Investigation And Preparation Of Nanobiomimetic Materials Modified With A Novel Peptide Derived From Bone Morphogenetic Protein-2

Objective In this research, a new biomimetic bone tissue engineering scaffold material, BMP-2-derived peptide P24 / PLGA-[ASP-PEG] composite was constructed. The effect of P24 to the adhesion, proliferation and osteodifferentiation of bone mesenchymal stem cells (BMSCs) were investigated.Methods PLGA was modified with polyethylene glycol (PEG) and asparagic acid (ASP), and tri-block polymer PLGA-[ASP-PEG] material was prepared. Then peptide P24 was introduced into PLGA-(PEG-ASP)n scaffolds by cross-linkers. Thus, BMP-2-derived peptide P24 / PLGA-[ASP-PEG] composite was synthesized. BMSCs were cultured on three kinds of material respectively, one was P24 / PLGA-[ASP-PEG] biomimetic material (Group A), one was PLGA-[ASP-PEG] material (Group B), the other was simple PLGA material (Group C). The lineage specificity of the cells on these materials were detected by Flow cytometry(FCM). The cell adhesion was assessed by precipitation method. The proliferative ability of BMSCs were measured by MTT assay, Coomassie brilliant blue staining and scanning electron microscope(SEM). And the measure of alkaline phosphatase(ALP) activity and osteocalcin(OCN) level and ALP staining and calcium tubercle staining were performed to assess the cells'differentiation towards osteoblasts.Results FCM confirmed the cells that isolated and cultured were BMSCs. The adhesive ratio measure suggested that adhesion and proliferation of BMSCs on the surface of P24 / PLGA-[ASP-PEG] is much higher than the control group (P<0.05). These were coincident with the cells'growth curve and the result of total protein assay and SEM. The ALP activity and osteocalcin(OCN) level of BMSCs in P24 / PLGA-[ASP-PEG] group were significant higher than in PLGA-[ASP-PEG] group and PLGA group. And in the assessment of ALP staining and calcium tubercle staining, positive staining was expressed firstly in P24 / PLGA-[ASP-PEG] group.Conclusion The PLGA-[ASP-PEG] material was confirmed to improve BMSCs'adhesion and proliferation, and maintain their morphology. The material and its degradation product didn't impact BMSCs'osteodifferentiation. And the BMP-2 derived peptide P24 could induce BMSCs'differentiation towards osteoblasts, its activity was equal to BMP-2.PartⅡDose-dependent of BMP-2-derived Peptied on Osteogentc Induction of BMSCs in vitroObjective To evaluate the capability of synthesized BMP2-derived peptide P24 on osteogenic induction of BMSCs, and investigate its dose-dependent and optimal dose in vitro.Methods BMSCs were separated from 4-week-old SD rats and cultured with normal medium. At 3rd generation the medium was changed to osteogenetic medium which contained different dose of BMP2-derived peptide. The concentrations of BMP2-derived peptide contained in medium were 300,200,100,50 and 0μg/ml, respectively. These groups were labeled with A～E by turns. The change of cell morphology was observed with microscopy. ALP activities and OCN levels were taken at different points of time. The mRNA expression level of several osteogenetic marker such as Type I collagen(Col-I), osteopontin(OPN) and osteocalcin(OCN) were measured using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) technique to detect the osteoinductivity of different concentration of BMP2-derived peptide,.Results Under inverted phase contrast microscope, MSCs had changed their shape from long fusiform to short fusiform or polygon with the osteogenetic medium for 3~4 days. With the increase of the concentration of BMP2-derived peptide in medium, the time of MSCs changed their shape into osteoblast-like had advanced accordingly. The detection of ALP activities and OCN levels suggested that group A and group B were significant higher than the rest three groups (P<0.05). But there was no statistical significance between group A and group B(P>0.05). FQ-PCR suggested that after cultured for 14d, Col-I, OPN and OCN were expressed in each group. And the value of cycle threshold (Ct) of Group A and Group B were significant higher than the rest three groups(P<0.05). But there has no statistical significance between group A and group B(P>0.05).Conclusion BMP2-derived peptide can induce BMSCs to differentiate into osteoblasts effectively. And among the PLGA-[ASP-PEG] material system, the optimal inductive concentration of P24 was 200μg/ml.PartⅢEctopic osteogenesis of BMP-2-derived peptide combined with PLGA-[ASP-PEG] scaffoldObjective Evaluated the ectopic osteogenetic capacity of synthesis BMP2-derived peptide combined with poly (lactic acid / glycolic acid / asparagic acid-co-polyethylene glycol) (PLGA-[ASP-PEG]) by animal experiment.Methods A total of 48 SD rats were allocated into three groups randomly. Three kinds of materials were respectively implanted into each side of superficial layer of erector spinae. Group A: BMP2-derived peptide P24 / PLGA-[ASP-PEG] complex, Group B: simple PLGA-[ASP-PEG], Groups C: simple gelatin sponge . The bone formation of the implants was detected by X-ray and CT three-dimensional imaging, further assessed by histological sections in different points of time. The osteogenic marker, Col-I and OPN were detected by western blot at protein level and by RT-PCR at mRNA level.Results X-ray and CT three-dimensional imaging showed obvious osteogenesis in group A, occasional osteogenesis in group B, and no bone formation in group C in the 8th week. Histological examination showed at 8th week, in group A, bone trabeculae were found to be robust and wide with some compact bone-like tissue formed on their surface, and plenty of new bone formation was found, some bone trabeculae and new vessels located around them; and the material was absorbed totally. In group B, fibroplasia was found around implant and chondrocytes and osteoblasts could be seen occasionally; and in Group C, implanted gelation sponge was absorbed, the area displayed muscle tissue-like appearance, no new bone structure could be found. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) suggested that Col-I mRNA and OPN mRNA were expressed at each groups at 8th week, and in group A the express levels were higher than others. Western blot obtained the same consequence.Conclusion BMP2-derived peptide P24 could induce ectopic osteogenesis, and its ectopic osteogenetic capacity and osteoinductivity were equal to nature BMP-2. Os endochondrale acted predominantly during osteogenesis.PartⅣAccelerated bone regeneration with a novel synthetic BMP-2-derived bioactive oligopeptide combined with a nano-biomimetic PLGA-[ASP-PEG] scaffoldObjective Furtherly investigate the osteogenetic capacity of biomimetic materials modified with a novel peptide P24 derived from Bone Morphogenetic Protein-2 combined with PLGA-[ASP-PEG] polymer by using them to repair critical-sized bone defect.Methods A total of 36 new zealand white rabbits were allocated into three groups randomly. A 1.5cm unilateral segmental critical-sized bone defect was created in the left femoral diaphysis in all rabbits. Three kinds of materials were used respectively. P24 combined with PLGA-[ASP-PEG] scaffold were implanted in group A, PLGA-[ASP-PEG] without P24 were implanted in group B, and simple gelatin sponge were implanted in group C. Five-hole plates and screws were applied to perform internal fixation routinely. Macroscopic observation, X-ray, histological investigation, ALP activity of repaired tissue and biomechanics measure were taken to evaluate the bone repair capability of each kind of material.Results Radiographic findings showed: In group A, at 8th week, the boundary between bone callus and host bone became obscure, bone callus appeared uneven in density, the profile of os integumentale was displayed, bone defect almost disappeared, typical symbol of bone healing was observed; at 12th week, bone callus moulding was found, the texture of new os integumentale became clear, and them united with the broken end naturally, and the medullary cavity was trend to be recanalization. According to the standardized radiographic scales of bone defect repair, 9 rabbits were assessed as Grade 4 or above (fine ratio was 64.28%). In group B, at 12th week, a little bone callus was formed in the defect area, the broken ends were absorbed and became small, the medullary cavity began to be atresic, and the defect was still presented. There were 5 rabbits that be assessed as Grade 0 (62.5%). In group C, at 12th week, ununion symbol was still observed, atresic medullary cavity and loosing plate were be found. All of the rabbits were assessed as grade 0. Histological observations showed: In group A, in 8th week, active osteoblasts could be found, plenty of new bone tissue was formed and fused with each other, and seemed like woven bone and bone trabecula, lamellar bone was formed by calcification of bone trabeculae. At 12th week, woven bone tissue became thick and intensive, and turned into typical lamellar bone, osteocytes were trend to be mature, and with angiopoiesis and its invasion, irregular medullary cavities were formed. In group B, at 8th week, fibroplasia could be found and the material was absorbed partly.; at 12th week, new bone formation was found in the juncture of implant and host bone tissue, and bone trabeculae and lamellar bone were appeared. The middle of material was absorbed subtotally, massive fibrous tissue could be seen, and chondrocytes were be occasionally seen. In group C, at 12th week, the defect area was filled by fibrous connective tissue, hyperplasy in broken ends were inconspicuous, and medullary cavities were blocked. Biomechanics measure suggested the ultimate bending load of group A was 122.14±12.83N, which reparative ratio was 87.42% when compared with the reference value of normal bone in the same group; and in group B, the ultimate bending load was 38.38±8.09N, the reparative ratio of it was 26.89% when compared with the reference value of normal bone in the same group. And there was statistical difference between group A and B (p<0.05).Conclusion This novel biomimetic material P24/PLGA-[ASP-PEG] is an ideal scaffold for bone tissue engineering, which can trigger typical os endochondrale procession and improve repair of bone defect.