| Objective:1. To explore the dynamic change of endogenous basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) in the course of injury and regeneration of rabbit sciatic nerve. To study the inducting differentiation with the sciatic nerve extracts on rabbit adipose-derived stem cells (ADSCs) in vitro.2. To explore the feasibility of fabricating a modified acelluar nerve scaffold by the technique of hypotonic buffer combined with freeze-drying.3. To explore the feasibility of construction of tissue engineered peripheral nerve based on improved acellular nerve scaffold compounding with ADSCs.Methods:1. Modeling the injury of rabbit sciatic nerve with forceps. ELISA was used to examine the content of NGF, bFGF and BDNF at normal and3,7,14days of injured sciatic nerve extract supernatant.ADSCs were isolated from rabbits. ADSCs at passage3were cultured with induction media containing the extracts of normal sciatic nerve and injured sciatic nerve. After7days, induced ADSCs were detected with RT-PCR to investigate Schwann cells differentiation ability.2. Improve the traditional acellularized nerve scaffold with the technique of hypotonic buffer combined with freeze-drying. After the aceilularization process, the structural changes of nerves in each group were observed by HE straining, immunofluorescent staining and field emission scanning electron microscope (SEM). The interval porosity and void diameter in each group were measured by the software of mimics. The biomechanical properties of nerves in each proup were tested by mechanical apparatus (Endura TEC ELF3200). Effects of scaffold extract on ADSCs proliferation was examined by MTT and viability of ADSCs seeded on scaffold was detected by Live/Dead staining.3. Micro-injection was used to construct the tissue engineered peripheral nerve. Cell viability was analysed using HE staining and SEM. ADSCs were labeled with PKH26dye and seeded onto the scaffold. The hybrids were cultured in vitro for1week, and then implanted subcutaneously into nude mice. After4weeks culture in vivo, evaluation of hybrids were carried out with in vivo small animal imaging system and fluorescence microscope.Results:1. In the injurying group, the content of bFGF, BDNF and NGF was markedly higher than that in the normal group. ADSCs cultured in vitro had a spindle shape, rapid proliferation and good atability. Induced ADSCs became Schwann-like in morphology. The gene expression of the cells in S-100displayed markedly strong time-dependent increases in the injuring3and7days.2. According to light and electron microscopy, the scaffold was cell-free. The scaffold had porosity of49.3%±9.8%with (17.6±3.7)μm pore diameter. Elestic modulus was2.45±0.65MPa. MTT indicated scaffold extract had no influence on cell proliferation. Live/Dead showed ADSCs had a good proliferation on the scaffold.3. PKH-26dye was good for labelling the ADSCs, marked validity was95.12%. The cellular vitality and differentiated abilities were not affected by the PKH-26dye. A lot of cells were viable even after4weeks in vivo culture as seen by in vivo small animal imaging system and fluorescence microscope.Conclusion:1. In the early recovery process after the the peripheral nerve injury, the nerve tissue can regulate the secretion of NGF, bFGF and BDNF. and the secretion of these three growth factors has relationship with the time of injury; The ADSCs can be induced to differentiate into Schwann-like cells or neuron-like cells with sciatic nerve extracts; And the early time (3-7days) after the injury is the best time for stem cell transplantation.2. The acellularized nerve scaffold of the improved chemical method group can be used as a new kind of nerve scaffold.3. Construction of tissue engineered peripheral nerve based on improved acellular nerve scaffold with ADSCs is feasible. |
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