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Bone homeostasis is the equilibrium between organic and inorganic components of the extracellular matrix (ECM) and cells. Alteration of this balance has consequences on bone mass and architecture, resulting in conditions such as osteoporosis (OP). Given ECM protein mutual regulation and their effects on bone structure and mineralization, further insight into their expression is crucial to understanding bone biology under normal and pathological conditions. This study focused on Type I Collagen, which is mainly responsible for structural properties and mineralization of bone, and selected proteins implicated in matrix composition, mineral deposition, and cell-matrix interaction such as Decorin, Osteocalcin, Osteopontin, Bone Sialoprotein 2, Osteonectin and Transforming Growth Factor beta. We developed a novel multidisciplinary approach in order to assess bone matrix in healthy and OP conditions more comprehensively by exploiting the Fourier Transform Infrared Imaging (FTIRI) technique combined with histomorphometry, Sirius Red staining, immunohistochemistry, and Western Blotting. This innovatory procedure allowed for the analysis of superimposed tissue sections and revealed that the alterations in OP bone tissue architecture were associated with warped Type I Collagen structure and deposition but not with changes in the total protein amount. The detected changes in the expression and/or cooperative or antagonist role of Decorin, Osteocalcin, Osteopontin, and Bone Sialoprotein-2 indicate the deep impact of these NCPs on collagen features of OP bone. Overall, our strategy may represent a starting point for designing targeted clinical strategies aimed at bone mass preservation and sustain the FTIRI translational capability as upcoming support for traditional diagnostic methods.

Artículo con numerosos autores, sólo se recogen el primero y los pertenecientes a la UAM, Background and study aim: Advanced therapy medicinal products (ATMP) frequently lack of clinical data on efficacy to substantiate a future clinical use. This study aims to evaluate the efficacy to heal long bone delayed unions and non-unions, as secondary objective of the EudraCT 2011-005441-13 clinical trial, through clinical and radiological bone consolidation at 3, 6 and 12 months of follow-up, with subgroup analysis of affected bone, gender, tobacco use, and time since the original fracture. Patients and methods: Twenty-eight patients were recruited and surgically treated with autologous bone marrow derived mesenchymal stromal cells expanded under Good Manufacturing Practices, combined to bioceramics in the surgical room before implantation. Mean age was 39 ±13 years, 57% were males, and mean Body Mass Index 27 ±7. Thirteen (46%) were active smokers. There were 11 femoral, 4 humeral, and 13 tibial non-unions. Initial fracture occurred at a mean ±SD of 27.9 ±31.2 months before recruit- ment. Efficacy results were expressed by clinical consolidation (no or mild pain if values under 30 in VAS scale), and by radiological consolidation with a REBORNE score over 11/16 points (value of or above 0.6875). Means were statistically compared and mixed models for repeated measurements estimated the mean and confidence intervals (95%) of the REBORNE Bone Healing scale. Clinical and radiological con- solidation were analyzed in the subgroups with Spearman correlation tests (adjusted by Bonferroni). Results: Clinical consolidation was earlier confirmed, while radiological consolidation at 3 months was 25.0% (7/28 cases), at 6 months 67.8% (19/28 cases), and at 12 months, 92.8% (26/28 cases including the drop-out extrapolation of two failures). Bone biopsies confirmed bone formation surrounding the bioce- ramic granules. All locations showed similar consolidation, although this was delayed in tibial non-unions. No significant gender difference was found in 12-month consolidation (95% confidence). Higher consoli- dation scale values were seen in non-smoking patients at 6 ( p = 0.012, t -test) and 12 months ( p = 0.011, t -test). Longer time elapsed after the initial fracture did not preclude the occurrence of consolidation. Conclusion: Bone consolidation was efficaciously obtained with the studied expanded hBM-MSCs com- bined to biomaterials, by clinical and radiological evaluation, and confirmed by bone biopsies, with lower consolidation scores in smokers, This research received funding from the European Union’s Sev- enth Framework Programme ( FP7/FP7-HEALTH-2009 ) with the RE- BORNE Project (under G.A. 241876), and the European Union’s Horizon 2020 Programme ( H2020-SC1 2016-2017 ), with the OR- THOUNION Project (under G.A. 7333288)

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Bone is an extremely dynamic tissue, undergoing continuous remodeling for its whole lifetime, but its regeneration or augmentation due to bone loss or defects are not always easy to obtain. Bone tissue engineering (BTE) is a promising approach, and its success often relies on a “smart” scaffold, as a support to host and guide bone formation through bone cell precursors. Bone homeostasis is maintained by osteoblasts (OBs) and osteoclasts (OCs) within the basic multicellular unit, in a consecutive cycle of resorption and formation. Therefore, a functional scaffold should allow the best possible OB/OC cooperation for bone remodeling, as happens within the bone extracellular matrix in the body. In the present work OB/OC co-culture models, with and without scaffolds, are reviewed. These experimental systems are intended for different targets, including bone remodeling simulation, drug testing and the assessment of biomaterials and 3D scaffolds for BTE. As a consequence, several parameters, such as cell type, cell ratio, culture medium and inducers, culture times and setpoints, assay methods, etc. vary greatly. This review identifies and systematically reports the in vitro methods explored up to now, which, as they allow cellular communication, more closely resemble bone remodeling and/or the regeneration process in the framework of BTE. Statement of significance Bone is a dynamic tissue under continuous remodeling, but spontaneous healing may fail in the case of excessive bone loss which often requires valid alternatives to conventional treatments to restore bone integrity, like bone tissue engineering (BTE). Pre-clinical evaluation of scaffolds for BTE requires in vitro testing where co-cultures combining innovative materials with osteoblasts (OBs) and osteoclasts (OCs) closely mimic the in vivo repair process. This review considers the direct and indirect OB/OC co-cultures relevant to BTE, from the early mouse-cell models to the recent bone regenerative systems. The co-culture modeling of bone microenvironment provides reliable information on bone cell cross-talk. Starting from improved knowledge on bone remodeling, bone disease mechanisms may be understood and new BTE solutions are designed.

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium-deficient hydroxyapatite (CDHA). The effect of heparin-functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (ß-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered ß-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and ß-TCP present OC at 21–28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption.

Background Safety and feasibility of a regenerative strategy based on the use of culture-expanded mesenchymal stromal cells (MSCs) have been investigated in phase 2 trials for the treatment of nonunion and osteonecrosis of the femoral head (ONFH). As part of the clinical study, we aimed to evaluate if bone turnover markers (BTMs) could be useful for predicting the regenerative ability of the cell therapy product. Materials and Methods The bone defects of 39 patients (nonunion: n = 26; ONFH: n = 13) were treated with bone marrow–derived MSCs, expanded using a clinical-grade protocol and combined with biphasic calcium phosphate before implantation. Bone formation markers, bone-resorption markers and osteoclast regulatory proteins were measured before treatment (baseline) and after 12 and 24 weeks from surgery. At the same time-points, clinical and radiological controls were performed to evaluate the bone-healing progression. Results We found that C-Propeptide of Type I Procollagen (CICP) and C-terminal telopeptide of type-I collagen (CTX) varied significantly, not only over time, but also according to clinical results. In patients with a good outcome, CICP increased and CTX decreased, and this trend was observed in both nonunion and ONFH. Moreover, collagen biomarkers were able to discriminate healed patients from non-responsive patients with a good diagnostic accuracy. Discussion CICP and CTX could be valuable biomarkers for monitoring and predicting the regenerative ability of cell products used to stimulate the repair of refractory bone diseases. To be translated in a clinical setting, these results are under validation in a currently ongoing phase 3 clinical trial.

Osteoporosis (OP) is a skeletal disorder responsible for the weakening of the bone structure and, consequently, for an increased fracture risk in the elderly population. In the past, bone mineral density (BMD) variation was considered the best OP indicator, but recently the focus has shifted toward the variation of microstructural bone parameters. This work is based on the characterisation of 8-mm cylindrical biopsies harvested from proximal humeral heads belonging to healthy and osteoporotic patients, in order to assess the OP-related variations of bone properties at different scale lengths. In particular, bone biopsies underwent micro-computed tomography analysis to study the most relevant features of bone architecture and extrapolate the tissue mineral density (TMD) value of bone trabeculae. Compression tests and nanoindentations were performed to investigate the macro- and micromechanical properties of bone biopsies, respectively. In addition, XRD analyses were performed to obtain the mean hydroxyapatite (HA) crystallite size, while Raman spectroscopy investigated the collagen secondary structure. Thermogravimetric analysis was performed to evaluate the ratio between organic and inorganic phases. From the obtained results, OP samples showed a more anisotropic and less interconnected structure responsible for reduced compression strength. From this, it can be supposed that OP caused an alteration of bone structure that led to inferior macroscopic mechanical properties. Furthermore, OP samples possessed higher TMD and bigger HA crystals that are correlated to an increase of the hardness value obtained by means of nanoindentation. This less controlled HA crystal growth is probably due to an alteration of the organic matrix structure, as revealed by the increase of the random coil contribution in the Raman spectra of the OP bone. This higher crystal content led to an increase in trabecular density and hardness. In conclusion, the obtained data showed that OP affects bone properties at different scale lengths causing an alteration of its morphological, structural and mechanical features.

The punctual analysis of bone Extracellular Matrix (ECM) proteins represents a pivotal point for medical research in bone diseases like osteoporosis. Studies in this field, historically done to appreciate bone biology, were mainly conducted on animal samples and, up to today, only a few studies on protein detection in human bone are present. The challenges in bone ECM protein extraction and quantitation protocols are related to both the separation of proteins from the mineral content (i.e. hydroxyapatite) and the difficulty of avoiding protein denaturation during the extraction processes. The aim of the present work was to define appropriate protocol(s) for bone ECM protein extraction that could be applied to investigate both normal and pathological conditions. We compared and optimised some of the most used protocols present in the literature, modifying the protein precipitation method, the buffer used for resuspension and/or the volume of reagent used. Bradford and BCA assays and Western Blotting were used to evaluate the variations in the total protein recovery and the amount of selected proteins (Type I Collagen, TGF-β, IGF-1, Decorin, Osteopontin, Bone Sialoprotein-2 and Osteocalcin). Collectively, we were capable to draw-up two single-extract protocols with optimal recovery and ideal protein content, that can be used for a detailed analysis of ECM proteins in pathological bone samples. Time-consuming multi-extract procedures, optimised in their precipitation methods, are however crucial for a precise detection of specific proteins, like osteocalcin. As the matter of fact, also the demineralization processes, commonly suggested and performed in several protocols, could hinder an accurate protein detection, thus inherently affecting the study of a pathological bone ECM. This study represents a starting point for the definition of appropriate strategies in the study of bone extracellular matrix proteins involved in the onset and maintenance of bone diseases, as well as a tool for the development of customized scaffolds capable to modulate a proper feedback loop in bone remodelling, altered in case of diseases like osteoporosis.

ORTHOUNION is a multicentre, open, comparative, three-arm, randomized clinical trial (EudraCT number 2015-000431-32) to compare the efficacy, at one and two years, of autologous human bone marrow-derived expanded mesenchymal stromal cell (hBM-MSC) treatments versus iliac crest autograft (ICA) to enhance bone healing in patients with diaphyseal and/or metaphysodiaphyseal fracture (femur, tibia, and humerus) status of atrophic or oligotrophic nonunion (more than 9 months after the acute fracture, including recalcitrant cases after failed treatments). The primary objective is to determine if the treatment with hBM-MSCs combined with biomaterial is superior to ICA in obtaining bone healing. If confirmed, a secondary objective is set to determine if the dose of 100 × 106 hBM-MSCs is noninferior to that of 200 × 106 hBM-MSCs. The participants (n=108) will be randomly assigned to either the experimental low dose (n=36), the experimental high dose (n=36), or the comparator arm (n=36) using a central randomization service. The trial will be conducted in 20 clinical centres in Spain, France, Germany, and Italy under the same clinical protocol. The confirmation of superiority for the proposed ATMP in nonunions may foster the future of bone regenerative medicine in this indication. On the contrary, absence of superiority may underline its limitations in clinical use.

Metals and metal alloys are the most used materials in orthopedic implants. The focus is on total hip arthroplasty (THA) that, though well tolerated, may be associated with local and remote adverse effects in the medium-long term. This review aims to summarize data on the biological consequences of the metal implant degradation that have been attributed predominantly to metal-on-metal (MoM) THA. Local responses to metals consist of a broad clinical spectrum ranging from small asymptomatic tissue lesions to severe destruction of bone and soft tissues, which are designated as metallosis, adverse reactions to metal debris (ARMD), aseptic lymphocytic vasculitis associated lesion (ALVAL), and pseudotumors. In addition, the dissemination of metal particles and ions throughout the body has been associated with systemic adverse effects, including organ toxicity, cancerogenesis, teratogenicity, and immunotoxicity. As proved by the multitude of studies in this field, metal degradation may increase safety issues associated with THA, especially with MoM hip systems. Data collection regarding local, systemic and long-term effects plays an essential role to better define any safety risks and to generate scientifically based recommendations.

Background: ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. Methods: Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5–10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. Findings: With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). Interpretation: Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. Funding: EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876)., The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/FP7-HEALTH-2009); REBORNE Project (GA: 241876)

The design of synthetic bone grafts to foster bone formation is a challenge in regenerative medicine. Understanding the interaction of bone substitutes with osteoclasts is essential, since osteoclasts not only drive a timely resorption of the biomaterial, but also trigger osteoblast activity. In this study, the adhesion and differentiation of human blood-derived osteoclast precursors (OCP) on two different micro-nanostructured biomimetic hydroxyapatite materials consisting in coarse (HA-C) and fine HA (HA-F) crystals, in comparison with sintered stoichiometric HA (sin-HA, reference material), were investigated. Osteoclasts were induced to differentiate by RANKL-containing supernatant using cell/substrate direct and indirect contact systems, and calcium (Ca++) and phosphorus (P5+) in culture medium were measured. We observed that OCP adhered to the experimental surfaces, and that osteoclast-like cells formed at a rate influenced by the micro- and nano-structure of HA, which also modulate extracellular Ca++. Qualitative differences were found between OCP on biomimetic HA-C and HA-F and their counterparts on plastic and sin-HA. On HA-C and HA-F cells shared typical features of mature osteoclasts, i.e. podosomes, multinuclearity, tartrate acid phosphatase (TRAP)-positive staining, and TRAP5b-enzyme release. However, cells were less in number compared to those on plastic or on sin-HA, and they did not express some specific osteoclast markers. In conclusion, blood-derived OCP are able to attach to biomimetic and sintered HA substrates, but their subsequent fusion and resorptive activity are hampered by surface micro-nano-structure. Indirect cultures suggest that fusion of OCP is sensitive to topography and to extracellular calcium. Statement of Significance The novelty of the paper is the differentiation of human blood-derived osteoclast precursors, instead of mouse-derived macrophages as used in most studies, directly on biomimetic micro-nano structured HA-based surfaces, as triggered by osteoblast-produced factors (RANKL/OPG), and influenced by chemistry and topography of the substrate(s). Biomimetic HA-surfaces, like those obtained in calcium phosphate cements, are very different from the conventional calcium phosphate ceramics, both in terms of topography and ion exchange. The role of these factors in modulating precursors’ differentiation and activity is analysed. The system is closely reproducing the physiological process of attachment of host cells and further maturation to osteoclasts toward resorption of the substrate, which occurs in vivo after filling bone defects with the calcium phosphate grafts.

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low- and high-temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.

Background aims Avascular necrosis of the femoral head (AVN) occurs as common result of various conditions or develops as a primary entity, with a high freqency in young adults. Because of its tendency toward osteoarthritis requiring total hip arthroplasty, alternative treatments are being advocated, including cell therapy with mesenchymal stromal cells (MSCs). Because osteonecrotic bone is a severely hypoxic tissue, with a 1–3% oxygen tension, the survival and function of multipotent cells is questionable. Methods In this study, the proliferative, immunophenotypic and osteogenic properties of bone marrow (BM)-derived MSCs from a clinical series of patients with AVN were evaluated under in vitro conditions mimicking the hypoxic milieu of AVN to verify the rationale for cell therapy. MSCs retrieved from the iliac crest (BM-MSC) were isolated, expanded and induced to osteogenic differentiation under a 2% pO2 atmosphere (hypoxia) in comparison with the standard 21% pO2 (normoxia) that is routinely used in cell culture assays. Results Both proliferation and colony-forming ability were significantly enhanced in hypoxia-exposed BM-MSCs compared with BM-MSCs under normoxia. The expression of bone-related genes, including alkaline phosphatase, Type I collagen, and osteocalcin was significantly increased under hypoxia. Moreover, mineral deposition after osteogenic induction was not hampered, but in some cases even enhanced under low oxygen tension. Conclusions These findings support autologous cell therapy as an effective treatment to stimulate bone healing in the hypoxic microenvironment of AVN.

Purpose For bone regeneration therapy using stem cells, well-defined ex vivo protocols to expand mesenchymal stromal cells (MSC), as well as assays to show their potential differentiation into the osteogenic lineage, are needed. Aim of this study was to analyze the role of the biochemical osteogenic inducers, i.e. ascorbic acid, dexamethasone, and β-glycerophosphate, employed in the current protocols for osteogenic differentiation of MSC in vitro, to address the requirements for reliable differentiation systems. Methods MSC were isolated from the bone marrow of donors (46–73 years of age) undergoing total hip replacement, and expanded in vitro. At confluence, MSC were cultured under four different conditions: α-MEM plus serum (basal medium or C1), basal medium plus ascorbate (C2), basal medium plus ascorbate and dexamethasone (C3), or basal medium plus ascorbate, dexamethasone and β-glycerophosphate (C4). Morphology, proliferation, mineralization, alkaline phosphatase, collagen and expression of bone-related genes of MSC under the different media were analyzed at fixed time points. Results MSC proliferation and the number of colony forming units were increased by ascorbic acid, whereas dexamethasone enhanced the proportion of ALP-positive CFU and was critical for mineral deposition. Runx-2 and type I collagen gene expression decreased along with additive-induced MSC differentiation, i.e. from C1 to C4, while ALP and osteocalcin were differently regulated. Conclusion Our findings support the role of different inducers on the sequential stages of MSC expansion and osteogenic differentiation in vitro, suggesting the addition of DEX following proliferation to ensure mineralization, as an index of in vivo osteogenic potency of human mesenchymal cells.

The design of composite scaffolds with slow degradation kinetics imposes the assessment of the time-course of degradation to predict the long-term in vitro behavior. In this work, the effect of hydroxyapatite (HA) particles on the hydrolytic degradation of poly epsilon-caprolactone composite scaffold was investigated. The study of accelerated degradation mechanisms in alkaline medium enabled analysing comparable degradation profiles at different times. The accurate qualitative and quantitative study of morphology by scanning electron microscopy supported by image analysis demonstrated only a negligible effect on the structural porosity, to be ascribed to the addition of micrometric HA as a filler. Moreover, by comparing the Raman spectra with thermal analysis(thermogravimetry and differential scanning calorimetry) the role of HA on the composite degradation mechanism was defined, by separately quantifying the contribution of HA particles in the bulk and on the surface, on the bone formation as a function of modifications induced in the pore morphology, as well as physical and chemical properties of the polymer matrix. Indeed, HA particles alter the poly epsilon-caprolactone crystallinity inducing a "shielding" effect of the polymer matrix. Meanwhile, the slight reduction of pore size as a function of the increasing HA content and the improvement of the effective hydrophilicity of the scaffolds also influence the degradation by faster mechanisms. Finally, it has been proven that the presence of HA enhances the scaffold bioactivity and human osteoblast cell response, remarking the active role of bioactive signals on the promotion of the surface mineralization and, as a consequence, on the cell-material interaction.

Background and purpose Ceramic-on-ceramic articulation is an attractive alternative to metal-on-polyethylene (PE) bearings, but little is known about the in vivo effects induced by dissemination of alumina wear debris in the periprosthetic tissues. We hypothesized that wear debris is not the main factor responsible for loosening and failure of the implant but that mechanical problems caused by incorrect surgical technique, prosthetic design, or trauma, may cause instability of the implants and result in production of wear debris. Patients and methods Clinical, radiographic, laboratory, and microbiological data from 30 consecutive patients with failed alumina-on-alumina arthroplasties, 19 with screwed socket and 11 with press-fit socket, were systematically collected and evaluated. Retrieved peri-implant tissues and prosthesis wear were also analyzed. Results and Interpretation Loosening was due to malpositioning, primary mechanical instability, trauma, or infection. Bone stock was generally preserved, even if screwed implants showed higher levels of osteolysis. Variable implant wear and tissue macrophage reaction were present but activation of giant cells/osteoclasts was not induced, and no correlation between histocytic reaction and the level of osteolysis was found. These findings indicate that, in contrast to the situation with metal-on-PE bearings, wear debris and occasional osteolysis were the effect rather than the cause of failure of ceramic-on-ceramic implants, and that press-fit socket fixation was the socket fixation design of preference.

The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(epsilon-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 microm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell-material interaction are simultaneously requested.

Current methods for the replacement of skeletal tissue involve the use of autografts, allografts and, recently, synthetic substitutes, which provide a proper amount of material to repair large bone defects. Engineered bone seems a promising approach, but a number of variables have to be set prior to any clinical application. In this study, four different poly caprolactone-based polymers (PCL) were prepared and tested in vitro using osteoblast-like Saos-2 cells. Differences among three-dimensional polymers include porosity, addition of hydroxyapatite (HA) particles, and treatment with simulated body fluid. Biochemical parameters to assess cell/material interactions include viability, growth, alkaline phosphatase release, and mineralization of osteoblastic cells seeded onto three-dimensional samples, while their morphology was observed using light microscopy and SEM. Preliminary results show that the polymers, though degrading in the medium, have a positive interaction with cells, as they support cell growth and functions. In the short-term culture (3-7 days) of Saos-2 on polymers, little differences were found among PCL samples, with the presence of HA moderately improving the number of cells onto the surfaces. In the long term (3-4 weeks), it was found that the HA-added polymers obtained the best colonization by cells, and more mineral formation was observed after coating with SBF. It can be concluded that PCL is a promising material for three-dimensional scaffold for bone formation, and the presence of bone-like components improves osteoblast activity.

Nineteen patients who had undergone hip revision surgery for aseptic loosening of joint prostheses were studied. Tissue samples were harvested at the interface between bone and implant, either at the stem or at the cotyle level. Immunohistochemistry was performed on tissue sections to detect nitric oxide synthase (NOS), the enzyme which enables the synthesis of nitric oxide (NO), a molecule which can activate bone resorption. Quantitative analysis of the positive cells and correlation with the presence of particulate wear debris and radiological data were performed. The authors observed a trend towards a moderate increase in positive cells due to inducible NOS in tissues containing particulate wear debris, especially of a plastic material. This increase, however, did not achieve statistical significance. On the contrary, there was a statistical correlation between iNOS (inducible NOS) and the severity of osteolysis around the prosthetic implant. Pharmacological control of the biosynthesis of NO may be considered in the prevention or treatment of loosening.

The expression of thrombomodulin after contact with CMW 1 bone cement extracts was studied in human umbilical vein endothelial cells. Cement extracts after 1 h and 7-day curing induced no significant variations in thrombomodulin antigen levels and in mRNA expression. Significant increase of thrombomodulin was observed when endothelial cells were treated with all-trans retinoic acid (ATRA). ATRA induced the increase of thrombomodulin also in cells incubated with cement extracts. These results suggest that CMW 1 bone cement does not impair the expression of thrombomodulin in endothelial cells.

The aim of the study was to evaluate some aspects of the immunocompatibility of 10 acrylic bone cements. Mononuclear cells harvested from healthy individuals were cultured with cement extracts which were tested to assess their effect on the viability of lymphocytes, unstimulated and phytohaemoagglutinin (PHA)-stimulated, activating resting lymphocytes, and changing the reactivity of PHA-stimulated lymphocytes. After 24 h the extracts did not increase the percentage of dead cells in unstimulated or PHA-stimulated lymphocytes. The early apoptotic events of culture were evaluated after 4 and 24 h in PHA-stimulated lymphocytes: at 4 h three cements, namely Zimmer-dough type, Palacos s R and CMW-1, increased significantly the percentage of apoptotic cells, while at 24 h no differences were found. Cement extracts did not activate the resting lymphocytes, whereas the response of the PHA-stimulated cells was significantly modified. All cements decreased the expression of the interleukin 2 receptor (CD25) and the lymphocyte proliferation, whereas only two materials (Zimmer-dough type, CMW 1) affected the expression of early activation antigen (CD69). These findings show that the products released from bone cement are not able, by themselves, to elicit a specific immune response; on the contrary they hamper the function of lymphocytes activated by an exogenous stimulus. r 2001 Elsevier Science Ltd. All rights reserved.

The purpose of this study was to investigate in vitro the apoptosis- and/or necrosis-inducing potential of polymethylmethacrylate (PMMA)-based bone cements for prosthetic surgery. Four bone cements widely used in orthopedics were tested as extracts onto osteoblast-like MG-63 cells and for comparison, HL-60 cells, which are remarkably sensitive to apoptotic stimuli. Neutral red uptake (NRU) was used to measure cell viability while Hoechst 33258 staining was used to detect DNA content. Apoptosis was characterized using a BrdU-based ELISA assay for DNA fragmentation and examined by fluorescence microscopy using acridine orange and propidium iodide staining of nuclei. The generation of reactive oxygen species (ROS), which could mediate apoptosis, was verified using dichlorofluorescein-diacetate (DCFH-DA) oxidation to DCF. After 24 h of challenge of the cells with the four cement extracts, the viability of either MG-63 or HL-60 cells was found to be unaltered, as recorded by NRU. Apoptotic cell death was induced by three cements in HL-60, whereas MG-63 cells were significantly affected by the four cements tested: the finding of DNA fragments both in the cytoplasm and supernatants of MG-63 after 24 h demonstrated that these cells underwent late-apoptosis secondary necrosis. Fluorescent staining of the nuclei confirmed the results obtained with the ELISA test. Oxygen free radicals were elicited by two cements in HL-60 cells, while MG-63 did not generate ROS in response to cements. This study helps to gain more insight into the mechanism of cell death induced by PMMA-based cements and suggests apoptosis of osteoblasts as a part of the tissue reaction around cemented prostheses.

Trace elements such as zinc (Zn) and selenium (Se) play an important role in maintaining the metabolic homeostasis in elderly people and the risk of deficiency seems to increase in proportion to the age. Zn and Se concentrations, as indices of the micronutrient status in healthy subjects over 90 years, are scarcely analyzed and could represent a model for studying the physiology of successful aging. Our aim was to investigate Zn and Se concentrations in the healthy persons over the age of 90 years. One hundred and fifty two subjects volunteered for the study. They were divided into two groups: 90 non-institutionalized nonagenarians/centenarians (91-110 years) (group A) and 62 elderly subjects (60-90 years) used for comparison (group B). Serum concentrations of Zn and Se were determined, respectively, by flame atomic absorption spectrophotometry (FAAS) and electrothermal atomic absorption spectrophotometry (ETAAS). The effect of age and sex on ion concentrations was investigated. Mean values+/-standard deviation of Zn and Se concentrations in the group A were 11.97+/-2.00 and 0.87+/-0.28 micromol/l, respectively. A significant decrease of Se and Zn values was demonstrated in group A, when compared with group B, in both males and females. However, 84.4% of the 'healthy' nonagenarians/centerians had both Zn and Se concentrations equal to or greater than the lowest values of the elderly group and only 3.3% of cases showed both Zn and Se deficiencies. Consequently, a prospective and follow-up evaluation of Zn and Se could be proposed as a good index for a correct monitoring of the micronutrient deficiencies, that could represent an early sign of disease.

In order to evaluate if carbon coated polyethylene terephthalate (C-PET) could favor inflammatory reactions, the expression of interleukin-6 (IL-6) by cultured human umbilical vein endothelial cells was tested in vitro. The cultures were put in contact with C-PET for 1, 24, 48 and 72 h. The same cells cultured on tissue culture-treated polystyrene without biomaterials were tested as the negative control; the same cells incubated with LPS were the positive control. The level of IL-6 in the conditioned medium was tested by enzyme immunoassay; the mRNA expression was evaluated by RT-PCR with specific primers. The cultures incubated with C-PET produced non significantly different amount of IL-6 compared to the negative control and did not induce the expression of IL-6 specific mRNA. LPS induced a significantly higher release of IL-6 in the medium and the expression of mRNA after 24, 48 and 72 h. We conclude that C-PET does not stimulate the synthesis of IL-6 and therefore does not favor inflammatory reaction through the release of this cytokine. © 2001 Kluwer Academic Publishers

Periprosthetic osteolysis is a major clinical problem in total hip and total knee arthroplasty and polymethylmethacrylate (PMMA) is a possible etiologic factor. Recently, increasing importance was ascribed to interleukin-6 (IL-6) as an agent favouring bone resorption. The aim of the present study was to investigate the role of bone cements on IL-6 production by MG63. The effect of four acrylic bone cements (Sulfix-60, CMW 1, CMW 2, and CMW 3) on the protein release and mRNA expression of IL-6 in osteoblast-like cell line MG63 was examined using IL-1beta (0.2 microg ml(-1)) as the positive control. The extracts in minimum essential medium (MEM) of the cements were tested, following 1-h and 7-day curing. CMW 1 and CMW 2 significantly increased the IL-6 release into the culture media (p < 0.01). The cells incubated with Sulfix-60 and CMW 3 produced no significantly different levels of IL-6 than the basal production. A positive correlation was found between the concentration of IL-6 and the contents of benzoylperoxide (p = 0.0003) and barium sulphate (p < 0.0001). MG63 expressed IL-6 mRNA constitutively, as demonstrated by the positivity of the negative controls too. We conclude that CMW 1 and CMW 2 increase the production of IL-6 in MG63 cells. The response to Sulfix-60 and CMW 3 is not significantly greater than the negative control.

The effect of three methacrylate-based cements used for the fixation of joint prostheses on tissue-factor production by human umbilical vein endothelial cells was evaluated in vitro. The extracts in the culture medium of the cements were tested after 1-h and 7-day curing. The endothelial cells were incubated with the cement extracts for 4 h, and then the tissue factor was determined in cell lysates with both the recalcification time and enzyme immuno assay. The cements did not induce significant production of tissue factor and, therefore, did not activate the extrinsic pathway of coagulation within the limits of the mechanism considered.

The compatibility of a methacrylate-based bone cement (CMW 1, DePuy International Ltd, England) used for the fixation of joint prostheses was evaluated on plasma, an erythrocyte suspension and cultured human endothelial cells. The extract of the cement was tested, following 1 hour and 7 days of curing. After the contact in vitro of the extract with plasma, activated partial thromboplastin time, antithrombin III, thrombin-antithrombin complexes and fibrin degradation products were assayed. Hemolytic activity was tested by adding the cement extracts to a suspension of erythrocytes. After 4 hours of incubation at 37 degrees C, the hemoglobin concentration was determined on the supernatants by the colorimetric method. The effect of the cement on tissue factor and thrombomodulin production was evaluated on human umbilical vein endothelial cell cultures. Tissue factor was determined in cell lysates by enzyme immunoassay, following 4 hours' incubation of cultures with the cement extract. Thrombomodulin was assayed in cell lysates by enzyme immuno assay, after 24 hours' incubation with the cement extract. The response to all trans-retinoic acid (ATRA) was tested. The cement caused no significant modifications of the coagulation tests, had no hemolytic activity, did not determine tissue factor production and did not modify thrombomodulin, compared to the negative control. The response to stimulation with ATRA was similar to that of the negative control. We conclude that the cement extract does not affect the plasmatic phase of coagulation, has no effect on erythrocytes, does not induce the expression of procoagulant activity by endothelial cells and does not impair their antithrombotic property, within the limits of the tests performed.

The aim of the study was to evaluate the sensitization to metals in patients with Co-Cr hip prosthesis. Peripheral blood mononuclear cells (PBMC) were collected from 14 healthy donors and three groups of patients: 10 candidates for primary total joint replacements, 11 patients with well-fixed implant and 13 patients with aseptic loosening of the hip prosthesis. PBMCs were cultured with the metal ions employed for implant manufacturing and the expression of CD69 activation antigen on CD3/T lymphocytes was detected by flow cytometry. Chromium extract increased significantly the expression of CD3/CD69 phenotype in patients with loosening of hip prosthesis. The chromium-induced 'activation index' was higher in patients with loosening of hip prosthesis than in healthy donors and in pre-implant patients. The cobalt-stimulated PBMC of patients with either well-fixed or loosened prosthesis had an 'activation index' significantly higher than healthy donors. The activation index values were used to graduate the PBMC-response as 'normal' (or = 0.9 and2), 'low' (0.9) and 'high' (or = 2): an high-activation index was observed only in chromium-exposed PBMC of patients with prosthesis. Our data show that chromium released from orthopedic implants could be responsible for the lymphocyte sensitization and flow cytometry is an easy and reliable method for monitoring the hypersensitivity state in patients with metal prostheses. Activated lymphocytes in the peri-implant tissue are likely to elicit a localized immune response and contribute to maintain the inflammatory process evolving in the implant failure.

The authors evaluated the ability of bone cement to modify the pro"le of pro-in#ammatory cytokines secreted by the immune cells. Peripheral blood mononuclear cells (PBMC) collected from healthy individuals were cultured with cement extracts and tested to assess the release of IL-1b, TNFa, GM-CSF and IL-6 in both unstimulated and PHA-stimulated PBMC. The cytokine release of unstimulated PBMC was very poor, and in particular the IL-1b was undetectable: the addition of cement extract increased both TNFa and GM-CSF release and decreased IL-6, sometimes signi"cantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1b and IL-6 release, while both the mean concentration and the index of release of TNFa and GM-CSF were changeable. In conclusion our results showed that leachable components of some bone cements can induce in vitro the release of pro-in#ammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These "ndings allowed us to identify materials endowed with the highest in#ammatory power. ( 2000 Elsevier Science Ltd. All rights reserved.

The authors examined 54 biopsies taken from the tissue surrounding loosened hip joint prostheses. In situ apoptotic cell identification was performed by the detection of single- and double-stranded DNA breaks that occurred in the early stages of apoptosis. Both types of breaks can be revealed by labeling the free 3′-OH termini with modified nucleotides (fluoresceine-dUTP) in an enzymatic reaction catalyzed by terminal deoxynucleotidyl transferase (TdT). Results were correlated with the presence of wear debris in the tissue and with the use of bone cement for prosthesis fixation. Apoptotic cells were present in a higher percentage in tissue sections where metal particles were present (24% apoptotic cells) if compared to areas where no wear (6%), or plastic wear (2.8%) or ceramic wear (1.5%) was observed. Apoptosis is neither related to bone cement, nor to the time it takes for the implant to fail. Cell death by apoptosis may be important in implants which release metal ions by corrosion or wear and may have been underestimated up to now, as it is a `clean’ way of cell death, leading to limited damage in the surrounding tissues.

The quantification of sister chromatid exchange (SCE) during mitosis is a useful index for evaluating genotoxic effects in subjects occupationally or incidentally exposed to potentially toxic substances. The authors investigated the hypothesis that ions released by corrosion from prosthetic components of fracture fixation devices are associated with change in SCE incidence. In the present study, ten patients with implants were examined, and fifteen subjects with no implants were used as controls. SCE and high frequency cell (HFC) numbers were evaluated in circulating lymphocytes. In addition, nickel (Ni) and chromium (Cr) ion values in the serum were measured because, after iron, these metals are major components of stainless steel. A significant increase in SCE numbers was observed in patients compared to the control population (4.9 ± 1.3 vs. 3.5 ± 1.4). Ni concentration was 1.71 ± 1.49 ng/mL in patients and 0.72 ± 0.52 ng/mL in control subjects; Cr concentration was, respectively, 1.01 ± 0.77 ng/mL and 0.19 ± 0.27 ng/mL. The increase of serum Cr and Ni was statistically significant. No correlation was found between the increased Cr concentrations and SCE number while Cr ion levels were found to be significantly correlated to HFC. An inverse correlation between Ni level and SCE numbers was observed. Our findings suggest that Cr release by stainless steel implants could have a genotoxic effect; thus it would be useful to carefully monitor implanted subjects with regard to serum ion dosage, SCE analysis, and HFC evaluation. In any case, it would be appropriate to remove the implant when fracture fixation is reached. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 50, 21–26, 2000.

The biocompatibility of two cyanoacrylate surgical glues (Glubran and Glubran 2), supplied by General Enterprise Marketing, Viareggio, Lucca, Italy, was tested through cytotoxicity and blood compatibility tests and the evaluation of antimicrobial activity. Cytotoxicity and blood compatibility tests were performed on the polymerized glues. Using the neutral red uptake test, the extracts from Glubran and Glubran 2 after polymerization were non-toxic to L929 cells only when diluted 1: 10 with culture medium. Glubran and Glubran 2 induced a significant decrease of activated partial thromboplastin time (APTT), which is favourable with regard to the desired haemostasis. The APTT shortening determines a haemostatic effect and therefore contribute to the tissue adhesion induced by the glues. Otherwise, no significant variation of prothrombin activity, fibrinogen, platelet number, total and differential leukocyte count was induced by the glues, which, in addition, did not show haemolytic effect. There was no difference between Glubran and Glubran 2 regarding haemocompatibility. The antimicrobial ability of the unpolymerized glues was tested onto Bacillus subtilis var. niger for 3 weeks: neither Glubran nor Glubran 2 were found effective in this respect. In conclusion, we can assume that cytotoxicity was severe with the undiluted glues, but was acceptable when glues were diluted. On the contrary, blood compatibility was acceptable for the intended use of the glues. No difference was found between Glubran and Glubran 2 after polymerization.

The ability of bone cements to modify the apoptotic program in activated immune cells and the mechanisms by which they act were evaluated. Mononuclear cells were collected from healthy individuals, cultured for 4 and 24 h with phytohemoagglutinina-P and cement extracts and then tested to assess: (a) cell viability; (b) early apoptotic events, by Annexin V/propidium iodide staining; and (c) the expression of pro- (p53, c-myc, ICE) and anti-apoptotic (bcl-2) genes. After 4 h three cements were able to increase significantly the percentage of apoptotic cells, while after 24 h no differences were found. The proportion of dead cells was not significantly changed at either culture time. The simultaneous expression of both pro-apoptotic (ICE, c-myc, p53) and antiapoptotic genes (bcl-2) was investigated only with regard to the materials which induced significant changes in apoptosis: two cements induced the p53 expression, while the third down-regulated bcl-2. As apoptosis regulates the balance of immune response, the authors recommend that the interaction between materials and immune cells should be assessed, so that the use of pro-apoptotic materials may be avoided in patients with immune defects.

In this study twenty-two patients who had revision surgery for aseptic loosening of joint prostheses were examined. The concentration in serum of soluble products of corrosion from the implant, that is, chromium (Cr), cobalt (Co), and nickel (Ni) ions, and the number of white blood cells (leucocytes, myeloid cells, lymphocyte subpopulations) were measured. Twenty patients with no implants were used as controls. The patients who had revision surgery showed normal Ni concentration whereas by statistical analysis that same patient group was shown to have serum Cr and Co levels significantly higher than those of the control. By flow cytometry, a significant decrease of leucocytes, myeloid cells, lymphocytes, and CD16 populations as found in patients versus controls whereas CD3, CD4, CD8, and CD20 positive cells were decreased, but not significantly. In the arthroplasty patients the Cr concentrations were inversely correlated with some of the immunologic parameters while no significant correlation was found between Co levels and decreased lymphocyte subpopulations. Only in revision surgery patients with high Cr concentrations did we find a significant decrease of lymphocytes, namely of CD4 and CD16 positive cells; revision surgery patients with normal Cr concentrations did not show a significant decrease of lymphocyte subpopulations. These data suggest that the presence of metal ions, especially chromium, released from prosthesis components could be associated with changes of lymphocyte subpopulations in patients with loosening of joint prostheses.

The aim of the present study was to examine: (1) the effects of nicotine on human gingival fibroblasts (HGF); (2) differences between smokers (or = 10 cigarettes/day at least for 5 years) and non-smokers; (3) differences between patients of different age. HGF were obtained, through biopsies during periodontal surgical procedures, from 15 patients which were divided in 4 groups: 4 patients, smokers agedor = 25 years; 4 patients, non-smokers agedor = 25 years; 3 patients, smokers agedor = 40 years; 4 patients, non-smokers agedor = 40 years. Nicotine has been tested in 3 different concentrations: 6 microg/ml; 60 microg/ml; 600 microg/ml. To assess cells viability, the neutral red (NR) test was used; to evaluate cell proliferation, the Hoechst test was employed. After 48 h of nicotine exposure, it was found that 600 microg/ml nicotine was strongly cytotoxic to HGF of all groups, with a significant reduction of both proliferation and viability of cells versus control. Comparison between groups of the same age: when comparing untreated HGF (i.e., control values) of smokersor = 25 years versus non-smokersor = 25 years, cell proliferation, but not viability, was found to be increased in smokers. Both viability and proliferation of control cells of smokersor = 40 years were increased versus non-smokersor = 40 years. HGF of non-smokersor = 25 years, when exposed to nicotine 600 microg/ml, have less viability and proliferation than HGF of smokers of the same age. Comparison between groups of different age: In the smoker group, untreated HGF (i.e., control values) had similar viability and proliferation, irrespective of age, but nicotine 600 microg/ml kills more HGF in smokersor = 25 years than in smokersor = 40 years. In non-smokers, untreated HGFor = 25 years replicate less, but are not less viable than HGFor = 40 years. When challenged with nicotine 600 microg/ml, HGFor = 25 years were less viable than HGFor = 40 years. From this study, it appears that the smoking history and the patient age could be relevant for final evaluation of the results, since HGF from smokers are less sensitive to nicotine than HGF from non-smokers, and cells from older donors are more resistant to nicotine than cells from younger donors.

In testing for the cytotoxicity of medical devices, crucial parameters are the type of cells, the duration of the exposure, the physical form of the device and the method of evaluation. Using cell cultures the changes of cell functions induced by artificial materials may be evaluated. In screening tests the effect of biomaterials on functions common to all cell types is investigated through both continuous lines and primary cultures. In supplementary tests the effect of biomaterials on specific functions is studied, employing cells of the same type of those which will be in contact with the device in vivo. Osteoblasts, either from primary culture or an established line, are used to study the interaction with materials for bone implants. We evaluated whether dental cements affect the replication cycle of osteoblast-like cells MG63. The S phase was inhibited by the cements with a phenolic group, even though to a different extent. Mononuclear cell cultures are used to investigate the immune response, which is important in the host reaction to implant. We investigated whether the chromium ions released in a biological fluid may induce PBMC to produce cytokines involved in bone resorption. Chromium extract did not impair TNFα release significantly, but inhibited IL-6 release significantly in stimulated PBMC. Endothelial cell cultures are useful to the evaluation of materials for vascular grafts. We studied the influence of some types of Dacron on endothelial functions. Collagen-coated Dacron decreased significantly the production of 6-keto-prostaglandin F1α. Knitted Dacron caused a significant increase of tissue factor, a significant reduction in PECAM-1 and a significant increase in ELAM-1.

The aim of this study was the evaluation of the release of bone-resorbing cytokines in peripheral blood mononuclear cells (PBMC) of patients with aseptic loosening of Co–Cr hip prostheses. TNF-α, IL-6, and GM-CSF were measured in both unstimulated and PHA-stimulated PBMC, and in PBMC cultured in the presence of chromium and cobalt extracts. Blood samples from healthy donors were used as controls. Serum samples of both patients and healthy donors were tested to determine the concentration of metal ions. The proportion of lymphocyte, monocyte and lympocyte subpopulation in cultured PBMC did not differ in patients and the control group. In unstimulated PBMC the release of TNF was significantly higher in patients than in the control group, while IL-6 was significantly decreased and no change was observed for GM-CSF. When the PBMC were challenged with chromium extract, all the ‘index of cytokine release’ resulted higher in patients than in the control group; cobalt extract increased both the TNF and GM-CSF index, but not the index of IL-6 release. Metal concentrations in serum from patients were significantly increased and correlated with the TNF release in PBMC stimulated with both metal extract. Our results suggest that a CoCr-implant releases a large amount of metal ions which could mediate the priming or the renewal of a cell-mediated hypersensitivity reaction. The prevalence of circulating lymphocytes responsible for the delayed hypersensitivity, namely Th1, would justify both the significant increase of TNF and the significant decrease of IL6 in unstimulated PBMC of patients, as well as the significant increase of the ‘index of cytokine release’ after the challenge with metal ions.

Tobacco smoke is considered to be a major risk factor in the development of cardiac diseases and lung cancer. It has also been shown that periodontitis is more prevalent and more severe in smokers than in non-smokers. Nicotine, the major pyridine alkaloid in tobacco, has been shown to participate in periodontal disease, exerting both local and systemic effects. In the present study, the effects of nicotine (6μg/ml, 60μg/ml and 600μg/ml) on human gingival fibroblasts (HGF) were assessed by using various exposure protocols. The responses of HGF cultures obtained from smokers and non-smokers were compared to those found when using a continuous cell line (L-929). Neutral red uptake (NRU) and the measurement of DNA content with bis-benzimide dye were used to assess cell viability and cell number, respectively. NRU was the most sensitive technique for the detection of cytotoxic effects. L-929 cells were found to be affected by nicotine in the NRU assay, with a strong cytotoxic effect with 600μg/ml nicotine, and a “response” with 60μg/ml nicotine when prolonged or double challenge was applied. Non-smoker HGF and smoker HGF reacted to nicotine in different ways, depending on the concentrations and the exposure times used, but had identical reactions following double exposure. With the Hoechst DNA assay, 600μg/ml nicotine was found to affect the growth of non-smoker HGF after long or repeated exposure, while smoker HGF were affected only by repeated exposure; growth of L-929 cells was not affected. It was concluded that HGF from smokers are able to sustain higher concentrations of nicotine without adverse effects than are non-smoker HGF and L-929 cells. If this occurs in vivo, nicotine would not be considered to be a major toxicant to HGF in smokers.

Silicones for dental impression largely are used to record the geometry of hard and soft dental tissues. They are considered to be medical devices, and the assessment of cytotoxicity is a necessary step in the evaluation of their biocompatibility. Extracts of six addition-type and six condensation-type silicones have been tested with L929 cells according to the ISO 10993-Part 5 standard. The cytotoxicity was evaluated by three different methods: neutral red uptake, propidium iodide (PI) staining, and amido black staining. According to the selected specific assay, contact between cells and material extracts was maintained for 24 h in the first series of experiments; then, considering that in vivo application of these materials is restricted to a few minutes, additional experiments were performed after 1 h of cell/extract contact. Analysis of the results showed that the addition-type silicones are nontoxic even when tested after prolonged exposure of the cells to the materials while the condensation-type silicones were cytotoxic at 24 h of incubation. Nevertheless, harm to the patient actually could be negligible, considering its very short time of exposure in vivo. This is supported by our finding that most are not toxic after 1 h. We suggest that the experimental conditions of cytotoxicity testing have to be relevant to the in vivo situation; accordingly, the time of exposure should be designed carefully.