Anna R. Tellegen

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.

Intradiscal application of a PCLA–PEG–PCLA hydrogel loaded with celecoxib for the treatment of back pain in canines : What's in it for humans?

Chronic low back pain is a common clinical problem in both the human and canine population. Current pharmaceutical treatment often consists of oral anti‐inflammatory drugs to alleviate pain. Novel treatments for degenerative disc disease focus on local application of sustained released drug formulations. The aim of this study was to determine safety and feasibility of intradiscal application of a poly(ε‐caprolactone‐co‐lactide)‐b‐poly(ethylene glycol)‐bpoly(ε‐caprolactone‐co‐lactide) PCLA–PEG–PCLA hydrogel releasing celecoxib, a COX‐2 inhibitor. Biocompatibility was evaluated after subcutaneous injection in mice, and safety of intradiscal injection of the hydrogel was evaluated in experimental dogs with early spontaneous intervertebral disc (IVD) degeneration. COX‐2 expression was increased in IVD samples surgically obtained from canine patients, indicating a role of COX‐2 in clinical IVD disease. Ten client‐owned dogs with chronic low back pain related to IVD degeneration received an intradiscal injection with the celecoxib‐loaded hydrogel. None of the dogs showed adverse reactions after intradiscal injection. The hydrogel did not influence magnetic resonance imaging signal at long‐term follow‐up. Clinical improvement was achieved by reduction of back pain in 9 of 10 dogs, as was shown by clinical examination and owner questionnaires. In 3 of 10 dogs, back pain recurred after 3 months. This study showed the safety and effectiveness of intradiscal injections in vivo with a thermoresponsive PCLA–PEG–PCLA hydrogel loaded with celecoxib. In this set‐up, the dog can be used as a model for the development of novel treatment modalities in both canine and human patients with chronic low back pain.

Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix : From bench towards bedside

The socioeconomic burden of chronic back pain related to intervertebral disc (IVD) disease is high and current treatments are only symptomatic. Minimally invasive strategies that promote biological IVD repair should address this unmet need. Notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) during IVD maturation and degeneration. The regenerative potential of NC-secreted substances on CLCs and mesenchymal stromal cells (MSCs) has already been demonstrated. However, identification of these substances remains elusive. Innovatively, this study exploits the regenerative NC potential by using healthy porcine NC-derived matrix (NCM) and employs the dog as a clinically relevant translational model. NCM increased the glycosaminoglycan and DNA content of human and canine CLC aggregates and facilitated chondrogenic differentiation of canine MSCs in vitro. Based on these results, NCM, MSCs and NCM+MSCs were injected in mildly (spontaneously) and moderately (induced) degenerated canine IVDs in vivo and, after six months of treatment, were analyzed. NCM injected in moderately (induced) degenerated canine IVDs exerted beneficial effects at the macroscopic and MRI level, induced collagen type II-rich extracellular matrix production, improved the disc height, and ameliorated local inflammation. MSCs exerted no (additive) effects. In conclusion, NCM induced in vivo regenerative effects on degenerated canine IVDs. NCM may, comparable to demineralized bone matrix in bone regeneration, serve as ‘instructive matrix’, by locally releasing growth factors and facilitating tissue repair. Therefore, intradiscal NCM injection could be a promising regenerative treatment for IVD disease, circumventing the cumbersome identification of bioactive NC-secreted substances.

Controlled release of celecoxib inhibits inflammation, bone cysts and osteophyte formation in a preclinical model of osteoarthritis

Abstract Major hallmarks of osteoarthritis (OA) are cartilage degeneration, inflammation and osteophyte formation. COX-2 inhibitors counteract inflammation-related pain, but their prolonged oral use entails the risk for side effects. Local and prolonged administration in biocompatible and degradable drug delivery biomaterials could offer an efficient and safe treatment for the long-term management of OA symptoms. Therefore, we evaluated the disease-modifying effects and the optimal dose of polyesteramide microspheres delivering the COX-2 inhibitor celecoxib in a rat OA model. Four weeks after OA induction by anterior cruciate ligament transection and partial medial meniscectomy, 8-week-old female rats (n = 6/group) were injected intra-articular with celecoxib-loaded microspheres at three dosages (0.03, 0.23 or 0.39 mg). Unloaded microspheres served as control. During the 16-week follow-up, static weight bearing and plasma celecoxib concentrations were monitored. Post-mortem, micro-computed tomography and knee joint histology determined progression of synovitis, osteophyte formation, subchondral bone changes, and cartilage integrity. Systemic celecoxib levels were below the detection limit 6 days upon delivery. Systemic and local adverse effects were absent. Local delivery of celecoxib reduced the formation of osteophytes, subchondral sclerosis, bone cysts and calcified loose bodies, and reduced synovial inflammation, while cartilage histology was unaffected. Even though the effects on pain could not be evualated directly in the current model, our results suggest the application of celecoxib-loaded microspheres holds promise as novel, safe and effective treatment for inflammation and pain in OA.

Intradiscal delivery of celecoxib-loaded microspheres restores intervertebral disc integrity in a preclinical canine model

ABSTRACT Low back pain, related to degeneration of the intervertebral disc (IVD), affects millions of people worldwide. Clinical studies using oral cyclooxygenase‐2 (COX‐2) inhibitors have shown beneficial effects, although side‐effects were reported. Therefore, intradiscal delivery of nonsteroidal anti‐inflammatory drugs can be an alternative treatment strategy to halt degeneration and address IVD‐related pain. In the present study, the controlled release and biologic potency of celecoxib, a selective COX‐2 inhibitor, from polyesteramide microspheres was investigated in vitro. In addition, safety and efficacy of injection of celecoxib‐loaded microspheres were evaluated in vivo in a canine IVD degeneration model. In vitro, a sustained release of celecoxib was noted for over 28 days resulting in sustained inhibition of inflammation, as indicated by decreased prostaglandin E2 (PGE2) production, and anti‐catabolic effects in nucleus pulposus (NP) cells from degenerated IVDs on qPCR. In vivo, there was no evidence of adverse effects on computed tomography and magnetic resonance imaging or macroscopic evaluation of IVDs. Local and sustained delivery of celecoxib prevented progression of IVD degeneration corroborated by MRI, histology, and measurement of NP proteoglycan content. Furthermore, it seemed to harness inflammation as indicated by decreased PGE2 tissue levels and decreased neuronal growth factor immunopositivity, providing indirect evidence that local delivery of a COX‐2 inhibitor could also address pain related to IVD degeneration. In conclusion, intradiscal controlled release of celecoxib from polyesteramide microspheres prevented progression of IVD degeneration both in vitro and in vivo. Follow‐up studies are warranted to determine the clinical efficacy of celecoxib‐loaded PEAMs in chronic back pain. Graphical abstract Figure. No caption available.

Intradiscal Injection of a Slow Release Formulation of Celecoxib for the Treatment of Dogs with Low Back Pain

Introduction Intervertebral disc (IVD) disease characterized by low back pain is common in both humans and large breed dogs. Inflammatory mediators such as prostaglandin E2 (PGE2) play a key role in IVD degeneration, causing structural changes of the IVD and low back pain. The current conservative and surgical treatment modalities do not reverse IVD degeneration and have certain drawbacks. Long term systemic administration of non-steroidal anti-inflammatory drugs can cause gastro-intestinal side effects. Decompressive surgery is effective in over 80% of the patients, but is rather costly and has a long recovery period. Injectable formulations that enable local sustained release of anti-inflammatory drugs aim at decreasing inflammation and thereby inhibiting degeneration and pain. In vitro, controlled release of celecoxib, a COX-2 inhibitor, from a thermoreversible acetyl-capped PCLA-PEG-PCLA hydrogel inhibited PGE2 production and enhanced matrix production for 28 days in 3D culture of canine nucleus pulposus cells that were subjected to the catabolic effects of TNF-α. In vivo, sustained release of celecoxib by the hydrogel for up to 60 days was shown and safely applied intradiscally in laboratory dogs with naturally occurring IVD degeneration. The aim of the present study is to report the safety and feasibility of intradiscal application in client-owned dogs with low back pain related to IVD degeneration. Material and Methods Client-owned dogs with low back pain were diagnosed by MRI with degenerative lumbosacral stenosis marked by mild to moderate IVD degeneration and protrusion. The dogs were surgical candidates but were offered intradiscal injection. The PCLA-PEG-PCLA thermogel, loaded with 0.013mg/ml celecoxib, was percutaneously injected into the nucleus pulposus of L7-S1 under fluoroscopy guidance. Follow-up consisted of clinical examination, owner questionnaires, and objective gait analysis by measurement of ground reaction forces (GRFs) using force plate analysis (FPA) at 6 weeks, and 3 and 6 months after injection. MRI was repeated after 3 months and included T2- and T1-weighted images and T2-mapping. Results Ten dogs with DLLS were injected with the celecoxib-loaded hydrogel. None of the dogs showed adverse reactions after intradiscal injection. Follow up MRI showed no worsening of the IVD degeneration. Clinical improvement was achieved by reduction of low back pain in 9/10 dogs, as was shown by clinical examination, force plate analysis and owner questionnaires. In 3/10 dogs low back pain recurred at 3 months and they subsequently underwent standard-of-care surgical treatment. Conclusion This study showed the safety and feasibility of intradiscal injections with a thermoresponsive hydrogel loaded with celecoxib. Ongoing studies concentrate on the long term clinical follow up of these patients. Future studies will determine the optimal loading dose of celecoxib for clinical efficacy. In this setup, the dog can be used as a model for the development of novel treatment modalities in both canine and human patients with chronic low back pain.

Pedicle Screw-Rod Fixation for Treatment of Lumbosacral Intervertebral Disc Degeneration in Dogs with Low Back Pain

Introduction Low back pain in nonchondrodystrophic large breed dogs is a common problem and the most common cause is degenerative lumbosacral stenosis (DLSS). DLSS is characterized by intervertebral disc (IVD) degeneration of L7-S1, disc bulging, disc herniation, spinal canal stenosis, compression of neuronal tissue (cauda equina), thickened nerve roots (neuritis), soft tissue and bony proliferations, and foraminal stenosis. This ultimately results in discogenic and neurogenic pain. DLSS in dogs maybe treated conservatively or surgically. The most common surgical treatment is decompressive dorsal laminectomy and partial discectomy but in severe degenerated IVDs or in cases with foraminal stenosis and neuritis this may not be sufficient to treat low back pain. In these cases, pedicle screw-rod fixation (PSRF) can be performed. The aim of PSRF was to increase the intervertebral foraminal space for the exiting nerves and to promote spinal fusion. Distraction of the peripheral joints is one of the most promising new techniques in allowing chondrocytes to initiate repair of damaged cartilage in osteoarthritis. IVD degeneration, such as osteoarthritis, is also characterized by a decreasing number of nucleus pulposus cells and disc matrix. PSRF allows distraction of the IVD and may initiate, in a similar fashion as in joints, repair of the disc matrix. This was investigated in a pilot study in a dog that underwent temporary distraction and follow-up with magnetic resonance imaging (MRI). The aim of the present study was to investigate the effectiveness of PSRF in dogs with severe lumbosacral disc degeneration, to assess the effect of PSRF on the intervertebral foramina and on spinal fusion and, in a pilot study, to investigate the regenerative role of temporary distraction on the degenerated lumbosacral disc. Materials and Methods Eleven dogs with confirmed diagnosis of DLSS and IVD degeneration underwent PSRF of the lumbosacral junction (Fig. 1). In 10 (out of the 11) dogs, PSRF was combined with dorsal laminectomy, disc fenestration, and various techniques to promote spinal fusion. In a subset of these patients (4 of 10 dogs), permanent distraction of the lumbosacral joint before PSRF was applied. In order to investigate the effect of temporary distraction of the degenerated lumbosacral joint, the PSRF was applied on the distracted joint for 3 months and removed thereafter. During follow-up, the dogs were monitored using questionnaires to owners and by clinical evaluation, diagnostic imaging with computed tomography and MRI, and measurement of ground reaction forces by force plate analysis. In the 4 dogs in which permanent distraction was applied, the increase in the intervertebral foramen was measured and in 1 dog the effect of temporary distraction on the disc matrix was evaluated on MRI. Results Clinical evaluation, responses of questionnaires to owners, and force plate data showed improvement or complete resolution of clinical signs after pedicle screw-rod fixation in 11 dogs during follow-up periods ranging from 5 months to 4 years. Diagnostic imaging showed no implant failures. In none of the 10 dogs was intervertebral body bone fusion of the lumbosacral joint achieved. In the dogs in which permanent distraction was applied, postoperative imaging showed significant enlargement of the intervertebral foramen between L7 and S1 (Fig. 1). In the dog with temporary distraction, MRI indicated no changes in the IVD signal 3 months after removal of the PSRF device. Fig. 1 Canine lumbosacral spine segment in a dog with degenerative lumbosacral stenosis before and after distraction plus pedicle screw-rod fixation (PSRF). Conclusion Pedicle screw-rod fixation offers a surgical treatment option for large dogs with severe degenerative lumbosacral stenosis. However, PSRF alone does not result in intervertebral body fusion between L7 and S1 and for that additional methods are necessary. Distraction and PSRF is effective in enlarging the intervertebral foramina and alleviating clinical signs. Temporary distraction alone, to initiate regeneration of the intervertebral disc matrix, did not have adverse effects and is an interesting concept for further investigation. Disclosure of Interest None declared