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). 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. 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. 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.

Degenerative disc disease, a painful pathology of the intervertebral disc (IVD), often causes disability and reduces quality of life. Although regenerative cell-based strategies have shown promise in clinical trials, none have been widely adopted clinically. Recent developments demonstrated that spheroid-based approaches might help overcome challenges associated with cell-based IVD therapies. Spheroids are three-dimensional multicellular aggregates with architecture that enables the cells to differentiate and synthesize endogenous ECM, promotes cell-ECM interactions, enhances adhesion, and protects cells from harsh conditions. Spheroids could be applied in the IVD both in scaffold-free and scaffold-based configurations, possibly providing advantages over cell suspensions. This review highlights areas of future research in spheroid-based regeneration of nucleus pulposus (NP) and annulus fibrosus (AF). We also discuss cell sources and methods for spheroid fabrication and characterization, mechanisms related to spheroid fusion, as well as enhancement of spheroid performance in the context of the IVD microenvironment.

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.

Lower back pain is a common condition with significant morbidity and economic impact. The pathophysiology is poorly understood but is in part attributable to degenerative disc disease (DDD). The healthy intervertebral disc ensures spine functionality by transferring the perceived load to the caudally adjacent vertebrae. The exposure to recurring mechanical load is mirrored in the mineralization pattern of the subchondral bone plate (SBP), where increased bone density is a sign of repetitive localized high stress. Computed tomography -osteoabsorptiometry (CT-OAM) is a technique based on conventional CT scans that displays the mineral density distribution in the SBP as a surface-color map. The objective of this study was to measure and analyze the SBP mineral density patterns of healthy lumbar intervertebral disc (IVDs) and those suffering DDD using CT-OAM densitograms. These findings should provide in vitro insight into the long-term morphological properties of the IVD and how these differ in the state of disc degeneration. The CT-data sets of spines from 17 healthy individuals and 18 patients displaying DDD in the lumbar spine were acquired. Individual vertebrae of both cohorts were 3D reconstructed, processed using image analysis software, and compared to one another. Maximum intensity projection of the subchondral mineralization provided surface densitograms of the SBP. The relative calcium concentration, the local maxima of mineralization, and a mean surface projection of level-defined SBPs were calculated from the densitogram and statistically compared. The inferior SBP, adjacent to degenerating disc, display an 18-41 % higher relative calcium concentration than their healthy counterparts. In the opposing superior SBPs the relative calcium content is significantly increased. Whereas it is reasonably consistent for L1-L3 (L1: 132 %, L2: 127 %, L3: 120 %), the increase grows in caudal direction (L4: 131 %, L5: 148 %, S1: 152 %). Furthermore, a change in the areal distribution of excessive mineralization can be differentiated between healthy and diseased motion segments. The acquired data provide in vitro proof of the mechanical and anatomical properties of the SBP in relation to the state of disc degeneration. In conjunction with the diagnostic use of CT-osteoabsorptiometry, our data provide a basis for a non-invasive and sensitive technique that correlates with disc functionality. This could be promising in various cases, from early identification of early stages of DDD, tracking disease progression, and assessing the repercussions of surgical procedures or experimental therapies.

Intervertebral disc (IVD) degeneration is the primary contributor to low back pain, the leading cause of disability worldwide. Although various triggers have been associated with IVD degeneration, its precise aetiology remains unclear. Consequently, current treatments fail to address the underlying degradative processes. Mechanical loading plays a critical role in IVD homeostasis, and aberrant mechanical stimulation has been identified as a key driver of extracellular matrix degradation in the proteoglycan-rich core of the IVD—the nucleus pulposus (NP). Elucidating the molecular mechanisms of IVD mechanotransduction could therefore be pivotal in identifying effective drug targets. However, we are lacking easy-to-use, reliable models to study IVD’s mechanobiological mechanisms in human cells. Here, we present the first mechanically active, microscale, human cell-based NP-on-a-Chip (NPoC) model that mimics the native NP microenvironment and enables controlled investigation of mechanically induced degenerative processes. Starting from primary human NP cells, we demonstrate that hypoxic culture (i.e. 2% O2) results in 3D constructs with gene expression levels of NP markers (ACAN, COL2A1, CDH2, OVOS2), and matrix composition (collagen type II and glycosaminoglycans) comparable with the native NP tissue. NPoC constructs respond to cyclic compression in an intensity- and duration-dependent manner. Physiological compression (10%) enhances glycosaminoglycan deposition, whereas hyperphysiological compression (30%), especially if prolonged in time (16 h/day), induces upregulation of inflammatory and catabolic markers (PTGS2, MMP13), matrix degradation, and increased apoptosis—thus recapitulating clinical hallmarks of NP degeneration. As a proof of concept for the platform’s perspective utility in therapeutic screening, we demonstrate that inhibition of the mechanoresponsive channel TRPV4 with GSK205 restores baseline expression levels of mechanosensitive and catabolic genes. The new NPoC is thus suitable for studying NP mechanobiology and screening mechanotransduction-targeting drugs, and it may facilitate the future discovery of disease modifying therapies for discogenic low back pain.

Der Begriff „Rückenschmerzen“ wird mit mehr als 65 Diagnosen in Verbindung gebracht. Entsprechend ist klar, dass es nicht „die“ Therapie für das Volksleiden Nummer 1 geben kann. Jeder Fall muss individuell evaluiert werden, um entsprechend einer fundierten Diagnose die richtige Therapie in die Wege zu leiten. Eine OP kommt bei Rückenschmerzpatienten nur relativ selten in Frage.