Background. Wear particles are considered to be the major culprit for the aseptic loosening. Their characterization is thus crucial for the understanding of their bioreactivity and contribution to the development of aseptic loosening. Methods. Metal wear debris particles were analyzed directly in periprosthetic tissue resins by scanning electron microscopy (SEM) combined with back-scattered electron imaging (BSE) and energy dispersive X-ray spectroscopy (EDS). Four groups of tissue samples retrieved at revision operations of loosened hip implants with different bearing surfaces (metal-on-metal, ceramic-on-polyethylene and metal-on-polyethylene), and different material of the femoral stem (Ti alloy, CoCrMo and polymer combined with stainless steel) were investigated. Tissue samples were first analyzed histologicaly. Sections from the same paraffin blocks were then carbon coated and analyzed using SEM/BSE/EDS method. Results. Metal particles were detected in all samples. Their composition corresponded to the composition of the implant components. The gradation of metal particles ranged from +1 to +3. A considerable number of big metal particles were actually agglomerates of submicron particles visible only at higher magnification. The clustering of particles was observed primarily for CoCrMo and, to a lesser extent, for stainless steels particles. The median sizes of CoCrMo clusters in two groups of samples were 2.9 1.8 m (range, 0.5 to 7.6 m) and 3.2 1.0 m (range, 1.9 to 5.4 m). The effect of clustering was not observed for Ti particles. The median sizes of individual Ti particles determined in two groups of samples were 2.5 3.6 m (range, 0.4 to 17.3 m) and 4.3 2.8 m (range, 0.8 to 11.0 m). Conclusion. Scanning electron microscopy combined with back-scattered electron imaging is an appropriate and selective method to recognize metal particles in tissue sections, without being destructive to specimens. When the size of the particles is considered, however, it should be differed between the size of individual particles and size of clusters of particles. Besides its benefits, this study has some limitations: the detection of particles smaller than 0.4 m is difficult, and this method cannot be used to identify polyethylene particles

INTRODUCTION. In order to address high failure rates following rotator cuff repairs, a greater understanding is required of the underlying structural changes so that treatments can be appropriately targeted and biomarkers of failure can be identified. As collagen is the primary constituent of tendon and determines force transmission, collagen structural changes may affect responses to loading. For example changes in collagen 1 and 5 are associated with the hyperelastic Ehlers-Danlos syndrome, which is diagnosed by looking for pathopneumonic altered collagen fibres or ‘collagen flowers’ in skin using transmission electron microscopy (TEM). To date no study has been performed on the microstructure of torn human rotator cuff tendons using TEM. It was hypothesized that normal, small and massive human rotator cuff tendons tears will have altered microscopic structures. The unique study aimed to use TEM to compare the ultrastructure of small and massive rotator cuff tears, to normal rotator cuff tendons. METHODS. Samples from 7 human rotator cuff tendons repairs were obtained, including 4 massive (>5 cm) and 3 small (< 1 cm) tears, and 3 matched normal controls with no history of connective tissue disorders. Specimens were fixed in 4% glutaraldehyde in 0.1M phosphate buffer, processed and examined blind using routine TEM examination. To assess whether changes in the relative expression of collagen 1 and 5 (COL1A1, COL5A1 and COL5A2) occurred in all tears, qPCR was performed on another 6 phenotypically matched patients. RESULTS. The basic structure of the normal tendon consisted of tightly packed clumps of dense packed parallel running collagen fibers with few fibroblasts and small amounts of fine filamentous material between clumps. In contrast, torn samples were more variable with areas of less dense packing of collagen fibers and larger areas of filamentous material plus variable numbers of lipid droplets both within the fibroblast and between the collagen bundles. There was also evidence of twisting and random orientation of individual collagen fibers. All torn tendons showed evidence of a proportion of the fibers within the collagen bundles being enlarged with a serrated outline, similar in appearance to ‘collagen flowers’. Clear differences between the small and massive tears were not identified. qRT-PCR of torn rotator cuff tendon specimens demonstrated no altered collagen expression compared to normal tendons. DISCUSSION. This novel study has identified the previously unreported presence of atypical collagen fibers with focal swelling resulting in the appearance of ‘collagen flowers’ in torn rotator cuff tendons only. This appearance is considered pathognomonic of Ehlers-Danlos syndrome, classical type 1 and 2. Torn tendons also showed an increase in filamentous material, and infiltration with fat droplets. These novel findings may offer insight into the mechanisms of structural damage that contribute to rotator cuff failure. Further examination is required, to evaluate the significance of these observations

Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce peri-operative bleeding. Increasingly, topical administration as an intra-articular injection or peri-operative wash is being administered at concentrations between 10–100mg/ml. This study investigated effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations. Tendon, synovium and cartilage obtained from routine orthopaedic surgeries were used ex vivo or cultured for in vitro studies using various concentrations of TXA. They were stained with 5-chloromethylfluorescein diacetate and propidium iodide and imaged using confocal microscopy to identify the proportion of live and dead cells. The in vitro effect of TXA on primary cultured tenocytes, synovial like fibroblast (FLS) cells and chondrocytes was investigated using cell viability assays (MTT), fluorescent microscopy and multi-protein apoptotic arrays for cell death. There was significant (p<0.01) increase in cell death in all tissue treated with 100mg/ml TXA, ex vivo. MTT assays revealed significant (p<0.05) decrease in cell viability following treatment with 50 or 100mg/ml of TXA within 4 hours of all cell types cultured in vitro. Additionally, there was significant (p<0.05) increase in cell apoptosis detected by fluorescent microscopy within 1 hour of exposure to TXA. Furthermore, multi-protein apoptotic arrays detected increased apoptotic proteins within 1 hour of TXA treatment in tenocytes and FLS cells. Our study provides evidence of TXA cytotoxicity to human peri-articular tissues ex vivo and in vitro at concentrations and durations of treatment routinely used in clinical environments. Clinicians should therefore show caution when considering use of topical TXA administration

Osteoporosis is a major healthcare burden, responsible for significant morbidity and mortality. Manipulating bone homeostasis would be invaluable in treating osteoporosis and optimising implant osseointegration. Strontium increases bone density through increased osteoblastogenesis, increased bone mineralisation, and reduced osteoclast activity. However, oral treatment may have significant side effects, precluding widespread use. We have recently shown that controlled disorder nanopatterned surfaces can control osteoblast differentiation and bone formation. We aimed to combine the osteogenic synergy of nanopatterning with local strontium delivery to avoid systemic side effects. Using a sol-gel technique we developed strontium doped and/or nanopatterned titanium surfaces, with flat titanium controls including osteogenic and strontium doped media controls. These were characterised using atomic force microscopy and ICP-mass spectroscopy. Cellular response assessed using human osteoblast/osteoclast co-cultures including scanning electron microscopy, quantitative immunofluorescence, histochemical staining, ELISA and PCR techniques. We further performed RNAseq gene pathway combined with metabolomic pathway analysis to build gene/metabolite networks. The surfaces eluted 800ng/cm2 strontium over 35 days with good surface fidelity. Osteoblast differentiation and bone formation increased significantly compared to controls and equivalently to oral treatment, suggesting improved osseointegration. Osteoclast pre-cursor survival and differentiation reduced via increased production of osteoprotegrin. We further delineated the complex cellular signalling and metabolic pathways involved including unique targets involved in osteoporosis. We have developed unique nanopatterned strontium eluting surfaces that significantly increase bone formation and reduce osteoclastogenesis. This synergistic combination of topography and chemistry has great potential merit in fusion surgery and arthroplasty, as well as providing potential targets to treat osteoporosis

Osteoinductive bone substitutes are in their developmental infancy and a paucity of effective grafts options persists despite clinical demand. Bone mineral substitutes such as hydroxyapatite cause minimal biological activity when compared to osteoinductive systems present biological growth factors in order to drive bone regeneration. We have previously demonstrated the in-vitro efficacy of a bioengineered system at presenting growth factors at ultra low-doses. This study aimed to translate this growth factor delivery system towards a clinically applicable implant. Osteoinductive surfaces were engineered using plasma polymerisation of poly(ethyl acrylate) onto base materials followed by adsorption of fibronectin protein and subsequently growth factor (BMP-2). Biological activity following ethylene oxide (EO) sterilisation was evaluated using ELISAs targeted against BMP-2, cell differentiation studies and atomic force microscopy. Scaffolds were 3D printed using polycaprolactone/hydroxyapatite composites and mechanically tested using a linear compression models to calculate stress/strain. In-vivo analysis was performed using a critical defect model in 23 mice over an 8 week period. Bone formation was assessed using microCT and histological analysis. Finally, a computer modelling process was developed to convert patient CT images into surface models, then formatted into 3D-printable scaffolds to fill critical defects. Following EO sterilisation, there was no change in scaffold surface and persistent availability of growth factors. Scaffolds showed adequate porosity for cell migration with mechanical stiffness similar to cancellous bone. Finally, the in vivo murine model demonstrated rapid bone formation with evidence of trabecular remodelling in samples presenting growth factors compared to controls

It is widely accepted by orthopaedic surgeons that antibiotics should be withheld until aspiration has been performed to increase the odds of identifying an organism in septic arthritis. Patients often present to other specialties that may not be as familiar with these principles. Twenty-five of forty-nine patients with septic arthritis of the native or prosthetic knee had received antibiotics prior to review by the orthopaedic service. Patients were significantly less likely to demonstrate an organism on initial microscopy (entire cohort p=0.001, native knees p=0.006, prosthetic knees p=0.033) or on subsequent culture (entire cohort p=0.001, native knees p=0.017, prosthetic knees p=0.012) of their aspirate if they had received antibiotics. The sensitivity of microscopy dropped from 0.58 to 0.12 when patients had received antibiotics (native knees 0.46 to 0, prosthetic knees 0.72 to 0.27). The sensitivity of the culture dropped from 0.79 to 0.28 when the patient had received antibiotics (native knees 0.69 to 0.21, prosthetic knees 0.91 to 0.36). Patients treated with empirical antibiotics are less likely to demonstrate an organism on microscopy and culture of their initial aspirate. There is a significantly high false negative rate associated with knee aspiration, particularly with prior administration of antibiotics

Aims. Compartment syndrome results from increased intra-compartmental pressure (ICP) causing local tissue ischaemia and cell death, but the systemic effects are not well described. We hypothesised that compartment syndrome would have a profound effect not only on the affected limb, but also on remote organs. Methods. Using a rat model of compartment syndrome, its systemic effects on the viability of hepatocytes and on inflammation and circulation were directly visualised using intravital video microscopy. Results. We found that hepatocellular injury was significantly higher in the compartment syndrome group (192 PI-labelled cells/10. -1 . mm. 3. , standard error of the mean (. sem. ) 51) compared with controls (30 PI-labelled cells/10. -1 . mm. 3. , . sem . 12, p < 0.01). The number of adherent venular white blood cells was significantly higher for the compartment syndrome group (5 leukocytes/30s/10 000 μm. 2. , . sem 1. ) than controls (0.2 leukocytes/30 s/10 000 μm. 2. , . sem . 0.2, p < 0.01). Volumetric blood flow was not significantly different between the two groups, although there was an increase in the heterogeneity of perfusion. Conclusions. Compartment syndrome can be accompanied by severe systemic inflammation and end organ damage. This study provides evidence of the relationship between compartment syndrome in a limb and systemic inflammation and dysfunction in a remote organ. Cite this article: Bone Joint J 2016; 98-B:1132–7

Antimicrobial resistance (AMR) is projected to result in 10 million deaths every year globally by 2050. Without urgent action, routine orthopaedic operations could become high risk and musculoskeletal infections incurable in a “post-antibiotic era.” However, current methods of studying AMR processes including bacterial biofilm formation are 2D in nature, and therefore unable to recapitulate the 3D processes within in vivo infection. Within this study, 3D printing was applied for the first time alongside a custom-developed bioink to bioprint 3D bacterial biofilm constructs from clinically relevant species including Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa. Bacterial viability and biofilm formation in bioprinted constructs was excellent, with confocal laser scanning microscopy (CSLM) used to demonstrate biofilm production and maturation over 28 days. Bioprinted 3D MRSA and MSSA biofilm constructs had greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Raman spectroscopy was also adapted in a novel approach to non-invasively diagnose 3D bioprinted biofilm constructs located within a joint replacement model. In conclusion, mature bacterial biofilm constructs were reproducibly 3D bioprinted for the first time using clinically relevant bacteria. This methodology allows the study of antimicrobial biofilm penetration in 3D, and potentially aids future antimicrobial research, replicating joint infection more closely than current 2D culture models. Furthermore, by deploying Raman spectroscopy in a novel fashion, it was possible to diagnose 3D bioprinted biofilm infections within a joint replacement model

Introduction. The concept of “bone graft expanders” has been popularised to increase the volume and biological activity of the implanted Material. HYPOTHESIS. Orthoss® granules support exogenously seeded MSCs and attract neighbouring host MSCs. Methods. In 3-D cultures’ Orthoss® granules were seeded with 2×10. 5. bone marrow MSCs/granule and maintained in MSC expansion or differentiation media for 21 days. In homing experiments’ bone autografts were placed in close proximity to Orthoss®. Scaffold colonisation and MSC differentiation were assessed by confocal microscopy’ standard electron microscopy’ and energy-dispersive X-ray spectroscopy. Results. Long-term incubation of MSC/scaffold resulted in formation of multiple cell-matrix layers lining the scaffold pores as well as outer surfaces. MSC differentiation to osteoblasts was evident as strong deposition of Calcium and Phosphorus was detected in both MSC expansion and osteogenic conditions. Cell egress experiments demonstrated the migration of cells from neighbouring autografts and their attachment and re-settlement on Orthoss®. Discussion & Conclusions. Orthoss® scaffolds support MSC attachment’ growth and osteogenic differentiation whereas resident bone subpopulations can rapidly migrate towards’ attach’ and expand on them. These results indicate that Orthoss® can serve as a graft expander for repairing large bone defects in trauma patients

A 7-day randomised controlled pre-clinical trial utilising an existing extremity war wound model compared the efficacy of saline soaked gauze to commercially available dressings. The Flexor Carpi Ulnaris of anaesthetised rabbits was exposed to high-energy trauma using a computer-controlled jig and inoculated with 10. 6. Staphylococcus aureus 3 hours prior to application of dressing. Quantitative microbiological assessment demonstrated reduced bacterial counts in INADINE (Iodine) and ACTICOAT (Nanocrystalline Silver) groups and an increase in ACTIVON TULLE (Manuka Honey) group (2-way ANOVA p<0.05). Clinical observations were made throughout the study. Haematology and plasma cytokines were analysed at intervals. Post-mortem histopathology included subjective semi-quantitative assessment of pathology severity using light microscopy to grade muscle injury and lymph node activation. Tissue samples were also examined using scanning electron microscopy (SEM). There were no bacteraemias, abscesses, purulent discharge or evidence of contralateral axillary lymph node activation. There were no significant differences in animal behaviour, weight change, maximum body temperature or white blood cell count elevation nor in pathology severity in muscle or lymph nodes (Kruskal-Wallis). There was no evidence of bacterial penetration or biofilm formation on SEM. Interleukin-4 and Tumour Necrosis Factor α levels were significantly higher in the ACTIVON TULLE group (1-way ANOVA p<0.05). This time-limited study demonstrated a statistically significant reduction in Staphylococcus aureus counts in wounds dressed with INADINE and ACTICOAT and an increase in wounds dressed with ACTIVON TULLE. There was no evidence that any of these dressings cause harm but nor have we established any definite clinical advantage associated with the use of the dressings tested in this study

Compartment syndrome, a devastating consequence of limb trauma, is characterised by severe tissue injury and microvascular perfusion deficits. We hypothesised that leucopenia might provide significant protection against microvascular dysfunction and preserve tissue viability. Using our clinically relevant rat model of compartment syndrome, microvascular perfusion and tissue injury were directly visualised by intravital video microscopy in leucopenic animals. We found that while the tissue perfusion was similar in both groups (38.8% (standard error of the mean (. sem). 7.1). , 36.4. % (. sem. 5.7), 32.0% (. sem. 1.7), and 30.5% (. sem. 5.35) continuously-perfused capillaries at 45, 90, 120 and 180 minutes compartment syndrome, respectively versus 39.2% (. sem. 8.6), 43.5% (. sem. 8.5). , . 36.6% (. sem. 1.4) and 50.8% (. sem. 4.8) at 45, 90, 120 and 180 minutes compartment syndrome, respectively in leucopenia), compartment syndrome-associated muscle injury was significantly decreased in leucopenic animals (7.0% (. sem. 2.0), 7.0%, (. sem. 1.0), 9.0% (. sem. 1.0) and 5.0% (. sem. 2.0) at 45, 90, 120 and 180 minutes of compartment syndrome, respectively in leucopenia group versus 18.0% (. sem. 4.0), 23.0% (. sem. 4.0), 32.0% (. sem. 7.0), and 20.0% (. sem. 5.0) at 45, 90, 120 and 180 minutes of compartment syndrome in control, p = 0.0005). This study demonstrates that the inflammatory process should be considered central to the understanding of the pathogenesis of cellular injury in compartment syndrome. Cite this article: Bone Joint J 2015;97-B:539–43

Introduction. The Precice nail is the latest intramedullary lengthening nail with excellent early outcomes. Implant complications have led to modification of the nail design. The aim of this study was to perform a retrieval study of Precice nails following lower limb lengthening. To assess macroscopic and microscopic changes to the implants and assess differences following design modification, with identification of potential surgical, implant and patient risk factors. Method. 15 nails were retrieved from 13 patients following lower limb lengthening. Macroscopic and microscopic surface damage to the nails were identified. Further analysis included radiology and micro-CT prior to sectioning. The internal mechanism was then analysed with Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy to identify corrosion. Results. 7 male and 3 females underwent 12 femoral lengthenings, 9 antegrade and 3 retrograde. 3 females underwent tibial lengthening. All patients obtained the desired length with no implant failure and full regenerate consolidation. Surface degradation was noted on the telescopic part of every nail design, less on the latest implants. Microscopic analysis confirmed fretting and pitting corrosion. Following sectioning black debris was noted in all implants. The early designs were found to have fractured actuator pins and the pin and bearings had evidence of corrosive debris. The latest designs had evidence of biological deposits suggestive of fluid ingress within the nail. Conclusion. This study suggests fluid ingress occurs with every generation of Precice nail despite modifications. The presence of biological fluid could be an early warning sign of potential corrosion. This in theory could lead to actuator pin fracture and implant failure. The clinical relevance is the potential re-use of a “dormant” nail in patients requiring secondary limb segment lengthening. Retraction of the nail in-situ and re-use for further lengthening requires careful consent for potential implant failure

Our unpublished data has indicated that the perivascular stem cells (PSCs) have increased chondrogenic potential compared to mesenchymal stem cells (MSCs) derived in culture. There has been a recent change in the theory that stem cells work by a paracrine effect rather than differentiation. There are minimal data demonstrating the persistence of implanted stem cells when used for engraftment. This study aimed to develop an autologous large animal model for perivascular stem cells as well as to determine if cells were retained in the articular cartilage defects. The reactivity of anti-human and anti-ovine antibodies was ascertained using immunohistochemistry and fluorescence-activated cell sorting (FACS). A panel of antibodies were combined and used to identify and purify pericytes (CD34-CD45-CD146+) and adventitial cells (CD34+CD45-CD146-) using FACS. The purified cells were cultured and their identity checked using FACS. These cultured cells demonstrated osteogenic, adipogenic and chondrogenic potential. Autologous ovine PSCs (oPSCs) were isolated, cultured and transfected using a GFP virus. The transfection rate was 88%. The cells were implanted into an articular cartilage defect on the medial femoral condyle using a hydrogel, four weeks following implantation the condyle was explanted and confocal laser scanning microscopy demonstrated the presence of oPSCs in the defect. Histology did not demonstrate any repair tissue at this early time point. These data have confirmed the viability our large animal model and that the implanted stem cells were retained in the defect four weeks following implantation

Articular cartilage has very poor repair potential, however it has an extraordinary capacity to withstand physiological mechanical loads in an intact joint. The nature and extent of chondrocyte death in articular cartilage following many forms of injury (trephine, scalpel, osteotome, sutures and drilling) has been characterised, but the ability to bear mechanical injury from iatrogenic surgical interventions is still unknown. A standard arthroscopic probe was moved at varying physiological pressures along the articular cartilage of joint before staining with fluorescent dyes to allow live/dead cell imaging using laser confocal scanning microscopy and imaging software, Image J. Bovine metatarsal phalangeal joints and fresh human cadaveric femoral condyles were used. The probe caused statistically significant chondrocyte death in bovine cartilage (p=0.02). Mild pressure 5% cell death, moderate (standard arthroscopic technique pressure) 22% and severe pressure 38%. A similar result was seen in human tissue with 24% cell death at moderate pressure compared to a control (p=0.0699). The widely assumed benign arthroscopic probe produces significant cell death in articular cartilage when used at standard operating pressures

In some centres, serial bedside aspirations, in association with intravenous antibiotics, are still an accepted treatment for septic arthritis (Mathews, Postgraduate Medical Journal, 2008). However, there is a risk that bacterial products remain in the joint, even when the bacteria have been destroyed. We have conducted a study to ascertain whether bacterial products alone have an effect on in situ chondrocyte viability. A hip aspirate (25μl), containing Staphylococcus aureus, from a patient with septic arthritis was added to 5ml culture medium and incubated (37°C) for 48hrs. The solution was then centrifuged (3400g for 10mins) and the supernatant removed. Cartilage explants were harvested from a bovine metacarpophalangeal joint, placed into the bacterial supernatant and incubated at 37°C. Explants were removed at hourly intervals over a 6-hour period and stained with the fluorescent probes chloromethylfluorescein di-acetate (10μM) and propidium iodide (10μM) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. Chondrocyte death increased markedly with time: 0.04% at 2hrs, 28% at 4hrs and 39% at 6hrs. This study shows that bacterial products rapidly penetrate the cartilage matrix and have a damaging effect on in situ chondrocyte viability. Further work will clarify the contributions made by the various toxic components in the culture supernatant, but these data support the need to remove the bacteria and their products aggressively as part of the treatment of septic arthritis

Staphylococcus aureus is a highly virulent pathogen and is implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections have suggested that alpha (Hla), beta (Hlb) and gamma (Hlg) toxins are key virulence factors. In particular, the ‘pore-forming’ alpha toxin is believed to be most potent. In this study, we have assessed the influence of alpha toxin on in situ chondrocyte viability. Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and placed into flasks containing Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with the following isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-) or DU1090 (Hla-Hlb+Hlg+). The explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death obtained using Volocity 4 software. The alpha toxin-producing S. aureus caused rapid cell death, with 24.8+/−3.7% at 18hrs and 44.6+/−7.2% at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%; p<0.001) compared to the alpha toxin knockout strain (4.1+/−1.7%; means +/− SEM; n=4). In situ chondrocyte viability was significantly compromised by alpha toxin, with beta and gamma toxins having minimal effect. Further work will clarify the exact mechanism through which this important toxin induces chondrocyte death. Thereafter, it is hoped that targeted treatments can be developed to reduce the extent of cartilage destruction during, and after, an episode of septic arthritis

Introduction. Nonunions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing, although the effects of different clinical ES waveforms available in clinical practice on BMMSCs cellular activities is unknown. Materials and Methods. We compared Direct Current (DC), Capacitive Coupling (CC), Pulsed Electromagnetic wave (PEMF) and Degenerate Wave (DW) by stimulating human-BMMSCs for 5 days for 3 hours a day. Cytotoxicity, cell proliferation, cell-kinetics and cell apoptosis were evaluated after ES. Migration and invasion were assessed using fluorescence microscopy and affected gene and protein expression were quantified. Results. DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms and unstimulated cells after 5 days of ES (p < 0.001). DC, DW and CC resulted in significantly more cells in S phase and G2/M phase (p < 0.01) compared to the unstimulated BMMSCs. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression (p < 0.001) compared to the other waveforms and unstimulated BMMSCs. DC increased cellular migration in a scratch-wound assay and all ES waveforms increased migration gene expression with DC having the greatest effect (p < 0.01). Conclusion. The ES waveform is vital in influencing BMMSCs cellular activities. Migration and invasion were increased by ES which suggests that the recruitment of BMMSCs to the healing site during a fracture could be increased by ES

Background. Continual implant stability is an important factor for the long-term success of cementless hip replacements. The increasing lifespan of patients causes a higher frequency of osteoporosis which may result in implant loosening due to bone loss. This study aimed to evaluate stability of long living implants in patients with advanced age. Patients and methods. Nine cementless stems made of Titanium-alloy including adjacent bone tissue obtained post mortem were evaluated by radiologic-microradigraphical, histological and morphometrical analysis. The percentage of the surface area covered by bone (BICI=bone implant contact index) was determined. The age of seven women and two men ranged between 81 and 92 years. The time in situ ranged between 10 and 20 years. From the entire length of the femora bearing implants 5 transverse segments were excised, dehydrated, embedded in methylmethacrylate. After the grinding procedure, the sections were evaluated by light microscopy and morphometrical analysis. The autopsy findings were recorded. Atherosclerosis and their related diseases were evident in all cases. Results. The femora of all female patients revealed features of high bony atrophy with concomitant transformation of the corticalis into spongy bone, whereas in male patients minor to moderate atrophic bone changes in the proximal femoral area without implication of the corticalis could be observed. All of the cementless stems made of Titanium-alloy showed osteointegration. The stabilization of the implant resulted in the forceps-like encasement of the edges of the implant within the cortical anchoring and by the development of compensatory bony hypertrophy. The BICI ranged between 35 und 63 percent. Conclusion. Elderly patients provided with cementless hip replacments revealed stable implants in spite of marked bone atrophy and an implantation period up to 20 years. Simultaneously, severe atherosclerosis and their related diseases, which may contribute to bone loss, were evident. The present findings may result from the favoring properties of cementless endoprostheses made of titanium alloy, cortical prosthesis anchoring, and self regulating bone processes. Pharmacologic and therapeutic consequences together with geriatric assessment should be required to preserve functionality and mobility

Engineered bone tissue to recreate the continuity of damaged skeletal segments is one of the field of interest of tissue engineering. Trabecular titanium has very good mechanical properties and high in vitro and in vivo biocompatibility: it can be used in biomedical applications to promote osteointegration demonstrating that it can be successfully used for regenerative medicine in orthopaedic surgery (1). Purpose of this investigation was to evaluate the behavior of adipose tissue derived stem cells (hASCs) cultured on scaffolds of Trabecular TitaniumTM (Lima-Lto) (TT). hASCs are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo (2). The hASCs were obtained from the subcutaneous adipose tissue of healthy donors during total hip replacement procedures after digestion with collagenase. They were seeded on monolayer and on the TT scaffolds, and incubated at 37 degrees C in 5% CO2 with osteogenic medium or control medium. The expression of bone-related genes using RT-PCR, time course of alkaline phosphatase activity and morphological investigation with Scanning Electron Microscopy (SEM) were performed to evaluate the osteogenic differentiation of hASCs. Alkaline phosphatase activity, marker of the differentiation toward the osteogenic pattern, was significantly higher in hASCs grown with osteogenic medium than in cells grown with control medium, both in monolayer and TT scaffolds; moreover, also alkaline phosphatase of hASCs grown on TT scaffolds in the presence of control medium increased with time, differently from that of cells grown on monolayer. The osteogenic differentiated hASCs expressed the bone-related genes type I collagen, osteocalcin, Runx-2 and alkaline phosphatase. SEM observations showed that hASCs differentiated toward osteoblast-like cells: they produced a big amount of extracellular matrix that covered the surface of the porous scaffolds with bridges between the pore walls. These data suggest that hASCs are able to adhere to TT scaffolds, to acquire an osteoblastic phenotype and to produce abundant extracellular matrix, with but also without osteogenic medium. We can therefore conclude that this material carries osteinductive properties being responsible of ostegenic differentiation; consequently, this scaffold/cells construct is effective to regenerate damaged tissue and to restore the function of bone tissue

Prosthetic joint infection is one of the most challenging complications of joint alloplasty and the diagnosis remains difficult. The aim of the study was to investigate the bacterial flora in surgical samples from 22 prosthetic patients using a panel of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, sequencing, phylogeny, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH). Concomitant samples were cultured by standard methods. Molecular methods detected presence of bacteria in samples from 12 of 22 patients. Using clone libraries a total of 40 different bacterial species were identified including known pathogens and species not previously described in association with prosthetic joint infections. The predominant species were Propionibacterium acnes and Staphylococcus epidermidis; polymicrobial infections were found in 9 patients. Culture-based methods showed bacterial growth in 8 cases with the predominant species being S. epidermidis. Neither anaerobic bacteria (including P. acnes) nor any of the species not previously described in implant infections were isolated. Additionally, 7 of the 8 culture positive cases were monomicrobial. Overall, the results of culture-based and molecular methods showed concordance in 11 cases (hereof 9 negative by both methods) and discrepancy in 6 cases. In the remaining 5 cases, culture-based methods identified only one species or a group of bacteria (e.g., coagulase negative staphylococci or coryneform rods), while culture-independent molecular methods were able to detect several distinct bacterial species including a species within the group identified by culture. A qPCR assay was developed to assess the abundance of Propionibacterium while S. aureus was quantified by a published S. aureus qPCR assay. These quantifications confirmed the findings from the clone library approach and showed the potential of qPCR for fast detection of bacteria in orthopedic samples. Additionally, both single cells and microcolonies were visualized using FISH and confocal scanning laser microscopy. In conclusion, the molecular methods detected a more diverse bacterial flora in prosthetic joint infections than revealed by standard culture-based methods, and polymicrobial infections were more frequently observed. The pathogenesis of these microorganisms and their role in implant-associated infections needs to be determined