Objectives

Surgical marking during tendon surgery is often used for technical and teaching purposes. This study investigates the effect of a gentian violet ink marker pen, a common surgical marker, on the viability of the tissue and cells of tendon.

Tissue engineering by self-assembly is a technique that consists of growing cells on surfaces made of thermoresponsive polymers, that allow the production of contiguous cell sheets by simply lowering the temperature below the polymer's low critical solution temperature. In this approach cell-cell junctions and deposited extracellular matrix (ECM) remain intact, which provides a better cell localisation at the site of injury. However, these systems lack the possibility to fabricate multi-layered and three-dimensional cell sheets that would better recapitulate native tissues. Moreover, the fabrication of ECM-rich cell sheets would be highly desirable. This limitation could be overcome by inducing macromolecular crowding (MMC) conditions. Herein we venture to fabricate electrospun thermoresponsive nanofibres to sustain the growth and detachment of ECM-rich tissue substitutes in the presence of a MMC microenvironment. A copolymer of 85% poly-N-isopropylacrylamide and 15% N-tert-butylacrylamide (pNIPAAm/NTBA) were used for all experiments. To create aligned nanofibers, the polymer was electrospun and collected on a mandrel rotating at 2000 rpm. Human adipose derived stem cells (hADSC) were treated with media containing macromolecular crowders to enhance matrix deposition. Cell viability and morphology were assessed, and immunocytochemistry was conducted in order to estimate matrix deposition and composition. Adipogenic, osteogenic and chondrogenic assays were performed both with and without the presence of MMC. Non-invasive cell detachment was enabled by decreasing the temperature of culture to 10 °C for 20 minutes. The electrospinning process resulted in the production of pNIPAm/NTBA fibres in the diameter range from 1 to 2 µm and an overall alignment of 80%. Cell viability, proliferation and metabolic activity revealed that hADSCs were able to grow on the thermoresponsive scaffold. The cells were able to detach as an intact cell sheet in presence of MMC. Moreover, it was demonstated that MMC, by a volume extrusion effect, enhances Collagen type I deposition, which is one of the main components of the ECM. Histological analysis revealed that in the presence of MMC the cells were able to self-assembled into three dimensional multi-layers. The cells were able to differentiate towards the osteogenic and adipogenic lineage in the presence of MMC. Interestingly we were able to fabricate three-dimensional chondrogenic cell sheet both with and without MMC. Collectively the pNIPAm/NTBA thermoresponsive fibres were able to sustain the growth and the detachment of ECM-rich multi-layered cell sheets. The pNIPAm/NTBA fibres were able to successfully sustain growth and detachment of ECM-rich tissue equivalents. We believe that replacement, repair and restoration of tissue function can be accomplished best using cells that create their own tissue-specific extracellular matrix with a precision and stoichiometric efficiency still unmatched by man-made devices

Tungsten has been increasing in demand for use in manufacturing and recently, medical devices, as it imparts flexibility, strength, and conductance of metal alloys. Given the surge in tungsten use, our population may be subjected to elevated exposures. For instance, embolism coils made of tungsten have been shown to degrade in some patients. In a cohort of breast cancer patients who received tungsten-based shielding for intraoperative radiotherapy, urinary tungsten levels remained over tenfold higher 20 months post-surgery. In vivo models have demonstrated that tungsten exposure increases tumor metastasis and enhances the adipogenesis of bone marrow-derived mesenchymal stem cells while inhibiting osteogenesis. We recently determined that when mice are exposed to tungsten [15 ppm] in their drinking water, it bioaccumulates in the intervertebral disc tissue and vertebrae. This study was performed to determine the toxicity of tungsten on intervertebral disc. Bovine nucleus pulposus (bNP) and annulus fibrosus (bAF) cells were isolated from bovine caudal tails. Cells were expanded in flasks then prepared for 3D culturing in alginate beads at a density of 1×10. ∧. 6 cells/mL. Beads were cultured in medium supplemented with increasing tungsten concentrations in the form of sodium tungstate [0, 0.5, 5, 15 ug/mL] for 12 days. A modified GAG assay was performed on the beads to determine proteoglycan content and Western blotting for type II collagen (Col II) synthesis. Cell viability was determined by counting live and dead cells in the beads following incubation with the Live/Dead Viability Assay kit (Thermo Fisher Scientific). Cell numbers in beads at the end of the incubation period was determined using Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific). Tungsten dose-dependently decreased the synthesis of proteoglycan in IVD cells, however, the effect was significant at the highest dose of 15 ug/mL. (n=3). Furthermore, although tungsten decreased the synthesis of Col II in IVD cells, it significantly increased the synthesis of Col I. Upregulation of catabolic enzymes ADAMTS4 and −5 were also observed in IVD cells treated with tungsten (n=3). Upon histological examination of spines from mice treated with tungsten [15 ug/mL] in their drinking water for 30 days, disc heights were diminished and Col I upregulation was observed (n=4). Cell viability was not markedly affected by tungsten in both bNP and bAF cells, but proliferation of bNP cells decreased at higher concentration. Surprisingly, histological examination of IVDs and gene expression analysis demonstrated upregulation of NGF expression in both NP and AF cells. In addition, endplate capillaries showed increases in CGRP and PGP9.5 expression as determined on histological sections of mouse IVDs, suggesting the development of sensory neuron invasion of the disc. We provide evidence that prolonged tungsten exposure can induce disc fibrosis and increase the expression of markers associated with pain. Tungsten toxicity may play a role in disc degeneration disease

Purpose. Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response. Method. Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism. Results. Cell viability in CEP isolated discs was evaluated after four weeks and four months of organ culture under high and physiological nutritional state. Previous studies have shown that high glucose levels are needed to maintain cell viability in organ culture, but in our model 96–98% live cells were present throughout the disc independent of FBS and glucose levels and the duration of culture tested. Western blot probing for the G1 domain of aggrecan showed no difference with the change of nutritional state across all regions indicating that low nutritional state had no detrimental effect on disc metabolism. Conclusion. We have developed a novel technique for isolation and culturing of intact IVDs. The described CEP system maintained sufficient nutrient supply and high cell survival in all regions of the disc for up to four months of culture also under physiological culturing condition. As the CEP system maintains high cell viability in long term cultures, it is a suitable model in which the regenerative effect of various bioactive peptides can be studied. The availability of an intact disc organ culture system has considerable advantage over the culture of isolated disc cells, as it maintains the cells in their unique microenvironment, so making any response to catabolic or anabolic agents more physiologically relevant

Purpose. Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response. Method. Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism. Results. Cell viability in CEP isolated discs was evaluated after four weeks and four months of organ culture under high and physiological nutritional state. Previous studies have shown that high glucose levels are needed to maintain cell viability in organ culture, but in our model 96–98% live cells were present throughout the disc independent of FBS and glucose levels and the duration of culture tested. Western blot probing for the G1 domain of aggrecan showed no difference with the change of nutritional state across all regions indicating that low nutritional state had no detrimental effect on disc metabolism. Conclusion. We have developed a novel technique for isolation and culturing of intact IVDs. The described CEP system maintained sufficient nutrient supply and high cell survival in all regions of the disc for up to four months of culture also under physiological culturing condition. As the CEP system maintains high cell viability in long term cultures, it is a suitable model in which the regenerative effect of various bioactive peptides can be studied. The availability of an intact disc organ culture system has considerable advantage over the culture of isolated disc cells, as it maintains the cells in their unique microenvironment, so making any response to catabolic or anabolic agents more physiologically relevant

Introduction. Local anaesthetic has been reported to have a detrimental effect on human chondrocytes both in vitro and in vivo. Magnesium, an NMDA-receptor antagonist, may be an alternative intra-articular analgesic agent following arthroscopy. We aimed to report the dose response effect of commonly used local anaesthteitc on chondrocyte viability and also report on the effect of adding magnesium to local anaesthetic. Methods. Human chondrocytes were grown under standard conditions. Cells were exposed to either lignocaine (0.5, 1, 2%), levobupivacaine (0.125, 0.25, 0.5%), bupivacaine (0.125, −.25, 0.5%) or ropivacaine (0.1875, 0.375, 0.75%) for 15 minutes. Cells were also exposed to a local anesthetic agent with the addition of magnesium (10, 20, or 50%). Cells exposed to media or saline served as controls. The MTS assay was used to assess cell viability 24-hours after exposure. Results. One-way ANOVA showed an expected dose response in all local anaesthetic groups with the exception of lignocaine. Magnesium alone was no more toxic than normal saline (P>0.3). 50% magnesium showed similar effect on cell viability to the least toxic local anaesthetic (lignocaine 1%, P=0.31). The addition of magnesium to the local anesthetic agents resulted in greater cell viability than when cells were treated with the respective local anaesthetic alone (lignocaine (P=0.033), levobupivacaine (P=0.007), bupivacaine (P<0.001), ropivacaine (P<0.001)). Conclusion. We have shown that cell viability is greater in the presence of magnesium than selected local anaesthetics and also with the addition of magnesium to local anaesthetic compared to the local anesthetic agent alone. We believe that these findings offer support to an alternative intra-articular analgesia following arthroscopy

Introduction:. Unicompartmental knee arthroplasty (UKA) has been used in the past decades to treat progressive cartilage degeneration in a single compartment. Concern has been raised over the rate of revision procedures for polyethylene wear and osteoarthritic progression into the adjacent compartment. Few studies have examined the pathology of cartilage degeneration in the setting of UKA. This study aims to investigate the viability of knee chondrocytes introduced to high and low concentrations of orthopaedic wear debris particulate. Methods:. Normal human articular chondrocytes (nHAC-Kn) were expanded in DMEM/F12 containing 10% FBS, 1% Penicillin/Streptomycin (Pen/Strp), and 50 μg/mL ascorbic acid (Asc). 24 hours prior to the start of the experiment, cells were seeded on 96-well plates at a density of 3500 cells/cm. 2. and exposed to DMEM/F12 containing 5% FBS, 1% Pen/Strp, and 50 μg/mL Asc. Particles (equivalent circle diameter range: 0.2–7 μm) at a low dose of 100: 1 (particles: cells) and high dose 1000: 1 (particles: cells) were introduced to treatment wells (n = 6). Control wells (n = 6) contained particles with no cells. Treatment groups included high and low doses of TiAl. 6. V. 4. alloy, 316L Stainless Steel, and Co-Cr-Mo alloy. At days 1, 3, 5, and 7, cells were assayed with a 3-(4,5-Dimethylthiazol-2-yl)-2,5-dyphenyltetrazolium bromide (MTT) assay for determination of cell viability. Light microscopy was performed at each timepoint to assess change in cell morphology. Results:. All groups displayed a minor decrease in cell viability after 24 hours of exposure to particles. Similarly, a second distinct decrease in viability occurred at the day 3 timepoint. Days 5 and 7 yielded little change in cell viability. Results are displayed in Figure 1. Observations of light microscopy revealed cells may actively engulf particles over time. Images show particle concentrations at the same locations as chondrocytes with few particles present between cells. Conclusions:. Wear debris has been implicated as a contributing source to osteolysis and component loosening. A potential effect on the cellular level can ultimate lead to effects on the entire tissue and complications on the clinical level. A decrease in chondrocyte viability has been shown in response to the presence of particulate wear debris. Our results showed decreases in cell viability were most noticeable between 24 and 72 hours after introduction to particles. Chondrocyte death may contribute to progression of cartilage degeneration into healthy compartments of the knee. Continued experiments are underway further characterizing chondrocyte response to wear debris particulate with respect to protein and gene expression in an extended 7 day in vitro culture

Testing potential therapeutics in the regeneration of the disc requires the use of model systems. Although several animal models have been developed to test intervertebral disc (IVD) regeneration, application becomes costly when used as a screening method. The bovine IVD organ culture system offers an inexpensive alternative, however, in the current paradigm, the bony vertebrae is removed to allow for nutrient diffusion to disc cells. This provides limitations on the conditions and strategies one can employ in investigating IVD regeneration and mechanisms in degenerative disc disease (i.e. complex loading). Although one method has been attempted to extend the survival of bovine vertebrae containing IVDs (vIVD) cell viability declined after two weeks in culture. Our goal was to develop and validate a long-term organ culture model with vertebral bone, which could be used subsequently for studying biological repair of disc degeneration and biomechanics. Preparation of vIVDs: Bovine IVDs from the tails of 22–28-month-old steers were prepared for organ culture by parallel cuts through the adjacent vertebral bodies at 1cm from the endplates using an IsoMet®1000 Buehler precision sectioning saw. vIVDs were split into two groups: IVDs treated with PrimeGrowth Media kit (developed by Intervertech and licensed to Wisent Bioproducts) and IVDs with DMEM. The PrimeGrowth group was incubated for 1h in PrimeGrowth Isolation Medium (Cat# 319–511-EL) and the DMEM group for 1h in DMEM. After isolation, IVDs were washed in PrimeGrowth Neutralisation Medium (Cat# 319–512-CL) while the other IVDs were washed in DMEM. The discs isolated with PrimeGrowth and DMEM were cultured for up to 5 months in sterile vented 60 ml Leakbuster™ Specimen Containers in PrimeGrowth Culture Medium (Cat# 319–510-CL) and DMEM with no mechanical load applied. Live/Dead Assay: vIVDs cultured for 1 or 5 months were dissected and cell viability was assessed in different regions by confocal microscopy using Live/Dead® (Invitrogen) fluorescence assay. Glucose Diffusion: After one month of culture, vIVDs were incubated for 72h in diffusion medium containing PBS (1x), CaCl2 (1mM), MgCl2 (0.5mM), KCl2 (5mM), 0.1% BSA and 150µM 2-NDBG, a D-glucose fluorescent analogue. Discs were dissected and IVD tissues were incubated in guanidinium chloride extraction buffer. Extracts were measured for fluorescence. After 5 months in culture, vIVDs prepared with PrimeGrowth kit demonstrated approximately 95% cell viability in all regions of the disc. However, dramatic reductions (∼90%) in vIVD viability were measured in DMEM group after 1 month. vIVD viability was related to the amount of 2-NDBG incorporated into the disc tissue. We have developed a novel method for isolating IVDs with vertebral bone capable of long-term viability. This method may not only help in the discovery of novel therapeutics in disc regeneration, but could also advance our understanding on complex loading paradigms in disc degeneration

Calcification of the intervertebral disc (IVD) has been correlated with degenerative disc disease (DDD), a common cause of low back pain. The appearance of calcium deposits has been shown to increase with age, and its occurrence has been associated with several other disorders such as hyperparathyroidism, chondrocalcinosis, and arthritis. Trauma, vertebral fusion and infection have also been shown to increase the incidence of IVD calcification. The role of IVD calcification in the development DDD is unknown. Our preliminary data suggest that ionic calcium content and expression of the extracellular calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR) and regulator of calcium homeostasis, are increased in the degenerated discs. However, its role in DDD remains unclear. IVD Cells: Bovine and normal human IVD cells were incubated in PrimeGrowth culture medium (Wisent Bioproducts, Canada; Cat# 319–510-CL, −S1, and S2) and supplemented with various concentrations of calcium (1.0, 1.5, 2.5, 5.0 mM), a CaSR agonist [5 µM], or IL-1β [10 ng/ml] for 7 days. Accumulated matrix protein was quantitated for aggrecan and type II collagen (Col II) by Western blotting. Conditioned medium was also collected from cells treated for 24h and measured for the synthesis and release of total proteoglycan using the DMMB assay and Western blotting for Col II content. IVD Cultures: Caudal IVDs from tails of 20–24 month old steers were isolated with the PrimeGrowth Isolation kit (Wisent Bioproducts, Canada). IVDs were cultured for 4 weeks in PrimeGrowth culture medium supplemented with calcium (1.0, 2.5, or 5.0 mM), or a CaSR agonist [5 µM]. Cell viability was measured in NP and AF tissue using Live/Dead Imaging kit (ThermoFisher, Waltham, MA), to determine if Ca2+ effects cell viability end the expression of aggrecan and Col II was evaluated in the IVD tissue by Western blotting. Histological sections were prepared to determine total proteoglycan content, alkaline phosphatase expression and degree of mineralisation by von Kossa staining. The accumulation of aggrecan and Col II decreased dose-dependently in IVD cells following supplementation with calcium or the CaSR agonist. Conditioned medium also demonstrated decreases in the synthesis and release of proteoglycan and collagen with increasing Ca2+ dose or direct activation of the CaSR with agonist. A similar phenomenon was observed for total proteoglycan and aggrecan and Col II in IVDs following calcium supplementation or the CaSR agonist. In addition to decreases in Col II and aggrecan, increases in alkaline phosphatase expression and mineralisation was observed in IVDs cultured in elevated Ca2+ concentrations without affecting cell viability. Our results suggest that changes in the local concentrations of calcium are not benign, and that activation of the CaSR may be a contributing factor in IVD degeneration. Determining ways to minimise Ca2+ infiltration into the disc may mitigate disc degeneration

Introduction. Silicon nitride (SiN) is a recently introduced bearing material for THR that has shown potential in its bulk form and as a coating material on cobalt-chromium (CoCr) substrates. Previous studies have shown that SiN has low friction characteristics, low wear rates and high mechanical strength. Moreover, it has been shown to have osseointegration properties. However, there is limited evidence to support its biocompatibility as an implant material. The aim of this study was to investigate the responses of peripheral blood mononuclear cells (PBMNCs) isolated from healthy human volunteers and U937 human histiocytes (U937s) to SiN nanoparticles and CoCr wear particles. Methods. SiN nanopowder (<50nm, Sigma UK) and CoCr wear particles (nanoscale, generated in a multidirectional pin-on-plate reciprocator) were heat-treated for 4 h at 180°C and dispersed by sonication for 10 min prior to their use in cell culture experiments. Whole peripheral blood was collected from healthy donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep® as a density gradient medium and incubated for 24 h in 5% (v/v) CO2at 37°C to allow attachment of mononuclear phagocytes. SiN and CoCr particles were then added to the phagocytes at a volume concentration of 50 µm3 particles per cell and cultured for 24 h in RPMI-1640 culture medium in 5% (v/v) CO2 at 37°C. Cells alone were used as a negative control and lipopolysaccharide (LPS; 200ng/ml) was used as a positive control. Cell viability was measured after 24 h by ATPLite assay and tumour necrosis factor alpha (TNF-α) release was measured by sandwich ELISA. U937s were co-cultured with SiN and CoCr particles at doses of 0.05, 0.5, 5 and 50 µm3 particles per cell for 24h in 5% (v/v) CO2 at 37 C. Cells alone were used as a negative control and camptothecin (2 µg/ml) was used as a positive control. Cell viability was measured after 0, 1, 3, 6 and 9 days. Results from cell viability assays and TNF-α response were expressed as mean ±95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. Results and Discussion. At a high volume concentration of particles (50µm3 per cell), SiN did not affect the viability of PBMNCs, while CoCr significantly reduced the viability over a 24 h period [Figure 1A]. Similarly, SiN particles had no effect on the viability of U937s up to 9 days with a range of particle doses (0.05–50 µm3 per cell) [Figure 2A]. In contrast, CoCr particles significantly reduced the viability of U937s after 6 days [Figure 2B]. Additionally, CoCr particles caused significantly elevated levels of pro-inflammatory cytokine TNF-α, whereas no inflammation was associated with SiN particles [Figure 1B]. Conclusion. This study has demonstrated the in-vitro biocompatibility of SiN nanoparticles. Therefore, SiN is a promising orthopaedic bearing material not only due to its suitable mechanical and tribological properties, but also due to its biocompatibility. Acknowledgements. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. GA-310477 LifeLongJoints

Introduction. Currently, there is a focus on the development of novel materials to articulate against cartilage. Such materials should either eliminate or delay the necessity of total joint replacement. While cobalt-chromium (CoCr) alloy is still a material of choice and used for hemi-arthroplasties, spacers, and repair plugs, alternative materials are being studied. Pyrolytic carbon (PyC) is a biocompatible material that has been available since the 1980s. It has been widely and successfully used in small joints of the foot and the hand, but its tribological effects in direct comparison to cobalt-chromium (CoCr) remain to be investigated. Methods. A four station simulator (Figure 1), mimicking joint load and motion, was used for testing. The simulator is housed in an incubator, which and provides the necessary environmental conditions for cartilage survival. Articular cartilage disks (14mm in diameter) were obtained from the trochleas of six to eight months old steer for testing and free-swelling controls. Disks (n=8 per material) were placed in porous polyethylene scaffolds within polypropylene cups and mounted onto the simulator to articulate against 28mm balls of either PyC or CoCr. Each ball was pressed onto the cartilage disk with 40N. In order to allow fluidal load support, the contact migrated over the biphasic cartilage with a 5.2 mm excursion. Concomitantly, the ball oscillated with ±30° at 1 Hz. Testing was conducted for three hours per day over 10 days in Mini ITS medium. Media samples were collected at the end of each three hour test. Upon test commencement, media was pooled (days 1, 4, 7, 10) and analyzed for proteoglycans/sGAGs and hydroxyproline. In addition, total material release into media was estimated by determining the dry weight increase of media samples. For this purpose, 1 ml aliquots of fresh and test media were dialyzed, lyophilized and weighed on a high precision balance. Disk morphology and cell viability were histologically examined. Results. During each day of testing, cartilage control, CoCr and PyC samples released an average of 0.236, 0.253, re 0.268 mg/mL of glycol-proteins into the medium. After running-in (day 1), the increase was highly linear (R. 2. >0.99) and similar for all three testing conditions. Proteoglycan/GAG (Figure 2) and hydroxyproline release (Figure 3) were also similar for both materials (p=0.46 re. p=0.12), but significantly different from control (p<0.01). Histological and cell viability images support the hypothesis of superficial zone damage of the cartilage disks for both materials. Cell viability was not different from control (p>0.33). Discussion. The performance of PyC and CoCr was comparable using this in vitro simulation model, however appears not optimal. The observed surface fibrillation may lead to tissue breakdown in the long-term. The wear mechanism has yet to be elucidated but appears to be of adhesive nature. The lack of proteins in the medium might have suppressed boundary lubrication and thus may have played a role in the non-optimal performance of these materials. In summary, a live tissue model of articular cartilage found no difference comparing pyrolytic carbon with the current clinical gold standard CoCr

Osteosarcoma (OS) is the most prevalent bone tumor in children and young adults. Most tumors arise from the metaphysis of the long bones and easily metastasize to the lungs. Current therapeutic strategies of osteosarcoma are routinely surgical resection and chemotherapy, which are limited to the patients suffering from metastatic recurrence. Therefore, to investigate molecular mechanisms that contribute to osteosarcoma progression is very important and may shed light on targeted therapeutic approach to improve the survival of patients with this disease. Several miRNAs have been found expressed differentially in osteosarcoma (OS), In this study, we found that miR-144 significantly suppresses osteosarcoma cell proliferation, migration andinvasion ability in vitro, and inhibited tumor growth and metastasisin vivo. The function and molecular mechanism of miR-144 in Osteosarcoma was further investigated. Tissue samples from fifty-one osteosarcoma patients were obtained from Shanghai Ninth People's Hospital. The in vitro function of miR-144 in Osteosarcoma was investigated by cell viability assay, wound healing assay, invasion assay, the molecular mechanism was identified by Biotin-coupled miRNA capture, Dual-luciferase reporter assays, etc. the in vivo function of miR-144 in osteosarcoma was confirmed by osteosarcoma animal model and miR-144−/− zebrafish model. Mechanically, we demonstrated that Ras homolog family member A (RhoA) and its pivotal downstream effector Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) were both identified as direct targets of miR-144. Moreover, the negative co-relation between downregulated miR-144 and upregulated ROCK1/RhoA was verified both in the osteosarcoma cell lines and clinical patients' specimens. Functionally, RhoA with or without ROCK1 co-overexpression resulted a rescue phenotype on the miR-144 inhibited cell growth, migration and invasion abilities, while individual overexpression of ROCK1 had no statistical significance compared with control in miR-144 transfected SAOS2 and U2-OS cells. This study demonstrates that miR-144 inhibited tumor growth and metastasis in osteosarcoma via dual-suppressing of RhoA and ROCK1, which could be a new therapeutic approach for the treatment ofosteosarcoma

Introduction. According to American Joint Replacement Registry, particle mediated osteolysis represents 13 % of the knee revision surgeries performed in the United States. The comprehension of mechanical and wear properties of materials envisioned for TJR is a key step in product development. Furthermore, the maintenance of UHMWPE mechanical properties after material modification is an important aspect of material success. Initial studies conducted by our research group demonstrated that the incorporation of ibuprofen in UHMWPE had a minor impact on UHMWPE physicochemical and mechanical properties. Drug release was also evaluated and resulted in an interesting profile as a material to be used as an anti-inflammatory system. Therefore, the present study investigated the effect of drug release on the mechanical and biological properties of ibuprofen-loaded UHMWPE. Experimental. UHMWPE resin GUR 1020 from Ticona was for sample preparation. Samples with drug concentrations of 3% and 5% wt were consolidated as well as samples without anti-inflammatory addition through compression molding at 150 °C and 5 MPa for 15 minutes. Mechanical properties were evaluated via the tensile strength experiment (ASTM D638) and dynamic mechanic tests. Wear resistance was measured using the pin on disc (POD) apparatus. Finally, cytotoxicity analysis was conducted based on ISO 10993–5. Results. Dynamic-mechanic analysis demonstrated no difference in flexion modulus and stress for all materials (Table 1). No difference was also verified during cyclical loading experiments (Table 1), which indicates that the drug concentration added to material composition did not affect these properties. POD experiments were proposed to evaluate wear resistance of ibuprofen-loaded UHMWPE samples considering the combination of materials similar to those employed in TJR. Results from POD tests are presented in Table 1. Volumetric wear was close to zero for all samples after 200 thousand cycles. Comprehension of the effect of drug release on mechanical properties is essential to estimate how the material will behave after implantation. Therefore, mechanical properties were assessed after 30 days of ibuprofen release and the results were compared with those obtained in samples as prepared (Table 2). Initial results demonstrated a decrease in elastic modulus in samples prepared with ibuprofen. However, no difference was verified between UHMWPE, UHMWPE 3% IBU and UHMWPE 5% IBU after ibuprofen release. Finally, cell viability of UHMWPE 3% IBU and UHMWPE 5% was found to be superior to 100% (Figure 1). Therefore, both materials can be considered nontoxic. Conclusions. Ibuprofen-loaded UHMWPE did not demonstrate a significant influence on the mechanical and biological behavior of UHMWPE. Dynamic-mechanical tests demonstrated constancy for all samples under analysis. Wear testing resulted in gravimetric wear close to zero, for all tested materials. Mechanical properties conducted after 30 days of ibuprofen release also had a positive outcome. Although presenting a difference in modulus prior and after release tests, modulus and tensile yield stress remained inside acceptable range indicated to UHMWPE used in orthopedic implants. Furthermore, after drug elution UHMWPE 3% IBU and UHMWPE 5% IBU recovered original UHMWPE properties. Cytotoxicity assessment was performed and both ibuprofen-based formulations were considered nontoxic according to ISO 10993–5. For any figures or tables, please contact the authors directly

Large cartilage lesions in younger patients can be treated by fresh osteochondral allograft transplantation, a surgical technique that relies on stable initial fixation and a minimum chondrocyte viability of 70% in the donor tissue to be successful. The Missouri Osteochondral Allograft Preservation System (MOPS) may extend the time when stored osteochondral tissues remain viable. This study aimed to provide an independent evaluation of MOPS storage by evaluating chondrocyte viability, chondrocyte metabolism, and the cartilage extracellular matrix using an ovine model. Femoral condyles from twelve female Arcott sheep (6 years, 70 ± 15 kg) were assigned to storage times of 0 (control), 14, 28, or 56 days. Sheep were assigned to standard of care [SOC, Lactated Ringer's solution, cefazolin (1 g/L), bacitracin (50,000 U/L), 4°C storage] or MOPS [proprietary media, 22-25°C storage]. Samples underwent weekly media changes. Chondrocyte viability was assessed using Calcein AM/Ethidium Homodimer and reported as percent live cells and viable cell density (VCD). Metabolism was evaluated with the Alamar blue assay and reported as Relative Fluorescent Units (RFU)/mg. Electromechanical properties were measured with the Arthro-BST, a device used to non-destructively compress cartilage and calculate a quantitative parameter (QP) that is inversely proportional to stiffness. Proteoglycan content was quantified using the dimethylmethylene blue assay of digested cartilage and distribution visualized by Safranin-O/Fast Green staining of histological sections. A two-way ANOVA and Tukey's post hoc were performed. Compared to controls, MOPS samples had fewer live cells (p=0.0002) and lower VCD (p=0.0004) after 56 days of storage, while SOC samples had fewer live cells (p=0.0004, 28 days; p=0.0002, 56 days) and lower VCD (p=0.0002, 28 days; p=0.0001, 56 days) after both 28 and 56 days (Table 1). At 14 days, the percentage of viable cells in SOC samples were statistically the same as controls but VCD was lower (p=0.0197). Cell metabolism in MOPS samples remained the same over the study duration but SOC had lower RFU/mg after 28 (p=0.0005) and 56 (p=0.0001) days in storage compared to controls. These data show that MOPS maintained viability up to 28 days yet metabolism was sustained for 56 days, suggesting that the conditions provided by MOPS storage allowed fewer cells to achieve the same metabolic levels as fresh cartilage. Electromechanical QP measurements revealed no differences between storage methods at any individual time point. QP data could not be used to interpret changes over time because a mix of medial and lateral condyles were used and they have intrinsically different properties. Proteoglycan content in MOPS samples remained the same over time but SOC was significantly lower after 56 days (p=0.0086) compared to controls. Safranin-O/Fast Green showed proteoglycan diminished gradually beginning at the articular surface and progressing towards bone in SOC samples, while MOPS maintained proteoglycan over the study duration (Figure 1). MOPS exhibited superior viability, metabolic activity and proteoglycan retention compared to SOC, but did not maintain viability for 56 days. Elucidating the effects of prolonged MOPS storage on cartilage properties supports efforts to increase the supply of fresh osteochondral allografts for clinical use. For any figures or tables, please contact the authors directly

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging (BLI) and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with increased expression of Runx-2 (p = 0.009), Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in partial osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), and did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

This study investigated confocal laser scanning microscopy (CLSM) as a novel method of imaging of chondrocytes on a collagen membrane used for articular cartilage repair. Cell viability and the effects of surgery on the cells were assessed. Cell images were acquired under four conditions: 1, Pre-operative 2, After handling 3, Heavily grasped with forceps 4, Cut around the edge. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 1.12–1.68 ± 0.22 × 10. 6. cells/cm. 2. in specimens without significant trauma (n=25 images), this decreased to 0.253 × 10. 6. cells/cm. 2. in the specimens that had been grasped with forceps (p <0.001) (5 images). Cell viability on delivery grade membrane was 86.8±2.1%. The viability dropped to 76.3 ± 1.6% after handling and 35.1 ± 1.7% after crushing with forceps. Where the membrane was cut with scissors, there was a band of cell death where the viability dropped to 17.3 ± 2.0% compared to 73.4 ± 1.9% in the adjacent area (p <0.001). Higher magnification revealed cells did not have the rounded appearance of chondrocytes. CLSM can quantify and image the fine morphology of cells on a MACI membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with significantly increased expression of the Osteoblast markers Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), but did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

Intervertebral disc (IVD) degeneration plays a major role in low back pain which is the leading cause of disability. Current treatments in severe cases require surgical intervention often leading to adjacent segment degeneration. Injectable hydrogels have received much attention in recent years as scaffolds for seeding cells to replenish disc cellularity and restore disc properties and function. However, they generally present poor mechanical properties. In this study, we investigated several novel thermosensitive chitosan hydrogels for their ability to mimic the mechanical properties of the nucleus pulposus (NP) while being able to sustain the viability of NP cells, and retain proteoglycans. CH hydrogels were prepared by mixing the acidic chitosan solution (2% w/v) with various combinations of three gelling agents: sodium hydrogen carbonate (SHC) and/or beta-glycerophosphate (BGP) and/or phosphate buffer (PB) (either BGP0.4M, SHC0.075M-BGP0.1M, SHC0.075M-PB0.02M or SHC0.075M-PB0.04M). The gelation speed was assessed by following rheological properties within 1h at 37°C (strain 5% and 1Hz). The mechanical properties were characterized and compared with that of human NP tissues. Elastic properties of the hydrogels were studied by evaluating the secant modulus in unconfined compression. Equilibrium modulus was also measured, using an incremental stress-relaxation test 24h after gelation in unconfined compression (5% strain at 5%/s followed by 5min relaxation, five steps). Cells from bovine IVD were encapsulated in CH-based gels and maintained in culture for 14 days. Cytocompatibility was assessed by measuring cell viability, metabolism and DNA content. Glycosaminoglycan (GAG) synthesis (retained in the gel and released) was determined using DMMB assay. Finally injectability was tested using human cadaveric discs. Unconfined compression confirmed drastically enhanced mechanical properties compared to conventional CH-BGP hydrogels (secant Young modulus of 105 kPa for SHC0.075PB0.02 versus 3–6 kPa for BGP0.04). More importantly, SHC0.075PB0.02 and SHC0.075BGP0.1 hydrogels exhibited mechanical properties very similar to NP tissue. For instance, equilibrium modulus was 5.2±0.6 KPa for SHC0.075PB0.02 and 8±0.8 KPa for SHC0.075BGP0.1 compared to 6.1±1.7 KPa for human NP tissue. Rheological properties and gelation time (G′=G″ after less than 15 s at 37°C, and rapid increase of G') of these hydrogels also appear to be adapted to this application. Cell survival was greater than 80% in SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels. Cells encapsulated in the new formulations also showed significantly higher metabolic activity and DNA content after 14 days of incubation compared to cells encapsulated in BGP0.4 hydrogel. Cells encapsulated in SHC0.075BGP0.1 and SHC0.075PB0.02 produced significantly higher amounts of glycosaminoglycans (GAG) compared to cells encapsulated in SHC0.075PB0.04 and BGP0.4 hydrogels. The total amount of GAG was higher in SHC0.075BGP0.1 hydrogel compared to SHC0.075PB0.02. Interestingly, both the SHC0.075BGP0.1 and SHC0.075PB0.02 hydrogels retained similar amounts of GAG. Injectability through a 25G syringe, filling of nuclear clefts and good retention in human degenerated discs was demonstrated for SHC0.075PB0.02 hydrogel. SHC0.075BGP0.1 appears to be a particularly promising injectable scaffold for IVD repair by providing suitable structural environment for cell survival, ECM production and mechanical properties very similar to that of NP tissue

Aim. Most orthopedic infections are due to the microbial colonization of abiotic surfaces, which evolves into biofilm formation. Within biofilms, persisters constitute a microbial subpopulation of cells characterized by a lower metabolic-activity, being phenotipically tolerant to high concentrations of antibiotics. Due to their extreme tolerance, persisters may cause relapses upon treatment discontinuation, leading to infection recalcitrance hindering the bony tissue regeneration. Using isothermal microcalorimetry (IMC), we aimed to evaluate in vitro the presence of persisters in a methicillin-resistant Staphylococcus aureus (MRSA) biofilm after treatment with high concentrations of vancomycin (VAN) and their ability to revert to a normal-growing phenotype during incubation in fresh medium without antibiotic. Moreover, the ability of daptomycin to eradicate the infection by killing persisters was also investigated. Method. A 24h-old MRSA ATCC 43300 biofilm was exposed to 1024 µg/ml VAN for 24h. Metabolism-related heat of biofilm-embedded cells, either during or after VAN-treatment, was monitored in real-time by IMC for 24 or 48h, respectively. To evaluate the presence of VAN-derived “persisters” after antibiotic treatment, beads were sonicated and detached free-floating bacteria were further challenged with 100xMIC VAN (100 µg/ml) in PBS+1% Cation Adjusted Mueller Hinton Broth (CAMHB).. Suspensions were plated for colony counting. The resumption of persister cells' normal growth was analysed by IMC on dislodged trated cells for 15h in CAMHB. Activity of 16 µg/ml daptomycin was assessed against persister cells by colony counting. Results. When incubated with 1024 µg/ml VAN, MRSA biofilm produced undetectable heat, suggesting a strong reduction of cell viability and/or cellular metabolism. However, the same samples re-inoculated in fresh medium produced a detectable and delayed metabolism-related heat signal, similarly to that generated by persister cells. The following exposure to 100xMIC VAN resulted in neither complete killing nor bacterial growth, strongly supporting the hypothesis of a persistent phenotype. IMC analysis indicated that VAN-treated biofilm cells resumed normal growth with a ∼3h-delay, as compared to the untreated growth control. Daptomycin treatment yielded a complete eradication of persister cells selected after VAN treatment. Conclusions. Hostile environmental conditions (e.g. high antibiotic bactericidal concentrations) select for persister cells in MRSA biofilm after 24h-treatment in vitro. A staggered treatment vancomycin/daptomycin allows complete biofilm eradication. These results support the use in clinical practice of a therapeutic regimen based on the combined use of antibiotics to kill persisters and eradicate MRSA biofilms. IMC represents a suitable technique to detect persisters and characterize in real-time their reversion to a metabolically-active phenotype

Currently, different techniques to evaluate the biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal/ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. Moreover, cell culture systems evaluate the biological responses of cells to a biomaterial as the combined effect of both particles and ions. We have developed a novel cell culture system suitable for testing the all three type of particles and ions, separately. The method was tested by evaluating the biological responses of human peripheral blood mononuclear cells (PBMNCs) to UHMWPE, cobalt-chromium alloy (CoCr), and Ti64 alloy wear particles. Methods. Clinically relevant sterile UHMWPE, CoCr, and Ti64 wear particles were generated in a pin-on-plate wear simulator. Whole peripheral blood was collected from healthy human donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell, UK) and seeded into the wells of 96-well and 384-well cell culture plates. The plates were then incubated for 24 h in 5% (v/v) CO. 2. at 37°C to allow the attachment of mononuclear phagocytes. Adherent phagocytes were incubated with UHMWPE and CoCr wear debris at volumetric concentrations of 0.5 to 100 µm. 3. particles per cell for 24 h in 5% (v/v) CO. 2. at 37°C. During the incubation of cells with particles, for each assay, two identical plates were set up in two configurations (one upright and one inverted). After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer, UK). Intracellular oxidative stress was measured using the DCFDA-based reactive oxygen species detection assay (Abcam, UK). TNF-α cytokine was measured using sandwich ELISA. DNA damage was measured by alkaline comet assay. The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. Results and Discussion. Cellular uptake of UHMWPE, CoCr and Ti64 particles was confirmed by optical microscopy. PBMNCs incubated with UHMWPE particles did not show any adverse responses except the release of significant levels of TNF-α cytokine at 100 µm. 3. particles per cell, when in contact with particles. PBMNCs incubated with CoCr wear particles showed adverse responses at high particle doses (100 µm. 3. particles per cell) for all the assays. Moreover, cytotoxicity was observed to be a combined effect of both particles and ions, whereas oxidative stress and DNA damage were mostly caused by ions. Ti64 wear particles did not show any adverse responses except cytotoxicity at high particle doses (100 µm. 3. particles per cell). Moreover, this cytotoxicity was mostly found to be a particle effect. In conclusion, the novel cell culture system is suitable for evaluating the biological impact of orthopaedic wear particles and ions, separately