Introduction. Primary cilia are organelles found singularly on almost every cell in the body, including tenocytes. Tendon is a hierarchical, composite structure, and previous work from our group has suggested that the cell populations in the inter-fascicular matrix (IFM) may be different from those within the fascicle matrix (FM). This study investigated how stress deprivation influenced the primary cilia of both cell types, and the mechanics of the IFM and the FM. Materials and Methods. Rat tail tendons were dissected and then either tested immediately (fresh), or maintained in media for 1 week, either stress deprived or at 4% static strain. Fascicles and IFM were then either, fixed and imaged to determine cilia length (n = 80–160 cilia per group from across 3 rats), or mechanically tested to determine the static and viscoelastic properties of both the fascicles and the IFM (n = 6–8 per group). Results. Cilia length in the IFM and FM of fresh samples were not significantly different. After 1 week of stress deprivation, the cilia had significantly increased in length in both the IFM and FM, however the increase in length in the IFM was significantly greater than that in the FM. Cilia in tissue maintained at 4% static strain were significantly shorter than those in stress deprived tissue, however they remained longer than those in fresh tissue. The tensile strength of the fascicles was not affected by stress deprivation or static strain conditions. However, the viscoelastic properties of the stress deprived fascicles were significantly reduced. By contrast, the tensile strength of the IFM was significantly reduced in the stress deprived samples, indicative of greater degradation in this region. Discussion. This is the first time differences in the cilia have been observed between tendon regions. Their different response to stress deprivation provides further evidence that these populations of cells respond differently to changes in mechanical stimulation. Cilia length increased more in the region where there was more mechanical degradation, suggesting that cilia are responding to their local mechanical environment

Background. Reduced bone mineral density is recognised as a risk factor for hip fractures and fragility fractures in general. Vitamin D is important in maintaining healthy bone mineral levels and can therefore affect risk of hip fracture. We investigated the correlation between vitamin D levels and bone mineral density, as well as fracture type, in neck of femur fractures and also assessed the relationship of vitamin D and social deprivation. Method. We included all patients admitted to our department, with a neck of femur fracture over one year (October 2013 to October 2014). We analysed vitamin D levels for all patients during admission and compared these to bone mineral density scores, based on DEXA scan results; hip fracture type & comminution, based on admission radiographs; and levels of social deprivation, based on the patient's address. Results. In total 360 patients were admitted over the study period, with a neck of femur fracture, of which 298 had vitamin D assessed and 76 had DEXA scans. Of these cohorts, 71% were found to be vitamin D deficient and 7% had osteoporosis. No significant correlation was found between vitamin D scores and bone density, or with level of vitamin D deficiency and fracture type or comminution. A significant correlation was however identified, between low vitamin D levels and decreasing levels of social deprivation (R=0.11, p=0.04). Conclusion. No relationship was identified between vitamin D levels and hip fracture type, suggesting that vitamin D cannot be used to predict patients at risk of more comminuted fractures. Although no relationship was also identified for bone mineral density and vitamin D, this may be because the sample size of DEXA scans was relatively small. Interestingly the relationship between vitamin D and social deprivation was the reverse of what was expected and suggests that affluent individuals may be at greater risk of low vitamin D

The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia. Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3). Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and COL2A1 expression analysis showed that MSC chondrogenesis was not compromised by any treatment. Importantly, hypertrophy markers (COL10A1, ALPL, SPP1, IBSP) were significantly reduced by PGE2 treatment, but increased by all inhibitors. Additionally, PGE2 significantly decreased ALP activity (2.9-fold), whereas the inhibitors caused a significant increase (1.3-fold, 1.7-fold, 1.8-fold). This identified PGE2 as an important inhibitor of chondrocyte hypertrophy. Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment

Introduction. Migration of bone cells and precursor cells to the site of a bone defect can accelerate bone regeneration. Therefore, guidance of these cells by direct current (DC) is an interesting approach to improve implant ingrowth or fracture healing. To allow a better understanding of DC-induced directed migration, a specific stimulation chamber was established and the influence of DC on calcium channel expression in osteoblasts was investigated. Methods. Human osteoblasts were isolated from femoral heads of patients undergoing total hip arthroplasty after patient”s consent. The study was approved by the local ethical committee (AZ: 2010–10). Differentiation into osteoblasts was ensured by cultivation in standard cell culture medium enriched with β-glycerophosphate, ascorbic acid and dexamethasone. 2×10. 3. osteoblasts were seeded into custom-made chambers for DC field application. After 12 h DC was applied to chambers via Ag/AgCl electrodes set into separate reservoirs coupled to cell culture area by 2% agarose bridges in order to prevent cytotoxic impact of electrochemical reactions proceeding at the electrodes. Electric fields ranging from 150 to 450 V/m were applied to cells for 7 h. Several cell images were taken over time and used for evaluation of migration direction and speed with ImageJ software. Subsequently, cells were lysed in Trizol for RNA isolation and semiquantitative real-time polymerase chain reaction of voltage-gated calcium channels Cav1.4 and Cav3.2 as well as stretch-activated magnesium and calcium channel TRPM7 was performed. Results. Migration velocity of DC stimulated bone cells was 6.4 ± 2.1 µm/h whereas unstimulated control cells migrated significantly slower with a velocity of 3.6 ± 1.1 µm/h (p<0.001). No correlation between magnitude of electric field and migration velocity was found. Migration of osteoblasts was directed towards the anode during DC application while unstimulated cells migrated undirectedly. Gene expression analysis showed significant correlation of electric field strength and TRPM7 expression (p<0.01) appearing in increased TRPM7 expression after exposure to higher electric fields. Voltage-gated calcium channels Cav1.4 and Cav3.2 were not regulated by DC fields. Conclusion. A chamber for DC field application on human osteoblasts was established and migration velocity and direction was found to be influenced by DC fields. Regulation of selected calcium channels by DC was observed for stretch-activated channel TPRM7 that is known to be involved in osteoblast differentiation and migration induced by platelet-derived growth factor. Future studies will concentrate on investigation of involvement of specific calcium channels in osteoblast migration by using specific calcium channel inhibitors and calcium deprivation from cell culture medium

Using in situ hybridisation and the terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labelling (TUNEL) reaction in rats with osteonecrosis of the femoral head we have studied the effect of ischaemia on the gene expression of the stress proteins oxygen-regulated protein 150 (ORP150) and haemoxygenase 1 (HO1) and the death mechanism of the cells involved in osteonecrosis. Both ORP150 and HO1 have been reported to have important roles in the successful adaptation to oxygen deprivation. ORP150 and HO1 mRNA expression was induced by ischaemia in osteoblasts and osteocytes. In proliferative chondrocytes, these signals were detected constitutively. During the development of ischaemic osteonecrosis, the mechanism of cell death was apoptosis as indicated by DNA fragmentation and the presence of apoptotic bodies in osteocytes, chondrocytes and bone-marrow cells. After the initial ischaemic event, expression of ORP150 and HO1 mRNA, the TUNEL-positive reaction and empty lacunae were found sequentially. These findings were exclusive and may be considered to be markers for each stage in the development of osteonecrosis

An understanding of the remodelling of tendon is crucial for the development of scientific methods of treatment and rehabilitation. This study tested the hypothesis that tendon adapts structurally in response to changes in functional loading. A novel model allowed manipulation of the mechanical environment of the patellar tendon in the presence of normal joint movement via the application of an adjustable external fixator mechanism between the patella and the tibia in sheep, while avoiding exposure of the patellar tendon itself. Stress shielding caused a significant reduction in the structural and material properties of stiffness (79%), ultimate load (69%), energy absorbed (61%), elastic modulus (76%) and ultimate stress (72%) of the tendon compared with controls. Compared with the material properties the structural properties exhibited better recovery after re-stressing with stiffness 97%, ultimate load 92%, energy absorbed 96%, elastic modulus 79% and ultimate stress 80%. The cross-sectional area of the re-stressed tendons was significantly greater than that of stress-shielded tendons.

The remodelling phenomena exhibited in this study are consistent with a putative feedback mechanism under strain control. This study provides a basis from which to explore the interactions of tendon remodelling and mechanical environment.

We report the effects of local administration of osteogenic protein-1 on the biomechanical properties of the overstretched anterior cruciate ligament in an animal model. An injury in the anterior cruciate ligament was created in 45 rabbits. They were divided into three equal groups. In group 1, no treatment was applied, in group II, phosphate-buffered saline was applied around the injured ligament, and in group III, 12.5 μg of osteogenic protein-1 mixed with phosphate-buffered saline was applied around the injured ligament. A control group of 15 rabbits was assembled from randomly-selected injured knees from among the first three groups. Each rabbit was killed at 12 weeks.

The maximum load and stiffness of the anterior cruciate ligament was found to be significantly greater in group III than either group 1 (p = 0.002, p = 0.014) or group II (p = 0.032, p = 0.025). The tensile strength and the tangent modulus of fascicles from the ligament were also significantly greater in group III than either group I (p = 0.002, p = 0.0174) or II (p = 0.005, p = 0.022).

The application of osteogenic protein-1 enhanced the healing in the injured anterior cruciate ligament, but compared with the control group the treated ligament remained lengthened. The administration of osteogenic protein-1 may have a therapeutic role in treating the overstretched anterior cruciate ligament.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.

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.