Evaluation of sorption capacity of antibiotics and antibacterial properties of a cyclodextrin-polymer functionalized hydroxyapatite-coated titanium hip prosthesis.

Infection still present as one of common complications after total hip replacement (∼2.5%), which may cause serious outcomes. For preventing such risk, loading antibiotics onto implants for increasing local drug concentration at targeted sites could be a solution. This study aims at modifying the surface of hydroxyapatite (HA) coated titanium hip implant material (Ti-HA) with polymer of cyclodextrin (polyCD) for loading antibiotics, to achieve a sustained local drug delivery. Two widely applied antibiotics (tobramycin and rifampicin) in orthopedic surgery were loaded alone or in combination. The drug adsorption isotherm, drug release kinetics and drug's efficacy were thoroughly investigated. The results proved that polyCD coating significantly improved the affinity of both drugs to Ti-HA surface, while the mechanism of drug-polyCD interaction varies from the nature of drug, courtesy of the structural complex of polyCD. The advantage of dual-drug loading was highlighted by its strong efficacy against both Staphylococcus aureus and Enterobacter cloacae, which overcomes the limitation of mono-drug loading for an effective treatment against both bacterial strains. The prolonged antibacterial activity of antibiotic loaded Ti-HA-polyCD samples confirmed that polyCD could be a promising drug-delivery system, for sustained antibiotics release or other potential applications e.g., antimitotic agent release.

Poly-cyclodextrin functionalized porous bioceramics for local chemotherapy and anticancer bone reconstruction.

The progress in bone cancer surgery and multimodal treatment concept achieve only modest improvement in the overall survival, due to failure in clearing out residual cancer cells at the surgical margin and extreme side-effects of adjuvant postoperative treatments. Our study aims to propose a new method based on cyclodextrin polymer (polyCD) functionalized hydroxyapatite (HA) for achieving a high local drug concentration with a sustained release profile and a better control of residual malignant cells via local drug delivery and promotion of the reconstruction of bone defects. PolyCD, a versatile carrier for therapeutic molecules, can be incorporated into HA (bone regeneration scaffold) through thermal treatment. The parameters of polyCD treatment on the macroporous HA (porosity 65%) were characterized via thermogravimetric analysis. Good cytocompatibility of polyCD functionalized bioceramics was demonstrated on osteoblast cells by cell vitality assay. An antibiotic (gentamicin) and an anticancer agent (cisplatin) were respectively loaded on polyCD functionalized bioceramics for drug release test. The results show that polyCD functionalization leads to significantly improved drug loading quantity (30% more concerning gentamicin and twice more for cisplatin) and drug release duration (7 days longer concerning gentamicin and 3 days longer for cisplatin). Conclusively, this study offers a safe and reliable drug delivery system for bioceramic matrices, which can load anticancer agents (or/and antibiotics) to reduce local recurrence (or/and infection).

Evaluation of a bio-based hydrophobic cellulose laurate film as biomaterial--study on biodegradation and cytocompatibility.

The study aims to validate an original bio-based material, obtained by grafting fatty chains, and more especially lauric chains (C12) onto cellulose, for medical applications. The mechanical properties of the synthesized cellulose laurate (C12) are close to those of petrochemical ones such as low density polyethylene. This cellulose-based polymer is transparent, flexible, and hydrophobic. To evaluate the stability of the cellulosic films in biological fluids the samples are soaked in simulated body fluid or blood plasma for a few hours to 6 months, and then submitted to mechanical and chemical analyses. The simultaneously performed cytocompatibility tests were the colony-forming viability, the vitality and cell proliferation tests using NIH 3T3 fibroblasts and MC 3T3 osteoblast-like cells. The results show the stability, the biocompatibility, and the noncytotoxicity of the synthesized cellulose laurate films. This biomaterial may so be considered for surgical applications.

Improving endothelial cell adhesion and proliferation on titanium by sol-gel derived oxide coating.

In-stent restenosis becomes increasingly prevalent as a difficult-to-treat disease. An alternative therapeutic strategy is enhancing endothelialization on metallic stent surfaces. This study attempted to modify surface chemistry and topography of commercial pure titanium (cp-Ti) by different sol-gel derived oxide coatings (TiO(2), SiO(2), SiO(2)/TiO(2), and Nb(2)O(5)) to improve endothelialization. The physiochemical properties of the modified surfaces were characterized by ellipsometry, atomic force microscope, and sessile-drop method. The cell adhesion/proliferation quantity, cell adhesion morphology, and focal adhesion protein expression were evaluated with human pulmonary microvascular endothelial cell line. The thickness of oxide coatings approximates to 100 nm; significantly rougher nanoporous structure was found in the TiO(2) and Nb(2)O(5) coatings than that of cp-Ti. SiO(2) coating possesses the highest surface energy (75.1 mJ/m(2)) and the lowest was for cp-Ti (45.7 mJ/m(2)). TiO(2) coating showed significantly higher endothelial cell adhesion rate than others; TiO(2), Nb(2)O(5), and TiO(2)/SiO(2) coatings exhibited higher endothelial proliferation in 3-day assays than noncoated Ti. In hemocompatible test, they also showed good hemocompatibility. These results offer the insight into that certain oxide coatings on titanium could significantly improve endothelial cell adhesion and proliferation especially in early period, which will favor reaching the endothelialization rapidly and suitable as matrix for "endothelial seeding" stent.

Prolonged local antibiotics delivery from hydroxyapatite functionalised with cyclodextrin polymers.

Per-operative infection is a common complication for bone-graft surgery. Combining antiseptic agents with graft materials may offer a solution by increasing local drug concentration at target sites. Aiming to achieve a sustained local antibiotic (ATB) delivery for a widely applied bone substitute material - hydroxyapatite (HA), we attempted incorporating hydroxypropyl-beta-cyclodextrin polymer (polyHPbetaCD) into microporous HA via impregnating either in a CD monomers mixture solution or a pre-synthesized CD polymer solution, followed by thermal fixation processing. In such functionalised material (CD-HA), polyHPbetaCD could entrap ATBs and release them progressively. Infrared-spectroscopic analysis confirmed the presence of polyHPbetaCD in functionalised HA via both processing pathways; polyHPbetaCD functionalisation yields were quantitated by thermogravimetric analysis for optimising the processing regime. Ciprofloxacin (CFX) and vancomycin (VCM), commonly applied in orthopaedics, have been respectively loaded on CD-HA by dip-coating. For both ATBs, kinetic release test in phosphate buffered saline showed significantly increased initial-burst amount and prolonged release from CD-HA compared with those from non-functionalised HA. Encouragingly, ATBs loaded CD-HA also revealed a prolonged bacteriostatic activity against Staphylococcus aureus and progressively increased cytocompatibility to osteoblasts (MC3T3-E1). Overall, polyHPbetaCD functionalisation on HA could be an effective drug-delivery model for loading different drug molecules in prevention of infection.

The corrosion and biological behaviour of titanium alloys in the presence of human lymphoid cells and MC3T3-E1 osteoblasts.

Corrosion behaviour of biomedical alloys is generally determined in mineral electrolytes: unbuffered NaCl 0.9% (pH 7.4) or artificial saliva (pH 6.8). The assays with exclusive utilization of these electrolytes are of low relevance for the biological condition, to which the alloys will be exposed once implanted in the human organism. As an approach to the biological situation regarding the interaction of proteins, electrolytes and metals, we added the RPMI cell culture medium containing foetal calf serum as a biological electrolyte (pH 7.0). The analysis of corrosion behaviour was also performed in the presence of human lymphoid cells (CEM). The rest potential (Er) and the global polarization were determined on cp-Ti, micro-arc oxidized cp-Ti (MAO-Ti), four different Ti-alloys (Ti6Al4V, Ti12Zr, Ti(AlMoZr), Ti(NbTaZr)) and 316L stainless steel. The 316L exhibited an appropriate Er and a good passive current density (Ip), but a high corrosion potential (Ec) and a very low breakdown potential (Eb) in all electrolytes. All Ti-alloys exhibited a much better electrochemical behaviour: better Er and Ec and very high Eb. No significant differences of the above parameters existed between the Ti-alloys, except for Zr-containing alloys that showed better corrosion behaviour. A remarkable difference, however, was stated with respect to the electrolytes. NaCl 0.9% induced strong variations between the Ti-alloys. More homogeneous results were obtained with artificial saliva and RPMI medium, which induced a favourable Ec and an increased Ip. The presence of cells further decreased these values. The unbuffered NaCl solution seems to be less appropriate for the analysis of corrosion of metals. Additional in vitro biological assessments with CEM cell suspensions and MC3T3-E1 osteoblasts confirmed the advantages of the Ti(AlMoZr) and Ti(NbTaZr) alloys with an improved cell proliferation and vitality rate.

Polyester vascular prostheses coated with a cyclodextrin polymer and activated with antibiotics: cytotoxicity and microbiological evaluation.

Polyester (PET) vascular grafts are used to replace or bypass damaged arteries. To minimize the risk of infection during and after surgical interventions, a PET vascular prosthesis (Polythese) was functionalized with cyclodextrin polymers (PolyCDs) in order to obtain the controlled release of antibiotics (ABs: ciprofloxacin, vancomcyin and rifampicin). An epithelial cell line (L132) was used to determine the viability of the antibiotics, and human pulmonary microvascular endothelial cells (HPMEC) were used for cell proliferation by cell counting and cell vitality with Alamar Blue fluorescent dye. Staphylococcus aureus, Escherichia coli and Enteroccocus sp. were used to determine the antimicrobial activity of AB-loaded virgin and PolyCD-grafted Polythese by the minimum inhibitory concentration method. The spectrophotometric titration results first showed that a larger amount of ABs was sorbed onto PolyCD-coated Polythese compared to virgin Polythese (26.7 vs. 35.3 mg g(-1), 51.1 vs. 72.4 mg g(-1) and 4.1 vs. 21.0 mg g(-1), respectively, for rifampicin, vancomycin and ciprofloxacin). These results were further confirmed by a microbiological test, which showed AB-loaded PolyCD-coated Polythese displayed better antimicrobial activity. The viability test revealed the toxicity of rifampicin (22 mg l(-1)) and ciprofloxacin (35 mg l(-1)), and the absence of toxicity of vancomycin. These tests allow us to further explain the lower vitality and proliferation of HPMEC on the AB-loaded PolyCD-coated Polythese, which was due not to the functionalization process of prostheses but to the cytotoxicity of certain ABs themselves. Moreover, such a property could be exploited to tackle intracellular bacteria, such as in tuberculosis and other diseases, and will not compromise further in vivo applications of our functionalized vascular prostheses.

Osteoblast interaction with DLC-coated Si substrates.

Diamond-like carbon (DLC) coating is a convenient means of modifying material surfaces that are sensitive to wear, such as titanium and silica substrates. This work aims to evaluate the osteoblast-like cells' response to DLC-coated Si (Si-DLC), which was treated under different conditions. DLC and deuterated DLC films were deposited by plasma-enhanced chemical vapor deposition to obtain a 200-nm-thick layer on all the samples. Three types of precursor gas were applied for deposition: pure methane (CH(4)), pure deuterated methane (CD(4)) and their half/half mixture. All surface treatments were performed under two different self-bias voltages (V(sb)): -400 and -600V. The modified surfaces were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, Rutherford backscattering spectroscopy, elastic recoil detection analysis, X-ray reflectometry and the sessile-drop method. MC3T3-E1 osteoblasts were cultured on the Si-DLC wafers for 3 and 6 days. Biological tests to measure cell proliferation, cell vitality, cell morphology and cell adhesion were performed. All DLC coatings produced a slightly more hydrophobic state than non-treated Si. Certain types of amorphous DLC coating, such as the surface treated under the V(sb) of -600V in pure methane (600CH(4)) or in pure deuterated methane (600CD(4)), offered a significantly higher cell proliferation rate to Si substrate. Scanning electron microscopy observations confirmed that the optimal cell adhesion behavior, among all the treated surfaces, occurred on the surface of the 600CH(4) and 600CD(4) groups, which showed increased amounts of filopodia and microvilli to enhance cell-environment exchange. In conclusion, DLC coating on Si could produce better surface stability and improved cellular responses.

Osteoblast responses to different oxide coatings produced by the sol-gel process on titanium substrates.

In order to improve the osseointegration of endosseous implants made from titanium, the structure and composition of the surface were modified. Mirror-polished commercially pure (cp) titanium substrates were coated by the sol-gel process with different oxides: TiO(2), SiO(2), Nb(2)O(5) and SiO(2)-TiO(2). The coatings were physically and biologically characterized. Infrared spectroscopy confirmed the absence of organic residues. Ellipsometry determined the thickness of layers to be approximately 100nm. High resolution scanning electron microscopy (SEM) and atomice force microscopy revealed a nanoporous structure in the TiO(2) and Nb(2)O(5) layers, whereas the SiO(2) and SiO(2)-TiO(2) layers appeared almost smooth. The R(a) values, as determined by white-light interferometry, ranged from 20 to 50nm. The surface energy determined by the sessile-drop contact angle method revealed the highest polar component for SiO(2) (30.7mJm(-2)) and the lowest for cp-Ti and 316L stainless steel (6.7mJm(-2)). Cytocompatibility of the oxide layers was investigated with MC3T3-E1 osteoblasts in vitro (proliferation, vitality, morphology and cytochemical/immunolabelling of actin and vinculin). Higher cell proliferation rates were found in SiO(2)-TiO(2) and TiO(2), and lower in Nb(2)O(5) and SiO(2); whereas the vitality rates increased for cp-Ti and Nb(2)O(5). Cytochemical assays showed that all substrates induced a normal cytoskeleton and well-developed focal adhesion contacts. SEM revealed good cell attachment for all coating layers. In conclusion, the sol-gel-derived oxide layers were thin, pure and nanostructured; consequent different osteoblast responses to those coatings are explained by the mutual action and coadjustment of different interrelated surface parameters.

[Influence of inorganic antibacterial agents on the systemic toxicity and cytotoxicity of a self-etching primer].

OBJECTIVE: To evaluate the influence of incorporating inorganic antibacterial agents on the systemic toxicity and cytotoxicity of an experimental self-etching primer (ESP). METHODS: Six kinds of inorganic agents were incorporated respectively into the primer. Systemic toxicity in vivo tests in rats and direct contact in vitro cytotoxicity tests with NIH fibrohiasts were conducted. RESULTS: Systemic toxicity tests revealed neither toxic manifestations nor significant differences in body weight gain hetween control and other groups. There were no significant differences between experimental groups and empty control on cell vitality and cell proliferation rates. Toxicity was only observed in areas heneath the specimens and/or in the direct vicinity of the specimen edge. There was no influence on the cell density over the limit of specimens. CONCLUSION: The incorporation of tested inorganic antibacterial agents with a proper concentration had no significant influence on the systemic toxicity and cytotoxicity of the tested self-etching primer.

On the use of ultrasounds to quantify the longitudinal threshold force to detach osteoblastic cells from a conditioned glass substrate.

Cell adhesion on a biomaterial is an important phase of the cell-material interactions and the quality of this phase governs the success of the biomaterial integration. Understanding of the phenomena of cell adhesion and in particular understanding of cell adhesion on biomaterials is of crucial importance for the development of new biomaterials with excellent biocompatibility. One of the physical quantitative indexes to evaluate the quality of cell-material adhesion is its strength. Determining the strength of adhesive bonds requires applying external forces to the cells. Thus, a few methods have been developed to evaluate the strength of cell-material adhesion (micropipette, microplates, microcantilever, ...). These methods apply shear forces on adherent cells. The aim of our work is the development of a new ultrasonic characterization method of cellular adhesion on substrates. With our method, longitudinal acoustic waves are applied on cell culture to impose a longitudinal strain on cells. Only the cells subjected to a sufficient level of strain will be detached from the substrate. The idea is to correlate cell detachment rate to the longitudinal strain threshold supported by cells. From this result, we can deduce the critical force just sufficient to detach the cell. This global method can be adapted for different cell types and for different substrates. This method can provide an evaluation of the effect of functionalization on substrates. The technique is investigated for the 200 kHz ultrasound frequency. An insonificator adapted to the use of cell culture boxes was developed and calibrated. Tests were carried out on a glass substrate with or without biological conditioning. We used the MC3T3-E1 osteoblastic cell line. Our results to date provide the value of the necessary force to detach with reproducibility osteoblastic cells from glass.

Ti-Cp functionalization by deposition of organic/inorganic silica nanoparticles.

In orthopaedics and cardiovascular surgery, titanium has become the metal of choice, due to its excellent mechanical properties and biocompatibility. In many surgical operations, chemicals and/or biomolecules (such as antibiotics or growth factors) are used in conjunction with prostheses, so as to avoid or stimulate targeted biological events. Often, immobilization instead of release of such molecules is preferred to optimize their effects, thus avoiding ectopic transformations. A versatile method for the functionalization of pure Ti is shown here, which allows the covalent immobilization of polypeptides. In order to avoid the hydrolysable Ti-O-Si bond found in directly silanized Ti, we use organic/inorganic silica colloids, derived from commercially available 25 nm Ludox silica nanoparticles. Prior to deposition onto Ti-Cp, the silica nanoparticles are functionalized by a propylsemicarbazide moiety by silanization. After spin-coating onto the Ti substrates, the colloids were shown by SEM to form a uniform layer, and to be very strongly adsorbed; the reactivity of the supported semicarbazide (Sc) functionalities being maintained. Chemoselective reaction of semicarbazide groups on the surface with aldehyde moieties present on the polypeptide of interest was chosen in this work due to its efficiency, to its compatibility with the proteinogenic amino acids and in particular cystein and to the use of mild experimental conditions. Aldehyde groups are also easily introduced onto polypeptides by synthesis, oxidation of N-terminal Ser residue or polysaccharide moieties of glycoproteins. Biological assays with MC3T3-E1 osteoblasts revealed an excellent cytocompatibility as shown by the assessment of cell viability, vitality and morphology.

Antibacterial functionalization of an experimental self-etching primer by inorganic agents: microbiological and biocompatibility evaluations.

Antibacterial activities have been demonstrated on oral bacteria with inorganic antibacterial agents (ABAs) after their incorporations into an experimental self-etching primer (ESP) before curing. This study was to assess their biocompatibility and antibacterial activity after curing. Six ABAs were incorporated respectively into ESP for treating specimens. After curing, their bactericidal activities on Streptococcus mutans and influences to the early bacterial colonization were assessed by direct contact and viable count. Systemic toxicity in rats after short-term oral exposure and direct contact cytotoxicity with NIH3T3 fibroblasts were tested. Incorporation of ZnOw AT-83, Longbei antibiotic, Antim-AMS2 or IONPURE-H significantly enhanced the antibacterial effect of ESP after curing, even after 1 month aging. Specimens treated by ESP with ZnOw AT-83, Longbei antibiotic or Antim-AMS2 showed slightly less bacterial adhesion than control. Animal experiments revealed neither toxic signs nor significant differences in body weight gain between control and other groups. Cell vitality or proliferation rates were ranged from 76% to 100% with respect to controls. Basic magnesium hypochlorite, ZnOw AT-83 and ZnOw AT-88 were less toxic. Toxicity only observed in areas beneath the specimens and/or in the direct vicinity of the specimen edge. From microbiological and biocompatibility aspects, the tested ABAs can be effectively incorporated in ESP to provide antibacterial activity against S. mutans. ZnOw AT-83 was the most promising one.

Antibacterial activities of inorganic agents on six bacteria associated with oral infections by two susceptibility tests.

The antibacterial effects of six inorganic antibacterial agents were assessed using broth dilution and agar dilution tests on six pathogenic bacteria associated with oral infectious diseases: Streptococcus mutans (ATCC 25175), S. mutans (Ingbritt), Actinomyces viscosus (ATCC 15987), Lactobacillus casei (ATCC 393), Staphylococcus aureus (ATCC 29213) and Candida albicans (ATCC 90028). The results of the broth dilution test were significantly lower than those of the agar dilution test (F=38.290; P<0.01). The six inorganic agents notably inhibited the growth of tested common oral bacteria in vitro. Among them, Longbei inorganic antibiotic powder was the strongest antibacterial agent, followed by ZnO whisker antibacterial complex (ZnOw) AT-83, IONPURE-H, basic magnesium hypochlorite, ZnOw AT-88 and Antim-AMS2. The broth dilution test appears to be more suitable for testing insoluble inorganic agents.

In vitro studies on the influence of precultural conditioning method on osteoblast reactions of a new type of injectable calcium cement material.

A new injectable dicalcium phosphate dehydrate (DCPD)-based cement material "PD" VitalOs Cement was studied to elucidate the process of equilibrium occurring in the early stage of implantation. The present study investigated the pH variations of the cement sample-immersing culture medium at determined intervals, time-dependent calcium/phosphate release, cell proliferation, and vitality in the cells-cement coculture milieu, after different preculture conditionings of the samples. Measurement of pH variation showed that without renewing the medium, pH value of sample lixiviate medium first dropped and, after 70 h, gradually balanced. When medium was renewed each day, pH value of lixiviate medium first descended and, after 24 h, gradually returned to pH 7.2. The cell viability revealed an excellent cytocompatibility of the cement. Both cell proliferation and vitality test showed that the preculture conditioning treatment is important at least for good performance of osteoblasts growing on the surface of calcium phosphate hydraulic cement (CPHC) samples in vitro. The results of calcium and phosphate assays clearly showed that this cement material can continuously dissolve to release calcium and phosphate in the liquid cell culture environment. The decrease of proliferation in some experimental groups with short conditioning is due to an excess of acid, which still can have some influence on cell growth after 24 h, since the biological milieu is not continuously renewed as in in vivo conditions.