Degenerative disc disease (DDD) is a common cause of lower back pain. Calcification of the intervertebral disc (IVD) has been correlated with DDD, and is especially prevalent in scoliotic discs. 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. Our data indicate that Ca²⁺ and expression of the extracellular calcium-sensing receptor (CaSR) are significantly increased in mild to severely degenerative human IVDs. In this study, we evaluated the effects of Ca²⁺ and CaSR on the degeneration and calcification of IVDs.

Human donor lumbar spines of Thompson grade 2, 3 and 4 through organ donations within 24 hs after death. IVD cells, NP and AF, were isolated from tissue by sequential digestion with Pronase followed by Collagenase. Cells were expanded for 7 days under standard cell culture conditions. Immunohistochemistry was performed on IVD tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting and RT-qPCR were performed on cultured NP and AF cells to demonstrate expression of CaSR, matrix proteins aggrecan and collagen, catabolic enzymes and calcification markers. IVD cells were cultured in increasing concentrations of Ca²⁺ [1.0-5.0 mM], CaSR allosteric agonist (cincalcet, 1 uM), and IL-1b [5 ng/mL] for 7 days. Ex vivo IVD organ cultures were prepared using PrimeGrowth Disc Isolation System (Wisent Bioproducts, Montreal, Quebec). IVDs were cultured in 1.0, 2.5 mM Ca²⁺ or with cinacalcet for 21 days to determine effects on disc degeneration, calcification and biomechanics. Complex modulus and structural stiffness of disc tissues was determined using the MACH-1 mechanical testing system (Biomomentum, Laval, Quebec).

Ca²⁺ dose-dependently decreased matrix protein synthesis of proteoglycan and Col II in NP and AF cells, similar to treatment with IL-1b. (n = 4). Contrarily to IL-1b, Ca²⁺ and cincalcet did not significantly increase the expression of catabolic enzymes save ADAMTS5. Similar effects were observed in whole organ cultures, as Ca²⁺ and cinacalcet decreased proteoglycan and collagen content. Although both Ca²⁺ and cinacalcet increased the expression of alkaline phosphatase (ALP), only in Ca²⁺-treated IVDs was there evidence of calcium deposits in NP and AF tissues as determined by von Kossa staining. Biomechanical studies on Ca²⁺ and cinacalcet-treated IVDs demonstrated decreases in complex modulus (p<0.01 and p<0.001, respectively; n=5), however, only Ca²⁺-treated IVDs was there significant increases stiffness in NP and AF tissues (p<0.001 and p<0.05, respectively; n=3).

Our results suggest that changes in the local concentrations of calcium and activation of CaSR affects matrix protein synthesis, calcification and IVD biomechanics. Ca²⁺ may be a contributing factor in IVD degeneration and calcification.

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.

Osteoarthritis (OA) is a chronic degenerative joint disorder that affects millions of people. There are currently no therapies that reverse or repair cartilage degradation in OA patients. Link N (DHLSDNYTLDHDRAIH) is a naturally occurring peptide that has been shown to increase both collagen and proteoglycan synthesis in chondrocytes and intervertebral disc cells [1,2]. Recent evidence indicates that Link N activates Smad1/5 signaling in cultured rabbit IVD cells presumably by interacting with the bone morphogenetic protein (BMP) type II receptor [3], however, whether a similar mechanism exists in chondrocytes remains unknown. In this study we determined whether Link N can stimulate matrix production and reverse degradation of human OA cartilage under inflammatory conditions.

OA cartilage was obtained from donors undergoing total knee arthroplasty with informed consent. OA cartilage/bone explants and OA chondrocytes were prepared from each donor. Cells were prepared in alginate beads (2×106 cells/mL) for gene expression analysis using qPCR. Cells and cartilage explants were exposed to IL-1β (10ng/ml), human Link N (hLN) (1μg/ml) or co-incubated with IL-1β+hLN for 7 and 21 days, respectively. Media was supplemented every three days. Cartilage/bone explants were measured for total glycosaminoglycan (GAG) content (retained and released) using the dimethylmethylene blue (DMMB) assay. Western blotting was performed to determine aggrecan and collagen expression in cartilage tissue. To determine NFκB activation, Western blotting was performed for detection of P-p65 in chondrocytes cultured in 2D following 10 min exposure of IL-1β in the presence of 10, 100, or 1000 ng/mL hLN.

Link N significantly decreased in a dose-dependent manner IL-1β-induced NFκB activation in chondrocytes. Gene expression profiling of matrix proteins indicated that there was a trend towards increased aggrecan and decreased collagen type I expression following hLN and IL-1β co-incubation. HLN significantly decreased the IL-1β-induced expression of catabolic enzymes MMP3 and MMP13, and the neuronal growth factor NGF (p < 0 .0001, n=3). In OA cartilage/bone explants, hLN reversed the loss of proteoglycan in cartilage tissue and significantly increased its synthesis whilst in the presence of IL-1β.

Link N stimulated proteoglycan synthesis and decreased MMP expression in OA chondrocytes under inflammatory conditions. One mechanism for Link N in preserving matrix protein synthesis may, in part, be due to its ability in rapidly suppressing IL-1β-induced activation of NF-κB. Further work is needed to determine whether Link N directly inhibits the IL-1β receptor or interferes with NFκB activation through an independent pathway(s).

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.

Osteoarthritis (OA) is a multifactorial debilitating disease that affects over four million Canadians. Although the mechanism(s) of OA onset is unclear, the biological outcome is cartilage degradation. Cartilage degradation is typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II) – partly due to the up-regulation of catabolic enzymes - aggrecanases a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). There is currently no treatment that will prevent or repair joint damage, and current medications are aimed mostly at pain management. When pain becomes unmanageable arthroplastic surgery is often performed. Interest has developed over the presence of calcium crystals in the synovial fluid of OA patients, as they have been shown to activate synovial fibroblasts inducing the expression of catabolic agents. We recently discovered elevated levels of free calcium in the synovial fluid of OA patients and raised the question on its role in cartilage degeneration.

Articular cartilage was isolated from 5 donors undergoing total hip replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase and expanded in DMEM supplemented with 10% heat-inactivated FBS. OA and normal human articular chondrocytes (PromoCell, Heidelberg, Germany) were transferred to 6-well plates in culture medium containing various concentrations of calcium (0.5, 1, 2.5, and 5 mM CaCl2), and IL-1β. Cartilage explants were prepared from the same donors and included cartilage with the cortical bone approximately 1 cm2 in dimension. Bovine articular cartilage explants (10 months) were used as a control. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. Immunohistochemistry was performed on cartilage explants to measure expression of Col X, MMP-13, and alkaline phosphatase. The sulfated glycosaminoglycan (GAG, predominantly aggrecan) content of cartilage was analyzed using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay, and aggregan fragmentation was determined by Western blotting using antibody targeted to its G1 domain. Western blotting was also performed on cell lysate from both OA and normal chondrocytes to measure aggrecan, Col II, MMP-3 and −13, ADAMTS-4 and −5.

Ca2+ significantly decreased the proteoglycan content of the cartilage explants as determined by the DMMB assay. The presence of aggrecan and Col II also decreased as a function of calcium, in both the human OA and bovine cartilage explants. When normal and OA chondrocytes were cultured in medium supplemented with increasing concentrations of calcium (0.5–5 mM Ca2+), aggrecan and Col II expression decreased dose-dependently. Surprisingly, increasing Ca2+ did not induce the release of MMP-3, and −13, or ADAMTS-4 and-5 in conditioned media from OA and normal chondrocytes. Interestingly, inhibition of the extracellular calcium-sensing receptor CaSR) reversed the effects of calcium on matrix protein synthesis.

We provide evidence that Ca2+ may play a direct role in cartilage degradation by regulating the expression of aggrecan and Col II through activation of CaSR.

Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects.

Articular cartilage was isolated from donors undergoing total hip or knee replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase, and expanded in DMEM-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1, COL2A1, COL10A1, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13, on an ABI 7500 fast qPCR system.

Although IL-1β did not significantly decrease the expression of matrix proteins, it did increase the expression of ADAMTS-4, −5, and MMP3 and −13 when compared to controls (Kruskal-Wallis, p < 0 .05, n=3). TUDCA treatment alone did not significantly increase the expression of catabolic enzymes but it did increase the expression of collagen type II. When IL-1β was coincubated with TUDCA, the expression of ADAMTS-4, ADAMTS-5, and MMP-13 significantly decreased by ∼40-fold, ∼10-fold, and ∼3-fold, respectfully.

We provide evidence that the catabolic activities of IL-1β on human cartilage can be abrogated through modulation of the ER stress response.

Osteoarthritis (OA) is a multifactorial disease that affects millions of Canadians. Although, there is not one specific mechanism that causes OA, the biological outcome is cartilage degradation. The articular cartilage in joints is composed primarily of the proteoglycan aggrecan and type II collagen (Col II) which together provide cartilage with functional properties. In OA, the imbalance of the anabolic and catabolic activities of chondrocytes favors cartilage catalysis. The main inflammatory cytokine involved in cartilage degradation is interleukin (IL) 1β. It has previously been demonstrated that Link N, a 16 residue peptide derived from proteolytic cleavage of link protein, can stimulate matrix proteins in normal cartilage and intervertebral discs (IVDs). Recently, we showed that a shorter sequence of Link N (sLink N), consisting of the first 8 residues of the peptide, has the potential to increase synthesis of matrix proteins in IVD cells in vitro and stimulate repair in ex vivo IVD organ culture. There are currently no treatments that actively repair cartilage in OA joints. In the present study, we aimed to evaluate the potential of sLink N as a therapeutic agent in the repair of OA cartilage.

OA cartilage was isolated from four donors undergoing total knee replacement (50–70 y). Cells were recovered from the cartilage of each knee by sequential digestion with Pronase followed by Collagenase, and expanded in PrimeGrowth culture medium (Wisent Bioproducts, Canada; Cat# 319–510-CL, −S1, and −S2). After 7 days in culture, cells were treated for 24h with sLink N (0.5, 5, 50, 500 or 5000 ng/ml) or sLink N in combination with IL-1β (1 ng/ml) to mimic an inflammatory milieu. Conditioned media was collected and measured for proteoglycan (GAG) release using the safranin O and for Col II synthesis by Western blotting. Human articular cartilage explants including cartilage with subchondral bone were prepared from the same donors using the PrimeGrowth Isolation kit (Wisent, Canada) and cultured for 21 days in presence of IL-1β (1ng/ml) and sLink N (0.5, 5, 50, 500 or 5000 ng/ml). Aggrecan and Col II were extracted with guanidine buffer and measured by Western blotting.

Treatment of OA chondrocytes significantly increased the GAG and Col II synthesis. The EC50 dose-response of sLink N on GAG synthesis was 67 ± 41 nM [65 ± 40 ng/ml] and the GAG synthesis reached a maximum of 194 ± 30% with the highest dose above control. When chondrocytes were cultured in the presence of IL-1β, GAG synthesis was also elevated by sLink N above control. Treatment of OA cartilage explants with sLink N increased the content of aggrecan and Col II even in the presence of IL-1β.

Our results suggest that sLink N is a growth factor supplement that can increase cartilage matrix protein synthesis, and a chondroprotective agent, by modulating the catabolic effects of IL-1β. sLink N is the first small-peptide to demonstrate potential in cartilage repair of OA joints.

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.

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.

Purpose

Developmental exposure to estrogens has been shown to affect a number of organ systems, including long and short bones. Epigenetic effects of DES exposure have been shown to affect the third generation of progeny. Furthermore, recent studies have shown that environmental exposure to estrogen-like compounds is much higher than originally anticipated. This study aims to discover the effect of in utero exposure to a well-known estrogen agonist, diethylstilbestrol (DES), on lumbar bone, intervertebral disc (IVD), and articular cartilage. Femoral bone was studied to determine the specificity of the effect.

Purpose

A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media.

Purpose

Whilst it is known that oxidative stress can cause early degenerative changes observed in experimental osteoarthritis and that a major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients express type X collagen, a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification), little is known whether the expression of type X collagen in MSCs from OA patients can be related to oxidative stress or inflammatory reactions that occur during this 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.

Purpose: Several studies have shown elevated levels of metal ions in blood of patients with metal-on-metal (MM) total hip arthroplasty (THA). The outstanding question that remains is the clinical impact of these elevated ion levels. Even though it is well known that exposure to heavy metals such as lead, copper, mercury, nickel, and cadmium) may lead to significant alterations in human sperm morphology and motility, less is known on the effect of Co and Cr on semen parameters. The aim of the present study was to investigate the effect of metal ions on the semen of males of child fathering age with MM hip arthroplasty.

Method: Semen was collected form 10 patients between 41 and 49 years old (mean = 45±6 years) by masturbation after 2–3 days of abstinence. Samples were examined within 1h after ejaculation for morphology, motility, and number of sperm cells following standard criteria from the World Health Organization (WHO). Co and Cr concentrations were measured in both the seminal plasma and in the blood of patients by inductively coupled plasma-mass spectroscopy (ICP-MS). Since spermatozoa membrane polyunsaturated fatty acids are vulnerable to attack by reactive oxygen species (leading to peroxide formation), peroxide concentrations were measured in both the seminal plasma and the blood of patients.

Results: Results showed that the concentration of both Co and Cr ions was significantly lower in the seminal plasma than in the blood of the patients. Results also showed that the levels of peroxides were lower in the seminal plasma than in the blood plasma of these patients. Importantly, the ejaculate volume, the sperm density, the total sperm count, the pH, and the percentage of cells with normal morphology were in the range of the WHO criteria for fertile population and also in the range of reference patients in the city of measurements. However, the viability was a little bit lower than what was observed in a fertile population without prosthesis.

Conclusion: The presence of Co and Cr ions in the blood of males of child fathering age with MM hip arthroplasty raised concerns about the quality of semen in these patients. Results of the present study strongly suggest that the raised of Co and Cr had no significant effect on sperm parameters of young patients with MM prosthesis. The methods used to identify potential normal and fertile semen samples are still contradictory and not exactly defined. Studies showed for example that only total numbers of sperm with progressive mobility are significantly different in the fertile than in sub-fertile men, while others suggested that the fertile population should be defined by sperm concentration or sperm morphology. In conclusion, results suggest that Co and Cr ions generated from MM prosthesis have no significant effect on the sperm parameters of young patients of child fathering age. Further longitudinal studies are however necessary to conclusively determine the effect of metal ions from MM prosthesis on sperm parameters.

Purpose: Several studies have been directed toward using mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients for cartilage or disc repair because these patients are the ones that will require a source of autologous stem cells if biological repair of tissue lesions is to be a therapeutic option. A major drawback of current cartilage and intervertebral disc tissue engineering repair is that these cells rapidly express type X collagen, a marker of late stage chondrocyte hyperthrophy implicated in endochondral ossification. However, a novel plasma-polymerized thin film material, named nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The specific aim of this study was to determine if the suppression of type X collagen by PPE:N is maintained when MSCs are transferred to pellet cultures in chondrogenic defined media.

Method: MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA using a protocol approved by the Research Ethics Committee of our institution. Cells were then expanded for 2–3 passages in DMEM high glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin, and finally cultured on polystyrene (PS) cell culture dishes or PPE: N surfaces for 3 and 7 days. Cells were transferred for 3 additional days in a chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mg/ml bovine serum albumin, 5 μg/ml insulin, 50 μg/ml ascorbic acid, 5 ng/ ml sodium selenite, 5 μg/ml transferrin) in pellet culture or on PS cell culture dishes. Cells were then lysed and proteins were separated on 4–20% acrylamide gels and transferred to nitrocellulose membranes. Type X collagen was detected by Western blot; GAPDH expression was used as an internal control for protein loading.

Results: Results showed that type X collagen protein was expressed in MSCs from OA patients cultured on polystyrene but was suppressed when cultured on PPE: N. Since defined chondrogenic medium are commonly used in pellet culture to promote in vitro chondrogenesis, we then investigated the effect of transferring cells pre-cultured on PPE:N into pellet culture on type X collagen expression. However, the decreased type X collagen expression was not maintained in these conditions and that the expression returned to control values. The decreased type X collagen expression was maintained when the cells were cultured on PS cell culture dishes.

Conclusion: The use of MSCs is promising for tissue engineering of cartilage and intervertebral disc. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. However, when MSCs stem cells are transferred to pellet cultures, type X collagen is rapidly re-expressed suggesting that pellet cultures may not be suitable for chondrogenesis of MSCs from OA patients.

Purpose: Mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients are not well characterized and little is known of how they are regulated. Recent evidence indicates that a major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human MSCs from OA patients express type X collagen (COL10), a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). However, the intracellular pathways for transducing signals that regulate hypertrophy in MSCs remain unclear. In chondrocytes, this pathway is mediated by mitogen activated protein kinase (MAPK) p38. The aim of this study was to determine the phosphorylation levels of ERK/p38 MAPK signaling molecules in MSCs from OA patients compared to those from normal patients.

Method: MSCs were obtained from aspirates from the intramedullary canal of donors (60–80 years of age) undergoing total hip replacement for OA. Cells were cultured in DMEM high glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin for 2–3 passages. Cells were then lysed and proteins were separated on 10% acrylamide gels and transferred to nitrocellulose membranes. Protein expression was determined by Western blot using specific antibodies directed against type X collagen, ERK, phosphorylated-ERK, p38, phosphorylated-p38, JNK, phosphorylated-JNK, AKT, and phosphorylated-AKT. GAPDH was used as a housekeeping gene. Proteins were detected using the West Pico Chemiluminescence substrates and analyzed using the Bio-Rad VersaDoc equipped with a cooled CCD 12 bit camera. Normal mesenchymal stem cells from a 22 years old woman were purchased from Lonza (Switzerland).

Results: Results show that the expression of COL10 was markedly increased in MSCs of OA patients compared to control patient. Results also shows that the phosphorylation of all the signal transduction proteins studied was induced in MSCs of patients with OA. Indeed, the phosphorylation of ERK (3.4±0.9 times the control), p38 (1.7±0.3 times the control), JNK (5.40±1.14 times the control), and AKT (4.3±0.8 times the control) was higher in MSCs of OA patients compared to control normal patients.

Conclusion: In the normal donor, MSCs continue to exhibit their in situ behavior in that they expressed very little or no COL10. This may relate to the fact that normal MSCs being multipotent in nature like to maintain an undifferentiated state. In contrast, MSCs from OA patients expressed COL10: this suggests that they are in a situation were they can be preprogrammed not only to replace the degraded articular cartilage but also the damaged subchondral bone. Since the phosphorylation of ERK/p38 MAPK signaling molecules is also lower in normal MSCs, our results also suggest that this signaling pathway is implicated in the control of COL10 expression. This finding is of great importance for the understanding of COL10 regulation in general and may lead to important advances in the comprehension of COL10 related diseases.

Purpose: A major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients express type X collagen (COL10), a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) regulate endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. In the present study, we investigated the effect of PTH on the expression of COL10 in MSCs from OA patients and analyzed the potential mechanisms related to its effect.

Method: MSCs were obtained from aspirates from the intramedullary canal of donors (60–80 years of age) undergoing total hip replacement for OA. Cells were cultured for 2–3 passages in DMEM high glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were then incubated for 0–24h without (Control) or with 100 nM PTH (1–34). Cells were lysed and proteins were separated on 10% acrylamide gels and transferred to nitrocellulose membranes. Protein expression was detected by Western blot using specific antibodies directed against COL10, p38, phosphorylated-p38 (p-p38), SAP/JNK, phosphorylated-SAP/JNK (p-JUNK). GAPDH was used as a housekeeping gene. Protein levels were analyzed using a Bio-Rad VersaDoc equipped with a cooled CCD 12 bit camera.

Results: Results showed that PTH inhibited in a time-dependent manner the expression of COL10 in MSCs from OA patients. The level of expression reached 21% of control (79% inhibition) after 24h. This inhibitory effect of PTH was reversed by Calphostin C, an inhibitor of protein kinase C. To further investigate the mechanism of action related to the effect of PTH on COL10 expression, we measured the phosphorylation of p38 and showed that PTH also inhibited this phosphorylation, which is an indicator of its activity. The level of phosphorylation reached 74% of control after 3h and stayed stable thereafter. Similarly, treatment of MSCs with PTH suppressed the phosphorylation of JNK, another major stress-activated MAP kinase. The level of phosphorylation reached 65% of control after 6h and returned to control values after 24h.

Conclusion: Results of the present study suggested that PTH may be a potential regulator of COL10 expression in MSCs from OA patients. Results also suggested a role for the protein kinase C and the p38/JNK pathways in this regulation. p38 and JNK are serine and threonine protein kinases that are activated by osmotic pressure, stress, and cytokines. It is therefore not surprising that their activities were elevated as OA (degenerative joint disease) is a result of trauma or infection to the joint and is characterized by an up-regulation of cytokines. Further studies are however necessary to better understand the role of these molecules in hypertrophy.

Purpose: Several studies have shown elevated levels of metal ions in blood of patients with metal-on-metal (MM) total hip arthroplasty (THA). To minimize wear, the elastohydrodynamic theory suggests wear reduction for larger diameter head bearings. This significant reduction in wear has been demonstrated in hip simulators for the 36 mm-head compared to the 28 mm-head prosthesis. However, the survival of larger head MM THAs and the levels of metal ions in the blood of patients having these implants are still to be determined.

Method: Fifty (50) patients (56 hips) who received a DePuy Ultamet™ MM hip bearing (40/44 mm-head) at our Institution between July 1st 2007 and August 31st 2008 were included in the study. Clinical and radiologic data were collected pre-operatively as well as at 6–8 weeks, 4–6 months, and 1 year postoperatively. Results were compared to those of subjects (65 patients, 71 hips) who received a 36 mm-head prosthesis. Cobalt (Co) and chromium (Cr) concentrations were measured at 1 year post-operatively in the blood of patients by ICP-MS. Since Co and Cr ions have the potential to induce irreversible biochemical damage to macromolecules, the levels of oxidative stress markers (total antioxidants and lipid peroxides) were measured in the plasma of these patients.

Results: At their 1-year post-operatively follow-ups, all patients were doing well and no sign of osteolysis was observed on X-rays. Harris Hip Score increased in both groups with a tendency to higher score in the 40–44 mm group compared to the 36 mm group. Activity score also increased in both groups after 1 year without statistical significant differences. Results also show that the levels of Co and Cr ions increased significantly in both groups compared to the Pre-OP control group. The levels of Co were also significantly higher in patients with large head arthroplasty (40 and 44 mm-head) compared to those of the 36 mm-head group (p=0.012). The levels of Cr were similar in both the large head and the 36 mm-head group (p=0.41). Finally, results show that there were no differences in the levels of total antioxidants and peroxides between the 40–44 mm group and the 36 mm group. Moreover, there was no increase in the level of these markers of oxidative stress compared to the Pre-OP control group.

Conclusion: The present study shows that at 1-year postoperatively, patients with large 40–44 mm-head THA had comparable clinical outcomes than those with 36 mm-head prosthesis. However, the levels of Co ions were significantly higher in these patients compared to patients with 36 mm-head THA. This suggests a higher health risk for these patients due to the presence of these ions. However, there were no effects on the levels of oxidative stress markers in the blood of these patients, suggesting that there is no increased risk at short-term. In conclusion, due to the high level of Co ions, longer follow-ups are required to conclusively determine the outcomes of the patients and the survivorship of these new bearings.

Purpose: Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of proteoglycan aggregates present within the extracellular matrix of the disc. Link-N peptide is the N-terminal peptide of link protein, which stabilizes the proteoglycan aggregates. It is generated in vivo by proteolytic degradation during tissue turnover. We have previously shown that this peptide can stimulate the synthesis of proteoglycans and collagens by IVD cells in vitro. However, to date, there have been no reports on the effect of Link-N on the IVD in vivo. The purpose of the present study was to determine the effect of intradiscally administration of Link-N peptide on disc cell survival and extracellular matrix synthesis using a rabbit annular needle puncture model of IVD degeneration.

Method: Twelve New Zealand white rabbits (~3.5 kg; 5–6 months old) received an annular puncture with an 18-gauge needle on 2 non-contiguous discs (L2–L3 and L4–L5). The disc (L3–L4) between the punctured discs and that above (L5–L6) was left intact as internal controls. Two weeks after the initial puncture, the anterior surfaces of the previously punctured discs (L2–L3 and L4–L5) were injected with either saline (10 μl/disc) or Link-N (100 μg in 10μl saline/disc) into the center of the NP. Disc height was radiographically monitored biweekly. After 12 weeks post-injection, all the rabbits were euthanized and the IVDs from both experimental groups were removed from each lumbar spine for biochemical analysis. The nucleus pulposus (NP) was separated from the annulus fibrosus (AF), the specimens weighed (wet weight), the content of DNA measured using PicoGreen, and the total contents of sulfated glycosaminoglycans (GAG) measured by the 1,9-dimethylmethylene blue (DMMB) assay.

Results: Following needle puncture that initiates disc degeneration, the disc height index (DHI) decreased by about 25%. By 6 weeks after Link-N injection, the mean percent DHI of injected discs in the Link-N group was higher than in the saline group. This difference in mean percent DHI was maintained during the rest of the follow-up. Puncturing the IVD also led to a decrease in proteoglycan content in both the NP and the AF in saline-treated discs. Treatment with Link-N stimulated proteoglycan synthesis (GAG) in both the NP and AF by about 20%. Link-N did not cause an increase in the DNA content of the discs.

Conclusion: Results of the present study show that Link-N can stimulate proteoglycan production in vivo when administered to degenerate disc. This stimulation occurs in both the NP and AF of the disc and in the absence of any effect on cell division. The changes observed with Link-N on proteoglycan synthesis are similar to those reported after injection of osteogenic protein-1 (OP-1) Thus, Link-N appears to be equally effective at stimulating repair of the IVD in vivo. One major advantage of Link-N over OP-1 for therapeutic use is the large saving in cost, Link-N being about 400 times cheaper than OP-1.

Purpose: The presence of metal ions in the blood of patients with a metal-on-metal (MM) bearing points to the importance of understanding the long-term effects of these ions. Metal ions have the potential to induce the production of reactive oxygen species (ROS), making them prime suspects for inducing molecular damage in circulating cells. The aim of this study was to analyze the levels of oxidative stress markers in the plasma of patients with hip surface replacement.

Method: Blood was collected up to 3 years after implantation from 66 patients with articular surface replacement (ASRÔ, DePuy Orthopaedics) and 54 patients with 36 mm-head MM THA. Forty (40) pre-operative patients were also assessed as control group. Total anti-oxidant levels were measured by the Oxford Biomedical total antioxidant power assay (Oxford, MI) to obtain an overview of the defense capacity of patient’s oxidative stress. Peroxide concentrations were measured by the Biomedica OxyStat assay (Medicorp, Montreal, QC) to quantify damage to lipids in the systemic circulation. Nitrototyrosine levels were quantified using the BIOXYTECH® Nitrotyrosine-EIA assay (OxisResearch™, Portland OR) to measure damage to proteins. The concentrations of metal ions were analyzed by inductively coupled plasma-mass spectroscopy.

Results: Results showed that there were no statistical differences in the concentrations of total antioxidants, lipid peroxides, and protein nitrotyrosines between the control, the ASR, and the 36 mm-head groups. Furthermore, there was no correlation between the concentrations of these markers and the concentrations of both Co and Cr ions (r2 £ 0.006).

Conclusion: The single most significant obstacle preventing a broader application of metal-on-metal hip arthroplasties and resurfacings continues to be the concerns regarding elevated metal ion levels in the blood and urine of patients. The present results showed that there were no changes in the levels of oxidative stress markers in patients with MM bearings compared to the control group. Given the possible latency periods related to metal ion exposure, longer follow-ups are required to conclusively determine the effects of elevated circulating ions on oxidative stress in the blood of patients with MM bearings.

Purpose: Recent evidence indicates that a major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic patients rapidly express type X collagen (COL10A1), a marker of late-stage chondrocyte hypertrophy associated with endochondral ossification. We recently discovered that a novel atmospheric-pressure plasma-polymerized thin film substrate, named “nitrogen-rich plasma-polymerized ethylene” (PPE:N), is able to inhibit COL10A1 expression in committed MSCs. However, the cellular mechanisms implicated in the inhibition of COL10A1 expression by PPE:N surfaces are unknown.

Method: Human mesenchymal stem cells (MSCs) were obtained from aspirates from the intramedullary canal of donors (60–80 years of age) undergoing total hip replacement for osteoarthritis. Bone marrow aspirates were processed and MSCs were cultured on commercial polystyrene (PS control) and on PPE:N surfaces in the presence of different kinases and cyclooxygenase inhibitors for 3 days. Total RNA was extracted with TRIzol reagent (Invitrogen, Burlington, ON) and the expression of COL10A1, cyclooxygenase-1 (COX-1), and 5-lipoxygenase (5-LOX) genes was measured by real-time quantitative RT-PCR.

Results: Results showed that a non-specific inhibitor of cyclooxygenases reduced the expression of COL10A1. In contrast, inhibitors of protein kinases stimulated the expression of COL10A1. Furthermore, potent and selective inhibitors of COX-1 and 5-LOX also reduced the expression of COL10A1. However, COX-2 and 12-LOX inhibitors had no significant effect on the expression of COL10A1. COX-1 gene expression was also decreased when MSCs were incubated on “S5” PPE:N surfaces. Interestingly, MSCs did not express 5-LOX.

Conclusion: PPE:N surfaces suppress COL10A1 expression through the inhibition of COX-1 which is directly implicated in the synthesis of prostaglandins. The decreased expression of COX-1 and COL10A1 in human MSCs cultured on PPE:N is therefore in agreement with the induction of the osteogenic capacity of rat bone marrow and bone formation by systemic or local injection of PGE2 in rats. However, PGE2 and other prostaglandins inhibited COL10A1 expression in chick growth plate chondrocytes. This suggests that the effect of prostaglandins on COL10A1 expression may be cell-specific or may be dependent on pre-existing patho-physiological conditions.

Purpose: Metal-on-metal articulations in total hip arthroplasty (THA) have been recently re-introduced for the treatment of osteoarthritis. There have been excellent short-term clinical outcomes reported. The long-term clinical results of these implants are still unknown. In this study, we examined the four to ten -year results of metal-on-metal total hip arthroplasty at our institution.

Method: All patients receiving a metal-on-metal prosthesis were prospectively registered in a computerized database. The period July 1997 – May 2003 was selected for analysis. This group contained 251 cases (207 patients) and had an average follow-up of 6.8 years. Of the 251 implants the femoral head components were either Metasul^™ (203) or Ultamet^™ (48) and the acetubular component Fitek^™ (12), Interop^™ (191) or Pinnacle^™ (48). All patients were assessed with the Harris hip and UCLA activity scores at routinely scheduled follow-up visits. The mean age of patients at surgery was 52 years (18 to 70 years old). The average follow-up was of 6.8 years (4.2–10 years). No patients were lost to follow-up.

Results: The mean UCLA and Harris hip score pre-operatively were 3.6 (1–8) and 42 (15–81), respectively. Values of the UCLA and the Harris hip scores on the most recent follow-up visit were 7.0 (60% of patients had a score equal to or greater than 7) and 85 (70% of patients had a score equal to or greater than 85), respectively. Thirteen hips (5.1%) were revised in total: 1 (0.4%) for early deep infection, 1 (0.4%) for revision of undersized stem and 11 (4.3%) for acetabular revisions due to manufacturing defects. Radiological results showed no osteolysis and 9 non-progressive radiolucency.

Conclusion: At four to ten year follow-up, metal-on-metal total hip replacement provides consistently good clinical and radiographic results with a low revision rate. They provide longstanding pain alleviation and improved function and activity levels in patients suffering from osteoarthritis.

Purpose: It has been shown that ischemic/hypoxic stress as well as shear stress and mechanical injury are linked to the pathogenesis of osteoarthritis. The aim of this study was to determine whether Egr-1 (Early Growth Response protein-1), a transcription factor induced by stress or injury, affects articular cartilage and intervertebral discs (IVDs).

Method: Experimental studies used 6- to 7-month-old adult female wild-type C57Bl/6 or Egr-1-deficient (knockout) mice. All animals were sacrificed at the same age interval (8- to 9-months) and stored at −20°C. Prior to dissection, posterior-anterior and lateral x-rays of whole mice were done. Right knee joint and cervical to lumbar spine were stained with hematoxylineosin (H& E), Safranin-O/Fast green, and Weigert’s hematoxylin/alcian blue/picrosirius red for histological analysis. Bone mineral density (BMD) was measured using a PIXImus Bone Densitometer System. Micro computed tomography (CT) data were acquired on a SkyScan T1072 X-ray Microscope-Microtomograph.

Results: Results showed that the articular cartilage of knee joints of Egr-1 knockout mice was more irregular and degenerative than in the wild type mice. Furthermore, a lower concentration of proteoglycans (predominantly aggrecan) was observed in articular cartilage of knockout mice. The nucleus pulposus (NP) of the knockout mice IVD also showed signs of degeneration and a loss of notochordal cells. The overall disc height was also reduced compared to wild type mice. The number of cells in the endplate was higher in the knockout mice than in the wild type animals. Furthermore, there was a trend for increased lumbar vertebrae BMD as well a significant increase in BMD in the femur of the Egr-1 knockout mice. The relative bone volume (BV/TV) was significantly smaller in Egr-1 knockout mice as was trabecular number and trabecular separation, while there was increased bone surface to bone volume.

Conclusion: Our findings showed signs of degeneration in both articular cartilage and IVD in Egr-1 knockout mice. Moreover, the loss of height in IVD and cellular components of the NP, as well as the increased cell numbers in cartilage of the endplate showed some resemblance to those of human degenerative disc diseases. However, further studies are needed to determine the mechanism by which Egr-1 leads to articular cartilage and IVD degeneration.

Purpose: Several studies have shown elevated levels of metal ions in the blood of patients with metal-on-metal (MM) total hip arthroplasty (THA). Even though there is no conclusive evidence that the elevated levels of ions have any detrimental effects on the patients, the presence of these ions is still a cause of concerns. The potential of metal ions released from MM implants for oxidative stress is unknown. In the present study, we measured the concentrations of oxidative stress markers in the plasma of patients with MM THAs.

Method: Blood from patients having MM THAs was collected up to 10 years post-operatively into Sarstedt Li-Heparin tubes. Plasma was prepared by centrifugation at 500 × g for 10 min. Plasma was chosen as opposed to whole blood because it is known that the assays for oxidative stress are not recommended for blood and can lead to erroneous data. Total antioxidant levels were measured by the Oxford Biomedical total antioxidant power assay to obtain an overview of the defense capacity of patients against oxidative stress. The activity of catalase and glutathione peroxidase, two antioxidant enzymes acting on specific reactive oxygen species, was measured by enzymatic assays. Peroxide concentrations were measured by the Biomedica Oxy-Stat assay to quantify damage to lipids in the systemic circulation. Nitrototyrosine levels were quantified using the BIOXYTECH® Nitrotyrosine-EIA assay to measure damage to proteins. Levels in patients without prostheses were used as control.

Results: There were no statistical differences in the concentrations of total antioxidants, lipid peroxides, and nitrotyrosines throughout the period of study. The activity of catalase and glutathione peroxidase was also stable over time. Moreover, there was no correlation between the concentrations of these markers and the concentrations of both cobalt and chromium ions.

Conclusion: Metal ions have the potential to induce the production of reactive oxygen species (free radicals) and cause oxidative stress in the plasma of patients with MM THAs. The present study showed that there were no changes in the levels of oxidative stress markers or antioxidant enzymes in these patients up to 10 years post-operatively. Taken together, the data strongly suggest that metal ions may not cause a significant oxidative stress in patients with MM THAs.

Purpose: Since the human intervertebral disc (IVD) is loaded in compression for approximately 16h per day, we investigated the effect of 16h compression loading on the magnetic resonance imaging (MRI) parameters, biochemical contents, and mechanical properties, of IVDs.

Method: Bovine caudal discs (2–3 years-old; non-degenerated) from 3-disc motion segments were injected in the NP with either 5 mg of trypsin in 40 μl Tris buffer or with Tris buffer only. The motion segments were placed in bags containing saline solution and antibiotics and were kept at 37°C throughout the experiment. The motion segments were subjected to either 16h of cyclic compression loading or were left unloaded for 16h. The motion segments were then paraffin embedded for MRI examinations, which were carried out in a 1.5T machine. The IVDs were dissected and the NP and AF were separated for biochemical and mechanical analyses. The NP and AF tissues were analyzed for contents of water, glycosaminoglycan (GAG), total collagen, and denatured collagen. Swelling pressure, compressive modulus HA, and hydraulic permeability were also measured.

Results: Loading had a significant effect on the MRI parameters (T1, T2, T1rho, MTR, ADC) of both the NP and AF tissues. Loading had a greater effect on the MR parameters and biochemical composition of the NP than trypsin. In contrast, trypsin had a larger effect on the mechanical properties. Localized trypsin injection predominantly affected the NP. T1rho was sensitive to loading and correlated with the water content of the NP and AF but not with their proteoglycan content.

Conclusion: Few studies have been directed towards developing an objective and accurate diagnostic tool in the detection and quantification of matrix and mechanical changes in early IVD degeneration. In this report, we demonstrated that MR parameters were influenced by compression loading. We also show showed specific correlations between T1rho and the structural and compositional changes in the disc. Further studies are required to determine the potential of the T1rho technique to be used as a non-invasive diagnostic tool of the biochemical and mechanical changes occurring in disc degeneration.

Purpose: Hormone replacement therapy for the menopause seems to be associated with a decrease in the prevalence of symptoms and radiological alterations related to hip and knee osteoarthritis. However, little is known on the effects of estrogen in articular cartilage and intervertebral disc (IVD). The aim of this study was to evaluate the developmental changes in mouse articular cartilage and intervertebral discs under estrogen deficiency.

Method: Experimental studies used 6- to 7-month-old adult female wild-type or bilaterally ovariectomized (OVX) C57Bl/6 mice. All animals were sacrificed at the same age interval (8- to 9-months) and stored at −20°C. Prior to dissection, posterior-anterior and lateral x-rays of whole mice were done. Right knee joint and cervical to lumbar spine were stained with hematoxylineosin (H& E), Safranin-O/Fast green, and Weigert’s hematoxylin/alcian blue/picrosirius red for histological analysis. Bone mineral density (BMD) was measured using a PIXImus Bone Densitometer System. Micro computed tomography (CT) data were acquired on a SkyScan T1072 X-ray Microscope-Microtomograph.

Results: Degeneration, including the loss of notochordal cells, was observed in the nucleus pulposus (NP) of the IVD of OVX mice. The annulus fibrosus (AF) showed marked thinning as compared to the wild type. Furthermore, the OVX group showed decreased IVD heights and trend of endplate ossification. Knee joints of OVX mice showed a trend towards having more gross degenerative changes, like areas of cartilage erosion. A decrease in articular cartilage thickness was also observed. Certain layers of cartilage were more affected than others, suggesting a specific role of estrogens in the developing cartilage. Also, the BMD was reduced in both the femur and lumbar vertebrae of the OVX group. Finally, MicroCT results showed a decrease in percent bone volume, trabecular thickness, trabecular number, and an increase in trabecular separation.

Conclusion: The present study showed AF thinning, decreased IVD height, NP degeneration, and loss of cellular components in the NP in ovariectomized mice. Likewise, the articular cartilage revealed more degenerative changes, including a decrease in articular thickness. Results suggest that estrogens play a role in maintaining healthy cartilage and IVD.

Despite a relentless search for adequate and effective treatment, low back pain is one of the most prevalent and costly illness in today’s society. While disc degeneration has been implicated as a major etiologic component of low back pain, there has been relatively little study in developing an objective, accurate, non-invasive diagnostic tool in the detection and quantification of matrix changes in early disc degeneration. The aim of the present study was to establish the correlations between magnetic resonance (MR) parameters and the biochemical and mechanical properties of the nucleus pulposus (NP) undergoing targeted trypsin digestion and axial compression.

Three-disc segments from bovine tails were either unloaded or loaded (cyclic compression: 50N-300N-50N at 1 Hz for 16h) to evaluate the effect of compression loading and the interactive effects of trypsin treatment and mechanical loading. The MR examinations were carried out in a 1.5-Tesla whole-body Siemens Avanto System (Siemens AG, Germany). The frozen NP and annulus fibrosus (AF) tissue sections reserved for mechanical analysis were tested under confined compression; swelling pressure was calculated based on the increase in measured force throughout the initial dwell period. Total water, proteoglycan, collagen, and denatured collagen contents were also measured.

Results showed that loading had a significant effect on the MR properties (T1, T2, T1ñ, MTR, ADC) of both disc tissues. Loading had a greater effect on the MR parameters and biochemical composition of the NP than trypsin. In contrast, trypsin had a larger effect on the mechanical properties. Results also indicated that localised trypsin injection predominantly affected the NP. T1ñ was sensitive to loading and correlated with the water content of the NP and AF but not with their proteoglycan content.

Results support the concept that physiologic loading is an important confounder and that T1ñ is an essential parameter in efforts to develop quantitative MRI as a non-invasive diagnostic tool to detect and quantify matrix and material changes in early disc degeneration. Further studies are required to determine the potential of the T1ñ technique to be used as a non-invasive diagnostic tool of the biochemical and mechanical changes occurring in disc degeneration.

Hip surface replacement is an alternative for young patients considered for hip replacement. The in vivo release of ions from these surfaces has yet to be well evaluated. In the present study, we compared the concentrations of metal ions in blood of patients with hip surface replacement and metal-on-metal (MM) total hip arthroplasty (THA).

Blood was collected six months and one year after implantation time into Sarstedt Monovette® tubes for trace metal analysis from patients having Articular Surface Replacement (ASR®, DePuy Orthopaedics; n=61), 28 mm-head MM THA (n=18), and 36 mm-head MM THA (n=25). The concentrations of cobalt (Co), chromium (Cr), and molybdenum (Mo) were analyzed by inductively coupled plasma-mass spectroscopy (ICP-MS). Since metal ions are potent inducers of oxidative stress, total antioxidant, peroxide, and nitrotyrosine levels (oxidative stress markers) were also measured in plasma of the patients.

The median Co and Cr levels progressively and significantly increased in the three groups during the first year post-operation (compared to patients without hip bearings (n=25)). After six months, the levels of Co and Cr were significantly higher in patients with ASR and 28 mm MM THA than in patients with 36 mm MM THA. There was no difference after one year. The level of activity, as measured by the UCLA activity score, was higher in ASR patients than in 28 and 36 mm MM THA after one year. No differences were observed for Mo levels in these patients when compared to our control group. There was no increase of oxidative stress marker levels in patients with ASR and 36 mm MM THA and no correlation between the concentrations of Co and Cr ions and the levels of oxidative stress markers.

Our results show that, at one year post-operation, the concentration of ions in patients with ASRs is similar than those in patients with MM THAs. Moreover, results suggest that metal ions liberated from MM bearings do not induce damage to macromolecules by oxidative stress in plasma of patients. Longer follow-ups are still required to characterise the concentration of ions in ASR and to determine conclusively the effects of elevated circulating ions.

Although the etiology of low back pain is unclear, it is believed that intervertebral disc (IVD) degeneration plays a major role. In the present study, we sought to determine if bovine IVD cells maintain their phenotype in a mouse subcutaneous injection model, while embedded or not in biocompatible matrices.

Nucleus pulposus (NP) cells were isolated from adult bovine tails. Ten million cells were resuspended either in 500 ƒÝl of DMEM or in a negatively (alginate) or positively (chitosan) charged matrix. The mixtures were then injected subcutaneously in Balc/c nude mice. After two weeks, the mice were sacrificed and the implants harvested. The implants were examined histologically with a hematoxylin and eosin stain. The implant size was measured and the cells were counted. Proteoglycan was assessed by the GAG assay. The expression of type I and II collagens, aggrecan, and CD24 genes was analyzed by reverse transcription ¡V polymerase chain reaction (RT-PCR).

Histologic evaluation confirms the presence of cells in all NP implants. The presence of alginate increased the implant size, the number of cells in the implants, and to a lesser extent, the proteoglycan content, compared to implants formed with cells injected alone. However, chitosan had no effect on the implant size, the number of cells and the aggrecan content. NP implants expressed the same pattern of genes as the native NP tissue (i.e. type I and II collagens, aggrecan, and CD24). The presence of alginate did not affect this expression pattern whereas chitosan decreased slightly their expression.

After injection in mice, bovine NP cells appeared to retain their native phenotype. The RT-PCR analysis revealed that NP cells expressed aggrecan, type I and type II collagens as well as CD24, a specific marker for the NP phenotype. Also, NP cells can be embedded in matrices to produce NP-like features in vivo. In conclusion, we have developed a simple mouse subcutaneous injection model that recreates the features of the native IVD and avoids the need to use a disc degeneration model.

The aim of this study was to analyze in human macrophages the effects of Co²⁺ and Cr³⁺ ions on the activity of caspase-8 and caspase-3, initiator and executioner of apoptosis, respectively. Caspase-3 and -8 activities were measured by colorimetric assays. Results show that Co²⁺ ions induced caspase-3 activity in a time-dependent manner. Co²⁺ had no effect on caspase-8 activity. The activation of caspase-3 by Cr³⁺ was time-dependent while caspase-8 activity reached a maximum after eight hours and decreased thereafter. Since caspase-8 is primarily activated by membrane-associated events, our results suggest that Cr³⁺ interacts with cell membrane components to induce macrophage apoptosis, whereas Co²⁺ seems to stimulate apoptosis most likely through intracellularly located mechanisms.

Because of their potential for improved wear performance, there has been a revived interest in metal-metal bearings, made of cobalt-chromium-molybdenum alloys, as an alternative to the use of conventional metal-polyethylene bearings. However, metal ion toxicity remains a major cause for concern. Previous studies suggested that both cobalt (Co²⁺) and chromium (Cr³⁺) ions induce macrophage apoptosis. The interest in apoptosis lies in the fact that it offers specific targets for therapeutic intervention.

The aim of this study was to analyze the effects in human macrophages of Co²⁺ and Cr³⁺ ions on the activity of caspase-8 and caspase-3, initiator and executioner of apoptosis, respectively.

U937 human macrophages were exposed to 0–10 ppm Co²⁺ (CoCl₂) and 0–500 ppm Cr³⁺ (CrCl₃). Caspase-3 and caspase-8 activities were measured by colorimetric assays based on the recognition of specific amino acid sequences (DEVD and IETD, respectively).

Results show that Co²⁺ ions induced caspase-3 activity with a significant increase after four hour incubation and a maximal 2.65-fold increase reached after twenty-four hour with 10 ppm. Co²⁺ had no effect on caspase-8 activity.

Cr³⁺ ions significantly stimulated caspase-3 activity after four hours with a maximal 1.75-fold stimulation reached after twenty-four hours, reaching only 50% of that observed with Co²⁺. Caspase-8 activity was significantly increased after two hours incubation, peaking at eight hours with a 2.2-fold increase, and decreasing thereafter.

Since caspase-8 is primarily activated by membrane-associated events, our results suggest that Cr³⁺ interacts with cell membrane components to induce macrophage apoptosis. On the other hand, Co²⁺ seems to stimulate caspase-3 activity and apoptosis most likely through intracellularly located mechanisms.

Purpose: Quantitative MRI is currently being tested as an early and non-invasive diagnostic tool of disc problems prior to the appearance of symptoms. The aim of the present study was to determine the effects of cyclic loading and enzymatic digestion on quantitative MRI, biochemical composition, and mechanical properties of intervertebral disc tissue.

Methods: Bovine tail segments consisting of three discs were subjected to 16h of cyclic compression loading (50N–300N–50N at 1Hz) or left unloaded for 16h while in saline solution at 37°C. Prior to loading, the nucleus pulposus were injected with either a trypsin or buffer solution. MR examinations were carried out in a 1.5T Siemens` Avanto system to measure T1 and T2 relaxation times, magnetization transfer ratio (MTR), and trace of the apparent diffusion coefficient (TrD). The nucleus pulposus and annulus fibrosus were dissected and analyzed for contents of water, glycosaminoglycan, total collagen, and denatured collagen. Cylindrical nucleus pulposus and annulus fibrosus tissue plugs were harvested, prepared, and tested under confined compression to measure compressive modulus (HA) and hydraulic permeability (k). ANOVA and linear regression analyses were performed (p< 0.05).

Results: Loading decreased the T1, T2, and TrD of NP while it increased MTR. Only water content in the nucleus pulposus was significantly influenced by loading. T1, water content, and k of the annulus fibrosus tissue were significantly reduced with loading.|Enzymatic treatment of the nucleus pulposus had no effect on its MR properties, but increased the percent of denatured collagen and thus decreased HA. None of the biochemical, mechanical, and MR parameters of the annulus fibrosus changed with trypsin treatment.

Conclusions: Dynamic loading of the disc segments for 16h decreased the permeability of both disc tissues. This was consistent with the measured drop in tissue hydration and was observed as a decrease in T1. Targeted trypsin digestion of the nucleus pulposus was confirmed with no detectable changes in the biochemical, biomechanical, or MR properties of the annulus fibrosus. Future studies will address additional quantitative MR parameters such as T1-rho, a higher strength magnet, and different enzymatic treatments. Funding: Other Education Grant Funding Parties: Canadian Institutes of Health Research, McGill William Dawson Scholar Award, and Whitaker Foundation

Purpose: To investigate the effect of amifostine and dexrazoxane on bone mass of the vertebrae and femurs of doxorubicin treated male rats.

Methods: Amifostine, Doxorubicin and Dexrazoxane were purchased from SMBD-Jewish General Hospital Pharmacy. Lactating Sprague Dawley dams with 14 male pups were purchased from Charles River Canada. At neonate day 10, rat pups were randomly divided into 4 groups of n=5. Pups were injected once intraperitoneally with either Phosphate Saline Buffer 1X (saline), or drugs, AMF (50 mg/kg), AMF + DOX (50 mg/kg +3 mg/ kg), or with AMF + DXR + DOX (50 mg/kg + 60 mg + 3 mg/kg, 20:1 DXR to DOX ratio). AMF and DXR were injected 30 minutes prior to the DOX injection. After injection, rat pups were returned to their mothers until weaning on neonate day 22. Rats were then sacrificed at day 38 (28 Post-Injection, PI). Bone mineral density (BMD) and micro computed tomography were analyzed.

Results: Dissection of male pups days 1, 5 or 9 post-injection did not reveal any intestinal or organ damage. AMF treatment alone led to a slight but not significant increase in the right femoral, left femoral and lumbar vertebral BMDs. Similarly, AMF + DOX or AMF + DXR + DOX treated rats had no significant change in either femoral and vertebrae BMD.

Conclusions: We recently showed that a single injection of DOX in young female rats is associated with low bone turnover resulting in vertebrae and femur bone growth deficits. However, no such a difference was detected when similarly treated males were examined. The role of sex steroid hormone at this age is unclear as sex hormones level are very low in neonates at the time of injection and the rats, male and female, were sacrificed prior to puberty. To define the role of sex hormone in the observed gender-specific drug susceptibility we plan on comparing the response of intact to ovariectomized female rats to the drug regimen.

Funding : Other Education Grant

Funding Parties : CIHR

Metal particles and ions are liberated from the articular interface of metal-metal (MM) total hip arthroplasties. To better understand their cellular effect, we analyzed the internalization of these metal particles and ions by macrophages in vitro. Macrophages were exposed to metal particles isolated from MM prostheses cycled in a hip simulator and to metal ions. Cells were processed for transmission electron microscopy analysis. Results reveal the internalization of metal particles and Cr³⁺ ions in specifically localized cytoplasmic areas. This study is the first to reveal that metal particles of clinically relevant size and Cr³⁺ ions are internalized by an apparently active process.

In order to minimize articular interface wear, metal-metal (MM) hip implants have been considered as an alternative to conventional metal-polyethylene bearings. While the local histological effects of the metallic particles and ions appear to be similar to that seen with metal-polyethylene hip replacements (i.e., a foreign-body macrophage response), little is known about the cellular effects of these metal particles and ions.

The purpose of this study was to better understand the cellular effect of metal particles and ions, we analyzed their internalization by macrophages in vitro.

J774 mouse macrophages were exposed to metal particles isolated from serum of MM prostheses cycled in a hip simulator and to Cr³⁺ (CrCl₃) and Co²⁺ (CoCl₂) ions. Cells were then processed for transmission electron microscopy analysis.

Micrographs revealed the internalization of metal particles and Cr³⁺ ions in specifically localized cytoplasmic areas, suggesting that they are phagocytosed via an active pathway. Energy disperse X-ray analysis spectra of macrophages incubated with Cr³⁺ revealed a chromium phosphate composition. The same structure and composition were also observed when Cr³⁺ ions were incubated in culture medium without cells, suggesting that they were formed outside the cells. Co²⁺ ions did not form visibly agglomerated structures.

This study is the first to reveal that metal particles of clinically relevant size are internalized by an apparently active process and that Cr³⁺ ions can be internalized by macrophages after binding to phosphorus or phosphoproteins. Kinetic studies are now necessary to better understand the mechanism of phagocytosis and the ultimate outcome of these particles and ions in macrophages.

Purpose: To develop an improved understanding of the in vivo behavior of intervertebral disc (IVD) cells for determining the phenotype of a differentiated stem cell in tissue engineering applications.

Methods: Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from adult bovine tails while notochordal cells were extracted from fetal bovine intervertebral disc. Ten million cells (of each cell type) in 500 & #61549;l of DMEM were then injected subcutaneously in C57Bl/6 mice. After 2 weeks, the mice were sacrificed and the specimens harvested. They were examined grossly, histologically and by scanning electronic microscopy (SEM) for the evidence of IVD-like structure formation. Proteoglycan was assessed by the GAG assay and PCR for analysis of gene expression. Control tissue (from bovine NP and AF) were directly fixed in glutaraldehyde, without any isolation technique and examined in SEM.

Results: After 2 weeks, SEM examination of specimens from AF and NP closely resembled normal bovine AF and NP. Of special interest here was the finding that some mice injected with cells from the AF developed an organized arrangement of parallel collagen fibres while NP cells injected mice had an amorphous structure with few collagen fibers. The GAG assay showed pro-teoglycan content for each samples, ranging from 3.8 microg to 26 microg. The morphology of the specimens retrieved from notochordal cells injected mice were also amorphous punctuated with thin collagen fibrils.

Conclusions: This study demonstrates that subcutaneous injection of bovine disc cells in mice can result in formation of disc structures similar to those of the bovine IVD. We believe that the cellular communication of the bovine disc cells is maintained in the mouse leading to architectural organization of the collagen fibers with the mouse as a source of nutrients. This technology may be useful in determining the phenotype of a differentiated stem cell for tissue engineering of IVD.

Hyaline cartilage and immature nucleus pulposus possess similar macromolecules in their extracellular matrix, and there is no unique molecular marker to distinguish the two tissues. We show that in normal disc (fifteen to twenty-five years old), the GAG to hydroxyproline ratio (proteoglycan to collagen ratio) within the nucleus pulposus is approximately 28:1. However, the GAG to hydroxyproline ratio within hyaline cartilage of the same group is 2.5:1. This information is important in identifying stem cell conversion to a nucleus pulposus cell phenotype rather than a chondrocyte phenotype for tissue engineering of intervertebral disc.

Tissue engineering of intervertebral discs (IVDs) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. Since there is no unique marker for disc tissue, and because cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. In this study, we compare the proteoglycan to collagen ratio in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals.

To distinguish between a normal NP and hyaline cartilage phenotype for tissue engineering of IVDs.

Human lumbar spine specimens were harvested from fresh cadavers, aged twelve week to seventy-nine year. Discs and endplates were examined for total collagen using the hydroxyproline assay and glycosaminoglycan (GAG) content using a standard assay.

In a mature disc with no degeneration (fifteen to twentyfive years), the GAG to hydroxyproline ratio within the NP is approximately 28:1. However, the ratio within the hyaline cartilage endplate of the same group is 2.5:1.

A high proteoglycan to collagen ratio can be used to distinguish NP cells from chondrocytes. The lower NP collagen content is probably responsible for its gelatinous nature rather than the firm texture of hyaline cartilage, and this is essential for normal disc function. This information is crucial in identifying a NP-like phenotype when MSCs are induced to differentiate into a disc cell as opposed to a chondrocyte, for tissue engineering of IVDs.

The in situ increased production of matrix metalloproteinases (MMPs) has been associated with the development of periprosthetic osteolysis. The aim of the study was to compare the effect of Co²⁺ and Cr³⁺ ions on macrophages matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP (TIMP-1) expression. Using reverse transcription-polymerase chain reaction (RT-PCR), we showed that both Co²⁺ and Cr³⁺ ions induce the expression of MMP-1 and TIMP-1 in a dose-dependent manner. Since MMP-1 and TIMP-1 participate in the extracellular matrix degradation and tissue remodeling, our results suggest that the modulation of MMP-1 and TIMP-1 may contribute to the development of periprosthetic osteolysis.

The in situ increased production of matrix metalloproteinases (MMPs) has been associated with the development of periprosthetic osteolysis. Aseptic loosening due to periprosthetic osteolysis is the major cause of total hip arthroplasty failure. Because of their potential for improved wear performance, there has been a revived interest in metal-metal bearings, made of cobalt-chromium-molybdenum alloys. However, metal particle and ion toxicity remains a major cause for concern.

The aim of the study was to determine the effects of Co²⁺ and Cr³⁺ ions on the expression of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1), two proteins participating in the extracellular matrix degradation and tissue remodeling.

Human U937 macrophages were incubated with Co²⁺ and Cr³⁺ ions. The expression of MMP-1 and TIMP-1 mRNAs was measured by reverse transcription-polymerase chain reaction (RT-PCR) and calculated as the ratio of the house keeping gene GAPDH expression.

Results show that both Co²⁺ and Cr³⁺ ions induced in a dose-dependent manner the expression of PCR products (mRNAs) of MMP-1 (135 bp) and TIMP-1 (328 bp). Co²⁺ ions were more effective in inducing MMP-1 and TIMP-1 expression than Cr³⁺ ions. The induction of MMP-1 and TIMP-1 paralleled the induction of TNF-α mRNA expression.

Our results demonstrate that the expression of MMP-1 and TIMP-1 were up regulated by incubating macrophages with Co²⁺ and Cr³⁺ ions, suggesting that metal ions contribute to tissue damage in the periprosthetic environment and that variations in MMP-1 and TIMP-1 expression may contribute to periprosthetic osteolysis.

Purpose: Knowledge of factors regulating the turnover, repair, and degeneration of the intervertebral disc (IVD) is lacking. Although type II collagen (CII) fragments accumulate in the degenerative IVD, little is known of how they affect the degenerative process. In this study, the effect of a CII fragment, CII-(245–270), known to be critical in arthritis was investigated on gene expression of proteinases, collagen, and proteoglycan by bovine disc cells to determine its role in matrix turnover.

Methods: Cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) of adult bovine tails were cultured in the absence (control) or presence of the fragment. The fragment CII-(245–270) (US Biological, Massachusetts) was dissolved in culture medium to a final concentration of 1& #956;g/ml. PCR was performed and products were visualized by ethidium bromide staining.

Results: Addition of the CII-(245–270) peptide at 1& #956;g/ml to NP and AF cells enhanced expression of genes for MMP-1, cathepsin K, and aggrecan after 48 hours compared with the control. MMP-13 was also upregulated in the NP. In contrast, the effect in the AF was time dependent. Type II collagen was upregulated throughout the culture time in the NP as opposed to the AF where its expression was enhanced only on day 2.

Conclusions: We have shown that the CII-(245–270) peptide can alter gene expression of proteinases, collagen, and proteoglycan in bovine disc cells. The present study reveals the complex interrelationships of gene expression in the disc that accompany fragmentation of type II collagen. This new information suggests that increased levels of these fragments, in degenerated discs, may stimulate disc breakdown but may also attempt to protect the disc, by unknown mechanisms Funding: Other Education Grant Funding Parties: AO foundation, Switzerland

Recent evidence indicates that link N can stimulate synthesis of proteoglycans and collagen by adult (2–4 years old) bovine disc tails. Here we sought to determine the effect of link N on the accumulation of disc matrix proteins from young (eight to twenty month old) bovine tails. We show that degradation products of link protein generated by matrix metalloproteinases cannot “feed-back” and stimulate matrix assembly of the disc matrix from young bovine tails but may have a regulatory role in cell proliferation. Link N may have value only in stimulating the growth and regeneration of the old damaged intervertebral disc.

To date, there have been no reports on the effect of the amino terminal peptide of link protein (DHLSD-NYTLDHDRAIH) (link N) on disc cells from young (eight to twenty month old) bovine coccygeal discs. Link N is produced when removed by proteolysis from the N-terminus of the link protein of cartilage proteoglycan aggregates. We recently showed that link N can act directly on disc cells from adult (two to four years old) bovine discs to stimulate matrix production (J Cell Biochem, 2003; 88:1202–13).

To examine whether link N can play a role in maintaining the matrix integrity of young bovine disc cells.

Nucleus pulposus (NP) and annulus fibrosus (AF) cells were isolated from fresh grade I discs from young steers, and cultured in pellets at 1 million cell per tube in 1 ml of DMEM-high glucose supplemented with 1% 100X Pen-Strep, 1% ITS, 1 mg/ml BSA, and 50 μg/ml ascorbic acid. Cell pellets were digested and then analysed for sulfated glycosaminoglycan, type II collagen, percent denatured type II collagen, type IX collagen, and DNA content, using specific assays.

A concentration of 100 ng/ml link N significantly increased the DNA content of AF cells. However, link N had no significant effect on proteoglycan, type II and type IX collagen accumulation.

This study demonstrates that link N at a concentration of 10 ng/ml and 100 ng/ml cannot stimulate matrix production but may increase cell division in young bovine disc tails.

Purpose: In vivo, intertervertebral disc cells exist in a low oxygen environment ranging from 5% O2 for the annulus fibrosus (AF) cells to 1% O2 for the nucleus pulpous (NP) cells. Various conditions have been used for in vitro cell culture and seem that AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply, O2 levels and biomechanical loading. The aim of this study was to determine how AF and NP cells respond to different O2 concentrations when cultured in a 3 dimensional system consisting of an alginate scaffold.

Methods: Bovine AF and NP cells were embedded in alginate beads and incubated in airtight polypropylene containers at different O2 concentration of 1%, 5% or 21%. Culture medium was changed every third day and the culture was carried out for 21 days. The pro-teoglycan content of the medium was analyzed using the DMMB assay. Cells were recovered from the alginate beads at two time points, day 8 and day 21 and RT-PCR was performed to amplify gene expression of GAPDH and aggrecan.

Results: In both cell types, the cumulative production of GAG increased with time in culture up to day 9, and then tended to plateau in the AF cells but continue to increase in the NP cells. At all time points, the level of GAG synthesis by NP cells was greater than by AF cells. All GAG synthesis trends were the similar at all O2 levels (1%, 5% and 21%).

Conclusions: In the alginate scaffold NP cells continue to exhibit their in situ behaviour by producing more proteoglycan than AF cells. Perhaps surprisingly, both cell types showed little change in GAG production with variations of O2 levels from 1–21%. This contrasts with other studies where GAG production is dependent on O2 concentrations. In the culture system used in this work, both cell types metabolize easily well at low oxygen as they do in normal conditions. Funding: Other Education Grant

Purpose: Articular surface replacement (ASR) is an alternative for young patients considered for hip replacement. The in vivo release of ions from these surfaces has yet to be well evaluated. The purpose of the present study was to compare the concentrations of metal ions in blood of patients with ASR and metal-on-metal (MM) total hip arthroplasty (THA).

Methods: Blood was collected 6 months after implantation time into Sarstedt Monovette® tubes for trace metal analysis from patients having ASR (n=61), 28 mm-head MM THA (n=18), and 36 mm-head MM THA (n=25). The concentrations of cobalt (Co), chromium (Cr), and molybdenum (Mo) were analyzed by inductively coupled plasma-mass spectroscopy (ICP-MS).

Results: The median Co level was not significantly different between the 3 groups (2.35 ppb, 2.00 ppb, and 2.50 ppb for the 28 mm MM THA, 36 mm MM THA, and the ASR patients, respectively). The median Cr level was significantly lower in the 36 mm MM THA patients (0.10 ppb) compared to the 28 mm MM THA (0.15 ppb) and the ASR (0.40 ppb) patients. The median Mo level was significantly lower in the 36 mm MM THA patients (1.30 ppb) compared to the 28 mm MM THA (2.00 ppb) and the ASR (1.50 ppb) patients.

Conclusions: Our results show that the level of ions in 36 mm MM THA patients was lower than in 28 mm MM THA patients. This can be explained by the fact that 28 mm MM bearings are resistant to microseparation during the normal gait cycle, which is theoretically accompanied by a reduction of fluid film lubrication and increased potential for the production of wear debris. Our results also show that the ion levels in patients having ASRs is similar to that observed in 28 mm MM THA patients but higher than in 36 mm MM THA patients. The diametric clearance of ASRs is typically much greater and the potential for a ‘suction fit’ may be less, leading to higher ion production. The concentration of ions in long-term follow-up remains however to be elucidated. Funding: Educational Grant from the Canadian Orthopaedic Foundation

Purpose: One of the major concerns regarding metal-on-metal prostheses is the biological and biochemical activities of chromium (Cr) ions. Previous studies showed that Cr3+ ions form nanostructures in cell culture media and to date, there has been little attempt to understand the nature of implant-derived metal ions in adjacent tissues or in biofluid. The aim of this work was to determine the nature of proteins present in serum involved in the formation of Cr nanostuctures

Methods: RPMI 1640 and DMEM media supplemented with 5% human serum (HS) or 5% foetal bovine serum (FBS) were incubated for 1h at 37°C in the presence of 50 ppm of Cr3+ (CrCl3). Structures were then isolated and separated by SDS-PAGE. Proteins were stained by Coomassie blue and analyzed by liquid chromatography-quadrupole-time of flight-mass spectrometer (LC-Q-Tof-MS). Data were submitted to Mascot software for a search against the NCBI nonredundant database

Results: Results show that Cr-nanostructures can interact with proteins from both human and bovine serums. On SDS-PAGE, the molecular weights of the proteins were between 40 to 90 kDa. The LC-Q-Tof-MS results suggest that Cr-nanostructures are the result of the interaction with numerous proteins present in serum. However, the complete analysis of results demonstrates that only two proteins (in both RPMI and DMEM) are implicated in these nanostructures: albumin and trans-ferrin. For both proteins, at least 40 peptides matched to the complete sequence of the proteins. The ion scores (“peptide identity score”) were between 79 and 108. Ion scores > 45 indicate identity or extensive homology

Conclusions: Human serum contains more than 400 different proteins. Albumin, the major protein of human serum, has been shown to play a scavenger role by binding and transporting injected and ingested Cr. Albumin could also play an immunological role by addressing signals to defense cells, such as macrophages. Trans-ferrin, known as an iron-carrying protein, also plays a scavenger role for Cr. This suggests that the binding of Cr to these proteins may protect cells from the cytotoxic effect of Cr ions. However, the relation with Cr nano-structures in vivo remains to be determined