Mouse Osteoblastic Cells Research Articles

Fluorine substituted nano hydroxyapatite: Synthesis, bio-activity and antibacterial response study

In the present investigation, nano hydroxyapatite (HAp) and fluorine substituted hydroxyapatite (FHAp) with varying fluorine concentrations have been successfully prepared by hydrothermal method. The as synthesized powders were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), high resolution transmission electron microscope (HRTEM) and ion chromatography (IC) for fluorine content analysis. XRD results showed shifting of HAp peaks with increasing fluorine substitution and detected calcium fluoride (CaF2) phase in the synthesized powders where theoretically 100% or more OH ions substituted by F ions. The crystallinity of HAp powder gradually increased and cell volume decreased with fluorine substitution. HRTEM of the synthesized powders showed the nano rod like morphology. The IC was carried out to understand the degree fluoridation in the synthesized powder. The IC result of in vitro fluorine ion leaching showed that fluorine concentration in aqueous medium depends on the degree of fluoridation in the synthesized powder. The as synthesized powders were checked for antimicrobial activity by using both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) strains. The powder having ~33% fluorine substitution showed bactericidal effect. The in vitro fluorine ion release study showed significantly high fluorine concentration in aqueous medium for the HAp powders having more than 50% fluorine substituted. The in vitro cell viability assay using mouse osteoblast cell line (MC3T3-E1) confirmed that the powders were non-cytotoxic.

TRPM3/TRPV4 regulates Ca2+-mediated RANKL/NFATc1 expression in osteoblasts

Mechanical stress plays an important role in the regulation of bone turnover. However, the mechanism underlying hypo-osmotic stress-induced cellular response in osteoblasts remains poorly understood. In this study, we investigated the effect of hypotonic stress on the expression of bone remodeling factors, including the receptor activator of nuclear factor-kappa B ligand (RANKL) and the nuclear factor of activated T cells type c1 (NFATc1) in primary mouse osteoblasts and MC3T3-E1 cells. Hypo-osmotic stress induced significant increases in RANKL mRNA expression and intracellular Ca2+ concentration ([Ca2+]i) from the extracellular space. Hypo-osmotic stress-induced effects on [Ca2+]i and RANKL and NFATc1 protein expression were decreased by antagonists of transient receptor potential melastatin 3 (TRPM3) and vanilloid 4 (TRPV4). Agonists of TRPM3 and TRPV4 activated [Ca2+]i and RANKL and NFATc1 protein expression. Furthermore, genetic suppression of Trpm3 and Trpv4 reduced hypo-osmotic stress-induced effects in mouse osteoblasts. These results suggest that hypo-osmotic stress induces increases in [Ca2+]i through TRPM3 and TRPV4 to regulate RANKL and NFATc1 expression in mouse osteoblastic cells and that mechanical stress-activated TRP channels may play a critical role in bone remodeling.

Effects of aspirin-loaded graphene oxide coating of a titanium surface on proliferation and osteogenic differentiation of MC3T3-E1 cells

Graphene oxide (GO) has attracted considerable attention for biomedical applications such as drug delivery because of its two-dimensional structure, which provides a large surface area on both sides of the nanosheet. Here, a new method for titanium (Ti) surface modification involving a GO coating and aspirin (A) loading (A/Ti-GO) was developed, and the bioactive effects on mouse osteoblastic MC3T3-E1 cells were preliminarily studied. The X-ray photoelectron spectrometry indicated new C-O-N, C-Si-O-C, and C-N=C bond formation upon GO coating. Remarkably, the torsion test results showed stable bonding between the GO coating and Ti under a torsional shear force found in clinical settings, in that, there was no tearing or falling off of GO coating from the sample surface. More importantly, through π-π stacking interactions, the release of aspirin loaded on the surface of Ti-GO could sustain for 3 days. Furthermore, the A/Ti-GO surface displayed a significantly higher proliferation rate and differentiation of MC3T3-E1 cells into osteoblasts, which was confirmed by a water-soluble tetrazolium salt-8 (WST-8) assay and alkaline phosphatase activity test. Consequently, Ti surface modification involving GO coating and aspirin loading might be a useful contribution to improve the success rate of Ti implants in patients, especially in bone conditions.

Inhibition of osteoblast differentiation by ritonavir.

Osteoporosis is one of the chronic complications seen in human immunodeficiency virus (HIV)-infected patients, and affects patients at high prevalence. The causes of osteoporosis in HIV-infected patients are multiple, and include chronic HIV infection, living habits such as smoking and alcohol consumption, and antiretroviral drug use. Among antiretroviral drugs, protease inhibitors have been reported to be associated with osteoporosis. However, it remains to be determined how anti-HIV drugs affect osteoblast differentiation. In the current study, MC3T3-E1 cells, a mouse osteoblastic cell line, were cultured in osteoblast differentiation medium with or without different protease inhibitors (ritonavir, lopinavir, darunavir or atazanavir), and alkaline phosphatase (ALP) activity and the expression of Runt-related transcription factor 2 (Runx2) were analyzed. The ALP activity in MC3T3-E1 cells cultured with ritonavir was significantly reduced compared with that in cells in only osteoblast inducer reagent, indicating that ritonavir inhibited osteoblast differentiation. Meanwhile, ALP activity was not reduced in cells cultured with any of the other inhibitors. In addition, ritonavir inhibited the expression of Runx2, a key regulator of osteoblast differentiation, in the early period of osteoblast differentiation. To the best of our knowledge, this is the first study to demonstrate that ritonavir inhibits osteoblast differentiation in vitro. The present findings may explain the mechanism of osteopenia induced by combination antiretroviral therapy involving protease inhibitors.

Controlled Drug Release Property of Nano-Porous Silica Micro Particles and their Cytocompatibility

In this study, we investigated in cytocompativility and controlled release behavior of a model drug from nanoporous silica microparticles (NPSM). When Mouse osteoblastic cells (MC3T3-E1) were exposed to NPMSs, they indicated excellent cell viability. From NPSM contained in dental glass ionomer cement (NPMS-GIC), they can release cationic model drug molecules gradually in water for 2 weeks when they were contained in GIC. In contrast, GIC without NPSM specimens released the molecule only a couple of days. In the case of inionic molecules, their release were alos determined only a couple of days. These results suggest that NPSM can be used as a sustainable drug-release system in dental and medical fields

Effect of pore geometry on the fatigue properties and cell affinity of porous titanium scaffolds fabricated by selective laser melting

Porous titanium scaffolds with different unit cell type (tetrahedron and octahedron) and pore size (500 µm and 1000 µm) were fabricated by selective laser melting (SLM), and the effects of unit cell type and pore size on their fatigue properties and cell affinity were studied. The fatigue properties were performed by static and dynamic mechanical testing, while the cell affinity was evaluated in vitro with mouse osteoblast cells. It was found that octahedron scaffolds exhibited superior static mechanical properties, longer fatigue lives and higher fatigue strength in comparison to those of tetrahedron ones. As expected, scaffolds with 1000 µm pore resulted in lower compressive properties and shorter fatigue lives compared to those with 500 µm pore. The differences were analyzed based on the unit cell structure, porosity, and manufacturing imperfections. Scanning electron microscopy (SEM) and immunofluorescence showed that cells spread better on octahedron scaffolds than those on tetrahedron ones. Meanwhile, the scaffolds with 1000 µm pore were more suitable for cell attachment and growth within the same unit cell owing to higher porosity. The comparison of different pore geometry on the mechanical and biological property provided further insight into designing an optimal porous scaffold.

Quantitative and Dynamic Phase Imaging of Biological Cells by the Use of the Digital Holographic Microscopy Based on a Beam Displacer Unit

The digital holographic microscopy can be used for the quantitative and dynamic phase imaging of biological cells, which has a very wide application in the fields of biological and medical sciences. To make the measurement system simple, compact, and low-cost, a common-path configuration based on a beam displacer unit is introduced in the digital holographic microscopy. The simple optical structure and common-path design reduces the system requirement for the light source coherence, realizes the convenient adjustment of the object and reference beams and achieves an excellent system temporal stability of 0.53 nm. The living mouse osteoblastic cells during mitotic phase are quantitatively measured to demonstrate the capability and applicability of the system.

Probing Cytocompatibility, Hemocompatibility, and Quantitative Inflammatory Response in Mus musculus toward Oxide Bioceramic Wear Particulates and a Comparison with CoCr.

Peri-prosthetic bone resorption and loosening of artificial joints have been widely recognized to limit the performance of the load-bearing implants. Therefore, the present study probes into the cyto-, hemo-, and histocompatibility of the small sized wear particulates of ZrO2-toughened Al2O3 (ZTA). In order to develop a comprehensive, yet clinically relevant understanding, a comparison is made with two baseline ceramics (Al2O3 and ZrO2) and metallic material (CoCr), all in particulateforms. Whilein vitro cytotoxicity assessment was carried out with mouse osteoblast cells, preclinical testing of intra-articularly injected particulates up to the concentration 25 wt % in PBS over the period of 12 weeks was performed in mouse model. Interestingly, mouse osteoblast cells, cultured in media with ZTA, Al2O3, and ZrO2 particulates of three different concentrations (0.25, 2.5, and 25 mg/mL) exhibited uncompromised cell viability and considerable cell spreading, up to the time frame of 72 h. In contrast, similar experiments with CoCr particulates demonstrated significant decrease in cellular growth with drastic change in osteoblast proliferation behavior. Furthermore, the decrease in RBC damage after contact with ZTA, Al2O3, and ZrO2 particulates at 25 mg/mL of dose level illustrated 4.4, 5.8, and 1.25% hemolysis, respectively, confirming clinically acceptable hemocompatibility. However, hematic activity of CoCr particles was reflected with 15.6% of hemolysis. In vivo, the absence of any significant effect of intra-articularly injected ceramic (Al2O3, ZrO2, ZTA) as well as metallic (CoCr) particulates on complete hemogram and serum biochemistry of Balb/C mice was recorded at all the time points up to 12 weeks. The extensive histological analysis confirmed the absence of any signature of the tissue-level toxicity at all time points. The pro-inflammatory cytokine analysis using TNF-α and IL-1β markers provided complementary evidence toward nongranulomatous and nonimmunogenic response of synovial membrane of knee joint and other vital organs of mice that were exposed to Al2O3, ZrO2, ZTA, and CoCr particulates. Taken together, our results establish the nontoxic nature of oxide ceramic particulates to bone cells, in vitro, as well as to periprosthetic tissue, in vivo.

Abstract 2136: Transcriptome analysis of osteoblasts fused with cancer-derived exosomes

Abstract Breast cancer tumors have a high rate of metastasis and certain aggressive subtypes favor the bone environment. Clinically, it is estimated that metastatic breast cancer has a prevalence of up to 70% of bone tumors. Exosomes are important in cell-cell communication, and are increasingly being recognized as key contributors for priming targeted tissues for metastasis. Despite numerous advances in exosome detection and cargo characterization, transcriptional effects of cancer exosomes on targeted cells remain difficult to characterize at physiologically relevant levels. This is largely due to a combination of limited exosome secretion/uptake and an inability to segregate affected cells from the population. Conventional approaches to circumvent these limitations include spiking cell environments with exosome quantities far in excess of physiological concentrations. We sought to analyze the transcriptional effect of cancer exosomes on cells that have uptaken cancer-derived exosomes in a dynamic co-culture environment. In order to accomplish this, we first engineered a human breast cancer cell line (MDA-MB-231) to express GFP labeled exosomes. This novel cell line expresses three GFP fusion proteins for tetraspanin exosome markers (CD9, CD63, and CD81) known to be found on exosomes derived from MDA-MB-231 cells. Stable, intrinsic, fluorescent labeling of three exosome markers allows for an improved, more sensitive method for the analysis of exosome transfer. We can now examine transfer representing a more diverse pool of exosomes requiring no manipulation post exosome isolation prior to visualization. To investigate breast cancer exosome-mediated alterations on gene expression in bone cells to mimic breast cancer bone metastasis, this GFP exosome expressing line (MDA-MB-231 exo-GFP) was co-cultured with a mouse osteoblast cell line (MC3T3) then analyzed via flow cytometry to quantify the uptake of cancer exosomes in target cell population over time. MC3T3 cells that become GFP positive, indicative of exosome mediated transfer, increased from 0.91% of the population on day 3 post co-culture to 17.3% on day 14. Subsequent cell sorting and RNA-seq of GFP positive MC3T3 populations yielded novel insights into the progressive transcriptional effect of cancer exosomes on target cells. Future research will expand upon this approach to examine the effects on bone cells non-metastatic and metastatic cancer cell lines have on the metastatic niche through exosome-mediated signaling. This approach introduces a novel method for prolonged transcriptional analysis of cancer and target cell co-culture allowing the segregation of exosome affected and unaffected subpopulations of target cells. This study was funded by DOD grant BC151687. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). Citation Format: Nicholas H. Hum, Kelly A. Martin, Gabriela G. Loots. Transcriptome analysis of osteoblasts fused with cancer-derived exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2136.

Determination of ideal Mg–35Zn–xCa alloy depending on Ca concentration for biomaterials

Mg is a potential material for orthopedic and craniofacial implants. However, the failure of peri-implant osseointegration often occurs because pure Mg corrodes rapidly and has low mechanical strength. This study aimed to overcome these limitations via the development of various Mg-35Zn-xCa alloys. Electrochemical and immersion corrosion of these alloys were examined in simulated body fluid, and their cytotoxicity (cell proliferation and morphology) was assessed in a mouse osteoblast cell (MC3T3-E1). Structural analysis revealed that the Mg–35Zn–xCa alloys had a dendritic composition. The volume fractions of the second phases (Mg2Ca and Ca2Mg6Zn3) in the alloys increased with an increase in Ca concentration. The Mg–35Zn–xCa (x = 2, 3) alloys displayed greater hardness values (p < 0.05) and better corrosion resistance than the others; the Mg–35Zn–2Ca displayed the highest Ecorr and lowest Icorr value. Cells of all groups had extended filopodia, thereby indicating that cell adhesion was intact. At each time point, the number of attached cells was the highest in the extracted media of Mg–35Zn–2Ca alloy, indicating that this material has good osteoconductivity. Based on the mechanical, corrosive, and biological properties, the Mg–35Zn–2Ca alloy can be a useful biomaterial for orthopedic implants.

Characterizations of hyaluronate-based terpolymeric hydrogel synthesized via free radical polymerization mechanism for biomedical applications.

In the present study, a novel terpolymeric hydrogel was developed using sodium hyaluronate (HA), 2-hydroxyethyl acrylate (2-HEA), and poly(ethylene glycol) diacrylate (PEGDA) via free radical polymerization for biomedical applications. To achieve elasticity, swelling ability, porous architecture and sufficient gel strength, hyaluronate was chemically modified by grafting and crosslinking methods using 2-HEA and PEGDA, respectively. The structure and compositions of the fabricated terpolymer (HA-g-p(2-HEA)-x-PEGDA) were verified by FTIR, 1H HR-MAS-NMR, and TGA analyses. The surface morphology and cross-section of the hydrogel was detected by SEM analysis. The gel nature of terpolymer in aqueous medium at 37 °C was confirmed from swelling study, and rheological experiment. Non-cytotoxicity and biocompatibility of the HA-g-p(2-HEA)-x-PEGDA hydrogel were ascertained by in vitro mouse osteoblastic cells (MC3T3) proliferation, and viability studies. Hematoxylin and eosin Y, and Masson's trichrome stainings were performed to show tissue regeneration ability on the prepared hydrogel. In vitro release results of proangiogenic drug-dimethyloxalylglycine (DMOG), and antibiotics-tetracycline (TCN) showed sustained release behaviour from the prepared hydrogel under different pHs at 37 °C. The mathematical models fitted data imply that both DMOG and TCN release follow first order kinetics, while, the release mechanism is primarily controlled by diffusion as well as erosion process. Finally, the novel biocompatible HA-g-p(2-HEA)-x-PEGDA gel, which showed sustained drugs release, and regeneration ability of extracellular matrix and collagen, could be employed in biomedical applications, especially, for the delivery of DMOG/TCN, and in tissue engineering.

A new insight into cystatin C contained in milk basic protein to bone metabolism: Effects on osteoclasts and osteoblastic MC3T3-E1 cells invitro.

A milk protein fraction possessing alkaline isoelectric points (milk basic protein [MBP]) improves bone metabolism invivo, and it inhibits bone resorption by osteoclasts and promotes mouse osteoblastic MC3T3-E1 cells proliferation invitro. Cystatin C (CysC) is a component of MBP and shows bone resorption inhibitory activity. Therefore, it is likely that MBP with higher CysC content improves bone metabolism more effectively. In this study, we prepared MBP with low and high contents of CysC and compared its effects on bone metabolism with standard MBP invitro. Our results showed that the CysC content in MBP was positively related to not only bone resorption inhibitory activity but also MC3T3-E1 cells proliferative activity. Furthermore, purified CysC stimulated MC3T3-E1 cells proliferation. These results indicate that CysC contributes to promotion of MC3T3-E1 cells proliferation, and MBP with higher CysC content shows enhanced bone resorption inhibitory activity and MC3T3-E1 cells proliferative activity. CysC is considered an important factor in the effect on bone metabolism of MBP.

Graphene Oxide-A Tool for the Preparation of Chemically Crosslinking Free Alginate-Chitosan-Collagen Scaffolds for Bone Tissue Engineering.

Developing a biodegradable scaffold remains a major challenge in bone tissue engineering. This study was aimed at developing novel alginate-chitosan-collagen (SA-CS-Col)-based composite scaffolds consisting of graphene oxide (GO) to enrich porous structures, elicited by the freeze-drying technique. To characterize porosity, water absorption, and compressive modulus, GO scaffolds (SA-CS-Col-GO) were prepared with and without Ca2+-mediated crosslinking (chemical crosslinking) and analyzed using Raman, Fourier transform infrared (FTIR), X-ray diffraction (XRD), and scanning electron microscopy techniques. The incorporation of GO into the SA-CS-Col matrix increased both crosslinking density as indicated by the reduction of crystalline peaks in the XRD patterns and polyelectrolyte ion complex as confirmed by FTIR. GO scaffolds showed increased mechanical properties which were further increased for chemically crosslinked scaffolds. All scaffolds exhibited interconnected pores of 10-250 μm range. By increasing the crosslinking density with Ca2+, a decrease in the porosity/swelling ratio was observed. Moreover, the SA-CS-Col-GO scaffold with or without chemical crosslinking was more stable as compared to SA-CS or SA-CS-Col scaffolds when placed in aqueous solution. To perform in vitro biochemical studies, mouse osteoblast cells were grown on various scaffolds and evaluated for cell proliferation by using MTT assay and mineralization and differentiation by alizarin red S staining. These measurements showed a significant increase for cells attached to the SA-CS-Col-GO scaffold compared to SA-CS or SA-CS-Col composites. However, chemical crosslinking of SA-CS-Col-GO showed no effect on the osteogenic ability of osteoblasts. These studies indicate the potential use of GO to prepare free SA-CS-Col scaffolds with preserved porous structure with elongated Col fibrils and that these composites, which are biocompatible and stable in a biological medium, could be used for application in engineering bone tissues.

Egg White Ovotransferrin Shows Osteogenic Activity in Osteoblast Cells.

Ovotransferrin, the major protein in egg white, is a member of transferrin family. The objective of this study was to study the effects of ovotransferrin on cell proliferation, differentiation, mineralization and osteoclastogenesis of bone osteoblast cells. Effect of ovotransferrin (concentrations ranging from 1 to 1000 μg/mL) on the proliferation, differentiation, and mineralization of mouse osteoblast cells MC3T3-E1 was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay, Western blot, immunofluorescence, and Alizarin-S red staining, respectively. Our results showed that ovotransferrin stimulated cell proliferation (enhanced BrdU incorporation), differentiation (enhanced expression of alkaline phosphatase and type-I collagen), and mineralization (increased calcium deposits) in a dose-dependent manner. Furthermore, ovotransferrin could increase the expression of osteoprotegerin (OPG) while decreasing the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), suggesting its role in inhibition of bone resorption. This study demonstrated for the first time that ovotransferrin might promote bone formation while preventing bone resorption, which might open up a new application of egg white protein ovotransferrin as a functional ingredient in bone health management.

TGF-β1 acts as mediator in fluoride-induced autophagy in the mouse osteoblast cells

It is well known that excess fluoride intake can result in fluorosis, which is a serious public health problem. TGF-β1 affects a wide variety of cellular activities and plays an important role in fluorosis. Recent literature proved that fluoride induces autophagy, however, the mechanism is still unclear, and the role of TGF-β1 in the fluoride-induced autophagy should be further illustrated. Therefore, in this study, plasmids and small interfering RNA (siRNA) were used to overexpress and silence the TGF-β1 expression in the osteoblasts cells. Our results demonstrated that sodium fluoride (NaF) (2.26 mg/L F-) exposure lead to a significant decrease in proliferation rate of mouse osteoblast cells in a dose-dependent manner with a concomitant increase in the expression levels of TGF-β1 and autophagic markers. Overexpression of TGF-β1 significantly increased the fluorescence intensity of MDCstained cells, the mRNA and protein expression levels of Beclin1 and LC3-II/I, with a concomitant decrease in p62. However, TGF-β1 silencing resulted in vice versa. In summary, we concluded that TGF-β1 plays a mediator role in NaF-induced autophagy of mouse osteoblast cells. In the future, TGF-β1-autophagy signaling network could be a new idea for the investigation of skeletal fluorosis.

Understanding the functional role of genistein in the bone differentiation in mouse osteoblastic cell line MC3T3-E1 by RNA-seq analysis

Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. To obtain insight into the effect of genistein on bone differentiation, RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell differentiation was monitored by measuring osteoblast differentiation factors (ALP production, bone mineralization, and expression of osteoblast differentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identified in genistein-treated cells (FDR q-value < 0.05 and fold change > 1.5). KEGG pathway and Gene Ontology (GO) enrichment analyses were performed to estimate the biological functions of DEGs and demonstrated that these DEGs were highly enriched in functions related to chemotactic cytokines. The functional relevance of DEGs to genistein-induced osteoblastic cell differentiation was further evaluated by siRNA-mediated knockdown in MC3T3-E1 cells. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation. These results imply their major functional roles in bone differentiation regulated by genistein.

Direct-writing of 3D periodic TiO2 bio-ceramic scaffolds with a sol-gel ink for in vitro cell growth

In this work, three-dimensional (3D) periodic TiO2 bio-ceramic scaffolds were created by continuous filament writing with TiO2 sol-gel ink followed by sintering. The TiO2 sol-gel ink has the advantage of creating 3D bio-ceramic scaffolds with finer feature size than commonly used ceramic powder based ink systems. The structure, pore sizes, and interconnectivity could be controlled through 3D structure design and printing parameter adjustment. To evaluate the potential of these 3D TiO2 bio-ceramic scaffolds for bone tissue engineering, the mouse osteoblastic cell line MC3T3-E1 was cultured on them. The observed good viability demonstrated that 3D TiO2 bio-ceramic scaffolds created by direct-writing technique provide a biocompatible environment that favors cell growth and attachment.

(Fe/Sr) Codoped Biphasic Calcium Phosphate with Tailored Osteoblast Cell Functionality.

Although doped bioceramics have been widely investigated for biomedical applications, the codoped bioceramics remain mostly unexplored for bone regeneration applications. For example, the impact of codoping of Sr2+ and Fe3+ ions on the phase stability and cytocompatibility is not explored so far. In this perspective, the objective of the present study is to quantitatively understand this aspect in case of Fe/Sr codoped biphasic calcium phosphate (BCP). Following sol-gel synthesis, codoped BCP samples with Sr/Fe dopant concentrations of 2, 10, 20, 30, and 40 mol % as well as doped BCPs with single dopant (Sr or Fe) with similar compositions were calcined at 800 °C in air. Using extensive Rietveld analysis, the dopant content dependent crystallographic properties (e.g lattice parameters) and phase stability of HA/TCP are quantitatively assessed. In vitro cytocompatibility of codoped samples has been assessed using mouse osteoblast cells. An important observation is that, while singular dopant of Sr/Fe at 20 mol % or higher amount reduces cell viability significantly, osteoblast viability is not compromised to any significant extent on Sr/Fe codoped BCP, compared to undoped BCP. Our results indicate that one can tailor osteoblast functionality by controlling the codopant content. More importantly, all the codoped BCPs support cell proliferation, when single doped BCP exhibits significant reductionin cell viability, at dopant content of 10 mol % or higher. Cell morphological analysis supports extensive cell spreading on codoped BCPs. An attempt has been made to correlate the variation in cellular response with HA/TCP ratio and ion dissolution behavior. Taken together, the present work establishes unique advantage of Sr/Fe codoping approach toward realizing their bone replacement application.

The potential use of thermosensitive chitosan/silk sericin hydrogels loaded with longan seed extract for bone tissue engineering.

In this study, hydrogels that were thermosensitive at body temperature were developed using chitosan (CS)/silk sericin (SS)/β-glycerophosphate (β-GP) loaded with longan seed extract (LE) for use in bone tissue engineering. These hydrogels were transformed into a gel at 37 °C within 10 min via interactions between CS and β-GP. The incorporation of SS resulted in a shorter gelation time of 5–7 min. The morphological structure of the thermosensitive CS/β-GP hydrogels exhibited an irregular pore structure, whereas the morphological structure of the thermosensitive CS/SS/β-GP hydrogels became more slender and porous. The incorporation of SS affected the network structure of the CS hydrogels, which degraded more rapidly. Moreover, the cumulative amounts of both gallic acid (GA) and ellagic acid (EA) released from the hydrogels loaded with LE increased with an increase in the SS content. Finally, these thermosensitive hydrogels were non-toxic to both a mouse fibroblast cell line (NCTC clone 929) and a mouse osteoblast cell line (MC3T3-E1) and promoted the attachment of MC3T3-E1 cells to the surface of the hydrogels. Therefore, these thermosensitive hydrogels might be a promising candidate for bone tissue engineering.

Selective Androgen Receptor Modulator, YK11, Up-Regulates Osteoblastic Proliferation and Differentiation in MC3T3-E1 Cells.

Androgens are key regulators that play a critical role in the male reproductive system and have anabolic effects on bone mineral density and skeletal muscle mass. We have previously reported that YK11 is a novel selective androgen receptor modulator (SARM) and induces myogenic differentiation and selective gene regulation. In this study, we show that treatment of YK11 and dihydrotestosterone (DHT) accelerated cell proliferation and mineralization in MC3T3-E1 mouse osteoblast cells. Further, YK11-treated cells increased osteoblast specific differentiation markers, such as osteoprotegerin and osteocalcin, compared to untreated cells. These observations were attenuated by androgen receptor (AR) antagonist treatment. To clarify the effect of YK11, we investigated rapid non-genomic signaling by AR. The phosphorylated Akt protein level was increased by YK11 and DHT treatment, suggesting that YK11 activates Akt-signaling via non-genomic signaling of AR. Because it is known Akt-signaling is a key regulator of androgen-mediated osteoblast differentiation, YK11 has osteogenic activity as well as androgen.