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

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.

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

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)

Locking compression plate fixation of critical-sized bone defects in sheep. Development of a model for veterinary bone tissue engineering

ABSTRACT Purpose To develop a segmental tibial bone defect model for tissue engineering studies in veterinary orthopedics using single locking compression plate (LCP) fixation and cast immobilization. Methods A 3-cm bone defect was created in the right tibia of 18 adult Suffolk sheep. A 10-hole, 4.5-mm LCP was applied to the dorsomedial aspect of the bone. Four locking screws were inserted into the proximal and three into the distal bone fragment. Operated limbs were immobilized with casts. Animals were submitted to stall rest, but were allowed to bear weight on the operated limb. During the recovery period, animals were checked daily for physiological parameters, behavior and lameness. Follow-up radiographs were taken monthly. Results Surgical procedures and postoperative recovery were uneventful. Animals adapted quickly to casts and were able to bear weight on the operated limb with no signs of discomfort or distress. No clinical or radiographic complications were detected over a 90-day follow-up period. Conclusions Surgical creation of tibial segmental bone defects followed by fixation with single LCP and cast immobilization was deemed a feasible and appropriate model for veterinary orthopedic research in tissue engineering.

Quantification of adhesion of mesenchymal stem cells spread on decellularized vein scaffold

ABSTRACT Purpose: To evaluate methods that improve adipose-derived stem cells (ASCs) population in decellularized biological venous scaffold for tissue engineering in blood vessels, a model in rabbits. Methods: The ASC was expanded until the third passage. Inferior vena cava (IVC) was submitted to the decellularization process using 1% sodium dodecyl sulfate (SDS) or 2% sodium deoxycholate (SD) to compose 12 study groups (G): pure SD or SDS, exposed or not to 1% TritonX-100 (TX-100) and exposed or not to poly-l'lysine and laminin (PL). Scaffolds were covered with 1 × 105 or 1 × 106 ASCs diluted in 10 μL Puramatrix™. The histological analysis was done by cell counting in hematoxylin and eosin (HE) and nuclei count in immunofluorescence (IF) with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Results: The study of groups in HE and IF showed similar results. For both analyses,IVC-SD-1 × 106 ASC and IVC-SD-PL-1 × 106 ASC provided the best results. The IF technique showed better sensitivity than HE, with a weak agreement between them. Conclusions: Decellularizing agent and the number of ASC influence scaffolds cellularization response and the best protocols as those ones using SD with or without the addition of PL.

In vitro and in vivo genotoxicity and cytotoxicity analysis of protein extract from Aplysina fulva sponges

This study evaluated the physicochemical and morphological properties of a marine sponge protein extract (PE) using scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), analysis of mass loss and pH and in vitro and in vivo. Scanning electron microscopy showed that PE fibers present a granular aspect and irregular structure and the element carbon followed by oxygen was detected in the EDS analysis. Moreover, a 29% of mass loss was observed after 14 days and the pH slightly modified after 14 days. Cell viability of fibroblast cells (L929) of control and PE at a concentration of 25% demonstrated higher values compared to the groups. Osteoblast cell viability of PE at 25 and 50% was significantly higher. Comet assay on day 1 showed higher values for PE at 25%. In addition, in vivo experiments demonstrated that in the treated animals, the bone defects were filled with biomaterial particles, granulation tissue and some areas of newly formed bone. Furthermore, similar immunoexpression of Runx-2 and Cox-2 was observed. Taken together, all results suggest that PE is biocompatible, present non-citotoxicity in the in vitro studies (at the lower concentration) and in the in vivo studies and it can be considered as an alternative source of collagen for tissue engineering proposals.

In vitro and in vivo genotoxicity and cytotoxicity analysis of protein extract from Aplysina fulva sponges

This study evaluated the physicochemical and morphological properties of a marine sponge protein extract (PE) using scanning electron microscopy (SEM), Energy dispersive X-ray spectroscopy (EDS), analysis of mass loss and pH and in vitro and in vivo. Scanning electron microscopy showed that PE fibers present a granular aspect and irregular structure and the element carbon followed by oxygen was detected in the EDS analysis. Moreover, a 29% of mass loss was observed after 14 days and the pH slightly modified after 14 days. Cell viability of fibroblast cells (L929) of control and PE at a concentration of 25% demonstrated higher values compared to the groups. Osteoblast cell viability of PE at 25 and 50% was significantly higher. Comet assay on day 1 showed higher values for PE at 25%. In addition, in vivo experiments demonstrated that in the treated animals, the bone defects were filled with biomaterial particles, granulation tissue and some areas of newly formed bone. Furthermore, similar immunoexpression of Runx-2 and Cox-2 was observed. Taken together, all results suggest that PE is biocompatible, present non-citotoxicity in the in vitro studies (at the lower concentration) and in the in vivo studies and it can be considered as an alternative source of collagen for tissue engineering proposals.(AU)

Histological analysis of elastic cartilages treated with alkaline solution / Análise histológica de cartilagens elásticas tratadas com solução alcalina

The elastic cartilage is composed by chondroblasts and chondrocytes, extracellular matrix and surrounded by perichondrium. It has a low regeneration capacity and is a challenge in surgical repair. One of obstacles in engineering a structurally sound and long-lasting tissue is selecting the most appropriate scaffold material. One of the techniques for obtaining biomaterials from animal tissues is the decellularization that decreases antigenicity. In this work, alkaline solution was used in bovine ear elastic cartilages to evaluate the decellularization and the architecture of the extracellular matrix. The cartilages were treated in alkaline solution (pH13) for 72 hours and lyophilized to be compared with untreated cartilages by histological analysis (hematoxylin-eosin, Masson's trichrome and Verhoeff slides). Areas of interest for cell counting and elastic fiber quantification were delineated, and the distribution of collagen and elastic fibers and the presence of non-fibrous proteins were observed. The results demonstrated that the alkaline solution caused 90% decellularization in the middle and 13% in the peripheral region, and maintenance of the histological characteristics of the collagen and elastic fibers and non-fibrous protein removal. It was concluded that the alkaline solution was efficient in the decellularization and removal of non-fibrous proteins from the elastic cartilages of the bovine ear.(AU)

A cartilagem elástica é composta por condroblastos e condrócitos, matriz extracelular e envolta por pericôndrio. Possui uma baixa capacidade de regeneração e é um desafio em reparos cirúrgicos. Um dos obstáculos na engenharia de tecido estruturalmente sólido e de longa duração é a seleção do material de arcabouço mais adequado. Uma das técnicas para obtenção de biomateriais oriundos de tecidos animais é a descelularização, que diminui a antigenicidade. Neste trabalho, foi utilizada solução alcalina em cartilagem elástica auricular bovina para avaliar a descelularização e a arquitetura da matriz extracelular. As cartilagens foram tratadas em solução alcalina (pH13) durante 72 horas e liofilizadas, e comparadas com cartilagens não tratadas por análise histológica (hematoxilina-eosina, tricrômio de Masson e Verhoeff). Foram determinadas as áreas de interesse para contagem celular e quantificação de fibras elásticas, observada a distribuição de colágeno e fibras elásticas e a presença de proteínas não fibrosas. Os resultados demonstraram que a solução alcalina causou 90% de descelularização na região central e 13% na região periférica, manutenção das características histológicas do colágeno e fibras elásticas e remoção das proteínas não fibrosas. Concluiu-se que a solução alcalina foi eficiente na descelularização e retirada de proteínas não fibrosas de cartilagens elásticas da orelha de bovinos.(AU)

Histological analysis of elastic cartilages treated with alkaline solution / Análise histológica de cartilagens elásticas tratadas com solução alcalina

The elastic cartilage is composed by chondroblasts and chondrocytes, extracellular matrix and surrounded by perichondrium. It has a low regeneration capacity and is a challenge in surgical repair. One of obstacles in engineering a structurally sound and long-lasting tissue is selecting the most appropriate scaffold material. One of the techniques for obtaining biomaterials from animal tissues is the decellularization that decreases antigenicity. In this work, alkaline solution was used in bovine ear elastic cartilages to evaluate the decellularization and the architecture of the extracellular matrix. The cartilages were treated in alkaline solution (pH13) for 72 hours and lyophilized to be compared with untreated cartilages by histological analysis (hematoxylin-eosin, Masson's trichrome and Verhoeff slides). Areas of interest for cell counting and elastic fiber quantification were delineated, and the distribution of collagen and elastic fibers and the presence of non-fibrous proteins were observed. The results demonstrated that the alkaline solution caused 90% decellularization in the middle and 13% in the peripheral region, and maintenance of the histological characteristics of the collagen and elastic fibers and non-fibrous protein removal. It was concluded that the alkaline solution was efficient in the decellularization and removal of non-fibrous proteins from the elastic cartilages of the bovine ear.(AU)

A cartilagem elástica é composta por condroblastos e condrócitos, matriz extracelular e envolta por pericôndrio. Possui uma baixa capacidade de regeneração e é um desafio em reparos cirúrgicos. Um dos obstáculos na engenharia de tecido estruturalmente sólido e de longa duração é a seleção do material de arcabouço mais adequado. Uma das técnicas para obtenção de biomateriais oriundos de tecidos animais é a descelularização, que diminui a antigenicidade. Neste trabalho, foi utilizada solução alcalina em cartilagem elástica auricular bovina para avaliar a descelularização e a arquitetura da matriz extracelular. As cartilagens foram tratadas em solução alcalina (pH13) durante 72 horas e liofilizadas, e comparadas com cartilagens não tratadas por análise histológica (hematoxilina-eosina, tricrômio de Masson e Verhoeff). Foram determinadas as áreas de interesse para contagem celular e quantificação de fibras elásticas, observada a distribuição de colágeno e fibras elásticas e a presença de proteínas não fibrosas. Os resultados demonstraram que a solução alcalina causou 90% de descelularização na região central e 13% na região periférica, manutenção das características histológicas do colágeno e fibras elásticas e remoção das proteínas não fibrosas. Concluiu-se que a solução alcalina foi eficiente na descelularização e retirada de proteínas não fibrosas de cartilagens elásticas da orelha de bovinos.(AU)

Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering / Associação de arcabouço de polipropileno e células tronco mesenquimais para uso em engenharia de tecido

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

A engenharia de tecidos substitui tecidos danificados com a manipulação de células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. As células-tronco mesenquimais (MSCs) são boas candidatas para engenharia de tecido, pois são um dos tipos celulares recrutadas para a reparação de tecidos lesionados. O arcabouço deve ser um dispositivo estrutural que forneça uma estrutura para o crescimento e a diferenciação celular no sítio, sendo a tela de polipropileno um exemplo. O objetivo deste estudo foi avaliar o cultivo de células-tronco mesenquimais de tecido de adiposo (ADSCs), isoladas de camundongos C57Bl/6 GFP+, em dois tipos de telas de polipropileno (macroporosa e microporosa) em placas de cultura convencionais e revestidas com metacrilato, durante quinze dias, para obter o melhor protocolo de interação entre a tela e as células. A escolha do melhor método foi baseada na adesão, manutenção da adesão e viabilidade durante cultivo. A quantidade de ADSCs aderidas foi verificada diariamente em contagem em Câmara de Neubauer e através de uma curva de crescimento realizada através de ensaio de MTT. As ADSCs aderidas nas telas foram visualizadas com a marcação de DAPI, panótico, hematoxilina e eosina, imumo-histoquímica (integrina) e imunofluorescência (actina). Nas duas formas de cultivo e nos dois tipos de telas de polipropileno houve aderência das ADSCs. Houve maior aderência na tela microporosa, no período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno oferece um bom arcabouço para as ADSCs se aderirem.

Associação de arcabouço de polipropileno e células tronco mesenqimais para uso em engenharia de tecido / Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering

A engenharia de tecidos tem como objetivo substituir tecidos danificados, manipulando células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. A proposta deste estudo foi avaliar duas técnicas de cultivo de células-tronco mesenquimais (MSC) em diferentes placas de cultura, utilizando dois tipos de telas de polipropileno (macroporoso e microporoso), para obter as melhores condições de interação entre a tela e as células, e definir uma proposta de protético para engenharia de tecidos. As telas de polipropileno foram cultivadas com células-tronco mesenquimais de tecido adiposo (ADSCs) isoladas de camundongos C57B1/6 GFP+ durante quinze dias em placas revestidas com metacrilato ou não revestidas com metacrilato. A quantidade de ADSCs aderidas foram verificadas diariamente em Câmara de Neubauer e através de uma curva de crescimento realizada pelo ensaio MTT. As ADSCs aderidas às malhas foram visualizadas com marcação de DAPI, panóticas, hematoxilina e eosina imuno-histoquímica e imunofluorescência. O melhor protocolo foi na tela microporosa, no o período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno fornece um bom suporte para as ADSCs se aderirem podendo ser utilizada na engenharia de tecidos.(AU)

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.(AU)

Associação de arcabouço de polipropileno e células tronco mesenqimais para uso em engenharia de tecido / Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering

A engenharia de tecidos tem como objetivo substituir tecidos danificados, manipulando células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. A proposta deste estudo foi avaliar duas técnicas de cultivo de células-tronco mesenquimais (MSC) em diferentes placas de cultura, utilizando dois tipos de telas de polipropileno (macroporoso e microporoso), para obter as melhores condições de interação entre a tela e as células, e definir uma proposta de protético para engenharia de tecidos. As telas de polipropileno foram cultivadas com células-tronco mesenquimais de tecido adiposo (ADSCs) isoladas de camundongos C57B1/6 GFP+ durante quinze dias em placas revestidas com metacrilato ou não revestidas com metacrilato. A quantidade de ADSCs aderidas foram verificadas diariamente em Câmara de Neubauer e através de uma curva de crescimento realizada pelo ensaio MTT. As ADSCs aderidas às malhas foram visualizadas com marcação de DAPI, panóticas, hematoxilina e eosina imuno-histoquímica e imunofluorescência. O melhor protocolo foi na tela microporosa, no o período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno fornece um bom suporte para as ADSCs se aderirem podendo ser utilizada na engenharia de tecidos.

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

Reconstruction of parietal bone defects with adiposederived mesenchymal stem cells: experimental study

Purpose: This study assessed the regeneration potential of mesenchymal stem cells (MSC) from adipose tissue associated with platelet-rich plasma (PRP) in bone regeneration. Methods: Thirty Wistar rats (Rattus norvegicus albinos) were divided into five groups (according to the grafting material and time to euthanasia): (1) autograft - 14 days (control), (2) autograft - 28 days (control), (3) MSC + PRP - 14 days, (4) MSC + PRP + papaverine - 14 days and (5) MSC + PRP + papaverine - 28 days. After euthanasia, the graft was removed and histological slides were prepared. They were assessed by a blinded pathologist using a previously published histological scale as parameter. Results: There was some degree of neoformed bone trabeculae (NBT) in 93.3% of the samples, as well as osteoblastic activity (OA). The autograft groups (14 and 28 days) had higher levels in the formation of bone trabeculae. Nonparametric data were analyzed using the Wilcoxon-Mann-Whitney test and proved not to be statistically significant at p 0.05. Conclusions: Experimental parietal bone reconstruction, combining MSC, PRP and papaverine presented regeneration in all groups with no significant difference among them.(AU)

Biocompatibilidade do gel de quitosana associado ao glicerol fosfato na reparação de defeitos ósseos induzidos experimentalmente no rádio de coelhos (Oryctolagus cuniculus) / [Biocompatibility of chitosan gel associated with glycerol phosphate for the healing of experimentally induced radial defects in rabbits (Oryctolagus cuniculus)]

A engenharia de tecidos caracteriza-se como ciência interdisciplinar, a qual vem desenvolvendo biomateriais para a regeneração do tecido ósseo no âmbito das medicinas humana e veterinária. O objetivo desta pesquisa foi avaliar a regeneração óssea obtida da aplicação do hidrogel de quitosana associado ao glicerol fosfato em falha óssea experimentalmente induzida no rádio de coelhos. Foram utilizados 15 coelhos adultos, distribuídos aleatoriamente em dois grupos, representados por cada um dos rádios de cada animal, sendo um grupo tratado com hidrogel de quitosana associado ao glicerol fosfato (grupo biomaterial - GB) e um grupo que não recebeu tratamento com o biomaterial (grupo controle - GC). Os animais foram avaliados radiograficamente, por densitometria óptica e análise histológica, nos períodos 30, 60 e 90 dias pós-operatórios. Houve superioridade estatística na média geral das avaliações radiográficas do GB (2,33±0,48) sobre o GC (1,77±0,06). As médias gerais de avaliação densitométrica do GB foram superiores às do GC, sendo 6,207±1,374 e 5,71±1,512, respectivamente. A avaliação histopatológica do GB foi superior à do GC nos períodos de 30, 60 e 90 dias. Assim, é possível afirmar que o hidrogel de quitosana constitui biomaterial de características desejáveis, promovendo consolidação óssea mais rápida e eficiente, sem causar reações adversas.(AU)

Tissue engineering is an interdisciplinary science that has been developing biomaterials for bone regeneration in medicine and veterinary medicine, following an imminent need. The aim of this study was to evaluate bone regeneration after use of chitosan hydrogel associated with glycerol phosphate in experimentally induced bone gap in the radius of rabbits. Fifteen adult rabbits were randomly distributed in two experimental groups, represented by each radius of every single animal. The animals in the Biomaterial Group (GB) were treated with a glycerol phosphate-associated chitosan hydrogel and in the Control Group (GC) they received no treatment with the biomaterial. The animals were evaluated clinically, radiographically, histologically and by optic densitometry at 30, 60 and 90 days postoperatively. There was statistical superiority in the general average of the radiographic estimates of GB (2.33 ± 0.48) over the CG (1.77 ± 0.06). The general averages of GB densitometric evaluation were higher than the CG, being 6.207 ± 1.374 and 5.71 ± 1.512, respectively. Histopathological evaluation of GB was superior to CG in periods of 30, 60 and 90 days. Chitosan hydrogel constitutes a biomaterial of desired characteristics, promoting faster and more efficient bone repair when compared to GC.(AU)

Bioengineering the ovary to preserve and reestablish female fertility

Different bioengineering strategies can be presently adopted and have been shown to have great potential in the treatment of female infertility and ovarian dysfunction deriving from chemotherapy, congenital malformations, massive adhesions as well as aging and lifestyle. One option is transplantation of fresh or cryopreserved organs/fragments into the patient. A further possibility uses tissue engineering approaches that involve a combination of cells, biomaterials and factors that stimulate local ability to regenerate/ repair the reproductive organ. Organ transplant has shown promising results in large animal models. However, the source of the organ needs to be identified and the immunogenic effects of allografts remain still to be solved before the technology may enter the clinical practice. Decellularization/ repopulation of ovary with autologous cells or follicles could represent an interesting, still very experimental alternative. Here we summarize the recent advancements in the bioengineering strategies applied to the ovary, we present the principles for these systems and discuss the advantages of these emerging opportunities to preserve or improve female fertility.

Bioengineering the ovary to preserve and reestablish female fertility

Different bioengineering strategies can be presently adopted and have been shown to have great potential in the treatment of female infertility and ovarian dysfunction deriving from chemotherapy, congenital malformations, massive adhesions as well as aging and lifestyle. One option is transplantation of fresh or cryopreserved organs/fragments into the patient. A further possibility uses tissue engineering approaches that involve a combination of cells, biomaterials and factors that stimulate local ability to regenerate/ repair the reproductive organ. Organ transplant has shown promising results in large animal models. However, the source of the organ needs to be identified and the immunogenic effects of allografts remain still to be solved before the technology may enter the clinical practice. Decellularization/ repopulation of ovary with autologous cells or follicles could represent an interesting, still very experimental alternative. Here we summarize the recent advancements in the bioengineering strategies applied to the ovary, we present the principles for these systems and discuss the advantages of these emerging opportunities to preserve or improve female fertility.(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)

Adding a dimension to cell fate

Cell fate specification, gene expression and spatial restriction are process finely tuned by epigenetic regulatory mechanisms. At the same time, mechanical forces have been shown to be crucial to drive cell plasticity and boost differentiation. Indeed, several studies have demonstrated that transitions along different specification states are strongly influenced by 3D rearrangement and mechanical properties of the surrounding microenvironment, that can modulate both cell potency and differentiation, through the activation of specific mechanosensing-related pathways. An overview of small molecule ability to modulate cell plasticity and define cell fate is here presented and results, showing the possibility to erase the epigenetic signature of adult dermal fibroblasts and convert them into insulin-producing cells (EpiCC) are described. The beneficial effects exerted on such processes, when cells are homed on an adequate substrate, that shows “in vivo” tissue-like stiffness are also discussed and the contribution of the Hippo signalling mechanotransduction pathway as one of the mechanisms involved is examined. In addition, results obtained using a genetically modified fibroblast cell line, expressing the enhanced green fluorescent protein (eGFP) under the control of the porcine insulin gene (INS) promoter (INS-eGFP transgenic pigs), are reported. This model offers the advantage to monitor the progression of cell conversion in real time mode. All these observations have a main role in order to allow a swift scale-up culture procedure, essential for cell therapy and tissue engineering applied to human regenerative medicine, and fundamental to ensure an efficient translation process from the results obtained at the laboratory bench to the patient bedside. Moreover, the creation of reliable in vitro model represents a key point to ensure the development of more physiological models that, in turn, may reduce the number of animals used, implementing non-invasive investigations and animal welfare and protection.