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Safety of intradiscal injection and biocompatibility of polyester amide microspheres in a canine model predisposed to intervertebral disc degeneration.

ABSTRACT: Repair of degenerated intervertebral discs (IVD) might be established via intradiscal delivery of biologic therapies. Polyester amide polymers (PEA) were evaluated for in vitro cytotoxicity and in vivo biocompatibility, and thereafter intradiscal application of PEA microspheres (PEAMs) in a canine model predisposed to IVD degeneration at long-term (6 months) follow-up. PEA extracts did not induce cytotoxicity in mouse fibroblast cells (microscopy and XTT assay), while a slight foreign body reaction was demonstrated by histopathology after intramuscular implantation in rabbits. Intradiscal injection of a volume of 40 µL through 26 and 27G needles induced no degenerative changes in acanine model susceptible to IVD disease. Although sham-injected IVDs showed increased CAV1 expression compared with noninjected IVDs, which may indicate increased cell senescence, these findings were not supported by immunohistochemistry, biomolecular analysis of genes related to apoptosis, biochemical and histopathological results. PEAM-injected IVDs showed significantly higher BAX/BCL2 ratio vs sham-injected IVDs suggestive of an anti-apoptotic effect of the PEAMs. These findings were not supported by other analyses (clinical signs, disc height index, T2 values, biomolecular and biochemical analyses, and IVD histopathology). PEAs showed a good cytocompatibility and biocompatibility. PEAMs are considered safe sustained release systems for intradiscal delivery of biological treatments. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 707-714, 2017.

SUBMITTER: Willems N

PROVIDER: S-EPMC6690078 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Intervertebral disc herniation (IVDH) is an important pathology in humans and also in dogs. While the molecular disease mechanisms are well investigated in humans, little is known about the inflammatory mediators in naturally occurring canine IVDH. The objective of this study was to investigate whether the involved proinflammatory cytokines in human IVDH are also key cytokines in canine IVDH and thus to elucidate the suitability of the dog as a model for human trials. 59 samples from 25 dogs with surgically confirmed thoracolumbar IVDH were collected and classified in three subgroups: herniated (H), affected non-herniated (NH) disc, and adjacent non-affected (NA) disc. Discs from 11 healthy dogs acted as controls (C). Samples were analyzed for IL-1, IL-6, IL-8, and TNF-? expression (qPCR/ELISA) as well as cell infiltration and activation of the MAP kinase pathways (immunohistochemistry). Gene and protein expression of all key cytokines could be detected in IVDH affected dogs. Canine IVDH was significantly associated with a higher gene expression of IL-6 (H?>?C, NH?>?C) and TNF-? (H?>?C, NH?>?C, NA?>?C) and a significant down-regulation of IL-1? (H?<?C). Dogs with spontaneous pain had significantly higher IL-6 mRNA compared to those with pain arising only upon palpation. An inter-donor comparison (H and HN relative to NA) revealed a significant increase of IL-6 gene expression (H?>?NA, NH?>?NA). IL-8 (H?>?C, NA?>?C) and TNF-? (NH?>?C) protein levels were significantly increased in diseased dogs while inversely, IL-6 protein levels were significantly higher in patients with better clinical outcome. Aside from resident IVD cells, mostly monocytes and macrophages were found in extruded material, with concomitant activation of extracellular signal-regulated kinase p38 in the majority of samples. Dogs with spontaneous IVDH might provide a useful model for human disc diseases. Although the expression of key cytokines found in human IVDH was also demonstrated in canine tissue, the inflammatory mechanisms accompanying canine IVDH diverges partially from humans, which will require further investigations in the future. In dogs, IL-6 seems to play an important pathological role and may represent a new potential therapeutic target for canine patients.

Project description:Background: Canine intervertebral disc disease (IVDD) represents a significant clinical problem in veterinary medicine, with similarities to the human pathology. Host-derived damage-associated molecular patterns like fibronectin fragments (FnF) that develop during tissue dysfunction may be of specific relevance to IVD pathologies by inducing an inflammatory response in resident cells. Aim: This project aimed to determine the presence and pathobiological role of FnF during IVD herniation in dogs, with a focus on inflammation. Methods: Herniated nucleus pulposus (NP) material from five dogs as well as non-herniated adjacent NP material from three dogs was collected during spinal surgery required due to acute IVD herniation. The presence of different types of FnF were determined by Western blot analysis. NP cells isolated from six herniated canine IVDs were then exposed to 30 kDa FnF. NP cell inflammation and catabolism was examined by investigating the expression of IL-1β, IL-6, IL-8, and COX-2, as well as MMP-1 and MMP-3 by qPCR (all targets) and ELISA (IL-6, PGE2). Results: Amongst multiple sized FnF (30, 35, 45, and >170kDa), N-terminal fragments at a size of ~30 kDa were most consistently expressed in all five herniated IVDs. Importantly, these fragments were exclusively present in herniated, but not in non-herniated IVDs. Exposure of canine NP cells to 500 nM 30 kDa FnF caused a significant upregulation of IL-6 (62.5 ± 79.9, p = 0.032) and IL-8 (53.0 ± 75.7, p = 0.031) on the gene level, whereas IL-6 protein analysis was inconclusive. Donor-donor variation was observed in response to FnF treatment, whereby this phenomenon was most evident for COX-2, with three donors demonstrating a significant downregulation (0.67 ± 0.03, p = 0.003) and three donors showing upregulation (6.9 ± 5.5, p = 0.21). Co-treatment with Sparstolonin B, a TRL-2/TRL-4 antagonist, showed no statistical difference to FnF treatment alone in all tested target genes. Conclusion: Given the presence of the 30 kDa FnF in canine herniated IVDs and the proinflammatory effect of 30 kDa FnF on NP cells, we concluded that the accumulation of FnF may be involved in the pathogenesis of canine IVDD. These results correspond to the findings in humans with IVDD.

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Safety of intradiscal injection and biocompatibility of polyester amide microspheres in a canine model predisposed to intervertebral disc degeneration.

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