Osteogenic Function Research Articles

Scutellarin enhances osteoblast proliferation and function via NF-κB-mediated CXCR4 induction

In the current study, we aimed to examine the function of scutellarin on human osteoblast proliferation and osteogenic function. The results indicated that scutellarin enhanced osteoblast proliferation over a seven day period. This increase in cell proliferation was associated with corresponding increases in osteoblast activity, as measured by alkaline phosphatase (ALP) secretion, intracellular calcium ion influx, and calcium deposition. These anabolic effects were associated with C-X-C chemokine receptor type 4 (CXCR4) mRNA levels and protein induction. Knockdown of CXCR4 reversed the scutellarin-induced increases in cell proliferation, ALP activity, and calcium deposition. Furthermore, scutellarin increased p65 phosphorylation in a dose-dependent manner, which resulted in the increased binding of phosphorylated p65 to the CXCR4 gene promoter region, to increase CXCR4 protein expression. p65 phosphorylation inhibition resulted in a decrease in CXCR4 protein expression. A p65 inhibitor blocked scutellarin-induced increases in osteoblast proliferation and function. Moreover, in a rat model of estrogen-deficient osteoporosis, scutellarin restored ovariectomy-induced bone loss in mice. Taken together, both cellular and animal models support the novel findings that scutellarin increased osteoblast proliferation and function through NF-κB/p65-mediated CXCR4 induction.

Identification of the effector domain of biglycan that facilitates BMP-2 osteogenic function

We have reported that recombinant biglycan (BGN) core protein accelerates bone formation in vivo by enhancing bone morphogenetic protein (BMP)-2 function. The purpose of the present study was to identify the specific domain (“effector”) within the BGN core protein that facilitates BMP-2 osteogenic function. Thus, we generated various recombinant and synthetic peptides corresponding to several domains of BGN, and tested their effects on BMP-2 functions in vitro. The results demonstrated that the leucine-rich repeats 2–3 domain (LRR2-3) of BGN significantly enhanced the BMP-2 induced Smad1/5/9 phosphorylation, osteogenic gene expression, and alkaline phosphatase activity in myogenic C2C12 cells. Furthermore, addition of LRR2-3 to osteoblastic MC3T3-E1 cells accelerated in vitro mineralization without compromising the quality of the mineral and matrix. These data indicate that LRR2-3 is, at least in part, responsible for BGN’s ability to enhance BMP-2 osteogenic function, and it could be useful for bone tissue regeneration.

CypD-mPTP axis regulates mitochondrial functions contributing to osteogenic dysfunction of MC3T3-E1 cells in inflammation.

Bone is a dynamic organ, the bone-forming osteoblasts and bone-resorbing osteoclasts form the physiological basis of bone remodeling process. During pathological process of numerous inflammatory diseases, these two aspects are uncoupled and the balance is usually tipped in favor of bone destruction. Evidence suggests that the inflammatory destruction of bone is mainly attributed to oxidative stress and is closely related to mitochondrial dysfunction. The mechanisms underlying osteogenic dysfunction in inflammation still need further investigation. Reactive oxygen species (ROS) is associated with mitochondrial dysfunction and cellular damage. Here, we reported an unexplored role of cyclophilin D (CypD), the major modulator of mitochondrial permeability transition pore (mPTP), and the CypD-mPTP axis in inflammation-induced mitochondrial dysfunction and bone damage. And the protective effects of knocking down CypD by siRNA interference or the addition of cyclosporin A (CsA), an inhibitor of CypD, were evidenced by rescued mitochondrial function and osteogenic function of osteoblast under tumor necrosis factor-α (TNF-α) treatment. These findings provide new insights into the role of CypD-mPTP-dependent mitochondrial pathway in the inflammatory bone injury. The protective effect of CsA or other moleculars affecting the mPTP formation may hold promise as a potential novel therapeutic strategy for inflammation-induced bone damage via mitochondrial pathways.

Using an Engineered Galvanic Redox System to Generate Positive Surface Potentials that Promote Osteogenic Functions.

Successful osseointegration of orthopaedic and orthodontic implants is dependent on a competition between osteogenesis and bacterial contamination on the implant-tissue interface. Previously, by taking advantage of the highly interactive capabilities of silver nanoparticles (AgNPs), we effectively introduced an antimicrobial effect to metal implant materials using an AgNP/poly(dl-lactic- co-glycolic acid) (PLGA) coating. Although electrical forces have been shown to promote osteogenesis, creating practical materials and devices capable of harnessing these forces to induce bone regeneration remains challenging. Here, we applied galvanic reduction-oxidation (redox) principles to engineer a nanoscale galvanic redox system between AgNPs and 316L stainless steel alloy (316L-SA). Characterized by scanning electron microscopy , energy-dispersive X-ray spectroscopy, atomic force microscopy, Kelvin probe force microscopy, and contact angle measurement, the surface properties of the yield AgNP/PLGA-coated 316L-SA (SNPSA) material presented a significantly increased positive surface potential, hydrophilicity, surface fractional polarity, and surface electron accepting/donating index. Importantly, in addition to its bactericidal property, SNPSA's surface demonstrated a novel osteogenic bioactivity by promoting peri-implant bone growth. This is the first report describing the conversion of a normally deleterious galvanic redox reaction into a biologically beneficial function on a biomedical metal material. Overall, this study details an innovative strategy to design multifunctional biomaterials using a controlled galvanic redox reaction, which has broad applications in material development and clinical practice.

Modulation of osteogenic and haemostatic activities by tuning cationicity of genipin-crosslinked chitosan hydrogels

Chitosan as a natural cationic polysaccharide has drawn wide interests as surface modification materials in orthopedic applications, with the potential to achieve combined osteogenic, antimicrobial and haemostatic functions. The cationicity of chitosan has been reported to play an important role in modifying the osteoblastic cell responses and the antibacterial activities, while its effect on the haemostatic properties has been rarely studied. To this aim, we prepared carboxymethyl chitosan hydrogels with different cationicity through crosslinking with different concentrations of genipin (1%, 2.5%, 5% and 10%). The genipin concentration strongly influenced both mesenchymal stem cell (MSC) responses and blood coagulation activity for chitosan-hydroxyapatite samples. Increasing genipin concentration overall enhanced the osteogenic and haemostatic potentials, and an optimum window of chitosan cationicity (5% genipin in our case) led to both the best MSC response and coagulant activities. In particular, the cationicity had demonstrated a profound modulation effect on the haemostatic activities of chitosan samples, through influencing three different aspects of the coagulation processes, including intrinsic coagulation pathway, aggregation and activation of platelet, and activation of erythrocyte. Tuning the crosslinking degree thus provides a simple and effective approach to achieving combined osteogenic and haemostatic functions, which has great potential in surface modification of surgical implants.

Polydopamine-assisted functionalization of heparin and vancomycin onto microarc-oxidized 3D printed porous Ti6Al4V for improved hemocompatibility, osteogenic and anti-infection potencies

Enhanced antiinfection activities, improved hemocompatibility and osteo-compatibility, and reinforced osseointegration are among the most important considerations in designing multifunctional orthopedic biomaterials. Hereby, anti-infective and osteogenic multifunctional 3D printed porous Ti6Al4V implant with excellent hemocompatibility was successfully designed and fabricated. In brief, osteogenic micro-arc oxidation (MAO) coatings with micro/nanoscale porous topography were generated in situ on 3D printed Ti6Al4V scaffolds, on which heparin and vancomycin were easily immobilized. The surface microstructure, morphology, and chemical compositions were characterized employing scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). High loading capacity and sustained vancomycin release profiles were revealed using high performance liquid chromatography (HPLC). Favorable antibacterial and antibiofilm performances against pathogenic Staphylococcus aureus ( S. aureus ) were validated in vitro through microbial viability assays, Live/Dead bacterial staining, and crystal violet staining. Human mesenchymal stem cells (hMSCs) were seeded on the scaffolds and their proliferation and viability were assessed using Cell Counting Kit and Live/Dead cell viability kit. Further, osteoblastic differentiation abilities were evaluated using alkaline phosphatase (ALP) activity as a hall marker. Additionally, the improved hemocompatibility of the heparinized scaffolds was confirmed by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT). Overall, our results show that the surface-modified 3D printed porous Ti6Al4V possesses balanced antibacterial and osteogenic functions while exhibiting extra anticlotting effects, boding well for future application in customized functional reconstruction of intricate bone defects.

Improved cell adhesion and osteogenesis using a PLTGA (poly l-lactide, 1,3-trimethylene carbonate, and glycolide) terpolymer by gelatin-assisted hydroxyapatite immobilization for bone regeneration.

A PLTGA (poly l-lactide, trimethylene carbonate, glycolide) terpolymer possesses great potential for orthopedic applications due to its excellent biocompatibility and controllable biodegradability. However, the unfavorable surface conditions of bare PLTGA such as its poor hydrophilicity and smooth morphology impede its clinical applications, highlighting the need for tailored surface modifications to improve its cytological behavior and osteogenic capacity. We herein develop a facile and effective strategy to deposit a gelatin/hydroxyapatite (GEL/HAP) hybrid coating onto the surface of PLTGA that involves consecutive chemical grafting and in situ reaction steps. Following the surface modification treatment, the resultant PLTGA scaffold with the GEL/HAP coating exhibited drastically improved hydrophilicity (79.1°vs. 48.2° in water contact angle) and increased surface roughness (18.4 vs. 267.9 nm, more than 14-fold, in root-mean-square roughness), respectively. In addition, preosteoblast (MC3T3-E1) cells were seeded onto the bare/modified PLTGA scaffold to evaluate biological performance, including cell adhesion, proliferation, mineralization and osteogenic differentiation. Based on these results, the GEL/HAP hybrid coating can endow the PLTGA terpolymer substrate with enhanced cell adhesion, proliferation and osteogenic functionality. Overall, post-treatment of PLTGA with the GEL/HAP hybrid coating may be a promising methodology in bone regeneration applications.

Mesenchymal Stem Cells Promote the Osteogenesis in Collagen-Induced Arthritic Mice through the Inhibition of TNF-α.

Objective To investigate the effects of umbilical cord mesenchymal stem cell (UC-MSC) transplantation on joint damage and osteoporosis in collagen-induced arthritis (CIA) mice and to explore the mechanisms by which UC-MSCs modulate the osteogenic differentiation. Methods CIA mice were divided into the following treated groups: UC-MSC transplantation group, antitumor necrosis factor- (TNF-) α group, and zoledronic acid (ZA) group. Microcomputed tomography (micro-CT) was used to analyze the bone morphology parameters. Osteogenic differentiation of treated CIA mice was determined. Bone marrow mesenchymal stem cells (BM-MSCs) from CIA mice were treated with TNF-α in vitro to explore their effects on osteogenesis. Results The arthritis score was significantly reduced in the UC-MSC transplantation and anti-TNF-α-treated CIA groups, compared with control mice (P < 0.001). Micro-CT showed that CIA mice developed osteoporosis at 12 weeks after immunization. The bone morphology parameters were partially improved in UC-MSC-treated CIA mice. Impaired osteogenic differentiation functions were indicated by decreased ALP activity (P < 0.001) and reduced mRNA and protein levels of osteogenic marker genes (P < 0.05) in CIA mice compared with DBA/1 mice. UC-MSC treatment significantly upregulated the impaired osteogenic differentiation ability in CIA mice. Meanwhile, the serum TNF-α level was decreased significantly in the UC-MSC group. The osteogenesis was reduced with the addition of TNF-α in vitro. Conclusion This study demonstrated that UC-MSC transplantation not only significantly improved the joint damage but also played a beneficial role in osteoporosis in CIA mice. Mechanistically, the improved osteogenic differentiation of CIA under UC-MSC treatment may be achieved by inhibition of TNF-α.

Oral epithelial cells inhibit the proliferation, mRNA expressions of collagen type I, RUNX2 and BGP, and ALP activity of osteoblast-like cells in coculture

Cell-to-cell interactions play important roles in regulating cell proliferation and differentiation, tissue production, organ construction and ontogenesis. The purpose of this study was to investigate the effects of oral epithelial (OE) cells on osteoblasts’ behavior for proliferation and osteogenic differentiation. Osteoblast-like cells were co-cultured in 6-well-dishes with or without OE cells growing on cell culture inserts. After 1, 3, 7 and 14 d, the characteristics of osteoblast-like cells were evaluated regarding cell proliferation, mRNA expression levels of collagen type I, RUNX2 and bone Gla protein (BGP), and alkaline phosphate (ALP) activity. In cell proliferation assays, the numbers of osteoblast-like cells cultured with or without OE cells increased over time for 2 w, but the proliferation of osteoblast-like cells co-cultured with OE cells plateaued after 7 d. The mRNA expression levels of type I collagen in osteoblast-like cells cultured with or without OE cells tended to decrease over time, but the decrease in osteoblast-like cells co-cultured with OE cells was more dramatic. The mRNA expression levels of RUNX2 and BGP in osteoblast-like cells cultured without OE cells tended to increase over time, but those increases in cells cultured without OE cells were more dramatic than in osteoblast-like cells cocultured with OE cells. The ALP activity of osteoblast-like cells cultured without OE cells increased until 7 d and then decreased at 14 d, but osteoblast-like cells cultured with OE cells underwent little change in ALP activity throughout the 14 d of culture. The ALP activity of osteoblast-like cells cultured without OE cells was significantly higher at 7 and 14 d than in osteoblast-like cells cultured with OE cells. In conclusion, these results indicate that OE cells inhibit the osteogenic differentiation and function of osteoblast-like cells.

Layered titanates with fibrous nanotopographic features as reservoir for bioactive ions to enhance osteogenesis

In this study, an osteogenic environment was constructed on Ti alloy implants by in-situ formation of nanosized fibrous titanate, Na2Ti6O13, loaded with bioactive ions, i.e. Sr, Mg and Zn, to enhance surface bioactivity. The bioactive ions were loaded by ion exchange with sodium located at inter-layer positions between the TiO6 slabs, and their release was not associated with the degradation of the structural unit of the titanate. In-vitro cell culture experiments using MC3T3-E1 cells proved that both bioactive ions and nanotopographic features are critical in promoting osteogenic differentiation of the cells. It was found that the osteogenic functions of the titanate can be modulated by the type and amount of ions incorporated. This study points out that nanosized fibrous titanate formed on the Ti alloy can be a promising reservoir for bioactive ions. The major advantage of this approach over other alternatives for bioactive ion delivery using degradable bioceramic coatings is its capacity of maintaining the structural integrity of the coating and thus avoiding structural deterioration and potential mechanical failure.

TGF-β1-Dependent Downregulation of HDAC9 Inhibits Maturation of Human Osteoblasts

Transforming growth factor β (TGF-β) is a key regulator of bone density. Recently, we have shown that TGF-β1 effectively blocks bone morphogenetic protein-induced maturation of human osteoblasts (hOBs) in a histone deacetylase (HDAC)-dependent manner. To better understand the underlying mechanisms and to identify possible therapeutic targets, the current study aimed at characterizing the expression changes of different HDACs in hOBs following recombinant human TGF-β1 treatment and investigating the effect of the altered HDACs on both the proliferation and maturation of hOBs and osteogenic cell lines. As expected from our previous work, exposure to rhTGF-β1 induced the expression of HDACs (HDAC1, -2, -3, -6). However, to our surprise, rhTGF-β1 treatment strongly suppressed the expression of HDAC9 during osteogenic differentiation. HDAC9 is reported to suppress osteoclastogenesis; however, little is known about the role of HDAC9 in osteogenesis. Chemical inhibition of HDAC9 with TMP269 increased cell numbers of hOBs, but significantly decreased their osteogenic function (alkaline phosphatase activity and matrix mineralization). In osteogenic cell lines (MG-63, CAL-72 and SAOS-2), the expression of HDAC9 negatively correlates with their proliferation capacity and positively correlates with their osteogenic differentiation potential. Being able to boost osteoclasts while inhibiting osteoblasts makes HDAC9 an interesting therapeutic target to support fracture healing and bone metabolisms.

Engineering vascularized and innervated bone biomaterials for improved skeletal tissue regeneration.

Blood vessels and nerve fibers are distributed throughout the entirety of skeletal tissue, and play important roles during bone development and fracture healing by supplying oxygen, nutrients, and cells. However, despite the successful development of bone mimetic materials that can replace damaged bone from a structural point of view, most of the available bone biomaterials often do not induce sufficient formation of blood vessels and nerves. In part, this is due to the difficulty of integrating and regulating multiple tissue types within artificial materials, which causes a gap between native skeletal tissue. Therefore, understanding the anatomy and underlying interaction mechanisms of blood vessels and nerve fibers in skeletal tissue is important to develop biomaterials that can recapitulate its complex microenvironment. In this perspective, we highlight the structure and osteogenic functions of the vascular and nervous system in bone, in a coupled manner. In addition, we discuss important design criteria for engineering vascularized, innervated, and neurovascularized bone implant materials, as well as recent advances in the development of such biomaterials. We expect that bone implant materials with neurovascularized networks can more accurately mimic native skeletal tissue and improve the regeneration of bone tissue.

A comparative study on the effect of promoting the osteogenic function of osteoblasts using isoflavones from Radix Astragalus.

Radix Astragalus has been shown to exert beneficial effects regarding the prevention postmenopausal osteoporosis. However, its mechanism of action remains to be investigated. Calycosin, formononetin, and calycosin-7-O-β-d-glucoside are the main isoflavone constituents of Astragalus. In this study, the abilities of these 3 compounds to promote osteogenic function of osteoblasts were compared, and the structure-activity relationships of these osteotrophic isoflavones were determined. Calycosin exhibited a greater effect than formononetin and calycosin-7-O-β-d-glucoside regarding improvements in osteogenic function of osteoblasts, as demonstrated by cell proliferation, alkaline phosphatase activity, collagen I and osteocalcin secretion, and the number and area of mineralized bone nodules. This suggests that calycosin may be better than formononetin and calycosin-7-O-β-d-glucoside at preserving bone mass. In addition, calycosin, formononetin, and calycosin-7-O-β-d-glucoside stimulate the expression of bone morphogenetic protein 2 and runt-related transcription factor 2 proteins, which indicates that all 3 agents may promote the osteogenesis of osteoblasts via regulation of bone morphogenetic protein 2 expression. In conclusion, calycosin may be the best candidate, with higher osteogenic activity than formononetin and calycosin-7-O-β-d-glucoside. The higher osteogenic activity of calycosin could be attributable to the superiority of its chemical structure (a hydroxyl group at position C3 of Ring B and no glucosyl group).

Promoting Effect of Pinostrobin on the Proliferation, Differentiation, and Mineralization of Murine Pre-osteoblastic MC3T3-E1 Cells.

Pinostrobin (PI), a natural flavonoid found in a variety of plants, is well known for its rich pharmacological activities. However, its osteogenic function remains unclear. The aim of this study is to evaluate the effect of PI on the proliferation, differentiation, and mineralization of murine pre-osteoblastic MC3T3-E1 cells in vitro using MTT, alkaline phosphatase (ALP) activity, the synthesis of collagen I (Col I) assay, and Von-Kossa staining, respectively. The expression of osteocalcin (OCN) mRNA in cells was detected by real-time PCR. The effect of PI on the differentiation of dexamethasone (DEX)-suppressed cells was also investigated. The results showed that PI greatly promoted the proliferation of MC3T3-E1 cells at 5–80 μg/mL (p < 0.05 or p < 0.01), and caused a significant elevation of ALP activity, Col I content, and mineralization of osteoblasts at 10–40 μg/mL (p < 0.05 or p < 0.01), and the expression levels of OCN gene were greatly upregulated after PI treatment (p < 0.01). Furthermore, PI could rescue the inhibition effect of cell differentiation induced by DEX. Taken together, these results indicated that PI could directly promote proliferation, differentiation, and mineralization of MC3T3-E1 cells and has potential for use as a natural treatment for osteoporosis.

Ontogenic Identification and Analysis of Mesenchymal Stromal Cell Populations during Mouse Limb and Long Bone Development.

SummaryBone-derived mesenchymal stromal cells (MSCs) differentiate into multiple lineages including chondro- and osteogenic fates and function in establishing the hematopoietic compartment of the bone marrow. Here, we analyze the emergence of different MSC types during mouse limb and long bone development. In particular, PDGFRαposSCA-1pos (PαS) cells and mouse skeletal stem cells (mSSCs) are detected within the PDGFRαposCD51pos (PαCD51) mesenchymal progenitors, which are the most abundant progenitors in early limb buds and developing long bones until birth. Long-bone-derived PαS cells and mSSCs are most prevalent in newborn mice, and molecular analysis shows that they constitute distinct progenitor populations from the earliest stages onward. Differential expression of CD90 and CD73 identifies four PαS subpopulations that display distinct chondro- and osteogenic differentiation potentials. Finally, we show that cartilage constructs generated from CD90pos PαS cells are remodeled into bone organoids encompassing functional endothelial and hematopoietic compartments, which makes these cells suited for bone tissue engineering.

RETRACTED ARTICLE: Tridax procumbens flavonoids: a prospective bioactive compound increased osteoblast differentiation and trabecular bone formation

BackgroundThe Tridax procumbens extracts (TPE) are known for their ethno-medicinal properties to increase osteogenic functioning in mesenchymal stem cells. Recently, we found that the T. procumbens flavonoids (TPF) significantly suppressed the RANKL-induced osteoclasts differentiation and bone resorption. The TPF also promoted osteoblasts differentiation and bone formation demonstrated by increasing bone formation markers in cultured mouse primary osteoblasts. However, the effects of the TPF on in vivo bone formation remain unclear. In this study, we investigated the effects of the TPF on in vivo bone formation, injected the TPF (20 mg/kg) twice a day in the low calcium diet mice and killed them after 21 day. Radiographic and histomorphometric analyses were performed on the dissected bones to determine the anabolic effects of the TPF.ResultsBone mineral density and bone mineral content of the TPF-treated mice were significantly increased compared to the control mice. Bone formation-related indices like osteoblast number, osteoblast surface, bone volume, mineralizing surface, mineral apposition rate and bone formation rate were significantly increased in the TPF-treated mice compared to the control mice.ConclusionOur findings point towards the stimulation of bone formation by TPF, suggested that the TPF could be a potential natural anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Gold nanoparticles in injectable calcium phosphate cement enhance osteogenic differentiation of human dental pulp stem cells

In this study, a novel calcium phosphate cement containing gold nanoparticles (GNP-CPC) was developed. Its osteogenic induction ability on human dental pulp stem cells (hDPSCs) was investigated for the first time. The incorporation of GNPs improved hDPSCs behavior on CPC, including better cell adhesion (about 2-fold increase in cell spreading) and proliferation, and enhanced osteogenic differentiation (about 2–3-fold increase at 14 days). GNPs endow CPC with micro-nano-structure, thus improving surface properties for cell adhesion and subsequent behaviors. In addition, GNPs released from GNP-CPC were internalized by hDPSCs, as verified by transmission electron microscopy (TEM), thus enhancing cell functions. The culture media containing GNPs enhanced the cellular activities of hDPSCs. This result was consistent with and supported the osteogenic induction results of GNP-CPC. In conclusion, GNP-CPC significantly enhanced the osteogenic functions of hDPSCs. GNPs are promising to modify CPC with nanotopography and work as bioactive additives thus enhance bone regeneration.

The prenylflavonoid Icaritin enhances osteoblast proliferation and function by signal transducer and activator of transcription factor 3 (STAT-3) regulation of C-X-C chemokine receptor type 4 (CXCR4) expression

In this study, we examined the effects of a natural prenylflavonoid Icaritin (ICT), on human osteoblast proliferation and osteogenic function. We observed that ICT dose-dependently enhanced osteoblast proliferation by ~15% over a 7day period. This increase in cell proliferation was associated with corresponding increases in osteoblast functions as measured by ALP secretion, intracellular calcium ions influx and calcium deposition. These anabolic effects were associated with a 4-fold increase in CXCR4 mRNA and protein expression. Silencing of CXCR4 protein expression using small interfering RNA reversed ICT-induced increase in cell proliferation, ALP activity and calcium deposition. Interestingly, we observed that ICT dose-dependently increased STAT-3 phosphorylation; and this resulted in increased binding of phosphorylated STAT-3 to the promoter region of the CXCR4 gene, to increase CXCR4 protein expression. Furthermore, we found that inhibition of STAT-3 phosphorylation resulted in a decrease in CXCR4 protein expression; whilst increasing phosphorylation of STAT-3 using a constitutive active STAT-3 vector significantly increased CXCR4 levels. Moreover, the chemical inhibition of STAT-3 phosphorylation annulled our previously observed ICT-induced increases of osteoblast proliferation and function. Finally, in a rat model of estrogen-deficient osteoporosis, ICT restored both osteoblasts numbers and CXCR4 expression. Taken together, both cellular and animal models support the novel findings that ICT; through the phosphorylation of STAT-3, up-regulated CXCR4, to increase osteoblast proliferation and function.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment.

LncRNA-H19 Modulates Wnt/β-catenin Signaling by Targeting Dkk4 in Hindlimb Unloaded Rat.

To investigate the biological functions of long noncoding RNA-H19 (H19) in the pathogenesis of disuse osteoporosis (DOP). Fifty-four male Sprague Dawley (SD) rats were randomly divided into three groups: baseline control (BC, 6), age-matched control (AC, 24), and hindlimb unloading (HLU, 24). The rats in the BC group were sacrificed at the beginning of the experiment, while the AC and HLU rats were sacrificed at different times (7, 14, 21 and 28 days after HLU). The DOP model was verified by micro-CT scan, and quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify the expression of osteogenic genes (OPG, RunX2 and OPG). Gene sequencing and bioinformatic analysis were performed to find H19 target genes and the associated signaling pathway, which were first verified on tissue samples. Further verification was performed by knocking down the H19 and related gene in rat osteoblast cell line (UMR106 cell). Then, the changes of associated signaling pathway and osteogenic function were examined to confirm the prediction of the bioinformatic analysis. Micro-CT scans and quantitative real-time polymerase chain reaction (qRT-PCR) tests showed progressively deteriorated trabecular bone and decreased level of osteogenic genes in the metaphysis of distal femur during HLU, indicating the successful establishment of a DOP model. According to RNA sequencing, 1351 mRNA and 464 lncRNA were abnormally expressed in response to mechanical unloading, in which the H19 decreased 2.86 fold in HLU rats. There were 1426 mRNA predicted to be the target genes of H19, and KEGG pathway analysis suggested that Wnt signaling pathway (Wnt signaling) was the top pathway responsible for these target genes. In the Wnt-associated genes targeted by H19, 11 were differentially expressed between HLU and AC rats, among which Dkk4 increased 2.44 fold in HLU rats when compared to normal controls. These results of sequencing and bioinformatic analysis were confirmed by the low expression of H19, overexpression of Dkk4 and inhibited Wnt signaling observed in DOP rats. Subsequent in vitro cell assay further demonstrated that knockdown of H19 led to upregulation of Dkk4, and inhibition of Wnt signaling and osteogenic function in UMR106 cell. These effects can be greatly reversed after application of knocking down Dkk4. Our findings demonstrated that low expression of H19, induced by mechanical unloading, leads to development of DOP through inhibition of Wnt signaling by promoting Dkk4 expression.