Change in FGF-2 circulating levels after arterial embolization in patients with bone metastases.

Arterial embolization, aimed at the mechanical occlusion of tumor-feeding vessels, represents a satisfactory palliative therapy for bone metastases. In this study, we evaluated if the circulating levels of three factors related to the metastatic process change in response to embolization. Seven patients who underwent embolization of a single skeletal metastasis from carcinomas were analyzed prospectively. Circulating levels of Vascular Endothelial Growth Factor A (VEGF-A), Fibroblast Growth Factor 2 (FGF-2), and Tartrate-Resistant Acid Phosphatase-5b Isoform (TRACP5b) were evaluated before and after embolization at 1, 3, and 6 months. According to morphological and clinical evaluations, all the embolizations were successful. VEGF-A and TRACP5b did not show significant changes after the treatment. On the contrary, FGF-2 signifi- cantly decreased 1 month after the treatment. FGF-2 appears as a promising candidate for monitoring the efficacy of emboli- zation in patients with osteolytic metastases.

Bone regeneration and stem cells.

This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Orthopaedic research in italy: state of the art.

The most significant results in experimental and clinical orthopaedic research in Italy within the last three years have been primarily in major congenital diseases, bone tumors, regenerative medicine, joint replacements, spine, tendons and ligaments. The data presented in the following discussion is comparable with leading international results, highlighting Italian orthopaedic research excellemce as well as its shortcomings.

Interferon-alpha inhibits in vitro osteoclast differentiation and renal cell carcinoma-induced angiogenesis.

Bone is a common site of osteolytic and richly vascularized metastases of renal cell carcinoma (RCC) and Interferon (IFN)-alpha based therapies have been considered for the treatment of patients affected by this disease. The effects of IFN-alpha on metastatic RCC patients have been related to its immunomodulatory, and cytotoxic activity on tumor cells, but there could be an effect also on tumor induced osteoclast differentiation and bone angiogenesis. When osteoclasts obtained from human peripheral blood mononuclear cells, cultured in the presence of receptor activator of nuclear factor-kappaB (RANKL) and macrophage-colony stimulating factor (M-CSF), were treated with IFN-alpha, the expression of bone tartrate resistant acid phosphatase (TRACP) type 5b was reduced, as well as calcium-phosphate resorption activity and expression of pro-osteoclatic transcription factor c-Fos. IFN-alpha modulation of angiogenesis was studied by analysis of proliferation, survival, and migration of a bone endothelial cell line (BBE), and by the analysis of pro-angiogenic factor expression in RCC cell lines. IFN-alpha inhibited bone endothelial cell proliferation and the expression of FGF-2, while the vascular endothelial growth (VEGF) did not show any significant variation. Moreover, IFN-alpha inhibited the migration induced by the RCC through the impairment of fibroblast growth factor-2 (FGF-2) secretion. These data demonstrate multiple activities of IFN-alpha on renal cancer-induced bone disease, in addition to its recognized role as a cytotoxic and immunomodulatory agent, because they indicate its ability to reduce bone resorption and to impair tumor-associated angiogenesis, and they also suggest the use of IFN-alpha to treat skeletal metastases of other carcinomas.

In vivo study on the healing of bone defects treated with bone marrow stromal cells, platelet-rich plasma, and freeze-dried bone allografts, alone and in combination.

The repair of confined trabecular bone defects in rabbits treated by autologous bone marrow stromal cells (BMSC), platelet-rich plasma (PRP), freeze-dried bone allografts (FDBA) alone and in combination (BMSC + PRP; FDBA + BMSC; FDBA + PRP; FDBA + PRP + BMSC) was compared. A critical size defect was created in the distal part of the femurs of 48 adult rabbits. Histology and histomorphometry were used in the evaluation of healing at 2, 4, and 12 weeks after surgery. The healing rate (%) was calculated by measuring the residual bone defect area. Architecture of the newly formed bone was compared with that of bone at the same distal femur area of healthy rabbits. The defect healing rate was higher in PRP + BMSC, FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC treatments, while lower values were achieved with PRP treatment at all experimental times. The highest bone-healing rate at 2 weeks was achieved with FDBA + PRP + BMSC treatment, which resulted significantly different from PRP (p < 0.05) and BMSC (p < 0.05) treatments. At 4 weeks, the bone-healing rate increased except for PRP treatment. Finally, the bone-healing rate of FDBA + PRP, FDBA + BMSC, and FDBA + PRP + BMSC was significantly higher than that of PRP at 12 weeks (p < 0.05). At 12 weeks, significant differences still existed between PRP, BMSC, and FDBA groups and normal bone (p < 0.05). These results showed that the combination of FDBA, BMSC and PRP permitted an acceleration in bone healing and bone remodeling processes.

Differences in ion release after ceramic-on-ceramic and metal-on-metal total hip replacement. Medium-term follow-up.

Modern metal-on-metal bearings produce less wear debris and osteolysis, but have the potential adverse effect of release of ions. Improved ceramic-on-ceramic bearings have the lowest wear of all, but the corrosion process has not been analysed. Our aim was to measure the serum ion release (ng/ml) in 23 patients having stable hip prostheses with a ceramic-on-ceramic coupling (group A) and to compare it with the release in 42 patients with a metal-on-metal bearing (group B) in the medium term. Reference values were obtained from a population of 47 healthy subjects (group C). The concentrations of chromium, cobalt, aluminium and titanium were measured. There was a significant increase of cobalt, chromium and aluminium levels (p < 0.05) in group B compared with groups A and C. Group A did not differ significantly from the control group. Despite the apparent advantage of a metal-on-metal coupling, especially in younger patients with a long life expectancy, a major concern arises regarding the extent and duration of ion exposure. For this reason, the low corrosion level in a ceramic-on-ceramic coupling could be advantageous.

Evaluation of bone healing enhancement by lyophilized bone grafts supplemented with platelet gel: a standardized methodology in patients with tibial osteotomy for genu varus.

Orthopedic practice may be adversely affected by an inadequate bone repair that might compromise the success of surgery. In recent years, new approaches have been sought to improve bone healing by accelerating the rate of new bone formation and the maturation of the matrix. There is currently great interest in procedures involving the use of platelet gel (PG) to improve tissue healing, with satisfactory results both in vitro and in maxillofacial surgery. Otherwise, to our knowledge, only a preliminary clinical study was undertaken in the orthopedic field [Kitoh et al., Bone 2004;35:892-898] and the efficacy of PG is still controversial. Our paper focuses on the effect on bone regeneration by adding PG to lyophilized bone chips used for orthopedic applications. The clinical model and the laboratory methodology were standardized. As a clinical model, we employed the first series of patients of a randomized case-control study undergoing high tibial osteotomy (HTO) for genu varus. Ten subjects were enrolled: in 5 patients lyophilized bone chips supplemented with PG were inserted during tibial osteotomy (group A); 5 patients were used as a control (group B) and lyophilized bone chips without gel were applied. Forty-five days after surgery, computed tomography scan guided biopsies of grafted areas were obtained and the bone maturation was evaluated by a standardized methodology: the osteogenic and angiogenic processes were semi-quantitatively characterized by using histomorphometry, and the mineral component of the lyophilized and host bone was analyzed by using X-ray diffraction technique with sample microfocusing and microradiography. Lyophilized bone with PG seems to accelerate the healing process, as shown by new vessel formation and deposition of newly formed bone, with no evidence of inflammatory cell infiltrate, when compared with lyophilized bone without gel. On the contrary, lyophilized bone undergo a resorption process, and a fibrous tissue often fills the spaces between chips. A histiocytic/giant-cell reaction is sometimes present. Otherwise, no differences have been found concerning microstructure. Our findings show the reliability of the methodology used to monitor early bone repair. The completion of the study and the evaluation of the ultimate clinical outcome are necessary in order to verify PG in vivo effects in orthopedic surgery.

Effects of activated platelet concentrates on human primary cultures of fibroblasts and osteoblasts.

BACKGROUND: Platelet alpha granules contain growth factors released into the surrounding environment during activation. This property has been used in clinical medicine to accelerate the repair process by activating in vitro autologous platelets with thrombin and has also been proposed to promote the proliferation of bone cells. The aim of this research was to assess the effect of platelet concentrates activated with thrombin on human gingival fibroblasts and human osteoblasts from trabecular bone. METHODS: Platelet concentrates, activated with bovine thrombin, were added to the cells in serum-free medium. The cultures were assessed for proliferation by vital stain and cell count after 72-hour incubation. Alkaline phosphatase activity was tested after 72-hour incubation on the osteoblast lysates by a colorimetric assay. After 21 days the formation of mineral nodules was tested in the osteoblast cultures by alizarin red staining. The effects of the activated platelet concentrates (APC) were compared with the serum-free medium (SF), or with platelet-poor plasma added medium (PPP). RESULTS: The fibroblast growth in the presence of APC was higher, though not significantly, than SF. APC resulted in a nonsignificant decrease in proliferation and alkaline phosphatase expression in osteoblasts, compared both to serum free medium, and PPP. Mineralization was only modestly increased after incubation with APC in comparison with serum-free medium. CONCLUSIONS: There were no statistical differences in fibroblast proliferation, or in osteoblast growth and functions between serum-free conditions and the platelet gel treatment. Therefore, neither fibroblast proliferation nor osteoblast growth and functions were affected by the activated platelet concentrates in vitro.

Systemic cross-linked N-terminal telopeptide and procollagen I C-terminal extension peptide as markers of bone turnover after total hip arthroplasty.

There is no diagnostic, non-invasive method for the early detection of loosening after total hip arthroplasty. In a pilot study, we have analysed two serum markers of bone remodelling, procollagen I C-terminal extension peptide (PICP) and cross-linked N-terminal telopeptide (NTx), as well as the diagnostic performance of NTx for the assessment of osteolysis. We recruited 21 patients with loosening (group I), 18 with a well-fixed prosthesis (group II) and 17 at the time of primary arthroplasty for osteoarthritis (OA) (group III). Internal normal reference ranges were obtained from 30 healthy subjects (group IV). The serum PICP level was found to be significantly lower in patients with OA and those with loosening, when compared with those with stable implants, while the NTx level was significantly increased only in the group with loosening, suggesting that collagen degradation depended on the altered bone turnover induced by the implant. This hypothesis was reinforced by the finding that the values in the pre-surgery patients and stable subjects were comparable with the reference range of younger healthy subjects.A high specificity and positive predictive value for NTx provided good diagnostic evidence of agreement between the test and the clinical and radiological evaluations. The NTx level could be used to indicate stability of the implant. However, further prospective, larger studies are necessary.

Serum concentrations of zinc and selenium in elderly people: results in healthy nonagenarians/centenarians.

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 vitro evaluation of cell/biomaterial interaction by MTT assay.

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

Platelet release of transforming growth factor-beta and beta-thromboglobulin after in vitro contact with acrylic bone cements.

Three methacrylate-based bone cements used for the fixation of joint prostheses were evaluated: Sulfix-60 (Sulzer Orthopedic Inc., Baar, Switzerland). CMW1 (DePuy International Ltd., England). and CMW2 (DePuy International Ltd., England). The cements after polymerization were put in contact in vitro with platelet-rich plasma. Plasma, in contact only with siliconized glass, was used as a negative control. After contact, platelet number. beta-thromboglobulin (beta-TG), and transforming growth factor-beta1 (TGF-beta1) were determined. The Student's paired t test showed that the ccments induced no significant modifications of platelet number. CMWI and Sulfix-60 determined a significant increase in beta-TG compared with the negative control. All cements determined a significant increase in TGF-beta1. Significant differences were also seen in the levels of beta-TG and TGF-beta1 between cements with a content of benzoyl peroxide < 1 (Sulfix-60) and those with a content > 1 (CMW1 and CMW2). The cement with zirconium dioxide (Sulfix-60) produced higher levels of beta-TG and TGF-beta1, compared to those with barium sulphate (CMW1 and CMW2). In conclusion, all the cements induced the secretion of TGF-beta1 CMW1 and Sulfix-60 determined also a significant release of beta-TG. Platelet activation induced by the cements from one side could contribute to the pathogenesis of deep venous thrombosis, that often occurs after prosthetic implant and is caused also by other factors, including surgical trauma and venous stasis. From the other side, activated platelets can release growth factors favoring bone formation.

Numerical and functional modifications in platelets induced by polyester coated by a hydrophilic polymer.

We evaluated the alterations in number, functionality and release reaction of the platelets contained in plasma, filtered through a polyester filter and coated with a hydrophilic polymer. The alterations in number were examined by counting before and after filtration. The morphological modifications were studied by determining the mean platelet volume. The functional alterations were analysed by a platelet aggregation test, induced by ADP and collagen. The presence of products from the release reaction in filtered plasma was studied using radioimmunoassays of beta-thromboglobulin, platelet factor 4 and thromboxane B2. The results obtained showed that the filtration of plasma through the material did not determine a significant platelet adhesion, did not alter the volume or the functionality of the platelets and induced no release reaction.

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

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

Seven surgical silicones retain Staphylococcus aureus differently in vitro.

In an in vitro study, we have quantitatively evaluated the capability of seven different types of silicone to retain a Staphylococcus aureus strain, isolated from a surgical wound. All the silicone specimens were taken from prostheses already used in plastic or ophthalmological surgery. Two polymers were used as controls: polystyrene, because of its known capability to favour in vitro bacterial recovery, and nylon, for its bacterial repellence. The results show that all silicones are suitable substrata for Staphylococcus aureus. However, there are some differences among silicone types. The amounts of bacteria retained from silicone oils are greater than or equal to those obtained from the positive control material.

Disposable contact lenses and bacterial adhesion. In vitro comparison between ionic/high-water-content and non-ionic/low-water-content lenses.

An in vitro quantitative study of the adhesion of a Staphylococcus aureus strain to two types of disposable contact lenses has been carried out. The first type was an ionic/high-water-content (I-HWC) lens (42% Etafilcon A, 58% water) and the second was a non-ionic/low-water-content (Nl-LWC) lens (61.4% poly(2-hydroxyethyl methacrylate), 38.6% water). Adhesion to the two lens types was evaluated both in basic conditions and after treatment with lysozyme. The results showed that I-HWC lenses are more prone to Staphylococcus aureus adhesion than NI-LWC lenses, both untreated (+15.4%) and treated with lysozyme (+20.5%). Lysozyme increased bacterial adhesion by 30.5% on the lenses with lower water content, and by 36.3% on those with higher water content.

In vitro biocompatibility of a polyurethane catheter after deposition of fluorinated film.

The in vitro biocompatibility of an experimental surface-treated polyurethane was compared with an untreated polyurethane already used for intravascular catheters. The experimental surface was coated with a fluorinated film using a glow discharge treatment. Neither of the catheters was cytotoxic for L929 murine fibroblasts, caused platelet adhesion or release reaction, or changed the mean platelet volume. The surface-treated polyurethane, however, caused a higher adhesion of Staphylococcus aureus than did the untreated one. Therefore, using in vitro testing, it has been ascertained that the examined material, though not being cytotoxic and not modifying platelet behaviour, could favour bacterial adherence.

In vitro cytokine production by mononuclear cells exposed to bone cement extracts.

The authors evaluated the ability of bone cement to modify the profile of pro-inflammatory 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-1beta, TNFalpha, 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-1beta was undetectable: the addition of cement extract increased both TNFalpha and GM-CSF release and decreased IL-6, sometimes significantly. The most recurrent observation in PHA-stimulated PBMCs exposed to bone cement extract was the increase in both IL-1beta and IL-6 release, while both the mean concentration and the index of release of TNFalpha 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-inflammatory cytokines which are known to be involved in the bone resorption associated with aseptic loosening of hip prostheses. These findings allowed us to identify materials endowed with the highest inflammatory power.

Cytotoxicity, blood compatibility and antimicrobial activity of two cyanoacrylate glues for surgical use.

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.

Toxicity of cyanoacrylates in vitro using extract dilution assay on cell cultures.

Comparative cytotoxicity testing of four cyanoacrylate adhesives suggested for orthopaedic applications was performed. These substances were placed in complete culture medium with serum and the resulting extraction fluids were tested on L 929 cells and human lymphocytes. Testing procedures include cell morphology assessment using light microscopy and vital dyes, cell counting using a computer-assisted image analysis system, cell growth measurement using total protein content assay and cell viability assessment using the MTT method. Quantitation of the toxicity of the degradation products released by cyanoacrylates in the extracts was achieved and differences in the cytopathic effect related to the chemical composition of the cyanoacrylates were found. A toxicity rating of the assayed cyanoacrylate adhesives was obtained as follows (in order of increasing toxicity): BCA < xCA < ECAg < ECAl.