A. Pizzoferrato

In vitro evaluation of cell/biomaterial interaction by MTT assay

The tetrazolium-based colorimetric assay (MTT test) measures only in vitro living cells and the results are directly related to the number of viable cultured cells. It has been adopted in immunological investigations, cancer research and, recently, biocompatibility evaluation. We used the MTT method with minor modifications to fit it to an in vitro study of biomaterial-cell interactions. The MTT assay was confirmed to be feasible, rapid and reproducible. Moreover, it showed a good correlation with other in vitro proliferation assays, such as the 3H-thymidine uptake assay. By using the MTT method and the ASTM procedure for extracting biomaterials, we quantified the in vitro cell compatibility of different metals and polymers.

Cell culture methods for testing biocompatibility.

Cell culture systems may be of value in testing the biocompatibility of prosthetic materials before they are introduced into clinical use. In recent years, in vitro methods for assaying biomaterials have gained in importance owing to the growing concern over the use of animals for biomaterials testing. Significant effort is therefore being focused toward developing predictive and quantitative, but also simple and reliable, methods of testing using cultured cells. At present, a number of methods for measuring both the cytotoxicity and the specific cytocompatibility of different materials are available. The usefulness of these systems is no longer confined to screening new materials; they can be used to study the mechanisms of action of various materials during tissue/material interaction. This paper reviews the published literature on the use of cell culture models in evaluating biocompatibility and reports on the personal experience of the authors, who have been using cell culture systems for many years and for different purposes.

Application of a combination of neutral red and amido black staining for rapid, reliable cytotoxicity testing of biomaterials

Cell viability and growth for cytotoxicity evaluation of materials for prosthetic devices has been tested using various methods. The aim of this study was to extend the choice of reliable methods to quantify cytotoxicity of materials in vitro. By measuring both viability and growth of cells exposed to biomaterials in vitro, two different parameters are analysed and quantified upon reading of the absorbance of coloured solutions in a spectrophotometer. Neutral red uptake and amido black staining of cells have been used for cell viability and cell number measurement, respectively: they have been found to be well correlated with the number of surviving cells. These methods have been adjusted to a 96-well microplate cell culture system and re-evaluated as simple and reliable methods for the quantitative assessment of biomaterial effect on cells.

Cytotoxicity testing of cyanoacrylates using direct contact assay on cell cultures

The use of a tissue adhesive for surgical procedures has prompted a large number of clinical and experimental studies. Alkyl-2-cyanoacrylate esters constitute a family of adhesives with good mechanical properties but their biological compatibility has to be assessed. In this study the cytotoxicity of three commercially available cyanoacrylates and one of unknown composition has been determined. The first part of the study deals with direct contact testing procedures using L 929 cells challenged with drops of adhesives: cell morphology, cell growth and bacterial growth inhibition were assayed. Testing methods included cell viability assay using vital dyes, cell growth measurement using crystal violet staining uptake and bacterial growth assay using S. aureus growth inhibition. All the cyanoacrylate adhesives tested were found to be cytotoxic and to inhibit cell proliferation: differences between the cyanoacrylates were found.

Cell death induced by metal ions: necrosis or apoptosis?

We have evaluated if the cytotoxic effects of metals released from implants are due to necrosis or apoptosis. Peripheral blood mononuclear cells were exposed to different concentrations of chromium, nickel and cobalt extracts and the characteristics of both apoptosis and necrosis were evaluated by flow-cytometry at different culture endpoints. In order to define the prevalence of apoptosis or necrosis, the ratio cell death/apoptosis was calculated. A ratio of ≤1 means the prevalence of apoptotic events; a ratio >1 indicates the acute toxicity of the tested substance (necrosis). The extracts of chromium, cobalt and nickel had a cytotoxic effect on the mononuclear cells; high concentrations of cobalt and nickel produced cell necrosis, whereas by lowering the extract concentration apoptotic phenomena were observed. High chromium concentrations can induce cell death by apoptosis. Our data suggest that when large amounts of nickel and cobalt are released from implanted metal devices, necrosis is produced and consequently a strong inflammatory tissue reaction is likely to occur. The release of either chromium or limited amounts of nickel and cobalt induces toxicity characterized by apoptotic phenomena, which allows an adaptation of the tissue to the implant.

Cellular events in the mechanisms of prosthesis loosening

The functional restoration of a joint damaged by trauma or disease is obtained by prosthetic surgery. In particular the implantation of hip prostheses is regarded as routine in orthopedic surgery and thorough research has been developed in this field. The prosthetic replacement of the knee and even more so the ankle and elbow occurs less frequently in clinical practice and has been studied less intensively. The results of artifical hip replacement are generally good, both in terms of pain relief and the restoration of satisfactory joint function. Nevertheless, as time passes, a high rate of failures have been recorded due to prosthesis infections, fracture and wearing of the prosthetic components and prosthesis loosening by various causes. The use of ultra-filtered air and laminar flow in operating theatres and antibiotic prophylaxis have dramatically reduced the incidence of infections in total hip arthroplasty. Thanks to the setting up of new stem configurations and the use of superalloys that are highly resistant to fatigue failure, the fracture of the femoral component has been virtually eliminated as a complication of total hip arthroplasty replacements. Loosening is thus the most frequent complication in total hip replacement.

In vitro adhesion of Staphylococcus epidermidis on heparin-surface-modified intraocular lenses

Abstract This quantitative study assessed the in vitro adhesion of Staphylococcus epidermidis on two types of intraocular lenses: conventional poly(methyl methacrylate) (PMMA) and heparin‐surface‐modified PMMA. Level adhesion was measured by microbiological turbidimetry. We also measured modifications in the bacterium’s structural fatty acids after adhesion using gas chromatography.

Hydroxyapatite-coated orthopaedic screws as infection resistant materials: in vitro study

The authors evaluated in vitro the adherence of a Staphylococcus epidermidis strain to hydroxyapatite-coated stainless-steel screws-used in orthopaedic surgery for external fracture fixation-in comparison with the adherence to uncoated screws. Evaluations were also performed on analogous groups of screws immersed for 72 and 168 h in a solution at 37 degrees C, in order to simulate the interstitial fluid in a simplified way. Adherence values on coated prostheses resulted significantly lower compared with those observed on metal prostheses, both in basal conditions and after immersion in saline solution. Moreover, both on coated and on uncoated screws a significant reduction in bacterial adherence was noted related to the duration of the prosthesis permanence in saline solution.

Evaluation of autologous cancellous bone grafting of mandibular discontinuity defects in dogs using a Dacron-urethane prosthesis.

Reconstruction of 2 cm discontinuity defects using autologous cancellous bone chips and a Dacron-urethane prosthesis was evaluated in four canine mandibles. Histologic studies were performed on specimens taken six and 12 months after bone grafting. All four grafts were completely incorporated, producing a reconstruction that conformed precisely to the desired shape. Remodeling resulted in a trabecular pattern similar to that of the normal mandible. The prosthesis was well tolerated and did not elicit an inflammatory response.

Heparin surface treatment of poly(methylmethacrylate) alters adhesion of a Staphylococcus aureus strain : utility of bacterial fatty acid analysis

Bacterial adhesion on biomaterials is an important cause of associated infection. Many authors have studied the adhesion mechanisms of bacteria on biomaterials. These studies were useful in making materials more and more refractory to bacterial adhesion. We analysed the gas chromatographic modifications of structural fatty acids of a Staphylococcus aureus strain after adhesion on two polymers, poly(methylmethacrylate) (PMMA), whose biological compatibility is known, and heparin-surface-modified PMMA (HSM-PMMA). We noted changes to the chromatographic peaks peculiar to the fatty acids of S. aureus for each tested material and particularly for HSM-PMMA.