| BackgroundBone defects have long been plagued by orthopedics in clinical effective treatment. Bone tumors, severe bone trauma may cause bone defects, bone repair materials have been invented by many scholars, including autologous bone, allograft bone and artificial bone. Since autologous bone can be resorpted quickly and obtain perfect clinical effect, It is limited by the source of the problem; Allogeneic bone, the xenograft are limited bone due to immune rejection in the clinical application, and the risk of infection is also one of the factors in restricting the development Artificial bone with have drawn wide, easy processing, good biocompatibility, low price, can be disinfected, conducive to clinical operation, etc., become the mainstream of the current artificial bone as repair materials.The artificial bone mainly includes two types of inorganic and organic materials. The hydroxyapatite (hydroxylapatite, HA) and tricalcium phosphate (tricalcium phosphate, TCP) represent the former and the representative of the latter are polylactic acid, polyglycolic acid or bothby a copolymer. With respect to the organic material, Inorganic materials have good mechanical strength, in which coralline hydroxyapatite (Coralline Hydroxyapatite, CHA) has been paid great attention due to the wide variety of sources, biocompatibility, bone conduction, harmless to human body. Despite the artificial bone has been used in clinical and achieved good results, but the implanted artificial bone often can not be absorbed by the body, resulting in bone graft failure. This is because the artificial bone (diameter>5mm) implanted in the human body, lack of artificial center of the supply of nutrients and low oxygen partial pressure, can not meet the artificial center of the seed cell growth, proliferation, differentiation, and osteogenic need, resulting in the seed cells stop growing or even death, so that fundamental reason about loss of osteoblasts effect lies in the weak inductive effect of tissue engineering bone. In addition, poor bone osteogenic effect is also an important reason for constraining the development of artificial bone. To this end, three ways are used to solve the artificial bone ossification activity by the domestic and foreign researchers. Firstly, composition of the cultured cells and artificial bone by culturing different types of cells all together.The different types of cells in the mixed culture, can promote cell growth and differentiation by the mutual regulatory relationships among cells. Youcheng fibroblasts, smooth muscle cells, and osteoblasts are commonly used to cultured in a mixed state. The living cells which can promote osteogenic effect are advantages of the joint cell culture, but the operation is cumbersome, long culture period, not easy to promote in clinical; Another approach which can promote artificial bone inductive effect is micro-surgery, prefabricated fascia and muscle flap with vascular pedicle are mainly used to wrap bone for gaining adequate blood supply, New composite flap have many advantages such as blood for reliable, bone viability, resistance to infection,but the disadvantages are also obvious, pre-surgery would be required to increase the suffering of the patients. Another method is to use a cell growth factor to promote the artificial bone induction effect, the method is simple, easy to promote in clinical. Relatively speaking, cell growth factor, due to promote the activity of bone ossification significantly in clinical application, has become a hot spot of research in this field, articles on cell growth factor promotes the artificial bone induction effect have been published on top magazines in this field. The growth factors such as bone morphogenetic protein (bone morphogenetic protein, BMPs), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), and transforming growth factor-β (transforming growth factor-β, TGF-P) are currently used to promote bone ossification. For the study of bone induction effect, mainly concentrated in the BMPs, the present study shows that the BMPs have a significant osteogenic effect. However, due to the short half-life, easy metabolic loss, BMPs can not maintain a sufficient concentration. Currently, load the active growth factor through the sustained release carrier can effectively solve this problem.In recent years, the study focused on sustained release carrier load active growth factor has being gained more and more attention and concern. From traditional carriers to a variety of drug-loaded microspheres carrier, the goal is to extend the sustained release of the growth factor, precisely control the release concentration, and expand its scope of application. But how to regulate the activity of cytokine release in accordance with the clinical requirements by preparing different sustained release microspheres, in order to maximize the promotion of artificial bone into bone-inducing activity and vascular activity, are the main issues that need to be addressed. The rapid development of controlled release technology and continued emergence of artificial biodegradable materials and their composites in recent years, provides an unprecedented opportunities and conditions for the controlled release of active cytokines and tissue-engineered artificial bone to promote osteoinductive activity. Microspheres controlled release system used in sustained release of the active growth factor, has opened a new research space for the development and utilization of exogenous growth factors. For the research on osteogenic inducing activity of sustained-release microspheres, many scholars at home and abroad have prepared sustained-release microspheres loaded the bone morphogenetic protein by the polymer synthesis and structure control technology, and the osteogenic effect is encouraging. However, the research on controlling the structure of sustained-release microspheres is not yet perfect. At the same time, how the interactions between microspheres molecules and growth factors affect the release process, how to regulate growth factor release by changing the structure of the microspheres, still need in-depth study. Specially, in order to improve the bone-inducing activity of composite microspheres loaded growth factor, the activity factors on how the microspheres promote ossification need to research deeply. The influencing factors include types of microspheres, the structure of the microspheres, the type of growth factor, and the interaction mechanism between the microspheres and growth factors, and the release process control on polymer microspheres load growth factors.The project has constructed a kind of polymer composite microspheres loaded recombinant human bone morphogenetic protein and has gained a good bone inductive effect. Because the performance of a carrier which is a polymer microsphere, is the key to allow cell growth factor controlled release according to clinical need. Chitosan (CS) of the item was selected as polymer microspheres for the synthesis support model. Chitosan is a natural polysaccharide, only basic polysaccharide in its molecule two free amino groups, the condensation reaction of the aldehyde-ammonia to form keybridge curing microspheres, the drug can be fixed in the skeleton. The microspheres having advantages such as non-toxic, good biocompatibility, and biodegradable, controlled drug release, improvement of the local retention of the drugs, addition of the bioavailability of the drug and other characteristics. The drug loading rate of recombinant human bone morphogenetic protein-2/Chitosan Microspheres is relatively low (33.437±2.290μg/mg).The reason may be only electrostatic interaction occur between the recombinant human bone morphogenetic protein and chitosan during the preparation of the microspheres, As a result,they could not bind together in a large number. Because dextran there are some heparin binding sites among sulfate and bone morphogenetic protein therefore, we have introduced dextran sulfate (DS) into microsphere as an intermediate carrier, and we have prepared recombinant human bone morphogenetic protein-2/chitosan/sulfuric acid Portugalpolysaccharide microspheres and gained high loading efficiency and stability release effect. Currently, more studies focus on sustained-release carrier loaded active growth factor and the clinical performance of these microspheres, but research on the mechanism of interaction between the relevant growth factor and carrier remains few, which is the main factors to determine the release process of the active growth factor. The influencing factors about Sustained-release body in the process of bone activity in promoting bone also need a system study.For this reason, the project has prepared single and composite microspheres with chitosan and dextran sulfate preparation. We have studied the interactions between growth factor (recombinant human bone morphogenetic protein-2) and the carrier polymer microspheres (the interaction mechanism between chitosan and dextran sulfate) using the resonance light scattering spectroscopy and fluorescence spectroscopy. At the same time, we have also and its recombinant human bone morphogenetic protein-2release process occurs clarify recombinant human bone morphogenetic protein-2release in thermodynamic and kinetic control process and compositemicrosphere sustained release mechanism; We also have explored the interactions between recombinant human bone morphogenetic protein-2and blank microspheres in the release process, furthermore, we have stated the thermodynamic and kinetic control process of recombinant human bone morphogenetic protein-2in the release process, and revealed the sustained release mechanism of the composite microsphere; We have studied the morphology, particle size and distribution control method of single and composite microsphere particles, as well as their impact on the the wrapped load rate of protein and release process. We have also revealed the regulation of the carrier morphology on controlling release of recombinant human bone morphogenetic protein-2; At the same time, we have studied the release process and its osteoinductive effect of recombinant human bone morphogenetic protein-2/chitosan/dextran sulfate composite microspheres. The research results of the project would provide new ideas and theoretical guidance manual repair of bone defects.Objectives:1. Preparation nanosphere of chitosan/dextran sulfate, study the polyelectrolyte complex process of dextran sulfate/chitosan nanospheres, investegate the interaction effect of the pH value, ion concentration of chitosan, sulfate dextranimpact, analysis of the interaction between the sugar contained protein blank microsphere diameter grain, provide a theoretical basis for the preparation of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.2. Preparation of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres, observe morphology of the microspheres and microspheres. Drive tablets the dispersity of study, to calculate the protein encapsulation efficiency, drug loading ratedraw the sustained-release curve.3. Study polyelectrolyte complex process of Dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, studies of the pH value, ion concentration of recombinant human bone morphogenetic protein-2, chitosanthe influence of the interaction between the sugars, dextran sulfate combined release curves, analysis of the sustained-release mechanism.4. Assess Biological safety of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres, provide a theoretical basis for clinical applications and Osteoinductive activity studies of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.5. Design cytology experiments, evaluate induction of osteogenic activity of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres in vitro on bone marrow mesenchymal stem cells, control with blank microsphere group, free of recombinant human bone morphogeneticprotein-2group, the groups were compared to assess similarities and differences of the two microspheres osteogenic effect, from the perspective of cytology.6. Statistical analysis:statistical description of the measurement data are expressed as mean±standard deviation, using SPSS13.0statistical software for analysis, the groups were compared using one-way ANOVA, multiple comparisons with LSD-t test method homogeneity of variance between the two groups,If heterogeneity of variance applications Games-Howell method. P<0.05was considered statistically significant.Methods:1. Prepare chitosan/dextran sulfate Nanosphere by the ionic crosslinking, study the pH and ion concentration on the interaction between dextran sulfate, chitosan, by resonance light scattering method, analyze contained protein blank microsphere diameter grain range by scane electron microscopy and Zeta potential, provide a theoretical basis for complex and dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres.2. Prepare dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres by ionic crosslinking, observe microspheres morphology using scanning electron microscopy and atomic force microscopy, study microsphere diameter grain and dispersitystudy of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres. Calculate protein encapsulation efficiency, drug loading efficiency, draw sustained-release curve recombinant human bone morphogenetic protein-2kit.3. Study complex poly electrolyte process of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres by Resonance light. To explore the pH, ion concentration of recombinantthe human bone morphogenetic protein-2, chitosan, dextran sulfate interaction between the combination of sustained-release curve analysis of slow-release mechanism in the application of resonance light scattering method.4. Assess biosafety of dextran sulfate/recombinant human bone morphogenetic protein-2/the chitosan nanospheres by experimental implant materials in accordance with the national standards biological toxicity assessment, recovery and subcultured rat fibroblasts sulfatedthe glucan/recombinant human bone morphogenetic protein-2/chitosan microspheres and dextran sulfate/chitosan blank microspheres cytotoxic experiment, a control group to assess their biological security.5. Design cytological experiments, to culture SD rat bone marrow mesenchymal stem cells in vitro by adding dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, dextran sulfate/chitosan blankmicrospheres, recombinant human bone morphogenetic protein-2by the MTT assay dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres in vitro bone marrow mesenchymal stem cells into bone-inducing activity, and blank microsphere group, recombinant human bone morphogenetic protein-2group, negative control group were analyzed.The results:1. Prepare dextran sulfate satisfied/chitosan microspheres in application of ionic crosslinking method, observe microspheres in scanning electron microscopy and atomic force microscopy, discover microsphere which is prompted into a ball, in a regular shape, at a good dispersity, average grain diameter about210nm, the sugar/chitosan nanospheres composite solution RLS intensity will change very rapidly by changing sulfuric acid glucan outside unconditioned stimulus, such as changing the pH of the solution, and the addition of metal ions,. State at room temperature, dextran sulfate sodium and chitosan can be combined in shape of a polyelectrolytecomplexes.2. Successfully prepare dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres in application of ionic crosslinking method. CS/DS/rh-BMP-2nanospheres evenly distributed, with an average particle size (217±8) nm, entrapment efficiency and drug loading of the microspheres were (85.6±3)%and47.245±3.321ug/mg. Microspheres release two hours after the a sudden release period in vitro, two days after the release reached a peak, followed by a slow decline in the release cycle is about28days.3. To study CS, DSS, and the relationship between the rhBMP-2in application of resonance light scattering method, the results show that the weak interaction between CS rhBMP-2, the main force for the van der Waals force, DSS with rhBMP-2exists between the heparin binding site, there is a strong interaction. In the process of preparation, rhBMP-2with DSS first combination in the sustained-release process, in combination with CS rhBMP-2will be preferentially released.4. Assess biosafety of experimental implant materials in accordance with the national standards on dextran sulfate/recombinant human bone morphogenetic protein-2/the chitosan nanospheres, recovery and subcultured rat fibroblast cells, cytotoxicThe test indicated that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan biocompatible, no significant cytotoxicity. Biocompatibility of chitosan/sodium dextran sulfate blank microspheres carrier reliable, non-cytotoxic is good protein carrier.5. culture SD rat bone marrow mesenchymal stem cells In vitro, add dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres, chitosan/dextran sulfate satisfied blank microspheres, restructuring cultivation of human bone morphogenetic protein-2, and found that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan microspheres and free recombinant human bone morphogenetic protein in the experimental concentration could not promote effect on the proliferation of bone marrow mesenchymal stem cells obviously. Sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres could promote the differentiation of mesenchymal stem cells significantly. In the bone marrow effects cultivate3d Sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres improve the role of bone marrow-derived mesenchymal stem cells ALP content below free recombinant human bone morphogenetic protein-2, but after5d, dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres effective control of recombinant human bone morphogenetic protein-2release, so that dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nanospheres could promote the differentiation of bone marrow mesenchymal stem cells.It was significantly stronger than free recombinant human bone morphogenetic protein-2group, there was significant difference (P<0.01). To observe the growth of calcium nodules on optical microscope found7d, dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan calcium nodules growth significantly better than free recombinant human bone morphogenetic protein-2group, promote bone marrow obvious role of mesenchymal stem cell differentiation.Conclusions:we have prepared by dextran sulfate satisfied/chitosan microspheres in application of ionic crosslinking method. The microspheres have many advantages such as into a ball, regular shape, disperse well, a good release properties. At the same time, we have also studied recombinant human bone morphogenetic protein-2release process occurs clarify recombinant human bone morphogenetic protein-2release in thermodynamic and kinetic control process and compositemicrosphere sustained release mechanism; We also have explored the interactions between recombinant human bone morphogenetic protein-2and blank microspheres in the release process, furthermore, we have stated the thermodynamic and kinetic control process of recombinant human bone morphogenetic protein-2in the release process, and revealed the sustained release mechanism of the composite microsphere; We have studied the morphology, particle size and distribution control method of single and composite microsphere particles, as well as their impact on the the wrapped load rate of protein and release process. We have also revealed the regulation of the carrier morphology on controlling release of recombinant human bone morphogenetic protein-2; At the same time, we have studied the release process and its osteoinductive effect of recombinant human bone morphogenetic protein-2/chitosan/dextran sulfate composite microspheres. The research results of the project would provide new ideas and theoretical guidance manual repair of bone defects. |
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