| Objective:(1).Preparation of chitosan hydrogel as release carrier of recombinant human bone morphogenetic protein 2(rhBMP-2),research its loading quantity,encapsulation efficiency and sustained release rate,in order to obtain steady release of rhBMP-2 cytokines.(2).Loaded the prepared recombinant human bone morphogenetic protein 2 chitosan hydrogel on novel HA/ZrO2 gradient biocomposite artificial vertebrae and prepared a new tissue engineering bone,then the novel artificial vertebrae were implanted into beagle dogs' spines defect models to study bone reparation effect of the novel artificial vertebrae.Method:(1).Prepared blank hydrogel by ion-crosslinked method,respectively crosslinked 1.0mg/ml,1.5mg/ml,2.0mg/ml concentration of chitosan(Chitosan CS)with 0.5mg/ml,0.7mg/ml,1.0mg/ml concentration of Sodium Tripolyphosphate(Sodium Tripolyphosphate,TPP),the crosslinked solution contained mass ratio of CS:TPP was 5:1,then lyophilized by freeze-drying apparatus.Scanning electron microscopy was used to observe lyophilized chitosan microscopic surface morphology of each group,and test swelling coefficient in each group,used the chitosan hydrogel with minimum swelling degree to be loaded with rhBMP-2.Enzyme-linked immunosorbent assay kit(ELISA)was employed to detect drug loading mass,encapsulation efficiency and sustained release characteristics of the lowest swelling degree group of chitosan hydrogel which loaded with rhBMP-2.(2).Loaded recombinant human bone morphogenetic protein 2 chitosan hydrogel on the new HA/ZrO2 gradient biocomposite artificial vertebrae and implanted it into animal's vertebral bone defect area.Selected 12 male beagle dogs,randomly divided into 3 groups as group A:implanted HA/ZrO2 gradient biocomposite artificial vertebrae loaded with recombinant human bone morphogenetic protein 2 chitosan hydrogel(n=4);group B:implanted pure HA/ZrO2 gradient biocomposite artificial vertebrae(n=4);group C:implanted autogenous iliac(n=4).Observed each group animals'activity cases at week 4,week 8 and week 12;Taken X-ray imaging observations respectively at week 6 and week 12 of each group;Animals were sacrificed at week 12 after surgery and taken specimens.In vitro Micro CT scan was used to detect the amount of new bone volume inside the material(Bone volume,BV),then the mechanical property was evaluated by extreme compression test.Finally,provided a comprehensive evaluation about repairing capability of the novel HA/ZrO2 gradient biocomposite artificial vertebrae loaded with bone morphogenetic protein-2 to vertebral bone defect.Results:(1).The hydrogel cross-linked by 1.5mg/ml concentration of CS and 0.5mg/ml concentration of TPP exhibited the lowest swelling degree,the swelling degree was 238.28%±28.06%,when compared with other groups,there were significantly statistical differences(P<0.05).SEM scanning showed the lyophilized chitosan was a three-dimensional network structure with uniform distribution of chitosan microspheres internal.When loaded with rhBMP-2,chitosan hydrogel encapsulation efficiency was 91.88%±1.53%,the loaded mass of drug was 39.84 ng/mg ± 2.34 ng/mg;The release rate was 28.32%±3.01%at day 1,and 48.92%±6.27%at day 3,while 74.40%±6.29%at day 12.(2).General observation of all animals showed the wounds healed with no obvious infection and purulent exudate.In addition to group B,there were no significant differences of each group compared the average number of jumps per minute(activity case)at week 12 with their preoperative(P>0.05).At week 6 and week 12,X-ray imaging observation showed:In group A,with the passage of time after vertebral replacement surgery,callus formation gradually increased around the material,since the gap between artificial vertebrae and autogenous bone was gradually filled with new bone,so boundary between the implant and host bone become increasingly blurred.While the artificial vertebrae and autologous bone had almost closely linked at week 12 with no obvious boundary between artificial vertebrae and autologous bone;In group C,there were apparently self-absorption and bone osteolysis at non-load bearing areas at week 12,and appeared rapidly autologous bone remodeling.Meanwhile the bone mineral density increased at load bearing areas when compared with week 6.The graft bone and autologous bone had almost completely fused at week 12;In group B,however,the repair to bone defect was slower than group A and group C after vertebral body replacement surgery.The specimens were taken at week 12,the in vitro Micro CT observation showed:three dimensional CT reconstruction could be seen there was a large number of new bone formation inside the artificial vertebral,and bone volume detection result at week 12 were 145.38 mm3±18.52 mm3 in group A and 86.30mm3±15.59mm3 in group B,there was significant difference between the two groups(P<0.05).Ultimate compressive test showed the maximum compressive strengths of vertebras were 14.03 MPa ±1.66 MPa in group A;8.62 MPa ±1.24 MPa in group B and 13.78 MPa ±1.43 MPa in group C.The group A and group C were significantly higher than that of group B in statistics analysis(P<0.05).Conclusion:The novel HA/ZrO2 gradient biocomposite artificial vertebrae loaded with recombinant human bone morphogenetic protein 2 can excellently repair vertebral defect and can achieve a similar result to autologous bone substitute,it may be an ideal bone tissue engineering alternative material. |
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