| Background:The treatment of bone defect was difficult to clinical workers,and autologous bone was thought to be the most considered solution.However,autologous bone transplantation had many disadvantages.Therefore,it’s quite necessary to look for some suitable tissue-engineered bone materials for substitution,which was popular in recent years.Three types of materials most often used in clinic were as follows:medical metal materials,organic polymer materials and bioceramic materials.Hydroxyapatite materials represented by Bio-Oss had widely proved their value in clinic.In this study,we developed a new hydroxyapatite material derived from oyster shells:natural calcined porous bone repair material(OHA).It had been proved to have good biocompatibility and osteoinductivity in prophase studies in vitro,but the osteogenic efficiency and safety evaluation in vivo were not completely clear.This study would assess the osteogenic efficiency and safety evaluation of this new material in animal experiment and clinical trial.Part Ⅰ:Animal experiment ⅠPurpose:To study the ability of new material to promote bone repair by establishing rabbit skull critical sized bone defect model.Materials and methods:Twelve experiment animals were divided into two groups randomly(six for each).Bone defects with a size of 15mm in diameter were made on calvaria top at both sides.The right sides were filled with OHA as experimental sides and the opposites were as controls.Six animals were euthanized at 6th and 12th week respectively.Cone beam CT(CBCT)was used for imaging detection,HE staining was used for histomorphology detection,then the results were analyzed by t test.Result:1)The rabbit skull critical sized bone defect model was successfully established,and all 12 experimental animals survived and completed the experiment.2)The results of CBCT imaging showed that at 6th or 12th week,the experimental sides showed high density filling image,which were higher than controls.At 6th week after operation,the image of experimental sides showed higher density compared to normal bone,and the connection with normal bone was smooth;At 12th week in experimental sides,the images continuously showed high density,but the thickness of whole layer was slightly thinner compared with 6th week;The control sides were still black or discontinuous.According to the gray value measured by Image J,the density of the experimental side decreased significantly at 12th week compared with 6th week(P<0.05).3)HE staining results showed that at 6th week,fibrous connective tissue crisscrossed around bone powder particles,no obvious new bone was observed.A small number of multinucleated macrophages and monocytes were seen on the experimental side;At 12th week after operation,the connective tissue between granules on the experimental side was significantly thicker and connected more closely.Several bone islands surrounded by new formed bone could be seen at the edge area,especially at the bottom,and new formed bone tissue started growing towards center.In the control side,a large number of tissue cells were seen at 6th week,and the fibrous tissue structure was loose.No obvious bone like tissue was found.At 12th week after operation,only thickened layered fibrous tissue was covered,and no new formed bone was found.Conclusion:1)OHA showed good osteoconductivity in vivo,and promoted the repair of critical sized bone defects,and showed the potential as bone grafts.2)OHA had certain degradability in animals.3)OHA didn’t cause inflammatory reaction.Part Ⅱ Clinical trialsPurpose:To evaluate the effectiveness and safety of OHA in the site preservation of alveolar.Method:27 volunteer patients participated in our medical center.After tooth extraction,certain materials were embedded in socket.OHA was applied as the experimental group,and hydroxyapatite of another brand was as control.CBCT was taken at 0th day and 24th week.The changes of alveolar ridge width were used to evaluate the effectiveness as the main index,and height changes and wound healing grades were as secondary indexes;The laboratory tests of blood routine,liver and kidney function and blood glucose before and 24 weeks after operation were used as the main indicators of safety evaluation,and adverse events should be recorded in details.Result:Effectiveness evaluation:1)Changes of alveolar ridge width:the width in the experimental group was with an average decrease of 1.34±1.03mm at 24th week.And the control was with an average decrease of 1.16±1.6mm.The changes showed no difference(P>0.05).2)The average height of alveolar ridge in the experimental group was with an average decrease of 1.27±1.65mm;The control group was with an average decrease of 2.29±1.29mm.The changes showed no difference(P>0.05).3)Two weeks after operation,the wounds of all cases in two groups healed well without obvious redness,induration,hematoma,effusion,infection and suppuration.Safety evaluation:1)No difference was observed in the exception value comparation(P>0.05).2)No adverse events occurred in the whole test cycle.Conclusion:1)OHA could effectively reduce the absorption of alveolar ridge after tooth extraction,and showed no significant statistical difference compared with the control group.The effectiveness was equivalent to that of the control group.2)OHA in the experimental group and hydroxyapatite in the control group both showed good safety in the clinical trial of the experimental center,with little stimulation to the human body,no impact on the healing of local wounds,no obvious rejection reaction,and little impact on various indexes of laboratory examination.Part Ⅲ Animal experiment ⅡPurpose:To approach whether OHA combined with concentrated growth factors(CGF)would gain better osteogenic effect in the repair of critical sized bone defects and CGF’s ability to promote vascularization in vitro.Method:The preparation of animal model was prepared as same as the first part.CGF from rabbit blood was prepared.It was mixed with OHA in equal proportion and implanted into the right defect areas.CGF was implanted into the left defect areas as control groups.Animal samples were collected at 6 weeks and 12 weeks respectively for CBCT detection and HE detection;After BMSCs were induced for 14 days by CGF conditioned medium prepared,angiogenesis was detected and data were analyzed.Result:1)Rabbit blood derived CGF was successfully prepared.2)The CBCT results showed that the bone density in the defect areas were significantly higher at 12th week than 6th week in OHA+CGF groups;At the 6th and 12th week of CGF sides,the images showed only intermittent white line image,while the density was significantly lower than that of OHA+CGF sides.3)The HE results showed that in the OHA+CGF groups,a large number of fibers and woven bones were interlaced around bone powder particles 6th week after operation,the thickness of woven bones and fibers was significantly increased,and the vascular tissue was more abundant;At 12th week,the woven bone between powders became thicker,more mature,and arranged more regularly.More homogeneous stained bone like tissues was observed.In CGF group,a large number of histiocytes were seen at 6th week after operation,and a large number of layered fibrous tissues were seen at 12th week,without obvious osteoid tissue formation.4)Angiogenesis experiment showed that the ability of BMSCs in lumen formation was significantly enhanced after the induction of CGF,and there was significant statistical difference between two groups.Conclusion:1)OHA combined with CGF could significantly improve the bone repair effect in the defect area.2)CGF could promote angiogenesis in vitro,which may promote osteogenesis by increasing vascularization in vivo. |
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