P. J. Doherty

Semisynthetic resorbable materials from hyaluronan esterification

In recent years, research on new, biocompatible, degradable materials has seen the development of a series of modified natural polymers. Among these, a new class of materials consisting of different hyaluronan derivatives promises to be useful in a whole range of clinical applications thanks to their varied biological properties. These new materials are obtained by chemical modification of purified hyaluronan consisting of the partial or total esterification of the carboxyl groups of this natural polymer. This review on the properties of the new materials reports some of their biocompatibility and characterization aspects based on findings from studies conducted on the ethyl and benzyl hyaluronan esters, two representative members of this new class of compounds, and is intended to arouse interest in the potential of other, as yet unexplored derivatives. From the results of a number of investigations, the various derivatives appear to possess different physico-chemical properties, especially as far as the degree of hydration and polymer stability are concerned. In addition, the type of esterification and extent of chemical esterification of hyaluronan considerably affects the biological properties of these materials, offering a range of polymers either favouring or, conversely, inhibiting the adhesion of certain types of cell.

A preliminary assessment of poly(pyrrole) in nerve guide studies

This study investigates the biocompatibility of polypyrrole, a conducting polymer, and comments on its potential as an effective guidance channel for the regeneration of nervous tissue. The polymer was prepared in our laboratories by an electro-polymerization process. Pyrrole is placed in an electrolyte and when a potential is applied polypyrrole is deposited at the anode. After polymerization the polypyrrole is easily removed from the anode. Extraction in methanol for a period of 1 week was carried out to remove residual electrolyte. The biocompatibility of the material was assessed in vitro and in vivo. The response of two cell lines growing in contact with the polymer was evaluated. L929 mouse fibroblast and neuro2a neuroblastoma cells contacted the polypyrrole in a specially constructed cell culture chamber which allowed a controlled current to pass through the material. In vivo, the material was evaluated following implantation into a rat model. furthermore, the effect of charge on the cell lines was examined using the same cell culture chamber, but substituting platinum wire for the polypyrrole. Finally, the polypyrrole was deposited directly onto the platinum wire and introduced to the cell culture chamber. The results demonstrate that the polypyrrole is cytocompatible in vitro if prepared by appropriate extraction techniques. In vivo there was only a minimal tissue response after 4 weeks in situ. The cell culture chamber model proved successful and allowed a current up to 1 mA to be applied across the polypyrrole or platinum wire while in contact with both cell lines. Some evidence of toxicity was evident when a current of 1 mA was applied across the polymer for periods up to 96 h. However, it is clear from these experiments that polypyrrole can be an effective medium for carrying current in a biological environment.

Biodegradation of hyaluronic acid derivatives by hyaluronidase

Hyaluronic acid (salt) (HA) has been chemically modified as a biomaterial for medical applications such as controlled drug release matrices, nerve guides and wound dressings. A series of HA derivatives, which include different ester types and different degrees of esterification, have been used to investigate the stability of these materials in testicular hyaluronidase. Gel permeation chromatography and capillary viscometer have been employed to determine the size of the molecules, the former used for the water insoluble derivatives that dissolve in dimethyl sulphoxide, the latter for the water soluble samples. The preliminary experimental results indicated that the molecular weight of fully esterified hyaluronic acid (both ethyl and benzyl esters) did not decrease after treatment in the enzyme for 7 and 14 days while the water soluble partially esterified HA were degraded by the enzyme producing a sharp reduction of viscosity within minutes. These observations tend to suggest that the carboxylic groups in the beta-glucoronic acid unit are the activation centre of this enzyme and the total blockage of these groups can restrict the cleavage of beta (1-->4) glycoside bonds by this enzyme.

A study of the reproducibility of the MTT test

The MTT test has been widely used as a rapid and sensitive method for screening anticancer drugs as well as for the assessment of cytotoxicity of materials. The reproducibility of the MTT test has been studied in this paper in three ways. First, the reproducibility of MTT assay itself has been investigated. The comparisons were performed within the plate, between plates as well as between flasks of the cultured cells. The Students t-test was used to analyse the data and statistically significant differences were found for all the groups compared. Second, the influence of random sampling was investigated. Statistically significant differences were found with non-random sampling but no differences were found with random sampling. The third part examined the stability of the optical density of the solution read by a spectrophotometer and its dependence on temperature. The stability of the optical density was examined at room temperature and 4°C. Plates were maintained at these temperatures between readings. The optical density of dissolved formazan solution was compared by analysing the result with ONEWAY and statistically significant differences were found for the group of data at room temperature while no differences were shown for the group of data at 4°C.

Quantitative assessment of the tissue response to films of hyaluronan derivatives

The aim of this study was to evaluate the in vivo response following implantation into a rat model of three innovative hyaluronan derivatives for clinical use: HYAFF 7, HYAFF 11 and HYAFF 11p75 (respectively, the 100% ethyl ester, 100% and 75% benzyl esters). The tissue reaction evoked by films of these new biomaterials implanted into the dorsolumbar musculature of rats was assessed quantitatively using a well established technique based upon an image analysis system. The number of inflammatory cells present and the patterns of cell distribution around the implant up to a distance of 642 microns were examined at different time periods after implantation. Since a well-delineated tissue-material interface was needed for this type of investigation, it was not possible to apply image analysis to sections once dissolution of the implanted materials had begun. Films of both the total esters, HYAFF 7 and HYAFF 11, were found to undergo a slow dissolution process and, after a month, films of these materials were still present at the site of implantation. Differences in response to the two materials were observed only during the first two weeks, particularly with respect to neutrophil distribution and total cellularity. HYAFF 7 was found to be more reactive, with higher numbers of neutrophils near the surface of the implant than HYAFF 11. Thereafter, the differences between the two materials were minimal and owing mainly to a faster dissolution of HYAFF 7 films. After 3 and 5 months, considerable degradation of films of both total esters had occurred. Significant quantities of material appeared inside numerous macrophages with an ED1-positive phenotype. Only a very thin layer of fibrous connective tissue, indicative of low reactivity, was found to surround the site of implantation, separating the dissolved material and the phagocytic cells from healthy muscular tissue. ED2-positive macrophages were primarily confined within the lining connective tissue. The partial benzyl ester, HYAFF 11p75, showed a different behaviour. In fact, evidence of film dissolution was already present a week after the implantation. After two weeks, the implanted films were completely dissolved and numerous ED1-positive macrophages phagocytosing the material were observed at the site of implantation. Therefore, in agreement with previous in vitro studies, which showed a greater susceptibility to degradation of hyaluronan derivatives with lower percentage of esterification, HYAFF 11p75 underwent resorption faster than the corresponding total ester.

EFFECTS OF A NANOPARTICULATE SILICA SUBSTRATE ON CELL ATTACHMENT OF CANDIDA ALBICANS

Aims:  To investigate the influence of silica nanoparticles on the attachment and growth of Candida albicans cells.

Evaluation of soft tissue response to a poly(urethane urea)

Variations in the performance of vascular prostheses constructed of polyurethanes, and some evidence which suggested that these variations could be due not to the properties of the polymer itself, but to differences in the cellular response to the various microstructures of porous polyurethanes require investigation. Experiments were performed to evaluate quantitatively the extent of the cell behaviour adjacent to a series of polyurethane samples. It was shown that, with Biomer, a polyurethane urea, the profile of cell behaviour as a function of distance from the implant surface and of time following implantation, the response of cells in general and macrophages in particular, varied considerably with different internal microstructure. This supports the suggestion that the cellular response to different structures and susceptibility to degradation are related.

A study of cell behaviour on the surfaces of multifilament materials.

Since many fibres are very strong, they are considered to have useful potential for fibre reinforcement of orthopaedic and dental implant materials. Fibres exposed on the surface of composites may significantly influence the cellular response not only due to the chemistry but also due to the fibre size and shape. This study has concentrated on investigation of cellular responses to fibre-specific aspects of fibre-reinforced composites. Four multifilament materials with diameter less than 20 μm were used: Kevlar 29(K), silicon carbide(SiC), nylon 66(N), and polyethylene terephthalate(PET). Established cell line L929 fibroblasts were used as the cell model. Cell behaviour on the surfaces of fibres was examined using direct cell counting (after 3, 5, 8 h and 1, 2, 3 days), scanning electron microscopy (SEM) (after 2 h and 2 days), and fluorescent staining of F-actin, which was analysed by confocal laser scanning microscopy (CLSM) (after 2 h and 2 days). The results showed that fibroblasts adhered and grew very well on all fibre surfaces, although less cells were observed on PET from direct cell counting. Significant orientational behaviour of cells was found on all fibre surfaces from the SEM and CLSM analysis, independent of the bulk chemistry of the fibres.

Biocompatibility evaluation of glass ionomer cement using cell culture techniques

Cell culture techniques have been employed to carry out a preliminary investigation into the biocompatibility of a glass ionomer cement (GIC). A modification of the commonly used Agar Overlay test and a rapid, colorimetric assay based on the reduction of tetrazolium salt were used. The GIC is shown to leach a cytotoxic agent, possibly fluoride. This agent is effectively removed by an extraction procedure, and re-examination of the GIC demonstrates its cytocompatibility.

Haemocompatiblity of controlled release glass

There are many medical applications which benefit from the use of soluble biomaterials, including the sustained release of drugs over a precise period of time, or temporary conduits for controlling nerve regrowth. We have manufactured a series of phosphate-based controlled release glasses (CRGs) in which the solubility could be controlled by varying the concentration of CaO and Na2O. Fibres of the CRG containing iron and cerium were placed into direct contact with human neutrophils and macrophages in tissue culture for 2.5 and 24 h respectively and the responses analysed by scanning electron microscopy (SEM) and confocal microscopy. The supernatants were analysed for the cytokine IL-1β by enzyme-linked immunosorbent assay (ELISA). Disks of CRG of various compositions were placed in contact with whole blood for 30 min and platelet adhesion assessed by SEM. Activation of platelets, granulocytes and complement were quantified by ELISA for β-thromboglobulin, elastase and iC3b. Intrinsic coagulation activation was measured by timing the clotting of recalcified plasma. Only the cerium fibre inhibited IL-1β release from macrophages. No platelet adhesion was observed to any disk composition. Three compositions containing MgO inhibited plasma clotting and showed an insignificant level of complement activation. This study has demonstrated the development of a number of compositions of CRG, which have great potential in a wide variety of biomedical applications.