Induced pluripotent stem cells from patients with focal cortical dysplasia and refractory epilepsy.

Focal cortical dysplasia (FCD) is caused by numerous alterations, which can be divided into abnormalities of the cortical architecture and cytological variations; however, the exact etiology of FCD remains unknown. The generation of induced pluripotent stem cells (iPSCs) from the cells of patients with neurological diseases, and their subsequent tissue­specific differentiation, serves as an invaluable source for testing and studying the initial development and subsequent progression of diseases associated with the central nervous system. A total of 2 patients demonstrating seizures refractory to drug treatment, characterized as FCD Type IIb, were enrolled in the present study. Fibroblasts were isolated from residual skin fragments obtained from surgical treatment and from brain samples obtained during surgical resection. iPSCs were generated following exposure of fibroblasts to viral vectors containing POU class 5 homeobox 1 (OCT4), sex determining region Y­box 2 (SOX2), Kruppel­like factor 4 and c­MYC genes, and were characterized by immunohistochemical staining for the pluripotent markers homeobox protein NANOG, SOX2, OCT4, TRA1­60 and TRA1­81. The brain samples were tested with antibodies against protein kinase B (AKT), phosphorylated­AKT, mechanistic target of rapamycin (mTOR) and phosphorylated­mTOR. Analysis of the AKT/mTOR pathway revealed a statistically significant difference between the cerebral tissues of the two patients, which were of different ages (45 and 12 years old). Clones with the morphological features of embryonic cells were detected on the 13th day and were characterized following three subcultures. The positive staining characteristics of the embryonic cells confirmed the successful generation of iPSCs derived from the patients' fibroblasts. Therefore, the present study presents a method to obtain a useful cellular source that may help to understand embryonic brain development associated with FCD.

Effect of Platelet Rich Plasma and Platelet Rich Fibrin on sciatic nerve regeneration in a rat model.

The aim of this study was to evaluate the effect of Platelet Rich Plasma (PRP) and Platelet Rich Fibrin (PRF) on peripheral nerve repair. Thirty-two Wistar rats were randomly divided into four equal treatments groups: autologous nerve grafts (ANG), silicon tube plus saline solution (SS), silicon tube plus PRP, and silicon tube plus PRF. In ANG group, 10 mm segment from sciatic nerve was excised and reimplanted between the nerve stumps. In the SS, PRP, and PRF groups, 5 mm segment from sciatic nerve was excised and bridged with a 12 mm silicone conduit to create a 10 mm nerve gap. The conduit was filled in accordance with the different treatments. Walking track analysis was performed periodically and on the 90th post-operative day histomorphometric analysis was performed. The ANG, PRF, and PRP groups presented a significant functional improvement in relation to the SS group (P = 0.001) on 90 days after surgery. Histomorphometric analysis demonstrated that the ANG group achieved a larger nerve fiber diameter in proximal stump while comparing with the SS group (P =0.037) and showed larger fiber diameter in median stump in comparison to the PRP group (P = 0.002) and PRF group (P = 0.001). Axonal diameter and myelin sheath thickness showed no statistical significant difference between the groups in the three stumps (P ≥ 0.05). This study suggests that PRP and PRF have positive effects on the functional nerve recovery; however, these groups don't achieve a significant improvement on the histomorphometric analysis.

VIABILITY OF VASCULARIZED BONE GRAFT FROM THE ILIAC CREST USING THE ILIAC BRANCH OF THE ILIOLUMBAR ARTERY: EXPERIMENTAL STUDY ON RATS.

OBJECTIVE: Through an experimental model, our aim was to create inferences about the viability of vascularized bone grafts from the iliac crest in rats and investigate their histological features. METHODS: Twenty-one rats were used, divided into two groups: the first consisted of animals that were subjected to the technique of vascularized bone graft pedicled onto the iliac branch of the iliolumbar artery; the second (control group) underwent the same procedure as performed on the first group, with the addition of ligation of the vascular pedicle. The viability of the bone grafts was observed for three weeks, by means of direct observation of the graft, histology and immunohistochemistry. RESULTS: All the vascularized grafts evaluated in the first week showed viability according to direct observation, histology and immunohistochemistry. However, in the second and third weeks, direct observation showed that 75% of the grafts were unviable, while histological analysis and immunohistochemistry showed that 50% were unviable. CONCLUSIONS: Some grafts that are designed to be vascularized became unviable and began to behave like non-vascularized grafts under direct observation and histology. Despite the possibility of failure, use of vascularized bone grafts should be encouraged, because descriptive histology shows greater cell density in the medullary bone portion, and osteocytes that function better regarding deposition of bone matrix, with preservation of the intraosseous vascular network.

COMPARATIVE STUDY ON USE OF PLATELET-RICH PLASMA ALONE AND IN COMBINATION WITH ALPHA-TRICALCIUM PHOSPHATE CEMENT FOR BONE REPAIR IN RATS.

OBJECTIVES: To evaluate the effect of alpha-tricalcium phosphate (α-TCP) cement combined with platelet-rich plasma (PRP) on osteogenesis, and to compare the results with use of PRP alone. METHODS: A bilateral defect was produced in rat femurs and was filled with one of two types of treatments (PRP or α-TCP + PRP). The outcomes were evaluated after four and eight weeks. Radiographic images provided values for the lesion area, and histology (picrosirius staining) indicated the area of new bone formation. RESULTS: The means relating to the lesion area of the α-TCP + PRP group (2.64 ± 2.07 and 1.91 ± 0.93 mm(2), after four and eight weeks, respectively) showed numerically better but non-significant results (p > 0.05) than those seen in the PRP group (5.59 mm 2 ± 2.69 and 3.23 ± 1.46 mm 2, after four and eight weeks, respectively). The mean new bone formation rates were 62.7% ± 12.1 and 79.01% ± 6.25 in the PRP group, and 73.3% ± 12.7 and 85.86% ± 10.45 in α-TCP + PRP group, after four and eight weeks, respectively (p > 0.05). CONCLUSION: The data from this study suggest that treatment with α-TCP cement combined with PRP does not show any significant difference in comparison with PRP alone. However, there is a possible early effect on bone regeneration when the two biomaterials are applied together.

Estudo comparativo do uso isolado de plasma rico em plaquetas e combinado com cimento de alfa-fosfato tricálcico no reparo ósseo em ratos / Comparative study on use of platelet-rich plasma alone and in combination with alpha-tricalcium phosphate cement for bone repair in rats

OBJETIVOS: Avaliar o efeito do cimento α-TCP combinado com PRP sobre a osteogênese, comparando os resultados com PRP utilizado isoladamente. MÉTODOS: Foi confeccionado defeito bilateral no fêmur de ratos e preenchido com um dos dois tipos de tratamentos (PRP ou α-TCP+PRP), sendo avaliado em quatro e oito semanas. As imagens radiográficas forneceram valores da área da lesão, e a histologia (coloração Picrosirius) indicou a área de neoformação óssea. RESULTADOS: As médias referentes à área de lesão do grupo α-TCP+PRP (2,64mm² ± 2,07 e 1,91mm² ± 0,93; quatro e oito semanas, respectivamente) demonstraram numericamente melhores resultados, porém não significativos (p > 0,05), em comparação com aqueles observados no grupo PRP (5,59mm² ± 2,69 e 3,23mm² ± 1,46; quatro e oito semanas, respectivamente). As médias de neoformação óssea foram de 62,7% ± 12,1% e 79,01% ± 6,25 no grupo PRP, e 73,3% ± 12,7 e 85,86% ± 10,45 no grupo α-TCP+PRP, em quatro e oito semanas, respectivamente (p > 0,05). CONCLUSÃO: Os dados deste estudo sugerem que o tratamento com cimento α-TCP combinado com PRP não demonstra diferença significativa quando comparado ao PRP isolado. Entretanto, há um possível efeito precoce sobre a regeneração óssea quando os dois biomateriais são aplicados em conjunto.

OBJECTIVES: To evaluate the effect of alpha-tricalcium phosphate (α-TCP) cement combined with platelet-rich plasma (PRP) on osteogenesis, and to compare the results with use of PRP alone. METHODS: A bilateral defect was produced in rat femurs and was filled with one of two types of treatments (PRP or α-TCP + PRP). The outcomes were evaluated after four and eight weeks. Radiographic images provided values for the lesion area, and histology (picrosirius staining) indicated the area of ​​new bone formation. RESULTS: The means relating to the lesion area of the α-TCP + PRP group (2.64 ± 2.07 and 1.91 ± 0.93 mm², after four and eight weeks, respectively) showed numerically better but non-significant results (p > 0.05) than those seen in the PRP group (5.59 mm ² ± 2.69 and 3.23 ± 1.46 mm ², after four and eight weeks, respectively). The mean new bone formation rates were 62.7% ± 12.1 and 79.01% ± 6.25 in the PRP group, and 73.3% ± 12.7 and 85.86% ± 10.45 in α-TCP + PRP group, after four and eight weeks, respectively (p > 0.05). CONCLUSION: The data from this study suggest that treatment with α-TCP cement combined with PRP does not show any significant difference in comparison with PRP alone. However, there is a possible early effect on bone regeneration when the two biomaterials are applied together.

Viabilidade do enxerto ósseo da crista ilíaca vascularizado pelo ramo ilíaco da artéria iliolombar: estudo experimental em ratos / Viability of vascularized bone graft from the iliac crest using the iliac branch of the iliolumbar artery: experimental study on rats

OBJETIVO: Através de um modelo experimental, pretendemos criar inferências sobre a viabilidade do enxerto ósseo vascularizado da crista ilíaca em ratos e verificar suas características histológicas. MÉTODO: Foram utilizados 21 ratos, que foram divididos em dois grupos: o primeiro consistindo por animais submetidos à técnica de enxerto ósseo vascularizado e pediculado sobre o ramo ilíaco da artéria iliolombar; o segundo (grupo controle) sofreu o mesmo procedimento do primeiro com a adição da ligadura do pedículo vascular. A viabilidade dos enxertos ósseos foi verificada durante três semanas, através da visualização direta do enxerto, histologia e imunoistoquímica. RESULTADOS: Todos os enxertos vascularizados avaliados na primeira semana apresentaram viabilidade segundo a observação direta, histologia e imunoistoquímica. Entretanto, na segunda e terceira semanas os enxertos mostraram-se inviáveis em 75% dos casos quando submetidos à avaliação segundo a observação direta e em 50% dos casos quando realizada a análise histológica e imunoistoquímica. CONCLUSÃO: Alguns enxertos vascularizados em sua concepção tornaram-se inviáveis e passaram a se comportar como enxertos não vascularizados sob a análise da observação direta e histológica. Apesar da possibilidade de falha, o uso de enxertos ósseos vascularizados deve ser incentivado, pois a histologia descritiva demonstrou maior densidade celular na porção óssea medular, osteócitos com maior funcionalidade na deposição de matriz óssea, com rede vascular intraóssea preservada.

OBJECTIVE: Through an experimental model, our aim was to create inferences about the viability of vascularized bone grafts from the iliac crest in rats and investigate their histological features. METHODS: Twenty-one rats were used, divided into two groups: the first consisted of animals that were subjected to the technique of vascularized bone graft pedicled onto the iliac branch of the iliolumbar artery; the second (control group) underwent the same procedure as performed on the first group, with the addition of ligation of the vascular pedicle. The viability of the bone grafts was observed for three weeks, by means of direct observation of the graft, histology and immunohistochemistry. RESULTS: All the vascularized grafts evaluated in the first week showed viability according to direct observation, histology and immunohistochemistry. However, in the second and third weeks, direct observation showed that 75% of the grafts were unviable, while histological analysis and immunohistochemistry showed that 50% were unviable. CONCLUSIONS: Some grafts that are designed to be vascularized became unviable and began to behave like nonvascularized grafts under direct observation and histology. Despite the possibility of failure, use of vascularized bone grafts should be encouraged, because descriptive histology shows greater cell density in the medullary bone portion, and osteocytes that function better regarding deposition of bone matrix, with preservation of the intraosseous vascular network.

Efeito de fatores neurotróficos sobre o reparo de nervo periférico: [revisão] / Effect of neurotrophic factors on peripheral nerve repair: [review]

Objetivos: esta revisão teve como objetivo analisar e discutir estudos sobre os principais fatores de crescimento testados in vitro e in vivo na regeneração de nervos periféricos.Fonte de dados: os artigos foram selecionados nas bases de dados LILACS, Medline e SciELO, utilizando os seguintes descritores: regeneração nervosa, fatores de crescimento, fator de crescimento neural, fator neurotrófico derivado do cérebro, neurotrofina 3, neurotrofina 4/5, fator neurotrófico ciliar, fator neurotrófico derivado de linhagem de célula glial, fator de crescimento do endotélio vascular, fator de crescimento similar à insulina, sistema nervoso periférico, lesões, neurônios sensoriais, neurônios motores, enxerto autólogo e ratos.Síntese dos dados: diversos fatores tróficos, também conhecidos como fatores de crescimento, são utilizados e testados in vitro e in vivo na regeneração de nervos periféricos. Essas proteínas atuam diretamente na proliferação e diferenciação de diferentes tipos celulares, sendo capazes de promover reparo tecidual e recuperação funcional. Geralmente o modelo ideal para a aplicação dessas substâncias é um sistema de entrega contínua através de condutos biodegradáveis.Conclusões: é possível concluir que a combinação de dois ou mais fatores de crescimento provavelmente exerça um efeito sinérgico na regeneração do nervo, principalmente quando associada a biomateriais absorvíveis com liberação controlada. Apesar do conhecimento obtido sobre essas proteínas apontar para um melhora da regeneração nervosa, ainda são necessários mais estudos experimentais antes de transpô-los para a aplicação clínica.

Aims: This review aimed to analyze and discuss studies about the main growth factors tested in vitro and in vivo on peripheral nerves regeneration.Source of data: Articles were selected from the databases LILACS, Medline, and SciELO, using the following key words: nerve regeneration, nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, neurotrophin 4/5, ciliary neurotrophic factor, glial cell line-derived neurotrophic factor, vascular endothelial growth factor, insulin-like growth factor, peripheral nervous system, injury, sensory neurons, motor neurons, autologous graft, and rats.Summary of findings: Several trophic factors, also known as growth factors, are used and tested in vitro and in vivo regeneration of peripheral nerves. These proteins act directly on the proliferation and differentiation of different cell types, being able to promote tissue repair and functional recovery. Usually the ideal model for the application of these substances is a continuous delivery system via biodegradable conduits.Conclusions: It is possible to conclude that the combination of two or more growth factors probably exercise a synergistic effect on nerve regeneration, especially when associated with absorble biomaterials with controlled release. Although the knowledge obtained about these proteins indicate an improvement in nerve regeneration, further experimental studies are needed before transpose them into clinical application.

PERIPHERAL NERVE REGENERATION: CELL THERAPY AND NEUROTROPHIC FACTORS.

Peripheral nerve trauma results in functional loss in the innervated organ, and recovery without surgical intervention is rare. Many surgical techniques can be used for nerve repair. Among these, the tubulization technique can be highlighted: this allows regenerative factors to be introduced into the chamber. Cell therapy and tissue engineering have arisen as an alternative for stimulating and aiding peripheral nerve regeneration. Therefore, the aim of this review was to provide a survey and analysis on the results from experimental and clinical studies that used cell therapy and tissue engineering as tools for optimizing the regeneration process. The articles used came from the LILACS, Medline and SciELO scientific databases. Articles on the use of stem cells, Schwann cells, growth factors, collagen, laminin and platelet-rich plasma for peripheral nerve repair were summarized over the course of the review. Based on these studies, it could be concluded that the use of stem cells derived from different sources presents promising results relating to nerve regeneration, because these cells have a capacity for neuronal differentiation, thus demonstrating effective functional results. The use of tubes containing bioactive elements with controlled release also optimizes the nerve repair, thus promoting greater myelination and axonal growth of peripheral nerves. Another promising treatment is the use of platelet-rich plasma, which not only releases growth factors that are important in nerve repair, but also serves as a carrier for exogenous factors, thereby stimulating the proliferation of specific cells for peripheral nerve repair.

Regeneração de nervos periféricos: terapia celular e fatores neurotróficos / Peripheral nerve regeneration: cell therapy and neurotrophic factors

Traumatismos em nervos periféricos resultam na perda de função do órgão inervado e raramente apresentam recuperação sem a intervenção cirúrgica. Diversas técnicas cirúrgicas são passíveis de serem empregadas para o reparo nervoso. Dentre elas, ressalta-se o uso da técnica de tubulização, podendo ser acrescentados fatores com capacidade regenerativa na câmara. A terapia celular e engenharia de tecidos surgem como uma alternativa para estimular e auxiliar a regeneração de nervos periféricos. Portanto, o objetivo desta revisão é fornecer um levantamento e uma análise de estudos experimentais e clínicos, quanto aos resultados obtidos, que utilizam a terapia celular e engenharia de tecidos como ferramentas para otimizar o processo de regeneração. Os artigos utilizados são oriundos de bases de dados científicas LILACS e Medline, através de pesquisas realizadas no PubMed e SciELO. Artigos sobre o uso de células-tronco, células de Schwann, fatores de crescimento, colágeno, laminina e plasma rico em plaquetas no reparo de nervos periféricos foram sintetizados ao longo da revisão. Com base nos diversos estudos pode-se concluir que a utilização de células-tronco derivadas de diferentes fontes apresentam resultados promissores na regeneração nervosa, pois estas possuem capacidade de diferenciação neuronal, demonstrando, assim, resultados funcionais eficazes. O uso de tubos acrescidos de elementos bioativos com liberação controlada também otimiza o reparo nervoso, promovendo uma maior mielinização e crescimento axonal dos nervos periféricos. Outro tratamento promissor é o uso de plasma rico em plaquetas, que, além de liberar fatores de crescimento importantes no reparo nervoso, ainda serve como um carreador para fatores exógenos estimulando a proliferação de células específicas no reparo de nervo periférico.

Peripheral nerve trauma results in functional loss in the innervated organ, and recovery without surgical intervention is rare. Many surgical techniques can be used for nerve repair. Among these, the tubulization technique can be highlighted: this allows regenerative factors to be introduced into the chamber. Cell therapy and tissue engineering have arisen as an alternative for stimulating and aiding peripheral nerve regeneration. Therefore, the aim of this review was to provide a survey and analysis on the results from experimental and clinical studies that used cell therapy and tissue engineering as tools for optimizing the regeneration process. The articles used came from the LILACS, Medline and SciELO scientific databases. Articles on the use of stem cells, Schwann cells, growth factors, collagen, laminin and platelet-rich plasma for peripheral nerve repair were summarized over the course of the review. Based on these studies, it could be concluded that the use of stem cells derived from different sources presents promising results relating to nerve regeneration, because these cells have a capacity for neuronal differentiation, thus demonstrating effective functional results. The use of tubes containing bioactive elements with controlled release also optimizes the nerve repair, thus promoting greater myelination and axonal growth of peripheral nerves. Another promising treatment is the use of platelet-rich plasma, which not only releases growth factors that are important in nerve repair, but also serves as a carrier for exogenous factors, thereby stimulating the proliferation of specific cells for peripheral nerve repair.