Expression Of Osteogenic Marker Genes Research Articles

Effects of Substitution Ratios of Zinc-Substituted Hydroxyapatite on Adsorption and Desorption Behaviors of Bone Morphogenetic Protein-2.

Understanding interactions between bone morphogenetic proteins (BMPs) and biomaterials is of great significance in preserving the structure and bioactivity of BMPs when utilized in clinical applications. Currently, bone morphogenetic protein-2 (BMP-2) is one of the most important growth factors in bone tissue engineering; however, atomistic interactions between BMP-2 and zinc-substituted hydroxyapatite (Zn-HAP, commonly used in artificial bone implants) have not been well clarified until now. Thus, in this work, the interaction energies, binding/debinding states, and molecular structures of BMP-2 upon a series of Zn-HAP surfaces (Zn-HAPs, 1 at%, 2.5 at%, 5 at%, and 10 at% substitution) were investigated by hybrid molecular dynamics (MD) and steered molecular dynamics (SMD) simulations. Meanwhile, cellular studies including alkaline phosphatase (ALP) activity and reverse transcription-polymerase chain reaction (RT-PCR) assay were performed to verify the theoretical modeling findings. It was found that, compared to pure HAP, Zn-HAPs exhibited a higher binding affinity of BMP-2 at the adsorption process; meanwhile, the detachment of BMP-2 upon Zn-HAPs was more difficult at the desorption process. In addition, molecular structures of BMP-2 could be well stabilized upon Zn-HAPs, especially for Zn10-HAP (with a 10 at% substitution), which showed both the higher stability of cystine-knots and less change in the secondary structures of BMP-2 than those upon HAP. Cellular studies confirmed that higher ALP activity and osteogenic marker gene expression were achieved upon BMP-2/Zn-HAPs than those upon BMP-2/HAP. These findings verified that Zn-HAPs favor the adsorption of BMP-2 and leverage the bioactivity of BMP-2. Together, this work clarified the interaction mechanisms between BMP-2 and Zn-HAPs at the atom level, which could provide new molecular-level insights into the design of BMP-2-loaded biomaterials for bone tissue engineering.

Activation of the pattern recognition receptor NOD1 in periodontitis impairs the osteogenic capacity of human periodontal ligament stem cells via p38/MAPK signalling.

Nucleotide oligomerization domain receptor 1 (NOD1) mediates host recognition of pathogenic bacteria in periodontium. However, the specific role of NOD1 in regulating osteogenesis is unclear. Therefore, this study focused on the activation status of NOD1 in periodontitis and its effect on the osteogenic capacity of human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanism. Histological staining and Western blot were utilized to assess NOD1 expression in the periodontium of people with or without periodontitis. HPDLSCs were cultured under NOD1 agonist or antagonist treatment. Q-PCR and Western blot were employed to assess the expression of osteogenic marker genes and proteins. Alizarin red staining and alkaline phosphatase staining were used to determine the osteogenic capability of hPDLSCs. The activation of downstream signalling was determined and specific inhibitors were utilized to confirm the signalling pathway in NOD1-regulated osteogenesis. NOD1 expression is significantly elevated in periodontitis. With NOD1 activated by particular agonist tri-DAP, the osteogenic potential of hPDLSCs was impaired. NOD1 antagonist co-incubation partially restored the decreased osteogenesis in hPDLSCs. P38/MAPK was phosphorylated in tri-DAP-induced NOD1 activation. The inhibitor of p38 rescued the suppression of osteogenesis induced by tri-DAP in hPDLSCs. Our study revealed the expression status of NOD1 in periodontitis. Its activation greatly decreased the osteogenic capacity of hPDLSCs which was mediated by the phosphorylation of p38 downstream signalling.

Use of collagenase to isolate adipose tissue-derived stem cells – substantial manipulation or not?

Background: Collagenase is commonly used to isolate the stromal vascular fraction (SVF) or adipose tissue-derived stem cells (ADSCs) from human adipose tissue. Enzymatic breakdowns may be a substantial manipulation according to the classifications of medical regulatory authorities. This study investigates the possible effects of human adipose tissue dissociation with collagenase on in vitro function and behavior of ADSCs. Methods and results: Adipose tissue from nine donors was divided into two equal fractions. SVF was then isolated either mechanically or with collagenase, respectively. The resulting cells were analyzed for their surface markers directly after isolation and at passage five. Proliferation, tri-lineage differentiation, and secretome markers were measured after passage four. Using collagenase compared to mechanical isolation did not alter the expression of typical surface markers of ADSCs. ADSCs isolated with collagenase showed a significantly shorter population doubling time (p < 0.001), a significantly higher mean specific GPDH-activity, a stronger intensity in perilipin staining (p = 0.005), and a significantly higher extracellular calcium deposition (p = 0.006) than mechanically isolated ADSCs. The expression of adipogenic and osteogenic marker genes was not different in mechanically versus enzymatically isolated ADSCs. There were no significant differences in proteoglcyan production (p > 0.05) and the concentration of type 2 collagen. Except for an increased CCL2 concentration in mechanically isolated ASDCs (p = 0.01), there were no significant differences in the concentration of secreted proteins between both isolation methods. Conclusions: The use of collagenase does not substantially impair central in vitro characteristics and functions of human adipose tissue-derived stem cells.

Zingerone stimulates osteoblast differentiation by increasing Smad1/5/9-mediated HO-1 expression in MC3T3-E1 cells and primary mouse calvarial cells.

Zingerone is a non-volatile compound found mainly in dried ginger. Zingerone increases the expression of osteogenic markers and has antioxidant effects. A previous study showed that zingerone accelerated osteoblast differentiation by suppressing the expression of Smad7, a member of the inhibitory Smad (I-Smad) family. However, it is not known if zingerone can induce osteoblast differentiation by regulating Smad1/5/9, a member of the receptor-regulated Smad (R-Smad) family. In addition, osteoblast differentiation induced by Smad1/5/9 mediated increases in the expression of heme oxygenase 1 (HO-1) has not been reported. This study investigated the effects of zingerone on osteoblast differentiation and confirmed the relationship between Smad1/5/9 and HO-1. Zingerone increased the expression of osteogenic genes including runt-related transcription factor 2 (Runx2), distal-less homeobox (Dlx5) and osteocalcin (OC) and also promoted Smad1/5/9 phosphorylation. Interestingly, HO-1 expression was also elevated by zingerone, and an inhibitor of HO-1 (Sn[IV] protoporphyrin IX dichloride [SnPP]) suppressed the zingerone-induced increase in HO-1 expression and expression of osteogenic marker genes such as Dlx5, Runx2 and OC. Protein phosphatase 2A Cα (PP2A Cα, an inhibitor of Smad1/5/9) suppressed the zingerone-induced increase in HO-1 expression and expression of osteogenic marker genes. The zingerone-induced increase in HO-1 luciferase activity was suppressed by PP2A Cα. Taken together; our data demonstrate that zingerone promotes osteoblast differentiation by increasing Smad1/5/9 mediated HO-1 expression.

Silicocarnotite: Novel Silicate Bioceramic With Osteogenic Property for Repairing Rat Cranial Critical-Sized Bone Defects

Critical-sized bone defects are an intractable orthopedic disease which often fails to regenerate spontaneously and requires additional intervention. Current therapies, including autografts and allografts, are not always satisfactory. Herein, the novel calcium phosphate bioceramic-containing silicon (CPS) with a carnotite structure was synthesized. In the present study, CPS was prepared for investigating the biocompatibility and bioactivity in vitro and in vivo in comparison to hydroxyapatite (HA). Our results showed that CPS bioceramics had favorable biocompatibility and rBMSCs could adhere on the surface well in vitro. Moreover, CPS could promote osteogenic differentiation of rBMSCs and the expression of osteogenic differentiation marker genes, including ALP, Runx-2, BSP, OCN, and OPN. In vivo, the results of micro-CT, histomorphometry, and histology analyses showed that CPS significantly enhanced critical-sized calvarial defects healing compared with HA. Overall, the present study demonstrated that CPS bioceramics had satisfactory bioactivities and osteogenic capacities, which could be a potential option for reconstructing critical-sized bone defects.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Bone Regeneration In vitro and In vivo.

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I. Two critical size bone defect models were used to analyze bone regeneration. BSP incorporated in collagen type I increased bone regeneration only marginally at one concentration in a calvarial defect model. To induce the mechanical stability, three-dimensional printing was used to produce a stable porous cylinder of polylactide. The cylinder was filled with collagen type I and immobilized BSP and implanted into a femoral defect of critical size in rats. This hybrid material was able to significantly induce bone regeneration. Our study clearly shows the osteogenic effect of BSP when combined with collagen type I as carrier and thereby offers various approaches and options for its use as bioactive molecule in bone substitute materials.

Excess vitamin a might contribute to submucous clefting by inhibiting WNT-mediated bone formation.

Cleft lip and/or palate (CLP) is a common craniofacial birth defect caused by genetic as well as environmental factors. The phenotypic spectrum of CLP also includes submucous clefts with a defect in the palatal bone. To elucidate the contribution of vitamin A, we evaluated the effects of the vitamin A metabolite all-trans retinoic acid (ATRA) on the osteogenic differentiation and mineralization of mouse embryonic palatal mesenchymal cells (MEPM). MEPM cells were isolated from the prefusion palates of E13 mouse embryos from three different litters. MEPM cells were cultured with and without 0.5μM ATRA in osteogenic medium. Differentiation was analysed by the expression of osteogenic marker genes and alkaline phosphatase (ALP) activity after 1, 2, and 7 days. The expression of Wnt marker genes was also analysed. Mineralization was assessed by alizarin red staining after 7, 14, 21, and 28 days. The bone marker genes Sp7, Runx2, Alpl, and Col1a1 were inhibited 10% ± 2%, 59% ± 7%, 79% ± 12% and 57% ± 20% (P < .05) at day 7. ALP activity was inhibited at days 1 and 7 by 35 ± 0% (P < .05) and 23 ± 6% (P < .001). ATRA also inhibited mineralization at 3 and 4 weeks. Finally, expression of the universal Wnt marker gene Axin2 was strongly reduced, by 31 ± 18% (P < .001), at day 7. Our data indicate that ATRA (vitamin A) inhibits bone formation by reducing Wnt signalling. This might contribute to the molecular aetiology of submucous clefting.

EGR1 plays an important role in BMP9-mediated osteoblast differentiation by promoting SMAD1/5 phosphorylation.

Bone morphogenetic proteins (BMPs) are essential regulators of skeletal homeostasis, and BMP9 is the most potently osteogenic among them. Here, we found that BMP9 and BMP2 rapidly induced early growth response 1 (EGR1) protein expression in osteoblasts through MEK/ERK pathway-dependent transcriptional activation. Knock-down of EGR1 using siRNA significantly inhibited BMP9-induced matrix mineralization and osteogenic marker gene expression in osteoblasts. Knock-down of EGR1 significantly reduced SMAD1/5 phosphorylation and inhibited the expression of their transcriptional targets in osteoblasts stimulated by BMP9. In contrast, forced EGR1 overexpression in osteoblasts enhanced BMP9-mediated osteoblast differentiation and SMAD1/5 phosphorylation. An intracellular association between EGR1 and SMAD1/5 was identified using immunoprecipitation assays. These results indicated that EGR1 plays an important role in BMP9-stimulated osteoblast differentiation by enhancing SMAD1/5 phosphorylation.

Bone cell-specific deletion of thyroid hormone transporter Mct8 distinctly regulates bone volume in young versus adult male mice.

Thyroid hormones are critical regulators of bone metabolism. Their cellular import is guided through transporter proteins, including the monocarboxylate transporter 8 (MCT8). Conditional Mct8 knockout in osteoblast and osteoclast precursors leads to trabecular bone gain in 12-week-old male mice. Given that thyroid hormones regulate both skeletal development and bone maintenance, we investigated the effect of bone cell-specific Mct8 deletion in 6-week-old (young) and 24-week-old (adult) male mice. Mct8 ablation in osteoclast precursors led to trabecular bone gain at the spine in 6-week-old animals compared to age-matched controls, whereas adult animals displayed a shift towards trabecular bone loss in both femur and vertebra. Mct8 deficiency in osteoprogenitors increased osteoblast numbers and trabecular bone mass at the spine of young mice, without skeletal differences between adult knockout mice and littermate controls. In contrast, young mice lacking Mct8 in late osteoblasts/osteocytes exhibited lower trabecular bone volume at the spine and femur compared to respective controls, but no differences were detected at 24weeks of age. In vitro studies of osteoblasts with Dmp1-Cre promotor driven Mct8 deletion showed no significant alterations of osteogenic marker gene expression and mineralization capacity suggesting that MCT8 is not crucial for osteoblast maturation. Overall, we observed mild effects with conditional Mct8 knockout on bone microarchitecture and bone turnover especially during growth implying a secondary role for MCT8 as a thyroid hormone transporter in bone.

Naringin Promotes Osteogenic/Odontogenic Differentiation of Dental Pulp Stem Cells via Wnt/β-Catenin.

Purpose This investigation intended to unravel the effect and mechanism of naringin on the proliferation and osteogenic differentiation of human dental pulp stem cells (hDPSCs). Methods hDPSCs were induced to differentiate, and the degree of cell differentiation was observed by alizarin red staining, Oil Red O staining, and Alcian blue staining. hDPSCs were treated with 0, 20, 40, and 80 μmol/L naringin for 48 h, respectively. The proliferation rate and chemotaxis of the cells were measured by MTT and transwell assay, alkaline phosphatase (ALP) activity and osteogenic differentiation degree by ALP staining and alizarin red staining, and gene expression of osteogenic markers by qRT-PCR. Additionally, western blot was performed to test the levels of Wnt/β-catenin signaling-related proteins in hDPSCs. Results The isolated hDPSCs with spindle-shaped morphology had good differentiation capability. Further experiments confirmed naringin-caused increases in the proliferation rate and migration ability of hDPSCs. In addition, compared with the control group, naringin-treated cells had strong ALP activity and ossification levels and higher expression of Runx2, OPN, DSPP, and DMP1. The western blot results showed that naringin significantly activated Wnt/β-catenin signaling in hDPSCs. Conclusion Taken together, naringin enhances the proliferation, migration, and osteogenesis of hDPSCs through stimulating Wnt/β-catenin signaling pathway.

In vitro and in ovo impact of the ionic dissolution products of boron-doped bioactive silicate glasses on cell viability, osteogenesis and angiogenesis

Due to the pivotal role of angiogenesis in bone regeneration, the angiogenic properties of biomaterials are of high importance since they directly correlate with the biomaterials’ osteogenic potential via ‘angiogenic-osteogenic coupling’ mechanisms. The impact of bioactive glasses (BGs) on vascularization can be tailored by incorporation of biologically active ions such as boron (B). Based on the ICIE16-BG composition (in mol%: 49.5 SiO2, 36.3 CaO, 6.6 Na2O, 1.1 P2O5, 6.6 K2O), three B-doped BGs have been developed (compositions in mol%: 46.5/45.5/41.5 SiO2, 36.3 CaO, 6.6 Na2O, 1.1 P2O5, 6.6 K2O, 3/4/8 B2O3). The influence of B-doping on the viability, cellular osteogenic differentiation and expression of osteogenic and angiogenic marker genes of bone marrow-derived mesenchymal stromal cells (BMSCs) was analyzed by cultivating BMSCs in presence of the BGs’ ionic dissolution products (IDPs). Furthermore, the influence of the IDPs on angiogenesis was evaluated in ovo using a chorioallantoic membrane (CAM) assay. The influence of B-doped BGs on BMSC viability was dose-dependent, with higher B concentrations showing limited negative effects. B-doping led to a slight stimulation of osteogenesis and angiogenesis in vitro. In contrast to that, B-doping significantly enhanced vascularization in ovo, especially in higher concentrations. Differences between the results of the in vitro and in ovo part of this study might be explained via the different importance of vascularization in both settings. The implementation of new experimental models that cover the ‘angiogenic-osteogenic coupling’ mechanisms is highly relevant, for instance via extending the application of the CAM assay from solely angiogenic to angiogenic and osteogenic purposes.

Electrospun Silk Fibroin/kappa-Carrageenan Hybrid Nanofibers with Enhanced Osteogenic Properties for Bone Regeneration Applications.

Simple SummaryBone tissue engineering has recently been considered as a potential alternative approach to treating patients with bone disorders/defects caused by tumors, trauma, and infection. Scaffolds play a crucial role in the field because they can serve as a template that can provide optimal structural and functional support for cells. In this study, we prepared a series of electrospun silk fibroin/kappa-carrageenan nanofibrous membranes with the aim of mimicking bone extracellular matrix structure and composition and improving the biological properties of silk-fibroin-based nanofibers. Our research found that a combinational approach blending kappa-carrageenan and silk fibroin could enhance the biological properties of the nanostructured scaffold. kappa-carrageenan could also enhance the osteogenic potential and bioactivity properties of silk fibroin nanofibers, while genipin crosslinking preserved the mechanical strength of hybrid nanofibrous mats, indicating that the electrospun hybrid scaffolds could be a potential candidate for bone regeneration applications.In this study, a novel nanofibrous hybrid scaffold based on silk fibroin (SF) and different weight ratios of kappa-carrageenan (k-CG) (1, 3, and 5 mg of k-CG in 1 mL of 12 wt% SF solution) was prepared using electrospinning and genipin (GP) as a crosslinker. The presence of k-CG in SF nanofibers was analyzed and confirmed using Fourier transform infrared spectroscopy (FTIR). In addition, X-ray diffraction (XRD) analysis confirmed that GP could cause SF conformation to shift from random coils or α-helices to β-sheets and thereby facilitate a more crystalline and stable structure. The ultimate tensile strength (UTS) and Young’s modulus of the SF mats were enhanced after crosslinking with GP from 3.91 ± 0.2 MPa to 8.50 ± 0.3 MPa and from 9.17 ± 0.3 MPa to 31.2 ± 1.2 MP, respectively. Notably, while the mean fiber diameter, wettability, and biodegradation rate of the SF nanofibers increased with increasing k-CG content, a decreasing effect was determined in terms of UTS and Young’s modulus. Additionally, better cell viability and proliferation were observed on hybrid scaffolds with the highest k-CG content. Osteogenic differentiation was determined from alkaline phosphatase (ALP) activity and Alizarin Red staining and expression of osteogenic marker genes. To this end, we noticed that k-CG enhanced ALP activity, calcium deposition, and expression of osteogenic genes on the hybrid scaffolds. Overall, hybridization of SF and k-CG can introduce a promising scaffold for bone regeneration; however, more biological evaluations are required.

Three-dimensional bioprinting of mesenchymal stem cells using an osteoinductive bioink containing alginate and BMP-2-loaded PLGA nanoparticles for bone tissue engineering.

Hydrogels mimicking the physicochemical properties of the native extracellular matrix have attracted great attention as bioinks for three-dimensional (3D) bioprinting in tissue engineering applications. Alginate is a widely used bioink with beneficial properties of fast gelation and biocompatibility; however, bioprinting using alginate-based bioinks has several limitations, such as poor printability, structural instability, and limited biological activities. To address these issues, we formulated various bioinks using bone morphogenetic protein-2 (BMP-2)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles and alginate for mesenchymal stem cell (MSC) printing and induction of osteogenic differentiation. Incorporation of PLGA nanoparticles into alginate could enhance the mechanical properties and printability of the bioink. In particular, Alg/NPN30 (30mg/mL PLGA nanoparticles and 3% w/v alginate) was most suitable for 3D printing with respect to printability and stability. BMP-2-loaded PLGA nanoparticles (NPBMP-2) displayed sustained in vitro release of BMP-2 for up to two weeks. Further in vitro studies indicated that bioinks composed of alginate and NPBMP-2 significantly induced osteogenesis of the MSCs compared with other controls, evidenced by enhanced calcium deposition, alkaline phosphatase activity, and gene expression of osteogenic markers. Our novel bioink consisting of widely used biocompatible components displays good printability, stability, and osteogenic inductivity, and holds strong potential for cell printing and bone tissue engineering applications.

Facilitative role of circPVT1 in osteogenic differentiation potentials of bone marrow mesenchymal stem cells from patients with osteoporosis through the miR-30d-5p/ITGB3 axis

ObjectiveThe critical role of circular RNAs (circRNAs) in osteoporosis (OP) has been highlighted. We tried to explore the role of circPVT1 in OP in relation to microRNA-30d-5p (miR-30d-5p) and ITGB3. MethodsAfter bone marrow collection, bone marrow mesenchymal stem cells (BMSCs) were isolated and identified. Then, Pearson coefficient was used to analyze the correlation among circPVT1, miR-30d-5p and ITGB3, and the binding sites were predicted and verified. Gain- and loss-of function assays in circPVT1, miR-30d-5p and ITGB3 were performed to analyze their effect on osteogenic differentiation of BMSCs. ResultsThe osteogenic differentiation of BMSCs from OP patients was significantly decreased, and reduced circPVT1 expression was found in the BMSCs from OP patients. Overexpression of circPVT1 stimulated the formation of calcified nodules, increased alkaline phosphatase activity, and enhanced the expression of osteogenic marker genes in the BMSCs from OP patients. Additionally, circPVT1 expression was negatively correlated with miR-30d-5p, and miR-30d-5p was negatively correlated with ITGB3 in OP patients. Mechanically, circPVT1 regulated the osteogenic differentiation potential of BMSCs by relieving the inhibition of miR-30d-5p on ITGB3 through the competitive endogenous RNA mechanism. ConclusionOur study highlighted a circPVT1/miR-30d-5p/ITGB3 axis in regulating osteogenic differentiation potential of BMSCs from OP patients.

Metformin Promotes Differentiation and Attenuates H2O2-Induced Oxidative Damage of Osteoblasts via the PI3K/AKT/Nrf2/HO-1 Pathway.

At present, the drug treatment of osteoporosis is mostly focused on inhibiting osteoclastogenesis, which has relatively poor effects. Metformin is a drug that can potentially promote osteogenic differentiation and improve bone mass in postmenopausal women. We aimed to detect the molecular mechanism underlying the osteogenic effect of metformin. Our study indicated that metformin obviously increased the Alkaline phosphatase activity and expression of osteogenic marker genes at the mRNA and protein levels. The PI3K/AKT signaling pathway was revealed to play an essential role in the metformin-induced osteogenic process, as shown by RNA sequencing. We added LY294002 to inhibit the PI3K/AKT pathway, and the results indicated that the osteogenic effect of metformin was also blocked. Additionally, the sequencing data also indicated oxidation-reduction reaction was involved in the osteogenic process of osteoblasts. We used H2O2 to mimic the oxidative damage of osteoblasts, but metformin could attenuate it. Antioxidative Nrf2/HO-1 pathway, regarded as the downstream of PI3K/AKT pathway, was modulated by metformin in the protective process. We also revealed that metformin could improve bone mass and oxidative level of OVX mice. In conclusion, our study revealed that metformin promoted osteogenic differentiation and H2O2-induced oxidative damage of osteoblasts via the PI3K/AKT/Nrf2/HO-1 pathway.

Clinical relevance of circulating angiogenic cells in patients with ischemic stroke

BackgroundEndothelial progenitor cells (EPCs) are circulating angiogenic cells with endothelial features associated with risk for stroke. We aimed to delve into their functional characteristics. EPCs were isolated and cultured from Ischemic Stroke (IS) patients and predictors of their variance evaluated.MethodsThis is a single-center observational study evaluating 187 consecutively hospitalized patients with IS. EPCs were isolated from blood samples. The number of circulating angiogenic cells (CACs), colony-forming units (CFU-ECs) and the emergence of late outgrowths endothelial cells (LOECs) were counted. We collected clinical variables and measured the stromal cell-derived factor 1 alpha (SDF1α) serum levels. We also examined the relative telomere length and the expression of osteogenic gene markers in CACs.ResultsCACs counts and CFU-ECs colony numbers were positively correlated (rho = 0.41, p < 0.001, n = 187). We found significant differences according to whether thrombolytic treatment was performed in the distribution of CFU-ECs (odds ratio (OR) = 2.5; 95% confidence interval (CI) 1.01–6.35; p = 0.042) and CACs (OR = 4.45; 95% IC 1.2–15.5; p = 0.012). The main determinants of CACs variation were the number of risks factors, thrombolysis treatment, arterial hypertension, LOECs occurrence, and the vascular endothelial growth factor expression, whereas CFU-ECs variations depended on hemoglobin content and the relative reduction in the National Institutes of Health Stroke Scale (NIHSS) criteria. The main predictors of LOECs appearance were thrombolysis and length of hospital stay.ConclusionsOur study supports the relevance of patient risk factors and treatments in the analysis of the functional properties of EPCs.

A multicellular bioprinted cell construct for vascularized bone tissue regeneration

• A bioprinting strategy using a biocompatible collagen/β-TCP bioink and the hASC/endothelial cells was proposed. • hASC/HUVEC-laden structures with cell-differentiation niches for bone regeneration were proposed. • The bioprinted structure induced significantly greater new bone and blood vessel formation. In the present study, a bioprinted cell-laden construct consisting of collagen, β-tricalcium phosphate, and two different cell types, human adipose stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs), was prepared for bone tissue regeneration in spinal fusion. By using the appropriate printing parameters and bioceramic/cell compositions in the bioink, a 3D cell-laden construct consisting of microscale struts was successfully generated. To evaluate the contribution of the laden stem cells and endothelial cells in promoting angiogenic and osteogenic activities, differentiation of the cells within the bioink was analyzed by measuring gene expression profiles and immunofluorescence imaging. The hASC/HUVEC-laden structure exhibited a strong angiogenic phenotype and upregulated angiogenic factors, as well as enhanced expression of osteogenic gene markers due to the synergistic crosstalk of the two cell types. The construct laden with hASCs/HUVECs was implanted in a mouse model of posterolateral lumbar spinal fusion, and the structure provided significantly higher new bone formation and angiogenesis compared with constructs laden with only hASCs. The in vitro and in vivo findings suggest that biomimetic cell-laden constructs could perform as potential bone tissue regenerating structures to encourage effective spinal fusion.

Fluoride induces hypomethylation of BMP2 and activates osteoblasts through the Wnt/β-catenin signaling pathway

BackgroundSkeletal fluorosis has become a public health issue in recent years as its serious impact on patients’ life expectancy. Bone morphogenetic protein 2 (BMP2) plays a key role in promoting osteogenesis. However, the mechanism of BMP2-Wnt/β-catenin axis in skeletal fluorosis needs further exploration. MethodsThe RT-qPCR and western blot assay were carried out to examine the mRNA and protein levels. Cell viability was measured by MTT assay. A commercial ALP assay kit was used to detect ALP activities. Alizarin Red staining was performed to measure the formation of mineralized nodules. Methylation-specific PCR (MSP) was performed to measure the methylation level of BMP2. ResultsFluoride promoted the expression of osteogenic marker genes (OPN, OCN, OSX and RUNX2) and induced the proliferation and differentiation of MC3T3-E1 cells. Fluoride induced hypomethylation and high expression of BMP2. Furthermore, knockdown of BMP2 reversed the promoting effect of fluoride on osteogenic differentiation of MC3T3-E1. The expression of β-catenin, glycogen synthase kinase 3β (GSK3β), wingless/integrated 3α (Wnt3α), low-density lipoprotein receptor-related protein 5 (LRP5) and dishevelled 1 (Dv1) were increased in osteoblasts treated with fluoride, however, knockdown of BMP2 reversed this phenomenon. Simultaneous knockdown of BMP2 and β-catenin significantly inhibited the differentiation of osteoblasts induced by fluoride. ConclusionFluoride contributed to proliferation and differentiation of osteoblasts through BMP2-Wnt/β-catenin axis, providing a feasible theoretical basis for the treatment of skeletal fluorosis.

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.

A comparative analysis of the osteogenic capacity of osteoblasts from newborn and two-week-old rats

AimTo compare the proliferation and osteogenic differentiation of osteoblasts between newborn rats (1d group) and two-week-old rats (14d group) and to clarify the mechanism underlying these effects. MethodThe endogenous expression of osteogenic marker genes was detected by qPCR, including ALP, OCN, Col1a1, and Runx2. The osteoblasts proliferation was evaluated by EdU assay and Western Blotting [PCNA and Cyclin D1]. ALP activities in osteoblasts were detected using a PNPP kit, ALP staining and qPCR. Mineralized nodule formation and intracellular calcium levels were assessed by Alizarin Red staining and calcium colorimetric assay respectively while OCN, Col1a1 and Runx2 levels in osteoblasts were analyzed by immunostaining. Osteogenesis-associated pathways including Wnt/β-Catenin, Akt/PPAR and Smad were analyzed via Western Blotting. ResultEndogenous ALP, OCN, Col1a1, and Runx2 expression levels were significantly higher in osteoblasts from 14d group than those from 1d group. After treatment with osteogenic induction medium, osteoblast proliferation, ALP activity, mineralized nodule formation, and intracellular calcium levels were markedly increased in osteoblasts from 1d group, with similar results also being observed for the expression of OCN, Col1a1, and Runx2. Wnt3a, β-catenin, p-Akt, p-Smad1/5/8, and p-Smad5 protein levels were also higher in osteoblasts from 1d group relative to those from 14d group, while the expression of PPARγ was lower. ConclusionThe superior osteogenic differentiation capacity in osteoblasts was associated with the higher activation levels of Wnt/β-Catenin, Akt/PPAR and Smad signaling pathways, and the enhanced proliferative activity in osteoblasts from 1d group.