Should you adjust that herniated disc? Thoughts from a chiropractor/molecular scientist.

Low back pain accounts for the most years lost to disability of any malady worldwide but most cases of disc herniation (DH) and degenerative disc disease (DDD) resolve with conservative methods. Numerous tissue sources of pain affecting the degenerative/herniated disc have been identified, with changes secondary to the influence of inflammation figuring prominently among them. Due to the proven linkage of inflammation to the pain and progression of disc degeneration, anti-inflammatory/anti-catabolic and pro-anabolic repair strategies are gaining prominence for novel therapeutic approaches. Current treatments include conservative therapies such as modified rest, exercise, anti-inflammatory treatments, and analgesics. There is no accepted proposed mechanism of action to support the direct role of spinal manipulation for the treatment of the degenerative and/or herniated disc. However, there are published accounts of very serious adverse events accompanying such treatments leading to the question; 'should a patient with suspected painful IVD be treated with manipulation?

Les douleurs lombaires sont responsables du plus grand nombre d'années perdues pour cause d'invalidité, toutes pathologies confondues, mais la plupart des cas de hernie discale (HD) et de discopathie dégénérative (DD) sont résolus par des méthodes conventionnelles. De nombreuses sources tissulaires de douleur affectant le disque dégénératif/herniaire ont été identifiées, les changements secondaires à l'influence de l'inflammation figurant en bonne place parmi elles. En raison du lien avéré entre l'inflammation et la douleur et la progression de la dégénérescence discale, les stratégies de réparation anti-inflammatoires/anticataboliques et pro-anaboliques sont de plus en plus utilisées comme nouvelles approches thérapeutiques. Les traitements actuels comprennent des thérapies conventionnelles telles que le « repos modifié ¼, l'exercice, les traitements anti-inflammatoires et les analgésiques. Il n'existe pas de mécanisme d'action proposé et accepté pour soutenir le rôle direct de la manipulation de la colonne vertébrale dans le traitement de la dégénérescence et/ou de la hernie discale. Toutefois, des comptes rendus publiés font état d'effets indésirables très graves liés à ces traitements, ce qui amène à se poser la question suivante : « Un patient soupçonné de souffrir d'une discopathie dégénérative douloureuse doit-il être traité par manipulation? ¼.

An update on animal models of intervertebral disc degeneration and low back pain: Exploring the potential of artificial intelligence to improve research analysis and development of prospective therapeutics.

Animal models have been invaluable in the identification of molecular events occurring in and contributing to intervertebral disc (IVD) degeneration and important therapeutic targets have been identified. Some outstanding animal models (murine, ovine, chondrodystrophoid canine) have been identified with their own strengths and weaknesses. The llama/alpaca, horse and kangaroo have emerged as new large species for IVD studies, and only time will tell if they will surpass the utility of existing models. The complexity of IVD degeneration poses difficulties in the selection of the most appropriate molecular target of many potential candidates, to focus on in the formulation of strategies to effect disc repair and regeneration. It may well be that many therapeutic objectives should be targeted simultaneously to effect a favorable outcome in human IVD degeneration. Use of animal models in isolation will not allow resolution of this complex issue and a paradigm shift and adoption of new methodologies is required to provide the next step forward in the determination of an effective repairative strategy for the IVD. AI has improved the accuracy and assessment of spinal imaging supporting clinical diagnostics and research efforts to better understand IVD degeneration and its treatment. Implementation of AI in the evaluation of histology data has improved the usefulness of a popular murine IVD model and could also be used in an ovine histopathological grading scheme that has been used to quantify degenerative IVD changes and stem cell mediated regeneration. These models are also attractive candidates for the evaluation of novel anti-oxidant compounds that counter inflammatory conditions in degenerate IVDs and promote IVD regeneration. Some of these compounds also have pain-relieving properties. AI has facilitated development of facial recognition pain assessment in animal IVD models offering the possibility of correlating the potential pain alleviating properties of some of these compounds with IVD regeneration.

A comparative study of mesenchymal stem cell transplantation and NTG-101 molecular therapy to treat degenerative disc disease.

Cellular replacement therapy using mesenchymal stem cells (MSCs) and/or the delivery of growth factors are at the forefront of minimally invasive biological treatment options for Degenerative Disc Disease (DDD). In this study, we compared the therapeutic potential of a novel drug candidate, NTG-101 to MSCs, including rat cartilage derived stem cells (rCDSCs), bone marrow stem cells (rBMSCs) and human Umbilical Cord Derived Mesenchymal Stem Cells (hUCMSCs) for the treatment of DDD. We induced DDD using a validated image-guided needle puncture injury in rat-tail IVDs. Ten weeks post-injury, animals were randomized and injected with MSCs, NTG-101 or vehicle. At the end of the study, histological analysis of the IVD-Nucleus Pulposus (NPs) injected with NTG-101 or rCDSCs showed a healthy or mild degenerative phenotype in comparison to vehicle controls. Immunohistochemical analysis revealed strong expression of aggrecan, collagen 2, brachyury and Oct4 in IVD-NPs injected with NTG-101. Our results also demonstrated suppression of inflammation induced p38 and NFκB resulting in inhibition of catabolic genes, but activation of Smad-2/3, Erk-1/2 and Akt-dependent signaling inducing anabolic genes in IVD-NP on treatment with NTG-101. In conclusion, a single injection of NTG-101 into the degenerative disc demonstrated superior benefits compared to stem cell transplantation.

Injectable Biologics for the Treatment of Degenerative Disc Disease.

PURPOSE OF REVIEW: Spinal pain and associated disability is a leading cause of morbidity worldwide that has a strong association with degenerative disc disease (DDD). Biologically based therapies to treat DDD face significant challenges posed by the unique milieu of the environment within the intervertebral disc, and many promising therapies are in the early stages of development. Patient selection, reasonable therapeutic goals, approach, and timing will need to be discerned to successfully translate potential therapeutics. This review provides a brief overview of the status of intradiscal biologic therapies. RECENT FINDINGS: Proposed systemic delivery of therapeutic agents has not progressed very much in large part due to the risk of adverse events in remote tissues plus the very limited vascular supply and therefore questionable delivery to the intervertebral disc nucleus pulposus. Intradiscal delivery of therapeutic proteins shows good potential for clinical trials and translation with encouraging results from large animal pre-clinical studies plus an enhanced understanding of the biology of DDD. There are a few cell-based therapies currently under pre-clinical and clinical trial investigation; however, these attempts continue to be hampered by unknown if any, mechanism of action, no downstream detection of transplanted cells, mixed results concerning efficacy, small sample numbers, and a lack of objective evidence of pain mediation. Treatment of DDD using biologically based therapeutics is a widely sought-after goal; however, potential therapies need to address pain and disability in larger, well-controlled studies.

NTG-101: A Novel Molecular Therapy that Halts the Progression of Degenerative Disc Disease.

The tremendous cost, pain and disability associated with degenerative disc disease (DDD) makes the development of a biological agent that can mitigate the course of DDD, a critical unmet need. We have identified and reported that a single injection of a combination of recombinant human (rh) Transforming growth factor beta 1 (TGF-ß1) and Connective tissue growth factor (CTGF) proteins into the injured intervertebral disc (IVD) nucleus pulposus (NP) can mediate DDD in a pre-clinical rodent model. In this study, we developed and evaluated the efficacy of a novel molecular therapy (NTG-101) containing rhTGF-ß1 and rhCTGF proteins suspended in an excipient solution using in vivo models of DDD including rat-tail and chondrodystrophic (CD) canines. Needle puncture injury in CD-canine NPs resulted in loss of hydration, disc height and showed radiographic evidence of DDD like humans. However, NTG-101-injected IVDs maintained disc height and demonstrated retention of viscoelastic properties as compared to IVDs injected with phosphate buffer saline (PBS, 1X, pH = 7.2). In addition, a single intra-discal injection of NTG-101 into the injured IVD-NPs resulted in sustained expression of healthy extra-cellular matrix (ECM) proteins (aggrecan, collagen 2A1) and reduced expression of inflammation associated proteins and molecules (IL-1ß, IL-6, IL-8, MMP-13, Cox-2 and PGE2) as compared to vehicle controls. In conclusion, we demonstrated that a single intra-discal injection of the novel formulation, NTG-101 confers a robust anti-inflammatory, anti-catabolic and pro-anabolic effects in pre-clinical models of DDD thereby restoring homeostasis. These findings suggest the therapeutic potential of NTG-101 for clinical use.

Molecular Therapy for Degenerative Disc Disease: Clues from Secretome Analysis of the Notochordal Cell-Rich Nucleus Pulposus.

Degenerative disc disease (DDD) is associated with spinal pain often leading to long-term disability. However, the non-chondrodystrophic canine intervertebral disc is protected from the development of DDD, ostensibly due to its retention of notochordal cells (NC) in the nucleus pulposus (NP). In this study, we hypothesized that secretome analysis of the NC-rich NP will lead to the identification of key proteins that delay the onset of DDD. Using mass-spectrometry, we identified 303 proteins including components of TGFß- and Wnt-signaling, anti-angiogeneic factors and proteins that inhibit axonal ingrowth in the bioactive fractions of serum free, notochordal cell derived conditioned medium (NCCM). Ingenuity Pathway Analysis revealed TGFß1 and CTGF as major hubs in protein interaction networks. In vitro treatment with TGFß1 and CTGF promoted the synthesis of healthy extra-cellular matrix proteins, increased cell proliferation and reduced cell death in human degenerative disc NP cells. A single intra-discal injection of recombinant TGFß1 and CTGF proteins in a pre-clinical rat-tail disc injury model restored the NC and stem cell rich NP. In conclusion, we demonstrate the potential of TGFß1 and CTGF to mitigate the progression of disc degeneration and the potential use of these molecules in a molecular therapy to treat the degenerative disc.

Bedside to bench and back to bedside: Translational implications of targeted intervertebral disc therapeutics.

Spinal pain and associated disability is a leading cause of morbidity worldwide that has a strong association with degenerative disc disease (DDD). DDD can begin in early-late adolescence and has a variable course. Biologically based therapies to treat DDD face significant challenges posed by the unique milieu of the environment within the intervertebral discs. Many potential promising therapies are still in the early stages of development with a hostile microenvironment within the disc presenting unique challenges. The translational potential of this article: Patient selection, reasonable therapeutic goals, approach, and timing will need to be discerned in order to successfully translate potential therapeutics.

A commentary on the implications of medication prescription rights for the chiropractic profession.

There is a growing desire within the chiropractic profession to expand the scope of practice to include limited medication prescription rights for the treatment of spine-related and other musculoskeletal conditions. Such prescribing rights have been successfully incorporated into a number of chiropractic jurisdictions worldwide. If limited to a musculoskeletal scope, medication prescription rights have the potential to change the present role of chiropractors within the healthcare system by paving the way for practitioners to become comprehensive specialists in the conservative management of spine / musculoskeletal disorders. However, if the chiropractic profession wishes to lobby to expand the scope of practice to include limited prescriptive authority, several issues must first be addressed. These would include changes to chiropractic education and legislation, as well as consideration of how such privileges could impact the chiropractic profession on a more theoretical basis. In this commentary, we examine the arguments in favour of and against limited medication prescription rights for chiropractors and discuss the implications of such privileges for the profession.

Notochordal cell conditioned medium (NCCM) regenerates end-stage human osteoarthritic articular chondrocytes and promotes a healthy phenotype.

BACKGROUND: Notochordal cell conditioned medium (NCCM) derived from non-chondrodystrophic dogs has pro-anabolic and anti-catabolic effects upon nucleus pulposus (NP) cells. Here, for the first time, we assessed the ability of NCCM to influence the production of extracellular matrix and inflammatory proteins by healthy and osteoarthritic human chondrocytes within engineered cartilage tissues. We hypothesized that, similar to its action on NP cells, NCCM exerts metabolic and anti-catabolic effects on human articular chondrocytes and has the potential to significantly counteract inflammatory mediators. METHODS: Chondrocytes from nine non-osteoarthritic patients and from six osteoarthritic (OA) donors at the time of total knee arthroplasty were chondro-differentiated in pellets for 2 weeks. Non-OA pellets were exposed for 72 hours to IL-1ß/TNF-α and then cultured up to 14 days in 2 % FBS-supplemented NCCM or 2 % FBS-supplemented medium (control (ctr)). OA pellets were cultured in NCCM or ctr medium without pro-inflammatory treatment. Tissues after each culture phase were analyzed biochemically (GAG/DNA), (immuno-) histologically (collagen I, II and GAG) and by Western blotting. Supernatants were analyzed by ELISA. RESULTS: Response to NCCM was age and disease dependent with healthy chondrocyte pellets (from donors >55 years of age) recovering their glycosaminoglycan (GAG) contents to baseline levels only with NCCM. OA pellets treated with NCCM significantly increased GAG content (1.8-fold) and levels of hyaluronic acid link protein (HAPLN), fibromodulin and SOX-9. The catabolic proteins (matrix metalloproteinase (MMP)-3 and MMP-13) and pro-inflammatory enzyme levels (cyclooxygenase-2 (COX-2)) were markedly reduced and there was significantly reduced secretion of pro-inflammatory chemokines (IL-6 and IL-8). CONCLUSIONS: NCCM restores cartilage matrix production of end-stage human OA chondrocytes towards a healthy phenotype and suppresses the production of inflammatory mediators. Harnessing the necessary and sufficient factors within NCCM that confers chondroprotection and regenerative effects could lead to a minimally invasive agent for treatment of degenerative and inflammatory joint diseases.

The cellular and molecular biology of the intervertebral disc: A clinician's primer.

Clinicians routinely encounter patients suffering from both degenerative and acute spinal pain, often as a consequence of pathology affecting the intervertebral disc (IVD). The IVD is a complex structure essential to spinal function and is subject to degenerative disease and injury. However, due to the complexity of spinal pain syndromes it is often difficult to determine the extent of the IVD's contribution to the genesis of spinal pain. The location of the IVD is within close proximity to vital neural elements and may in the event of pathological change or injury compromise those structures. It is therefore important that clinicians performing manual therapy understand the cellular and molecular biology of the IVD as well as its clinical manifestation of degeneration/injury in order to safely manage and appreciate the role played by the disc in the development of mechanical spinal pain syndromes.

Les cliniciens voient régulièrement des patients souffrant de douleurs vertébrales à la fois dégénératives et aiguës, souvent une conséquence d'une pathologie affectant le disque intervertébral (DIV). Le DIV est une structure complexe essentielle à la fonction rachidienne et peut être touché par des maladies dégénératives et des blessures. Toutefois, en raison de la complexité des syndromes de douleurs vertébrales, il est souvent difficile de déterminer la part de contribution du DIV à la genèse de cette douleur. L'emplacement du DIV est à proximité d'éléments neuronaux vitaux et peut, en cas de changement pathologique ou d'une blessure, compromettre ces structures. Il est donc important que les cliniciens administrant une thérapie manuelle comprennent la biologie cellulaire et moléculaire du DIV ainsi que la manifestation clinique de la dégénérescence et des blessures de celui-ci, afin de gérer en toute sécurité et d'apprécier le rôle joué par le disque dans le développement des syndromes de douleurs vertébrales mécaniques.

Chiropractors as Primary Spine Care Providers: precedents and essential measures.

Chiropractors have the potential to address a substantial portion of spinal disorders; however the utilization rate of chiropractic services has remained low and largely unchanged for decades. Other health care professions such as podiatry/chiropody, physiotherapy and naturopathy have successfully gained public and professional trust, increases in scope of practice and distinct niche positions within mainstream health care. Due to the overwhelming burden of spine care upon the health care system, the establishment of a 'primary spine care provider' may be a worthwhile niche position to create for society's needs. Chiropractors could fulfill this role, but not without first reviewing and improving its approach to the management of spinal disorders. Such changes have already been achieved by the chiropractic profession in Switzerland, Denmark, and New Mexico, whose examples may serve as important templates for renewal here in Canada.

Les chiropraticiens ont la possibilité de traiter une partie importante des affections vertébrales, mais le taux d'utilisation des services de chiropratique est resté faible et largement inchangé depuis des décennies. D'autres professions de la santé telles que la podologie, la physiothérapie et la naturopathie ont réussi à gagner la confiance du public et des professionnels, améliorer leur champ de pratique et développer des créneaux distincts au sein des systèmes de soins de santé dominants. En raison de la lourde charge que placent les soins de la colonne vertébrale sur le système de soins de santé, la création d'un « prestataire de soins primaires de la colonne vertébrale ¼ pourrait combler un créneau utile pour satisfaire les besoins de la société. Les chiropraticiens pourraient assumer ce rôle, mais non sans avoir d'abord examiné et amélioré leur approche de la gestion des affections vertébrales. Ces transformations ont déjà été réalisées par la profession chiropratique en Suisse, au Danemark et au Nouveau-Mexique. Leurs exemples peuvent servir de modèles importants pour la réforme au Canada.

Pathophysiology and natural history of cervical spondylotic myelopathy.

STUDY DESIGN: This study is a combination of narrative and systematic review. OBJECTIVE: Clinicians who deal with cervical spondylotic myelopathy (CSM) should be up-to-date with the emerging knowledge related to the cascade of pathobiological secondary events that take place under chronic cervical spinal cord compression. Moreover, by performing a systematic review, we aim to (1) describe the natural history and (2) determine potential risk factors that affect the progression of CSM. SUMMARY OF BACKGROUND DATA: The pathophysiology, natural history, as well as the factors associated with clinical deterioration have not been fully described in CSM. METHODS: For the first part of the study, a literature review was performed. To answer key questions 1 and 2 of the second goal, a systematic search was conducted in PubMed and the Cochrane Collaboration Library for articles published between January 1, 1956, and November 7, 2012. We included all articles that described the progression and outcomes of CSM for which no surgical intervention was given. RESULTS: By performing a narrative literature review, we found that the assumption that acute traumatic spinal cord injury and CSM share a similar series of cellular and molecular secondary injury events was made in the past. However, recent advances in basic research have shown that the chronic mechanical compression results in secondary injury mechanisms that have distinct characteristics regarding the nature and the temporal profile compared with those of spinal cord injury. For the purpose of the systematic review, 10 studies yielding 16 publications met inclusion criteria for key questions 2 and 3. Moderate-strength evidence related to the natural history of CSM suggests that 20% to 60% of patients will deteriorate neurologically over time without surgical intervention. Finally, there is low-strength evidence indicating that the area of circumferential compression is associated with deteriorating neurological symptoms. CONCLUSION: CSM has unique pathobiological mechanisms that mainly remain unexplored. Although the natural history of CSM can be mixed, surgical intervention eliminates the chances of the neurological deterioration. EVIDENCE-BASED CLINICAL RECOMMENDATIONS: RECOMMENDATION: Evidence concerning the natural history of CSM suggests that 20% to 60% of patients will deteriorate neurologically over time without surgical intervention. Therefore, we recommend that patients with mild CSM be counseled regarding the natural history of CSM and have the option of surgical decompression explained. OVERALL STRENGTH OF EVIDENCE: Moderate. STRENGTH OF RECOMMENDATION: Strong. SUMMARY STATEMENTS: Chronic compression of the spinal cord results in progressive neural cell loss related to secondary mechanisms including apoptosis, neuroinflammation, and vascular disruption.

Nonoperative management of cervical myelopathy: a systematic review.

STUDY DESIGN: Systematic review. OBJECTIVE: To conduct a systematic review investigating the evidence of (1) efficacy, effectiveness, and safety of nonoperative treatment of patients with cervical myelopathy; (2) whether the severity of myelopathy affects outcomes of nonoperative treatment; and (3) whether specific activities or minor injuries are associated with neurological deterioration in patients with myelopathy or asymptomatic stenosis being treated nonoperatively. SUMMARY OF BACKGROUND DATA: Little is known about the appropriate role of nonoperative treatment in the management of cervical myelopathy, which is typically considered a surgical disorder. METHODS: A systematic search was conducted in PubMed and the Cochrane Collaboration Library for articles published between January 1, 1956, and November 20, 2012. We included all articles that compared nonoperative treatments or observation with surgery for patients with cervical myelopathy or asymptomatic cervical cord compression to determine their effects on clinical outcomes, including myelopathy scales (Japanese Orthopaedic Association, Nurick), general health scores (36-Item Short Form Health Survey), and pain (neck and arm). Nonoperative treatments included physical therapy, medications, injections, orthoses, and traction. We also searched for articles evaluating the effect of specific activities or minor trauma in neurological outcomes. Case reports and studies with less than 10 patients in the exposure group were excluded. RESULTS: Of 54 citations identified from our search, 5 studies reported in 6 articles met inclusion criteria. In 1 randomized controlled study, there was low evidence that nonoperative treatment may yield equivalent or better outcomes than surgery in those with mild myelopathy. For moderate to severe myelopathy, nonoperative treatment had inferior outcomes versus surgery in 2 cohort studies, despite the fact that surgically treated patients were worse at baseline. There was insufficient evidence to determine whether specific activities or minor trauma is a risk factor for neurological deterioration in those with myelopathy or asymptomatic cord compression. CONCLUSION: There is a paucity of evidence for nonoperative treatment of cervical myelopathy, and further studies are needed to determine its role more definitively. In particular, for the patient with milder degrees of myelopathy, randomized studies comparing nonoperative with surgical treatment would be particularly helpful, as would trials comparing specific types of nonoperative treatments with the natural history of myelopathy. EVIDENCE-BASED CLINICAL RECOMMENDATIONS: RECOMMENDATION 1: Because myelopathy is known to be a typically progressive disorder and there is little evidence that nonoperative treatment halts or reverses its progression, we recommend not routinely prescribing nonoperative treatment as the primary modality in patients with moderate to severe myelopathy. OVERALL STRENGTH OF EVIDENCE: Low. STRENGTH OF RECOMMENDATION: Strong. RECOMMENDATION 2: If there is a role for nonoperative treatment as a primary treatment modality, it may be in the patient with mild myelopathy. However, it is not clear which specific forms of nonoperative treatment provide any benefit compared with the natural history. If nonoperative treatment is selected, we suggest care be taken to observe for neurological deterioration. OVERALL STRENGTH OF EVIDENCE: Low. STRENGTH OF RECOMMENDATION: Weak. RECOMMENDATION 3: In those with asymptomatic spondylotic cord compression but no clinical myelopathy, the available literature neither supports nor refutes the notion that minor trauma is a risk factor for neurological deterioration. We suggest that patients should be counseled about this uncertainty. OVERALL STRENGTH OF EVIDENCE: Low. STRENGTH OF RECOMMENDATION: Weak. Recommendation 4: In those with a clinical diagnosis of cervical spondylotic myelopathy but no ossification of the posterior longitudinal ligament, the available studies did not specifically address the issue of neurological deterioration secondary to minor trauma. However, in those with underlying ossification of the posterior longitudinal ligament, trauma may be more likely to cause worsening of existing myelopathy or even initiate symptoms in those who were previously asymptomatic. We suggest that patients should be counseled about these possibilities. OVERALL STRENGTH OF EVIDENCE: Low. STRENGTH OF RECOMMENDATION: Weak.

Intervertebral disc-derived stem cells: implications for regenerative medicine and neural repair.

STUDY DESIGN: An in vitro and in vivo evaluation of intervertebral disc (IVD)-derived stem/progenitor cells. OBJECTIVE: To determine the chondrogenic, adipogenic, osteogenic, and neurogenic differentiation capacity of disc-derived stem/progenitor cells in vitro and neurogenic differentiation in vivo. SUMMARY OF BACKGROUND DATA: Tissue repair strategies require a source of appropriate cells that could be used to replace dead or damaged cells and tissues such as stem cells. Here we examined the potential use of IVD-derived stem cells in regenerative medicine approaches and neural repair. METHODS: Nonchondrodystrophic canine IVD nucleus pulposus (NP) cells were used to generate stem/progenitor cells (NP progenitor cells [NPPCs]) and the NPPCs were differentiated in vitro into chondrogenic, adipogenic, and neurogenic lineages and in vivo into the neurogenic lineage. NPPCs were compared with bone marrow-derived mesenchymal (stromal) stem cells in terms of the expression of stemness genes. The expression of the neural crest marker protein 0 and the Brachyury gene were evaluated in NP cells and NPPCs. RESULTS: NPPCs contain stem/progenitor cells and express "stemness" genes such as Sox2, Oct3/4, Nanog, CD133, Nestin, and neural cell adhesion molecule but differ from mesenchymal (stromal) stem cells in the higher expression of the Nanog gene by NPPCs. NPPCs do not express protein 0 or the Brachyury gene both of which are expressed by the totality of IVD NP cells. The percentage of NPPCs within the IVD is 1% of the total as derived by colony-forming assay. NPPCs are capable of differentiating along chondrogenic, adipogenic, and neurogenic lineages in vitro and into oligodendrocyte, neuron, and astroglial specific precursor cells in vivo within the compact myelin-deficient shiverer mouse. CONCLUSION: We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.

Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration.

INTRODUCTION: The relative resistance of non-chondrodystrophic (NCD) canines to degenerative disc disease (DDD) may be due to a combination of anabolic and anti-catabolic factors secreted by notochordal cells within the intervertebral disc (IVD) nucleus pulposus (NP). Factors known to induce DDD include interleukin-1 beta (IL-1ß) and/or Fas-Ligand (Fas-L). Therefore we evaluated the ability of notochordal cell conditioned medium (NCCM) to protect NP cells from IL-1ß and IL-1ß +FasL-mediated cell death and degeneration. METHODS: We cultured bovine NP cells with IL-1ß or IL-1ß+FasL under hypoxic serum-free conditions (3.5% O2) and treated the cells with either serum-free NCCM or basal medium (Advanced DMEM/F-12). We used flow cytometry to evaluate cell death and real-time (RT-)PCR to determine the gene expression of aggrecan, collagen 2, and link protein, mediators of matrix degradation ADAMTS-4 and MMP3, the matrix protection molecule TIMP1, the cluster of differentiation (CD)44 receptor, the inflammatory cytokine IL-6 and Ank. We then determined the expression of specific apoptotic pathways in bovine NP cells by characterizing the expression of activated caspases-3, -8 and -9 in the presence of IL-1ß+FasL when cultured with NCCM, conditioned medium obtained using bovine NP cells (BCCM), and basal medium all supplemented with 2% FBS. RESULTS: NCCM inhibits bovine NP cell death and apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1ß and IL-1ß+Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen type 2, CD44, link protein and TIMP-1) and down-regulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4, which encodes a protein for aggrecan remodeling, is increased. NCCM also protects against IL-1+FasL-mediated down-regulation of Ank expression. Furthermore, NP cells treated with NCCM in the presence of IL-1ß+Fas-L down-regulate the expression of IL-6 by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells. CONCLUSIONS: Notochordal cell-secreted factors suppress NP cell death by inhibition of activated caspase-9 and -3/7 activity and by up-regulating genes contributing anabolic activity and matrix protection of the IVD NP. Harnessing the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies in the treatment of DDD.

Aberrant axial mineralization precedes spinal ankylosis: a molecular imaging study in ank/ank mice.

INTRODUCTION: The diagnosis of ankylosing spondylitis is made from a combination of clinical features and the presence of radiographic evidence that may be detected only after many years of inflammatory back pain. It is not uncommon to have a diagnosis confirmed 5 to 10 years after the initial onset of symptoms. Development of a more-sensitive molecular imaging technology to detect structural changes in the joints would lead to earlier diagnosis and quantitative tracking of ankylosis progression. Progressive ankylosis (ank/ank) mice have a loss of function in the Ank gene, which codes for a regulator of PPi transport. In this study, we used these ank/ank mutant mice to assess a noninvasive, quantitative measure of joint ankylosis with near-infrared (NIR) molecular imaging in vivo. METHODS: Three age groups (8, 12, and 18 weeks) of ank/ank (15 mice) and wild-type littermates (12 +/+ mice) were assessed histologically and radiographically. Before imaging, OsteoSense 750 (bisphosphonate pamidronate) was injected i.v. Whole-body images were analyzed by using the multispectral Maestro imaging system. RESULTS: OsteoSense 750 signals in the paw joints were higher in ank/ank mice in all three age groups compared with controls. In the spine, significantly higher OsteoSense 750 signals were detected early, in 8-week-old ank/ank mice compared with controls, although minimal radiographic differences were noted at this time point. The molecular imaging changes in the ank/ank spine (8 weeks) were supported by histologic changes, including calcium apatite crystals at the edge of the vertebral bodies and new syndesmophyte formation. CONCLUSIONS: Changes in joint pathology of ank/ank mice, as evaluated by histologic and radiographic means, are qualitative, but only semiquantitative. In contrast, molecular imaging provides a quantitative assessment. Ankylosis in ank/ank mice developed simultaneously in distal and axial joints, contrary to the previous notion that it is a centripetal process. NIR imaging might be feasible for early disease diagnosis and for monitoring disease progression in ankylosing spondylitis.

The enigma that is the nucleus pulposus cell: the search goes on.

The development of an effective treatment for degenerative disc disease has been hampered for many years by what seems a fundamental problem; what exactly defines a nucleus pulposus (NP) cell? The paper by Gilson and colleagues elegantly re-opens the debate concerning the lineage and identity of NP cells that are alike yet different from chondrocytes. As we pursue novel investigations and treatment strategies for degenerative disc disease, how do we isolate these unique cells and what is the role of the primordial notochordal cell that may well linger within the NP far longer and perhaps in a different phenotypic appearance than previously thought? The paper by Gilson and colleagues that is the subject of the present editorial presents compelling data concerning the heterogeneity of the cells of the NP, and their origin, development, maturation and function.

The regenerative capacity of the notochordal cell: tissue constructs generated in vitro under hypoxic conditions.

OBJECT: The intervertebral disc (IVD) is a highly avascular structure that is occupied by highly specialized cells (nucleus pulposus [NP] cells) that have adapted to survive within an O(2) concentration of 2-5%. The object of this study was to investigate the effects of long-term hypoxic and normoxic tissue cultures of nonchondrodystrophic canine notochordal cells-cells that appear to protect the disc NP from degenerative change. METHODS: The authors obtained notochordal cells from nonchondrodystrophic canines according to their established methods and placed them into monolayer and 3D culture using sodium alginate globules under either hypoxic (3.5% O(2)) or normoxic (21% O(2)) conditions. Histological, immunohistochemical, scanning electron microscopy, and histomorphometric methods were used to evaluate the cells within the globules after 5 months in culture. RESULTS: Notochordal cells under in vitro hypoxic tissue culture conditions produced a highly complex, organized, 3D cellular construct that was strikingly similar to that observed in vivo. In contrast, traditional normoxic tissue culture conditions resulted in notochordal cells that failed to produce an organized matrix. Hypoxia resulted in a matrix rich in aggrecan and collagen II, whereas normoxic cultured cells did not produce any observable aggrecan or collagen II after 5 months of culture. CONCLUSIONS: Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition-robust aggrecan, and type II collagen production. Normoxic tissue culture conditions, however, lead to a failure of these cells to thrive and a lack of extracellular matrix production and significantly smaller cells. The authors suggest that future studies of NP cells and, in particular, notochordal cells should utilize hypoxic tissue culture conditions to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions.

The Notochord, Notochordal cell and CTGF/CCN-2: ongoing activity from development through maturation.

The growth regulating factor CTGF/CCN-2 is an integral factor in growth and development, connective tissue maintenance, wound repair and cell cycle regulation. It has recently been reported that CTGF/CCN-2 is involved in very early development having been detected in early notochord formation in zebrafish using CTGF/CCN-2 promoter-driven green fluorescent protein (GFP) plasmids. In these studies fluorescence was detected early in the developing embryos, a finding of considerable significance in that CTGF/CCN-2 deficient mutant mice die early after birth due to severe cartilage and skeletal dysplasia and respiratory failure. Such findings confirm the importance of CTGF/CCN-2 in development and of the necessary and sufficient role of this molecule in formation of the skeleton, extracellular matrix and chondrogenesis. Of particular relevance to the relationship between the notochordal cell and CTGF/CCN-2 there is a remarkable sub-species of canine, the 'non-chondrodystrophic' canine that is protected from developing degenerative disc disease (DDD). These animals are unique in that they preserve the population of notochordal cells within their disc nucleus (NP) and these cells secrete CTGF/CCN-2. We have detected CTGF/CCN-2 within conditioned medium developed from the notochordal cells of these animals (NCCM) and used this conditioned medium to demonstrate robustly increased proteoglycan production. The addition of recombinant human CTGF/CCN-2 to totally serum-free media containing cultures of bovine NP cells replicated the robustly increased aggrecan gene expression found with NCCM alone strongly suggesting the importance of the effect of CTGF/CCN-2 in notochordal cell biology within the disc nucleus of non-chondrodystrophic canines. The chondrodystrophic canine, another sub-species on the other hand are almost totally devoid of notochordal cells and they develop DDD profoundly and early. These two sub-species of canine reflect a naturally occurring animal model that is an excellent example of differential notochordal cell survival and possible associated developmental differences in extracellular maintenance.