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Osteoarthritis (OA) and chronic low back pain due to degenerative (intervertebral) disc disease (DDD) are two of the major causes of disabilities worldwide, affecting hundreds of millions of people and leading to a high socioeconomic burden. Although OA occurs in synovial joints and DDD occurs in cartilaginous joints, the similarities are striking, with both joints showing commonalities in the nature of the tissues and in the degenerative processes during disease. Consequently, repair strategies for articular cartilage (AC) and nucleus pulposus (NP), the core of the intervertebral disc, in the context of OA and DDD share common aspects. One of such tissue engineering approaches is the use of injectable hydrogels for AC and NP repair. In this review, the state-of-the-art and recent developments in injectable hydrogels for repairing, restoring, and regenerating AC tissue suffering from OA and NP tissue in DDD are summarized focusing on cell-free approaches. The various biomaterial strategies exploited for repair of both tissues are compared, and the synergies that could be gained by translating experiences from one tissue to the other are identified. Impact statement Joints affected by osteoarthritis (OA) and degenerative (intervertebral) disc disease (DDD) share similarities in tissue composition and in the degenerative disease processes. This has led to the development of similar tissue engineering approaches to repair the articular cartilage (AC) and the nucleus pulposus (NP), in the context of OA and DDD, such as injectable hydrogels. In this review, recent developments in injectable hydrogels for repair of AC and NP tissues are summarized, biomaterial strategies are compared, and synergies are identified focusing on cell-free approaches. The summarized developments are expected to inspire more cross talk between both research fields.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

Low back pain related to intervertebral disk (IVD) degeneration has a major socioeconomic impact on our aging society. Therefore, stem cell therapy to activate self-repair of the IVD remains an exciting treatment strategy. In this respect, tissue-specific progenitors may play a crucial role in IVD regeneration, as these cells are perfectly adapted to this niche. Such a rare progenitor cell population residing in the nucleus pulposus (NP) (NP progenitor cells [NPPCs]) was found positive for the angiopoietin-1 receptor (Tie2+), and was demonstrated to possess self-renewal capacity and in vitro multipotency. Here, we compared three sorting protocols; that is, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and a mesh-based label-free cell sorting system (pluriSelect), with respect to cell yield, potential to form colonies (colony-forming units), and in vitro functional differentiation assays for tripotency. The aim of this study was to demonstrate the efficiency of three widespread cell sorting methods for picking rare cells (

Preclinical studies involving large animal models aim to recapitulate the clinical situation as much as possible and bridge the gap from benchtop to bedside. To date, studies investigating intervertebral disc (IVD) degeneration and regeneration in large animal models have utilized a wide spectrum of methodologies for outcome evaluation. This paper aims to consolidate available knowledge, expertise, and experience in large animal preclinical models of IVD degeneration to create a comprehensive tool box of anatomical and functional outcomes. Herein, we present a Large Animal IVD Scoring Algorithm based on three scales: macroscopic (gross morphology, imaging, and biomechanics), microscopic (histological, biochemical, and biomolecular analyses), and clinical (neurologic state, mobility, and pain). The proposed algorithm encompasses a stepwise evaluation on all three scales, including spinal pain assessment, and relevant structural and functional components of IVD health and disease. This comprehensive tool box was designed for four commonly used preclinical large animal models (dog, pig, goat, and sheep) in order to facilitate standardization and applicability. Furthermore, it is intended to facilitate comparison across studies while discerning relevant differences between species within the context of outcomes with the goal to enhance veterinary clinical relevance as well. Current major challenges in pre‐clinical large animal models for IVD regeneration are highlighted and insights into future directions that may improve the understanding of the underlying pathologies are discussed. As such, the IVD research community can deepen its exploration of the molecular, cellular, structural, and biomechanical changes that occur with IVD degeneration and regeneration, paving the path for clinically relevant therapeutic strategies., The basic tool box entailing clinical, macroscopic, and microscopic scale outcomes was developed to guide preclinical large animal studies in the field of disc‐related spinal pain. In depth understanding of the intricate relationship between molecular, cellular, structural, and biomechanical changes that occur with IVD degeneration and regeneration will help pave the path for clinically relevant therapeutic strategies in the field.

Low back pain is a leading cause of disability worldwide. Intervertebral disc (IVD) degeneration is often associated with low back pain but is sometimes asymptomatic. IVD calcification is an often overlooked disc phenotype that might have considerable clinical impact. IVD calcification is not a rare finding in ageing or in degenerative and scoliotic spinal conditions, but is often ignored and under-reported. IVD calcification may lead to stiffer IVDs and altered segmental biomechanics, more severe IVD degeneration, inflammation and low back pain. Calcification is not restricted to the IVD but is also observed in the degeneration of other cartilaginous tissues, such as joint cartilage, and is involved in the tissue inflammatory process. Furthermore, IVD calcification may also affect the vertebral endplate, leading to Modic changes (non-neoplastic subchondral vertebral bone marrow lesions) and the generation of pain. Such effects in the spine might develop in similar ways to the development of subchondral marrow lesions of the knee, which are associated with osteoarthritis-related pain. We propose that IVD calcification is a phenotypic biomarker of clinically relevant disc degeneration and endplate changes. As IVD calcification has implications for the management and prognosis of degenerative spinal changes and could affect targeted therapeutics and regenerative approaches for the spine, awareness of IVD calcification should be raised in the spine community.

Biomaterials for regeneration of the intervertebral disc must meet complex requirements conforming to biological, mechanical and clinical demands. Currently no consensus on their characterization exists. It is crucial to identify parameters and their method of characterization for accurate assessment of their potential efficacy, keeping in mind the translation towards clinical application. This review systematically analyses the characterization techniques of biomaterial systems that have been used for nucleus pulposus (NP) restoration and regeneration. Substantial differences in the approach towards assessment became evident, hindering comparisons between different materials with respect to their suitability for NP restoration and regeneration. We have analysed the current approaches and identified parameters necessary for adequate biomaterial characterization, with the clinical goal of functional restoration and biological regeneration of the NP in mind. Further, we provide guidelines and goals for their measurement. Statement of significance: Biomaterials intended for restoration of regeneration of the nucleus pulposus within the intervertebral disc must meet biological, biomechanical and clinical demands. Many materials have been investigated, but a lack of consensus on which parameters to evaluate leads to difficulties in comparing materials as well as mostly partial characterization of the materials in question. A gap between current methodology and clinically relevant and meaningful characterization is prevalent. In this article, we identify necessary methods and their implementation for complete biomaterial characterization in the context of clinical applicability. This will allow for a more unified approach to NP-biomaterials research within the field as a whole and enable comparative analysis of novel materials yet to be developed.

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.

Notochordal cells (NCs) reside in the core of the healthy disc and produce soluble factors that can stimulate nucleus pulposus cells (NPCs). These NC-derived factors may be applied in intervertebral disc regeneration for treatment of low-back pain. However, identification of the active soluble factors is challenging. Therefore a novel approach to directly use porcine NC-rich NP matrix (NCM) is introduced. We explored porcine NCM's anabolic effects on bovine NPCs harvested from caudal discs of adolescent and adult (2-2.5 vs. 4-6 year old) cows. NC-conditioned medium (NCCM) and NCM were produced from porcine NC-rich NP tissue. Bovine NPCs were cultured in alginate beads for 4 weeks in base medium (BM), NCCM, and NCM to investigate NCM's regenerative potential. Porcine NCM increased glycosaminoglycan (GAG) content of both adolescent and adult bovine NPCs. This was through increased proliferation of adolescent bovine NPCs, whereas in adult bovine NPCs, it was mostly through increased GAG production per NPC. Furthermore, adolescent bovine NPCs were cultured in BM and porcine NCM treated with interleukin (IL)-1β to investigate NCM's potential in an inflammatory environment. Addition of IL-1β enhanced IL1β and CXCL8 (IL8) gene expression, while NCM diminished IL1β gene expression. IL-1β reduced GAG and DNA content, but the addition of NCM relative to BM improved GAG and DNA content. Altogether, porcine NCM exerts bovine NPC-age dependent effects, and NCM's anabolic effect on adult NPCs is stronger compared with NCCM. Furthermore, porcine NCM induced an anabolic response of bovine NPCs in an inflammatory environment and may have anti-inflammatory properties. Therefore, NCM has potential in a regenerative therapy for disc degeneration, and warrants additional in vivo studies.

The intervertebral disc (IVD) is a highly hydrated tissue, the rich proteoglycan matrix imbibes water, enabling the disc to withstand compressive loads. During ageing and degeneration increased matrix degradation leads to dehydration and loss of function. Aquaporins (AQP) are a family of transmembrane channel proteins that selectively allow the passage of water in and out of cells and are responsible for maintaining water homeostasis in many tissues. Here, the expression of all 13 AQPs at gene and protein level was investigated in human and canine non‐degenerate and degenerate IVDs to develop an understanding of the role of AQPs during degeneration. Furthermore, in order to explore the transition of notochordal cells (NCs) towards nucleus pulposus (NP) cells, AQP expression was investigated in canine IVDs enriched in NCs to understand the role of AQPs in IVD maturation. AQP0, 1, 2, 3, 4, 5, 6, 7 and 9 were expressed at gene and protein level in both non‐degenerate and degenerate human NP tissue. AQP2 and 7 immunopositivity increased with degeneration in human NP tissue, whereas AQP4 expression decreased with degeneration in a similar way to AQP 1 and 5 shown previously. All AQP proteins that were identified in human NP tissue were also expressed in canine NP tissue. AQP2, 5, 6 and 9 were found to localise to vacuole‐like membranes and cell membranes in NC cells. In conclusion, AQPs were abundantly expressed in human and canine IVDs. The expression of many AQP isotypes potentially alludes to multi‐faceted functions related to adaption of NP cells to the conditions they encounter within their microenvironment in health and degeneration. The presence of AQPs within the IVD may suggest an adaptive role for these water channels during the development and maintenance of the healthy, mature IVD.

Objective To report the long‐term outcome of nine dogs treated for caudal cervical spondylomyelopathy (CCSM) with surgical spinal fusion. Study design Short case series. Animals Nine large‐breed dogs. Methods Medical records of dogs treated for disc‐associated CCSM (2013‐2016) were reviewed. The surgery objective was spinal distraction by implantation of a SynCage and fixation with two Unilock plates. Follow‐up included the Helsinki pain score questionnaire, neurological grading, radiography, computed tomography (CT), and micro‐CT (μCT) with subsequent histopathology (two dogs). Results Clinical follow‐up was obtained between 9 and 51 months (27.4 ± 13.4 months). The Helsinki pain score and neurological Griffith score improved (P

BACKGROUND CONTEXT Local corticosteroids have been used to relieve symptoms of chronic low back pain, although treatment effects have been shown to wear off relatively fast. Prolonging corticosteroid presence by controlled release from biomaterials may allow for longer pain relief while circumventing adverse effects such as high bolus dosages. PURPOSE The purpose of this study was to evaluate the safety and efficacy of intradiscal controlled release of triamcinolone acetonide (TAA) by poly(esteramide) microspheres in a canine degenerated intervertebral disc (IVD) model. STUDY DESIGN In a preclinical experimental large animal model, the effect of prolonged glucocorticoid exposure on disc degeneration was evaluated. METHODS Degeneration was accelerated by nucleotomy of lumbar IVDs of Beagle dogs. After 4 weeks, microspheres loaded with 8.4 µg TAA, and 0.84mg TAA were administered to the degenerated IVDs by intradiscal injection (n=6 per group). Empty microspheres (n=6) and all adjacent non-nucleotomized noninjected IVDs were included as controls (n=24). Immediately prior to TAA administration and after 12 weeks, magnetic resonance imaging was performed. Degenerative changes were evaluated by disc height index, Pfirrmann grading, T1ρ and T2 mapping values, postmortem CT scans, macroscopic and microscopic grading, and biochemical/immunohistochemical analysis of inflammation and extracellular matrix content. In addition, nerve growth factor (NGF) protein expression, a biomarker for pain, was scored in nucleus pulposus (NP) tissues. The study was funded by a research grant from Health Holland (1.3million euros = 1.5million US dollars). RESULTS Macroscopic evaluation and CT images postmortem were consistent with mild disc degeneration. Other abnormalities were not observed. Nucleotomy-induced degeneration and inflammation was mild, reflected by moderate Pfirrmann grades and PGE2 levels. Regardless of TAA dosage, local sustained delivery did not affect disc height index nor Pfirrmann grading, T1ρ and T2 mapping values, PGE2 tissue levels, collagen, GAG, and DNA content. However, the low dosage of TAA microspheres significantly reduced NGF immunopositivity in degenerated NP tissue. CONCLUSIONS This is the first in vivo application in a preclinical large animal model of a controlled release formulation of corticosteroids in mild IVD degeneration. Sustained release of TAA locally in the IVD appeared safe and reduced NGF expression, suggesting its potential applicability for pain relief, although beneficial effects were absent on tissue degeneration. CLINICAL SIGNIFICANCE The present platform seems to be promising in extending the local controlled delivery of TAA with the potency to provide long-standing analgesia in the subset of LBP patients suffering from discogenic pain.

Matrix production by nucleus pulposus (NP) cells, the cells residing in the center of the intervertebral disc, can be stimulated by growth factors. Bone morphogenetic proteins (BMPs) hold great promise. Although BMP2 and BMP7 have been used most frequently, other BMPs have also shown potential for NP regeneration. Heterodimers may be more potent than single homodimers, but it is not known whether combinations of homodimers would perform equally well. In this study, we compared BMP2, BMP4, BMP6, and BMP7, their combinations and heterodimers, for regeneration by human NP cells. The BMPs investigated induced variable matrix deposition by NP cells. BMP4 was the most potent, both in the final neotissue glysosaminoglycan content and incorporation efficiency. Heterodimers BMP2/6H and BMP2/7H were more potent than their respective homodimer combinations, but not the BMP4/7H heterodimer. The current results indicate that BMP4 might have a high potential for regeneration of the intervertebral disc. Moreover, the added value of BMP heterodimers over their respective homodimer BMP combinations depends on the BMP combination applied.

Blood vessel and neurite ingrowth into the degenerating intervertebral disc (IVD) are related to pain. In reported studies, notochordal cell (NC)‐conditioned medium (NCCM) induced a regenerative response of nucleus pulposus (NP) cells, but also inhibition of neurite and vessel formation. NC matrix (NCM) derived from NC‐rich NP tissue, induced even stronger anabolic effects than NCCM. Thus, the aim was to investigate whether NCM has similar anti‐neurogenic and ‐angiogenic properties as NCCM. NCM and NCCM where produced from porcine NC‐rich NP tissue. Human umbilical vein endothelial cells (HUVECs) were cultured in base medium (BM, 300 mOsm), NCCM (produced at 300 and 400 mOsm), NCM, or with chondroitin sulfate (CS, positive control) in angiogenesis‐inducing medium, after which vessel length was measured. Although CS alone inhibited vessel growth, NCCM (both osmolarities) stimulated vessel formation by HUVECs. NCM did not affect vessel growth relative to BM. SH‐SY5Y cells were cultured in BM, NCCM, and NCM on poly‐D‐lysine coated and polystyrene surfaces, and analyzed for neurite length and percentage of neurite expressing cells. On coated surfaces, neither NCCM nor NCM affected neurite growth. On a polystyrene surface, NCCM and NCM induced a higher number of neurite‐expressing cells. NCCM's previously reported anti‐angiogenic and ‐neurogenic effects were not observed in this study. Although addition of CS inhibited HUVEC vessel formation, other factors may be present in NCCM and NCM that affect neurite and vessel growth. Therefore, future studies testing an NC‐based regenerative strategy should carefully assess the risk of such adverse effects in an in vivo setting. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 36:3188–3195, 2018.

Chronic back and neck pain is a prevalent disability, often caused by degeneration of the intervertebral disc. Because current treatments for this condition are less than satisfactory, a great deal of effort is being applied to develop new solutions, including regenerative strategies. However, the path from initial promising idea to clinical use is fraught with many hurdles to overcome. Many of the keys to success are not necessarily linked to science or innovation. Successful translation to clinic will also rely on planning and awareness of the hurdles. It will be essential to plan your entire path to clinic from the outset and to do this with a multidisciplinary team. Take advice early on regulatory aspects and focus on generating the proof required to satisfy regulatory approval. Scientific demonstration and societal benefits are important, but translation cannot occur without involving commercial parties, which are instrumental to support expensive clinical trials. This will only be possible when intellectual property can be protected sufficiently to support a business model. In this manner, commercial, societal, medical, and scientific partners can work together to ultimately improve patient health. Based on literature surveys and experiences of the co-authors, this opinion paper presents this pathway, highlights the most prominent issues and hopefully will aid in your own translational endeavors.

Objectives Degenerative lumbosacral stenosis (DLSS) is characterized by intervertebral disc degeneration and causes lower back pain in dogs. Temporary distraction in rabbit models with induced intervertebral disc degeneration showed signs of intervertebral disc repair. In the present study, we assessed safety and efficacy of temporary segmental distraction in a dog with clinical signs of DLSS. Methods Distraction of the lumbosacral junction by pedicle screw–rod fixation was applied in a 5-year-old Greyhound with DLSS and evaluated by radiography, magnetic resonance imaging, and force plate analysis before and after distraction. Results Safe distraction of the lumbosacral junction was demonstrated, with improvement of clinical signs after removal of the distraction device. Signal intensity of the intervertebral disc showed no changes over time. T2 value was highest directly after removal of the distraction device but decreased by 10% of the preoperative value at 9 months of follow-up. Disc height decreased (8%) immediately after removal of the distraction device, but recovered to the initial value. A decrease in the pelvic/thoracic propulsive force during pedicle screw–rod fixation and distraction was demonstrated, which slowly increased by 4% compared with the initial value. Clinical significance Temporary pedicle screw–rod fixation in combination with distraction in a dog with DLSS was safe, improved clinical signs and retained disc height at 9 months of follow-up.

Since the seminal work by Hans-Jörgen Hansen in 1952, it has been assumed that intervertebral disc (IVD) degeneration in chondrodystrophic (CD) dogs involves chondroid metaplasia of the nucleus pulposus, whereas in nonchondrodystrophic (NCD) dogs, fibrous metaplasia occurs. However, more recent studies suggest that IVD degeneration in NCD and CD dogs is more similar than originally thought. Therefore, the aim of this study was to compare the histopathology of IVD degeneration in CD and NCD dogs. IVDs with various grades of degeneration (Thompson grade I–III, n = 7 per grade) from both CD and NCD dogs were used (14 CD and 18 NCD dogs, 42 IVDs in total). Sections were scored according to a histological scoring scheme for canine IVD degeneration, including evaluation of the presence of fibrocyte-like cells in the nucleus pulposus. In CD dogs, the macroscopically non-degenerated nucleus pulposus contained mainly chondrocyte-like cells, whereas the non-degenerated nucleus pulposus of NCD dogs mainly contained notochordal cells. The histopathological changes in degenerated discs were similar in CD and NCD dogs and resembled chondroid metaplasia. Fibrocytes were not seen in the nucleus pulposus, indicating that fibrous degeneration of the IVD was not present in any of the evaluated grades of degeneration. In conclusion, intervertebral disc degeneration was characterized by chondroid metaplasia of the nucleus pulposus in both NCD and CD dogs. These results revoke the generally accepted concept that NCD and CD dogs suffer from a different type of IVD degeneration, in veterinary literature often referred to as chondroid or fibroid degeneration, and we suggest that chondroid metaplasia should be used to describe the tissue changes in the IVD in both breed types.

During intervertebral disc ageing, chondrocyte-like cells (CLCs) replace notochordal cells (NCs). NCs have been shown to induce regenerative effects in CLCs. Since vesicles released by NCs may be responsible for these effects, we characterized NC-derived extracellular vesicles (EVs) and determined their effect on CLCs.EVs were purified from porcine NC-conditioned medium (NCCM) through size exclusion chromatography, ultracentrifugation or density gradient centrifugation. Additionally, the EVs were quantitatively analyzed by high-resolution flow cytometry. The effect of NCCM-derived EVs was studied on canine and human CLC micro-aggregates in vitro and compared with NCCM-derived proteins and unfractionated NCCM.Porcine NCCM contained a considerable amount of EVs. NCCM-derived EVs induced GAG deposition in canine CLCs to a comparable level as NCCM-derived proteins and unfractionated NCCM, and increased the DNA and glycosaminoglycan (GAG) content of human micro-aggregates, although to a lesser extent than unfractionated NCCM. The biological EV effects were not considerably influenced by ultracentrifugation compared with size exclusion-based purification. Upon ultracentrifugation, interfering GAGs, but not collagens, were lost. Nonetheless, collagen type I or II supplemented to CLCs in a concentration as present in NCCM induced no anabolic effects.Porcine NCCM-derived EVs exerted anabolic effects comparable to NCCM-derived proteins, while unfractionated NCCM was more potent in human CLCs. GAGs and collagens appeared not to mediate the regenerative EV effects. Thus, NC-derived EVs have regenerative potential, and their effects may be influenced by the proteins present in NCCM. The optimal combination of NC-secreted factors needs to be determined to fully exploit the regenerative potential of NC-based technology.

Introduction: Degeneration of the intervertebral disc (IVD) is a frequent cause for back pain in humans and dogs. Link-N stabilizes proteoglycan aggregates in cartilaginous tissues and exerts growth factor-like effects. The human variant of Link-N facilitates IVD regeneration in several species in vitro by inducing Smad1 signaling, but it is not clear whether this is species specific. Dogs with IVD disease could possibly benefit from Link-N treatment, but Link-N has not been tested on canine IVD cells. If Link-N appears to be effective in canines, this would facilitate translation of Link-N into the clinic using the dog as an in vivo large animal model for human IVD degeneration. Materials and methods: This study’s objective was to determine the effect of the human and canine variant of Link-N and short (s) Link-N on canine chondrocyte-like cells (CLCs) and compare this to those on already studied species, i.e. human and bovine CLCs. Extracellular matrix (ECM) production was determined by measuring glycosaminoglycan (GAG) content and histological evaluation. Additionally, the micro-aggregates’ DNA content was measured. Phosphorylated (p) Smad1 and -2 levels were determined using ELISA. Results: Human (s)Link-N induced GAG deposition in human and bovine CLCs, as expected. In contrast, canine (s)Link-N did not affect ECM production in human CLCs, while it mainly induced collagen type I and II deposition in bovine CLCs. In canine CLCs, both canine and human (s)Link-N induced negligible GAG deposition. Surprisingly, human and canine (s) Link-N did not induce Smad signaling in human and bovine CLCs. Human and canine (s) Link-N only mildly increased pSmad1 and Smad2 levels in canine CLCs. Conclusions: Human and canine (s)Link-N exerted species-specific effects on CLCs from early degenerated IVDs. Both variants, however, lacked the potency as canine IVD regeneration agent. While these studies demonstrate the challenges of translational studies in large animal models, (s)Link-N still holds a regenerative potential for humans.

Background Intervertebral 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. Results PGE2 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. Conclusions PGE2 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. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0635-6) contains supplementary material, which is available to authorized users.

Background Preceding intervertebral disc (IVD) degeneration, the cell phenotype in the nucleus pulposus (NP) shifts from notochordal cells (NCs) to chondrocyte-like cells (CLCs). Microarray analysis showed a correlation between caveolin-1 expression and the phenotypic transition of NCs to CLCs. With a clinical directive in mind, the aim of this study was to determine the role of caveolin-1 in IVD degeneration. As a scaffolding protein, caveolin-1 influences several signaling pathways, and transforming growth factor (TGF)-β receptors have been demonstrated to colocalize with caveolin-1. Therefore, the hypothesis of this study was that caveolin-1 facilitates repair by enhancing TGF-β signaling in the IVD. Methods Protein expression (caveolin-1, apoptosis, progenitor cell markers, extracellular matrix, and phosphorylated Smad2 [pSmad2]) was determined in IVDs of wild-type (WT) and caveolin-1-null mice and canine IVDs of different degeneration grades (immunofluorescence, immunohistochemistry, and TUNEL assay). Canine/human CLC microaggregates were treated with chondrogenic medium alone or in combination with caveolin-1 scaffolding domain (CSD) peptide and/or caveolin-1 silencing RNA. After 28 days, gene and protein expression profiles were determined. Results The NP of WT mice was rich in viable NCs, whereas the NP of caveolin-1-null mice contained more collagen-rich extracellular matrix and fewer cells, together with increased progenitor cell marker expression, pSmad2 TGF-β signaling, and high apoptotic activity. During canine IVD degeneration, caveolin-1 expression and apoptotic activity increased. In vitro caveolin-1 silencing decreased the CLC microaggregate glycosaminoglycan (GAG) content, which could be rescued by CSD treatment. Furthermore, CSD increased TGF-β/pSmad2 signaling at gene and protein expression levels and enhanced the anabolic effects of TGF-β1, reflected in increased extracellular matrix deposition by the CLCs. Conclusions Caveolin-1 plays a role in preservation of the NC phenotype. Additionally, it may be related to CLC apoptosis, given its increased expression in degenerated IVDs. Nevertheless, CSD enhanced CLC GAG deposition in vitro, and hence the increased caveolin-1 expression during IVD degeneration may also facilitate an ultimate attempt at repair. Further studies are needed to investigate how caveolin-1 modifies other signaling pathways and facilitates IVD repair. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0960-y) contains supplementary material, which is available to authorized users.

Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3 · 10 6 cells/mL) and NPCs + BMSCs (6 · 10 6 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10ng/mL transforming growth factor- b 1 (TGF- b 1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9 , A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.

Introduction Strategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain. Methods Canine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue. Results In vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7. Conclusions An intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.

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. © 2014 Bach et al.; licensee BioMed Central Ltd.

Chronic low back pain (CLBP) affects a large population worldwide. Intervertebral disc (IVD) degeneration is a leading case of CLBP. To date, the clinical treatments are limited to pain relief strategies, and to date, no efficient therapy was developed aiming at IVD regeneration. This thesis aimed to understand the degenerative processes of IVD and propose potential therapeutic routes to halt degeneration and achieve regeneration. Blocking inflammation is considered a viable strategy to relieve pain and delay disc degeneration. Pro-inflammatory cytokine, TNF-α, is highly associated with inflammation and pain in IVD degeneration. Therefore, in Chapter 2, a TNF-α-induced ex vivo IVD degenerative model was established on the bovine whole caudal IVD culture system under mechanical loading. This model can be used to further elucidate the mechanisms of TNF-α-induced IVD degeneration, and importantly, can serve as a model for screening of bioactives targeting TNF-a inhibition. A previous study reported that bone morphogenetic protein- 4 (BMP-4) has potent effects on NP matrix regeneration, as shown in a co-culture of human NP cells and mesenchymal stem cells. In Chapter 3, the effects of BMP-4 on IVD regeneration were evaluated in vitro and in vivo sheep models. The regenerative effects of BMP-4 on NP and AF cells were shown by improved cell proliferation and ECM production. However, no regeneration in the NP nor AF was observed in vivo. These results suggest that direct intradiscal injection of BMP-4 may not be a suitable strategy for IVD regeneration. More factors should be considered before attempting translation BMP-4 for IVD regeneration, for instance, the delivery method. To restore the function of the IVD, keeping its structural integrity is essential. Although the AF is a crucial component of IVD, this structure is often overlooked in therapy development. In Chapter 4, AF analog scaffolds were explored and evaluated in vitro and ex vivo. Our findings demonstrate that the combination of cellular, biomaterial, and bioactive strategies has excellent potential for AF regeneration. In Chapter 5, we found that CD146 is essential for the contractility of AF cells. Furthermore, we partly elucidated the underlying signaling pathway of CD146 upregulation by TGF-β in AF cells and suggest that CD146 can potentially be used as a functional marker in AF repair strategies. Intradiscal administration has been proposed as a desirable strategy for drug delivery to increase local drug concentration, prolong drug retention, and reduce systemic side effects because of the isolated nature of IVD. In Chapter 6, this study provides evidence of drug release and retention profiles in the IVD by using different methods (nuclear magnetic resonance spectroscopy, near-infrared imaging) and models (ex vivo, and in vivo on sheep and rats). Results showed that drug retention in IVD can be affected by degeneration and that a proper drug release system could prolong drug retention in IVD, which theoretically may improve treatment outcomes.

Nearly three-quarters of the human population will be affected by low back pain at some stage in their lives. While this condition is multifactorial, intervertebral disc (IVD) degeneration is one of its major causes, involved in at least 40% of chronic (low) back pain cases. Like humans, also dogs suffer from spontaneous IVD degeneration with similar characteristics. As such, within the concept of One Health, regenerative medicine can benefit both patient populations. In this respect, the dog is considered to be a suitable model for its own species and for human IVD degeneration. As no effective therapies to retard or reverse IVD degeneration have yet been devised, there is huge interest in potential regenerative treatments for both human and veterinary patients. For this reason, new cell- and growth factor-based regenerative strategies for the treatment of human and canine IVD degeneration were investigated in this thesis. In this respect, the value of studying developmental biology was demonstrated, as interesting targets were identified, e.g. caveolin-1, parathyroid hormone related peptide (PTHrP) and indian hedgehog (IHH). Not all proposed agents appeared to exert a positive effect in vitro (mesenchymal stromal cells, Link-N), and promising in vitro results did not always translate to beneficial effects in vivo (bone morphogenetic protein 7; BMP7). However, notochordal cell-based therapies induced regenerative effects across both species susceptible for IVD disease. While identifying the bioactive factors secreted by notochordal cells (e.g. extracellular vesicles) is challenging, it is valuable from a fundamental perspective and will provide innovative insights for the improvement of regenerative treatment strategies. Nonetheless, a more straightforward and promising alternative for bench to (veterinary) bedside translation is the application of notochordal cell-derived matrix (NCM), which exerted regenerative effects on IVD cells and in dogs with IVD degeneration.

Low back pain is a common type of pain restricting daily activity in man and dogs. Degeneration of the intervertebral disc (IVD), is strongly associated with back pain. Patients with late stage IVD disease and pain refractory to medication can only be surgically treated. Such surgical treatments demand an extended period of revalidation but do not lead to full recovery. Therefore, the aim of this thesis was to develop innovative treatments to regenerate early degenerated IVDs in order to treat back pain and regain disc function. First of all safety of intradiscal injection was shown in a canine model predisposed for IVD degeneration. In contrast to the regenerative effects of the growth factor rhBMP-7 in vitro in canine IVD cells, intradiscal injection in vivo in a canine model showed no regeneration of the IVD, but extensive extradiscal new bone formation. Biomaterials loaded with bioactive substances could function as local sustained release systems and thereby provide a solution to this problem. Hence, safety and biocompatibility of injectable microspheres and a thermoreversible hydrogel were investigated and demonstrated in the previously mentioned canine model. Intradiscal application of sustained release of the anti-inflammatory drug celecoxib, to decrease levels of the inflammatory mediator PGE2, seemed most beneficial in dogs with clinical signs of IVD degeneration. In canine patients with late stage IVD disease surgical stabilization of the spinal segment is indicated, and can be provided by pedicle screw-rod fixation (PSRF). A good long term clinical outcome of PSRF was described in 12 client-owned dogs with back pain due to severe degenerative lumbosacral IVD disease. Subsequently, PSRF in combination with temporary distraction, thereby unloading the IVD, was applied in a dog with mild clinical signs of back pain related to IVD degeneration. Although the dog improved from a clinical perspective, it remains to be determined whether temporary distraction of a spinal segment results in regeneration and restoration of IVD function.