The Application Of NHAC/PLLA Seeked Novel Short Peptide From BMP-2 In Bone Repair

Research backgroundWorldwide,bone defects caused by trauma,tumor and infection are increasing year by year.There are about 4 million people in China each year because of various problems need to treat bone defects.In addition to bone defects caused by disease,bone grafting is often needed in plastic surgery.Bone graft material has become the second largest graft after blood transfusion,and has a trend of increasing year by year.So far,autogenous bone graft was used as the gold standard widely used in clinic,but its source is limited and for the deletion region function,and bone can cause patients with persistent pain,nerve injury and new fracture,limiting its clinical application;bone allograft for dangerous immune reaction and the spread of disease.Also limit its clinical application.Therefore,it is an important subject in the field of medical and biomaterial science to develop an ideal bone graft to repair bone defects.Besides good biocompatibility and biodegrade ability,the ideal bone graft material should have good bone conduction and bone induction.In the past 10 years,the research focus has been focused on the design and application of bone graft materials.Natural bone is mainly composed of apatite and collagen.Therefore,the bone repair material composed of hydroxyapatite and collagen is widely studied.The nano hydroxyapatite/collagen(nHAC)material developed by Cui Fuzhai,Professor of Tsinghua University,has been proved to be very close to the natural bone tissue in composition and structure.Nature Materials has commented that his experiments have demonstrated the theory of hierarchical structure of mineralized collagen,and opened up a new way to produce biological materials by biomimetic self-assembly.Polylactic acid(PLLA)is non-toxic and biodegradable,has been widely used in the research of bone tissue engineering scaffold materials,and by the U.S.Food and Drug Administration(FDA)approved are widely used in medical suture,temporary stent and drug delivery carrier.NHAC,a new bone tissue engineering material,nHAC/PLLA and PLLA,has been successfully prepared.The microstructure of its structure and classification is similar to that of natural bone tissue,which can improve the adhesion of cells,stimulate cell proliferation and distribution.Since it was approved by the food and Drug Administration in 2011,it has been used in thousands of cases,and the clinical data show that it has broad clinical application value.BMPs(bone)protein(morphogenetic)was first discovered and named by Urist.BMPs belongs to the transforming growth factor beta(TGF-beta)superfamily,is a group with a similar structure of highly conserved proteins,can induce bone and cartilage formation in vivo,and in body growth,endochondral ossification and fracture,the early expression of cartilage repair,development and regeneration plays an important role in the embryonic bone.BMP-2 is one of the most widely studied and one of the most active BMPs.Although BMP-2 has osteoinductive activity,but natural BMP-2 not only a limited number of expensive,and mixed with the carrier has many side effects,such as protein adsorption on the surface of the material after random folding,the active sites are not fully exposed to its biological activity is not high;in a biological environment in early explosive release then,the concentration quickly dropped to below the level of treatment and its protein degradation,it is difficult to continue to play a role.At present,domestic and foreign scholars using gene recombinant technology for preparation of recombinant human bone morphogenetic protein 2(rhBMP-2),but the complex preparation process,long production cycle,low yield,quality control is not easy,so the cost is high,the price is also very expensive,difficult to mass production,at the same time there is also genetic engineering product safety issues.Materials science and Engineering College of Tsinghua University,according to the active nucleotide sequence of BMP2,a new type of short peptide containing 17 amino acids was synthesized by chemical synthesis of P17-BMP-2.It can play BMP2 like unique osteoinductive activity,and solid phase synthesis method,the process is relatively simple,easy to use peptide synthesizer,large-scale preparation,effectively overcome the current domestic external gene engineering preparation process is complicated,expensive and safety risks and other shortcomings.Therefore,this topic will be the new peptide P17-BMP-from Tsinghua University and 2 high induced collagen base nano hydroxyapatite(nHAC/PLLA)composite materials,through in vitro experiments,experimental rabbit mandibular defect,the osteoinductive capacity of novel peptide P17-BMP-2 in vitro,and the load on the nHAC/PLLA,in promoting the role and change of the repair process bone defect,provide experimental data for its clinical application.Research objective1 Synthesis of a novel short peptide containing 17 amino acids by solid phase peptide synthesis P17-BMP-2.2To study the ability of P17-BMP-2 to induce osteogenesis3 The short peptide P17-BMP-2 was loaded onto the composite material of nano hydroxyapatite(nHAC/PLLA)by the adsorption method.4 To investigate the controlled release effect of P17-BMP-2 loaded peptide in vitro.5.In vitro and in vivo experiments were carried out to verify the osteogenic ability of the loaded P17-BMP-2 short peptide in vitro and in vivo.Research method1 Preparation of a novel peptide P17-BMP-2 The synthesized peptide was purified by gel filtration chromatography(FMOC/tBU)and purified by high performance liquid chromatography(HPLC).2 P17-BMP-2 induced activity test in vitro The contents of AKP in each group were detected by alkaline phosphatase(AKP)kit at different time points.3.The combination of new peptide P17-BMP2 and scaffold nHAC/PLLA The scaffold materials were observed under scanning electron microscope and transmission electron microscope(nHAC/PLLA)Under aseptic conditions,P17-BMP-2 was dissolved in deionized water and adjusted to the desired concentration(2 mg/ml and 10mg/ml).Nano hydroxyapatite/collagen/polylactic acid material(nHAC/PLLA)(5 x 5 x 5mm)was dipped into P17-BMP-2 solution,and gently stirred for 2 h at 200 rpm.Vacuum drying,drying,freezing and sterilizing.4.P17-bmp-2 was loaded on nHAC/PLLA,allograft bone,gelatin sponge,and the BCA protein concentration kit was used to compare the activity of sustained release.5.Detection of the activity of composite peptide nHAC/PLLA on cultured cellsMSC of rats were planted in each group,and the proliferation of MSC in each group was detected by MTT method after 2,4 and 6 days,respectively.Cell proliferation was detected by PI staining and confocal laser scanning microscopy.The cells were observed by SEM.6 Animal experimentsAll the experimental animals were randomly divided into four groups,5 in each group:A:blank control,no implants,B:nHAC/PLLA,C:nHAC/PLLA/,P17-BMP-2(2mg/g),D:nHAC/PLLA/,P17-BMP-2(10mg/g).The non-penetrating bone defect(10mm x 5mm x 5mm)was retained in the mandible,and the buccal lingual bone cortex was preserved.7 Imaging analysisX-ray observations were performed in 2 and 4 weeks after surgery.According to the Lane-Sandhu X-ray scoring standard,the amount of bone volume,bone connection and bone remodeling in the process of bone healing were comprehensively evaluated.8 Histological analysisBone material after implantation respectively 2,4 weeks were taken under the animal,complete mandible operation area,4 degrees C fixed in 4%paraformaldehyde containing picric acid and 0.2%,0.1M pH= 7.4 phosphate buffer in 7 days,and in pH=7.5,decalcified in 20%EDTA solution for 8 weeks,after the rinse water is more than 12h.After dehydration,transparent,paraffin embedded,continuous cutting 5 m tissue sheet of 10,for histological staining(H.E.).Lane and Sandhu development of histological scoring system for statistical processing.Research results1 The purity of the synthetic peptide was 96%according high pressure liquid chromatography,mass spectra showed that the average molecular weight of the synthetic peptide was 2023.67m/z,the average molecular weight of consistent polypeptide and designed in theory,short peptide sequence is correct,satisfy the requirement of the experiment.2 Alkaline phosphatase showed that the content of alkaline phosphatase increased with time.At second and third weeks,the new short peptide containing 10mg/ml was lower than the positive control group.The content of alkaline phosphatase in the experimental group was significantly higher than that in the negative control group.At the same time,the group containing 30mg/ml P17-BMP-2 was significantly better than that of other groups on the content of alkaline phosphatase.3.Electron microscope observation nHAC/PLLA,we can see the irregular arrangement of pore structure4.The delayed release effect of P17-BMP-2 on nHAC/PLLA was better than that of allograft bone5.The results of MTT showed that the number of cells increased gradually and the activity increased with time.The cells containing 10mg/g short peptide P17-BMP-2 were the most active.After cell staining,confocal laser scanning showed that 10mg/g short peptide P17-BMP-2 had the strongest activity in the materials group.6.The analysis results show 4 images:2 weeks after operation,the blank control group no bone density,bone repair materials were found in low density,and the composite model of peptide material group density than the blank control group density group and simple material to deep.After 4 weeks,each group was better than the density at 2 weeks after operation are deep,and the area increased.In the blank control group,the density shadow was deepened but not continuous.The area with the highest density of the new compound short peptide is completely filled with the defect area.According to the results of Lane-Sandhu X ray score,the score of 4 weeks after operation was significantly higher than that of the latter 2 weeks.With the increase of the content of the new compound short peptide P17-BMP-2,the higher the score.7 Histological analysis showed that:at the end of the 2 week after operation,A group(blank control group)had fiber binding at the edge of the defect area,and fibroblasts were observed.Group B(simple material group)visible rabbit host bone implant materials and binding sites began to blur the fiber connection,the formation of a large number of connective tissue.And connective tissue,fibroblasts into the deep material,but not full of materials.The formation of small vessels and inflammatory cell infiltration material at the junction between the material and the host bone.Slight degradation of material.C group and D group(Material Group implanted a novel short peptide P17-BMP-2),there are a lot of inflammatory cell infiltration,fibroblast formation,connective tissue,fiber connection is more obvious,there are a large number of small angiogenic and osteogenic cells,with novel peptides and increase the content of visible early bone formation and formation the new active ossification organization,slight degradation of visible material.4 weeks after operation,A group(blank control group),visible fiber connection,active new bone formation.B group(implanted nHAC/PLLA group)with a small amount of inflammatory reaction,connective tissue,fibrous connection is formed at the junction of the host bone and implant materials,a large number of small vessels formed,and active new bone formation,bone aggregation.Visible material degradation.C group and D group(Material Group implanted a novel short peptide P17-BMP-2)visible inflammatory cell infiltration was less than 2 weeks,there are a large number of active new bone formation,and with the new peptide content increased new bone formation increased,and the new bone formed a new structure of ossification.The boundary between the host bone and the newly formed bone is blurred,and the early cortical bone formation can be seen.Obvious degradation of implant material.According to the results of Lane-Sandhu histological score,the results showed that 4 weeks after the operation,bone defect repair was significantly better than the repair at the end of the week after surgery,the mean score of histological score was statistically significant()(p<0.001).At 4 weeks after operation,D group was significantly better than other groups.Conclusion1,A novel peptide P17-BMP-2 with 17 amino acids was synthesized by solid-phase peptide synthesis.2 New peptide P17-BMP-2 can promote the proliferation of mouse MSC in vitro and induce MSC to differentiate into bone in vitro.3.P17-BMP-2 loaded on nHAC/PLLA has better release effect.4.nHAC/PLLA combined with a novel peptide P17-BMP-2 can induce accelerated bone repair,which can provide experimental data and theoretical support for its clinical application.