| Chapter1Study on the Feasibility of Defatting Bone Graft Material with LipaseObjective:To evaluate the feasibility of deffatting bone graft material with lipase, lipids content, surface structure, microstructure, effects on the proliferation of osteogenic precursor cells, and biocompatibility of processed bone graft were investigated.Method:The fresh pig femurs were frozen at-76℃for two weeks. The soft tissues were removed from the bone. Then the bones were sawed into pieces and cleaned with ultrasonic waves for48h in the deionized water,divided into four groups for degreasing. Lipase treated group:the bone materials were soaked in the lipase solution with concentration of1%for4h, the PH value was adjusted to10. NaHCO3/Na2CO3treating group:the bone materials were soaked in NaHCO3/Na2CO3buffer solution with PH=10for4h. Acetone treating group (positive control group):the bone materials were extracted in acetone for24h and soaked in absolute alcohol for another36h to remove acetone. Deionized water treating group (negative control group):the bone materials soaked in the deionized water for4h. All the degreasing treatments were performed at40℃. After degreasing, the bone materials were cleaned for another12h. After the bone materials were lyophilized, the lipids content of each group of treated bone materials was calculated according to the weight difference before and after the Soxhlet extraction. The surface structure of each group of bone materials was observed by scanning electron microscope. The histological sections of bone materials were prepared, stained with HE and Masson trichrome stain. After being sterilized by irradiation, the extract liquids of the different groups were prepared respectively at a concentration of0.2g of bone samples per ml of H-DMEM medium and incubated at37℃for72h. Besides the experimental group, nontoxic polyethylene disks was referred as the negative control group,toxic phenol as the positive control group and medium as the reagent control group. MC3T3-E1cells at the exponential phase were seeded on a96-well culture plate with100μl of medium with1×104cells per ml. Extract liquids were changed after incubated in a humidified atmosphere of5%CO2at37℃for24h. The cell viability was determined using CCK-8assay at1st,4th and7th day after treatment respectively. At the end of the culture period,10μ1of the CCK-8solution was added to each well of the culture plate. After6h incubation, absorbance at450nm was measured with a micro-plate reader. Cell relative proliferation rate was calculated and the cytotoxic grade was evaluated. Different groups of bone materials were put in the6-well tissue culture plate and the exponential phase of MC3T3-E1cells were seeded on the bone materials at a concentration of2×105/ml with500μL cell suspension per material. After3h co-culturing incubation the medium was added to be sufficient. At7th day after co-culturing, the bone material-cell complexes were harvested and observed by scanning electron microscopy. Different groups of bone materials were embedded in rat subcutaneous. At the1nd,4th,12th week after implantation, three rats were sacrificed randomly respectively. The implanted materials and surrounding tissue were harvested. The histological sections were prepared and stained with HE. Then the histological changes were observed under light microscope to evaluate histocompatibility.Results:After treatment, the surfaces of bone materials defatted with lipase, acetone or Na2CO3/NaHCO3were clean, while there was some grease on the surface of bone materials treated with deionized water. After being sterilized by irradiation, bone materials defatted with lipase and acetone appeared white, the bone material treated with Na2CO3/NaHCO3solution was slightly yellow and bone materials treated with deionized water turned to be deep yellow. There was no significant difference between the lipids content of the bone materials treated by lipase (0.46±0.16%) and the bone materials treated with acetone (1.11±0.13%)(P=0.065). Both of them were lower than that of the bone materials treated with Na2CO3/NaHCO3(3.46±0.69%), the difference was of statistical significance (P<0.001). The lipids content of the bone materials treated with deionized water (8.88±0.18%) was higher than that of other groups, the difference was of statistical significance (P<0.001). Observed under scanning electron microscope, on the surface of bone materials defatted with lipase or acetone, the collagen fibers were intact and oriented in a classical fern-like organization, only a little amount of cell debris attached on the trabecular. On the surface of bone materials treated with Na2CO3/NaHCO3solution, there were some small lipid droplets and cell debris attached on the trabecular. On the surface of bone materials treated with deionized water, the lipid droplets bulked over the surface of the trabecula with the bone matrix unexposed. Observed from sections of materials with HE staining, no cells and cellular debris in the center of the bone cubes defatted with lipase were left. Bone treated with acetone was similar with that with lipase. Bone marrow residues, such as cell debris could be observed in the bone materials treated with Na2CO3/NaHCO3. There were some bone marrow residues in the medullar cavities of the bone grafts processed by water. On images of sections with Masson’s trichrome staining, the bone collagen of all groups of materials was stained red. The bone matrix kept the intact collagen which appeared organized in classical lamellar-like. The result of CCK-8assay showed the cytotoxic grade of the bone materials defatted with lipase or acetone was0or1at the1st,4th,7th day after treatment; The cytotoxic grade of the bone materials defatted with Na2CO3/NaHCO3was1at all three time points; The cytotoxic grade of the bone materials treated with deionized water was grade2～4. Observed under the scanning electron microscope, MC3T3-E1cells attached and grew on bone trabecular of the bone materials defatted with lipase or acetone. The cells appeared fusiform or polygonal, fusing together and almost covered the bone trabecular; Only a few cells were observed on the bone processed by Na2CO3/NaHCO3. On the surface of bone materials treated with deionized water, cells dispersed, globular in shape. The histocompatibility evaluation in vivo showed that all the bone materials had no obvious immune rejection and other adverse reactions at the1st,4th,12th week after implantation. Conclusion:The effect of lipase on defatting the bone tissue is obvious and advisable since the defatting procedure has no damages on the surface structure of bone tissue and no toxic residue. Therefore it is a potential choice to use lipase for bone graft material defatting.Chapter2Collagens Extracted from Allogeneic Bone and their Physicochemical PropertiesObjective:To prepare viscous carrier for the malleable allogeneic bone reconstructive material, the bones were defatted by lipase and digested with pepsin at 4℃to extract the bone collagen, followed by that, physicochemical properties, viscosity, BMP content, molecular structure, cytocompatibility of the bone collagen at different extracting stages were measured.Methods:The fresh bone of rat limbs were frozen at-76℃for two weeks. The soft tissues were removed from the bone. Then the bones were ultrasonically cleaned, degreased by lipase, lyophilized, and crushed to particles. After being decalcified by0.6mol/L HCL, the bone particles were immersed in acetic acid solution with the pepsin concentration of2.5g/L at4℃for72h. After the enzymolysis, the bone collagen mixture was centrifuged at a speed of15000rounds per minute for20minutes and the supernatant was harvested. One third of the supernatant was taken out and marked as group1(the crude bone collagen). NaCl was added in the supernatant to precipitate collagen overnight. Centrifugated again, the precipitate was harvested and dissolved by acetic acid solution. Then the bone collagen was centrifuged again and the supernatant was harvested. Half of the supernatant was taken out and marked as group2(the initial purified bone collagen). The PH value of the collagen solution was adjusted to7with the NaOH solution. NaCl was added in the supernatant to precipitate collagen overnight. Centrifuged again, the precipitate was harvested and dissolved by acetic acid solution. Then the bone collagen was centrifuged again. The supernatant was harvested and marked as group3(the purified bone collagen). All three groups of collagen solution were dialyzed in Na2HPO4solution to inactivate the pepsin for8h. Then the collagens were dialyzed in acetic acid solution for another48h. All operations above are carried out at4℃. At last, all three groups of collagen solution were lyophilized. Collagen solution with a concentration of0.2%of the three groups was prepared and their viscosity was measured respectively at37℃. Collagen solution with a concentration of1%of the three groups was prepared1%, and BMP content in bone collagen was detected respectively by the rat BMP ELISA kit. Collagen solution with a concentration of0.2%of the three groups was prepared and the molecular weight distribution of them was detected respectively by SDS-PAGE electrophoresis. Collagen solution with a concentration of0.4%of the three groups was prepared and analyzed respectively by the UV-Vis spectrophotometer. The standard solution of Ⅰ-type collagen was used as the control. The collagens were sterilized by irradiation. The extract liquids of the three groups of collagen were prepared respectively according to the ratio of1.25cm2collagen membrane surface area to1ml L-DMEM medium at37℃for72h. L929cells at the exponential phase were seeded on a96-well tissue culture plate with100μl of medium with1×104cells per ml. Extract liquids were changed after incubated in a humidified atmosphere of5%CO2at37℃for24h. L-DMEM medium was used as reagent control. The cytotoxicity was detected respectively by CCK-8assay at1st,4th and7th day after treatment. The collagen sponge membranes of the three groups were put in the6-well tissue culture plate. Then the exponential phase of L929cells were seeded on collagen sponge membranes at a concentration of2×105/ml and incubated in the incubator.The morphological changes of L929cells were observed under phase contrast microscope.Results:The bone collagen mixture solution was colorless, transparent, viscous liquid. It turned to be more transparent after purification. The lyophilized bone collagen appeared a white fluffy sponge. The viscosity of collagen solutions with a concentration of0.2%was2383.33±202.07mpa.s,1643.33±80.208mpa.s,1480±80.00mpa.s respectively. The viscosity of the crude bone collagen was higher than the collagen of other two groups, with a significant difference (P<0.01). The BMP content of bone collagens were3.364±0.076ng/g,2.953±0.054ng/g,2.841±0.043ng/g respectively. The BMP content of the crude bone collagen is higher than the other two groups, the difference was statistically significant (P<0.001). The BMP content of the initial purified bone collagen is higher than that of the purified bone collagen, the difference was statistically significant (P<0.05). Electrophoresis of the bone collagens revealed that the bone collagens were composed of several proteins with apparent molecular weights of about100kDa and200kDa. Spectral analysis showed, absorption spectrum of the bone collagens were similar with the standard rat type I collagen, the maximum absorption of the bone collagen was about232nm. The crude bone collagen had the most absorption peaks. The absorption peaks of the initial purified bone collagen and the purified bone collagen were less. CCK-8assay showed the relative proliferation rate of the bone collagen at different extracting procedure was more than95%, cytotoxic grades were1at1st,4th and7th day. Morphology of L929cells cultured on the bone collagen membranes appeared no significant difference compared with the normal L929cells.Conclusion:The bone collagens extracted from allogeneic bone with pepsin enzymolysis have slight cytotoxicity, high viscosity, and contain some BMP. They are the potential choices as viscous carrier of the malleable allogeneic bone reconstructive materials. The cytocompatibility of the crude bone collagen is similar with the initial purified bone collagen and the purified bone collagen, while the viscosity and the BMP content of the crude bone collagen was higher than that of purified collagen. So the crude bone collagen was chosen as viscous carrier for the preparation of malleable allogeneic bone reconstructive materials.Chapter3Preparation and Biocompatibility of the Malleable Allogeneic Bone Reconstructive MaterialsObjective:To prepare malleable allogeneic bone reconstructive materials by compounding the allogeneic DBM with the bone collagen, the DBM particles and the bone collagen compounded at the different ratios and then the malleable ability and disintegration of these composites were tested to determine the optimal ratio of the DBM particles and the bone collagen, finally the cytotoxicity and biocompatibility of composites were evaluated.Methods:DBM particles with the size of250～750μm and the crude bone collagen were prepared according to the method in the chapter2and sterilized by irradiation. The bone collagen solutions with concentration of0.75%,1.5%,3%,4.5%,6%were prepared respectively with sterile acetic acid in a sterile environment. The pH value of the bone solutions were adjusted to7by NaOH solution. Then the DBM particles were compounded with the bone collagen at a ratio of450mg DBM particles to1ml bone collagen solutions. The malleable ability and disintegration of the composites were tested. Each composite for the same volume were immersed in the sterile PBS solution in the6-well tissue culture plate at37℃. The disintegration was observed at different time points. The optimal ratio of the DBM particles and the bone collagen in the composites was determined by the malleable ability and disintegration of the composites. The extract liquid of optimal composite was prepared according to the ratio of O.lg composite to1ml L-DMEM medium at37℃for72h. Then the cytotoxicity of the composite was detected by using L929cells with CCK-8assay at1st,4th and7th day after treatment. The DBM particles and optimal composite were embedded in rat subcutaneous with non-toxic polyethylene film as negative control. At the1nd,4th,12th week after implantation, three rats were sacrificed randomly respectively. The implanted materials and surrounding tissues were harvested. The histological sections were prepared and stained with HE. Then the histological changes were observed under light microscope to evaluate histocompatibility.Results:All the composites prepared with DBM particle and different concentrations of bone collagen were malleable and could be formed to certain shape. The composite prepared with the bone collagen at a concentration of0.75%was loose and friable; The plasticity of the composite prepared with the bone collagen at a concentration of1.5%was improved; The composites prepared with the bone collagen at a concentration of3%or4.5%had the best malleable ability. The DBM particles in the composite bonded tightly. The composite prepared with the bone collagen at a concentration of6%turned to be hard and less malleable. All the composites can keep integrated in the PBS solution at37℃in a certain time. The composite prepared with the bone collagen at a concentration of0.75%was fully disintegrated at2nd hour. The composite prepared with the bone collagen at a concentration of1.5%or3%was fully disintegrated at4th hour. All the composites were fully disintegrated at6th hour. According to the malleable ability and disintegration of the composites the optimal ratio of the DBM particles and the bone collagen in the composites was450mg DBM particles to1ml the bone collagen with a concentration of4.5%. So the optimal ratio of DBM/collagen was10/1. CCK-8assay showed the relative proliferation rate of the composite was more than95%, cytotoxic grades were1at1st,4th and7th day. The histocompatibility evaluation in vivo showed that DBM particles and the composite had no obvious immune rejection and other adverse reactions at the1st,4th,12th week after implantation. The DBM particles single or compounded was absorbed and the mesenchymal cells appeared around the DBM particles from the4th week.Conclusion:The malleable allogeneic bone reconstructive material prepared by the allogeneic DBM compounding with the bone collagen has ability of good plasticity, disintegration, slight cytotoxicity, good biocompatibility and biodegradability. The optimal ratio of the DBM particles and the bone collagen in the composites was10/1. Chapter4Study on Bone Regeneration in Rat Calvarial Defects with the Malleable Allogeneic Bone Reconstructive MaterialObjective:To investigate the bone regeneration of the malleable allogeneic bone reconstructive material, the rat calvaria bone defect model was established and implanted with the malleable allogeneic bone reconstructive material. The bone regeneration of the defects was evaluated by histological observation (HE staining and Masson trichrome staining), calcein fluorescence labeling, and Micro-CT scanning.Method:The malleable allogeneic bone reconstructive material was prepared by compounded DBM particles with the bone collagen solution at concentration of4.5%at a ratio of450mg to1ml in a sterile environment. Forty-eight SD rats (250g) were used to investigate the bone regeneration of the malleable allogeneic bone reconstructive material in this study. The animals were divided into3groups randomly and anesthetized. The animals were prepared using a sterile technique in a sterile environment. An anterior-posterior midline incision was made through the skin and muscle to expose cranium. Then a full-thickness6×6mm2parietal defect was created and filled respectively with material according to group. The experimental groups:The DBM Group, bone defect filled with the DBM particles with size of250～750μm; The malleable allogeneic bone reconstructive material group:bone defect filled with the malleable allogeneic bone reconstructive material; The blank control group (the negative control group), nothing was implanted into the bone defect. Four rats of each group were sacrificed randomly at the1st,4th,12th week after surgery. The cranial vault with the attached tissue was removed from each animal and fixed, decalcified, and embedded in paraffin. The sections were made and stained with HE and Masson trichrome stain for light microscopy. Four rats were chosen randomly to label new bone formation with intramuscular injection of calcein solution at11th week after surgery. The labeled animals were sacrificed and1week later. The cranial vault with the attached tissue was removed, fixed, embedded in methylmethacrylate.10μm of histological sections were obtained using a special microtome and analyzed by fluorescent microscopy. At12th week after surgery, one specimen of each group was selected randomly after fixation and scanned in the Micro-CT scanner. Following micro-CT analysis, the specimens were taken for histological sections.Results:Gross observation showed that the DBM particles removed obviously from the bone defect in the DBM group. Bulk of the malleable allogeneic bone reconstructive material remained at the site of implantation except a small amount of that removed in the malleable allogeneic bone reconstructive material group. The bone defects in the blank control group were soft to palpation. Histological observation showed that, at the4th week after surgery, the mesenchymal cells arranged around the scattered DBM particles, fibrous tissue also appeared at interspace of DBM particles in the bone defects of the DBM group. In the malleable allogeneic bone reconstructive material group, a large number of mesenchymal cells arranged around or at the interspace of DBM particles and a small amount of new bone was also seen the bone defect while the bone defect was filled with fibrous tissue in the blank control group. At the8th week after surgery, DBM particles were partially absorbed companied by a large number of mesenchymal cells attaching the DBM particles, a small amount of new bone appearing and fibrous tissue still visible in the bone defects of the DBM group. In the malleable allogeneic bone reconstructive material group, the bone defect filled with woven bone and DBM particles, the woven bone surrounded with the absorbed DBM particles. The bone defect was still filled with fibrous tissue in the blank control group. At the12th week after surgery, woven bone and absorbed DBM particles scattered in bone defect, fibrous tissue was still seen in the bone defects of the DBM group. In the the malleable allogeneic bone reconstructive material group, the bone defect was filled with the mature lamellar bone and the DBM particles which had not yet fully degraded, new bone marrow cavity appeared. The bone defects in the blank control group were still filled with fibrous tissue, a small amount of new bone formed at the edge. Histological observation of section stained with Masson trichrome stain showed that at the4th week after surgery, the bone defects were filled with DBM particles and loose connective tissue fibers in the DBM group. In the malleable allogeneic bone reconstructive material group, a large amount of mesenchymal cells distributed around and interspace of the DBM particles in the bone defects. In blank control group, the bone defects were filled with the loose connective tissue. At the8th week after surgery, the DBM particles were further degraded, the dyed red areas of them increased, the dyed green loose connective tissue was still seen in the defects of the DBM group. In the malleable allogeneic bone reconstructive material group, the bone defects were filled with woven bones which were dyed green and absorbed DBM. In blank control group, the bone defects were still filled with the loose connective tissue. At the12th week after surgery, the defects of the DBM group were filled with the DBM particles, loose connective tissue, and a small mount of woven bone. In the malleable allogeneic bone reconstructive material group, the bone defects were filled with absorbed DBM and the lamellar bones which were dyed red. In the blank control group, bone defects were still filled with fibrous tissue. At the12th week after surgery, observed under the fluorescence microscopy, a small amount of green fluorescence appeared in the defects of the DBM group. The visible green fluorescence bands were seen in the defects of the malleable allogeneic bone reconstructive material group. Only little green fluorescent appeared at the edge of host bone in the bone defect of the blank control group. At the12th week after surgery, high resolution Micro-CT detection and3D reconstruction revealed new bone formation in all groups. High density of mineralized new bone tissue appeared at the edge and center of the defect of the DBM group, but the majority of the bone defect had not yet been completely bone repaired. Multiple mineralized bone tissue distributed in the bone defect of the malleable allogeneic bone reconstructive material group, but the bone defect had been not completely bone repaired. The high density point of mineralized bone tissue was also seen at the edge of host bone in the bone defect of the blank control group. Volume of bone, bone mineral content, bone mineral density, tissue mineral content, tissue mineral density and bone volume fraction in the bone defect of the malleable allogeneic bone reconstructive material group were higher than that of the DBM group and the blank control group.Conclusion:The malleable allogeneic bone reconstructive material prepared in this study has the ability of good bone regeneration, good biocompatibility and biodegradability. |
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