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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) Dulbecco's modified Eagle's medium (DMEM)/F12 (300 mOsm/L), (2) a-MEM (300 mOsm/L), and (3) a-MEM adjusted to 400mOsm/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 a-MEM adjusted to 400mOsm/L showed the highest gene expression of brachyury, cytokeratin 18, and aggrecan, the highest GAG production, and the lowest collagen 1a1 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. © 2014, Mary Ann Liebert, Inc. 2014.

Degeneration of the intervertebral disc (IVD) is a common problem in dogs and humans. IVD degeneration can lead to herniation of the IVD with subsequent compression of neural structures and various clinical signs, including back pain. Current treatment of IVD disease is conservative or surgical. However, these therapies do not restore health or functionality to the IVD and may lead to spinal instability and recurrence of clinical signs. Therefore, the aim of this thesis was to investigate the process of IVD degeneration and develop new treatment strategies for dogs with IVD degenerative disease, as well as for humans with IVD degenerative disease using the dog as a research model for translational research purposes. The key findings of the thesis were: Surgical treatments for canine IVD disease that involve partial removal of the IVD result in a significant loss of disc height and spinal stability. Additional stabilization and restoration of IVD functionality are required for optimal treatment of dogs with IVD degenerative diseases. Pedicle screw-rod fixation of the lumbosacral junction can successfully stabilize spinal segments affected by IVD degeneration in middle-sized and large-breed dogs. Although this procedure does not result in interbody fusion of the spinal segment, the surgical outcome is successful and appears to restore the propulsive function of the hind limbs in patients affected by degenerative lumbosacral stenosis. Ways to improve the surgical technique to achieve spinal fusion have been identified. Functional repair of the nuclectomized IVD can be achieved by inserting a nucleus pulposus prosthesis into the nuclear cavity created after nuclectomy. The success of prosthesis insertion can be evaluated by radiography, computed tomography, and magnetic resonance imaging. When intact, in situ, and with adequate closure of the annulus fibrosus, the prosthesis significantly restores disc height and spinal stiffness to the nuclectomized spinal segment, and therefore may serve as a valuable future treatment for dogs with IVD disease. IVD degeneration alters the biomechanics of the spine. The spinal biomechanics of chondrodystrophic dogs (with nucleus pulposus rich in chondrocyte-like cells) and non-chondrodystrophic dogs (with nucleus pulposus rich in notochordal cells) are markedly different, probably as a result of differences in the cell population and the extracellular matrix of the nucleus pulposus, and other biomechanical factors. The notochordal cell, the main cell type of the healthy canine nucleus pulposus, exhibits basal canonical Wnt signaling. Early IVD degeneration, which involves the replacement/differentiation of notochordal cells by/into chondrocyte-like cells, is associated with down-regulation of canonical Wnt signaling and with a decreased expression of caveolin-1, a protein that appears to be fundamental to the preservation of notochordal cells. Early IVD degeneration is accompanied by significant changes in the expression of genes involved in numerous biomolecular signaling pathways, including extracellular matrix remodeling and bone morphogenetic protein signaling. These findings might facilitate the design of strategies for IVD regeneration and help clarify several points debated in the current scientific literature.

INTRODUCTION: Early 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. METHODS: Dual 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 ex pression 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.

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) 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 remodelling, and DNA- and GAG analyses were performed on 1, 7, 14, and 28 days in culture. Large, vesicle-containing cells organised 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 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 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. PMID:24304309[PubMed - as supplied by publisher]

Vet Surg. 2012 Oct;41(7):829-37. doi: 10.1111/j.1532-950X.2012.01021.x. Soft tissue artifact in canine kinematic gait analysis. Schwencke M, Smolders LA, Bergknut N, Gustås P, Meij BP, Hazewinkel HA. Source Department of Clinical Sciences of Companion Animals,, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Abstract OBJECTIVE: To investigate, noninvasively, the soft tissue artifact (STA) in canine kinematic gait analysis. STUDY DESIGN: Experimental study. ANIMALS: Labrador retrievers (n = 4). METHODS: Kinematic study: Reflective markers were glued to the skin over bony landmarks, with the distance between 2 markers representing the length of the underlying scapula, humerus, ulna, femur, and crus. The distance between these markers (marker distance [MD]) was measured with infrared cameras while the dogs stood still or walked on a treadmill. Fluoroscopy study: Radiopaque markers were glued on the skin over the spinous process of the L6 vertebra and the stifle to allow fluoroscopic observation of the markers and underlying skeletal segments while the dogs walked on the treadmill. The position of the markers was compared with the position of the underlying skeletal segments during different phases of the step cycle. RESULTS: Kinematic study: Significant differences were found between MD during standing and walking for all bones investigated. Mean percentage differences in MD ranged from -18% to +6%. Fluoroscopy study: Significant displacements relative to the bony landmarks were found ranging from 0.4 to 1.2 cm. CONCLUSIONS: Analysis of the motion of skeletal structures with the use of markers attached to the skin showed that the skin moves relative to underlying skeletal structures. When working with a 3-D motion-capture system using skin markers, researchers should be aware that the STA could significantly influence their results. © Copyright 2012 by The American College of Veterinary Surgeons. PMID: 23075438 [PubMed - in process]

A nucleus pulposus prosthesis (NPP) made of the hydrogel N-vinyl-2-pyrrolidinone copolymerized with 2-(4′-iodobenzoyl)-oxo-ethyl methacrylate has recently been developed. The special features of this NPP, i.e. intrinsic radiopacity and its ability to swell in situ to fill the nucleus cavity and restore disc height, were investigated ex vivo in canine spinal specimens. L7-S1 intervertebral discs were isolated from three canine spinal specimens, and the dimensions of the nuclei pulposi were measured. Based on these averaged measurements, the NPP prototype was made and inserted in its dry form (xerogel) into a canine cadaveric spinal segment and allowed to swell overnight at 38 °C. The integrity of the NPP and the filling of the nucleus cavity were assessed before and after swelling, using radiography, computed tomography, and magnetic resonance imaging. The ability of the NPP to restore disc height was assessed on radiographs of 10 spinal specimens. Thereafter the NPP was macroscopically assessed in situ by dissection of the spinal specimen. Both on imaging and macroscopically, 9/10 NPPs appeared to have a near perfect fit and disc height was restored in 8/10 spinal segments. The NPP may thus be an acceptable treatment option for low back patients meeting the requirements for NPP treatment.