A modified hydrogel production protocol to decrease cellular content

Purpose: To analyze the cytotoxicity and cell in porcine-derived decellularized skin matrix. Methods: We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation. Results: After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days. Conclusions: The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.

Tissue healing changes on wounds in rats after treatment with Hancornia speciosa latex in cream-gel formulation

Purpose: Hancornia speciosa latex has shown pharmacological potential in wound healing processes due to its angiogenic, osteogenic, and anti-inflammatory activities. The aims of this study were to carry out a cream-gel formulation with 5, 10 and 25% of H. speciosa serum latex and to evaluate its potential to stimulate the skin regeneration in rats' wounds. Methods: One hundred and twenty rats were divided into five groups: neutral control with saline (G1), cream-gel based on H. speciosa latex serum at 5% m/v (G2), cream-gel at 15% m/v (G3), cream-gel at 25% m/v (G4), and cream-gel (G5). The animals were euthanized at three, seven, 14 and 21 days after the injury induction, and some parameters were analyzed: wound contraction, necrosis, fibrin, polymorphonuclear and mononuclear infiltrates, fibroblast, angiogenesis, hemorrhage, and collagen. Results:The therapeutic treatment with cream-gel at 15 and 25% is beneficial in the inflammatory phase of healing processes since it increased the angiogenesis and proliferation of mononuclear infiltrations in wounds. Regarding wound contraction, the treatment with cream-gel (5 and 15%) induced a higher rate of contraction in the proliferative phase. The 15% cream-gel formulation stimulated a greater production of collagen in the injured tissues. Conclusions: H. speciosa cream-gel is a low-cost herbal medicine which can aid in tissue repair.

A pilot study on the use of 3D printers in veterinary medicine

Manufatura aditiva (AM) é o processo de juntar materiais para criar objetos tridimensionais camada por camada usando uma impressora 3D a partir de um modelo digital. A grande vantagem da Manufatura Aditiva é permitir um design mais livre do que os processos tradicionais. O desenvolvimento de processos de manufatura aditiva tem permitido otimizar a produção do produto customizado através da modelagem da geometria e do conhecimento dos parâmetros morfométricos das estruturas corporais. A impressão 3D revolucionou o campo da Medicina Regenerativa porque, partindo das imagens de tomografia computadorizada (TC) e utilizando materiais tradicionais como plásticos e metais, pode fornecer próteses customizadas para cada paciente, que se adaptam perfeitamente às necessidades do sujeito e atuam como suporte de estruturas. A impressão 3D permite imprimir andaimes porosos tridimensionais com uma forma precisa e composição química adequada para uso médico e veterinário. Alguns desses andaimes são biodegradáveis ​​e parecem ser ideais para engenharia de tecido ósseo. Na verdade, eles são capazes de simular propriedades da matriz extracelular que permitem suporte mecânico, favorecendo as interações mecânicas e fornecendo um modelo para fixação celular e estimulação in vivo da formação do tecido ósseo.

A pilot study on the use of 3D printers in veterinary medicine

Manufatura aditiva (AM) é o processo de juntar materiais para criar objetos tridimensionais camada por camada usando uma impressora 3D a partir de um modelo digital. A grande vantagem da Manufatura Aditiva é permitir um design mais livre do que os processos tradicionais. O desenvolvimento de processos de manufatura aditiva tem permitido otimizar a produção do produto customizado através da modelagem da geometria e do conhecimento dos parâmetros morfométricos das estruturas corporais. A impressão 3D revolucionou o campo da Medicina Regenerativa porque, partindo das imagens de tomografia computadorizada (TC) e utilizando materiais tradicionais como plásticos e metais, pode fornecer próteses customizadas para cada paciente, que se adaptam perfeitamente às necessidades do sujeito e atuam como suporte de estruturas. A impressão 3D permite imprimir andaimes porosos tridimensionais com uma forma precisa e composição química adequada para uso médico e veterinário. Alguns desses andaimes são biodegradáveis ​​e parecem ser ideais para engenharia de tecido ósseo. Na verdade, eles são capazes de simular propriedades da matriz extracelular que permitem suporte mecânico, favorecendo as interações mecânicas e fornecendo um modelo para fixação celular e estimulação in vivo da formação do tecido ósseo.(AU)

Potential application of aminiotic stem cells in veterinary medicine

In regenerative medicine stem cell biology has become one of the most interesting and more often studied subject. The amniotic membrane is the innermost layer of the fetal membranes and is considered a potential tool to treat many pathologies. It is used because it can be collected from discarded fetal material and is a rich source of stem cells with high proliferation and plasticity ratio capable of proliferating and differentiate in vitro. We propose to elucidate the characteristics and potencial clinical application of cells derived of amniotic membrane in veterinary medicine.

Potential application of aminiotic stem cells in veterinary medicine

In regenerative medicine stem cell biology has become one of the most interesting and more often studied subject. The amniotic membrane is the innermost layer of the fetal membranes and is considered a potential tool to treat many pathologies. It is used because it can be collected from discarded fetal material and is a rich source of stem cells with high proliferation and plasticity ratio capable of proliferating and differentiate in vitro. We propose to elucidate the characteristics and potencial clinical application of cells derived of amniotic membrane in veterinary medicine.(AU)

Immunophenotyping, plasticity tests and nanotagging of stem cells derived from adipose tissue of wild rodent agouti (Dasyprocta prymnolopha) / Imunofenotipagem, testes de plasticidade e marcação com nanocristais de céluas-tronco derivadas de tecido adiposo em cutias

There is a growing interest in the study of unspecialized mesenchymal stem cells, for there are still some discussions about their in vitro behavior. Regenerative medicine is a science undergoing improvement which develops treatments as cell therapy using somatic stem cells. In several studies, adipose tissue is presented as a source of multipotent adult cells that has several advantages over other tissue sources. This study aimed to characterize and evaluate the tagging of mesenchymal stem cells from the agoutis adipose tissue (Dasyprocta prymonolopha), with fluorescent intracytoplasmic nanocrystals. Fibroblast cells were observed, plastic adherent, with extended self-renewal, ability to form colonies, multipotency by differentiation into three lineages, population CD90 + and CD45 - expression, which issued high red fluorescence after the tagging with fluorescent nanocrystals by different paths and cryopreserved for future use. It is possible to conclude that mesenchymal stem cells from agouti adipose tissue have biological characteristics and in vitro behavior that demonstrate its potential for use in clinical tests.(AU)

Há um interesse crescente no estudo das células estaminais mesenquimais, não especializadas, pois ainda existem algumas discussões sobre seu comportamento in vitro. A medicina regenerativa é uma ciência em fase de crescimento que desenvolve tratamentos como terapia celular utilizando células estaminais somáticas. Em vários estudos, o tecido adiposo é apresentado como uma fonte de células adultas multipotentes que tem várias vantagens em relação a outras fontes de tecido. Este estudo teve como objetivo caracterizar e avaliar a marcação de células estaminais mesenquimais do tecido adiposo de cutias (Dasyprocta prymnolopha) com nanocristais intracitoplasmáticos fluorescentes. Observaram-se células fibroblásticas, aderentes ao plástico, com autorrenovação prolongada, capacidade de formar colônias, diferenciação em três linhagens, população CD90 + e expressão CD45, que emitiram alta fluorescência vermelha após a marcação com nanocristais fluorescentes por diferentes vias, e criopreservadas para uso futuro. É possível concluir que as células estaminais mesenquimais do tecido adiposo de cutias têm características biológicas e comportamentos in vitro que demonstram seu potencial para uso em testes clínicos.(AU)

Immunophenotyping, plasticity tests and nanotagging of stem cells derived from adipose tissue of wild rodent agouti (Dasyprocta prymnolopha) / Imunofenotipagem, testes de plasticidade e marcação com nanocristais de céluas-tronco derivadas de tecido adiposo em cutias

There is a growing interest in the study of unspecialized mesenchymal stem cells, for there are still some discussions about their in vitro behavior. Regenerative medicine is a science undergoing improvement which develops treatments as cell therapy using somatic stem cells. In several studies, adipose tissue is presented as a source of multipotent adult cells that has several advantages over other tissue sources. This study aimed to characterize and evaluate the tagging of mesenchymal stem cells from the agoutis adipose tissue (Dasyprocta prymonolopha), with fluorescent intracytoplasmic nanocrystals. Fibroblast cells were observed, plastic adherent, with extended self-renewal, ability to form colonies, multipotency by differentiation into three lineages, population CD90 + and CD45 - expression, which issued high red fluorescence after the tagging with fluorescent nanocrystals by different paths and cryopreserved for future use. It is possible to conclude that mesenchymal stem cells from agouti adipose tissue have biological characteristics and in vitro behavior that demonstrate its potential for use in clinical tests.(AU)

Há um interesse crescente no estudo das células estaminais mesenquimais, não especializadas, pois ainda existem algumas discussões sobre seu comportamento in vitro. A medicina regenerativa é uma ciência em fase de crescimento que desenvolve tratamentos como terapia celular utilizando células estaminais somáticas. Em vários estudos, o tecido adiposo é apresentado como uma fonte de células adultas multipotentes que tem várias vantagens em relação a outras fontes de tecido. Este estudo teve como objetivo caracterizar e avaliar a marcação de células estaminais mesenquimais do tecido adiposo de cutias (Dasyprocta prymnolopha) com nanocristais intracitoplasmáticos fluorescentes. Observaram-se células fibroblásticas, aderentes ao plástico, com autorrenovação prolongada, capacidade de formar colônias, diferenciação em três linhagens, população CD90 + e expressão CD45, que emitiram alta fluorescência vermelha após a marcação com nanocristais fluorescentes por diferentes vias, e criopreservadas para uso futuro. É possível concluir que as células estaminais mesenquimais do tecido adiposo de cutias têm características biológicas e comportamentos in vitro que demonstram seu potencial para uso em testes clínicos.(AU)

Associação, de laserterapia de baixa potência e celulas-tronco mesenquimais / Association of low-level laser therapy and mesenchymal stem cells / Asociación de la terapia com láser de baja intensidad y células madre mesenquimales

A laserterapia de baixa potência e as células-tronco mesenquimais constituem estratégias promissoras em medicina regenerativa. Entretanto, os efeitos da interação dessas terapias ainda são pouco discutidos. Assim, o objetivo desse trabalho foi verificar, com base em dados da literatura, os possíveis efeitos favoráveis ou adversos da associação do laser de baixa potência e células-tronco mesenquimais. É descrito que a laserterapia leva ao aumento da viabilidade e proliferação celular e induz a diferenciação das células-tronco em condições específicas in vitro e in vivo. Ainda, não causa danos nucleares, não havendo efeitos adversos relatados. Dessa forma, a associação da laserterapia de baixa potência e células-tronco mesenquimais é segura e aumenta a qualidade da regeneração tecidual.(AU)

Low-Ievel laser therapy and mesenchymal stem cells are promising strategies in regenerative medicine. However, the effects of the interaction of these therapies are still little discussed. The objective of this study was to determine, based on literature data, possible favorable or adverse effects of the combination of low-Ievel laser and mesenchymal stem cells. It is reported that the lasertherapy leads to increased cell viability and proliferation and induces the differentiation of stem cells under specific conditions in vitro and in vivo. Although does not cause nuclear damage, with no adverse effects reported. Thus, the association of low-Ievel laser therapy and mesenchymal stem cells is safe and increases the quality of tissue regeneration.(AU)

La terapia con láser de baja intensidad y las células madre mesenquimales son prometedoras estrategias en medicina regenerativa. Sin embargo, son aún poco discuten los efectos de la interacción de estas terapias. El objetivo de este estudio fue determinar, con base en datos de la literatura, los posibles efectos favorables o desfavorables de la combinación de láser de baja potencia y las células madre mesenquimales. Se ha informado de que la láserterapia conduce a aumento de la viabilidad y proliferación celular e induce la diferenciación de las células madre en condiciones específicas in vitro y in vivo. Aunque no causa dano nuclear, con no efectos adversos reportados. Por tanto, la asociación de la terapia láser de baja intensidad y las células madre mesenquimales es seguro y aumenta la calidad de la regeneración de tejidos.(AU)

Associação, de laserterapia de baixa potência e celulas-tronco mesenquimais / Association of low-level laser therapy and mesenchymal stem cells / Asociación de la terapia com láser de baja intensidad y células madre mesenquimales

A laserterapia de baixa potência e as células-tronco mesenquimais constituem estratégias promissoras em medicina regenerativa. Entretanto, os efeitos da interação dessas terapias ainda são pouco discutidos. Assim, o objetivo desse trabalho foi verificar, com base em dados da literatura, os possíveis efeitos favoráveis ou adversos da associação do laser de baixa potência e células-tronco mesenquimais. É descrito que a laserterapia leva ao aumento da viabilidade e proliferação celular e induz a diferenciação das células-tronco em condições específicas in vitro e in vivo. Ainda, não causa danos nucleares, não havendo efeitos adversos relatados. Dessa forma, a associação da laserterapia de baixa potência e células-tronco mesenquimais é segura e aumenta a qualidade da regeneração tecidual.

Low-Ievel laser therapy and mesenchymal stem cells are promising strategies in regenerative medicine. However, the effects of the interaction of these therapies are still little discussed. The objective of this study was to determine, based on literature data, possible favorable or adverse effects of the combination of low-Ievel laser and mesenchymal stem cells. It is reported that the lasertherapy leads to increased cell viability and proliferation and induces the differentiation of stem cells under specific conditions in vitro and in vivo. Although does not cause nuclear damage, with no adverse effects reported. Thus, the association of low-Ievel laser therapy and mesenchymal stem cells is safe and increases the quality of tissue regeneration.

La terapia con láser de baja intensidad y las células madre mesenquimales son prometedoras estrategias en medicina regenerativa. Sin embargo, son aún poco discuten los efectos de la interacción de estas terapias. El objetivo de este estudio fue determinar, con base en datos de la literatura, los posibles efectos favorables o desfavorables de la combinación de láser de baja potencia y las células madre mesenquimales. Se ha informado de que la láserterapia conduce a aumento de la viabilidad y proliferación celular e induce la diferenciación de las células madre en condiciones específicas in vitro y in vivo. Aunque no causa dano nuclear, con no efectos adversos reportados. Por tanto, la asociación de la terapia láser de baja intensidad y las células madre mesenquimales es seguro y aumenta la calidad de la regeneración de tejidos.

Fetal membranes stem cells application in pets

Background: Stem cells are precursor cells that have the capacity for self-renewal and could generate cells with characteristics similar to cells and differentiation, generating varied cell lines. Considering the plasticity of cells can be classified into totipotent, pluripotent or multipotent. According to the isolation period, the stem cells can be classified as embryonic, fetal and adult. In the embryo stage are considered totipotent because they can rebuild any tissue in the body and adulthood are considered multipotent, since they have a more limited plasticity. The fetal tissues and the fetus is a potential source for stem cells, since they expand more rapidly compared to the cells after birth. Stem cells of fetal membranes are derived from extraembryonic tissues with high capacity to differentiate into various tissues. The cord blood stem cells have mesenchymal and hematopoietic, and mesenchymal cells have the potential to proliferate and differentiate into multiple cell lineages. The yolk sac in dogs is morphologically composed of three layers: a single layer of endoderm, a simple mesothelium, and intermediate to them, the vascular mesenchyme. Work identified a population of pluripotent cells in the yolk sac can differentiate into hematopoietic cells, however, can be isolated mesenchymal stem cells. In this review we aim to focus new isolations of cells from umbilical cord blood and yolk sac of dogs, reviewing the main literature on this species. The importance of using dogs out of work has intensified in recent years, since many diseases can manifest itself in a similar way to humans. Additionally, the dog is a pet, and interest in the treatment of diseases and improved quality of life of this species has been accentuated in veterinary medicine. Thus, identifying the cellular sources in the dog opens new horizons for preclinical studies and new therapies for veterinary medicine. Review: This study is related to morphological biology multipotent stem cells, focusing its expansion and use in cell therapy in animal models that have different pathologies. A widely studied model for muscular dystrophy is the GRMD (Golden Retriever Muscular Dystrophy), which is homologous to DMD (Duchenne Muscular Dystrophy) that affects humans. It is a recessive genetic disease, X chromosome which affects approximately 1 in every 3500 boys. It is characterized by a progressive muscle degeneration, resulting from the absence or reduction in the production of dystrophin protein present in the sarcoplasmic membrane of muscle fibers. Conclusion: The use of cells derived from fetal tissue are strong candidates for veterinary regenerative medicine, since they have high capacity for cellular differentiation. The use of fetuses and fetal tissues of humans still has limitations, so the dog is a viable alternative for studies of fetal stem cells. Thus, it is extremely important to know the characteristics of morphology and proliferation of cells derived from fetuses and fetal annexes canines, including yolk sac and umbilical cord as well as know the feasibility of clinical application of these cells in preclinical testing in animal models and eventually in human medicine, thus contributing to regenerative medicine.

Derivation and potential applications of pluripotent stem cells for regenerative medicine in horses

Background: The ability to create tissues using pluripotent stem cells to repair or replace tissue lost due to damage, i.e. regenerative medicine, is developing very rapidly in many fields of human medicine. For veterinarians, regenerative medicine has focused mainly in the use of stem cells for arthritis and tendon ligament repair, indicating a need for treating musculoskeletal injuries. Our objective is to review the available approaches being used to derive pluripotent stem cells and discuss their potential use for regenerative medicine in the horse. Review: Adult adipose- and bone marrow-derived mesenchymal stem cells (MSC) are being used in practice to treat injuries in horses. However, there is scarce scientific evidence of their effectiveness and little is known of the mechanisms by which such cell preparations improve the healing process. For instance, although early healing response of articular cartilage injury was improved by treatment with injection of MSC, they did not enhance the long-term tissue response, indicating that cell proliferation was attenuated. Better protocols for the isolation and clinical testing of equine MSC are required to confirm healing properties. In contrast to MSC, embryonic stem cells (ESC) derived from the inner-cell- mass (ICM) of blastocyst stage embryos carry the ability to proliferate indefinitely in vitro and, given appropriate and favorable conditions, can differentiate into any tissue in the body. Parthenogenesis (PG) and somatic cell nuclear transfer (SCNT) are used to obtain a genetic match to the host animal and, thereby, eliminate the risk of inducing immune rejection of the grafted tissue. However, apart from the typical markers of pluripotency, equine ESC also express markers of trophoblastic tissues, indicating that they are different and possibly less able to differentiate than the ESC lines obtained in other species. Consequently, further studies are underway to identify conditions to obtain fully pluripotent ESC lines from equine SCNT embryos. To overcome the limitations of ESC lines derived from equine embryos, induced pluripotent stem cells (iPSC) were derived using a piggyBac transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally controlled fashion. Our established fetal-derived iPSC lines express hallmark pluripotency markers, display a stable karyotype after prolonged culture, and are able to form teratomas in immunodeficient mice containing tissues from all three embryonic layers. By establishing a protocol for deriving stable iPSC lines in the horse, we expect that new opportunities will be shortly developed for regenerative therapies in this species. Conclusion: It is possible to derive autologous pluripotent stem cells in horses by using both ESC and iPSC-derived approaches. Although ESC lines are generally the gold standard of pluripotency, further research is required to improve the proliferative and pluripotency characteristics for clinical applications. On the other hand, equine iPSC show excellent stability during prolonged in vitro culture and have the capacity to differentiate into the three germ layers in vivo, suggesting that they could soon be used in pre-clinical trials. Therefore, further studies need to be performed to establish reliable protocols for assessing the regenerative properties of iPS and ESC for equine muscle-skeletal injuries.

Cell detachment rates and confluence of fibroblast and osteoblast cell culture using different washing solutions / Taxas de desprendimento celular e confluência de cultura celular de fibroblastos e osteoblastos a partir de diferentes soluções de lavagem

The advancements in the cell culture studies have led to the development of regenerative medicine concept. The aim of this study is to compare the effectiveness of some washing solutions, including phosphate buffered saline (PBS), sodium chloride (NaCl), and ringer's lactate (RL) on the rate of detachment and confluency in fibroblast and osteoblast cell culture. Baby Hamster Kidney 21 clone 13 (BHK21/C13) fibroblast cells and 7F2 osteoblast were cultured on T25 flasks for 3-4 days. Three treatment groups were classified on the basis of different washing solutions used in the moment before trypsinization: PBS, 0.9% NaCl, and RL. Each group was measured for the detachment rate and cell confluence. The measurement was done in 2 passage numbers. The use of PBS, NaCl, and RL washing solution showed that detachment time was less than 5 minutes for the fibroblasts and 3 minutes for the osteoblasts. There was a significant difference in the rate of fibroblast cell detachment (p=0.006) and osteoblast (p=0.016). The capability of fibroblasts and osteoblasts to achieve a confluence of 106 cells/well on the first and second measurements was almost the same between the washing solution groups. The use of physiological 0.9% NaCl solution as a washing solution in fibroblast and osteoblast cell culture has almost the same effectiveness as PBS to help accelerate cell detachment in less than 5 minutes without influencing the capability of cells to proliferate.

Os avanços nos estudos de cultura de células levaram ao desenvolvimento do conceito de medicina regenerativa. O objetivo deste estudo é comparar a eficácia de algumas soluções de lavagem, incluindo solução salina tamponada com fosfato (PBS), cloreto de sódio (NaCl) e lactato de ringer (RL) na taxa de desprendimento e confluência em cultura de células de fibroblastos e osteoblastos. Células de fibroblastos Baby Hamster Kidney 21 clone 13 (BHK21/C13) e osteoblastos 7F2 foram cultivadas em frascos T25 por 3-4 dias. Três grupos de tratamento foram classificados com base nas diferentes soluções de lavagem utilizadas no momento anterior à tripsinização: PBS, NaCl 0,9% e RL. Cada grupo foi medido para a taxa de desprendimento e confluência celular. A medição foi feita em 2 números de passagem. O uso de solução de lavagem PBS, NaCl e RL mostrou que o tempo de descolamento foi inferior a 5 minutos para os fibroblastos e 3 minutos para os osteoblastos. Houve uma diferença significativa na taxa de desprendimento de células fibroblásticas (p=0.006) e osteoblasto (p=0.016). A capacidade de fibroblastos e osteoblastos de atingir uma confluência de 106 células/well na primeira e segunda medições foi quase a mesma entre os grupos de solução de lavagem. O uso de solução fisiológica de NaCl (0.9%) como solução de lavagem em cultura de células de fibroblastos e osteoblastos tem quase a mesma eficácia que o PBS para ajudar a acelerar o desprendimento celular em menos de 5 minutos sem influenciar a capacidade das células de proliferar.