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2.
Journal of the Korean Association of Oral and Maxillofacial Surgeons ; : 199-218, 2005.
Artigo em Coreano | WPRIM | ID: wpr-61733

RESUMO

PURPOSE OF STUDY: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. MATERIALS AND METHODS: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with beta-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells(1x10(6)) or BDNF-Ad infected Schwann cells(1x10(6)) were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. RESULTS: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were 1.54+/-4.0*10(6) and 9.66+/-9.6*10(6). 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell 0.69 microgram/microliter of DNA was detected and in BDNF-Adenovirus transfected Schwann cell 0.795 microgram/microliter of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. CONCLUSION: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.


Assuntos
Animais , Humanos , Ratos , Adenoviridae , beta-Galactosidase , Fator Neurotrófico Derivado do Encéfalo , Cálcio , Adesão Celular , Técnicas de Cultura de Células , Citarabina , DNA , DNA Complementar , Fibroblastos , Marcha , Gânglios Espinais , Biblioteca Gênica , Terapia Genética , Vetores Genéticos , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Rim , Filtros Microporos , Fatores de Crescimento Neural , Regeneração Nervosa , Neurônios , Nervos Periféricos , Reação em Cadeia da Polimerase , Regeneração , RNA Mensageiro , Células de Schwann , Nervo Isquiático
3.
Journal of the Korean Association of Oral and Maxillofacial Surgeons ; : 100-107, 2004.
Artigo em Coreano | WPRIM | ID: wpr-193874

RESUMO

Schwann cell, one of important components of peripheral nervous system, interact with neurons to mutually support the growth and replication of embryonal nerves and to maintain the different functions of adult nerves. The Ara-C, known as an antimitotic agent, have been used to have high effectiveness in eliminating fibroblasts during Schwann cell culture period. This enrichment effect is also known to be cummulative with each successive pulse of Ara-C applied and is due to a progressive loss of fibroblasts. But the cytotoxicity by Ara-C is also cummulative and noticeable over the period. To determine the most effective application time and interval of Ara-C in the Schwann cell culture, we observed the Schwann cell purity and density with the Ara-C treatment in plain and three-dimensional culture from dorsal root ganglion of new born rat. By culturing dispersed dorsal root ganglia, we can repeatedly generate homogenous Schwann cells, and cellular morphology and cell count with mean percentages were evaluated in the plain culture dishes and in the immunostainings of S-100 and GFAP in the three-dimensional culture. The Ara-C treated cultures showed a higher Schwann cell percentage (31.0%+/-8.09% in P4 group to 65.5%+/-24.08% in P2 group), compared with that obtained in the abscence of Ara-C (17.6%+/-6.03%) in the plain culture after 2 weeks. And in the three-dimensional culture, S-100 positive cells increased to 56.22%+/-0.67% and GFAP positive cells to 66.46%+/-1.83% in G2 group (p<0.05), higher yield than other groups with Ara-C application. Therefore, we concluded that the Ara-C treatment is effective for the proliferation of Schwann cells contrast to the fibroblasts in vitro culture, and the first application after 24 hours from cell harvesting and subsequent 2 pulse treatment (P2 group in plain culture and G2 group in three-dimensional culture) was more effective than other application protocols.


Assuntos
Adulto , Animais , Humanos , Ratos , Contagem de Células , Técnicas de Cultura de Células , Citarabina , Fibroblastos , Gânglios Espinais , Neurônios , Sistema Nervoso Periférico , Células de Schwann , Raízes Nervosas Espinhais
4.
Journal of the Korean Association of Oral and Maxillofacial Surgeons ; : 465-473, 2004.
Artigo em Coreano | WPRIM | ID: wpr-13074

RESUMO

PURPOSE: The essential triad for nerve regeneration is nerve conduit, supporting cell and neurotrophic factor. In order to improve the peripheral nerve regeneration, we used polyglycolic acid(PGA) tube and brain-derived neurotrophic factor(BDNF) gene transfected Schwann cells in sciatic nerve defects of SD rat. MATERIALS AND METHODS: Nerve conduits were made with PGA sheet and outer surface was coated with poly(lactic-co-glycolic acid) for mechanical strength and control the resorption rate. The diameter of conduit was 1.8mm and the length was 17mm. Schwann cells were harvested from dorsal root ganglion(DRG) of SD rat aged 1 day. Schwann cells were cultured on the PGA sheet to test the biocompatibility adhesion of Schwann cell. Human BDNF gene was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into E1 deleted region of adenovirus shuttle vector, pAACCMVpARS. BDNF-adenovirus was multiplied in 293 cells and purified. The BDNF-Adenovirus was then infected to the cultured Schwann cells. Left sciatic nerve of SD rat (250g weighing) was exposed and 14mm defects were made. After bridging the defect with PGA conduit, culture medium(MEM), Schwann cells or BDNFAdenovirus infected Schwann cells were injected into the lumen of conduit, respectively. 12 weeks after operation, gait analysis for sciatic function index, electrophysiology and histomorphometry was performed. RESULTS: Cultured Schwann cells were well adhered to PGA sheet. Sciatic index of BDNF transfected group was -53.66+/-13.43 which was the best among three groups. The threshold of compound action potential was between 800 to 1000microA in experimental groups which is about 10 times higher than normal sciatic nerve. Conduction velocity and peak voltage of action potential of BDNF group was the highest among experimental groups. The myelin thickness and axonal density of BDNF group was significantly greater than the other groups. CONCLUSION: BDNF gene transfected Schwann cells could regenerate the sciatic nerve gap(14mm) of rat successfully.


Assuntos
Animais , Humanos , Ratos , Potenciais de Ação , Adenoviridae , Axônios , Fator Neurotrófico Derivado do Encéfalo , Eletrofisiologia , Marcha , Biblioteca Gênica , Vetores Genéticos , Bainha de Mielina , Regeneração Nervosa , Nervos Periféricos , Ácido Poliglicólico , Reação em Cadeia da Polimerase , Regeneração , Células de Schwann , Nervo Isquiático , Raízes Nervosas Espinhais
5.
Journal of the Korean Association of Oral and Maxillofacial Surgeons ; : 151-156, 2003.
Artigo em Coreano | WPRIM | ID: wpr-120887

RESUMO

This study was performed to develop a useful nerve conduit which provides favorable environment for Schwann cell viability and proliferation. Milipore membrane of 0.45um pore size was selected because it permits nutritional inflow from the outside of the conduit and prevents from invading the fibrotic tissue into the conduit. The membrane was rolled and sealed to form a conduit of 2mm diameter and 20mm length. To improve the axonal regeneration and to render better environment for endogenous and exogenous Schwann cell behaviour, the microgeometry and surface of conduit was modified by coating with thin film of calcium phosphate. Cellular viability within the conduit and attachment to its wall were assessed with MTT assay and SEM study. Milipore filter conduit showed significantly higher rate of Schwann cell attachment and viability than the culture dish. However, the reverse was true in case of fibroblast. Coating with thin film of low crystalline calcium phosphate made more favorable environment for both cells with minimal change of pore size. These findings means the porous calcium phosphate coated milipore nerve conduit can provide much favorable environment for endogenous Schwann cell proliferation and exogenous ones, which are filled within the conduit for the more advanced strategy of peripheral nerve regeneration, with potential of reducing fibrotic tissue production.


Assuntos
Axônios , Cálcio , Proliferação de Células , Sobrevivência Celular , Cristalinas , Fibroblastos , Membranas , Regeneração Nervosa , Nervos Periféricos , Regeneração , Células de Schwann
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