Marianna A. Tryfonidou

Intervertebral disc degeneration in the dog. Part 2: Chondrodystrophic and non-chondrodystrophic breeds

Dogs can be grouped into two distinct types of breed based on the predisposition to chondrodystrophy, namely, non-chondrodystrophic (NCD) and chondrodystrophic (CD). In addition to a different process of endochondral ossification, NCD and CD breeds have different characteristics of intravertebral disc (IVD) degeneration and IVD degenerative diseases. The anatomy, physiology, histopathology, and biochemical and biomechanical characteristics of the healthy and degenerated IVD are discussed in the first part of this two-part review. This second part describes the similarities and differences in the histopathological and biochemical characteristics of IVD degeneration in CD and NCD canine breeds and discusses relevant aetiological factors of IVD degeneration.

Intervertebral disc degeneration in the dog. Part 1: Anatomy and physiology of the intervertebral disc and characteristics of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is common in dogs and can give rise to a number of diseases, such as IVD herniation, cervical spondylomyelopathy, and degenerative lumbosacral stenosis. Although there have been many reports and reviews on the clinical aspects of canine IVD disease, few reports have discussed and reviewed the process of IVD degeneration. In this first part of a two-part review, the anatomy, physiology, histopathology, and biochemical and biomechanical characteristics of the healthy and degenerated IVD are described. In Part 2, the aspects of IVD degeneration in chondrodystrophic and non-chondrodystrophic dog breeds are discussed in depth.

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

IntroductionEarly degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.MethodsDual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.ResultsEarly IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.ConclusionsEarly IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte‐like cells (CLCs). Wnt/β‐catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non‐chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non‐chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/β‐catenin signaling in the healthy, NC‐rich NP and early degenerated, CLC‐rich NP of both breed types by immunohistochemistry of β‐catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c‐myc. Both NCs and CLCs showed nuclear and cytoplasmic β‐catenin protein expression and axin2 gene expression, but β‐catenin signal intensity and Wnt target gene expression were higher in the CLC‐rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/β‐catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c‐myc gene expression. In conclusion, Wnt/β‐catenin signaling activity in the NC within the NC‐rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/β‐catenin signaling activity in early IVD degeneration may be a reflection of its dual role.

Vitamin D3 metabolism in dogs

Plasma concentrations of the main vitamin D(3) metabolites (i.e., 25(OH)D(3), 1,25(OH)(2)D(3), and 24,25(OH)(2)D(3)) were measured in 14 weeks old large- and small-breed dogs (adult body weight 60 kg vs. 6 kg), raised under the same conditions. Levels of 25(OH)D(3) (approx. 22 microg/l) and 1,25(OH)(2)D(3) (approx. 40 ng/l) were similar in both groups, whereas plasma 24,25(OH)(2)D(3) concentrations were lower in large-breed dogs (7 microg/l vs. 70 microg/l, large- vs. small-breed dogs, respectively). The lower plasma 24,25(OH)(2)D(3) concentrations could be explained by the higher plasma GH and IGF-I concentrations in the large- vs. small-breed dogs, and these hormones are known to suppress 24-hydroxylation. Plasma 24,25(OH)(2)D(3) concentrations increased during Ca supplementation in small-breed but not in large-breed dogs (100 microg/l vs. 7 microg/l, respectively). Hypophosphatemia induced by a high dietary Ca content was only seen together with increased plasma 1,25(OH)(2)D(3) concentrations in euparathyroid dogs and not in hypoparathyroid dogs. Hyperparathyroidism due to Ca deficiency was accompanied by increased plasma 1,25(OH)(2)D(3) concentrations and decreased plasma 24,25(OH)(2)D(3) concentrations in both large- and small-breed dogs, together with generalized osteoporosis. Large-breed pups fed on a standard diet supplemented with Ca and P had decreased plasma concentrations of both 25(OH)D(3) and 1,25(OH)(2)D(3), which may indicate an increased clearance of these metabolites; the low plasma concentrations of the di-hydroxylated vitamin D metabolites were considered responsible for the disturbance in cartilage maturation (i.e., osteochondrosis) in these dogs. Even lower concentrations of all vitamin D(3) metabolites were seen in young dogs raised on a vitamin D(3)-deficient diet, and led to disturbed osteoid and cartilage mineralization (i.e., rickets). These studies indicate that there is a hierarchy of factors regulating vitamin D(3) metabolism in dogs, i.e., GH and IGF-I suppress 24-hydroxylase more than hypercalcemia or hypophosphatemia does; 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3) are only reciprocally related in hyperparathyroidism; excessive Ca and P intake increases the turnover of vitamin D(3) metabolites; and the synergism between parathyroid hormone and 1,25(OH)D(3) seems to play a role in skeletal mineralization. The low plasma 24,25(OH)(2)D(3) concentrations in large-breed dogs raised on standard dog food may play a role in the etiology of disturbances in endochondral ossification during the rapid growth phase.

Increased osmolarity and cell clustering preserve canine notochordal cell phenotype in culture

Degeneration of the intervertebral disc (IVD) is associated with a loss of notochordal cells (NCs) from the nucleus pulposus (NP) and their replacement by chondrocyte-like cells. NCs are known to maintain extracellular matrix quality and stimulate the chondrocyte-like NP cells, making NCs attractive for designing new tissue engineering approaches for IVD regeneration. However, optimal conditions, such as osmolarity and other characteristics of the culture media, for long-term culture of NCs are not known. The purpose of this study was to investigate the effects of different culture media and osmolarity on the physiology of NCs in vitro. NC clusters isolated from canine IVDs were suspended in alginate beads and cultured at 37°C under normoxic conditions for 28 days. Three different culture conditions were investigated; (1) Dulbeccos modified Eagles medium (DMEM)/F12 (300 mOsm/L), (2) α-MEM (300 mOsm/L), and (3) α-MEM adjusted to 400 mOsm/L to mimic a hyperosmolar environment. NC morphology, expression of genes related to NC markers, matrix production and remodeling, and DNA- and glycosaminoglycan (GAG) analyses were performed on 1, 7, 14, and 28 days in culture. Large, vesicle-containing cells organized in clusters, characterized as NCs, remained present during 28 days for all culture conditions. However, the proportion of the NC clusters decreased over time, whereas the proportion of spindle-shaped cells increased. Gene expression profiling at 7, 14, and 28 days in culture compared to day 1 indicated a initial loss of NC phenotype followed by some recovery of brachyury and aggrecan gene expression after 28 days of culture supporting a potential recovery of NC phenotype. NCs cultured in α-MEM adjusted to 400 mOsm/L showed the highest gene expression of brachyury, cytokeratin 18, and aggrecan, the highest GAG production, and the lowest collagen 1α1 gene expression. In conclusion, NCs cultured in alginate in native cell clusters, partially retained their characteristic morphology and recovered their phenotype in long-term culture. The type of culture medium and medium osmolarity appear to be important factors for culturing NC clusters. These findings provide additional information concerning the maintenance of NCs in vitro that may aid further mechanistic inquiry into the biology of NCs.

Inflammatory profiles in canine intervertebral disc degeneration

BackgroundIntervertebral disc (IVD) disease is a common spinal disorder in dogs and degeneration and inflammation are significant components of the pathological cascade. Only limited studies have studied the cytokine and chemokine profiles in IVD degeneration in dogs, and mainly focused on gene expression. A better understanding is needed in order to develop biological therapies that address both pain and degeneration in IVD disease. Therefore, in this study, we determined the levels of prostaglandin E2 (PGE2), cytokines, chemokines, and matrix components in IVDs from chondrodystrophic (CD) and non-chondrodystrophic (NCD) dogs with and without clinical signs of IVD disease, and correlated these to degeneration grade (according to Pfirrmann), or herniation type (according to Hansen). In addition, we investigated cyclooxygenase 2 (COX-2) expression and signs of inflammation in histological IVD samples of CD and NCD dogs.ResultsPGE2 levels were significantly higher in the nucleus pulposus (NP) of degenerated IVDs compared with non-degenerated IVDs, and in herniated IVDs from NCD dogs compared with non-herniated IVDs of NCD dogs. COX-2 expression in the NP and annulus fibrosus (AF), and proliferation of fibroblasts and numbers of macrophages in the AF significantly increased with increased degeneration grade. GAG content did not significantly change with degeneration grade or herniation type. Cytokines interleukin (IL)-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, immune protein (IP)-10, tumor necrosis factor (TNF)-α, and granulocyte macrophage colony-stimulating factor (GM-CSF) were not detectable in the samples. Chemokine (C-C) motif ligand (CCL)2 levels in the NP from extruded samples were significantly higher compared with the AF of these samples and the NP from protrusion samples.ConclusionsPGE2 levels and CCL2 levels in degenerated and herniated IVDs were significantly higher compared with non-degenerated and non-herniated IVDs. COX-2 expression in the NP and AF and reactive changes in the AF increased with advancing degeneration stages. Although macrophages invaded the AF as degeneration progressed, the production of inflammatory mediators seemed most pronounced in degenerated NP tissue. Future studies are needed to investigate if inhibition of PGE2 levels in degenerated IVDs provides effective analgesia and exerts a protective role in the process of IVD degeneration and the development of IVD disease.

Biocompatibility and intradiscal application of a thermoreversible celecoxib-loaded poly-N-isopropylacrylamide MgFe-layered double hydroxide hydrogel in a canine model.

IntroductionChronic low back pain due to intervertebral disc (IVD) degeneration is associated with increased levels of inflammatory mediators. Current medical treatment consists of oral anti-inflammatory drugs to alleviate pain. In this study, the efficacy and safety of a novel thermoreversible poly-N-isopropylacrylamide MgFe-layered double hydroxide (pNIPAAM MgFe-LDH) hydrogel was evaluated for intradiscal controlled delivery of the selective cyclooxygenase (COX) 2 inhibitor and anti-inflammatory drug celecoxib (CXB).MethodsDegradation, release behavior, and the ability of a CXB-loaded pNIPAAM MgFe-LDH hydrogel to suppress prostaglandin E2 (PGE2) levels in a controlled manner in the presence of a proinflammatory stimulus (TNF-α) were evaluated in vitro. Biocompatibility was evaluated histologically after subcutaneous injection in mice. Safety of intradiscal application of the loaded and unloaded hydrogels was studied in a canine model of spontaneous mild IVD degeneration by histological, biomolecular, and biochemical evaluation. After the hydrogel was shown to be biocompatible and safe, an in vivo dose–response study was performed in order to determine safety and efficacy of the pNIPAAM MgFe-LDH hydrogel for intradiscal controlled delivery of CXB.ResultsCXB release correlated to hydrogel degradation in vitro. Furthermore, controlled release from CXB-loaded hydrogels was demonstrated to suppress PGE2 levels in the presence of TNF-α. The hydrogel was shown to exhibit a good biocompatibility upon subcutaneous injection in mice. Upon intradiscal injection in a canine model, the hydrogel exhibited excellent biocompatibility based on histological evaluation of the treated IVDs. Gene expression and biochemical analyses supported the finding that no substantial negative effects of the hydrogel were observed. Safety of application was further confirmed by the absence of clinical symptoms, IVD herniation or progression of degeneration. Controlled release of CXB resulted in a nonsignificant maximal inhibition (approximately 35 %) of PGE2 levels in the mildly degenerated canine IVDs.ConclusionsIn conclusion, this study showed biocompatibility and safe intradiscal application of an MgFe LDH-pNIPAAM hydrogel. Controlled release of CXB resulted in only limited inhibition of PGE2 in this model with mild IVD degeneration, and further studies should concentrate on application of controlled release from this type of hydrogel in animal models with more severe IVD degeneration.

Genome wide analysis indicates genes for basement membrane and cartilage matrix proteins as candidates for hip dysplasia in Labrador Retrievers

Hip dysplasia, an abnormal laxity of the hip joint, is seen in humans as well as dogs and is one of the most common skeletal disorders in dogs. Canine hip dysplasia is considered multifactorial and polygenic, and a variety of chromosomal regions have been associated with the disorder. We performed a genome-wide association study in Dutch Labrador Retrievers, comparing data of nearly 18,000 single nucleotide polymorphisms (SNPs) in 48 cases and 30 controls using two different statistical methods. An individual SNP analysis based on comparison of allele frequencies with a χ2 statistic was used, as well as a simultaneous SNP analysis based on Bayesian variable selection. Significant association with canine hip dysplasia was observed on chromosome 8, as well as suggestive association on chromosomes 1, 5, 15, 20, 25 and 32. Next-generation DNA sequencing of the exons of genes of seven regions identified multiple associated alleles on chromosome 1, 5, 8, 20, 25 and 32 (p<0.001). Candidate genes located in the associated regions on chromosomes 1, 8 and 25 included LAMA2, LRR1 and COL6A3, respectively. The associated region on CFA20 contained candidate genes GDF15, COMP and CILP2. In conclusion, our study identified candidate genes that might affect susceptibility to canine hip dysplasia. These genes are involved in hypertrophic differentiation of chondrocytes and extracellular matrix integrity of basement membrane and cartilage. The functions of the genes are in agreement with the notion that disruptions in endochondral bone formation in combination with soft tissue defects are involved in the etiology of hip dysplasia.

Delayed endochondral ossification in early medial coronoid disease (MCD): A morphological and immunohistochemical evaluation in growing Labrador retrievers

Medial coronoid disease (MCD) is a common joint disease of dogs. It has a multifactorial aetiology, but the relationship between known causal factors and the disease has yet to be elucidated. As most of the published literature is clinical and it reports changes associated with advanced disease, it is not known whether the changes reflect the cause or consequences of the condition. The aim of this study was to investigate early micromorphological changes occurring in articular cartilage and to describe the postnatal development of the medial coronoid process (MCP) before MCD develops. Three litters of MCD-prone young Labrador retrievers were purpose-bred from a dam and two sires with MCD. Comparisons of the micromorphological appearance of the MCP in MCD-negative and MCD-positive joints demonstrated that MCD was initially associated with a disturbance of endochondral ossification, namely a delay in the calcification of the calcifying zone, without concurrent abnormalities in the superficial layers of the joint cartilage. Cartilage canals containing patent blood vessels were only detected in dogs <12 weeks old, but the role of these channels in impaired ossification requires further investigation. Retained hyaline cartilage might ossify as the disease progresses, but weak areas can develop into cracks between the retained cartilage and the subchondral bone, leading to cleft formation and fragmentation of the MCP.