Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37ºC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.(AU)

As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a [...].(AU)

Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor)

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.

Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor)

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.(AU)

Fibrin biopolymer as scaffold candidate to treat bone defects in rats

Background:Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs.Methods:Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis.Results:It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation.Conclusions:FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.(AU)

Fibrin biopolymer as scaffold candidate to treat bone defects in rats

Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. Methods: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. Results: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. Conclusions: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.(AU)

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin

ABSTRACT The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.

RESUMO O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin / Diferenciação osteogênica de células tronco mesenquimais do tecido adiposo cultivadas com diferentes concentrações de prolactina

The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.(AU)

O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.(AU)

Osteogenic differentiation of adipose tissue-derived mesenchymal stem cells cultured with different concentrations of prolactin / Diferenciação osteogênica de células tronco mesenquimais do tecido adiposo cultivadas com diferentes concentrações de prolactina

The objective was to evaluate the in vitro effect of prolactin in osteogenic potential of adipose tissue-derived mesenchymal stem cells (ADSCs) in female rats. ADSCs were cultured in osteogenic medium with and without the addition of prolactin and distributed into three groups: 1) ADSCs (control), 2) ADSCs with addition of 100ng/mL of prolactin and 3) ADSCs with addition of 300ng/mL of prolactin. At 21 days of differentiation, the tests of MTT conversion into formazan crystals, percentage of mineralized nodules and cells per field and quantification of genic transcript for alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I by real-time RT-PCR were made. The addition of prolactin reduced the conversion of MTT in group 3 and increased the percentage of cells per field in the groups 2 and 3, however without significantly increasing the percentage of mineralized nodules and the expression of alkaline phosphatase, osteopontin, osteocalcin, bone sialoprotein, BMP-2 and collagen I. In conclusion, the addition of prolactin in concentrations of 100ng/mL and 300ng/mL does not change the osteogenic differentiation to the ADSCs of female rats despite increase in the cellularity of the culture.(AU)

O objetivo do presente trabalho foi avaliar o efeito in vitro da prolactina sobre o potencial osteogênico de células-tronco mesenquimais do tecido adiposo (CTM-TA) em ratas. CTM-TA foram cultivadas em meio osteogênico com e sem adição de prolactina e distribuídas em três grupos: 1) CTM-TA (controle), 2) CM-TA com adição de 100ng/mL de prolactina e 3) CTM-TA com adição de 300ng/mL de prolactina. Aos 21 dias de diferenciação, foram realizados os testes de conversão do MTT em cristais de formazan, porcentagem de nódulos mineralizados e células por campo e quantificação dos transcritos gênicos para fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. A adição de prolactina reduziu a conversão do MTT no grupo 3 e aumentou a porcentagem de células por campo nos grupos 2 e 3, sem alterar significativamente a porcentagem de nódulos mineralizados e a expressão de fosfatase alcalina, osteopontina, osteocalcina, sialoproteína óssea, BMP-2 e colágeno I. Conclui-se que a adição de prolactina nas concentrações de 100ng/mL e 300ng/mL não altera a diferenciação osteogênica das CTM-TA de ratas, apesar do aumento de celularidade da cultura.(AU)

ISOLAMENTO E CULTIVO DE CÉLULAS TRONCO MESENQUIMAIS EXTRAÍDAS DO TECIDO ADIPOSO E DA MEDULA ÓSSEA DE CÃES

Abstract The objective of this study was to establish a protocol for the isolation and culture of mesenchymal stem cells (MSC) from bone marrow and adipose tissue of dogs. Three 6-month-old male dogs were used. Approximately 3.0 cm3 of adipose tissue and 3.0 mL of bone marrow were collected. The samples were processed and the isolated cells were cultured in DMEM. The cells were subjected to phenotypic characterization and to osteogenic, adipogenic, and condrogenic differentiation to confirm the isolation of the MSC. The cells showed elongated and fusiform morphology and they were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Phenotypic characterization revealed high expression of the MSC markers CD90(80.04%) and CD29(96%) in the cells from bone marrow and high expression of CD90(60.94%) and CD29(77.08%) in the cells from adipose tissue. In addition, phenotypic characterization revealed low expression of hematopoietic markers CD45(1.45%) and CD34(1.53%) in the cells from bone marrow and low expression of CD45(1.45%) and CD34(1.53%) in the cells from adipose tissue. Based on these results, the modifications applied to classical protocols simplified the process and proved to be efficient in the isolation, culture, and expansion of MSC isolated from the bone marrow and adipose tissue of dogs.

Resumo Objetivou-se estabelecer um protocolo para extração, cultivo e expansão de células tronco mesenquimais (CTM), utilizando-se 3,0 mL da medula óssea e 3,0 cm3 de tecido adiposo do subcutâneo de três cães machos com seis meses de idade. As amostras foram processadas e as células extraídas e cultivadas em DMEM. Para comprovação do isolamento de CTM, procedeu-se a caracterização fenotípica e a diferenciação osteogênica, adipogênica e condrogênica. As células isoladas apresentaram morfologia alongada e fusiforme e capacidade de se diferenciar em osteoblastos, adipócitos e condrócitos. A caracterização fenotípica revelou alta expressão de marcadores de CTM CD90 (80,04%) e CD29 (96%) nas células de origem medular e CD90 (60,94%) e CD29 (77,08%) nas de origem adiposa. A expressão de marcadores hematopoiéticos foi baixa tanto nas células de origem medular CD45 (1,45%) e CD34 (1,53%), quanto nas de origem adiposa CD45 (1,45%) e CD34 (1,53%). As modificações e adaptações realizadas nos protocolos clássicos simplificaram o processo e foram eficientes, permitindo o isolamento e cultivo de CTM da medula óssea e do tecido adiposo de cães.

Isolamento e cultivo de células tronco mesenquimais extraídas do tecido adiposo e da medula óssea de cães / Isolation and culture of mesenchymal stem cells derived from adipose tissue and bone marrow of dogs

Objetivou-se estabelecer um protocolo para extração, cultivo e expansão de células tronco mesenquimais (CTM), utilizando-se 3,0 mL da medula óssea e 3,0 cm3 de tecido adiposo do subcutâneo de três cães machos com seis meses de idade. As amostras foram processadas e as células extraídas e cultivadas em DMEM. Para comprovação do isolamento de CTM, procedeu-se a caracterização fenotípica e a diferenciação osteogênica, adipogênica e condrogênica. As células isoladas apresentaram morfologia alongada e fusiforme e capacidade de se diferenciar em osteoblastos, adipócitos e condrócitos. A caracterização fenotípica revelou alta expressão de marcadores de CTM CD90 (80,04%) e CD29 (96%) nas células de origem medular e CD90 (60,94%) e CD29 (77,08%) nas de origem adiposa. A expressão de marcadores hematopoiéticos foi baixa tanto nas células de origem medular CD45 (1,45%) e CD34 (1,53%), quanto nas de origem adiposa CD45 (1,45%) e CD34 (1,53%). As modificações e adaptações realizadas nos protocolos clássicos simplificaram o processo e foram eficientes, permitindo o isolamento e cultivo de CTM da medula óssea e do tecido adiposo de cães. (AU)

The objective of this study was to establish a protocol for the isolation and culture of mesenchymal stem cells (MSC) from bone marrow and adipose tissue of dogs. Three 6-month-old male dogs were used. Approximately 3.0 cm3 of adipose tissue and 3.0 mL of bone marrow were collected. The samples were processed and the isolated cells were cultured in DMEM. The cells were subjected to phenotypic characterization and to osteogenic, adipogenic, and condrogenic differentiation to confirm the isolation of the MSC. The cells showed elongated and fusiform morphology and they were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Phenotypic characterization revealed high expression of the MSC markers CD90(80.04%) and CD29(96%) in the cells from bone marrow and high expression of CD90(60.94%) and CD29(77.08%) in the cells from adipose tissue. In addition, phenotypic characterization revealed low expression of hematopoietic markers CD45(1.45%) and CD34(1.53%) in the cells from bone marrow and low expression of CD45(1.45%) and CD34(1.53%) in the cells from adipose tissue. Based on these results, the modifications applied to classical protocols simplified the process and proved to be efficient in the isolation, culture, and expansion of MSC isolated from the bone marrow and adipose tissue of dogs.(AU)

Isolamento e cultivo de células tronco mesenquimais extraídas do tecido adiposo e da medula óssea de cães / Isolation and culture of mesenchymal stem cells derived from adipose tissue and bone marrow of dogs

Objetivou-se estabelecer um protocolo para extração, cultivo e expansão de células tronco mesenquimais (CTM), utilizando-se 3,0 mL da medula óssea e 3,0 cm3 de tecido adiposo do subcutâneo de três cães machos com seis meses de idade. As amostras foram processadas e as células extraídas e cultivadas em DMEM. Para comprovação do isolamento de CTM, procedeu-se a caracterização fenotípica e a diferenciação osteogênica, adipogênica e condrogênica. As células isoladas apresentaram morfologia alongada e fusiforme e capacidade de se diferenciar em osteoblastos, adipócitos e condrócitos. A caracterização fenotípica revelou alta expressão de marcadores de CTM CD90 (80,04%) e CD29 (96%) nas células de origem medular e CD90 (60,94%) e CD29 (77,08%) nas de origem adiposa. A expressão de marcadores hematopoiéticos foi baixa tanto nas células de origem medular CD45 (1,45%) e CD34 (1,53%), quanto nas de origem adiposa CD45 (1,45%) e CD34 (1,53%). As modificações e adaptações realizadas nos protocolos clássicos simplificaram o processo e foram eficientes, permitindo o isolamento e cultivo de CTM da medula óssea e do tecido adiposo de cães.

The objective of this study was to establish a protocol for the isolation and culture of mesenchymal stem cells (MSC) from bone marrow and adipose tissue of dogs. Three 6-month-old male dogs were used. Approximately 3.0 cm3 of adipose tissue and 3.0 mL of bone marrow were collected. The samples were processed and the isolated cells were cultured in DMEM. The cells were subjected to phenotypic characterization and to osteogenic, adipogenic, and condrogenic differentiation to confirm the isolation of the MSC. The cells showed elongated and fusiform morphology and they were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Phenotypic characterization revealed high expression of the MSC markers CD90(80.04%) and CD29(96%) in the cells from bone marrow and high expression of CD90(60.94%) and CD29(77.08%) in the cells from adipose tissue. In addition, phenotypic characterization revealed low expression of hematopoietic markers CD45(1.45%) and CD34(1.53%) in the cells from bone marrow and low expression of CD45(1.45%) and CD34(1.53%) in the cells from adipose tissue. Based on these results, the modifications applied to classical protocols simplified the process and proved to be efficient in the isolation, culture, and expansion of MSC isolated from the bone marrow and adipose tissue of dogs.

Goat umbilical cord cells are permissive to small ruminant lentivirus infection in vitro

Small ruminant lentiviruses isolated from peripheral blood leukocytes and target organs can be propagated in vitro in fibroblasts derived from goat synovial membrane cells. These cells are obtained from tissues collected from embryos or fetuses and are necessary for the establishment of the fibroblast primary culture. A new alternative type of host cells, derived from goat umbilical cord, was isolated and characterized phenotypically with its main purpose being to obtain cell monolayers that could be used for the diagnosis and isolation of small ruminant lentiviruses in cell culture. To accomplish this goal, cells were isolated from umbilical cords; characterized phenotypically by flow cytometry analysis; differentiate into osteogenic, chondrogenic and adipogenic lineage; and submitted to viral challenge. The proliferation of goat umbilical cord cells was fast and cell monolayers formed after 15 days. These cells exhibited morphology, immunophenotype, growth characteristics, and lineage differentiation potential similar to mesenchymal stem cells of other origins. The goat umbilical cord derived cells stained positive for vimentin and CD90, but negative for cytokeratin, CD34 and CD105 markers. Syncytia and cell lysis were observed in cell monolayers infected by CAEV-Cork and MVV-K1514, showing that the cells are permissive to small ruminant lentivirus infection in vitro. These data demonstrate the proliferative competence of cells derived from goat umbilical cords and provide a sound basis for future research to standardize this cell lineage.(AU)

Células osteoprogenitoras podem aumentar a formação óssea precoce em defeitos ósseos críticos de cães / Osteoprogenitor cells can enhance early bone formation in critical bone defects in dogs

The aim of this study was to evaluate the effect of osteoprogenitor cells derived from mesenchymal stem cells from adipose tissue (OC-AD-MSCs), and differentiated into osteoblasts, in the treatment of critical bone defects in dogs. Adipose tissue derived mesenchymal stem cells (AD-MSCs) were subjected to osteogenic differentiation for 21 days and used in the treatment of bone defects in dogs radius. Either three experimental groups were bone defects treated with OC-AD-MSCs (OC), defects filled with autogenous bone (Control- C +), or empty defects (Control- C -). Bone regeneration was assessed by radiology, densitometry, and histomorphometry. The area of new bone formation was higher in the OC group compared to the control group (C-) on postoperative day 15. Defects treated with OC-AD-MSCs showed greater neovascularization than the other two groups at 90 days. We concluded that treatment with OC-AD-MSCs increased the area of new bone formation 15 days after surgery; however, it didn’t complete the bone union in critical bone defects in the radius of dogs at 90 days.

O objetivo deste estudo foi avaliar o efeito das células osteoprogenitoras derivadas de células tronco mesenquimais do tecido adiposo (CO-CTM-AD) no tratamento de defeitos ósseos críticos de cães. As células tronco mesenquimais do tecido adiposo (CTM-AD) foram submetidas à diferenciação osteogênica por 21 dias e usadas no tratamento de defeitos ósseos em rádios de cães. Foram constituídos três grupos experimentais: defeitos ósseos tratados com CO-CTM-AD (OC), defeitos preenchidos com osso autógeno (C+) e defeitos não preenchidos (C-). A regeneração óssea foi avaliada por meio de exames radiográficos, densitométricos e histomorfométricos. A área de neoformação óssea foi maior no grupo OC em relação ao grupo C- no 15o dia de pós-operatório. Os defeitos tratados com CO-CTM-AD mostraram maior neovascularização que os demais grupos aos 90 dias de avaliação. Conclui-se que o tratamento com CO-CTM-AD aumentou a área de osso neoformado no 15o dia de pós-operatório, mas não foi suficiente para que houvesse a completa união óssea em defeitos ósseos críticos no rádio de cães aos 90 dias.

Detection of SNPs in the BMP6 Gene and Their Association with Carcass and Bone Traits in Chicken

BMP6, a member of the subfamilies of the morphogenetic proteins (BMPs), plays a crucial role in osteogenic and chondrocyte differentiation in vitro and stimulates chondrogenesis, making chondrocytes differen-tiate on their terminal stage. The objective of this study is to explore the relationship between polymorphism of BMP6 gene and slaughter traits in chicken respectively. We screened the exonic and intronic regions of BMP6 gene by DNA pool construction and amplified DNA fragment by PCR, and finally, we got nine SNPs. Association analysis revealed that BMP6 had no significant association among all slaughter traits in Yellow bantam chicken. However, BMP6 had a significant difference with femur weight, tibia weight, femur length (p 0.05), and was extremely significant with tibia length (p 0.01) in Avian chicken. Moreover, femur perimeter also had significant correlation with BMP6 in Avian chicken. These results provide useful information for further investigation on the function of chicken BMP6 gene.(AU)

Células osteoprogenitoras podem aumentar a formação óssea precoce em defeitos ósseos críticos de cães / Osteoprogenitor cells can enhance early bone formation in critical bone defects in dogs

The aim of this study was to evaluate the effect of osteoprogenitor cells derived from mesenchymal stem cells from adipose tissue (OC-AD-MSCs), and differentiated into osteoblasts, in the treatment of critical bone defects in dogs. Adipose tissue derived mesenchymal stem cells (AD-MSCs) were subjected to osteogenic differentiation for 21 days and used in the treatment of bone defects in dogs radius. Either three experimental groups were bone defects treated with OC-AD-MSCs (OC), defects filled with autogenous bone (Control- C +), or empty defects (Control- C -). Bone regeneration was assessed by radiology, densitometry, and histomorphometry. The area of new bone formation was higher in the OC group compared to the control group (C-) on postoperative day 15. Defects treated with OC-AD-MSCs showed greater neovascularization than the other two groups at 90 days. We concluded that treatment with OC-AD-MSCs increased the area of new bone formation 15 days after surgery; however, it didnt complete the bone union in critical bone defects in the radius of dogs at 90 days.(AU)

O objetivo deste estudo foi avaliar o efeito das células osteoprogenitoras derivadas de células tronco mesenquimais do tecido adiposo (CO-CTM-AD) no tratamento de defeitos ósseos críticos de cães. As células tronco mesenquimais do tecido adiposo (CTM-AD) foram submetidas à diferenciação osteogênica por 21 dias e usadas no tratamento de defeitos ósseos em rádios de cães. Foram constituídos três grupos experimentais: defeitos ósseos tratados com CO-CTM-AD (OC), defeitos preenchidos com osso autógeno (C+) e defeitos não preenchidos (C-). A regeneração óssea foi avaliada por meio de exames radiográficos, densitométricos e histomorfométricos. A área de neoformação óssea foi maior no grupo OC em relação ao grupo C- no 15o dia de pós-operatório. Os defeitos tratados com CO-CTM-AD mostraram maior neovascularização que os demais grupos aos 90 dias de avaliação. Conclui-se que o tratamento com CO-CTM-AD aumentou a área de osso neoformado no 15o dia de pós-operatório, mas não foi suficiente para que houvesse a completa união óssea em defeitos ósseos críticos no rádio de cães aos 90 dias.(AU)