Objectives

The purpose of this study was to evaluate in vivo biocompatibility of novel single-walled carbon nanotubes (SWCNT)/poly(lactic-co-glycolic acid) (PLAGA) composites for applications in bone and tissue regeneration.

Biocompatibility of Co-Cr alloy was significantly improved by forming rough TiO2 layer on the surface. The TiO2 layer was formed by coating the Co-Cr alloy with Ti through electron beam deposition followed by micro-arc oxidation (MAO) of the Ti. Biocompatibility of Co-Cr alloy was enhanced by coating with titanium, and it was improved further by micro-arc oxidation treatment. MAO process was dependent on the thickness of coated titanium layer and applied voltage. There were close relationships between the phase, morphology and thickness of TiO2 layer and the applied voltage. Biocompatibility of the specimens coated with Ti and MAO treated after Ti coating were evaluated by in vitro ALP activity tests

Direct metal printed (DMP) porous iron implants possess promising mechanical and corrosion properties for various clinical application. Nevertheless, there is a requirement for better co-relation between in vitro and in vivo corrosion and biocompatibility behaviour of such biomaterials. Our present study evaluates absorption of porous iron implants under both static and dynamic conditions. Furthermore, this study characterizes their cytocompatibility using fibroblastic, osteogenic, endothelial and macrophagic cell types. In vitro degradation was performed statically and dynamically in a custom-built set-up placed under cell culture conditions (37 °C, 5% CO2 and 20% O2) for 28 days. The morphology and composition of the degradation products were analysed by scanning electron microscopy (SEM, JSM-IT100, JEOL). Iron implants before and after immersion were imaged by μCT (Quantum FX, Perkin Elmer, USA). Biocompatibility was also evaluated under static and dynamic in vitro culture conditions using L929, MG-63, HUVEC and RAW 264.7 cell lines. According to ISO 10993, cytocompatibility was evaluated directly using live/dead staining (Live and Dead Cell Assay kit, Abcam) in dual channel fluorescent optical imaging (FOI) and additionally quantified by flow cytometry. Furthermore, cytotoxicity was indirectly quantified using ISO conform extracts in proliferation assays. Strut size of DMP porous iron implants was 420 microns, with a porosity of 64% ± 0.2% as measured by micro-CT. After 28 days of physiological degradation in vitro, dynamically tested samples were covered with brownish degradation products. They revealed a 5.7- fold higher weight loss than statically tested samples, without significant changes in medium pH. Mechanical properties (E = 1600–1800 MPa) of these additively manufactured implants were still within the range of the values reported for trabecular bone, even after 28 days of biodegradation. Less than 25% cytotoxicity at 85% of the investigated time points was measured with L929 cells, while MG-63 and HUVEC cells showed 75% and 60% viability, respectively, after 24 h, with a decreasing trend with longer incubations. Cytotoxicity was analysed by two-way ANOVA and post-hoc Tukey's multiple comparisons test. Under dynamic culture conditions, live-dead staining and flow cytometric quantification showed a 2.8-fold and 5.7-fold increase in L929 and MG-63 cell survival rates, respectively, as compared to static conditions. Therefore, rationally designed and properly coated iron-based implants hold potential as a new generation of absorbable Orthopaedic implants

Biodegradable metals as orthopaedic implant materials receive substantial scientific and clinical interest. Marketed cardiovascular products confirm good biocompatibility of iron. Solid iron biodegrades slowly in vivo and has got supra-physiological mechanical properties as compared to bone and porous implants can be optimized for specific orthopaedic applications. We used Direct Metal Printing (DMP)3 to additively manufacture (AM) scaffolds of pure iron with fine-tuned bone-mimetic mechanical properties and improved degradation behavior to characterize their biocompatibility under static and dynamic 3D culture conditions using a spectrum of different cell types. Atomized iron powder was used to manufacture scaffolds with a repetitive diamond unit cell design on a ProX DMP 320 (Layerwise/3D Systems, Belgium). Mechanical characterization (Instron machine with a 10kN load cell, ISO 13314: 2011), degradation behavior under static and dynamic conditions (37ºC, 5% CO2 and 20% O2) for up of 28 days, with μCT as well as SEM/energy-dispersive X-ray spectroscopy (EDS) (SEM, JSM-IT100, JEOL) monitoring under in vivo-like conditions. Biocompatibility was comprehensively evaluated using a broader spectrum of human cells according to ISO 10993 guidelines, with topographically identical titanium (Ti-6Al-4V, Ti64) specimen as reference. Cytotoxicity was analyzed by two-way ANOVA and post-hoc Tukey's multiple comparisons test (α = 0.05). By μCT, as-built strut size (420 ± 4 μm) and porosity of 64% ± 0.2% were compared to design values (400 μm and 67%, respectively). After 28 days of biodegradation scaffolds showed a 3.1% weight reduction after cleaning, while pH-values of simulated body fluids (r-SBF) increased from 7.4 to 7.8. Mechanical properties of scaffolds (E = 1600–1800 MPa) were still within the range for trabecular bone, then. At all tested time points, close to 100% biocompatibility was shown with identically designed titanium (Ti64) controls (level 0 cytotoxicity). Iron scaffolds revealed a similar cytotoxicity with L929 cells throughout the study, but MG-63 or HUVEC cells revealed a reduced viability of 75% and 60%, respectively, already after 24h and a further decreased survival rate of 50% and 35% after 72h. Static and dynamic cultures revealed different and cell type-specific cytotoxicity profiles. Quantitative assays were confirmed by semi-quantitative cell staining in direct contact to iron and morphological differences were evident in comparison to Ti64 controls. This first report confirms that DMP allows accurate control of interconnectivity and topology of iron scaffold structures. While microstructure and chemical composition influence degradation behavior - so does topology and environmental in vitro conditions during degradation. While porous magnesium corrodes too fast to keep pace with bone remodeling rates, our porous and micro-structured design just holds tremendous potential to optimize the degradation speed of iron for application-specific orthopaedic implants. Surprisingly, the biological evaluation of pure iron scaffolds appears to largely depend on the culture model and cell type. Pure iron may not yet be an ideal surface for osteoblast- or endothelial-like cells in static cultures. We are currently studying appropriate coatings and in vivo-like dynamic culture systems to better predict in vivo biocompatibility

Summary Statement. A novel biomimetic polydioxanone tendon patch with woven and electrospun components is biocompatible, recapitulates native tendon architecture and creates a tissue-healing microenvironment directed by a subpopulation of regenerative macrophages. The woven component provides tensile strength while the tendon heals. Introduction. There is great interest in the use of biomimetic devices to augment tendon repairs. Ideally, implants improve healing without causing adverse local or systemic reactions. Biocompatibility remains a critical issue prior to implantation into humans, as some implants elicit a foreign body response (FBR) involving inflammation, poor wound healing and even fistulae formation. Additionally, the effect on articular cartilage locally or systemically with placement of a juxta-articular implant has not been examined. The purpose of this study is to test the in vivo biocompatibility of a novel hybrid woven and electrospun polydioxanone patch in a rat tendon transection model. Patients and Methods. Sixty Lewis rats were divided into 4 groups in which the infraspinatus was surgically transected 3 mm from its insertion. Tendons were repaired with a woven and electrospun polydioxanone patch (PDOe) and 5-0 Prolene sutures. Vicryl and Silk patches or a simple Prolene suture repair served as comparators. Animals were sacrificed at 1, 2, 4, 6 and 12 weeks to examine the biocompatibility of the implants. Immunohistochemistry was used to examine macrophage subpopulations and hematoxylin and eosin staining was used to assess foreign-body giant cells and both analyzed with a one-way ANOVA with significance set at p<.05. Articular cartilage was scrutinised with semi-quantitative analysis. Hind paw inflammatory indices were used to determine the systemic effects and biomechanical testing the tensile strength of the materials over time. Results. The PDOe patch remained grossly quiescent at all time-points. There was a severe inflammatory reaction to Vicryl at one and 2-week time-points with gross exudate. Silk patches were associated with larger fibrous capsules at each time point. There were no adverse systemic effects and articular cartilage remained normal with no differences between materials to controls. Immunohistochemistry showed a significantly higher ratio of regenerative to inflammatory macrophages for the PDOe patch compared to other constructs at each time-point and similar to controls. Silk and Vicryl patches had a greater than 10-fold increase in foreign-body giant cells compared to the PDOe patch and controls (p<.05) suggesting incorporation rather than rejection and walling off of the biomaterial. Tensile strength of the PDOe patch increased in the first 2 weeks to greater than 90 N and gradually declined to a mean of 22 N at 12 weeks. Discussion/Conclusion. The novel PDOe patch appears to be biocompatible and illicit very little FBR in this rat tendon injury model. Importantly, there was no joint reaction to the biomaterial which has not been addressed previously. We believe the electrospun component of the patch recapitulates native tendon architecture creating a tissue healing microenvironment directed by a regenerative macrophage subpopulation. These results corroborate earlier in vitro work that showed incorporation of tenocytes within the electrospun scaffold. The woven component of the scaffold provides tensile strength as the tendon heals and begins to degrade after healing is underway making it less likely to elicit a FBR. Based on these and earlier in vitro data we believe this implant shows excellent biocompatibility and is ready to proceed to human trials

Introduction. Currently, different techniques to evaluate biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal and ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. We have developed a single method using 3D agarose gels that is suitable to test the biocompatibility of all three types of wear debris simultaneously. Moreover, stimulation of the cells by wear particles embedded in a 3D gel better mimics the in vivo environment. Materials and Methods. Clinically relevant sterile UHMWPE and CoCr wear particles were generated using methodologies described previously [1,2]. Commercially available nanoscale and micron-sized silicon nitride (Si. 3. N. 4. ) particles (<50 nm and <1 μm, Sigma UK) were sterilised by heat treatment for 4h at 180°C. Agarose-particle suspensions were prepared by mixing warm 2% (w/v) low-melting-point agarose solution with the particles dispersed by sonication in DMEM culture media. The suspensions were then allowed to set at room temperature for 10 min in 96 well culture plates. Sub-confluent L929 murine fibroblasts were cultured on the prepared gels for up to 6 days in 5% (v/v) CO. 2. at 37°C. After incubation, the viability of cells was measured using the ATP-lite assay. The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. Results and Discussion. The gels were observed to ensure uniform distribution of particles and migration of cells into the gel. No significant reduction in viability was observed for nanoscale and micron-sized Si. 3. N. 4. particles at low doses (0.5 μm. 3. per cell) and high doses (50 μm. 3. per cell), or for UHMWPE wear debris at high doses (100 μm. 3. per cell) [Figure1]. Moreover, the viability was significantly reduced for high doses of CoCr wear debris (50 μm. 3. per cell) and the positive control, camptothecin (2 μg.ml. −1. ) at day 6 [Figure1]. These results are consistent with the literature [2,3] and therefore validate our 3D agarose cell culture method for comparing cytotoxicity of polymer, metal and ceramic particles in a single assay, simultaneously. Conclusion. Biocompatibility ofpolymer, metal and ceramic wear debris can be tested simultaneously by using 3D particle embedded agarose gels. Acknowledgements. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. GA-310477 LifeLongJoints

Summary Statement. Innovative nanocomposite carbon coating doped with Si can significantly improve the osseintegration of orthopaedics implants. Additionally, this kind of coating increases the mechanical resistance of the implants, what is especially important on case of joints (frictional pairs). Introduction. Use of layers of carbon-doped silicon, which leads to the synthesis of layers improving mechanical and biological characteristics, let obtain good strength by volume features. Suitable introduction to the structure of amorphous silicon dioxide layer allow for the production of higher adhesion to metallic substrates and consequently the increased thickness and hardness. The increased thickness of the layer leads to a stronger diffusion barrier to harmful metal ions from the implant material and thus consequently improving the biocompatibility of the implant. Moreover, a silicon beneficial effect on stress relaxation layer formed during the synthesis. This allows for improved biocompatibility, also affects other property obtained in the case of silicon carbide layers, the bacteriastability. This further protects the surface of the implant against the risk of bacterial colonization in both the implantation and subsequent use in the body, and preferably suppressing inflammation and faster healing of surgical wounds. The thus obtained product is much better than the biological and mechanical parameters of currently offered. Patients & Methods. In order to evaluate the fabricated coatings conditions examination of the basic physicochemical and mechanical properties were conducted (AFM, Raman, XPS, nanoindentation technique). The in vitro and in vivo tests were also conducted. As a biological material osteoblast Saos-2 cells and endothelial cells line EA. 926 were used. For the evaluation of proliferation and cytotoxicity a “live/dead” test was used. For testing bactericidal activity of the C/Si coatings, an exponential growth phase of E. coli strain DH5 α was used. Test of bacterial immediate toxicity and bacterial colonization were performed. A model of rabbits and guinea pigs were used to obtained results with reference to irritation, intradermal reactivity, sensitization, local effects after implantation with the histopathological examination, cytotoxicity test. Results. XPS results have shown that the silicon content for each group of samples, both steel and titanium alloy is about 3, 4 and 5 percent. Increasing the concentration of silicon above 5% results in the weakening of the mechanical properties of the layer and lead to delamination of the sterilization process. Addition of silicon in the range of 3–5% does not negatively affect the mechanical and structural properties of the modified surface and from this point of view, all the criterion of strength. Performed studies confirmed very good mechanical properties of C/Si coatings. In vitro studies have indicated the optimal concentration of silicon in the coating, where the material is biocompatible and also has good antibacterial properties. Biocompatibility of silicon coatings was also confirmed by irritation and sensitization testing in the in vivo model. Discussion/Conclusion. Final result of the surface modification C/Si coating depends on modification of two effects, i.e. the formation of the transition layer of the substrate material and the synthesis of the outer carbon coating. Results of in vitro and in vivo tests confirmed very good biological properties of coatings which proved the fact that it is possible to improve the parameters of the implant work at the same time adding to the intrinsic the antibactericidal properties

Introduction

Stryde® lengthening nail has been recently withdrawn because of concerns about osteolysis and other bone lesions that have been observed early after implantation. The present study analyses the incidence and features of these bone lesions in our patients.

Aim

The utilization of silver as an anti-infective agent is a subject of debate within the scientific community, with recurring discussions surrounding its biocompatibility. Presently, galvanic silver coating finds widespread clinical application in mitigating infection risks associated with large joint arthroplasties. While some instances have linked this coating to sporadic cases of localized argyria, these occurrences have not exhibited systematic or functional limitations. To address concerns regarding biocompatibility, a novel approach has been devised for anti-infective implant coatings: encapsulating silver nitrate within a biopolymer reservoir for non-articulating surfaces. This poly-L-lactic acid layer releases silver ions gradually, thereby circumventing biocompatibility concerns.

Bone fractures are highly observed clinical situation in orthopaedic treatments. In some cases, there might be non-union problems. Therefore, recent studies have focused on tissue engineering applications as alternative methods to replace surgical procedures. Various biopolymer based scaffolds are produced using different fabrication techniques for bone tissue engineering applications.

In this study, hydroxyapatite (HAp) and loofah containing carboxymethyl chitosan (CMC) scaffolds were prepared. In this regard, first 4 ml of CMC solution, 0.02 g of hydroxyapatite (HAP) and 0.06 g of poly (ethylene glycol) diglycidyl ether (PEGDE) were mixed in an ultrasonic bath until the HAp powders were suspended. Next, 0.04 g of loofah was added to the suspension and with the help of PEGDE as the cross-linking agent, then, the mixture was allowed to cross-link at 40^oC overnight. Finally, the three-dimensional, porous and sponge-like scaffolds were obtained after lyophilization (TELSTAR - LyoQuest −85) at 0.1 mbar and −25°C for 2 days.

Morphologies, chemical structures and thermal properties of the scaffolds were characterized by scanning electron microscopy (SEM), Fourier Transform infrared spectroscopy (FT-IR) and thermogravimetric differential thermal analysis (TGA/DTA), respectively. In addition, swelling behavior and mechanical properties of the scaffolds under compression loading were determined.

In order to investigate biocompatibility of the scaffolds, WST-1 colorimetric assay at days 0, 1, 3, 5 and 7 was conducted by using human dermal fibroblast. Also, histological and morphological analysis were performed for cell attachment at day 7.

In conclusion, the produced scaffolds showed no cytotoxic effect. Therefore, they can be considered as a candidate scaffold for bone tissue regeneration. Further studies will be performed by using bone marrow and periosteum derived mesenchymal stem cells with these scaffolds.

Abstract

Introduction

Because of its high strength and allowance for bone integration, Ti-6Al-4V is the most commonly used material for load bearing bone implants. Compared to conventional production methods, 3D printing Ti-6Al-4V introduces advantages as (near-) net-shape manufacturing of complex geometries, and optimization of utilization rate of the material. However, as result of the additively production procedure, microstructure and surface properties differ from those manufactured using conventional techniques. Therefore, the resulting mechanical properties and biocompatibility of the 3D printed Ti-6Al-4V are investigated in this study. First, it was aimed to reveal the tensile properties of the material and verify if these depend on build orientation. Second, it was determined which post process method provides the best osteoconductivity.

Since 2010, there has been a sharp decline in the use of metal-on-metal joint replacement devices due to adverse responses associated with the release of metal wear particles and ions in patients. Surface engineered coatings offer an innovative solution to this problem by covering metal implant surfaces with biocompatible and wear resistant materials. The present study tests the hypothesis whether surface engineered coatings can reduce the overall biological impact of a device by investigating recently introduced silicon nitride coatings for joint replacements. Biological responses of peripheral blood mononuclear cells (PBMNCs) to Si3N4 model particles, SiNx coating wear particles and CoCr wear particles were evaluated by testing cytotoxicity, inflammatory cytokine release, oxidative stress and genotoxicity.

Clinically relevant wear particles were generated from SiNx-on-SiNx and CoCr-on-CoCr bearing combinations using a multidirectional pin-on-plate tribometer. All particles were heat treated at 180°C for 4 h to destroy endotoxin contamination. Whole peripheral blood was collected from healthy donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell) and incubated with particles at various volumetric concentrations (0.5 to 100 µm3 particles/cell) for 24 h in 5% (v/v) CO2 at 37°C. After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer); TNF-alpha release was measured by ELISA (Invitrogen); oxidative stress was measured using H2DCFDA (Abcam); and DNA damage was measured by comet assay (Tevigen). The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis.

No evidence of cytotoxicity, oxidative stress, TNF-alpha release, or DNA damage was observed for the silicon nitride particles at any of the doses. However, CoCr wear particles caused cytotoxicity, oxidative stress, TNF-alpha release and DNA damage in PBMNCs at high doses (50 µm3 particles per cell). This study has demonstrated the in-vitro biocompatibility of SiNx coatings with primary human monocytic cells. Therefore, surface engineered coatings have potential to significantly reduce the biological impact of metal components in future orthopaedic devices.

Introduction

We have previously demonstrated that peroxide crosslinked vitamin E-blended UHMWPE maintains its clinically-required wear and mechanical properties [1]. This material can potentially be used as an irradiation-free bearing surface for TJA. However, using organic peroxides in medical devices requires a thorough examination of tissues in contact with the implant. For this study we crosslinked polyethylene using five times the needed concentration of peroxide (2,5-Dimethyl-2,5-di(t-butylperoxy)-hexyne-3 or P130), followed by implantation to determine implant biocompatibility, and pre and post implant peroxide residual contents.

The osseointegration of implants is related to the early interactions between osteoblastic cells and titanium surfaces. The behavior of osteoblast cells was compared on four different titanium surfaces in vitro and in vivo: machined, blasted, plasma spray and micro-arc oxidation.

X-ray diffraction and scanning electron microscope investigations were performed in order to assess the structure and morphology. Biologic and morphologic responses to the osteoblast cell lines (Saos-2) were then examined, using Promega proliferation assay, alkaline phosphatase activity, vβ3 integrin expression and cytoskeleton staining (Rhodamine-Phallodine). The analysis of gene expression for osteocalcin and collagen I was done through RT-PCR. In addition, differential histologic evaluation and interfacial strength at the bone-implant interfaces were then evaluated in the distal femur of four beagle dogs.

In conclusion, micro-arc oxidation of titanium appears to exhibit more favorable osteoblast adhesion and stronger interfacial strength than the compared groups in vitro and in vivo as well.

The steric and electrostatic complementarity of natural proteins and other macromolecules are a result of evolutionary processes. The role of such complementarity is well established in protein-protein interactions, accounting for the known protein complexes. To our knowledge, non-biological systems have not been a part of such evolutionary processes. Therefore, it is desirable to design and develop nonbiological surfaces, such as implant devices (e.g. bone growth for non-cemented fixation), that exhibit such complementarity effects with the natural proteins.

Cell attachment and spreading in vitro is generally mediated by adhesive proteins such as fibronectin and vitronectin [1]. The primary interaction between cells and adhesive proteins occurs through integrin and an RGD amino acid sequence. The adsorption of adhesive proteins plays an important role in cell adhesion and bone formation to an implant surface [1]. The ability of the implant surface to adsorb these proteins determines its aptitude to support cell adhesion and spreading and its biocompatibility. For example, the enhancement of osteoblast precursor attachment on hydroxyapatite (HA) as compared to titanium and stainless steel was related to increased fibronectin and vitronectin absorption [2].

The role of surface characteristics, such as topography, has been studied in recent years without the emergence of a comprehensive and consistent model [1]. For example, while no statistically significant influence of surface roughness on osteoblast proliferation and cell viability was detected in the study of metallic titanium surfaces [3], the TiO2 film enhances osteoblast adhesion, proliferation and differentiation upon an increase in roughness [4].

We designed and produced ceramic [5] and metallic coatings via an ion beam assisted deposition process with spatial dispersion (roughness) comparable to the size of proteins (3–20nm). Our ceramic and cobaltchrome (CoCr) coatings exhibit high hardness and contact angles with serum of 0° and 40° to 50°, respectively. Furthermore, our theoretical calculations and quantum-mechanical modeling clearly indicate that the spatial electric potential variation across our designed ceramic surfaces is comparable to the electrostatic potential variation of proteins such as fibronectin, promoting increased absorption on these surfaces. Therefore, an increase in the concentration of adhesive proteins on the designed surfaces results in the enhancement of the focal adhesion of cells. Our experimental results of the adhesion and proliferation of osteoblast-like stromal cells from mouse bone marrow indicate that our nanostructured coatings are three to five times better than growing on HA and orthopaedic grades of titanium and CoCr. Our results are consistent with the steric and electrostatic complementarity of nanostructured surfaces and adhesive proteins. This paper presents the adhesion and proliferation of osteoblast-like cells on micro-and nanostructured surfaces and provides new models describing the mechanism responsible for the enhancement of cell adhesion on nanostructured ceramic and metallic surfaces compared with orthopaedic materials.

Aim

Silver is known for its excellent antimicrobial activity, including activity against multiresistant strains. The aim of the current study was to analyze the biocompatibility and potential influence on the fracture healing process a silver-coating technology for locking plates compared to silver-free locking plates in a rabbit model.

Introduction

Silicon nitride (SiN) is a recently introduced bearing material for THR that has shown potential in its bulk form and as a coating material on cobalt-chromium (CoCr) substrates. Previous studies have shown that SiN has low friction characteristics, low wear rates and high mechanical strength. Moreover, it has been shown to have osseointegration properties. However, there is limited evidence to support its biocompatibility as an implant material. The aim of this study was to investigate the responses of peripheral blood mononuclear cells (PBMNCs) isolated from healthy human volunteers and U937 human histiocytes (U937s) to SiN nanoparticles and CoCr wear particles.

Impaction grafting with morsellised allograft is becoming the treatment of choice for revision arthroplasty, especially in the younger patient. The optimum treatment of the graft prior to impaction has not been determined. Some groups wash the graft prior to impaction and others do not. Washing of graft has been shown to enhance bone ingrowth in an animal model, however the reasons for this remain unclear. The aim of this study was to identify any underlying cellular cytotoxicty of fresh frozen allograft bone before and after washing.

Paired samples of washed and unwashed morcellised FFH allograft were taken during revision hip arthroplasties. Washing was performed by 4 consecutive rinses in 300ml warmed saline, the bone being filtered between each exchange of saline. Contact cytotoxicity assays involved culture of cell lines in direct contact with bone samples. Quantitative cytotoxicity assays utilised culture media conditioned with the bone samples and subsequent assessment of cell metabolism and viability using both dimethylthiazol (MTT) and neutral red (NR) assays. Assays were performed with human osteoblastic (MG63) and fibroblastic (HSF) cell lines.

Nine pairs of samples were analysed. Contact assays demonstrated a clear zone of cellular inhibition around the unwashed bone samples. Quantitative assays were performed in triplicate for each cell type and both MTT and NR assays giving 108 paired assay results. 88.9% of pairs (92/108) showed cytotoxicity in the unwashed sample. No washed samples demonstrated cytotoxicity. When grouped by assay and cell type, analysis of means showed statistically significant differences between washed and unwashed samples in MG63-NR (p=0.0025), HSF-NR (p=0.0004) and MG63-MTT (p=0.008). The difference observed in the HSF-MTT assays did not reach statistical significance (p=0.06).

Unwashed FFH allograft can be cytotoxic to human osteoblastic and fibroblastic cell lines in vitro. This suggests that allograft should be washed prior to impaction in order to optimise the biological compatibility.

The release of metallic elements from the surfaces of orthopaedic prostheses, either by leaching or breakdown of the device into the body, is a potential health problem to patients. During the endurance of this bio-material metal alloys undergo to corrosion with the possibility that metal ions interact with biofluids and tissues inducing adverse biological effects (local or systemic damage such as cytotoxicity, delayed hypersensitivity, effects on angiogenesis and mutagenicity). Indeed, as the metal ions are released over a period of time, they will continue to stimulate the inflammatory response and to influence those cells within the chronic response.

The safe evaluation of biocompatibility of metallic-based orthopaedic prostheses is a prerequisite for their use in medicine. In this context, the use of in vitro model systems is of growing importance, not only as components of the initial phases of the safe evaluation process, but also as alternative (non-animal) methods for regulatory purpose through the cycle of research, development, validation and acceptance by regulatory authorities.

The aim of this work is to review the activity of IMETOX (In vitro Metal Toxicology) project of ECVAM in relation to biomaterials for hard tissue substitute. The research is aiming at integrating aspects of metal toxicity in different toxicological areas (e.g. systemic toxicity, reproductive toxicity, immunotoxicity, carcinogenicity).

Examples of tier testing as basis for new in vitro strategies for the evaluation of immunotoxicity, basal cyto-toxicity, and carcinogenic potential of metals of interest as components of medical devices (e.g. Ag, Co, Cr, Pt, Ti, V) investigated by cell cultures (mouse fibroblasts Balb/3T3, human keratinocytes HaCaT) are presented.

Key factors influencing cellular metal toxicity (low dose exposure, speciation, interaction among elements) and the new emerging problems of the cytotoxic potential of metal-nanoparticles are discussed.