| I. BACKGROUND:The peripheral nerve defect and reconstruction is worldwide problems. Autologous nerve grafts is the gold standard for clinical therapy, But donor source is limited and the damage for the area of sensory and motor function. So exploring new method for repair of peripheral nerve injury is of great significance. Tissue engineering research provides a new idea for solving this problem. Seed cells and duct stent made of complex is the core of the construction of tissue engineering. Now, Seed cells in tissue engineering is widely applied:schwann cells and bone marrow mesenchymal stem cells, skeletal muscle stem cells, neural stem cells and embryonic stem cells, etc. Bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs) have the potential of self-renewal and multidirectional differentiation. Bone marrow mesenchymal stem cells which under the appropriate culture conditions, can be stable in vitro survival and reproduction, and keep the multidirectional differentiation ability, it is the ideal operating cell in vitro. Which differentiation of bone marrow mesenchymal stem cells into schwann cells and neurons cells can secrete NGF, has promotionly effect for nerve repair. In addition, the materials of bone marrow mesenchymal stem cells get easily, and can rapid proliferation and differentiation in vitro, after transplantation it can survive in the immune exemption area, avoid immune rejection, which is used for repair of peripheral nerve injury treatment research, has a broad application prospectThe catheter materials mainly include:biological nerve conduits, synthetic catheter tube, composite tissue. Catheter materials as on which artificial extracellular matrix for cell adhesion, growth, proliferation, differentiation, and favorable for normal metabolism. Many studies have shown that nerve regeneration process, break the nerve regeneration, and the role of neurotrophic factor and nerve growth factor, when nerve defect after more than a certain distance, the lack of supply of nutrients, resulting in distal and proximal can’t reconnect. Prophase research designed and synthesized by l-arginine glycine-aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid/beta tricalcium phosphate sustained-release catheter/nerve growth factor, and is used to repair the10mm sciatic nerve defects of rats, the results show that the catheter on rat sciatic nerve defect with good bridge and promote the role of nerve growth, and animal experiments proved that the new composite nerve conduit materials no cytotoxicity.This experiment through directional induced bone marrow mesenchymal stem cells differentiate into nerve cells, identification of the specific markers of expression, as seed cells in nerve conduits and made into new catheter composite materials.Ⅱ. OBJECTIVE:1. Preliminary research from the perspective of bionics design, nerve basement membrane structure and composition, on the basis of the analysis and research to biological absorbable PDLLA as basic skeleton material, adding PRGD components and can promote nerve growth can induce nerve growth model NGF, add can adjust PH beta TCP component placement area, successfully build PRGD/PDLLA/NGF plays/beta TCP new nerve conduits composite material, the purpose aims to evaluate its in vitro cell studies cell affinity and slow-release performance.2, Under the condition of in vitro separation and purification of rat bone marrow mesenchymal stem cells (ipads marrow mesenchymal stem cells, BMSCs) and subculture, add alkaline fibroblast growth factor, Basic fibroblast growth factor, bFGF) and epidermal growth factor, epidermal growth factor, EGF) was carried out on the third generation of BMSCs induced, observation of cell morphology change, using immunohistochemical method to detect after the differentiation of neural cells markers; Cells differentiate into nerve cells, the purpose aims to test evaluation that bone marrow mesenchymal stem cells differentiation into neural cells is feasible. As the basic research of peripheral nerve injury repair:3, After verifying the new nerve conduit has good cell affinity and slow-release performance and Basic fibroblast growth factor, Basic fibroblast growth factor, bFGF) and epidermal growth factor, epidermal growth factor, EGF) can induce bone marrow mesenchymal stem cells differentiate into nerve cells, on the basis of the new type of nerve conduits made complex composite materials and bone marrow mesenchymal stem cells, in vitro cell biology was used to evaluating the biocompatibility and evaluate the possibility of large animals long peripheral nerve repair, for the next phase of animal experimental research and theoretical basis for clinical application.III. METHODS:1. In New nerve conduits composite material design and preparationFrom bionic design point of view, first of all to nerve basement membrane structure and composition, on the basis of the analysis and study on its own design and RGD peptide was synthesized by grafting polymer poly (glycolic acid-L lysine-lactic acid)(PRGD), with ethyl acetate as solvent, respectively in concentration of85%, relative molecular mass of10x25*104～104of polylactic acid (PDLLA), the concentration of10%PRGD, concentration was5%, the average particle size of less than500nm beta tricalcium phosphate (beta TCP), and traces of nerve growth factor (NGF plays), by ultrasonic dispersion, magnetic stirring, PRGD prepared by solvent volatilization method/PDLLA/beta TCP/NGF plays composite membrane2. Rat bone marrow mesenchymal stem cell separation, purification and batchesSD rats by intraperitoneal injection of10%chloral hydrate, the volume fraction of75%ethanol immersion30min after anesthesia, placed in a super clean bench, under the strict aseptic operation take bilateral femur and tibia of rats, the epiphyseal end cut off, on both ends of the exposed bone marrow cavity, with a5ml syringe pump DMEM liquid rinse marrow cavity, repeatedly to cortical bone white, collect rinses in sterile centrifuge tube,1000r/min, the centrifugal10min, abandon to supernatant fluid, heavy suspension, counting, adjust the cell inoculation density of107L-1in culture bottle, add broth (containing10%fetal bovine serum,100u/ml of penicillin, streptomycin100g/L), at37℃and5%CO2rope rope, rope and humidity incubator incubation. After24h in liquid and remove adherent cells, not once every3days in liquid, after basic covered bottle to cells with0.25%trypsin digestion, according to the proportion of1:2to extend, amplification and purification. Daily use inverted phase contrast microscope observation of cell morphology and growth conditions.3. BMSCs differentiation and identification3.1Proliferation and differentiation of BMSCsThe third generation of cells which grow to be in good condition with0.25%trypsin digestion, and adjust the density of1x105/l vaccination within the24hole culture plate, divided into induction group and the control group, each set after six holes, at37℃and5%CO2saturated humidity incubation. Abandoned after24h of culture medium, induction group to join inducers (including20ng/mlEGF,20ng/mlbFGF DMEM culture), control continues to use containing10%fetal bovine serum DMEM culture, in liquid once every3days, continuous culture for7days, daily use inverted phase contrast microscope observation of cell morphology change and growth.3.2Cell immunohistochemical identificationInduced after7days, make immunohistochemical assay culture plate, a rabbit anti rat NSE respectively and beta Tubulin and rabbit anti rat GFAP;2fight for fluorescein isothiocyanate)(fluorescein5(6)-isothiocyanate, FITC). Steps as follows:abandon culture absorption,4%paraformaldehyde fixed10min;0.3%Triton-X100membrane for10min, phosphate buffer (phosphate buffer saline, PBS) wash3times, goat serum closed20min, add a resistance (10)4℃overnight incubation, PBS wash3times, plus two drops (1:100)37℃resistance incubation for30min, randomly selected10vision observation induced cell staining positive cell number. Main observation indexes:the morphological features of bone marrow mesenchymal stem cells;(2) the bone marrow mesenchymal stem cells into neural cells morphological changes during the differentiation;(3) NSE, beta Tubulin, the expression of GFAP.4. New neural tube composite material preparation of leaching solutionWeigh and sterilized1-arginine glycine-aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid/beta tricalcium phosphate/nerve growth factor composites100mg loading15mL centrifuge tube, then add the3mL cell culture. Put centrifugal tube at37℃constant temperature oscillation incubator (100r/min), removed after24h of leaching solution, according to the proportion of1:9dilution to join alpha blending in MEM broth, ensure that aseptic leaching solution preparation process 5. New type of nerve conduits compatibility with BMSCs composite materials research5.1Determined by MTT method to detect activity of bone marrow mesenchymal stem cellsThe third generation of bone marrow mesenchymal stem cells Will grow well in with5x104/ml inoculation density on96-well culture plate, the experimental group and control group and blank group (only add broth, excluding cells to zero), the experimental group add to digest-glycine-aspartic acid polypeptide amino acid grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid)/beta tricalcium phosphate/nerve growth factor composite membrane0.2mL leaching solution, the control volume fraction containing10%fetal bovine serum culture medium of0.2mL, at37℃and5%volume fraction of CO2saturated humidity incubation. The next day and a half amount in liquid, continuous culture for7d. Respectively take cultivating hc-positie1d cells were determined by MTT method to detect (reference detailed steps), on the enzyme league immune detector select570-nm wavelength determination of absorbance values, record test data5.2Flow cytometry instrument-FITC Annexin V/PI double staining to detect cell apoptosis rateThe third generation of bone marrow mesenchymal stem cells which grow well inoculated in6holes culture plate, divided into experimental group and the control group, experimental group add to digest-glycine-aspartic acid polypeptide amino acid grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid)/beta tricalcium phosphate/nerve growth factor of composite2mL leaching solution, the control volume fraction containing10%fetal bovine serum2mL of the culture medium,37℃and5%volume fraction of CO2saturated humidity incubation. Every2d in liquid one,7d continuous culture, cultivate hc-positie1d cells were taken to Annexin V-FITC/PI staining to detect cell apoptosis rate, record the experiment data and streaming scatterplot, left upper quadrant (FITC/PI+) for mechanical damage cells, the bottom left quadrant (FITC-/-) PI for normal living cells, upper right quadrant (FITC+/+) PI late apoptosis or cell death and lower right quadrants (FITC+/-) PI for early apoptotic cells,(cell apoptosis rate calculated by the instrument).5.3Scanning electron microscopy (sem) observation of BMSCsGlycine-aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid/beta tricalcium phosphate/nerve growth factor composite film culture plate holes cut into12hole size, and cell concentration to1x1091-1bone marrow mesenchymal stem cell culture plate placed in the12hole together in37℃and5%volume fraction of CO2and incubation under saturated humidity conditions. Will develop after7d, the material from PBS wash3times, volume fraction of2.5%glutaraldehyde4℃for1h. Volume fraction of30%,30%,30%,50%,90%,100%tert-butyl alcohol dehydration, level4dehydration before each1times, each time10min,2levels of dehydration after each2times, each time10min. Retain samples of tert-butyl alcohol-20℃ice cold storage within4h,12h dry; Metal spraying, observed under scanning electron microscopy (sem) material surface cell growth conditions6Main observation indexes6.1the expression of NSE, beta Tubulin, GFAP6.2BMSCs in leaching solution growth, cell viability and cell apoptosis rate7Statistical methodsResults expressed in x±s, using SPSS13.0statistical software for statistical analysis, comparison between groups using independent sample t test, P<0.05for significant differences IV. RESULTS:1.The successful preparation of PRGD/PDLLA/beta-TCP/NGF plays nerve conduits, appearance of white, odorless, tasteless. Material surface smooth, no cracks, no burr, texture soft, can be bent, easy for stitching. In vitro cultivation and RSC96cells has good cell affinity, and sustainable release NGF30days or more.2.The cultivation of the bone marrow mesenchymal stem cells and induced:the original generation of BMSCs vaccination began to appear phenomenon of wall stickers within24h, the cells mostly circular. Adherent cells after72h has parapodium stretched out, and most cells were fusiform accompanied by2-3bump, the nucleus is larger, round flat,1～2nucleoli. After three generations of purification culture of BMSCs form for long spindle, to form a multiple bumps and comes in contact with each other, completely merged into spiral shape, mesh, radial structure. BMSCs induced by bFGF and EGF after24h, part of the cell proliferation in clusters (figure3). Continue to develop after72h visible part from two or more processes, cytoplasmic retraction, cell body show polygonal or irregular, refraction significantly increased. Cultivate visible stellate cells the number gradually increased after7days, network connection between bumps into cells, can be found in the nuclei and nucleoli3Cell immunohistochemical identification of NSE, beta Tubulin, GFAP expression:BMSCs differentiation after7days, part of the nucleated cells showed typical change of neuron and the expression of NSE and beta Tubulin positive, show that the neural stem cell characteristics, GFAP dyeing results are negative4Arginine, glycine and aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid)/beta tricalcium phosphate/nerve growth factor leaching solution of bone marrow mesenchymal stem cells:the influence of the experiment determined by MTT method is used to detect absorbance values, the experimental group and control group cells were found,5,7d after cocultivation, absorbance values of the experimental group was obviously higher than that of control group (P<0.05). According to describe the growth curve of absorbance values, with the extension of incubation time, the experimental group and control group absorbance value increases gradually, showed that cell activity increased gradually, the experimental group absorbance values are significantly greater than the control group, the experimental cell activity is higher than the control group, as shown in figure3. Show that1-arginine glycine-aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid/beta tricalcium phosphate/nerve growth factor leaching solution of bone marrow mesenchymal stem cells not only non-toxic effect, also can increase the cell activity5. Arginine, glycine and aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid)/beta tricalcium phosphate/nerve growth factor extract on apoptosis of bone marrow mesenchymal stem cells:the influence of flow cytometry instrument-FITC Annexin V/PI double staining results showed that the experimental group (four groups all take9o’clock in the morning time test) apoptosis rate was (6.43+3.02)%; Compared to control group, apoptosis rate was (16.36+6.88)%, compared two groups have significant difference (P<0.05); Cells trained7d, the test results show that the experimental group upper right quadrant late apoptotic cells was significantly less than the control group, and cell activity significantly greater than the control group. Proves that1-arginine glycine-aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid/beta tricalcium phosphate/nerve growth factor on leaching solution non-toxic role of bone marrow mesenchymal stem cells, the cells can prolong survival time, has the good cell compatibility6.Arginine glycine-aspartic acid polypeptide grafted poly (glycolic acid-L lysine-lactic acid)/poly (lactic acid/beta tricalcium phosphate/nerve growth factor composite material impact on the growth of the bone marrow mesenchymal stem cells:see7d training on scanning electron microscopy (sem) observation, arginine, glycine-aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid/beta tricalcium phosphate/nerve growth factor of composite part of the degradation, the surface of the cell number is more, cell growth condition is good, the volume is bigger, the cell body make multiple projections, and the connections between cells in a woven mesh, its axon is longer and coarser, the sample is typical of the neuron cell performance, and its degradation products have no cytotoxicity, release of nerve growth factor can promote bone marrow mesenchymal stem cells into neurons in the role of cell differentiation, cell compatibility is goodV. CONCLUSION:1. PRGD/PDLLA/β-TCP/NGF nerve conduit was successfully prepared through the bionic basement membrane components of natural nerve. In vitro experiments showed that the material has good biocompatibility and cell affinity, and can be sustained release of NGF up to30days.2. Bone marrow mesenchymal stem cells with strong proliferation and differentiation of self potential, the suitable condition can be kind differentiation into neural cells in vitro, for repair of peripheral nerve injury research provides a new way of thinking.3. experiment1-arginine glycine-aspartic acid polypeptide grafted poly (glycolic acid-L-lysine-lactic acid)/poly (lactic acid)/beta tricalcium phosphate/nerve growth factor composites with bone marrow mesenchymal stem cells has good compatibility, can be used as a good carrier for building artificial bionic nerve. Laid a theoretical foundation for the next phase of animal experiment... |
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