Cigarette smoking has a negative impact on the skeletal system by reducing bone mass and increasing the risk of fractures through its direct or indirect effects on bone remodeling. Recent evidence shows that smoking causes an imbalance in bone turnover, making bone vulnerable to osteoporosis and fragility fractures. In addition, cigarette smoking is known to have deleterious effects on fracture healing, as a positive correlation has been shown between the daily number of cigarettes smoked and years of exposure to smoking, although the underlying mechanisms are not fully understood. Smoking is also known to cause several medical and surgical complications responsible for longer hospital stays and a consequent increase in resource consumption. Smoking cessation is, therefore, highly advisable to prevent the onset of metabolic bone disease. However, some of the consequences appear to continue for decades. Based on this evidence, the aim of our work was to assess the impact of smoking on the skeletal system, particularly bone fractures, and to identify the pathophysiological mechanisms responsible for the impairment of fracture healing. Because smoking represents a major public health problem, understanding the association between cigarette smoking and the occurrence of bone disease is necessary in order to identify potential new targets for intervention

Advances in our understanding of skeletal stem cells and their role in bone development and repair, offer the potential to open new frontiers in bone regeneration. However, the ability to harness these cells to replace or restore the function of traumatised or lost skeletal tissue as a consequence of age or disease remains a significant challenge. We have developed protocols for the isolation, expansion and translational application of skeletal cell populations with cues from developmental biology informed by in vitro and ex vivo models as well as, nanoscale architecture and biomimetic niche development informing our skeletal tissue engineering approaches. We demonstrate the importance of biomimetic cues and delivery strategies to directly modulate differentiation of human adult skeletal cells and, central to clinical application, translational studies to examine the efficacy of skeletal stem and cell populations in innovative scaffold compositions for orthopaedics. While a number of challenges remain multidisciplinary approaches that integrate developmental and engineering processes as well as cell, molecular and clinical techniques for skeletal tissue engineering offer significant promise. Harnessing such approaches across the hard tissue interface will ultimately improve the quality of life of an increasing ageing population

Recent approaches have sought to harness the potential of stem cells to regenerate bone lost as a consequence of trauma or disease. Bone marrow aspirate (BMA) provides an autologous source of skeletal stem cells (SSCs) for such applications, however previous studies have demonstrated that the concentration of SSCs present in iliac crest BMA is below that required for robust bone regeneration. Here we present a novel acoustic-facilitated filtration strategy to concentrate BMA for SSCs, clinically applicable for intra-operative orthopaedic use. The aim of this study was to demonstrate the efficacy of this strategy in concentrating SSCs from iliac crest bone marrow, as well as femoral canal BMA from older patients. Iliac crest BMA (Lonza, Rockville, MD, USA) and femoral canal BMA was obtained with informed consent from older patients during total hip replacement. 5 to 40ml of BMA was processed via the acoustically-aided exclusion filtration process to obtain 2-8 fold volume reductions. SSC concentration and function was assessed by flow-cytometry, assays for fibroblastic colony-forming units (CFU-F) and multi-lineage differentiation along chondrogenic, osteogenic and adipogenic pathways examined. Seeding efficiency of enriched and unprocessed BMA (normalised to cell number) onto allograft was assessed. Iliac crest BMA from 15 patients was enriched for SSCs in a processing time of only 15 minutes. Femoral BMA from 15 patients in the elderly cohort was concentrated up to 5-fold with a corresponding enrichment of viable and functional SSCs, confirmed by flow cytometry and assays for CFU-F. Enhanced osteogenic (P<0.05) and chondrogenic (P<0.001) differentiation was observed using concentrated aspirate, as evidenced by biochemical assay and semi-quantitative histological analysis. Furthermore, enhanced cell seeding efficiency onto allograft was seen as an effect of SSC concentration per ml of aspirate (P<0.001), confirming the utility of this approach for application to bone regeneration. The ability to rapidly enrich BMA demonstrates potential for intra-operative application to enhance bone healing and offers immediate capacity for clinical application to treat many scenarios associated with local bone stock loss. Further in vivo analysis is ongoing prior to clinical tests

Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect. Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1

Introduction and Objective. Calcium phosphates are among the most commonly used bone graft substitute materials. Compositions containing predominantly monetite (∼84.7%) with smaller additions of beta-tricalcium phosphate (β-TCP; ∼8.3%) and calcium pyrophosphate (Ca-PP; ∼6.8%) have previously been demonstrated to exhibit osteoinductive properties. Such a multi-component calcium phosphate bioceramic was fashioned in the form of hollowed-out, dome-shaped devices (15 mm diameter, 4 mm height), each reinforced with a 3D printed Ti6Al4V ELI frame. With the aim to induce bone formation beyond the skeletal envelope, these devices were investigated in vivo using a sheep (Ovis aries) occipital bone model. Materials and Methods. The bioceramic composition was prepared from a mixture of β-TCP/dicalcium pyrophosphate and monocalcium phosphate monohydrate powders mixed with glycerol. The Ti6Al4V ELI frame was positioned into a dome-shaped mould and bioceramic paste was poured over the frame and allowed to set, in sterile water, prior to removal from the mould. In adult female sheep (n=7), the devices were positioned directly over the bone and stabilised using self-drilling screws. After 52 weeks, the devices were retrieved, resin embedded, and used for X-ray micro-computed tomography (micro-CT), histology, backscattered electron scanning electron microscopy (BSE-SEM), energy dispersive X-ray spectroscopy (EDX), micro-Raman spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Results. The bioceramic composition (Ca/P: ∼0.85 at. %) transforms to carbonated apatite (Ca/P: ∼1.2 at. %, Mg/Ca: ∼0.03 at. %), in vivo, largely at the expense of monetite and Ca-PP whereas β-TCP remains detectable. Discrete particles of Ca-PP are identified by correlative BSE-SEM and micro-Raman spectroscopy. Together with chemical transformation, physical degradation is evident within the bulk of the bioceramic. Beyond the confines of the skeletal envelope, de novo bone occupies ∼53–84% (∼73 ± 11%; mean ± standard deviation) of the hollowed-out space. Low porosity and the arrangement of remodelled bone into a concentric lamellar pattern is indicative of cortical-like structure. Such areas are typically surrounded by yet unremodelled, and microstructurally disordered, woven bone that stains intensely with blue cationic dyes, owing to relatively higher acid phosphate content. This pattern indicates a recurring sequence of woven bone formation followed by remodelling. Bone formation is also visible within the bioceramic. Recently remodelled and areas of ongoing remodelling are identified by relatively lower mineral density than the surrounding woven bone. Dendritic extensions of osteocytes appear to extend into the bioceramic surface. Both micro-Raman spectroscopy and FTIR reveal little, if any, detectable difference between the mineral and organic phases of the extracellular matrix, between de novo and native bone. Conclusions. The bioceramic composition undergoes physical degradation, but remains largely intact by 52 weeks in vivo, and only partially transforms to carbonated apatite. In addition to very high bone volume within the hollowed-out bioceramic device, the overall composition and microstructure of de novo bone are similar to native bone. Notably, the mineral phase of bone in response to, and in direct contact with the β-TCP, monetite, and Ca-PP, remains exclusively carbonated apatite

Tissue engineering is a promising approach to regenerate damaged skeletal tissues. In particular, the use of injectable hydrogels alleviates common issues of poor cell viability and engraftment. However, uncontrolled cell fate, resulting from unphysiological environments and degradation rates, still remain a hurdle and impedes tissue healing. We thus aim at developing a new platform of injectable hyaluronic acid (HA) hydrogels with a large panel of properties (stiffness, degradation…) matching those of skeletal tissues. Hence, HA with different molecular weights were functionalized with silylated moieties. Upon injection, these hydrogels formed through a sol-gel chemistry within 5 to 20 minutes in physiological conditions, as demonstrated by rheological characterization. By varying the crosslinking density and concentration, we obtained hydrogels spanning a large range of elastic moduli (E = 0.1–20 kPa), similar to those of native ECMs, with tunable biodegradation rates (from 24 hours to > 50 days) and swelling ratios (500 to 5000% (w/w)). Cell viability was confirmed by Live/Dead assays and will be completed by in vivo subcutaneous implantations in mice to study the foreign body reaction and degradation rate. We further developed hybrid HA/biphasic calcium phosphate granules hydrogels and demonstrated a strong mechanical reinforcement (E = 0.1 MPa) and a faster relaxation behaviour (τ. 1/2. < 400s), with similar degradation rates. Ongoing in vitro differentiation assays and in vivo implantations in a rabbit femur model will further assess their ability to drive bone regeneration. Collectively, these results suggest that this hydrogel platform offers promising outcomes for improved strategies in skeletal tissue engineering

TGF-beta signaling has a well established role not only in adult organ homeostasis but also in skeletal development. Follistatin-like 3 (FSTL3), related to follistatin, is an inhibitor of TGF-beta ligands, with an established role in glucose and fat metabolism. However it has not previously been studied in skeletal development. Using a FSTL3 knock-out (KO) mouse model we have studied both embryonic skeletal development and adult bone phenotypes. Staining for skeletal and cartilage markers during development shows acceleration of skeletal tissue differentiation, with an eventual normalization at E18.5 (which is just prior to birth). Acceleration of bone mineralization occurs during both endochondral and intramembranous ossification. Use of micro-CT imaging highlighted the development of a scoliosis in the KO animals, along with abnormal shape of cranium and cranial sutures. Further investigation of the cranial phenotype in adult KO mice reveals craniosynastosis, with atypical fusion of the frontal suture. These mice have a change in overall cranial shape with shortening of the anterior head and a compensatory expansion of the posterior cranial bones, in a similar fashion to brachyencephaly. Our study therefore highlights a significant role of FSTL3 in skeletal tissue development and mineralization, as well as the development of clinically significant skeletal developmental disorders such as scoliosis, craniosynastosis and brachyencephaly

Osteoporosis affects millions globally and current anti-catabolic treatments are limited by significant side-effects. Osteoporosis arises when skeletal stem cells (SSC) no longer sufficiently replenish osteoblasts, leading to net bone loss. A key regulator of SSC behaviour is physical loading, yet the mechanisms by which SSCs sense and respond to changes in their mechanical environment are virtually unknown. Primary cilia are nearly ubiquitous ‘antennae-like’ cellular organelles that have very recently emerged as extracellular chemo/mechano-sensors and thus, are strong candidates to play an important role in regulating SSC responses in bone. This paper will demonstrate that the SSC primary cilium plays an important role in loading-induced bone formation via initial chemosensation and transduction of the potent chemokine TGFβ1 regulating SSC recruitment to the bone surface and secondly it will be shown that the primary cilium is a cAMP responsive mechanosensor directly regulating SSC mechanotransduction via localisation of adenylyl cyclase 6 to the ciliary microdomain. Finally, it will be shown that targeting the cilium therapeutically can be an effective approach to enhance both biochemical and biophysically induced SSC osteogenesis contributing to bone formation, demonstrating a novel anabolic therapy for bone loss diseases such as osteoporosis

Osteoarthritis is characterised by the loss and damage of cartilage in synovial joints. Whilst joint replacement is the gold standard for end stage disease, repair or regenerative strategies aim to slow disease progression, maintain joint function and defer the need for joint replacement. One approach seeks to target endogenous repair after drilling or microfracture (a type of trauma induced repair) in the area of cartilage loss – connecting the defect to the underlying bone marrow niche. The rationale of this approach is that cells delivered to the defect site, from the bone marrow, will bring about cartilage repair. Bone marrow contains multipotent cells, including stem and stromal populations, of both the haematopoietic and skeletal systems. Bone marrow mesenchymal stromal cells (BMSCs) are characterised by tri-lineage differentiation (bone, cartilage and adipose tissue) and contribute to the formation of the bone marrow niche, which maintains haematopoietic stem cell quiescence. This quiescence ensures life-long haematopoiesis and the supply of mature blood cells to the haematopoietic system. In this study we investigate the interactions between haematopoietic and BMSCs (in both human and mouse cultures) specifically to understand the consequences on BMSCs during tissue repair. A murine MSC cell-line model was co-cultured with enriched fractions of primary murine haematopoietic progenitor cells isolated based on c-Kit, Sca-1, and lineage markers. Similarly, human bone marrow derived MSCs were co-cultured with primary bone marrow haematopoietic fractions isolated based on CD34, CD38 and lineage markers. Using confocal microscopy, we demonstrated that the two cell populations directly interact through cell-cell contact with haematopoietic cells located above and below the MSC monolayer. Cultures were then pushed to differentiate down the osteogenic lineage. Results indicate that MSCs co-cultured with haematopoietic cells exhibited significant inhibition of osteogenesis when analysed by functional assay of matrix mineralisation and gene expression analysis for transcripts including Runx2, Osterix and type I collagen. These data support the hypothesis that hematopoietic progenitor cells influence both the local homeostasis of the bone marrow as well as the repair potential of stromal cells. Such interactions could be important for the resolution of injury after trauma induced repair. Furthermore, manipulation of these interactions, such as the administration of haematopoietic cell stimulating agents, could be used to improve treatment outcomes

Osteoporosis (OP) and osteoarthritis (OA) are leading causes of musculoskeletal dysfunction in elderly, with chondrocyte senescence, inflammation, oxidative stress, subcellular organelle dysfunction, and genomic instability as prominent features. Age-related intestinal disorders and gut dysbiosis contribute to host tissue inflammation and oxidative stress by affecting host immune responses and cell metabolism. Not surprisingly, the development of OP and OA correlate with dysregulations of the gut microflora in rodents and humans. Intestinal microorganisms produce metabolites, including short-chain fatty acids, bile acids, trimethylamine N-oxide, and liposaccharides, affecting mitochondrial function, metabolism, biogenesis, autophagy, and redox reactions in chondrocytes to regulate joint homeostasis. Modulating the abundance of specific gut bacteria, like Lactobacillus and Bifidobacterium, by probiotics or fecal microbiota transplantation appears to suppress age-induced chronic inflammation and oxidative damage in musculoskeletal tissue and holds potential to slow down OP development. The talk will highlight treatment options with probiotics or metabolites for modulating the progression of OA and OP.

Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

Bone tissue experiences continued remodelling in response to changes in its biochemical and biophysical environment. Given the finite lifespan of osteoblasts, this continued bone formation requires replenishment from a progenitor population. Although this is largely believed to be from a skeletal stem cell population, given the limitation in in-vivo markers for this cell type, progress in demonstrating this mechanism is limited. Therefore, we characterized the LepR-Cre mouse strain and evaluated whether LepR positive cells are the progenitor population and if they contribute to the osteoblast population over time and in mechanically-induced bone formation in-vivo. Transgenic mouse strains; B6.129(Cg)-Leprtm2(cre)Rck/J to study LepR-expressing cells and B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J as a reporter strain were obtained from Jackson Laboratories. Characterization studies were performed on LepR:tdTomato mice at embryonic stage (19.5dpc), 8 and 12 weeks old. Mice (12 weeks old) were subjected to compressive tibia loading with a 11N peak load for 40 cycles, every other day for 2 weeks. Histological analysis reveal that LepR is expressed from the embryonic stage in various organs including bones. LepR positive cells are found around blood vessels and on bone surfaces. Flow cytometry analysis show the amount of LepR positive cells negative for CD45 and Ter-119 markers inside the bone marrow increases over time and following tibial loading. Mechanical loading induces an increase in bone mass and bone parameters. This model allows us to track and evaluate the role of LepR positive cells as bone forming cells, and to decipher the role of these cells in mechanically-induced bone formation

Background. Following endosteal uncemented orthopaedic device implantation, the initial implant/bone interface retains spaces and deficiencies further exacerbated by pressure necrosis and resultant bone resorption. This implant-bone space requires native bone infill through the process of de novo osteogenesis. New appositional bone formation on the implant surface is known as contact osteogenesis and is generated by osteogenic cells, including skeletal stem cells (SSCs), colonising the implant surface and depositing the extracellular bone matrix. Surface nanotopographies provide physical cues capable of triggering SSC differentiation into osteoblasts, thus inducing contact osteogenesis, translated clinically into enhanced osseointegration and attainment of secondary stability. The current study has investigated the in vitro and in vivo effects of unique nanotopographical pillar substrates on SSC phenotype and function. Methods. Adult human SSCs were immunoselected, enriched using STRO-1 antibody and cultured on control and test surfaces for 21 days in vitro. The test groups comprised Ti-coated substrates with planar or modified surfaces with nanopillar. Osteoinductive potential was analysed using qPCR and immunostaining to examine gene expression and protein synthesis. Results. Following in vitro (n=5) culture on nanopillars, the expression of osteogenic genes (ALP, Collagen 1, OPN and OCN) and of Osteopontin protein (a bone matrix protein), on Ti pillars were both significantly enhanced when compared to control or Ti planar surfaces. Conclusions. Discrete raised surface nanopillars modulate adult SSC populations in the absence of any chemical cues and enhance their osteogenic properties, an effect not observed on planar Ti constructs. Hence, these findings herald exciting opportunities to improve the implant surface design, implant osseointegration, and, ultimately, implant survival. Level of evidence. Original experimental study

Angiogenesis and the ability to provide appropriate vascular supply are crucial for skeletal tissue engineering. The aim of this study was to investigate the angiogenic potential of human dental pulp stromal cells (HDPSCs) and stro-1 positive populations as well as their role in tissue regeneration (the clinical reality). HDPSC were isolated from the pulp tissues of human permanent teeth by collagenase digestion. STRO-1 positive cells were enriched using monoclonal anti- STRO-1 and anti- CD45 PE conjugated antibodies together with and fluorescence activated cell sorting (FACS). Cells isolated by FACS were grown to passage4 and cultured as monolayers or on 3D Matrigel scaffold in endothelial cell growth medium-2 (EGM-2) with/without 50ng/mL of vascular endothelial growth factor (VEGF). Cells cultured in alpha MEM supplemented with 10% FCS were used as controls. After 24, 48 and 72 hours angiogenic marker expression (CD31, CD34, vWF and VEGFR-2) was determined by qRT-PCR and immuno-histochemistry. Using three different donors, 0.5-1.5% of total HDPSCs population was characterized as STRO-1+/CD45- cells At each time point cells cultured as monolayer in EGM-2 with VEGF showed up regulation of CD31 and VEGFR-2 expression compared to the control group while expression of CD34 and vWF remained unaffected. However on Matrigel, all four genes were up regulated to different extents. CD31 and VEGFR-2 were up regulated to a greater degree compared to CD34 and vWF. Changes in gene expression in both cell types were time dependent. Immuno-histochemical staining confirmed that the HDPSCs cultured in the test group showed positive staining for the four angiogenic markers (CD31, CD34 vWF and VEGFR-2) when grown in both monolayer and 3D Matrigel culture compared to control cultures. When cultured on Matrigel (but not Monolayer) for 7 days, HDPSC formed tube-like structures in the VEGF treated group. This indicates the potential of use HDPSCs and their STRO-1 positive population for angiogenesis to enhance skeletal tissue repair and/or regeneration toward translational research for clinical benefit

AIM. Avascular necrosis (AVN) of the femoral head is a potentially debilitating disease of the hip in young adults. Impaction bone grafting (IBG) of morcellised fresh frozen allograft is used in a number of orthopaedic conditions. This study has examined the potential of skeletal stem cells (SSC) to augment the mechanical properties of impacted bone graft and we translate these findings into clinical practice. STUDY DESIGN. We have examined the effect of SSC density on augmentation of bone formation. An in vitro model was developed to replicate the surgical IBG process. Plain allograft was used as the control, and the SSC's seeded at a density of 5×103, 5×104 and 2×105 cells per cc of allograft for the experimental groups. All samples were cultured for 2 weeks and mechanically tested to determine shear strength using the Mohr Coulomb failure curve. The approach was translated to 3 patients with early avascular necrosis (AVN) of the femoral head. The patient's bone marrow was concentrated in theatre using a centrifugation device and the concentrated fraction of SSC's were seeded onto milled allograft. The patient's necrotic bone was drilled, curetted and replaced with impacted allograft seeded with SSC's. Osteogenic potential of concentrated and unconcentrated marrow was simultaneously compared in vitro by colony forming unit assays. RESULTS. The mechanical properties of the impacted allograft was significantly improved as a function of increasing SSC density. The difference compared to the control plain allograft was highly significant at the 2×105 level (p=0.001). Autologous SCC's on impacted bone allograft was subsequently applied in 3 patient cases and up to two year follow up demonstrates no deleterious effect. Critically the analysis of concentrated marrow demonstrated a higher SSC count in vitro than plain marrow aspirate. DISCUSSION. We have demonstrated the potential of skeletal stem cells to augment the mechanical properties of impacted bone allograft in a laboratory model and subsequently translated these findings into a new technique for the treatment of AVN of the femoral head. Such an approach provides not only improved mechanical support to the overlying cartilage but critically improved biology for new bone formation. The early clinical results are encouraging and indicate potential use also in fracture non-unions and void filling of bone defects

Introduction. The proteoglycan aggrecan is a major component of the cartilaginous matrices which provides resistance against compressive forces. Spontaneously occurring functional null mutations in the aggrecan gene (Acan) in various species lead to perinatal chondrodysplasia. The aim of the present study was to investigate the cellular and biomechanical properties of the cartilaginous growth plate, and the development of intervertebral disc in a novel, experimentally induced aggrecan mutant mouse strain carrying an insertion in exon 5 of the Acan gene. Methods. The novel aggrecan mutant mice were generated by inserting a loxP site into exon 5 (E5i) by homologous recombination in ES cells. Wild type and homozygous mutant (Acan-E5i/E5i) mice were analyzed by skeletal staining, histology and immunohistochemistry. Proliferation and survival were assessed by phosphorylated histone H3 immunostaining and TUNEL assay, respectively. Shape index (SI) in the proliferative zone (PZ) of the growth plate (GP) was calculated as a ratio of the long and short axes of the cells. Orientation of the PZ chondrocytes was characterized by the angle between the cell long axis and longitudinal direction of the bone growth. Imaging and stiffness measurements were performed by atomic force microscopy (AFM). Results. Acan-E5i/E5i mice are characterized by severe dwarfism, short snout, protruding tongue, cleft palate, and die at birth due to respiratory failure. On sections the cartilage of mutant mice appeared as tightly packed chondrocytes surrounded by a compressed matrix. At E18.5 and E14.5, the mutant PZ consisted of rounded (SI=1.71 at E18.5; SI=1.72 at E14.5) non-oriented chondrocytes, compared to the wild type PZ with flattened (SI=3.92 at E18.5; SI=3.90 at E14.5), columnar cells oriented with right angle to the longitudinal axis of the growth. At E13, the shape and orientation of mutant chondrocytes were similar to control. AFM at E14.5 and E18.5 demonstrated a stiffer matrix with denser collagen network in the mutant compared to wild type. The mutant cartilage had increased apoptosis and reduced proliferation rate at E18.5. The IVDs development appeared normal at E13.5-E14.5, however, the IVD was severely malformed at E18.5. Discussion. We have shown that aggrecan deficiency impairs cartilage biomechanics and results in a stiffer matrix. The altered mechanical properties might be responsible for the disorganization of mutant GP and compression of the IVD at around birth. Interestingly, the altered matrix mechanics is dispensable for early flattening and orientation of GP proliferative chondrocytes. In summary, aggrecan is essential for proper cartilage cytoarchitecture and morphogenesis by ensuring the suitable mechanical environment

Background. Metastatic bone patients who require surgery needs to be evaluated in order to maximise quality of life and avoiding functional impairment, minimising the risks connected to the surgical procedures. The best surgical procedure needs to be tailored on survival estimation. There are no current available tool or method to evaluate survival estimation with accuracy in patients with bone metastasis. We recently developed a clinical decision support tool, capable of estimating the likelihood of survival at 3 and 12 months following surgery for patients with operable skeletal metastases. After making it publicly available on . www.PATHFx.org. , we attempted to externally validate it using independent, international data. Methods. We collected data from patients treated at 13 Italian orthopaedic oncology referral centers between 2008 and 2012, then applied to PATHFx, which generated a probability of survival at three and 12-months for each patient. We assessed accuracy using the area under the receiver-operating characteristic curve (AUC), clinical utility using Decision Curve Analysis DCA), and compared the Italian patient data to the training set (United States) and first external validation set (Scandinavia). Results. The Italian dataset contained 287 records with at least 12 months follow-up information. The AUCs for the three-month and 12-month estimates was 0.80 and 0.77, respectively. There were missing data, including the surgeon's estimate of survival that was missing in the majority of records. Physiologically, Italian patients were similar to patients in the training and first validation sets. However notable differences were observed in the proportion of those surviving three and 12-months, suggesting differences in referral patterns and perhaps indications for surgery. Conclusions. PATHFx was successfully validated in an Italian dataset containing missing data. This study demonstrates its broad applicability to European patients, even in centers with differing treatment philosophies from those previously studied

Background. Lengthening over nail (LON) and the use of internal lengthening nails have been developed to minimize patients' time in a frame during femur lengthening. This study compares the outcomes of two techniques of femur lengthening, LON and Intramedullary Skeletal Kinetic Distraction (ISKD). Methods. In this retrospective study, 12 consecutive ISKD procedures were performed for femoral lengthening and followed for an average of 76 months. After the ISKD group, 20 consecutive femoral lengthening procedures were performed as an LON technique and followed for an average of 27 months. Results. There was no significant difference in achieving the lengthening goals between the two procedures. The healing index for the LON group averaged 1.4 months/cm, while the ISKD group was 3.2 months/cm (p=0.242). The distraction rates for the ISKD had a fast group (>1mm/day) with an average distraction rate of 1.7 mm/day and a slow group (<1mm/day) with a distraction rate of 0.84 mm/day. The LON group had an average distraction rate of 0.88 mm/day (p<0.001). The incidence of complications that required further unanticipated surgeries for the LON group was 1/20 (5%), while the ISKD group had complications in 6/12 femurs (50%, p=0.004). Conclusions. We concluded that the LON technique is a more predictable and reliable method for femoral lengthening than the ISKD

Osteoporosis (OP) results in a reduction in the mechanical competence of the bone tissue of the sufferers. In skeletal sites such as the proximal femur and the vertebrae, OP manifests itself in low trauma fragility fractures which are debilitating for the patient. The relationships between the compressive strength of cancellous tissue and its apparent density are well established in studies of the past. Recently the authors have presented a method able to assess the fracture toughness properties of cancellous bone (1), a challenging cellular material which can exhibit large elasto-plastic deformations. The in-vitro measurement of fracture toughness alongside the customary compressive strength can provide a comprehensive assessment of the mechanical capacity of cancellous bone, which will reflect closer its ability to resist crack initiation. The aims of the present study were: (1) to examine whether the observed fracture toughness deterioration can also be detected by non-invasive quantitative ultrasound (QUS); and (2) to provide rational evidence for the well proven ability of QUS to predict directly ‘risk of fracture’. 20 femoral heads were obtained from donors undergoing emergency surgery for a fractured neck of femur. QUS investigations of the calcaneus, proximal phalanx and distal radius were undertaken within 72 hours of surgery. 128 fracture toughness samples and 20 compression cores were manufactured and tested. Two clinical QUS systems were used to obtain in-vivo scan data and then directly compared those to the density, porosity and the fracture mechanics of tissue extracted from the same individuals. The results demonstrated not only that there was a significant link between in-vivo determined QUS values for the calcaneus and finger to the density of the density of the femoral head; but that there was also a significant link between the QUS results from the calcaneus and the fracture toughness of the cancellous bone from the femoral head. These results point towards a systemic effect of osteoporosis which affects similarly different parts of the skeleton and supports the use of clinical QUS systems as a diagnostic tool for the prediction of fracture risk

With around 20–40% of our bodyweight, skeletal muscles are the biggest organ complex of the human body. Being a metabolic active tissue, muscle mass, function and fibertype composition is highly regulated in a tight spatial-temporal manner. In geriatric patients, it is essentially important to understand the underlying mechanisms of the age related losses of fiber size and total number of fibers, as well as fibertype shifting.

To date, there have been few studies dealing with gene expression profiling of skeletal muscles, mostly focusing on age related differences in whole-muscle specimen. Being carried out on mouse or rat limb muscles, most other studies do not represent the conditions of human muscle, due to the differences in fibertype composition. Our study provides a fibertype-specific approach for whole-genome expression analysis in human skeletal muscle.

22 fresh frozen biceps brachii and quadriceps femoris muscle samples were acquired from the muscle bank of the Friedrich-Baur-Institut, Department of Neurology, Ludwig-Maximilians-University, Munich, Germany. Consecutive cross-sections were used for immunohistochemical myosine-heavy-chain-staining and individual fibers were acquired by laser-capture-microdissection. Around 100 cells of each fibertype of each biopsy were dissected, reversely transcribed, pre-amplified and labeled for microarray analysis. Fiber type-specific gene expression was analyzed with ANOVA. Correction for multiple testing was performed using the Benjamini-Hochberg procedure with a conservative threshold and the pathway analysis was carried out using the Ingenuity Pathway Analysis program (QIAGEN).

By comparing type I vs. type IIa, type I vs. type IIx and type IIa vs. type IIx, we could identify 2855, 2865 and 510 differentially expressed genes. As expected, many differentially regulated genes belong to functional groups like cytoskeleton, muscle contraction and energy metabolism, proving the feasibility of our study. However, many genes that are involved in the response to oxidative stress were also differently regulated, showing distinct mechanisms of the different fiber types, of coping with oxidative stress. In consensus with available literature, the relative proportion of type I fibers seemed to increase with age. Despite higher levels of oxidative stress, type I fibers seem to have more efficient antioxidative mechanisms in comparison to type IIa and IIx fibers, which might explain the higher vulnerability of members of the type II family to oxidative stress. Furthermore, genes that are involved in fibertype specification were also regulated differently. However, we could not verify an age-specific activation of pathways involved in fibertype shifting. Whether fibertype shifting is solely due to disproportionate loss of type II fibers, or also in vivo - transdifferentiation of fibers, has to be investigated further.