Dehydrocostus lactone (DHC) promotes osteoblastic differentiation and mineralization through p38/RUNX-2 signaling.

Dysregulation of osteoblastic differentiation is an important risk factor of osteoporosis, the therapy of which is challenging. Dehydrocostus lactone (DHC), a sesquiterpene isolated from medicinal plants, has displayed anti-inflammatory and antitumor properties. In this study, we investigated the effects of DHC on osteoblastic differentiation and mineralization of MC3T3-E1 cells. Interestingly, we found that DHC increased the expression of marker genes of osteoblastic differentiation, such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Additionally, DHC increased the expressions of collagen type I alpha 1 (Col1a1) and collagen type I alpha 2 (Col1a2). We also demonstrate that DHC increased ALP activity. Importantly, the Alizarin Red S staining assay revealed that DHC enhanced osteoblastic differentiation of MC3T3-E1 cells. Mechanistically, it is shown that DHC increased the expression of Runx-2, a central regulator of osteoblastic differentiation. Treatment with DHC also increased the levels of phosphorylated p38, and its blockage using its specific inhibitor SB203580 abolished the effects of DHC on runt-related transcription factor 2 (Runx-2) expression and osteoblastic differentiation, suggesting the involvement of p38. Based on these findings, we concluded that DHC might possess a capacity for the treatment of osteoporosis by promoting osteoblastic differentiation.

Relaxin-2 promotes osteoblastic differentiation mediated by epidermal growth factor and epidermal growth factor receptor signaling.

Loss of osteogenic differentiation potential of osteoblasts has been associated with the pathogenesis of osteoporosis. Thus, stimulation of osteoblastic differentiation is a therapeutic strategy for osteoporosis. Relaxin-2 is a peptide hormone with potent biological functions. However, the effects of Relaxin-2 in osteoblastic differentiation and osteoporosis have not been reported before. Here, we report a novel physiological role of Relaxin-2 in promoting osteoblastic differentiation and mineralization of MC3T3-E1 cells. Our results indicate that exposure to Relaxin-2 upregulated the expression, and elevated the activity of alkaline phosphatase (ALP) when MC3T3-E1 cells were cultured in osteogenic differentiation medium (OM). Additionally, Relaxin-2 upregulated the mRNA levels of osteocalcin (ocn), osteopontin (opn), and collagen type I alpha 1 (Col1a1). The alizarin red S staining assay revealed that Relaxin-2 promoted the mineralization of MC3T3-E1 cells. We also found that Relaxin-2 increased the expression of Runx-2 as well as the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR). Importantly, silencing of EGF abolished the effects of Relaxin-2 in osteoblastic differentiation and related gene expression. These findings suggest that Relaxin-2 stimulates osteogenic differentiation through activating EGF/EGFR signaling.

AEBP1 restores osteoblastic differentiation under dexamethasone treatment by activating PI3K/AKT signalling.

Adipocyte enhancer-binding protein 1 (AEBP1) is closely implicated in osteoblastic differentiation and bone fracture; this research aimed to investigate the effect of AEBP1 on restoring osteoblastic differentiation under dexamethasone (Dex) treatment, and its interaction with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. Pre-osteoblastic MC3T3-E1 cells were cultured in osteogenic medium and treated by Dex to mimic steroid-induced osteonecrosis cellular model. They were then further transfected with control or AEBP1-overexpressed lentiviral vectors. Finally, cells were treated with the PI3K inhibitor LY294002, with or without AEBP1-overexpressed lentiviral vectors. AEBP1 expression showed a downward trend in MC3T3-E1 cells under Dex treatment in a dose-dependent manner. AEBP1-overexpressed lentiviral vectors increased relative cell viability, alkaline phosphatase (ALP) staining, Alizarin red staining and osteoblastic differentiation markers including osteocalcin (OCN), osteopontin (OPN), collagen type I alpha 1 (COL1A1), runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), but decreased cell apoptosis rate in MC3T3-E1 cells under Dex treatment; besides, AEBP1-overexpressed lentiviral vectors positively regulated p-PI3K and p-AKT expressions. Furthermore, LY294002 treatment decreased relative cell viability, Alizarin red staining, osteoblastic differentiation markers including OCN, OPN, RUNX2 and BMP, increased cell apoptosis rate and did not affect ALP staining in MC3T3-E1 cells under Dex treatment; meanwhile, LY294002 treatment weakened the effect of AEBP1 overexpression vectors on the above cell functions. AEBP1 restores osteoblastic differentiation under Dex treatment by activating the PI3K/AKT pathway.

FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis.

Bone defects have remained a clinical problem in current orthopedics. Bone marrow mesenchymal stem cells (BM-MSCs) with multi-directional differentiation ability have become a research hotspot for repairing bone defects. In vitro and in vivo models were constructed, respectively. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to detect osteogenic differentiation ability. Western blotting (WB) was used to detect the expression of osteogenic differentiation-related proteins. Serum inflammatory cytokine levels were detected by ELISA. Fracture recovery was evaluated by HE staining. The binding relationship between FOXC1 and Dnmt3b was verified by dual-luciferase reporter assay. The relationship between Dnmt3b and CXCL12 was explored by MSP and ChIP assays. FOXC1 overexpression promoted calcium nodule formation, upregulated osteogenic differentiation-related protein expression, promoted osteogenic differentiation, and decreased inflammatory factor levels in BM-MSCs, and promoted callus formation, upregulated osteogenic differentiation-related protein expression, and downregulated CXCL12 expression in the mouse model. Furthermore, FOXC1 targeted Dnmt3b, with Dnmt3b knockdown decreasing calcium nodule formation and downregulating osteogenic differentiation-related protein expression. Additionally, inhibiting Dnmt3b expression upregulated CXCL12 protein expression and inhibited CXCL12 methylation. Dnmt3b could be binded to CXCL12. CXCL12 overexpression attenuated the effects of FOXC1 overexpression and inhibited BM-MSCs osteogenic differentiation. This study confirmed that the FOXC1-mediated regulation of the Dnmt3b/CXCL12 axis had positive effects on the osteogenic differentiation of BM-MSCs.

Supplementation with carnosine, a food-derived bioactive dipeptide, alleviates dexamethasone-induced oxidative stress and bone impairment via the NRF2 signaling pathway.

BACKGROUND: Carnosine, a natural bioactive dipeptide derived from meat muscle, possesses strong antioxidant properties. Dexamethasone, widely employed for treating various inflammatory diseases, raises concerns regarding its detrimental effects on bone health. This study aimed to investigate the protective effects of carnosine against dexamethasone-induced oxidative stress and bone impairment, along with its underlying mechanisms, utilizing chick embryos and a zebrafish model in vivo, as well as MC3T3-E1 cells in vitro. RESULTS: Our findings revealed that carnosine effectively mitigated bone injury in dexamethasone-exposed chick embryos, accompanied by reduced oxidative stress. Further investigation demonstrated that carnosine alleviated impaired osteoblastic differentiation in MC3T3-E1 cells and zebrafish by suppressing the excessive production of reactive oxygen species (ROS) and enhancing the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPX). Moreover, mechanistic studies elucidated that carnosine promoted the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), thereby facilitating the transcription of its downstream antioxidant response elements, including heme oxyense-1 (HO-1), glutamate cysteine ligase modifier (GCLM), and glutamate cysteine ligase catalytic (GCLC) to counteract dexamethasone-induced oxidative stress. CONCLUSION: Overall, this study underscores the potential therapeutic efficacy of carnosine in mitigating oxidative stress and bone damage induced by dexamethasone exposure, shedding light on its underlying mechanism of action by activating the NRF2 signaling pathway. © 2024 Society of Chemical Industry.

Arl4c is involved in tooth germ development through osteoblastic/ameloblastic differentiation.

Murine tooth germ development proceeds in continuous sequential steps with reciprocal interactions between the odontogenic epithelium and the adjacent mesenchyme, and several growth factor signaling pathways and their activation are required for tooth germ development. The expression of ADP-ribosylation factor (Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby involved in epithelial morphogenesis and tumorigenesis. In contrast, the other functions of Arl4c (in addition to cellular growth) are largely unknown. Although we recently demonstrated the involvement of the upregulated expression of Arl4c in the proliferation of ameloblastomas, which have the same origin as odontogenic epithelium, its effect on tooth germ development remains unclear. In the present study, single-cell RNA sequencing (scRNA-seq) analysis revealed that the expression of Arl4c, among 17 members of the Arf-family, was specifically detected in odontogenic epithelial cells, such as those of the stratum intermedium, stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in non-ameloblast and inner enamel epithelium, which include immature cells, of P7 mouse incisors. In the mouse tooth germ rudiment culture, treatment with SecinH3 (an inhibitor of the ARNO/Arf6 pathway) reduced the size, width and cusp height of the tooth germ and the thickness of the eosinophilic layer, which would involve the synthesis of dentin and enamel matrix organization. In addition, loss-of-function experiments using siRNAs and shRNA revealed that the expression of Arl4c was involved in cell proliferation and osteoblastic cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis with a gene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that osteoblastic differentiation-related gene sets and/or GO terms were downregulated in shArl4c-expressing odontogenic epithelial cells. These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes to tooth germ development through osteoblastic/ameloblastic differentiation.

IPO7 Promotes Odontoblastic Differentiation and Inhibits Osteoblastic Differentiation Through Regulation of RUNX2 Expression and Translocation.

RUNX2, an important transcriptional factor for both odontoblastic and osteoblastic differentiation, is upregulated during osteoblastic differentiation, but downregulated during late odontoblastic differentiation. However, the specific mechanism of the different RUNX2 expression in bone and dentin remains largely unknown. Importin 7 (IPO7), a member of the karyopherin ß-superfamily, mediates nucleocytoplasmic transport of proteins. In this study, we found that IPO7 was increasingly expressed from pre-odontoblasts to mature odontoblasts. IPO7 expression was increased with odontoblastic differentiation of mouse dental papilla cells (mDPCs) and knockdown of IPO7-inhibited cell differentiation. While in MC3T3-E1 cells, IPO7 was decreased during osteoblastic differentiation and knockdown of IPO7-promoted cell differentiation. In mPDCs, IPO7 was able to bind with some odontoblastic transcription factors, and imported them into the nucleus, but not with RUNX2. Furthermore, IPO7 inhibited the total RUNX2 expression by promoting HDAC6 nuclear localization during odontoblastic differentiation. However, in MC3T3-E1 cells, IPO7 inhibited the nuclear distribution of RUNX2 but did not affect the total protein level of RUNX2. In conclusion, we found that IPO7 promotes odontoblastic differentiation and inhibits osteoblastic differentiation through regulating RUNX2 expression and translocation differently.

Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

A nonobstructive condition: Medial arterial calcification. / ä¸å¼•èµ·ç®¡è ”ç‹­çª„çš„è¡€ç®¡ä¸­è†œé’™åŒ–.

Vascular calcification, including intimal and medial calcification, is closely associated with a significant increase in cardiovascular diseases. Although increased understandings were achieved, people still know much more about intimal calcification than medial calcification because the latter doesn't obstruct the arterial lumen, commonly considered as a non-significant finding. We clarified the pathologic characteristic of medial calcification, its difference from intimal calcification, principally focused on its clinical relevance, such as diagnosis, nosogenesis, and hemodynamics. We underline the importance of identifying and distinguishing medial calcification, understanding its effect to local/systematic arterial compliance, and relationship to diabetic neuropathy. Recent studies emphasize do not ignore its predictive role in cardiovascular mortality. It is of great clinical significance to summarize the mechanisms of occurrence, lesion characteristics, diagnostic methods, pathogenic mechanisms, hemodynamic changes, and the distinction as well as association of intimal calcification with intimal calcification.

Involvement of Rab11 in osteoblastic differentiation: Its up-regulation during the differentiation and by tensile stress.

Rab GTPases, the largest group of small monomeric GTPases, have been shown to participate in membrane trafficking involving many cellular processes. However, their roles during osteoblastic differentiation remain to be elucidated. In this study, we investigated Rab GTPase involvement in osteoblastic differentiation. Protein levels of a series of Rabs (Rab4, Rab5, Rab7, Rab9a, Rab11a/b, and Rab27) were increased during osteoblastic differentiation of MC3T3-E1 cells, and the Rab11a/b levels were particularly pronounced in the presence of Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, an activator of osteoblastogenesis. We subsequently investigated the functional contribution of Rab11a and Rab11b during osteoblastic differentiation. The alkaline phosphatase (ALP) levels were reduced by Rab11b depletion but not by Rab11a depletion. Because our result suggested that Rab11a and Rab11b could be regulated downstream of Runx2 (Runt-related transcription factor 2), a key transcription factor for osteoblastic differentiation, we investigated the effects of the double knockdown of Runx2 and Rab11a or Rab11b on osteoblastic phenotypes. The double knockdown significantly reduced ALP activity as well as collagen deposition compared with single Runx2 knockdown. Furthermore, the Rab11a and Rab11b response to mechanical stress in vivo was investigated using a mouse orthodontic tooth movement model. Rab11a and Rab11b expression was enhanced in the periodontal ligament, where bone formation is activated by tensile stress. This study shows that Rab11a and Rab11b are regulated downstream of Runx2 in osteoblastic differentiation, and their expressions are also controlled by tensile stress.

CBX4-dependent regulation of HDAC3 nuclear translocation reduces Bmp2-induced osteoblastic differentiation and calcification in adamantinomatous craniopharyngioma.

BACKGROUND: Calcification of adamantinomatous craniopharyngioma (ACP) often causes problems with tumor resection, leading to a high incidence of deadly complications and tumor recurrence. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are 2 key enzymes that regulate histone acetylation and play important roles in tumor development. However, the roles of HAT and HDAC in the calcification and osteoblastic differentiation of ACP are not known. METHODS: In this study, primary cells were isolated from ACP tissues, and calcification was induced with bone morphogenetic protein 2 (Bmp2). HDAC3 expression was assessed in 12 tissue samples by Western blotting and immunohistochemistry. ACP calcification was assessed by Alizarin red staining. A luciferase reporter assay was performed to examine the interaction between miR-181b and the 3'-untranslated region of the polycomb chromobox 4 (CBX4) gene. RESULTS: Our results showed that the expression of HDAC3 was increased in the calcified ACP samples, but inhibition of HDAC3 promoted ACP cell calcification and osteoblastic differentiation. Mechanistically, HDAC3 nuclear translocation was suppressed by Bmp2, leading to Runx2 protein expression and Osterix, osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) mRNA expression. In addition, this process was suppressed by CBX4, which stabilized the nuclear localization of HDAC3. miR-181b, the expression of which was increased in Bmp2-induced ACP cells, directly targeted and decreased CBX4 expression and inhibited the nuclear localization of HDAC3. CONCLUSIONS: Our results demonstrate that Bmp2 increases miR-181b levels to directly target and inhibit CBX4 expression, leading to a reduction in the CBX4-dependent regulation of HDAC3 nuclear translocation, which results in Runx2 activation/osteoblastic differentiation and calcium deposition in ACP. Further studies targeting these cascades may contribute to therapeutic interventions used for recurrent ACP. Video Abstract.

Differential regulation of NPY and SP receptor expression in STRO-1+ve PDLSCs by inflammatory cytokines.

OBJECTIVES: The aims of this study were to investigate neuropeptide receptor expression regulation on STRO-1 +ve periodontal ligament stem cells (PDLSCs) in response to inflammatory cytokines and to investigate a potential osteogenic effect of neuropeptides. BACKGROUND: Nerve fibres innervating the periodontal tissues in humans contain several neuropeptides including neuropeptide Y and substance P. The role of neuropeptide receptors on PDLSCs, including their response to the local inflammatory environment of periodontitis, is currently unknown. METHODS: A homogenous population of STRO-1 +ve PDLSCs was prepared by immunomagnetic separation of cells obtained by the tissue out-growth method from healthy premolar teeth from a single donor. Regulation of gene expression of the neuropeptide Y Y1 receptor and substance P receptor tachykinin receptor 1 was investigated. A potential osteogenic effect of neuropeptide Y and substance P was also investigated by measuring alkaline phosphatase (ALP) activity, Alizarin red staining and quantifying osteogenic gene expression. RESULTS: Treatment of STRO-1 +ve PDLSCs with tumour necrosis factor-alpha or interleukin 1-beta up-regulated the expression of the neuropeptide Y's Y1 receptor, but down-regulated substance P's receptor. Significantly increased ALP activity was observed in STRO-1 +ve PDLSCs treated with neuropeptide Y but not substance P. Further studies showed that neuropeptide Y had a modest osteogenic effect on cells at both a functional level and a gene level. CONCLUSIONS: Expression of the neuropeptide Y Y1 receptor gene on STRO-1 +ve PDLSCs was sensitive to local inflammatory cytokines. Treatment of cells with neuropeptide Y was found to produce a modest enhanced osteogenic effect.

circSKIL promotes osteoblastic differentiation of periodontal ligament cells by sponging miR-532-5p to activate Notch signaling.

BACKGROUND AND OBJECTIVE: Periodontal ligament cells (PDLCs) possess the capacity to differentiate into a variety of cell types to benefit periodontal regeneration. In this study, we examined the circSKIL/miR-532-5p/Notch1 axis in controlling the osteoblastic differentiation of PDLCs. METHODS: Primary human PDLCs (hPDLCs) were isolated and induced to differentiate into osteoblasts. Osteogenic responses were assessed for the expressions of osteoblast-related marker proteins (including alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (RUNX2) by RT-PCR. The formation of mineralized nodules was examined by Alizarin Red S (ARS) staining and ALP activity. Expressions of circSKIL, miR-532-5p, and Notch1 were measured by RT-PCR and western blotting, and their regulations by combining bioinformatic analysis and luciferase reporter assay. Notch signaling was assessed for the expressions of hairy and enhancer of split-1 (HES1) and Notch intracellular domain (NICD). RESULTS: During osteoblastic differentiation of hPDLCs, circSKIL, and Notch1 were up-regulated, while miR-532-5p down-regulated. By sponging miR-532-5p, circSKIL activated Notch signaling, increasing levels of Notch1, HES1, and NICD. Functionally, knocking down circSKIL or overexpressing miR-532-5p inhibited osteoblastic differentiation of PDLCs, down-regulating ALP, OCN, BMP2, and RUNX2, and reducing ARS staining or ALP activity. The impacts of circSKIL knockdown were rescued by miR-532-5p inhibitor or overexpressing Notch1, while those caused by up-regulating miR-532-5p were reversed by overexpressing Notch1. CONCLUSION: By targeting miR-532-5p and up-regulating Notch1, circSKIL critically controls osteoblastic differentiation of hPDLCs. Therefore, modulating this axis may maximize the differentiation of PDLCs into osteoblasts and benefit periodontal regeneration.

Tiron Has Negative Effects on Osteogenic Differentiation via Mitochondrial Dysfunction in Human Periosteum-Derived Cells.

Tiron is a potent antioxidant that counters the pathological effects of reactive oxygen species (ROS) production due to oxidative stress in various cell types. We examined the effects of tiron on mitochondrial function and osteoblastic differentiation in human periosteum-derived cells (hPDCs). Tiron increased mitochondrial activity and decreased senescence-associated ß-galactosidase activity in hPDCs; however, it had a detrimental effect on osteoblastic differentiation by reducing alkaline phosphatase (ALP) activity and alizarin red-positive mineralization, regardless of H2O2 treatment. Osteoblast-differentiating hPDCs displayed increased ROS production compared with non-differentiating hPDCs, and treatment with tiron reduced ROS production in the differentiating cells. Antioxidants decreased the rates of oxygen consumption and ATP production, which are increased in hPDCs during osteoblastic differentiation. In addition, treatment with tiron reduced the levels of most mitochondrial proteins, which are increased in hPDCs during culture in osteogenic induction medium. These results suggest that tiron exerts negative effects on the osteoblastic differentiation of hPDCs by causing mitochondrial dysfunction.

Cellular and Biochemical Characterization of Mesenchymal Stem Cells from Killian Nasal Polyp.

Killian's (antrochoanal) polyp is a unilateral nasal polypoid lesion of the maxillary sinus especially affecting children and young adults with unilateral nasal obstruction, pus discharge, and headache. Although its etiology is unclear, chronic inflammation, autoreactivity, allergies, and viral infections are implicated in its formation and development, causing nasal tissue remodeling. In this context, we isolated and cultured mesenchymal stem cells from surgical biopsies of three patients with Killian nasal polyp (KNP-MSCs) while healthy nasal tissue (HNT-MSCs) was used as control. Our results demonstrated that KNP-MSCs exhibited reduced cell proliferation compared to HNT-MSCs, and migrated less than the control, showing a partial epithelial phenotype with low mRNA levels of I-CAM and a significant increase of E-cad. Subsequently, both MSCs were induced to osteoblastic or adipocyte differentiation for up to 20 days. KNP-MSCs underwent to differentiate into osteoblasts but exhibited reduced ALP activity and calcium deposits and low mRNA levels of osteogenesis-associated genes compared to osteogenic induced-HNT-MSCs. Conversely, KNP-MSCs and HNT-MSCs have shown the same adipogenic differentiation potential, with a similar lipid droplet amount, adipocyte gene expression, and triacylglycerols content. Taken together, these results first demonstrated the cellular and molecular characterization of MSCs derived from the Killian nasal polyp.

Decorin Promotes Osteoblastic Differentiation of Human Periodontal Ligament Stem Cells.

The aim of this study is to clarify the biological functions of decorin (DCN) in the healing and regeneration of wounded periodontal tissue. We investigated the expression pattern of DCN during the healing of wounded periodontal tissue in rats by immunohistochemistry and the effects of DCN on the osteoblastic differentiation of human periodontal ligament (PDL) stem cells (HPDLSCs) and preosteoblasts by Alizarin red S staining, quantitative reverse transcription-polymerase chain reactions, and western blotting. The expression of DCN was increased around the wounded PDL tissue on day 5 after surgery compared with the nonwounded PDL tissue, whereas its expression was not changed in the osteoblastic layer around the wounded alveolar bone. Furthermore, DCN promoted the osteoblastic differentiation of HPDLSCs, but it did not affect the osteoblastic differentiation of preosteoblasts. ERK1/2 phosphorylation was upregulated during the DCN-induced osteoblastic differentiation of HPDLSCs. DCN did not affect proliferation, migration, or the PDL-related gene expression of HPDLSCs. In conclusion, this study demonstrates that DCN has a role in the healing of wounded periodontal tissue. Furthermore, DCN secreted from PDL cells may contribute to bone healing by upregulating osteoblastic differentiation through ERK1/2 signaling in HPDLSCs, indicating a therapeutic effect of DCN in periodontal tissue regeneration.

Promotion effect of apical tooth germ cell-conditioned medium on osteoblastic differentiation of periodontal ligament stem cells through regulating miR-146a-5p.

BACKGROUND: MicroRNAs (miRNAs) play an important role in gene regulation that controls stem cells differentiation. Periodontal ligament stem cells (PDLSCs) could differentiate into osteo-/cementoblast-like cells that secretes cementum-like matrix both in vitro and in vivo. Whether miRNAs play key roles in osteoblastic differentiation of PDLSCs triggered by a special microenviroment remains elusive. In this study, we aimed to investigate potential miRNA expression changes in osteoblastic differentiation of PDLSCs by the induction of apical tooth germ cell-conditioned medium (APTG-CM). METHODS AND RESULTS: First, we analyzed the ability of APTG-CM to osteogenically differentiate PDLSCs. The results exhibited an enhanced mineralization ability, higher ALP activity and increased expression of osteogenic genes in APTG-CM-induced PDLSCs. Second, we used miRNA sequencing to analyze the miRNA expression profile of PDLSCs derived from three donors under 21-day induction or non-induction of APTG-CM. MiR-146a-5p was found to be up-regulated miRNA in induced PDLSCs and validated by RT-qPCR. Third, we used lentivirus-up/down system to verify the role of miR-146a-5p in the regulation of osteoblastic differentiation of PDLSCs. CONCLUSIONS: In conclusion, our results demonstrated that miR-146a-5p was involved in the promotion effect of APTG-CM on osteoblastic differentiation of PDLSCs, and suggested that miR-146a-5p might be a novel way in deciding the direction of PDLSCs differentiation.

Stromal cell-derived factor-1 promotes osteoblastic differentiation of human bone marrow mesenchymal stem cells via the lncRNA-H19/miR-214-5p/BMP2 axis.

BACKGROUND: Stromal cell-derived factor-1 (SDF-1) plays an important role in the osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs), but the specific mechanism remains unclear. Our study aimed to clarify the role of the lncRNA-H19/miR-214-5p/BMP2 axis in the osteoblastic differentiation of hBMMSCs induced by SDF-1. METHODS: We used reverse-transcriptase polymerase chain reaction, western blotting, alkaline phosphatase activity test, and Alizarin red staining to evaluate the osteoblastic differentiation of primary hBMMSCs and the luciferase reporter assay to determine if lncRNA-H19 binds with miR-214-5p. RESULTS: Our results indicated that SDF-1 (50 ng/mL) promotes the osteoblastic differentiation of hBMMSCs, significantly upregulates osteoblastogenic genes (OCN, OSX, RUNX2, and ALP), and increases Alizarin red staining, alkaline phosphatase activity, and lncRNA-H19 expression. Luciferase reporter assay verified that lncRNA-H19 binds with and represses miR-214-5p, thereby upregulating BMP2 expression. Use of miR-214-5p inhibitor or overexpression of lncRNA-H19 can promote the osteoblastic differentiation of hBMMSCs, but miR-214-5p or shH19 inhibits the osteoblastic differentiation of hBMMSCs. Treatment with an miR-214-5p inhibitor could rescue the inhibitory effect of shH19 on the osteoblastic differentiation of hBMMSCs. CONCLUSIONS: Taken together, SDF-1 promotes the osteoblastic differentiation of hBMMSCs through the lncRNA-H19/miR-214-5p/BMP2 axis. Increased osteoblastic differentiation by an miR-214-5p inhibitor reveals a new possible strategy for the treatment of bone defect and osteoporosis.

The PPAR-γ/SFRP5/Wnt/ß-catenin signal axis regulates the dexamethasone-induced osteoporosis.

BACKGROUND: The inhibition of glucocorticoid (GC) on osteoblastic differentiation of bone marrow stromal stem cells (BMSC) is an important pathway for GC to reduce bone formation. Recent studies implicated an important role of peroxisome proliferator-activated receptor-gamma (PPAR-γ) in GC-mediated cell proliferation and differentiation. Thus, our purpose is to investigate the role of PPAR-γ in regulating rat BMSC (rBMSC) osteoblastic differentiation. METHODS: The rBMSC treated with dexamethasone (Dex) was used to construct an in vitro cell model of GC-induced osteoporosis. The expressions of PPAR-γ, RUNX2, ALP, OPN and SFRP5 in cells were detected by RT-qPCR and western blot assays. Osteogenic differentiation of rBMSC was measured by Alizarin Red S (ARS) staining analysis. Lentivirus-delivered shRNA was used to knock down PPAR-γ or SFRP5, and lentivirus-delivered constructs were used to overexpress SFRP5 in rBMSC to verify the effect of PPAR-γ or SFRP5 on cell osteogenic differentiation. RESULTS: Dex significantly reduced rBMSC osteoblastic differentiation. The expression of PPAR-γ was enhanced in Dex treated rBMSC. PPAR-γ down-regulation improved Dex inhibition of rBMSC osteogenic differentiation. Moreover, PPAR-γ knockdown promoted protein levels of RUNX2, ALP, OPN and Dex-decreased rBMSC osteogenic differentiation. The expression of SFRP5 was reduced while Wnt and ß-catenin were increased in PPAR-γ knockdown and Dex treated rBMSC. Moreover, the up-regulation of SFRP5 reversed the osteogenic differentiation of rBMSC induced by PPAR-γ knockdown. CONCLUSION: These data indicated that in GC-induced osteoporosis, PPAR-γ/SFRP5 affects osteogenic differentiation by regulating the Wnt/ß-catenin signaling pathway.

Activin A Promotes Osteoblastic Differentiation of Human Preosteoblasts through the ALK1-Smad1/5/9 Pathway.

Activin A, a member of transforming growth factor-ß superfamily, is involved in the regulation of cellular differentiation and promotes tissue healing. Previously, we reported that expression of activin A was upregulated around the damaged periodontal tissue including periodontal ligament (PDL) tissue and alveolar bone, and activin A promoted PDL-related gene expression of human PDL cells (HPDLCs). However, little is known about the biological function of activin A in alveolar bone. Thus, this study analyzed activin A-induced biological functions in preosteoblasts (Saos2 cells). Activin A promoted osteoblastic differentiation of Saos2 cells. Activin receptor-like kinase (ALK) 1, an activin type I receptor, was more strongly expressed in Saos2 cells than in HPDLCs, and knockdown of ALK1 inhibited activin A-induced osteoblastic differentiation of Saos2 cells. Expression of ALK1 was upregulated in alveolar bone around damaged periodontal tissue when compared with a nondamaged site. Furthermore, activin A promoted phosphorylation of Smad1/5/9 during osteoblastic differentiation of Saos2 cells and knockdown of ALK1 inhibited activin A-induced phosphorylation of Smad1/5/9 in Saos2 cells. Collectively, these findings suggest that activin A promotes osteoblastic differentiation of preosteoblasts through the ALK1-Smad1/5/9 pathway and could be used as a therapeutic product for the healing of alveolar bone as well as PDL tissue.