Viability Of Human Osteoblasts Research Articles

Biodegradable and Biocompatible Thermoplastic Poly(Ester-Urethane)s Based on Poly(ε-Caprolactone) and Novel 1,3-Propanediol Bis(4-Isocyanatobenzoate) Diisocyanate: Synthesis and Characterization.

A series of non-toxic biodegradable and biocompatible polyurethanes bearing p-aminobenzoate moieties are presented. The introduction of this attractive motif was carried out by the synthesis of a novel isocyanate. These biodegradable polymers were chemically and physically characterized by several techniques and methods including bioassay and water uptake measurements. The molecular weight of the soft segment (poly-ε-caprolactone, PCL) and hard segment crystallinity dictated the mechanical behavior and water uptake. The behavior of short PCL-based polyurethanes was elastomeric, whilst increasing the molecular weight of the soft segment led to plastic polyurethanes. Water uptake was hindered for long PCL due to the crystallization of the soft segment within the polyurethane matrix. Furthermore, two different types of chain extender, hydrolyzable and non-hydrolyzable, were also evaluated: polyurethanes based on hydrolyzable chain extenders reached higher molecular weights, thus leading to a better performance than their unhydrolyzable counterparts. The good cell adhesion and cytotoxicity results demonstrated the cell viability of human osteoblasts on the surfaces of these non-toxic biodegradable polyurethanes.

Improvement of physicochemical properties of ternary nanocomposites based on hydroxyapatite/CuO/graphene oxide for biomedical usages

The nanocomposites (NCs) based on hydroxyapatite (HAP) could be improved into biologically safe and effective antibacterial materials. Combining HAP with copper oxide (CuO) and graphene oxide (GO) in a ternary nanocomposite (TNC) increased the human osteoblast viability in vitro to about 98.3% ± 3%. The structure was also analyzed with XRD, FTIR, XPS. The average roughness (Ra) decreased from 68.2 to 42.4 nm, while the root mean square roughness (Rq) changed slightly from 55.5 to 54.4 nm. The TEM investigation showed a decrease in the diameter and length of nanorod particles from 14 to 37 nm to 11 and 34 nm. In addition, the nano strip particles decreased from 17 to 14 nm in diameter, while a change in length was from 151 to 139 nm in diameter and length, respectively. The nanorods belong to HAP, while nanostrips belong to CuO. The polarization resistance decreased from 108 to around 44.5 (ohm.cm2). The antibacterial activity was done against the gram-negative E. coli, and the gram-positive S. aureus where inhibition zones reached 17.1 ± 1.3 and 14.3 ± 0.7 mm, respectively. The enhancement in cell viability and antibacterial behavior indicate the biocompatibility of the biomaterials for biomedical applications.

Cell viability of novel composite hydrogels loaded with hydroxyapatite for oral and maxillofacial bone regeneration.

The development of hydrogels for maxillofacial bone regeneration holds vast potential. However, some challenges need to be addressed to further their application in clinical settings. One challenge is optimizing cell viability. To improve mechanical strength, various materials have been investigated; however, incorporation of these materials within the hydrogel network may affect cell viability. The purpose of this study was to evaluate the cell viability of novel gelatin-alginate composite hydrogels loaded with hydroxyapatite (HA) and nano-hydroxyapatite (n-HA) for maxillofacial bone regeneration. Nine different hydrogels were prepared: three loaded with 0.5%, 1%, and 3% w/v HA; three loaded with 0.25%, 0.5%, and 1% w/v n-HA; one not loaded as a control and two HA and n-HA hydrogels with a lower concentration of the EDC crosslinker. Cell viability of human osteoblasts exposed to the hydrogels as affected by the HA type, size, and concentration, as well as to the crosslinker concentration, was investigated. An Alamar Blue assay was used to evaluate cell viability in the presence of hydrogel extracts and in aqueous solutions (without the hydrogel). A qualitative model was developed for explaining cell viability and growth. Higher percentages of cell viability were observed in the hydrogels loaded with hydroxyapatite as compared with the control. The effect of HA-related parameters, i.e., particle size and concentration, was found to increase the cytotoxic effect, as expressed in lower cell viability. The most favorable composites were the n-HA hydrogels. The incorporation of n-HA in the hydrogel to form a composite seems to be a very promising approach for maxillofacial bone regeneration applications.

Antibacterial composite membranes of polycaprolactone/gelatin loaded with zinc oxide nanoparticles for guided tissue regeneration

The bacterial colonization of absorbable membranes used for guided tissue regeneration (GTR), as well as their rapid degradation that can cause their rupture, are considered the major reasons for clinical failure. To address this, composite membranes of polycaprolactone (PCL) and gelatin (Gel) loaded with zinc oxide nanoparticles (ZnO-NPs; 1, 3 and 6 wt% relative to PCL content) were fabricated by electrospinning. To fabricate homogeneous fibrillar membranes, acetic acid was used as a sole common solvent to enhance the miscibility of PCL and Gel in the electrospinning solutions. The effects of ZnO-NPs in the physico-chemical, mechanical and in vitro biological properties of composite membranes were studied. The composite membranes showed adequate mechanical properties to offer a satisfactory clinical manipulation and an excellent conformability to the defect site while their degradation rate seemed to be appropriate to allow successful regeneration of periodontal defects. The presence of ZnO-NPs in the composite membranes significantly decreased the planktonic and the biofilm growth of the Staphylococcus aureus over time. Finally, the viability of human osteoblasts and human gingival fibroblasts exposed to the composite membranes with 1 and 3 wt% of ZnO-NPs indicated that those membranes are not expected to negatively influence the ability of periodontal cells to repopulate the defect site during GTR treatments. The results here obtained suggest that composite membranes of PCL and Gel loaded with ZnO-NPs have the potential to be used as structurally stable GTR membranes with local antibacterial properties intended for enhancing clinical treatments.

Influence of the Surface Roughness of PEEK GRF30 and Ti6Al4V SLM on the Viability of Primary Human Osteoblasts Determined by the MTT Test.

The aim of the study was to clearly determine whether selected modern medical materials and three dimensional printing allow for satisfactory viability of human osteoblasts, which is important from the point of view of the subsequent osseointegration process. Moreover, as implants are produced with various topography, the influence of surface roughness on viability of bone cells was evaluated. To conduct the research, primary human osteoblasts (PromoCell) were used. Cells were seeded on samples of glass-reinforced polyetheretherketone (30% of the filling), Ti6Al4V manufactured with the use of selective laser melting technology and forged Ti6Al4V with appropriately prepared variable surface roughness. To assess the viability of the tested cells the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used. Results showed that all evaluated materials do not exhibit cytotoxic properties. Moreover, on their basis it can be concluded that there is a certain surface topography (designated i.a. as roughness equal to approx. Ra = 0.30 μm), which ensures the highest possible viability of human osteoblasts. On the basis of the received data, it can also be concluded that modern glass-reinforced polyetheretherketone or Ti6Al4V produced by rapid prototyping method allow to manufacture implants that should be effectively used in clinical conditions.

Activation of the Wnt Pathway by Small Peptides: Rational Design, Synthesis and Biological Evaluation.

A computational analysis of the X-ray structure of the low-density lipoprotein receptor-related protein 6 (LRP6) with the Dickkopf-1 (DKK1) C-terminal fragment has allowed us to rationally design a small set of decapeptides. These compounds behave as agonists of the canonical Wnt pathway in the micromolar range when tested on a dual luciferase Wnt functional assay in glioblastoma cells. Two of the oligopeptides showed a lack of cytotoxicity in human primary osteoblasts isolated from sponge bone tissue (femoral heads or knees of elderly patients). According to the mechanism of action, the studies revealed a dose- and time-dependent increase in the viability of human osteoblasts. These results may indicate a potential therapeutic application of this class of compounds in the treatment of bone diseases related to aging, such as osteoporosis.

Cytoprotective effects of melatonin on zoledronic acid-treated human osteoblasts

ObjectiveTo evaluate the cytoprotective effects of melatonin (MLT) on zoledronic acid (ZA)-treated human osteoblasts. MethodsHuman osteoblasts were exposed to ZA (1, 5, 10, 50, 100 and 300 μM) and MLT (1, 10, 50, 100 y 200 μM) for 24, 48 and 72 h of incubation, to evaluate their effects on cell viability. ResultsAs ZA concentration increased, greater reductions in cell viability of human osteoblasts were induced whether at 24, 48 or 72 h incubation. At 24 h incubation with MLT, greatest cell viability was obtained when low dose of MLT was applied (without significant differences); 48 and 72 h incubation presented the greatest cell viability with the highest MLT concentrations (100 and 200 μM). MLT at concentrations of 100 and 200 μM would appear to have a certain cytoprotective effect on ZA-treated human osteoblasts with low concentrations of ZA (1 y 5 μM), whether at 24, 48 or 72 h; however, at ZA concentrations ≥10 μM the possible cytoprotective effects of MLT were low at 24 h incubation. ConclusionsMLT has a cytoprotective effect on ZA-treated human osteoblasts and could represent a promising preventative alternative for patients at risk of bisphosphonate-related osteonecrosis of the jaw.

Laser Surface Pattering of Titanium for Improving the Biological Performance of Dental Implants.

Direct laser interference patterning (DLIP) is used to produce periodic line-like patterns on titanium surfaces. An Nd:YAG laser operating at 532 nm wavelength with a pulse duration of 8 ns is used for the laser patterning process. The generated periodic patterns with spatial periods of 5, 10, and 20 µm are produced with energy densities between 0.44 and 2.6 J cm-2 with a single laser pulse. With variation of energy density, different shapes of the arising topography are observed due to the development of the solidification front of the molten material at the maxima positions. Characterization of the surface chemistry shows that the DLIP treatment enhances the content of nitrogen of the titanium reactive layer from 3.9% up to 23.4%. The structural analysis near the titanium surface shows no changes in microstructure after the laser treatment. Contact angles between 65° and 79° are measured on both structured and turned reference surfaces. Cell viability of human osteoblasts on line-like patterned surfaces after 7 d in cultivation medium is 16% higher compared to the grit-blasted and acid-etched references. Finally, the possibility of patterning complex 3D dental implants is shown.

Antibacterial and Biocompatible Titanium-Copper Oxide Coating May Be a Potential Strategy to Reduce Periprosthetic Infection: An In Vitro Study.

Periprosthetic infections are devastating for patients and more efficacious preventive strategies are needed. Surface-modified implants using antibacterial coatings represent an option to cope with this problem; however, manufacturing limitations and cytotoxicity have curbed clinical translation. Among metals with antibacterial properties, copper has shown superior in vitro antibacterial performance while maintaining an acceptable cytotoxicity profile. A thin film containing copper could prevent early biofilm formation to limit periprosthetic infections. This pilot study presents the in vitro antibacterial effect, cytotoxicity, and copper ion elution pattern of a thin film of titanium-copper oxide (TiCuO). (1) Do titanium alloy (Ti6Al4V) discs coated with a thin film of TiCuO reduce Staphylococcus epidermidis biofilm and planktonic cell density compared with uncoated discs? (2) Do Ti6Al4V discs coated with a thin film of TiCuO affect normal human osteoblast viability compared with untreated cells? (3) Is copper ion concentration generated by coated discs lower than previously published copper ion concentrations that cause 50% toxicity in similar human cell lines in vitro (TC50)? Ninety Ti6Al4V discs (12.5 mm diameter; 1.25 mm thick) were used in this study. Seventy-two Ti6Al4V discs were coated with a thin film of either titanium oxide (TiO) or TiCuO containing 20%, 40%, or 80% copper using high-power impulse magnetron sputtering (HiPIMS). Eighteen Ti6Al4V discs remained uncoated for control purposes. We tested antibacterial properties of S epidermidis grown on discs in wells containing growth medium. After 24 hours, planktonic bacteria as well as biofilms removed by sonication were quantitatively cultured. Annexin/Pi staining was used to quantify in vitro normal human osteoblast cell viability at 24 hours and Day 7, respectively. Copper elution was measured at Days 1, 2, 3, 7, 14, and 28 using an inductively coupled plasma mass spectrometer to analyze aliquots of culture medium. Copper ion concentration achieved at 24 hours was compared with previously published TC50 for gingival fibroblast, a phenotypically similar cell line with available data regarding copper ion exposure. Discs coated with TiCuO 80% copper showed greater biofilm and planktonic cell density reduction when compared with other tested compositions (analysis of variance [ANOVA]; p < 0.001). Discs coated with TiCuO 80% copper showed mean biofilm and planktonic cell density of 4.0 log10 (SD = 0.4) and 5.7 log10 (SD = 0.2). Discs coated with TiCuO 80% showed a mean difference in biofilm and planktonic cell density of 2.5 log10 (95% confidence interval [CI], 1.9-3.1 log10; p < 0.001) and 1.2 (95% CI, 0.6-1.8; p < 0.001), respectively, when compared with uncoated discs. Normal human osteoblast viability did not differ among all groups at 24 hours (ANOVA; p = 0.2) and Day 7 (ANOVA; p = 0.7). Discs coated with TiCuO 80% copper showed a mean difference (95% CI) in relative cell viability (%) at 24 hours and Day 7 of 31.1 (95% CI, -19.4 to 81.7; p = 0.4) and -5.0 (95% CI, -7.8 to 17.9; p = 0.9), respectively, when compared with untreated cells. For all TiCuO-coated discs, copper ion elution peaked at 24 hours and slowly decreased in a curvilinear fashion to nearly undetectable levels by Day 28. Discs coated with TiCuO 80% copper showed mean copper ion concentration at 24 hours of 269.4 µmol/L (SD = 25.2 µmol/L) and this concentration was lower than previously published TC50 for similar human cell lines at 24 hours (344 µmol/L, SEM = 44 µmol/L). This pilot study demonstrates a proof of concept that a thin-film implant coating with TiCuO can provide a potent local antibacterial environment while remaining relatively nontoxic to a human osteoblast cell line. Further research in an animal model will be necessary to establish efficacy and safety of this technique and whether it might be useful in the design of implants. A thin film coating with TiCuO demonstrates high antibacterial activity and low cellular cytotoxicity to human osteoblasts in vitro. Taken together, these properties represent a potential strategy for preventing periprosthetic infection if further work in animal models can confirm these results in vivo.

The influence of metallic ions from CoCr28Mo6 on the osteogenic differentiation and cytokine release of human osteoblasts

Abstract Inflammatory reactions associated with osteolysis and implant loosening can be the result of generated CoCr particles and the simultaneous release of ions, which are the consequence of wear at the articulating surfaces of metallic implants. By now, there is little knowledge about the influence of CoCr ions on the viability of human osteoblasts (hOB). Therefore, metallic ions were generated from solid CoCr28Mo6 alloys using electric potentials against hydrogen bridge electrodes. For the cell experiments, hOBs (n ≥ 4) in the third passage were used. Cells were cultured for 24 h before incubated with the generated ion solution in two different concentrations (100 µg/l and 500 µg/l in DMEM with 10 % FCS). Cells incubated without ions served as controls. After 48 h and 96 h cell viability was determined (WST-1 assay, live-dead-stainings). Additionally, gene expression levels of osteogenic markers and cytokines were analysed. A significant increase of metabolic activity and vital cells could be shown for both ion concentrations. Gene expression of alkaline phosphatase and Col1 was highly induced after 96 h for 100 µg/l whereas Col1 protein was significantly reduced on both time points (48 h: p=0.008 (100 µg/l, 500 µg/l); 96 h: p=0,029 (500 µg/l)) compared to the control. Additionally, MMP1 expression was decreased after 48 h and 96 h. IL6 mRNA was concentration-dependent increased after 48 h whereas a significant decrease could be shown for the highest ion concentration (p=0.029). Compared to the lower ion concentration, IL8, MCP1 (p=0.029) and TNF mRNA were clearly reduced for the higher ion solution after 96 h. Our results suggest that ions from CoCr28Mo6 alloys have only marginal effects on osteoblast viability whereas Col1 expression is reduced and cytokine release is induced. In further studies, the effects of metallic ions and particles from CoCr28Mo6 on other cells will be examined.

Human endogenous peptide p33 inhibits detrimental effects of LL‐37 on osteoblast viability

High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types. Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation. Cell number was assessed by counting cells in a Bürker chamber. Intracellular Ca(2+) was monitored by recording Fluo 4-AM fluorescence using a laser scanning confocal microscope. Cellular expression of p33 was determined by western blotting. LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50 ) of 4μm and a rapid and sustained rise in the intracellular Ca(2+) concentration of osteoblasts, suggesting that LL-37 forms pores in the cell membrane. p33 (10μm) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca(2+) concentration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonstimulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts. The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteoblasts from LL-37-induced cell damage in patients suffering from chronic periodontitis associated with high levels of LL-37 locally.

Tribological properties of biocompatible Ti–10W and Ti–7.5TiC–7.5W

This study investigates and compares the microstructure, biocompatibility, and tribological properties of two different Ti-based composites, Ti–10W and Ti–7.5TiC–7.5W, with those of pure Ti for their potential use in biomedical applications. In particular, cold and hot isostatic-pressing and arc-melting methods were utilized and compared for the microstructure of the composites. Nano-scratch measurements and pin-on-disk wear tests were employed to understand their tribological behavior. As compared to pure Ti, Ti–10W and Ti–7.5TiC–7.5W showed significantly improved nano-scratch resistance (by 85 and 77%, respectively) and wear resistance (by 64 and 66%, respectively), in good agreement with hardness measurements. For biocompatibility examination, both microculture tetrazolium test (MTT) and water soluble tetrazolium (WST-1) test were used to quantify the cell viability of human osteoblasts and mouse fibroblasts on the materials. Both of the Ti-based composites showed acceptable biocompatibility in comparison with the pure Ti control.

Comparative Analysis of the Oxygen Supply and Viability of Human Osteoblasts in Three-Dimensional Titanium Scaffolds Produced by Laser-Beam or Electron-Beam Melting.

Synthetic materials for bone replacement must ensure a sufficient mechanical stability and an adequate cell proliferation within the structures. Hereby, titanium materials are suitable for producing patient-individual porous bone scaffolds by using generative techniques. In this in vitro study, the viability of human osteoblasts was investigated in porous 3D Ti6Al4V scaffolds, which were produced by electron-beam (EBM) or laser-beam melting (LBM). For each examination, two cylindrical scaffolds (30 mm × 10 mm in size, 700 µm × 700 µm macropores) were placed on each other and seeded with cells. The oxygen consumption and the acidification in the center of the structures were investigated by means of microsensors. Additionally, the synthesis of pro-collagen type 1 was analyzed. On the LBM titanium scaffolds, vital bone cells were detected in the center and in the periphery after 8 days of cultivation. In the EBM titanium constructs, however, vital cells were only visible in the center. During the cultivation period, the cells increasingly produced procollagen type 1 in both scaffolds. In comparison to the periphery, the oxygen content in the center of the scaffolds slightly decreased. Furthermore, a slight acidification of the medium was detectable. Compared to LBM, the EBM titanium scaffolds showed a less favorable behavior with regard to cell seeding.

Neuropeptides stimulate human osteoblast activity and promote gap junctional intercellular communication

Neuropeptides released from the skeletal nerve fibers have neurotransmitter and immunoregulatory roles; they exert paracrine biological effects on bone cells present close to the nerve endings expressing these signaling molecules. The aims of this study were a systematic investigation of the effects of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), Neuropeptide Y (NPY) and tyrosine hydroxylase (TH) on the cell viability and function of the human osteoblasts, and comparing their difference in the role of regulating bone formation. Cultures of normal human osteoblasts were treated with SP, CGRP, VIP, NPY or TH at three concentrations. We found that each of the five neuropeptides induced increases in cell viability of human osteoblasts. The stimulatory action of NPY was the highest, followed by VIP, SP and TH, while CGRP had the lowest stimulatory effect. The viability index of osteoblasts was inversely associated with the concentration of neuropeptides, and positively with the time of exposure. Moreover, the five neuropeptides increased the ALP activity and osteocalcin to different extents in a dose-dependent manner. The GJIC of osteoblasts was significantly promoted by neuropeptides. The results demonstrated that neuropeptides released from skeletal nerve endings after a stimulus appeared to be able to induce the proliferation and activity of osteoblasts via enhancing GJIC between cells, and further influence the bone formation. These findings may contribute toward a better understanding of the neural influence on bone remodeling and improving treatments related to bone diseases.

Osteoblast viability on hydroxyapatite with well-adjusted submicron and micron surface roughness as monitored by the proliferation reagent WST-1

The impact of the cell surface roughness on titanium alloys used for biomedical implants has been extensively studied, whereas the dependency of human osteoblast viability on hydroxyapatite (HA) submicron and micron surface roughness has hitherto not yet been investigated in detail. Therefore, we investigate in this study the effect of HA substrates with different well-adjusted surface roughness on human osteoblast proliferation using the standard colorimetric reagent WST-1. By grinding, we obtained HA surfaces with six levels of well-defined surface roughness ranging from Sa = 3.36 µm down to 0.13 µm, resulting in hydrophilic contact angles from 11° to 27°. Energy dispersive X-ray spectroscopy, X-ray diffraction, and X-ray fluorescence measurements confirmed that neither grinding paper residues nor changes of the crystal structure were introduced to the HA substrates by the grinding process. By applying this simple surface treatment, we were able to isolate other effects from surface chemistry, crystal structure, and relative density. The changes of the osteoblast proliferation (WST-1 assay) on these different roughened HA surfaces after 7 days were found to be insignificant (p > 0.05), evaluated by one-way analysis of variance and Tuckey's Multiple Comparison Method. The results of this study show that all roughened HA surfaces, regardless of the microtopography, are biocompatible and allow osteoblast attachment, proliferation, and collagen type I production. The comparison with surface roughness used for standard Ti-based implants yielded that for HA no finishing process is necessary to ensure a sound human osteoblast cell proliferation in vitro.

Effects of bisphosphonates on human osteoblasts as an important constituent of the bone marrow microenvironment.

e13620 Background: Bisphosphonates (BPs) are widely used for the treatment in bone metastatic disease and for the management of osteoporosis. Their antiresorptive effects can be explained by inhibition of osteoclastic resorption, promoting osteoclast apoptosis. However, recent evidence also points to a direct action of bisphosphonates on bone-forming osteoblasts. Despite its clinical importance, the mechanisms of BPs on osteoblast function still remain poorly characterized. Methods: In this study, the influence of BPs zoledronate (zol), ibandronate (iban), and pamidronate (pam) on molecular and cellular functions of human osteoblasts was assessed by focusing on osteogenic marker gene expression, cell proliferation and viability. Results: The regulation of osteogenic markers was analyzed by quantitative real-time RT-PCR after exposure of primary osteoblasts (pOB) to different concentrations (0.01-100 μM) of zol and iban for 48 h and 72 h. The gene-expression analyses revealed an important increase in osteopontin and in osteocalcin expression with 100 μM, decrease in type I collagen alpha-2 with 0.01-100 μM zol and iban and an unchanged expression of bone sialoprotein, runt-related transcription factor 2 and osteonectin after 48 h and 72 h. Further, exposure of pOB to different zol but not to iban concentrations for 72 h resulted in a significant overexpression of alkaline phosphatase relative to control cells, except for the treatment with 100 μM. Proliferation assays demonstrated that treatment with iban and pam for 72 h did not affect the osteoblast number at concentrations of 0.01-10 μM, whereas 100 μM resulted in a notably cell growth reduction. Zol showed an inhibitory proliferation action already by 1 μM. Moreover, a significant decrease of osteoblast viability was noted after treatment with ≥ 1 μM zol after 48 h, which could be enhanced after 72 h. Conclusions: Our results demonstrated that bisphosphonates revealed prominent differences in the mode of action on human osteoblast functions. Zol, recognized as the most potent BP, reduced both the expression of osteogenic markers and the proliferation as well as the viability of human osteoblasts at concentrations in the low micro-molar range. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis

Polymer-ceramic nanocomposites for applications in the bone surgery

The subject of this work was preparation and investigation of properties of a nanocomposite material based on polymer matrix modified with nanometric silica particles (SiO2). The composite matrix consisted of resorbable P(L/DL)LA polymer with certified biocompatibility. Nanometric silica was introduced into the matrix by means of ultrasonic homogenisation and/or mechanical stirring. The silica was introduced directly e.g. as nanoparticles or inside calcium alginate fibres which contained 3 wt.% of amorphous SiO2. Proper dispersion of nano-filliers was confirmed by means of thermal analysis (TG/DTA, DSC). It was observed, that the presence of inorganic nanoparticles influenced several surface parameters of the nanocomposites i.e. hydrophility (a decrease of surface energy) and topography (both in micro- and nano-scale). Additionally, the nanocomposites exhibited enhanced mechanical properties (Young's modulus, tensile strength) compared to the pure polymer. The nanocomposites were bioactive materials (SBF/3 days/37oC). Biological tests (MTT test) showed a good viability of human osteoblasts (hFOB 1.19) in contact with the nanocomposites surface. Results of preliminary biological tests carried out with the use of mother cells extracted from human bone marrow showed that the nanocomposites may provide differenation of bone cells.

Influence of Catalyst Type on Biocompatibility of Polyurethanes

Segmented polyurethanes (PUR) based on poly(ε-caprolactone)diol (PCL) as soft segments and cycloaliphatic diisocyanate were obtained. Two different types of catalysts were used for synthesis: dibutyltin dilaurate (DBTDL) and iron (III) acetylacetonate Fe(acac)3. Structure, thermal analysis, water absorption of obtained polyurethanes were studied. Polyurethanes catalyzed by Fe(acac)3 have lower glass transition temperature (Tg2) and melting temperature (Tm2) of hard domains in comparison with polyurethanes synthesized with DBTDL. Amount of absorbed water is in the range of 1,0-2,0% and is higher for PUR catalyzed by DBTDL. SEM observations show that type of catalyst used in synthesis affect structure of polyurethanes. Biological test XTT was done in order to determine influence of various metallic compounds used as catalyst on viability of human bone cell culture. The XTT results indicate no influence on viability of human osteoblasts cultured on the obtained PUR.

Porous hydroxyapatite–tricalcium phosphate bioceramics

Porous bioceramics hydroxyapatite–tricalcium phosphate (HA–TCP), aimed to be applied in clinic, was fabricated by powder metallurgy and evaluated using both in vitro and in vivo models. Porous HA–TCP was supposed as a partially biodegradable material and designed as a scaffold for bone reconstruction or regeneration. The material processing was proposed and the physical properties as well as the microstructure feature were characterised here. Biological postulation of the relationship between seeding density, proliferation and viability of human osteoblasts cultured on the porous HA–TCP was quantitatively measured. Bone reconstruction was investigated both in vitro and in vivo by use of these biodegradable scaffolds with pore sizes ranged from 200 to 400 μm in diameter. The degradable bioceramic supported cellular proliferation seeded on the scaffold and showed normal differentiated function in vitro. Suitable pore size of the porous bioceramic was required if promotion of bone reconstruction was desired. Clinical trials showed that the bioceramics were successfully applied for bone reconstruction and regeneration and could be partially degraded in human body in 18 months. This approach suggests the feasibility of using porous HA–TCP bioceramics for the transplantation of autogenous osteoblasts to regenerate bone tissue.

Osteoblast viability and differentiation with Me 2SO as cryoprotectant compared to osteoblasts from fresh human iliac cancellous bone

The aim of this study was to compare the viability of human osteoblasts cryopreserved with Me 2SO to that of fresh human iliac cancellous bone using cell culture techniques. Osteoblasts were obtained by spontaneous outgrowth of human iliac cancellous bone specimens in experiment I. In experiment II, human iliac cancellous bone was frozen with 10% Me 2SO at −80 °C for 2 weeks and osteoblasts grew spontaneously after thawing at 37 °C by removing Me 2SO with sucrose. The cells were grown in culture flasks containing DMEM as a culture medium, supplemented with 10% fetal calf serum. They were kept at 37 °C in a humidified atmosphere of 95% air and 5% CO 2. Cells from the second passage were plated at a density of 5 times 10 3 cells/cm 2 in 24-well plates. For detection of viability and differentiation, WST-1 assay, determination of alkaline phosphatase activity, concentration of procollagen I peptide, concentration of osteocalcin, and indirect immunofluorescence for osteopontin, collagen type I, integrin β 1, and fibronectin were applied. Experiments were conducted at four stages of confluence (days 4, 7, 14, and 21 after plating the cells). Based on the results of this study, we conclude that osteoblast-like cells survived cryopreservation and synthesized a range of markers that were consistent with this cell type.