Expression Levels Of Osteogenic Genes Research Articles

Oriented Surface Nanotopography Promotes the Osteogenesis of Mesenchymal Stem Cells

Stem cells could regulate their fate by responding to physical signals from their microenvironment. Recently, a few studies have shown that the spontaneous osteogenic differentiation of mesenchymal stem cells (MSCs) could occur on surfaces with nanoscaled structures (e.g., nanotubes and nanopits), which is essentially useful when designing biomaterial scaffolds for bone tissue engineering applications. However, the osteogenic nanotopography has not been widely investigated and the osteogenic mechanism of surfaces is still not well understood. In this study, oriented nanograting-like surfaces are fabricated by stretching the nanoholed inverse opal films, which are made through a convenient bottom-up approach of the self-assembly of colloidal nanoparticles. The effect of different surface nanotopography on the osteogenic differentiation of rat bone marrow-derived MSCs (rBMSCs) is examined in the absence of osteogenic inducing media and the underlying mechanisms are investigated using immunofluorescence staining and quantitative real-time polymerase chain reaction (PCR) analysis. It is found that rBMSCs on the oriented nanograting-like surfaces show significantly higher expression levels of osteogenic genes and more phosphorylation of Smad1/5, extracellular signal-regulated kinases (ERKs), and p38, confirming the activation of Smad and mitogen-activated protein kinase signaling pathways in the nanotopography-induced osteogenesis. Thus, the oriented nanograting-like surface nanotopography may provide a new strategy for the surface design of biomaterial scaffolds in bone tissue engineering.

Effects of inactivated Enterococcusfaecalis on the proliferation and osteogenic induction of osteoblasts.

The present study aimed to evaluate the effects of Enterococcus faecalis, inactivated by the common intracanal irrigants sodium hypochlorite (NaOCl) and chlorhexidine (CHX), on osteoblasts. E. faecalis was inactivated with 2% CHX or 5.25% NaOCl. Subsequently, the Cell Counting kit-8 assay was used to examine the effects of CHX- and NaOCl-inactivated E. faecalis on MC3T3-E1 osteoblast cell proliferation. Alizarin red staining was used to determine osteoblast mineralization, and osteogenic induction was quantified by determining the optical density of the dye solution. The relative expression levels of osteogenic genes were detected after 1, 4, 7 and 14 days of stimulation with CHX- and NaOCl-inactivated E. faecalis by reverse transcription-quantitative polymerase chain reaction. The results indicated that CHX-inactivated E. faecalis inhibited osteoblast proliferation, whereas NaOCl-inactivated E. faecalis did not suppress cell proliferation. Various concentrations of CHX- and NaOCl-inactivated E. faecalis induced different degrees of osteoblast mineralization. The expression levels of osteocalcin, alkaline phosphatase, runt-related transcription factor 2, osteopontin and osterix were upregulated in cells following stimulation with 107 and 105 colony-forming units/ml E. faecalis inactivated by CHX and NaOCl; the upregulation of these osteogenic genes occurred at various time points. In conclusion, the present study demonstrated that CHX-inactivated E. faecalis exerted more of an effect on osteoblast proliferation compared with NaOCl-inactivated E. faecalis. In addition, CHX- and NaOCl-inactivated E. faecalis was able to induce mineralization and relevant osteogenic gene expression in osteoblast cells.

Fabrication of gelatin methacrylate/nanohydroxyapatite microgel arrays for periodontal tissue regeneration.

IntroductionPeriodontitis is a chronic infectious disease and is the major cause of tooth loss and other oral health issues around the world. Periodontal tissue regeneration has therefore always been the ultimate goal of dentists and researchers. Existing fabrication methods mainly focused on a top–down tissue engineering strategy in which several drawbacks remain, including low throughput and limited diffusion properties resulting from a large sample size. Gelatin methacrylate (GelMA) is a kind of photocrosslinkable and biocompatible hydrogel, with the capacities of enabling cell encapsulation and regeneration of functional tissues. Here, we developed a novel method to fabricate GelMA/nanohydroxylapatite (nHA) microgel arrays using a photocrosslinkable strategy. The viability, proliferation, and osteogenic differentiation and in vivo osteogenesis of human periodontal ligament stem cells (hPDLSCs) encapsulated in microgels were evaluated. The results suggested that such microgels provide great potential for periodontal tissue repair and regeneration.MethodsMicrogel arrays were fabricated by blending different weight ratios of GelMA and nHA. hPDLSCs were encapsulated in GelMA/nHA microgels of various ratios for a systematic evaluation of cell viability, proliferation, and osteogenic differentiation. In vivo osteogenesis in nude mice was also studied.ResultsThe GelMA/nHA microgels exhibited appropriate microarchitecture, mechanical strength, and surface roughness, thus enabling cell adhesion and proliferation. Additionally, the GelMA/nHA microgels (10%/2% w/v) enhanced the osteogenic differentiation of hPDLSCs by elevating the expression levels of osteogenic biomarker genes, such as ALP, BSP, OCN, and RUNX2. In vivo ectopic transplantation results showed that GelMA/nHA microgels (10%/2% w/v) increased mineralized tissue formation with abundant vascularization, compared with the 1%, 3%, and the pure GelMA group.ConclusionThe GelMA/nHA microgels (10%/2% w/v) facilitated hPDLSCs viability, proliferation, and osteogenic differentiation in vitro and further promoted new bone formation in vivo, suggesting that the GelMA/nHA microgels (10%/2% w/v) provide great potential for periodontal tissue regeneration.

Insulin-like growth factor 1 promotes the proliferation and committed differentiation of human dental pulp stem cells through MAPK pathways

ObjectivesInsulin-like growth factor 1 (IGF-1) is a broad-spectrum growth-promoting factor that plays a key role in natural tooth development. Human dental pulp stem cells (hDPSCs) are multipotent and can influence the reparative regeneration of dental pulp and dentin. This study was designed to evaluate the effects of IGF-1 on the proliferation and differentiation of human dental pulp stem cells. MethodsHDPSCs were isolated and purified from human dental pulps. The proliferation and osteo/odontogenic differentiation of hDPSCs treated with 100ng/ml exogenous IGF-1 were subsequently investigated. ResultsMTT assays revealed that IGF-1 enhanced the proliferation of hDPSCs. ALP activity in IGF-1-treated group was obviously enhanced compared to the control group from days 3 to 9. Alizarin red staining revealed that the IGF-1-treated cells contained a greater number of mineralization nodules and had higher calcium concentrations. Moreover, western blot and qRT-PCR analyses demonstrated that the expression levels of several osteogenic genes (e.g., RUNX2, OSX, and OCN) and an odontoblast-specific marker (DSPP) were significantly up-regulated in IGF-1-treated hDPSCs as compared with untreated cells (P<0.01). Interestingly, the expression of phospho-ERK and phospho-p38 were also up-regulated, indicating that the MAPK signaling pathway is activated during the differentiation of hDPSCs. ConclusionsIGF-1 can promote the proliferation and osteo/odontogenic differentiation of hDPSCs by activating MAPK pathways.

Biomimetic coatings enhance tribocorrosion behavior and cell responses of commercially pure titanium surfaces.

Biofunctionalized surfaces for implants are currently receiving much attention in the health care sector. Our aims were (1) to create bioactive Ti-coatings doped with Ca, P, Si, and Ag produced by microarc oxidation (MAO) to improve the surface properties of biomedical implants, (2) to investigate the TiO2 layer stability under wear and corrosion, and (3) to evaluate human mesenchymal stem cells (hMSCs) responses cultured on the modified surfaces. Tribocorrosion and cell experiments were performed following the MAO treatment. Samples were divided as a function of different Ca/P concentrations and treatment duration. Higher Ca concentration produced larger porous and harder coatings compared to the untreated group (p < 0.001), due to the presence of rutile structure. Free potentials experiments showed lower drops (-0.6 V) and higher coating lifetime during sliding for higher Ca concentration, whereas lower concentrations presented similar drops (-0.8 V) compared to an untreated group wherein the drop occurred immediately after the sliding started. MAO-treated surfaces improved the matrix formation and osteogenic gene expression levels of hMSCs. Higher Ca/P ratios and the addition of Ag nanoparticles into the oxide layer presented better surface properties, tribocorrosive behavior, and cell responses. MAO is a promising technique to enhance the biological, chemical, and mechanical properties of dental implant surfaces.

Promotive Effect of Zinc Ions on the Vitality, Migration, and Osteogenic Differentiation of Human Dental Pulp Cells.

Zinc is an essential trace element for proper cellular function and bone formation. However, its exact role in the osteogenic differentiation of human dental pulp cells (hDPCs) has not been fully clarified before. Here, we speculated that zinc may be effective to regulate their growth and osteogenic differentiation properties. To test this hypothesis, different concentrations (1×10-5, 4×10-5, and 8×10-5M) of zinc ions (Zn2+) were added to the basic growth culture medium and osteogenic inductive medium. Cell viability and migration were measured by cell counting kit-8 (CCK-8) and transwell migration assay in the basic growth culture medium, respectively. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expression levels of selective osteogenic differentiation markers and zinc transporters. Alkaline phosphatase (ALP) activity analysis and alizarin red S staining were used to investigate the mineralization of hDPCs. Exposure of hDPCs to Zn2+ stimulated their viability and migration capacity in a dose- and time-dependent manner. RT-qPCR assay revealed elevated expression levels of osteogenic differentiation-related genes and zinc transporters genes in various degrees. ALP activity was also increased with elevated Zn2+ concentrations and extended culture periods, but enhanced matrix nodules formation were observed only in 4×10-5 and 8×10-5M Zn2+ groups. These findings suggest that specific concentrations of Zn2+ could potentiate the vitality, migration, and osteogenic differentiation of hDPCs. We may combine optimum zinc element into pulp capping materials to improve their biological performance.

A bioceramic with enhanced osteogenic properties to regulate the function of osteoblastic and osteocalastic cells for bone tissue regeneration

Bioceramics for regenerative medicine applications should have the ability to promote adhesion, proliferation and differentiation of osteoblast and osteoclast cells. Osteogenic properties of the material are essential for rapid bone regeneration and new bone formation. The aim of this study was to develop a silicate-based ceramic, gehlenite (GLN, Ca2Al2SiO7), and characterise its physiochemical, biocompatibility and osteogenic properties. A pure GLN powder was synthesised by a facile reactive sintering method and compacted to disc-shaped specimens. The sintering behaviour and degradation of the GLN discs in various buffer solutions were fully characterised. The cytotoxicity of GLN was evaluated by direct and indirect methods. In the indirect method, primary human osteoblast cells (HOBs) were exposed to diluted extracts (100, 50, 25, 12.5 and 6.25 mg ml−1) of fine GLN particles in culture medium. The results showed that the extracts did not cause any cytotoxic effect on the HOBs with the number of cells increasing significantly from day 1 to day 7. GLN-supported HOB attachment and proliferation, and significantly enhanced osteogenic gene expression levels (Runx2, osteocalcin, osteopontin and bone sialoprotein) were compared with biphasic calcium phosphate groups (BCP, a mixture of hydroxyapatite (60wt.%) and β-tricalcium phosphate(40wt.%)). We also demonstrated that in addition to supporting HOB attachment and proliferation, GLN promoted the formation of tartrate-acid resistance phosphatase (TRAP) positive multinucleated osteoclastic cells (OCs) derived from mouse bone marrow cells. Results also demonstrated the ability of GLN to support the polarisation of OCs, a prerequisite for their functional resorptive activity which is mainly influenced by the composition and degradability of biomaterials. Overall, the developed GLN is a prospective candidate to be used in bone regeneration applications due its effective osteogenic properties and biocompatibility.

Room temperature housing results in premature cancellous bone loss in growing female mice: implications for the mouse as a preclinical model for age-related bone loss.

Room temperature housing (22°C) results in premature cancellous bone loss in female mice. The bone loss was prevented by housing mice at thermoneutral temperature (32°C). Thermogenesis differs markedly between mice and humans and mild cold stress induced by standard room temperature housing may introduce an unrecognized confounding variable into preclinical studies. Female mice are often used as preclinical models for osteoporosis but, in contrast to humans, mice exhibit cancellous bone loss during growth. Mice are routinely housed at room temperature (18-23°C), a strategy that exaggerates physiological differences in thermoregulation between mice (obligatory daily heterotherms) and humans (homeotherms). The purpose of this investigation was to assess whether housing female mice at thermoneutral (temperature range where the basal rate of energy production is at equilibrium with heat loss) alters bone growth, turnover and microarchitecture. Growing (4-week-old) female C57BL/6J and C3H/HeJ mice were housed at either 22 or 32°C for up to 18weeks. C57BL/6J mice housed at 22°C experienced a 62% cancellous bone loss from the distal femur metaphysis during the interval from 8 to 18weeks of age and lesser bone loss from the distal femur epiphysis, whereas cancellous and cortical bone mass in 32°C-housed mice were unchanged or increased. The impact of thermoneutral housing on cancellous bone was not limited to C57BL/6J mice as C3H/HeJ mice exhibited a similar skeletal response. The beneficial effects of thermoneutral housing on cancellous bone were associated with decreased Ucp1 gene expression in brown adipose tissue, increased bone marrow adiposity, higher rates of bone formation, higher expression levels of osteogenic genes and locally decreased bone resorption. Housing female mice at 22°C resulted in premature cancellous bone loss. Failure to account for species differences in thermoregulation may seriously confound interpretation of studies utilizing mice as preclinical models for osteoporosis.

Betulinic acid synergically enhances BMP2-induced bone formation via stimulating Smad 1/5/8 and p38 pathways.

BackgroundHealing of bone defects is a dynamic and orchestrated process that relies on multiple growth factors and cell types. Bone morphogenetic protein 2 (BMP2) is a key growth factor for bone healing, which stimulates mesenchymal stem cells to differentiate into osteoblasts. Betulinic acid (BetA) is a natural pentacyclic triterpenoid from plants. This study aimed to examine combinatory effects of BetA and BMP2 on ectopic bone generation in mice.ResultsIn MC3T3-E1 preosteoblast culture, 10–15 μM of BetA increased the alkaline phosphatase (ALP) activity and expression levels of osteogenic marker genes without the decreased cell viability. In addition, BetA synergistically enhanced BMP2-induced gene expressions and mineralization with the enhancement of phosphorylation of Smad1/5/8 and p38. In an in vivo ectopic bone formation model, combination of BetA (50 μg) and BMP2 (3 μg) resulted in increases in the amount of new bone generation, compared with treatment with BMP2 alone. Histological studies showed that bone generation with cortical and trabecular structures was resulted from the combination of BetA and BMP2.ConclusionBetA can enhance in vivo osteogenic potentials of BMP2, possibly via stimulating Smad 1/5/8 and p38 pathways, and combination of both agents can be considered as a therapeutic strategy for bone diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-016-0260-5) contains supplementary material, which is available to authorized users.

Comparison of the Osteogenic Potential of Mineral Trioxide Aggregate and Endosequence Root Repair Material in a 3-dimensional Culture System

IntroductionThe ability to promote osteoblast differentiation is a desirable property of root-end filling materials. Several in vitro studies compare the cytotoxicity and physical properties between mineral trioxide aggregate (MTA) and Endosequence root repair material (ERRM), but not their osteogenic potential. Three-dimensional cultures allow cells to better maintain their physiological morphology and better resemble in vivo cellular response than 2-dimensional cultures. Here we examined the osteogenic potential of MTA and ERRM by using a commercially available 3-dimensional Alvetex scaffold. MethodsMandibular osteoblasts were derived from 3-week-old male transgenic reporter mice where mature osteoblasts express green fluorescent protein (GFP) driven by a 2.3-kilobase type I collagen promoter (Col(I)-2.3). Mandibular osteoblasts were grown on Alvetex in direct contact with MTA, ERRM, or no material (negative control) for 14 days. Osteoblast differentiation was evaluated by expression levels of osteogenic genes by using quantitative polymerase chain reaction and by the spatial dynamics of Col(I)-2.3 GFP-positive mature osteoblasts within the Alvetex scaffolds by using 2-photon microscopy. ResultsERRM significantly increased alkaline phosphatase (Alp) and bone sialoprotein (Bsp) expression compared with MTA and negative control groups. Both MTA and ERRM increased osterix (Osx) mRNA significantly compared with the negative control group. The percentage of Col(I)-2.3 GFP-positive cells over total cells within Alvetex was the highest in the ERRM group, followed by MTA and by negative controls. ConclusionsERRM promotes osteoblast differentiation better than MTA and controls with no material in a 3-dimensional culture system. Alvetex scaffolds can be used to test endodontic materials.

Enhanced proliferation and differentiation effects of a CGRP- and Sr-enriched calcium phosphate cement on bone mesenchymal stem cells.

Because of its good osteoconductivity, strontium (Sr) ranelate has been extensively used as a bone substitute for the treatment of bone disorders. To facilitate treatment, Sr is also incorporated into calcium phosphate cement (Sr-CPC); however, the Sr from Sr-CPC is not sufficient to induce a significant increase of bone mass in an ovariectomized rat model. To improve the efficiency of Sr-CPC, we developed a calcitonin gene-related peptide (CGRP)- and Sr-enriched CPC (CGRP-Sr-CPC). We used X-ray diffraction and Fourier transform infrared spectroscopy to measure properties of CGRP-Sr-CPC. We also employed a cell proliferation assay, alkaline phosphatase (ALP) assay and real-time PCR to assess the effects of CPC implants on proliferation and differentiation of bone mesenchymal stem cells (BMSCs) from an ovariectomized rat model. CGRP did not change the composition, pore sizes and compressive strength of the cement body as compared with Sr-CPC. Meanwhile, CGRP-Sr-CPC did not show cell cytotoxicity to BMSCs. Further, CGRP and Sr released from CGRP-Sr-CPC significantly enhanced the cell proliferation of BMSCs and increased the activity of ALP during differentiation of BMSCs, compared with CGRP- or Sr-CPC. Moreover, CGRP-Sr-CPC significantly up-regulated the expression levels of osteogenic differentiation-related genes including Alp, Bmp2, Osteonectin and Runx2 during differentiation. These findings demonstrate the optimized effects of CGRP- and Sr-enriched CPC in promoting proliferation and osteogenic differentiation of BMSCs, suggesting the potential ability of this novel cement to assist the formation of new bone during osteoporosis-induced bone disorders.

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has been as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes.

A Novel Hybrid‐Structured Titanium Surface Promotes Adhesion of Human Dermal Fibroblasts and Osteogenesis of Human Mesenchymal Stem Cells while Reducing S. epidermidis Biofilm Accumulation

We provide a comparative analysis of protein adsorption, primary human cell behavior, and biofilm formation on modified titanium substrates of either micro‐, nano‐, or hybrid micro/nano‐scale feature sizes. While studies revealed that nano‐scale structures initially decreased the attachment and spreading of both human fibroblasts (hDFs) and mesenchymal stem cells (hMSCs), hMSC differentiation studies revealed that hybrid structures promoted the highest levels of osteogenic gene expression and attenuated biofilm formation by Staphylococcus epidermidis. Taken together, this novel approach of generating a hybrid topographical feature results in a potential implant material capable of enhanced dermal cell adhesion and osteogenic differentiation while limiting biofilm accumulation.

Enhanced in vitro biological activity generated by surface characteristics of anodically oxidized titanium--the contribution of the oxidation effect.

Anodically oxidized titanium surfaces, prepared by spark discharge, have micro-submicron surface topography and nano-scale surface chemistry, such as hydrophilic functional groups or hydroxyl radicals in parallel. The complexity of the surface characteristics makes it difficult to draw a clear conclusion as to which surface characteristic, of anodically oxidized titanium, is critical in each biological event. This study examined the in vitro biological changes, induced by various surface characteristics of anodically oxidized titanium with, or without, release of hydroxyl radicals onto the surface. Anodically oxidized titanium enhanced the expression of genes associated with differentiating osteoblasts and increased the degree of matrix mineralization by these cells in vitro. The phenotypes of cells on the anodically oxidized titanium were the same with, or without, release of hydroxyl radicals. However, the nanomechanical properties of this in vitro mineralized tissue were significantly enhanced on surfaces, with release of hydroxyl radicals by oxidation effects. In addition, the mineralized tissue, produced in the presence of bone morphogenetic protein-2 on bare titanium, had significantly weaker nanomechanical properties, despite there being higher osteogenic gene expression levels. We show that enhanced osteogenic cell differentiation on modified titanium is not a sufficient indicator of enhanced in vitro mineralization. This is based on the inferior mechanical properties of mineralized tissues, without either being cultured on a titanium surface with release of hydroxyl radicals, or being supplemented with lysyl oxidase family members.

MiR-124 negatively regulates osteogenic differentiation and in vivo bone formation of mesenchymal stem cells.

MicroRNAs are novel key regulators of cellular differentiation. Dlx transcription factors play an important role in osteoblast differentiation, and Dlx5 and Dlx2 are known targets of miR-124. Therefore, in the present study, we investigated the regulatory effects of miR-124 on the osteogenic differentiation and in vivo bone formation of mesenchymal stem cells (MSCs). During osteogenic induction by BMP2, the expression levels of miR-124 were inversely correlated with those of osteogenic differentiation marker genes in human and mouse bone marrow-derived MSCs, MC3T3-E1 cells and C2C12 cells. The overexpression of a miR-124 mimic significantly decreased the expression levels of Dlx5, Dlx3, and Dlx2, whereas the silencing of miR-124 with hairpin inhibitors significantly increased the expression of these Dlx genes. Luciferase reporter assays demonstrated that miR-124 directly targets the 3'UTRs of Dlx3, Dlx5, and Dlx2. The overexpression of a miR-124 mimic suppressed the osteogenic marker gene expression levels, alkaline phosphatase activity and matrix mineralization, which were all significantly increased by the overexpression of a miR-124 inhibitor. When ectopic bone formation was induced by the subcutaneous transplantation of human bone marrow-derived MSCs in nude mice, MSCs overexpressing a miR-124 inhibitor significantly enhanced woven bone formation compared with control MSCs. However, MSCs overexpressing a miR-124 mimic exhibited increased adipocyte differentiation at the expense of ectopic bone formation. These results suggest that miR-124 is a negative regulator of osteogenic differentiation and in vivo bone formation and that the targeting of Dlx5, Dlx3, and Dlx2 genes partly contributes to this inhibitory effect exerted by miR-124.

Bone formation by human umbilical cord perivascular cells.

We investigated the possibility of employing human umbilical perivascular cells (HUCPVCs) within the context of finding an alternative source of mesenchymal stromal cells (MSC) for bone tissue engineering. Since it has previously been reported that conditioned medium (CM) from osteogenic bone marrow (BM) MSCs can potentiate osteogenic differentiation in a secondary cell population, we also employed BM-MSCs to generate CM to stimulate osteogenesis in the HUCPVCs. The BM-MSCs were a commercially available immortalized human cell line. In vitro assays showed negligible levels of osteogenic gene expression in HUCPVCs compared to BM-MSC, but alkaline phosphatase was detected when HUCPVC were cultured in osteogenic medium in the presence of CM from BM-MSC. An in vivo assay employing a rat calvarial osteotomy defect, together with a collagen sponge scaffold, showed that HUCPVCs provided statistically significant bony repair compared to controls. BM-MSC loaded scaffolds were not statistically different from either controls or HUCPVCs. The addition of BM-MSC CM to HUCPVCs also produced no statistically significant difference to the bone formed by HUCPVCs alone. Our results demonstrate that the in vitro assays employed did not predict in vivo outcomes, and that the BM-MSC cell line employed, or CM from such cells, provided no osteogenic advantage over the use of HUCPVCs alone.

Factors circulating in the blood of type 2 diabetes mellitus patients affect osteoblast maturation – Description of a novel in vitro model

Type 2 diabetes mellitus (T2DM) is one of the most frequent metabolic disorders in industrialized countries. Among other complications, T2DM patients have an increased fracture risk and delayed fracture healing. We have demonstrated that supraphysiological glucose and insulin levels inhibit primary human osteoblasts׳ maturation. We aimed at developing a more physiologically relevant in vitro model to analyze T2DM-mediated osteoblast changes. Therefore, SCP-1-immortalized pre-osteoblasts were differentiated with T2DM or control (non-obese and obese) sera. Between both control groups, no significant changes were observed. Proliferation was significantly increased (1.69-fold), while AP activity and matrix mineralization was significantly reduced in the T2DM group. Expression levels of osteogenic marker genes and transcription factors were altered, e.g. down-regulation of RUNX2 and SP-7 or up-regulation of STAT1, in the T2DM group. Active TGF-β levels were significantly increased (1.46-fold) in T2DM patients׳ sera. SCP-1 cells treated with these sera showed significantly increased TGF-β signaling (2.47-fold). Signaling inhibition effectively restored osteoblast maturation in the T2DM group. Summarizing our data, SCP-1 cells differentiated in the presence of T2DM patients׳ serum exhibit reduced osteoblast function. Thus, this model has a high physiological impact, as it can identify circulating factors in T2DM patients׳ blood that may affect bone function, e.g. TGF-β.

Tenascin-C mimetic Peptide nanofibers direct stem cell differentiation to osteogenic lineage.

Extracellular matrix contains various signals for cell surface receptors that regulate cell fate through modulation of cellular activities such as proliferation and differentiation. Cues from extracellular matrix components can be used for development of new materials to control the stem cell fate. In this study, we achieved control of stem cell fate toward osteogenic commitment by using a single extracellular matrix element despite the contradictory effect of mechanical stiffness. For this purpose, we mimicked bone extracellular matrix by incorporating functional sequence of fibronectin type III domain from native tenascin-C on self-assembled peptide nanofibers. When rat mesenchymal stem cells (rMSCs) were cultured on these peptide nanofibers, alkaline phosphatase (ALP) activity and alizarin red staining indicated osteogenic differentiation even in the absence of osteogenic supplements. Moreover, expression levels of osteogenic marker genes were significantly enhanced revealed by quantitative real-time polymerase chain reaction (qRT-PCR), which showed the remarkable bioactive role of this nanofiber system on osteogenic differentiation. Overall, these results showed that tenascin-C mimetic peptides significantly enhanced the attachment, proliferation, and osteogenic differentiation of rMSCs even in the absence of any external bioactive factors and regardless of the suitable stiff mechanical properties normally required for osteogenic differentiation. Thus, these peptide nanofibers provide a promising new platform for bone regeneration.

Cartilage oligomeric matrix protein gene multilayers inhibit osteogenic differentiation and promote chondrogenic differentiation of mesenchymal stem cells.

There are still many challenges to acquire the optimal integration of biomedical materials with the surrounding tissues. Gene coatings on the surface of biomaterials may offer an effective approach to solve the problem. In order to investigate the gene multilayers mediated differentiation of mesenchymal stem cells (MSCs), gene functionalized films of hyaluronic acid (HA) and lipid-DNA complex (LDc) encoding cartilage oligomeric matrix protein (COMP) were constructed in this study via the layer-by-layer self-assembly technique. Characterizations of the HA/DNA multilayered films indicated the successful build-up process. Cells could be directly transfected by gene films and a higher expression could be obtained with the increasing bilayer number. The multilayered films were stable for a long period and DNA could be easily released in an enzymatic condition. Real-time polymerase chain reaction (RT-PCR) assay presented significantly higher (p < 0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. These results demonstrated that HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing.

Propranolol, a β-adrenergic antagonist, attenuates the decrease in trabecular bone mass in high calorie diet fed growing mice

We investigated the effects of high calorie and low calorie diets on skeletal integrity, and whether β-adrenergic blockade (BB) attenuates bone loss induced by dietary calorie alteration. Male 6-week-old C57BL/6 mice were assigned to either an ad-lib fed control diet (CON), a high calorie diet (HIGH), or a low calorie diet (LOW) group. In each diet group, mice were treated with either vehicle (VEH) or propranolol, a β-adrenergic antagonist. Over 12-weeks, β-blockade mitigated body weight and fat mass increases induced by the high calorie diet. Femoral trabecular bone mineral density and the expression levels of osteogenic marker genes in bone marrow cells were reduced in HIGHVEH and LOWVEH mice, and BB significantly attenuated this decline only in HIGH mice. In summary, the magnitude of bone loss induced by low calorie diet was greater than that caused by high calorie diet in growing mice, and β-blockade mitigated high calorie diet-induced bone loss. [BMB Reports 2014; 47(9): 506-511]