| Peripheral nerve injury is a serious complication that might happen during the implant surgery. If that happens, patients will suffer from sensory dysfunction in mucosa, chin and lower lip. Schwann cells(SCs) are the main glial cells in peripheral nerve system. They play a critical and substantial role in the peripheral nerve regeneration. How to promote the proliferation, secretion and migration of SCs has profound meaning. It has been found that certain growth factors,such as vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF) and insulin-like growth factor(IGF) have the potential to promote nerve regeneration. However, the clinical application of traditional growth factors has many disadvantages, such as the complexity of the extraction process, low purity, risk of immunological rejection, instability in vivo, and the need for repeated local injections in order to keep effective concentration. In addition, the injection of a single kind of growth factor could hardly provide an environment as complex as in vivo. Therefore, an alternative method to deliver growth factors is needed.Concentrated growth factor(CGF), as a new generation of platelet concentrate, is a reliable source of growth factors. It contains a variety of growth factors, such as transforming growth factor-β(TGF-β), platelet-derived growth factor-BB(PDGF-BB), FGF, IGF and VEGF. CGF is obtained by centrifuging the self-venous blood. It has gained considerable popularity for its autologous nature, easy collection, simple and cost-effective preparation, and safe clinical application without the risks associated with immunological rejection. Additionaly, compared to a single kind of growth factor, CGF contains several kinds of growth factors. It can provide a more complex and suitable environment for nerve growth. All those advantages make it possible for the application of CGF to the treatment of peripheral nerve injury.In this experiment, the effects of CGF on proliferation, neurotrophic secretion and migration of SCs in vitro were studied, and the mechanisms of CGF induced cell migration were investigated. The impact of CGF on functional recovery and micro-structure of nervers after injury in vivo were detected as well. The purpose of the study is to provide a theoretical basis for the clinical applications of CGF for treatment of peripheral nerve injury during implant surgery. 1. Characteristic study of CGF and the growth factor release from CGFThe surface structure of CGF was observed by a scanning electron microscopy(SEM). The release of transforming growth factor-β1(TGF-β1) form CGF was measured by an Enzyme-linked immunosorbent assay(ELISA). SEM analysis showed that CGF had a fiber-like structure. These fibers intertwined and formed a three-dimensional network, in which a large number of of biconcave disk-like red blood cells and platelets with multiple projections were trapped. The fiber diameter of CGF was 0.36±0.14 μm and the porosity was(40.44±2.97) %. ELISA assay showed that CGF could release TGF-β1 for at least 13 days. On the first day, the release of TGF-β1 was 75 pg/ml. Then it increased gradually to the peak of 130 pg/ml on the 5th day. After that the release of TGF-β1 decreased slowly to 60pg/ml on the 13 th day. The structural features and the ability to release growth factors make it possible for CGF to become an ideal bioscaffold and a growth factor provider. 2. The effect of CGF on SCs proliferationThe impact of CGF on SCs proliferation was investigated by a Cell Counting Kit-8(CCK-8) assay and cell cycle analysis. The CCK-8 analysis revealed that all the CGF medium with different concentrations(200%, 100% and 50 %) were able to promote the proliferation of SCs, whereas CGF with the concentration of 100% had the most obvious effect. Therefore, 100% CGF was chosen as the optimal concentration for subsequent experiments. Cell cycle analysis showed that, the proliferation index(PI) in CGF treated SCs were significantly higher than DMEM treated SCs, which provided further evidence of the proliferative effect of CGF on the SCs. 3. The effect of CGF on neurotrophic secretion of SCsRT-q PCR and Western blot were employed to detect the effect of CGF on m RNA and protain expressions of NGF and GDNF. RT-q PCR analysis demonstrated that the NGF and GDNF m RNA expressions in CGF treated SCs were significantly higher than those in DMEM treated cells at each time point. In detail,NGF m RNA expression in CGF group was 1.3 fold of that in DMEM group on day 1, 1.6 fold on day 2, and 1.8 fold on day 3. GDNF m RNA expression in CGF group was 1.8 fold of that in DMEM group on day 1, 1.6 fold on day 2, and 1.6 fold on day 3. Werstern blot showed that, NGF protein expressions in CGF groups were significantly increased to 1.1 fold and 1.5 fold of those in DMEM groups on days 2 and 3. GDNF protein expression in CGF groups was significantly increased to 1.3 fold of that in DMEM group on day 3. These results suggested that CGF could promote the neurotrophic secretion of SCs. 4. The effect of CGF on SCs migration and the mechanismsA scratch assay was employed to analyze the influence of CGF on SCs migration. The migration of SCs is regulated by many signaling pathways, among which integrin β1 signaling pathway occupies an important position. In this experiment, integrin β1 and its downstream signal molecule p-FAK were detected by Western blot, in order to unveil whether the augmentation of migration was mediated by this signaling pathway. Then integrin β1 was scienced by RNA interfering technique and the changes with cell migration after integrin β1 sciencing was investigated.The scratch assay showed that the migration rate in CGF treated group was 4.25 fold of that in the DMEM treated group, suggesting that CGF promoted the migration of SCs. Western blot showed that the expressions of integrin β1 and p-FAK were increased by CGF, indicating integrin β1 and p-FAK signal pathways were involved in CGF induced SC migration. Different si RNA plasmids(si RNA-N, si RNA-1, si RNA-2 and si RNA-3) were constructed and used to transfect SCs, among which si RNA-2 came up with the best effect on integrin β1 silencing. RT-q PCR and Western blot indicated that, after si RNA-2 transfection, the integrin β1 m RNA expression was decreased by 70-80%, and integrin β1 protein expression was reduced by 70%. After β1 integrin was silenced by si RNA-2, Transwell cell migration assay showed that CGF could still promote SCs migration.In conclusion, CGF promoted SCs migration partially through integrin β1 patyway. Integrin β1 did not play an essential role during this process. There might be some other signal pathways involved. 5. The effect of CGF on nerve regeneration in vivoA model of sciatic nerve injury crush injury was established to observe the effect of CGF on nerve regeneration after peripheral nerve injury in vivo. Footprint analysis showed that after CGF treatment, the sciatic functional index(SFI) was significantly increased compared to the untreated group, indicating CGF could promote functional recovery after nerve injury. Toluidine blue staining showed that, after nerve injury, the distortion of myelin sheath was obvious, and few newly generated myelinated fibers were observed. In the CGF treated group, the normal appearance of myelin sheath was maintained, and lots of newly generated myelinated fibers were observed. TEM displayed that nerve injury could cause the splits of myelin sheath from the axon, the distortion of myelin sheath, the twisting of myelin lamellae, and the irregular arrangement of nerve fibers. In the CGF treated group, twisting of myelin lamellae was not visible. Regular nerve arrangement was observed, and lots of newly generated myelinated fibers and SCs as well. These data suggested that CGF could promote functional recovery and improve the microstructure of peripheral nerve after nerve injury. |
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