Osteoblast Induction Research Articles

3D printed zirconia ceramic tool for bone repair with multifunction of drug release, drilling and implantation

Ceramics is a promising material that has been widely used as artificial bones. However, most available investigations were devoted to new material development and implant structure design, while few of studies focused on innovative hybrid implants that can act not only as implants but also as surgery tools for solving specific health issues. This paper innovatively designed and additively-manufactured a zirconia ceramic surgery tool, which can not only act as a drilling tool, but also retain itself in the drilled hole after the surgery as bone scaffold, at the meantime delivery active ingredient (Vitamin C) for fast recovery. To achieve early intervention, the proposed zirconia ceramic tool was additively manufactured with inter-connected pore structures that can benefit the recovery. The zirconia ceramic surgery tools (with 50 %, 70 % and 80 % porosity) showed the required mechanical properties to act as a drilling tool. The tool also showed drug releasing function with the different diffusion rates based on drug diffusion experiments. The zirconia ceramic tool with Vitamin C coating also showed enhanced cell adhesion and accelerated cell growth based on the osteoblast induction assessment. Based on above, the proposed zirconia ceramic tool is expected to bring new possibilities for wide applications of ceramic tools in surgeries.

Silencing of Jumonji domain-containing 1C inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells via nuclear factor-κB signaling.

Osteoporosis is a common metabolic bone disorder induced by an imbalance between osteoclastic activity and osteogenic activity. During osteoporosis, bone mesenchymal stem cells (BMSCs) exhibit an increased ability to differentiate into adipocytes and a decreased ability to differentiate into osteoblasts, resulting in bone loss. Jumonji domain-containing 1C (JMJD1C) has been demonstrated to suppress osteoclastogenesis. To examine the effect of JMJD1C on the osteogenesis of BMSCs and the potential underlying mechanism. BMSCs were isolated from mouse bone marrow tissues. Oil Red O staining, Alizarin red staining, alkaline phosphatase staining and the expression of adipogenic and osteogenic-associated genes were assessed to determine the differentiation of BMSCs. Bone marrow-derived macrophages (BMMs) were incubated with receptor activator of nuclear factor-kappa Β ligand to induce osteoclast differentiation, and osteoclast differentiation was confirmed by tartrate-resistant acid phosphatase staining. Other related genes were measured via reverse transcription coupled to the quantitative polymerase chain reaction and western blotting. Enzyme-linked immunosorbent assays were used to measure the levels of inflammatory cytokines, including tumor necrosis factor alpha, interleukin-6 and interleukin-1 beta. The osteogenic and adipogenic differentiation potential of BMSCs isolated from mouse bone marrow samples was evaluated. JMJD1C mRNA and protein expression was upregulated in BMSCs after osteoblast induction, while p-nuclear factor-κB (NF-κB) and inflammatory cytokines were not significantly altered. Knockdown of JMJD1C repressed osteogenic differentiation and enhanced NF-κB activation and inflammatory cytokine release in BMSCs. Moreover, JMJD1C expression decreased during BMM osteoclast differentiation. The JMJD1C/NF-κB signaling pathway is potentially involved in BMSC osteogenic differentiation and may play vital roles in the pathogenesis of osteoporosis.

Recombinant human secretory leukocyte protease inhibitor (rhSLPI) coated titanium enhanced human osteoblast adhesion and differentiation

Osseointegration is vital to success in orthopedic and dental reconstructions with implanted materials. The bone matrix or cells—particularly osteoblasts—are required to achieve functional contact on the implant surface. Osteoblast induction is therefore essential for osteogenesis to occur. Enhancement of osteoblast adhesion, proliferation, and differentiation, particularly by implant surface modifications, have been found challenging to develop. Secretory Leukocyte Protease Inhibitor (SLPI), a cation ionic protein with anti-inflammatory and anti-bacterial activities, showed activation in osteoblast proliferation and differentiation. However, the effects of coating recombinant human (rh) SLPI on a titanium alloy surface on human osteoblast adhesion, proliferation, and differentiation has never been investigated. In this study, titanium alloys (Ti–6Al–4V) were coated with rhSLPI, while human osteoblast adhesion, proliferation, differentiation, actin cytoskeletal organization, and gene expressions involved in cell adhesion and differentiation were investigated. The results indicate that coating titanium with 10–100 µg/ml rhSLPI enhanced the physical properties of the Ti surface and enhanced human osteoblast (hFOB 1.19) cell adhesion, activated actin dynamic, enhanced adhesive forces, upregulated integrins α1, α2, and α5, enhanced cell proliferation, mineralization, alkaline phosphatase activity, and upregulated ALP, OCN, and Runx2. This is the first study to demonstrate that coating SLPI on titanium surfaces enhances osseointegration and could be a candidate molecule for surface modification in medical implants.

Alendronate-derived carbon dots for bioimaging and effective osteoblastic differentiation induction and promotion

Carbon dots (CDs) belong to zero-dimensional carbon-based nanomaterials, which are promising in various biomedical applications including imaging, biosensing and drug delivery. In this work, we have synthesized novel fluorescent CDs (Alen-CDs) derived from alendronate, an anti-osteoporosis drug, through a one-step hydrothermal method. The ability of Alen-CDs for fluorescent imaging was evaluated via in vitro cellular bioimaging. In addition, the capability of Alen-CDs to induce in vitro osteoblastic differentiation was investigated by alkaline phosphatase staining, activity test and alizarin red staining. As demonstrated, Alen-CDs was effective for in vitro fluorescent imaging without obvious toxicity. Also, Alen-CDs promoted the survival of rat bone marrow mesenchymal stem cells and stimulated osteoblastic differentiation. In conclusion, Alen-CDs are a new type of nanomaterial with good biocompatibility and fluorescent properties. Alen-CDs could be applied as a potential probe for bioimaging and a promising nanodrug for bone regeneration as well as the combination to be used as a theranostic agent.

Evaluation of polyetheretherketone composites modified by calcium silicate and carbon nanotubes for bone regeneration: mechanical properties, biomineralization and induction of osteoblasts.

Desired orthopedic implant materials must have a good biological activity and possess appropriate mechanical property that correspond to those of human bone. Although polyetheretherketone (PEEK) has displayed a promising application prospect in musculoskeletal and dentistry reconstruction thanks to its non-biodegradability and good biocompatibility in the body, the poor osseointegration and insufficient mechanical strength have significantly limited its application in the repair of load-bearing bones and surgical operations. In this study, carbon nanotubes (CNT)/calcium silicate (CS)/polyetheretherketone ternary composites were fabricated for the first time. The addition of CS was mainly aimed at improving biological activities and surface hydrophilicity, but it inevitably compromised the mechanical strength of PEEK. CNT can reinforce the composites even when brittle CS was introduced and further upgraded the biocompatibility of PEEK. The CNT/CS/PEEK composites exhibited higher mechanical strengths in tensile and bending tests, 64% and 90% higher than those of brittle CS/PEEK binary composites. Besides, after incorporation of CNT and CS into PEEK, the hydrophilicity, surface roughness and ability to induce apatite-layer deposition were significantly enhanced. More importantly, the adhesion, proliferation, and osteogenic differentiation of mouse embryo osteoblasts were effectively promoted on CNT/CS/PEEK composites. In contrast to PEEK, these composites exhibited a more satisfactory biocompatibility and osteoinductive activity. Overall, these results demonstrate that ternary CNT/CS/PEEK composites have the potential to serve as a feasible substitute to conventional metal alloys in musculoskeletal regeneration and orthopedic implantation.

Mangiferin-Enriched Mn-Hydroxyapatite Coupled with β-TCP Scaffolds Simultaneously Exhibit Osteogenicity and Anti-Bacterial Efficacy.

Biphasic calcium phosphate (BCP) containing β-tricalcium phosphate and manganese (Mn)-substituted hydroxyapatite (HAP) was synthesized. Biomedical scaffolds were prepared using this synthesized powder on a sacrificial polyurethane sponge template after the incorporation of mangiferin (MAN). Mn was substituted at a concentration of 5% and 10% in HAP to examine the efficacy of Mn at various concentrations. The phase analysis of the as-formed BCP scaffold was carried out by X-ray diffraction analysis, while the qualitative observation of morphology and the osteoblast cell differentiation were carried out by scanning electron microscopy and confocal laser scanning microscopy techniques. Gene expressions of osteocalcin, collagen 1, and RUNX2 were carried out using qRT-PCR analyses. Significantly higher (p < 0.05) levels of ALP activity were observed with extended osteoblast induction on the mangiferin-incorporated BCP scaffolds. After characterization of the specimens, it was found that the scaffolds with 10% Mn-incorporated BCP with mangiferin showed better osteogenicity and simultaneously the same scaffolds exhibited higher anti-bacterial properties as observed from the bacterial viability test. This study was carried out to evaluate the efficacy of Mn and MAN in BCP for osteogenicity and antibacterial action.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Crosstalk between microRNA-21–5p and the transcription factor Dec1 maintains osteoblast function

MicroRNAs are associated with pivotal post-transcriptional gene regulation in bone formation. Human differentiated embryonic chondrocyte expressed gene 1 (Dec1) is also involved in regulating osteoblastogenesis. In the present study, we aimed to investigate the distinctive role of miR-21–5p and Dec1 in osteoblast function and to determine their biological functions. MC3T3-E1 pre-osteoblastic cells were used for in vitro analyses. miR-21–5p knockout (KO) mice, Dec1KO mice and age-matched wild-type (WT) mice were used to characterize the influence of miR-21–5p and Dec1 deficiencies on bone formation. Morphological analyses [micro-computed tomography (micro-CT)] were performed, and measurements were collected to validate miR-21-5pKO mice. Histopathological changes in mouse femur tissues were assessed by H-E staining, Azan staining, Masson's Trichrome staining, and Toluidine Blue staining. Quantitative real-time RT-PCR, western blotting and immunohistochemical staining were used to characterize the expression levels of Alkaline Phosphatase, Runx2, Osterix, Osteopontin, Dec1 and miR-21–5p. Bioinformatics analyses and dual-luciferase reporter assays were performed to confirm Dec1 as a target of miR-21–5p. Dec1 expression was gradually increased from day 7 of osteoblast induction, while miR-21–5p showed a peak at day 21. In non-induced osteoblasts, a mechanistically gain-of-function transfection study with a miR-21–5p mimic enhanced Runx2 and Osterix expression but suppressed Dec1. miR-21-5pKO mice had reduced bone growth. Dec1-deficient mice showed advanced bone formation at the age of 12 weeks compared to WT mice. The Dec1 deficiency upregulated Runx2 and Osterix expression in Dec1KO mouse femurs. Those changes, however, were reversed in miR-21-5pKO mouse femurs compared to WT mouse femurs. Dual-luciferase reporter assays showed that Dec1 is a possible downstream target of miR-21–5p. These findings showed that the reduced osteogenic potential due to a miR-21–5p deficiency is achieved by enhanced Dec1 expression and that the miR-21–5p/Dec1 axis is involved in regulating osteoblast function.

Electrospun polylactic acid scaffolds with strontium- and cobalt-doped bioglass for potential use in bone tissue engineering applications.

The development of nanoscale biomaterials associated with polymers has been growing over the years, due to their important structural characteristics for applications in biological systems. The present study aimed to produce and test polymeric scaffolds composed of polylactic acid (PLA) fibers associated with a 58S bioglass doped with therapeutic ions for use in tissue engineering. Three 58S Bioglass was obtained by the sol-gel route, pure and doped with 5% strontium and cobalt ions. Solutions of 7% PLA was used as control and added the three different bioglass, 4% of 58S bioglass (PLA-BG), 4% bioglass-doped strontium (PLA-BGSr) and 4% bioglass-doped cobalt (PLA-BGCo). Scaffolds were produced through electrospinning process, and was characterized chemical and morphologically. The in vitro tests were performed using mesenchymal cells cultures from femurs of nine rats, grown in osteogenic supplemented total culture medium. After osteoblastic differentiation induction cell viability, alkaline phosphatase activity, total protein content quantification, and visualization of mineralization nodule tests were performed. Analysis of normal distribution used the Shapiro-Wilk test (nanofibers diameter and biological assay). Data were compared using the Kruskal-Wallis nonparametric test (p= 0.05). The bioglasses produced proved to be free of nitrate, chlorinated and nano-sized, with effective incorporation of therapeutic ions in their structure. All materials showed cell viability (>70%), total protein production, and alkaline phosphatase activity. It was possible to develop polylactic acid scaffolds associated with 58S bioglass doped with therapeutic ions without cytotoxicity. Scaffolds characteristics appear to sustain its application in bone tissue engineering.

Comparison of Osteoblastic Differentiation of Human Periodontal Ligament Stem Cells through Application of Two β-tricalcium Phosphate Products: An in vitro Study.

Osteoblastic differentiation of periodontal ligament stem cells (PLSCs) is essential for alveolar bone regeneration. The purpose of this study was to compare the potential of two β-tricalcium phosphate (βTCP) products to induce osteoblastic differentiation of human PLSCs. In this in vitro study, human PLSCs were cultured in mediums supplemented with Guidor Easy-Graft [βTCP+polylactide-co-glycolide (PLCG)+n-methyl-2-pyrrolidone (NMP)] [Sunstar Company, Swiss] or Sorbone [βTCP] [Meta Company, South Korea] as two alloplasts experimental groups, mesenchymal cells differentiated into osteoblasts without alloplast as positive control group, and mesenchymal cells without osteoblastic induction as negative control group. Osteoblastic differentiation was evaluated using Alizarine Red staining and spectrophotometry to assay calcium deposits and real-time polymerase chain reaction to examine expression of alkaline phosphatase (ALP) and osteopontin (OPN) antigens on day 21. Data were analyzed by using SPSS 22 software and one-way ANOVA and Bonferoni tests (p< 0.05). Spectrophotometry confirmed that calcium deposits were higher in Guidor Easy-Graft group compared to Sorbone group (p< 0.001) and higher in two experimental groups than controls (p< 0.05). According to real-time polymerase chain reaction, level of ALP expression was higher in Sorbone than Guidor and the levels of Guidor, positive control and negative control were equal; OPN levels of the positive control were more than the other groups. OPN levels of Sobone, Guidor and negative control were the same. These findings indicated that Guidor Easy-Graft and Sorbone enhanced differentiation of human PLSCs to osteoblasts, and could be employed as appropriate bone-graft materials.

Comparison between hydroxyapatite and polycaprolactone in inducing osteogenic differentiation and augmenting maxillary bone regeneration in rats.

BackgroundThe selection of appropriate scaffold plays an important role in ensuring the success of bone regeneration. The use of scaffolds with different materials and their effect on the osteogenic performance of cells is not well studied and this can affect the selection of suitable scaffolds for transplantation. Hence, this study aimed to investigate the comparative ability of two different synthetic scaffolds, mainly hydroxyapatite (HA) and polycaprolactone (PCL) scaffolds in promoting in vitro and in vivo bone regeneration.MethodIn vitro cell viability, morphology, and alkaline phosphatase (ALP) activity of MC3T3-E1 cells on HA and PCL scaffolds were determined in comparison to the accepted model outlined for two-dimensional systems. An in vivo study involving the transplantation of MC3T3-E1 cells with scaffolds into an artificial bone defect of 4 mm length and 1.5 mm depth in the rat’s left maxilla was conducted. Three-dimensional analysis using micro-computed tomography (micro-CT), hematoxylin and eosin (H&E), and immunohistochemistry analyses evaluation were performed after six weeks of transplantation.ResultsMC3T3-E1 cells on the HA scaffold showed the highest cell viability. The cell viability on both scaffolds decreased after 14 days of culture, which reflects the dominant occurrence of osteoblast differentiation. An early sign of osteoblast differentiation can be detected on the PCL scaffold. However, cells on the HA scaffold showed more prominent results with intense mineralized nodules and significantly (p < 0.05) high levels of ALP activity with prolonged osteoblast induction. Micro-CT and H&E analyses confirmed the in vitro results with bone formation were significantly (p < 0.05) greater in HA scaffold and was supported by IHC analysis which confirmed stronger expression of osteogenic markers ALP and osteocalcin.ConclusionDifferent scaffold materials of HA and PCL might have influenced the bone regeneration ability of MC3T3-E1. Regardless, in vitro and in vivo bone regeneration was better in the HA scaffold which indicates its great potential for application in bone regeneration.

Targeting Sirt1, AMPK, Nrf2, CK2, and Soluble Guanylate Cyclase with Nutraceuticals: A Practical Strategy for Preserving Bone Mass.

There is a vast pre-clinical literature suggesting that certain nutraceuticals have the potential to aid the preservation of bone mass in the context of estrogen withdrawal, glucocorticoid treatment, chronic inflammation, or aging. In an effort to bring some logical clarity to these findings, the signaling pathways regulating osteoblast, osteocyte, and osteoclast induction, activity, and survival are briefly reviewed in the present study. The focus is placed on the following factors: the mechanisms that induce and activate the RUNX2 transcription factor, a key driver of osteoblast differentiation and function; the promotion of autophagy and prevention of apoptosis in osteoblasts/osteoclasts; and the induction and activation of NFATc1, which promotes the expression of many proteins required for osteoclast-mediated osteolysis. This analysis suggests that the activation of sirtuin 1 (Sirt1), AMP-activated protein kinase (AMPK), the Nrf2 transcription factor, and soluble guanylate cyclase (sGC) can be expected to aid the maintenance of bone mass, whereas the inhibition of the serine kinase CK2 should also be protective in this regard. Fortuitously, nutraceuticals are available to address each of these targets. Sirt1 activation can be promoted with ferulic acid, N1-methylnicotinamide, melatonin, nicotinamide riboside, glucosamine, and thymoquinone. Berberine, such as the drug metformin, is a clinically useful activator of AMPK. Many agents, including lipoic acid, melatonin, thymoquinone, astaxanthin, and crucifera-derived sulforaphane, can promote Nrf2 activity. Pharmacological doses of biotin can directly stimulate sGC. Additionally, certain flavonols, notably quercetin, can inhibit CK2 in high nanomolar concentrations that may be clinically relevant. Many, though not all, of these agents have shown favorable effects on bone density and structure in rodent models of bone loss. Complex nutraceutical regimens providing a selection of these nutraceuticals in clinically meaningful doses may have an important potential for preserving bone health. Concurrent supplementation with taurine, N-acetylcysteine, vitamins D and K2, and minerals, including magnesium, zinc, and manganese, plus a diet naturally high in potassium, may also be helpful in this regard.

Involvement of MiRNA-211-5p and Arhgap11a Interaction During Osteogenic Differentiation of MC3T3-E1 Cells.

ObjectiveMicroRNAs (miRNAs) are well-recognized for their abilities to regulate gene expression post-transcriptionally in plants and animals. Recently, miRNA-messenger RNA (mRNA) regulatory relationships have been confirmed during biological processes, including osteogenic differentiation. This study aimed to find out more candidate miRNA-mRNA pairs involved in the osteogenic differentiation of MC3T3-E1 cells.MethodsAn MC3T3-E1-based microarray dataset (accessioned as GSE46400) downloaded from the Gene Expression Omnibus included MC3T3-E1 cells with or without 14-day osteoblast differentiation osteoblast induction. Multiple miRNA-mRNA prediction databases were searched by differentially expressed genes (DEGs) to obtain pairs of a miRNA-DEG regulatory network. The MC3T3-E1 cells were cultured and incubated in the osteogenic differentiation medium for 14 days. The expressions of candidate miRNAs and mRNAs were determined by real-time quantitative PCR(RT-qPCR) in MC3T3-E1 cells. The miRNA-mRNA interactions were verified by dual-luciferase reporter gene assays and experiments using mimics miRNA or their inhibitors.ResultsWe identified 715 upregulated DEGs and 603 downregulated DEGs between MC3T3-E1 cells with and without osteoblast induction by analyzing the raw data of the GSE46400 dataset. There were 7 overlapped miRNA-mRNA pairs identified during osteogenic differentiation of MC3T3-E1 cells, including mmu-miR-204-5p-Arhgap11a, mmu-miR-211-5p-Arhgap11a, mmu-miR-24-3p-H2afx, mmu-miR-3470b-Chek2, mmu-miR-3470b-Dlgap5, mmu-miR-466b-3p-Chek1, and mmu-miR-466c-3p-Chek1. The Arhgap11a, H2afx, Chek2, Dlgap5, and Chek1 were hub genes downregulated in MC3T3-E1 cells after osteogenic differentiation, verified by RT-qPCR results. The RT-qPCR also determined declined expressions of miR-204-5p and miR-24-3p concomitant with elevated expressions of miR-211-5p, miR-3470b, miR-466b-3p, and miR-466c-3p in the MC3T3-E1 cells, with osteoblast induction compared with undifferentiated MC3T3-E1 cells. Dual-luciferase reporter gene assays demonstrated Arhgap11a as the target of miR-211-5p. MiR-211-5p upregulation by its mimic increased Arhgap11a expression in MC3T3-E1 cells.ConclusionOur study characterizes miR-211-5p targeting Arhgap11a promotes the osteogenic differentiation of MC3T3-E1 cells, which provides novel targets to promote the osteogenesis process during bone repair.

Biocompatibility of platinum-based bulk metallic glass in orthopedic applications

Bulk metallic glasses (BMGs) are a class of amorphous metals that exhibit high strength, ductility paired with wear and corrosion resistance. These properties suggest that they could serve as an alternative to conventional metallic implants that suffer wear and failure. In the present study, we investigated Platinum (Pt)-BMG biocompatibility in bone applications. Specifically, we investigated osteoclast formation on flat and nanopatterned Pt57.5Cu14.7Ni5.3P22.5 (atomic percent) as well as titanium (control). Specifically, receptor activator of NF-κB (RANK) ligand-induced murine bone marrow derived mononuclear cell fusion was measured on multiple nanopatterns and was found to be reduced on nanorods (80 and 200 nm in diameter) and was associated with reduced tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase (MMP9) expression. Evaluation of mesenchymal stem cell (MSC) to osteoblast differentiation on nanopatterned Pt-BMG showed significant reduction in comparison to flat, suggesting that further exploration of nanopatterns is required to have simultaneous induction of osteoblasts and inhibition of osteoclasts.In vivo studies were also pursued to evaluate the biocompatibility of Pt-BMG in comparison to titanium. Rods of each material were implanted in the femurs of mice and evaluated by x-ray, mechanical testing, micro-computed tomography (micro-CT), and histological analysis. Overall, Pt-BMG showed similar biocompatibility with titanium suggesting that it has the potential to improve outcomes by further processing at the nanoscale.

Transforming growth factor-\u03b21-induced N-cadherin drives cell\u2013cell communication through connexin43 in osteoblast lineage

Gap junction (GJ) has been indicated to have an intimate correlation with adhesion junction. However, the direct interaction between them partially remains elusive. In the current study, we aimed to elucidate the role of N-cadherin, one of the core components in adhesion junction, in mediating connexin 43, one of the functional constituents in gap junction, via transforming growth factor-β1(TGF-β1) induction in osteoblasts. We first elucidated the expressions of N-cadherin induced by TGF-β1 and also confirmed the upregulation of Cx43, and the enhancement of functional gap junctional intercellular communication (GJIC) triggered by TGF-β1 in both primary osteoblasts and MC3T3 cell line. Colocalization analysis and Co-IP experimentation showed that N-cadherin interacts with Cx43 at the site of cell–cell contact. Knockdown of N-cadherin by siRNA interference decreased the Cx43 expression and abolished the promoting effect of TGF-β1 on Cx43. Functional GJICs in living primary osteoblasts and MC3T3 cell line were also reduced. TGF-β1-induced increase in N-cadherin and Cx43 was via Smad3 activation, whereas knockdown of Smad3 signaling by using siRNA decreased the expressions of both N-cadherin and Cx43. Overall, these data indicate the direct interactions between N-cadherin and Cx43, and reveal the intervention of adhesion junction in functional gap junction in living osteoblasts.

SLPI is a critical mediator that controls PTH-induced bone formation

Osteoclastic bone resorption and osteoblastic bone formation/replenishment are closely coupled in bone metabolism. Anabolic parathyroid hormone (PTH), which is commonly used for treating osteoporosis, shifts the balance from osteoclastic to osteoblastic, although it is unclear how these cells are coordinately regulated by PTH. Here, we identify a serine protease inhibitor, secretory leukocyte protease inhibitor (SLPI), as a critical mediator that is involved in the PTH-mediated shift to the osteoblastic phase. Slpi is highly upregulated in osteoblasts by PTH, while genetic ablation of Slpi severely impairs PTH-induced bone formation. Slpi induction in osteoblasts enhances its differentiation, and increases osteoblast–osteoclast contact, thereby suppressing osteoclastic function. Intravital bone imaging reveals that the PTH-mediated association between osteoblasts and osteoclasts is disrupted in the absence of SLPI. Collectively, these results demonstrate that SLPI regulates the communication between osteoblasts and osteoclasts to promote PTH-induced bone anabolism.

Exosomal miR-335 derived from mature dendritic cells enhanced mesenchymal stem cell-mediated bone regeneration of bone defects in athymic rats

BackgroundTransplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) embedded in a bio-compatible matrix has been demonstrated as a promising strategy for the treatment of bone defects. This study was designed to explore the effect and mechanism of exosomes derived from mature dendritic cells (mDC-Exo) on the BM-MSCs-mediated bone regeneration using the matrix support in an athymic rat model of femoral bone defect.MethodsThe BM-MSCs were isolated from rats and incubated with osteoblast induction medium, exosomes derived from immature DCs (imDC-Exo), mDC-Exo, and miR-335-deficient mDC-Exo. BM-MSCs treated without or with mDC-Exo were embedded in a bio-compatible matrix (Orthoss®) and then implanted into the femoral bone defect of athymic rats.ResultsmDC-Exo promoted the proliferation and osteogenic differentiation of BM-MSCs by transferring miR-335. Mechanistically, exosomal miR-335 inhibited Hippo signaling by targeting large tongue suppressor kinase 1 (LATS1) and thus promoted the proliferation and osteogenic differentiation of BM-MSCs. Animal experiments showed that mDC-Exo enhanced BM-MSCs-mediated bone regeneration after bone defect, and this effect was abrogated when miR-335 expression was inhibited in mDC-Exo.ConclusionmDC-Exo promoted osteogenic differentiation of BM-MSCs and enhanced BM-MSCs-mediated bone regeneration after femoral bone defect in athymic rats by transferring miR-335.

Osteoinductive effect of soluble transforming growth factor beta receptor 3 on human osteoblast lineage.

The development of bone requires carefully choregraphed signaling to bone progenitors to form bone. Our group recently described the requirement of transforming growth factor beta receptor 3 (TGFβR3), a receptor involved in TGFβ pathway signaling, during osteoblast lineage commitment in mice. The TGFβ pathway is known to play multiple osteo-inductive and osteo-inhibitory roles during osteoblast development and TGFβR3 human mutations are associated with reduced bone mineral density, making TGFβR3 a unique target for bone inductive therapy. In this article, we demonstrated increased mineralization of human pediatric bone-derived osteoblast-like cells (HBO) when treated with soluble TGFβR3 (sR3) using Alizarin Red staining. Osteogenic commitment of HBO cells was demonstrated by induction of osteogenic genes RUNX2, osteocalcin, osteopontin, and osterix. Evaluation of the canonical TGFβ pathway signaling demonstrated that sR3 was able to induce bone formation in HBO cells, mainly through activation of noncanonical targets of TGFβ pathway signaling including AKT, ERK, and p38 MAP kinases. Inhibition of these osteogenic noncanonical pathways in the HBO cells also inhibited mineralization, suggesting they are each required. Although no induction of SMAD1, 5, and 9 was observed, there was the activation of SMAD2 and 3 suggesting that sR3 is primarily signaling via the noncanonical pathways during osteogenic induction of the HBO. Our results highlight the important role of TGFβR3 in osteoblast induction of mineralization in human bone cells through noncanonical targets of TGFβ signaling. Future studies will focus on the ability of sR3 to induce bone regeneration in vivo using animal models.

Osteogenic Response of MC3T3-E1 and Raw264.7 in the 3D-Encapsulated Co-Culture Environment.

Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix® 3D culture system, which provides a physiologically relevant environment. The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. Treatment with osteoblastic induction factors (+3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D co-culture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK-RANKL signaling, which is important in osteoblast regulation of osteoclasts. In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

Effects of inhibiting PDK‑1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation invitro.

Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3-phosphoinositide-dependent protein kinase-1 (PDK-1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK-1 in the process of mouse osteoblast differentiation in vitro. In the BX-912 group, BX-912, a specific inhibitor of PDK-1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1flox/flox mice were designed and generated, and were used to obtain BMSCsPDK1flox/flox. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd-cre-EGFP) was used to disrupt the PDK-1 gene in BMSCsPDK1flox/flox; this served as the pHBAd-cre-EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)-PDK1 and p-AKT were gradually increased during the osteoblast differentiation process. Notably, BX-912 treatment and disruption of the PDK-1 gene with pHBAd-cre-EGFP effectively reduced the number of alkaline phosphatase (ALP)-positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK-1 in the pHBAd-cre-EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3–7. The mRNA expression levels of the osteoblast-related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX-912 and pHBAd-cre-EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX-912 and disruption of the PDK-1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK-1 in osteoblasts.