Avaliação da viabilidade do selante de fibrina derivado de veneno de serpente como arcabouço biológico para células-tronco mesenquimais de medula óssea de ratos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Células-tronco mesenquimais de equinos: isolamento, cultivo e caracterização

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Função cardíaca de cães submetidos ao transplante autólogo de células-tronco hematopoiéticas em dois modelos experimentais de cardiomiopatia

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Tratamento com minociclina e transplante intraestriatal de células mononucleares da medula óssea após acidente vascular experimental encefálico

Diversos estudos sugerem que a tetraciciclina semi-sintética minociciclina e o transplante de células mononucleares da medula óssea (CMMOs) induzem neuroproteção em modelos experimentais de acidente vascular encefálico (AVENC). No entanto, poucos investigaram, comparativamente, os efeitos destas duas abordagens terapêuticas após AVENC induzido por microinjeções de endotelina – 1 (ET -1). Nesta dissertação, objetivou-se comparar os efeitos do bloqueio microglial com minociclina com os obtidos pelo transplante intraestriatal de CMMOs na fase aguda após acidente vascular encefálico experimental, sobre a área de lesão, neuroproteção, apoptose de recuperação funcional. Ratos machos adultos, da raça Wistar, pesando entre 250 e 350g, foram distribuídos em quatro grupos experimentais: controle (chamado de Salina) - isquêmico tratado com salina (N=4), isquêmico tratado com minociclina (N=4), isquêmico tratado com CMMOs (N=3) e doador de CMMOs (N=2). Testes comportamentais foram realizados em 1, 3 e 7 dias pós-isquemia para avaliar a recuperação funcional entre os grupos. Animais tratados com minociclina receberam 2 doses diárias de 50mg/kg nos 2 primeiros dias, e 5 aplicações únicas de 25mg/kg (i.p) nos dias subsequentes até o sexto dia após a indução isquêmica. 1x106 de CMMOs foram obtidas de ratos da mesma linhagem e transplantadas diretamente no estriato, 24h após a lesão isquêmica. Todos os animais foram perfundidos 7 dias após a indução isquêmica. Secções coronais foram coradas por violeta de cresila para análise histopatológica geral, e por imunohistoquímica para a identificação de corpos neuronais (neuN), microglia/macrófagos ativados (ED1) e células apoptóticas (Caspase-3). A análise histopatológica geral mostrou grande palor, perda tecidual e intensa ativação microglial/ macrofágica no estriato de animais tratados com solução salina estéril. O tratamento com CMMO foi mais eficaz do que a minociclina (P<0,05, ANOVA-Tukey) na redução do número de microglia/macrófagos ativados (salina 276,3 ± 9,3); CMMOs 133,8 ± 6,8) e minociclina 244,6 ± 7,1). CMMOs e minociclina reduziram a área de lesão, em 67,75% e 69,1%, respectivamente. Os dois tratamentos promoveram o mesmo nível de preservação neuronal (p< 0,05) em relação ao controle, 61,3 ± 1,5); 86,8 ± 3,4) e 81 ± 3,4). As CMMOs reduziram de forma mais eficaz (p<0,01) o número de células apoptóticas em relação à minociclina e grupo controle (26,5 ± 1,6); 13,1 ± 0,7) e 19,7 ± 1,1). Ambas as abordagens terapêuticas promoveram recuperação funcional dos animais isquêmicos. Os resultados sugerem que o tratamento com CMMOs é mais eficaz na modulação da resposta microglial e na diminuição da apoptose do que o tratamento com minociclina, apesar de ambos serem igualmente eficazes para indução da neuroproteção. Estudos futuros devem investigar se o tratamento com minociclina associado ao transplante de CMMOs produzem efeitos sinérgicos, o que poderia amplificar os níveis de neuroproteção observados.

Utilização de células tronco mesenquimais autólogas para o tratamento de éguas com endometrite crônica degenerativa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparação entre enxerto ósseo autógeno e rhMP-2 (infuse Bone Graft) na reconstrução de maxila atrófica anterior

Pós-graduação em Odontologia - FOAR

Do progenitor cells from different tissue have the same phenotype?

The purpose of this work was to validate isolation methods for sheep mesenchymal stem cells (MSC) from different sources and to explore the hypothesis that MSC exhibit markers of the same phenotype independent from tissue source. Cells derived from ovine bone marrow, synovial membrane and adipose tissue were characterized using the following markers: CD44, CD45, CD11b and MHC-I. The isolated MSC were cultivated, went through osteogenic, chondrogenic and adipogenic differentiation, and were characterized by flow cytometry using mouse anti-ovine CD44, CD45 and MHC-I monoclonal antibody (mAb), and mouse anti-bovine CD11b mAb. Ovine MSC from all three sources differentiated under chondorgenic, osteogenic and adipogenic conditions. Also, MSC from the three tissues were found to express CD44 and MHC-I but lack of CD11b and CD45. The results obtained revealed that our isolation methods for the different tissues tested are valid and that MSC from the three sources studied have same immunophenotic characteristics. (C) 2014 Published by Elsevier Ltd. All rights reserved.

Mesenchymal stem cells modulate release of matrix proteins from tendon surfaces in vitro: a potential beneficial therapeutic effect

Aim: Injury of tendons contained within a synovial environment, such as joint, bursa or tendon sheath, frequently fails to heal and releases matrix proteins into the synovial fluid, driving inflammation. This study investigated the effectiveness of cells to seal tendon surfaces and provoke matrix synthesis as a possible effective injectable therapy. Materials & methods: Equine flexor tendon explants were cultured overnight in suspensions of bone marrow and synovium-derived mesenchymal stems cells and, as controls, two sources of fibroblasts, derived from tendon and skin, which adhered to the explants. Release of the most abundant tendon extracellular matrix proteins into the media was assayed, along with specific matrix proteins synthesis by real-time PCR. Results: Release of extracellular matrix proteins was influenced by the coating cell type. Fibroblasts from skin and tendon appeared less capable of preventing the release of matrix proteins than mesenchymal stems cells. Conclusion: The source of cell is an important consideration for cell therapy.

Benzophenone guttiferone A from Garcinia achachairu Rusby (Clusiaceae) Presents Genotoxic Effects in Different Cells of Mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Peripheral Blood Mononuclear Phagocytes From Patients With Chronic Periodontitis Are Primed for Osteoclast Formation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

M-CSF Priming of Osteoclast Precursors Can Cause Osteoclastogenesis-Insensitivity, Which Can be Prevented and Overcome on Bone

Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. J. Cell. Physiol. 229: 210-225, 2014. (c) 2014 Wiley Periodicals, Inc.

Uso de células-tronco para o tratamento de tendinite em eqüinos

Brazil has the fourth largest horse herd in the world, this is due the recognition and appreciation that the different equestrian games are having within the country. Injuries of the tendon, especially in the digital flexor tendon, are the main cause of athletic life reduction among horses. The treatment of tendinitis in horses seeks full recovery of the damage tissue reestablishing the function previously lost, however conventional treatments have proven to be ineffective when considered the quality of the scar tissue and the rate of recurrence. Due to this, the use of adult stem cells to the treatment of musculoskeletal injuries of horses has been studied for some time. This method of treatment consists of aspiration of bone marrow or removal of subcutaneous fat tissue and implantation of these cells in the injured tissue. After obtaining the bone marrow the implantation can be performed with total bone marrow, with the mononuclear fraction of MSC or with cells cultured in vitro. From the fat tissue is used the stromal vascular fraction obtained by collagenase digestion, followed or not by cell culture. According to some studies, cell therapy with material obtained from bone marrow or adipose tissue has shown to be viable, given that these materials are abundant in repair components such as mesenchymal stem cells (MSC), growth factors and other components of the collagen matrix. Several studies using both types of cells have shown great potential and promising clinical results. However, knowledge of the biology and characterization of these cells remain largely unknown, and therefore is needed great care and caution when using stem cells for the treatment of musculoskeletal disorders in horses

Anti-inflammatory effect of MAPK phosphatase-1 local gene transfer in inflammatory bone loss

Alveolar bone loss associated with periodontal diseases is the result of osteoclastogenesis induced by bacterial pathogens. The mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) is a critical negative regulator of immune response as a key phosphatase capable of dephosphorylating activated MAPKs. In this study, rat macrophages transduced with recombinant adenovirus (Ad.)MKP-1 specifically dephosphorylated activated MAPKs induced by lipopolysaccharide (LPS) compared with control cells. Bone marrow macrophages from MKP-1 knockout (KO) mice exhibited higher interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and select chemokine compared with wild-type (WT) mice when stimulated by LPS. In addition, bone marrow cultures from MKP-1 KO mice exhibited significantly more osteoclastogenesis induced by LPS than when compared with WT mice. Importantly, MKP-1 gene transfer in bone marrow cells of MKP-1 KO mice significantly decreased IL-6, IL-10, TNF-α and chemokine levels, and formed fewer osteoclasts induced by LPS than compared with control group of cells. Furthermore, MKP-1 gene transfer in an experimental periodontal disease model attenuated bone resorption induced by LPS. Histological analysis confirmed that periodontal tissues transduced with Ad. MKP-1 exhibited less infiltrated inflammatory cells, less osteoclasts and less IL-6 than compared with rats of control groups. These studies indicate that MKP-1 is a key therapeutic target to control of inflammation-induced bone loss.

Diabetes reduces mesenchymal stem cells in fracture healing through a TNF alpha-mediated mechanism

Diabetes interferes with bone formation and impairs fracture healing, an important complication in humans and animal models. The aim of this study was to examine the impact of diabetes on mesenchymal stem cells (MSCs) during fracture repair.Fracture of the long bones was induced in a streptozotocin-induced type 1 diabetic mouse model with or without insulin or a specific TNF alpha inhibitor, pegsunercept. MSCs were detected with cluster designation-271 (also known as p75 neurotrophin receptor) or stem cell antigen-1 (Sca-1) antibodies in areas of new endochondral bone formation in the calluses. MSC apoptosis was measured by TUNEL assay and proliferation was measured by Ki67 antibody. In vitro apoptosis and proliferation were examined in C3H10T1/2 and human-bone-marrow-derived MSCs following transfection with FOXO1 small interfering (si)RNA.Diabetes significantly increased TNF alpha levels and reduced MSC numbers in new bone area. MSC numbers were restored to normal levels with insulin or pegsunercept treatment. Inhibition of TNF alpha significantly reduced MSC loss by increasing MSC proliferation and decreasing MSC apoptosis in diabetic animals, but had no effect on MSCs in normoglycaemic animals. In vitro experiments established that TNF alpha alone was sufficient to induce apoptosis and inhibit proliferation of MSCs. Furthermore, silencing forkhead box protein O1 (FOXO1) prevented TNF alpha-induced MSC apoptosis and reduced proliferation by regulating apoptotic and cell cycle genes.Diabetes-enhanced TNF alpha significantly reduced MSC numbers in new bone areas during fracture healing. Mechanistically, diabetes-enhanced TNF alpha reduced MSC proliferation and increased MSC apoptosis. Reducing the activity of TNF alpha in vivo may help to preserve endogenous MSCs and maximise regenerative potential in diabetic patients.

Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)