Spinal diseases such as unstable fractures, infections, primary or secondary tumors or deformities require surgical stabilization with implants. The long-term success of this treatment is only ensured by a solid bony fusion. The size of the bony defect, the often poor bone quality and metabolic diseases increase the risk of non-union and make the case a great burden for the patient and a challenge for the surgeon. The goal of spinal fusion can only be achieved if the implants used offer sufficient mechanical stability and the local biological regeneration potential is large enough to form sufficient bone. The lecture will present challenging clinical cases. In addition, implant materials and new surgical techniques are discussed. Local therapeutic effects are achieved through the release of osteopromotive or anti-resorbtive drugs, growth factors and antibiotics. By influencing biological pathways, basic orthopedic research has strong potential to further positively change future spinal surgery

Bioactive glasses, such as 45S5 Bioglass (BG), have been shown to promote bone ingrowth both in vitro and in vivo. The goal of this study was to analyze the effect of a high dose of BG (20%) in Direct Ink Writing (DIW)-produced controlled-geometry PCL-BG composite scaffolds in both their mechanical and biological performance. Porous cubes of 5 × 5 × 5 mm, 50% porosity and pore size and strut diameter of 400 µm were fabricated in a 3D-Bioplotter (EnvisionTec) to investigate their biological performance (n = 3). Additionally, cylindrical specimens (10 mm diameter; 15 mm height) with same internal structure were fabricated for mechanical testing (n = 6). The cylindrical specimens were tested by compression in a universal testing machine (ZwickRoell) with a 10 kN load cell. The tests were performed at 1.00 mm/min with extensometers in both sides. For biological characterization, scaffolds were sterilized in 70% ethanol overnight and pre-incubated with DMEM for 1 hour at room temperature. 1×10. 5. human gingival mesenchymal stem cells (hGMSCs) in 50 µl DMEM were seeded on the scaffolds using agarose molds to improve cell adhesion, and cultured in standard cell-culture conditions for 3, 7 and 14 days. To measure cell proliferation, the reagent CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega) was added to the cell-seeded scaffolds at each time point, using non-seeded scaffolds as blank controls. The OD (490 nm) was measured in a BioTek 800 TS plate reader. Both the apparent elastic modulus and yield stress were significantly lower in the scaffolds with 20% BG than their PCL control counterparts (p < 0.0001 for elastic modulus and p < 0.005 for yield stress, t-test). Cell proliferation in the scaffolds by MTS was variable, with the 20% BG scaffolds showing a significantly higher signal after seven days in culture (p < 0.05 by t-test), but a significantly lower signal after 14 days in culture (p < 0.05 by t-test). In conclusion, scaffolds with 20% BG showed a lower mechanical performance than their PCL counterparts in terms of both their apparent elastic modulus and yield stress. Additionally, scaffolds with 20% BG showed variable cell proliferation rates in terms of their metabolic activity over a two-week period. The decrease in proliferation rate after week 2 after an initial increase at the end of week 1 could be due to cytotoxic effects of the BG at this high dose (20%) after long term exposure. These results suggest that a dosage of 20% BG may not necessarily improve the mechanical and biological performance of scaffolds, so future experiments are required in order to characterize the optimum BG dosage in PCL scaffolds for tissue engineering applications

Majority of osteoporosis related fractures are treated surgically using metallic fixation devices. Anchorage of fixation devices is sometimes challenging due to poor osteoporotic bone quality that can lead to failure of the fracture fixation. Using a rat osteoporosis model, we employed neutron tomography and histology to study the biological effects of implant augmentation using an isothermally setting calcium sulphate/hydroxyapatite (CaS/HA) biomaterial with synthetic HA particles as recruiting moiety for systemically administered bisphosphonates. Using an osteoporotic sawbones model, we then provide a standardized method for the delivery of the CaS/HA biomaterial at the bone-implant interface for improved mechanical anchorage of a lag-screw commonly used for hip fracture fixation. As a proof-of-concept, the method was then verified in donated femoral heads and in patients with osteoporosis undergoing hip fracture fixation. We show that placing HA particles around a stainless-steel screw in-vivo, systemically administered bisphosphonates could be targeted towards the implant, yielding significantly higher peri-implant bone formation compared to un-augmented controls. In the sawbones model, CaS/HA based lag-screw augmentation led to significant increase (up to 4 times) in peak extraction force with CaS/HA performing at par with PMMA. Micro-CT imaging of the CaS/HA augmented lag-screws in cadaver femoral heads verified that the entire length of the lag-screw threads and the surrounding bone was covered with the CaS/HA material. X-ray images from fracture fixation surgery indicated that the CaS/HA material could be applied at the lag-screw-bone interface without exerting any additional pressure or risk of venous vascular leakage.: We present a new method for augmentation of lag-screws in fragile bone. It is envisaged that this methodcould potentially reduce the risk of fracture fixation failure especially when HA seeking “bone active” drugs are used systemically

Introduction. Non-union is agonising for patients, complex for surgeons and a costly burden to our healthcare service; as such, its management must be well defined. There is debate as to the requirements for the successful treatment of such patients, in particular, the need for additional biological therapies to ensure union. This study's primary aim was to determine if operative treatment alone was an effective treatment for the non-union of long bones in the upper and lower limbs compared to the pre-existing literature using biological therapies. Materials and Methods. A single-centre retrospective cohort study using prospectively collected data was performed. Inclusion was defined as patients 16 years or older with a radiologically confirmed non-union of the upper or lower limb long bones managed with surgical treatment alone between 2014–2019, with at least a 12 month follow up. Patients with bone defects or whose non-unions were treated with biological therapies were excluded from this study. The primary aim was assessed via the outcomes of union, time to union and RUST score. Results. 82 patients were included, 43 receiving percutaneous interventions and 39 receiving open interventions. Overall, a union rate of 97.56% was achieved with a mean time to union of 6.43 months. The mean RUST score increased from 6.09 at diagnosis to a final RUST score of 11.36 (p < 0.0001). Surgical factors showed that percutaneous interventions were most successful with a union rate of 100.00% with a mean time to union of 6.29 months. Augmentation surgery was associated with the shortest time to union of 4.47 months. Binary regression showed no statistically significant influence of patient factors. In 16 patients, complications were observed, including limb length discrepancy, ongoing pain and subsequent ankle problems. Conclusions. These results show non-inferior outcomes using operative treatment alone in non-union management as compared to the pre-existing literature on using biological therapies. Percutaneous interventions showed the most successful results and patient factors seemed to have little influence on this method's success. The continued use of biological therapies as a first line treatment should be questioned

While cell morphology has been recognized as a fundamental regulator of cell behavior, few studies have measured the complex cell morphological changes of chondrocytes using quantitative cell morphometry descriptors in relation to inflammation and phenotypic outcome. Acute vs. persistent exposure to IL-1β and how IL-1β modulated dynamic changes in cell morphology in relation to the phenotype, donor and OA grade in healthy and osteoarthritis (OA) chondrocytes was investigated. A panel of quantitative cell morphometry descriptors was measured using an automated high-throughput method. Absolute quantification of gene expression was measured by ddPCR followed by correlation analyses. In OA chondrocytes, chronic IL-1β significantly decreased COL2A1, SOX9, and ACAN, increased IL-6 and IL-8 levels and caused chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype. In healthy chondrocytes, 3 days after acute (72 h) IL-1β exposure, COL1A2 and IL-6 significantly increased but had minor effects on cell morphology. However, in healthy chondrocytes, persistent IL-1β led to more profound effects in all cell morphology descriptors and chondrocytes expressed significantly less COL2A1 and more IL-6 and IL-8 vs. controls and acutely-stimulated chondrocytes. In both OA and healthy chronically-stimulated chondrocytes, area, width and circularity were sensitive to the persistent presence of the IL-1β cytokine. Moreover, there were many significant and strong correlations among the measured parameters, with several indications of an IL-1β-mediated mechanism. Cell morphology combined with gene expression analysis could guide researchers interested in understanding inflammatory effects in the complex domain of cartilage/chondrocyte biology. Use of quantitative cell morphometry could complement classical approaches by providing numerical data on a large number of cells, thereby providing a biological fingerprint for describing chondrocyte phenotype, which could help to understand how changes in cell morphology lead to disease progression

After anterior cruciate ligament (ACL) rupture, reconstructive surgery with a hamstring tendon autograft is often performed. Despite overall good results, ACL re-rupture occurs in up to 10% of the patient population, increasing to 30% of the cases for patients aged under 20 years. This can be related to tissue remodelling in the first months to years after surgery, which compromises the graft's mechanical strength. Resident graft fibroblasts secrete matrix metalloproteinases (MMPs), which break down the collagen I extracellular matrix. After necrosis of these fibroblasts, myofibroblasts repopulate the graft, and deposit more collagen III rather than collagen I. Eventually, the cellular and matrix properties converge towards those of the native ACL, but full restoration of the ACL properties is not achieved. It is unknown how inter-patient differences in tissue remodelling capacity contribute to ACL graft rupture risk. This research measured patient-specific tissue remodelling-related properties of human hamstring tendon-derived cells in an in vitro micro-tissue platform, in order to identify potential biological predictors for graft rupture. Human hamstring tendon-derived cells were obtained from remnant autograft tissue after ACL reconstructions. These cells were seeded in collagen I gels on a micro-tissue platform to assess inter-patient cellular differences in tissue remodelling capacity. Remodelling was induced by removing the outermost micro-posts, and micro-tissue compaction over time was assessed using transmitted light microscopy. Protein expression of tendon marker tenomodulin and myofibroblast marker α-smooth muscle actin (αSMA) were measured using Western blot. Expression and activity of remodelling marker MMP2 were determined using gelatin zymography. Cells were obtained from 12 patients (aged 12–51 years). Patient-specific variations in micro-tissue compaction speed or magnitude were observed. Up to 50-fold differences in αSMA expression were found between patients, although these did not correlate with faster or stronger compaction. Surprisingly, tenomodulin was only detected in samples obtained from two patients. Total MMP2 expression varied between patients, but no large differences in active fractions were found. No correlation of patient age with any of the remodelling-related factors was detected. Remodelling-related biological differences between patient tendon-derived cells could be assessed with the presented micro-tissue platform, and did not correlate with age. This demonstrates the need to compare this biological variation in vitro - especially cells with extreme properties - to clinical outcome. Sample size is currently increased, and patient outcome will be determined. Combined with results obtained from the in vitro platform, this could lead to a predictive tool to identify patients at risk for graft rupture

Titanium alloys are one of the most used for orthopaedic implants and the fabrication of them by 3D printing technology is a raising technology, which could effectively resolve existing challenges. Surface modification of Ti surfaces is often necessary to improve biocorrosion resistance, especially in inflammatory conditions. Such modification can be made by coatings based on hydrogels, like alginate (Alg) - a naturally occurring anionic polymer. The properties of the hydrogel can be further enhanced with calcium phosphates like octacalcium phosphate (OCP) as a precursor of biologically formed hydroxyapatite. Formed Alg-OCP matrices have a high potential in wound healing, delivery of bioactive agents etc. but their effect on 3D printed Ti alloys performance was not well known. In this work, Alg-OCP coated 3D printed samples were studied with electrochemical measurements and revealed significant variations of corrosion resistance vs. composition of the coating. The potentiodynamic polarization test showed that the Alg-OCP-coated samples had lower corrosion current density than simple Alg-coated samples. Electrochemical impedance spectroscopy indicated that OCP incorporated hydrogels had also a high value of the Bode modulus and phase angle. Hence Alg-OCP hydrogels could be highly beneficial in protecting 3D printed Ti alloys especially when the host conditions for the implant placement are inflammatory. AcThis work was supported by the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions GA860462 (PREMUROSA). The authors also acknowledge the access to the infrastructure and expertise of the BBCE – Baltic Biomaterials Centre of Excellence (European Union Horizon 2020 programme under GA857287)

Introduction. According to American Joint Replacement Registry, particle mediated osteolysis represents 13 % of the knee revision surgeries performed in the United States. The comprehension of mechanical and wear properties of materials envisioned for TJR is a key step in product development. Furthermore, the maintenance of UHMWPE mechanical properties after material modification is an important aspect of material success. Initial studies conducted by our research group demonstrated that the incorporation of ibuprofen in UHMWPE had a minor impact on UHMWPE physicochemical and mechanical properties. Drug release was also evaluated and resulted in an interesting profile as a material to be used as an anti-inflammatory system. Therefore, the present study investigated the effect of drug release on the mechanical and biological properties of ibuprofen-loaded UHMWPE. Experimental. UHMWPE resin GUR 1020 from Ticona was for sample preparation. Samples with drug concentrations of 3% and 5% wt were consolidated as well as samples without anti-inflammatory addition through compression molding at 150 °C and 5 MPa for 15 minutes. Mechanical properties were evaluated via the tensile strength experiment (ASTM D638) and dynamic mechanic tests. Wear resistance was measured using the pin on disc (POD) apparatus. Finally, cytotoxicity analysis was conducted based on ISO 10993–5. Results. Dynamic-mechanic analysis demonstrated no difference in flexion modulus and stress for all materials (Table 1). No difference was also verified during cyclical loading experiments (Table 1), which indicates that the drug concentration added to material composition did not affect these properties. POD experiments were proposed to evaluate wear resistance of ibuprofen-loaded UHMWPE samples considering the combination of materials similar to those employed in TJR. Results from POD tests are presented in Table 1. Volumetric wear was close to zero for all samples after 200 thousand cycles. Comprehension of the effect of drug release on mechanical properties is essential to estimate how the material will behave after implantation. Therefore, mechanical properties were assessed after 30 days of ibuprofen release and the results were compared with those obtained in samples as prepared (Table 2). Initial results demonstrated a decrease in elastic modulus in samples prepared with ibuprofen. However, no difference was verified between UHMWPE, UHMWPE 3% IBU and UHMWPE 5% IBU after ibuprofen release. Finally, cell viability of UHMWPE 3% IBU and UHMWPE 5% was found to be superior to 100% (Figure 1). Therefore, both materials can be considered nontoxic. Conclusions. Ibuprofen-loaded UHMWPE did not demonstrate a significant influence on the mechanical and biological behavior of UHMWPE. Dynamic-mechanical tests demonstrated constancy for all samples under analysis. Wear testing resulted in gravimetric wear close to zero, for all tested materials. Mechanical properties conducted after 30 days of ibuprofen release also had a positive outcome. Although presenting a difference in modulus prior and after release tests, modulus and tensile yield stress remained inside acceptable range indicated to UHMWPE used in orthopedic implants. Furthermore, after drug elution UHMWPE 3% IBU and UHMWPE 5% IBU recovered original UHMWPE properties. Cytotoxicity assessment was performed and both ibuprofen-based formulations were considered nontoxic according to ISO 10993–5. For any figures or tables, please contact the authors directly

The ATTUNE™ Knee System (DePuy Synthes) comprises of a tibial insert that is made from AOX™, an antioxidant-stabilized polyethylene. The antioxidant used in AOX is pentaerythritol tetrakis [3-(3, 5-di-tertiary butyl-4-hydroxyphenyl)] propionate (PBHP). A biological risk assessment of the degradation products arising from PBHP has been performed. This assessment focuses on the requirements of ISO 10993–1:2009, ISO 14971:2007, and the Medical Device Directive 93/42/EEC. Because the orthopedic implant is a permanent implant, consideration has been given to all relevant endpoints defined by ISO 10993–1 Biological evaluation of medical devices – Part 1: Evaluation and testing within a risk management process. Comprehensive biocompatibility testing including long-term (26 weeks) subcutaneous implantation has been conducted which confirms the biosafety of the polyethylene compound[1]. In addition to the biological safety testing completed, the overall safety and the associated toxicological risk of exposure to degradation products of PBHP has been given due consideration. The guidelines for the Threshold for Toxicological Concern (TTC) provided by The Product Quality Research Institute (PQRI) Leachables and Extractables Working Group were used in the assessment[2]. This working group is a collaboration of chemists and toxicologists from the U.S. Food and Drug Administration (FDA), industry, and academia. The TTC principle allows safety assessment in the absence of substance-specific hazard data, based on very low levels of exposure to that substance. A Margin of Safety (MOS) is calculated as the ratio of the threshold safety value to the actual exposure quantities determined and used in the assessment. A MOS value greater than 1 is typically judged by risk assessors and regulatory bodies to be unlikely to cause harm and the risk may be considered low. The identity of the degradation products as well as the corresponding 30-day leachable quantities from a water:acetone extraction media has been previously reported [3] and provided here (Table 1). The amount of leachables determined from Table 1 for all products were well below the TTC of 150 ng/device and hence no toxicological risks were identified for these compounds. In order to further examine the toxicological risk assessment, aggressive extraction using Dynamic Head Space (DHS) extraction was done and analytical testing was performed on the degradation products of PBHP using gas chromatography/mass spectrometry (GC /MS). These estimated quantities along with literature information from biological safety studies of the chemicals that were identified from the quantitative GC/MS analysis of degradation products of PBHP were used in the review and toxicological assessment per the methodology described in ISO 14971 and ISO 10993–18. The extraction and analysis confirmed the same sixteen compounds previously identified. The quantities and the calculated margins of safety are summarized (Table 2). In conclusion, upon review of actual test results of PBHP degradation products (Table 1), there is little probability that these organic degradation products would cause a systemic reaction and not be safe. Thus, the potential biological hazards identified in ISO 10993–1:2009 due to the quantified leachables have been verified to be minimal with a high Margin of Safety relative to the Threshold of Toxicological Concern

The growth in the popularity of tissue engineering principles in the treatment of musculoskeletal disorders has been complemented greatly with research investment into tissue specific scaffolds. Biological scaffolds produced by means of decellularising native tissues have the advantage of providing the natural complex hierarchical matrix and, in doing so, replicating the specific biomechanical and biological functions of the tissue in question. Decellularisation treatments are multi-faceted, vary considerably between different processes and may involve many lengthy treatment steps. Some of these bio-processes may cause undesirable structural changes to the extracellular matrix of tissues and, by association, their mechanical properties. Thus, it is of paramount importance to ensure that the properties of the scaffolds are not affected to the extent of reducing their integration, biomechanical performance and longevity. This talk consists of a body of work detailing investigations into bio-process optimisation, sterilisation strategies and the regenerative and functional capacity of decellularised xenogeneic and allogeneic tendon, ligament and bone scaffolds. In addition, on-going work concerning advanced pre-clinical assessment, stratification of these products to particular patient populations and the importance of the manufacturing value chain in their translation will be discussed

Currently, different techniques to evaluate the biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal/ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. Moreover, cell culture systems evaluate the biological responses of cells to a biomaterial as the combined effect of both particles and ions. We have developed a novel cell culture system suitable for testing the all three type of particles and ions, separately. The method was tested by evaluating the biological responses of human peripheral blood mononuclear cells (PBMNCs) to UHMWPE, cobalt-chromium alloy (CoCr), and Ti64 alloy wear particles. Methods. Clinically relevant sterile UHMWPE, CoCr, and Ti64 wear particles were generated in a pin-on-plate wear simulator. Whole peripheral blood was collected from healthy human donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell, UK) and seeded into the wells of 96-well and 384-well cell culture plates. The plates were then incubated for 24 h in 5% (v/v) CO. 2. at 37°C to allow the attachment of mononuclear phagocytes. Adherent phagocytes were incubated with UHMWPE and CoCr wear debris at volumetric concentrations of 0.5 to 100 µm. 3. particles per cell for 24 h in 5% (v/v) CO. 2. at 37°C. During the incubation of cells with particles, for each assay, two identical plates were set up in two configurations (one upright and one inverted). After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer, UK). Intracellular oxidative stress was measured using the DCFDA-based reactive oxygen species detection assay (Abcam, UK). TNF-α cytokine was measured using sandwich ELISA. DNA damage was measured by alkaline comet assay. The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. Results and Discussion. Cellular uptake of UHMWPE, CoCr and Ti64 particles was confirmed by optical microscopy. PBMNCs incubated with UHMWPE particles did not show any adverse responses except the release of significant levels of TNF-α cytokine at 100 µm. 3. particles per cell, when in contact with particles. PBMNCs incubated with CoCr wear particles showed adverse responses at high particle doses (100 µm. 3. particles per cell) for all the assays. Moreover, cytotoxicity was observed to be a combined effect of both particles and ions, whereas oxidative stress and DNA damage were mostly caused by ions. Ti64 wear particles did not show any adverse responses except cytotoxicity at high particle doses (100 µm. 3. particles per cell). Moreover, this cytotoxicity was mostly found to be a particle effect. In conclusion, the novel cell culture system is suitable for evaluating the biological impact of orthopaedic wear particles and ions, separately

Biphasic calcium phosphates (BCP) are the most frequently used materials because of their mineral analogy with bio-mineral part of bones. Their chemical synthesis can be modulated by doping, in order to respond to the biological needs. We present here the biological responses induced by copper ions in solution, to characterize its cytotoxicity and antibacterial activity. We also investigate the antibacterial property of Cu-doped BCP (Ca10 Cu0.1 (PO4)6 (OH)1.8 O0.2) on a strain of clinical interest: S. aureus, compared to undoped BCP. The sol-gel route has been used to prepare the BCP ceramics. Human BMC (Bone Marrow Cells) were obtained from metaphysal cancellous bone collected during hip arthroplasty and used for cytotoxicity evaluations. A strain of Staphylococcus aureus isolated from an osteoarticular infection after total knee arthroplasty was used to evaluate antibacterial activities. Results indicate that 3 ppm of copper ions leads to the death of all cultured bacteria in 24 hours and 25 ppm caused the death of all cells in 15 days. Regarding BCP, the undoped bioceramics increased the bacterial growth compared to a control without bioceramic. After 16 hours of contact, the copper ions released by the Cu-doped BCP induced a significant decrease of the bacterial concentration, indeed no viable bacteria were found. These materials seem to be a promising alternative for the preparation of multifunctional bone substitutes

Modification of ordinary jig (angle guide) used for DCS fixation so as to make it more suitable for biological DCS. We have modified the jig used for ordinary DCS fixation so as to make it more suitable for biological DCS. In ordinary DCS jig, the hole for guide wire lies towards one end and the handle is attached at the other end. We have removed the handle and attached it adjacent to hole for guide pin so that the other end is free and can be slided in submuscular plane without actually exposing the whole length of femur. Subsequently, we beveled the free end and removed the sharp points and this helps in making sub muscular plane easily and with minimum soft tissue trauma. The modified jig was applied in a patient with fracture subtrochanteric femur in submuscular plane through 2 cm long incision and its position confirmed by c-arm. Position was found to be similar to that observed with ordinary DCS jig. The idea of making this presentation is that we can modify classical instrumentation used for internal fixation to make them suitable for biological fixation. This is a small innovation in that direction

Introduction: Nanometre sized UHMWPE particles have recently been isolated from periprosthetic tissues and hip simulator lubricants [. 1. ,. 2. ]. The biological response to UHMWPE particles of 0.1 μm and above has been well characterised, with particles in the 0.1–1.0 μm size range having the highest biological activity [. 3. ]. The purpose of the study was to determine the biological activity of nanometre-sized particles in terms of osteolytic cytokine release from primary human monocytes. Methods: Monocytes were isolated from peripheral blood from 5 healthy donors by density gradient centrifugation over Lymphoprep. Cells were cultured using the agarose gel technique [. 3. ] at particle volume (μm3):cell number ratios of 10:1 and 100:1. The particles used were:. 1 Polystyrene FITC-conjugated FluoSpheres (FS; Invitrogen) in 20 nm, 40 nm, 0.2 μm and 1.0 μm sizes. 2a Complete Ceridust® 3615 (CD), a low MW polyethylene powder (size range 15 nm – 53 μm). 2b Nanometresized Ceridust® (fractionated by filtration using 10, 1, 0.1, 0.05 & 0.015 μm filters). 3 Clinically relevant GUR 1120 UHMWPE debris produced aseptically using a multidirectional wear rig. All particles were tested for the presence of endotoxin prior to culture with cells. Cells without particles were used as a negative control and 200 ng/ml LPS was used as a positive control. Cell viability was assessed using the ATP Lite assay (Perkin Elmer) and ELISA was used to determine TNF-alpha, IL-1beta, IL-6 and IL-8 release at 3, 6, 12 and 24 h. Results: FluoSpheres and CD had no effect on cell viability at 10 or 100:1. Clinically relevant UHMWPE particles had no effect on cell viability at 10:1, however, at 100:1 significant differences (P< 0.05) were seen at 3, 12 and 24 h for Donors 1 and 3. The 40 nm, 0.2μm and 1.0 μm FS caused significant elevation of TNF-α release at the 12 and 24 h time points at 100:1. There was no significant increase in TNF-α release for the 20 nm FS (3/5 donors). Particle volume and particle size showed correlation with cellular response, with the 20 nm FS showing the lowest biological activity. Clinically relevant UHMWPE particles and nanometre sized CD produced significantly higher quantities of TNF-alpha at 100:1. Release of interleukins IL-1beta, IL-6 and IL-8 followed a similar trend to TNF-alpha release. Discussion: This study found that all nanometre-sized particles had the potential to provoke inflammatory cytokine release from macrophages. Particle volume and particle size played critical roles in initiating cellular responses. There was a lower particle size limit, with the 20 nm FS showing the lowest activity. Nanometre-sized polyethylene particles (CD) caused elevated TNF-α release, and since it has been shown that nanometre-sized UHMWPE particles are produced in large numbers in vivo [. 2. ], the relative contribution of these particles to osteolysis should be considered. The biological response to nanometre-sized clinically relevant UHMWPE particles is currently under investigation

Improvements in arthroplasty design and materials led to superior lifetime of the implants. Nevertheless, aseptic loosening due to particulate debris is still one of the most frequent late reasons for revision of hip and knee replacements. The complex process of inflammation and osteolysis due to wear particles is not understood in detail so far. A cellular and receptor mediated response to wear particles results in a release of pro-inflammatory cytokines and induces an inflammatory reaction causing periprosthetic osteolysis. The overall cellular response is influenced by particle volume as well as characteristics. But there is still a lack of data concerning all signalling pathways that are involved. To answer some open questions appropriate in vivo models are shown closing the loop between wear simulation, particle analysis, generation of sterile particles and biological evaluation. Beyond that, new aspects of particle effects and deposits in retrieved human tissue are given

In recent years, novel therapies for intervertebral disc (IVD) regeneration have been developed that are based on the delivery of cells, biomaterials or bioactive molecules. The efficacy of these biological therapies depends on the type and degree of IVD degeneration. Whole organ culture bioreactors provide an attractive platform for pre-clinical testing of IVD therapeutics, since the cells are maintained within their native extracellular matrix, and the endplate remains intact to fulfil its function. Moreover, defined regimes of mechanical stress are applied to the IVD, representing either physiological or degenerative, detrimental loading. Different degrees of degeneration can be induced by high load, low nutrition, enzyme injection, and/or mechanical damage; while recent organ culture models also implement an inflammatory component. Using whole organ culture models, we found that mesenchymal stem cell injection into nucleotomized IVDs had an anabolic effect on the IVD cells. Furthermore, hyaluronan hydrogels were beneficial for cell delivery and mechanical support. We also found that anti-inflammatory treatment could partially prevent the induction of cytokines in an inflammatory model. However, chemokine delivery did not induce a significant repair response in an annulus fibrosus defect. In line with 3R principles, relevant ex-vivo models are essential to reliably test biological IVD treatments

There are numerous studies in the current literature that have demonstrated altered levels of various biomarkers in the serum of patients with implant loosening. Despite increasing interest in the biology of implant incorporation there are no studies investigating the changes in biological marker (of either osteoblastic or osteoclastic activity) levels during the integration of the bone-implant interface. Such a study would provide data about the biological profile of normal integration and would be helpful for future monitoring of implant prosthetic performance (either normal or abnormal). We present data from a study performed on 100 osteoarthritic patients, who underwent cementless THA (Synergy, Reflexion Interfit, Smith & Nephew) and 100 non arthritic volunteers. Serial measurements of serum biochemical markers (bone formation and resorption), of cytokines and of other biological mediators and growth factors were evaluated at regular intervals over the course of six years. Curves of per cent changes from baseline and marker variability curves have been created for each marker which are indicative of the incorporation process. Evaluating markers of osteoblastic activity, a first response, with average values below baseline, was observed at the level of the seventh day (perhaps as a response to local trauma). A second osteo-productive response was observed between the third week and 9 months (peak average values at the level of the 6. th. month). At the 1st year time interval, average values reached baseline and remained at this level up to the 6th postoperative year. Evaluating markers of osteoclastic activity, a first response, with average values above baseline, was observed at the level of the seventh day (perhaps as a response to local trauma). A second osteoclastic response was observed between the third week and 3 months (perhaps a coupling response to enhanced osteoblastic activity). At 6 months, average values reached baseline and remained at this level up to the 6th postoperative year. It seems that bone implant interface in cementless total hip arthroplasty remains active up to the 9. th. postoperative month. Possible future deviation from such ‘individual normal’ curves will be indicative of the initiation of the osteolysis process and loss of fixation

INTRODUCTION. Ultra-High Molecular Weight Polyethylene (UHMWPE) wear debris is thought to be a main factor in the development of osteolysis (1). However, the method for the evaluation of the biological response to UHMWPE particles has not yet been standardized. In this study, four different types of UHMWPE particles were generated using a mechanized pulverizing method and the biological responses of macrophages to the particles were investigated using an inverted cell culturing process (2). MATERIALS & METHODS. Virgin samples were manufactured via Direct Compression Molding (DCM) technique from UHMWPE GUR1050 resin powder (Ticona, USA). For vitamin E (VE)-blended sample, the resin was mixed with VE at 0.3 wt% and the mixture was then molded using DCM. The crosslinked virgin samples were made by gamma ray irradiation to UHMWPE GUR1020 resin sheet (Meditech, USA) with doses of 95kGy ±10% and annealed. The VE-blended crosslinked samples were made by electron beam irradiation to VE-blended samples with doses of 300kGy and annealed. The material conditions were summarized in Figure 1. To pulverize the samples, the Multi-Beads Shocker (Yasui Kikai, Japan) was used. After pulverization, samples were dispersed in an ethanol solution and sequentially filtered through polycarbonate filters. Over 100 sections of the filter were selected randomly and images of the particles were analyzed using scanning electron microscope (SEM). To analyze the macrophage biological response, an inverted cell culturing process was used (2). The mouse macrophage-like cells were seeded at densities of 4×105cells per well in a 96-well culture plate and incubated for 1h. UHMWPE particles suspended in the culture medium were then added to each well in the appropriate amount. After that, fresh medium was added to fill the wells, and a sealing film was used to cover the culture plate. The culture plate was then inverted to cause the UHMWPE particles interact with the adhered macrophages. The inverted culture plate was incubated for 8h. The amount of TNF-α was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS & DISCUSSION. Geometric measurements showed no significant difference in the UHMWPE particles (Figure 2). The amount of TNF-α released stimulated by the crosslinked virgin particles showed significantly higher relative to the other UHMWPE particles (Figure 3). During crosslinking irradiation, the carbon free radicals are generated in the main chain (3). In the presence of oxygen, these radicals can react to form peroxy radicals and when the peroxy free radicals react with hydrogen they form hydroperoxides, which can further degrade into other oxidation products (4). It has been reported that VE hinders this cascading in UHMWPE (5). Therefore, it is possible that oxidation of the crosslinked virgin UHMWPE was involved in the cytokine response observed in this study. However resin material, molding technique and the irradiation method were different between crosslinked virgin and VE-blended crosslinked samples. Further consideration will be needed to examine the relationship between residual radicals, hydroperoxides and biological response

Purpose of the study: Few data are available concerning the proper management of patients with a periprosthetic fracture of the hip who presents biological signs of inflammation (increased WBC, sedimentation rate, or C-reactive protein). The purpose of this work was to determine the prevalence of elevated biological markers in this type of patient and to determine the reliability of such markers for the diagnosis of periprosthetic infection. Material and methods: A periprosthetic hip fracture was diagnosed in 204 patients from 2000 to 2006. The WBC count, the sedimentation rate and the serum CRP level were noted at admission to the emergency ward. The diagnosis of infection was confirmed by at least two positive bacteriological samples of tissue biopsy and/or joint fluid collected at surgery. A statistical analysis was conducted to determine the prevalence of elevated biological markers of inflammation, the sensitivity, their specificity and their positive predictive value for deep infection. Results: Twenty-one patients (11.6%) developed a periprosthetic infection. Among the 204 patients, the WBC count increased in 16.2%, sedimentation rate in 33.3% and CRP in 50.5%. The sensitivity was 24% (WBC), 50% (sedimentation rate) and 83% (CRP). The specificity was 85% (WBC), 69% (sedimentation rate) and 56% (CRP). The positive predictive value was low (18, 21 and 29% respectively). Discussion: Markers of inflammation are frequently ordered before surgery to search for infection but can be elevated for various reasons. Most often, these markers are elevated because of the patient’s general status and are thus related to other co-morbid conditions and/or reaction to the new fracture. In this population, the WBC count did not contribute to the diagnosis of infection as only 24% of the infected patients had a high count. CRP and sedimentation rate and the WBC count had low positive predictive values. Conclusion: This study shows that an isolated elevation of biological markers of inflammation in a patient with a periprosthetic fracture is not a good indicator of infection

Purpose of the study: Bone morphogenetic proteins (BMPs) are osteoinducing proteins which play a primordial role in bone repair. To obtain optimal mineralization in vivo, high doses of heparin binding growth factor must be used. Studies have demonstrated that functionalized dextranes (FD) present affinity for heparin binding growth factor. We studied the capacity of dextrane derivatives to interact with BMP-2 and potentialize its biological activity in vitro. Material and methods: Different soluble FD were obtained by random substitution of carbosymethyl (CM), benzylamide (B) and sulfate (Su) groups on native dextrane chains. Gel electrophoresis was used to study the affinity of the anionic FDs for BMP-2. The effect of polymers on osteoinduction activity of BMP-2 was evaluated by histochemistry. ALP (an early marker) synthesized by mypoblasts C2C12 were dosed seven days after injection in presence of BMP-2 associated or not with polymers. IN addition, expression of osteocalcin (late marker) was quantified by RT-PCR. Results: Electrophoresis demonstrated that DMCB and DMCBSu interacted with BMP-2. These interactions appeared to increase with B level but decreased with Su level. We worked with FD1, a DMCB with a high affinity for BMP-2. The ALP activity was clearly potentialized when BMP-2 was associated with heparin and even better with FD1. Expression of osteocalcin was also amplified with the FD1-BMP-2 association. The influence on the biological activity of BMP-2 of FD, presenting different degrees of substitution, was also tested. Only FDs containing a high concentration of B expressed affinity for BMP-2, potentializing the biological activity of the protein. Discussion: Dextanes functionalized with a high rate of benzylamide substitution interact with BMP-2 while sulfate substitution limits such interaction. Only FDS which interact with BMP-2 can potentialize the protein’s biological activity in vitro. Two hypotheses can be put forward: i) FD presents BMP-2 to its receptor cell, ii) FD protects BMP-2 from proteolytic degradation or capture by antagonists. The capacity of FD1 to potentialize the biological activity of BMP-2 could be a way of reducing the quantity of growth factor needed for optimal bone repair