair the radius bone defect of the rabbits. The osteogenesis of bone defect repairwere studied through gross observation, X-ray check, alkaline phosphatase activityassay, biomechanics test, histological observation and quantitative analysis in eachtime point after the operation.Results 1. Optical microscope observing and electron microscope scanningshowed BMSC sticking on the surface in three dimensional with hypso-density and secreting great amounts of Matrix Granule to form mass in experiment group. TheDNA content from implants developed from experiment group was greater than thatfrom control group at all time points (p＜0.05 at 4,7 days). The calcium nodusstaining of BMSC could be seen in experiment group after 7days culture. BMSC Thecell attachment, growth, cell cycle and differentiation in the experiment group wereobviously superior to the control group. 2. The general state of health of the animal ineach group were normal and the wound healed with I stage after operation. Alkalinephosphatase activity assay showed it was more stronger in experiment group than thatin control group at the same time point by t-test(p＜0.01). X-ray check showed that thebaffle rate was higher in experiment group than that in control group at 4,8weeksafter operation. The compression stiffness of experiment group exceed that of controlgroup in biomechanics test. Experiment group and control group could form cartilagetissue and bone formation in vivo. The contents of ossification in experiment groupwere obviously more than that in control group at 4,8weeks.However,in the blankgroup there was no bone formation. Quantitative analysis through the image softwareshowed the new bone formation quantity in experiment group was higher than that incontrol group(p＜0.01). More new bone tissue formed gradually as the time went by inboth two groups. Statistics software SPSS was used for analysis. Lymphocytequantity of the histology and analysis of CD4⁺,CD8⁺ cell of body showed that therewas no immune response after implantation. 3. The animal's general state was normalafter the repair operation. The materials draw from the animal showed the bony callusabundant in experiment group and the bone defect was almost repaired at 4weeksafter operation while the defect was partly repaired in control group. At 8weeks afterthe operation the bone defect of both group was both repaired. X-ray check showedthat the baffle rate of the bone defect was higher in experiment group than that incontrol group at 4,8weeks after operation and alkaline phosphatase activity assay showed it was more higher in experiment group than that in control group at the sametime point by (p＜0.01). The three point bending test of experiment group exceed thatof control group in biomechanics test. In the histological examine, the mature bone inexperiment group was more than that in control group at 4weeks after operation. Thebone defect was instead of mature lamellar bone at 8weeks after operation while therewere also cartilage at the edge of bone defect in control group. Quantitative analysisthrough the image software showed the bone repairing quantity in experiment groupwas higher than that in control group(p＜0.01). SPSS software was also used forstatistics analysis. More mature bone tissue formed gradually as the time went by inboth two groups, however there was no bone repairing in the blank group.Conclusions 1. Allogenic deproteinized bone combined fibrin glue is moresuitable forgrowing, differentiation of BMSC than allogenic deproteinized bonealone, and it can be used as scaffold for BMSC. 2. The speed of new bone formationis more fast when allogenic deproteinized bone combined fibrin glue as scaffold forBMSC and the quantity of bone formation is more than allogenic deproteinized bonealone in earlier period. 3. The bone defect repairing speed and quality of Allogenicdeproteinized bone combined fibrin glue is obviously superior than allogenicdeproteinized bone alone, and it can be used as a combination scaffold for bone tissueengineering.