| BackgroundThe management of bone defects has always been a challenge in clinicalperiodontics. The traditional autogenous and allograft always are first selections,but there are many limitations with the materials used in the operation. calciumphosphate cements,(CPCs) is a new bone substitute material with manyadvangtanges,but it's widely use was set back by slow degradation. rhBMP-2 caninduce the regeneration of bone,Therefore,the application of rhBMP-2 is expectedto obtain more realistic bone regeneration.When BMP is transplanted into the body,too rapid diffusion and hydrolyzation restricts its osteoinduction and its applicationon the target cells. Therefore,the clinical application of BMP must have sustainedrelease carrier. Normal bone defects can heal fast with rhBMP-2/CPC,but therelease of the CPC factor capacity is limited. The study of microspheres as drugcarriers begun in the middle term of 1970s,after that,microspheres as drug delivery system have draw much attention in pharmaceutical field and have beensuccessfully applied in some clinical cases. When degradable micropheres loadedwith growth factors are mixed with CPC to construct a macroporous composite,therelease ability and degradation rate of the composite can be enhanced. At presentpeople mainly use PLGA material as microspheres,but it has some limitations: slowdecomposition rate and degradation products can damage the surrounding tissue andcells.GMs are biocompatible and controlled-degradable.GMs may replace PLGAmicrospheres as good modified materials.ObjectiveThe carrier material for preparation of microspheres is gelatin,which holdssuch characteristic as excellent biodegradability,low or no toxicity andbiodegradability. On the basis of gelatin's self-crosslinking and its solidification byaldehyde,rhBMP-2/GMs were prepared by improved emulsificationchemical-crosslinking method according to orthogonal factorization optimizationmethod. Many other methods were used to evaluate morphologic property,encapsulation rate,ectopia osteogenesis,drug release property of rhBMP-2/GMs.To evaluate the healing effects of the new artificial composite bone in bone defectmodels, using the "biomechanical characteristics - X-ray observations - theorganization form" comprehensive evaluation methods.Methods1 Preparation and quality examination of rhBMP-2/GMs1.1 On the basis of gelatin's self-crosslinking and its solidification by aldehyde,rhBMP-2/GMs were prepared by improved emulsification chemica crosslinkingmethod according to orthogonal factorization optimization method.1.2 Evaluate the drug content,encapsulation rate,morphologic property and microsphere-size with a scanning electron microscope.1.3 Investigation of rhBMP-2-releaseing characters in vitro. Phosphate buffer salinesolution was regard as rhBMP-2-releaseing medium,and numerical data wererecorded at different time.The curve of accumulative rhBMP-2-releaseing ofrhBMP-2-GMS in vitro was describeed.The leaching liquor of rhBMP-2/GMs/CPCwas collected after the composites had been soaked in the physiological saline for1,4,7,14,21 and 28 days,and the release of rhBMP-2 was measured by ELISAmethod.2 Evaluate the biological effects of rhBMP-2/GMs loaded CPC in vivio.Macroporous CPC was developed using GMs with 5 wt% weight ratios. ThreeCPC loaded rhBMP-2/GMs,GMS,rhBMP-2 were fabricated and implanted intothe bone defect of rabbits radius. After 6,12 weeks,the defects were detected byX-ray and dual energy X-rayabsorption meter. After 12weeks,the rabbits werekilled. rhBMP-2/GMs/CPC,GMS/CPC and rhBMP-2/CPC were obtained andhistopathological method was used to research the bone ingrowth and the materialdegradation.Osteo inductive ability and degradation rate of three scaffolds wasevaluated histologically.3 Statistical methods: Measurement data were presented as mean x±s standarddeviation and analyzed by q test and ANOVA,respectively. Significance differencedefined,as P value is less than 0.05Results1.Quality examination of rhBMP-2/GMs1.1 The microspheres were spherical in shape and easily dispersible in character,with a diameter of 78±20μm,(average diameter 87.2μm). 1.2 The drug content and encapsulation rate were 9.8% and 80.5%,respectively.1.3 The stability and redispersity of GMs were fairly excellent.1.4 rhBMP-2-releasing kinetics could be divided into two stages: the fast-releasestage and the sustained-release stage. 70% of total loaded rhBMP-2 was released inthe first ten days. The release of different time points of rhBMP-2/GMs/CPCwashigher than rhBMP - 2 / CPC group.2.Evaluate the biological effects of rhBMP-2/GMs loaded CPC in vitro.X-ray analysis showed that bone defect healing of group A was the fastest,andremiain-volume of group A was smaller than these of group B and C.Both CPCloaded rhBMP-2 and rhBMP-2/GMs could induce bone formation. Significantlymore bone was found in rhBMP-2/GMs/CPC group comparing with GMS/CPCgroup and rhBMP-2/CPC group.Histomorphologically,the amount of new bonewas observed deeper into the rhBMP-2/GMs/CPC composite,but little resorption ofrhBMP-2/CPC could be detected.Mineralization rate and bone density showed nodifference between three groups after 6 weeks,but after 12 weeks the values werehightest in the group A. New bones grew into the cores of the rhBMP-2/GMs/CPCwhich resulted from GMs degradation and rhBMP-2 bone inductionConclusion1 The best method of preparation of rhBMP-2/GMs was worked out,and thisstudy indicated that the technology of preparing rhBMP-2/GMs was stable andrepeatable.2 The experiments of rhBMP-2-releasing kinetics indicated that rhBMP-2/GMs andrhBMP-2/GMs/CPC possessed excellent sustained-release character.3 The degradation performance and bone induction abilities of CPC loadedrhBMP-2/GMs were stronger than those of application of rhBMP-2/CPC and GMs/CPC.4 Microspheres that of CPC loaded rhBMP-2. sustained-release system will bepossible to apply in bone tissue engineering in the future.
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