Evaluation of the osteogenic potential of crocin-incorporated collagen scaffold on the bone marrow mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for use in regenerative medicine. Several studies have shown that low-level laser irradiation (LLLI) could affect the differentiation and proliferation of MSCs. The aim of this study was to examine the influence of LLLI at different energy densities on BMSCs differentiation into neuron and osteoblast. Human BMSCs were cultured and induced to differentiate to either neuron or osteoblast in the absence or presence of LLLI. Gallium aluminum arsenide (GaAlAs) laser irradiation (810 nm) was applied at days 1, 3, and 5 of differentiation process at energy densities of 3 or 6 J/cm(2) for BMSCs being induced to neurons, and 2 or 4 J/cm(2) for BMSCs being induced to osteoblasts. BMSCs proliferation was evaluated by MTT assay on the seventh day of differentiation. BMSCs differentiation to neurons was assessed by immunocytochemical analysis of neuron-specific enolase on the seventh day of differentiation. BMSCs differentiation to osteoblast was tested on the second, fifth, seventh, and tenth day of differentiation via analysis of alkaline phosphatase (ALP) activity. LLLI promoted BMSCs proliferation significantly at all energy densities except for 6 J/cm(2) in comparison to control groups on the seventh day of differentiation. LLLI at energy densities of 3 and 6 J/cm(2) dramatically facilitated the differentiation of BMSCs into neurons (p < 0.001). Also, ALP activity was significantly enhanced in irradiated BMSCs differentiated to osteoblast on the second, fifth, seventh, and tenth day of differentiation (p < 0.001 except for the second day). Using LLLI at 810 nm wavelength enhances BMSCs differentiation into neuron and osteoblast in the range of 2-6 J/cm(2), and at the same time increases BMSCs proliferation (except for 6 J/cm(2)). The effect of LLLI on differentiation and proliferation of BMSCs is dose-dependent. Considering these findings, LLLI could improve current in vitro methods of differentiating BMSCs prior to transplantation.

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. We reported previously that glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor gamma-2 (Ppargamma2) expression and blocks adipocyte differentiation. Here we show that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppargamma2 and C/ebpalpha were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, our results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair.

Matrine enhances osteogenic differentiation of bone marrow-derived mesenchymal stem cells and promotes bone regeneration in rapid maxillary expansion

Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.

Background: Cav 1.3 can affect the classical osteoclast differentiation pathway through calcium signaling pathway. However, its effect and mechanism in osteoporosis (OS) are not clear. Methods: We performed cell transfection, real-time fluorescence quantitative PCR (qPCR), flow cytometry, SA-β-Gal staining, Alizarin Red S staining, ALP activity test, immunofluorescence, western blot, and cell viability assay to analyze cell viability, cell cycle, osteogenesis differentiation and autophagy activities in vitro. Meanwhile, GST-pull down and CHIP experiments were conducted to explore the influence of Cav 1.3 and Sprouty-related EVH1 domain 2 (Spred 2) on bone marrow-derived mesenchymal stem cells (BMSCs). Results: OS lead to the decreased of bone mineral density and differentiation ability of BMSCs in rats. Cav 1.3 was upregulated in OS rats. Overexpression of Cav 1.3 inhibited the activity of BMSCs, the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN), as well as promoted the cell cycle arrest and senescence. Furthermore, the negative correlation between Cav 1.3 and Spred 2 was found through GST-pull down and CHIP. Overexpression of Spred 2 increased the expressions of microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 of BMSCs, which ultimately promoted the cell activity of BMSCs and ALP, RUNX2, OCN expression. Conclusion: Cav 1.3 negatively regulates Spred 2-mediated autophagy and cell senescence, and damages the activity and osteogenic differentiation of BMSCs in OS rats. Funding Statement: National Natural Science Foundation of China (No.81900800); the Science and Technology Project of Shaanxi (2019JM-560). Declaration of Interests: All authors declare no conflict of interest. Ethics Approval Statement: The ethic approval was obtained from the Ethic Committee of the First Affiliated Hospital of Xi’an Jiaotong University School of Medicine.

Historical Perspectives and Advances in Mesenchymal Stem Cell Research for the Treatment of Liver Diseases

The present study investigated the effect of connexin 43 (Cx43) on the regulation of osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) using low-frequency-pulsed electromagnetic fields (LPEMF). The BMSCs were isolated and cultured in vitro using adherent whole-bone marrow cultures. CCK-8 assay was used to detect the effects of LPEMF on the proliferation ability of BMSCs and alkaline phosphatase (ALP) activity and the levels of osteogenic marker genes were detected to evaluate the osteogenic ability change following LPEMF treatment. Lentiviral vector-mediated RNA interference was transfected into BMSCs to inhibit the expression of Cx43 and western blotting was used to detect Cx43 expression. The BMSCs showed the highest proliferation following LPEMF treatment at 80 Hz for 1 h. The results of ALP activity, osteogenic marker genes and Alizarin Red S staining showed that the osteogenic ability was notably increased following LPEMF treatment at 80 Hz for 1 h. Cx43 expression increased during the osteogenic differentiation of BMSCs following LPEMF treatment at 80 Hz. The enhanced osteogenic differentiation of the LPEMF-treated BMSCs were partially reversed when Cx43 expression was inhibited. LPEMF may promote the osteogenic differentiation of BMSCs by regulating Cx43 expression and enhancing osteogenic ability.

Mohawk homeobox (MKX) has been demonstrated as a tendon/ligament specific transcription factor. The aim of this study was to investigate the role of MKX in ligament/tenogenic differentiation of bone marrow derived mesenchymal stem cells (BMMSCs). Human BMMSCs were treated with 50 ng/ml BMP-12 or transduced with MKX or scleraxis (SCX) adenoviral vector. Gene expression analysis was performed by quantitative reverse transcribed polymerase chain reaction (qRT-PCR). Rat BMMSCs were seeded in a collagen scaffold and transplanted into a rat Achilles tendon defect model. Tenogenesis related gene expressions and histological features were analyzed. BMP-12 induced tenogenesis in BMMSCs as indicated by increased COL1a1, TNXB, DCN and SCX mRNA, and MKX expression increased simultaneously. Rat BMMSCs enhanced defect repair and were still detectable 3 weeks after transplantation. Increased expressions of COL1a1, TNC and TNMD in vivo were also correlated with upregulated MKX. Adenoviral MKX promoted expression of COL1a1, TNXB, and TNMD in BMMSCs. This study demonstrated that MKX gene expression is enhanced during the tenogenic differentiation of BMMSCs in vitro and in vivo, and the adenoviral overexpression of MKX increases tendon extracellular matrix gene expression and protein production. Thus, MKX is a key factor for tenogenic differentiation of MSCs.

MicroRNAs (miRNAs) are novel key regulators of cellular differentiation. miR‑124 has been reported to regulate osteogenic differentiation of bone marrow‑derived mesenchymal stem cells (BMSCs). However, the specific mechanisms involved have not yet been fully elucidated. The present study aimed to investigate the effect of miR‑124 on osteogenic differentiation of BMSCs and its underlying mechanisms. In the present study, it was found that alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and the protein levels of osterix (Sp7) and runt‑related transcription factor2 (Runx2) were significantly increased, whereas the expression of miR‑124 was decreased in a time‑dependent manner during osteogenic differentiation of BMSCs. Following overexpression of miR‑124 via transfection of miR‑124 mimics in BMSCs, Runx2 protein expression and ALP activity were significantly decreased. By contrast, inhibition of miR‑124 expression led to an increase in ALP activity and Runx2 expression. Sp7 expression was suppressed in BMSCs transfected with miR‑124 mimics while increased when miR‑124 expression was inhibited, indicating that miR‑124 regulates the expression of Sp7. Moreover, a luciferase reporter assay further verified that Sp7 is the direct target of miR‑124. Finally, the effect of miR‑124 inhibitor on promoting the differentiation of BMSCs was abolished following treatment with a small interfering RNA targeting Sp7. Taken together, the present study demonstrates that miR‑124 inhibits the osteogenic differentiation of BMSCs by targeting Sp7.

Objective To investigate the effects of double gene recombinant vector on the proliferation and osteogenesis of alcohol-induced rabbit stem cells. Methods The rabbit bone marrow mesenchymal stem cells (BMSCs) at the third-generation were randomly divided into 6 groups: (1) normal group [bone marrow stem cells (BMSCs) without special treatment]; (2)model group (BMSCs without gene transfection); (3) unrelated sequence group (10 μl unrelated sequence transfected BMSCs); (4) siPPARγ group (10 μl siPPARγ gene vector transfected BMSCs); (5) exCGRP group (10 μl CGRP gene vector transfected BMSCs); (6) double gene group (10 μl double gene recombinant vector transfected BMSCs). The final mass concentration of 0.09 mol/L alcohol was added to all groups except the normal group. BMSCs proliferation was detected by methyl thiazol tetrazolium (MTT) method. Alkaline phosphatase (ALP) activity, laminin content, collagen type I content, and osteocalcin content in medium and ALP staining were detected at 14th day later. Results The proliferation in double gene group was obvious, and the proliferation curve was close to the normal group. The ALP activity, laminin content, collagen type I content and osteocalcin content in medium of the cells in the double gene group were the highest [(18.593±2.350) U/L, (24.422±3.110) ng/ml, (5.951±0.650) μg/L, (5.836±0.630) ng/ml respectively], which were significantly higher than those in model group [(6.528±0.830) U/L, (9.422±1.250) ng/ml, (1.733±0.230) μg/L, (2.016±0.240) ng/ml], unrelated sequence group [(7.011±0.850) U/L, (9.693±1.260) ng/ml, (1.663±0.220) μg/L, (1.913±0.230) ng/ml], obviously higher than those in siPPARγ group [(13.536±1.710) U/L, (17.103±2.230) ng/ml, (3.486±0.410) μg/L, (3.686±0.420) ng/ml], exCGRP group [(13.692±1.760) U/L, (17.185±2.240) ng/ml, (3.525±0.420) μg/L, (3.833±0.430) ng/ml], normal group [(15.056±1.910) U/L, (18.528±2.430) ng/ml, (4.035±0.460) μg/L, (4.012±0.460) ng/ml], and the difference was statistically significant (all P=0.000). The contents in model group and unrelated sequence group were significantly lower than those in normal group (all P=0.000). The contents in siPPARγ and exCGRP groups were slightly lower than those in normal group: for ALP activity (P=0.222, 0.274), for laminin (P=0.362, 0.390), for collagen type I (P=0.082, 0.105), and for osteocalcin (P=0.276, 0.543). The contents in double gene group were significantly higher than those in normal group: for ALP activity (P=0.031), for laminin (P=0.010), for collagen type I and osteocalcin (all P=0.001). The contents in double gene group were significantly higher than those in siPPARγ and exCGRP groups: for ALP activity (P=0.005, 0.006), for laminin (all P=0.003), for collagen type I and osteocalcin (all P=0.000). ALP staining showed that the cytoplasm in a large number of cells in double gene group was dyed dark blue and more than that in normal group. Conclusion The double gene recombinant vector can maintain the normal proliferation of rabbit stem cells induced by ethanol, and significantly promote osteogenic activity, which is superior to the single gene effect. Key words: Double gene recombinant vector; Ethanol; Stem cells; Proliferation; Osteogenic activity; Rabbit

This study examined the effect of wild-type Smad3 gene on the osteoblastic differentiation of rat bone marrow derived mesenchymal stem cells in vitro. Bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the complexes of pcDNA3.0-Myc-Smad3 or pcDNA3.0-Myc-Smad3△C and Lipofectamine reagent. Immunofluorescence staining was performed to evaluate the c-Myc signal in MSCs. The cell proliferation was detected by MTT method. To clarify the osteoblastic characteristics in stably transfected MSCs, alkaline phosphatase (ALP) mRNA and core binding factor α1 (Cbfa1) mRNA were investigated by RT-PCR, and ALP activity and mineralization were examined by p-nitrophenolphosphate method and alizarin red staining respectively. PD98059, a specific inhibitor of the ERK signaling pathway, was used to determine the role of ERK in Smad3-MSCs osteoblastic differentiation. c-Myc signal was detected in Smad3-MSCs and Smad3△C-MSCs. The proliferation of Smad3-MSCs was slower than that of Smad3△C-MSCs or V-MSCs.The relative levels of ALP mRNA and Cbfa1 mRNA in Smad3-MSCs, as well as ALP activity and mineralization, were markedly higher than those in Smad3△C-MSCs or V-MSCs. Although ALP activity and mineralization were slightly lower in Smad3-MSCs.treated with PD98059 than in those without PD98059 treatment, no significant difference was found between them (P＞0.05). It is concluded that the wild-type Smad3 gene, which is a crucial component promoting bone formation,can inhibit the proliferation of MSCs and enhance the osteoblastic differentiation of uncommitted MSCs and the maturation of committed MSCs independent of the ERK signaling pathway.

In this study, we sought to investigate the expression of microRNA (miR)-214 on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and explore the possible underlying mechanisms. We found that the overexpression of miR-214 effectively promoted the adipocyte differentiation of BMSCs in vitro, reduced alkaline phosphatase (ALP) activity and the gene expression of collagen type I (Col I), osteocalcin (OCN) and osteopontin (OPN) in the BMSCs. We further found that the overexpression of miR-214 suppressed the protein expression of fibroblast growth factor (FGF), phosphorylated c-Jun N-terminal kinase (p-JNK) and phosphorylated p38 (p-p38) in the BMSCs. The downregulation of miR-214 promoted the osteogenic differentiation of BMSCs, and increased ALP activity and Col I, OCN and OPN gene expression in the BMSCs. It also increased FGF p-JNK and p-p38 protein expression in the BMSCs. The use of JNK inhibitor (SP600125) enhanced the inhibitory effects of miR-214 overexpression on osteogenic differentiation, ALP activity, and Col I, OCN and OPN gene expression in the BMSCs. Lastly, the use of p38 inhibitor (SB202190) also enhanced the inhibitory effects of miR-214 overexpression on ALP activity, and Col I, OCN and OPN gene expression in the BMSCs. These results provide a mechanism responsible for the suppressive effects of miR-214 on the osteogenic differentiation of BMSCs involving the inhibition of the JNK and p38 pathways.

Bone tissue engineering requires scaffolds that mimic the native extracellular matrix and provide sustained delivery of osteoinductive factors. This study focuses on developing a multifunctional scaffold using a green electrospinning process to combine the biocompatibility of silk fibroin (SF) with a non-viral gene delivery system for sustained expression of Bone Morphogenetic Protein 2 (BMP2). A green electrospinning technique, using an aqueous SF and polyethylene oxide (PEO) solution, was employed to fabricate nanofibrous scaffolds, eliminating the use of harsh organic solvents. Polyethylenimine (PEI) modified liposomes (LipoPEI) were used to encapsulate a BMP2-encoding plasmid (pDNA Bmp2 ). These gene-loaded nanoparticles were incorporated into the SF-PEO nanofibers. The resulting scaffolds were characterized for morphology (SEM), structure (FTIR, XRD), and drug release kinetics. Biological performance was evaluated by assessing cell viability (MTT assay), cell attachment (SEM), gene transfection efficiency (confocal microscopy), and osteogenic differentiation (alkaline phosphatase (ALP) activity, Alizarin Red S staining) using bone marrow mesenchymal stem cells (BMSCs). Physicochemical characterization confirmed the successful formation of uniform pDNABmp2@LipoPEI nanocomplexes with a particle size of approximately 266 nm and a positive surface charge of +16.9 mV. These nanocomplexes were homogeneously incorporated into smooth, bead-free SF-PEO nanofibers with average diameters ranging from 460 to 541 nm. The composite scaffold demonstrated a highly sustained release of pDNABmp2 over 14 days. In vitro studies using rat bone marrow-derived mesenchymal stem cells (BMSCs) revealed that the scaffold possesses excellent biocompatibility, promoting robust cell adhesion, spreading, and proliferation. Furthermore, the gene-loaded scaffold successfully mediated the transfection of BMSCs, leading to significant upregulation of osteogenic markers, including alkaline phosphatase (ALP) activity and extensive calcium mineral deposition over 21 days. The novel composite scaffold combines the structural advantages of SF with a sustained BMP2 gene delivery system, showing remarkable potential to promote osteogenic differentiation. This work presents a promising, environmentally friendly, and effective platform for bone tissue engineering and regenerative medicine.

Cell therapy has been proposed as an effective treatment method for bone regeneration using stem cells and osteogenic inducting mediators. In the present study, the ability of homeopathic remedy Symphytum officinale to induce osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rat BM-MSCs) was studied MSCs were isolated from the rat’s bone morrow. Cytotoxic effect of Symphytum on the MSCs was evaluated by MTT assay. The osteogenic induction ability of Symphytum has been assessed by alizarin red staining (ALZ) and quantitative alkaline phosphatase (ALP) activity assays and compared with the osteogenic standard medium. MTT assay results illustrated that Symphytum 200 C had a high cytotoxicity (to about 75.2 ± 3.12), so that lower potencies were used for the next experiments. Symphytum 6 C had the most color intensity in ALZ and ALP staining assays. Also, quantitative analyses of ALP activity revealed that Symphytum 6 C caused the most increase in the ALP activity up to 4.04 ± 0.34 and 7.86 ± 1.38 on day 7 and 21, respectively. According to this finding, Symphytum could enhance osteogenic differentiation of MSCs and can be considered as a safe, low cost, and highly effective drug for bone regeneration. Cell therapy has been proposed as an effective treatment method for bone regeneration using stem cells and osteogenic inducting mediators. In the present study, the ability of homeopathic remedy Symphytum officinale to induce osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rat BM-MSCs) was studied. The results illustrated that Symphytum 6 C had the most osteogenic induction effect compare with osteogenic standard medium. According to this finding, Symphytum officinale as a natural compound can significantly induce osteogenic differentiation of rat BM-MSCs. So, it could be considered as a safe and more cost-effective therapeutic agent for clinical treatment of bone disorders.

In orthodontics, mechanical stress plays an important role in the process of bone remodeling. Mechanical stress has an effect on osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). However, the mechanism remains to be studied. The aim of this study is to investigate the effects of demethyltransferase fat mass and obesity-associated (FTO) on osteogenic differentiation of BMSCs under mechanical stress condition. The rat BMSCs were cultured in vitro, followed by flow cytometry to identify the cell surface antigens. Osteogenic differentiation of BMSCs was induced by mechanical stress by using the flexcell tension system for 6h every day and 3days in total. BMSCs were transfected by using plasmid for FTO knockdown. The expression level of FTO, hypoxia-inducible factor (HIF)-1α, runt-related transcription factor 2(RUNX2), bone morphogenetic proteins(BMPs) and alkaline phosphatase (ALP) were measured by real-time qPCR, western blotting. ALP activity were determined by ALP staining assays. The expression of FTO and HIF-1α in BMSCs with mechanical stress were significantly higher than BMSCs without mechanical stress, also, the expression of osteogenic differentiation markers were higher in BMSCs with mechanical stress. Knockdown of FTO decreased expression of osteogenic differentiation marker and ALP activity in stretched BMSCs. In addition, the expression of HIF-1α was decreased after knocking down FTO. FTO promotes the expression of HIF-1α and osteogenic differentiation under the condition of mechanical stress. This finding may facilitate the clinical application of orthodontics and the mechanism research of mechanical stress-induced osteogenesis.