Follicular unit grafting in chronic ulcers: a valuable technique for integrated management

Abstract Chronic ulcers significantly affect the quality of life of patients and impose a high cost on the healthcare system. The therapeutic management should be comprehensive, taking into consideration the etiological diagnosis of the wound and the characteristics of the wound bed when deciding on a therapeutic proposal appropriate to the healing phase, correcting factors that delay healing. During the epithelialization phase, repair techniques with grafts are recommended to shorten re-epithelialization time, improve the quality of scar tissue, and achieve adequate pain management. Currently, due to the reported benefits of skin appendages, the technique of follicular unit auto-grafting obtained with a scalp punch is among the chosen strategies for wound repair. This is a minimally invasive, outpatient practice, whose technique has advantages over the donor site, patients recovery and well-being.

Organoides en Odontología. Avances en este Nuevo Enfoque de Investigación: Revisión Sistemática Exploratoria

Los Organoides (O) son un tipo de cultivo celular 3D, que reproducen las características morfológicas y funcionales de diversos órganos o tejidos en un entorno in vivo. Se logran a través de la proliferación y diferenciación de Células Madres (CM) en distintas líneas celulares con capacidad de autoorganizarse. Son capaces de reproducir forma, función, expresión génica o repuesta a estímulos de la misma forma que el órgano original. Esto le ha permitido servir de base para múltiples investigaciones en el ámbito médico y odontológico. En los últimos años, se ha podido recrear con éxito, prácticamente, todos los órganos de nuestro cuerpo, como pulmones, hígado, tracto reproductivo, cerebro y muchos otros (Bartfeld, 2021). De la misma forma, son varias las líneas de investigación odontológicas desarrolladas. En específico, la creación de O de órganos orales como dientes y glándulas salivales, son las más reportadas (Oshima et al., 2017). Sin embargo, no son del común conocimiento del odontólogo general. Esta revisión sistemática exploratoria, tiene como objetivo presentar una visión general de la evidencia acumulada, determinado las áreas odontológicas de investigación, así como sus resultados. La investigación odontológica, en base al uso de O, es de alta calidad y de vanguardia, mostrando resultados prometedores, que auguran un gran futuro, tanto para la odontología como para los pacientes.

Organoids (O) are a type of 3D cell culture, which reproduce the morphological and functional characteristics of various organs or tissues in an in vivo environment. They are achieved through the proliferation and differentiation of Stem Cells (SC) into different cell lines with the ability to self-organize. They are capable of reproducing form, function, gene expression, or responses to stimuli in the same way as the original organ. This has allowed it to serve as the basis for multiple investigations in the medical and dental field. In recent years, it has been possible to successfully recreate practically all human organs, such as the lungs, liver, reproductive tract, brain and many others (Bartfeld, 2021). In the same way, there are several lines of dental research developed, specifically, the creation of O from oral organs such as teeth and salivary glands, are the most reported (Oshima et al., 2017). However, they are not common knowledge of the general dentist. This exploratory systematic review aims to present an overview of the accumulated evidence, determining the dental research areas, as well as their results. Dental research, based on the use of O, is of high quality and cutting-edge, showing promising results and a favorable future, both for dentistry and for patients.

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.

Lipotransferencia por decantación asistida con células madre del tejido adiposo para el rejuvenecimiento facial

Introducción: El mito de rejuvenecer o ser bello eternamente es un sueño que la humanidad siempre ha compartido en muchas leyendas. Objetivo: Evaluar los resultados de la lipotransferencia por decantación asistida con células madre del tejido adiposo para el rejuvenecimiento facial. Métodos: Se realizó un estudio descriptivo, prospectivo y longitudinal de 35 pacientes seleccionados por muestreo aleatorio simple, en el Servicio de Cirugía Plástica del Hospital Hermanos Ameijeiras de La Habana, desde septiembre de 2019 hasta igual periodo de 2022. Resultados: En la casuística, la edad media fue de 46,5 ± 11,5 años con valores mínimo de 34 y máximo de 57 años; 48,6 % se encontraban en el grupo etario de 50-59 años. Se constató un predominio del fototipo cutáneo II (60,0 %); en pacientes sanos, el mayor porcentaje con grado de envejecimiento fue el de tipo III (57,1 %). Prevalecieron las arrugas finas en reposo y líneas más profundas con expresión facial (40,0 %) en quienes recibirían lipotransferencia asistida. Posterior al tratamiento se constató mejoría en todos los pacientes; ninguno presentó complicación. La evaluación de este procedimiento resultó ser buena (94,3 %). Conclusiones: La lipotransferencia es un procedimiento mínimamente invasivo con ventajas en cuanto a histocompatibilidad, durabilidad y menor número de complicaciones; tiene una elevada tasa de aceptación. El resultado final favorable, la seguridad y la efectividad se observan en la satisfacción del paciente.

Introduction: The myth to rejuvenate or to be eternally beautiful is a dream that the humanity has always shared in many legends. Objective: To evaluate the results of lipotransference by assisted decantation with stem cells of adipose tissue for the facial rejuvenation. Methods: A descriptive, prospective and longitudinal study was carried out with 35 patients selected by simple random sampling in the Plastic Surgery Service of Hermanos Ameijeiras Hospital in Havana city, from September, 2019 to the same month in 2022. Results: In the case material, the mean age was of 46,5 ± 11,5 years with minimum values of 34 and maximum 57 years; 48.6% was in the 50-59 age group. A prevalence of the II cutaneous phototype was verified (60.0%); in healthy patients, the highest percentage with aging degree was that of type III (57.1%). There was a prevalence of fine wrinkles in rest and deeper lines with facial expression (40.0%) in those who would receive assisted lipotransference. After the treatment improvement was verified in all the patients; none presented complication. The evaluation of this procedure was good (94.3%). Conclusions: Lipotransference is a minimumly invasive procedure with advantages as for histocompatibility, durability and smaller number of complications; it has a high rate of acceptance. The favorable final result, security and effectiveness are observed in the patient's satisfaction.

Neural and Neural Crest-Derived Stem Cell Distribution in the Adult Human Dental Pulp / Distribución de Células Madre Neurales y Derivadas de la Cresta Neural en la Pulpa Dental Humana Adulta

SUMMARY: Despite comprehensive studies and reports about the properties of dental pulp stem cells (DPSCs) in vitro, we still need to confirm whether these in vitro characteristics coincide with the nature of DPSCs in situ. The anatomical location of DPSCs populations in the dental pulp has yet to be investigated. Moreover, the mesenchymal DPSCs have been much more studied than the neural crest-derived DPSCs. In this study, well-recognized neural/neural crest stem cell markers NCAM1, Nestin, SNAIL/SLUG, SOX9, and S100 are being investigated by immunohistochemistry to localize the precise location of these populations of DPSCs within the human adult dental pulp.All previously mentioned markers were expressed in the dental pulp, and their intensity and location of expression were reported.

A pesar de estudios e informes exhaustivos sobre las propiedades de las células madre de la pulpa dental (DPSC) in vitro, todavía necesitamos confirmar si estas características in vitro coinciden con la naturaleza de las DPSC in situ. La ubicación anatómica de las poblaciones de DPSC en la pulpa dental aún no se ha investigado. Además, las DPSC mesenquimales han sido mucho más estudiadas que las DPSC derivadas de la cresta neural. En este estudio, se están investigando mediante inmunohisto química marcadores de células madre de la cresta neural/ neural NCAM1, Nestin, SNAIL/SLUG, SOX9 y S100 para localizar la ubicación precisa de estas poblaciones de DPSC dentro de la pulpa dental humana adulta. Todos los marcadores mencionados anteriormente se expresaron en la pulpa dental y se informó su intensidad y ubicación de expresión.

Células tronco e endodontia regenerativa / Stem cells and regenerative endodontics

Desde o incremento das pesquisas das células-tronco em 1961, por cientistas canadenses, os avanços em estudos, pesquisas e o desenvolvimento de novos tratamentos com esse tipo de recurso se mostram promissores. O uso de células-tronco é uma grande aposta tanto para a medicina quanto para a odontologia regenerativa. Os tratamentos com essa terapia podem oferecer mais qualidade de vida para as pessoas. O potencial dessas células tão especiais se encontra em duas características peculiares: elas são capazes de se multiplicarem e de se diferenciarem em outros tipos de células, como de tecidos, cartilagens e neurônios. É dessa maneira que elas têm um papel fundamental para estudos e tratamentos relacionados à regeneração. O uso de células-tronco na Odontologia torna possível diferentes processos odontológicos que oferecem mais qualidade de vida ao paciente. Isso porque fatores como defeitos genéticos, hábitos nocivos, cáries dentárias e perdas precoces dos dentes contribuem com a perda de dentes ao longo da vida. No início do século XXI, por volta dos anos de 2005, 2006, pesquisadores começaram a publicar em revistas internacionais da área uma nova técnica baseada no uso de célulastronco existentes no osso de sustentação dos dentes e na articulação dento alveolar. Esta técnica, chamada de Revascularização, promove o aparecimento de um novo tecido pulpar sadio, devolvendo ao dente sua vitalidade e higidez(AU)

Since the increase in stem cell research in 1961 by Canadian scientists, advances in studies, research and the development of new treatments with this type of resource have shown promise. The use of stem cells is a big bet for both medicine and regenerative dentistry. Treatments with this therapy can offer more quality of life for people. The potential of these very special cells lies in two peculiar characteristics: they are able to multiply and differentiate into other types of cells, such as tissues, cartilage and neurons. It is in this way that they play a key role for studies and treatments related to regeneration. The use of stem cells in dentistry makes possible different dental processes that offer more quality of life to the patient. That's because factors such as genetic defects, harmful habits, tooth decay, and early tooth loss all contribute to lifelong tooth loss. At the beginning of the twenty-first century, around the years 2005, 2006, researchers began to publish in international journals of the area a new technique based on the use of existing stem cells in the supporting bone of the teeth and in the alveolar tooth joint. This technique, called Revascularization, promotes the appearance of a new healthy pulp tissue, returning to the tooth its vitality and hygiene(AU)

Criopreservação de células-tronco mesenquimais do tecido adiposo: Uma alternativa para lipoenxertia seriada na reconstrução mamária / Cryopreservation of adipose tissue-derived mesenchymal stem cells: An alternative to serial fat grafting in breast reconstruction

Introdução: A lipoenxertia é um enxerto autólogo de células do tecido celular subcutâneo, que pode ser utilizada como técnica complementar na reconstrução mamária. Diante disso, a criopreservação de células-tronco mesenquimais provenientes de tecido adiposo (CTDAs) poderia ser uma maneira de realizar a coleta em um tempo cirúrgico e após realizar a lipoenxertia de forma fracionada. O dimetilsulfóxido (DMSO) é um criopreservante utilizado em pesquisas com células, porém é potencialmente tóxico, o que impossibilitaria a utilização de CTDAs criopreservadas na prática clínica. Novos criopreservantes celulares, sem toxicidade, vêm sendo descritos na literatura científica experimental, como as substâncias L-prolina e trealose. Com isso, esse trabalho teve como objetivo avaliar a viabilidade de CTDAs criopreservadas com a combinação de L-prolina e trealose, em um período de até 90 dias. Método: Estudo experimental, no qual foram obtidas amostras de lipoaspirado provenientes de 9 pacientes. A fração celular foi processada e congelada com L-prolina (1,5M) + trealose (0,2M), ou com DMSO + soro fetal bovino (SFB), como controle. Após 30 e 90 dias, as amostras foram descongeladas e a viabilidade celular foi avaliada pela técnica de MTT. Resultados: A análise das CTDAs, após 1 e 3 meses de congelamento, indicou que as amostras tratadas com L-prolina + trealose apresentaram viabilidade semelhante àquelas preservadas com DMSO e SFB (p=0,444). Conclusão: A associação de L-prolina e trealose manteve CTDA viáveis por 30 e 90 dias de congelamento, podendo ser uma alternativa como criopreservante celular sem toxicidade e viabilizando o uso de lipoenxertia seriada.

Introduction: Fat grafting is an autologous graft of cells from subcutaneous tissue, which can be used as a complementary technique in breast reconstruction. Given this, the cryopreservation of adipose tissue-derived mesenchymal stem cells (ADMSCs) could be a way to collect them in one surgical procedure and after performing fractional fat grafting. Dimethyl sulfoxide (DMSO) is a cryopreservative used in cell research, but it is potentially toxic, which would make it impossible to use cryopreserved ADMSCs in clinical practice. New cellular cryopreservatives, without toxicity, have been described in the experimental scientific literature, such as the substances L-proline and trehalose. Therefore, this work aimed to evaluate the viability of ADMSCs cryopreserved with the combination of L-proline and trehalose over up to 90 days. Method: Experimental study in which lipoaspirate samples were obtained from 9 patients. The cellular fraction was processed and frozen with L-proline (1.5M) + trehalose (0.2M) or with DMSO + fetal bovine serum (FBS) as control. After 30 and 90 days, the samples were thawed, and cell viability was assessed using the MTT technique. Results: The analysis of ADMSCs, after 1 and 3 months of freezing, indicated that samples treated with L-proline + trehalose showed similar viability to those preserved with DMSO and SFB (p=0.444). Conclusion: The association of L-proline and trehalose kept ADMSC viable for 30 and 90 days of freezing, and could be an alternative as a cellular cryopreservative without toxicity and enabling the use of serial fat grafting.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

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.

Research progress on mesenchymal stem cell products and their exosomes in the treatment of inflammatory bowel disease / 中国药科大学学报

@#Abstract: Inflammatory bowel disease (IBD), whose pathogenesis remains elusive, is a group of autoimmune diseases characterized by chronic, progressive, and lifelong inflammation of the digestive tract. The pathogenesis of IBD remains elusive. Although a number of drugs have been developed to treat IBD, their effects are merely anti-inflammatory. In addition, current treatments for IBD are easily susceptible to resistance in clinical practice. Mesenchymal stem cells (MSCs) have been reported to have the ability to migrate to the site of inflammation, with potent immunoregulatory effects, and to rebalance the immune microenvironment and restore the integrity of the epithelial barrier with significant value of application, particularly for patients who are refractory to classic medicines. In this paper, we reviewed the clinical applications, mechanisms and engineerable properties of MSC products and their exosomes to provide some reference for the use of MSCs and their exosomes in the treatment of IBD.

Mechanism of Wenfei Huaxian Decoction-containing Serum in Delaying Inflammatory Senescence of Lung Mesenchymal Stem Cells Based on NAMPT/SIRT1 / 中国实验方剂学杂志

ObjectiveThe lung mesenchymal stem cells （LMSCs） induced by D-galactose （D-gal） were intervened by Wenfei Huaxian decoction-containing serum to explore the mechanism of Wenfei Huaxian decoction in delaying the senescence of LMSCs through the nicotinamide phosphoribosyltransferase/silent information regulator 1 （NAMPT/SIRT1） signaling pathway. MethodWenfei Huaxian decoction-containing serum was prepared. LMSCs were isolated by gradient density centrifugation， and they were cultured and identified in vitro. The senescence model in vitro was established by stimulating cells via D-gal for 24 h. LMSCs cells were modeled after being treated with different volume fractions （5%， 10%， 20%， 40%， and 80%） of Wenfei Huaxian decoction-containing serum for 24 h， and the cell proliferation level was detected by methyl thiazolyl tetrazolium （MTT） method. The cells were randomly divided into blank serum group， model group， and high， medium， and low dose groups of Wenfei Huaxian decoction-containing serum. Senescence-associated β-galactosidase （SA-β-gal） staining was used to detect the senescence of LMSCs in each group. The content of NAD + was detected by colorimetry. The levels of senescence-associated factors （p16 and p53）， interleukin-6 （IL-6）， and tumor necrosis factor-α （TNF-α） in cell culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the relative expression of senescence-associated proteins and NAMPT/SIRT1 signaling pathway-related proteins. ResultCompared with the blank serum group， the proliferation of LMSCs was significantly inhibited after D-gal stimulation for 24 h （P<0.01）. Compared with the model group， the proliferation of LMSCs could be promoted after intervention with the corresponding Wenfei Huaxian decoction-containing serum （P<0.05， P<0.01）. Compared with the blank serum group， the SA-β-gal staining of LMSCs in the model group after D-gal stimulation was enhanced， and the content of NAD+ was increased （P<0.01）. The expression levels of senescence factors p16 and p53， as well as SASP pro-inflammatory factors IL-6 and TNF-α in the cell culture supernatant， were significantly increased （P<0.01）. The expression of senescence-associated proteins p16， p21， and p53 increased （P<0.01）， and the protein expression of NAMPT， SIRT1， peroxisome proliferator-activated receptor γ coactivator 1α （PGC-1α）， and forkhead box family transcription factor O1 （FoxO1） decreased （P<0.01）. Compared with the model group， the SA-β-gal staining of LMSCs in each group of Wenfei Huaxian decoction-containing serum was significantly reduced， and the content of NAD+ was decreased （P<0.01）. The senescence factors （p16 and p53） and inflammatory factors （IL-6 and TNF-α） in the cell culture supernatant were significantly decreased （P<0.01）. The expression of senescence-associated proteins （P16， P21， and P53） decreased （P<0.05， P<0.01）. The protein expressions of NAMPT， SIRT1， PGC-1α， and FoxO1 were significantly up-regulated （P<0.05， P<0.01）. ConclusionWenfei Huaxian decoction can alleviate senescence and inflammatory response damage of D-gal-induced LMSCs， and its mechanism may be related to the regulation of the NAMPT/SIRT1 signaling pathway.

Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells / 北京大学学报(医学版)

Objective:To explore the effect of ubiquitin-specific protease 42(USP42)on osteogenic differentiation of human adipose-derived stem cells(hASCs)in vivo and in vitro.Methods:A combina-tion of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy.Alkaline phosphatase(ALP)staining and quantification,alizarin red S(ARS)staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group(knockdown group and overexpression group)and the control group.Quantitative re-verse transcription PCR(qRT-PCR)was used to detect the expression levels of osteogenesis related genes in the experimental group and control group,and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group.Nude mice ectopic im-plantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo.Results:The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group,and those in overexpression group were significantly higher than those in control group.After 7 days of osteogenic induction,the ALP activity in the knockdown group was sig-nificantly higher than that in the control group,and ALP activity in overexpression group was significantly lower than that in control group.After 14 days of osteogenic induction,ARS staining was significantly deeper in the knockdown group than in the control group,and significantly lighter in overexpression group than in the control group.The results of qRT-PCR showed that the mRNA expression levels of ALP,os-terix(OSX)and collagen type Ⅰ(COL Ⅰ)in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction,and those in overexpression group were signifi-cantly lower than those in control group.The results of Western blotting showed that the expression levels of runt-related transcription factor 2(RUNX2),OSX and COL Ⅰ in the knockout group were significant-ly higher than those in the control group at 14 days after osteogenic induction,while the expression levels of RUNX2,OSX and COL Ⅰ in the overexpression group were significantly lower than those in the control group.Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group.Conclusion:Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vi-vo,and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs,and USP42 can provide a potential therapeutic target for bone tissue engineering.

Developmental toxicity of Cry1Ab protein in the embryonic stem-cell model / 北京大学学报(医学版)

Objective:To evaluate the developmental toxicity of Cry1Ab protein by studying its effects on cell proliferation and differentiation ability using a developmental toxicity assessment model based on embryonic stem-cell.Methods:Cry1Ab protein was tested in seven dose groups(31.25,62.50,125.00,250.00,320.00,1 000.00,and 2 000.00 μg/L)on mouse embryonic stem cells D3(ES-D3)and 3T3 mouse fibroblast cells,with 5-fluorouracil(5-FU)used as the positive control and phos-phate buffer saline(PBS)as the solvent control.Cell viability was detected by CCK-8 assay to calculate the 50％inhibitory concentration(IC50)of the test substance for different cells.Additionally,Cry1 Ab protein was tested in five dose groups(125.00,250.00,320.00,1 000.00,and 2 000.00 μg/L)on ES-D3 cells,with PBS as the solvent control and 5-FU used for model validation.After cell treatment,cardiac differentiation was induced using the embryonic bodies(EBs)culture method.The growth of EBs was observed under a microscope,and their diameters on the third and fifth days were measured.The proportion of EBs differentiating into beating cardiomyocytes was recorded,and the 50％inhibition con-centration of differentiation(ID50)was calculated.Based on a developmental toxicity discrimination func-tion,the developmental toxicity of the test substances was classified.Furthermore,at the end of the cul-ture period,mRNA expression levels of cardiac differentiation-related markers(Oct3/4,GATAA-4,Nkx2.5,and β-MHC)were quantitatively detected using real-time quantitative polymerase chain reaction(qPCR)in the collected EBs samples.Results:The IC50 of 5-FU was determined as 46.37 μg/L in 3T3 cells and 32.67 μg/L in ES-D3 cells,while the ID50 in ES-D3 cells was 21.28 μg/L.According to the discrimination function results,5-FU was classified as a strong embryotoxic substance.There were no sta-tistically significant differences in cell viability between different concentrations of Cry 1 Ab protein treat-ment groups and the control group in both 3T3 cells and ES-D3 cells(P＞0.05).Moreover,there were no statistically significant differences in the diameter of EBs on the third and fifth days,as well as their morphology,between the Cry1Ab protein treatment groups and the control group(P＞0.05).The cardi-ac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group(P＞0.05).5-FU significantly reduced the mRNA expression levels of β-MHC,Nkx2.5,and GATA-4(P＜0.05),showing a dose-dependent trend(P＜0.05),while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend(P＜0.05).However,there were no statistically significant differences in the mRNA expression levels of mature cardiac marker β-MHC,early cardiac differentiation marker Nkx2.5 and GATA-4,and pluripotency-associated marker Oct3/4 between the Cry1Ab protein treatment groups and the control group(P＞0.05).Conclusion:No developmental toxicity of Cry1Ab protein at concen-trations ranging from 31.25 to 2 000.00 μg/L was observed in this experimental model.

Honokiol inhibits the proliferation of human adipose-derived mesenchymal stem cells / 基础医学与临床

Objective To investigate the effects of honokiol on proliferation and apoptosis of human adipose-derived mesenchymal stem cells(hADSCs),and to investigate the effect of the drug on the tumor microenvironment.Methods hADSCs were incubated with different concentrations of honokiol,the proliferation of hADSCs was detec-ted by MTS and Trypan blue staining,and cell apoptosis was assessed by annexin V/PI double staining.In the meantime,expression of mRNA and protein related to cell proliferation and apoptosis were detected by qPCR and Western blot,respectively.The expression of total MEK,phosphorylated MEK,total ERK and phosphorylated ERK proteins in the MEK-ERK1/2 signaling pathway were detected by Western blot.Results The effect of honokiol on inhibiting proliferation and promoting apoptosis of hADSCs was significantly enhanced with the increase of concen-tration.The expressions of proliferation-related genes CCND1,MKI67 and PCNA were down-regulated.The expres-sions of pro-apoptotic genes BAX and TP53 was up-regulated,and the expressions of anti-apoptotic gene BCL2 was down-regulated.Honokiol inhibited MEK and ERK1/2 phosphorylation in a concentration-dependent manner.Conclusions Honokiol inhibits proliferation and promotes apoptosis of hADSCs,and the specific mechanism is po-tentially related to the inhibition of MEK-ERK1/2 pathway.

Effect and mechanism of transplantation bone marrow stromal stem cells transfected NRG1 gene on repair of hemi-transected spinal cord injury in rats / 解放军医学杂志

Objective To investigate the effect and mechanism of transplantation of neuregulin1(NRG1)gene-modified bone marrow mesenchymal stem cells(BMSCs)on the repair of hemi-transected spinal cord injury(SCI)in rats.Methods Isolated and cultured rat BMSCs,followed by transfection with the NRG1 gene.The levels of NRG1 in BMSCs lysate and culture supernatant was deected by ELISA method,and the proliferation activity of the BMSCs was detected by cell counting method.Forty-three healthy 8-week-old SD rats were randomly divided into control group(n=10),SCI model group(n=10),BMSCs group(n=10),and NRG1-BMSCs group(n=13).After establishing the spinal cord hemisection model,animals received in-situ transplantation of BMSCs or NRG1-BMSCs.On the 1,7,14,21,and 28 days after transplantation,the hind limb motor function was evaluated using BBB score and inclined plate test;on the 7th day after transplantation,the migration and distribution of transplanted cells was monitored using a fluorescence microscope;on the 28th day after transplantation,the pathological changes of rat spinal cord tissues was examined using HE staining and Nissl staining;cell apoptosis using TUNEL staining,and levels of endoplasmic reticulum stress-related proteins[X-box binding protein 1(XBP1),C/EBP homologous protein(CHOP),activating transcription factor 4(ATF4),ATF6,glucose-regulated protein 78(GRP78)]and apoptosis-related proteins[B-cell lymphoma-2(Bcl-2)and Bcl-2-associated protein X(Bax)]in rat spinal cord tissues using Western blotting.Results BMSCs were successfully isolated,cultured,and transfected with the NRG1 gene.ELISA method results showed that the NRG1 contents in the NRG1-BMSCs lysate and culture supernatant were significantly higher than that of BMSCs in a time-dependent manner(P<0.05).The proliferation activity of NRG1-BMSCs was significantly higher than that of BMSCs(P<0.05).On the 21 and 28 days after transplantation,the BBB score and the slope angle of the inclined plate in NRG1-BMSCs group were higher than those in SCI model group or BMSCs group(P<0.05).However,it did not reverse to the level in control group(P<0.05).On the 28th day after transplantation,compared with the SCI model group and BMSCs group,neuronal pyknosis reduced,the Nissl body density increased,the expression levels of XBP1,CHOP,ATF4,ATF6,GRP78,and Bax,and the rate of TUNEL-positive cells significantly reduced in NRG1-BMSCs group(P<0.05),and the expression level of Bcl-2 significantly increased(P<0.05).Conclusion Transplantation of NRG1 gene-modified BMSCs can alleviate SCI and improve the recovery of motor function in rats.The mechanism may be related to promoting the proliferation activity of BMSCs,inhibiting cell apoptosis,and mitigating endoplasmic reticulum stress.

Deubiquitinase Mysm1 regulates differentiation of neural stem cells into astrocytes by regulating expressions of glial fibrillary acidic protein / 军事医学

Objective To investigate the effect of Mysm1 on the differentiation of neural stem cells(NSCs)into astrocytes and the possible mechanism.Methods NSCs were prepared from E12.5 cortices of wild-type C57BL/6 mice,cultured in vitro and induced to differentiate into astrocytes.Immunofluorescence staining,real-time quantitative PCR and Western blot assay were used to detect the expressions of Mysm1 during the differentiation of NSCs into astrocytes in vitro.Lentivirus was used to knock down Mysm1 expressions in NSCs before real-time quantitative PCR and Western blot assay were used to detect the knockdown efficiency.Immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after Mysm1 knockdown in vitro.Transcriptomics was adopted to detect the differential gene after knockdown of Mysm1 in NSCs in vitro.Western blot assay was used to verify the changes of proteins associated with the differential gene.Cut-Tag was used to detect the enrichment of Mysm1 in the promoter region of glial fibrillary acidic protein(GFAP)genes during the differentiation of NSCs into astrocytes in vitro.After overexpression of GFAP following knockdown of Mysm1,immunofluorescence staining and Western blot assay were used to compare the differentiation of NSCs into astrocytes before and after overexpression in vitro.Results The expression of Mysm1 was gradually increased when NSCs were induced to differentiate into astrocytes in vitro.Mysm1 knockdown inhibited the differentiation of NSCs into astrocytes in vitro.Mysm1 affected the differentiation of NSCs into astrocytes by regulating the expression of GFAP.Overexpression of GFAP after Mysm1 knockdown partially rescued the ability of NSCs to differentiate into astrocytes.Conclusion Mysm1 regulates the differentiation of NSCs into astrocytes by epigenetically controlling GFAP transcription.

Zoledronic acid affects osteogenic differentiation and bone formation by regulating the expression of SIRT3/P53 / 中华内分泌外科杂志

Objective:To explore the mechanism of zoledronic acid (ZOL) affects osteogenic differentiation and bone formation through the regulation of sirtuin 3 (SIRT3) / P53 expression.Methods:Bone marrow mesenchymal stem cells (BMSCs) were induced to differentiate into osteogenic cells, the expression of SIRT3 in the cells was detected, and the targeting regulation relationship between SIRT3 and P53 was analyzed. The intracellular expressions of SIRT3 and P53 were intervened and ZOL was used to treat the cells. MTT method, Western blot method and kit were used to detect cell viability, osteogenesis-related genes Osteoprotegerin (OPG), runt-related transcription factor 2 (Runx2) expression, alkaline phosphatase (ALP) activity and alizarin red S (ARS) staining, respectively. Ovariectomy (OVX) was used to construct a rat model and explore the effect of ZOL on the progression of osteoporosis (OP) in vivo.Results:ZOL promoted osteogenic differentiation of BMSCs. The expression of SIRT3 was down-regulated in the serum of OP patients (0.78±0.23) compared with that of healthy subjects (1.00±0.26 vs. 0.78±0.23. t=3.85, P<0.001). During the osteogenic differentiation of BMSCs, the expression level of SIRT3 gradually increased with the prolonged induction of osteogenesis. Compared with the p53 protein expression and BMSCs activity in the control group, SIRT3 knockout could increase the expression level of p53 protein (0.59±0.05 vs. 1.01±0.11. t=6.02, P=0.004) but inhibited the activity of BMSCs (100.00±8.41 vs. 51.26±5.59. t=8.36, P=0.001). After ZOL treatment, the inhibitory effect of SIRT3 on cell viability (49.61±5.11 vs. 87.61±7.31. t=7.38, P=0.002) and osteogenesis was relieved, and the level of P53 was inhibited (1.10±0.10 vs. 0.69±0.04. t=6.59, P=0.003). P53 overexpression partially offseted the effects of ZOL on cell viability (84.61±6.52 vs. 66.54±5.47. t=3.68, P=0.021) and osteogenesis. Compared with the sham surgery group, the OVX group showed inhibition of osteogenesis in rats, and ZOL treatment significantly improved osteogenic inhibition. ZOL treatment increased the expression level of SIRT3 protein in bone tissue of OVX rats, but inhibited the expression level of P53. Conclusion:ZOL promoted osteogenic differentiation and bone formation of BMSCs by promoting the ubiquitination and degradation of P53 by SIRT3.

Research progress on immunomodulatory properties of periodontal ligament stem cells / 口腔疾病防治

@#Periodontal ligament stem cells (PDLSCs) have the potential for multidirectional differentiation and are the preferred seed cells for periodontal tissue regeneration. In recent years, a large number of studies have confirmed that PDLSCs also possess broad immunomodulatory properties. Therefore, in-depth exploration of their specific molecular mechanisms is of great significance for the treatment of periodontitis. The aim of this paper is to summarize the research progress on the regulation of PDLSCs on various immune cells and the effect of the inflammatory environment on the immune characteristics of PDLSCs to provide an important theoretical basis for the allotransplantation of PDLSCs and improve the therapeutic effect of periodontal tissue regeneration. Studies have shown that PDLSCs possess a certain degree of immunosuppressive effect on both innate and acquired immune cells, and inflammatory stimulation may lead to the impairment of the immunoregulatory properties of PDLSCs. However, current studies are mainly limited to in vitro cell tests and lack in-depth studies on the immunomodulatory effects of PDLSCs in vivo. In vivo studies based on cell lineage tracing and conditional gene knockout technology may become the main directions for future research.

Potential of human induced pluripotent stem cells differentiating into corneal epithelial cells in simulated limbal stem cell microenvironment / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the potential of human induced pluripotent stem cells(hiPSCs)differentiating into corneal epithelial cells in the simulated limbal stem cells(LSCs)microenvironment.METHODS: The hiPSC cell lines were established in vitro, and hiPSCs were co-cultured with corneal stromal cells in transwell system, which simulated the LSC microenvironment. Bone morphogenetic protein 4(BMP4)and a specific transforming growth factor β inhibitor(SB431542)were added to improve the differentiation efficacy. The expression of corneal epithelial cell-specific markers CK3 and CK12, corneal epithelial cell precursor CK15, and the limbal stem cell markers ABCG5 were determined by immunofluorescence staining and flow cytometry.RESULTS: The hiPSCs were actively proliferated in vitro, and immunofluorescence staining showed positive stem cell-specific markers OCT4, SOX2, TRA-1-60 and NANOG. Furthermore, hiPSCs co-cultured with corneal stromal cells exhibited LSCs markers ABCG5 and corneal epithelial cell precursor markers CK15 were positive; however, corneal epithelial cell markers CK3 and CK12 were negative. With the addition of BMP4 and SB431542, hiPSCs showed positive expression of CK3, and the CK3 expression increased over the time.CONCLUSION: With the addition of SB431542 and BMP4, hiPSCs cultured in simulated LSCs microenvironment could differentiate into corneal epithelial cells.

Research progress of mesenchymal stem cell-derived exosomes in the treatment of dry eye / 国际眼科杂志(Guoji Yanke Zazhi)

Exosomes are extracellular vesicles that facilitate cellular communication by transmitting biomolecules and altering the biochemical characteristics of receptor cells. Mesenchymal stem cell-derived exosomes(MSC-Exos)are lipid bilayer vesicles secreted by mesenchymal stem cells(MSCs). These exosomes have similar functions to MSCs and contain bioactive substances such as proteins, lipids, and nucleic acids. MSC-Exos play a vital role in intercellular communication and are involved in essential physiological processes including immune regulation, tissue damage repair, and angiogenesis promotion. Consequently, they have gained significant attention in research, particularly in the treatment of immune inflammatory diseases, ischemic diseases, and other related fields. This article provides an in-depth analysis of the potential treatment mechanisms for dry eye, focusing on the pathogenesis of the condition, including inflammatory reactions, nerve regeneration, and tissue repair. The objective is to establish a foundation for the application of MSC-Exos in the treatment of dry eye, thereby offering a valuable reference for the future clinical applications.