Summary

Prosthetic UHMWPE added with vitamin E and crosslinked UHMWPE are able to decrease significantly the adhesion of various bacterial and fungal strains limiting biomaterial associated infection and consequent implant failure.

Giant cell tumor (GCT) of bone is an aggressive tumor with high rate of recurrence. Bad prognosis factors were inquired, without a definite identification: type of treatment, soft tissue invasion, high proliferation rate at histology, pathologic fracture.

From January 2000 to February 2008, 38 patients affected by GCT were treated in a regional reference centre, 17 male, mean age 32 (range 16–69, median 29); one patient had 2 localizations (tarsal bone and proximal tibia); 3 were recurrences previously treated in other hospitals. Seven cases were in upper limb, 1 case in the sacrum, 30 in lower limb (20 around the knee); fracture at presentation was present in 6 cases; bone aneurismal cyst (ABC) was associated in 4 cases. Five cases in stage 3 were treated by bone resection followed in 4 cases by allograft and/or prosthesis (no reconstruction in 1 proximal fibula excision); 33 cases were treated by curettage, local chemical (phenole) and mechanical adjuvants (burring), filling with bone grafts in 13 cases, cement in 8 cases, cement and allografts in subchondral area in 11 cases. The sacral lesion was only curetted.

Seven patients developed a local recurrence, in 2 patients twice, for a total of 9 recurrences (19% of treatments). Recurrences occurred in 2 proximal tibia, in 2 distal femurs, in 1 proximal femur, in 1 distal radius and in 1 proximal fibula. The first treatment was bone grafts in 3 cases (23% of recurrence), bone cement and grafts in 2 cases (18% of recurrence), cement in 1 case (12% of recurrence), resection in the proximal fibula with severe soft tissue invasion. Two patients with associated ABC developed a recurrence and two with fracture at presentation.

In this study, increased rate of recurrences occurred with pathologic fracture at presentation, soft tissue invasion and ABC association.

Aim: Wear of the UHMWPE component is responsible for many TJR failures. It is now well known that oxidation of UHMWPE, induced by radiation sterilisation in the presence of oxygen, dramatically increases the wear rate. Vitamin E is already used as biocompatible antioxidant in a number of applications, thus it has been suggested as a suitable stabiliser for orthopaedic UHMWPE. This work investigates the role of the vitamin E on the oxidation process of a gamma-sterilized material.

Methods: GUR 1050 (Meditech, Fort Wayne, IN) resins were blended with 0.05 wt% to 0.5wt% vitamin E and compression molded into billets. Gamma irradiation to 30 and 100 kGy was carried out in an industrial plant. This material was then sectioned using a microtome into 180 micron-thick specimens in preparation for the accelerated ageing.

The samples were treated in an air circulating oven at 90°C. Every 20 hours they were analyzed with FTIR and the carbonyl concentration was recorded.

The CL-imaging measurements were run at 180°C under oxygen in a Differential Scanning Calorimetry (DSC) coupled to a CCD camera. The Oxidation Induction Time (OIT) has been measured as the starting time of oxidation, extrapolated from the CL curve in the function of time.

Results: The results obtained from both techniques clearly evidence that 0.05 wt % of Vitamin E it is enough to stabilize even the material irradiated to the highest dose (100 kGy). Irradiation of UHMWPE leads to the formation of alkyl radicals. When irradiation is carried out in air, macroalkyl radicals can react with oxygen to form hydroperoxides, which in turn decompose giving other oxidation species, mainly ketones and acids. The overall result of irradiation in air is the formation of oxidation products and a decrease in the molecular mass, due to chain fragmentation.

The ability of α-tocopherol as a free radicals scavenger during gamma irradiation prevents the reaction of polymer radicals with oxygen. While performing this role, α-tocopherol is consumed and transformed into a variety of by-products. Nevertheless, higher OIT for the doped and irradiated specimens compared to the control (0% Vit.E, 0 kGy) suggest even a stabilising effect of these by-products.

Conclusions: Vitamin E has been shown to be highly efficient against radiation-induced oxidation and therefore it should be recommended as biocompatible stabilizer for orthopaedic UHMWPE, in order to preserve good mechanical properties.

Background: Chromosomal translocation are frequently observed in leukemias and sarcomas; these translocations break specific genes in the involved chromosomes and create novel chimeric genes that encode a fusion protein. Advances in these techniques have increased knowledge of the genes involved in tumoral development; molecular techniques have enabled more precise diagnosis as well as identification of new prognostic factors.

Aims: To explore the use of Reverse-Transcriptase Poly-merase Chain Reaction (RT-PCR) assay for detecting fusion transcripts in a series of Soft Tissue Sarcomas (STS) and compare the results with histopathologic diagnosis.

Material and methods: We studied 80 biopsies performed at Orthopaedic Oncology and Reconstructive Surgery Department, CTO-CFR-M.Adelaide Hospital Turin Italy, with clinical suspect of STS. Histological diagnosis was obtained contemporary to evaluation of chimeric transcripts detected by RT-PCR. cDNA were PCR amplified using primer specific for each sarcoma. Paraffin-embedded tissue samples were not used because the poor quality of the extracted RNA may give wrong positive results. Results Histology confirmed 21 Ewing Sarcoma (ES), 14 Synovial sarcoma (SS), 7 Mixoid liposarcoma (M-LPS), 4 Extraskeletal Myxoid Chondrosarcoma (E-MCDS), 4 Dermatofibrosarcoma protuberans (DFSP), 10 Rhabdo-myosarcoma, 10 Leiomyosarcoma. Of the 21 tumors diagnosed as ES, 21 (100%) expressed EWS-FLI1 chimeric transcripts. All 14 SS were positive for SYT-SSX fusion transcripts. Of the 7 cases with diagnosis of M-LPS, six were positive for EWS-CHOP transcripts; of the four cases of E-MCS 3 were positive for EWS-CHN fusion transcripts. All 4 DFSP were positive for COL1A1-PDGFB transcripts. Expression of Myo-D1, tested in ten cases of Rhabdomyosarcoma, was positive while in ten cases of Leiomyosarcoma no expression of Myo-D1 was detected by RT-PCR Ten cases were non sarcoma and negative for molecular biology.

Conclusion These results demonstrate a strong concordance between the standard histopathological diagnosis and molecular results. These techniques could be a useful method to increase the quality of histologic diagnosis in difficult cases.

Introduction: Packaging of Muscolo-Skeletal Tissues (MST) stored at −80°C must assure safety and sterility in order to minimize any risk of bacterial contamination and/or mechanical failure. Polymeric bags demonstrated problem of integrity at −80°C; gamma ray sterilisation induce oxidation decreasing mechanical properties, whereas Ethylene Oxide (EtO) does not. Antioxidant biocompatible additive, as Vitamin E, could improve mechanical resistance.

Objectives: Based on a previous paper presented at EATB 2005 congress, to analyse mechanico-chemical properties of plastic bags routinely used in MST Banks and new samples in order to identify and solve possible problems arising from the chemical composition and/or sterilisation.

Materials and Methods: Five different polymeric sterile bags used in three International Banks (three gamma and two EtO sterilised) and four experimental sample, manufactured on purpose from a Linear Low Density PolyEthylene (LLDPE) 150 microns thick films (EtO and e-beam sterilised), two added of Vitamin E, were analysed. Impact resistance was evaluated both on frozen and unfrozen material (in oven at 37°C); results were related to chemical composition, Tg, sterilisation and Fourier Transformed InfraRed Spectroscopy (FTIR).

Results: Three samples routinely used (one gamma and one EtO) showed severe macroscopic modification (glassy behaviour) at frozen temperature with no resistance to any mechanical stress Two samples (EtO) did not resist to mechanical tests at frozen state. The four experimental LLDPE, EtO and e-beam sterilised, resisted to mechanical tests. FTIR analysis confirmed the chemical composition declared by the commercial film: pure LLDPE, without any toxic additive and LLDPE with vitamin E.

Discussion: Packaging must use polymers with adeguate glass transition temperature (Tg) in order to maintain at −80° the rubbery state, not stiff nor fragile (not to pass to glassy state). High energy radiation oxidize polymer and decrease their mechanical resistance. LLDPE combine low Tg of the amorphous phase and low crystallinity, resulting in good mechanical properties at working temperature and at −80°C. Addition of Vitamin E protect against oxidation. EtO sterilisation does not modify the structure.

Conclusion: A LLDPE added of Vitamin E, sterilised by EtO and e-beam could improved packaging and storage of tissues at −80°C, with increased resistance to oxidation.

Aims: Angiogenesis is a multistep phenomenon, critical for tumor growth and prognosis in many solid neoplasms. Microvessels density (MVD) is a method of assessing angiogenesis and adversely affects DFS and OS in breast, lung and colorectal cancer. Few data are available in STS in which stage and grading are until now fundamental.

Methods: Our perspective study determined the level of MVD in a series of STS and correlated these results with DFS and OS, comparing its prognostic value with grading and stage. MVD was determined with CD 31 immunostains in formalin fixed, paraffin embedded tissues. Intratumoral MVD was assessed by light microscopic analysis. Hot spots defined the positive areas. The study included 45 patients, 35 with localized and 10 with metastatic disease at diagnosis. All tumors were located in upper or lower extremities. Histology were: 13 liposarcoma,11 MFH, 5 leiomiosarcoma, 5 PNST, 3 rabdo, 3 synovialsarcoma, 3 undifferentiated and 2 fibrosarcoma. Following Coindre classification 23 pts had low grade and 22 high grade STS.

Results: median follow up is 23 months (2–84). At present 20 pts (44.4%) are alive and DF, 11 (24.4%) alive with disease, 14 (31.1%) dead. Median survival is 75 months. Median MVD of all specimens is 62 microvessels/mm² (7–161). 32 pts (71.1%) have low MVD (group A) and 13 pts (29.9%) high MVD (group B). Mean survival is 62.7 mo in group A (median 75) and 36 mo in group B (median not reached) (p 0.01); median DFS respectively 24 mo and 15 mo (p 0.01). There is also a significant association with histological grade and survival: 75 mo in low grade and 34 mo in high grade tumors (p 0.05) and presence of metastasis at diagnosis (median survival: M+23 mo, M−75 mo). Unfortunately no relationship between angiogenesis and grading is found.

Conclusions: Our study confirms the prognostic importance of grade and staging in pts with STS. Moreover the role of MVD in prognosis is well defined and should be used as a routine marker in STS histological diagnosis.

Backgroud: Leiomyosarcoma (LMS) is characterized by malignant smooth muscle cells. LMS are principally tumours of adult life, children rarely develop these tumours. The cause of LMS is not understood, however the genetic alterations are thought to be important, if not inciting, in the formation and progression of sarcoma. The silencing of tumour suppressor genes by promoter hypermethylation is a common feature among many types of malignancies; it has been proposed that DNA methylation provides an alternative pathway to gene mutation.

Aims: To evaluate the promoter methylation in LMS: two tumour-related genes (MGMT and RASSF1A) were studied.

Materials and methods: 14 LMS specimens were obtained from patients treated at Orthopaedic Oncology and Reconstructive Surgery Department, CTO-CFR-M.Adelaide Hospital, Turin. Genomic DNA was extracted from paraffin sections and frozen samples according to standard protocols. Transformation of the methylations patterns in the CpG island of RASSF1A and MGMT of DNA were determined by chemical modification. To analyzed the role of aberrant DNA in LMS, methylation status by SP-PCR was evaluated. PCR products were amplified by unmethylated (U) and methylated (M)-specific primers.

Results: The RASSF1A promoter was methylated in 4 (29%) LMS, MGMT promoter was methylated in 2 (15 %); 1 of these patients with methylation had both RASSF1A and MGMT. Eight LMS samples did not show any methylation.

Conclusions: Our data indicate that inactivation of RASSF1A is a common event in LMS. Aberrant methylation of the RASSF1A promoter region is one of the most frequent epigenetic inactivation events detected in human cancer and leads to silencing of RASSF1A; moreover inactivation of RASSF1A was usually associated to poor prognosis. According to recent reports, that demonstrated that promoter hypermethylation of the MGMT gene is not a frequent event in LMS, the present study detected MGMT in 2 (15%) tumour samples.

During the last 15 years we have had the opportunity of analysing more than 700 UHMWPE prosthetic components (hip, knee and shoulder). Among them, about 500 were retrieved during revision surgery, while the remaining were new, ready-to implant, variably shelf-aged samples. The analysis of such a large, representative sample provided several important insights into the variables which influence the behaviour of UHMWPE in vivo; moreover, a long period of observation gave us the opportunity to follow changes and improvements in the field over time. All samples dated back to the nineties or before and sterilized with high energy radiation, either shelf-aged or retrieved, showed variable, but generally high, oxidation levels. Starting from the observation of these samples and with the aid of specimens irradiated on purpose under controlled conditions, some improvement has been achieved in the knowledge of radiation-induced oxidation process. The importance of the determination of hydroperoxides on the oxidation potential has been highlighted and the influence of variables such as sterilisation atmosphere, packaging, temperature and dose rate on the oxidation process has been clarified. The need for a suitable stabilizer to minimize oxidation arises during these studies.

We also had the opportunity of analysing a large number of EtO-sterilised samples, both new and retrieved. A small amount of them, all manufactured in the nineties, showed some bulk-oxidation which has been related to the presence of calcium stearate into the pristine resin. None of the newly produced, calcium stearate-free samples showed any oxidation and this group allowed to explore the behaviour of undegraded UHMWPE in vivo and in the shelf. Diffusion of polar compounds from the synovial fluid into polyethylene was observed in the majority of the retrieved samples. The nature of these products have been investigated along with their possible influence on the mechanical properties of the polymer.

In the last five years, we had the opportunity to study a significant number of crosslinked polyethylenes, both new and retrieved. The results of this study indicate that the variables of the crosslinking process can greatly influence final material properties and that not all cross-linked polyethylenes are the same.

Introduction Sterilisation of UHMWPE prosthetic components by high-energy radiation in air induces an oxidative degradation of the polymer, which may compromise the mechanical performances of the whole implant. Many manufacturers shifted to gas sterilization with EtO and gas plasma or to radiation sterilization in inert atmosphere and with barrier packaging. Aim of the present study was to investigate the relationship between sterilisation method, packaging, oxidation and mechanical properties of current orthopaedic UHMWPE.

Materials 100 sterilised UHMWPE hip, knee, and shoulder components by 19 orthopedic manufacturers were studied. The components were cut in half and sectioned using a microtome into slices of controlled thickness (0,1–0,3mm) which were analysed by FTIR. The UHMWPE packaging was also evaluated by FTIR, in order to correlate the extent of oxidation to the storage conditions. Mechanical properties were evaluated using the small punch test, as described in ASTM F2183-02.

Results The UHMWPE packaging was classified, when possible, into one of the following types; A: PET blister(s) with Tyvek® gas-permeable cover; B: packaging involving a polymeric multilayer bag; C: packaging involving at least one Aluminium foil. Using Type A, air permeable packaging for radiation-sterilized UHMWPE is the equivalent to irradiation in air. Many radiation sterilized implants packaged using Type A materials were severely oxidized. In the case of packaging type B, we detected moderately low oxygen index (OI) in the majority of samples, but an average high hydroperoxide level, even though type B packaging has well-documented oxygen barrier properties. UHMWPE components contained in packaging type C exhibit low OI and hydroperoxide level, due to the impermeable Al foil. The small punch test measurements showed that the oxidised sample exhibit generally diminished mechanical properties. Reductions in the yield load (up to 15%), in the ultimate load (up to 33%) and in the ultimate displacement (up to 21%), compared to the original or EtO sterilised material, have been measured on the majority of the oxidised samples.

Discussion The present results confirm that oxidation has a negative effect on the mechanical properties of UHMWPE. It remains difficult to generalise about the overall effectiveness of barrier packaging at protecting UHMWPE from oxidation, but it is our opinion that a complete absence of sterilisation-induced oxidation can only be guaranteed by gas sterilisation.

Aims: to investigate the mechanical properties of a new nanocomposite bone cement radiopacified with Barium Sulfate (BaSu) nanoparticles added at different concentrations, compared to a control cement with the classical BaSu microparticles.

Methods: the starting material was Endurance (J& J/ DePuy, USA) bone cement without BaSu; the radi-opacifier particles have been mixed into the cement powder in several different concentrations of 5, 10, 20, 30, 40% of the weight respectively. Two groups were studied: controls, with classical medical grade BaSu particles (average size 1000 nm) and nanocomposites, with nanoparticles (av. size 100 nm). In accordance with the ASTM, an Instron 4201 machine tested a minimum of 6 specimens for each concentration. Tensile tests were performed at cross-head speeds of 1mm/sec, while compression tests were performed at 25,4 mm/sec. Results were statistically analysed.

Results: nanocomposites had higher compressive Yield strength in all groups except 30 and 40% and lower compressive Modulus in all but 5% group (no significant difference). Nanocomposites had higher tensile values in 5%, 10%, and 40% concentrations for Strain-to-failure, yield stress, and Work-of-Fracture, and no significant differences in the other concentrations. Tensile modulus had not statistically significant variations. Higher BaSu concentrations give increases in tensile modulus and decreases in the other tensile properties for both the groups. The nanocomposite outperformed the control in the 5, 10, and 20% groups, while the 30 and 40% groups had no significant differences; all the results were above ASTM requirements.

Conclusions: bone cement has several uses, like joint replacement, filling defects in tumour or revision surgery, and more recently vertebroplasty. These applications require different properties and would have benefits from the possibility to change viscosity, radiopacity, time of polymerisation, mechanical features. Previous studies have demonstrated the improved performances of the new nanocomposite cement at the clinical used concentration of 10%. This study investigated the possibility to augment the concentration of BaSu and therefore the radiopacity and their relative effect on the mechanical properties; the results demonstrated the good compliance of the nanoparticles cement in this field. This would be useful in particular for specific applications such vertebroplasty. Further studies are needed to investigate and determine the ideal fatigue, handling and mixing properties, viscosity and radiopacity.