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In this study, nanostructured hydroxyapatite (HAp) was successfully extracted from the bones of Priacanthus macracanthus (Red Big Eye), a species commonly processed in the fish industry, generating significant waste. The extraction process utilized an alkaline hydrolysis method optimized with 2 M sodium hydroxide at 250 °C for 5 h, producing high-purity HAp. Fourier transform infrared spectroscopy (FT-IR) confirmed the presence of characteristic phosphate peaks at 1044 cm⁻¹ and 963 cm⁻¹, and hydroxyl peaks at 632 cm⁻¹. Powder X-ray diffraction (XRD) analysis showed prominent peaks at 2θ values of 25.9°, 32.2°, 39.8°, and 46.7°, corresponding to the crystalline planes of HAp. Field-emission scanning electron microscopy (FESEM) revealed spherical HAp particles with sizes ranging from 50 to 80 nm. Biocompatibility was assessed using human osteoblast-like MG-63 cells, showing a proliferation rate of 92 % compared to the control. Cytotoxicity tests indicated no significant adverse effects, supporting the potential use of this HAp in biomedical applications. Importantly, this method offers a sustainable solution for managing fish bone waste, contributing to pollution control by reducing environmental burdens associated with discarded bone wastes. Future research will focus on in vivo biocompatibility studies and exploring applications in pollution mitigation and tissue engineering. This study highlights the dual benefits of utilizing biowaste for valuable HAp production and addressing environmental pollution challenges, making it a promising approach for sustainable material synthesis and environmental management. The cost-effective and environmentally friendly process further underscores the feasibility of scaling up this method for industrial applications, providing a greener alternative to conventional HAp synthesis.

Objectives: Our study aimed to clarify the role of mitogen-activated protein kinases (MAPKs) in transforming growth factor (TGF)-β1-stimulated mineralization in the human osteoblast-like MG63 cells. The viability of MG63 cells under TGF-β1 stimulation was assessed by MTS assay. Western blotting determined TGF-β1-mediated activation of extracellular signal-related protein kinase (ERK), p38, and c-Jun amino-terminal kinase (JNK). Mineralization-related gene expression was examined by quantitative real-time PCR, and mineral deposition levels were evaluated by alizarin red S staining. TGF-β1 had no effect on MG63 cell proliferation. Activation of p38 was observed at 3 h post TGF-β1 stimulation. Moreover, JNK phosphorylation was upregulated by TGF-β1 from 1 to 6 h post stimulation, but had no activation on ERK phosphorylation throughout the experimental period. Treatment with JNK inhibitor diminished the alizarin red S-stained area in a dose-dependent manner. Mineral deposition was unaffected by MEK inhibitor, whereas p38 inhibitor increased the red-stained area. Gene expression levels of ALP and BSP were significantly decreased under treatment with JNK inhibitor and p38 inhibitor. The MEK inhibitor had no effect on the TGF-β1–mediated upregulation of ALP and BSP. Although all three inhibitors suppressed expression of COL I , none were found to stimulate expression of OCN. Human osteoblast-like MG63 cells maturation and mineralization are induced through JNK activation of MAPK signaling in response to TGF-β1. [ABSTRACT FROM AUTHOR]

Different signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to the calcium-related pathway of phosphoinositide specific phospholipase C (PLC) enzyme family. PLC enzymes were demonstrated to be involved in the differentiation of osteoblasts and differently localize in the nucleus, cytoplasm or both depending on the isoform. The amino-steroid molecule U-73122 inhibits the enzymes belonging to the PLC family. In addition to the temporary block of the enzymatic activity, U-73122 promotes off-target effects, including modulation of the expression of selected PLC genes and different localization of PLC enzymes, depending on the cell line, in different cell lines. In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of PLC genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122. Our results confirm that all PLC genes are transcribed in hOBs, that probably splicing variants of selected PLC genes are expressed and that all PLC enzymes are present in hOBs, except for PLC δ3 in quiescent hOBs at seeding. Our results confirm literature data excluding toxicity of U-73122 on cell survival. Our results indicate that U-73122 did not significantly affect the transcription of PLC genes. It acts upon the localization of PLC enzymes, as PLC enzymes are detected in cell protrusions or pseudopodia-like structures, at the nuclear or the plasma membrane, in membrane ruffles and/or in the endoplasmic reticulum.

Different signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to the calcium-related pathway of phosphoinositide specific phospholipase C (PLC) enzyme family. PLC enzymes were demonstrated to be involved in the differentiation of osteoblasts and differently localize in the nucleus, cytoplasm or both depending on the isoform. The amino-steroid molecule U-73122 inhibits the enzymes belonging to the PLC family. In addition to the temporary block of the enzymatic activity, U-73122 promotes off-target effects, including modulation of the expression of selected PLC genes and different localization of PLC enzymes, depending on the cell line, in different cell lines. In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of PLC genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122. Our results confirm that all PLC genes are transcribed in hOBs, that probably splicing variants of selected PLC genes are expressed and that all PLC enzymes are present in hOBs, except for PLC ?3 in quiescent hOBs at seeding. Our results confirm literature data excluding toxicity of U-73122 on cell survival. Our results indicate that U-73122 did not significantly affect the transcription of PLC genes. It acts upon the localization of PLC enzymes, as PLC enzymes are detected in cell protrusions or pseudopodia-like structures, at the nuclear or the plasma membrane, in membrane ruffles and/or in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Synergy between biomaterial surfaces and cells is known to be important due to the direct and inevitable interactions that mediate cell behavior. Thus, the design of biomimetic surfaces with proper topography and chemistry is crucial for optimization of cellular responses. Herein, we report surface topography mimicking ability of chitosan (CH) biopolymer and its promising application as a platform for osteoblast cell culture. CH is frequently used in bone tissue engineering applications. For this reason, anisotropic bone surface was chosen to demonstrate its surface mimicking skill. Initially, bone surface topography is replicated by using soft lithography and polydimethylsiloxane (PDMS) molds. Subsequently, solvent casting by CH is performed on the replicated molds, and then polymer membranes with bone surface topography are obtained. To prepare nanocomposite, graphene oxide (GO) is blended into CH membranes to enhance biocompatibility. It is observed that CH and CH/GO nanocomposite membranes are both eligible to mimic anisotropic bone surface. Considering the surface of bone tissue, hydroxyapatite (HA) modification is also conducted using ultraviolet/ozone method. Following that, human osteoblasts are chosen to evaluate the cell responses on mimicked surfaces. The results indicate that surface mimicking has a positive impact on osteoblast viability and morphology. [ABSTRACT FROM AUTHOR]

Bacterial internalization is a strategy that non-intracellular microorganisms use to escape the host immune system and survive inside the human body. Among bacterial species, Staphylococcus aureus showed the ability to interact with and infect osteoblasts, causing osteomyelitis as well as bone and joint infection, while also becoming increasingly resistant to antibiotic therapy and a reservoir of bacteria that can make the infection difficult to cure. Despite being a serious issue in orthopedic surgery, little is known about the mechanisms that allow bacteria to enter and survive inside the osteoblasts, due to the lack of consistent experimental models. In this review, we describe the current knowledge about S. aureus internalization mechanisms and various aspects of the interaction between bacteria and osteoblasts (e.g., best experimental conditions, bacteria-induced damages and immune system response), focusing on studies performed using the MG-63 osteoblastic cell line, the best traditional (2D) model for the study of this phenomenon to date. At the same time, as it has been widely demonstrated that 2D culture systems are not completely indicative of the dynamic environment in vivo, and more recent 3D models—representative of bone infection—have also been investigated.

(1) In order to enable a more widespread use of uncemented titanium-based endoprostheses to replace cobalt-containing cemented endoprostheses for joint replacement, it is essential to achieve optimal osseointegrative properties and develop economic fabrication processes while retaining the highest biomedical quality of titanium materials. One approach is the usage of an optimized form of Ti6Al4V-precision casting for manufacturing. Besides the chemical and physical properties, it is necessary to investigate possible biological influences in order to test whether the new manufacturing process is equivalent to conventional methods. (2) Methods: Primary human osteoblasts were seeded on discs, which were produced by a novel Ti6Al4V centrifugal-casting process in comparison with standard machined discs of the same titanium alloy. In a second step, the surfaces were modified by calcium or phosphorus ion beam implantation. In vitro, we analyzed the effects on proliferation, differentiation, and apoptotic processes. (3) Results: SEM analysis of cells seeded on the surfaces showed no obvious differences between the reference material and the cast material with or without ion implantation. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) proliferation assay also did not reveal any significant differences. Additionally, the osteogenic differentiation process tested by quantitative polymerase chain reactions (PCR), Alizarin red S assay, and C-terminal collagen type I propeptide (CICP) Elisa was not significantly modified. No signs of induced apoptosis were observed. (4) Conclusions: In this study, we could show that the newly developed process of centrifugal casting generated a material with comparable surface features to standard machined Ti6Al4V material. In terms of biological impact on primary human osteoblasts, no significant differences were recognized. Additional Ca- or P-ion implantation did not improve or impair these characteristics in the dosages applied. These findings indicate that spin casting of Ti6Al4V may represent an interesting alternative to the production of geometrically complex orthopedic implants.

Objective This study aimed to investigate the effect of calcium ion (Ca2+) on the migration and osteogenic differentiation of human osteoblasts and explore the proper concentration and correlation mechanism. Methods A series of Ca2+ solutions with different concentrations was prepared. Osteoblast migration was assessed by Transwell assay, and proliferation was studied via the CCK-8 colorimetric assay. The mRNA expression of osteogenic genes was examined via reverse transcription-polymerase chain reaction (RT-PCR), and the mineralized nodule was examined by alizarin red-S method. After calcium sensitive receptor (CaSR) antagonism, Ca2+-induced migration and osteogenic differentiation were analyzed. Results In the migration experiment, 2, 4, and 6 mmol.L-1 Ca2+ could promoted osteoblast migration at three timepoints (8, 16, and 24 h), whereas 10 mmol.L-1 Ca2+ considerably inhibited migration at 8 h. The Ca2+ concentration range of 2-10 mmol-L'1 could promote proliferation, osteogenic differentiation, and mineralization of human osteoblasts. Moreover, mineralization was predominantly induced by 8 and 10 mmol.L-1 Ca2+. CaSR antagonism could reduce Ca2+-induced migration and osteogenic differentiation of human osteoblasts. Conclusion Low Ca2+ concentration favored osteoblast migration, whereas high Ca2+ concentration favored osteogenic differentiation. The Ca2+ concentrations of 4 and 6 mmol.L-1 could substantially induce osteoblast migration and osteogenic differentiation, and the Ca2+-CaSR pathway participated in signal transduction. [ABSTRACT FROM AUTHOR]

The objective of this study is to develop chitosan/montmorillonite (C/MMT) composite scaffolds based on improved properties for bone tissue engineering applications. With the freeze-drying technique, strontium (Sr 2+ ) modified C/MMT composite scaffold with an interconnected porous structure was produced. X-ray diffraction, fourier transform infrared spectroscopy, thermal gravimetric analysis, and scanning electron microscopy (SEM) were employed to investigate the structural properties, surface morphology and porosity of the composite scaffold. One of the aims of this study was to document the release of Sr 2+ from the non-modified and modified scaffolds into the cell culture medium. The biocompatibility of composite scaffolds was evaluated in cell cultures. Human osteoblasts (hOBs) were cultured, expanded and seeded on Sr 2+ -modified and non-modified C/MMT scaffolds. In-vitro cell viability and proliferation were investigated using MTT (3‑(4,5‑dimethylthiasol‑2‑yl)‑2,5‑diphenyltetrazolium bromide) assay and DNA content analysis. Live/dead cell staining assay and SEM were used for evaluating the cell-laden constructs. In-vitro studies showed that C/MMT scaffolds had no negative effects on osteoblasts. Ions present in the MMT were released into the cell culture medium, to induce osteoblast activity in the C/MMT scaffold system. Findings indicate that Sr 2+ modification of MMT-chitosan improves scaffold properties, suggesting Sr 2+ -modified C/MMT composite may be a promising biomaterial for bone tissue engineering. [ABSTRACT FROM AUTHOR]

Thermal trauma can irreversibly damage bone cells; however, the mechanisms by which thermal trauma affects thebone microenvironment are poorly characterized. Heat shock protein-60 (HSP60) can be induced by stresses, including hyperthermia, and released from cells as an endogenous danger signal. The aim of this study was to determine the effects of heatstress on HSP60 release by human osteoblasts. Normal human osteoblasts (NHOst) were exposed to heat stress at 40°C to46°C for 5-15 min and then cultured for 24 h. Cell viability was analyzed using the MTT assay. HSP60 protein expressionand release were analyzed by Western blotting of cell lysates and conditioned medium. HSP60 subcellular localization wasanalyzed using immunocytochemistry. Annexin-V-FITC/propidium iodide staining and the lactate dehydrogenase (LDH) assaywere used to investigate the mechanisms of cell death. We found that heat-stress significantly reduced NHOst cell viabilityin a dose- and time-dependent manner (p

We evaluated the cytotoxic effects of the debris apically extruded during root canal retreatment on primary human osteoblast (HOb) cells in vitro. TNF-α and IL-1β levels were also measured. We examined three different techniques: conventional hand-files, and Mtwo and Reciproc retreatments. Filled mandibular incisors were prepared for a cytotoxicity assay in an experimental root model. The material was divided into three groups according to the technique used. Ten teeth were used as control. HOb cells were exposed to the extruded content and cytotoxicity was evaluated using the MTT test (assessing cell metabolic activity). TNF-α and IL-1β production was also analyzed using enzyme-linked immunosorbent assay. Then, all the teeth were radiographed and the residual filling material was quantified. Data were subjected to analysis of variance (ANOVA) and Tukey's test (P < 0.05). The conventional hand-file technique was significantly more cytotoxic than the other methods (P < 0.05). Reciproc was less cytotoxic than Mtwo retreatment (P < 0.05). All endodontic retreatment techniques led to a significant upregulation of IL-1β levels (P < 0.05). However, only the conventional hand-file technique caused a significant increase in TNF-α levels (P < 0.05). Root-filling removal did not affect the levels of these proteins (P > 0.05). The Reciproc system required less time than the other two methods to remove the root-filling materials (P < 0.01). The endodontic retreatment with Reciproc was the least cytotoxic and the least time-consuming method of gutta-percha and sealer removal. (J Oral Sci 58, 211-217, 2016). [ABSTRACT FROM AUTHOR]

Objective: To observe the influence of puerarin on the differentiation of osteoblasts in patients with senile women with osteoporosis in vitro. Methods: The cancellous bone of elderly women with osteoporosis artificial femoral head replacement patients was collected. The osteoblasts were cultured by bone tissue block method. To observe ALP and OC expressed by osteoblasts with different concentrations(0, 0.01, 0.1, 1 mol/L) puerarin. Results: With the increase of the puerarin concentration(0.01~1 mol/L), the expression of ALP and OC in osteoblasts were increased, the concentration of two groups were statistically significant(P<0.05). With the increase of the concentration of puerarin, osteoblast numbers of calcified nodules increased, there were significant differences between the groups(P<0.05). Conclusion: 0.01, 0.1, 1 mol/L puerarin dose dependently stimulated osteoblasts express ALP and OC, and can improve the calcification capacity of osteoblasts. [ABSTRACT FROM AUTHOR]

Hypothesis Plasma polymerization has been vastly employed for bringing changes in the surface functionality of the biomaterials by changing the surface chemistry without affecting the bulk properties. Therefore, we wanted to analyze the effect of plasma polymerization on the cellular response towards biocomposite cryogel surface and expected a positive response as found with the previous biomaterials. Experiments We performed experiments for the incorporation of polyallylamine plasma on the polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogel surface. After plasma modification, the plasma coated/modified surfaces were studied at physicochemical level via methods like X-ray photoelectron spectroscopy, scanning electron microscopy, contact angle and rheology analysis. At biological level, analysis was performed through scanning electron microscopy, energy dispersive X-ray spectroscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and alkaline phosphatase (ALP) assay. Findings In this paper we highlight a different aspect of plasma modification where both physical and chemical characteristics of surface gets altered on modification of PTAC biocomposite cryogels with plasma polymerized allylamine (ppAAm). This further affected the response of human osteoblasts on these modified/coated cryogels when compared to the unmodified/uncoated cryogels, wherein, the human osteoblasts on coated cryogel surface show higher alkaline phosphatase production and delayed mineralization as compared to the uncoated cryogels. [ABSTRACT FROM AUTHOR]

Highlights: [•] Calcium effects on human osteoblast mineralisation and differentiation were tested. [•] Calcium induced matrix mineralisation and osteoblast to osteocyte transition. [•] Calcium induced osteoprotegerin (OPG) expression and secretion. [•] MEPE–ASARM peptides inhibited calcium-mediated mineralisation. [•] Human osteoblast to osteocyte transition is sensitive to extracellular calcium. [Copyright &y& Elsevier]

The influence of single-layer graphene produced by chemical vapor deposition on human osteoblast cells under different conditions was studied. Measurements probed the ability of cells to adhere and proliferate on graphene compared with SiO2/Si substrates and standard tissue culture plastic when cells were incubated for the first 2 h in the presence or the absence of fetal bovine serum (FBS), thus influencing the initial, direct interaction of cells with the substrate. It was found that after 48 h of human osteoblast incubation on graphene films, there were a comparable number of cells of a similar size irrespective of the presence or the absence of serum proteins. On the other hand, a strong initial influence through the presence of FBS proteins on cell number and cell size was observed in the case of the SiO2/Si substrate and control plastic. Thus, our study showed that the initial presence/absence of FBS in the medium does not determine cell fate in the case of a graphene substrate, which is very unusual and different from the behavior of cells on other materials. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3001-3007, 2012. [ABSTRACT FROM AUTHOR]

Abstract: The effect of calcium phosphate-based microchannels on the growth and orientation of human osteoblast cells is investigated in this study. As substrates, hydroxyapatite-based microchannels with high contouring accuracy were fabricated by a novel micro-moulding technique. Microchannels obtained by this method featured widths ranging from 16.0±0.7 to 76.6±1.4μm and depths from 7.9±0.8 to 15.5±1.3μm. Surface and contour characterization was carried out using X-ray diffraction analysis, scanning electron microscopy imaging and 3D-confocal profilometry. Cell activity and alignment on microchannels with different widths were determined after 1 and 3days by photometric spectroscopy and fluorescence microscopic imaging and statistically analysed by Tukey’s multiple comparison test. On days 1 and 3 for microchannels of width 16 and 30μm, 70–80% of the osteoblasts oriented within an angular range of 0–15° relative to the microchannel direction. Interestingly, only 20% of the cells grew inside the microchannels for channel widths of 16 and 30μm. Substrates with channel widths of 45, 65 and 76μm allowed ∼40% of the cells to grow inside. The depth of the microchannel showed hardly any significant impact. All micropatterned surfaces provoked a good cell attachment, as flat and spread cell morphologies with lamellipodiae and filopodiae could already be observed after 1day. The effect of the microchannels on osteoblast activity was determined using the colorimetric WST-1 assay. In addition, the cell differentiation was assessed by collagen type I staining. The cell activity obtained by WST-1 assay differed insignificantly for all micropatterned samples of various widths and depths. The assessment of collagen type I yielded the same amounts for all micropatterned samples after 1, 3 and 7days. In summary, the microchannel width of HA-based patterns has a distinct effect on the directed growth of human osteoblast cells, allowing novel design strategies for surfaces such as dental implants. [Copyright &y& Elsevier]

Abstract: Metal-on-metal hip replacement (MOMHR) using large diameter bearings has become a popular alternative to conventional total hip arthroplasty, but is associated with elevated local tissue and circulating levels of chromium (Cr) and cobalt (Co) ions that may affect bone health. We examined the effects of acute and chronic exposure to these metals on human osteoblast and osteoclast formation and function over a clinically relevant concentration range previously reported in serum and within hip synovial fluid in patients after MOMHR. SaOS-2 cells were cultured with Co2+, Cr3+ and Cr6+ for 3days after which an MTS assay was used to assess cell viability, for 13days after which alkaline phosphatase and cell viability were assessed and for 21days after which nodule formation was assessed. Monocytes were isolated from human peripheral blood and settled onto dentine disks then cultured with M-CSF and RANKL plus either Co2+, Cr3+ or Cr6+ ions for 21days from day 0 or between days 14 and 21. Cells were fixed and stained for TRAP and osteoclast number and amount of resorption per dentine disk determined. Co2+ and Cr3+ did not affect osteoblast survival or function over the clinically equivalent concentration range, whilst Cr6+ reduced osteoblast survival and function at concentrations within the clinically equivalent serum range after MOMHR (IC50 =2.2μM). In contrast, osteoclasts were more sensitive to metal ions exposure. At serum levels a mild stimulatory effect on resorption in forming osteoclasts was found for Co2+ and Cr3+, whilst at higher serum and synovial equivalent concentrations, and with Cr6+, a reduction in cell number and resorption was observed. Co2+ and Cr6+ within the clinical range reduced cell number and resorption in mature osteoclasts. Our data suggest that metal ions at equivalent concentrations to those found in MOMHR affect bone cell health and may contribute to the observed bone-related complications of these prostheses. [Copyright &y& Elsevier]

Abstract: Nel-like protein 1 (NELL-1) is an osteoinductive molecule associated with premature calvarial suture closure. Here we identified apoptosis related protein 3 (APR3), a membrane protein known as a proliferation suppressor, as a binding protein of NELL-1 by biopanning. NELL-1 and APR3 colocalized on the nuclear envelope of human osteoblasts. NELL-1 significantly inhibited proliferation of osteoblasts co-transfected with APR3 through further down-regulation of Cyclin D1. The co-expression of NELL-1 and APR3 enhanced Ocn and Bsp expression and mineralization. RNAi of APR3 significantly reduced the differentiation effect of NELL-1. These findings suggest that the effects of NELL-1 on osteoblastic differentiation and proliferation are partly through binding to APR3. Structured summary of protein interactions: APR3 physically interacts with NELL1 by pull down (View interaction) APR3 and NELL1 colocalize by fluorescence microscopy (View interaction) NELL1 physically interacts with SMEK1 by phage display (View interaction) NELL1 physically interacts with EIF4B by phage display (View interaction) NELL1 physically interacts with APR3 by anti bait coimmunoprecipitation (View interaction) NELL1 physically interacts with EIF5B by phage display (View interaction) NELL1 physically interacts with DNTTIP2 by phage display (View interaction) NELL1 physically interacts with EEF1A1 by phage display (View interaction) NELL1 physically interacts with Nexilin by phage display (View interaction) NELL1 physically interacts with PLCG1 by phage display (View interaction) NELL1 physically interacts with APR3 by phage display (View interaction) NELL1 physically interacts with SLTM by phage display (View interaction) NELL1 physically interacts with SSRP1 by phage display (View interaction) NELL1 physically interacts with NOL10 by phage display (View interaction) [Copyright &y& Elsevier]

Abstract: Recent studies have demonstrated the involvement of the sympathetic nerve system in bone metabolism. We have previously demonstrated the expression of adrenergic receptors in the human osteoblast SaM-1 cell line. The aim of this study was to reveal the function of these receptors in osteoblasts using electrophysiological methods. During whole-cell patch clamp recording, the application of noradrenaline reduced the currents induced by a voltage ramp, and the effect was larger in the positive potential range. Pretreatment with the selective α1B-adrenergic receptor antagonist chloroethylclonidine eliminated the inhibitory effect of noradrenaline. Using Cs-based pipette solution to block potassium channels, the inhibitory effect of noradrenaline disappeared. In contrast, the effect was unaffected in the presence of tetraethylammonium (TEA), a potassium channel blocker. These results suggest that noradrenaline suppresses Cs-sensitive and TEA-insensitive potassium channels via the α1B-adrenergic receptor in human osteoblasts. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the in vitro cytotoxicity and cytocompatibility of the developed aliphatic polyester co-polymer scaffolds: poly(L-lactide-co-ε-caprolactone) and poly(L-lactide-co-1,5-dioxepan-2-one). The scaffolds were produced by solvent casting and particulate leaching, and tested by direct and indirect contact cytotoxicity assays on human osteoblast-like cells and mouse fibroblasts. Cell morphology was documented by light and scanning electron microscopy. Viability was assessed by the MTT, neutral red uptake, lactic dehydrogenase and apoptosis assays. Extraction tests confirmed that the scaffolds did not have a cytotoxic effect on the cells. The cells grew and spread well on the test scaffolds with good cellular attachment and viability. The scaffolds are noncytotoxic and biocompatible with the two cell types and warrant continued investigation as potential constructs for bone tissue engineering. [ABSTRACT FROM PUBLISHER]

Abstract: In this study, influence of coating chemistry, channel width and tortuosity of various two-dimensional micro-channels were explored on micromachined Si using osteoblast precursor cells line 1 (OPC1). The rationale for our study is to delineate the influence of different porosity parameters on bone cell attachment and proliferation in vitro. Channel widths of 100, 200, 300, 400, and 600μm; channel bends of 0, 1, and 2 right angles; and gold and silicon dioxide coatings on single-crystal Si were studied. Experiments were conducted with channel tops under glass covered and uncovered conditions keeping the channel depth at 220μm. Independent samples were evaluated using SEM imaging and MTT assay to measure bone cell morphology and quantity. Images were taken of micro-channels and exterior chambers at 50×, 500×, 1000×, and 5000× magnifications. Channel and chamber cell densities were scored as follows: bare (score=0), scattered (1), limited (2), abundant (3), and overflowing (4). Samples were then scored and statistically analyzed for major differences. In general, OPC1 cells proliferated at least 5% or better based on cell numbers under uncovered conditions than glass covered. Channel widths of 100μm largely prohibited cell proliferation and diffusion by narrow path inhibition with the lowest average score of 1.17. Among channel bends of 0, 1, and 2 right angles, an increase in micro-channel tortuosity from 0–2 bends amplified OPC1 cell growth upwards of ~6.6%. A one-way ANOVA showed significant differences in cell quantity for alternating channel tortuosity at a significance level of p <0.05. No preference was found for gold or silicon dioxide coatings on Si for bone cell proliferation. [Copyright &y& Elsevier]

Ultramicrotomy, focused ion beam scanning electron microscopy (FIBSEM) and cryogenic FIBSEM (cryo-FIBSEM) techniques, as developed for the controlled cross-sectioning of mesenchymal stem cells (MSCs) and human osteoblasts (HObs) on titanium (Ti) substrates for transmission electron microscopy (TEM) investigation, are compared. Conventional ultramicrotomy has been used to section cells on Ti-foil substrates embedded in resin, but significant problems with cell detachment using this technique restricted its general applicability. Conventional FIBSEM ‘lift-out’ procedures were found to be effective for the preparation of uniform sections of fixed and dehydrated cell/Ti specimens, but the control of cell staining remains an issue. Cryo-FIBSEM procedures used with an ‘H-bar’ sample geometry enabled the sectioning of fixed and hydrated cell/Ti specimens, but issues remain over ion beam-induced artefacts and control of frost on the sample foils. [ABSTRACT FROM AUTHOR]

Any biomaterial implanted within the human body is influenced by the interactions that take place between its surface and the surrounding biological milieu. These interactions are known to influence the tissue interface dynamic, and thus act to emphasize the need to study cell-surface interactions as part of any biomaterial design process. The work described here investigates the relationship between human osteoblast attachment, spreading and focal contact formation on selected surfaces using immunostaining and digital image processing for vinculin, a key focal adhesion component. Our observations show that a relationship exists between levels of cell attachment, the degree of vinculin-associated plaque formation and biocompatibility. It also suggests that cell adhesion is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation. [ABSTRACT FROM AUTHOR]

Abstract: Carbon nanotubes (CNT) possess excellent mechanical properties to play the role as reinforcement for imparting strength and toughness to brittle hydroxyapatite (HA) bioceramic coating. However, lack of processing technique to uniformly distribute multiwalled CNTs in HA coating and limited studies and sparse knowledge evincing toxicity of CNTs has kept researchers in dispute for long. In the current work, we have addressed these issues by (i) successfully distributing multiwalled CNT reinforcement in HA coating using plasma spraying to improve the fracture toughness (by 56%) and enhance crystallinity (by 27%), and (ii) culturing human osteoblast hFOB 1.19 cells onto CNT reinforced HA coating to elicit its biocompatibility with living cells. Unrestricted growth of human osteoblast hFOB 1.19 cells has been observed near CNT regions claiming assistance by CNT surfaces to promote cell growth and proliferation. [Copyright &y& Elsevier]

Abstract: The cellular response to mineral trioxide aggregate (MTA) is important for the repair and regeneration of periradicular tissues. The purpose of this study was to analyze the response of human alveolar bone cells to MTA. A human alveolar bone chip was obtained from an oral surgical procedure and explant cultures harvested after 3 to 4 weeks of outgrowth in α-minimum essential medium supplemented with fetal calf serum. Cells in early passage were seeded onto preset ProRoot (gray) MTA, tooth-colored (white) MTA, and MTA prepared with local anesthetic solution. Scanning electron microscopy showed cells were attached and spread out onto MTA within 24 hours, and proliferated to form a matrix-like layer within 7 days. Cell attachment and cell-surface interactions with the gray and white MTA, and with the MTA prepared with local anesthetic were comparably propagated for 14 days. The surgically derived human alveolar bone cells provided a clinically relevant model that demonstrated the capacity of both ProRoot and tooth-colored MTA to support cell attachment, proliferation, and matrix formation. [Copyright &y& Elsevier]

Summary: Alveolar bone cells have a unique origin and functionality, but may resemble skeletal osteoblasts. Osteoblast differentiation and gene expression are regulated by the Runx2/Cbfa1 transcription factor. However, most studies on Runx2/Cbfa1 expression have been on rodent cells and the few studies on human osteoblasts have had differing results. The purpose of this study was to characterize Runx2/Cbfa1 expression in primary cell cultures derived from human alveolar bone. An alveolar bone chip was incubated in α-minimum essential medium (α-MEM) supplemented with fetal calf serum (10% FCS). Explant cultures were harvested after 3–4 weeks of outgrowth and grown in α-MEM with FCS. This media was supplemented with ascorbate, β-glycerophosphate and dexamethasone to promote osteoblast differentiation over 14 days. RT-PCR analysis and Western blots showed a rapid increase in Runx2/Cbfa1 mRNA (2.1-fold) and protein (2.3-fold) levels in 3 days, followed by a slight decline. There was also a rapid increase in bone sialoprotein expression (2.9-fold) in 3 days, followed by a further increase (3.6-fold) at 14 days. There was a slower increase in alkaline phosphatase expression (1.6-fold) and activity (3.1-fold) over 7 days, followed by a gradual decline. In contrast, collagen mRNA levels showed little change over 14 days. These findings attest to the osteogenic potential of primary cell cultures derived from human alveolar bone. Osteoblastic differentiation in human alveolar bone involves an increase in Runx2/Cbfa1 expression that may be an important component of the differentiation process. [Copyright &y& Elsevier]

Abstract: Intercellular calcium waves (ICW) are calcium transients that spread from cell to cell in response to different stimuli. We previously demonstrated that human osteoblast-like cells in culture propagate ICW in response to mechanical stimulation by two mechanisms. One mechanism involves autocrine activation of P2Y receptors, and the other requires gap junctional communication. In the current work we ask whether long-term culture of osteoblast-like cells affects the propagation of ICW by these two mechanisms. Human osteoblast-like cells were isolated from bone marrow. Mechanically induced ICW were assessed by video imaging of Fura-2 loaded cells after 1, 2 and 4 months culture. The P2Y2 receptor and the gap junction protein Cx43 were assessed by Western blot and real-time PCR. In resting conditions, P2Y mediated ICW prevailed and spread rapidly to about 13 cells. P2Y receptor desensitization by ATP disclosed gap junction-mediated ICW which diffused more slowly and involved not more than five to six cells. After 2 months in culture, ICW appeared slower and wave propagation was much less inhibited by P2Y desensitization, suggesting an increase in gap junction-mediated ICW. After 4 months in culture cells still responded to addition of ATP, but P2Y desensitization did not inhibit ICW propagation. Our data indicate that the relative role of P2Y-mediated and gap junction-mediated ICW changes during osteoblast differentiation in vitro. In less differentiated cells, P2Y-mediated ICW predominate, but as cells differentiate in culture, gap-junction-mediated ICW become more prominent. These results suggest that P2Y receptor-mediated and gap junction-mediated mechanisms of intercellular calcium signaling may play different roles during differentiation of bone-forming cells. [Copyright &y& Elsevier]

Non-resorbable thermoplastic polymers have become more important for reconstructive surgery due to their excellent chemical and physical properties. Polyetheretherketone-β-tricalcium phosphate (βTCP-PEEK) composites were developed as alternative materials for load-bearing applications. This study presents the effect of polyetheretherketone (PEEK) specimens incorporated with 5, 10, 20 and 40 wt% β-tricalcium phosphate (βTCP) and processed by injection molding on cultivated osteoblast cells. Normal human osteoblast (NHOst) cells were seeded onto polymer discs to evaluate cell viability and proliferation after 24, 72 and 120 h of cultivation by employing the WST-1 assay. Standard tissue culture plastic was used as a control. The osteoblast cells were found to be viable in all PEEK groups, while the cell proliferation was progressively inhibited due to the incorporated β-tricalcium phosphate. βTCP-PEEK showed concentration independent decrease of cell proliferation compared to the unfilled PEEK and the control group. In summary, this study confirms the non-toxic nature of pure PEEK, whereas this could not definitely be verified for βTCP-PEEK as a composite material in chosen concentrations of β-tricalcium phosphate in vitro. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the effect of extracellular adenosine triphosphate (ATP) on the production of interleukin (IL)-6, whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as are involved in signal transduction systems in human osteoblastic SaM-1 cells. These human osteoblasts constitutively expressed P2X4, P2X5, P2X6, P2Y2, P2Y5, and P2Y6 purinergic receptors. ATP increased gene- and protein-expression of IL-6 in SaM-1 cells. The expression of the IL-6 mRNA was maximal at 1h, and the increase in IL-6 synthesis in response to ATP (10–100μM) occurred in a concentration-dependent manner. Over the same concentration range of the nucleotide that was effective for IL-6 synthesis, ATP caused an increase in the intracellular Ca2+ concentration ([Ca2+]i), which increase was inhibited by pretreatment with suramin, a P2Y receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker, but not by the extracellular Ca2+-chelating agent EGTA. The pretreatment of SaM-1 cells with suramin or 2-APB also inhibited the increase in IL-6 synthesis in response to ATP. These findings suggest that extracellular ATP-induced IL-6 synthesis occurs through P2Y receptors and mobilization of Ca2+ from internal stores in human osteoblastic cells. [Copyright &y& Elsevier]

It is important to elucidate whether the leptin receptor, especially the long signal-transducing form of the leptin receptor (OB-Rb) is expressed in human osteoblasts. We detected the expression of human OB-Rb in cultured commercially available human osteoblasts (NHOst cells) using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). After confirming the expression of OB-Rb, we investigated the effect of leptin on NHOst cells. Leptin enhanced cell proliferation of the cells shown by the MTT assay. Furthermore, leptin changed the copy numbers of Bax and Bcl-2 mRNAs in the cultured cells as shown by real-time quantitative RT-PCR, although the effect was not consistent. Leptin did not change the production of osteocalcin and osteopontin by the cells. Leptin did not change the expression of OB-Rb mRNA in the cells. In conclusion, OB-Rb mRNA is expressed in cultured commercially available human osteoblasts. Leptin may have some effects on bone metabolism by directly modulating cell proliferation and apoptosis of osteoblasts in humans. [ABSTRACT FROM AUTHOR]

Objective: The present study was designed to evaluate the pharmacological characteristics of Emdogain (EMD) on cell growth and cell activity in human osteoblasts. Methods: Cell proliferation as well as several gene and protein expressions were examined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) procedures in human osteoblastic cells (SaM-1) treated with EMD (30 μ g ml-1 ). Results: Treatment of osteoblasts with EMD significantly stimulated cell proliferation and fibroblast growth factor (FGF)-2 expression but decreased alkaline phosphatase expression. In addition, increases in cyclooxygenase (COX)-2 expression and decreases in matrix metalloproteinases (MMP)-1 expression were observed in osteoblasts treated with EMD. The effects of EMD on FGF-2 and MMP-1 expressions were not observed in osteoblasts treated with NS-398, an inhibitor of COX-2. The decrease in MMP-1 mRNA by EMD was prevented by treatment with antisense oligodeoxynucleotide (AS-ODN) for FGF-2. Conclusion: Emdogain showing both stimulation of cell proliferation and inhibition of cell differentiation has been shown to increase FGF-2 expression in the mediation of prostaglandin E2 and to decrease MMP-1 mRNA expression through the activation of FGF-2. FGF-2 may underlie in the action of EMD on osteoblasts during periodontal regeneration. [ABSTRACT FROM AUTHOR]

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. DU possesses both a radiological (alpha particle) and a chemical (metal) component. Since DU has a low-specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. In the current study we demonstrate that DU can generate oxidative DNA damage and can also catalyze reactions that induce hydroxyl radicals in the absence of significant alpha particle decay. Experiments were conducted under conditions in which chemical generation of hydroxyl radicals was calculated to exceed the radiolytic generation by 106-fold. The data showed that markers of oxidative DNA base damage, thymine glycol and 8-deoxyguanosine could be induced from DU-catalyzed reactions of hydrogen peroxide and ascorbate similarly to those occurring in the presence of iron catalysts. DU was 6-fold more efficient than iron at catalyzing the oxidation of ascorbate at pH 7. These data not only demonstrate that DU at pH 7 can induced oxidative DNA damage in the absence of significant alpha particle decay, but also suggest that DU can induce carcinogenic lesions, e.g. oxidative DNA lesions, through interaction with a cellular oxygen species. [Copyright &y& Elsevier]

Recent studies have indicated a critical role for vascular endothelial growth factor (VEGF) during the process of endochondral ossification, in particular in coupling cartilage resorption with bone formation. Therefore, we studied the chemoattractive and proliferative properties of human VEGF-A on primary human osteoblasts (PHO) and compared these data with the effects of human basic fibroblast growth factor (bFGF) and human bone morphogenetic protein-2 (BMP-2). Furthermore, initial experiments were carried out to characterize VEGF-binding proteins on osteoblastic cells possibly involved in the response. For the first time, to our knowledge, we could demonstrate a chemoattractive effect of VEGF-A, but not VEGF-E, on primary human osteoblasts. The effect of VEGF-A was dose-dependent and did not reach a maximum within the concentration range tested (up to 10 ng/mL). The maximal effect observed was a chemotactic index (CI) of 2 at a concentration of 10 ng/mL. bFGF and BMP-2 exhibited maxima at 1.0 ng/mL with CI values of 2.5 and 2, respectively. In addition to its effect on cell migration, VEGF-A stimulated cell proliferation by up to 70%. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of VEGF receptors VEGFR-1 (Flt-1), VEGFR-2 (Kdr), and VEGFR-3 (Flt-4), as well as neuropilin-1 and -2. An in vitro kinase assay failed to demonstrate activation of VEGFR-2 upon stimulation with either VEGF-E or VEGF-A, consistent with the idea that the effect of VEGF-A on primary human osteoblasts is mediated via VEGFR-1. Taken together, our data establish that human osteoblasts respond to VEGF-A, suggesting a functional role for this growth factor in bone formation and remodeling. [Copyright &y& Elsevier]

The use of biodegradable polymers in the field of orthopaedic surgery has gained increased popularity, as surgical pins and screws, and as potential biological scaffolds for repairing cartilage and bone defects. One such group of polymers that has gained considerable attention are the polyesters, poly(lactide-co-glycolide) (PLAGA) and polylactic acid (PLA), because of their minimal tissue inflammatory response, favorable biocompatibility and degradation characteristics. The objective of this study was to evaluate human osteoblastic cell adherence and growth on PLAGA and PLA scaffolds by examining integrin receptor (α2,α3,α4,α5,α6 and β1) expression. Primary human osteoblastic cells isolated from trabecular bone adhered efficiently to both PLAGA and PLA, with the rate of adherence on PLAGA comparable to that of control tissue culture polystyrene (TCPS), and significantly higher than on PLA polymers at 3, 6 and 12 h. Human osteoblastic phenotypic expression, alkaline phosphatase (ALP) activity was positive on both degradable matrices, whereas osteocalcin levels were significantly higher on cells grown on PLAGA than on PLA composites. Interestingly, the integrin subunits, α2,α3,α4,α5,α6 and β1 were all expressed at higher levels by osteoblasts cultured on PLAGA than those on PLA as analyzed by westerns blots and by flow cytometry. Among the integrins, α2,α5 and β1 showed the greatest difference in levels between the two surfaces. Thus, both PLA and PLAGA support osteoblastic adhesion and its accompanying engagement of integrin receptor and expression of osteocalcin and ALP. However PLAGA consistently appeared to be a better substrate for osteoblastic cells based on these parameters. This study is one of the first to investigate the ability of primary human osteoblastic cells isolated from trabecular bone to adhere to the biodegradable polymers PLAGA and PLA, and to examine the expression of their key adhesion receptors (integrins) on these substrates. [Copyright &y& Elsevier]

A biomaterial that is both bioactive and capable of controlled drug release is highly attractive for bone regeneration. In previous works, we demonstrated the possibility of combining activated carbon fiber cloth (ACC) and biomimetic apatite (such as calcium-deficient hydroxyapatite (CDA)) to develop an efficient material for bone regeneration. The aim to use the adsorption properties of an activated carbon/biomimetic apatite composite to synthetize a biomaterial to be used as a controlled drug release system after implantation. The adsorption and desorption of tetracycline and aspirin were first investigated in the ACC and CDA components and then on ACC/CDA composite. The results showed that drug adsorption and release are dependent on the adsorbent material and the drug polarity/hydrophilicity, leading to two distinct modes of drug adsorption and release. Consequently, a double adsorption approach was successfully performed, leading to a multifunctional and innovative ACC-aspirin/CDA-tetracycline implantable biomaterial. In a second step, in vitro tests emphasized a better affinity of the drug (tetracycline or aspirin)-loaded ACC/CDA materials towards human primary osteoblast viability and proliferation. Then, in vivo experiments on a large cortical bone defect in rats was carried out to test biocompatibility and bone regeneration ability. Data clearly highlighted a significant acceleration of bone reconstruction in the presence of the ACC/CDA patch. The ability of the aspirin-loaded ACC/CDA material to release the drug in situ for improving bone healing was also underlined, as a proof of concept. This work highlights the possibility of bone patches with controlled (multi)drug release features being used for bone tissue repair. [ABSTRACT FROM AUTHOR]

Expression of progesterone receptors (PR) was studied in human osteoblast-like cell lines and primary human osteoblast cultures at the molecular level. Using the sensitive reverse transcriptase polymerase chain reaction (RT-PCR) and oligonucleotide primers which flank the progesterone-binding domain of human PR, progesterone receptor (PR) mRNA was detected in three osteoblast-like cell lines--HOS-TE85, MG-63, and SAOS-2. When compared with beta-actin gene expression, levels of PRmRNA transcripts varied between cell lines (PRmRNA in HOS-TE85 > MG-63 >> SAOS-2). In addition, RT-PCR confirmed the presence of PRmRNA transcripts in primary human osteoblast cells cultured from collagenase-treated bone. Immunostaining was used to visualize PR protein in cells. All osteoblast-like cell lines showed specific staining for PR. Immunoreactivity was distributed equally in the nucleus and cytoplasm. The level of staining was significantly lower than that detected in PR-positive MCF-7 breast cancer cells though well above background levels obtained for PR-negative HeLa cells. The finding that PR is expressed at both the level of mRNA and protein in several osteoblast-like cell lines as well as in human primary osteoblast cultures indicates that bone-forming osteoblast cells are direct targets for progesterone action. [ABSTRACT FROM AUTHOR]

• FNDC5/irisin is expressed in hPDL cells, hDPCs, hOBs, rodent periodontium and pulp. • FNDC5/irisin expression is differently regulated in hPDL cells, hDPCs, and hOBs. • Odontoblast-like differentiation may affect FNDC5/irisin expression and secretion. To examine the expression and regulation of fibronectin type III domain-containing protein 5/irisin (FNDC5/irisin) in primary human periodontal ligament (hPDL) cells, dental pulp stem cells (hDPCs) and osteoblasts (hOBs). FNDC5/irisin was identified in sections of paraffin embedded rat maxillae, cryo-sections of 3D cultured spheroids hPDL cells, hDPCs and hOBs, 2D cultured hPDL cells, hDPCs and hOBs by immunohistochemistry. The expression of FNDC5/irisin was identified by qPCR, followed by sequencing of the qPCR product. Regulation of FNDC5/irisin expression in hPDL cells, hDPCs and hOBs were evaluated after administration of different concentrations of irisin and all-trans retinoic acid (ATRA). qPCR and ELISA were used to identify expression and secretion of FNDC5/irisin in odontoblast-like differentiation of hDPCs. FNDC5/irisin was confirmed to be present in rat periodontium and dental pulp regions, as well as in 2D and 3D cultured hPDL cells, hDPCs and hOBs. BLAST analyses verified the generated nucleotide alignments matched human FNDC5/irisin. FNDC5/irisin gene expression was enhanced during odontoblast-like differentiation of hDPCs whereas the secretion of the protein was decreased compared to control. The protein signals in rat periodontal and pulpal tissues were higher than that of alveolar bone, and the expression of FNDC5/irisin was differently regulated by recombinant irisin and ATRA in hPDL cells and hDPCs compared to hOBs. FNDC5/irisin expression was verified in rodent periodontium and dental pulp, and in hPDL cells, hDPCs and hOBs. The FNDC5/irisin expression was regulated by recombinant irisin and ATRA. Finally, expression and secretion of FNDC5/irisin were affected during odontoblast-like differentiation of hDPCs. [ABSTRACT FROM AUTHOR]