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 mechanism of traumatic brain injury promoting fracture healing / 中国修复重建外科杂志

OBJECTIVE@#To summarize the research progress on the mechanism related to traumatic brain injury (TBI) to promote fracture healing, and to provide theoretical basis for clinical treatment of fracture non-union.@*METHODS@#The research literature on TBI to promote fracture healing at home and abroad was reviewed, the role of TBI in fracture healing was summarized from three aspects of nerves, body fluids, and immunity, to explore new ideas for the treatment of fracture non-union.@*RESULTS@#Numerous studies have shown that fracture healing is faster in patients with fracture combined with TBI than in patients with simple fracture. It is found that the expression of various cytokines and hormones in the body fluids of patients with fracture and TBI is significantly higher than that of patients with simple fracture, and the neurofactors released by the nervous system reaches the fracture site through the damaged blood-brain barrier, and the chemotaxis and aggregation of inflammatory cells and inflammatory factors at the fracture end of patients with combined TBI also differs significantly from those of patients with simple fracture. A complex network of humoral, neural, and immunomodulatory networks together promote regeneration of blood vessels at the fracture site, osteoblasts differentiation, and inhibition of osteoclasts activity.@*CONCLUSION@#TBI promotes fracture healing through a complex network of neural, humoral, and immunomodulatory, and can treat fracture non-union by intervening in the perifracture microenvironment.

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

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

Sema3A secreted by sensory nerve induces bone formation under mechanical loads / 国际口腔科学杂志·英文版

Bone formation and deposition are initiated by sensory nerve infiltration in adaptive bone remodeling. Here, we focused on the role of Semaphorin 3A (Sema3A), expressed by sensory nerves, in mechanical loads-induced bone formation and nerve withdrawal using orthodontic tooth movement (OTM) model. Firstly, bone formation was activated after the 3rd day of OTM, coinciding with a decrease in sensory nerves and an increase in pain threshold. Sema3A, rather than nerve growth factor (NGF), highly expressed in both trigeminal ganglion and the axons of periodontal ligament following the 3rd day of OTM. Moreover, in vitro mechanical loads upregulated Sema3A in neurons instead of in human periodontal ligament cells (hPDLCs) within 24 hours. Furthermore, exogenous Sema3A restored the suppressed alveolar bone formation and the osteogenic differentiation of hPDLCs induced by mechanical overload. Mechanistically, Sema3A prevented overstretching of F-actin induced by mechanical overload through ROCK2 pathway, maintaining mitochondrial dynamics as mitochondrial fusion. Therefore, Sema3A exhibits dual therapeutic effects in mechanical loads-induced bone formation, both as a pain-sensitive analgesic and a positive regulator for bone formation.

Bioceramic scaffolds with two-step internal/external modification of copper-containing polydopamine enhance antibacterial and alveolar bone regeneration capability / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Magnesium-doped calcium silicate (CS) bioceramic scaffolds have unique advantages in mandibular defect repair; however, they lack antibacterial properties to cope with the complex oral microbiome. Herein, for the first time, the CS scaffold was functionally modified with a novel copper-containing polydopamine (PDA(Cu2+‍)) rapid deposition method, to construct internally modified (*P), externally modified (@PDA), and dually modified (*P@PDA) scaffolds. The morphology, degradation behavior, and mechanical properties of the obtained scaffolds were evaluated in vitro. The results showed that the CS*P@PDA had a unique micro-/nano-structural surface and appreciable mechanical resistance. During the prolonged immersion stage, the release of copper ions from the CS*P@PDA scaffolds was rapid in the early stage and exhibited long-term sustained release. The in vitro evaluation revealed that the release behavior of copper ions ascribed an excellent antibacterial effect to the CS*P@PDA, while the scaffolds retained good cytocompatibility with improved osteogenesis and angiogenesis effects. Finally, the PDA(Cu2+)-modified scaffolds showed effective early bone regeneration in a critical-size rabbit mandibular defect model. Overall, it was indicated that considerable antibacterial property along with the enhancement of alveolar bone regeneration can be imparted to the scaffold by the two-step PDA(Cu2+) modification, and the convenience and wide applicability of this technique make it a promising strategy to avoid bacterial infections on implants.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process / Atividade enzimática de marcadores ósseos no crescimento de Lithobates catesbeianus (Shaw, 1802) durante o processo de ossificação

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.

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

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

Comparative Assessment of Contact Osteogenesis at the Titanium Implant-Bone Junction in Male Rabbits with Dissimilar Femoral Defects / Evaluación Comparativa de la Osteogénesis de Contacto en la Unión entre el Hueso y el Implante de Titanio en Conejos Macho con Defectos Femorales Diferentes

SUMMARY: Traumatized bone tissue has the capacity to repair itself so that it eventually regains its almost original form, even in the case of artificially inserted implants. The process that stays at the base of the regeneration is represented by osteogenesis or remote osteogenesis. The major difference between the two types of bone formation is the location of the cement line, which is located on the surface of the implant for contact osteogenesis and on the surface of the bone defect for remote osteogenesis. The aim of the present study was to assess the contact osteogenesis in the case of inserted titanium screws in holes with diameters of 1.8 mm and 1 mm respectively. The obtained results show, in the case of the groove with 1.8 mm that the newly proliferated bone represents 73.85 % of the total area, while in the case of the groove with 1 mm in diameter the value of the newly proliferated bone is 26.15 %. In conclusion, the insertion of titanium screws by self-tapping into the hole smaller than the core of the screw is accompanied by bone proliferation by contact osteogenesis much more modest than in the case of insertion into the hole larger than the core of the screw.

El tejido óseo traumatizado tiene la capacidad de reparar en forma espontánea, de modo que eventualmente recupera su forma casi original, incluso en el caso de implantes insertados artificialmente. El proceso que queda en la base de la regeneración está representado por la osteogénesis u osteogénesis a distancia. La principal diferencia entre los dos tipos de formación ósea es la ubicación de la línea de cemento, que se encuentra en la superficie del implante para la osteogénesis de contacto y en la superficie del defecto óseo para la osteogénesis remota. El objetivo del presente estudio fue evaluar la osteogénesis de contacto en el caso de tornillos de titanio insertados en forámenes con diámetros de 1,8 mm y 1 mm respectivamente. Los resultados obtenidos muestran, en el caso del surco de 1,8 mm que el hueso neoproliferado representa el 73,85 % del área total, mientras que en el caso del surco de 1 mm de diámetro el valor del hueso neoproliferado es del 26,15 %. En conclusión, la inserción de tornillos de titanio por autorroscantes en el foramen menor que el núcleo del tornillo se acompaña de una proliferación ósea por osteogénesis de contacto mucho más modesta que en el caso de la inserción en el foramen mayor que el núcleo del tornillo.

Estudio de biocompatibilidad de matrices poliméricas combinadas, responsivas al pH, con aplicación en Ingeniería de Tejido Óseo / Biocompatibility study of combined pH-responsive polymeric matrices with application in Bone Tissue Engineering

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Unveiling the role of osteoblastic micro RNAs in the skeleton: from biological functions to therapeutic potential / Rol de los micro-ARN osteoblásticos en el esqueleto: desde las funciones biológicas al potencial terapéutico

MicroRNAs (miRNAs) are small non-coding RNA molecules that play critical roles in post-transcriptional gene regulation. They function by binding to target messenger RNA (mRNA) molecules, leading to their degradation or inhibiting their translation into proteins. In the context of skeletal diseases, such as osteoporosis, osteoarthritis, and bone metastasis, there is growing evidence osteoblastic miRNAs, are involved in the regulation of bone formation and maintenance.Osteoblasts are bone-forming cells responsible for synthesizing and depositing the extracellular matrix, which ultimately mineralizes to form bone tissue. Osteoblastic miRNAs modulate various aspects of osteoblast function, including proliferation, differentiation, mineralization, and apoptosis. Dysregulation of these miRNAs can disrupt the balance between bone formation and resorption, leading to skeletal diseases.The therapeutic implications of targeting osteoblastic miRNAs in skeletal diseases are significant. Modulating the expression levels of specific miRNAs holds promise for developing novel therapeutic strategies to enhance bone formation, prevent bone loss, and promote bone regeneration. Potential therapeutic approaches include the use of synthetic miRNA mimics to restore miRNA expression in diseases associated with miRNA downregulation or the use of anti-miRNA oligonucleotides to inhibit miRNA function in diseases associated with miRNA upregulation.miRNA-based therapies are still in the early stages of development, and further research is needed to fully understand the complexity of miRNA networks. Additionally, the delivery of miRNAs to specific target tissues and cells remains a challenge that needs to be addressed for effective clinical translation. Nonetheless, targeting osteoblastic miRNAs represents a promising avenue for future therapeutic interventions in skeletal diseases. (AU)

Los micro-ARNs (miARNss) son pequeños ARN no codificantes que desempeñan un papel fundamental en la regulación génica postranscripcional. Ejercen su función al unir-se a moléculas de ARN mensajero (ARNm), promoviendo su degradación e inhibiendo su traducción en proteínas. En el contexto de las enfermedades esqueléticas, como la osteoporosis, la osteoartritis y la metástasis ósea existe evidencia de que los miARNs osteoblásticos están involucrados en la regulación de la formación y del mantenimiento óseo. Los osteoblastos son células formadoras de hueso responsables de sintetizar y depositar la matriz extracelular, que finalmente se mineraliza para formar el hueso. Los miARNs derivados de osteoblastos modulan varios aspectos de la función de estas células, incluida la proliferación, diferenciación, mineralización y la apoptosis. La desregulación de estos miARNs puede alterar el equilibrio entre la formación y la resorción ósea, lo que lleva a enfermedades óseas. Las implicaciones terapéuticas de los miARNs osteoblásticos en enfermedades esqueléticas son significativas. La modulación de los niveles de expresión de miARNs específicos es prometedora para desarrollar nuevas estrate-gias terapéuticas a fin de mejorar la formación, prevenir la pérdida y promover la regeneración ósea. Los enfoques terapéuticos potenciales incluyen el uso de miméticos de miARNs para restaurar la expresión de miARNs o el uso de oligonucleótidos anti-miARNs para inhibir su función. Las terapias basadas en miARNs aún se encuentran en las primeras etapas de desarrollo. La administración de miARNs a las células y los tejidos específicos sigue siendo un desafío para lograr una aplicación clínica eficaz. (AU)

Establishment and evaluation of rabbit model of closed tibial fracture / 中国骨伤

OBJECTIVE@#To explore the effect of a modified three-point bending fracture device for establishing a rabbit model of closed tibial fracture.@*METHODS@#The model of closed tibial fracture was established in 40 6-month-old male New Zealand white rabbits with a body weight of 2.5 to 3.0 kg, and the model was verified at 6 weeks after operation. Five rabbits underwent pre modeling without temporary external fixation before modeling, and then were fractured with a modified three-point bending fracture device;35 rabbits underwent formal modeling. Before modeling, needles were inserted, and splints were fixed externally, and then the fracture was performed with a modified three-point bending fracture device. The fracture model and healing process were evaluated by imaging and histopathology at 2 hours, 4 weeks, and 6 weeks after operation.@*RESULTS@#Two hours after modeling, the prefabricated module showed oblique fracture in varying degrees and the broken end shifted significantly;Except for 1 comminuted fracture, 2 curved butterfly fractures and 2 without obvious fracture line, the rest were simple transverse and oblique fractures without obvious displacement in formal modeling group. According to the judgment criteria, the success rate of the model was 85.71%. Four weeks after modeling, the fixed needle and splint of the experimental rabbits were in good position, the fracture alignment was good, the fracture line was blurred, many continuous callus growths could be seen around the fracture end, and the callus density was high. Six weeks after modeling, many thick new bone trabeculae at the fracture, marginal osteoblasts attached, and a small number of macrophages were seen under the microscope. The intramembrane osteogenesis area was in the preparation bone stage, the medullary cavity at the fracture had been partially reopened, the callus was in the absorption plastic stage, and many osteoclasts were visible. The X-ray showed that the fracture line almost disappeared, part of the medullary cavity had been opened, the external callus was reduced around, the callus was in the plastic stage, and the bone cortex was continuous. It suggests that the fracture model showed secondary healing.@*CONCLUSION@#The improved three-point bending fracture device can establish a stable rabbit model of closed tibial fracture, and the operation is simple, which meets the requirements of closed fracture model in basic research related to fracture healing.

Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification / 中国骨伤

OBJECTIVE@#To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification.@*METHODS@#Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins.@*RESULTS@#The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01).@*CONCLUSION@#Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.

Advances on pentraxin 3 in osteoporosis and fracture healing / 中国骨伤

Pentaxin 3 (PTX3), as a multifunctional glycoprotein, plays an important role in regulating inflammatory response, promoting tissue repair, inducing ectopic calcification and maintaining bone homeostasis. The effect of PTX3 on bone mineral density (BMD) may be affected by many factors. In PTX3 knockout mice and osteoporosis (OP) patients, the deletion of PTX3 will lead to decrease of BMD. In Korean community "Dong-gu study", it was found that plasma PTX3 was negatively correlated with BMD of femoral neck in male elderly patients. In terms of bone related cells, PTX3 plays an important role in maintaining the phenotype and function of osteoblasts (OB) in OP state;for osteoclast (OC), PTX3 in inflammatory state could stimulate nuclear factor κ receptor activator of nuclear factor-κB ligand (RANKL) production and its combination with TNF-stimulated gene 6(TSG-6) could improve activity of osteoclasts and promote bone resorption;for mesenchymal stem cells (MSCs), PTX3 could promote osteogenic differentiation of MSCs through PI3K/Akt signaling pathway. In recent years, the role of PTX3 as a new bone metabolism regulator in OP and fracture healing has been gradually concerned by scholars. In OP patients, PTX3 regulates bone mass mainly by promoting bone regeneration. In the process of fracture healing, PTX3 promotes fracture healing by coordinating bone regeneration and bone resorption to maintain bone homeostasis. In view of the above biological characteristics, PTX3 is expected to become a new target for the diagnosis and treatment of OP and other age-related bone diseases and fracture healing.

Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide / 华西口腔医学杂志

OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

Exercise regulates bone metabolism via microRNAs / 生理学报

It has been well documented that exercise can improve bone metabolism, promote bone growth and development, and alleviate bone loss. MicroRNAs (miRNAs) are widely involved in the proliferation and differentiation of bone marrow mesenchymal stem cells, osteoblasts, osteoclasts and other bone tissue cells, and regulation of balance between bone formation and bone resorption by targeting osteogenic factors or bone resorption factors. Thus miRNAs play an important role in the regulation of bone metabolism. Recently, regulation of miRNAs are shown to be one of the ways by which exercise or mechanical stress promotes the positive balance of bone metabolism. Exercise induces changes of miRNAs expression in bone tissue and regulates the expression of related osteogenic factors or bone resorption factors, to further strengthen the osteogenic effect of exercise. This review summarizes relevant studies on the mechanism whereby exercise regulates bone metabolism via miRNAs, providing a theoretical basis for osteoporosis prevention and treatment with exercise.

Study on in vitro differentiation of human adenoid-derived mesenchymal stem cells into olfactory sensory neurons / 中华耳鼻咽喉头颈外科杂志

Objective: To investigate the feasibility of isolation and culture of human adenoid-derived mesenchymal stem cells (aMSCs) in vitro, and to observe the differentiation of aMSCs into olfactory sensory neurons. Methods: Adenoid tissues surgically removed from children with adenoid hypertrophy in the Second Xiangya Hospital of Central South University from September to November of 2020 were collected. The adenoid tissues were digested and isolated by trypsin and then cultured with adhesion method. The expressions of cell surface antigens CD45, CD73 and CD90 on aMSCs of P5 generation were tested by flow cytometry, and the ability of osteogenic and adipogenic induction were used to identify cell differentiation ability. Then, aMSCs were induced into differentiation by retinoic acid (RA), sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), RA+SHH, RA+bFGF, SHH+bFGF and RA+SHH+bFGF, respectively. The morphology of differentiated cells was observed under inverted microscope. The expression of β-tubulin 3, which was the specific marker of sensory neuron, the expressions of growth associated protein-43 (GAP43) and olfactory maker protein (OMP), which were the specific markers of olfactory sensory neuron, were detected by immunofluorescence antibody assay. The expression intensities were compared by Chi-square test of four-grid table data. Results: aMSCs were successively isolated and cultured from human adenoid tissues. P0 cells generation had good adhesion and proliferation performance. P2 cells were basically purified. P5 cells expressed CD73 and CD90 with the purity of 99.3% and 99.75% respectively, without CD45 expression. P5 cells had a good ability of osteogenic differentiation and adipogenic differentiation. Neuron-like morphology and expression of β-tubulin 3 were found in differentiated cells after induced by RA, SHH, or bFGF, respectively. An induction of expression of GAP43 was found in differentiated cells of bFGF+SHH group and RA+SHH+bFGF group, without expression of OMP of each group. The intensity of GAP43 expression of RA+SHH+bFGF group was stronger than that of bFGF+SHH group (χ2=17.48, P<0.005). Conclusions: aMSCs can be cultured from human adenoid tissues, with the stably passaged and good differentiation ability. As a new population of mesenchymal stem cells, aMSCs have the neuroregenerative properties and could differentiate into immature olfactory sensory neurons under the induction of RA+SHH+bFGF in vitro.

Effect of PKM2 on Osteogenic and Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Myeloma Bone Disease / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs.@*METHODS@#BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs.@*RESULTS@#Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2.@*CONCLUSION@#The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.

Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM.@*METHODS@#Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor.@*RESULTS@#The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells.@*CONCLUSION@#The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.

Advances in surgical strategies for ossification of posterior longitudinal ligament involving the C 2 segment / 中国修复重建外科杂志

OBJECTIVE@#To evaluate the application of surgical strategies for the treatment of cervical ossification of the posterior longitudinal ligament (OPLL) involving the C 2 segment.@*METHODS@#The literature about the surgery for cervical OPLL involving C 2 segment was reviewed, and the indications, advantages, and disadvantages of surgery were summarized.@*RESULTS@#For cervical OPLL involving the C 2 segments, laminectomy is suitable for patients with OPLL involving multiple segments, often combined with screw fixation, and has the advantages of adequate decompression and restoration of cervical curvature, with the disadvantages of loss of cervical fixed segmental mobility. Canal-expansive laminoplasty is suitable for patients with positive K-line and has the advantages of simple operation and preservation of cervical segmental mobility, and the disadvantages include progression of ossification, axial symptoms, and fracture of the portal axis. Dome-like laminoplasty is suitable for patients without kyphosis/cervical instability and with negative R-line, and can reduce the occurrence of axial symptoms, with the disadvantage of limited decompression. The Shelter technique is suitable for patients with single/double segments and canal encroachment >50% and allows for direct decompression, but is technically demanding and involves risk of dural tear and nerve injury. Double-dome laminoplasty is suitable for patients without kyphosis/cervical instability. Its advantages are the reduction of damage to the cervical semispinal muscles and attachment points and maintenance of cervical curvature, but there is progress in postoperative ossification.@*CONCLUSION@#OPLL involving the C 2 segment is a complex subtype of cervical OPLL, which is mainly treated through posterior surgery. However, the degree of spinal cord floatation is limited, and with the progress of ossification, the long-term effectiveness is poor. More research is needed to address the etiology of OPLL and to establish a systematic treatment strategy for cervical OPLL involving the C 2 segment.

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.