Strontium-doping promotes bone bonding of titanium implants in osteoporotic microenvironment

To improve the biological activities of titanium implant surfaces and promote efficient early bone formation, the formation of bioactive coatings that promote bone formation on titanium implant surfaces is currently an important area of research. In this study, we prepared a zinc-doped porous coating (Zn-PEO) on a titanium alloy surface via plasma electrolytic oxidation. The surface characteristics of the samples were evaluated with field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), profilometry, static contact angles and nanoscratch studies. The slow release of zinc from the coating was detected with inductively coupled plasma‒mass spectrometry (ICP‒MS). The corrosion resistances of the samples were evaluated via electrochemical studies. On this basis, we evaluated the biocompatibility and biological activity of the sample through in vitro cell experiments. The results confirmed that a Zn-PEO coating was successfully prepared on the surface of the titanium alloy; it exhibited a porous micro/nanomorphology, and the coating and the substrate were tightly bound. The coating increased the roughness of the titanium surface and improved the wear and corrosion resistance. More importantly, the coating promoted adhesion, proliferation, differentiation and mineralization of bone marrow mesenchymal stem cells and had good biological activity. Therefore, Zn-PEO coatings with porous structures were prepared on the surfaces of titanium implants through plasma electrolytic oxidation. The Zn-PEO coating exhibited good surface properties and biological activity and has good application prospects.

Titanium (Ti) implants are widely used in the clinic as bone substitutes and dental implants, but further improvements are needed to obtain high osteogenic ability and consequent osseointegration. Knockdown of long noncoding RNA MIR31HG promotes osteogenic differentiation and bone formation. In this study, we fabricated a Ti surface functionalized with siRNA targeting MIR31HG (siMIR31HG) and accelerated osteogenesis of bone marrow mesenchymal stem cells (BMSCs). Chitosan/siRNA complex was loaded onto the thermal alkali-treated Ti surface to fabricate the siMIR31HG-functionalized Ti surface. The surface morphology, siRNA loading and release efficiency, and transfection efficacy were investigated, and the biological effects, such as cell proliferation, cell morphology, and osteogenic activity, were determined. The results showed that the siMIR31HG-functionalized Ti implant generated an ∼50% knockdown of MIR31HG, with no apparent cytotoxicity, which consequently enhanced osteogenic differentiation of BMSCs, as indicated by the increase of ALP production, extracellular matrix mineralization, osteogenic gene expression, and ectopic bone formation in vivo. The siMIR31HG biofunctionalization can be used to obtain better osseointegration of Ti implant in the clinic.

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein‐protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up‐regulated genes and 93 down‐regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule‐related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose‐derived stem cells. In summary, the present results provide novel insights into the microtubule‐ and cytoskeleton‐related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.

The neuronal differentiation of mesenchymal stem cells offers a new strategy for the treatment of neurological disorders. Thus, there is a need to identify a noninvasive and sensitive in vivo imaging approach for real-time monitoring of transplanted stem cells. Our previous study confirmed that magnetic resonance imaging, with a focus on the ferritin heavy chain 1 reporter gene, could track the proliferation and differentiation of bone marrow mesenchymal stem cells that had been transduced with lentivirus carrying the ferritin heavy chain 1 reporter gene. However, we could not determine whether or when bone marrow mesenchymal stem cells had undergone neuronal differentiation based on changes in the magnetic resonance imaging signal. To solve this problem, we identified a neuron-specific enolase that can be differentially expressed before and after neuronal differentiation in stem cells. In this study, we successfully constructed a lentivirus carrying the neuron-specific enolase promoter and expressing the ferritin heavy chain 1 reporter gene; we used this lentivirus to transduce bone marrow mesenchymal stem cells. Cellular and animal studies showed that the neuron-specific enolase promoter effectively drove the expression of ferritin heavy chain 1 after neuronal differentiation of bone marrow mesenchymal stem cells; this led to intracellular accumulation of iron and corresponding changes in the magnetic resonance imaging signal. In summary, we established an innovative magnetic resonance imaging approach focused on the induction of reporter gene expression by a neuron-specific promoter. This imaging method can be used to noninvasively and sensitively detect neuronal differentiation in stem cells, which may be useful in stem cell-based therapies.

CXC chemokines are essential for osteogenic differentiation of bone mesenchymal stem cells (BMSCs) for use in bone tissue engineering and regenerative medicine in clinical settings. However, an accurate understanding of the underlying mechanisms is still needed. In this study, we analyzed the effects of CXC chemokine ligand-13 (CXCL13) on osteogenic differentiation of rat BMSCs and initiated a preliminary discussion on possible mechanisms. BMSCs were isolated from bone marrow of rat and incubated with CXCL13 recombinant protein in differentiation medium. The main osteogenesis indexes were alkaline phosphatase (ALP) activity and calcium nodes. Expression of Runx2 and CXCR5 was determined using western blot, while miRNAs were determined with quantitative-RT-PCR. Si-CXCR5 was transfected into MSCs to silence CXCR5. A miRNA-23a mimic was transfected into BMSCs for overexpression of miRNA-23a. Recombinant CXCL13 induced ALP activity, deposition of calcium salts, and formation of calcium nodes, and it also increased expression of Runx2. The expression of recombinant CXCL13 suppressed expression of miRNA-23a. Overexpression of miR-23a reversed CXCL13 induced-osteogenic differentiation of BMSCs and expression of Runx2. Recombinant CXCL13 attenuated the interaction of miRNA-23a with the Runx2 3′UTR. Silencing of CXCR5 abrogated recombinant CXCL13-induced downregulation of miRNA-23a expression. In summary, CXCL13 promotes osteogenic differentiation of BMSCs by inhibiting miR-23a expression.

Functionalization of a clustered TiO2 nanotubular surface with platelet derived growth factor-BB covalent modification enhances osteogenic differentiation of bone marrow mesenchymal stem cells

Iron overload inhibits osteogenic commitment and differentiation of mesenchymal stem cells via the induction of ferritin

ncRNA involves in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). WNT5A participates in the growth and development of osteogenic differentiation. This study aims to investigate whether lncRNA MALAT1 regulates BMSCs osteogenesis through WNT5A. qRT-PCR was done to detect the lncRNA MALAT1 level and osteogenic markers in osteoporosis patients and control groups. The above markers and WNT5A protein levels were detected by Western blot. The degree of osteogenic differentiation was detected by ALP activity assay and ALP staining. The differentiation ability of BMSCs after lncRNA MALAT1 overexpression was detected by ARS staining. The binding site of lncRNA MALAT1 to WNT5A was determined by dual luciferase reporter assay. lncRNA MALAT1 expression was higher in patients with osteoporosis, and decreased significantly with increased osteogenic induction. Overexpression of lncRNA MALAT1 in BMSCS reduced WNT5A level, while interference with lncRNA MALAT1 increased WNT5A levels. In cells with overexpression of lncRNA MALAT1, transfection of si-WNT5A can significantly downregulate the RUNX2, OSX, ALP, OCN, OPN, and COL1A1, thereby inhibiting osteogenic differentiation, interfering with the regulation of WNT signaling pathway and regulating BMSCs osteogenic differentiation. lncRNA MALAT1 and WNT5A can regulate BMSCs osteogenesis, thus accelerating the progression of osteoporosis.

Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is closely associated with bone diseases. Circular RNAs are reported to be involved in BMSC differentiation. CircSmg5 (circ_0001145) has been identified to be downregulated in an osteoporosis mouse model. In this study, we aimed to explore the function and regulatory mechanism of circSmg5 in BMSC osteogenic differentiation. The Alizarin Red staining and alkaline phosphatase staining assays were performed to explore the osteogenic differentiation of BMSCs. The interaction between circ_0001145, miR-194-5p, and frizzled class receptor 6 (Fzd6) was analyzed by luciferase reporter assay. The nuclear translocation of β-catenin was assessed using immunofluorescence staining. CircSmg5 is in stable circular structure. CircSmg5 expression was elevated in the process of BMSC osteogenic differentiation. CircSmg5 overexpression promoted the osteogenic differentiation of BMSCs. CircSmg5 bound with miR-194-5p, whose expression was decreased in the osteogenic differentiation of BMSCs. MiR-194-5p directly targeted the 3'UTR of Fzd6. The mRNA and protein levels of Fzd6 were positively modulated by circSmg5 and negatively regulated by miR-194-5p in BMSCs. CircSmg5 was demonstrated to promote the BMSC osteogenic differentiation by targeting the miR-194-5p/Fzd6 axis to activate the Wnt/β-catenin signaling.

Patients with spinal cord injury frequently develop neurogenic heterotopic ossification, whose pathogenesis remains incompletely understood. Existing research models struggle to accurately simulate the complex pathological process. To establish a reliable neurogenic heterotopic ossification research model, elucidate its pathogenesis, and explore early intervention strategies, this study successfully developed a spinal cord injury-induced neurogenic heterotopic ossification mouse model. Significant ectopic bone formation, restricted hip and knee joint mobility, and motor dysfunction were observed, accompanied by elevated expression of the osteogenic markers alkaline phosphatase, runt-related transcription factor 2, sex-determining region Y-box 9, and osteocalcin . Proteomics and quantitative polymerase chain reaction analysis revealed upregulation of chemokine (C-X-C motif) ligand (CXCL)12, C-X-C chemokine receptor type 4 (CXCR4), and LYN proto-oncogene, whereas CXCL1 was downregulated in the neurogenic heterotopic ossification group. In vivo experiments confirmed abnormal accumulation of M1 macrophages in muscles surrounding early ectopic bone tissue, with markedly elevated CXCL12 expression. In vitro studies further revealed that M1 macrophages are the primary source of CXCL12 secretion, and their cell culture supernatants promote the proliferation, migration, and osteoblastic differentiation potential of bone marrow mesenchymal stem cells. Mechanistically, CXCL12 activates the phosphatidylinositol 3-kinase/protein kinase B pathway by binding to the CXCR4 receptor, thereby driving the osteogenic differentiation of bone marrow mesenchymal stem cells. These findings indicate a causal relationship between the pathological process of neurogenic heterotopic ossification following spinal cord injury and the activation of the CXCL12-CXCR4-phosphatidylinositol 3-kinase-protein kinase B signaling axis, which modulates bone marrow mesenchymal stem cell function through M1 macrophage polarization. This study reveals a key mechanism driving the osteogenic differentiation of bone marrow mesenchymal stem cells, providing new directions for early warning and targeted treatment of neurogenic heterotopic ossification following spinal cord injury.

Hypermethylation-mediated downregulation of long non-coding RNA MEG3 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells and promotes pediatric aplastic anemia

It has been previously reported that psoralen, one of the active ingredients in Psoralea corylifolia, could induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), suggesting its potential to treat osteoporosis. Additionally, runt-related transcription factor 2 (Runx2) is a transcription factor that plays vital roles in BMSC osteogenic differentiation. However, whether and how microRNAs (miRNAs/miRs) modulate osteogenic differentiation induced by psoralen have not yet been examined, to the best of the authors' knowledge. The present study aimed to identify the miRNA target genes that regulate osteogenic differentiation of BMSCs induced by psoralen. A Cell Counting Kit-8 assay and alizarin red staining were used to detect the viability and osteogenic differentiation of BMSCs, respectively, under treatment with psoralen. miRNA microarray analysis was performed to identify the differentially expressed miRNAs under treatment with psoralen. A bioinformatics analysis and a luciferase reporter assay were conducted to identify the targets of miR-488. Finally, the mechanisms of miR-488 in psoralen-induced BMSC osteogenic differentiation were investigated using overexpression or inhibition methods in vitro. Cell viability was elevated and osteogenic differentiation of BMSCs was improved under treatment with psoralen. miRNA microarray analysis and further validation by reverse transcription-quantitative PCR revealed that miR-488 was downregulated during psoralen-induced BMSC osteogenic differentiation. Bioinformatics analysis and experimental validation by a luciferase reporter assay identified Runx2 as a potential target of miR-488. Overexpression of miR-488 by transfection with miR-488 mimics markedly inhibited the expression of Runx2, Osterix and alkaline phosphatase, whereas, the inhibition of miR-488 expression by the miR-488 inhibitor promoted their expression compared with the control. Rescue assays demonstrated that Runx2 overexpression partially rescued the inhibitory effect of miR-488 on BMSC osteogenic differentiation. The present results suggested that miR-488 is a negative regulator of psoralen-induced BMSC osteogenic differentiation by targeting Runx2, providing a possible therapeutic target for osteoporosis.

Objective To investigate the effects of miR-144-3p-targeted regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene on proliferation, apoptosis, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under retraction force. Methods The BMSCs of rats were randomly divided into the tension MSC group with detrusor stimulation and the MSC group without detrusor stimulation, after which osteogenic differentiation of BMSCs was induced in both groups. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic differentiation ability of the two groups of cells. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression of miR-144-3p and PTEN in the two groups of cells after osteogenic differentiation. Bioinformatics website and dual luciferase reporter were used to detect the relationship between miR-144-3p and PTEN. The tension MSC group was used as a control group, and miR-144-3p mimics (miR-144-3p mimic group), mimic controls (mimic-NC group), PTEN interferers (si-PTEN group), and interference controls (si-NC group) were transfected into BMSCs. The BMSCs were then continuously stimulated for 24 h using a Flexercell in vitro cellular mechanics loading device, applying a draft force at a frequency of 1 Hz and a deformation rate of 18%. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) colorimetric assay; the expression levels of cyclin, cyclin-dependent kinases (CDK), BCL2-associated X (BAX), B-cell lymphoma-2 (BCL-2), and other cell cycle and apoptosis related proteins were detected by western blot (WB); and the osteogenic differentiation ability of MSC cells was detected by ALP staining and alizarin red staining. Results Compared with the MSC group, the level of miR-144-3p was significantly lower and the level of PTEN was significantly higher in the tension MSC group. ALP staining showed normal activity in the MSC group and decreased ALP activity in the tension MSC group compared to the MSC group. Alizarin red staining in the MSC group showed scattered calcium nodule formation, and alizarin red staining showed red nodules with a more uniform color distribution. Compared to the MSC group, the tension MSC group showed fewer, smaller, and lighter staining mineralized nodules. Compared with the tension group and mimic-NC group (si-NC group), the proliferation rate of cells in the miR-144-3p mimic group (si-PTEN group) was significantly higher; the expression levels of PTEN and BAX were significantly lower; and the expression levels of cyclin, CDK, and BCL-2 protein were significantly higher. ALP staining results revealed that the miR-144-3p mimic group (si-PTEN group) showed significantly higher osteogenic differentiation ability and ALP activity of MSC than the tension group and mimic-NC group (si-NC group). Conclusion miR-144-3p may inhibit apoptosis and promote proliferation and osteogenic differentiation of BMSCs under tension by targeting and regulating PTEN.

The aim of this study was to improve the cytocompatibility and differentiation of human bone marrow-derived mesenchymal stem cells on the surface of titanium implants by immobilizing biofunctional molecules on their surface. Gly-Arg-Gly-Asp-Ser (GRGDS) peptides, human plasma fibronectin (pFN), or type I collagen from calf skin (Col) was covalently immobilized on the titanium surfaces. Twice as many cells attached to the Col- and pFN-immobilized titanium surfaces than attached to the as-polished surface control. The ALP activity of the cells, as well as the mineralized nodule formation, was significantly higher on the Col- and pFN-immobilized titanium surfaces than on the as-polished surfaces. These results indicate that the immobilization of biofunctional molecules such as Col and pFN on titanium surfaces enhances the attachment, spreading, proliferation, and differentiation of human bone marrow-derived mesenchymal stem cells, which may lead to a more rapid bone-titanium integration.

Vitamin D metabolites are essential for bone regeneration and mineral homeostasis. The vitamin D precursor 7-dehydrocholesterol can be used after UV irradiation to locally produce active vitamin D by osteoblastic cells. Furthermore, UV-irradiated 7-dehydrocholesterol is a biocompatible coating for titanium implants with positive effects on osteoblast differentiation. In this study, we examined the impact of titanium implants surfaces coated with UV-irradiated 7-dehydrocholesterol on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. First, the synthesis of cholecalciferol (D3) was achieved through the incubation of the UV-activated 7-dehydrocholesterol coating for 48 h at 23℃. Further, we investigated invitro the biocompatibility of this coating in human umbilical cord mesenchymal stem cells and its potential to enhance their differentiation towards the osteogenic lineage. Human umbilical cord mesenchymal stem cells cultured onto UV-irradiated 7-dehydrocholesterol-coated titanium implants surfaces, combined with osteogenic supplements, upregulated the gene expression of several osteogenic markers and showed higher alkaline phosphatase activity and calcein blue staining, suggesting increased mineralization. Thus, our results show that the use of UV irradiation on 7-dehydrocholesterol -treated titanium implants surfaces generates a bioactive coating that promotes the osteogenic differentiation of human umbilical cord mesenchymal stem cells, with regenerative potential for improving osseointegration in titanium-based bone anchored implants.