Joint registries suggest a downward trend in the use of uncemented Total Knee Replacements (TKR) since 2003, largely related to reports of early failures of uncemented tibial and patella components. Advancements in uncemented design such as trabecular metal may improve outcomes, but there is a scarcity of high-quality data from randomised trials.

319 patients <75 years of age were randomised to either cemented or uncemented TKR implanted using computer navigation. Patellae were resurfaced in all patients. Patient outcome scores, re-operations and radiographic analysis of radiolucent lines were compared.

Two year follow up was available for 287 patients (144 cemented vs 143 uncemented). There was no difference in operative time between groups, 73.7 v 71.1 mins (p= 0.08). There were no statistical differences in outcome scores at 2 years, Oxford knee score 42.5 vs 41.8 (p=0.35), International Knee Society 84.6 vs 84.0 (p=0.76), Forgotten Joint Score 66.7 vs 66.4 (p=0.91). There were two revisions, both for infection one in each group (0.33%). 13 cemented and 8 uncemented knees underwent re-operation, the majority of these being manipulation under anaesthetic (85.7%), with no difference (8.3% vs 5.3%, 95% CI -2.81% to 8.89%, p = 0.31). No difference was found in radiographic analysis at 2 years, 1 lucent line was seen in the cemented group and 3 in the uncemented group (0.67% v 2.09%, 95%CI -4.1% to 1.24%, p = 0.29).

We found no difference in clinical or radiographic outcomes between cemented and uncemented TKR including routine patella resurfacing at two years.

Early results suggest there is no difference between cemented and uncemented TKR at 2 years with reference to survivorship, patient outcomes and radiological parameters.

Hamstring grafts have been associated with reduced strength, donor site pain and muscle strains following Anterior Cruciate Ligament Reconstruction (ACLR). Traditional graft fixation methods required both semitendinosus and gracilis tendons to achieve a graft of sufficient length and diameter, but newer techniques allow for shorter, broad single tendon grafts.

This study seeks to compare the outcomes between Single Tendon (ST) and Dual Tendon (DT) ACLR, given there is no prospective randomised controlled trial (RCT) in the literature comparing outcomes between these options.

In this ongoing RCT: (ANZ Clinical Trials Registry ACTRN126200000927921) patients were recruited and randomised into either ST or DT groups. All anaesthetic and surgical techniques were uniform aside from graft technique and tibial fixation. 13 patients were excluded at surgery as their ST graft did not achieve a minimum 8mm diameter. 70 patients (34 ST, 36DT) have been assessed at 6 months, using PROMS including IKDC2000, Lysholm and Modified Cincinnati Knee, visual analog scale for pain frequency (VAS-F) and severity (VAS-S), dedicated donor site morbidity score, KT-1000 assessment, and isokinetic strength.

Graft diameters were significantly lesser in the ST group compared to the DT group (8.44mm/9.11mm mean difference [MD],-0.67mm; P<0.001). There was a significant and moderate effect in lower donor site morbidity in the ST group compared to the DT group (effect size [ES], 0.649; P = .01). No differences between groups were observed for knee laxity in the ACLR limb (P=0.362) or any of the patient-reported outcome measures (P>0.05). Between-group differences were observed for hamstrings strength LSI favouring the ST group, though these were small-to-moderate and non-significant (ES, 0.351; P = .147).

ST (versus DT) harvest results in significantly less donor site morbidity and this is the first prospective RCT to determine this. There were no differences between ST and DT hamstring ACLR were observed in PROMs, knee laxity and hamstring strength. Younger female patients tend to have inadequate single tendon size to produce a graft of sufficient diameter, and alternative techniques should be considered. Further endpoints include radiological analysis, longer term donor site morbidity, revision rates and return to sport and will continue to be presented in the future.

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.

Introduction

Infection rates following arthroplasty surgery are reported between 1–4%, with considerably higher rates in revision surgery. The associated costs of treating infected arthroplasty cases are over 4 times the cost of primary arthroplasties, with significantly worse functional and satisfaction outcomes. In addition, multiple antibiotic resistant bacteria are developing, so to reduce the infection rates and costs associated with arthroplasty surgery, new preventative methods are required. HINS-light is a novel blue light inactivation technology which kills bacteria through a photodynamic process, and is proven to have bactericidal activity against a wide range of species. The aim of this study was to investigate the efficacy of HINS-light for the inactivation of bacteria isolated from infected arthoplasty cases.