Osteogenic Differentiation Of hBM-MSCs Research Articles

Surfactant-assisted photo-crosslinked silk fibroin sponges: A versatile platform for the design of bone scaffolds

Critical size bone defects cannot heal without aid and current clinical approaches exhibit some limitations, underling the need for novel solutions. Silk fibroin, derived from silkworms, is widely utilized in tissue engineering and regenerative medicine due to its remarkable properties, making it a promising candidate for bone tissue regeneration in vitro and in vivo. However, the clinical translation of silk-based materials requires refinements in 3D architecture, stability, and biomechanical properties.In earlier research, improved mechanical resistance and stability of chemically crosslinked methacrylate silk fibroin (Sil-Ma) sponges over physically crosslinked counterparts were highlighted. Furthermore, the influence of photo-initiator and surfactant concentrations on silk properties was investigated. However, the characterization of sponges with Sil-Ma solution concentrations above 10 % (w/V) was hindered by production optimization challenges, with only cell viability assessed.This study focuses on the evaluation of methacrylate sponges' suitability as temporal bone tissue regeneration scaffolds. Sil-Ma sponge fabrication at a fixed concentration of 20 % (w/V) was optimized and the impact of photo-initiator (LAP) concentrations and surfactant (Tween 80) presence/absence was studied. Their effects on pore formation, silk secondary structure, mechanical properties, and osteogenic differentiation of hBM-MSCs were investigated. We demonstrated that, by tuning silk sponges' composition, the optimal combination boosted osteogenic gene expression, offering a strategy to tailor biomechanical properties for effective bone regeneration. Utilizing Design of Experiment (DoE), correlations between sponge composition, porosity, and mechanical properties are established, guiding tailored material outcomes. Additionally, correlation matrices elucidate the microstructure's influence on gene expressions, providing insights for personalized approaches in bone tissue regeneration.

MiR-126 mitigates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting the ERK1/2 and Bcl-2 pathways.

Human bone marrow mesenchymal stem cells (hBMMSCs) are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The objective of the present study is to assess microRNA-126 (miR-126) and examine its effects on the osteogenic differentiation of hBMMSCs. In this study, we investigate the role of miR-126 in the progression of osteogenic differentiation (OD) as well as the apoptosis and inflammation of hBMMSCs during OD induction. OD is induced in hBMMSCs, and matrix mineralization along with other OD-associated markers are evaluated by Alizarin Red S (AR) staining and quantitative PCR (qPCR). Gain- and loss-of-function studies are performed to demonstrate the role of miR-126 in the OD of hBMMSCs. Flow cytometry and qPCR-based cytokine expression studies are performed to investigate the effect of miR-126 on the apoptosis and inflammation of hBMMSCs. The results indicate that miR-126 expression is downregulated during the OD of hBMMSCs. Gain- and loss-of function assays reveal that miR-126 upregulation inhibits the differentiation of hBMMSCs into osteoblasts, whereas the downregulation of miR-126 promotes hBMMSC differentiation, as assessed by the determination of osteogenic genes and alkaline phosphatase activity. Furthermore, the miR-126 level is positively correlated with the production of inflammatory cytokines and apoptotic cell death. Additionally, our results suggest that miR-126 negatively regulates not only B-cell lymphoma 2 (Bcl-2) expression but also the phosphorylation of extracellular signal‑regulated protein kinase (ERK) 1/2. Moreover, restoring ERK1/2 activity and upregulating Bcl-2 expression counteract the miR-126-mediated suppression of OD in hBMMSCs by promoting inflammation and apoptosis, respectively. Overall, our findings suggest a novel molecular mechanism relevant to the differentiation of hBMMSCs into osteoblasts, which can potentially facilitate bone formation by counteracting miR-126-mediated suppression of ERK1/2 activity and Bcl-2 expression.

Demineralized bone matrix combined with cytotoxic T-lymphocyte-associated protein 4 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells and suppresses the activation of T lymphocytes in vitro.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) can promote osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs), and CTLA4-modified bone marrow mesenchymal stem cells possess immunoregulatory effects. In the present study, we aimed to construct a new tissue engineering bone using demineralized bone matrix and CTLA4 protein, designated as DBM-CTLA4 (+). The effects of DBM-CTLA4 (+) on the osteogenic differentiation of hBMMSCs and T lymphocyte activation were evaluated through in vitro experiments. The cumulative release of CTLA4 from DBM-CTLA4 (+) was determined using enzyme-linked immunosorbent assay. DBM-CTLA4 (+) was co-cultured in a Transwell chamber with either phytohemagglutinin-treated hBMMSCs or human peripheral blood mononuclear cells (hPBMCs). Osteogenic differentiation of hBMMSCs was assessed by calcium deposition, ALP activity, and the protein levels of COL1A1, RUNX2, BMP2, and OPN. T lymphocyte activity was assessed by measuring the protein levels of IL-2, L-17, HLA-DRA1, IFN-γ, and RANKL. Our results showed that the cumulative release rates of CTLA4 at 7, 14, 21, and 28days were 12.6%±1.4%, 30.2%±2.3%, 49.8%±3.8%, and 60.5%±2.7%, respectively. Compared to the negative control, DBM-CTLA4 (+) promoted the proliferation of hBMMSCs, and enhanced calcium deposition, ALP activity, and protein levels of COL1A1, RUNX2, BMP2, and OPN. Moreover, DBM-CTLA4 (+) decreased the levels of IL-2, IL-17, HLA-DR, IFN-γ, and RANKL in hPBMCs treated with phytohemagglutinin. In conclusion, DBM-CTLA4 (+) promoted proliferation and osteogenic differentiation of hBMMSCs and suppressed T lymphocyte activation.

Regulatory effects of miR-28 on osteogenic differentiation of human bone marrow mesenchymal stem cells

ABSTRACT We aimed to assess the regulatory effects of miR-28 on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs). HBMMSCs isolated, cultured and induced (at P3) to undergo osteogenic induction. The expressions of miRNAs were detected by gene microarray, and differentially expressed miRNAs in hBMMSCs compared with induced cells were obtained by significance analysis of microarrays. The microarray findings were confirmed by RT-PCR. TargetScan showed that signal transducer and activator of transcription 1 (STAT1) was the downstream target gene of miR-28. The relationship between miR-28 and STAT1 was validated using dual-luciferase reporter gene assay. HBMMSCs were transfected with miR-28 mimics and STAT1 siRNA, respectively. Samples were collected on day 10 after osteogenic differentiation, and the alkaline phosphatase (AKP) activity, Runt-related transcription factor 2 (RUNX2, a key regulator of osteogenic differentiation) and STAT1 expressions were determined using kits, PCR and Western blotting, respectively. Cell proliferation and migration were detected through CCK-8 and Transwell assays, respectively. During the osteogenic differentiation of hBMMSCs, the expression level of miR-28 increased. MiR-28 specifically bound the 3’-untranslated region (3’UTR) of STAT1 mRNA. It inhibited STAT1 expression in a targeted manner during osteogenic differentiation. Interference with STAT1 partially mimicked the regulatory effects of miR-28 overexpression on the osteogenic differentiation of hBMMSCs. Interference with STAT1 or overexpression of miR-28 did not affect proliferation or migration. MiR-28 has gradually increased expression during the osteogenic differentiation of hBMMSCs, which can directly bind STAT1 3’UTR and inhibit its expression, thereby up-regulating AKP and RUNX2, and promoting osteogenic differentiation.

The effect of local steroid application on bony fusion in a rat posterolateral spinal arthrodesis model.

IntroductionLocal steroid administration during anterior cervical spine surgery has been shown to improve postoperative dysphagia. However, concerns over potential complications remain. This study aims to evaluate the effect of local steroid administration on bone regeneration and spine fusion in a preclinical model, as well as the impact on osteogenic differentiation of human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) in a 3D culture system.Materials and methodsForty‐five rats underwent bilateral L4‐L5 posterolateral lumbar fusion (PLF) utilizing local delivery of low‐dose recombinant human bone morphogenetic protein‐2 (rhBMP‐2; 0.5 μg/implant). Rats were divided into three groups: no steroid (control), low dose (0.5 mg/kg), and high dose (2.5 mg/kg) of triamcinolone. Bone growth and fusion were assessed using radiography, blinded manual palpation, and micro‐CT analysis and were visualized by histology. The impact of triamcinolone exposure on osteogenic differentiation of hBM‐MSCs was evaluated by gene expression analysis, alkaline phosphatase activity assay, and alizarin red staining.ResultsNo significant differences in fusion scores or rates were seen in the low‐ or high‐dose steroid treatment groups relative to untreated controls. Quantification of new bone formation via micro‐CT imaging revealed no significant between‐group differences in the volume of newly regenerated bone. Triamcinolone also had no negative impact on pro‐osteogenic gene transcript levels, and ALP activity was enhanced in the presence of triamcinolone. Mineral deposition appeared comparable in cultures grown with and without triamcinolone.ConclusionsLocal steroid application does not seem to inhibit rhBMP‐2‐mediated spine fusion in rats, though our study may not be adequately powered to detect differences in fusion as measured by manual palpation or bone volume as measured by micro‐CT. These findings suggest that local triamcinolone may not increase pseudarthrosis in spine fusion procedures. Further large animal and clinical studies to verify its safety and efficacy are warranted.

Circular RNA Hsa_circ_0006766 targets microRNA miR-4739 to regulate osteogenic differentiation of human bone marrow mesenchymal stem cells

ABSTRACT Circular RNAs (circRNAs) are emerging as important regulators in bone metabolism, which is mediated by microRNA (miRNA) sponges. However, it is not clear how circRNA regulates osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs).Therefore, based on the previous circRNA chip results, hsa_circ_0006766, which is differentially expressed in the osteogenic differentiation of hBM-MSCs, was screened out, and bioinformatics analysis was performed to predict potential target miRNAs. During osteogenic differentiation of hBM-MSCs, hsa_circ_0006766 and its target miRNAs (miR-4739, miR-619-5p, miR-5787, miR-7851-3p, and miR-3192-5p) were detected by quantitative Real Time-PCR (qRT-PCR). Target gene prediction for the differentially expressed target miRNAs was performed, and target genes were validated by dual-luciferase reporter gene assay and qRT-PCR. It is shown that hsa_circ_0006766 was up-regulated and miR-4739 was down-regulated during osteogenic differentiation of hBM-MSCs.Moreover, the target gene Notch2 was predicted to be highly expressed during osteogenic differentiation. And dual-luciferase assay proved that Notch2 was the gene targeting to miR-4739. Taken together, our finding confirmed that hsa_circ_0006766 may act as a major regulatory part in osteogenic differentiation of hBM-MSCs via an hsa_circ_0006766–miR-4739–Notch2 regulatory axis. Accordingly, hsa_circ_0006766 may affect the development of osteoporosis and may thus become a therapeutic target.

LncRNA LOXL1-AS1 controls osteogenic and adipocytic differentiation of bone marrow mesenchymal stem cells in postmenopausal osteoporosis through regulating the miR-196a-5p/Hmga2 axis.

Exploring molecular mechanisms of human bone marrow mesenchymal stem cells (hBMMSCs) differentiation, a crucial step for bone formation, is a new direction for treating postmenopausal osteoporosis. LncRNAs are involved in lots of biological processes including hBMMSCs differentiation. The present study aimed to explore the effect of LOXL1-AS1 on hBMMSCs differentiation. We examined the expression levels of LOXL1-AS1, miR-196a-5p and Hmga2 in peripheral blood from postmenopausal osteoporosis patients by RT-qPCR, and detected their changes during the osteogenic differentiation of hBMMSCs by RT-qPCR. RT-qPCR and western blot measured the level of biomarkers of bone formation and osteogenic differentiation (osteopontin, OPN; Alkaline phosphatase, ALP; Runt-related transcription factor-2, Runx-2). The effects of LOXL1-AS1 on the osteogenic and adipocytic differentiation of hBMMSCs were, respectively, determined by ALP, ARS staining assays and oil red O staining assay. The abnormal high expression of LOXL1-AS1 was found in patients. LOXL1-AS1 expression showed a gradual decrease during the osteogenic differentiation of hBMMSCs. However, LOXL1-AS1 overexpression inhibited the hBMMSCs osteogenic differentiation but promoted adipocytic differentiation. Furthermore, LOXL1-AS1 was identified to be a sponge of miR-196a-5p and Hmga2 as a target gene of miR-196a-5p. Besides, LOXL1-AS1 sponged miR-196a-5p to mediate Hmga2 expression, which played contrary effects on regulating osteogenic and adipocytic differentiation of hBMMSCs. Moreover, LOXL1-AS1/miR-196a-5p/Hmga2 axis regulated hBMMSCs differentiation through controlling C/EBPβ-mediated PPARγ expression. These findings facilitate understanding the molecular mechanism of hBMMSCs differentiation and bring up a novel sight for postmenopausal osteoporosis therapy.

Synergistic Effect of Cell-Derived Extracellular Matrices and Topography on Osteogenesis of Mesenchymal Stem Cells.

Cell-derived matrices (CDMs) are an interesting alternative to conventional sources of extracellular matrices (ECMs) as CDMs mimic the natural ECM composition better and are therefore attractive as a scaffolding material for regulating the functions of stem cells. Previous research on stem cell differentiation has demonstrated that both surface topography and CDMs have a significant influence. However, not much focus has been devoted to elucidating possible synergistic effects of CDMs and topography on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). In this study, polydimethylsiloxane (PDMS)-based anisotropic topographies (wrinkles) with various topography dimensions were prepared and subsequently combined with native ECMs produced by human fibroblasts that remained on the surface topography after decellularization. The synergistic effect of CDMs combined with topography on osteogenic differentiation of hBM-MSCs was investigated. The results showed that substrates with specific topography dimensions, coated with aligned CDMs, dramatically enhanced the capacity of osteogenesis as investigated using immunofluorescence staining for identifying osteopontin (OPN) and mineralization. Furthermore, the hBM-MSCs on the substrates decorated with CDMs exhibited a higher percentage of (Yes-associated protein) YAP inside the nucleus, stronger cell contractility, and greater formation of focal adhesions, illustrating that enhanced osteogenesis is partly mediated by cellular tension and mechanotransduction following the YAP pathway. Taken together, our findings highlight the importance of ECMs mediating the osteogenic differentiation of stem cells, and the combination of CDMs and topography will be a powerful approach for material-driven osteogenesis.

TNF-α-induced NF-κB activation upregulates microRNA-150-3p and inhibits osteogenesis of mesenchymal stem cells by targeting β-catenin.

Although systemic or local inflammation, commonly featured by cytokine activation, is implicated in patients with bone loss, the underlying mechanisms are still elusive. As microRNAs (miR), a class of small non-coding RNAs involved in essential physiological processes, have been found in bone cells, we aimed to investigate the role of miR for modulating osteogenesis in inflammatory milieu using human bone marrow mesenchymal stem cells (hBM-MSCs). Induced by proinflammatory cytokine TNF-α, miR-150-3p was identified as a key player in suppressing osteogenic differentiation through downregulating β-catenin, a transcriptional co-activator promoting bone formation. TNF-α treatment increased the levels of miR-150-3p, which directly targeted the 3′-UTR of β-catenin mRNA and in turn repressed its expression. In addition, we observed that miR-150-3p expression was increased by TNF-α via IKK-dependent NF-κB signalling. There are three putative NF-κB binding sites in the promoter region of miR-150, and we identified −686 region as the major NF-κB binding site for stimulation of miR-150 expression by TNF-α. Finally, the osteogenic differentiation of hBM-MSCs was inhibited by either miR-150-3p overexpression or TNF-α treatment, which was prevented by anti-miR-150-3p oligonucleotides. Taken together, our data suggested that miR-150-3p integrated inflammation signalling and osteogenic differentiation and may contribute to the inhibition effects of inflammation on bone formation, thus expanding the pathophysiological functions of microRNAs in bone diseases.

Culture on a 3,4-Dihydroxy-l-Phenylalanine-Coated Surface Promotes the Osteogenic Differentiation of Human Mesenchymal Stem Cells

The culture surface can affect the in vitro differentiation of stem cells. In this study, we investigated whether modifying the culture surface with 3,4-dihydroxy-l-phenylalanine (DOPA), an element of mussel adhesion protein, could enhance the in vitro osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMMSCs). hBMMSCs cultured on DOPA-coated plates exhibited better cell adhesion and spreading compared with noncoated conventional tissue culture plates. The DOPA coated did not affect the apoptosis or viability of the cultured hBMMSCs. Also, hBMMSCs cultured on DOPA-coated plates exhibited a higher degree of osteogenic differentiation than did hBMMSCs cultured on noncoated plates, as evaluated with alkaline phosphate (ALP) activity, calcium content, and the mRNA expression of runt-related transcription factor 2, ALP, and osteocalcin. Further, hBMMSCs cultured on DOPA-coated plates demonstrated a higher capability of ectopic bone formation in vivo following implantation in the subcutaneous space of athymic mice compared with hBMMSCs cultured on noncoated plates, as evaluated with microcomputer topography analysis and histomorphometry. These results indicate that modifying the culture surface with DOPA can enhance the in vitro osteogenic differentiation of hBMMSCs.

Effects of imatinib mesylate in osteoblastogenesis

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 microM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p<0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p<0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis.

Role of New Tyrosine Kinase Inhibitors in Osteoblastogenesis

It has been reported that imatinib mesylate (IM) may affect bone tissue remodeling mainly by both an inhibitory activity on osteoclastogenesis and an induction of osteoblastogenesis. Dasatinib (DA) and Nilotinib (NI) are new generation tyrosine kinase inhibitors presently approved for chronic myeloid leukemia patients after imatinib failure. We therefore evaluated possible effects of DA and NI on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BM-MSCs). BM-MSCs are multipotent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Mesenchymal stem cells (hBM-MSCs) were obtained from bone marrow samples of normal healthy adult bone marrow donors, isolated by density gradient (mononuclear fraction) and cultured either in standard medium (SM) or in osteogenic medium (OM) (0.2 mM ascorbic acid, 0.1 μm dexamethasone and 10 mM β-glycerophosphate) with or without DA 2nM or NI 100nM. Osteogenic differentiation of hBM-MSCs was evaluated by changes in morphology, presence of mineralized nodules (evidenced by Alizarin red) and expression of osteoblast-associated genes such as osteocalcin (OCN), RUNX2 and Bone morphogenetic protein (BMP-2) evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by Scion Image. After 21days of culture, in comparison to control cultures, hBM-MSCs placed in OM, DA, NI and DA+OM, NI+OM exhibited changes in cell morphology from a spindle-shaped fibroblastic appearance to a rounder more cuboidal shape and the cells formed an extensive network of dense multilayered nodules (extracellular mineralization).Table I indicates mRNA expression of osteogenic markers in different culture conditions and shows that both DA and NI alone or in combination with OM, increase RUNX2, OCN, and BMP-2 expression.SMDANIOMDA + OMNI + OMRUNX21,59 0,202,09 0,164,2 0,312,86 0,254,41 0,414,18 0,24OCN2,57 0,283,2 0,143,14 0,093,59 0,173,6 0,283,62 0,25BMP-21,55 0,192,27 0,174,16 0,272,84 0,284,43 0,304,21 0,30SM= standard medium, OM= osteogenic medium, DA= dasatinib, NI= nilotinib In summary, our data show that both DA and NI, as already reported IM, may induce osteogenic differentiation of mesenchymal cells thus indicating that they potentially favour osteoblastogenesis.