Pulsed laser deposition temperature effects on strontium-substituted hydroxyapatite thin films for biomedical implants.

Substituting small molecule drugs with abundant and easily affordable ions may have positive effects on the way countless disease treatments are approached. The interest in strontium cation in bone therapies soared in the wake of the success of strontium ranelate in the treatment of osteoporosis. A new method for producing thin strontium-containing hydroxyapatite (Sr-HA, Ca9Sr(PO4)6(OH)2) films as coatings that render bioinert titanium implant bioactive is reported here. The method is based on the combination of a mechanochemical synthesis of Sr-HA targets and their deposition in form of thin films on top of titanium with the use of laser ablation at low pressure. The films were 1-2 µm in thickness and their formation was studied at different temperatures, including 25, 300, and 500 °C. Highly crystalline Sr-HA target transformed during pulsed laser deposition to a fully amorphous film, whose degree of long-range order recovered with temperature. Particle edges became somewhat sharper and surface roughness moderately increased with temperature, but the (Ca+Sr)/P atomic ratio, which increased 1.5 times during the film formation, remained approximately constant at different temperatures. Despite the mostly amorphous structure of the coatings, their affinity for capturing atmospheric carbon dioxide and accommodating it as carbonate ions that replace both phosphates and hydroxyls of HA was confirmed in an X-ray photoelectron spectroscopic analysis. As the film deposition temperature increased, the lattice voids got reduced in concentration and the structure gradually "closed," becoming more compact and entailing a linear increase in microhardness with temperature, by 0.03 GPa/°C for the entire 25-500 °C range. Biocompatibility and bioactivity of Sr-HA thin films deposited on titanium were confirmed in an interaction with dental pulp stem cells, suggesting that these coatings, regardless of the processing temperature, may be viable candidates for the surface components of metallic bone implants.

Improved Healing by Adjuvant Osteoconductive Therapy Using a Novel Cotton-Like Hydroxyapatite Sheet After Median Sternotomy.

The union rate of wire fixation after median sternotomy remains unsatisfactory. We developed a novel osteoconductive sheet composed of hydrophilized hydroxyapatite and evaluated its osteogenetic effect when interposed between sternal halves in a canine model. Eighteen canines were divided equally into groups based on the hemostatic agent used: osteoconductive sheet (S), none (C), and bone wax (BW). After median sternotomy, the sternal halves were closed by wire fixation. In each group, 3 canines were euthanized at 1 month, while 3 were euthanized at 2 months. Resected sternums were mechanically assessed by the 3-point bending test, radiographically assessed by micro-CT, and pathologically assessed to quantify the osteogenesis between sternal halves. Compared with the BW group, the S group had a greater maximum stress at 1 month (S: 322.9 ± 107.7 N, C: 233.0 ± 62.7 N, BW: 124.9 ± 88.4 N; P = 0.025), and greater maximum shear force at 1 month (S: 1.92 ± 0.67 N/m2; C: 1.23 ± 0.28 N/m2; BW: 0.68 ± 0.41 N/m2; P = 0.025). Micro-CT revealed that the S group had more osteogenesis than the BW group at 1 month (25.7% ± 9.8% vs 6.9% ± 9.2%), and 2 months (34.0% ± 15.1% vs 14.8% ± 9.4%); the respective values in the C group were 17.1% ± 7.2% and 29.7% ± 9.3%. Pathologic examination revealed that the S group had the greatest osteogenetic area at 2 months (S: 38.8% ± 18.8%; C: 24.5% ± 6.9%; BW: 24.7% ± 18.6%). Adjuvant osteoconductive therapy using a cotton-like hydroxyapatite sheet in addition to wire fixation significantly improved sternal healing compared with BW. This new material also showed relatively better outcome than the C group.

Interaction of Folic Acid with Nanocrystalline Apatites and Extension to Methotrexate (Antifolate) in View of Anticancer Applications.

Nanocrystalline apatites mimicking bone mineral represent a versatile platform for biomedical applications thanks to their similarity to bone apatite and the possibility to (multi)functionalize them so as to provide "à la carte" properties. One relevant domain is in particular oncology, where drug-loaded biomaterials and engineered nanosystems may be used for diagnosis, therapy, or both. In a previous contribution, we investigated the adsorption of doxorubicin onto two nanocrystalline apatite substrates, denoted HA and FeHA (superparamagnetic apatite doped with iron ions), and explored these drug-loaded systems against tumor cells. To widen their applicability in the oncology field, here we examine the interaction between the same two substrates and two other molecules: folic acid (FA), often used as cell targeting agent, and the anticancer drug methotrexate (MTX), an antifolate analogue. In a first stage, we investigated the adsorptive behavior of FA (or MTX) on both substrates, evidencing their specificities. At low concentration, typically under 100 mmol/L, adsorption onto HA was best described using the Sips isotherm model, while the formation of a calcium folate secondary salt was evidenced at high concentration by Raman spectroscopy. Adsorption onto FeHA was instead fitted to the Langmuir model. A larger adsorptive affinity was found for the FeHA substrate compared to HA; accordingly, a faster release was noticed from HA. In vitro tests carried out on human osteosarcoma cell line (SAOS-2) allowed us to evaluate the potential of these compounds in oncology. Finally, in vivo (subcutaneous) implantations in the mouse were run to ascertain the biocompatibility of the two substrates. These results should allow a better understanding of the interactions between FA/MTX and bioinspired nanocrystalline apatites in view of applications in the field of cancer.

Injectable TEMPO-oxidized nanofibrillated cellulose/biphasic calcium phosphate hydrogel for bone regeneration.

Nanofibrillated cellulose, obtained from rice straw agricultural wastes was used as a substrate for the preparation of a new injectable and mineralized hydrogel for bone regeneration. Tetramethyl pyridine oxyl (TEMPO) oxidized nanofibrillated cellulose, was mineralized through the incorporation of a prepared and characterized biphasic calcium phosphate at a fixed ratio of 50 wt%. The TEMPO-oxidized rice straw nanofibrillated cellulose was characterized using transmission electron microscopy, Fourier transform infrared, and carboxylic content determination. The injectability and viscosity of the prepared hydrogel were evaluated using universal testing machine and rheometer testing, respectively. Cytotoxicity and alkaline phosphatase level tests on osteoblast like-cells for in vitro assessment of the biocompatibility were investigated. Results revealed that the isolated rice straw nanofibrillated cellulose is a nanocomposite of the cellulose nanofibers and silica nanoparticles. Rheological properties of the tested materials are suitable for use as injectable material and of nontoxic effect on osteoblast-like cells, as revealed by the positive alkaline phosphate assay. However, nanofibrillated cellulose/ biphasic calcium phosphate hydrogel showed higher cytotoxicity and lower bioactivity test results when compared to that of nanofibrillated cellulose.

Bone regeneration with micro/nano hybrid-structured biphasic calcium phosphate bioceramics at segmental bone defect and the induced immunoregulation of MSCs.

Adequate bone regeneration has been difficult to achieve at segmental bone defects caused by disease. The surface structure and phase composition of calcium phosphate bioceramic are crucial for its bioactivity and osteoinductivity. In the present study, biphasic calcium phosphate (BCP) bioceramics composed of micro-whiskers and nanoparticles hybrid-structured surface (hBCP) were fabricated via a hydrothermal reaction. The in vivo long bone defect model of beagle dogs implanted with hBCP bioceramics achieved a higher quality regenerated bone as compared to the traditional smooth-surface BCP control group. After a 12-week implantation period, more new bone formation within the implanted material and a higher fracture load were observed in the hBCP group (p < 0.05 vs. control). In addition, the local bone integration efficacy, as determined by nanoindentation, showed a significantly closer elastic modulus of the implanted hBCP bioceramics to that of the natural bone adjacent. Finally, in vitro gene microarray analysis of the mesenchymal stem cells (MSCs) co-cultured with two bioceramics showed that the hBCP group induced a drastic downregulation of the genes associated with inflammatory response, which was never documented in previous studies regarding biomaterials with a micro/nano hybrid structure. The tumor necrosis factor (TNF) signalling pathway was the most involved and preferentially inhibited by the hBCP material. Collectively, the findings suggested that the micro/nano hybrid-structured bioceramics augmented local bone regeneration at segmental bone defects and presented a potential alternative to autologous bone grafts.

Development of Sr-incorporated biphasic calcium phosphate bone cement.

To follow the design strategy of traditional biphasic calcium phosphate (BCP) ceramic, in the present study, strontium-doped biphasic calcium phosphate bone cement (Sr-BCPC) composites comprising Sr-ß-tricalcium phosphate (TCP)/Sr-hydroxyapatite (HAP) had been prepared for the first time using Sr x -ß-TCP/tetracalcium phosphate (TTCP) as a cement powder and diluted phosphoric acid as a cement liquid. The phase composition, setting time, compressive strength, washout resistance, in vitro degradation rate, microstructure evolutions, hydration dynamics and cytotoxicity of Sr-BCPC at various Sr contents were intensively investigated. It was found that the final cement product was composed of entangled Sr-HAP nano-needles and cobblestone-like Sr-ß-TCP sub-micron particles, and the weight percentages in the final cement product after hydration in simulated body fluid for 24 h were in the ranges of 60 wt%-70 wt% Sr-HAP and 30 wt%-40 wt% Sr-ß-TCP, respectively. Sr and the concentration of Sr exhibit significant effects on the phase compositions, compressive strength, setting time, in vitro degradation rate and cytotoxicity of the biphasic bone cement. In particular, the degradation rate increased considerably with the increase of the Sr-ß-TCP phase. It is anticipated that the introduction of the 'biphasic' design into calcium phosphate bone cements is an effective strategy to improve their degradation properties.

Effect of BioAggregate on Receptor Activator of Nuclear Factor-Kappa B Ligand-induced Osteoclastogenesis from Murine Macrophage Cell Line In Vitro.

INTRODUCTION: This study aimed to investigate the effect of BioAggregate, a calcium silicate-based nanoparticulate bioceramic, on the regulation of receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation and bone resorption in vitro, as well as to delineate the underlying molecular mechanism. The performance of BioAggregate was compared with that of ProRoot mineral trioxide aggregate (MTA). METHODS: Cells of a murine macrophage cell line RAW 264.7 were treated with various concentrations of BioAggregate and MTA extracts. Cytotoxicity of material extracts was evaluated with Cell Counting Kit-8 assay. RANKL-induced osteoclast differentiation and function were assessed with tartrate-resistant acid phosphatase staining, F-actin staining, and lacunar resorption pits assay. The mRNA expression associated with osteoclast function was detected with quantitative real-time polymerase chain reaction. Related molecular signaling pathways were investigated with Western blot and immunofluorescence. RESULTS: BioAggregate extracts dose-dependently inhibited RANKL-induced osteoclast formation and resorption capacity without evident cytotoxicity. RAW 264.7 cells exposed to BioAggregate extracts also presented a decrease in RANKL-stimulated mRNA expression of osteoclast-related genes and transcription factors. Moreover, cells treated with BioAggregate extracts exhibited attenuated TRAF6 expression, suppressed mitogen-activated protein kinase signaling, and decreased nuclear translocation of NFATc1 and c-Fos in the presence of RANKL. Comparable effects were induced by MTA. CONCLUSIONS: BioAggregate and MTA exhibit comparable inhibitory effect on osteoclast differentiation and function in vitro, and our findings provide valuable insights into the mechanism of bioceramic-mediated anti-osteoclastogenic activity.

Hydroxyapatite-based materials of marine origin: a bioactivity and sintering study.

Single phase hydroxyapatite (HAp) and biphasic material hydroxyapatite/ß-tricalcium phosphate (HAp/ß-TCP) were obtained from a marine source (Atlantic cod fish bones). Here we report a study on the biological properties of these materials, including cytotoxicity, bioactivity and haemocompatibility. Results showed that the materials are not cytotoxic, neither in their powder nor in pellet form; indeed growth of Saos-2 cells was comparable to that of commercial. The haemolysis rate was lower than 2%; hence the materials can be classified as non-haemolytic. Moreover, when immersed in Simulated Body Fluid (SBF), crystal formation was observed on the surface of both materials. The sintering behaviour of the samples was also studied; both powders showed very high sinterability (density higher than 95% of the theoretical value). Overall, these results confirm the suitability of these materials for biomedical applications.

Effects on growth and osteogenic differentiation of mesenchymal stem cells by the strontium-added sol-gel hydroxyapatite gel materials.

In the present study, strontium-modified hydroxyapatite gels (Sr-HA) at different concentrations were prepared using sol-gel approach and their effect on human-bone-marrow-derived mesenchymal stem cells, were evaluated. The effect of Strontium on physico-chemical and morphological properties of hydroxyapatite gel were evaluated. Morphological analyses (SEM and TEM) demonstrate that an increasing in the amount of Sr ions doped into HA made the agglomerated particles smaller. The substitution of large Sr2+ for small Ca2+ lead to denser atomic packing of the system causing retardation of crystals growth. The biological results demonstrated that hydroxyapatite gel containing from 0 to 20 mol% of Sr presented no cytotoxicity and promote the expression of osteogenesis related genes including an early marker for osteogenic differentiation ALP; a non-collagen protein OPN and a late marker for osteogenic differentiation OCN. Finally, the Sr-HA gels could have a great potential application as filler in bone repair and regeneration and used in especially in the osteoporotic disease.

In vitro evaluation of human osteoblast-like cell proliferation and attachment on nanostructured fluoridated hydroxyapatite.

The effect of the fluorine content and nano-structure of fluoridated hydroxyapatite (FHA) on human osteoblast-like (HO) cell behavior were investigated. FHA nanopowders and bulk nanostructured FHA, produced via mechanical alloying and two-step sintering, respectively, were used. The cytotoxicity of FHA nanopowders was assessed by MTT. Cell attachment to the surface of the bulk nanostructured FHA was evaluated by culturing of HO cells. Although HO cells proliferated 10 % more in contact with FHA nanopowders compared to culture medium without FHA nanopowders, an increase in the fluorine content of FHA caused a delay in the cell proliferation by about 2 days. Cell attachment on the bulk nanostructured FHA did not change the fluorine content.

Biphasic calcium phosphate loading on polycaprolactone/poly(lacto-co-glycolic acid) membranes for improved tensile strength, in vitro biocompatibility, and in vivo tissue regeneration.

Electrospun polycaprolactone and poly(lacto-co-glycolide) membranes were loaded with biphasic calcium phosphate powder to facilitate osteoconductivity. Different concentrations of biphasic calcium phosphate powder were added to the polymer solution, and successful loading was confirmed by X-ray diffraction analysis, transmission electron microscope, and scanning electron microscope with energy-dispersive spectroscopy visualization. The effect of the added biphasic calcium phosphate on the polymer membrane was investigated in terms of the material's tensile strength and strain, in vitro cytocompatibility, and in vivo tissue regeneration. It was observed that the tensile strength of the membranes increased with the addition of the biphasic calcium phosphate powder. Immersion in simulated body fluid solution for seven days leads to the formation of apatite-like deposits in the fibers, which further improved the mechanical stability. Moreover, proliferation and adhesion of osteoblast-like cells were more apparent upon the addition of the biphasic calcium phosphate powder as seen with the increasing cell density from (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and micrographs from scanning electron microscope and confocal microscopy. Sample membranes were also implanted to investigate the membrane's ability to regenerate bone in a rat calvarium. Histological staining and micro-CT histomorphometric analyses showed neo-bone formation in the implanted rat skull.

The cytotoxic evaluation of mineral trioxide aggregate and bioaggregate in the subcutaneous connective tissue of rats.

OBJECTIVES: The purpose of this study was to evaluate and compare the cytotoxic effects of ProRoot MTA and DiaRoot BA, a bioceramic nanoparticulate cement, on subcutaneous rat tissue. STUDY DESIGN: Fifty Sprouge Dawley rats were used in this study. Polyethylene tubes filled with ProRoot MTA and DiaRoot BioAggregate, along with a control group of empty, were implanted into dorsal connective tissue of rats for 7, 15, 30, 60, and 90 days. After estimated time intervals the rats were sacrificed. The specimens were fixed, stained with hematoxylin and eosin, and then evaluated under a light microscope for inflammatory reactions and mineralization. RESULTS: All groups evoked a severe to moderate chronic inflammatory reaction at 7 and 15 days, which decreased with time. Both the MTA and BioAggregate groups showed similar inflammatory reactions, except at 90 days when MTA showed statistically significant greater inflammation (p>0.05). The MTA group showed foreign body reaction at all times. Compared to BioAggregate, MTA showed significantly more foreign body reaction at 60 and 90 days (p<0.0001). After 30 days foreign body reaction of BioAggregate decreased significantly. Both MTA and BioAggregate groups showed similar necrosis at 7 and 15 days (p=0.094 and p=0.186 respectively). No necrosis was observed after 15 days. Similarly there was no fibrosis after 30 days for both MTA and BioAggregate groups (p>0.05). CONCLUSIONS: Since DiaRoot BioAggregate showed significantly better results than MTA, we can conclude that it is more biocompatible. However, further studies are required to confirm this result.

Biocompatibility of BioAggregate and mineral trioxide aggregate on the liver and kidney.

AIM: To investigate and compare the systemic toxic effect of DiaRoot BioAggregate and grey ProRoot Mineral trioxide aggregate (MTA) on the liver and kidney after 7 and 30 days. METHODOLOGY: Forty-two white albino rats were divided into two main groups. Group (1), considered the control group (n = 18), was further divided into two subgroups. The negative control subgroup (n = 6) received no treatment. The empty tube subgroup (n = 12) received empty sterile Teflon tubes. In Group (2), considered the experimental group (n = 24), the rats were divided equally into two subgroups. One subgroup received MTA, whilst the other received BioAggregate. The materials in the Teflon tubes were implanted subcutaneously in the dorsal side of the rats. Blood samples were taken to investigate the change of kidney and liver functions on day 7 and day 30. The liver and kidney organs were subjected to histopathological examination and calculation of the number of inflammatory cells. Data analysis was performed using one-way anova with post hoc multiple comparisons with the Tukey's test. Student's t-test was used to compare the changes in liver and kidney functions amongst the groups. RESULTS: On day 7, a significantly more severe inflammatory reaction was observed in both experimental subgroups compared with the control (P < 0.05); the severity decreased after 30 days. The kidney functions were not affected after 7 days but had subsequently increased after 30 days (P < 0.001). Liver functions increased after 7 days and had decreased in the BioAggregate subgroup after 30 days, whilst in the MTA subgroup, a continuous increase in the level of liver function was observed. CONCLUSIONS: Mineral trioxide aggregate had adverse effects on the liver and kidney that were significantly more severe than BioAggregate but with no permanent damage.

Preparation of a novel anorganic bovine bone xenograft with enhanced bioactivity and osteoconductivity.

A novel anorganic bovine bone xenograft with enhanced bioactivity and osteoconductivity was prepared by an ion substitution method using sodium hypochlorite. Bovine bone granules were defatted, washed, and then soaked in sodium hypochlorite solution at room temperature. Subsequently, the granules were dried and then heat-treated at 1000°C with sodium hypochlorite. As a control, bovine bone granules were prepared with the same conditions but without sodium hypochlorite treatment. Phase, functional group, and elemental analyses by XRD, FTIR, and EPMA showed that the granules heat-treated without and with sodium hypochlorite were pure hydroxyapatite and sodium-chlorine-bearing hydroxyapatite, respectively. After soaking in simulated body fluid (SBF) for 1 week, low crystalline hydroxyl carbonate apatite fully covered the surface of sodium-chlorine-bearing hydroxyapatite, whereas it formed little on the hydroxyapatite surface. After soaking in SBF and deionized water, ICP-AES and IC analyses showed that the dissolutions of calcium, sodium, chlorine, and hydroxyl ions from sodium-chlorine-bearing hydroxyapatite notably increased compared with those from hydroxyapatite. This resultantly increased the ionic activity product of apatite in SBF and induced new formation of low crystalline hydroxyl carbonate apatite. The cytotoxicity test by BCA assay showed that there were no statistically significant differences between hydroxyapatite and sodium-chlorine-bearing hydroxyapatite. In addition, sodium-chlorine-bearing hydroxyapatite showed better osteoconductivity in the calvarial defects of New Zealand white rabbits within 4 weeks compared with that of hydroxyapatite. The results suggest that this novel anorganic bovine bone xenograft possesses encouraging potential for use as a bone grafting material due to better bioactivity and osteoconductivity than hydroxyapatite.

The chemical composition of synthetic bone substitutes influences tissue reactions in vivo: histological and histomorphometrical analysis of the cellular inflammatory response to hydroxyapatite, beta-tricalcium phosphate and biphasic calcium phosphate ceramics.

Bone substitute material properties such as granule size, macroporosity, microporosity and shape have been shown to influence the cellular inflammatory response to a bone substitute material. Keeping these parameters constant, the present study analyzed the in vivo tissue reaction to three bone substitute materials (granules) with different chemical compositions (hydroxyapatite (HA), beta-tricalcium phosphate (TCP) and a mixture of both with a HA/TCP ratio of 60/40 wt%). Using a subcutaneous implantation model in Wistar rats for up to 30 days, tissue reactions, including the induction of multinucleated giant cells and the extent of implantation bed vascularization, were assessed using histological and histomorphometrical analyses. The results showed that the chemical composition of the bone substitute material significantly influenced the cellular response. When compared to HA, TCP attracted significantly greater multinucleated giant cell formations within the implantation bed. Furthermore, the vascularization of the implantation bed of TCP was significantly higher than that of HA implantation beds. The biphasic bone substitute group combined the properties of both groups. Within the first 15 days, high giant cell formation and vascularization rates were observed, which were comparable to the TCP-group. However, after 15 days, the tissue reaction, i.e. the extent of multinucleated giant cell formation and vascularization, was comparable to the HA-group. In conclusion, the combination of both compounds HA and TCP may be a useful combination for generating a scaffold for rapid vascularization and integration during the early time points after implantation and for setting up a relatively slow degradation. Both of these factors are necessary for successful bone tissue regeneration.

Evaluation of hydroxyapatite-bioglass and hydroxyapatite-ethyl vinyl acetate composite extracts on antioxidant defense mechanism and genotoxicity: an in vitro study.

Hydroxyapatite-bioglass (HA BG) and hydroxyapatite-ethyl vinyl acetate (HA EVA) are two composite materials that have been developed for bone substitution. Their activity on antioxidant defense mechanism and genotoxicity has not been investigated before. To further confirm its biocompatibility, the present study was undertaken to investigate the effect of HA BG and HA EVA on mice liver antioxidant mechanism along with chromosomal aberrations in human lymphocytes. Physiological saline extract of HA BG and HA EVA showed no adverse effect on liver antioxidant mechanism compared to the cyclophosphamide (CP)-induced toxicity on mice liver homogenate. The results were judged from the in vitro studies made on reduced glutathione, glutathione reductase and lipid peroxidation. These results were well supported by CP- and mytomycin C (MC)-induced genotoxicity studies on human lymphocytes in the presence and absence of a metabolic activator (S9). Hence, it was suggested that these tests could be considered for preliminary toxicological screening of materials intended for clinical applications ahead of in vivo animal model evaluation.

Direct cytotoxicity evaluation of 63S bioactive glass and bone-derived hydroxyapatite particles using yeast model and human chondrocyte cells by microcalorimetry.

In this study, the cytotoxicity evaluation of prepared 63S bioactive glass and bone-derived hydroxyapatite particles with yeast and human chondrocyte cells was carried out using isothermal micro-nano calorimetry (IMNC), which is a new method for studying cell/biomaterial interactions. Bioactive glass particles were made via sol-gel method and hydroxyapatite was obtained from bovine bone. Elemental analysis was carried out by XRF and EDXRF. Amorphous structure of the glass and completely crystalline structure of HA were detected by XRD analysis. Finally, the cytotoxicity of bioactive glass and bone-derived HA particles with yeast and cultured human chondrocyte cells was evaluated using IMNC. The results confirmed the viability, growth and proliferation of human chondrocyte cells in contact with 63S bioactive glass, and bone-derived HA particles. Also the results indicated that yeast model which is much easier to handle, can be considered as a good proxy and can provide a rapid primary estimate of the ranges to be used in assays involving human cells. All of these results confirmed that IMNC is a convenient method which caters to measuring the cell-biomaterial interactions alongside the current methods.

Cytotoxicity and genotoxicity property of hydroxyapatite-mullite eluates.

Long-term biomedical applications of implant materials may cause osteolysis, aseptic losing and toxicity. Therefore, we investigated the cytotoxic and genotoxic potential of hydroxyapatite (HA) mullite eluates in L929 mouse fibroblast cells. The spark plasma sintered HA-20% mullite biocomposite (HA20M) were ground using mortar and pestle as well as ball milling. The cells were exposed for 6 h to varying concentrations (10, 25, 50, 75 and 100%) of the eluates of HA-20% mullite (87 nm), HA (171 nm) and mullite (154 nm). The scanning electron microscopy and MTT assay revealed the concentration dependent toxicity of H20M eluate at and above 50%. The analysis of the DNA damaging potential of HA, mullite and HA20M eluates using Comet assay demonstrated a significant DNA damage by HA20M which was largely related to the presence of mullite. The results collectively demonstrate the cytotoxic and genotoxic potential of HA20M eluate in L929 cells is dependent on particle size, concentration and composition.

Cytotoxicity evaluation of 63s bioactive glass and bone-derived hydroxyapatite particles using human bone-marrow stem cells.

BACKGROUND: In recent years, bioceramics have been favored by biomaterials scientists and researchers. Due to their special and distinctive features, bioactive glass and hydroxyapatite possess a higher place among different types of bioceramics. METHOD: In this study, the effect of 63S bioactive glass and bone-derived hydroxyapatite particles on the proliferation of human bone-marrow stem cells (hMSCs) was investigated. Bioactive glass particles were made via sol-gel method and hydroxyapatite was obtained from bovine bone. The particle size and morphology were investigated by scanning electron microscope (SEM). Then the in vitro cytotoxicity of particles was evaluated using MTT assay. SEM showed that bioactive glass particles were in the nanoscale range and had tendency towards agglomeration. It was also confirmed that the hydroxyapatite particles were agglomerations of crystals cca 50-500 nm across. RESULTS: The results of MTT assay confirmed the viability and proliferation of hMSCs in contact with bioactive glass and bone-derived HA particles. The fabricated particles in combination with stem cells were shown to hold promising potential for further applications in tissue engineering and regenerative medicine.

Effect of bioaggregate on differentiation of human periodontal ligament fibroblasts.

AIM: To investigate the cytotoxicity of bioaggregate (BA; Innovative Bioceramix, Vancouver, BC, Canada) to human periodontal ligament (PDL) fibroblasts and its effect on differentiation of human PDL fibroblasts and to compare its performance to that of mineral trioxide aggregate (MTA; Dentsply, Tulsa, OK, USA). METHODOLOGY: Cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on days 1, 2, and 3 after incubation with BA or MTA. The influence of BA and MTA on differentiation of human PDL fibroblasts on days 3, 5, and 7 was evaluated by gene expression of alkaline phosphatase (ALP) and collagen type I (COLI) via quantitative real-time polymerase chain reaction (PCR). The data were analysed by one-way analysis of variance followed by Tukey's test. RESULTS: Cell numbers in the BA group were similar to that of the control group throughout the culture period, whereas MTA suppressed the proliferation of fibroblasts. ALP expression was significantly increased in the BA group on day 7, whilst it was enhanced by MTA on day 3. Gene expression of COLI was induced by both BA and MTA compared to the control group. CONCLUSIONS: Bioaggregate was nontoxic to human PDL fibroblasts and appeared to induce the differentiation of human PDL fibroblasts.