Objectives. During open orthopaedic surgery, joints may be exposed to air, potentially leading to cartilage drying and chondrocyte death, however, the long-term effects of joint drying in vivo are poorly understood. We used an animal model to investigate the subsequent effects of joint drying on cartilage and chondrocytes. Methods. The patellar groove of anaesthetised rats was exposed (sham-operated), or exposed and then subjected to laminar airflow (0.25m/s; 60 minutes) before wounds were sutured and animals recovered. Animals were monitored for up to eight weeks and then sacrificed. Cartilage and chondrocyte properties were studied by histology and confocal microscopy, respectively. Results. Joint drying caused extensive chondrocyte death within the superficial regions of cartilage. Histology of dried cartilage demonstrated a loss of surface integrity at four weeks, fibrillations at eight weeks, and an increased modified Mankin score (p < 0.001). Cartilage thickness increased (p < 0.001), whereas chondrocyte density decreased at four weeks (p < 0.001), but then increased towards sham-operated levels (p < 0.01) at eight weeks. By week eight, chondrocyte pairing/clustering and cell volume increased (p < 0.05; p < 0.001, respectively). Conclusions. These in vivo results demonstrated for the first time that as a result of laminar airflow, cartilage degeneration occurred which has characteristics similar to those seen in early osteoarthritis. Maintenance of adequate cartilage hydration during open orthopaedic surgery is therefore of paramount importance. Cite this article: Dr A. Hall. Drying of open animal joints in vivo subsequently causes cartilage degeneration. Bone Joint Res 2016;5:137–144. DOI: 10.1302/2046-3758.54.2000594

Objectives. The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. Methods. Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. Results. In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. Conclusion. This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes. Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362–372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2

This in vivo controlled laboratory study was performed to evaluate various intra-articular clinical injection regimes that might be less toxic than some in vitro studies suggest. We hypothesised that low-concentration, preservative-free, pH-balanced agents would be less toxic than high-concentration non-pH-balanced agents with preservatives, and that injections of individual agents are less toxic than combined injections. The left knees of 12- to 13-week-old Sprague–Dawley rats were injected once with eight different single agents, including low and high concentrations of ropivacaine and triamcinolone, alone and in combination, as well as negative and positive controls. The rats were killed at one week or five months, and live–dead staining was performed to quantify the death of chondrocytes. All injections except pH-balanced 0.2% ropivacaine combined with preservative-free 1 mg/ml triamcinolone acetonide resulted in statistically significant decreases in chondrocyte viability, compared with control knees, after one week and five months (p < 0.001). After one week there was no significant difference in viability between 0.2% and 0.5% ropivacaine; however, 4 mg/ml triamcinolone resulted in a lower viability than 1 mg/ml triamcinolone. Although many agents commonly injected into joints are chondrotoxic, in this in vivo study diluting preservative-free 10 mg/ml triamcinolone 1:9 in 0.2% pH-balanced ropivacaine resulted in low toxicity. Cite this article: Bone Joint J 2015; 97-B:933–8

Polyethylene particulate wear debris continues to be implicated in the aetiology of aseptic loosening following knee arthroplasty. The Oxford unicompartmental knee arthroplasty employs a spherical femoral component and a fully congruous meniscal bearing to increase contact area and theoretically reduce the potential for polyethylene wear. This study measures the in vivo ten-year linear wear of the device, using a roentgenstereophotogrammetric technique. In this in vivo study, seven medial Oxford unicompartmental prostheses, which had been implanted ten years previously were studied. Stereo pairs of radiographs were acquired for each patient and the films were analysed using a roentgen stereophotogrammetric analysis calibration and a computer-aided design model silhouette-fitting technique. Penetration of the femoral component into the original volume of the bearing was our estimate of linear wear. In addition, eight control patients were examined less than three weeks post-insertion of an Oxford prosthesis, where no wear would be expected. The control group showed no measured wear and suggested a system accuracy of 0.1 mm. At ten years, the mean linear wear rate was 0.02 mm/year. The results from this in vivo study confirm that the device has low ten-year linear wear in clinical practice. This may offer the device a survival advantage in the long term

Aims

Higher osteoblastic bone activity is expected in aseptic loosening and painful unicompartmental knee arthroplasty (UKA). However, insights into normal bone activity patterns after medial UKAs are lacking. The aim of this study was to identify the evolution in bone activity pattern in well-functioning medial mobile-bearing UKAs.

Aims

Massive endoprostheses rely on extra-cortical bone bridging (ECBB) to enhance fixation. The aim of this study was to investigate the role of selective laser sintered (SLS) porous collars in augmenting the osseointegration of these prostheses.

We have examined whether primary human muscle-derived cells can be used in ex vivo gene therapy to deliver BMP-2 and to produce bone in vivo. Two in vitro experiments and one in vivo experiment were used to determine the osteocompetence and BMP-2 secretion capacity of cells isolated from human skeletal muscle. We isolated five different populations of primary muscle cells from human skeletal muscle in three patients. In the first in vitro experiment, production of alkaline phosphatase by the cells in response to stimulation by rhBMP-2 was measured and used as an indicator of cellular osteocompetence. In the second, secretion of BMP-2 was measured after the cell populations had been transduced by an adenovirus encoding for BMP-2. In the in vivo experiment, the cells were cotransduced with a retrovirus encoding for a nuclear localised β-galactosidase gene and an adenovirus encoding for BMP-2. The cotransduced cells were then injected into the hind limbs of severe combined immune-deficient (SCID) mice and analysed radiographically and histologically. The nuclear localised β-galactosidase gene allowed identification of the injected cells in histological specimens. In the first in vitro experiment, the five different cell populations all responded to in vitro stimulation of rhBMP-2 by producing higher levels of alkaline phosphatase when compared with non-stimulated cells. In the second, the five different cell populations were all successfully transduced by an adenovirus to express and secrete BMP-2. The cells secreted between 444 and 2551 ng of BMP-2 over three days. In the in vivo experiment, injection of the transduced cells into the hind-limb musculature of SCID mice resulted in the formation of ectopic bone at 1, 2, 3 and 4 weeks after injection. Retroviral labelling of the cell nuclei showed labelled human muscle-derived cells occupying locations of osteoblasts in the ectopic bone, further supporting their osteocompetence. Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in BMP-2 gene therapy approaches

Human bone-marrow mesenchymal stem cells have an important role in the repair of musculoskeletal tissues by migrating from the bone marrow into the injured site and undergoing differentiation. We investigated the use of autologous human serum as a substitute for fetal bovine serum in the ex vivo expansion medium to avoid the transmission of dangerous transfectants during clinical reconstruction procedures. Autologous human serum was as effective in stimulating growth of bone-marrow stem cells as fetal bovine serum. Furthermore, medium supplemented with autologous human serum was more effective in promoting motility than medium with fetal bovine serum in all cases. Addition of B-fibroblast growth factor to medium with human serum stimulated growth, but not motility. Our results suggest that autologous human serum may provide sufficient ex vivo expansion of human bone-marrow mesenchymal stem cells possessing multidifferentiation potential and may be better than fetal bovine serum in preserving high motility

We have undertaken an in vivo assessment of the tissue metabolism and cellular activity in torn tendons of the rotator cuff. Cellular oxygen consumption was measured in 13 patients undergoing mini-open repair of small, medium, large and massive full-thickness tears. Measurements were also taken from three control patients who were undergoing open stabilisation of the shoulder with grossly normal tendons. The level of oxygen and nitrous oxide was measured amperometrically using silver needle microelectrodes at the apex of the tear and 1.5 cm from its edge. With nitrous oxide indicating the degree of perfusion, oxygen consumption was calculated at each location to reflect cellular activity. All of the torn tendons had lower levels of cellular activity than the control group. This activity was lower still in the tissue nearest to the edge of the tear with the larger tears showing the lowest activity. This indicated reduced levels of tissue metabolism and infers a reduction in tendon viability. Our findings suggest that surgical repair of torn tendons of the rotator-cuff should include the more proximal, viable tissue, and may help to explain the high rate of re-rupture seen in larger tears

We used three-dimensional movement analysis by computer modelling of knee flexion from 0° to 50° in 14 knees in 12 patients with recurrent patellar dislocation and in 15 knees in ten normal control subjects to compare the in vivo three-dimensional movement of the patella. Flexion, tilt and spin of the patella were described in terms of rotation angles from 0°. The location of the patella and the tibial tubercle were evaluated using parameters expressed as percentage patellar shift and percentage tubercle shift. Patellar inclination to the femur was also measured and patellofemoral contact was qualitatively and quantitatively analysed. The patients had greater values of spin from 20° to 50°, while there were no statistically significant differences in flexion and tilt. The patients also had greater percentage patellar shift from 0° to 50°, percentage tubercle shift at 0° and 10° and patellar inclination from 0° to 50° with a smaller oval-shaped contact area from 20° to 50° moving downwards on the lateral facet. Patellar movement analysis using a three-dimensional computer model is useful to clearly demonstrate differences between patients with recurrent dislocation of the patella and normal control subjects

We examined the mechanical properties of Vicryl (polyglactin 910) mesh in vitro and assessed its use in vivo as a novel biomaterial to attach tendon to a hydroxyapatite-coated metal implant, the interface of which was augmented with autogenous bone and marrow graft. This was compared with tendon re-attachment using a compressive clamp device in an identical animal model. Two- and four-ply sleeves of Vicryl mesh tested to failure under tension reached 5.13% and 28.35% of the normal ovine patellar tendon, respectively. Four-ply sleeves supported gait in an ovine model with 67.05% weight-bearing through the operated limb at 12 weeks, without evidence of mechanical failure. Mesh fibres were visible at six weeks but had been completely resorbed by 12 weeks, with no evidence of chronic inflammation. The tendon-implant neoenthesis was predominantly an indirect type, with tendon attached to the bone-hydroxyapatite surface by perforating collagen fibres

We have used in vivo microdialysis to monitor postoperative physiological events in the synovial membrane after arthroscopy. The levels of lactate were significantly higher in the synovial membrane than in the reference tissue (subcutaneous fat) and there was a significant increase in lactate after operation. Blood flow, measured as the ethanol ratio, was stable in both tissues. Our findings show that there was an increase in the local production of lactate since the levels of lactate in blood and the reference tissue were comparable and did not show a significant increase. There was also a consumption of glucose in the synovial membrane which was not observed in the reference tissue. The levels of pyruvate were higher in the synovial membrane. A state of reperfusion occurs in the synovial membrane after moderate trauma such as standard arthroscopy of the knee. Microdialysis should be further evaluated in studies of the in vivo physiology of the synovial membrane

Polyethylene acetabular cups retrieved at revision surgery were measured by a shadowgraph technique to determine linear wear, and the values were compared with those obtained from radiographs. There was a close correlation between them, although the radiographic measurements slightly underestimated the true wear. Average linear wear rates for surface-replacement components were much greater than those for conventional prostheses with femoral heads up to 32 mm in diameter. Volumetric wear, calculated using a new formula, was found to be less than previously reported in vivo, and similar in magnitude to the results of experimental wear tests in vitro. The volumetric wear rates were greatest for the surface-replacement components and, for conventional components, were found to increase in a linear manner with component diameter

We used demineralised bone matrix (DBM) to augment re-attachment of tendon to a metal prosthesis in an in vivo ovine model of reconstruction of the extensor mechanism at the knee. We hypothesised that augmentation of the tendon-implant interface with DBM would enhance the functional and histological outcomes as compared with previously reported control reconstructions without DBM. Function was assessed at six and 12 weeks postoperatively, and histological examination was undertaken at 12 weeks. A significant increase of 23.5% was observed in functional weight-bearing at six weeks in the DBM-augmented group compared with non-augmented controls (p = 0.004). By 12 weeks augmentation with DBM resulted in regeneration of a more direct-type enthesis, with regions of fibrocartilage, mineralised fibrocartilage and bone. In the controls the interface was predominantly indirect, with the tendon attached to the bone graft-hydroxyapatite base plate by perforating collagen fibres

Nanometre-sized particles of ultra-high molecular weight polyethylene have been identified in the lubricants retrieved from hip simulators. Tissue samples were taken from seven failed Charnley total hip replacements, digested using strong alkali and analysed using high-resolution field emission gun-scanning electron microscopy to determine whether nanometre-sized particles of polyethylene debris were generated in vivo. A randomised method of analysis was used to quantify and characterise all the polyethylene particles isolated. We isolated nanometre-sized particles from the retrieved tissue samples. The smallest identified was 30 nm and the majority were in the 0.1 μm to 0.99 μm size range. Particles in the 1.0 μm to 9.99 μm size range represented the highest proportion of the wear volume of the tissue samples, with 35% to 98% of the total wear volume comprised of particles of this size. The number of nanometre-sized particles isolated from the tissues accounted for only a small proportion of the total wear volume. Further work is required to assess the biological response to nanometre-sized polyethylene particles

Objectives

The aim of this study was to determine the polyethylene wear rate of Phase 3 Oxford Unicompartmental Knee Replacement bearings and to investigate the effects of resin type and manufacturing process.

Objective. In ex vivo hip fracture studies femoral pairs are split to create two comparable test groups. When more than two groups are required, or if paired femurs cannot be obtained, group allocation according to bone mineral density (BMD) is sometimes performed. In this statistical experiment we explore how this affects experimental results and sample size considerations. Methods. In a hip fracture experiment, nine pairs of human cadaver femurs were tested in a paired study design. The femurs were then re-matched according to BMD, creating two new test groups. Intra-pair variance and paired correlations in fixation stability were calculated. A hypothetical power analysis was then performed to explore the required sample size for the two types of group allocation. . Results. The standard deviation (. sd. ) of the mean paired difference in fixation stability increased from 2 mm in donor pairs to 5 mm in BMD-matched pairs. Intra-pair correlation was 0.953 (Pearson’s r) in donor pairs and non-significant at -0.134 (Pearson’s r) in BMD-matched pairs. Required sample size to achieve a statistical power of 0.8 increased from ten pairs using donor pairs to 54 pairs using BMD-matched pairs. Conclusion. BMD cannot be used to create comparable test groups unless sample size is increased substantially and paired statistics are no longer valid. Cite this article: Bone Joint Res 2014;3:317–20

Injury to muscles is very common. We have previously observed that basic fibroblast growth factor (b-FGF), insulin growth factor type 1 (IGF-1) and nerve growth factor (NGF) are potent stimulators of the proliferation and fusion of myoblasts in vitro. We therefore injected these growth factors into mice with lacerations of the gastrocnemius muscle. The muscle regeneration was evaluated at one week by histological staining and quantitative histology. Muscle healing was assessed histologically and the contractile properties were measured one month after injury. Our findings showed that b-FGF, IGF and to a less extent NGF enhanced muscle regeneration in vivo compared with control muscle. At one month, muscles treated with IGF-1 and b-FGF showed improved healing and significantly increased fast-twitch and tetanus strengths. Our results suggest that b-FGF and IGF-1 stimulated muscle healing and may have a considerable effect on the treatment of muscle injuries

Chondrocytes at the lower zone of the growth plate must be eliminated to facilitate longitudinal growth; this is generally assumed to involve apoptosis. We attempted to provide definitive electron-microscopic evidence of apoptosis in chondrocytes of physes and chondroepiphyses in the rabbit. We were, however, unable to find a single chondrocyte with the ultrastructure of ‘classical’ apoptosis in vivo, although such a cell was found in vitro. Instead, condensed chondrocytes had a convoluted nucleus with patchy chromatin condensations while the cytoplasm was dark with excessive amounts of endoplasmic reticulum. These cells were termed ‘dark chondrocytes’. A detailed study of their ultrastructure combined with localisation methods in situ suggested a different mechanism of programmed cell death. In addition, another type of death was identified among the immature chondrocytes of the chondroepiphysis. These cells had the same nucleus as dark chondrocytes, but the lumen of the endoplasmic reticulum had expanded to fill the entire non-nuclear space, and all cytoplasm and organelles had been reduced to dark, worm-like inclusions. Since these cells appeared to be ‘in limbo’, they were termed ‘paralysed’ cells. It is proposed that ‘dark chondrocytes’ and ‘paralysed cells’ are examples of physiological cell death which does not involve apoptosis. It is possible that the confinement of chondrocytes within their lacunae, which would prevent phagocytosis of apoptotic bodies, necessitates different mechanisms of elimination

Mechanical loading during physical activity produces strains within bones. It is thought that these forces provide the stimulus for the adaptation of bone. Tibial strains and rates of strain were measured in vivo in six subjects during running, stationary bicycling, leg presses and stepping and were compared with those of walking, an activity which has been found to have only a minimal effect on bone mass. Running had a statistically significant higher principal tension, compression and shear strain and strain rates than walking. Stationary bicycling had significantly lower tension and shear strains than walking. If bone strains and/or strain rates higher than walking are needed for tibial bone strengthening, then running is an effective strengthening exercise for tibial bone