Experimental Study On Bioactivity Of Bone-Seeking BMP-2-Related Oligopeptide In Combination With Biomimetic Bone Repair Materials

Part Ⅰ Evaluation on Bone-Seeking Properties of two Different synthetic oligopeptides derived from BMP-2Objective This paper aims to compare the bone-seeking properties of two different synthetic oligopeptides derived from BMP-2, so as to explore the relevant factors to influence the bone-seeking properties of the BMP2-related oligopeptide.Method Two different oligopeptides P20and P24were synthesized by solid phase synthesis process, and the P24contained a repeating sequence of Asp (aspartic acid) and phosphorylated serine. The nano-HAP crystals were used to simulate bone tissues, then the bone-seeking properties of two different oligopeptides were preliminary studied through traditional hydroxyapatite binding assay so as to determine the adsorption rates of these two peptides under different absorption time with different HAP quantity.Results Both oligopeptides could achieve rapid adsorption of HAP within30min, then their adsorption speed decreased with the increase of time, P20achieved its peak of adsorption rate at60min, P24achieved its peak of adsorption rate which was twice as much as that of P20at90min; the adsorption rates of both oligopeptides increased along with the increase in the quantity of HAP, and the adsorption rate of P24was significantly higher than that of P20for the different quantity of HAP such as5mg,10mg or15mg.(P<0.05), the adsorption rate of P24to15mg HAP was nearly3times as much as that of P20.Conclusion The addition of the active sites of repeating sequence of Asp and phosphorylated serine can significantly enhance the bone-seeking properties of the P24, and P24has a better bone-seeking property. The best adsorption time of the bone-seeking oligopeptides is90min, and their adsorption rates were positively correlated to the HAP quantity. Part Ⅱ Adhesion, Proliferation and Osteogenic Differentiation of MC3T3-E1cells on Controlled Release Biomimetic Bone Matrix Materials Loaded with Different BMP2-related oligopeptideObjective This paper aims to investigate the release kinetics of two kinds of BMP-2-related oligopeptide in vitro, so as to evaluate the effect of the different biomimetic bone matrix materials loaded with BMP-2-related oligopeptides on the adhesion, proliferation, and osteogenic differentiation of MC3T3-E1cell.Methods Two different BMP-2-related oligopeptides P20and P24were loaded within ture bone ceramics (TBC) or nHAC/PLA scaffold materials respectively, so as to construct4composites (P20/TBC, P20/nHAC/PLA, P24/TBC and P24/nHAC/PLA), and the P24/Absorbable collagen sponges(ACS) were used as control material. Then their in vitro release kinetics were measured. To evaluate the biologic activity of these materials, they were divided into3groups including Control, P20and P24group, and each group was divided into TBC and nHAC/PLA subgroup. The MC3T3-E1cells were cultured on six kinds of materials respectively. The cell adhesion rate were assessed by precipitation method. The proliferative ability of MC3T3-E1cells were determined by MTT assay. And the measure of alkaline phosphatase (ALP) activity were performed to assess the differentiation towards osteoblasts of MC3T3-E1cells Results The in vitro experiment showed that the release of P24or P20in nHAC/PLA or TBC scaffold material was slower than that of P24in ACS, and the release characteristics of P24was more striking than that of P20in TBC or nHAC/PLA material. While the cell adhesion rate, results of MTT experiments and ALP activity determination suggested that adhesion and proliferation of MC3T3-E1cells on the surfaces of all biomaterials containing BMP-2-related oligopeptides were higher than those on the surface of control groups, and those in P24group were significantly higher than those in P20group, and those in P24/TBC group were significantly higher than the P24/nHAC/PLA group (P<0.05).Conclusion TBC and nHAC/PLA scaffold materials are suitable as controlled release carrier for BMP-2-related oligopeptide delivery, and TBC material is particularly ideal. The bone-seeking properties of P24can markedly enhance the performance of the sustained-release of P24in the TBC and nHAC/PLA scaffold materials, and can significantly promote the MC3T3-E1cells adhesion, proliferation, and osteodifferentiation on the biomaterials. Part Ⅲ The effect of different BMP-2-related oligopeptides on ectopic bone formation in sintered bone or nHAC/PLA scaffold.Objective This paper aims to compare the ectopic osteogenesis capacities of two different BMP-2-related oligopeptides, P20and P24, combined with sintered bone (TBC) or nano-hydroxyapatite-collagen PLA (nHAC/PLA) scaffold material respectively, in order to explore the osteoinductive activity of bone-seeking BMP-2-related oligopeptide.Methods A total of30Sprague Dawley rats were divided into control group, P20group, and P24group randomly, each group was divided into TBC and nHAC/PLA subgroup (each n=10). Each rat was processed to prepare bilateral quadriceps muscle pouches model. Subsequently, the TBC material was implanted in left side and the nHAC/PLA material was implanted in the right side according to the experimental group. Radiographic and computer tomographic examinations were performed at2and8weeks post implantation, so as to evaluate the bone density. At2and8weeks after surgery the implanted materials were explanted, and then the total volume of the HAP in specimens (BV) were determined by high-resolution Micro CT, further histological examination were taken to determine the bone formation.Results The X-ray detection at8th weeks showed the densities of implants in P24groups were higher than those at2th weeks, the changes in densities of implants of P20and the control groups were not significant. The bone density results showed that, the density of implants in P20/TBC groups, P24/TBC groups at8weeks were significantly higher than those at2th week (P<0.05). While the density of implants in control groups, P20/nHAC/PLA groups, P24/nHAC/PLA groups were slightly higher than those at2th week (P>0.05). BV quantitative results showed:in TBC groups, the BV value of P24groups at2weeks or8weeks was significantly higher than that in other groups, while the that the BV value of P20only at8th week was significantly higher than that in control groups (P<0.05); in nHAC/PLA groups, the BV values of P20and P24groups at2th week were slightly higher than that in other groups(P>0.05), the BV value of P24groups at8th week was significantly higher than that in other groups (P<0.05), and the BV value of P20groups at8th week was significantly higher than that in control groups (P<0.05). The observation of histomorphology showed that sporadic osteoid could be observed in P24/TBC groups at2weeks and a large number of woven bone formation and many active osteoblasts and neovascularization structures could be seen in P24/TBC groups at8th week. Significantly less woven bone formation was seen for the P24/nHAC/PLA groups at8weeks compared to the P24/TBC groups. There were only sporadic osteoid could be seen in the P20groups; and in the control groups, no new bone structure could be found.Conclusion Both sintered bone and nHAC/PLA are ideal candidate for bone tissue engineering scaffold materials; the ectopic osteogenetic capacity and osteoinductivity of BMP-2-related oligopeptide was significantly improved by modified with bone-seeking moieties when coupled with TBC or nHAC/PLA scaffold material. Part IV Experimental Study on Repair of Critical-size Defects in Canine Radius Using Bone-Seeking BMP-2-related Oligopeptide Combined with Sintered Bone Scaffold MaterialObjective To investigate the efficacy of bone-seeking BMP-2-related oligopeptide/sintered bone (TBC) composite scaffold material for enhancing healing of critical-size defects in canine radius, so as to explore the feasibility of their further applications.Methods A total of6beagle dogs were processed to prepare the critical-size defects (20mm) in middle section of bilateral radials, these dogs were divided into4groups according to their preparation sides randomly:there was no implant placing into the control bone defect group, pure TBC material was implanted into TBC group, the bone-seeking P24/TBC material was implanted into P24group and the rhBMP2/T material was implanted into BMP2group. After12weeks and24weeks of implantation, the X-ray detection was performed for them.3D-CT and histomorphometric evaluations were performed to evaluate the bone repair capability of each kind of material at24weeks after implantation.Results The results of the radiographic and histomorphometric examination suggested that no new bone was found in the control group. In TBC group, the radiographic examination result at each time point showed that no callus could be found obviously and no obvious fusion was seen in bone-material interface; A little osteoid and woven bone, but no lamellar bone structure were observed in defects histomorphologically. In P24group, the bone-material interface was unclear and there was a little callus formation at12week; There was much callus formation with good osteointegrated at bone-material interface at24week. Histomorphometric examination showed much new bone formation, a large number of inteconnected lamellar bones, numerous active osteoblasts can be found, the bone-material interface had been integrated. The results of BMP2group were similar to those of P24group. And the differences between the new bone area percentage of P24group or BMP2group and that of TBC group were extremely significant (P<0.01), while the differences between that of P24group and that of BMP2group were not significant (P>0.05). Conclusion The bone-seeking P24/TBC composite scaffold material has good osteoinductive activity, it’s an ideal bone repair material for tissue engineering and deserve further pre-clinical research.