Low-frequency pulsed electromagnetic fields promote osteoblast mineralization and maturation of rats through the PC2/sAC/PKA/CREB signaling pathway / 南方医科大学学报

OBJECTIVE@#To explore whether the effect of low-frequency pulsed electromagnetic fields (PEMFs) in promoting osteoblast mineralization and maturation is related to the primary cilia, polycystin2 (PC2) and sAC/PKA/CREB signaling pathway.@*METHODS@#We detected the expression levels of PC2, sAC, PKA, CREB and their phosphorylated proteins in primary rat calvarial osteoblasts exposed to 50 Hz 0.6 mT PEMFs for 0, 5, 15, 30, 60, 90, and 120 min. We blocked PC2 function with amiloride hydrochloride and detected the changes in the activity of sAC/PKA/CREB signal pathway and the mineralization and maturation of the osteoblasts. These examinations were repeated in the osteoblasts after specific knockdown of PC2 via RNA interference and were the co-localization of PC2, sAC, PKA, CREB and their phosphorylated proteins with the primary cilia were using immunofluorescence staining. The expressions of PC2 and the signaling proteins of sAC/PKA/CREB pathway were detected after inhibition of primary ciliation by RNA interference.@*RESULTS@#The expression levels of PC2, sAC, p-PKA and p- CREB were significantly increased in the osteoblasts after exposure to PEMFs for different time lengths (P < 0.01). Blocking PC2 function or PC2 knockdown in the osteoblasts resulted in failure of sAC/PKA/CREB signaling pathway activation and arrest of osteoblast mineralization and maturation. PC2, sAC, p-PKA and p-CREB were localized to the entire primary cilia or its roots, but PKA and CREB were not detected in the primary cilia. After interference of the primary cilia, PEMFs exposure no longer caused increase of PC2 expression and failed to activate the sAC/PKA/CREB signaling pathway or promote osteoblast mineralization and maturation.@*CONCLUSION@#PC2, located on the surface of the primary cilia of osteoblasts, can perceive and transmit the physical signals from PEMFs and promote the mineralization and maturation of osteoblasts by activating the PC2/ sAC/PKA/CREB signaling pathway.

circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis / 国际口腔科学杂志·英文版

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue regeneration. Circular RNAs (circRNAs) have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis. However, how circRNAs regulate hASCs in osteogenesis is still unclear. Herein, we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs. Knockdown of circ_0003204 by siRNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs. We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p. We predicted and confirmed that miR-370-3p had targets in the 3'-UTR of HDAC4 mRNA. The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis. Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model, while overexpression of circ_0003204 inhibited bone defect repair. Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

Co-regulation of circadian clock genes and microRNAs in bone metabolism / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Mammalian bone is constantly metabolized from the embryonic stage, and the maintenance of bone health depends on the dynamic balance between bone resorption and bone formation, mediated by osteoclasts and osteoblasts. It is widely recognized that circadian clock genes can regulate bone metabolism. In recent years, the regulation of bone metabolism by non-coding RNAs has become a hotspot of research. MicroRNAs can participate in bone catabolism and anabolism by targeting key factors related to bone metabolism, including circadian clock genes. However, research in this field has been conducted only in recent years and the mechanisms involved are not yet well established. Recent studies have focused on how to target circadian clock genes to treat some diseases, such as autoimmune diseases, but few have focused on the co-regulation of circadian clock genes and microRNAs in bone metabolic diseases. Therefore, in this paper we review the progress of research on the co-regulation of bone metabolism by circadian clock genes and microRNAs, aiming to provide new ideas for the prevention and treatment of bone metabolic diseases such as osteoporosis.

Dental pulp stem cells as a promising model to study imprinting diseases / 国际口腔科学杂志·英文版

Parental imprinting is an epigenetic process leading to monoallelic expression of certain genes depending on their parental origin. Imprinting diseases are characterized by growth and metabolic issues starting from birth to adulthood. They are mainly due to methylation defects in imprinting control region that drive the abnormal expression of imprinted genes. We currently lack relevant animal or cellular models to unravel the pathophysiology of growth failure in these diseases. We aimed to characterize the methylation of imprinting regions in dental pulp stem cells and during their differentiation in osteogenic cells (involved in growth regulation) to assess the interest of this cells in modeling imprinting diseases. We collected dental pulp stem cells from five controls and four patients (three with Silver-Russell syndrome and one with Beckwith-Wiedemann syndrome). Methylation analysis of imprinting control regions involved in these syndromes showed a normal profile in controls and the imprinting defect in patients. These results were maintained in dental pulp stem cells cultured under osteogenic conditions. Furthermore, we confirmed the same pattern in six other loci involved in imprinting diseases in humans. We also confirmed monoallelic expression of H19 (an imprinted gene) in controls and its biallelic expression in one patient. Extensive imprinting control regions methylation analysis shows the strong potential of dental pulp stem cells in modeling imprinting diseases, in which imprinting regions are preserved in culture and during osteogenic differentiation. This will allow to perform in vitro functional and therapeutic tests in cells derived from dental pulp stem cells and generate other cell-types.

Application of RUNX2 gene over expression vector modified exosomes from BMSC combined with calcium carbonate scaffold system in bone defect / 中国骨伤

OBJECTIVE@#To investigate the effect of RUNX2 gene overexpression vector modified exosomes derived from bone marrow mesenchymal stem cells (BMSCs) combined with calcium carbonate scaffold system in bone defect.@*METHODS@#Rabbit BMSCs were used as the research object, and BMSCs were identified by flow cytometry. Construct RUNX2 gene overexpression vector, transfect BMSCs with lentivirus, and collect exosomes by ultracentrifugation. The morphology of exosomes was observed by transmission electron microscope, the expression of exosome marker CD63 was detected by Western blot, and the calcium carbonate scaffold was constructed by three chamber parallel automatic temperature control reaction system. According to whether the RUNX2 gene overexpression vector was transfected or not, the complex of BMSCs and calcium carbonate scaffold was divided into three groups, namely BMSCs group, RUNX2 overexpression group and exosome group. The osteogenic differentiation of BMSCs was detected by oil red O staining and RT-PCR. There were 9 clean adult healthy male New Zealand white rabbits, aged (12.97±1.21) months, with a body weight of (19.3±3.6) kg, with 3 rabbits in each group. The animal model of skull defect was constructed by surgical method, and the repair of bone defect was evaluated by imaging, he staining and Masson staining.@*RESULTS@#The results of flow cytometry showed that the expression of CD29 protein, CD44 protein, CD11b protein and CD45 protein on the surface of BMSCs were 99.5%, 100%, 0.1% and 0.1%, respectively. Transmission electron microscopy showed that the exosomes were bilayer vesicles with a diameter of 50 to 150 nm. Western blot showed that the molecular marker CD63 of exosomes was positive. Oil red O staining showed that the osteogenic differentiation of BMSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group (P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group (P<0.05). MSCs in exosome group was significantly higher than that in RUNX2 overexpression group and BMSCs group. The results of RT-PCR showed that the relative expressions of RUNX2, BMP-2 and ALP mRNA in BMSCs in exosome group were significantly higher than those in RUNX2 overexpression group and BMSCs group(P<0.05). The imaging results showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group. HE staining and Masson staining showed that the repair effect of skull defect in exosome group was better than that in RUNX2 overexpression group(P<0.05).@*CONCLUSION@#Compared with RUNX2 gene overexpression vector transfection, extraction of exosomes directly can promote the differentiation of BMSCs into osteoblasts more efficiently, and the combination with calcium carbonate scaffold can better promote the healing of bone defects. So as to provide new ideas and methods for the clinical treatment of bone defects.

Effect of polycystin2 on differentiation and maturation of osteoblasts promoted by low-frequency pulsed electromagnetic fields / 生物工程学报

It is known that low-frequency pulsed electromagnetic fields (PEMFs) can promote the differentiation and maturation of rat calvarial osteoblasts (ROBs) cultured in vitro. However, the mechanism that how ROBs perceive the physical signals of PEMFs and initiate osteogenic differentiation remains unknown. In this study, we investigated the relationship between the promotion of osteogenic differentiation of ROBs by 0.6 mT 50 Hz PEMFs and the presence of polycystin2 (PC2) located on the primary cilia on the surface of ROBs. First, immunofluorescence staining was used to study whether PC2 is located in the primary cilia of ROBs, and then the changes of PC2 protein expression in ROBs upon treatment with PEMFs for different time were detected by Western blotting. Subsequently, we detected the expression of PC2 protein by Western blotting and the effect of PEMFs on the activity of alkaline phosphatase (ALP), as well as the expression of Runx-2, Bmp-2, Col-1 and Osx proteins and genes related to bone formation after pretreating ROBs with amiloride HCl (AMI), a PC2 blocker. Moreover, we detected the expression of genes related to bone formation after inhibiting the expression of PC2 in ROBs using RNA interference. The results showed that PC2 was localized on the primary cilia of ROBs, and PEMFs treatment increased the expression of PC2 protein. When PC2 was blocked by AMI, PEMFs could no longer increase PC2 protein expression and ALP activity, and the promotion effect of PEMFs on osteogenic related protein and gene expression was also offset. After inhibiting the expression of PC2 using RNA interference, PEMFs can no longer increase the expression of genes related to bone formation. The results showed that PC2, located on the surface of primary cilia of osteoblasts, plays an indispensable role in perceiving and transmitting the physical signals from PEMFs, and the promotion of osteogenic differentiation of ROBs by PEMFs depends on the existence of PC2. This study may help to elucidate the mechanism underlying the promotion of bone formation and osteoporosis treatment in low-frequency PEMFs.

Lentivirus mediated siRNA hsa-circ-0000885 transfection of BMSCs and osteoclast co-culture system on cell differentiation, proliferation and apoptosis / 中国骨伤

OBJECTIVE@#To explore the effects of siRNA hsa-circ-0000885 modified bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation, cell proliferation and apoptosis in order to provide new ideas and methods for the clinical treatment of osteoporosis (OP).@*METHODS@#From September 2018 to February 2020, 13 patients with osteoporosis admitted to our hospital were selected as the research objects, including 11 females and 2 males, with an age of (65.45±10.77) years old. After obtaining the informed consent of patients, peripheral blood tissues were extracted. Then the expression level of cir-cRNA in peripheral blood mononuclear cells(PBMC) was detected by circ RNA chip. The expression of circ RNA was silenced by siRNA technology. The BMSCs were transfected with lentivirus. According to the siRNA interference plasmid hsa-circ-0000885, the cells were divided into the blank group, the empty vector group and the siRNA interference group. After 72 hours of treatment, the cell cycle was detected by flow cytometry, the apoptosis level was detected by AV-PI kit, and the osteogenic differentiation ability of BMSCs was detected by ALP staining.@*RESULTS@#The expression of hsa-circ-0000885 in PBMC of patients with osteoporosis was significantly higher than that of healthy controls (@*CONCLUSION@#The lentivirus mediated siRNA hsa-circ-0000885 plasmid transfected into BMSCs and osteoclast co culture system can promote cell proliferation, inhibit apoptosis and promote osteogenic differentiation of BMSCs, which can be used as a potential therapeutic target for OP patients.

External morphology, postcranial and appendicular osteology of three southwestern Atlantic flatfishes ( Paralichthys, Paralichthyidae), and comparisons with other congeneric species

Comparisons of the external morphology and analysis of osteological features of the postcranial and appendicular skeletons of three southwestern Atlantic flatfish species of the genus Paralichthys (P. isosceles, P. orbignyanus and P. patagonicus) were carried out. Bones are described, and detailed morphological, morphometric and meristic characteristics of these flounders are given in order to provide information about the external and internal morphology of three species of Paralichthys occurring in the south-west Atlantic waters that add new information and will help regarding within the framework of a phylogenetic study of the group. Interspecific differences were found in the number of vertebrae and intermuscular bones, as well as in the morphology and morphometry of vertebrae, caudal skeletons, pectoral and pelvic girdle bones. Relationships between bones are discussed and bone characteristics compared with those found in other species of Paralichthys and in other pleuronectiform species. The position of Paralichthys isosceles within Paralichthys is discussed, along with other congeners such as P. triocellatus and P. oblongus.(AU)

Se llevaron a cabo comparaciones de la morfología externa y el análisis de las características osteológicas de los esqueletos postcraneal y apendicular de tres especies de peces planos del Atlántico sudoccidental del género Paralichthys (P. isosceles, P. orbignyanus y P. patagonicus). Se describen los huesos, y se proporcionan características morfológicas, morfométricas y merísticas detalladas de estos lenguados con el fin de aportar información sobre la morfología externa e interna de tres especies de Paralichthys presentes en el Atlántico sudoccidental. Esta nueva información contribuirá al marco de un estudio filogenético del grupo. Se encontraron diferencias interespecíficas en el número de vértebras y huesos intermusculares, así como en la morfología y morfometría de las vértebras, los esqueletos caudales, los huesos de las cinturas pectoral y pélvica. Se discuten las relaciones entre los huesos y las características óseas en comparación con las encontradas en otras especies de Paralichthys y de otros Pleuronectiformes. Se discute la posición de Paralichthys isosceles dentro del género Paralichthys, junto con otros congéneres como P. oblongus y P. triocellatus.(AU)

MLO-Y4 osteocytic cell clones express distinct gene expression patterns characteristic of different stages of osteocyte differentiation / Clones de las células osteocíticas MLO-Y4 tienen diferentes patrones de expresión génica característicos de diferentes estadíos de diferenciación osteocítica

Osteocytes are the most abundant bone cell and are formed when osteoblasts become embedded in the bone matrix. Through changes in gene expression and paracrine effects, osteocytes regulate the number of osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, which are needed to maintain bone mass. MLO-Y4 is the better characterized osteocytic cell line; however, lacks expression of sclerostin, the product of the SOST gene, which is fundamental for osteocyte function and blocks bone formation. With the objective to isolate MLO-Y4 clones with different gene expression profiles, we performed cultures at very low density of MLO-Y4 cells stably transfected with nuclear green fluorescent protein (MLOnGFP). Cell morphology was visualized under a fluorescence microscope. Once the cells reached 80% confluency, RNA was extracted and quantitative real time PCR was performed. Clones exhibit different sizes and morphology, with some cells showing a spindle-like shape and others with abundant projections and a star-like shape. Gene expression also differed among clones. However, none of the clones examined expressed SOST. We conclude that the MLO-nGFP clones constitute a useful tool to study osteocyte differentiation and the role of osteocytes in the control of bone formation and resorption in vitro. (AU)

Los osteocitos son las células más abundantes del hueso y se forman cuando los osteoblastos se encuentran rodeados de matriz ósea. A través de cambios en la expresión génica y efectos paracrinos, los osteocitos controlan el número de osteoblastos que forman el hueso, y osteoclastos que resorben el hueso, células necesarias para mantener la masa ósea. Las células MLO-Y4 son la línea celular osteocítica más investigada; sin embargo, no expresan esclerostina, el pro esclerostina, el producto del gen SOST que bloquea la formación ósea y es indispensable para la función de los osteocitos. Con el objetivo de aislar clones de las células MLO-Y4 con diferentes perfiles de expresión génica, realizamos cultivos a muy baja densidad de las células transfectadas en forma estable con proteína verde fluorescente nuclear (MLO-nGFP). La morfología celular fue evaluada utilizando un microscopio de fluorescencia. Una vez que las células alcanzaron el 80% de confluencia, el ARN fue extraído y analizado por PCR cuantitativa en tiempo real. Las células de los diferentes clones tienen diferentes tamaños y morfología, algunas células son fusiformes y otras con proyecciones citoplasmáticas abundantes y en forma de estrella. La expresión de los genes también varió en los distintos clones. Sin embargo, ninguno de ellos expresó SOST. En conclusión, los clones de las células MLO-nGFP constituyen una herramienta útil para estudiar la diferenciación de los osteocitos y el rol de estas células en el control de la formación y resorción ósea in vitro. (AU)

Displasia fibrosa ósea / Fibrous dysplasia of bone

La displasia fibrosa ósea es un trastorno no hereditario del desarrollo esquelético caracterizado por una proliferación anormal de fibroblastos y diferenciación deficiente de osteoblastos que conduce a un reemplazo del tejido óseo esponjoso por tejido conectivo fibroso. Es producida por una mutación somática activadora del gen GNAS1 que induce una activación y proliferación de células mesenquimales indiferenciadas con formación de tejido fibroso y trabéculas óseas anómalas. Existen formas monostóticas, poliostóticas y craneofaciales con diversos grados de dolor, deformidades y fracturas óseas, aunque muchos casos son asintomáticos. En ocasiones se producen quistes óseos aneurismáticos, hemorragias, compromisos neurológicos y raramente osteosarcomas. Algunos casos se asocian a síndrome de McCune-Albright, síndrome de Mazabraud y a osteomalacia por hipofosfatemia por pérdida tubular renal inducida por el FGF23 producido por el tejido displásico. Los hallazgos en las radiografías convencionales son característicos, aunque variables y de carácter evolutivo. La gammagrafía ósea es la técnica de imagen con mayor sensibilidad para determinar la extensión de la enfermedad. El diagnóstico diferencial incluye múltiples lesiones óseas de características similares y en raras ocasiones se requiere biopsia ósea o estudio genético para confirmarlo. No existe un consenso unánime acerca del abordaje terapéutico de estos pacientes, razón por la cual es necesario un enfoque multidisciplinario. La conducta puede ser expectante o quirúrgica según el tipo de lesiones y es importante el manejo del dolor y de las endocrinopatías asociadas. La mayor experiencia publicada se refiere al uso de bifosfonatos y, más recientemente, denosumab. Los tratamientos actuales son insuficientes para modificar el curso de la enfermedad y es necesario el desarrollo de nuevas moléculas que actúen específicamente en el gen GNAS1 o sobre las células mesenquimales afectadas. (AU)

Fibrous dysplasia of bone is a noninherited developmental anomaly of bone characterized by abnormal proliferation of fibroblasts and differentiation of osteoblasts that cause a replacement of trabeculous bone by fibrous connective tissue. It is caused by a somatic mutation in the GNAS1 gene, which induces an undifferentiated mesenquimal cells activation and proliferation with formation of fibrous tissue and abnormal osseous trabeculae. There are monostotic, polyostotic and craniofacial variants with different grades of bone pain, deformities and fractures, although many cases remain asymptomatic. Aneurysmal bone cysts, bleeding, neurological compromise and infrequently osteosarcoma are possible complications. Some cases are associated to McCune-Albright syndrome, Mazabraud syndrome or hypophosphatemia and osteomalacia due to to renal tubular loss induced by FGF23 produced by dysplastic tissue. The findings on conventional radiography are characteristic although variable and evlolve with time. Bone scintigraphy is the most sensitive technique to evaluate the extent of disease. Differential diagnosis include several osseous lesions of similar appearance and, in some cases, bone biopsy or genetic testing may be necessary. Today, there is no consensus regarding the therapeutic approach for these patients and it is necessary a multidisciplinary medical team. Watchful waiting or surgical interventions can be indicated, depending on the type of bone lesions. Bone pain and associated endocrinopathies management are very important. Most published experience refers to the use of bisphosphonates and, more recently, denosumab. Current treatments are insufficient to modify the natural curse of the disease and therefore, new molecules with specific action on GNAS1 gene or affected mesenchymal cells are necessary. (AU)

Expression regulation and functional analysis of RGS2 and RGS4 in adipogenic and osteogenic differentiation of human mesenchymal stem cells

BACKGROUND: Understanding the molecular basis underlying the formation of bone-forming osteocytes and lipid-storing adipocytes will help provide insights into the cause of disorders originating in stem/progenitor cells and develop therapeutic treatments for bone- or adipose-related diseases. In this study, the role of RGS2 and RGS4, two members of the regulators of G protein signaling (RGS) family, was investigated during adipogenenic and osteogenenic differentiation of human mesenchymal stem cells (hMSCs). RESULTS: Expression of RGS2 and RGS4 were found to be inversely regulated during adipogenesis induced by dexamethasone (DEX) and 3-isobutyl-methylxanthine, regardless if insulin was present, with RGS2 up-regulated and RGS4 down-regulated in response to adipogenic induction. RGS2 expression was also up-regulated during osteogenesis at a level similar to that induced by treatment of DEX alone, a shared component of adipogenic and osteogenic differentiation inducing media, but significantly lower than the level induced by adipogenic inducing media. RGS4 expression was down-regulated during the first 48 h of osteogenesis but up-regulated afterwards, in both cases at levels similar to that induced by DEX alone. Expression knock-down using small interfering RNA against RGS2 resulted in decreased differentiation efficiency during both adipogenesis and osteogenesis. On the other hand, expression knock-down of RGS4 also resulted in decreased adipogenic differentiation but increased osteogenic differentiation. CONCLUSIONS: RGS2 and RGS4 are differentially regulated during adipogenic and osteogenic differentiation of hMSCs. In addition, both RGS2 and RGS4 play positive roles during adipogenesis but opposing roles during osteogenesis, with RGS2 as a positive regulator and RGS4 as a negative regulator. These results imply that members of RGS proteins may play multifaceted roles during human adipogenesis and osteogenesis to balance or counterbalance each other's function during those processes.

Avaliação histométrica e histoquímica de vidro bioativo no processo de reparação óssea / Histométrica assessment and histochemistry of bioactive glass in the process of bone repair

Os enxertos de origem autógena são osteogênicos e possuem como vantagens a produção de tecido ósseo originário do próprio organismo. A sua limitação é a grande morbidade cirúrgica. O biovidro é uma cerâmica bioativa com disponibilidade ilimitada que leva a uma cirurgia com menor morbidade. O presente estudo teve como objetivo avaliar o biovidro teste durante o reparo ósseo e compará-lo com outros substitutos ósseos, em defeitos cirúrgicos experimentais em tíbia de ratos. O biovidro teste (CEELBIO, Belo Horizonte, Brasil), previamente caracterizado foi comparado com o Biogran® (Biomet 3i Inovattions Inc., Palm Beach Gardens, USA), através de espectroscopia na região do infravermelho, microscopia eletrônica de varredura (MEV), espectroscopia de dispersão em energia (EDS) e fluorescência de Rx. Após as análises estruturais, deu-se início ao estudo in vivo. Foram utilizados 120 ratos (rattus norvegicus albinus, Wistar) machos, com aproximadamente 7 a 8 semanas. Os defeitos ósseos foram realizados na tíbia direita dos animais e preenchidos de acordo com a seguinte divisão: Grupo I- controle negativo, sem preenchimento; Grupo II- foi preenchido por Biogran®; Grupo III- preenchido por biovidro teste; Grupo IVcontrole positivo, com preenchimento com osso autógeno. Nos períodos de 7, 14, 21, 28, 49 e 70 dias pós-operatório, os animais foram eutanasiados e os processos de mineralização óssea e reparo foram analisados através de histomorfometria (% de osso neoformado no sítio do defeito). Níveis de BMP-2 foram mensurados através de ensaio de ELISA. Análise estatística foi realizada utilizando programa SPSS (versão 20.0, SPSS Inc., Chicago, USA). Os resultados da análise histológica demonstraram que, no controle negativo, houve neoformação óssea até os 14 dias (20,40%, p<0,001) e depois houve reabsorção em até 21 dias (6,60%, p<0,001). No grupo de Biogran®, houve uma neoformação óssea junto aos grãos em 7 dias (34,20%, p= 0,019) e que se manteve enquanto o material estava presente nos defeitos em 70 dias (15,67%, p= 0,048). O biovidro teste foi reabsorvido totalmente até 21 dias e os picos de osso neoformado foram observados em 7 (21,00%, p= 0,019) e 49 dias (15,60%, p= 0,036). Nesse grupo, células semelhantes a macrófagos, dispostas em lençol, foram visualizadas junto a tecido ósseo neoformado. Quanto ao controle positivo, o osso autógeno foi totalmente reabsorvido em até 14 dias e o pico de formação óssea se deu nesse 9 mesmo momento, em 14 dias, (40,80%, p<0,001), mostrando-se, nos tempos subsequentes, similar ao controle negativo. Células gigantes multinucleadas foram encontradas em áreas de remodelação óssea, junto ao Biogran® e ao biovidro teste. Os níveis de BMP-2 no grupo controle negativo se mostraram maiores nos tempos de 7 (418,80pg/mL, p= 0,871) e 28 dias (346,36 pg/mL, p= 0,035). No grupo Biogran® o pico de BMP-2 se deu em 7 dias (471,95 pg/mL, p= 0,871). O biovidro teste teve seu pico de liberação de BMP-2 em 7 dias também (471,39 pg/mL, p= 0,871). Já o controle positivo apresentou nível de BMP-2 em maior quantidade nos tempos de 7 (346,55 pg/mL, p= 0,871) e 21 dias (407,57 pg/mL, p= 0,300). O biovidro é um material degradável, e com características biológicas de osteoindução e osteocondução

The autogenous origin grafts are osteogenic and have the advantage of producing bone tissue originated from the body itself. Its limitation is the great surgical morbidity. The bioglass is a bioactive ceramic with unlimited availability that leads to a surgery with less morbidity. This study aimed to evaluate the bioglass test during bone repair and to compare it with other bone substitutes in experimental surgical defects in the tibia of rats. The bioglass test (CEELBIO, Belo Horizonte, Brazil), has been characterized and compared to the Biogran® (Biomet 3i Inovattions Inc., Palm Beach Gardens, USA) by spectroscopy in the infrared, scanning electron microscopy (SEM), EDS spectroscopy and Rx fluorescence. The study in vivo has been started after the structural analysis. 120 rats (Rattus norvegicus albinus, Wistar rats), with approximately 7 to 8 weeks, were used. The bone defects were performed in the right tibia of animals and filled according to the following breakdown: Group I- negative control without filling; Group II- filled by Biogran®; Group III- filled with bioglass test; IV- positive control group, with filling of autogenous bone. At 7, 14, 21, 28, 49 and 70 postoperative days, the animals were euthanized and bone mineralization processes and repair were analyzed by histomorphometry (% of newly formed bone in the defect site). BMP-2 levels were measured by ELISA assay. Statistical analysis was performed using SPSS (version 20.0, SPSS Inc., Chicago, USA). The results of the histological analysis showed that in the negative control there was no bone growth up to 14 days (20.40%, p <0.001) and after reabsorption, within 21 days (6.60%, p <0.001). In Biogran® group, there was new bone formation along the grain in 7 days (34.20%, p = 0.019) and that was maintained as the material was present in shortcomings in 70 days (15.67%, p = 0.048) . The test bioglass was completely reabsorbed within 21 days; new bone formation and peaks were observed in 7 (21.00%, p = 0.019) and 49 days (15.60%, p = 0.036). In this group, macrophage-like cells arranged in sheets, were viewed with the newly formed bone. On the positive control, autologous bone was completely reabsorbed within 14 days, and the peak bone formation occurred at the same moment, within 14 days (40.80%, p <0.001), showing, in subsequent occasions, similar to control negative. Multinucleated giant cells were found in areas of bone remodeling, with the Biogran® and test bioglass. BMP-2 levels in the negative control group were higher in 7 (418,80pg / ml, p = 11 0.871) and 28 days (346.36 pg / ml, p = 0.035). In Biogran® group peak BMP-2 occurred within 7 days (471.95 pg / ml, p = 0.871). The test bioglass had its peak BMP-2 release in 7 days as well (471.39 pg / ml, p = 0.871). The positive control showed BMP-2 level in greater quantities in 7 (346.55 pg / ml, p = 0.871) and 21 days (407.57 pg / ml, p = 0.300). The test bioglass is a biodegradable material, with biological characteristics of osteoinduction and osteoconduction

Utilização de células-tronco mesenquimais em procedimentos visando o crescimento ósseo maxilomandibular û revisão de literatura / Use of mesenchymal stem cells in procedures for maxilo-mandibular bone growth û literature review

As células-tronco mesenquimais são uma população de células adultas obtidas através da medula óssea, sangue do cordão umbilical, e estão cada vez mais sendo utilizadas para tratamentos regenerativos e pesquisas de engenharia tecidual nas mais diversas áreas da medicina, isto devido ao fato de essas células serem multipotenciais, que conseguem se diferenciar em quase todos os tipos de tecidos humanos, com exceção da placenta e anexos embrionários. Quando coletadas são criopreservadas a temperaturas abaixo de 180°C para manterem o seu potencial de proliferação e diferenciação osteogênica in vitro. Elas possuem capacidade de se autoproliferar e se diferenciar ao longo de várias linhagens, incluindo osso, cartilagem, tecido adiposo, células musculares e neurais. O objetivo deste artigo é revisar a literatura sobre a capacidade que as células-tronco possuem em se diferenciar e proporcionar o crescimento ósseo através de seu potencial osteogênico (contendo células formadoras de osso), osteoindutor (contendo substâncias osteoindutoras) e osteocondutor (servem como uma base para formação de osso). Uma variedade de opiniões a respeito do tipo de materiais que deve ser utilizado para aplicações clínicas em defeitos ósseos torna ampla a discussão sobre resultados de traumas e ressecções de tumores ósseos. Para isso, os biomateriais fornecem uma excelente base para o crescimento ósseo quando combinado com um aspirado de concentrado de medula óssea. Apesar de estar comprovado cientificamente que as células estromais podem formar ossos, ainda não há estudos clínicos suficientes.

The mesenchymal stem cells are a population of mature cells obtained from bone marrow and umbilical cord blood. These cells are being widely used on regenerative treatment and tissue engineering research in several areas of medicine. That is occurring due to the fact that these cells are multipotent, being able to differentiate themselves into almost every type of human tissues, with the exception of the placenta and embryonic annexes. When collected these cells, are cryopreserved at temperatures below -180°C to preserve their potential for proliferation and osteogenic differentiation in vitro. They also have the ability to self-proliferate and differentiate along various cell lineages, including bone, cartilage, adipose tissue, muscle, and nerve cells. The aim of this study was to review the literature on the ability that mesenchymal cells have to differentiate themselves and provide bone growth through its osteogenic potential (containing bone-forming cells), osteoinductivity potential (containing osteoinductive substances) and osteoconductive (serve as a basis for bone formation). A variety of opinions about the type of materials that should be used for clinical application in bone defects amplifies the discussion around trauma and bone tumors resections.Biomaterials provide an excellent foundation for new bone growth when combined with an aspirated bone marrow concentrate. Although, it has been scientifically proven that stromal cells can form new bones, there are not enough clinical studies about it.

Regulation of osteogenic differentiation of human adipose-derived stem cells by controlling electromagnetic field conditions

Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation.

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino / Potential of neural transdifferentiation of mesenchymal stem cells from equine bone marrow

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1. Com relação a análise imunofenotípica foi observado uma maior (P<0,001) expressão dos marcadores GFAP e β3 tubulina ao término do P2 quando comparado ao P1. A habilidade das CTMs em gerar tipos celulares relacionados a linhagem neural é complexa e multifatorial, dependendo não só dos agentes indutores, mas também do ambiente no qual estas células são cultivadas. Desta forma um maior número de estudos é necessário para o melhor entendimento do processo de transdiferenciação neural a partir de CTMs de equinos.

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension. It was also evidenced with the use of this protocol, lower cellular adhesion after exposure to media when compared to P1. Regarding the immunophenotyping analysis it was observed a higher (P<0.001) expression of the markers GFAP and β3 tubulin at the end of P2 compared to P1. The ability of MSCs to generate cell types related to neural lineage is complex and multifactorial, depending not only of inducing agents, but also the environment in which these cells will be cultivated. Thus a greater number of studies are necessary to better understand the process of neural transdifferentiation of MSCs from equine.

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos / In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.

The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and β-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12.Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Nasal profile assessment using geometric morphometrics in a sample of chilean population: clinical and forensic implications / Evaluación del perfil nasal mediante morfometría geométrica en unamuestra de población chilena. Implicancias clínicas y forenses

The search of morphological patterns of nasal profile using traditional morphometrics has been the goal of several studies aiming to orient therapeutical planning and forensic techniques such as facial reconstruction. The present study aims to find and describe such patterns with geometric morphometric tools in a sample of Chilean population with geometric morphometric tools. We used the lateral X-rays of 156 individuals (men and women) and 14 landmarks in bone and soft tissues. Procrustes analysis was performed followed by principal component analysis to assess general shape variation, regression of shape components against centroid size to study to allometric effect and discriminant analysis by sex with cross-validation test. Our results show the lack of shape patterns, and that the size, followed by sex, explains within a limited scope the shape changes which suggests the presence of uncontrolled variables and a high effect of inter-individual variation. In general terms bigger profiles, more frequent in men, show a lower Pronasal point, a more prominent nasal dorsum with a thicker layer of soft tissue, and a vertically expanded upper lip. We suggest the reassessment of the importance of individual aesthetic evaluation for clinical purposes, and recommend caution in concluding results based on forensic reconstruction techniques.

La descripción de patrones morfológicos del perfil nasal en la población que orienten la toma de decisiones y evaluación terapéutica, y que aseguren el éxito de técnicas como la reconstrucción facial forense, ha sido el objetivo de numerosos estudios basados en el uso de morfometría tradicional. El objetivo de este trabajo es el estudio del perfil nasal en una muestra de población chilena utilizando herramientas de la morfometría geométrica. Se utilizaron las radiografías de una muestra mixta de 156 individuos adultos y 14 hitos en perfil duro y blando. Se realizó el análisis de Procusto, seguido de análisis de componentesprincipales para el estudio exploratorio de la forma, regresión de los componentes de la forma contra el tamaño de centroide para el estudio del efecto alométrico en la forma del perfil nasal, análisis discriminante para la variable sexo y prueba de validación cruzada. Los resultados muestran que de las variables controladas, el tamaño y luego el sexo serían las que más explican la variabilidad observada, sin embargo el peso general de estas variables es bajo, sugiriendo la presencia de otras variables no controladas, y un gran componente de variación entre los individuos. Morfológicamente, se observa que los perfiles de mayor tamaño, de mayor frecuencia en hombres, presentan un punto nasal más descendido y un dorso nasal más curvo, así como un labio más expandido verticalmente. Desde el punto de vista estético, se sugiere dar importancia a la evaluación individual con fines terapéuticos. En antropología forense, se sugiere la prudencia en las conclusiones basadas en técnicas de reconstrucción debido a la falta de patrones morfológicos que orienten la técnica de manera certera.

Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN) was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

Human skeletal dysplasia caused by a constitutive activated transient receptor potential vanilloid 4 (TRPV4) cation channel mutation

The transient receptor potential vanilloid 4 (TRPV4) cation channel, a member of the TRP vanilloid subfamily, is expressed in a broad range of tissues where it participates in the generation of Ca2+ signals and/or depolarization of the membrane potential. Regulation of TRPV4 abundance at the cell surface is critical for osmo- and mechanotransduction. Defects in TRPV4 are the cause of several human diseases, including brachyolmia type 3 (MIM:113500) (also known as brachyrachia or spondylometaphyseal dysplasia Kozlowski type [MIM:118452]), and metatropic dysplasia (MIM:156530) (also called metatropic dwarfism or parastremmatic dwarfism [MIM:168400]). These bone dysplasia mutants are characterized by severe dwarfism, kyphoscoliosis, distortion and bowing of the extremities, and contractures of the large joints. These diseases are characterized by a combination of decreased bone density, bowing of the long bones, platyspondyly, and striking irregularities of endochondral ossification with areas of calcific stippling and streaking in radiolucent epiphyses, metaphyses, and apophyses. In this review, we discuss the potential effect of the mutation on the regulation of TRPV4 functions, which are related to human diseases through deviated function. In particular, we emphasize how the constitutive active TRPV4 mutant affects endochondral ossification with a reduced number of hypertrophic chondrocytes and the presence of cartilage islands within the zone of primary mineralization. In addition, we summarize current knowledge about the role of TRPV4 in the pathogenesis of several diseases.

Evidence for estrogen receptor expression during medullary bone formation and resorption in estrogen-treated male Japanese quails (Coturnix coturnix japonica)

The temporal expression of estrogen receptor (ER)-alpha and ER-beta mRNA was examined in male Japanese quails. Femurs of quails receiving 17beta-estradiol underwent RTPCR and histochemical analysis 1 to 15 days after treatment. Untreated quails were used as controls (day 0). Between days 0 and 5, cells lining the bone endosteal surface differentiated into osteoblasts, which in turn formed medullary bone. Expression of ER-alpha was already observed on day 0 and increased slightly during bone formation whereas ER-beta was hardly detected throughout this process. After osteoclasts appeared on the medullary bone surface, this type of bone disappeared from the bone marrow cavity (days 7~15). ER-alpha expression simultaneously decreased slightly and ER-beta levels remained very low. These results suggest that estrogen activity mediated by ER-alpha not only affects medullary bone formation but also bone resorption.