Background. Following an anterior cruciate ligament (ACL) injury, the affected knee is known to experience bone loss and is at significant risk of becoming osteoporotic. Surgical reconstruction is performed to attempt to restore the function of the knee and theoretically restore this bone density loss. Cross-sectional analysis of the proximal tibia using peripheral quantitative computed tomography (pQCT) enables localised analysis of bone mineral density (BMD) changes. The aim of this study was to establish the pattern of bone density changes in the tibia pre- and post- ACL reconstruction using pQCT image analysis. Methods. Eight patients who underwent ACL reconstruction were included. A cross sectional analysis of the proximal tibia was performed using a pQCT scanner pre-operatively and one to two years post-operatively on both the injured and contralateral (control) knee. The proximal two and three percent slices [S2 and S3] along the tibia were acquired. These were exported to Matlab(tm) and automated segmentation was performed to remove the tibia from its surrounding structures. Cross correlation was applied to co-register pairs of images and patterns of change in BMD were mapped using a t-test (p<0.05). Connected components of pixels with significant change in BMD were created and used to assess the impact of ACL injury & reconstruction on the proximal tibial BMD. Results. Prior to surgical ACL reconstruction, the BMD in the injured leg was significantly reduced relative to the control leg [S2: p=0.002, S3: p=0.002]. Post surgery, the proximal tibial BMD did not change in either leg [Control S2: p=0.102, S3: p=0.181; Injured S2: p=0.093, S3: p=0.439]. The post surgical images demonstrated patterns of increasing BMD surrounding the tunnel in the form of compact bone. Discussion. A significant reduction in proximal tibial BMD was observed in the ACL injured legs relative to control legs. The pattern of pre-operative bone loss was generally observed to be global across the entire slice. No change in BMD was observed following ACL reconstruction, in either injured or control leg. These results indicate that proximal tibial BMD is reduced and does not change after ACL reconstruction

Introduction and Objective. Type 2 diabetes mellitus (T2DM), and the often concurrent obesity, causes metabolic changes that affect many organs and tissues, including bone. Despite a normal or even higher bone mineral density (BMD), T2DM has often been associated with a higher fracture risk, indicating a compromised bone quality. In this work, we use a novel congenic leptin receptor-deficient BioBreeding Diabetes Resistant rat (BBDR.cg.lepr.cp) to investigate the impact of T2DM and obesity on bone morphology and architecture at the microscale. Materials and Methods. Two different anatomical locations, i.e., femur and cranium, were studied combining micro-computed X-ray tomography (micro-CT) with scanning electron microscopy (SEM). Micro-CT data were examined using advanced image analysis tools in three-dimensions (3D). Results. Both parietal bones and femurs were smaller, i.e., thinner and shorter, respectively, in diabetic animals compared to healthy controls. Image analysis of the sagittal suture revealed a reduced suture width and length in diabetic animals, suggesting an altered bone apposition rate. Histomorphometry analysis from micro-CT data highlighted differences in microstructure of both trabecular and cortical femur between diabetic and healthy rats. In particular, bone volume fraction (BV/TV) was lower in the T2DM group, while trabecular spacing (Tb.Sp) was increased, overall indicating a higher porosity in diabetic trabecular bone. SEM revealed the presence of extended portions of hyper-mineralized cartilage in the distal femur of the diabetic animals. Conclusions. Micro-CT analyses, combined with SEM imaging, suggest that T2DM impacts bone growth and remodelling, in turn leading to differences in the structural organization at the microscale

The most important outcome predictor of Legg-Calvé-Perthes disease (LCPD) is the shape of the healed femoral head. However, the deformity of the femoral head is currently evaluated by non-reproducible, categorical, and qualitative classifications. In this regard, recent advances in computer vision might provide the opportunity to automatically detect and delineate the outlines of bone in radiographic images for calculating a continuous measure of femoral head deformity. This study aimed to construct a pipeline for accurately detecting and delineating the proximal femur in radiographs of LCPD patients employing existing algorithms. To detect the proximal femur, the pretrained stateof-the-art object detection model, YOLOv5, was trained on 1580 manually annotated radiographs, validated on 338 radiographs, and tested on 338 radiographs. Additionally, 200 radiographs of shoulders and chests were added to the dataset to make the model more robust to false positives and increase generalizability. The convolutional neural network architecture, U-Net, was then employed to segment the detected proximal femur. The network was trained on 80 manually annotated radiographs using real-time data augmentation to increase the number of training images and enhance the generalizability of the segmentation model. The network was validated on 60 radiographs and tested on 60 radiographs. The object detection model achieved a mean Average Precision (mAP) of 0.998 using an Intersection over Union (IoU) threshold of 0.5, and a mAP of 0.712 over IoU thresholds of 0.5 to 0.95 on the test set. The segmentation model achieved an accuracy score of 0.912, a Dice Coefficient of 0.937, and a binary IoU score of 0.854 on the test set. The proposed fully automatic proximal femur detection and segmentation system provides a promising method for accurately detecting and delineating the proximal femoral bone contour in radiographic images, which is necessary for further image analysis

Anterior vertebral body tethering (AVBT) is a growth modulating procedure used to manage idiopathic scoliosis by applying a flexible tether to the convex surface of the spine in skeletally immature patients. The purpose of this study is to determine the preliminary clinical outcomes for an adolescent patient cohort. 18 patients with scoliosis were selected using a narrow selection criteria to undergo AVBT. Of this cohort, 11 had reached a minimum follow up of 2 years, 4 had reached 18 months, and 3 had reached 6 months. These patients all demonstrated a primary thoracic deformity that was too severe for bracing, were skeletally immature, and were analysed in this preliminary study of coronal plane deformity correction. Using open-source image analysis software (ImageJ, NIH) PA radiographs taken pre-operatively and at regular follow-up visits post-operatively were used to measure the coronal plane deformity of the major and compensatory curves. Pre-operatively, the mean age was 12.0 years (S.D. 10.7 – 13.3), mean Sanders score 2.6 (S.D. 1.8-3.4), all Risser 0 and pre-menarchal, with mean main thoracic Cobb angle of 52° (S.D. 44.2-59.8°). Post-operatively the mean angle decreased to 26.4° (S.D. 18.4-32°) at 1 week, 30.4° (S.D. 21.3-39.6°) at 2 months, 25.7° (S.D. 18.7-32.8°) at 6 months, 27.9° (S.D. 16.2-39.6°) at 12 months, and 36.8° (S.D. 22.6– 51.0°) at 18 months and 38.2° (S.D. 27.6-48.7°) at 2 years. The change in curve at 2 years post-operative was statistically significant (P=0.004). There were 4 tether breakages identified that did not require return to theatre as yet, one patient underwent a posterior spinal instrumented fusion due to curve progression. AVBT is a promising new growth modulation technique for skeletally immature patients with progressive idiopathic scoliosis. This study has demonstrated a reduction in scoliosis severity

Abstract. Objective. The preparation of host degenerate cartilage for repair typically requires cutting and/or scraping to remove the damaged tissue. This can lead to mechanical injury and cartilage cell (chondrocytes) death, potentially limiting the integration of repair material. This study evaluated cell death at the site of cutting injury and determined whether raising the osmotic pressure (hyper-osmolarity) prior to injury could be chondroprotective. Methods. Ex vivo human femoral head cartilage was obtained from 13 patients (5 males and 8 females: 71.8 years old) with Ethical Permission and Patient consent. Cartilage wells were created using 3 or 5mm biopsy punches. Cell death at the wounded edge of the host cartilage and the edge of the extracted explants were assessed by quantifying the percentage of cell death (PCD) and measuring the width of the cell death zone at identified regions of interest (ROI) using the confocal laser scanning microscopy and image analysis software. To assess the chondroprotective effect of hyper-osmolarity, cartilage specimens were incubated in 340 or 600mOsm media, five minutes prior to injury to allow the chondrocytes to respond to the altered osmolarity. Wounded cartilage explants and cartilage wells were then cultured for a further 150 minutes following injury. Results. In 340mOsm media, the PCD around the 3mm cartilage wells was significantly less compared to the corresponding explants (20.05±10.24% vs 35.25±4.86%; P=0.0003). When using the 5mm biopsy punch, the PCD at the wound edges was significantly lower when compared to the 3mm cartilage wells (13.33±7.80% vs 20.05±10.24%; P=0.0121) at the same osmolarity. The width of the cell death zone for the well edges for both 3 and 5mm punches was significantly narrower when compared to their corresponding harvested cartilage explants in 340mOsm media (P<0.0001; P=0.0218, respectively). Exposing cartilage to raised osmolarity (600mOsm) prior to injury significantly reduced the PCD for cartilage wells produced by the 3mm biopsy punches (from 20.05±10.24% to 12.24±6.00%; P=0.0025). In addition, the zone of cell death was marginally reduced at the edges of the 5mm cartilage wells (19.25±15.78mm to 12.72±9.09mm; P=0.0499). Conclusions. The choice of biopsy punch and the osmolarity of the incubation medium prior to cartilage injury markedly affected the extent of chondrocyte death both at the edges of the cartilage wells and the explants. The smaller biopsy punch caused more chondrocyte death in the native cartilage wells compared to the larger punch, but this could be compensated for by the chondroprotective effect of raising the osmotic pressure. In general, there was less cell death at the wounded edges of the cartilage wells, compared to the explants. These results suggest that there is scope for further optimising the cutting implements used to create the cartilage wells and protecting chondrocytes by hyper-osmolarity in order to minimize cell death at cut edges and potentially enhance integration between cartilage repair material and host cartilage. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Introduction and Objective. Chronic tendinopathy is a multifactorial disease and a common problem in both, athletes and the general population. Mechanical overload and in addition old age, adiposity, and metabolic disorders are among the risk factors for chronic tendinopathy but their role in the pathogenesis is not yet unequivocally clarified. Materials and Methods. Achilles tendons of young (10 weeks) and old (100 weeks) female rats bred for high (HCR) and low (LCR) intrinsic aerobic exercise capacity were investigated. Both Achilles tendons of 28 rats were included and groups were young HCR, young LCR, old HCR, and old LCR (n = 7 tendons per group/method). In this rat model, genetically determined aerobic exercise capacity is associated with a certain phenotype as LCR show higher body weight and metabolic dysfunctions in comparison to HCR. Quantitative real-time PCR (qPCR) was used to evaluate alterations in gene expression. For histological analysis, semi-automated image analysis and histological scoring were performed. Results. Age-related downregulation of tenocyte marker genes (Tenomodulin), genes related to matrix modelling and remodeling (Collagen type 1, Collagen type 3, Elastin, Biglycan, Fibronectin, Tenascin C), and Transforming growth factor beta 3 (Tgfb3) were detected in tendons from HCR and LCR. Furthermore, inflammatory marker Cyclooxygenase 2 (Cox2) was downregulated, while Microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in tendons from old HCR and old LCR. No significant alteration was seen in Interleukin 6 (Il6), Interleukin 1 beta (Il1b), and Tumor necrosis factor alpha (Tnfa). Histological analysis revealed that Achilles tendons of old rats had fewer and more elongated tenocyte nuclei compared to young rats, indicating a reduced metabolic activity. Even though higher content of glycosaminoglycans as a sign of degeneration was found in tendons of old HCR and LCR, no further signs of tendinopathy were detectable in histological evaluation. Conclusions. Overall, aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue, while low intrinsic exercise capacity did not cause any changes. Even though tendinopathy was not present in any of the groups, some of the shown age-related changes correspond to single characteristics of chronic tendon disease. This study gives an insight into tendon aging and its contribution to molecular and cellular changes in Achilles tendon tissue

Joint surface restoration of deep osteochondral defects represents a significant unmet clinical need. Moreover, untreated lesions lead to a high rate of osteoarthritis. The current strategies to repair deep osteochondral defects such as osteochondral grafting or sandwich strategies combining bone autografts with ACI/MACI fail to generate long-lasting osteochondral interfaces. Herein, we investigated the capacity of juvenile Osteochondral Grafts (OCGs) to repair osteochondral defects in skeletally mature animals. With this regenerative model in view, we set up a new biological, bilayered, and scaffold-free Tissue Engineered (TE) construct for the repair of the osteochondral unit of the knee. Skeletally immature (5 weeks old) and mature (11 weeks old) Lewis rats were used. Cylindrical OCGs were excised from the intercondylar groove of the knee of skeletally immature rats and transplanted into osteochondral defects created in skeletally mature rats. To create bilayered TE constructs, micromasses of human periosteum-derived progenitor cells (hPDCs) and human articular chondrocytes (hACs) were produced in vitro using chemically defined medium formulations. These constructs were subsequently implanted orthotopically in vivo in nude rats. At 4 and 16 weeks after surgery, the knees were collected and processed for subsequent 3D imaging analysis and histological evaluation. Micro-computed tomography (µCT), H&E and Safranin O staining were used to evaluate the degree of tissue repair. Our results showed that the osteochondral unit of the knee in 5 weeks old rats exhibit an immature phenotype, displaying active subchondral bone formation through endochondral ossification, the absence of a tidemark, and articular chondrocytes oriented parallel to the articular surface. When transplanted into skeletally mature animals, the immature OCGs resumed their maturation process, i.e., formed new subchondral bone, partially established the tidemark, and maintained their Safranin O-positive hyaline cartilage at 16 weeks after transplantation. The bilayered TE constructs (hPDCs + hACs) could partially recapitulate the cascade of events as seen with the immature OCGs, i.e., the regeneration of the subchondral bone and the formation of the typical joint surface architecture, ranging from non-mineralized hyaline cartilage in the superficial layers to a progressively mineralized matrix at the interface with a new subchondral bone plate. Cell-based TE constructs displaying a hierarchically organized structure comprising of different tissue forming units seem an attractive new strategy to treat osteochondral defects of the knee

Osteocytes are terminally differentiated long-lived cells and account for greater than 95% of the bone cell population. It has been established that osteocytes are connected through their highly developed dendritic network, which is necessary for the maintenance of optimal bone homeostasis. However, little is known on how osteocytes use the network to coordinate their cellular function and communication that requires energy and protein turnover. Here using super-resolution confocal imaging on both live and fixed osteocytes, we demonstrated conclusively that mitochondria are widely distributed and dynamically shared between osteocytes. Using confocal live cell imaging analysis we showed that inhibiting the contact between mitochondria and endoplasmic reticulum (ER) by the knockdown of MFN2 in osteocytes impedes the transfer of mitochondria suggesting the involvement of ER contact with mitochondria in the transfer process. Moreover, we showed that transport of mitochondria between osteocytes within the network enables rescue of osteocytes with dysfunction of mitochondria. Using the 3D tetraculture system with confocal imaging, we identify the transfer of mitochondria from healthy osteocytes enables recovery of mitochondria activities in osteocytes that devoid of mitochondrial DNA by ethidium bromide. The results indicated that when osteocytes are depleted of functional mitochondria, normal parental osteocytes can transfer mitochondria to these stressed osteocytes to provide them with energy. Collectively we show for the first time that the utilisation of mitochondrial transfer enables osteocytes to function with a network and coordinate their cellular activities in response to different energy demands

Objectives. Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied. Methods. We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis. Results. Our results show that clinical strains adhere to the material surface at lower concentrations than collection strains. A destructive effect of bacteria on preosteoblastic cells was also detected, especially with higher concentrations of bacteria. Conclusions. The method described herein can be used to evaluate the effect of surface modifications on bacterial adherence more accurately than conventional monoculture studies. Clinical strains behave differently than collection strains with respect to bacterial adherence. Cite this article: M. Martinez-Perez, C. Perez-Jorge, D. Lozano, S. Portal-Nuñez, R. Perez-Tanoira, A. Conde, M. A. Arenas, J. M. Hernandez-Lopez, J. J. de Damborenea, E. Gomez-Barrena, P. Esbrit, J. Esteban. Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the “race for the surface” theory. Bone Joint Res 2017;6:315–322. DOI: 10.1302/2046-3758.65.BJR-2016-0226.R2

Poly-ether-ether-ketone (PEEK) is a biomaterial commonly used for spinal implants and screws. It is often desirable for orthopaedic implants to osseointegrate, but as PEEK is biologically inert this will not occur. The aim of this project was to determine if injection mould nanopatterning can be used to create a make PEEK bioactive and stimulate osteogenesis in vitro. PEEK substrates were fabricated by injection mould nanopatterning to produce near-square (NSQ) nanopatterned PEEK and planar (FLAT) PEEK samples. Atomic force microscopy (AFM) and scanning electron microscopy were used to characterize the surface topography. Human bone marrow stromal cells (hBMSCs) were isolated from patients undergoing primary hip replacement operations and seeded onto the PEEK substrates. After 6 weeks the cells were stained using alizarin red S (ARS) stain (to detect calcium) and the von Kossa technique (to detect phosphate) and analyzed using CellProfiler image analysis software to determine: surface coverage; cell number; and expression of either calcium (ARS stain) or phosphate (von Kossa technique). ARS stain showed calcium expression (quantified relative to the number of cells) was increased on NSQ PEEK compared to FLAT PEEK (not statistically significant) and the surface coverage was similar. Von Kossa staining revealed more surface coverage on FLAT PEEK (69.1% cf. 31.9%), cell number was increased on FLAT PEEK (9803 ± 4066 cf. 4068 ± 1884) and phosphate expression relative to cell number was also increased (seven-fold) on NSQ PEEK (P < 0.05) compared to FLAT PEEK. Although hBMSCs may adhere to NSQ PEEK in smaller numbers, the cells expressed a relatively larger amount of calcium and phosphate. This indicates that the cells adopted a more osteoblastic phenotype and that nanopatterning PEEK induces hBMSC differentiation and stimulates osteogenesis. Injection mould nanopatterning therefore has the potential to improve osseointegration of PEEK implants in vivo

Summary Statement. The peripheral neuronal phenotype is significantly altered in rotator cuff tendinopathy (RCT) with a clear upregulation of the Glutaminergic system being present in disease. Introduction. Shoulder pain is the third most frequent cause of chronic musculoskeletal pain in the community and is usually caused by rotator cuff tendinopathy (RCT). The central and peripheral nervous system play an important role in both tissue homoeostasis and tendon healing. The Glutaminergic system is of key importance in driving the peripheral and central neuronal changes which increase the body's sensitivity to pain (1, 2). No study to date has investigated the role of the glutaminergic system in human RCT. We hypothesised that the peripheral neuronal phenotype would be altered in RCT, and would vary according to disease stage as measured by size of tear. The term ‘peripheral neuronal phenotype’ is used to refer to refer to specific characteristics of the peripheral nervous system, neuronal mediators and the receptors for these mediators in peripheral tissue. Methods. Rotator cuff tendon specimens were obtained from 64 patients undergoing the surgical repair of rotator cuff tears. Control supraspinatus tendon was obtained from 10 patients undergoing surgery for anterior instability using an ultrasound guided biopsy technique. Patients with rotator cuff tears were divided into 2 groups: the small/medium group (≤ 3cm size) and the large/massive group (>3cm size). The tendon tissue was histologically stained using Haematoxylin and Eosin, and immunohistochemically stained with primary antibodies visualised using 3, 3′-diaminobenzidine (DAB). Image analysis was performed blindly by 2 observers using Image-J to quantify the amount of DAB positive staining. Data was non-parametric in distribution and Mann-Whitney U tests were carried out using SPSS with significance levels set at a minimum of p<0.025. Results. There were significant changes in the peripheral neuronal phenotype in RCT. The Glutaminergic system was significantly up-regulated with an increase in Glutamate and changes in several related receptors in disease versus control (p<0.01). The standard deviation in nuclei count and mean cell nuclear area were both increased in disease (p<0.01) compared to controls. Tendon vascularity and cell proliferation were reduced in disease vs control (p<0.01). There were no significant correlations between pain scores and the peripheral tissue markers. Discussion/Conclusion. The peripheral neuronal phenotype is significantly altered in rotator cuff tendinopathy (RCT) with clear changes in the Glutaminergic system in disease. These findings are novel and improve our understanding of pain and tissue healing in RCT, potentially providing novel therapeutic targets

Introduction. Lesion location and volume are critical factors to select patients with osteonecrosis for whom resurfacing arthroplasty is appropriate. However, no reliable surgical planning system which can assess relationship between necrotic lesions and the femoral component has been established. We have developed a 3D-MRI-based planning system for resurfacing arthroplasty. The purpose of the present study was to evaluate its feasibility. Methods. The subjects included five patients with osteonecrosis of ARCO stage 3 or 4 who had undergone resurfacing THA at our institute. All patients had an MRI before surgery using 3D-SPGR sequences and fat suppression 3D-SPGR sequencea. In cases where it was difficult to distinguish bone marrow edema and reparative zone on 3D-SPGR images, fat suppression 3D-SPGR sequences were used. Simulation of resurfacing arthroplasty was performed on image analysis software where multidirectional oblique views could be reconstructed. The femoral neck axis was determined by drawing line through centers of two spheres which were fitted to the normal portion of the femoral head and the mid-portion of femoral neck. A femoral component was virtually implanted to align the femoral neck axis and match the implant center and femoral head center. Results. Planning could be performed within 10 minutes in every case. In all cases, size selection of acetabular and femoral component was within 1 size of actually implanted components. This 3D-MRI based planning system was useful to assess proportion and location of necrotic lesion in the preserved portion of femoral head in resurfacing THA. Conclusion. This preliminary study demonstrated that a 3D-MRI based planning system was useful in surgical planning of resurfacing arthroplasty for patients with osteonecrosis

Finite element models of the musculoskeletal system have the possibility of describing the in vivo situation to a greater extent than a single in vitro experimental study ever could. However these models and the assumptions made must be validated before they can be considered truly useful. The object of this study was to validate, using digital image correlation (DIC) and strain gauging, a novel free boundary condition finite element model of the femur. The femur was treated as a complete musculoskeletal construct without specific fixed restraint acting on the bone. Spring elements with defined force-displacement relationships were used to characterize all muscles and ligaments crossing the hip and knee joints. This model was subjected to a loading condition representing single leg stance. From the developed model muscle, ligament and joint reaction forces were extracted as well as displacement and strain plots. The muscles with the most influence were selected to be represented in the simplified experimental setup. To validate the finite element model a balanced in vitro experimental set up was designed. The femur was loaded proximally through a construct representative of the pelvis and balanced distally on a construct representing the tibio-femoral joint. Muscles were represented using a cabling system with glued attachments. Strains were recorded using DIC and strain gauging. DIC is an image analysis technique that enables non-contact measurement of strains across surfaces. The resulting strain distributions were compared to the finite element model. The finite element model produced hip and knee joint reaction forces comparable to in vivo data from instrumented implants. The experimental models produced strain data from both DIC and strain gauging; these were in good agreement with the finite element models. The DIC process was also shown to be a viable method for measuring strain on the surface of the specimen. In conclusion a novel approach to finite element modeling of the femur was validated, allowing greater confidence for the model to be further developed and used in clinical settings

Summary. Increased lateral ulnotrochlear joint space due to improper sizing in radial head arthroplasty may result in medial collateral ligament laxity, leading to increased osteophytes and arthritis. Introduction. Radial head (RH) arthroplasty is a common response to comminuted RH fractures. Typical complications include improper sizing, leading to changes in joint kinematics. Evidence of these changes should be visible through fluoroscopic images of affected joints. The two examined changes in this study are the ulnar deviation from distal radial translation (DRT), and the widening of the lateral ulnotrochlear joint space (LUT). Methods. Eight fresh-frozen cadaver arms were used. Initial images were taken with the native RH intact. The Kocher approach exposed the radiocapitellar (RC) joint capsule, preserving all ligaments. The RH was excised and Integra Katalyst CoCr (Plainsboro, NJ) telescoping, bipolar, RH inserted. Images were taken with implant sizings: −2mm, 0mm, +2mm, and +4mm, (from native) using 1mm washers preventing implant bipolarity. AP fluoroscopic images of the elbow were taken at full extension. Joint spaces were measured using image analysis, normalised using known radio-opaque lengths. Four LUT measurements were made, two medially and two laterally, and normalised by measuring the RH implant diameter. Each set (medial and lateral) were averaged together and the resulting value used for all comparisons. Images of distal ulnar deviation at the wrist were taken with the wrist in supination, the hand rotated medially. Measurements were from the distal medial radial tip to the distal lateral ulnar tip. Images were normalised by placing a scalpel in the same plane as measurement. Results. DRT values were difference paired for each arm using the 0mm values as baselines. One-way ANOVA of the paired values resulted in significant DT with sizing increases (p<0.01). The quotient of DRT and sizing determined comparative impact with the LUT increase. LUT joint gap measurements were percentage paired, with natives as the baseline, and One-way ANOVA used. A significant increase in LUT spacing occurred with increased sizings (p<0.01). Discussion. Increased ulnar deviation can increase loading on the TFCC, leading to possible TFCC tear, increased articular cartilage wear from carpal misalignment, and eventual wrist instability and arthritis. The percentage of the radial lengthening is represented in DRT. Over-sizing results in small percentages of increased radial length at the wrist, therefore deviation at the elbow must take place, either through rotation of the ulna, or translation. Either of these can be seen through LUT measurements. Previous measurements of the LUT space were made by Frank (2009), with similar results. This was being used as a method of improper sizing detection using radiographs. The percentage difference of LUT space for corresponding sizing: there is an increase in LUT space for every sizing; maybe due to loosening of the soft tissue from arthroplasty. Increased LUT space indicates the medial translation of proximal ulna. This can result in Medial Collateral Ligament laxity, leading to increased osteophytes, and arthritis. Use and non-treatment, can create a chronic, painful, disorder

Summary Statement. O-terminated nanocrystalline diamond films proposed as bone implant coatings are promising for adhesion and growth of osteoblasts, as well as for osteogenic cell differentiation and extracellular matrix production. Nanocrystalline diamond (NCD) films are promising materials for tissue engineering, especially for bone implants coating, due to their biocompatibility, chemical resistance and mechanical hardness. Nanostructure and morphology of the NCD films can efficiently mimic the properties of natural tissues, and thus they support the cell adhesion, proliferation and differentiation. In addition, the NCD wettability can be tailored by grafting specific atoms and functional chemical groups (e.g., oxygen, hydrogen, amine groups, etc.) which influence the adsorption and final geometry of proteins, and thus the behaviour of cultivated cells. Therefore, the NCD films are proposed as multifunctional materials for fundamental studies on the growth and adhesion of osteoblasts on bone implants, which is particularly our interest. The NCD films used in this study were grown on silicon substrates by microwave plasma-enhanced chemical vapor deposition. The quality of the grown NCD films was investigated by Raman spectroscopy, scanning electron microscopy and atomic force microscopy. In order to control the hydrophobic or hydrophilic character, the NCD film surfaces were grafted by hydrogen (H-termination) or oxygen (O-termination) atoms. The influence of surface termination on the surface wettability (wetting contact angle) was characterised by reflection goniometry using droplet of deionised water. The primary human osteoblasts and osteoblast-like Saos-2 cells were used for biological studies on H- and O-terminated NCD films. The cell adhesion and spreading was analysed by the visualisation of focal adhesion proteins (talin, paxillin) and actin fibers. Expression of markers of osteogenic cell differentiation (alkaline phosphatase, osteocalcin, collagen I) was monitored by the reverse transcription and Real-time PCR method, and also by immunostaining of expressed proteins and image analysis. The extracellular matrix production and composition, i.e. collagen content, calcium content and activity of alkaline phosphatase, were also quantified. Native type I collagen fibres were visualised by two-photon excitation microscopy and second harmonic generation imaging, together with immunostaining and fluorescence microscopy. We found that primary human osteoblasts cultivated on the O-terminated NCD films exhibited better adhesion compared to the H-terminated NCD films. Also the expression of osteogenic cell markers such as collagen and osteocalcin was higher on the O-terminated films. The mature collagen fibers were detected in Saos-2 cells on both H- and O-terminated NCD films; however, the quantity of collagen in extracellular matrix was higher on O-terminated NCD films. The amount of calcium and alkaline phosphatase activity were also significantly higher in Saos-2 cell layers on O-terminated NCD films. In conclusion, the higher wettability of the O-terminated NCD films (contact angle < 20°) is promising for adhesion and growth of osteoblasts. Besides, the O-terminated surface also seems to support the osteogenic differentiation of the cultivated cells, production of extracellular matrix proteins and subsequent extracellular matrix mineralization. This work was supported within the project “The Centre of Biomedical Research” (CZ.1.07/2.3.00/30.0025). This project is co-funded by the European Social Fund and the state budget of the Czech Republic. Other supports were provided by the Grant Agency of the Czech Republic (grant No. P108/11/0794)

Porous collagen-glycosaminoglycan (Col/GAG) scaffolds have previously been used clinically as regeneration templates for peripheral nerves and skin. [1]. For defects involving even minimal load-bearing applications however, these scaffolds do not possess the required stiffness. Calcium phosphates (CaPs) are often used as bone-graft substitutes due to their biocompatibility and direct bone-bonding ability. While CaPs have sufficient stiffness for bone-defect applications, unlike Col/GAG they lack elasticity and are very brittle. Combining these two materials produces a composite with enhanced material properties and chemical similarity to natural bone. The addition of CaP nanocrystallites into the Col/GAG matrix produces a 3-dimensional structure that maintains its structural integrity even when wet. In this study, the in vivo performance of mineralised Col/GAG composites was evaluated by implantation into a six-week ovine bone-defect model. Four different materials were implanted; Col/GAG alone, Col/GAG with octacalcium phosphate, Col/GAG with hydroxyapatite and Col/GAG with brushite. Implants with a diameter of 9mm and length of 9mm, were placed bilaterally into the distal femoral condyle of the hind legs of thirteen sheep. This site was selected due to the large volume of load-bearing cancellous bone. Cancellous autograft was harvested from the tibial tuberosity and placed in the defect sites of two sheep as a positive control. All animals were sacrificed after 6 weeks and tissue containing the implants was prepared for histological evaluation. Image analysis of Von Kossa stained sections showed that all mineralised Col/GAG implants had significantly more bone in the implant site than unmineralised Col/GAG but were not significantly different between CaPs. Interestingly, new bone formation often followed the structure of the porous material struts which acted as a template. The defect containing the autograft contained the greatest amount of new bone. Conclusions. The inclusion of mineral substantially improves the osteoconductivity of Col/GAG. No significant difference between the different calcium phosphates was seen. Whilst these materials did not stimulate bone formation to the same extent as autograft, many bone graft procedures are carried out with allograft which performs less favourably

Metal and their alloys have been widely used as implantable materials and prostheses in orthopaedic surgery. However, concerns exist as the metal nanoparticles released from wear of the prostheses cause clinical complications and in some cases result in catastrophic host tissue responses. The mechanism of nanotoxicity and cellular responses to wear metal nanoparticles are largely unknown. The aim of this study was to characterise macrophage phagocytosed cobalt/chromium metal nanoparticles both in vitro and in vivo, and investigate the consequent cytotoxicity. Two types of macrophage cell lines, murine RAW246.7 and human THP-1s were used for in vitro study, and tissues retrieved from pseudotumour patients caused by metal-on-metal hip resurfacing (MoMHR) were used for ex vivo observation. Transmission electron microscopy (TEM), scanning electron microscopy (SEM) in combination with backscatter, energy-disperse X-ray spectrometer (EDS), focused ion beam (FIB) were employed to characterise phagocytosed metal nanoparticles. Alamar blue assay, cell viability assays in addition to confocal microscopy in combination with imaging analysis were employed to study the cytotoxiticy in vitro. The results showed that macrophages phagocytosed cobalt and chromium nanoparticles in vitro and the phagocytosed metal particles were confirmed by backscatter SEM+EDS and FIB+EDS. these particles were toxic to macrophages at a dose dependent manner. The analysis of retrieved tissue from revision of MoMHR showed that cobalt/chromium metal nanoparticles were observed exclusively in living macrophages and fragments of dead macrophages, but they were not seen within either live or dead fibroblasts. Dead fibroblasts were associated with dead and disintegrated macrophages and were not directly in contact with metal particles; chromium but not cobalt was the predominant component remaining in tissue. We conclude that as an important type of innate immune cells and phagocytes, macrophages play a key role in metal nanoparticles related cytotoxicity. Metal nanoparticles are taken up mainly by macrophages. They corrode in an acidic environment of the phagosomes. Cobalt that is more soluble than chromium may release inside macrophages to cause death of individual nanoparticle-overloaded macrophages. It is then released into the local environment and results in death of fibroblasts and is subsequently leached from the tissue

Long term, secondary implant fixation of Total Disc Replacements (TDR) can be enhanced by hydroxyapatite or similar osseo-conductive coatings. These coatings are routinely applied to metal substrates. The objective of this in vivo study was to investigate the early stability and subsequent bone response adjacent to an all polymer TDR implant over a period of six months in an animal model. Six skeletally mature male baboons (Papio annubis) were followed for a period of 6 months. Using a transperitoneal exposure, a custom-sized Cadisc L device was implanted into the disc space one level above the lumbo-sacral junction in all subjects. Radiographs of the lumbar spine were acquired prior to surgery, and post-operatively at intervals up to 6 months to assess implant stability. Flourochrome markers (which contain molecules that bind to mineralization fronts) were injected at specified intervals in order to investigate bone remodeling with time. Animals were humanely euthanized six months after index surgery. Test and control specimens were retrieved, fixed and subjected to histological processing to assess the bone-implant-bone interface. Fluorescence microscopy and confocal scanning laser microscopy were utilized with BioQuant image analysis to determine the bone mineral apposition rates and gross morphology. Radiographic evaluation revealed no loss of disc height at the operative level or adjacent levels. No evidence of subsidence or significant migration of the implant up to 6 months. Heterotopic ossification was observed to varying degrees at the operated level. Histology revealed the implant primary fixation features embedded within the adjacent vertebral endplates. Flourochrome distribution revealed active bone remodeling occurring adjacent to the polymeric end-plate with no evidence of adverse biological responses. Mineral apposition rates of between 0.7 and 1.7 microns / day are in keeping with literature values for hydroxyapatite coated implants in cancellous sites of various species. Radiographic assessment demonstrates that the Cadisc L implant remains stable in vivo with no evidence of subsidence or significant migration. Histological analysis suggests the primary fixation features are engaged, and in close apposition with the adjacent vertebral bone. Flourochrome markers provide evidence of a positive bone remodelling response in the presence of the implant

Introduction. Histology remains the gold standard in morphometric and pathological analyses of osteochondral tissues in human and experimental bone and joint disease. However, histological tissue processing is laborious, destructive and only provides a two-dimensional image in a single anatomical plane. Micro computed tomography (μCT) enables non-destructive three-dimensional visualization and morphometry of mineralized tissues and, with the aid of contrast agents, soft tissues. In this study, we evaluated phosphotungstic acid-enhanced (PTA) μCT to visualize joint pathology in spine osteoarthritis. Methods. Lumbar facet joint specimens were acquired from six patients (5 female, age range 31–78) undergoing decompression surgery. Fresh osteochondral specimens were immediately fixed in formalin and scanned in a benchtop μCT scanner (65 kV, 153 mA, 25 μm resolution). Subsequently, samples were completely decalcified in 5% formic acid, equilibrated in 70% ethanol and stained up to ten days in 1% PTA (w/v) in 70% ethanol. PTA-stained specimens were scanned at 70 kV, 140 mA, 15 μm resolution. Depth-dependent analysis of X-ray attenuation in cartilage tissues was performed using ImageJ. Bone structural parameters of undecalcified and PTA-stained specimens were determined using CT Analyser and methods were compared using correlation and Bland-Altman analysis. Results. The maximal penetration depth of PTA in decalcified facet joint was 5 mm. Bone tissue showed strong and uniformly distributed X-ray attenuation, while mild to moderate and differentially distributed attenuation was observed in articular cartilage and subchondral marrow spaces. Measurements of bone volume (r=0.90, p=0.01) and bone surface (r=0.95, p=0.004) were strongly correlated between undecalcified and PTA-stained samples. Compared with PTA-stained samples, measurements in undecalcified specimens were consistently higher (∼14%). PTA-enhanced μCT visualization of cartilage tissues enabled the identification of individual chondrocytes and their pericellular microenvironment (chondrons). Owing to loss of collagen lower X-ray attenuation was observed in the middle and deep cartilage layers at the central, but not peripheral, regions of the degenerated facet joint specimens. Depth-dependent analysis of PTA-staining intensity suggested that the extent of collagen loss in articular cartilage might correlate with the thickness of the subchondral cortical plate. Conclusion. PTA-enhanced μCT is a low-cost, non-toxic and highly feasible method for ex vivo 3D-visualization of osteochondral pathology in human osteoarthritis. The method enables bone morphometric analysis, as well as collagen distribution in all anatomical planes. Contrast enhanced μCT has several applications in bone and osteoarthritis research including 3D histopathological grading, tissue stratification, and imaging and analysis of aberrant collagen metabolism in osteochondral disease

Introduction. This study investigated the binding agent Calcium/Sodium Alginate fibre gel and the addition of autogenic bone marrow aspirate (BMA) on bone growth into a porous HA scaffold implanted in an ovine femoral condyle critical-sized defect. Our hypothesis was that Alginate fibre gel would have no negative effect on bone formation and osteoconduction within the scaffold and that BMA would augment the incorporation of the graft with the surrounding bone at 6 and 12 weeks post implantation. Methods. 24, 8mm x 15mm defects were filled with either porous HA granules, porous HA granules + Alginate fibre gel (HA putty) or porous HA granules + Alginate fibre gel + BMA (HA putty +BMA) and remained in vivo for 6 and 12 weeks (n=4). 1ml of bone marrow aspirate per cm3 of graft was used. Image analysis quantified bone apposition rates, bone ingrowth, bone-implant contact and quantity of graft. Mann Whitney U tests were used for statistical analysis where p<0.05 was considered significant. Results. Highest bone formation were measured in the 12 week HA putty+BMA group (1.57±0.24(micromillimetres/day). HA granules at 12 weeks encouraged the greatest increase in bone formation (33.56±3.53%). Smaller amounts of bone was measured in the 6 week HA putty+BMA group (8.57±2.86%). Bone formation in the HA group at 12 weeks was significantly higher when compared with the HA putty (p= 0.043) and the HA putty+BMA group (p= 0.043). At both the 6 and 12 week time point, highest bone-implant contact was seen in the HA granules group (59.34±10.89% and 72.65±3.38% respectively) when compared with both the HA putty (p=0.018) and HA putty+BMA (p=0.047). Results showed no significant difference in the amount of implant remaining when each group was compared. Conclusions. Results from this study showed that the inclusion of BMA did not augment bone growth to the scaffold or increase its osteoconductive capacity when combined with Calcium/Sodium Alginate fibre gel. Further research is necessary to optimise Calcium/Sodium Alginate fibre gel when used to bind HA granules and to investigate the effect of BMA with this type of HA alone