Introduction. Metabolic disorders are among known risk factors for tendinopathies or spontaneous tendon ruptures. However, the underlying cellular and molecular mechanisms remain unclear. We have previously shown that human and rat tendon cells produce and secrete insulin upon glucose stimulation. Therefore, we hypothesize that nutritional glucose uptake affects tendon healing in a rat model. Materials and Methods. Unilateral full-thickness Achilles tendon defects were created in 60 female rats. Animals were randomly assigned to three groups receiving different diets for 2 weeks (high glucose diet, low glucose/high fat diet, control diet). Gait analysis was performed at three time points (n=20/group). In addition, tendon thickness, biomechanical (n=14/group), and histological and immunohistochemical analysis was conducted. Subsequently, a subtractive-suppression-hybridization (SSH) screen comparing cDNA pools (n=5) prepared from repair tissues of the high glucose and the control diet group was conducted to identify differentially expressed genes. Results. Newly formed repair tissue of the high-glucose and high-fat group was significantly thicker compared to the control group (p<0.001). Gait analysis revealed a significantly increased Intermediate Toe Spread for animals receiving high glucose diet one week p.o. compared to the control and high fat diet group (p<0.01). Maximum tensile load was significantly reduced in the control diet and the fat diet group, compared to intact tendons (p<0.05). Interestingly, there was no reduction evident for the glucose diet group. A similar trend was observed for tendon stiffness, with glucose diet repair tissue being significantly stiffer compared to the control diet (p<0.05). The proportion of Ki67+ cells in the repair tissue was 3,3% in the control, 9,8% in the glucose and 8,4% in the fat diet group, indicating an increased cell proliferation rate for the glucose and fat diet groups (p<0.001). Finally, the SSH screen revealed 48 candidate genes to be differentially expressed among the diet groups. Discussion. Tendon repair tissue quality is moderately affected by nutritional glucose. The molecular mechanisms underlying these effects on cells and matrix are currently under investigation and may be helpful in developing a dietary intervention scheme to support tendon regeneration after trauma or tendon disease

Scoliosis correction surgery is one of the longest and most complex procedures of all orthopedic surgery. The complication rate is therefore not negligible and is particularly high when the surgery is performed in patients with neuromuscular or connective tissue disease or complex genetic syndromes. In fact, these patients have various comorbidities and organ deficits (respiratory capacity, swallowing / nutrition, heart function, etc.), which can compromise the outcome of the surgery. In these cases, an accurate assessment and preparation for surgery is essential, also making use of external consultants. To make this phase simpler, more effective and homogeneous, a multidisciplinary path of peri-operative optimization is being developed in our Institute, which also includes the possibility of post-operative hospitalization for rehabilitation and recovery. The goal is to improve the basic functional status as much as possible, in order to ensure faster functional recovery and minimize the incidence of peri-operative complications, to be assessed by clinical audit. The path model and the preliminary results on the first patients managed according to the new modality are presented here. The multidisciplinary path involves the execution of the following assessments / interventions: • Pediatric visit with particular attention to the state of the upper airways and the evaluation of chronic or frequent inflammatory states • Cardiological Consultation with Echocardiogram. • Respiratory Function Tests, Blood Gas Analysis and Pneumological Consultation to evaluate indications for preoperative respiratory physiotherapy cycles, Non-Invasive Ventilation (NIV) cycles, Cough Machine. Possible Polysomnography. • Nutrition consultancy to assess the need for nutritional preparation in order to improve muscle trophism. • Consultation of the speech therapist in cases of dysphagia for liquids and / or solids. • Electroencephalogram and Neurological Consultation in epileptic patients. • Physiological consultation in patients already being treated with a cough machine and / or NIV. • Availability of postoperative hospitalization in the rehabilitation center (with skills in respiratory and neurological rehabilitation) for the most complex cases. When all the appropriate assessments have been completed, the anesthetist in charge at our Institute examines the clinical documentation and establishes whether the path can be considered complete and whether the patient is ready for surgery. At the end of the surgery, the patient is admitted to the Post-operative Intensive Care Unit of the Institute. If necessary, a new program of postoperative rehabilitation (respiratory, neuromotor, etc.) is programmed in a specialist reference center. To date, two patients have been referred to the preoperative optimization path: one with Ullrich Congenital Muscular Dystrophy, and one with 6q25 Microdeletion Syndrome. In the first case, the surgery was performed successfully, and the patient was discharged at home. In the second case, after completing the optimization process, the surgery was postponed due to the finding of urethral malformation with the impossibility of bladder catheterization, which made it necessary to proceed with urological surgery first. The preliminary case series presented here is still very limited and does not allow evaluations on the impact of the program on the clinical practice and the complication rate. However, these first experiences made it possible to demonstrate the feasibility of this complex multidisciplinary path in which a network of specialists takes part

Fractures and related complications are a common challenge in the field of skeletal tissue engineering. Vitamin D and calcium are the only broadly available medications for fracture healing, while zinc has been recognized as a nutritional supplement for healthy bones. Here, we aimed to use polaprezinc, an anti-ulcer drug and a chelate form of zinc and L-carnosine, as a supplement for fracture healing. Polaprezinc induced upregulation of osteogenesis-related genes and enhanced the osteogenic potential of human bone marrow-derived mesenchymal stem cells and osteoclast differentiation potential of mouse bone marrow-derived monocytes. In mouse experimental models with bone fractures, oral administration of polaprezinc accelerated fracture healing and maintained a high number of both osteoblasts and osteoclasts in the fracture areas. Collectively, polaprezinc promotes the fracture healing process efficiently by enhancing the activity of both osteoblasts and osteoclasts. Therefore, we suggest that drug repositioning of polaprezinc would be helpful for patients with fractures

As high incidences of tendinopathies are observed particularly in those who intensively use their tendons, we assume that pathological changes are caused, at least partially, by mechanical overload. This has led to the so-called overload hypothesis, explaining the development of tendinopathies by structural failure resulting from excessive load. At the same time, tendon loading is an important part in tendon rehabilitation. Currently, exercise treatment approaches such as eccentric training or heavy load resistance training are widely applied in tendinopathy rehabilitation, with good clinical results such as an improvement in function and a reduction in pain. Particularly those rehabilitative approaches which impose high strains on the tendon may induce an adaptation of the tendon's mechanical properties such as increased tendon stiffness. An increased tendon stiffness is often interpreted as desirable, as it may protect the tendon from overloading and thus prevent future strain injuries. However, the tendinopathic tendon is not necessarily less stiff than the tendon in the contralateral leg and an improvement in tendon stiffness is not necessarily accompanied by an improvement in tendon pain or function. In addition, metabolic factors, resulting e.g. in low-level systemic inflammation, may contribute to pathological tendon tissue changes and are not necessarily affected by an exercise program, while nutritional interventions or dietary supplements may potentially affect tendon cell metabolism. Indeed, dietary supplements have been introduced as an additional therapeutic approach in the treatment of tendinopathies in recent years, and their positive curative effects have been reported for both the general population and athletes. In the management of tendinopathies, it may thus be advisable if therapeutic approaches aim to address both tendon mechanics and tendon metabolism for better treatment effectiveness and a sustainable improvement in pain and function

Traumatic acute or chronic tendon injuries are a wide clinical problem in modern society, resulting in important economic burden to the health system and poor quality of life in patients. Due to the low cellularity and vascularity of tendon tissue the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Tissue engineering and regenerative medicine are promising alternatives to the natural healing process for tendon repair, especially in the reconstruction of large damaged tissues. The aim of TRITONE project is to develop a smart, bioactive implantable 3D printed scaffold, able to reproduce the structural and functional properties of human tendon, using FDA approved materials and starting from MSC and their precursor, MPC cell mixtures from human donors. Total cohort selected in the last 12 months was divided in group 1 (N=20) of subjects with tendon injury and group 2 (N=20) of healthy subject. Groups were profiled and age and gender matched. Inclusion criteria were age>18 years and presence of informed consent. Ongoing pregnancy, antihypertensive treatment, cardiovascular diseases, ongoing treatment with anti-aggregants, acetylsalicylic-acid or lithium and age<18 years were exclusion criteria. Firstly, we defined clinical, biological, nutritional life style and genetic profile of the cohort. The deficiency of certain nutrients and sex hormonal differences were correlated with tendon-injured patients. It was established the optimal amount of MPC/MSC human cell (collected from different patients during femoral neck osteotomy). Finally, most suitable biomaterials for tendon regeneration and polymer tendon-like structure were identified. Hyaluronic acid, chemical surface and soft-molecular imprinting (SOFT-MI) was used to functionalize the scaffold. These preliminary results are promising. It will be necessary to enroll many more patients to identify genetic status connected with the onset of tendinopathy. The functional and structural characterization of smart bioactive tendon in dynamic environment will represent the next project step

Background. Fractured neck of femurs cause substantial morbidity and mortality in elderly patients and represent a huge financial burden to the NHS. Hip fracture patients are generally malnourished on admission, often having poor nutritional inpatient intake, hindering recovery and increasing chances of “unfavourable outcome.” Nutritional care is included in intercollegiate guidelines for management of fractured neck of femur patients, but is nutrition a management priority in clinical practice?. Study Aim. To evaluate protein and energy intake of acute fractured neck of femur patients depending on admission MMSE, and compare these to department of health targets. Method. 40 acute fractured neck of femur admissions were recruited between December 08-March 09 and put into three groups depending on admission MMSE. Initial nutrition screening information (mid-arm circumference, grip strength, MUST score) was recorded and through food charts daily kcal and protein intake were calculated for a three day period. Results. 100% of patients recruited were high risk of malnutrition on admission. Overall average daily calorie intake over 3 days was 385.9 kCal, average protein intake was 14.1g. Intake for each group was well below recommended target intake of 1810kCal and 46.5g, p<0.05. Discussion. On admission all 40 patients included were classified high risk for malnutrition, therefore in need of dietetic and nutritional intervention. Two patients received dietician input and oral supplementation. Recorded nutritional intake was very low, well below target nutrient intakes for this population even before extra requirement due to the stress response is accounted for. Whilst there is a paucity of hard evidence linking poor nutrition to clinical outcome, this is likely to be detrimental to rehabilitation from surgery. Conclusion. Despite attempted adherence to NICE nutrition guidance, involving screening tools, care plans and protected mealtimes; acutely unwell, malnourished patients are not receiving their basic nutritional requirements

Introduction and Objective. Hyaluronic acid (HA) is an effective option for the treatment of osteoarthritis (OA) patients due to several properties such as normalization of the mechanical and rheological properties of the synovial fluid and amelioration of OA symptoms and joints function by promoting cartilage nutrition. Since OA progression is also significantly related to oxidative stress and reactive oxygen species (ROS), sodium succinate (SS) is envisioned as a promising compound for cartilage treatment by providing antioxidant defense able to normalize intracellular metabolism and tissue respiration via mitochondrial mechanism of action. The scope of this study was to investigate on an in vitro inflammatory model the efficacy of Diart. ®. product, a combination of HA and SS. Materials and Methods. Donor-matched chondrocytes and synoviocytes were obtained from KL 3–4 OA patients undergoing total knee replacement. At passage 4, inflammation was promoted with 1 ng/ml IL-1B for 48 hours in absence and presence of Diart. ®. at 1:3 dilution rate. Nitric oxide (NO) from cell culture supernatant was measured by Griess reaction. Mitochondrial and cytoplasmatic ROS evaluation was assessed by flow cytometry with MitoSox and dichlorodihydrofluorescein diacetate (DCFDA) assays. Gene expression of inflammation/oxidative stress-related transcripts (MMP1/MMP3/INOS/COX2) was evaluated by qRT-PCR using TBP as reference. Results. NO was detected only in inflamed chondrocytes and Diart® was able to abolish its levels. NO was not detected in synoviocytes in all conditions. IL-1B reduced both cytoplasmic (−66%) and mitochondrial (−68%) ROS in chondrocytes, with Diart® partially restoring (+40%) mitochondrial levels. In synoviocytes, IL-1B did not alter ROS, with Diart® modestly increasing (+27%) mitochondrial levels. Inflammation was able to increase transcript levels of all tested markers, with the exception of INOS in synoviocytes. In chondrocytes, Diart® significantly (p < 0.05) reduced COX2 (−75%) and MMP1 (−33%). In synoviocytes, Diart® significantly reduced COX2 (−77%) and MMP3 (−84%), with MMP1 53% decreased albeit without reaching statistical significance. Conclusions. Diart. ®. biochemical and physiologic properties in the tested in vitro model of inflammation on donor-matched chondrocytes and synoviocytes allowed reducing inflammation and oxidative stress-related markers, prompting the use of this combination as successful strategy to manage OA-related symptoms

Introduction and Objective. Zone 2 flexor tendon injuries are still one of the challenges for hand surgeons. It is not always possible to achieve perfect results in hand functions after these injuries. There is no consensus in the literature regarding the treatment of zone 2 flexor tendon injuries, tendon repair and surgical technique to be applied to the A2 pulley. The narrow fibro-osseous canal structure in zone 2 can cause adhesions and loss of motion due to the increase in tendon volume due to surgical repair. Different surgical techniques have been defined to prevent this situation. In our study, in the treatment of zone 2 flexor tendon injuries; Among the surgical techniques to be performed in addition to FDP tendon repair; We aimed to compare the biomechanical results of single FDS slip repair, A2 pulley release and two different pulley plasty methods (Kapandji and V-Y pulley plasty). Materials and Methods. In our study, 12 human upper extremity cadavers preserved with modified Larssen solution (MLS) and amputated at the mid ½ level of the arm were used. A total of 36 fingers (second, third and the fourth fingers were used for each cadaver) were divided into four groups and 9 fingers were used for each group. With the finger fully flexed, the FDS and FDP tendons were cut right in the middle of the A2 pulley and repaired with the cruciate four-strand technique. The surgical techniques described above were applied to the groups. Photographs of fingers with different loads (50 – 700 gr) were taken before and after the application. Proximal interphalangeal (PIP) joint angle, PIP joint maximum flexion angle and bowstring distance were measured. The gliding coefficient was calculated by applying the PIP joint angle to the single-phase exponential association equation. Results. Gliding coefficient after repair increased by %21.46 ± 44.41, %62.71 ± 116.9, %26.8 ± 35.35 and %20.39 ± 28.78 in single FDS slip repair, A2 pulley release, V-Y pulley plasty and Kapandji plasty respectively. The gliding coefficient increased significantly in all groups after surgical applications (p<0.05). PIP joint maximum flexion angle decreased by %3.17 ± 7.92, %12.82 ± 10.94, %8.33 ± 3.29 and %7.35 ± 5.02 in single FDS slip repair, A2 pulley release, V-Y pulley plasty and Kapandji plasty respectively. PIP joint maximum flexion angle decreased significantly after surgery in all groups (p<0.05). However, there was no statistically significant difference between surgical techniques for gliding coefficient and PIP joint maximum flexion angle. Bowstring distance between single FDS slip repair, kapandji pulley plasty and V-Y pulley plasty showed no significant difference in most loads (p>0.05). Bowstring distance was significantly increased in the A2 pulley release group compared to the other three groups (p<0.05). Conclusion. Digital motion was negatively affected after flexor tendon repair. Similar results were found in terms of gliding coefficient and maximum flexion angle among different surgical methods. As single FDS slipe repair preserves the anatomical structure of the A2 pulley therefore we prefer it as an ideal method for zone 2 flexor tendon repair. However, resection of FDS slip may jeopardizes nutrition to the flexor digitorum profundus tendon which weakens the repair site. Therefore the results must be confirmed by an in vivo study before a clinical recommendation can be made. Keywords: Flexor tendon; injury; pulley plasty; cadaver;

Sarcopenia is a progressive and generalized skeletal muscle disorder that involves loss of muscle mass and function. It is associated with increased adverse outcomes including falls, functional decline, frailty and mortality and affects 65% of people over the age of 65 more than half of people aged 80 and above. The factors that cause and worsen sarcopenia are categorised into two groups. The primary aetiological factor is ageing and the secondary factors include disease, physical inactivity, and poor nutrition. Sarcopenia is considered to be ‘primary' (or age-related) when no other specific cause is evident. However, a number of ‘secondary' factors may be present in addition to ageing. Sarcopenia can occur secondary to a systemic or inflammatory disease, including malignancy and organ failure. Physical inactivity is one of the major contributors to the development of sarcopenia, whether due to a sedentary lifestyle or to disease related immobility or disability. Furthermore, sarcopenia can develop as a result of inadequate protein consumption. Biomarkers are objective and quantifiable characteristics of physiological and pathophysiological processes. Biomarkers can be used to predict the development of sarcopenia in older susceptible adults and enable early interventions that can reduce the risk of physical disability, the co-morbidities associated with the loss of muscle mass and the poor health outcomes that result from sarcopenia. Non-invasive imaging technologies can be used as biomarkers to detect loss of skeletal muscle mass in sarcopenia include bone densitometry, computed tomography, ultrasound and magnetic resonance imaging. However, imaging requires sophisticated and expensive equipment that is not available in a resource poor setting. Therefore, markers of skeletal muscle strength and fitness and soluble biochemical markers in blood may be used as alternative biomarkers. Studies on sarcopenia have identified numerous soluble biochemical biomarkers. These biomarkers can be divided into two groups: “muscle-specific” and “non-muscle-specific” biomarkers. Since sarcopenia is associated with rapid skeletal muscle wasting, the skeletal muscle-specific isoform of troponin T may be considerate a useful biomarker of sarcopenia, since high troponin levels in blood are an expression of muscle wasting. Peptides derived from collagen type VI turnover may be potential biomarkers of sarcopenia. We have recently conducted a systematic review to summarize the data from recent mass-spectrometry based proteomic studies of the secretome of skeletal muscle cells in response to disease, exercise or metabolic stress in order to identify the proteins involved in muscle breakdown. Developing robust in vitro models for the study of sarcopenia using primary muscle cells is a high priority as is exploiting the in vitro models to understand catabolic and inflammatory processes and molecular mechanisms involved in sarcopenia. Co-cultures with adipose-derived and other cells may be used to screen for small molecules and biologicals capable of inhibiting the catabolic and inflammatory pathways involved in sarcopenia. This presentation reviews recent progress in this area and outlines opportunities for future research on sarcopenia

Intervertebral disc degeneration (IDD) affects more than 80% of the population and is often linked to a reduction of the proteoglycan content within the nucleus pulposus (NP). The nutritional decline and accumulation of degraded matrix products promote the inflammatory process favoring the onset of disease. Several regenerative approaches based on cell therapy have been explored. Recently, paracrine factors and extracellular vesicles (EVs) such as exosomes have been described to play a fundamental role in the cross-talk between mesenchymal stem cells (MSCs) and NP in the microenvironment. EVs vehicule different molecules: proteins, nucleic acids and lipids involved in intercellular communication regulating the homeostasis of recipient cells. Therefore, MSCs-derived exosomes are an interesting emerging tool for cell-free therapies in IDD. The aim of this study was to evaluate the in vitro effects of MSCs derived exosomes on human NP cells (hNPCs). Exosomes were isolated through a multistep ultracentrifugation of bone marrow-MSCs (BM-MSCs) conditioned media (CM), obtained by culturing BM-MSCs without fetal bovine serum (FBS) for 48 hours. Exosomal morphology was characterized by transmission electron microscope (TEM). The exosomes were quantified by bicinchoninic acid assay (BCA) and cryopreserved at –80 °C. hNPCs derived from surgical speciments digested with type II collagenase. After culture expansion in vitro, hNPCs in alginate beads (three-dimensional culture system) were treated with growth medium (controls), exosomes, CM, interleukin-1 beta (IL-1b), IL-1b plus exosomes, IL-1b plus CM. After 24 hours, total RNA was extracted and reverse-transcribed. Gene expression levels of catabolic and anabolic genes were analyzed through real time-polymerase chain reaction (qPCR). TEM analysis confirmed the cup-shaped vescicles in our preparations. Gene expression levels resulted to be modulated by both exosomes and CM compared to controls. In addition, both treatments were capable to alter the inflammatory stimuli of IL-1b. Interestingly, exosomes were able to change anabolic and catabolic gene expression levels differently from CM. In our experimental conditions, both exosomes and CM from BM-MSCs could be an interesting alternative strategy in intervertebral disc regeneration, overcoming the costs and translational limits of cell therapy to the clinical practice

Summary Statement. An organ culture experiment was simulated to explore the mechanisms that can link cell death to mechanical overload in the intervertebral disc. Coupling cell nutrition and tissue deformations led to altered metabolic transport that largely explained cell viability measurements. Introduction. Part of intervertebral disc (IVD) maintenance relies on limited nutrient availability to the cells and on mechanical loads, but effective implication of these two factors is difficult to quantify. Theoretical models have helped to understand the link between solute transport and cell nutrition in deforming IVD, but omitted the direct link between tissue mechanics and cell metabolism. Hence, we explored numerically the relation between disc mechanics and cell death in relation to an organ culture experiment. Methods. A finite element model of a caudal bovine IVD was created to reproduce an organ culture experiment. All subtissues were modelled, and coupled to cell metabolism in two ways: (i) mechanical strains and metabolic reactions were simply coupled to the diffusions of oxygen, lactate and glucose through a mechano-transport algorithm (IND model). (ii), a hypermetabolism model based on in vitro data involved a 30% increase in glucose consumption by the cells, activated either as a Step or as a Gaussian function over 15% strain (DIR model). Exponential decays of cell density occurred below 0.5 mM of glucose and/or below pH 6.78. Concentrations of 21 kPa oxygen and 4.5 mM glucose were imposed at the boundary, and a combination of 0.2 MPa compression and 10° bending was applied over 7 days. Results. The highest hypermetabolic response was given by the Step activation. For all models, cell death mostly occurred in the compressed area of the flexed IVD, and steady-state cell viability was reached in about two days of load. In the outer annulus fibrosus (AF), the DIR model with Step activation led to increased cell death, in line with the cell viability measured in vitro. In the inner AF, all cell viability results matched the reported measurements. Discussion/Conclusion. This study focused on elucidating the links between mechanical stimulation and cell survival in the IVD, and simulation of nutrition issues allowed reproducing the results of an organ culture experiment. Results suggest that mechano-regulated metabolism can play a significant role in the nutrition-related cell death. Truly, the IND model gave both low glucose and low pH, and altered metabolic transport represented the main cell death mechanism. Yet, the role of hypermetabolism was increased nearby the nutrient supply at the outer AF, meaning that cell death could occur, even in regions where nutrient supply seems ensured by short diffusion distances. Though further mechanistic developments must be considered, this novel mechano-regulated metabolism model permits mechano-transport models to be used to explore important interactions between tissue biophysics and multiphysics. In particular, the extracellular matrix degradation along degeneration and cell death can be coupled to the poromechanical parameters introduced, e.g. initial porosity and osmotic pressure values that largely depend on the proteoglycan concentration

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine. Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2

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

Introduction. Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy affecting approximately 3 million people in China (Stone R, 2009). The precise aetiology of KBD is not clear, but the lack of selenium and the pollution of mycotoxins in food are a suspected cause of KBD. In this pilot study, we use a rat model to investigate the effect of low selenium and T-2 toxin on articular cartilage metabolism. Methods. 140 male Sprague-Dawley rats were fed with selenium-deficient or normal diet for 4 weeks to produce a low selenium or normal nutrition status. The rats were then fed for a further 4 weeks with low selenium or normal diets with or without T-2 toxin (100ng per gram body weight per day). The rat knee joints were fixed and paraffin embedded and histological and immunohistochemical staining was performed to analyse the metabolism of articular cartilage. Results. There was increased cell cluster formation in the middle and/or deep zones in rats fed with both diets. However, an apparent cell loss was observed in the low selenium + T-2 toxin group with an apparent increase in caspase-3 staining, indicating the increased cell apoptosis. Moreover, toluidine blue staining was reduced in the low selenium + T-2 toxin group, suggesting a loss of sulphated glycosaminoglycans. Similarly, there was reduced 2B6 and 6C3 staining in the territorial matrix of chondrocytes, indicating a reduced synthesis in 4-sulhated and native CS motifs. In contrast, increased 1B5 staining was observed in the articular cartilage from the low selenium + T-2 toxin group, suggesting a lack of CS sulphatransferase activity. Interestingly, there was increased 7D4 staining in the superficial zone of articular cartilage from low selenium + T-2 toxin group, suggesting an initiation of an osteoarthritis-like lesion. Discussion. These results indicated that low selenium nutrition and T-2 toxin could promote cell apoptosis and disrupt CS-GAG metabolism in ECM of rat articular cartilage in this animal model, which is similar to that observed in KBD patients. Collectively, our results support the hypothesis that low selenium and T-2 toxin are the cause of KBD

Although success has been achieved with implantation of bone marrow mesenchymal stem cells (bMSCs) in degenerative discs, its full potential may not be achieved if the harsh environment of the degenerative disc remains. Axial distraction has been shown to increase hydration and nutrition. Combining both therapies may have a synergistic effect in reversing degenerative disc disease. In order to evaluate the effect of bMSC implantation, axial distraction and combination therapy in stimulating regeneration and retarding degeneration in degenerative discs, we first induced disc degeneration by axial loading in a rabbit model. The rabbits in the intervention groups performed better with respect to disc height, morphological grading, histological scoring and average dead cell count. The groups with distraction performed better than those without on all criteria except the average dead cell count. Our findings suggest that bMSC implantation and distraction stimulate regenerative changes in degenerative discs in a rabbit model

Introduction. Cell-based therapies become more and more prominent for the treatment of intervertebral disc (IVD) injuries. Different strategies are under current development and address the restoration of either annulus fibrosus (AF) or nucleus pulposus (NP). Application of such Advanced Therapy Medicinal Products (ATMPs) is strictly regulated. One requirement is to show the identity of the cells, to make sure the cells are indeed AF or NP cells and retained their IVD cell character during manufacturing process before injection to the site of injury. Therefore, we recently identified novel marker genes that discriminate AF and NP cells on tissue level. However, expression of these AF and NP tissue markers has not been investigated in cultured cells, yet. The aim of this study was to proof the tissue marker”s specificity to discriminate cultured AF and NP cells. Furthermore, we evaluated the tissue markers robustness to different cell culture conditions. Materials & Methods. AF and NP tissue was obtained from human lumbal IVD of five donors (31–45 years) with mild to moderate degenerative changes (Pfirrmann≤3). Cells were isolated by enzymatic digestion and expanded in culture medium containing 10% human serum and 1% antibiotics. To address specificity, AF and NP cells were cultured separately. To address robustness, 1) cells were cultured up to passage P2, 2) cell culture was performed using two different cell culture media and 3) cells were cryopreserved in an optional intermediate step. Gene expression analysis was performed for 11 novel AF and NP tissue marker: LDB2, ADGRL4, EMCN, ANKRD29, OLFML2A, SPTLC3, DEFB1, DSC3, FAM132B, ARAP2, CDKN2B (patent pending). Results & Discussion. In cell culture, AF and NP cells were indistinguishable by eye. Both AF and NP cells showed same cell morphology and cell growth through monolayer expansion. For most of the tested novel AF and NP tissue marker genes no difference was seen in cultured cells AF and NP cells on mRNA level. Overall marker expression was lower in cultured cells compared to tissue level. Hence, cultured AF and NP cells lost distinct characteristics that they showed before on tissue level. However, three tissue marker genes showed distinct expression in cultured AF and NP cells: LDB2, ARAP2 and DSC3. Furthermore, expression level was not changed by serial monolayer passaging, intermediate cryopreservation or different nutrition supplied by culture media. Hence, cell marker gene expression was robust to different cell culture conditions. Conclusion. We defined three markers to discriminate cultured AF and NP cells. Gene expression was specific for either AF or NP cells and robust. These novel AF and NP cell markers can be used to test cell identity and to show preservation of cell character in quality control of cell-therapeutic products. Morever markers are of high value for development of new ATMPs for targeted treatment of eigher AF or NP, as well as tissue engineered discs

Metabolic disorders are frequently associated with tendon degeneration and impaired healing after acute injury. However, the underlying cellular and molecular mechanisms remain largely unclear. We have previously shown that human and rat tendon cells responde to glucose stimulation in vitro by secretion of insulin. Therefore, we now hypothesize that nutritional glucose uptake affects tendon healing in a rat model. In female rats (n=30/group), unilateral full-thickness Achilles tendon defects were created. Immediately after surgery animals were either fed a glucose rich- or a control diet for up to 4 weeks. Gait analysis (Catwalk, Noldus) was performed at three time points. In addition, tendon thickness measurements, biomechanical testing and immunohistochemical analysis were conducted. Subsequently, gene expression analysis, comparing cDNA pools (n=5) prepared from repair tissues of both groups was performed. The repair tissues of the high glucose group were significantly thicker compared to the control group (p<0.001). The intermediate toe spread, an indicator of pain, were significantly improved in the high glucose group one and two weeks post surgery. Biomechanical analysis revealed that the repair tissues of the high glucose group were significantly stiffer (p<0.05) compared to the control group, no significant difference was detected for maximum tensile load…. The proportion of Ki67+ cells in the repair tissue was 3.3% in the control diet group and 9,8% in the high glucose group, indicating increased cell proliferation (p<0.001). Finally, gene expression analysis revealed the chondrogenic marker genes Collagen II, Aggrecan, COMP and SOX9 to be upregulated and genes involved in lipid metabolism like PPARgamma and Fabp2 to be downregulated in the glucose diet group. Here we show fort he first time that a high-glucose diet affects gait pattern and tendon biomechanics, influences tendon thickness and cell proliferation. Gene expression analysis reveals a regulation of chondrogenic as well as adipogenic marker genes. The molecular mechanisms underlying these effects on cells and extracellular matrix are currently under investigation, potentially revealing targets for developing a dietary intervention scheme to support tendon regeneration after trauma or tendon disease

At our district general hospital in the southwest of England, around 694 total knee replacements (TKR) are performed annually. Since spring 2013 we have been using an enhanced recovery protocol for all TKR patients, yet we have neither assessed compliance with the protocol nor whether its implementation has made a discernible and measurable difference to the delivery of care in this patient population. Enhanced recovery after surgery (ERAS) protocols are multi-modal care pathways designed to aid recovery. They are based on best evidence and promote a multi-disciplinary approach which standardises care and encompasses nutrition, analgesia and early mobilisation throughout the pre, intra and postoperative phases of an inpatient stay. ERAS has been found to reduce length of stay (LOS), readmission rates and analgesic requirements following surgery. 1, 2, 3. Additionally, they have been shown to improve range of knee movement following TKR and improve mobility, patient satisfaction whilst reducing mortality and morbidity. 4, 5, 6. With these benefits in mind, we sought to investigate how well our trauma and orthopaedic department was complying with a local ERAS protocol and whether we could replicate the benefits seen within the literature. Following approval from our local audit office in September 2015 we generated a patient list of elective TKR patients under the same surgeon before and after the implementation of the ERAS protocol. Using discharge summaries and patient notes we extracted data for 39 patients operated on prior to the ERAS implementation between January 2011 and December 2012 and 27 patients following its introduction between January 2014 and September 2015. Data collected included length of stay, time to discharge from inpatient physiotherapy and use of analgesia and antiemetics. Alongside this we audited the compliance with all facets of the local ERAS protocol. There was no statistically significant difference between the 2 groups in terms of demographics or pre-operative morbidity. Overall compliance with the ERAS protocol was good but there was some variability, especially with intraoperative medication and type of anaesthesia which was likely due to individual patient factors. Compliance with postoperative analgesia was especially good with 93% of patients receiving all 4 suggested analgesics within the ERAS group. Length of stay (LOS) was significantly reduced by 0.5 days per patient (p value < 0.4). Overall compliance with the ERAS protocol was good but there was some variability, especially with intraoperative medication and type of anaesthesia, which was likely due to individual patient factors. Compliance with postoperative analgesia was especially good with 93% of patients receiving all suggested analgesics within the ERAS group. In terms of LOS, we found a statistically significant difference between the pre-ERAS and ERAS group of 0.5 days per patient. Within the context of our DGH, a 0.5 day reduction in LOS translates to around 350 bed days per year and a potential saving of GBP 105,000 (EUR 132,000) making this a clinically significant finding

Ontogeny of long bone cross-sectional geometry has lasting effects on adult bone structure. Growth and development of bone is influenced by biological and mechanical factors but the importance of these factors is poorly understood. A study of prenatal, neonatal and infant development in a bone with simple loading patterns, may improve our understanding. Five vertebral columns aged between 6 months prenatal to 2.5 years postnatal, were analysed to quantify the changes in trabecular architecture before and after birth. Several measures were collected including trabecular: thickness, bone volume fraction, connectivity density, number, structure model index and anisotropy. The findings show that in the first year after birth there is a substantial loss of bone volume via decreasing trabecular thickness and number, which tends to increase after 1.2 years. This sequential pattern of development may be a functional response to the initial requirement for calcium mineral homeostasis before birth, followed by the need for trabecular architecture to adapt to mechanical loading after birth. Calcium is essential for growing neonates and therefore osteoclastic resorbtion is up regulated by increasing parathyroid hormone levels. This may account for the loss of bone between 0–1 year. At one year infants begin to walk bipedally, thus weight bearing and ground reaction forces increase. The stable bone volume and increase in organisation of trabecular architecture after one year may reflect increasing weight bearing and ground reaction forces. These findings suggest that nutritional requirements after birth may have a stronger influence on vertebral trabeculae architecture than learning to walk

We present a novel use for an adult proximal humeral locking plate. In our case an 18-year-old female with cerebral palsy sustained a peri-prosthetic fracture of a blade plate previously inserted for a femoral osteotomy. Treatment was revision using a long proximal humeral locking plate. She had a successful outcome. We present the history and operative management. The female had a history of quadriplegic cerebral palsy, asthma, diabetes mellitus and congenital heart disease. She had a gastrostomy tube for enteral feeding. She was on nutritional supplements, baclofen, Omeprazole and movicol. She is looked after by her parents and requires a wheelchair for mobility. She is unable to communicate. Surgical History: Right adductor tenotomy, aged 11. Femoral Derotation Osteotomy & Dega Acetabular Osteotomy, aged 13. Right distal hamstring and knee capsule release, aged 14. Admitted to A&E (aged 18); unwitnessed fall. Painful, swollen, deformed thigh with crepitus. Xrays demonstrated peri- prosthetic fracture below blade plate. No specific equipment available to revise. Decision made to use PHILOS (Synthes, UK). GA, antibiotics, supine on table. Lateral approach. Plate removed after excising overgrown bone. Reduced and held. 10hole PHILOS applied. Near anatomical reduction. Secure fixation with locking screws proximally away from blade plate defect. Blood loss 800ml. 5 days in hospital. Sequential fracture clinic review. Wound healed well. Fracture healed on Xray at 11 months and discharged. To our knowledge this is the first reported use of a PHILOS plate for this specific fracture. The complexity of this case and underlying neurological disorder deemed long blade plate revision unsuitable. Fracture rates after femoral derotation osteotomies rare. 5/157 and 1/58 in the two largest studies to date. Conservative measures were the main recommendation. We have demonstrated a straightforward method for revision fixation with an excellent outcome. It would be recommended as an alternative to other surgeons in this position