Human Osteoblast-like Cell Line Research Articles

Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes.

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.

Medical-grade polycaprolactone scaffolds made by melt electrospinning writing for oral bone regeneration \u2013 a pilot study in vitro

BackgroundThe spectrum of indications for the use of membranes and scaffolds in the field of oral and maxillofacial surgery includes, amongst others, guided bone regeneration (GBR). Currently available membrane systems face certain disadvantages such as difficult clinical handling, inconsistent degradation, undirected cell growth and a lack of stability that often complicate their application. Therefore, new membranes which can overcome these issues are of great interest in this field.MethodsIn this pilot study, we investigated polycaprolactone (PCL) scaffolds intended to enhance oral wound healing by means of melt electrospinning writing (MEW), which allowed for three-dimensional (3D) printing of micron scale fibers and very exact fiber placement. A singular set of box-shaped scaffolds of different sizes consisting of medical-grade PCL was examined and the scaffolds’ morphology was evaluated via scanning electron microscopy (SEM). Each prototype sample with box sizes of 225 μm, 300 μm, 375 μm, 450 μm and 500 μm was assessed for cytotoxicity and cell growth by seeding each scaffold with human osteoblast-like cell line MG63.ResultsAll scaffolds demonstrated good cytocompatibility according to cell viability, protein concentration, and cell number. SEM analysis revealed an exact fiber placement of the MEW scaffolds and the growth of viable MG63 cells on them. For the examined box-shaped scaffolds with pore sizes between 225 μm and 500 μm, a preferred box size for initial osteoblast attachment could not be found.ConclusionsThese well-defined 3D scaffolds consisting of medical-grade materials optimized for cell attachment and cell growth hold the key to a promising new approach in GBR in oral and maxillofacial surgery.

Effects of Nd:YAG low-level laser irradiation on cultured human osteoblasts migration and ATP production: in vitro study.

Low-level laser therapy has become one of the fastest growing fields of medicine in recent years. Many in vivo and in vitro studies have shown that laser irradiation activates a range of cellular processes in a variety of cell types and can promote tissue repair. However, few in vitro experiments have evaluated the effects of laser irradiation on cells in real time. The purpose of this study was to examine the effects of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on the migration of cultured human osteoblasts. A dedicated 96-well plate was used, and confluent cultures of the human osteoblast-like cell line, Saos-2, were injured with a wound maker. The wounded cells were then exposed to the Nd:YAG laser (wavelength of 1064nm) for 60s at 0.3W (10pps, 30mJ). The total energy density was about 10.34J/cm2. Images of the wounds were automatically acquired inside the CO2 incubator by the IncuCyte ZOOM™ software. In addition, after laser irradiation, the production of adenosine triphosphate (ATP) was measured using the CellTiter-Glo™ Luminescent Cell Viability Assay. Migration of cells from the border of the original scratch zone was accelerated by laser irradiation. In addition, compared with the control group, significant enhancement of ATP production was observed in the irradiated group. The present study showed that Nd:YAG laser irradiation (wavelength of 1064nm, 0.3W, 10 pps, 30mJ, 10.34J/cm2, irradiation time 60s) may contribute to the regeneration of bone tissues owing to enhanced osteoblast cell migration.

Plasma ion implantation enabled bio-functionalization of PEEK improves osteoblastic activity.

Slow appositional growth of bone in vivo is a major problem associated with polyether ether ketone (PEEK) based orthopaedic implants. Early stage promotion of osteoblast activity, particularly bone nodule formation, would help to improve contact between PEEK implantable materials and the surrounding bone tissue. To improve interactions with bone cells, we explored here the use of plasma immersion ion implantation (PIII) treatment of PEEK to covalently immobilize biomolecules to the surface. In this study, a single step process was used to covalently immobilize tropoelastin on the surface of PIII modified PEEK through reactions with radicals generated by the treatment. Improved bioactivity was observed using the human osteoblast-like cell line, SAOS-2. Cells on surfaces that were PIII-treated or tropoelastin-coated exhibited improved attachment, spreading, proliferation, and bone nodule formation compared to cells on untreated samples. Surfaces that were both PIII-treated and tropoelastin-coated triggered the most favorable osteoblast-like responses. Surface treatment or tropoelastin coating did not alter alkaline phosphatase gene expression and activity of bound cells but did influence the expression of other bone markers including osteocalcin, osteonectin, and collagen I. We conclude that the surface modification of PEEK improves osteoblast interactions, particularly with respect to bone apposition, and enhances the orthopedic utility of PEEK.

Involvement of bone morphogenetic protein–related pathways in the effect of aucubin on the promotion of osteoblast differentiation in MG63 cells

Aucubin, an iridoid glycoside found in several plants, such as Eucommia ulmoide and Rehmannia, has various pharmacological effects. Bone formation is a complex process in which osteoblast differentiation plays an important role. This study aimed to investigate the promotion effects of aucubin on osteoblast differentiation in MG63 cells, a human osteoblast-like cell line. Aucubin not only improved osteoblast differentiation, as shown by enhanced ALP (alkaline phosphatase) concentration and mineralization in cells, but increased the expression of various cytokines, including collagen I, osteocalcin, osteopontin, integrin β1, and Osterix. Aucubin strongly enhanced the levels of BMP2 (bone morphogenetic proteins-2) in MG63 cells, which play a central role during osteoblast differentiation. Further data show that aucubin exposure after 1 day, 7 days, and 14 days enhanced the expression of Smad1, 5, and 8, and the phosphoresced levels of MAPKs (mitogen-activated protein kinases) family Erk (extracellular signal–regulated kinases), JNK (c-Jun-NH2-terminal kinases), P38, and Akt (serine/threonine protein kinase)/mTOR (mammalian target of rapamycin)/p70s6k in MG63 cells. This study shows the improved effects of aucubin on osteoblast differentiation in MG63 cells, related to the signaling of BMP2-mediated Smads (drosophila mothers against decapentaplegic proteins), MAPKs, and Akt/mTOR/p70S6K. This study indicates the potential of aucubin for osteoporosis treatment.

Aryl Hydrocarbon Receptor Antagonists Mitigate the Effects of Dioxin on Critical Cellular Functions in Differentiating Human Osteoblast-Like Cells

The inhibition of bone healing in humans is a well-established effect associated with cigarette smoking, but the underlying mechanisms are still unclear. Recent work using animal cell lines have implicated the aryl hydrocarbon receptor (AhR) as a mediator of the anti-osteogenic effects of cigarette smoke, but the complexity of cigarette smoke mixtures makes understanding the mechanisms of action a major challenge. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a high-affinity AhR ligand that is frequently used to investigate biological processes impacted by AhR activation. Since there are dozens of AhR ligands present in cigarette smoke, we utilized dioxin as a prototype ligand to activate the receptor and explore its effects on pro-osteogenic biomarkers and other factors critical to osteogenesis using a human osteoblast-like cell line. We also explored the capacity for AhR antagonists to protect against dioxin action in this context. We found dioxin to inhibit osteogenic differentiation, whereas co-treatment with various AhR antagonists protected against dioxin action. Dioxin also negatively impacted cell adhesion with a corresponding reduction in the expression of integrin and cadherin proteins, which are known to be involved in this process. Similarly, the dioxin-mediated inhibition of cell migration correlated with reduced expression of the chemokine receptor CXCR4 and its ligand, CXCL12, and co-treatment with antagonists restored migratory capacity. Our results suggest that AhR activation may play a role in the bone regenerative response in humans exposed to AhR activators, such as those present in cigarette smoke. Given the similarity of our results using a human cell line to previous work done in murine cells, animal models may yield data relevant to the human setting. In addition, the AhR may represent a potential therapeutic target for orthopedic patients who smoke cigarettes, or those who are exposed to secondhand smoke or other environmental sources of aryl hydrocarbons.

Silicon bioceramic loaded with vancomycin stimulates bone tissue regeneration.

Porous ceramics doped with silicon and pure β-TCP were analyzed in terms of internal microstructure, cell behavior, and the percentage of newly formed bone. Additionally the materials were tested to determine which of the two had better properties to load and release vancomycin hydrochloride. Internal pore distribution and porosity were determined through high pressure mercury porosimetry and the specific surface area was measured by the Brunauer Emmet-Teller method. The proliferation and viability of the human osteoblast-like cell line MG-63 was studied to validate both materials. The materials were tested on eight New Zealand rabbits which created defects, 10 mm in diameter, in the calvaria bone. After 8 and 12 weeks a histological and histomorphometric analysis was performed. Si-β-TCP showed a higher porosity and specific surface area. The cytocompatibility test revealed acceptable results in terms of proliferation and viability whereas the percentage of new bone was higher in Si-β-TCP with a two-time study being statistically significant with 12 weeks of healing (p < 0.05).The vancomycin loaded within the ceramic scaffolds were burst released and the material had the ability to inhibit bacterial growth. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2307-2315, 2018.

Zoledronic Acid, Bevacizumab and Dexamethasone-Induced Apoptosis, Mitochondrial Oxidative Stress, and Calcium Signaling Are Decreased in Human Osteoblast-Like Cell Line by Selenium Treatment.

Increased intracellular free calcium ion (Ca2+) concentration induces excessive oxidative stress and apoptosis. Medical procedures such as zoledronic acid (Zol), bevacizumab (Bev), and dexamethasone (Dex) are usually used in the treatment of bone diseases (osteoporosis, Paget's disease, etc.) and to prevent metastasis in the bone although the procedures induce osteonecrosis of the jaw through excessive production of reactive oxygen species (ROS). Recently, we observed regulator roles of selenium (Se) on apoptosis and Ca2+ entry through transient receptor potential vanilloid 1 (TRPV1) channels in the cancer cell lines. Therefore, Se may modulate Zol, Bev, and Dex-induced oxidative stress and apoptosis through regulation of TRPV1 channel. In the current study, we investigated the protective effects of Se on apoptosis and oxidative stress through TRPV1 in Zol, Bev, and Dex-induced osteoblast-like cell line. We used human osteoblast-like cell line (Saos-2), and the cells were divided into 12 groups as control, Zol, Bev, Dex, Se, Zol+Se, Bev+Se, Dex+Se, Zol+Dex, Zol+Dex+Se, Zol+Bev, and Zol+Bev+Se which were incubated with drugs (Zol, Bev, Dex, and Se) for 24h. The cytosolic free Ca2+ concentration was increased by Zol, Bev, Dex, Zol+Bev, and Zol+Dex, although it was reduced by Se treatment. However, Zol, Bev, and Dex-induced increase in apoptosis, caspase 3, caspase 9, poly (ADP-ribose) polymerase 1 expression levels, and intracellular ROS production values in the cells were decreased by Se treatments. In conclusion, we observed that Zol, Bev, and Dex-induced apoptosis, mitochondrial oxidative stress, and calcium signaling are decreased in human osteoblast-like cell line by the Se treatment. Our findings may be relevant to the etiology and treatment of Zol, Bev, and Dex-induced osteonecrosis by Se.

Evaluation of selective laser sintered polyamide/hydroxyapatite composite compositions in vitro and in vivo

Objective: To study the suitability of the developed polyamide/hydroxyapatite (PA/HA) composite orthopaedic scaffold in vitro and in vivo. Methods: The fabricated scaffolds were seeded with human osteoblast-like cell line (MG 63) and tested for its viability & cytotoxicity, proliferation and calcium mineralization through MTT assay, ALP assay and Alizarin red S staining respectively. Finally, the scaffolds were implanted in non-immunocompromised male wistar rats subcutaneously and pathological tests were carried out. Results: The results of in vitro studies indicate that PA/HA (90:10) composite was non-cytotoxic and supported calcium mineralization relatively better than the other compositions of PA/HA. From the Hematoxylin & Eosin staining, inflammation was observed in the tissues surrounding the scaffold 7 weeks post implantation in rats. However, the scaffolds were found to be incorporated into the subcutaneous tissue at the end of one year without any rejection. Conclusion: PA/HA (90:10) composite scaffold fabricated using SLS is biocompatible when tested in rats, and could more likely be used for orthopedic applications.

Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines.

The aim of this study was to evaluate the cytotoxicity and bioactivity of calcium silicate-based cements combined with niobium oxide (Nb2O5) micro and nanoparticles, comparing the response in different cell lines. This evaluation used four cell lines: two primary cultures (human dental pulp cells - hDPCs and human dental follicle cells - hDFCs) and two immortalized cultures (human osteoblast-like cells - Saos-2 and mouse periodontal ligament cells - mPDL). The tested materials were: White Portland Cement (PC), mineral trioxide aggregate (MTA), white Portland cement combined with microparticles (PC/Nb2O5µ) or nanoparticles (PC/Nb2O5n) of niobium oxide (Nb2O5). Cytotoxicity was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue exclusion assays and bioactivity by alkaline phosphatase (ALP) enzyme activity. Results were analyzed by ANOVA and Tukey test (a=0.05). PC/Nb2O5n presented similar or higher cell viability than PC/Nb2O5µ in all cell lines. Moreover, the materials presented similar or higher cell viability than MTA. Saos-2 exhibited high ALP activity, highlighting PC/Nb2O5µ material at 7 days of exposure. In conclusion, calcium silicate cements combined with micro and nanoparticles of Nb2O5 presented cytocompatibility and bioactivity, demonstrating the potential of Nb2O5 as an alternative radiopacifier agent for these cements. The different cell lines had similar response to cytotoxicity evaluation of calcium silicate cements. However, bioactivity was more accurately detected in human osteoblast-like cell line, Saos-2.

P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts

The ionotropic ATP-gated P2X7 receptor (P2X7R) is involved in the regulation of many physiological functions including bone metabolism. Several studies on osteoblasts from rodents and human osteoblast-like cell lines have addressed the expression and function of P2X7R on these bone-forming cells however; its role in human primary osteoblasts has not yet been reported. The aim of this study was to assess the expression of the P2X7R in bone marrow-derived stromal cells and in primary human trabecular osteoblasts and to determine the function in bone formation and cell signaling. We report that osteoblasts derived from human trabecular explants express a functional P2X7R capable of agonist-induced increase in intracellular calcium concentration and a positive permeability to fluorescent dyes. These osteoblasts are fully differentiated cells with alkaline phosphatase activity and the ability to form mineralized nodules. We show that the transcriptional regulation of osteoblastic markers can be modulated by P2X7R activity or blockade thereby influencing the differentiation, proliferation and bone matrix formation by these primary human osteoblasts. Finally, we demonstrate that the P2X7R is involved in propagation of mechanically-induced intercellular signaling in addition to the known mechanisms involving calcium signaling via P2Y2 receptors and gap junction.

Potential of l-thyroxine to differentiate osteoblast-like cells via Angiopoietin1

Angiogenesis is closely associated with osteoblast differentiation. Previously, we demonstrated that bone formation can be accelerated by treatment with COMP-Angiopoietin1, a known angiogenic factor. Angiopoietin1 (Ang1) is a specific growth factor that generates stable and mature vasculature through the Tie2 receptor. In this study, we aimed to identify a novel drug that can activate endogenous Ang1 expression as a pharmacological treatment for bone formation. Therefore, Ang1 expression was examined in U2OS osteoblast-like cells treated with 770 drugs from a library of Food and Drug Administration (FDA)-approved drugs by using ELISA for Ang1. l-thyroxine was selected as a novel drug candidate. l-Thyroxine is a synthetic form of the hormone thyroxine, which is used to treat patients with hypothyroidism. Enzyme-linked immunosorbent assays (ELISAs) were performed to test whether Ang1 is induced in a dose-dependent manner in human osteoblast-like cell lines, U2OS and MG63. The effects of l-thyroxine on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and Alizarin red s staining. To determine the molecular mechanism, the expression of proteins related to bone formation and differentiation, such as type I collagen (COL1A1), osteocalcin (OC), bone sialoprotein (BSP), distal-less homeobox 5 (Dlx5), Runt-related transcription factor 2 (Runx2), osterix (OSX), and ALP, was tested by Western blotting analysis. Consequently, l-thyroxine induced Ang1 expression in a dose-dependent manner in both U2OS and M63 cells, which was confirmed by ELISA and Western blotting. Also, l-thyroxine activated ALP activity in U2OS and MG63 cells as well as ALP expression. Furthermore, l-thyroxine enhanced the expression of COL1A1, Runx2, OC, BSP, Dlx5, and OSX mRNA and proteins. Taken together, we demonstrated that l-thyroxine increased Ang1 expression and induces bone formation, differentiation, and mineralization in U2OS and MG63 cell lines, which suggests that l-thyroxine could be a potential bone production agent.

Pleiotropic roles of Ca+2/calmodulin-dependent pathways in regulating cadmium-induced toxicity in human osteoblast-like cell lines

The heavy metal cadmium is a widespread environmental contaminant that has gained public attention due to the global increase in cadmium-containing electronic waste. Human exposure to cadmium is linked to the pathogenesis of osteoporosis. We previously reported cadmium induces apoptosis and decreases alkaline phosphatase mRNA expression via extracellular signal-regulated protein kinase (ERK) activation in Saos-2 bone-forming osteoblasts. This study examines the mechanisms of cadmium-induced osteotoxicity by investigating roles of Ca+2/calmodulin-dependent protein kinase (CAMK) pathways. Saos-2 or MG-63 cells were treated for 24 or 48h with 5μM CdCl2 alone or in combination with calmodulin-dependent phosphodiesterase (PDE) inhibitor CGS-9343β; calmodulin-dependent kinase kinase (CAMKK) inhibitor STO-609; or calmodulin-dependent kinase II (CAMKII) inhibitor KN-93. CGS-9343β protected against cadmium-induced toxicity and attenuated ERK activation; STO-609 enhanced toxicity and exacerbated ERK activation, whereas KN-93 had no detectable effect on cadmium-induced toxicity. Furthermore, CGS-9343β co-treatment attenuated cadmium-induced apoptosis; but CGS-9343β did not recover cadmium-induced decrease in ALP activity. The major findings suggest the calmodulin-dependent PDE pathway facilitates cadmium-induced ERK activation leading to apoptosis, whereas the CAMKK pathway plays a protective role against cadmium-induced osteotoxicity via ERK signaling. This research distinguishes itself by identifying pleiotropic roles for CAMK pathways in mediating cadmium’s toxicity in osteoblasts.

Effect of physicochemical properties of a cement based on silicocarnotite/calcium silicate on in vitro cell adhesion and in vivo cement degradation

A silicon calcium phosphate cement (Si-CPC) was developed to produce a composite of calcium phosphate and calcium silicate. The silicon cements prepared with low silicon (Si) content were composed of crystalline phases of brushite and silicocarnotite. However, the cements prepared with high Si content were mainly composed of amorphous phases of silicocarnotite, hydroxyapatite and calcium silicate. The cement porosity was about 40% with a shift of the average pore diameter to the nanometric range with increasing Si content. Interestingly, this new cement system provides a matrix with a high specific surface area of up to 29 m2 g−1. The cytocompatibility of the new Si-doped cements was tested with a human osteoblast-like cell line (MG-63) showing an enhancement of cell proliferation (up to threefold) when compared with unsubstituted material. Cements with a high silica content also improved the cell attachment. The in vivo results indicated that Si-CPCs induce the formation of new bone tissue, and modify cement resorption. We conclude that this cement provides an optimal environment to enhance osteoblast growth and proliferation that could be of interest in bone engineering.

Biological influence of Ca/P ratio on calcium phosphate coatings by sol-gel processing

The objective of this work has been to develop low temperature sol-gel glass coatings to modify the substrate surface and to evaluate their bioactivity and biocompatibility. Glasses, based on SiO2·CaO·P2O5, were synthesized by the sol-gel technique using tetraethyl orthosilicate, calcium nitrate tetrahydrate and triethyl phosphate as precursors of SiO2, CaO and P2O5, respectively. Those materials, still in the sol phase, have been used to coat substrates by means of the dip-coating technique. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) has been used for characterize coatings and a microstructural analysis has been obtained using scanning electron microscopy (SEM). The potential applications of the coatings in the biomedical field were evaluated by bioactivity and biocompatibility tests. The coated substrate was immersed in simulated body fluid (SBF) for 21days and the hydroxyapatite deposition on its surface was subsequently evaluated via SEM-EDXS analysis, as an index of bone-bonding capability. In order to study the cell behavior and response to our silica based materials, prepared via the sol-gel method, with various Ca/P ratio and coating substrate, we have used the human osteoblast-like U2OS cell line.

Protective Effects of Black Hoof Medicinal Mushroom from Korea, Phellinus linteus (Higher Basidiomycetes), on Osteoporosis In Vitro and In Vivo.

The protective effect of Phellinus linteus (ethanol extract) against osteoporosis was investigated using the Saos-2 human osteoblast-like cell line and osteoclasts. A nontoxic concentration of Ph. linteus (10-2 to 10-8 mg mL-1) increased alkaline phosphatase (ALP) (EC 3.1.3.1) activity to a larger extent than soy did. Ph. linteus also attenuated the number and the activity of tartrate-resistant acid phosphatase-positive multinucleated osteoclasts. These results indicate that Ph. linteus likely regulated both osteoblasts and osteoclasts, contributing to the protection against osteoporosis. The protective effect of Ph. linteus was examined in ovariectomized (OVX) rats. Histological analysis indicates that Ph. linteus improved trabecular bone mass and reduced osteoclast frequency without affecting lipid droplet accumulation in the femur of OVX rats. A Ph. linteus supplementation for 12 weeks also significantly increased serum ALP activity and reduced urinary deoxypyridinoline level in OVX rats. Ultimately, we found that 12-week Ph. linteus supplementation increased the bone accumulation of minerals, including calcium, magnesium, and phosphorus. Collectively, Ph. linteus protected against osteoporosis by balancing the number of osteoblasts and osteoclasts, which was particularly associated with increased ALP activity in vitro and in vivo and mineral accumulation in bone.

Chitosan and carboxymethyl-chitosan capping ligands: Effects on the nucleation and growth of hydroxyapatite nanoparticles for producing biocomposite membranes

Synthetic biomaterials based on calcium phosphates (CaP) have been widely studied for bone tissue reconstruction therapies, but no definitive solution that fulfills all of the required properties has been identified. Thus, this study reports the synthesis of composite membranes based on nanohydroxyapatite particles (nHA) embedded in chitosan (CHI) and O-carboxymethyl chitosan (CMC) matrices produced using a one-step co-precipitation method in water media. Biopolymers were used as capping ligands for simultaneously controlling the nucleation and growth of the nHA particles during the precipitation process and also to form the polymeric network of the biocomposites. The bionanocomposites were extensively characterized using light microscopy (LM), scanning and transmission electron microscopy (SEM/TEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), atomic force microscopy (AFM), X-ray micro-CT analysis (μCT), and MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) cell proliferation assays for cell cytotoxicity. The results demonstrated that the ligands used during the synthesis highly affected the composites produced, primarily due the changes in the mechanisms and kinetics of nucleation and growth of the HA particles at the nanoscale level. The SEM images revealed that the use of carboxyl-functionalized chitosan (CMC) ligands significantly reduced the average size of the HA nanoparticles and caused the formation of a narrower size distribution (90±20nm) compared to the HA nanoparticles produced with chitosan ligands (220±50nm). The same trend was verified by the AFM analysis, where the nHA particles were formed evenly dispersed in the polymer matrix. However, the CMC-based composites were more homogeneously distributed, which was endorsed by the images collected via X-ray micro-CT. The FTIR spectra and the XRD analysis indicated that nanosized hydroxyapatite was the predominant calcium phosphate phase produced during the co-precipitation aqueous process for both the chitosan and CMC biocomposites. These novel hybrid systems based on chitosan and chitosan-derivatives with nHA composites were non-cytotoxic to a human osteoblast-like model cell line (SAOS) according to MTT in vitro assays. Moreover, the CMC-nHA biocomposites revealed a striking improvement in the cell viability response compared to the CHI-nHA biocomposite, which was attributed to the much higher surface area caused by the refinement of the nanoparticles size. Thus, the results of this study demonstrate that these novel bionanocomposite membranes offer promising perspectives as biomaterials for potential repair and replacement of cartilage and bone tissues.

A new iron calcium phosphate material to improve the osteoconductive properties of a biodegradable ceramic: a study in rabbit calvaria

β-tricalcium phosphate (β-TCP) is an osteoconductive and biodegradable material used in bone regeneration procedures, while iron has been suggested as a tool to improve the biological performance of calcium phosphate-based materials. However, the mechanisms of interaction between these materials and human cells are not fully understood. In order to clarify this relationship, we have studied the iron role in β-TCP ceramics. Iron-containing β-TCPs were prepared by replacing CaCO3 with C6H5FeO7 at different molar ratios. X-ray diffraction analysis indicated the occurrence of β-TCP as the sole phase in the pure β-TCP and iron-containing ceramics. The incorporation of iron ions in the β-TCP lattice decreased the specific surface area as the pore size was shifted toward meso- and/or macropores. Furthermore, the human osteoblastlike cell line MG-63 was cultured onto the ceramics to determine cell proliferation and viability, and it was observed that the iron-β-TCP ceramics have better cytocompatibility than pure β-TCP.Finally, in vivo assays were performed using rabbit calvaria as a bone model. The scaffolds were implanted for 8 and 12 weeks in the defects created in the skullcap with pure β-TCP as the control. The in vivo behavior, in terms of new bone formed, degradation, and residual graft material were investigated using sequential histological evaluations and histomorphometric analysis. The in vivo implantation of the ceramics showed enhanced bone tissue formation and scaffold degradation for iron-β-TCPs. Thus, iron appears to be a useful tool to enhance the osteoconductive properties of calcium phosphate ceramics.

Mesoporous Silica Nanoparticles Decorated with Carbosilane Dendrons as New Non-viral Oligonucleotide Delivery Carriers.

A novel nanosystem based on mesoporous silica nanoparticles covered with carbosilane dendrons grafted on the external surface of the nanoparticles is reported. This system is able to transport single-stranded oligonucleotide into cells, avoiding an electrostatic repulsion between the cell membrane and the negatively charged nucleic acids thanks to the cationic charge provided by the dendron coating under physiological conditions. Moreover, the presence of the highly ordered pore network inside the silica matrix would make possible to allocate other therapeutic agents within the mesopores with the aim of achieving a double delivery. First, carbosilane dendrons of second and third generation possessing ammonium or tertiary amine groups as peripheral functional groups were prepared. Hence, different strategies were tested in order to obtain their suitable grafting on the outer surface of the nanoparticles. As nucleic acid model, a single-stranded DNA oligonucleotide tagged with a fluorescent Cy3 moiety was used to evaluate the DNA adsorption capacity. The hybrid material functionalised with the third generation of a neutral dendron showed excellent DNA binding properties. Finally, the cytotoxicity as well as the capability to deliver DNA into cells, was tested in vitro by using a human osteoblast-like cell line, achieving good levels of internalisation of the vector DNA/carbosilane dendron-functionalised material without affecting the cellular viability.

The endocrine disruptor cadmium alters human osteoblast-like Saos-2 cells homeostasis in vitro by alteration of Wnt/β-catenin pathway and activation of caspases

The pollutant Cadmium (Cd) is widespread in the environment and causes alterations of human health by acting as an endocrine disruptor. Bone tissue seems to be a crucial target of Cd contamination. Indeed, we have previously demonstrated that this endocrine disruptor induces osteoblast apoptosis and necrosis. Thus, aim of this study was to further evaluate the effect of Cd on osteoblasts homeostasis, investigating potential modification of the Wnt/β-catenin intracellular pathway, the intracellular process involved in programmed cellular death and the cytoskeletal alterations. To this purpose, human osteoblastic Saos-2 cells, a human osteosarcoma osteoblast-like cell line, were cultured and treated with Cd. Osteoblastic cells were treated for 6 h with 10μM Cd, which induced nuclear translocation of β-catenin and increased expression of Wnt/β-catenin target genes. Longer exposure to the same Cd concentration induced osteoblastic cell apoptosis. To better characterize the intracellular events involved in these Cd-induced alterations, we evaluated the effect of Cd exposure on actin filaments and proteins associated to cytoskeletal actin, characterized by the presence of LIM domains. Long (15, 24 h) exposure of osteoblasts to Cd reduced LIM proteins expression and induced actin filaments destruction and a significant caspase-3 activation after 24 h. In addition, to prove that Cd induces osteoblastic cells apoptosis after long exposure, we performed TUNEL assay which demonstrated increase of cell apoptosis after 24 h. In conclusion, our study shows that osteoblasts exposed to Cd for short intervals of time demonstrated an increase in cell proliferation through a Wnt/β-catenin dependent mechanism, likely as a compensatory mechanism in response to cell injury. Longer exposure to the same Cd concentration induced cells apoptosis through cytoskeleton disruption-mediated mechanisms and caspase activation.