Characterization and plasticity of wharton's jelly mesenchymal stem cells of goat

Mesenchymal stem cells (MSCs), obtained from several anatomical sites, have already been described, characterized and used in therapeutic models for tissue repair. The umbilical cord mesenchymal stem cells, represented by cells from arteries and veins walls, as well as Wharton's jelly are easy to be obtained, highly available, require no invasive procedure, do not present risk to donors and do not present ethical limitation. The aim of this research was to analyze the plasticity of Wharton's jelly mesenchymal stem cells (WJ-MSCs) of goat, evaluating their behavior in vitro and characterizing them immunophenotypically. Thus, tests were performed on colony forming units, viability and cell growth curve, flow cytometry analysis and plasticity potential. Goat umbilical cord matrix cells exhibited fibroblastoid morphology with colony formation and self-renewal ability, always maintaining their undifferentiated state up to the eighth passage (P8). The growth curve kinetics exhibited the LAG, LOG, and DECAY phases, without displaying a PLATEAU phase. The plasticity assay demonstrated positive differentiation for osteogenic, adipogenic and chondrogenic lines, characterized by the synthesis of intracytoplasmic granules or extracellular matrix with the presence of calcium, lipids and proteoglycans. Flow cytometry demonstrated the expression of CD90 and CD105; absence of CD14 expression. It is concluded that the cell population isolated from the Wharton's jelly of goat constitutes a representative sample of mesenchymal stem cells, with great possibilities in the field of regenerative and reproductive medicine.

Role of the autologous mesenchymal stem cells compared with platelet rich plasma on cicatrization of cutaneous wounds in diabetic mice / Papel das células-tronco mesenquimais autólogas em comparação com plasma rico em plaquetas na cicatrização de feridas cutâneas em camundongos diabéticos

Chronic cutaneous lesions affect 15% of diabetic human patients and represent a risk 15 to 46 times larger of limb amputations compared to people with normal glycemia. It is assumed that half of these amputations could be prevented by early treatment of wounds, for example, with proper cell therapy. Objectives: In this study, the action of the autologous transplant of mesenchymal stem-cells (MSC) was evaluated compared to the treatment with autologous platelet rich plasma (PRP) in the cicatrization of cutaneous lesions induced in diabetic mice. These animals were previously treated with streptozootocin to induce diabetes mellitus and round wounds of 1.5cm in diameter were created in the posterior region. Diameters of the wounds and healing time were evaluated during 30 days and the results were submitted to variance analysis and Tukey's test average. It was noticed that the animals treated with MSC presented a more accelerated cicatrization of the cutaneous lesion than the animals treated with PRP. However, the treatment with PRP presented better results than just the daily asepsis of the lesions with saline or covering them with semi-permeable bandage. Besides, the use of semi-permeable bandage kept the cutaneous lesions of diabetic mice did not interfere negatively with cicatrization, proved to be harmless to use, but kept the cutaneous lesions more hydrated than the ones exposed to the environment.(AU)

Lesões cutâneas crônicas afetam 15% dos pacientes diabéticos e humanos representam um risco 15 a 46 vezes maior de amputações de membros em comparação com as pessoas com a glicemia normal. Supõe-se que a metade destas amputações poderia ser evitada por meio do tratamento precoce das feridas cutâneas com, por exemplo, uma adequada terapia celular. Objetivos: Neste estudo, a ação do transplante autólogo de células estaminais mesenquimais (MSC) foi avaliada em comparação com o tratamento com plasma rico em plaquetas autólogo (PRP) na cicatrização de lesões cutâneas induzidas em camundongos diabéticos. Estes animais foram previamente tratados com estreptozotocina para induzir diabetes mellitus e feridas redondas de 1,5 cm de diâmetro foram criadas na região posterior. Os diâmetros dos ferimentos e tempo de cicatrização foram avaliados durante 30 dias e os resultados foram submetidos à análise de variância e média pelo teste de Tukey. Verificou-se que os animais tratados com MSC apresentam uma cicatrização mais acelerada da lesão cutânea que do que os animais tratados com PRP. No entanto, o tratamento com PRP apresentou melhores resultados do que apenas a assepsia das lesões diariamente com solução salina ou cobrindo-os com atadura semi-permeável. Além disso, a utilização de atadura semi-permeável mantidas as lesões cutâneas de camundongos diabéticos não interfere negativamente com a cicatrização, provou ser inofensiva para usar, mas manteve as lesões cutâneas hidratadas mais do que os expostos ao meio ambiente.(AU)

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831) / Isolamento, expansão e diferenciação de progenitors celulares obtidos da polpa dentária de cutia (Dasyprocta prymnolopha Wagler, 1831)

The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831). Este estudo teve como objetivo isolar, expandir, diferenciar e caracterizar células progenitoras existentes na polpa dentária de cutia. O material foi lavado em solução de PBS e dissociado mecanicamente, com o auxílio de uma lâmina de bisturi, em placas contendo meio de cultura D-MEM/F-12, e incubadas em 5% de CO2-37⁰C. A curva de crescimento, o ensaio de CFU, a diferenciação osteogênica/adipogênica e a caracterização foram obtidas a partir do isolamento. As células começaram a ser liberadas, a partir do explante, em torno do sétimo dia de cultura. A partir do 22o dia, as células atingiram 80% de confluência. No teste para UFC, 81 colônias foram contadas aos 12 dias de cultivo. As curvas de crescimento pré- e pós-congelamento apresentaram crescimento regular, com intensa proliferação e potencial clonogênico. A diferenciação das células mostrou a formação de osteoblastos e de células de gordura, a partir de 15 dias de cultura em meio específico. A citometria de fluxo (FACS) apresentou-se como segue: CD34 (positivo), CD14 (negativo), CD45 (negativo), CD73 (positivo), CD79 (negativo), CD90 (positivo), CD105 (positivo), demonstrando a grande especificidade e comprometimento das células isoladas com linhagens de células-tronco mesenquimais. Estes resultados sugerem a existência de uma população de células-tronco mesenquimais isolada a partir de explantes da polpa dentária cutia, um modelo potencial para o estudo de linhagens de células-tronco obtidas a partir do tecido pulpar.

Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil

PURPOSE: To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. METHODS: Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) - Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers - Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). RESULTS: Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 106 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. CONCLUSIONS: Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied. .

Association of mesenchymal stem cells with platelet rich plasma on the repair of critical calvarial defects in mice / Associação de células-tronco mesenquimais com plasma rico em plaquetas na reparação de defeitos críticos em calvária de camundongos

PURPOSE: To evaluate the effects of mesenchymal stem cells (MSC) from eight mice C57BL/6 gfp+ bone marrows expanded in cultures associated with platelets rich plasma (PRP) deriving from another eight mice, in the repair of critical defects in calvarial bone produced in twenty-four adult isogenic mice C57BL/6. METHODS: The animals were submitted to a cranial defect of 6.0mm in diameter and divided into two equal experimental groups. Control group did not receive treatment and the treated group received a MSC pellet containing 1.0 x 10(7) cells/mL associated with 50.0µL of plasma gel containing 1.0 x 10(9) autologous platelets within the defect. RESULTS: In the treated group was observed process of angiogenesis and bone repair better than control group. CONCLUSION: Mesenchymal stem cells derived from bone marrow of C57BL/6 gfp+ mice associated with PRP gel applied in bone critical defects produced in calvarial contributes positively to the process of bone repair.

OBJETIVO: Avaliar os efeitos da associação das células-tronco mesenquimais (MSC) oriundas da medula óssea de oito camundongos jovens C57BL/6 gfp+ e expandidas em culturas, com Plasma Rico em Plaquetas (PRP) provenientes de outros oito camundongos, na reparação de defeitos críticos confeccionados em calvária de 24 camundongos adultos C57BL/6. MÉTODOS: Os animais foram submetidos a um defeito craniano de 6,0mm de diâmetro e separados em dois grupos experimentais iguais. O grupo controle não recebeu tratamento e no grupo tratado foi administrado, no interior do defeito, pellet de MSC contendo 1,0 x 10(7) células/mL associado com 50,0µL de plasma em gel autólogo contendo 1,0 x 10(9) plaquetas. RESULTADOS: No grupo tratado verificou-se processo de angiogênese e reparação óssea superior ao grupo controle. CONCLUSÃO: A associação das células-tronco mesenquimais (MSC) derivadas da medula óssea de camundongos C57BL/6 gfp+ com gel de PRP aplicadas em defeitos ósseos críticos confeccionadas em calvária de camundongos C57BL/6 jovens, contribuiu positivamente para o processo de reparação óssea.

Contribuição do plasma rico em plaquetas na reparação óssea de defeitos críticos criados em crânios de camundongos / Platelet-rich plasma contribute to the process of bone repair of critical defects created in the calvaria of mice

No presente estudo, foram avaliados, de forma macro e microscópica, os resultados da aplicação do PRP em defeitos ósseos críticos de 6,0mm de diâmetro confeccionados em calvária de 24 camundongos isogênicos C57BL/6 jovens, separados em dois grupos experimentais. O grupo controle não recebeu tratamento, e no grupo tratado foram depositados, no interior do defeito, 50,0µL plasma em gel contendo 1,0x10(9) plaquetas. Constatou-se que o gel de PRP autólogo depositado em defeitos críticos contribuiu positivamente para o processo de reparação óssea, mormente na fase inicial.

This study aimed to evaluate macro and microscopic results after PRP application in bone critical defects of 6.0mm in diameter realized in cavarial of twenty-four young isogenic mice C57BL/6. Control group didn't receive treatment and in the treated group it was deposited 50.0µL of plasma gel containing 1.0x10(9) platelets in the defects. It was found that autologous PRP gel contributed positively to the bone repair process, especially in initial phase.

Células-tronco mesenquimais / Mesenchymal stem cell

Dentre todas as células-tronco estudadas até o presente momento, as mesenquimais (MSC) destacam-se por sua elevada plasticidade, podendo originar tecidos mesodermais e não mesodermais. Além disso, possuem características imunomoduladoras e imunossupressoras que ampliam as possibilidades de utilização terapêutica. As MSC secretam uma grande variedade de citocinas pró e anti-inflamatórias e fatores de crescimento e, por meio dessas moléculas bioativas, proporcionam a modulação da resposta inflamatória, o restabelecimento do suprimento vascular e a reparação adequada do tecido, contribuindo para a homeostasia tissular e imunológica sob condições fisiológicas. Também podem induzir as demais células presentes no nicho tecidual a secretarem outros fatores solúveis que estimulam a diferenciação dessas células indiferenciadas, favorecendo o processo de reparação. A terapia celular com MSC é uma alternativa terapêutica promissora, porém a compreensão da biologia dessas células ainda é uma ciência em formação. Este artigo tem por objetivo realizar uma breve revisão sobre as células mesenquimais indiferenciadas.

Of all the stem cells studied so far, the mesenchymal stem cells (MSC) stand out for their high plasticity and capacity of generating mesodermal and non-mesodermal tissues. In addition, immunomodulatory and immunosuppressive features that expand possibilities for therapeutic use are present in these cells. A variety of pro and anti-inflammatory cytokines and growth factors are secrete for MSC and provide a modulation of inflammatory response, re-establishment of vascular supply and adequate repair of the tissue, contributing to tissue homeostasis under physiologic conditions. Therefore, they can induce secretion of soluble factors that stimulate their differentiation by other cells present at the niche's tissue, promoting the repair process. Cell therapy using MSC is a promises therapeutic alternative, but understanding the biology of these cells is still under construction. The aim of the article is to conduct a short review of these undifferentiated mesenchymal cells.

Parâmetros bioquímicos e hemogasométricos do sangue total canino armazenado em bolsas plásticas contendo CPDA-1 e CPD/SAG-M / Parameter biochemical and hemogasometric of the total blood stored of dogs in plastic stock markets I contend CPDA-1 and CPD/SAG-M

O presente trabalho teve como objetivo avaliar e comparar as alterações bioquímicas e hemogasométricas do sangue total canino armazenado em bolsas CPDA-1 e CPD/SAG-M. Foram utilizados 14 cães machos, adultos e saudáveis, distribuídos em dois grupos com sete animais cada, dos quais foi obtido sangue para o estudo. No primeiro grupo (G1), o sangue foi armazenado em bolsas CPDA-1 e, no segundo grupo (G2), o sangue foi armazenado em bolsas CPD/SAG-M. As amostras foram analisadas para: potássio, sódio, glicose, proteína plasmática total, DPG, pH sangüíneo, pO2 , pCO2 e bicarbonato. Os momentos estabelecidos para as análises laboratoriais foram: D0: imediatamente após a coleta do sangue dos animais; D7: sete dias após a coleta; D14: quatorze dias após a coleta; D21: vinte e um dias após a coleta; D31: trinta e um dias após a coleta; D41: quarenta e um dias após coleta. A análise dos resultados permitiu concluir que a bolsa CPD/SAG-M apresentou melhor desempenho quando comparada à CPDA-1 após 41 dias de estocagem. O DPG, mesmo com pH inferior a sete, continua sendo produzido por, pelo menos, uma semana após a estocagem do sangue na bolsa.

The present study was aimed at evaluating and comparing the biochemical and haemogasometrics alterations of the total blood of dogs stored in bags CPDA-1 and CPD/SAG-M. 14 male's dogs, adult and healthful dogs, were divided in two groups, which one blood was removed for the study. The blood of the first group (G1) was stored in bag CPDA-1 and that of the second group (G2) in bag CPD/SAG-M. The samples were analyzed for: potassium, sodium, glucose, total plasmatic protein, DPG, pH, pO2 , pCO2 , and bicarbonate. The moments for the laboratory analyses were established: D0: immediately after the collection of blood; D7: seven days after the collection; D14: fourteen days after the collection; D21: twenty and one days after the collection; D31: thirty and one days after the collection; D41: forty one days after collect. The evaluation of the results allowed including that: the bag CPD/SAG-M presented advantage when compared with the CPDA-1 after 41 days of stored. The DPG, even with pH lower then 7, continues being produced for, at least, one week after the stocking of the blood in the bag.