RESUMO
Cellular therapy (CT) can be defined as the transference into a person of healthy cells to correct defective functions. Yesterday (1950-2010), CT consisted mostly of hematopoietic transplants for the treatment of a variety of hematological disorders. Interestingly, during that period of time other cell types with therapeutic potential-including certain lymphoid populations and other nonhematopoietic cells-were discovered and characterized; thus, CT became a promising discipline for the treatment of a broader diversity of diseases. Today (2011-2023), CT has significantly grownup through preclinical studies and clinical trials, and it is currently progressing toward its consolidation as one of the pillars of medicine in the 21st century. Indeed, different types of stem cells (e.g., hematopoietic, mesenchymal, neural, and pluripotent), as well as different lymphoid and myeloid cell populations (e.g., TILs, CAR-Ts, CAR-NKs, and DUOC-01) are being used in clinical settings or are being tested in clinical trials. For the past decade, several CT modalities have been developed, and today, many of them are being used in the clinic. Tomorrow (2024-2040), already established CT modalities will surely be improved and applied more frequently, and novel therapies (that will include cell types such as iPSCs) will enter and expand within the clinical ground. It is noteworthy, however, that despite significant advancements and achievements, problems still need to be solved and obstacles need to be overcome. Technical, ethical, and economic issues persist and they need to be addressed. Undoubtedly, exciting times of challenges and opportunities are coming ahead in the CT arena.
Assuntos
Doenças Hematológicas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/transplanteRESUMO
Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O2â¢-) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O2â¢- formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O2â¢- formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O2â¢- formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O2â¢- formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.
Assuntos
Injúria Renal Aguda , Vesículas Extracelulares , Traumatismo por Reperfusão , Injúria Renal Aguda/metabolismo , Tecido Adiposo/metabolismo , Animais , Vesículas Extracelulares/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Mitocôndrias/metabolismo , Ratos , Ratos Wistar , Reperfusão , Traumatismo por Reperfusão/metabolismo , Superóxidos/metabolismoRESUMO
Chemical and topographical surface modifications on dental implants aim to increase the bone surface contact area of the implant and improve osseointegration. This study analyzed the cellular response of undifferentiated mesenchymal stem cells (MSC), derived from senile rats' femoral bone marrow, when cultured on a bioactive coating (by plasma electrolytic oxidation, PEO, with Ca2+ and P5+ ions), a sandblasting followed by acid-etching (SLA) surface, and a machined surface (MSU). A total of 102 Ti-6Al-4V discs were divided into three groups (n = 34). The surface chemistry was analyzed by energy dispersive spectroscopy (EDS). Cell viability assay, gene expression of osteoblastic markers, and mineralized matrix formation were investigated. The cell growth and viability results were higher for PEO vs. MSU surface (p = 0.001). An increase in cell proliferation from 3 to 7 days (p < 0.05) and from 7 to 10 days (p < 0.05) was noted for PEO and SLA surfaces. Gene expression for OSX, ALP, BSP, and OPN showed a statistical significance (p = 0.001) among groups. In addition, the PEO surface showed a higher mineralized matrix bone formation (p = 0.003). In conclusion, MSC from senile female rats cultured on SLA and PEO surfaces showed similar cellular responses and should be considered for future clinical investigations.
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Acute skin wound healing is a multistage process consisting of a plethora of tightly regulated signaling events in specialized cells. The Thy-1 (CD90) glycoprotein interacts with integrins and the heparan sulfate proteoglycan syndecan 4, generating a trimolecular complex that triggers bi-directional signaling to regulate diverse aspects of the wound healing process. These proteins can act either as ligands or receptors, and they are critical for the successful progression of wound healing. The expression of Thy-1, integrins, and syndecan 4 is controlled during the healing process, and the lack of expression of any of these proteins results in delayed wound healing. Here, we review and discuss the roles and regulatory events along the stages of wound healing that support the relevance of Thy-1, integrins, and syndecan 4 as crucial regulators of skin wound healing.
RESUMO
Tissue regeneration is often impaired in patients with metabolic disorders such as diabetes mellitus and obesity, exhibiting reduced wound repair and limited regeneration capacity. We and others have demonstrated that wound healing under normal metabolic conditions is potentiated by the secretome of human endothelial cell-differentiated mesenchymal stem cells (hMSC-EC). However, it is unknown whether this effect is sustained under hyperglycemic conditions. In this study, the wound healing effect of secretomes from undifferentiated human mesenchymal stem cells (hMSC) and hMSC-EC in a type-2 diabetes mouse model was analyzed. hMSC were isolated from human Wharton's jelly and differentiated into hMSC-EC. hMSC and hMSC-EC secretomes were analyzed and their wound healing capacity in C57Bl/6J mice fed with control (CD) or high fat diet (HFD) was evaluated. Our results showed that hMSC-EC secretome enhanced endothelial cell proliferation and wound healing in vivo when compared with hMSC secretome. Five soluble proteins (angiopoietin-1, angiopoietin-2, Factor de crecimiento fibroblástico, Matrix metallopeptidase 9, and Vascular Endothelial Growth Factor) were enriched in hMSC-EC secretome in comparison to hMSC secretome. Thus, the five recombinant proteins were mixed, and their pro-healing property was evaluated in vitro and in vivo. Functional analysis demonstrated that a cocktail of these proteins enhanced the wound healing process similar to hMSC-EC secretome in HFD mice. Overall, our results show that hMSC-EC secretome or a combination of specific proteins enriched in the hMSC-EC secretome enhanced wound healing process under hyperglycemic conditions.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Células-Tronco Mesenquimais/citologia , Proteínas Recombinantes/farmacologia , Cicatrização/efeitos dos fármacos , Angiopoietina-1/metabolismo , Angiopoietina-1/farmacologia , Angiopoietina-2/metabolismo , Angiopoietina-2/farmacologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados/química , Diabetes Mellitus Tipo 2/induzido quimicamente , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Geleia de Wharton/citologia , Geleia de Wharton/metabolismoRESUMO
O granuloma central de células gigantes (GCCG) dos maxilares é uma lesão intraóssea benigna, que pode apresentar um curso localmente agressivo. Setenta por cento dessas lesões apresentam mutações em TRPV4, KRAS ou FGFR1. Alguns estudos apontam que a população de células mononucleares parece ser o componente proliferativo da lesão. Essa população de células mononucleares é uma população mista, composta tanto por células mononucleares de origem monocítica quanto por células mesenquimais indiferenciadas. Assim, este estudo avaliou se, quando colocadas em cultura, o componente proliferativo da lesão é composto por células de origem mesenquimais ou de natureza monocítica. Foi estabelecida a cultura de células primárias de GCCG, a partir de uma amostra de conveniência composta por uma linhagem oriunda de uma lesão em mandíbula em paciente do sexo masculino, 20 anos. E, as amostras foram incubadas com os anticorpos CD14 e CD51/CD61, e triplicadas amostrais foram submetidas a citometria de fluxo para identificação das subpopulações de células presentes. Foi observado que somente as células mononucleares permaneciam ao longo das passagens. Pela citometria de fluxo, observou-se predominância de células CD14-CD51-CD61- triplamente negativas, compatível com o perfil esperado para células estromais/mesenquimais. Com base nos resultados, reforça-se a ideia de que as células mononucleares CD14- CD51-CD61- são centrais na patogênese dos GCCG, enquanto as células mononucleares de origem monocítica (CD14+) e as células gigantes semelhantes a osteoclastos (CD51+CD61 +) são reativas.
Central giant cell granuloma (CGCG) of the jaws is a benign intraosseous lesion, which may present an aggressive course. Seventy per cent of these lesions present mutations in TRPV4, KRAS or FGFR1. The population of mononuclear cells seems to be the proliferative component of the lesion. This mononuclear cell population is a mixed population, composed of both mononuclear cells of monocytic origin and undifferentiated mesenchymal cells. Thus, this study evaluated whether, when placed in culture, the proliferative component of (CGCG) is composed of mesenchymal cells or monocytic cells. The culture of primary CGCG cells was established from a convenience sample composed of a lineage originated from a mandibular lesion in 20 y.o. male patient. The samples were incubated with CD14 and CD51/CD61 antibodies, and triplicate samples were submitted to flow cytometry to identify the subpopulations of cells. It was observed that only mononuclear cells remained along the passages. By flow cytometry, a predominance of triple negative CD14-CD51-CD61- cells was observed, compatible with the expected profile for stromal/mesenchymal cells. Our results reinforce the idea that the mesenchymal cells (CD14-CD51-CD61-) have central importance in the CGCG pathogenesis, while the mononuclear cells of monocytic origin (CD14+) and the osteoclast-like giant cells (CD51+CD61+) are reactive.
Assuntos
Osteoclastos , Granuloma de Células Gigantes , Células Gigantes , Células-Tronco Mesenquimais , Citometria de FluxoRESUMO
Somatic cell nuclear transfer (SCNT) is a method with unique ability to reprogram the epigenome of a fully differentiated cell. However, its efficiency remains extremely low. In this work, we assessed and combined two simple strategies to improve the SCNT efficiency in the bovine. These are the use of less-differentiated donor cells to facilitate nuclear reprogramming and the embryo aggregation (EA) strategy that is thought to compensate for aberrant epigenome reprogramming. We carefully assessed the optimal time of EA by using in vitro-fertilized (IVF) embryos and evaluated whether the use of adipose-derived mesenchymal stem cells (ASCs) as donor for SCNT together with EA improves the blastocyst rates and quality. Based on our results, we determined that the EA improves the preimplantation embryo development per well of IVF and SCNT embryos. We also demonstrated that day 0 (D0) is the optimal aggregation time that leads to a single blastocyst with uniform distribution of the original blastomeres. This was confirmed in bovine IVF embryos and then, the optimal condition was translated to SCNT embryos. Notably, the relative expression of the trophectoderm (TE) marker KRT18 was significantly different between aggregated and nonaggregated ASC-derived embryos. In the bovine, no effect of the donor cell is observed on the developmental rate, or the embryo quality. Therefore, no synergistic effect of the use of both strategies is observed. Our results suggest that EA at D0 is a simple and accessible strategy that improves the blastocyst rate per well in bovine SCNT and IVF embryos and influence the expression of a TE-related marker. The aggregation of two ASC-derived embryos seems to positively affect the embryo quality, which may improve the postimplantation development.
Assuntos
Blastocisto/citologia , Clonagem de Organismos/veterinária , Técnicas de Cultura Embrionária/métodos , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Células-Tronco Mesenquimais/citologia , Animais , Bovinos , Embrião de Mamíferos/química , Feminino , Fertilização in vitro , GravidezRESUMO
In vitro 3D culture models have emerged in the cancer field due to their ability to recapitulate characteristics of the in vivo tumor. Herein, we described the establishment and characterization of 3D multicellular spheroids using ovarian cancer cells (SKOV-3) in co-culture with mesenchymal cells (MUC-9) or fibroblasts (CCD27-Sk). We demonstrated that SKOV-3 cells in co-culture were able to form regular and compact spheroids with diameters ranging from 300 to 400 µm and with a roundness close to 1.0 regardless of the type of stromal cell used. In the 3D culture an increase was not observed in spheroid diameter nor was there significant cell growth. What is more, the 3D co-cultures presented an up regulation of genes related to tumorigenesis, angiogenesis and metastases (MMP2, VEGFA, SNAI1, ZEB1 and VIM) when compared with 2D and 3D monoculture. As expected, both 3D cultures (mono and co-cultures) exhibited a higher Paclitaxel chemoresistance when compared to 2D condition. Although we did not observe differences in the Paclitaxel resistance between the 3D mono and co-cultures, the gene expression results indicate that the presence of mesenchymal cells and fibroblasts better recapitulate the in vivo tumor microenvironment, being able, therefore, to more accurately evaluate drug efficacy for ovarian cancer therapy.
Assuntos
Detecção Precoce de Câncer , Neoplasias Ovarianas , Linhagem Celular Tumoral , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Esferoides Celulares , Microambiente TumoralRESUMO
AIMS: The present study was designed to investigate whether the antinociceptive effect of bone marrow-derived mesenchymal stem/stromal cells (MSC) during oxaliplatin (OXL)-induced sensory neuropathy is related to antioxidant properties. MAIN METHODS: Male mice C57BL/6 were submitted to repeated intravenous administration of OXL (1 mg/kg, 9 administrations). After the establishment of sensory neuropathy, mice were treated with a single intravenous administration of MSC (1 × 106), vehicle or gabapentin. Paw mechanical and thermal nociceptive thresholds were evaluated through von Frey filaments and cold plate test, respectively. Motor performance was evaluated in the rota-rod test. Gene expression profile, cytokine levels, and oxidative stress markers in the spinal cord were evaluated by real-time PCR, ELISA and biochemical assays, respectively. KEY FINDINGS: OXL-treated mice presented behavioral signs of sensory neuropathy, such as mechanical allodynia and thermal hyperalgesia, which were completely reverted by a single administration of MSC. Repeated oral treatment with gabapentin (70 mg/kg) induced only transient antinociception. The IL-1ß and TNF-α spinal levels did not differ between mice with or without sensory neuropathy. MSC increased the levels of anti-inflammatory cytokines, IL-10 and TGF-ß, in the spinal cord of neuropathic mice, in addition to increasing the gene expression of antioxidant factors SOD and Nrf-2. Additionally, nitrite and MDA spinal levels were reduced by the MSC treatment. SIGNIFICANCE: MSC induce reversion of sensory neuropathy induced by OXL possibly by activation of anti-inflammatory and antioxidant pathways, leading to reestablishment of redox homeostasis in the spinal cord.
Assuntos
Transplante de Células-Tronco Mesenquimais , Oxaliplatina/toxicidade , Oxirredução , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Células Receptoras Sensoriais/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Animais , Interleucina-1beta/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Nociceptividade , Oxirredução/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/terapia , Reação em Cadeia da Polimerase em Tempo Real , Teste de Desempenho do Rota-Rod , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Medula Espinal/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
RESUMO Objetivo A escassez de pulmões viáveis ainda é um grande obstáculo para o transplante. As vítimas de trauma, que constituem potenciais doadores de pulmão, comumente apresentam choque hipovolêmico que acarreta inflamação e deterioração pulmonar e rejeição após o transplante. Buscando melhorar o enxerto pulmonar, testaram-se novas abordagens ao tratamento do doador. Este estudo foca o tratamento com células-tronco mesenquimais (CTMs) ou fatores solúveis produzidos pelas CTMs (FS-CTMs), usando um modelo com ratos para doadores de pulmão após choque hemorrágico. Métodos Quarenta e oito ratos foram divididos em quatro grupos: Controle (n=12), animais sem indução de choque hipovolêmico; Choque (n=12), animais submetidos a choque hipovolêmico (pressão arterial média de 40 mmHg); CTM (n=12), animais submetidos a choque hipovolêmico e tratados com CTMs; e FS (n=12), animais submetidos a choque hipovolêmico e tratados com FS-CTMs. Os animais foram submetidos a um procedimento de choque hipovolêmico (40 mmHg) com 50 minutos de duração. Os animais tratados foram monitorados por 115 minutos. Realizamos análise histopatológica do tecido pulmonar e quantificação dos marcadores inflamatórios (TNF-α, IL-1β, IL-6, IL-10, iCAM e vCAM) no tecido pulmonar e leucócitos no sangue periférico (LSPs). Resultados O choque hemorrágico resultou em taxas mais altas de LSPs e infiltrado de neutrófilos nos pulmões. Os animais do grupo FS apresentaram menor densidade de neutrófilos em comparação com os animais dos grupos Choque e CTM (p<0,001). Não foram observadas diferenças entre os grupos quanto aos níveis de citocinas no tecido pulmonar. Conclusão Os pulmões dos ratos submetidos a choque hemorrágico e tratados com FS-CTM apresentaram inflamação reduzida indicada por uma diminuição do infiltrado de neutrófilos nos pulmões.
ABSTRACT Objective The shortage of viable lungs is still a major obstacle for transplantation. Trauma victims who represent potential lung donors commonly present hypovolemic shock leading to pulmonary inflammation and deterioration and rejection after transplantation. Seeking to improve lung graft, new approaches to donor treatment have been tested. This study focuses on treatment with mesenchymal stem cells (MSCs) or soluble factors produced by MSCs (FS-MSC) using a rat model for lung donors after hemorrhagic shock. Methods Forty-eight rats were divided into four groups: Sham (n=12), animals without induction of hypovolemic shock; Shock (n=12), animals submitted to hypovolemic shock (mean arterial pressure 40 mmHg); MSC (n=12), animals submitted to hypovolemic shock and treated with MSCs, and FS (n=12), animals submitted to hypovolemic shock and treated with FS-MSC. The animals were subjected to a 50-minute hypovolemic shock (40 mmHg) procedure. The treated animals were monitored for 115 minutes. We performed histopathology of lung tissue and quantification of inflammatory markers (TNF-α, IL-1β, IL-6, IL-10, iCAM and vCAM) in lung tissue and peripheral blood leukocytes (PBLs). Results Hemorrhagic shock resulted in higher PBLs and neutrophil infiltrate in the lungs. FS animals had lower neutrophil density comparing with Shock and MSC animals (p<0.001). No differences in the cytokine levels in lung tissue were observed between the groups. Conclusions The lungs of rats submitted to hemorrhagic shock and treated with FS-MSC showed reduced inflammation indicated in a decrease in lung neutrophil infiltrate.
Assuntos
Animais , Ratos , Choque Hemorrágico/terapia , Transplante de Pulmão , Células-Tronco Mesenquimais , Modelos Animais de Doenças , Inflamação , PulmãoRESUMO
La terapia celular basada en células mesenquimales/estromales se aplica ampliamente en la medicina moderna, aun cuando no todos los mecanismos de supervivencia y diferenciación están identificados. Sin embargo, hace pocos años se comenzaron a encontrar elementos extracelulares que generan nuevos paradigmas. En el presente trabajo se explican las principales características y funciones atribuidas a los exosomas, nanopartículas constituidas por microvesículas secretadas por las células con efecto en la matriz extracelular, y su repercusión como alternativa hacia una medicina regenerativa libre de células. Estas estructuras participan de forma notoria y crucial en la comunicación intercelular, lo que ha supuesto un cambio en el concepto de las funciones y el papel que desempeñan estas vesículas en los organismos vivos, en particular en la restauración de tejidos dañados y la respuesta inflamatoria e inmunológica. Se comentan algunos ejemplos de la repercusión biotecnológica de los exosomas en empresas y el mercado biofarmaceútico(AU)
Mesenchymal/stromal cell ;based therapy is widely applied in modern medicine, even though not all survival and differentiation mechanisms are identified. However, a few years ago, extracellular elements began to be found that generate new paradigms. The present work explains the main characteristics and functions attributed to exosomes, nanoparticles made up of microvesicles secreted by with an effect on the extracellular matrix, and their impact as an alternative towards cell-free regenerative medicine. These structures participate, notoriously and critically, in intercellular communication, which has led to a change in the concept of the functions and role that these vesicles play within living organisms, particularly in the restoration of damaged tissues and the inflammatory and immunological response. Some examples of the exosomes' biotechnological impact on companies and the biopharmaceutical market are discussed(AU)
Assuntos
Humanos , Masculino , Feminino , Medicina Regenerativa/métodos , Exossomos/fisiologia , Células-Tronco Mesenquimais/fisiologiaRESUMO
Inherited metabolism disorders are serious childhood diseases that lead to significant cognitive impairment and regression of psychomotor development. The pathophysiology of the neural progressive deterioration is usually associated with severe neuroinflammation and demyelination, and as a consequence, neurodegeneration. At the moment they have no adequate treatment and require early and aggressive therapeutic approaches, which entail high mortality rates and, very frequently, low degrees of functional improvement and survival. Bone marrow transplantation and bone marrow mesenchymal cells grafts are therapeutic and experimental therapies that improve the course of these diseases through different mechanisms of action: enzyme replacement, membrane exchange and regulation of the inflammatory process.
Los trastornos heredados del metabolismo son enfermedades graves de la infancia que cursan con un gran deterioro cognitivo y del desarrollo psicomotor. La fisiopatología del progresivo deterioro del sistema nervioso suele estar asociada a una severa neuroinflamación y desmielinización, y como consecuencia, neurodegeneración. Por el momento no tienen cura y precisan de actitudes terapéuticas precoces y agresivas, que conllevan altas tasas de mortalidad y, muy frecuentemente, escasos grados de mejoría funcional y supervivencia. El trasplante de médula ósea y de células mesenquimales de médula ósea son terapias de elección y experimentales que consiguen mejorar el curso de estas enfermedades mediante diferentes mecanismos de acción: remplazo de enzima deficiente, intercambio de membranas y regulación del proceso inflamatorio.
Assuntos
Transplante de Medula Óssea/métodos , Doenças por Armazenamento dos Lisossomos/terapia , Transtornos Peroxissômicos/terapia , Humanos , Doenças por Armazenamento dos Lisossomos/fisiopatologia , Transplante de Células-Tronco Mesenquimais/métodos , Transtornos Peroxissômicos/fisiopatologiaRESUMO
Los trastornos heredados del metabolismo son enfermedades graves de la infancia que cursan con un gran deterioro cognitivo y del desarrollo psicomotor. La fisiopatología del progresivo deterioro del sistema nervioso suele estar asociada a una severa neuroinflamación y desmielinización, y como consecuencia, neurodegeneración. Por el momento no tienen cura y precisan de actitudes terapéuticas precoces y agresivas, que conllevan altas tasas de mortalidad y, muy frecuentemente, escasos grados de mejoría funcional y supervivencia. El trasplante de médula ósea y de células mesenquimales de médula ósea son terapias de elección y experimentales que consiguen mejorar el curso de estas enfermedades mediante diferentes mecanismos de acción: remplazo de enzima deficiente, intercambio de membranas y regulación del proceso inflamatorio.
Inherited metabolism disorders are serious childhood diseases that lead to significant cognitive impairment and regression of psychomotor development. The pathophysiology of the neural progressive deterioration is usually associated with severe neuroinflammation and demyelination, and as a consequence, neurodegeneration. At the moment they have no adequate treatment and require early and aggressive therapeutic approaches, which entail high mortality rates and, very frequently, low degrees of functional improvement and survival. Bone marrow transplantation and bone marrow mesenchymal cells grafts are therapeutic and experimental therapies that improve the course of these diseases through different mechanisms of action: enzyme replacement, membrane exchange and regulation of the inflammatory process.
Assuntos
Humanos , Transplante de Medula Óssea/métodos , Doenças por Armazenamento dos Lisossomos/terapia , Transtornos Peroxissômicos/terapia , Doenças por Armazenamento dos Lisossomos/fisiopatologia , Transtornos Peroxissômicos/fisiopatologia , Transplante de Células-Tronco Mesenquimais/métodosRESUMO
Manipulation of implant surface characteristics constitutes a promising strategy for improving cell growth and tissue response on a variety of materials with different surface topographies. Mesenchymal progenitor cells with a capacity to respond to titanium surface stimuli and differentiate into osteoblasts were used to perform comparative tests between two different implant topographies, including their functional interaction with pre-osteoblasts directly seeded onto the implants. Functional analysis of nanostructured implant surfaces was performed by in vitro assay analysis. The machined surface of titanium implants (mach group) was used as a control and compared with a nanoparticle HA activated surface implant (nano group), developed by the deposition of pure crystalline hydroxyapatite. Cell culture on the nano group surface resulted in higher cell adhesion and cultured osteoblast viability compared with the mach group. Scanning electron microscope (SEM) images revealed a stable interaction, indicated by the presence of focal cell adhesion formation. These results together with positive mineralization assays showed the nano group to be an excellent scaffold for bone-implant integration.
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O objetivo deste estudo foi avaliar um novo método de texturização por PEO com incorporação de Ca e P na superfície do Ti-6Al-4V em ossos de baixa densidade, por meio de avaliação in vitro, ex-in vivo e in vivo, em função de parâmetros topográficos e reparacionais. 57 ratas Wistar (Rattus novergicus), sendo 38 ratas com 6 meses de idade (Grupos OXV - submetidas à ovariectomia e SHAM - cirurgia fictícia) e 19 ratas senis (18 meses de idade: Grupo SENIL), foram divididas para realização do estudo ex-in vivo (n=9) e in vivo (n=48). Os grupos para análise ex-in vivo foram submetidos à eutanásia e os fêmures foram removidos e transportados em meio de cultura contendo meio essencial mínimo modificação alfa (α- MEM) suplementado com 500 µg/mL de gentamicina e 3 µg/mL de fungisona. As células-tronco mesenquimais de medula óssea (CTMsMO) dos fêmures, foram isoladas e cultivadas em meio de crescimento para manterem-se como CTMs. Após alcançar a subconfluência, as células foram cultivadas em 3 superfícies de discos de Ti-6Al-4V, grupo CONTROLE (superfície usinada) grupo AC (superfície tratada por Ataque Ácido e Jateamento) e grupo PEO (superfície tratada por Oxidação de Plasma Eletrolítico com associação de Cálcio e Fosforo). Para avaliação das respostas celulares foram realizados ensaios de viabilidade celular, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteina óssea (BSP) e osteopontina (OPN), atividade da fosfatase alcalina (ALP) e formação de matriz mineralizada. Os dados foram submetidos ao teste ANOVA 1 fator ou Kruskal-Wallis (p < 0,05). Nos experimentos in vivo, após 90 dias, foi instalado um implante em cada tíbia, sendo um implante pertencente ao grupo PEO e o outro implante do grupo AC. Após 42 dias da instalação dos implantes, 8 animais de cada grupo foram submetidos à eutanásia e suas tíbias passaram pela descalcificação, para a análise histológica e imunoistoquímica (OPG, RANKL, OC e TRAP). As demais ratas, após a eutanásia, tiveram suas tíbias coletadas e analisadas em microtomografia computadorizada (BV.TV, Tb.Th, Tb.N, Tb.Sp e Po(tot)) e, em seguida os implantes submetidos ao torque reverso em torquímetro digital (N.cm). A outra metade das tíbias foram processadas com inclusão em resina fotopolimerizadas para cortes calcificados e assim, a análise por microscopia confocal (calceina e alizarina) e em seguida, análise histométrica. Os dados foram submetidos ao teste ANOVA 1 fator ou Kruskal-Wallis, seguido de pós teste Tukey; p < 0,05). A análise da viabilidade celular mostrou que em todos os grupos testes de CTMs-MO para SHAM, OVX e SENIL apresentaram um crescimento progressivo nos diferentes tempos de 3, 7 e 10 dias. Avaliação da expressão gênica através dos genes Runx2, SP7/Osterix, ALP, BSP, OC e OPN e analise pela imunofluorecência apresentaram uma leve tendência de melhores respostas nas CTMs-MO SHAM para o grupo AC, CTMs-MO OVX para o grupo PEO e CTMsMO SENIS características semelhantes nos grupos AC e PEO. A atividade da fosfatase alcalina ocorreu maior expressão na superfície PEO no grupo SHAM, maior expressão na superfície CONTROLE no grupo OVX e no grupo SENIL houve um equilíbrio em todas as superfícies. A superfície PEO apresentou maior formação de nódulos de mineralização (21º dia) em todos os grupos. Nos experimentos in vivo, as análises histológicas mostrou maior neoformação óssea no grupo PEO quando comparado ao grupo AC nos grupos SHAM e OVX, e no grupo SENIL foram similares os resultados. A avaliação imunoistoquímica demonstrou um equilíbrio em todos os grupos na comparação de superfícies na proteína TRAP, para o processo de remodelação (OPG e RANKL) e mineralização (OC) nos grupos SHAM e SENIL (p>0,05), ocorrendo uma diminuição no grupo OVX, no qual os resultados do PEO foram mais favoráveis que AC nessa conformação. Na análise microtomográfica, BV.TV os resultados foram semelhantes em ambos os grupos, porém em OVX AC mostrou menor porcentagem de volume ósseo e uma maior porosidade (Po(tot)). A análise biomecânica por torque-reverso (N.cm) mostrou que os maiores valores pertenciam ao grupo PEO. A dinâmica do tecido ósseo representada pelo turnover ósseo, observado através dos fluorocromos (Calceína e Alizarina) mostrou-se similares nos grupos experimentais. As superficies PEO E AC nesse estudo demonstram que possuem uma grande capacidade de promoção da formação óssea independente dos tipos ósseos experimentais (SHAM, OVX e SENIL), tanto na área de contato osso e implante (ELCOI), quanto para a área de osso neoformado (AON). Diante daslimitações do estudo in vitro e in vivo, os resultados foram esclarecedores para acreditar que o método de texturização aqui testado, por meio da Oxidação por Plasma Eletrolítico (PEO), favoreceu à formação óssea, principalmente nos ossos mais críticos (OVX), inclusive evidenciando maior maturação óssea nos períodos mais tardios aqui analisados(AU)
The objective of this study was to evaluate a new PEO texturing method with Ca and P incorporation on the Ti-6Al-4V surface in low bone density, by means of in vitro, ex vivo and in vivo evaluation through topographic and repairment parameters. 57 Wistar rats (Rattus novergicus), being 38 at 6 months of age (OXV Groups - submitted to ovariectomy and SHAM surgery) and 19 senile rats (18 months of age: SENIL Group) were divided into three subgroups: ex-in vivo (n = 9) and in vivo (n = 48). The Groups for ex-in vivo analysis were euthanized and femurs were removed and transported in culture medium containing minimal alpha modification (α- MEM) medium supplemented with 500 µg / ml gentamicin and 3 µg / ml fungizone. The mesenchymal stem cells from bone marrow (MSC-M) of the femur were isolated and cultured in growth medium to remain as MSCs. After reaching the subconfluence, the cells were grown on 3 surfaces of Ti-6Al-4V discs, CONTROL group (machined surface) group AC (surface treated by etched-acid) and PEO group (surface treated by Electrolytic Plasma Oxidation with the association of Calcium and Phosphorus). Cell viability assays, gene expression of osteoblastic markers, bone sialoprotein (BSP) and osteopontin (OPN), alkaline phosphatase (ALP) activity, and mineralized matrix formation were performed to evaluate cellular responses. Data were submitted to ANOVA 1 factor test or Kruskal-Wallis test (P <0.05). In the groups for the in vivo study, after 90 days, an implant was installed on each tibia, one implant to the PEO group and another implant of the AC group. After 42 days of implant implantation, eight animals from each group underwent euthanasia and their tibiae underwent decalcification for histological and immunohistochemical analysis (OPG, RANKL, OC, and TRAP). The other rats, after euthanasia, had their tibiae collected and analyzed in computerized microtomography (BV.TV, Tb.Th, Tb.N, Tb.Sp and Po (tot)), and then implants submitted to reverse torque in Digital torque wrench (N.cm). Another half of the tibiae were processed with inclusion in photopolymerized resin for calcified cuts and thus the analysis by confocal microscopy (calcein and alizarin) and then histometric analysis. Data were submitted to 1-factor ANOVA or Kruskal-Wallis test, followed by Tukey test; p <0.05). The cell viability analysis showed that in all groups, MOH tests for SHAM, OVX, and SENIL showed a progressive growth in the different times of 3, 7 and 10 days. Evaluation of gene expression through the Runx2, SP7 / Osterix, ALP, BSP, OC and OPN genes and immunofluorescence analysis showed a slight tendency for better responses in the CTMs- MO SHAM for the AC group, CTMs-MO OVX for the PEO group and CTMs-MO SENIS features similar in AC and PEO groups. The alkaline phosphatase activity was higher on the PEO surface in the SHAM group, the greater expression on the CONTROL surface in the OVX group and the SENIL group showed a balance on all surfaces. The PEO surface presented a higher formation of mineralization nodules in all groups (21st day). For the in vivo analyzes, the histological analysis showed greater bone neoformation in the PEO group when compared to the AC group in the SHAM and OVX groups, and in the SENIL group, the results were similar. The immunohistochemical evaluation showed a balance in all groups in the comparison of surfaces in the TRAP protein, for the remodeling process (OPG and RANKL) and mineralization (OC) in the SHAM and SENIL groups (p> 0.05). OVX group, in which PEO results were more favorable than AC in this confirmation. In the microtomographic analysis, BV.TV the results were similar in both groups, but in OVX AC showed a lower percentage of bone volume and a higher porosity (Po (tot)). Biomechanical analysis by torque-reverse (N.cm) showed that the highest values belonged to the PEO group. The dynamics of the bone tissue represented by the bone turnover observed through the fluorochromes (Calcein and Alizarin) were similar in the experimental groups. The PEO and AC surfaces in this study demonstrate that they have a great ability to promote bone formation independent of experimental bone types (SHAM, OVX, and SENIL), both in the area of bone and implant contact (ELCOI) and in the area of newly formed bone (AON). Considering the limitations of the in vitro and in vivo study, the results were enlightening to believe that the texturing method tested here, through Electrolytic Plasma Oxidation (PEO), favored bone formation, mainly in the most critical bones (OVX), including evidence of increased bone maturation in the later periods analyzed here(AU)
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Animais , Ratos , Osteoporose , Implantes Dentários , Cultura Primária de Células , Ratos Wistar , Oxidação , Células-Tronco MesenquimaisRESUMO
RAC3 is a coactivator of steroid receptors and NF-κB. It is usually overexpressed in several tumors, contributes to maintain cancer stem cells and also to induce them when is overexpressed in non-tumoral cells. In this work, we investigated whether the inflammatory cytokine TNF may contribute to the transforming effects of RAC3 overexpression in the non-tumoral HEK293 cell line. The study model included the HEK293 tumoral transformed cell line constitutively overexpressing RAC3 by stable transfection and control non-tumoral cells transfected with an empty vector. The HeLa and T47D tumoral cells that naturally overexpress RAC3 were used as positive control. We found that TNF potentiated RAC3-induced mesenchymal transition, involving an increased E-Cadherin downregulation, Vimentin and SNAIL upregulation and enhanced migratory behavior. Moreover, concerning the molecular mechanisms by which TNF potentiates the RAC3 transforming action, they involve the IKK activation, which in addition induced the ß-Catenin transactivation. Our results demonstrate that although RAC3 overexpression could be a signal strong enough to induce cancer stem cells, the inflammatory microenvironment may be playing a key role contributing to the migratory and invasive phenotype required for metastasis and cancer persistence.
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BACKGROUND: A promising novel cell-free bioactive formulation for articular cartilage regeneration, called BIOF2, has recently been tested in pre-clinical trials. The aim of the present study was to evaluate the efficacy and safety of BIOF2 for intra-articular application in patients with severe osteoarthritis of the knee. METHODS: A prospective, randomized, 3-arm, parallel group clinical trial was conducted. It included 24 patients with severe osteoarthritis of the knee (WOMAC score 65.9 ± 17). Before they entered the study, all the patients were under osteoarthritis control through the standard treatment with nonsteroidal anti-inflammatory drugs (NSAIDs), prescribed by their family physician. Patients were distributed into three groups of 8 patients each (intra-articular BIOF2, total joint arthroplasty, or conservative treatment with NSAIDs alone). The WOMAC score, RAPID3 score, and Rasmussen clinical score were evaluated before treatment and at months 3, 6, and 12. BIOF2 was applied at months 0, 3, and 6. Complete blood count and blood chemistry parameters were determined in the BIOF2 group before treatment, at 72 h, and at months 1, 3, 6, and 12. In addition, articular cartilage volume was evaluated (according to MRI) at the beginning of the study and at month 12. RESULTS: The NSAID group showed no improvement at follow-up. Arthroplasty and BIOF2 treatments showed significant improvement in all the scoring scales starting at month 3. There were no statistically significant differences between the BIOF2 group and the arthroplasty group at month 6 (WOMAC score: 19.3 ± 18 vs 4.3 ± 5; P = 0.24) or month 12 (WOMAC score: 15.6 ± 15 vs 15.7 ± 17; P = 1.0). Arthroplasty and BIOF2 were successful at month 12 (according to a WOMAC score: ≤ 16) in 75% of the patients and the daily use of NSAIDs was reduced, compared with the group treated exclusively with NSAIDs (RR = 0.33, 95% CI 0.12-0.87, P = 0.02. This result was the same for BIOF2 vs NSAIDs and arthroplasty vs NSAIDs). BIOF2 significantly increased the articular cartilage by 22% (26.1 ± 10 vs 31.9 ± 10 cm2, P < 0.001) and produced a significant reduction in serum lipids. BIOF2 was well tolerated, causing slight-to-moderate pain only upon application. CONCLUSIONS: The intra-articular application of the new bioactive cell-free formulation (BIOF2) was well tolerated and showed no significative differences with arthroplasty for the treatment of severe osteoarthritis of the knee. BIOF2 can regenerate articular cartilage and is an easily implemented alternative therapy for the treatment of osteoarthritis. Trial registration Cuban Public Registry of Clinical Trials (RPCEC) Database RPCEC00000250. Registered 08/15/2017-Retrospectively registered, http://rpcec.sld.cu/en/trials/RPCEC00000250-En .
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Cartilagem Articular/efeitos dos fármacos , Células-Tronco Mesenquimais/química , Osteoartrite do Joelho/tratamento farmacológico , Esteroides/administração & dosagem , Adulto , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/farmacologia , Artroplastia do Joelho , Contagem de Células Sanguíneas , Cartilagem Articular/crescimento & desenvolvimento , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Feminino , Humanos , Injeções Intra-Articulares , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Osteoartrite do Joelho/sangue , Osteoartrite do Joelho/fisiopatologia , Osteoartrite do Joelho/cirurgia , Regeneração/efeitos dos fármacos , Esteroides/farmacologia , Resultado do TratamentoRESUMO
RAC3 is a transcription coactivator, usually overexpressed in several tumors and required to maintain the pluripotency in normal stem cells. In this work we studied the association between RAC3 overexpression on cancer cell stemness and the capacity of this protein to induce cancer stem properties in non tumoral cells. We performed in vitro and in vivo experiments using two strategies: by overexpressing RAC3 in the non tumoral cell line HEK293 and by silencing RAC3 in the human colorectal epithelial cell line HCT116 by transfection. Furthermore, we analysed public repository microarrays data from human colorectal tumors in different developmental stages. We found that RAC3 overexpression was mainly associated to CD133+ side-population of colon cancer cells and also to early and advanced stages of colon cancer, involving increased expression of mesenchymal and stem markers. In turn, RAC3 silencing induced diminished tumoral properties and cancer stem cells as determined by Hoechst efflux, tumorspheres and clonogenic growth, which correlated with decreased Nanog and OCT4 expression. In non tumoral cells, RAC3 overexpression induced tumoral transformation; mesenchymal phenotype and stem markers expression. Moreover, these transformed cells generated tumors in vivo. Our results demonstrate that RAC3 is required for maintaining and induction of cancer cell stemness.
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ABSTRACT A cytotoxicity study is performed on a poly(methyl methacrylate) polymer (PMMA) to be used for the fabrication of bone tissue by Rapid Prototyping (RP). The solution polymerization is conducted in a pilot plant reactor using more appropriated reagents in consideration of the medical application. Moreover, the polymer is efficiently handled to avoid the side effect of the monomer, reducing the concentration of this specie to 287,731 µg MMA/kg PMMA. The cytotoxicity of the polymer is determined through growth monitoring, adherence and morphology of L-929 cells. Additionally, MTT and LIVE/DEAD tests are performed. The results showed continuous and progressive growth of the cells on the surface of the specimens. Moreover, the material did not influence on the viability of mesenchymal cells and inverted fluorescence microscopy images showed a polyanionic dye calcein well retained in the cells in contact with the PMMA as well as the negative control after 72 hours. Thus, the polymer was efficiently synthesized and handled for the expected demands.
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Biologically active biomaterials as biopolymers and hydrogels have been used in medical applications providing favorable results in tissue engineering. In this research, a wound dressing device was designed by integration of an autologous clot hydrogel carrying mesenchymal stem-cells onto a biopolymeric scaffold. This hybrid biomaterial was tested in-vitro and in-vivo, and used in a human clinical case. The biopolymeric scaffold was made with gelatin, chitosan and hyaluronic acid, using a freeze-drying method. The scaffold was a porous material which was designed evaluating both physical properties (glass transition, melting temperature and pore size) and biological properties (cell viability and fibronectin expression). Two types of chitosan (120 and 300kDa) were used to manufacture the scaffold, being the high molecular weight the most biologically active and stable after sterilization with gamma irradiation (25kGy). A clot hydrogel was formulated with autologous plasma and calcium chloride, using an approach based on design of experiments. The optimum hydrogel was used to incorporate cells onto the porous scaffold, forming a wound dressing biomaterial. The wound dressing device was firstly tested in-vitro using human cells, and then, its biosecurity was evaluated in-vivo using a rabbit model. The in-vitro results showed high cell viability after one week (99.5%), high mitotic index (19.8%) and high fibronectin expression. The in-vivo application to rabbits showed adequate biodegradability capacity (between 1 and 2weeks), and the histological evaluation confirmed absence of rejection signs and reepithelization on the wound zone. Finally, the wound dressing biomaterial was used in a single human case to implant autologous cells on a skin surgery. The medical examination indicated high biocompatibility, partial biodegradation at one week, early regeneration capacity at 4weeks and absence of rejection signs.