| Bone defection and nonunion induced by trauma or tumor are still the puzzles of reconstructive orthopaedic surgery nowadays. Although autologous and allogeneic bone substitute materials, as the common replaceable materials, play important role in treatments, they still have many intrinsic disadvantages and do not meet the demands in all situations. Autologous bone grafts are considered to be the optimal selection, but the morbidity at donor sites, limited shape, size and amount of graft are the major drawbacks of this technique. Furthermore, the additional surgery makes patient undergo more body injuries, including pain and infection. Allogeneic bone material can be prepared with any shape, size needed in surgery without the limits of amount, however the inevitable immune response and the risk of virus disease transmission make it limit with application. In order to overcome the limitation of the two methods, it is important task to develop the better man-made biological material to repair bone defect.Hydroxyapatite (HA), a major in component of bone organic, has been used extensively for biomedical implant applications and bone regeneration dueing to its bioactive, biodegradable and osteoconductive properties. The microstructure of nano Hydroxyapatite (n-HA)is the same as natural bone matrix, it can be adsorbed by bone matrix, therefore it can enhance the biological activity. The n-HA can release more calcium and phosphor for inducing the formation of new bone. The mechanical properties of n-HA can not be fixed and repaired to meet the orthopedic needs, limiting its clinical application. The biodegradable material of poly(lactic-co-glycolic acid)(PLGA) have been permitted by FDA of USA for clinical application. PLGA used in tissue engineering bone and cartilage of the building and tissue repairing, there must be a prerequisite for uniform stent planted in a sufficient number of seed cells. Construction of tissue engineering will take some time and conditions, which limit its clinical application.In order to overcome the above shortcomings, a new type of n-HA and PLGA composite materials will be prepared in this study. The n-HA particles will be transplanted to the surface of grafted oligomeric lactic acid (LAc oligomer), then,modified n-HA (op-HA) blends with composite material made of PLGA. Thus ,op-HA/PLGA. is formatted. Modified op-HA particles in the PLGA matrix has more uniform dispersion, and op-HA surface oligomeric lactic acid chains into the PLGA matrix can be internal, it can enhance both the organic and inorganic phase of adhesion, increase the mechanical strength of composite materials. PLGA matrix material can be adjusted degradation of the material by adjusting the the ratio of the composition of lactic acid (LA) and glycolic acid (GA). The biological activity of the op-HA and PLGA degradation characteristics of the combination of polymer materials will enhance the biocompatibility and osteoinductive capabilities, making it more suitable for clinical need for bone defection repairing.To further enhance the biological activity and intelligent of materials, on the baseis on op-HA/PLGA materials, a conductive polymer materia will be introduced. Polyaniline (PA) is a synthetic polymer materials, commonly known as conductive plastics. As a medical biological materials, there is poor biocompatibility, non-degradable and difficult to process, poor solubility of defects, thus affecting its medical applications. A biodegradable aliphatic polyester and aniline oligomer block copolymer of (PAP) will be prepared in this study. By aliphatic polyester and the introduction of natural bio-polymer, can not only greatly improve the electrical activity of the soluble material, but also improve the biocompatibility of materials. The introduction of the aniline oligomer, which has the similar electrical activity as Polyaniline.Combined with pulsed electrical stimulation, a new type of electrical activity of nano-composite bone repairing material PAP/op-HA/PLGA, reflects the intelligence, induces bone formation and shortens the bone healing process, provides a basis for clinical applications.â… . Nanocomposite of op-HA/PLGA bioabsorbable bone repairing material1. Preparation and Characterization of MaterialsWe will grafte Lactic acid oligomer to the surface of n-HA,then blend with the PLGA matrix, thus op-HA/PLGA will be prepared. Op-HA grafting yield will be analyzed through Fourier transform infrared spectrometer (FT-IR), thermogravimetric instrument (TGA); op-HA/PLGA dispersion of surface particles will be observed through field emission scanning electron microscope (ESEM). By adjusting the ratio of porogen, different op-HA/PLGA porosity of composite materials, microstructure of materials, pore structure and porosity will be detected through ESEM, automatic surface area and porosity analyzer; the compressive strength of scaffold materials and bending strength will be tested by electronic Instron 1121 universal testing machine.The results showed that the graft ratio of op-HA was 8.3%(w/w), when blended with PLGA, op-HA has good dispersion. Preparation of the porous scaffolds. The porous scaffolds were prepared with the porogen of salt particles with certain size by melt-molding/particle leaching method. The mechanical strength, pore size and porosity were tested.2. Biocompatibility evaluationCulture osteoblasts vaccinated in op-HA/PLGA membrane, persisting 7d. At different time points we will stain osteoblasts by using fluorescein isothiocyanate (FITC), observe cells form and quantity in the material by Inverted fluorescence microscope, analyze the percentage of single-cell area by NIH Image J software, detect osteoblast proliferation in the surface of material by MTT; detect the expression of Collagen-I, BMP-2 and Osteonectin by RT-PCR. ESEM and EDX will be used to analyze calcium deposition of the composites. The results show that it can exhibite better cell adhesion, spreading and proliferation of rabbit osteoblasts compared with pure PLGA. Its gene expression of Collagen-I, Bone Morphogenetic Protein 2 (BMP-2) and Osteonectin are higher than that of PLGA. It indicate that the biocompatibility and osteogenic bioactivity of the op-HA/PLGA nanocomposite will be improved obviously than that of the pure PLGA.3. The repairing of rabbit radius defects of op-HA/PLGAThe rabbit's 20 mm radius defection is made and implanted with the composites. At 6 and12 weeks after operation, samples are harvested and examined by radiograph to evaluate the bone repairing property of the novel materials. The results show that at 12 weeks postoperatively, there is evident healing in 85% bone defection op-HA/PLGA groups.â…¡. New types of electrical activity of biodegradable nano-composite bone repairing material PAP/op-HA/PLGA1. Preparation and Characterization of MaterialsIn order to prepare the electric activity of biodegradable nano-composite bone repairing material PAP/op-HA/PLGA, we will use solution method. It will be prepared by compositing conductive polymer aniline oligomer (AP) and polylactic acid (PLA) block copolymer of (PAP) and op-HA/PLGA by a certain percentage. To analyze the electrochemical properties by ultraviolet spectrophotometer, cyclic voltammetry and electrochemical analyzer; to prepare PAP/op-HA/PLGA porous membrane by supercritical CO2 foam method, to observe membrane surface morphology by ESEM. The results showed that PAP/op-HA/PLGA has electrochemical properties; surface covered with porous.It can meet the requirements of tissue engineering scaffold material .2. Biocompatibility evaluationIn accordance with the biological materials compatibility testing methods and requirements, the International Organization for Standardization (ISO10993) and China (GB/T 16886). We detect PAP/op-HA/PLGA composites cytotoxicity by using vitro experiments, animal acute toxicity and pyrogen experiments in order to evaluate the biocompatibility of materials. The results show that PAP/op-HA/PLGA material has no cytotoxicity, no acute toxicity, no heat reaction, has better biocompatibility.3. Evaluation of cell adhesion, proliferation and osteogenic activity Under the pulse of electrical stimulation, we will culture cell vaccinated in PAP/op-HA/PLGA membrane, persisting 7d. At different time points we will stain osteoblasts by using fluorescein isothiocyanate (FITC), observe cells form and quantity in the material by Inverted fluorescence microscope, analyze the percentage of single-cell area by NIH Image J software, detect osteoblast proliferation in the surface of material by flow cytometry; detect the expression of Collagen-I, BMP-2 and Osteonectin by RT-PCR. The results show that PAP/op-HA/PLGA osteoblast adhesion and proliferation will be evaluated, gene expression of Collagen-I, BMP-2 and Osteonectin will be promoted under the pulse electrical stimulation.4. Experiment of implanted animals repairingPreparing rabbit radial defection model to evaluate the capacity of bone repairing materials under the pulse of electrical stimulation. We will put to death rabbit 12w old, take muscle tissue from implanting site. By RT-PCR, we will detect gene expression Type I collagen (Collagen-I), bone morphogenetic protein -2 (BMP-2) and bone connexin (Osteonectin). Under the pulse of electrical stimulation, PAP/op-HA/PLGA will play electrical activity , promot the repairing of bone defect, enhance bone healing process, promote bone plasticity, functional recovery, promot gene expression of BMP-2, Collagen-I and osteonectin.Conclusion: Op-HA/PLGA bone repair composite material has better cell adhesion, proliferation and osteogenic activity. The proportion of 85% porogen scaffolds has the best bone repairing. PAP/op-HA/PLGA has better biocompatibility, osteoblast adhesion, proliferation and osteogenic activity of gene expression. Under the pulse electrical stimulation, material properties has a better reflection. |
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