Osteoblast Differentiation Process Research Articles

A Composite Hydrogel Functionalized by Borosilicate Bioactive Glasses and VEGF for Critical-Size Bone Regeneration.

Critical-size bone defects pose a formidable challenge in clinical treatment, prompting extensive research efforts to address this problem. In this study, an inorganic-organic multifunctional composite hydrogel denoted as PLG-g-TA/VEGF/Sr-BGNPs is developed, engineered for the synergistic management of bone defects. The composite hydrogel demonstrated the capacity for mineralization, hydroxyapatite formation, and gradual release of essential functional ions and vascular endothelial growth factor (VEGF) and also maintained an alkaline microenvironment. The composite hydrogel promoted the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), as indicated by increased expression of osteogenesis-related genes and proteins in vitro. Moreover, the composite hydrogel significantly enhanced the tube-forming capability of human umbilical vein endothelial cells (HUVECs) and effectively inhibited the process of osteoblastic differentiation of nuclear factor kappa-B ligand (RANKL)-induced Raw264.7 cells and osteoclast bone resorption. After the implantation of the composite hydrogel into rat cranial bone defects, the expression of osteogenic and angiogenic biomarkers increased, substantiating its efficacy in promoting bone defect repair in vivo. The commendable attributes of the multifunctional composite hydrogel underscore its pivotal role in expediting hydrogel-associated bone growth and repairing critical bone defects, positioning it as a promising adjuvant therapy candidate for large-segment bone defects.

Relationship between arginine methylation and vascular calcification

In patients on maintenance hemodialysis (MHD), vascular calcification (VC) is an independent predictor of cardiovascular disease (CVD), which is the primary cause of death in chronic kidney disease (CKD). The main component of VC in CKD is the vascular smooth muscle cells (VSMCs). VC is an ordered, dynamic activity. Under the stresses of oxidative stress and calcium‐‑phosphorus imbalance, VSMCs undergo osteogenic phenotypic transdifferentiation, which promotes the formation of VC. In addition to traditional epigenetics like RNA and DNA control, post-translational modifications have been discovered to be involved in the regulation of VC in recent years. It has been reported that the process of osteoblast differentiation is impacted by catalytic histone or non-histone arginine methylation. Its function in the osteogenic process is comparable to that of VC. Thus, we propose that arginine methylation regulates VC via many signaling pathways, including as NF-B, WNT, AKT/PI3K, TGF-/BMP/SMAD, and IL-6/STAT3. It might also regulate the VC-related calcification regulatory factors, oxidative stress, and endoplasmic reticulum stress. Consequently, we propose that arginine methylation regulates the calcification of the arteries and outline the regulatory mechanisms involved.

Gallic acid induces osteoblast differentiation and alleviates inflammatory response through GPR35/GSK3β/β-catenin signaling pathway in human periodontal ligament cells.

Gallic acid (GA) possesses various beneficial functions including antioxidant, anticancer, anti-inflammatory as well as inhibiting osteoclastogeneis. However, effects on osteogenic differentiation, especially in human ligament periodontal (hPDL) cells, remain unclear. Thus, the aim of this study was to evaluate the function of GA on osteogenesis and anti-inflammation in hPDL cells and to explore the involved underlying mechanism. Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) treatment was used as a model for periodontitis. ROS production was determined by H2DCFDA staining. Trans-well and wound healing assays were performed for checking the migration effect of GA. Alizarin red and alkaline phosphatase activity (ALP) assays were performed to evaluate osteogenic differentiation. Osteogenesis and inflammatory-related genes and proteins were measured by real-time PCR and western blot. Our results showed that GA-treated hPDL cells had higher proliferation and migration effect. GA inhibited ROS production-induced by Pg-LPS. Besides, GA abolished Pg-LPS-induced inflammation cytokines (il-6, il-1β) and inflammasome targets (Caspase-1, NLRP3). In addition, GA promoted ALP activity and mineralization in hPDL cells, lead to enhance osteoblast differentiation process. The effect of GA is related to G-protein-coupled receptor 35 (GPR35)/GSK3β/β-catenin signaling pathway. GA attenuated Pg-LPS-induced inflammatory responses and periodontitis in hPDL cells. Taken together, GA may be targeted for therapeutic interventions in periodontal diseases.

Склеростин-залежні механізми формування остеопорозу у хворих цукровим діабетом 2 типу та ожирінням

при обстеженні 103 пацієнтів на цукровий діабет 2 типу (ЦД), що у 83 випадкахперебігав на тлі збільшеної маси тіла або ожиріння, було встановлено вірогідне збільшеннявмісту склеростину у сироватці крові, яке мало пряму кореляційну залежність з індексоммаси тіла. Висловлено припущення, що зміни даного глікопротеїну, який входить до групиантагоністів кісткових морфогенетичних білків, відбуваються внаслідок багатьох процесів ворганізмі: збільшення синтезу ФНП-α адипоцитами, ендотелієм судин, клітинами нейроглії натлі пре- та менопаузи, що сприяє порушенню мінеральної щільності кісткової тканини, тобтоформуванню остеопорозу. Ендокринний остеопороз становить найбільшу групу вторинногоостеопорозу, провідною причиною якого є цукровий діабет 2 типу. В разі наявності ЦД 2 типу,який перебігає на тлі ожиріння, формуються умови до розвитку остеопоротичних станів, одниміз механізмів розвитку яких може виступати глікопротеїн склеростин. Метою дослідженнястало визначення структурно-функціонального стану кісткової тканини шляхом дослідженнявмісту склеростину у хворих на цукровий діабет 2 типу, що перебігає на тлі топографічнихособливостей розподілу жирової тканини. До роботи було залучено 103 хворих на ЦД 2 типу,який у 83 випадках перебігав в поєднанні з надлишковою вагою або ожирінням (основна група). Догрупи порівняння увійшло 20 хворих на ЦД 2 типу, що був діагностований у пацієнтів з незмінениміндексом маси тіла (ІМТ). Вік хворих коливався від 31 до 55 років та в середньому по групах складав43±4,6 років та 44,1±2,1 роки відповідно. В обох групах хворих переважали чоловіки 44 (53%) та11 (55%) осіб. Тривалість захворювання реєструвалась в межах від 1 до 13 років. Визначеннярозподілу жирової тканини в організмі досліджували за допомогою ваг для характеристикискладу тіла - OMRON BF511 (Японія). Ідентифікували вміст вісцеральної жирової тканини(ВЖТ) та підшкірної жирової тканини (ПЖТ). Активність склеростину в сироватці крові
 визначали імуноферментним методом з використанням комерційних тест-систем «Biomedi-ca» (США) згідно інструкції на імуноферментному аналізаторі «Labline-90» (Австрія). Обробку
 отриманих даних здійснювали методом варіаційної статистики за допомогою ліцензійногопрограмного забезпечення Stata 10 («StatSoft Inc.», США). Перевірка на нормальність розподілуданих виконувалася за допомогою критерію Шапіро-Уілка. Для параметрів з нормальнимрозподілом розраховували середню величину (M) ± стандартну помилку середньої (m). Отриманірезультати розглядалися як статистично значущі при p<0,05. В результаті було визначено, щозбільшення ІМТ супроводжувалось підвищенням показника склеростину. Тобто, виявлені зміниглікопротеїну, який входить до групи антагоністів кісткових морфогенетичних білків, дають змогу припустити порушення процесу диференціації остеобластів. Тобто, як ЦД 2 типу, такі кількісна зміна жирової тканини з її перерозподілом, негативно впливають на метаболізмкісткової тканини. Так, перебіг цукрового діабету супроводжується збільшенням активностіпрозапальних цитокінів, а саме, ФНП-α – складового цитокінового каскаду жирової тканини.Цей феномен також пов’язують із залученням до процесу судин (ендотеліальні клітинипродукують даний цитокін), нервової тканини (залучення клітин нейроглії) та метаболічнимизсувами в організмі. Окрім того F.Xu та співавт. в експерименті доведено, що остеокластогенезпригнічується при гіперглікемічних та гіперінсулінемічних станах. Тобто, у хворих на ЦД призбільшенні ІМТ складається негативний «тандем» через додаткове надходження з багатьохклітин ФНП-α та збільшення синтезу склеростину, що не тільки «запускає» процес порушеннямінеральної щільності кісткової тканини (МЩКТ), але й підтримує його впродовж всьогоперебігу захворювання. Також певна роль відводиться віковому аспекту обстежених хворихна ЦД (середній вік менопаузи у здорових жінок нашої країни становить 45 років), що такожсприяє пригніченню кісткового метаболізму. В такому разі збільшення активності склеростинув поєднанні з негативним фоном (ожиріння, період менопаузи) можна розглядати як один ізмеханізмів формування остеопоротичних змін. При цьому поєднуються механізми первинного тавторинного ОП, що посилює і прискорює процес порушення архітектоніки кістки. Тому можназробити висновки, що у хворих на цукровий діабет 2 типу у поєднанні з ожирінням виникаютьумови до зниження якості кісткової тканини через уповільнення кісткового метаболізму, щосприяє формуванню остеопоротичних станів.

Increased Cellular Expression of Interleukin-6 in Patients With Ossification of the Posterior Longitudinal Ligament.

We performed histologic, immunohistochemical, immunoblot examination and suspension array analyses of cytokine expression in cultured cells derived from human cervical ossification of the posterior longitudinal ligament (OPLL). To determine the roles of interleukin-6 (IL-6) during the maturation of osteoblasts and chondrocytes associated with the development of OPLL. Ectopic OPLL affects ~3% of the general population, with a higher incidence in Asian ethnic groups. Alterations in cytokine profiles may influence osteoblast differentiation, but the mechanisms and signaling pathways associated with the ossification process remain unclear. Samples were collected from 14 patients with OPLL who had undergone spinal surgery and seven with cervical spondylotic myelopathy without OPLL. Tissue sections were used for histologic and immunohistochemical studies, and primary cells from ligamentum samples were used for cytokine array and immunoblotting. A suspension array was used to measure the concentrations of 27 inflammatory cytokines or growth factors. Suspension array and immunoblot analyses revealed significantly elevated levels of IL-6 in OPLL patients. Alterations in IL-6 concentrations were found to alter the expression of the genes Sox9 , Runx2 , and SIRT1 . In addition, immunohistochemical analysis revealed that these factors are present in mesenchymal cells within the degenerative portion of the ligament matrix that is adjacent to the ossification front. IL-6 plays a profound role in the osteoblast differentiation process along with the induction of chondrocyte hypertrophy and cell apoptosis in the early stages of ossification in OPLL. These changes in cytokine profiles are essential factors for regulation of the ectopic ossified plaque in OPLL.

TCDD alters essential transcriptional regulators of osteogenic differentiation in multipotent mesenchymal stem cells.

Differentiation of multipotent mesenchymal stem cells (MSCs) into bone-forming osteoblasts requires strict coordination of transcriptional pathways. Aryl hydrocarbon receptor ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been shown to alter osteoblast differentiation in vitro and bone formation in multiple developmental in vivo models. The goal of the present study was to establish a global transcriptomic landscape during early, intermediate, and apical stages of osteogenic differentiation in vitro in response to TCDD exposure. Human bone-derived mesenchymal stem cells (hBMSCs) were cultured in growth media (GM), osteogenic differentiation media (ODM), or ODM containing 10 nM TCDD (ODM + TCDD), thus enabling a comparison of the transcriptomic profiles of undifferentiated, differentiated, and differentiated-TCDD-exposed hBMSCs, respectively. In this test system, exposure to TCDD attenuated the differentiation of hBMSCs into osteoblasts as evidenced by reduced alkaline phosphatase activity and mineralization. At various timepoints, we observed altered expression of genes that play a role in the Wnt, fibroblast growth factor, bone morphogenetic protein/transforming growth factor beta developmental pathways, as well as pathways related to extracellular matrix organization and deposition. Reconstruction of gene regulatory networks with the interactive dynamic regulatory event miner (iDREM) analysis revealed modulation of transcription factors (TFs) including POLR3G, NR4A1, RDBP, GTF2B, POU2F2, and ZEB1, which may putatively influence osteoblast differentiation and the requisite deposition and mineralization of bone extracellular matrix. We demonstrate that the combination of RNA-Seq data in conjunction with the iDREM regulatory model captures the transcriptional dynamics underlying MSC differentiation under different conditions in vitro. Model predictions are consistent with existing knowledge and provide a new tool to identify novel pathways and TFs that may facilitate a better understanding of the osteoblast differentiation process, perturbation by exogenous agents, and potential intervention strategies targeting those specific pathways.

Metabolic features of myeloma cells in the context of bone microenvironment: Implication for the pathophysiology and clinic of myeloma bone disease.

Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasma cells (PCs) into the bone marrow (BM). The complex interaction between the BM microenvironment and MM PCs can lead to severe impairment of bone remodeling. Indeed, the BM microenvironment exerts a critical role in the survival of malignant PCs. Growing evidence indicates that MM cells have several metabolic features including enhanced glycolysis and an increase in lactate production through the upregulation of glucose transporters and enzymes. More recently, it has been reported that MM cells arehighly glutamine addicted. Interestingly, these metabolic changes in MM cells may affect BM microenvironment cells by altering the differentiation process of osteoblasts from mesenchymal stromal cells. The identification of glutamine metabolism alterations in MM cells and bone microenvironment may provide a rationale to design new therapeutic approaches and diagnostic tools. The osteolytic lesions are the most frequent clinical features in MM patients, often characterized by pathological fractures and acute pain. The use of the newer imaging techniques such as Magnetic Resonance Imaging (MRI) and combined Positron Emission Tomography (PET) and Computerized Tomography (CT) has been introduced into clinical practice to better define the skeletal involvement. Currently, the PET/CT with 18F-fluorodeoxyglucose (FDG) is the diagnostic gold standard to detect active MM bone disease due to the high glycolytic activity of MM cells. However, new tracers are actively under investigation because a portion of MM patients remains negative at the skeletal level by 18F-FDG. In this review, we will summarize the existing knowledge on the metabolic alterations of MM cells considering their impact on the BM microenvironment cells and particularly in the subsequent formation of osteolytic bone lesions. Based on this, we will discuss the identification of possible new druggable targets and the use of novel metabolic targets for PET imaging in the detection of skeletal lesions, in the staging and treatment response of MM patients.

Sodium Selenite Promotes Osteoblast Differentiation via The WNT/ß-Catenin Signaling Pathway.

ObjectivesOsteoporosis is regarded as a silent disorder affecting bone slowly, leading to an increased risk of fractures. Lately, selenium has been found to be associated with the acquisition and maintenance of bone health by affecting the bone remodeling process. However, the mechanism of action of selenium on bone is poorly understood. Here, the objective of this study is to examine the protective effects and mechanism of sodium selenite on the differentiation process of osteoblasts as well as under oxidative stress-induced conditions by evaluating the expression of osteoblast differentiation markers in the sodium selenite and/or hydrogen peroxide (H2O2)-treated MC3T3-E1 cell line.Materials and Methods In this experimental study, we confirmed the inducible osteogenic effect of sodium selenite on MC3T3-E1 cells. Moreover, we investigated the recovery of expression levels of osteogenic markers of sodium selenite in H2O2-treated MC3T3-E1 cells. Results It was observed that sodium selenite could promote alkaline phosphatase (ALP) activity and collagen synthesis in pre-osteoblasts. Also, sodium selenite enhanced the mRNA expression levels of osteogenic transcriptional factors, like osterix (OSX) and runt-related transcription factor 2 (Runx2). In addition, the terminal differentiation markers, such as osteocalcin (OCN) and collagen 1α (Col1α) were also increased after the treatment of sodium selenite. Also treatment of sodium selenite recused the H2O2-induced inhibition of osteoblastic differentiation of pre-osteoblasts cells via the WNT signaling pathway, implicating its antioxidant activity. Furthermore, sodium selenite restored the H2O2 repressed β-catenin stability and axin-2 reporter activity in MC3T3-E1 cells. ConclusionIt may be concluded that sodium selenite can stimulate bone formation and rescue the oxidative repression of osteogenesis by activating WNT signaling pathways. Further detailed studies on the role of selenium and its ability to stimulate bone formation via the WNT signaling pathway may project it as a potential therapeutic intervention for osteoporosis.

Exposure of primary osteoblasts to combined magnetic and electric fields induced spatiotemporal endochondral ossification characteristic gene- and protein expression profiles

PurposeMolecular processes in primary osteoblasts were analyzed in response to magnetic and electric field exposure to examine its potential impact on bone healing.MethodsPrimary osteoblasts were exposed to a combination of a magnetic field and an additional electric field (EFMF) (20 Hz, 700 mV, 5 mT, continuous sinusoids) in vitro. mRNA- and protein-expressions were assessed during a time interval of 21 days and compared with expression data obtained from control osteoblasts.ResultsWe observed an autonomous osteoblast differentiation process in vitro under the chosen cultivation conditions. The initial proliferative phase was characterized by a constitutively high mRNA expression of extracellular matrix proteins. Concurrent EFMF exposure resulted in significanly increased cell proliferation (fold change: 1.25) and reduced mRNA-expressions of matrix components (0.5–0.75). The following reorganization of the extracellular matrix is prerequisite for matrix mineralization and is characterised by increased Ca2+ deposition (1.44). On molecular level EFMF exposure led to a significant decreased thrombospondin 1 (THBS1) mRNA- (0.81) and protein- (0.54) expression, which in turn reduced the TGFß1-dependent mRNA- (0.68) and protein- (0.5) expression of transforming growth factor beta induced (ßIG-H3) significantly, an inhibitor of endochondral ossification. Consequently, EFMF exposure stimulated the expression of genes characteristic for endochondral ossification, such as collagen type 10, A1 (1.50), osteopontin (1.50) and acellular communication network factor 3 (NOV) (1.45).ConclusionsIn vitro exposure of osteoblasts to EFMF supports cell differentiation and induces gene- and protein-expression patterns characteristic for endochondral ossification during bone fracture healing in vivo.

PTH1R Actions on Bone Using the cAMP/Protein Kinase A Pathway.

After the initial signaling action of parathyroid hormone (PTH) on bone was shown to be activation of adenylyl cyclase, its target was found to be cells of the osteoblast lineage, to the exclusion of osteoclasts and their precursors. This led to the view that the osteoblast lineage regulated osteoclast formation, a proposal that was established when the molecular mechanisms of osteoclast formation were discovered. This is in addition to the effect of PTH1Rv signaling throughout the osteoblast differentiation process to favour the formation of bone-forming osteoblasts. Initial signaling in the PTH target cells through cAMP and protein kinase A (PKA) activation is extremely rapid, and marked by an amplification process in which the later event, PKA activation, precedes cAMP accumulation in time and is achieved at lower concentrations. All of this is consistent with the existence of “spare receptors”, as is the case with several other peptide hormones. PTH-related protein (PTHrP), that was discovered as a cancer product, shares structural similarity with PTH in the amino-terminal domain that allows the hormone, PTH, and the autocrine/paracrine agent, PTHrP, to share actions upon a common G protein coupled receptor, PTH1R, through which they activate adenylyl cyclase with equivalent potencies. Studies of ligand-receptor kinetics have revealed that the PTH/PTH1R ligand-receptor complex, after initial binding and adenylyl cyclase activation at the plasma membrane, is translocated to the endosome, where adenylyl cyclase activation persists for a further short period. This behavior of the PTH1R resembles that of a number of hormones and other agonists that undergo such endosomal translocation. It remains to be determined whether and to what extent the cellular effects through the PTH1R might be influenced when endosomal is added to plasma membrane activation.

Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process

Staphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.

PTX3 Effects on Osteogenic Differentiation in Osteoporosis: An In Vitro Study.

Pentraxin 3 (PTX3) is a glycoprotein belonging to the humoral arm of innate immunity that participates in the body’s defence mechanisms against infectious diseases. It has recently been defined as a multifunctional protein, given its involvement in numerous physiological and pathological processes, as well as in the pathogenesis of age-related diseases such as osteoporosis. Based on this evidence, the aim of our study was to investigate the possible role of PTX3 in both the osteoblastic differentiation and calcification process: to this end, primary osteoblast cultures from control and osteoporotic patients were incubated with human recombinant PTX3 (hrPTX3) for 72 h. Standard osteinduction treatment, consisting of β-glycerophosphate, dexamethasone and ascorbic acid, was used as control. Our results showed that treatment with hrPTX3, as well as with the osteogenic cocktail, induced cell differentiation towards the osteoblastic lineage. We also observed that the treatment not only promoted an increase in cell proliferation, but also the formation of calcification-like structures, especially in primary cultures from osteoporotic patients. In conclusion, the results reported here suggest the involvement of PTX3 in osteogenic differentiation, highlighting its osteoinductive capacity, like the standard osteoinduction treatment. Therefore, this study opens new and exciting perspectives about the possible role of PTX3 as biomarker and therapeutic agent for osteoporosis.

Berberine encapsulated nanoparticles stimulate osteoblast differentiation in vitro

Berberine has been known as a traditional component for treatment of intestinal-related diseases in Asian countries. Additionally, it possesses a variety of pharmacological properties, which were studied for treating tumor, diabetes, cardiovascular disease, hyperlipidemia, inflammation, bacterial and viral infections, cerebral ischemia trauma, and mental disease. Moreover, berberine has been known as an anti-osteoporotic agent by controlling both osteoclast (bone resorption cells) and osteoblast (bone-forming cells) functions. Beside the beneficial effects of berberine, it has some drawbacks that hindered its applications and resulted in low bioavailability. One of the most drawback characteristics of berberine is that it has poor watery solubility. To overcome these limits, nanotechnology has been used as the primary approach to deliver berberine in different nano-formulations. In this study, a novel berberine nanoparticle (nanoberberine, NBB) with good water dispersion was synthesized to enhance its bioavailability. The result showed that NBB was successfully developed in spherical shape and approximately 559 nm of mean size. Besides, in vitro release study revealed that berberine content release from NBB was 3 to 4 times higher than that from free berberine. Moreover, no cytotoxicity was observed for both NBB and berberine on osteoblast MC3T3-E1 cells at the tested concentrations. Additionally, alkaline phosphatase (ALP) activity, a marker for osteoblast differentiation process, was significantly higher in NBB compared to free berberine at the same test concentrations. This result indicated that NBB could be a potential biological agent for inducing bone formation. Overall, our data indicated that NBB could improve bioavailability, especially osteogenesis activity in vitro compared to free berberine.

Modulation of Endocannabinoid Tone in Osteoblastic Differentiation of MC3T3-E1 Cells and in Mouse Bone Tissue over Time.

Bone is a highly complex and metabolically active tissue undergoing a continuous remodeling process, which endures throughout life. A complex cell-signaling system that plays role in regulating different physiological processes, including bone remodeling, is the endocannabinoid system (ECS). Bone mass expresses CB1 and CB2 cannabinoid receptors and enzymatic machinery responsible for the metabolism of their endogenous ligands, endocannabinoids (AEA and 2-AG). Exogenous AEA is reported to increase the early phase of human osteoblast differentiation in vitro. However, regarding this cell context little is known about how endocannabinoids and endocannabinoid-related N-acylethanolamines like PEA and OEA are modulated, in vitro, during cell differentiation and, in vivo, over time up to adulthood. Here we characterized the endocannabinoid tone during the different phases of the osteoblast differentiation process in MC3T3-E1 cells, and we measured endocannabinoid levels in mouse femurs at life cycle stages characterized by highly active bone growth (i.e., of juvenile, young adult, and mature adult bone). Endocannabinoid tone was significantly altered during osteoblast differentiation, with substantial OEA increment, decline in 2-AG and AEA, and consistent modulation of their metabolic enzymes in maturing and mineralized MC3T3-E1 cells. Similarly, in femurs, we found substantial, age-related, decline in 2-AG, OEA, and PEA. These findings can expand existing knowledge underlying physiological bone cell function and contribute to therapeutic strategies for preventing bone-related metabolic changes accruing through lifespan.

Agastache rugosa ethanol extract suppresses bone loss via induction of osteoblast differentiation with alteration of gut microbiota

Osteoporosis is a metabolic skeletal disease characterized by bone loss and an increased risk of fractures. This study aimed to investigate the therapeutic effect of Agastache rugosa on postmenopausal osteoporosis and elucidate its mechanisms in modulating the bone status. In the osteoblast differentiation process with MC3T3-E1 pre-osteoblasts, ethanol extract of Agastache rugosa (EEAR) and its compounds increased the expression of the proteins and genes of the osteoblast differentiation-related markers such as Runt-related transcription factor 2 (RUNX2) and β-catenin along with the elevation of calcium deposits. An ovariectomized mouse model was utilized to determine the impact of EEAR extract on postmenopausal osteoporosis. Twelve weeks of AR treatment suppressed the loss of bone strength, which was observed through micro-computed tomography. AR elevated osteogenic markers in the bone marrow cells, and collagen type 1 alpha 1 in the distal femoral bone. The results of the 16S rRNA gene sequencing analysis of cecal gut microbiomes demonstrated that AR reversed the ovariectomy-induced changes in the gut microbiomes. Ethanol extract of Agastache rugosa has a therapeutic effect on postmenopausal osteoporosis via bone morphogenic protein, transforming growth factor β, and Wnt signaling pathway. It also increases the diversity of gut microbiota. Therefore, these data suggest that EEAR could be a potential candidate to treat postmenopausal osteoporosis.

Trelagliptin stimulates osteoblastic differentiation by increasing runt-related transcription factor 2 (RUNX2): a therapeutic implication in osteoporosis

ABSTRACT Osteoporosis, an aging-associated bone metabolic disease, is affecting millions of people worldwide. The deregulated process of osteoblastic differentiation has been linked with the progression of osteoporosis. Trelagliptin is a long-acting inhibitor of DPP-4 used for the management of type 2 diabetes mellitus. However, it is unknown whether Trelagliptin possesses a beneficial effect in osteoblastic differentiation. Interestingly, we found that treatment with Trelagliptin enhanced differentiation and promoted the mineralization of MC3T3-E1 cells. Firstly, Trelagliptin increased the activity of alkaline phosphatase (ALP) and promoted osteoblastic calcium deposition. Additionally, treatment with Trelagliptin upregulated ALP, osteocalcin (OCN), osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2). Notably, Trelagliptin increased RUNX2, a major regulator of osteoblastic differentiation. Mechanistically, Trelagliptin upregulated the levels of p-AMPKα. Blockage of AMPK with compound C abolished the effects of Trelagliptin in RUNX2 and osteoblastic differentiation, suggesting the involvement of AMPK. Our findings suggest that Trelagliptin might possess a potential for the treatment of osteoporosis.

Effects of inhibiting PDK‑1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation invitro.

Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3-phosphoinositide-dependent protein kinase-1 (PDK-1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK-1 in the process of mouse osteoblast differentiation in vitro. In the BX-912 group, BX-912, a specific inhibitor of PDK-1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1flox/flox mice were designed and generated, and were used to obtain BMSCsPDK1flox/flox. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd-cre-EGFP) was used to disrupt the PDK-1 gene in BMSCsPDK1flox/flox; this served as the pHBAd-cre-EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)-PDK1 and p-AKT were gradually increased during the osteoblast differentiation process. Notably, BX-912 treatment and disruption of the PDK-1 gene with pHBAd-cre-EGFP effectively reduced the number of alkaline phosphatase (ALP)-positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK-1 in the pHBAd-cre-EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3–7. The mRNA expression levels of the osteoblast-related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX-912 and pHBAd-cre-EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX-912 and disruption of the PDK-1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK-1 in osteoblasts.

Endogenous GDF11 regulates odontogenic differentiation of dental pulp stem cells.

Dental stem cell‐based tooth regeneration is the futuristic treatment for missing teeth. Growth differentiation factor 11 (GDF11), a novel member of the TGF‐beta superfamily, has been reported to play a critical role in regulating stem cell differentiation. However, the role of endogenous GDF11 during dental stem cell differentiation remains unknown. Here, we have shown that GDF11 was highly expressed in dental pulp tissues in both mouse and human. Knockdown of endogenous GDF11 in human dental pulp stem cells (hDPSCs) led to comparable proliferation and migration but attenuated odontogenic differentiation as evidenced by alkaline phosphatase and Alizarin Red S staining. In addition, transcriptional levels of odontogenic‐related genes were significantly down‐regulated according to real‐time polymerase chain reaction. Mechanistically, we performed RNA sequencing analysis and found that silencing of endogenous GDF11 compromised the process of ossification and osteoblast differentiation, especially down‐regulated transcription expression of Wnt pathway‐specific genes. Immunofluorescence staining also showed diminished β‐catenin expression and nuclei accumulation after knockdown of endogenous GDF11 in hDPSCs. In summary, our results suggested that endogenous GDF11 positively regulate odontogenic differentiation of hDPSCs through canonical Wnt/β‐catenin signalling pathway.

Nbr1-regulated autophagy in Lactoferrin-induced osteoblastic differentiation

The molecular mechanism of autophagy in Lactoferrin (LF) induced osteoblast differentiation is not fully demonstrated. In this study, alkaline phosphatase (ALP) activity, alizarin red S staining and ELISA were used to study N-terminal propeptide of type I procollagen (PINP) expression. mRFP-GFP-LC3 adenoviruses, mono-dansylcadaverine (MDC) staining, scanning electron microscopy, and western blot analysis was employed to probe the LF induced autophagy. The interaction between autophagy receptor Neighbor of Brca1 gene (Nbr1) and pp38 was studied. 3-methyladenine (3-MA) and chloroquine (CQ) could inhibit the activity of ALP, PINP and the autophagy in LF group. LF treatment could up-regulate and down-regulate the expressions of pp38 and Nbr1with a dose-dependent manner, respectively. LF could inhibit the recognition of pp38 and Nbr1. In addition, LF can prompt Nbr1-medicated autophagy and prevent pp38 degradation by autophagy. LF can induce Nbr1-mediated autophagy and inhibit pp38 entering into autophagy flux in the physiological process of osteoblast differentiation.Abbreviations: CQ:chloroquine；LF: Lactoferrin; 3-MA: 3-methyladenine; ALP: Alkaline phosphatase; ANOVA: Analysis of variance; CCK-8: Cell Counting Kit-8; LC3: Microtubule-associated protein light chain3; MDC: Monodansylcadaverine; Nbr1: neighbor of Brca1 gene; PINP: N-terminal propeptide of type I procollagen; PVDF: Polychlorotrifluoroethylene; pp38: phosphorylation p38; RAPA: Rapamycin; SDS: sodium dodecyl sulfate.

Lysine-Specific Demethylase 4A Regulates Osteogenic Differentiation via Regulating the Binding Ability of H3K9me3 with the Promoters of Runx2, Osterix and Osteocalcin.

A well-studied subject of epigenetics, the histone methylation located at lysine and arginine is overseen via methyltransferases and demethylases. Lysine-specific demethylase 4A (KDM4A) comprises a lysine demethylase and possesses specificity for H3K9me3 and H3K36me3, which is capable of being used in order to activate histone transcription. Our team examined the expression of KDM4A within Sprague Dawley (SD) rats and further investigated the mechanism via which this phenomena regulates osteogenic variation within the present study. The overexpression of KDM4A facilitated the process of osteoblast differentiation in bone mesenchymal stem cells (BMSC), while the knocking down differentiation via osteoblast was restrained via the suppression of the expression of Runx2, Osterix, alkaline phosphatase (ALP), and osteocalcin (OCN). Knocking down KDM4A lowered levels of the promoter expression of Runx2, osterix, and OCN, and raised levels of H3K27me3 expression. The results demonstrated that KDM4A possesses a crucial role within the differentiation of osteoblasts and furthermore regulates the expression of Runx2, Osterix, and OCN via H3K9me3. The present research may provide new insights into the treatment of bone healing.