Up-regulation of WISP2 by astragaloside activates the Wnt/β-catenin signaling pathway to promote osteogenic differentiation of BMSCs in patients with congenital scoliosis

Steroid-induced osteonecrosis of femoral head (SONFH) is a common and serious complication caused by long-term and/or excessive use of glucocorticoids (GCs). The decreased activity and abnormal differentiation of bone marrow mesenchymal stem cells (BMSCs) are considered to be one of the major reasons for the onset and progression of this disease. Periostin (POSTN) is a matricellular protein which plays an important role in regulating osteoblast function and bone formation. Sclerostin (SOST) is a secreted antagonist of Wnt signaling that is mainly expressed in osteocytes to inhibit bone formation. However, the exact role of POSTN and SOST in SONFH has not been reported yet. Therefore, we detected the differential expression of POSTN and SOST in BMSCs of SONFH Group patients, and Control Group was patients with traumatic ONFH (TONFH) and developmental dysplasia of the hip (DDH). Furthermore, we used lentiviral transfection to knockdown POSTN expression in BMSCs of patients with SONFH to study the effect of POSTN knockdown on the SOST expression and osteogenic differentiation of BMSCs. The results indicated that the endogenous expression of POSTN and SOST in BMSCs of SONFH Group was upregulated, compared with Control Group. POSTN was upregulated gradually while SOST was downregulated gradually at days 0, 3, and 7 of osteogenic differentiation of BMSCs in Control Group. Contrarily, POSTN was gradually downregulated while SOST was gradually upregulated during osteogenic differentiation of BMSCs in SONFH Group. This could be due to increased expression of SOST in BMSCs, which was caused by excessive GCs. In turn, the increased expression of POSTN in BMSCs may play a role in antagonizing the continuous rising of SOST during the osteogenic differentiation of BMSCs in patients with SONFH. POSTN knockdown significantly attenuated osteo-specific gene expression, alkaline phosphatase activity, and calcium nodule formation in vitro; thus inhibiting the osteogenic differentiation of BMSCs in patients with SONFH. Besides, POSTN knockdown upregulated SOST expression, increased GSK-3β activity, and downregulated β-catenin. These findings suggest that POSTN have an essential role in regulating the expression of SOST and osteogenic differentiation of BMSCs in patients with SONFH, and POSTN knockdown suppresses osteogenic differentiation by upregulating SOST and partially inactivating Wnt/β-catenin signaling pathway. Therefore, targeting POSTN and SOST may serve as a promising therapeutic target for the prevention and treatment of SONFH.

Bone formation, metabolism, and the stability of the bone marrow microenvironment are all impacted by the imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs). Despite this, it is unknown how BMSCs affect congenital scoliosis (CS). As a result, our research now focuses on explaining its associated impact and mechanism. In eukaryotic cells, N6-methyladenosine (m6A) is the most prevalent post-transcriptional alteration. The role of fat mass and obesity-related genes (FTO), an m6A demethylase, in regulating the differentiation of BMSCs is still unknown. We assessed alterations in the mRNA and protein levels of genes linked to the differentiation of BMSCs using samples taken from CS. According to our findings, According to our findings, FTO inhibits osteogenic differentiation and promotes the adipogenic differentiation of BMSCs. MMP1 knockdown has an inhibitory effect on BMSC osteogenic differentiation, whereas MMP1 overexpression promotes it. A specific ERG inhibitor called PD98059 prevents MMP1-mediated promotion. Additionally, our research revealed that FTO affects how BMSCs can differentiate in CS patients by regulating MMP1 levels. FTO-mediated MMP1 m6A modification underlies MMP1's promotion of osteogenic differentiation via the ERK pathway, implying that it could be a viable treatment target for CS.

To investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. Three lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining. After lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced ( P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups ( P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant ( P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased. Silencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.

The aim of this in vitro study was to evaluate the effects of low-level laser therapy (LLLT) at different energy intensities on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under healthy and inflammatory microenvironments. Human BMSCs and BMSCs from inflammatory conditions (i-BMSCs, BMSCs treated with tumor necrosis factor α; TNF-α) were subject to LLLT (Nd:YAG;1064nm) at different intensities. We designed one control group (without irradiation) and four testing groups (irradiation at 2, 4, 8, and 16J/cm2) for both BMSCs and i-BMSCs. Cell proliferation was measured using colony-forming unit fibroblast assay and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic capacity of cells was determined by alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin Red S staining and the mRNA transcript levels of genes runt-related transcription factor 2 (Runx2), ALP, and osteocalcin. Moreover, the effects of LLLT on secretion of TNF-α in BMSCs and i-BMSCs were measured by enzyme-linked immunosorbent assay. Our results demonstrated LLLT could significantly promote BMSC proliferation and osteogenesis at densities of 2 and 4J/cm2. LLLT at density of 8J/cm2 could promote the proliferation and osteogenesis of i-BMSCs. However, LLLT at 16J/cm2 significantly suppressed the proliferation and osteogenesis of BMSCs both in healthy and in inflammatory microenvironment. Moreover, we also found that the expression of TNF-α was obviously inhibited by LLLT at 4, 8, and 16J/cm2, in an inflammatory microenvironment. Considering these findings, LLLT could improve current in vitro methods of differentiating BMSCs under healthy and inflammatory microenvironments prior to transplantation.

Heath-related quality of life and functional outcomes in patients with congenital or juvenile idiopathic scoliosis after an average follow-up of 25 years: a cohort study

The polypeptide OP3-4 induced osteogenic differentiation of bone marrow mesenchymal stem cells via protein kinase B/glycogen synthase kinase 3β/β-catenin pathway and promoted mandibular defect bone regeneration

Background: Osteogenesis greatly depends on the differentiation of bone marrow mesenchymal stem cells (BMSCs). CKIP-1is considered to be a negative regulator of BMSCs. Methods: We established aCKIP-1 knockout mouse model, then isolated and cultured BMSCs from wild-type and knockout groups. Results: Our data demonstrated that CKIP-1 knockout significantly increased bone structure in theexperimental mousemodel and enhanced BMSCproliferation. CKIP-1 knockout contributed to osteoblastic and adipogenicdifferentiation. Furthermore, CKIP-1 regulated osteogenesis in BMSCs via the MAPK signaling pathway, and BMSCs from theCKIP-1 knockout mice wereeffective in repairing the skull defect null mice. Conclusion: Our results concluded that silencingof CKIP-1 promoted osteogenesis in experimental mice and increased BMSCs differentiation via upregulation of the MAPK signaling pathway.

Shear stress-mediated downregulation of miR-423-5p in M2 macrophage exosomes promotes osteogenic differentiation of bone marrow mesenchymal stem cells.

Secreted frizzled-related protein 1 (SFRP1) is associated with cell differentiation, and its expression can be modulated by resveratrol. However, their impacts on bone marrow mesenchymal stem cells (BMSCs)-induced osteogenesis and ovariectomy-triggered bone loss remain unclear. Therefore, we in this study aimed to dissect the regulation of resveratrol on SFRP1, along with its sequential effects on differentiation and osteoporosis prevention of BMSCs. The SFRP1 expression in the ovariectomized (OVX) mice-originated bone tissues, BMSCs and bone marrow-derived macrophages (BMMs), during their differentiation towards osteoblasts and chondrocytes, was quantified by qRT-PCR and Western-blot. SFRP1-siRNA was applied for studying its influence on osteogenesis of BMSCs. Additionally, we evaluated the impacts of resveratrol on OVX mice and SFRP1 expression. SFRP1 was significantly up-regulated in the OVX mice-derived bone tissues and BMSCs, but gradually decreased during osteogenesis. Its expression was not significantly changed in BMSCs during their differentiation towards osteoclasts or in BMMs. The knockout of SFRP1 significantly improved mineralization potentiality, alkaline phosphatase activity and expression of several osteoblast-specific genes. Moreover, the bone loss was ameliorated in OVX mice treated with resveratrol, whose therapeutic effects were achieved by facilitating the expression of osteogenesis-associated genes while suppressing the SFRP1 expression. We also observed that the SFRP1 exerted a negative effect on osteogenesis of BMSCs and estrogen deficiency-induced osteoporosis, enabling itself to be an indicator of osteogenesis and also a molecular target for PMOP treatment. Resveratrol is a suppressor of SFRP1that can be applied as an active ingredient for treating PMOP.

Objectives: High glucose (HG)–induced oxidative stress affects implant osseointegration through various pathways. Oxidative stress is widely recognized to suppress the Wnt/β-catenin signaling pathway, thereby impairing bone metabolism and homeostasis. However, there are few reports on whether excessive reactive oxygen species (ROS) influence osteogenic differentiation of stem cells via the noncanonical Wnt/calmodulin-dependent protein kinase II (CaMK II) pathway. An investigation of the mechanism by which ROS/CaMK II/β-catenin signaling axis influences the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) on titanium surfaces is being carried out in this investigation.Materials and Methods: In this study, titanium plates were specially treated to simulate implant surfaces. An osteogenic medium containing HG was used to cultivate BMSCs on titanium surfaces. The effects of excessive ROS induced by HG on the osteogenic differentiation of BMSCs, as well as on the expression of β-catenin and CaMK II, were examined using methods such as alkaline phosphatase (ALP) activity assay, quantitative real-time PCR (qRT-PCR), and immunofluorescence staining. Additionally, the effects of the Wnt/β-catenin and Wnt/CaMK II pathways on the osteogenesis of BMSCs on the titanium surface were observed by separately adding activators or inhibitors of β-catenin and CaMK II.Results: Excessive ROS induced by HG inhibited osteogenic differentiation. In a HG environment, β-catenin expression decreased, while CaMK II expression increased. Moreover, we observed that activation of the Wnt/β-catenin pathway promoted osteogenesis, whereas activation of the Wnt/CaMK II pathway inhibited it.Conclusions: In summary, BMSC osteogenesis on titanium surfaces is suppressed by HG–induced oxidative stress via the ROS/CaMK II/β-catenin signaling axis, which may subsequently impair implant osseointegration.

TGF-β2 and TGF-β1 differentially regulate the odontogenic and osteogenic differentiation of mesenchymal stem cells

Objective To investigate the effect of angiotensinⅡ(AngⅡ)on the differentiation of bone marrow mesenchymal stem cells(BMSCs) to keratinocytes and the signal mechanism. Methods The BMSCs from Wistar rats were isolated. The expression of BMSCs surface antigens was detected by flow cytometry. The secretion concentration of AngⅡwas detected by enzyme linked immunosorbent assay(ELISA).The expression of keratin 10(K10)was detected by immunocytochemistry after keratinocyte induction. The effects of AngⅡ, Losartan, PD123319 and their downstream signal molecule blockers on the number of K10 positive cells after keratinocyte induction by BMSCs were observed. Results The positive rate of BMSCs surface markers CD29 and CD90 was 99% or above.The positive rate of CD34 and CD45 was less than 2%.The concentration of AngⅡin cell supernatant showed an increasing trend with the increase of incubation time(P< 0.05).The K10 positive rate of BMSCs in keratinocyte induction group given AngⅡin 7 and 14 days was 69.02% and 82.10% respectively, which was obviously higher than that in control group(P< 0.05).The number of positive K10 cells treated with Losartan, SB203580, AG490, or SP600125 was reduced. Conclusion AngⅡcould significantly increase the conversion rate of BMSCs to keratinocytes.p38 mitogen activated protein kinase(p38MAPK), janus kinase 2/3(JAK2/3)and c- Jun N- terminal kinase(JNK) signal pathways were closely related with the role mediated by AngⅡ. AngⅡpromoted BMSCs to convert into keratinocytes, which might be one of the mechanisms of promoting wound epithelialization. Key words: AngiotensinⅡ; Mesenchymal stem cells; Keratinocyte; Wound healing

PurposeIn osteoporosis, the balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) is disrupted. The osteogenic differentiation of bone marrow MSCs (BMSCs) is important for improving osteoporosis. The aim of this study was to explore the role and molecular mechanism of miR-210 in the balance of osteogenic/adipogenic differentiation of BMSCs in postmenopausal osteoporosis.MethodsPostmenopausal osteoporosis rat models were constructed by ovariectomy (OVX). BMSCs were isolated from the femur in rats of Sham and OVX groups. MiR-210 was overexpressed and suppressed by miR-210 mimics and inhibitor, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA expression of miR-210, ephrin type-A receptor 2 (EPHA2), alkaline phosphatase (ALP), osterix (OSX), osteocalcin (Bglap), Runt-related transcription factor 2 (Runx2), peroxisome proliferator activated receptor gamma, and fatty acid binding protein 4 (FABP4) in each group of rat femoral tissues or BMSCs. Western blot was applied to detect the protein expression level of EPHA2 in rat femoral tissues and cells. Alizarin red S staining and oil red O staining were performed to assess the osteogenic and adipogenic differentiation of BMSCs, respectively. In addition, the targeting relationship between miR-210 and EPHA2 was verified by a dual luciferase gene reporter assay.ResultsThe expression of miR-210 was significantly reduced in femoral tissues and BMSCs of OVX rats, and its low expression was associated with reduced bone formation. The osteogenic differentiation was enhanced in OVX rats treated with miR-210 mimic. Overexpression of miR-210 in transfected BMSCs was also found to significantly promote osteogenic differentiation and even inhibit adipogenic differentiation in BMSCs, while knockdown of miR-210 did the opposite. Further mechanistic studies showed that miR-210 could target and inhibit the expression of EPHA2 in BMSCs, thus promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs.ConclusionMiR-210 promotes osteogenic differentiation and inhibits adipogenic differentiation of BMSCs by down-regulating EPHA2 expression. As it plays an important role in the osteogenic/adipogenic differentiation of osteoporosis, miR-210 can serve as a potential miRNA biomarker for osteoporosis.

Patients with type 2 diabetes mellitus (T2DM) are at high risk for osteoporosis. SIRT1 plays an important regulatory role in the occurrence and development of diabetes mellitus; however, it is still not clear whether SIRT1 is directly related to the osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) in T2DM patients. We obtained BMSCs from patients with T2DM and healthy volunteers to determine the effect of SIRT1 expression on the osteogenic capacity of BMSCs. As a result, SIRT1 expression in BMSCs in T2DM was significantly lower compared to healthy volunteers, but the proliferative capacity of BMSCs in the T2DM group was not significantly different from that of healthy volunteers. During osteogenic differentiation, the expression of SIRT1 in MSCs from T2DM patients was significantly decreased, and the osteogenic differentiation ability of MSCs from T2DM patients was significantly lower than healthy volunteers. After intervention with resveratrol, the expression of SIRT1 increased significantly, and the apoptotic rate of MSCs in T2DM patients decreased significantly. Moreover, resveratrol promoted osteoblast differentiation of MSCs. Our study confirmed that the expression of SIRT1 is directly related to the osteogenic potential of BMSCs in patients with T2DM. Resveratrol promoted the osteogenic differentiation of BMSCs by increasing the expression of SIRT1. The increased expression of SIRT1 significantly reduced BMSC apoptosis during osteogenic differentiation, which is one of the important mechanisms by which SIRT1 regulates the osteogenic ability of BMSCs. Our data also provide strong evidence that resveratrol may be used in the treatment of osteoporosis in patients with T2DM.

microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3