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Long-term culture of bovine nucleus pulposus explants in a native environment

Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, PO. Box 513, GEM-Z 4.115, 5600 MB, Eindhoven, The Netherlands.
The spine journal: official journal of the North American Spine Society (Impact Factor: 2.8). 01/2013; 13(4). DOI: 10.1016/j.spinee.2012.12.006
Source: PubMed

ABSTRACT BACKGROUND CONTEXT: Chronic low back pain is a disease with tremendous financial and social implications, and it is often caused by intervertebral disc degeneration. Regenerative therapies for disc repair are promising treatments, but they need to be tested in physiological models. PURPOSE: To develop a physiological in vitro explant model that incorporates the native environment of the intervertebral disc, for example, hypoxia, low glucose, and high tissue osmolarity. STUDY DESIGN: Bovine nucleus pulposus (NP) explants were cultured for 42 days in conditions mimicking the native physiological environment. Two different approaches were used to balance the swelling pressure of the NP: raised medium osmolarity or an artificial annulus. METHODS: Bovine NP explants were either cultured in media with osmolarity balanced at isotonic and hypertonic levels compared with the native tissue or cultured inside a fiber jacket used as an artificial annulus. Oxygen and glucose levels were set at either standard (21% O(2) and 4.5 g/L glucose) or physiological (5% O(2) and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents and fixed charge density), tissue histology, cell viability, and cellular behavior with messenger RNA (mRNA) expression. RESULTS: Both the hypertonic culture and the artificial annulus approach maintained the tissue matrix composition for 42 days. At Day 3, mRNA expressions of aggrecan, collagen Type I, and collagen Type II in both hypertonic and artificial annulus cultures were not different from Day 0; however, at Day 42, the artificial annulus preserved the mRNA expression closer to Day 0. Gene expressions of matrix metalloprotease 13, tissue inhibitor of matrix metalloprotease 1, and tissue inhibitor of matrix metalloprotease 2 were downregulated under physiological O(2) and glucose levels, whereas the other parameters analyzed were not affected. CONCLUSIONS: Although the hypertonic culture and the artificial annulus approach are both promising models to test regenerative therapies, the artificial annulus was better able to maintain a cellular behavior closer to the native tissue in longer term cultures.

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    • "However, a drawback of NP explants is that swelling pressure is no longer balanced by the AF and hyperosmolarity within the NP is difficult to maintain, which results in NP tissue swelling. A special fibre jacket (artificial annulus) has been developed to mimic the AF in the so-called NP explant system, creating a physiological model to test the efficacy of regenerative therapies for IVD degeneration [15,17]. To mimic the in vivo situation even closer, whole organ culture bioreactor systems, e.g. the loaded culture disc system, have been developed, in which intact IVD explants, including AF, NP, and EPs, are kept alive under loading conditions for as long as 6 weeks with preservation of biological and structural integrity [18,19]. "
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    ABSTRACT: Pain due to spontaneous intervertebral disc (IVD) disease is common in dogs. In chondrodystrophic (CD) dogs, IVD disease typically develops in the cervical or thoracolumbar spine at about 3-7 years of age, whereas in non-chondrodystrophic (NCD) dogs, it usually develops in the caudal cervical or lumbosacral spine at about 6-8 years of age. IVD degeneration is characterized by changes in the biochemical composition and mechanical integrity of the IVD. In the degenerated IVD, the content of glycosaminoglycan (GAG, a proteoglycan side chain) decreases and that of denatured collagen increases. Dehydration leads to tearing of the annulus fibrosus (AF) and/or disc herniation, which is clinically characterized by pain and/or neurological signs. Current treatments (physiotherapy, anti-inflammatory/analgesic medication, surgery) for IVD disease may resolve neurological deficits and reduce pain (although in many cases insufficient), but do not lead to repair of the degenerated disc. For this reason, there is interest in new regenerative therapies that can repair the degenerated disc matrix, resulting in restoration of the biomechanical function of the IVD. CD dogs are considered a suitable animal model for human IVD degeneration because of their spontaneous IVD degeneration, and therefore studies investigating cell-, growth factor-, and/or gene therapy-based regenerative therapies with this model provide information relevant to both human and canine patients. The aim of this article is to review potential regenerative treatment strategies for canine IVD degeneration, with specific emphasis on cell-based strategies.
    BMC Veterinary Research 01/2014; 10(1):3. DOI:10.1186/1746-6148-10-3 · 1.74 Impact Factor
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    • "Such a system allowed us to characterize and explore the mechanism of cellular transition in NP tissue without the influence of surrounding spinal structures or other systemic metabolic changes ex vivo. Culture models with ex vivo explants are useful tools to study the effects of micro-environmental conditions on native NP tissue [34]. The second objective was to measure the expression of the novel structure and symptom-modifying factors SHH, Sema3A and Noggin in response to daily dynamic loading during the maturation process. "
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    ABSTRACT: Notochordal cells (NCs) pattern aneural and avascular intervertebral discs (IVDs), and their disappearance, is associated with onset of IVD degeneration. This study induced and characterized the maturation of nucleus pulposus (NP) tissue from a gelatinous NC-rich structure to a matrix-rich structure populated by small NP cells using dynamic pressurization in an ex vivo culture model, and also identified soluble factors from NCs with therapeutic potential. Porcine NC-rich NP tissue was cultured and loaded with hydrostatic pressure (0.5 to 2 MPa at 0.1 Hz for 2 hours) either Daily, for 1 Dose, or Control (no pressurization) groups for up to eight days. Cell phenotype and tissue maturation was characterized with measurements of cell viability, cytomorphology, nitric oxide, metabolic activity, matrix composition, gene expression, and proteomics. Daily pressurization induced transition of NCs to small NP cells with 73.8%, 44%, and 28% NCs for Control, 1 Dose and Daily groups, respectively (P < 0.0002) and no relevant cell death. Dynamic loading matured NP tissue by significantly increasing metabolic activity and accumulating Safranin-O-stained matrix. Load-induced maturation was also apparent from the significantly decreased glycolytic, cytoskeletal (Vimentin) and stress-inducible (HSP70) proteins assessed with proteomics. Loading increased the production of bioactive proteins Sonic Hedgehog (SHH) and Noggin, and maintained Semaphorin3A (Sema3A). NP tissue maturation was induced from dynamic hydrostatic pressurization in a controlled ex vivo environment without influence from systemic effects or surrounding structures. NCs transitioned into small nonvacuolated NP cells probably via differentiation as evidenced by high cell viability, lack of nitric oxide and downregulation of stress-inducible and cytoskeletal proteins. SHH, Sema3A, and Noggin, which have patterning and neurovascular-inhibiting properties, were produced in both notochordal and matured porcine NP. Results therefore provide an important piece of evidence suggesting the transition of NCs to small NP cells is a natural part of aging and not the initiation of degeneration. Bioactive candidates identified from young porcine IVDs may be isolated and harnessed for therapies to target discogenic back pain.
    Arthritis research & therapy 09/2013; 15(5):R122. DOI:10.1186/ar4302 · 3.75 Impact Factor
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    ABSTRACT: Study Design Preclinical in vitro culture of human degenerated nucleus pulposus (NP) tissue. Objective Cyclooxygenase 2 inhibitors (e.g., celecoxib) inhibit prostaglandin E2 (PGE2) production, and they have been shown to upregulate regeneration of articular cartilage. In this study, we developed an explant culture system for use with human tissue and tested the potential of celecoxib. Methods NP explants were cultured with or without 1 μM of celecoxib and were analyzed at days 0 and 7 for biochemical content (water, sulfated glycosaminoglycans, hydroxyproline, and DNA), gene expression (for disk matrix anabolic and catabolic markers), and PGE2 content. Results Water and biochemical contents as well as gene expression remained close to native values after 1 week of culture. PGE2 levels were not increased in freshly harvested human NP tissue and thus were not reduced in treated tissues. Although no anabolic effects were observed at the dosage and culture duration used, no detrimental effects were observed and some specimens did respond by lowering PGE2. Conclusions Human degenerated NP explants were successfully cultured in a close to in vivo environment for 1 week. Further research, especially dosage-response studies, is needed to understand the role of PGE2 in low back pain and the potential of celecoxib to treat painful disks.
    02/2014; 4(1):33-40. DOI:10.1055/s-0033-1359724
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