Alkaline Phosphatase mRNA Levels Research Articles

Effects of i-PRF, A-PRF+, and EMD on Osteogenic Potential of Osteoblasts on Titanium.

The study evaluates three biologically active substances with known bone-inductive potential on previously decontaminated titanium (Ti) discs. Rough and smooth Ti surfaces were contaminated with a multispecies biofilm and cleaned with a chitosan brush. Discs were treated either with injectable-platelet-rich fibrin (i-PRF), advanced platelet-rich fibrin (A-PRF+), or enamel matrix derivatives (EMDs) before osteoblast seeding. Biocompatibility, adhesion, migration, and gene expression of runt-related transcription factor 2 (RUNX2), collagen Type I Alpha 2 (COL1a2), alkaline phosphatase (ALP), osteocalcin (OC), and osteonectin (ON) were performed. All the tested biologic agents similarly increased cell viability. Specifically, osteoblasts seeded over i-PRF and EMD-treated surfaces showed improvement in adhesion and migration and significantly increased ALP, OC, ON, RUNX-2, and COL1a2 mRNA levels up to 2.8 fold (p < 0.05) with no differences between Ti surfaces. i-PRF and EMD possess beneficial bioactive properties that enhance tissue healing and promote regeneration on thoroughly sterilized surfaces. Biologically active materials may hold the potential to influence the process of implant re-osseointegration, which warrants more research since sterilization of the affected surfaces under clinical conditions is still not reliably possible and remains one of the greatest challenges.

High-frequency low-intensity semiconductor laser irradiation enhances osteogenic differentiation of human cementoblast lineage cells

PurposeLaser irradiation activates a range of cellular processes in the periodontal components and promotes tissue repair. However, its effect on osteogenic differentiation of human cementoblast lineage cells remains unclear. This study aimed to examine the effects of high-frequency semiconductor laser irradiation on the osteogenic differentiation of human cementoblast lineage (HCEM) cells.MethodsHCEM cells were cultured to reach 80% confluence and irradiated with a gallium-aluminum-arsenide (Ga-Al-As) semiconductor laser with a pulse width of 200 ns and wavelength of 910 at a dose of 0–2.0 J/cm2. The outcomes were assessed by analyzing the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and type I collagen (COLL1) using real-time polymerase chain reaction (PCR) analysis 24 h after laser irradiation. Cell mineralization was evaluated using ALP activity, calcium deposition, and Alizarin Red staining.ResultsThe laser-irradiated HCEM cells showed significantly enhanced gene expression levels of ALP, RUNX2, and COLL1 as well as ALP activity and calcium concentration in the culture medium compared with the non-irradiated cells. In addition, enhanced calcification deposits were confirmed in the laser-irradiated group compared with the non-irradiated group at 21 and 28 days after the induction of osteogenic differentiation.ConclusionHigh-frequency semiconductor laser irradiation enhances the osteogenic differentiation potential of cultured HCEM cells, underscoring its potential utility for periodontal tissue regeneration.

Exosomal lncRNA HCP5 derived from human bone marrow mesenchymal stem cells improves chronic periodontitis by miR-24-3p/HO1/P38/ELK1 pathway

ObjectiveThe present study aimed to explore the function of human bone marrow mesenchymal stem cells (hBMMSCs)-derived exosomal long noncoding RNA histocompatibility leukocyte antigen complex P5 (HCP5) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to improve chronic periodontitis (CP). MethodsExosomes were extracted from hBMMSCs. Alizarin red S staining was used to detect mineralised nodules. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure HCP5 and miR-24-3p expression. The mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin, osterix, runt-related transcription factor 2, bone morphogenetic protein 2, osteopontin, fibronectin, collagen 1, heme oxygenase 1 (HO1), P38, and ETS transcription factor ELK1 (ELK1) were detected using RT-qPCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the HO1 and carbon monoxide concentrations. Heme, biliverdin, and Fe2+ levels were determined using detection kits. Micro-computed tomography, hematoxylin and eosin staining, ALP staining, tartrate-resistant acid phosphatase staining, ELISA, and RT-qPCR were conducted to evaluate the effect of HCP5 on CP mice. Dual luciferase, RNA immunoprecipitation, and RNA pulldown experiments were performed to identify the interactions among HCP5, miR-24-3p, and HO1. ResultsThe osteogenic ability of hPDLSCs significantly increased when co-cultured with hBMMSCs or hBMMSCs exosomes. Overexpression of HCP5 and HO1 in hBMMSCs exosomes promoted the osteogenic differentiation of hPDLSCs, and knockdown of HCP5 repressed the osteogenic differentiation of hPDLSCs. HCP5 knockdown enhanced the inflammatory response and repressed osteogenesis in CP mice. MiR-24-3p overexpression diminished the stimulatory effect of HCP5 on the osteogenic ability of hPDLSCs. Mechanistically, HCP5 acted as a sponge for miR-24-3p and regulated HO1 expression, and HO1 activated the P38/ELK1 pathway. ConclusionHBMMSCs-derived exosomal HCP5 promotes the osteogenic differentiation of hPDLSCs and alleviates CP by regulating the miR-24-3p/HO1/P38/ELK1 signalling pathway.

CA1 Modulates the Osteogenic Differentiation of Dental Follicle Stem Cells by Activating the BMP Signaling Pathway In Vitro.

Carbonic anhydrase 1 (CA1) has been found to be involved in osteogenesis and osteoclast in various human diseases, but the molecular mechanisms are not completely understood. In this study, we aim to use siRNA and lentivirus to reduce or increase the expression of CA1 in Dental follicle stem cells (DFSCs), in order to further elucidate the role and mechanism of CA1 in osteogenesis, and provide better osteogenic growth factors and stem cell selection for the application of bone tissue engineering in alveolar bone fracture transplantation. The study used RNA interference and lentiviral vectors to manipulate the expression of the CA1 gene in DFSCs during in vitro osteogenic induction. The expression of osteogenic marker genes was evaluated and changes in CA1, alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and Bone morphogenetic proteins (BMP2) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). The osteogenic effect was assessed through Alizarin Red staining. The mRNA and protein expression levels of CA1, ALP, RUNX2, and BMP2 decreased distinctly in the si-CA1 group than other groups (p < 0.05). In the Lentivirus-CA1 (LV-CA1) group, the mRNA and protein expressions of CA1, ALP, RUNX2, and BMP2 were amplified to varying degrees than other groups (p < 0.05). Apart from CA1, BMP2 (43.01%) and ALP (36.69%) showed significant upregulation (p < 0.05). Alizarin red staining indicated that the LV-CA1 group produced more calcified nodules than other groups, with a higher optical density (p < 0.05), and the osteogenic effect was superior. CA1 can impact osteogenic differentiation via BMP related signaling pathways, positioning itself upstream in osteogenic signaling pathways, and closely linked to osteoblast calcification and ossification processes.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 μM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Zuo Gui Wan Promotes Osteogenesis via PI3K/AKT Signaling Pathway: Network Pharmacology Analysis and Experimental Validation.

Osteogenesis is vitally important for bone defect repair, and Zuo Gui Wan (ZGW) is a classic prescription in traditional Chinese medicine (TCM) for strengthening bones. However, the specific mechanism by which ZGW regulates osteogenesis is still unclear. The current study is based on a network pharmacology analysis to explore the potential mechanism of ZGW in promoting osteogenesis. A network pharmacology analysis followed by experimental validation was applied to explore the potential mechanisms of ZGW in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs). In total, 487 no-repeat targets corresponding to the bioactive components of ZGW were screened, and 175 target genes in the intersection of ZGW and osteogenesis were obtained. And 28 core target genes were then obtained from a PPI network analysis. A GO functional enrichment analysis showed that the relevant biological processes mainly involve the cellular response to chemical stress, metal ions, and lipopolysaccharide. Additionally, KEGG pathway enrichment analysis revealed that multiple signaling pathways, including the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) signaling pathway, were associated with ZGW-promoted osteogensis. Further experimental validation showed that ZGW could increase alkaline phosphatase (ALP) activity as well as the mRNA and protein levels of ALP, osteocalcin (OCN), and runt related transcription factor 2 (Runx 2). What's more, Western blot analysis results showed that ZGW significantly increased the protein levels of p-PI3K and p-AKT, and the increases of these protein levels significantly receded after the addition of the PI3K inhibitor LY294002. Finally, the upregulated osteogenic-related indicators were also suppressed by the addition of LY294002. ZGW promotes the osteogenesis of BMSCs via PI3K/AKT signaling pathway.

Lidocaine inhibits osteogenic differentiation of human dental pulp stem cells in vitro

Lidocaine is an amide local anaesthetic commonly used for pain control, however, few studies have investigated the effect of lidocaine on the osteogenic differentiation of human dental pulp stem cells (HDPSCs). The present study aimed to determine the effect of lidocaine on HDPSC viability and osteogenic differentiation. HDPSCs were incubated with 0, 0.05, 0.2, 0.5, and 1 mM lidocaine for 24, 48 and 72 h, after which, MTT assays were performed. HDPSCs cultured with the above lidocaine concentrations and osteogenic differentiation medium for 7 and 14 days were stained for alkaline phosphatase (ALP). Protein and mRNA levels of relevant osteogenic factors (bone morphogenetic protein-2 [BMP-2] and runt-related transcription factor 2 [RUNX2]) were examined using western blotting and real-time reverse-transcription polymerase chain reaction, respectively. Lidocaine did not affect the viability of HDPSCs, however, lidocaine reduced ALP activity in HDPSCs. Levels of ALP, BMP-2, and RUNX2 mRNA were reduced with lidocaine, and levels of BMP-2 and RUNX2 proteins were decreased, versus controls. Lidocaine inhibits osteogenic differentiation markers in HDPSCs in vitro, even at low concentrations, without cytotoxicity. This study suggests that lidocaine may inhibit osteogenic differentiation in HDPSC-mediated regenerative medicine, including pulp regeneration and repair.

Humoral and cellular factors inhibit phosphate-induced vascular calcification during the growth period.

Hyperphosphatemia is an independent and non-classical risk factor of cardiovascular disease and mortality in patients with chronic kidney disease (CKD). Increased levels of extracellular inorganic phosphate (Pi) are known to directly induce vascular calcification, but the detailed underlying mechanism has not been clarified. Although serum Pi levels during the growth period are as high as those observed in hyperphosphatemia in adult CKD, vascular calcification does not usually occur during growth. Here, we have examined whether the defence system against Pi-induced vascular calcification can exist during the growth period using mice model. We found that calcification propensity of young serum (aged 3 weeks) was significantly lower than that of adult serum (10 months), possibly due to high fetuin-A levels. In addition, when the aorta was cultured in high Pi medium in vitro, obvious calcification was observed in the adult aorta but not in the young aorta. Furthermore, culture in high Pi medium increased the mRNA level of tissue-nonspecific alkaline phosphatase (TNAP), which degrades pyrophosphate, only in the adult aorta. Collectively, our findings indicate that the aorta in growing mouse may be resistant to Pi-induced vascular calcification via a mechanism in which high serum fetuin-A levels and suppressed TNAP expression.

Extracellular vesicles secreted from mouse muscle cells improve delayed bone repair in diabetic mice.

Humoral factors that are secreted from skeletal muscles can regulate bone metabolism and contribute to muscle-bone relationships. Although extracellular vesicles (EVs) play important roles in physiological and pathophysiological processes, the roles of EVs that are secreted from skeletal muscles in bone repair have remained unclear. In the present study, we investigated the effects of the local administration of muscle cell-derived EVs on bone repair in control and streptozotocin-treated diabetic female mice. Muscle cell-derived EVs (Myo-EVs) were isolated from the conditioned medium from mouse muscle C2C12 cells by ultracentrifugation, after which Myo-EVs and gelatin hydrogel sheets were transplanted on femoral bone defect sites. The local administration of Myo-EVs significantly improved delayed bone repair that was induced by the diabetic state in mice 9 days after surgery. Moreover, this administration significantly enhanced the ratio of bone volume to tissue volume at the damaged sites 9 days after surgery in the control mice. Moreover, the local administration of Myo-EVs significantly blunted the number of Osterix-positive cells that were suppressed by the diabetic state at the damage sites after bone injury in mice. Additionally, Myo-EVs significantly blunted the mRNA levels of Osterix and alkaline phosphatase (ALP), and ALP activity was suppressed by advanced glycation end product 3 in ST2 cells that were treated with bone morphogenetic protein-2. In conclusion, we have shown for the first time that the local administration of Myo-EVs improves delayed bone repair that is induced by the diabetic state through an enhancement of osteoblastic differentiation in female mice.

The effects of Twinlight laser treatment on the titanium surface proliferation and osteogenic differentiation of mesenchymal stem cells

BackgroundThe study aimed to observe the effects of a Twinlight laser on the titanium surface proliferation of inflammatory Mesenchymal stem cells (MSCs), inflammatory cytokine expression, and osteogenic differentiation.MethodsThe MSCs were collected from bone tissue of healthy individuals.The cellular inflammatory model was established with 1 μg/mL lipopolysaccharide (LPS).Under the cellular inflammatory model,divided into five groups: the normal control group (C); the inflammatory control group (L); Er:YAG laser group (L + E); Nd:YAG laser group (L + N); Er:YAG laser and Nd:YAG laser group (L + E + N). The treated cells were inoculated onto titanium disks.The normal and inflammatory MSCs on the surface of titanium surface were examined by CCK-8, scanning election microscopy (SEM), quantitative real-time polymerase chain reaction (qRT‑PCR) and other methods for their proliferation, growth pattern, expression of inflammatory factors Interleukin-6 (IL-6), Interleukin-8 (IL-8) and osteogenic genes Runx2 (Runt-related transcription factor 2) and alkaline phosphatase (ALP), providing the theoretical basis and experimental data for the Twinlight laser-assisted treatment of peri-implantitis. Statistical analyses were performed using a Student's t test with SPSS 17.0 software.ResultsThrough observation using SEM, the cell densities of the L + E + N, L + E, and L + N groups were similar, but cell bodies in the L + E + N group were fuller and each had more than two pseudopodia. The expression level of IL-6 mRNA in the L, L + N, L + E, and L + E + N groups was higher than in group C (P < 0.05), and the expression level of IL-8 mRNA in the L + E + N group was significantly lower than in group L (P < 0.0001). On day 7, the expression level of ALP mRNA in the L, L + N, L + E, and L + E + N groups was lower than in group C (P < 0.05). On day 14, there was no significant difference in the expression level of ALP mRNA among the L + N, L + E + N, and C groups (P > 0.05). On day 7, the expression level of RUNX2 mRNA in the L + E + N group was higher than in group L (P < 0.001). On day 14, the expression level of RUNX2 mRNA in the L + E + N group was higher than in group L (P < 0.01).ConclusionTwinlight laser treatment promoted cell proliferation, inhibited the expression of inflammatory cytokines, and effectively enhanced the osteogenic differentiation of cells on a titanium surface.

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway

BackgroundBone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms.MethodshDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria.ResultsThe calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture.ConclusionsThe stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.

Strontium Regulates the Proliferation and Differentiation of Isolated Primary Bovine Chondrocytes via the TGFβ/SMAD Pathway.

The present study evaluated the effects of strontium (Sr) on proliferation and differentiation of chondrocytes isolated from dairy cows, and whether Sr exerts its effects via transforming growth factor β (TGFβ) signaling. The chondrocytes were isolated from patellar cartilage from newborn Holstein bull calves (n = 3, 1 day old, 38.0 ± 2.8 kg, fasting) within 15 min after euthanasia, and treated with different concentrations of Sr (0, 0.1, 1, and 10 μg/ml, as SrCl2·6H2O). After pretreatment with or without activin receptor-like kinase 5 (ALK5) inhibitor (10 μM SB-505124) for 4 h, chondrocytes were incubated with Sr for another 4 h. Overall effects of Sr were evaluated relative to NaCl as the control. In contrast, the 1 μg/ml Sr-treated group served as the control to determine effects of preincubating with SB-505124. Western blot and qRT-PCR were used for measuring expression of proliferation-, differentiation-, and TGFβ1-responsive factors. Data were analyzed using one-way ANOVA in GraphPad Prism 7.0. Incubation with all doses of Sr increased TGFβ1/ALK5-induced SMAD3 phosphorylation, and at 10 μg/ml it inhibited ALK1-induced SMAD1/5/9 phosphorylation. Expression of mRNA and protein of the proliferation-responsive factors type Ⅱ Collagen α1 (COL2A1) and aggrecan (ACAN) was induced by Sr at 1 μg/ml. In contrast, Sr at 10 μg/ml inhibited the expression of differentiation-responsive factors type Ⅹ Collagen α1 (COL10A1) and secreted phosphoprotein 1 (SPP1), and at 1 μg/ml it had the same effect on alkaline phosphatase (ALPL) mRNA and protein levels. Cells were stained with PI/RNase Staining buffer to assess cell cycle activity using flow-cytometry. Incubation with Sr at 1 and 10 μg/ml induced an increase in the number of cells in the S-phase, leading to an increase in the proliferation index. Incubation with SB-505124 inhibited phosphorylation of SMAD3. Abundance of ACAN and COL2A1 mRNA and protein was lower when cells were pre-incubated with SB-505124. Overall, data indicated that Sr promotes proliferation and inhibits differentiation of primary chondrocytes by directing TGFβ1 signaling towards SMAD3 phosphorylation rather than SMAD1/5/9 phosphorylation. Whether these effects occur in vivo remains to be determined and could impact future application of Sr as an experimental tool in livestock.

Protective Effect of Iron Oxide Nanoparticles on Periodontal Injury in Rats by Inhibiting Collagenase-1 and Alkaline Phosphatase Expression

This study intends to assess whether iron oxide nanoparticles affect periodontal injury and collagenase-1 (COL-1), and alkaline phosphatase (ALP) in rats. In this study, the ALP activity and Col-1 concentration in rats with periodontal injury were determined.We detected the periodontal histopathological changes and expression of periodontal pocket depth (PD) and attachment loss (AL) by Hematoxylin and eosin (HE) staining.We also detected Col-1 and ALP proteins in periodontal tissues by Western blot. Real-time reverse transcription-polymerase chain reaction (RT-PCR) detected Col-1 and ALP mRNA level in periodontal tissues of rats in each group, while ALP activity and Col-1 concentration in gingival crevicular fluid in model group increased compared to sham group (P < 0.05). After intervention by iron oxide nanoparticles, ALP activity and Col-1 concentration in the gingival crevicular fluid of model rats decreased greatly (P < 0.05). The gingival atrophy was more serious in model group, and many inflammatory cells infiltrated into the tissue and destroyed the alveolar tissue. Meanwhile, the periodontal tissue from rats in intervention group was greatly improved, and the degree of alveolar bone destruction was also significantly reduced, while the PD and AL periodontal indexes were significantly inhibited (P < 0.05). The protein and relative expression showed that the protein and mRNA expressions of ALP and Col-1 in periodontal tissue from model group were lower than those in sham group (P < 0.05). After intervention by iron oxide nanoparticles, the protein and mRNA expressions of ALP and Col-1 in the periodontal tissues in intervention group increased (P < 0.05). Iron oxide nanoparticles can thus inhibit the expression of ALP and COL-1 in periodontal injury rats, and improve the periodontal injury tissue.

BMP-2 and asporin expression regulate 5-aza-dC-mediated osteoblast/cementoblast differentiation of periodontal dental ligament mesenchymal progenitor cells

Periodontal dental ligament (PDL) is composed of heterogeneous population of mesenchymal progenitor cells. The mechanisms that regulate the differentiation of these cells towards osteoblast/cementoblast phenotype are not fully understood. Some studies have demonstrated that is possible to change the pattern of cell differentiation via epigenetic mechanisms. The proposal of this study was to investigate whether 5-aza-2'-deoxycytidine (5-aza-dC) treatment would stimulate the osteoblast/cementoblast differentiation of periodontal ligament mesenchymal progenitor cells (PDL-CD105+ enriched cells), characterized as low osteoblast potential, through bone morphogenetic protein-2 (BMP-2) modulation. PDL-CD105+ cells from a single donor were cloned and characterized in two populations as high osteoblast/cementoblast potential (HOP) and low osteoblast/cementoblast potential (LOP) by mineralization in vitro and expression of osteogenic gene markers, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein 2 (BMP-2) and asporin (ASPN). Next, two LOP clones (L1 and L2) were pretreated with 5-aza-dC (10 μM) for 48 h, cultured under osteogenic condition and evaluated for mineralized matrix in vitro, transcription modulation of osteogenic gene markers, methylated and hydroxymethylated DNA levels of BMP-2 and ASPN and intracellular/extracellular expression of BMP-2 protein. LOP clones showed high expression of ASPN transcripts associated with low mRNA levels of BMP-2, RUNX2, ALP, and OCN. 5-aza-dC treatment raised hydroxymethylated DNA levels of BMP-2 and increased the expression of BMP-2 transcripts in both LOP clones. However, BMP-2 protein (intracellular and secreted forms) was detected only in L1 cell clones, in which it was observed an increased expression of osteoblast/cementoblast markers (RUNX2, ALP, OCN) associated with higher mineralization in vitro. In L2 cell clones, 5-aza-dC increased gene expression of ASPN, with no great change in for osteoblast/cementoblast differentiation potential. These data show that 5-aza-dC improves osteoblast/cementoblast differentiation of PDL-CD105+ cells via BMP-2 secretion, and this effect depends on low levels of ASPN expression.

Engineer a pre-metastatic niched microenvironment to attract breast cancer cells by utilizing a 3D printed polycaprolactone/nano-hydroxyapatite osteogenic scaffold - An in vitro model system for proof of concept.

Breast cancer bone metastasis is not a random process. It is affected by the local microenvironment which determines the propensity of cancer cells to invade and colonize into the secondary sites. This microenvironment is termed a pre-metastatic niche. With the flexibility to incorporate different biofactors, tissue-engineering scaffolds provide an advantageous environment to promote "designed" osteogenesis that may mimic the bony pre-metastatic niche. In the current study, designed polycaprolactone (PCL) scaffolds enriched with nano-hydroxyapatite (nHA) were fabricated through three-dimensional (3D) printing. Subsequently, human mesenchymal stem cells (hMSCs) were seeded onto PCL-nHA scaffolds for osteogenic differentiation to establish the pre-metastatic niched microenvironment. Furthermore, transwell migration assay was used to investigate recruitment of MDA-MB-231, MCF-7, and MDA-MB-453 breast cancer cells to the osseous PCL-nHA scaffolds. Our results showed that the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN) of hMSCs on the PCL-nHA scaffolds were dramatically increased compared those with the PCL scaffolds (control) at day 7, 14, and 28. Meanwhile, the migration analysis showed that the higher maturation of osteogenesis and bone metabolism collectively contributed to the creation of a more favorable niched site for the cancerous invasion. Moreover, one of the hypothesized key mediators for the promoted migration, CXCL12, was confirmed using an assay of antagonist LIT-927. This early study demonstrated that a designed tissue engineering scaffold can be utilized to create a bone-mimicking environment that serves as a novel platform to recapitulate the pre-metastatic niche and help interrogate the scheme of bone metastasis by breast cancer.

αCGRP Regulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Through ERK1/2 and p38 MAPK Signaling Pathways

As a typical neuropeptide richly distributed in central and peripheral nervous systems, α-calcitonin-gene-related peptide (αCGRP) has recently been found to play a crucial role in bone development and metabolism, but the mechanisms involved are not fully uncovered. Here, this study aimed to investigate the effects and underlying molecular mechanisms of αCGRP in regulating the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Using microarray technology, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses revealed that osteogenic properties of BMSCs were facilitated and mitogen-activated protein kinase (MAPK) signaling pathway was upregulated by αCGRP in this process. Through western blot assay, we proved that αCGRP led to an increased phosphorylation level of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 MAPK signaling cascades in a time-dependent manner. And αCGRP could promote differentiative capacity of BMSCs, showing upregulated mRNA and protein expression level of alkaline phosphatase (Alp), collagen type 1 (Col-1), osteopontin (Opn), and runt-related transcription factor 2 (Runx2), as well as increased ALP activity and calcified nodules. The addition of ERK1/2 or p38 MAPK inhibitor—U0126 or SB203580, resulted in an impaired osteogenic differentiation of BMSCs. Besides, inactivation of this signal transduction had negative impacts on proliferative activity and apoptotic process of αCGRP-mediated BMSCs. Our findings demonstrated that MAPK signaling pathway, at least in part, was responsible for the enhanced BMSCs’ osteogenesis induced by αCGRP, which might offer us promising strategies for bone-related disorders.

Fluid shear stress and endothelial cells synergistically promote osteogenesis of mesenchymal stem cells via integrin β1-FAK-ERK1/2 pathway.

Prevascularization and mechanical stimulation have been reported as effective methods for the construction of functional bone tissue. However, their combined effects on osteogenic differentiation and its mechanism remain to be explored. Here, the effects of fluid shear stress (FSS) on osteogenic differentiation of rat bone-marrow-derived mesenchymal stem cells (BMSCs) when cocultured with human umbilical vein endothelial cells (HUVECs) were investigated, and underlying signaling mechanisms were further explored. FSS stimulation for 1–4 h/day increased alkaline phosphatase (ALP) activity and calcium deposition in coculture systems and promoted the proliferation of cocultured cells. FSS stimulation for 2 h/day was selected as the optimized protocol according to osteogenesis in the coculture. In this situation, the mRNA levels of ALP, runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and protein levels of OCN and osteopontin (OPN) in BMSCs were upregulated. Furthermore, FSS and coculture with HUVECs synergistically increased integrin β1 expression in BMSCs and further activated focal adhesion kinases (FAKs) and downstream extracellular signal-related kinase (ERK), leading to the enhancement of Runx2 expression. Blocking the phosphorylation of FAK abrogated FSS-induced ERK phosphorylation and inhibited osteogenesis of cocultured BMSCs. These results revealed that FSS and coculture with HUVECs synergistically promotes the osteogenesis of BMSCs, which was mediated by the integrin β1-FAK-ERK signaling pathway.

Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 regulates the proliferation and osteogenic differentiation of human periodontal ligament stem cells by targeting miR-24-3p.

This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs). The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment. The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (P<0.05). The down-regulation of lncRNA KCNQ1OT1 inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). LncRNA KCNQ1OT1 targeted and regulated miR-24-3p. The overexpression of miR-24-3p inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Inhibition of miR-24-3p reversed the effect of the down-regulation of lncRNA KCNQ1OT1 on cell proliferation and mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.

Anti-Osteoporosis Effect of Heme on Osteoblast Pre-Cells and Rat Model

Objectives: Osteoporosis is a metabolic bone disease caused by various factors. As a prosthetic group of haemoglobin, heme can promote the formation of blood vessels and the regeneration of related cells by increasing the proliferation level of endothelial cells and reticulocytes. This study observed the anti-osteoporosis effect of heme on preosteoblast (POB) cells and rat models. Methods: Heme mimics were transfected into POB cells of patients in stable culture, and the effect of heme transfection on the activity of POB cells was detected via the cell scratch test and cell migration assay. 45 SD female rats were randomly divided into group A, the rats were only treated with surgery (n = 15); group B, the rats were given 30 mg/kg of heme chloride by tail vein injection on the 1st, 7th, and 14th days after surgery (n = 15); group C, rats were injected with the same amount of saline in the tail vein (n = 15). The mRNA and protein expressions of alkaline phosphatase (ALP), bone-specific alkaline phosphatase (B-ALP), and type I collagen C-terminal peptide (CTX) in rats serum were detected via western blot and real-time PCR (real-time polymerase chain reaction). The expressions of IL-6 and TNF-α in rats serum were detected via Elisa. The pathological changes in the morphology of distal femur were observed by Hematoxylin-eosin staining. Results: After transfected with heme mimics, the proliferation, migration and invasion ability of the patient’s cells increased significantly, and the apoptosis rate of the cells decreased significantly. The relative expression levels of ALP and CTX mRNA and protein in serum of osteoporosis patients and rat significantly decreased 24 hours after transfection of heme mimics (p &lt; 0.05), while B-ALP significantly increased. The expression of IL-6 and TNF-α in the rat model of osteoporosis group significantly increased (p &lt; 0.05), but after transfection with heme mimic, the expression of IL-6 and TNF-α in the rat reduced significantly. HE showed that, after transfection, the femoral trabeculae were substantially broader and thicker, and the number also increased significantly. The number of trabecular fractures and fat cells was decreased, the trabecular bones were smoother and close to the control group. Conclusion: Heme can significantly promote the proliferation, migration and invasion of POB cells in the body, and also can achieve the anti-osteoporosis effect by adjusting the expression of ALP, B-ALP, and CTX, IL-6 and TNF-α in osteoporosis model of rat.

Decellularized matrix could affect the proliferation and differentiation of periodontal ligament stem cells in vitro.

Recently, decellularized matrix (DCM) is considered as a new biomaterial for tissue regeneration. To explore the possible application of DCM in periodontal regeneration, the effect of DCM from three different cells on the proliferation and differentiation of human periodontal ligament stem cells (PDLSCs) was investigated. DCM derived from human periodontal ligament cells (PDLCs), dental pulp cells (DPCs), and gingival fibroblasts (GFs) were fabricated using Triton X-100/NH4 OH combined with DNase I. Allogeneic PDLSCs were cultured on PDLC-DCM, DPC-DCM, and GF-DCM, respectively. The proliferative capacity of PDLSCs was evaluated by PicoGreen assay kit. The expression of alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), osteocalcin (OCN), collagen I (COL1), periostin (POSTN), and cementum protein 1 (CEMP1) were detected by qRT-PCR and western blotting. PDLC-DCM, DPC-DCM, and GF-DCM had similar and integrated networks of extracellular matrix, as well as significantly decreased DNA content. Compared with control group in which PDLSCs were directly seeded in culture plates, PDLC-DCM, DPC-DCM, and GF-DCM promoted the proliferation of re-seeded PDLSCs. Additionally, PDLSCs on DCM exhibited higher mRNA and protein expression levels of ALP, RUNX2, OCN, and COL1. The expression of POSTN in PDLC-DCM group was significantly higher than control group at both mRNA and protein levels. PDLC-DCM, DPC-DCM, and GF-DCM could enhance the proliferation of PDLSCs. PDLC-DCM facilitated osteogenic differentiation and periodontal ligament differentiation of PDLSCs, while DPC-DCM and GF-DCM promoted osteogenic differentiation.