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OBJECTIVE: To identify the components of conditioned medium obtained from intervertebral disc nucleus pulposus-derived canine notochord cells, and to evaluate the capacity of such factors to affect disc-derived chondrocyte gene expression of aggrecan, versican, and hyaluronic acid synthase 2 (HAS-2) as a function of culture conditions. METHODS: Canine notochord cells obtained from nonchondrodystrophic dogs were cultured within alginate beads under conditions of serum deficiency (Dulbecco's modified Eagle's medium [DMEM]) to produce notochord cell-conditioned medium (NCCM). NCCM was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectroscopy. Bovine disc-derived chondrocytes were cultured with serum-deficient medium (DMEM) and NCCM and assayed for the effect of tissue culture conditions on aggrecan, versican, and HAS-2 gene expression. Next, chondrocyte gene expression for aggrecan was evaluated using DMEM containing recombinant connective tissue growth factor (rCTGF), and the results compared with those obtained using NCCM and DMEM. RESULTS: NCCM contained aggrecan, Cu/Zn superoxide dismutase, fibronectin, and CTGF precursor. Culture with NCCM caused an up-regulation of aggrecan, versican, and HAS-2 gene expression. NCCM induced aggrecan gene expression in chondrocytes at a level similar to that induced by 100-200 ng/ml rCTGF. Nonchondrodystrophic and chondrodystrophic canine notochord cells exhibited similar levels of CTGF gene expression. CONCLUSION: Nucleus pulposus-derived notochord cells secrete CTGF (CCN2), a recently discovered multifunctional growth factor. There is no difference between CTGF gene expression in nonchondrodystrophic and chondrodystrophic canine notochord cells, suggesting a possible role of CTGF as an anabolic factor within the disc nucleus that is, to at least some degree, dependent on the population of notochord cells within the disc nucleus. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare no conflict interest., (© 2023 The Authors. JOR Spine published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Background: Tissue sources of pain emanating from degenerative discs remains incompletely understood. Canine intervertebral discs (IVDs) were needle puncture injured, 4-weeks later injected with either phosphate-buffered saline (PBS) or NTG-101, harvested after an additional fourteen weeks and then histologically evaluated for the expression of NGFr, BDNF, TrkB and CALCRL proteins. Quantification was performed using the HALO automated cell-counting scoring platform. Immunohistochemical analysis was also performed on human IVD tissue samples obtained from spinal surgery. Immunohistochemical analysis and quantification of neurotrophins and neuropeptides was performed using an in vivo canine model of degenerative disc disease and human degenerative disc tissue sections. Discs injected with NTG-101 showed significantly lower levels of Nerve Growth Factor receptor (NGFr/TrkA, p = 0.0001), BDNF ( p = 0.009), TrkB ( p = 0.002) and CALCRL ( p = 0.008) relative to PBS injections. Human IVD tissue obtained from spinal surgery due to painful DDD show robust expression of NGFr, BDNF, TrkB and CALCRL proteins. A single intradiscal injection of NTG-101 significantly inhibits the expression of NGFr, BDNF, TrkB and CALCRL proteins in degenerative canine IVDs. These results strongly suggest that NTG-101 inhibits the development of neurotrophins that are strongly associated with painful degenerative disc disease and may have profound effects upon the management of patients living with discogenic pain.

Numerous publications over the past 22 years, beginning with a seminal paper by Aguiar et al., have demonstrated the ability of notochordal cell-secreted factors to confer anabolic effects upon intervertebral disc (IVD) cells. Since this seminal paper, other scientific publications have demonstrated that notochordal cells secrete soluble factors that can induce anti-inflammatory, pro-anabolic and anti-cell death effects upon IVD nucleus pulposus (NP) cells in vitro and in vivo, direct human bone marrow-derived mesenchymal stem cells toward an IVD NP-like phenotype and repel neurite ingrowth. More recently these factors have been characterized, identified, and used therapeutically to induce repair upon injured IVDs in small and large pre-clinical animal models. Further, notochordal cell-rich IVD NPs maintain a stable, healthy extracellular matrix whereas notochordal cell-deficient IVDs result in a biomechanically and extracellular matrix defective phenotype. Collectively this accumulating body of evidence indicates that the notochordal cell, the cellular originator of the intervertebral disc holds vital instructional cues to establish, maintain and possibly regenerate the intervertebral disc.

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., (© 2021. The Author(s).)

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.

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-β1) 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-β1 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-1β, 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.

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.

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 TGFβ- 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 TGFβ1 and CTGF as major hubs in protein interaction networks. In vitro treatment with TGFβ1 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 TGFβ1 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 TGFβ1 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.

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.

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-1β/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.

Introduction: In the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP)., Methods: We used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines., Results: We detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion., Conclusions: The CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

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.

In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from Thermotoga maritima in complex with a truncated tRNA. The RNA is mainly bound by the N-terminal ferredoxin-like domain (NFLD) and the THUMP domain of one subunit within the ThiI homo-dimer thereby positioning the U8 close to the catalytic center in the pyrophosphatase domain of the other subunit. The recognition of the 3'-CCA end by the THUMP domain yields a molecular ruler defining the specificity for U8 thiolation. This first structure of a THUMP/NFLD-RNA complex might serve as paradigm for the RNA recognition by THUMP domains of other proteins. The ternary ThiI-RNA-ATP complex shows no significant structural changes due to adenosine triphosphate (ATP) binding, but two different states of active site loops are observed independent of the nucleotide loading state. Thereby conformational changes of the active site are coupled with conformational changes of the bound RNA. The ThiI-RNA complex structures indicate that full-length tRNA has to adopt a non-canonical conformation upon binding to ThiI., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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.

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.

Introduction Effective therapies that may stop or even reverse disc degeneration remain elusive. A minimally invasive method through which nucleus pulposus (NP) cell viability could be achieved would revolutionize the treatment of degenerative disc disease (DDD). With the presented work, we have investigated if nonchondrodystrophic (NCD) canine intervertebral disc (IVD)-derived notochordal cell conditioned medium (NCCM) and chondrodystrophic (CD) canine IVD-derived conditioned medium (CDCM) are able to protect murine and human NP cells from apoptosis. Materials and Methods We developed NCCM and CDCM from hypoxic culture of freshly isolated NPs from NCD and CD canines, respectively. We obtained murine NP cells from nine different C57BL/6 mice and human NP cells from four patients who underwent surgery for discectomy. The cells were cultured with ADMEM/F-12 (control media), NCCM, or CDCM under hypoxic conditions (3.5% O2) and treated with IL-1β + FasL or Etoposide. All media were supplemented with 2% fetal bovine serum. We then determined the expression of specific apoptotic pathways in the murine and human NP cells by recording activated caspase-8, caspase-9, and caspase-3/7 activity. Results In the murine NP cells, NCCM inhibits IL-1β + FasL- and Etoposide-mediated apoptosis via suppression of activated caspase-9 and caspase-3/7, CDCM demonstrated an inhibitory effect on IL-1β + FasL-mediated apoptosis via caspase-3/7 (Fig. 1A). In the human NP cells, NCCM inhibits Etoposide- mediated apoptosis via suppression of activated caspase-8, caspase-9, and mainly caspase-3/7. CDCM demonstrated an inhibitory effect on Etoposide-mediated apoptosis via suppression of activated caspase-8, caspase-9, and mainly caspase-3/7, though not as effective as NCCM (Fig. 1B). Conclusion IL-1β + FasL are known key molecules in the progression of DDD. Here, we demonstrate that soluble factors secreted by the NCD IVD NP strongly protect murine NP cells not only from IL-1β + FasL but also from Etoposide-induced apoptosis via suppression of activated caspase-9 and caspase-3/7. In the human samples, addition of IL-1β + FasL did not increase cell death. Because the human cell samples were obtained from herniated discs that are probably already undergoing a degenerative process, it is likely that there was already some degree of activation by the endogenously secreted prodegenerative factors such as IL-1β + FasL. It may be that the NP cells, once they have reached a pivotal point of the degenerative cascade, no longer respond to exogenously applied IL-1β + FasL in contrast to the otherwise "healthy" discs obtained from the mice. Interestingly, the rescue effect of NCCM in the etoposide-treated cells (murine and human) suggests that NCCM is capable of influencing the signaling pathways known to be relevant to etoposide-induced cell death. A better understanding and harnessing of the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies for the treatment of DDD.

The intervertebral disk (IVD) is a fascinating and resilient tissue compartment given the myriad of functions that it performs as well as its unique anatomy. The IVD must tolerate immense loads, protect the spinal cord, and contribute considerable flexibility and strength to the spinal column. In addition, as a consequence of its anatomical and physiological configuration, a unique characteristic of the IVD is that it also provides a barrier to metastatic disease. However, when injured and/or the subject of significant degenerative change, the IVD can be the source of substantial pain and disability. Considerable efforts have been made over the past several decades with respect to regenerating or at least modulating degenerative changes affecting the IVD through the use of many biological agents such as growth factors, hydrogels, and the use of plant sterols and even spices common to Ayurvedic medicine. More recently stem/progenitor and autologous chondrocytes have been used mostly in animal models of disk disease but also a few trials involving humans. At the end of the day if biological therapies are to offer benefit to the patient, the outcomes must be improved function and/or less pain and also must be improvements upon measures that are already in clinical practice. Here some of the challenges posed by the degenerative IVD and a summary of some of the regenerative attempts both in vitro and in vivo are discussed within the context of the vital question: "Who is the patient?"

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.

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.

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.

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 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.

Study Design: Non-chondrodystrophic dog notochord cell conditioned medium was used to evaluate chondrocyte proteoglycan production and cell proliferation., Objectives: To evaluate the responsiveness of bovine disc-derived chondrocytes to notochord-cell conditioned medium with respect to proteoglycan and cell proliferation. In addition, to examine phenotypic changes of notochord cells cultured in monolayered as compared to 3-dimensional culture., Summary of Background Data: Non-chondrodystrophic dogs maintain their intervertebral disc notochord cells into adulthood and are protected from having degenerative disc disease develop. The chondrodystrophic breeds such as beagles do not preserve these cells and have disc disease develop much earlier in life. The role of the notochord cell within the disc nucleus is poorly understood., Methods: Canine notochord cells were cultured within alginate beads in serum-deficient conditions using Dulbecco modified Eagle medium to produce notochord cell conditioned medium (NCCM). NCCM was used to stimulate bovine disc chondrocytes from which we evaluated proteoglycan production and cell proliferation as compared to chondrocytes grown in DMEM alone. In addition, parallel cultures of notochord cells were seeded within alginate beads as well as in monolayer and cultured in order to examine for differences in phenotype between the 2 culture conditions., Results: The morphologic aspects of the intervertebral disc between the species differed markedly. A dose- dependent relationship was seen between proteoglycan production and NCCM concentration across various concentrations of NCCM in repeated experiments. Although there was a 4-fold increase in cell proliferation under all NCCM concentrations, this increase in cell proliferation was not dose dependent in the concentrations tested. Unlike chondrocytes, notochord cells do not adhere to tissue culture plate (monolayer) until at least day 4-6, do not markedly alter their phenotype, and rapidly assume masses of cells while floating within tissue culture medium., Conclusions: The biology of the disc-derived chondrocyte is profoundly affected by NCCM in that various concentrations of NCCM activate proteoglycan production in a dose-dependent fashion. However, in the doses tested in our study, cell proliferation was increased but in a nondose-dependent fashion. Notochord cells retain their phenotype even in monolayer and through the development of floating intimately associated masses of cells suggest the development and maintenance of cell-cell interaction. These masses of cells are retained even after 6 days in culture when they do attach to the tissue plate surface. The persistence of notochord cells in non-chondrodystrophic dog species suggests that these in vitro studies may mirror the milieu of the disc in vivo, in which the notochord cell may play a key role in disc homeostasis.

The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s4U. In this work, we show through deletion analysis of unmodified E. coli tRNA(Phe) that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the TpsiC primary sequence is not a recognition element. An unmodified tRNA(Phe) transcript in which the 3'-terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3' terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s4U modification is limited in the cell to tRNA.

Previous work has shown a progressive, age-related loss of knee joint innervation in the C57BL6Nia mouse. We did three experiments to describe further the loss and determine whether it might contribute to the development of knee osteoarthrosis in this model. Immunocytochemistry showed that the percentage of neurons expressing substance P and calcitonin gene-related peptide increased with age, indicating a relatively selective loss of mechanoreceptors. Histological examination of knee joints of mice at various ages showed that loss of joint innervation always preceded histological changes of cartilage degeneration. The mice usually developed a mild form of osteoarthrosis, but surgical ablation of joint innervation caused the development of severe patellofemoral osteoarthrosis. The findings are consistent with the hypothesis that an age-related loss of joint innervation may contribute to the development of osteoarthrosis.

Background: The diagnosis of pain in the upper back, shoulder, chest, and arm is often made with considerable confusion and may be accompanied by needless expense and suffering by the patient. Despite the paucity of evidence concerning the tissues and mechanisms responsible for interscapular and atypical chest pain or "pseudo-angina," practitioners of manual therapy maintain that manipulation of the costovertebral elements and associated soft tissues may be helpful in the treatment of these painful conditions., Objective: We have examined the costovertebral complex in humans with respect to the presence of immune-like reactivity to neurofilament protein and the neuropeptide substance P and calcitonin gene-related peptide, markers that reveal the presence of axons in peripheral tissues., Design: Human costovertebral complexes obtained at autopsy were processed with standard histologic examination and immunocytochemical methods to detect the presence of neurofilaments, substance P, and calcitonin gene-related peptide., Main Outcome Measures: Outcomes were descriptive and did not require statistical methods., Results: All costovertebral joints contained innervation within the anterior capsule and synovial tissues. In 4 separate cases, the costovertebral joints contained large intraarticular synovial inclusions or "meniscoids" found to contain small bundles of axons with immune-like reactivity to substance P. Axon bundles were identified in serial section with monoclonal antibodies to neurofilaments as well as with urea-silver nitrate staining., Conclusions: The costovertebral joint has been considered a candidate for producing back pain and/or pseudo-angina that may be ameliorated by spinal manipulation. This study has demonstrated that the costovertebral joint has the requisite innervation for pain production in a similar manner to other joints of the spinal column.

Benzylic organozinc reagents generated by insertion of zinc metal into benzyl-bromine bonds react with alkenyl(phenyl)iodonium triflates to provide single stereoisomers of trisubstituted olefins. The extremely high reactivity of the phenyliodonio moiety allows these reactions to be performed in the absence of copper salts or palladium catalysts. The reaction is performed from -40 degrees C to room temperature in THF. Excellent yields of the desired cross-coupled products have been obtained despite the occurrence of a competing electron-transfer-induced fragmentation.