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.

Simultaneous bilateral total hip arthroplasty (THA) in patients with bilateral hip osteoarthritis is gradually becoming attractive, as it requires a single anesthesia and hospitalization. However, there are concerns about the potential complications following this surgical option. The purpose of this study is to compare the short-term major and minor complications and assess the readmission rate, between patients treated with same-day bilateral THA and those with staged procedures within a year.

We retrospectively reviewed the charts of all patients with bilateral hip osteoarthritis that underwent simultaneous or staged (within a year) bilateral total THA in our institution, between 2016-2020. Preoperative patient variables between the two groups were compared using the 2-sample t-test for continuous variables, the Fisher's exact test for binary variables, or the chi-square test for multiple categorical variables. Similarly, differences in the 30-day major and minor complications and readmission rates were assessed. A logistic regression model was also developed to identify potential risk factors.

A total of 160 patients (mean age: 64.3 years, SD: ±11.7) that underwent bilateral THA was identified. Seventy-nine patients were treated with simultaneous and eighty-one patients with staged procedures. There were no differences in terms of preoperative laboratory values, gender, age, Body Mass Index (BMI), or American Society of Anesthesiologists Scores (ASA) (p>0.05) between the two groups. Patients in the simultaneous group were more likely to receive general anesthesia (43% vs 9.9%, p0.05). After controlling for potential confounders, the multivariable logistic regression analysis showed similar odds of having a major (odds ratio 0.29, 95% confidence interval [0.30-2.88], p=0.29) or minor (odds ratio 1.714, 95% confidence interval [0.66-4.46], p=0.27) complication after simultaneous compared to staged bilateral THA. No differences in emergency department visits or readmission for reasons related to the procedure were recorded (p>0.05).

This study shows that similar complication and readmission rates are expected after simultaneous and staged THAs. Simultaneous bilateral THA is a safe and effective procedure, that should be sought actively and counselled by surgeons, for patients that present with radiologic and clinical bilateral hip disease.

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).

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.

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.