Hartmut F. Hildebrand

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.

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.

Nickel, chromium, cobalt dental alloys and allergic reactions: an overview

Dental alloys containing nickel, cobalt and/or chromium are widely used for dental restorations. Ni-Cr alloys contain generally 69–81% nickel, whereas Co-Cr alloys contain mostly less than 1% of nickel. The main components of these alloys are Co (60–65%), Cr (27–30%) and Mo (5–6%). Another group of non-precious dental alloys are stainless steels with ca. 18% Cr and 8% Ni.

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.

Bone implants modified with cyclodextrin: Study of drug release in bulk fluid and into agarose gel

The aim of this work was to better understand the importance of the type of experimental setup used to monitor antibiotic release from functionalized hydroxyapatite implants. Microporous hydroxyapatite discs were prepared by sintering and subsequently functionalized with hydroxypropyl-β-cyclodextrin (HPβCD) polymer crosslinked with butanetetracarboxylic acid. On one hand, polymerization was performed within the implant after its impregnation with the monomers (CD-HA-M implant). On the other hand, a pre-synthesized HPβCD polymer was loaded and fixed onto the HA discs (CD-HA-P implant). Both types of implants were soaked with ciprofloxacin hydrochloride or vancomycin hydrochloride solution and dried at 37°C. The DSC study highlighted that the cyclodextrin polymer could interfere with both drugs, due to the carboxylic groups carried by the crosslinks. Drug release was measured into phosphate buffered saline pH 7.4 in agitated vials, or into agarose gels to more realistically mimic in vivo conditions. Importantly, in all cases, drug release into agarose gels was much slower than into well-agitated phosphate buffer. Non-functionalized discs displayed faster drug release because no complex could be formed and/or due to the absence of the HPβCD polymer network hindering drug diffusion within the implant pores. In the case of ciprofloxacin hydrochloride, drug release from the CD-HA-M implants was faster than drug release from the CD-HA-P implants due to the different polymer structures resulting in different complexation strengths, whereas in the case of vancomycin hydrochloride the release patterns were similar because vancomycin hydrochloride was not included into the cyclodextrin. The agarose gel method seems more biorelevant and discriminatory than the vial method for drug release measurements from bone implants.

Methyl-β-cyclodextrin modified vascular prosthesis: Influence of the modification level on the drug delivery properties in different media

A textile polyester vascular graft was modified with methyl-β-cyclodextrin (MeβCD) to obtain a new implant capable of releasing antibiotics directly in situ at the site of operation over a prolonged period and thereby prevent post-operative infections. We investigated the influence of the curing parameters (time and temperature) that allow control of the degree of functionalization (DF) of the support by MeβCD. The inclusion of ciprofloxacin (CFX) in the MeβCD cavity was observed in solution by two-dimensional (1)H NMR spectroscopy. The amount of CFX loaded on the modified graft increased with DF. Depending on the release medium (water, phosphate-buffered saline, or human plasma) and the DF of the prostheses, different kinetic profiles of release of CFX were obtained. The sustained release of CFX in human plasma was shown by microbiological assays that indicated prolonged antimicrobial activity against Staphylococcus aureus and Escherichia coli. Viability tests demonstrated the non-toxicity of MeβCD to an epithelial cell line (HPMEC), although a decrease in endothelial cell number was observed on the functionalized prosthesis, probably due to the roughness of the coating and also to the nature of the MeβCD polymer present on the surface of the fibers.

Comparative study of vascular prostheses coated with polycyclodextrins for controlled ciprofloxacin release

A textile polyester vascular graft was modified with cyclodextrins to obtain a new implant capable of releasing antibiotics (here ciprofloxacin, CFX) over prolonged time periods and thereby reducing the risk of post-operative infections. In this study, we compared samples modified with native and modified cyclodextrins, presenting different cavity sizes (β or γ cyclodextrins) and different substituent groups (hydroxypropyl and methyl). Drug release was measured in water, phosphate buffer pH 7.4 and blood plasma. The inclusion of CFX in the cyclodextrins cavities was observed in solution by two-dimensional (1)H NMR spectroscopy and confirmed by (1)F NMR measurements. Grafts modification with all cyclodextrins induced an increase of their sorption capacity towards CFX whose extent depended on the nature of the cyclodextrin: a 4-fold and 10-fold increase was observed in the cases of hydroxypropyl cyclodextrins and methylated β-cyclodextrin, respectively. Depending on the type of release medium and nature of CD, different CFX release kinetics were obtained. The discussion highlighted not only the role of the host guest complexation, but also that of the electrostatic interactions that occur between the anionic crosslinks of the cyclodextrins polymers, and CFX that presents a zwitterionic character. The microbiological assessment confirmed sustained CFX release in human plasma and demonstrated antibacterial efficiency of CD modified prostheses against Staphylococcus aureus and Escherichia coli for at least 24 h (compared to 4 h in the case of virgin grafts).

A chlorhexidine-loaded biodegradable cellulosic device for periodontal pockets treatment

Absorbent points widely used in endodontic therapy were transformed into bioresorbable chlorhexidine delivery systems for the treatment of the periodontal pocket by preventing its recolonization by the subgingival microflora. These paper points (PPs) were first oxidized to promote their resorption, then grafted with β-cyclodextrin (CD) or maltodextrin (MD) in order to achieve sustained delivery of chlorhexidine. We investigated the oxidation step parameters through the time of reaction and the nitric and phosphoric acid ratios in the oxidizing mixture, and then the dextrin grafting step parameters through the time and temperature of reaction. A first selection of the appropriate functionalization parameters was undertaken in relation to the degradation profile kinetics of the oxidized (PPO) and oxidized-grafted samples (PPO-CD and PPO-MD). Samples were then loaded with chlorhexidine digluconate (digCHX), a widely used antiseptic agent in periodontal therapy. The release kinetics of digCHX from PPO-CD and PPO-MD samples were compared to PP, PPO and to PerioChip(®) (a commercial digCHX containing gelatine chip) in phosphate buffered saline (pH 7.4) by ultraviolet spectrophotometry. The cytocompatibility of the oxidized-grafted PP was demonstrated by cell proliferation assays. Finally, the disc diffusion test from digCHX loaded PPO-MD samples immersed in human plasma was developed on pre-inoculated agar plates with four common periodontal pathogenic strains: Fusobacterium nucleatum, Prevotella melaninogenica, Aggregatibacter actinomycetem comitans and Porphyromonas gingivalis. To conclude, the optimized oxidized-dextrin-grafted PPs responded to our initial specifications in terms of resorption and digCHX release rates and therefore could be adopted as a reliable complementary periodontal therapy.

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.

Visceral mesh modified with cyclodextrin for the local sustained delivery of ropivacaine

The aim of the study was to develop a polyester visceral implant modified with a cyclodextrin polymer for the local and prolonged delivery of ropivacaine to reduce post operatory pain. Therefore, we applied a coating of an inguinal mesh with a crosslinked polymer of hydroxypropyl-β-cyclodextrin (HPβCD) whose specific host-guest complex forming properties were expected to improve the adsorption capacity of the implant toward anesthetic, and then to release it within a sustained period. The modification reaction of the textile with cyclodextrin was explored through the study of the influence of the pad/dry/cure process parameters and the resulting implant (PET-CD) was characterized by solid state NMR and SEM. Besides, the inclusion complex between ropivacaine and CD was studied by NMR and capillary electrophoresis in PBS medium. Finally, ropivacaine sorption test showed that a maximum of 30 mg/g of ropivacaine could be adsorbed on the functionalized samples. In dynamic batch tests in PBS at pH 7.4, the release could be observed up to 6h. The cytocompatibility of the PET-CD loaded with ropivacaine was also studied and reached 65% cell vitality after 6 days.