Phenotypic characteristics and differentiation potential of gingival mesenchymal stem cells in hyperglycemia ­ an ex vivo exploratory study / Características fenotípicas y potencial de diferenciación de las células madre mesenquimales gingivales en la hiperglucemia: un estudio exploratorio ex vivo

Background: The therapeutic use of gingival mesenchymal stem cells (GMSCs) as autologous cells may pose the challenge of alterations inflicted by the hyperglycemic environment. Objective: This study aims to assess the effects of hyperglycemia on the characteristics of GMSCs in diabetics. Materials and Methods: 10 patients who consented and fulfilled the criteria for inclusion and exclusion were recruited and categorized as test (HbA1c > 6.5) and control (HbA1c < 6.0). Gingival explants were obtained from gingival collar of teeth, washed, digested and cultured. The cells were subjected to microscopic observation to assess phenotype characteristics, and flow cytometry and qRT-PCR to assess differentiation potential. Stem cell markers CD90, CD73, CD105, CD34, CD45, HLA DR & HLA ABC, osteogenic differentiation markers RUNX2 & OCN, adipogenic differentiation markers PPARG2 & FABP4 and chondrogenic differentiation markers SOX9 & AGCN were evaluated. Results: Microscopic appearance of spindle shaped cells was found to be comparable in both groups. Flow cytometry results demonstrated comparable expressions with both groups, samples being positive for CD90, CD73, CD105, HLA ABC and negative for CD34, CD45 & HLA DR. There were variations in the expression of markers when assessed for differentiation potentials. Conclusions: The hyperglycemic environment did not manifest any changes in the phenotypic characteristics of GMSCs among diabetics. However, the expression of certain differentiation markers was significantly altered in the diabetic test population included. Further research is being conducted to understand the GMSCs in a hyperglycemic environment with an aim to develop strategies to optimize its clinical implications. Keywords: Gingiva; Mesenchymal stem cells; Diabetes mellitus; Cell Differentiation; Hyperglycemia; Flow cytometry.

Antededentes: El uso terapéutico de células madre mesenquimales gingivales(GMSC) como células autólogas puede plantear el desafío de las alteraciones infligidas por el entorno hiperglucémico. Objetivo: Este estudio tiene como objetivo evaluar los efectos de la hiperglucemia sobre las características de las GMSC en diabéticos. Materiales y Métodos: Se reclutaron y categorizaron 10 pacientes que dieron su consentimiento y cumplieron los criterios de inclusión y exclusión como prueba (HbA1c > 6,5) y control (HbA1c < 6,0). Los explantes gingivales se obtuvieron del cuello gingival de los dientes, se lavaron, digirieron y cultivaron. Las células se sometieron a observación microscópica para evaluar las características fenotípicas y a citometría de flujo y qRT-PCR para evaluar el potencial de diferenciación. Se evaluaron los marcadores de células madre CD90, CD73, CD105, CD34, CD45, HLA DR y HLA ABC, marcadores de diferenciación osteogénica RUNX2 y OCN, marcadores de diferenciación adipogénica PPARG2 y FABP4 y marcadores de diferenciación condrogénica SOX9 y AGCN. Resultados: Se encontró que la apariencia microscópica de las células fusiformes era comparable en ambos grupos. Los resultados de la citometría de flujo demostraron expresiones comparables en ambos grupos, siendo las muestras positivas para CD90, CD73, CD105, HLA ABC y negativas para CD34, CD45 y HLA DR. Hubo variaciones en la expresión de los marcadores cuando se evaluaron los potenciales de diferenciación. Conclusiones: El entorno hiperglucémico no manifestó ningún cambio en las características fenotípicas de las GMSC entre los diabéticos. Sin embargo, la expresión de ciertos marcadores de diferenciación se alteró significativamente en la población de prueba de diabetes incluida. Se están realizando más investigaciones para comprender las GMSC en un entorno hiperglucémico con el objetivo de desarrollar estrategias para optimizar sus implicaciones clínicas.

Potencial das membranas de células-tronco mesenquimais na regeneração periodontal: uma revisão sistemática de estudos pré-clínicos em modelo animal / Potential of mesenchymal stem cell sheets on periodontal regeneration: a systematic review of pre-clinical animal studies

A periodontite é uma doença inflamatória crônica multifactorial caracterizada pela destruição progressiva do aparelho de suporte periodontal. Atualmente, as técnicas convencionais para regeneração desses tecidos periodontais perdidos tiveram sucesso limitado. A tecnologia de membranas de células usando células-tronco mesenquimais apareceu como uma estratégia promissora na medicina regenerativa periodontal. Embora estudos recentes tenham mostrado o papel das membranas de células-tronco mesenquimais (MSCSs) no aumento dos tecidos de suporte dentário e ósseo, não há uma revisão sistemática focada especificamente na avaliação da regeneração periodontal em modelos animais ortotópicos. Esta revisão tem como objetivo avaliar o potencial das MSCSs na regeneração periodontal em comparação ao controle, em modelos animais experimentais. Estudos pré-clínicos em defeitos periodontais de modelos animais foram considerados elegíveis. A busca eletrônica incluiu as bases de dados MEDLINE, Web of Science, EMBASE e LILACS. Além disso, uma busca manual avaliou as revistas científicas na área de periodontia/regeneração. A revisão sistemática foi conduzida de acordo com as diretrizes de Preferred Reporting Item for Systematic Reviews and Meta-Analyses statement guidelines. A ferramenta do Centro de Revisão Sistemática para Experimentação com Animais de Laboratório (SYRCLE) foi usada para avaliar o risco de viés. Dos 3989 estudos obtidos a partir da busca no banco de dados eletrônicos foram incluídos 17 artigos. Foram empregados MSCSs autólogos, alógenos e xenógenos para melhorar a regeneração periodontal. Estes incluíram MSCSs do folículo dentário (DF), MSCSs do ligamento periodontal (PDL), MSCSs da polpa dentária (DP), MSCSs da medula óssea (BM), MSCSs periosteais alveolares (AP) e MSCSs gengivais (G). Em relação ao protocolo de indução de células, a maioria dos estudos utilizou ácido ascórbico (52,94%), outros utilizaram placas de cultura com polímero termo responsivo (47,06%). Os efeitos adversos, em relação à utilização das MSCSs no sítio doador, não foram identificados na maioria dos estudos, mesmo com o uso adjunto de scaffolds, membranas ou ambos. Meta-análise não foi considerada devido a heterogeneidades metodológicas. PDL-MSCSs demonstrou ser superior para aumento da regeneração periodontal em comparação ao controle, mas em um microambiente inflamatório induzido, DF-MSCSs foram melhores. Os DF-MSCSs parecem estar relacionados à espessura do cemento e dimensão periodontal. Além disso, DP-MSCSs e BM-MSCSs mostraram resultados melhores em comparação com o controle. Em contraste, AP-MSCSs não foram associados a melhorias na regeneração periodontal. A avaliação do risco de viés com a ferramenta da SYRCLE revelou que 44,12% dos estudos apresentavam baixo risco de viés, 55,29% foram incertos e 0,59%, alto risco. A presente revisão sistemática mostrou que as MSCSs podem aumentar a regeneração periodontal em modelos animais de defeito periodontal, fornecendo uma estratégia promissora para aumentar a regeneração periodontal.

Resveratrol Alleviates Osteoporosis by Promoting Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells via SITR1/PI3K/AKT Pathway / El Resveratrol Alivia la Osteoporosis al Promover la Diferenciación Osteogénica de las Células Madre Mesenquimales de la Médula Ósea a Través de la Vía SITR1/PI3K/AKT

SUMMARY: Senile osteoporosis is mainly caused by reduced osteoblast differentiation and has become the leading cause of fractures in the elderly worldwide. Natural organics are emerging as a potential option for the prevention and treatment of osteoporosis. This study was designed to study the effect of resveratrol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in osteoporosis mice. A mouse model of osteoporosis was established by subcutaneous injection of dexamethasone and treated with resveratrol administered by gavage. In vivo and in vitro, we used western blot to detect protein expression, and evaluated osteogenic differentiation of BMSCs by detecting the expression of osteogenic differentiation related proteins, calcium deposition, ALP activity and osteocalcin content. Resveratrol treatment significantly increased the body weight of mice, the level of serum Ca2+, 25(OH)D and osteocalcin, ration of bone weight, bone volume/total volume, trabecular thickness, trabecular number, trabecular spacing and cortical thickness in osteoporosis mice. In BMSCs of osteoporosis mice, resveratrol treatment significantly increased the expression of Runx2, osterix (OSX) and osteocalcin (OCN) protein, the level of calcium deposition, ALP activity and osteocalcin content. In addition, resveratrol treatment also significantly increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT in BMSCs of osteoporosis mice. In vitro, resveratrol increased the expression of SIRT1, p-PI3K / PI3K and p-AKT / AKT, Runx2, OSX and OCN protein, the level of calcium deposition, ALP activity and osteocalcin content in BMSCs in a concentration-dependent manner, while SIRT1 knockdown significantly reversed the effect of resveratrol. Resveratrol can attenuate osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells, and the mechanism may be related to the regulation of SIRT1/PI3K/AKT pathway.

La osteoporosis senil es causada principalmente por una diferenciación reducida de osteoblastos y se ha convertido en la principal causa de fracturas en las personas mayores en todo el mundo. Los productos orgánicos naturales están surgiendo como una opción potencial para la prevención y el tratamiento de la osteoporosis. Este estudio fue diseñado para estudiar el efecto del resveratrol en la diferenciación osteogénica de las células madre mesenquimales de la médula ósea (BMSC) en ratones con osteoporosis. Se estableció un modelo de osteoporosis en ratones mediante inyección subcutánea de dexametasona y se trató con resveratrol administrado por sonda. In vivo e in vitro, utilizamos Western blot para detectar la expresión de proteínas y evaluamos la diferenciación osteogénica de BMSC detectando la expresión de proteínas relacionadas con la diferenciación osteogénica, la deposición de calcio, la actividad de ALP y el contenido de osteocalcina. El tratamiento con resveratrol aumentó significativamente el peso corporal de los ratones, el nivel sérico de Ca2+, 25(OH)D y osteocalcina, la proporción de peso óseo, el volumen óseo/ volumen total, el espesor trabecular, el número trabecular, el espaciado trabecular y el espesor cortical en ratones con osteoporosis. En BMSC de ratones con osteoporosis, el tratamiento con resveratrol aumentó significativamente la expresión de las proteínas Runx2, osterix (OSX) y osteocalcina (OCN), el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina. Además, el tratamiento con resveratrol también aumentó significativamente la expresión de SIRT1, p-PI3K/PI3K y p-AKT/AKT en BMSC de ratones con osteoporosis. In vitro, el resveratrol aumentó la expresión de las proteínas SIRT1, p-PI3K/PI3K y p- AKT/AKT, Runx2, OSX y OCN, el nivel de deposición de calcio, la actividad de ALP y el contenido de osteocalcina en BMSC de manera dependiente de la concentración, mientras que La caída de SIRT1 revirtió significativamente el efecto del resveratrol. El resveratrol puede atenuar la osteoporosis al promover la diferenciación osteogénica de las células madre mesenquimales de la médula ósea, y el mecanismo puede estar relacionado con la regulación de la vía SIRT1/PI3K/AKT.

Impact of lithocholic acid on the osteogenic and adipogenic differentiation balance of bone marrow mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To Investigate the effects of lithocholic acid (LCA) on the balance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#Twelve 10-week-old SPF C57BL/6J female mice were randomly divided into an experimental group (undergoing bilateral ovariectomy) and a control group (only removing the same volume of adipose tissue around the ovaries), with 6 mice in each group. The body mass was measured every week after operation. After 4 weeks post-surgery, the weight of mouse uterus was measured, femur specimens of the mice were taken for micro-CT scanning and three-dimensional reconstruction to analyze changes in bone mass. Tibia specimens were taken for HE staining to calculate the number and area of bone marrow adipocytes in the marrow cavity area. ELISA was used to detect the expression of bone turnover markers in the serum. Liver samples were subjected to real-time fluorescence quantitative PCR (RT-qPCR) to detect the expression of key genes related to bile acid metabolism, including cyp7a1, cyp7b1, cyp8b1, and cyp27a1. BMSCs were isolated by centrifugation from 2 C57BL/6J female mice (10-week-old). The third-generation cells were exposed to 0, 1, 10, and 100 μmol/L LCA, following which cell viability was evaluated using the cell counting kit 8 assay. Subsequently, alkaline phosphatase (ALP) staining and oil red O staining were conducted after 7 days of osteogenic and adipogenic induction. RT-qPCR was employed to analyze the expressions of osteogenic-related genes, namely ALP, Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), as well as adipogenic-related genes including Adiponectin (Adipoq), fatty acid binding protein 4 (FABP4), and peroxisome proliferator-activated receptor γ (PPARγ).@*RESULTS@#Compared with the control group, the body mass of the mice in the experimental group increased, the uterus atrophied, the bone mass decreased, the bone marrow fat expanded, and the bone metabolism showed a high bone turnover state. RT-qPCR showed that the expressions of cyp7a1, cyp8b1, and cyp27a1, which were related to the key enzymes of bile acid metabolism in the liver, decreased significantly ( P<0.05), while the expression of cyp7b1 had no significant difference ( P>0.05). Intervention with LCA at concentrations of 1, 10, and 100 μmol/L did not demonstrate any apparent toxic effects on BMSCs. Furthermore, LCA inhibited the expressions of osteogenic-related genes (ALP, Runx2, and OCN) in a dose-dependent manner, resulting in a reduction in ALP staining positive area. Concurrently, LCA promoted the expressions of adipogenic-related genes (Adipoq, FABP4, and PPARγ), and an increase in oil red O staining positive area.@*CONCLUSION@#After menopause, the metabolism of bile acids is altered, and secondary bile acid LCA interferes with the balance of osteogenic and adipogenic differentiation of BMSCs, thereby affecting bone remodelling.

Advances in mesenchymal stem cells therapy for tendinopathies / 中华创伤杂志(英文版)

Tendinopathies are chronic diseases of an unknown etiology and associated with inflammation. Mesenchymal stem cells (MSCs) have emerged as a viable therapeutic option to combat the pathological progression of tendinopathies, not only because of their potential for multidirectional differentiation and self-renewal, but also their excellent immunomodulatory properties. The immunomodulatory effects of MSCs are increasingly being recognized as playing a crucial role in the treatment of tendinopathies, with MSCs being pivotal in regulating the inflammatory microenvironment by modulating the immune response, ultimately contributing to improved tissue repair. This review will discuss the current knowledge regarding the application of MSCs in tendinopathy treatments through the modulation of the immune response.

The expression mechanism of programmed cell death 1 ligand 1 and its role in immunomodulatory ability of mesenchymal stem cells / 中华创伤杂志(英文版)

Programmed cell death 1 ligand 1 (PD-L1) is an important immunosuppressive molecule, which inhibits the function of T cells and other immune cells by binding to the receptor programmed cell death-1. The PD-L1 expression disorder plays an important role in the occurrence, development, and treatment of sepsis or other inflammatory diseases, and has become an important target for the treatment of these diseases. Mesenchymal stem cells (MSCs) are a kind of pluripotent stem cells with multiple differentiation potential. In recent years, MSCs have been found to have a strong immunosuppressive ability and are used to treat various inflammatory insults caused by hyperimmune diseases. Moreover, PD-L1 is deeply involved in the immunosuppressive events of MSCs and plays an important role in the treatment of various diseases. In this review, we will summarize the main regulatory mechanism of PD-L1 expression, and discuss various biological functions of PD-L1 in the immune regulation of MSCs.

Exosomes derived from miR-133a-3p engineered mesenchymal stem cells promote myocardial repair in rats after acute myocardial infarction / 中华心血管病杂志

Objective: To investigate the effects of exosome derived from miR-133a-3p engineered human umbilical cord blood mesenchymal stem cells (ucMSC) on myocardial repair after acute myocardial infarction (AMI) in rats. Methods: UcMSC was amplified and cultured in vitro. Lentiviral carrying miR-133a-3p and negative control vectors were transfected into ucMSC. Exosomes secreted by the transfected ucMSC were named miR-133a-3p-Exo and miR-NC-Exo, respectively. The AMI model of rats was established by ligation of the left anterior descending coronary artery. MiR-133a-3p-Exo or miR-NC-Exo were then injected into the border zone of the infarct area. Cardiac function was assessed by echocardiography after twenty-eight days of intervention, and Masson staining was used to evaluate the area of myocardial fibrosis post-AMI. The myocardial apoptosis after infarction was evaluated by TUNEL staining and the angiogenesis after infarction was evaluated by immunofluorescence staining in the current study. Results: Compared with the miR-NC-Exo group, the left ventricular ejection fraction in the miR-133a-3p-Exo group was significantly increased ((47.4%±9.8%) vs. (64.2%±8.9%), P<0.05). While the myocardial fibrosis area ((31.2%±7.3%) vs. (18.0%±1.5%), P<0.01) and the percentage of apoptotic cardiomyocytes ((25.6%±3.6%) vs. (15.1%±4.4%), P<0.05) was significantly reduced in the miR-133a-Exo group. Besides, the expression of CD31 and α-smooth muscle actin (α-SMA) were also increased significantly in the miR-133a-3p-Exo group compared to the miR-NC-Exo group (CD31: (2.9±0.9) vs. (13.9±2.0), P<0.000 1, α-SMA: (3.5±0.9) vs. (11.0±1.6), P<0.000 1). Conclusion: Exosome derived from miR-133a-3p engineered ucMSC effectively inhibited myocardial apoptosis and promoted angiogenesis, thus improving the cardiac function after myocardial infarction in rats.

Exosomes derived from miR-133a-3p engineered mesenchymal stem cells promote myocardial repair in rats after acute myocardial infarction / 中华心血管病杂志

Objective: To investigate the effects of exosome derived from miR-133a-3p engineered human umbilical cord blood mesenchymal stem cells (ucMSC) on myocardial repair after acute myocardial infarction (AMI) in rats. Methods: UcMSC was amplified and cultured in vitro. Lentiviral carrying miR-133a-3p and negative control vectors were transfected into ucMSC. Exosomes secreted by the transfected ucMSC were named miR-133a-3p-Exo and miR-NC-Exo, respectively. The AMI model of rats was established by ligation of the left anterior descending coronary artery. MiR-133a-3p-Exo or miR-NC-Exo were then injected into the border zone of the infarct area. Cardiac function was assessed by echocardiography after twenty-eight days of intervention, and Masson staining was used to evaluate the area of myocardial fibrosis post-AMI. The myocardial apoptosis after infarction was evaluated by TUNEL staining and the angiogenesis after infarction was evaluated by immunofluorescence staining in the current study. Results: Compared with the miR-NC-Exo group, the left ventricular ejection fraction in the miR-133a-3p-Exo group was significantly increased ((47.4%±9.8%) vs. (64.2%±8.9%), P<0.05). While the myocardial fibrosis area ((31.2%±7.3%) vs. (18.0%±1.5%), P<0.01) and the percentage of apoptotic cardiomyocytes ((25.6%±3.6%) vs. (15.1%±4.4%), P<0.05) was significantly reduced in the miR-133a-Exo group. Besides, the expression of CD31 and α-smooth muscle actin (α-SMA) were also increased significantly in the miR-133a-3p-Exo group compared to the miR-NC-Exo group (CD31: (2.9±0.9) vs. (13.9±2.0), P<0.000 1, α-SMA: (3.5±0.9) vs. (11.0±1.6), P<0.000 1). Conclusion: Exosome derived from miR-133a-3p engineered ucMSC effectively inhibited myocardial apoptosis and promoted angiogenesis, thus improving the cardiac function after myocardial infarction in rats.

Ortobiológicos 2024: definición, elaboración y mecanismo de acción de las alternativas más utilizadas hoy en Ortopedia / Orthobiologics 2024. Definition, manufacturing, and mechanism of action of the most commonly used alternatives currently used in Orthopedics

La ortobiología está emergiendo como una nueva subespecialidad de la Ortopedia, con una aceptación gradual. Si bien el primer impulso del uso masivo de las terapias biológicas en Ortopedia vino de la mano del plasma rico en plaquetas y el concentrado de médula ósea por su elaboración y aplicación fáciles; en la última década, se han producido avances importantes y ha surgido numerosa evidencia clínica sobre los resultados de otras terapias biológicas prometedoras, como el lisado plaquetario, las células mesenquimales derivadas del tejido adiposo y los cultivos celulares. Este artículo tiene como objetivo describir las terapias biológicas más utilizadas actualmente en Ortopedia, con especial énfasis en su proceso de elaboración, su composición y mecanismo de acción. Nivel de Evidencia: V

Orthobiologics is emerging as a new subspecialty of orthopedics, with gradual acceptance. While platelet-rich plasma (PRP) and bone marrow concentrate (BMC) provided the initial catalyst for the widespread use of biological therapies in orthopedics due to their ease of preparation and application, there have been significant advances in the last decade, with numerous clinical evidence emerging on the outcomes of other promising biological therapies such as platelet lysate, adipose-derived stromal vascular fraction cells (SVF), and cell cultures. The following article aims to describe the most widely used biological therapies currently used in orthopedics, with special emphasis on their manufacturing process, composition, and mechanism of action. Level of Evidence: V

Endometrial mesenchymal stromal/stem cells improve regeneration of injured endometrium in mice

BACKGROUND: The monthly regeneration of human endometrial tissue is maintained by the presence of human endometrial mesenchymal stromal/stem cells (eMSC), a cell population co-expressing the perivascular markers CD140b and CD146. Endometrial regeneration is impaired in the presence of intrauterine adhesions, leading to infertility, recurrent pregnancy loss and placental abnormalities. Several types of somatic stem cells have been used to repair the damaged endometrium in animal models, reporting successful pregnancy. However, the ability of endometrial stem cells to repair the damaged endometrium remains unknown. METHODS: Electrocoagulation was applied to the left uterine horn of NOD/SCID mice causing endometrial injury. Human eMSC or PBS was then injected into the left injured horn while the right normal horn served as controls. Mice were sacrificed at different timepoints (Day 3, 7 and 14) and the endometrial morphological changes as well as the degree of endometrial injury and repair were observed by histological staining. Gene expression of various inflammatory markers was assessed using qPCR. The functionality of the repaired endometrium was evaluated by fertility test. RESULTS: Human eMSC successfully incorporated into the injured uterine horn, which displayed significant morphological restoration. Also, endometrium in the eMSC group showed better cell proliferation and glands formation than the PBS group. Although the number of blood vessels were similar between the two groups, gene expression of VEGF-α significantly increased in the eMSC group. Moreover, eMSC had a positive impact on the regeneration of both stromal and epithelial components of the mouse endometrium, indicated by significantly higher vimentin and CK19 protein expression. Reduced endometrial fibrosis and down-regulation of fibrosis markers were also observed in the eMSC group. The eMSC group had a significantly higher gene expression of anti-inflammatory factor Il-10 and lower mRNA level of pro-inflammatory factors Ifng and Il-2, indicating the role of eMSC in regulation of inflammatory reactions. The eMSC group showed higher implantation sites than the PBS group, suggesting better endometrial receptivity with the presence of newly emerged endometrial lining. CONCLUSIONS: Our findings suggest eMSC improves regeneration of injured endometrium in mice.

Fibroblasts inhibit osteogenesis by regulating nuclear-cytoplasmic shuttling of YAP in mesenchymal stem cells and secreting DKK1

BACKGROUND: Fibrous scars frequently form at the sites of bone nonunion when attempts to repair bone fractures have failed. However, the detailed mechanism by which fibroblasts, which are the main components of fibrous scars, impede osteogenesis remains largely unknown. RESULTS: In this study, we found that fibroblasts compete with osteogenesis in both human bone nonunion tissues and BMP2-induced ectopic osteogenesis in a mouse model. Fibroblasts could inhibit the osteoblastic differentiation of mesenchymal stem cells (MSCs) via direct and indirect cell competition. During this process, fibroblasts modulated the nuclear-cytoplasmic shuttling of YAP in MSCs. Knocking down YAP could inhibit osteoblast differentiation of MSCs, while overexpression of nuclear-localized YAP-5SA could reverse the inhibition of osteoblast differentiation of MSCs caused by fibroblasts. Furthermore, fibroblasts secreted DKK1, which further inhibited the formation of calcium nodules during the late stage of osteogenesis but did not affect the early stage of osteogenesis. Thus, fibroblasts could inhibit osteogenesis by regulating YAP localization in MSCs and secreting DKK1. CONCLUSIONS: Our research revealed that fibroblasts could modulate the nuclear-cytoplasmic shuttling of YAP in MSCs, thereby inhibiting their osteoblast differentiation. Fibroblasts could also secrete DKK1, which inhibited calcium nodule formation at the late stage of osteogenesis.

Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

ABSTRACT Objective: To assess the effects of cobalt chloride (CoCl2) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry. Material and Methods: Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl2 on 5th passage cells in a conventional incubator (37°C; 5CO2). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey's HSD. Results: Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group. Conclusion: Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

Tratamiento con metformina previene la adiposidad de la médula ósea femoral inducida por un síndrome metabólico experimental en ratas / Metformin treatment prevents experimental metabolic syndrome-induced femoral bone marrow adiposity in rats

RESUMEN Objetivo. Determinar el efecto de un tratamiento con metformina (MET) sobre la predisposición adipogénica de células progenitoras de médula ósea (CPMO), adiposidad de la médula ósea y propiedades biomecánicas óseas. Materiales y métodos. 20 ratas Wistar machos adultos jóvenes fueron separados en cuatro grupos, recibiendo en agua de bebida: 100% agua (C); 20% de fructosa (F); metformina 100 mg/kg peso/día (M); o fructosa más metformina (FM). Tras cinco semanas se sacrificaron los animales, se diseccionaron ambos húmeros para obtener CPMO, y ambos fémures para evaluar adiposidad medular (histomorfometría) y propiedades biomecánicas (flexión a 3 puntos). Las CPMO se cultivaron in vitro en medio adipogénico para evaluar expresión de RUNX2, PPAR-γ y RAGE por RT-PCR, actividad de lipasa y acumulación de triglicéridos. Resultados. La dieta rica en fructosa (grupo F) produjo un aumento tanto de triglicéridos in vitro, como de la adiposidad medular in vivo; siendo parcial o totalmente prevenido por un co-tratamiento con metformina (grupo FM). No se observaron diferencias en las pruebas biomecánicas femorales in vivo, ni en actividad de lipasa y relación RUNX2/PPAR-γ in vitro. La DRF aumentó la expresión de RAGE en CPMO, siendo prevenido por co-tratamiento con MET. Conclusiones. El síndrome metabólico inducido por una dieta rica en fructosa aumenta la adiposidad medular femoral y, en parte, la predisposición adipogénica de las CPMO. A su vez, esto puede ser prevenido total o parcialmente por un co-tratamiento oral con MET.

ABSTRACT Objective. To determine the effect of metformin (MET) treatment on adipogenic predisposition of bone marrow progenitor cells (BMPC), bone marrow adiposity and bone biomechanical properties. Materials and methods. 20 young adult male Wistar rats were sorted into four groups. Each of the groups received the following in drinking water: 100% water (C); 20% fructose (F); metformin 100 mg/kg wt/day (M); or fructose plus metformin (FM). After five weeks the animals were sacrificed. Both humeri were dissected to obtain BMPC, and both femurs were dissected to evaluate medullary adiposity (histomorphometry) and biomechanical properties (3-point bending). BMPC were cultured in vitro in adipogenic medium to evaluate RUNX2, PPAR-γ and RAGE expression by RT-PCR, lipase activity and triglyceride accumulation. Results. The fructose-rich diet (group F) caused an increase in both triglycerides in vitro, and medullary adiposity in vivo; being partially or totally prevented by co-treatment with metformin (group FM). No differences were found in femoral biomechanical tests in vivo, nor in lipase activity and RUNX2/PPAR-γ ratio in vitro. DRF increased RAGE expression in BMPC, being prevented by co-treatment with MET. Conclusions. Metabolic syndrome induced by a fructose-rich diet increases femoral medullary adiposity and, in part, the adipogenic predisposition of BMPC. In turn, this can be totally or partially prevented by oral co-treatment with MET.

Injection of freshly collected adipose tissue for the treatment complex cryptoglandular anal fistula: case report

Introduction: Perianal fistula is a common colorectal disease which is caused mainly by cryptoglandular disease. Although most cases are treated successfully by surgery, management of complex perianal fistulas (CPAF) remains a challenge with limited results in recurrence and sometimes associated with fecal incontinence. The CPAF treatment with autologous adipose-derived mesenchymal stem cells (ASCs) had become a research hotspot. The technique started to be used in the treatment of Crohn's disease (CD) fistulas, where the studies showed safe and goods result from the procedure. Cultured ASCs have been used but this approach requires the preceding collection of adipose tissue, time for isolation of ASCs and subsequent in vitro expansion, need for laboratory facilities, and expertise in cell culturing. These factors have been getting over by using the commercially available alternative, allogenic ASCs. Treatment with allogeneic ASCs has shown good results in patients with CD fistulas, however with the disadvantage of being expensive. Objective: To show that the injection with freshly collected adipose tissue is an alternative to treatment with autologous or allogenic ASCs with several advantages. Methods: In this case report, we show our first experience in the treatment of CPAF with the application of collected adipose tissue in a tertiary referral hospital from Belo Horizonte, Brazil. Results The patient had a good postoperative recuperation with a complete fistula healing after 8 months without adverse effects. Conclusion: Injection with freshly collected adipose tissue is a promising and apparently safe sphincter-sparing technique in the treatment of CPAF. (AU)

Avaliação in vitro da fotobiomodulação das SHEDs semeadas em um scaffold de osso bovino mineral desproteinizado e colagenado / In vitro evaluation of photobiomodulation of Stem cells from human exfoliated deciduous teeth seeded in a deproteinized bovine bone mineral with 10% collagen scaffold

A regeneração óssea é um processo importante para oferecer tratamentos reconstrutivos mais rápidos e eficientes, no entanto, limitações técnicas continuam sendo um desafio, assim como a velocidade de formação e maturação óssea. Portanto, as pesquisas têm se voltado para técnicas alternativas na regeneração óssea e atualmente, a engenharia tecidual tem estudado o uso de células tronco para tratamento de perdas ósseas. A eficácia e a taxa de sucesso das diferentes técnicas e scaffolds foram avaliadas. Porém, há pouca informação sobre a eficácia combinada de carreadores xenógenos, células tronco de dentes decíduos esfoliados humano (SHEDs) e a terapia de fotobiomodulação (PBMT) na regeneração de defeitos ósseos. Baseado em estudos prévios, a proposta deste estudo foi avaliar, in vitro, a ação da PBMT, uma técnica com propriedades imunomodulatórias, angiogênicas e com capacidade de aumentar a adesão, proliferação e migração celular ao biomaterial tridimensional de osso bovino mineralizado desproteinizado com colágeno suíno a 10% (OBMDC), semeado com SHEDs, para acelerar e aumentar a taxa de formação óssea. Foi utilizado o laser de diodo, com comprimento de onda de 660nm; 40mW de potência; 3J/cm2 de densidade de energia e 2 segundos de tempo de aplicação após 24h e 72h do plaqueamento. Para avaliar a proliferação, as SHEDs foram descongeladas cultivadas, plaqueadas, semeadas no scaffold de OBMDC e divididas em 8 grupos: 1) Controle 15%; 2) Controle 5%; 3) OBMDC 15%; 4) OBMDC 5%; 5) Laser 15%; 6) Laser 5%; 7) OBMDC-L 15%; 8) OBMDC-L 5% e a análise de proliferação foi realizada por MTT. Para avaliar diferenciação celular, as amostras foram divididas em quatro grupos: 1) Grupo Controle clonogênico: SHEDs cultivadas em meio clonogênico; 2) Grupo Controle mineralizante: SHEDs cultivadas em meio mineralizante; 3) Grupo laser clonogênico: SHEDs cultivadas em meio clonogênico com aplicação de laser; 4) Grupo laser mineralizante: SHEDs cultivada em meio mineralizante com aplicação de laser. Para o grupo laser, as células foram irradiadas no período de 24h e 72h após o plaqueamento e todas as amostras fixadas para análise da formação dos depósitos de cálcio, através do ensaio de vermelho de alizarina após 23 dias de cultivo celular e os dados foram tratados estatisticamente (p0,05). Para avaliar a morfologia celular das SHEDs em todos os grupos, utilizou-se o microscópio invertido de fase em 24h e 72h após o plaqueamento. O grupo OBMDC-L 5% SFB em 72h, demonstrou maior proliferação celular que o grupo Controle (p=0.0286). O grupo laser no meio mineralizante apresentou maior formação de depósito de matriz mineralizada em comparação ao grupo controle em meio clonogênico, controle em meio mineralizante e laser em meio clonogênico (p<0,0001). Considerando as condições experimentais deste estudo, concluiu-se que, in vitro, as SHEDs, semeadas em scaffold OBMDC, proliferaram mais após 2 aplicações de PBMT e houve diferenciação osteogênica das células após 23 dias em meio mineralizante.

Effects of 970 nm diode laser irradiation on morphology, proliferation, and differentiation of gingival mesenchymal stem cells / Efectos de la irradiación con láser de diodo de 970 m sobre la morfología, la proliferación y la diferenciación de las células madre mesenquimales gingivales

Objective: The objective of this study was to investigate the morphology, proliferation, and differentiation of gingival mesenchymal stem cells (GMSCs) irradiated with a 970 nm Diode Laser (LLLT). It is essential to validate the efficacy of treatment, optimize irradiation conditions and guarantee the safety and quality of stem cells for future use in dental applications. Materials and Methods: GMSCs were cultured in standard conditions and irradiated with a Diode laser (970 nm, 0.5W) with an energy density of 9J/cm2. Cell proliferation was assessed with the WST-1 proliferation kit. GMSCs were differentiated into chondrogenic and osteogenic lineages. Cell morphology was performed with Hematoxylin/eosin staining, and quantitative nuclear analysis was done. Cell viability was monitored with trypan blue testing. Results: GMSCs subjected to irradiation demonstrated a significant increase in proliferation at 72 hours compared to the non-irradiated controls (p=0.027). This indicates that the 970 nm diode laser has a stimulatory effect on the proliferation of GMSCs. LLLT-stimulated GMSCs exhibited the ability to differentiate into chondrogenic and osteogenic lineages. A substantial decrease in cell viability was observed 24 hours after irradiation (p=0.024). However, after 48 hours, the cell viability recovered without any significant differences. This indicates that there might be a temporary negative impact on cell viability immediately following irradiation, but the cells were able to recover and regain their viability over time. Conclusions: This study support that irradiation with a 970 nm diode laser could stimulate the proliferation of GMSCs, maintain their ability to differentiate into chondrogenic and osteogenic lineages, and has minimal impact on the mor- phological characteristics of the cells. These results support the potential use of NIR Lasers in combination with GMSCs as a promising strategy for dental treatments.

Objetivo: El objetivo de este estudio fue investigar la morfología, proliferación y diferenciación de las células madre mesenquimatosas (GMSC) irradiadas con un láser de diodo de 970 nm (LLLT). Es fundamental validar la eficacia del tratamiento, optimizar las condiciones de irradiación y garantizar la seguridad y calidad de las células madre para su uso futuro en aplicaciones dentales.Materiales y Métodos: Las GMSC se cultivaron en condiciones estándar y se irradiaron con un láser de diodo (970 nm, 0,5 W) con una densidad de energía de 9 J/cm2. La proliferación celular se evaluó con el kit de proliferación WST-1. Las GMSC se diferenciaron en linajes condrogénicos y osteogénicos. La morfología celular se realizó con tinción de hematoxilina/eosina y se realizó un análisis nuclear cuantitativo. La viabilidad celular se controló con prueba de azul de tripano. Resultados: Las GMSC sometidas a irradiación demostraron un aumento significativo en la proliferación a las 72 horas en comparación con los controles no irradiados (p=0,027). Esto indica que el láser de diodo de 970 nm tiene un efecto estimulante sobre la proliferación de GMSC. Las GMSC estimuladas con LLLT exhibieron la capacidad de diferenciarse en linajes condrogénicos y osteogénicos. Se observó una disminución sustancial de la viabilidad celular 24 horas después de la irradiación (p=0,024). Sin embargo, después de 48 horas, la viabilidad celular se recuperó sin diferencias significativas. Esto indica que podría haber un impacto negativo temporal en la viabilidad de las células inmediatamente después de la irradiación, pero las células pudieron recuperarse y recuperar su viabilidad con el tiempo. Conclusión: En conclusión, este estudio respalda que la irradiación con un láser de diodo de 970 nm podría estimular la proliferación de GMSC, mantener su capacidad para diferenciarse en linajes condrogénicos y osteogénicos y tiene un impacto mínimo en las características morfológicas de las células. Estos resultados respaldan el uso potencial de láseres NIR en combinación con GMSC como una estrategia prometedora para tratamientos dentales.

Scientific basis of acupuncture on mesenchymal stem cells for treating ischemic stroke / 中国针灸

The scientific basis of acupuncture on mesenchymal stem cells (MSCs) for treating ischemic stroke (IS) is discussed. MSCs transplantation has great potential for the treatment of tissue damage caused by early stage inflammatory cascade reactions of IS, but its actual transformation is limited by various factors. How to improve the homing efficiency of MSCs is the primary issue to enhance its efficacy. As such, the possible mechanisms of acupuncture and MSCs transplantation in inhibiting inflammatory cascade reactions induced by IS are explored by reviewing literature, and a hypothesis that acupuncture could promote the secretion of stromal cell-derived factor-1α (SDF-1α) from ischemic foci to regulate SDF-1α/CXC chemokine receptor 4 (CXCR4) axis, thereby improving the homing efficiency of MSCs transplantation, exerting its neuroprotective function, and improving the bed transformation ability, is proposed.

Human hair follicle-derived mesenchymal stem cells promote tendon repair in a rabbit Achilles tendinopathy model / 中华医学杂志(英文版)

BACKGROUND@#Hair follicles are easily accessible and contain stem cells with different developmental origins, including mesenchymal stem cells (MSCs), that consequently reveal the potential of human hair follicle (hHF)-derived MSCs in repair and regeneration. However, the role of hHF-MSCs in Achilles tendinopathy (AT) remains unclear. The present study investigated the effects of hHF-MSCs on Achilles tendon repair in rabbits.@*METHODS@#First, we extracted and characterized hHF-MSCs. Then, a rabbit tendinopathy model was constructed to analyze the ability of hHF-MSCs to promote repair in vivo . Anatomical observation and pathological and biomechanical analyses were performed to determine the effect of hHF-MSCs on AT, and quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical staining were performed to explore the molecular mechanisms through which hHF-MSCs affects AT. Furthermore, statistical analyses were performed using independent sample t test, one-way analysis of variance (ANOVA), and one-way repeated measures multivariate ANOVA as appropriate.@*RESULTS@#Flow cytometry, a trilineage-induced differentiation test, confirmed that hHF-derived stem cells were derived from MSCs. The effect of hHF-MSCs on AT revealed that the Achilles tendon was anatomically healthy, as well as the maximum load carried by the Achilles tendon and hydroxyproline proteomic levels were increased. Moreover, collagen I and III were upregulated in rabbit AT treated with hHF-MSCs (compared with AT group; P  < 0.05). Analysis of the molecular mechanisms revealed that hHF-MSCs promoted collagen fiber regeneration, possibly through Tenascin-C (TNC) upregulation and matrix metalloproteinase (MMP)-9 downregulation.@*CONCLUSIONS@#hHF-MSCs can be a treatment modality to promote AT repair in rabbits by upregulating collagen I and III. Further analysis revealed that treatment of AT using hHF-MSCs promoted the regeneration of collagen fiber, possibly because of upregulation of TNC and downregulation of MMP-9, thus suggesting that hHF-MSCs are more promising for AT.

Application of hydrogel-loaded stem cell exosomes in the field of tissue regeneration / 生物工程学报

In recent years, mesenchymal stem cell (MSCs)-derived exosomes have attracted much attention in the field of tissue regeneration. Mesenchymal stem cell-derived exosomes are signaling molecules for communication among cells. They are characterized by natural targeting and low immunogenicity, and are mostly absorbed by cells through the paracrine pathway of mesenchymal stem cells. Moreover, they participate in the regulation and promotion of cell or tissue regeneration. As a scaffold material in regenerative medicine, hydrogel has good biocompatibility and degradability. Combining the two compounds can not only improve the retention time of exosomes at the lesion site, but also improve the dose of exosomes reaching the lesion site by in situ injection, and the therapeutic effect in the lesion area is significant and continuous. This paper summarizes the research results of the interaction of exocrine and hydrogel composite materials to promote tissue repair and regeneration, in order to facilitate research in the field of tissue regeneration in the future.

Establishment and characterization of bone marrow mesenchymal stem cell lines stably synthesizing high-level dopamine / 生物工程学报

A triple-transgenic (tyrosine hydroxylase/dopamine decarboxylase/GTP cyclohydrolase 1, TH/DDC/GCH1) bone marrow mesenchymal stem cell line (BMSCs) capable of stably synthesizing dopamine (DA) transmitters were established to provide experimental evidence for the clinical treatment of Parkinson's disease (PD) by using this cell line. The DA-BMSCs cell line that could stably synthesize and secrete DA transmitters was established by using the triple transgenic recombinant lentivirus. The triple transgenes (TH/DDC/GCH1) expression in DA-BMSCs was detected using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Moreover, the secretion of DA was tested by enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). Chromosome G-banding analysis was used to detect the genetic stability of DA-BMSCs. Subsequently, the DA-BMSCs were stereotactically transplanted into the right medial forebrain bundle (MFB) of Parkinson's rat models to detect their survival and differentiation in the intracerebral microenvironment of PD rats. Apomorphine (APO)-induced rotation test was used to detect the improvement of motor dysfunction in PD rat models with cell transplantation. The TH, DDC and GCH1 were expressed stably and efficiently in the DA-BMSCs cell line, but not expressed in the normal rat BMSCs. The concentration of DA in the cell culture supernatant of the triple transgenic group (DA-BMSCs) and the LV-TH group was extremely significantly higher than that of the standard BMSCs control group (P < 0.000 1). After passage, DA-BMSCs stably produced DA. Karyotype G-banding analysis showed that the vast majority of DA-BMSCs maintained normal diploid karyotypes (94.5%). Moreover, after 4 weeks of transplantation into the brain of PD rats, DA-BMSCs significantly improved the movement disorder of PD rat models, survived in a large amount in the brain microenvironment, differentiated into TH-positive and GFAP-positive cells, and upregulated the DA level in the injured area of the brain. The triple-transgenic DA-BMSCs cell line that stably produced DA, survived in large numbers, and differentiated in the rat brain was successfully established, laying a foundation for the treatment of PD using engineered culture and transplantation of DA-BMSCs.