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Several studies have shown that cold atmospheric plasma (CAP) treatment can favourably modify titanium surfaces to promote osteoblast colonization. The aim of this study was to investigate the initial attachment of primary human osteoblasts to plasma-treated titanium. Micro-structured titanium discs were treated with cold atmospheric plasma followed by the application of primary human osteoblasts. The microwave plasma source used in this study uses helium as a carrier gas and was developed at the Leibniz Institute for Surface Modification in Leipzig, Germany. Primary human osteoblasts were analyzed by fluorescence and cell biological tests (alkaline phosphatase activity and cell proliferation using WST-1 assay). The tests were performed after 4, 12, and 24 h and showed statistically significant increased levels of cell activity after plasma treatment. The results of this study indicate that plasma treatment improves the initial attachment of primary human osteoblasts to titanium. For the first time, the positive effect of cold atmospheric plasma treatment of micro-structured titanium on the initial colonization with primary human osteoblasts has been demonstrated. Overall, this study demonstrates the excellent biocompatibility of micro-structured titanium. The results of this study support efforts to use cold atmospheric plasmas in implantology, both for preimplantation conditioning and for regeneration of lost attachment due to peri-implantitis.

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Biodegradable metal alloys may be successfully used to support bone repair, avoiding second surgery commonly needed when inert metal alloys are used. Combining a biodegradable metal alloy with a suitable pain relief agent could improve patient quality of life. AZ31 alloy was coated using a poly(lactic-co-glycolic) acid (PLGA) polymer loaded with ketorolac tromethamine using the solvent casting method. The ketorolac release profile from the polymeric film and the coated AZ31 samples, the PLGA mass loss of polymeric film, and the cytotoxicity of the optimized coated alloy were assessed. The coated sample showed a ketorolac release that was prolonged for two weeks, which was slower than that of just the polymeric film, in simulated body fluid. PLGA mass loss was complete after a 45-day immersion in simulated body fluid. The PLGA coating was able to lower AZ31 and ketorolac tromethamine cytotoxicity observed in human osteoblasts. PLGA coating also prevents AZ31 cytotoxicity, which was identified in human fibroblasts. Therefore, PLGA was able to control ketorolac release and protect AZ31 from premature corrosion. These characteristics allow us to hypothesize that the use of ketorolac tromethamine-loaded PLGA coating on AZ31 in the management of bone fractures can favor osteosynthesis and relief pain.

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PURPOSE: Molecular processes in primary osteoblasts were analyzed in response to magnetic and electric field exposure to examine its potential impact on bone healing. METHODS: Primary osteoblasts were exposed to a combination of a magnetic field and an additional electric field (EFMF) (20 Hz, 700 mV, 5 mT, continuous sinusoids) in vitro. mRNA- and protein-expressions were assessed during a time interval of 21 days and compared with expression data obtained from control osteoblasts. RESULTS: We observed an autonomous osteoblast differentiation process in vitro under the chosen cultivation conditions. The initial proliferative phase was characterized by a constitutively high mRNA expression of extracellular matrix proteins. Concurrent EFMF exposure resulted in significanly increased cell proliferation (fold change: 1.25) and reduced mRNA-expressions of matrix components (0.5-0.75). The following reorganization of the extracellular matrix is prerequisite for matrix mineralization and is characterised by increased Ca2+ deposition (1.44). On molecular level EFMF exposure led to a significant decreased thrombospondin 1 (THBS1) mRNA- (0.81) and protein- (0.54) expression, which in turn reduced the TGFß1-dependent mRNA- (0.68) and protein- (0.5) expression of transforming growth factor beta induced (ßIG-H3) significantly, an inhibitor of endochondral ossification. Consequently, EFMF exposure stimulated the expression of genes characteristic for endochondral ossification, such as collagen type 10, A1 (1.50), osteopontin (1.50) and acellular communication network factor 3 (NOV) (1.45). CONCLUSIONS: In vitro exposure of osteoblasts to EFMF supports cell differentiation and induces gene- and protein-expression patterns characteristic for endochondral ossification during bone fracture healing in vivo.

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Ti6Al4V as a common implant material features good mechanical properties and corrosion resistance. However, untreated, it lacks bioactivity. In contrast, coatings with calcium phosphates (CaP) were shown to improve cell-material interactions in bone tissue engineering. Therefore, this work aimed to investigate how to tailor biomimetic CaP coatings on Ti6Al4V substrates using modified biomimetic calcium phosphate (BCP) coating solutions. Furthermore, the impact of substrate immersion in a 1 M alkaline CaCl2 solution (pH = 10) on subsequent CaP coating formation was examined. CaP coatings were characterized via scanning electron microscopy, x-ray diffraction, energy-dispersive x-ray spectroscopy, and laser-scanning microscope. Biocompatibility of coatings was carried out with primary human osteoblasts analyzing cell morphology, proliferation, collagen type 1, and interleukin 6 and 8 release. Results indicate a successful formation of low crystalline hydroxyapatite (HA) on top of every sample after immersion in each BCP coating solution after 14 days. Furthermore, HA coating promoted cell proliferation and reduced the concentration of interleukins compared to the uncoated surface, assuming increased biocompatibility.

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ETHNOPHARMACOLOGICAL RELEVANCE: Postmenopausal osteoporosis is a major bone health issue worldwide. There is an unmet medical need for osteoporosis treatments, a disease which disproportionately impacts women. Exploring botanicals to prevent or treat osteoporosis is currently an interest of investigations. Rhizomes of Davallia mariesii T. Moore ex Baker (Davalliacea) are used an indigenous herbal medicine in Asia for injuries due to fractures, contusions, and strains. AIM OF THE STUDY: In the present study, we investigated the osteogenic effect of the water extract of rhizomes of D. mariesii (DMH) on bone loss induced by an ovariectomy (OVX) in mice and also its impact on osteogenesis in primary human osteoblasts (HObs). Additionally, we performed a quantitative analysis of compounds in the DMH extract. MATERIALS AND METHODS: OVX C57BL/6J mice were orally administrated DMH extract for 12 weeks, and microarchitecture parameters were examined by microcomputed tomography. DMH extract was fractionated in a bio-guided manner, and fractions were isolated to obtain active compounds using HObs. Cell viability was evaluated by an MTT assay. Characteristics of early and late osteogenesis were analyzed by alkaline phosphatase activity and a mineralization assay. Molecular mechanisms were explored by a real-time quantitative PCR. Compounds in the DMH extract were identified and quantified using liquid chromatography tandem mass spectroscopy (LC-MS/MS). RESULTS: DMH improved bone mineral densities of vertebrae and the femur. Through microarchitectural observations, DMH significantly decreased the bone surface/volume ratio and trabecular separation, and also increased the connectivity density in the OVX group. Additionally, DMH inhibited osteoclast differentiation in receptor activator of nuclear factor-κB ligand-induced osteoclasts and increased bone formation in HObs. After bio-guided fractionation and isolation, we found that eriodictyol-7-O-ß-d-glucuronide (2) significantly increased alkaline phosphatase activity, and 5-O-ß-d-(6-O-vanilloylglucopyranosyl)gentisic acid (3) substantially enhanced mineral deposition. In HObs, compound 3 was more potent in upregulating expressions of bone morphogenetic protein-2, bone sialoprotein, osteopontin, osterix, and estrogen receptor-α. The amount of bioactive compound 3 in DMH was 5.68 ±â€¯0.64 mg/g of dry weight according to LC-MS/MS. CONCLUSION: For the first time we report that D. mariesii and its isolated compounds demonstrated potent osteogenic activities. Quantitative results of D. mariesii could be a reference for phytochemical analyses.

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Titanium has been used for biomedical devices due to its excellent biocompatibility, which is based partly on its 2-8 nm thick titanium oxide layer. However, the relatively poor surface hardness, wear resistance and metal release of these layers may cause some problems in clinical application. In this study, titanium surfaces were modified using a TiO2 sol-gel coating, in order to improve surface properties and osteoblast function. No significant difference in surface roughness was observed between titanium and TiO2 sol-gel discs. The surface of TiO2 sol-gel discs possessed more wettability than titanium discs. The X-ray diffraction results showed amorphous TiO2 phase on titanium discs, whereas TiO2 sol-gel surfaces presented TiO2 rutile and anatase phase. After 4 hours, the number of osteoblasts seeded on TiO2 surface was significantly higher than those on titanium discs. The mRNA expression of bone sialoprotein and osteocalcin were also higher on day 5 and 7, respectively. Enzyme-linked immunosorbent assay(ELISA) analysis confirmed the increase of osteocalcin protein synthesis in osteoblasts grown on the TiO2 sol-gel surface. Alizarin red-S staining showed higher amount of calcium deposition from osteoblasts cultured on TiO2 surface than those on titanium discs at day 20. In conclusion, TiO2 sol-gel coated-titanium could enhance osteoblasts differentiation and promote mineralization, indicating its potential in improving osseointegration for clinical application.

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Coculturing of bone-forming and blood vessel-forming cells is a strategy aimed at increasing vascularity of implanted bone constructs in tissue-engineering applications. We previously described that the coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves the differentiation of both cell types, leading to the formation of functional blood vessels and enhanced bone regeneration. The objective of this study was to further delineate the multifaceted interactions between both cell types. To investigate the proteome of hOBs after cocultivation with HUVECs we used stable isotope labeling by amino acids in cell culture, revealing 49 significantly upregulated, and 54 significantly downregulated proteins. Amongst the highest regulated proteins, we found the proteins important for osteoblast differentiation, cellular adhesion, and extracellular matrix function, notably: connective tissue growth factor, desmoplakin, galectin-3, and cyclin-dependent kinase 6. The findings were confirmed by enzyme-linked immunosorbent assays. We also investigated whether the mRNA transcripts correlate with the changes in protein levels by quantitative real-time reverse transcription polymerase chain reaction. In addition, the data was compared to our previous microarray analysis of hOB transcriptome. Taken together, this in-depth analysis delivers reliable data suggesting the importance of coculturing of hOBs and HUVECs in tissue engineering.

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The cytokines secreted by immune cells have a large impact on the tissue, surrounding a fracture, e.g., by attraction of osteoprogenitor cells. However, the underlying mechanisms are not yet fully understood. Thus, this study aims at investigating molecular mechanisms of the immune cell-mediated migration of immature primary human osteoblasts (phOBs), with transforming growth factor beta (TGF-ß), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and focal adhesion kinase (FAK) as possible regulators. Monocyte- and macrophage (THP-1 cells ± phorbol 12-myristate 13-acetate (PMA) treatment)-conditioned media, other than the granulocyte-conditioned medium (HL-60 cells + dimethyl sulfoxide (DMSO) treatment), induce migration of phOBs. Monocyte- and macrophage (THP-1 cells)-conditioned media activate Smad3-dependent TGF-ß signaling in the phOBs. Stimulation with TGF-ß promotes migration of phOBs. Furthermore, TGF-ß treatment strongly induces NOX4 expression on both mRNA and protein levels. The associated reactive oxygen species (ROS) accumulation results in phosphorylation (Y397) of FAK. Blocking TGF-ß signaling, NOX4 activity and FAK signaling effectively inhibits the migration of phOBs towards TGF-ß. In summary, our data suggest that monocytic- and macrophage-like cells induce migration of phOBs in a TGF-ß-dependent manner, with TGF-ß-dependent induction of NOX4, associated production of ROS and resulting activation of FAK as key mediators.

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Human adipose-derived mesenchymal stem cells (Ad-MSCs) have been proposed as suitable option for cell-based therapies to support bone regeneration. In the bone environment, Ad-MSCs will receive stimuli from resident cells that may favor their osteogenic differentiation. There is recent evidence that this process can be further improved by extremely low frequency pulsed electromagnetic fields (ELF-PEMFs). Thus, the project aimed at (i) investigating whether co-culture conditions of human osteoblasts (OBs) and Ad-MSCs have an impact on their proliferation and osteogenic differentiation; (ii) whether this effect can be further improved by repetitive exposure to two specific ELF-PEMFs (16 and 26 Hz); (iii) and the effect of these ELF-PEMFs on human osteoclasts (OCs). Osteogenic differentiation was improved by co-culturing OBs and Ad-MSCs when compared to the individual mono-cultures. An OB to Ad-MSC ratio of 3:1 had best effects on total protein content, alkaline phosphatase (AP) activity, and matrix mineralization. Osteogenic differentiation was further improved by both ELF-PEMFs investigated. Interestingly, only repetitive exposure to 26 Hz ELF-PEMF increased Trap5B activity in OCs. Considering this result, a treatment with gradually increasing frequency might be of interest, as the lower frequency (16 Hz) could enhance bone formation, while the higher frequency (26 Hz) could enhance bone remodeling.

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The combination of the two techniques of rapid prototyping 3D-plotting and bioactive surface functionalization is presented, with emphasis on the in vitro effect of Bone Sialoprotein (BSP) on primary human osteoblasts (hOBs). Our primary objective was to demonstrate the BSP influence on the expression of distinctive osteoblast markers in hOBs. Secondary objectives included examinations of the scaffolds' surface and the stability of BSP-coating as well as investigations of cell viability and proliferation. 3D-plotted calcium phosphate cement (CPC) scaffolds were coated with BSP via physisorption. hOBs were seeded on the coated scaffolds, followed by cell viability measurements, gene expression analysis and visualization. Physisorption is an effective method for BSP-coating. Coating with higher BSP concentrations leads to enhanced BSP release. Two BSP concentrations (50 and 200 µg/mL) were examined in this study. The lower BSP concentration (50 µg/mL) decreased ALP and SPARC expression, whereas the higher BSP concentration (200 µg/mL) did not change gene marker expression. Enhanced cell viability was observed on BSP-coated scaffolds on day 3. hOBs developed a polygonal shape and connected in an intercellular network under BSP influence. Quantitative cell morphology analyses demonstrated for BSP-coated CPCs an enhanced cell area and reduced circularity. The strength of the above-mentioned effects of BSP-coated scaffolds in vivo is unknown, and future work is focusing on bone ingrowth and vascularization in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2565-2575, 2018.

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In this study, we investigated the effect of the two flavonoids, baicalin (baicalein 7-O-[Formula: see text]- d-glucuronic acid) and its aglycone, baicalein (5,6,7-trihydroxyflavone), after encapsulation into amorphous calcium polyphosphate (Ca-polyP) microparticles on mineralization of primary human osteoblasts (phOSB). Both flavonoids, which come from root extracts of Scutellaria baicalensis Georgi, are used in Traditional Chinese Medicine, and are nontoxic in cells up to a concentration of 3[Formula: see text][Formula: see text]g/ml. The morphogenetically active, energy-rich Ca-polyP particles with a stoichiometric P:Ca ratio of 1:2 are degraded by cellular alkaline phosphatase (ALP) to ortho-phosphate used for bone hydroxyapatite formation. Here we show that the flavone-loaded Ca-polyP microparticles are readily taken up by phOSB, resulting in the accumulation of polyP around the nuclei and the formation of intracellular vesicles containing the ALP. In addition, we demonstrate that baicalin/baicalein causes a rise of the intracellular calcium [Ca[Formula: see text]]i a level which markedly is augmented after encapsulation into Ca-polyP, through activation of the phospholipase C. Moreover, both flavones, either alone or associated with Ca-polyP microparticles, upregulate the expression of the osteoblast calcium efflux channel, the plasma membrane Ca[Formula: see text]-ATPase (PMCA), while the expression of ALP, which promotes bone mineralization, is induced by Ca-polyP and by the flavones only if present in the Ca-polyP-microparticle-associated form. As a result, the extent of bone mineralization is markedly enhanced. Based on the two-armed activating function, new applications of baicalin/baicalein as a component of nutriceuticals for osteoporosis prevention or bone implants can be envisaged.

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Transforming growth factor ß (TGF-ß) is a critical regulator of bone density owing to its multiple effects on cell growth and differentiation. Recently, we have shown that TGF-ß1 effectively blocks bone morphogenetic protein (BMP) induced maturation of osteoblasts by upregulating histone deacetylase (HDAC) activity. The current study aimed at investigating the effect of rhTGF-ß1 treatment on the expression of specific HDACs and their cellular effects, e.g., microtubule structures (primary cilia) and mechanosensation. Exposure to TGF-ß1 most significantly induced expression of HDAC6 both on gene and protein level. Being most abundant in the cytoplasm HDAC6 effectively deacetylates microtubule structures. Thus, TGF-ß1-induced expression of HDAC6 led to deformation and shortening of primary cilia as well as to reduced numbers of ciliated cells. Primary cilia are described to sense mechanical stimuli. Thus, fluid flow was applied to the cells, which stimulated osteoblast function (AP activity and matrix mineralization). Compromised primary cilia in TGF-ß1-treated cells were associated with reduced osteogenic function, despite exposure to fluid flow conditions. Chemical inhibition of HDAC6 with Tubacin restored primary cilium structure and length. These cells showed improved osteogenic function especially under fluid flow conditions. Summarizing our results, TGF-ß1 impairs human osteoblast maturation partially via HDAC6-mediated distortion and/or shortening of primary cilia. This knowledge opens up new treatment options for trauma patients with chronically elevated TGF-ß1-levels (e.g., diabetics), which frequently suffer from delayed fracture healing despite adequate mechanical stimulation. KEY MESSAGES: Exposure to TGF-ß1 induces expression of HDAC6 in human osteoblasts. TGF-ß1 exposed human osteoblasts show less and distorted primary cilia. TGF-ß1 exposed human osteoblasts are less sensitive towards mechanical stimulation. Mechanosensation can be recovered by HDAC6 inhibitor Tubacin in human osteoblasts.

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The development of new approaches leading to fast and successful vascularization of tissue-engineered constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Recently, TLR4 activation and LPS stimulation of endothelial cells have been reported to promote angiogenesis in a variety of settings. In this study, we demonstrate that TLR4 activation by Ultrapure LPS Escherichia coli 0111:B4 (LPS-EB) significantly enhances microvessel formation in a co-culture system consisting of outgrowth endothelial cells (OECs) and primary human osteoblasts (pOBs). The precise modes of TLR4 action on the process of angiogenesis have also been investigated in this study. Using quantitative fluorescence microscopy in monocultures of OECs and pOBs, it was found that TLR4 activation through LPS-EB upregulates the expression level of TLR4/MYD88 and enhances both angiogenesis and osteogenesis. Furthermore, ELISA and qRT-PCR have shown that the level of two adhesion molecules (ICAM-1 and E-selectin), two cytokines (IL-6 and IL-8) and two growth factors (VEGF and PDGF-BB) related to angiogenesis increase significantly after LPS-EB treatment. This increased understanding of the role of TLR4 in angiogenesis could be of value in various settings related to tissue repair and tissue engineering. Moreover, since LPS and TLR4 agonists improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for angiogenesis intervention in vivo and therefore could be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.

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Vascularization is essential for bone development, fracture healing, and bone tissue engineering. We have previously described that coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves differentiation of both cell types. Investigating the role of microRNAs (miRNAs) in this system, we found that miR-126 is highly upregulated in hOBs following coculturing with HUVECs. In this study we performed miR-126 gain-of-function and loss-of-function experiments in hOBs followed by microarray analysis in order to identify targets of miR-126. The transcript cluster IDs were sieved by applying cut-off criteria and by selecting transcripts which were upregulated following miR-126 downregulation and vice versa. The calmodulin regulated spectrin associated protein 1 (CAMSAP1) mRNA was confirmed to be differentially regulated by miR-126. Using the luciferase reporter assay it was demonstrated that CAMSAP1 is directly targeted by miR-126. In this study, we show that miR-126 and CAMSAP1 directly interact in hOBs. This finding has potential implications for tissue engineering applications. J. Cell. Biochem. 118: 1756-1763, 2017. © 2016 Wiley Periodicals, Inc.

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The enzyme 1α-hydroxylase (gene CYP27B1) catalyzes the synthesis of 1,25(OH)2D in both renal and bone cells. While renal 1α-hydroxylase is tightly regulated by hormones and 1,25(OH)2D itself, the regulation of 1α-hydroxylase in bone cells is poorly understood. The aim of this study was to investigate in a primary human osteoblast culture whether parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), calcitonin, calcium, phosphate, or MEPE affect mRNA levels of CYP27B1. Our results show that primary human osteoblasts in the presence of high calcium concentrations increase their CYP27B1 mRNA levels by 1.3-fold. CYP27B1 mRNA levels were not affected by PTH1-34, rhFGF23, calcitonin, phosphate, and rhMEPE. Our results suggest that the regulation of bone 1α-hydroxylase is different from renal 1α-hydroxylase. High calcium concentrations in bone may result in an increased local synthesis of 1,25(OH)2D leading to an enhanced matrix mineralization. In this way, the local synthesis of 1,25(OH)2D may contribute to the stimulatory effect of calcium on matrix mineralization.

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The metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D) is synthesized from its precursor 25-hydroxyvitamin D (25(OH)D) by human osteoblasts leading to stimulation of osteoblast differentiation in an autocrine or paracrine way. Osteoblast differentiation is also stimulated by mechanical loading through activation of various responses in bone cells such as nitric oxide signaling. Whether mechanical loading affects osteoblast differentiation through an enhanced synthesis of 1,25(OH)2D by human osteoblasts is still unknown. We hypothesized that mechanical loading stimulates the synthesis of 1,25(OH)2D from 25(OH)D in primary human osteoblasts. Since the responsiveness of bone to mechanical stimuli can be altered by various endocrine factors, we also investigated whether 1,25(OH)2D or 25(OH)D affect the response of primary human osteoblasts to mechanical loading. Primary human osteoblasts were pre-incubated in medium with/without 25(OH)D3 (400 nM) or 1,25(OH)2D3 (100 nM) for 24h and subjected to mechanical loading by pulsatile fluid flow (PFF). The response of osteoblasts to PFF was quantified by measuring nitric oxide, and by PCR analysis. The effect of PFF on the synthesis of 1,25(OH)2D3 was determined by subjecting osteoblasts to PFF followed by 24h post-incubation in medium with/without 25(OH)D3 (400 nM). We showed that 1,25(OH)2D3 reduced the PFF-induced NO response in primary human osteoblasts. 25(OH)D3 did not significantly alter the NO response of primary human osteoblasts to PFF, but 25(OH)D3 increased osteocalcin and RANKL mRNA levels, similar to 1,25(OH)2D3. PFF did not increase 1,25(OH)2D3 amounts in our model, even though PFF did increase CYP27B1 mRNA levels and reduced VDR mRNA levels. CYP24 mRNA levels were not affected by PFF, but were strongly increased by both 25(OH)D3 and 1,25(OH)2D3. In conclusion, 1,25(OH)2D3 may affect the response of primary human osteoblasts to mechanical stimuli, at least with respect to NO production. Mechanical stimuli may affect local vitamin D metabolism in primary human osteoblasts. Our results suggest that 1,25(OH)2D3 and mechanical loading, both stimuli of the differentiation of osteoblasts, interact at the cellular level.

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The purpose of bone tissue engineering is to employ scaffolds, cells, and growth factors to facilitate healing of bone defects. The aim of this study was to assess the viability and osteogenic differentiation of primary human osteoblasts and adipose tissue-derived mesenchymal stem cells from various donors on titanium dioxide (TiO2) scaffolds coated with an alginate hydrogel enriched with enamel matrix derivative. Cells were harvested for quantitative reverse transcription polymerase chain reaction on days 14 and 21, and medium was collected on days 2, 14, and 21 for protein analyses. Neither coating with alginate hydrogel nor alginate hydrogel enriched with enamel matrix derivative induced a cytotoxic response. Enamel matrix derivative-enriched alginate hydrogel significantly increased the expression of osteoblast markers COL1A1, TNFRSF11B, and BGLAP and secretion of osteopontin in human osteoblasts, whereas osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells seemed unaffected by enamel matrix derivative. The alginate hydrogel coating procedure may have potential for local delivery of enamel matrix derivative and other stimulatory factors for use in bone tissue engineering.

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BACKGROUND: Oxidative stress is involved in the pathogenesis of bone diseases such as osteoporosis, which has a high coincidence with fractures in elderly. Several studies showed positive effects of herbal bioactive substances on oxidative stress. This study analyses the effect of green tea extract (GTE) Sunphenon 90LB on primary human osteoblasts differentiation and viability during H2O2-induced oxidative stress. Moreover, it was analyzed, whether GTE acts during the HO-1 signaling pathway. METHODS: Human osteoblasts were isolated from femoral heads of patients undergoing total hip replacement. Beneficial effects of GTE on osteoblasts were examined in a dose- and time-dependent manner. Furthermore, GTE was given before, simultaneous with and after induction of oxidative stress with 1 mM H2O2 to simulate prophylactic, acute and therapeutic use, respectively. Cell damage was measured by LDH leakage and cell viability by MTT assay. Flow cytometry was applied to measure formation of Reactive Oxygen Species by using 2`7`-dichlorofluorescein-diacetate. The formation of Extracellular Matrix after differentiation with GTE supplementation during oxidative stress was visualized with von Kossa and Alizarin Red staining. Last one was additionally photometrically quantified. To assess the effects of H2O2 and GTE on the osteogenic genes, RT-PCR was performed. To evaluate the intramolecular influence of GTE after the stimulation the protein levels of HO-1 were analyzed. RESULTS: Stimulation of primary human osteoblasts with low doses of GTE during oxidative stress over 21 days improved mineralization. Furthermore, GTE supplementation in combination with H2O2 leads to a higher gene expression of osteocalcin and collagen1α1 during osteoblasts differentiation. Both are important for bone quality. Pre-incubation, co-incubation and post-incubation of osteoblasts with high doses of GTE protect the osteoblasts against acute oxidative stress as shown by increased cell viability, decreased LDH leakage, and reduced production of intracellular free radicals. Functional analysis revealed an increased HO-1 protein synthesis after stimulation with GTE. CONCLUSIONS: Incubation of human primary osteoblasts with GTE significantly reduces oxidative stress and improves cell viability. GTE also has a beneficial effect on ECM production which might improve the bone quality. Our findings suggest that dietary supplementation of GTE might reduce inflammatory events in bone-associated diseases such as osteoporosis.

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CaSiO3 (CS) ceramic has been extensively studied for biomedical applications. The main advantages are its ability to induce bone-like apatite formation and the beneficial effects of the dissolution products on the bone cells, resulting from high reactivity of CS in liquid solutions. However, the high reactivity also results in a rapid degradation rate and accordingly leads to a high pH value in the body fluid, adversely affecting bone cell responses, especially when CS is used as a highly porous scaffold. In this study, we provide an approach to minimize this pH-dependent cell damage and maximize the beneficial effects of the dissolution products of the CS scaffold by adding chemically stable and biocompatible Zn-containing hardystonite (Ca2ZnSi2O7, HT) into the CS scaffold, the resultant composite scaffold is referred to as HT-CS. We investigated the responses of primary human osteoblasts (HOBs) to the CS, HT and the HT-CS scaffolds. HOBs on HT and HT-CS scaffolds attached better than on the CS scaffold. HOBs cultured on the HT-CS scaffolds expressed higher gene expression levels for Runx-2, osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen type I (Col-I) and enhanced alkaline phosphatase (ALP) activity compared to those on the CS and HT scaffolds. The higher activity of the HOBs cultured on the HT-CS scaffold was ascribed to the moderate pH variation and the dissolution products containing Ca, Si and Zn.

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Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS). The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM) rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO-) 1 and superoxide-dismutase- (SOD-) 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers.

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