| PART1STUDY ON THE PREPARATION AND APPLICATION OFNANO CALCIUM-DEFICIENT HYDROXYAPATITE/MULTI(AMINO ACID) COPOLYMER/CALCIUMSULFATE HEMIHYDRATE COMPOSITEObjectiveAn injectable nano calcium-deficient hydroxyapatite/multi(amino acid)copolymer/calcium sulfate hemihydrate composite was prepared by usingin-situ polymerization method. The composition and structure of thecomposite was analyzed, and how the different ratio of n-CDHA/MAC andCSH impacted on the injectability, setting time and mechanical property ofthe composite was studied. In order to select the optimal ratio ofn-CDHA/MAC and CSH.MethodsThe n-CDHA/MAC compound was synthesized by an in-situpolymerization method. Then physically mixed with the powder of CSH at different ratios. The mixture was extruded at170°C using a twin-screwextruder, air-cooled, and pulverized using a mill into a powder of particlesize in the range of38to125μm. The n-CDHA/MAC/CSH compositeswith different weight percentages of n-CDHA/MAC (ie,10%,20%,30%,and40%) were obtained and moistened by distilled water with aappropriate liquid to powder ratio.FT-IR and XRD was used to measurethe composite’s composition and structure; The microstructure wasobserved by the scanning electron microscope; The injectability and settingtime was tested by the Vicat needle test according to the internationalstandard ISO9579-1989E, and the compressive strength of the compositewas tested by the REGER30–50mechanical testing machine.ResultsThe FT-IR and XRD result indicated that the n-CDHA/MAC/CSHcomposite is composed of n-CDHA, MAC and CSH, and each componentin the basic crystal structure does not change. There was a connection ofchemical bond in each component, not a mechanical bond;The composite’sSEM images showed that the n-CDHA, MAC and CSH bond with eachother evenly, no cavity was observed in the composite,the surface of thecomposite was density, rough and porosity. The setting time and injectabiltyof the n-CDHA/MAC/CSH composite increased with the elevation inweight percentages of n-CDHA/MAC. The biomechanical test showed thatcompressive strength of composite were21.24±1.92Mpa,28.42± 2.63Mpa,34.34±2.52Mpa,43.92±2.40Mpa and30.77±3.37Mpa with0wt%,10wt%,20wt%,30wt%and40wt%n-CDHA/MAC. Elevatedpercentages (up to30%) of the n-CDHA/MAC composition markedlyincreased the compressive strength of the n-CDHA/MAC/CSH compositeafter setting. However, when the weight percentage of the n-CDHA/MACcomponent reached40%, the compressive strength of the composite wassignificantly decreased, the composite even ruptured and collapsed.ConclusionThe n-CDHA/MAC/CSH composite was synthesized successfully by thein-situ polymerization method. There was a connection of chemical bond ineach component, not a mechanical bond; The injectability，settingtime and compressive strength of composite could be adjusted the ratio ofn-CDHA、MAC and CSH. Through the above screening, the compositecontained20-30wt%n-CDHA/MAC was in accordance with therequirement of orthopedic surgery.PART2STUDY ON THE CELLULAR COMPATIBILITY OFNANO CALCIUM-DEFICIENT HYDROXYAPATITE/MULTI(AMINO ACID) COPOLYMER/CALCIUMSULFATE HEMIHYDRATE COMPOSITEObjectiveTo evaluate the cellular compatibility of nano calcium-deficient hydroxyapatite/multi(amino acid) copolymer/calcium sulfate hemihydratecomposite in vitro and the impact on MC3T3-E1osteoblast’s biologybehaviors.MethodsExperiment one: The L929cytotoxicity test of n-CDHA/MAC/CSHcomposite.The L929cells were co-cultured with n-CDHA/MAC/CSH discs or theirextract liquid. The changes of L929cells’ morphology were observed bythe inverted phase contrast microscope, MTT assay was performed toevaluation the cell proliferation at different times, and the cells’ apoptosiswas detected by flow cytometer Annexin V-PE/7AAD double stainingmethod.Experiment two: Evaluate the effect of n-CDHA/MAC/CSH compositeon MC3T3-E1osteoblast’s biology behaviors.The MC3T3-E1osteoblasts were implanted both on the surface ofn-CDHA/MAC/CSH and pure CSH discs, respectively, the blank controlgroup was set up. The cells adhered and grew on the surface of biomaterialswere observed by the scanning electron microscope; Elisa assay wasperformed to test the effect of n-CDHA/MAC/CSH composite on alkalinephosphatase and osteocalcin secretion of MC3T3-E1cells; Transwellmigration test was selected to test the effects of n-CDHA/MAC/CSH composite on cell migration.ResultsExperiment one: The direct contact experiment indicated that then-CDHA/MAC/CSH composite had no obvious cytotoxicity, L929cellsattached and proliferated well and good cell morphology could be observed;MTT assay revealed that the toxic level of n-CDHA/MAC/CSH compositewas ranked as1;Flow cytometer test results confirmed that then-CDHA/MAC/CSH composite had no effect on the cells apoptosis（P>0.05）.Experiment two: The scanning electron microscope observed that theMC3T3-E1cells grew well on the surface of n-CDHA/MAC/CSH andCSH, had a normal morphology and proliferated dramatically and formedstratified cells layer as the time growth. The Elisa assay results showed thatthe ALP activity had no statistical difference among the three groups at1day（P>0.05）, at the4and7days, the ALP activity in n-CDHA/MAC/CSHgroup was higher than the CSH group and blank control group, thedifference was statistically（P<0.05）.The OCN content had no significantdifferences among the three groups at3,5and7days（P>0.05）,at10days,the OCN content in n-CDHA/MAC/CSH group and CSH group wassignificant higher than blank control group, at14days, the OCN content inn-CDHA/MAC/CSH group was significant higher than the CSH group andblank control group, the difference was statistically(P<0.05). Transwell migration test results indicated that there was no statistical differenceamong the three groups（P>0.05）.ConclusionThe n-CDHA/MAC/CSH composite had no obvious cytotoxicity, andhad satisfactory biocompatibility with MC3T3-E1osteoblast, which hadpositive regulatory effects on the cell adhesion, growth, proliferation andsecretion.PART3IN VIVO STUDY ON THE INJECTABILITY NANOCALCIUM-DEFICIENT HYDROXYAPATITE/MULTI(AMINO ACID) COPOLYMER/CALCIUMSULFATE HEMIHYDRATE COMPOSITE FORREPAIRING BONE DEFECT OF RABBIT FEMORALCONDYLEObjectiveTo investigate the ability of injectable nano calcium-deficienthydroxyapatite/multi(amino acid)/calcium sulfate hemihydrate compositerepaired the bone defect of rabbit femoral condyle.MethodsThe bone defects of6.5mm in diameter and1cm in depth were createdon the both sides of the rabbits’ femoral condyles. The n-CDHA/MAC/ CSH composites were implanted into the defects as experimental group, thecontrol group was implanted with the CSH, and the blank control groupwere subjected to no disposal. The general observation, Micro-CT scanningand Ven Gioeson histological staining were performed at4,8and16weeksafter operation. The biocompatibility in animals, ability of bone defectrepair and biodegradability of the n-CDHA/MAC/CSH composite wereinvestigated.ResultsThe incisions of animals in three groups were healed well. No obviousinflammation and infection signs were observed. Through the generalobservation, the cortical bone of bone defect in experimental group andcontrol group at16weeks after operation, the defect was still obvious inblank control group at16weeks after operation; The Micro-CT scanningimages showed that the experimental group and control group all had theability to repair the bone defect, but the trabecular quantity and density asdetermined was higher in the experimental group than in the control group,the bland control group did not find signs of new bone formation. The VenGioeson histological staining indicated the n-CDHA/MAC/CSH compositecoupled with generation of immature bone among it, the materials werebiodegraded mostly at8weeks after operation; The implanted CSH cementwas divided into several small pieces at week4and almost completelydegraded at week8. At week16, the number and density of trabecular bone were markedly lower in the CSH-treated group than in then-CDHA/MAC/CSH-treated group.ConclusionThe n-CDHA/MAC/CSH composite had excellen biocompatibility inanimals, and had better biodegrade rate and Ability to repair bone defectcompared to the pure CSH, had a good prospect of clinical application. |
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