Fabrication And Biological Effects Investigation Of The RhBMP-2-loaded Gelatin Microspheres

BackgroundThe management of periodontal defects, including destruction of the periodontal ligament, cementum and the formation of intrabony defects, has always been a challenge in clinical periodontics. At least four criteria must be met in order for regeneration to have occurred, these include all the features of the normal dentogingival complex that would equate to restoration of these tissues to their original form, function and consistency: (1) A functional epithelial seal must be re-established at the most coronal portion of the tissues and be no more than 2 mm in length; (2) new connective tissue fibers (Sharpey's fibers) must be inserted into the previously exposed root surface to reproduce both the periodontal ligament and the dentogingival fiber complex; (3) new acellular, extrinsic fiber cementum must be reformed on the previously exposed root surface; (4) Alveolar bone height must be restored to within 2 mm of the cementoenamel junction. The basis of periodontal regeneration is periodontal ligament cells (PDLCs). However, limited cells can't provide the effective condition for the repair of periodontal lesion. BMP is found in periodontal tissue, which can stimulate the proliferation, DNA synthesis, and the ALPactivity of PDLCs. Recent studies suggest rhBMP-2 not only induce the regeneration of alveolar bone, but induce the regeneration of cementum and periodontal fibers. Therefore, the application of rhBMP-2 is expected to obtain more realistic periodontal regeneration.When BMP is transplanted into the body, too rapid diffusion and hydrolyzation restricts its osteoinduction and its application on the target cells. Therefore, the clinical application of BMP must have sustained release carrier. Recent advances in growth factor biology and biodegradable polymer constructs have set the stage for successful tissue engineering of cartilage, bone and related tissues of which the periodontium could be considered a prime candidate for such procedures.The study of microspheres as drug carriers begun in the middle term of 1970s, after that, microspheres as drug delivery system have draw much attention in pharmaceutical field and have been successfully applied in some clinical cases. At present, people have realized that microspheres as drug carriers have the advantages of sustained or controlled release, passive or active drug targeting to specific tissues and so on, which will notably reduce the side effects of drug and improve their bioavailability as well as patient compliance. However, the report of drug delivery system on periodontal regeneration field was very limited, so we tried to introduce this system into periodontal field as to find a possible entrance that can control the release of rhBMP-2 in periodontal regeneration.ObjectiveThe carrier material for preparation of microspheres is gelatin, which holds such characteristic as excellent biodegradability, low or no toxicity and biodegradability. On the basis of gelatin's self-crosslinking and its solidification by aldehyde, rhBMP-2-GMS were prepared by improved emulsification chemical-crosslinkingmethod according to orthogonal factorization optimization method. Many other methods were used to evaluate morphologic property, encapsulation rate, ectopia osteogenesis, drug release property, stability and redispersity of rhBMP-2-GMS. MTT method and enzyme dynamics method were used to observe the proliferation and ALP expression of PDLCs after different doses of rhBMP-2 and rhBMP-2-GMS were added into the culture medium. At last, experiments on ectopia osteogenesis by coral scaffold loaded rhBMP-2-GMS were done so as to discuss the foreground of its application in periodontal tissue engineering.Methods1 Preparation and quality examination of rhBMP-2-GMS? On the basis of gelatin's self-crosslinking and its solidification by aldehyde, rhBMP-2-GMS were prepared by improved emulsification chemical-crosslinking method according to orthogonal factorization optimization method.? Evaluate the drug content, encapsulation rate, morphologic property and microsphere size with a scanning electron microscope.? Observe the stability and redispersity of rhBMP-2-GMS after a store in the refrigerator for 6 months. Ectopic osteogenesis effectiveness was evaluated by histological methods after the implantation into muscular pouch of thigh of nude mice.? Investigation of rhBMP-2-releasing characters in vitro. Physiological salt solution was regard as rhBMP-2-releasing medium, and numerical data were recorded at different time. The curve of accumulative rhBMP-2-releasing of rhBMP-2-GMS in vitro was described.2 Evaluate the biological effects of rhBMP-2-GMS on the proliferation and alkaline phosphatase (ALP) expression of PDLCsMTT method and enzyme dynamics method were used to observe the proliferation and ALP expression of PDLCs after different doses of rhBMP-2 and rhBMP-2-GMS were added into the culture medium.3 Two coral scaffolds loaded rhBMP-2 and rhBMP-2-GMS were fabricated and implanted subcutaneously into the nude mice. Osteoinductive ability of two scaffolds was evaluated histologically.4 Statistical methods: Measurement data were presented as mean± standard deviation and analyzed by T test and ANOVA, respectively. Significance difference defined, as P value is less than 0.05Results1 Quality examination of rhBMP-2-GMS? The microspheres were spherical in shape and easily dispersible in character, with a diameter span form 12 to 210 u m(average diameter 68.6 u m).? The drug content and encapsulation rate were 5.8% and 78.5%, respectively.? The stability and redispersity of rhBMP-2-GMS were fairly excellent and rhBMP-2-GMS could still induce bone formation after a store for 6 months.? rhBMP-2-releasing kinetics could be divided into two stages: the fast-release stage and the sustained-release stage. 75% of total loaded rhBMP-2 was released in the first ten days.2 The biological effects of rhBMP-2-GMS on the proliferation and alkaline phosphatase (ALP) expression of PDLCs? Cytomorphologic observationPDLCs ordered actinomorphicly and circinately, and periodontal fibroblast process is slim, with symmetrical cytoplasm and elliptoid or cycloidal nucleus. PDLCs were identified by positive stain for vimentin and negative for keratin.? Both rhBMP-2 and rhBMP-2-GMS could promote the proliferation andincrease the ALP activity of PDLCs in a concentration-related manner.Significantly greater proliferation and expression were found in rhBMP-2-GMsgroup comparing with rhBMP-2 group.3 Both coral scaffolds loaded rhBMP-2 and rhBMP-2-GMS could induce boneformation. Significantly more bone was found in rhBMP-2-GMS group comparingwith rhBMP-2 group.Conclusion1 The best method of preparation of rhBMP-2-GMS was worked out, and this study indicated that the technology of preparing rhBMP-2-GMS was stable and repeatable.2 The stability and redispersity of rhBMP-2-GMS were fairly excellent and rhBMP-2-GMS could still induce bone formation after a store for 6 months.3 The experiment of rhBMP-2-releasing kinetics indicated that rhBMP-2-GMS possessed excellent sustained-release character.4 The biological effects of rhBMP-2-GMS on the proliferation and ALP expression of PDLCs were better than that of application of rhBMP-2-GMS solely.5 Ectopia osteogenesis abilities of coral scaffolds loaded rhBMP-2-GMS were stronger than that of coral scaffolds loaded rhBMP-2.6 Microspheres sustained-release system will be possible to apply in periodontal tissue engineering in the future.