Disc cell clusters in pathological human intervertebral discs are associated with increased stress protein immunostaining

Intervertebral disc (IVD) cells within the annulus fibrosus (AF) and nucleus pulposus (NP) maintain distinct functional extracellular matrices and operate within a potentially noxious and stressful environment. How disc cells respond to stress and whether stress is responsible for triggering degeneration is unknown. Disc cell proliferation and cluster formation are most marked in degenerate IVDs, possibly indicating attempts at matrix repair. In other tissues, stress proteins increase rapidly after stress protecting cell function and, although implicated in degeneration of articular cartilage, have received little attention in degenerative IVD pathologies. We have compared the distribution of stress protein immunolocalization in pathological and control IVDs. Disc tissues were obtained at surgery from 43 patients with degenerative disc disease (DDD) and herniation, and 12 controls at postmortem. Tissues were immunostained with a polyclonal antibody for heat shock factor 1 (HSF-1) and monoclonal antibodies for the heat shock proteins, Hsp27 and Hsp72, using an indirect immunoperoxidase method. Positively stained cells were expressed as a percentage of the total. Cell cluster formation was also assessed. The proportion of cells in clusters was similar in the AF (both 2%) and NP (8 and 9%) of control and DDD samples, whereas in herniated tissues this was increased (AF 12%, NP 14%). Stress antigen staining tended to be more frequent in clustered rather than in single/doublet cells, and this was significant (P < 0.005) in both the AF and NP of herniated discs. Clustered cells, which are most common in herniated discs, may be mounting a protective response to abnormal environmental factors associated with disc degeneration. A better understanding of the stress response in IVD cells may allow its utilization in disc cell therapies.     

Nucleus pulposus degeneration alters properties of resident progenitor cells

Background contextThe intervertebral disc (IVD) possesses a minimal capability for self-repair and regeneration. Changes in the differentiation of resident progenitor cells can represent diminished tissue regeneration and a loss of homeostasis. We previously showed that progenitor cells reside in the nucleus pulposus (NP). The effect of the degenerative process on these cells remains unclear.PurposeWe sought to explore the effect of IVD degeneration on the abundance of resident progenitor cells in the NP, their differentiation potential, and their ability to give rise to NP-like cells. We hypothesize that disc degeneration affects those properties.Study designNucleus pulposus cells derived from healthy and degenerated discs were methodically compared for proliferation, differentiation potential, and ability to generate NP-like cells.MethodsIntervertebral disc degeneration was induced in 10 skeletally, mature mini pigs using annular injury approach. Degeneration was induced in three target discs, whereas intact adjacent discs served as controls. The disc degeneration was monitored using magnetic resonance imaging for 6 to 8 weeks. After there was a clear evidence of degeneration, we isolated and compared cells from degenerated discs (D-NP cells [NP-derived cells from porcine degenerated discs]) with cells isolated from healthy discs (H-NP cells) obtained from the same animal.ResultsThe comparison showed that D-NP cells had a significantly higher colony-forming unit rate and a higher proliferation rate in vitro. Our data also indicate that although both cell types are able to differentiate into mesenchymal lineages, H-NP cells exhibit significantly greater differentiation toward the chondrogenic lineage and NP-like cells than D-NP cells, displaying greater production of glycosaminoglycans and higher gene expression of aggrecan and collagen IIa.ConclusionsBased on these findings, we conclude that IVD degeneration has a meaningful effect on the cells in the NP. D-NP cells clearly go through the regenerative process; however, this process is not powerful enough to facilitate full regeneration of the disc and reverse the degenerative course. These findings facilitate deeper understanding of the IVD degeneration process and trigger further studies that will contribute to development of novel therapies for IVD degeneration.     

Alpha-smooth muscle actin in pathological human disc nucleus pulposus cells in vivo and in vitro

That a contractile actin isoform has been found in cells of other cartilage tissues in healing and disease states prompted this investigation of the presence of alpha-smooth muscle actin (alpha-SMA) in pathological human intervertebral disc tissue. The presence of this isoform has been reported in human intervertebral disc specimens obtained at autopsy from subjects for whom there were no reported symptoms. An objective of this study was to evaluate the cell density and percentage of alpha-SMA-containing cells in pathological nucleus pulposus tissue obtained from lumbar disc surgery from 17 patients. Additionally, explants of nucleus pulposus material were cultured to determine how alpha-SMA expression changed with time in vitro. Seventy-six 5-mm diameter explants (approximately 2 mm thick) pooled from six lumbar surgeries were cultured for 1, 2, 4, or 6 weeks. Microtomed sections of paraffin-embedded specimens were stained with hematoxylin and eosin or a monoclonal antibody to alpha-SMA. Histologically, cells were categorized as to alpha-SMA phenotype (positive or negative), and the areal cell density was determined. The evaluation of the cultured nucleus pulposus explants also included documentation of the percentage of cells that were round or elongated and the percentage of the cells that were part of a group (group: >/= 2 cells). Every nucleus pulposus section exhibited the presence of alpha-SMA-containing cells, which accounted for approximately 24 percent of the cells in vivo. In vivo, the cell density was significantly higher in older individuals (p = 0.02). The average time for cell outgrowth from the explants was 8.6 days. Approximately 10-15 percent of the cells in the explants stained positive for alpha-SMA. The time in culture had no significant effect on any of the outcome measures except the percentage of alpha-SMA-containing cells that were round (p = 0.008), with values decreasing through 4 weeks and then slightly rising at 6 weeks. The role of alpha-SMA in intervertebral disc pathology warrants further investigation.     

Intervertebral disc and stem cells cocultured in biomimetic extracellular matrix stimulated by cyclic compression in perfusion bioreactor

Background contextIntervertebral disc (IVD) degeneration often causes back pain. Current treatments for disc degeneration, including both surgical and nonsurgical approaches, tend to compromise the disc movement and cannot fully restore functions of the IVD. Instead, cell-based IVD tissue engineering seems promising as an ultimate therapy for IVD degeneration.PurposeTo tissue-engineer an IVD ex vivo as a biological substitute to replace degenerative IVD.Study designAn extracellular matrix (ECM) structure-mimetic scaffold, cocultured human IVD cells and human mesenchymal stem cells (hMSCs), and mechanical stimulation were used to biofabricate a tissue-engineered IVD.MethodsAn optimal ratio of human annulus fibrosus (hAF) cells to hMSCs for AF generation within aligned nanofibers, and that of human nucleus pulposus (hNP) cells to hMSCs for NP generation within hydrogels were first determined after comparing different coculture ratios of hAF or hNP cells to hMSCs. Nanofibrous strips seeded with cocultured hAF cells/hMSCs were constructed into multilayer concentric rings, enclosing an inner core of hydrogel seeded with hNP cells/hMSCs. A piece of nonwoven nanofibrous mat seeded with hMSC-derived osteoblasts was assembled on the top of the cellular nanofiber/hydrogel assembly, as an interface layer between the cartilagenous end plate and vertebral body. The final assembled construct was then maintained in an osteochondral cocktail medium and stimulated with compressive loading to further enhance the hAF and hNP cells differentiation and increase the IVD ECM production.ResultsAmong all cocultured groups, hAF cells and hMSCs in the ratio of 2:1 cultured in nanofibers showed the closest mRNA expression levels of AF-related markers to positive control hAF cells, whereas hNP cells and hMSCs in the ratio of 1:2 cultured in hydrogels showed the closest expression levels of NP-related markers to positive control hNP cells. The effects of compressive loading on chondrogenesis of hAF or hNP cell and hMSC coculture were dependent on the scaffold structure; the expression of cartilage-related markers in AF nanofibers was downregulated, whereas that in NP hydrogel was upregulated. Interestingly, we found that hMSC-derived osteogenic cells in the interface layer were turned into chondrogenic lineage cells, with decreased expression of osteogenic markers and increased expression of chondrogenic markers.ConclusionsWe demonstrate a unique approach using a biomimetic scaffold, IVD and stem cell coculture, and mechanical stimulation to tissue-engineer a biological IVD substitute. The results show that our approach provides both favorable physical and chemical cues through cell-matrix and cell-cell interactions and mechanobiological induction to enhance IVD generation ex vivo. Our findings may lead to viable tissue engineering applications of generating a functional biological IVD for the treatment of disc degeneration.     

Abundance of calpain and aggrecan-cleavage products of calpain in degenerated human intervertebral discs

Objective

To assess the expression of calpains and calpain-induced aggrecan fragmentation in early and advanced stages of degeneration of human intervertebral discs (IVDs).

Design

Disc tissue samples of 55 patients (mean age, 51.2 ± 22.3 years) who underwent intervertebral fusion were divided into groups with early and advanced degeneration based on the Thompson magnetic resonance imaging (MRI) scale. In advanced degeneration group, five patients (mean age, 35.5 ± 11.4 years) of lumbar disc herniation (LDH) were included. Protein levels of m- and μ-calpains and their inhibitor calpastatin were assayed, and immunohistochemical techniques were used to localize and quantify the production of the enzymes. To investigate calpain activity, we assayed purified aggrecan fragmentation in disc tissue by Western blotting and immunohistochemistry with VPGVA antibody, which recognizes the m-calpain generated neo-epitope GVA.

Results

Discs at early stages of degeneration expressed low levels of m- and μ-calpains and calpastatin, and few cells expressed degenerative enzymes. At more advanced stages of degeneration, the expression and number of cells immunopositive for m-calpain, μ-calpain and calpastatin were significantly higher. Further finding showed that anti-GVA-reactive aggrecan fragments were significantly higher in discs at advanced compared with early stages of degeneration. Herniated disc samples showed stronger expression and more cells immunopositive for calpains, calpastatin and GVA in the nucleus pulposus than in the annulus fibrous.

Conclusions

The expression of calpains, together with m-calpain-induced degradation products of extracellular matrix, was correlated with the degree of disc degeneration in human IVD tissue. These findings suggest that calpains may be involved in IVD degeneration via proteoglycan (PG) cleavage.     

The effects of recombinant human bone morphogenetic protein-2, recombinant human bone morphogenetic protein-12, and adenoviral bone morphogenetic protein-12 on matrix synthesis in human annulus fibrosis and nucleus pulposus cells

BACKGROUND CONTEXT: Bone morphogenetic proteins (BMPs) are potential therapeutic factors for degenerative discs, and BMP-12 does not have the osteogenic potential of BMP-2, making it better suited for intradiscal injection. However, no reports have compared the actions of BMP-2 and -12 on human annulus fibrosus (AF) and nucleus pulposus (NP) cells nor evaluated adenoviral-mediated gene therapy in human AF cells. PURPOSE: To evaluate and compare the effects of recombinant human (rh) BMP-2, rhBMP-12, and adenoviral BMP-12 (Ad-BMP-12) on nucleus pulposus and annulus fibrosis cell matrix protein synthesis. STUDY DESIGN: In vitro study using rhBMP-2 and -12 and adenoviral BMP-12 with human intervertebral disc (IVD) cells. METHODS: Human NP and AF IVD cells were isolated, maintained in monolayer, and incubated with BMP-2 or -12 for 2 days. AF and NP cells were transduced with Ad-BMP-12, pellets formed, and incubated for 6 days. Growth factor-treated cells were labelled with either 35-S or 3H-proline to assay matrix protein synthesis. RESULTS: rhBMP-2 increased NP proteoglycan, collagen, and noncollagen protein synthesis to 355%, 388%, and 234% of control. RhBMP-12 increased the same NP matrix proteins' synthesis to 140%, 143%, and 160% of control. Effects on AF matrix protein synthesis were minimal. Ad-BMP-12 significantly increased matrix protein synthesis and DNA content of AF and NP cells in pellet culture. NP synthesis of all matrix proteins and AF synthesis of proteoglycans was increased when the data were normalized to pellet DNA. AF synthesis of noncollagen protein and collagen was not modulated by Ad-BMP-12 if the data are normalized to pellet DNA content. CONCLUSIONS: Both rhBMP-2 and -12 increase human NP cell matrix protein synthesis while having minimal effects on AF cells. However, Ad-BMP-12 did increase matrix protein synthesis in both NP and AF cells, making it a potential therapy for enhancing matrix production in the IVD. These responses plus the proliferative action of Ad-BMP-12 seen in the current studies, and the lack of an osteogenic action noted in other studies justifies future studies to determine if gene therapy with BMP-12 could provide protective and/or reparative actions in degenerating discs.     

Histological and reference system for the analysis of mouse intervertebral disc

A new scoring system based on histo‐morphology of mouse intervertebral disc (IVD) was established to assess changes in different mouse models of IVD degeneration and repair. IVDs from mouse strains of different ages, transgenic mice, or models of artificially induced IVD degeneration were assessed. Morphological features consistently observed in normal, and early/later stages of degeneration were categorized into a scoring system focused on nucleus pulposus (NP) and annulus fibrosus (AF) changes. “Normal NP” exhibited a highly cellularized cell mass that decreased with natural ageing and in disc degeneration. “Normal AF” consisted of distinct concentric lamellar structures, which was disrupted in severe degeneration. NP/AF clefts indicated more severe changes. Consistent scores were obtained between experienced and new users. Altogether, our scoring system effectively differentiated IVD changes in various strains of wild‐type and genetically modified mice and in induced models of IVD degeneration, and is applicable from the post‐natal stage to the aged mouse. This scoring tool and reference resource addresses a pressing need in the field for studying IVD changes and cross‐study comparisons in mice, and facilitates a means to normalize mouse IVD assessment between different laboratories. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:233–243, 2018.     

NF-κB inhibitor,NEMO-binding domain peptide attenuates intervertebral disc degeneration

BACKGROUND CONTEXTNonphysiological mechanical loading and inflammation are both critically involved in intervertebral disc (IVD) degeneration, which is characterized by an increase in cytokines and matrix metalloproteases (MMPs) in the nucleus pulposus (NP). This process is known to be mediated by the NF-κB pathway.CLINICAL SIGNIFICANCECurrent clinical treatments for IVD degeneration focus on the alleviation of symptoms rather than targeting the underlying mechanism. Injection of an NF-κB inhibitor may attenuate the progression of IVD degeneration.PURPOSETo investigate the ability of the NF-κB inhibitor, NEMO binding domain peptide (NBD), to alter IVD degeneration processes by reducing IL-1β- and mechanically-induced cytokine and MMP levels in human nucleus pulposus cells in vitro, and by attenuating IVD degeneration in an in vivo rat model for disc degeneration.STUDY DESIGNExperimental in vitro and animal model.PATIENT SAMPLEDiscarded specimens of lumbar disc from 21 patients, and 12 Sprague Dawley rats.OUTCOME MEASURESGene and protein expression, cell viability, µMRI and histology.METHODSIL-1β-prestimulated human nucleus pulposus cells embedded into fibrin constructs were loaded in the Flexcell FX-5000 compression system at 5 kPa and 1 Hz for 48 hours in the presence and absence of NBD. Unloaded hNPC/fibrin constructs served as controls. Cell viability in loaded and unloaded constructs was quantified, and gene and protein expression levels determined. For in vivo testing, a rat needle disc puncture model was employed. Experimental groups included injured discs with and without NBD injection and uninjured controls. Levels of disc degeneration were determined via µMRI, qPCR and histology. Funding sources include $48,874 NASS Young Investigator Research Grant and $119,174 NIH 5K01AR071512-02. There were no applicable financial relationships or conflicts of interest.RESULTSMechanical compression of hNPC/fibrin constructs resulted in upregulation of MMP-3 and IL-8. Supplementation of media with 10 μM NBD during loading increased cell viability, and decreased MMP-3 gene and protein levels. IVD injury in rat resulted in an increase in MMP-3, IL-1β and IL-6 gene expression. Injections of 250 µg of NBD during disc injury resulted in decreased IL-6 gene expression. µMRI analysis demonstrated a reduction of disc hydration in response to disc needle injury, which was attenuated in NBD-treated IVDs. Histological evaluation showed NP and AF lesion in injured discs, which was attenuated by NBD injection.CONCLUSIONSThe results of this study show NBD peptide's capacity to reduce IL-1β- and loading-induced MMP-3 levels in hNPC/fibrin constructs while increasing the cells’ viability, and to attenuate IVD degeneration in rat, involving downregulation of IL-6. Therefore, NBD may be a potential therapeutic agent to treat IVD degeneration.     

Increased cell senescence is associated with decreased cell proliferation in vivo in the degenerating human annulus

Background contextDuring disc degeneration, there is a well-recognized loss of cells. This puts the remaining cell population at high risk for any further decrease in cell function or cell numbers. Cell senescence has recently been shown to be present in the aging/degenerating human disc. Senescent cell are viable, metabolically active, persist, and accumulate over time, but cannot divide. Little is known about the relationship between renewal of the disc cell population via cell proliferation and disc cell senescence.PurposeTo determine the percentage of senescent cells and proliferating cells in the human annulus in vivo.Study design/settingHuman annulus specimens were obtained from surgical subjects and control donors in a study approved by the authors' Human Subjects Institutional Review Board.Patient sampleOne Thompson Grade I disc, 4 Grade II discs, 9 Grade III discs, and 12 Grade IV discs were studied.Outcome measuresThe percentages of senescent cells and the percentage of proliferating cells.MethodsImmunohistochemistry was used to detect senescent cells using an antisenescence-associated beta-galactosidase antibody, and an antiproliferation antibody (Ki67). An average of 410 cells/specimens was counted to determine the percent senescence, and an average of 229 cells was counted to determine the percent proliferation.ResultsCell proliferation was low in both surgical and control normal donor annulus tissue (4.09%+1.77 (26), mean+SD (n)). There was no significant difference in the percentage of proliferating cells for more degenerate discs versus healthier discs (4.7%+1.6 (21) for Grades III and IV vs. 5.3%+1.9 (5) for Grades I and II). More degenerated Grades III and IV discs contained significantly greater percentages of senescent annulus cells than did the healthier Grades I and II discs (44.4%+20.0 (21) vs. 18.8%+11.0 (5), respectively; p=.011). A significant negative correlation was present between the percentage of senescent cells versus the percentage of proliferating cells, r=?0.013, p=.013. No correlation was present between age and the percentage of senescent cells or age and the percentage of proliferating cells.ConclusionsBecause senescent cells cannot divide, senescence may reduce the disc's ability to generate new cells to replace cells lost to necrosis or apoptosis. Senescent cells also accumulate in the disc over time, such that their metabolic patterns may contribute to the pathologic changes seen in degenerating discs. Novel data presented here show a significant negative correlation between the percentage of senescent cells and the percentage of proliferating cells during disc degeneration. Molecular work is underway in our lab to help us determine whether senescent cells in the disc secrete factors that can result in decreased proliferation in neighboring cells.     

Intradiscal injections of osteogenic protein-1 restore the viscoelastic properties of degenerated intervertebral discs

BACKGROUND CONTEXT: Using biochemical, histological, and radiological parameters in a rabbit model of intervertebral disc (IVD) degeneration, the intradiscal injection of a growth factor, such as osteogenic protein-1 (OP-1), has been shown to regenerate the IVD. However, very little is known about how such a biological therapeutic approach affects the biomechanical properties of the degenerated IVD. PURPOSE: To investigate the effects of an intradiscal injection of OP-1 on the biomechanical properties of IVDs in the rabbit annular-puncture disc degeneration model and to determine their relationship to biochemical properties. STUDY DESIGN/SETTING: In vivo study on the effects of intradiscally administered OP-1 on the biomechanical and biochemical properties of IVDs in the rabbit annular-puncture disc degeneration model. METHODS: New Zealand White rabbits (n=16) underwent annulus fibrosus (AF) puncture, using an 18-gauge needle, at L2-L3 and L4-L5 (L3-L4: nonpunctured control). Four weeks later, the punctured discs received an injection of either 5% lactose (10 microL) or OP-1 (100 microg/10 microL of 5% lactose) into the nucleus pulposus (NP). The disc height was radiographically monitored biweekly. After sacrifice and removal of bone-disc-bone complexes 8 weeks postinjection, the dynamic viscoelastic properties of the IVDs were tested by applying a cycle of sinusoidal strain in uniaxial compression at six loading frequencies (0.05 to 2 Hz). The biochemical properties of the dissected IVDs were then analyzed and correlated with the biomechanical properties. RESULTS: A single injection of OP-1 significantly restored disc height when compared with the lactose-injected discs (OP-1 vs. lactose, p<.001). The elastic modulus of the IVDs in the OP-1-injected discs was significantly higher than that in the lactose-injected discs at all frequencies (mean: +43%, p<.001). The viscous modulus in the OP-1-injected discs was significantly higher at 0.05, 0.2, 0.5, and 1 Hz (mean: +55%, p<.001) and showed higher tendencies at other frequencies (p=.08-.09). For both moduli, no significant differences were observed between the OP-1-injected and the nonpunctured control discs. The OP-1 injection significantly increased the proteoglycan (PG) content in the NP and AF, and the collagen content in the NP (p<.001-.05). Both elastic and viscous moduli showed significant positive correlations with PG content in the NP and collagen content in the NP and AF (Rho=.357-.466, p=.010-.047). CONCLUSIONS: We have shown for the first time that an injection of the growth factor, OP-1, restored the biomechanical properties of IVDs in a rabbit model of IVD degeneration. Comparing biomechanical with biochemical data suggests that the OP-1-induced biomechanical restoration was a consequence of increased activities of anabolic pathways that resulted in biochemical changes in the IVD.     

Mitochondrial bioenergetics,mass, and morphology are altered in cells of the degenerating human annulus

Back pain and intervertebral disc degeneration have a growing socioeconomic healthcare impact. Information on mitochondrial function in human intervertebral disc cells, however, is surprisingly sparse. We assessed mitochondrial bioenergetics, mass, and ultrastructure in annulus cells cultured from human discs of varying degenerative stages. Citrate synthase activity (reflecting mitochondrial mass) declined significantly with increasing Thompson grade (p < 0.0001). Both mitochondrial (p = 0.009) and non‐mitochondrial (p = 0.0029) respiration showed significant changes with increasing stages of disc degeneration. No significant relationships were found for the association of respiration data with herniated or non‐herniated status, or with subject age. Examination of mitochondrial ultrastructure in cultured annulus cells revealed unusual features which included mitochondrial inclusion bodies, poorly defined cristae and dark staining. Findings reported here are novel and document biochemical, metabolic, and morphologic abnormalities in mitochondria in cells from more degenerated annulus cells. Data suggest that the disc degenerative, not age, is a major factor associated with mitochondrial impairment, and also implicate oxidative stress, driven by mitochondrial dysfunction, as a major component within the degenerating disc. Findings have relevance to advancements in cell‐based therapies to treat disc degeneration. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1270–1275, 2013     

An in vitro study of dynamic cyclic compressive stress on human inner annulus fibrosus and nucleus pulposus cells

Background contextIntervertebral disc (IVD) cells exhibit diverse biologic responses to compressive stress depending on the compressive stress type, magnitude, duration, and anatomic zone of IVD cell origin. The previous studies mainly focused on the effects of compressive stress on animal IVD tissue. Few studies have investigated the response of human IVD tissue to compressive stress.PurposeTo assess the effect of dynamic cyclic compressive stress on biosynthesis of collagen and glycosaminoglycan of human inner annulus fibrosus (AF) and nucleus pulposus (NP) cells.Study design/settingObservation of moderate and high magnitudes of compressive stress on human IVD cell biosynthesis.Patient sampleHuman IVD of adolescent idiopathic scoliosis case undergoing thoracoscopic discectomy and fusion was collected.Outcome measuresCell morphology, cell proliferation assay, as well as collagen and glycosaminoglycan content were examined in vitro.MethodsIntervertebral discs were cultured under 0.2 or 0.4 MPa of compressive stress at 1 Hz for 2 hours twice a day up to 7 days. These were compared with samples unloaded. The analysis was done via electron microscopy examination, cell proliferation assay, as well as collagen and glycosaminoglycan content analysis.ResultsCollagen and glycosaminoglycan content in the inner AF and NP cells cultured under 0.2 MPa of compressive stress was significantly higher than that in the control cells but was significantly lower than that in the control cells under 0.4 MPa of compressive stress. The number of endoplasmic reticulum in the inner AF and NP cells cultured under 0.2 MPa of compressive stress was significantly higher than that in the control cells but was significantly lower than that in the control cells under 0.4 MPa of compressive stress.ConclusionThese findings imply that biosynthetic characteristics of human inner AF and NP cells may vary under varying degrees of compressive stresses, which may result in varying amounts of extracellular matrix being secreted.     

A small-molecule inhibitor of the Wnt pathway,lorecivivint (SM04690), as a potential disease-modifying agent for the treatment of degenerative disc disease

BACKGROUND CONTEXTAbnormal Wnt signaling in intervertebral discs (IVDs) progresses degenerative disc disease (DDD) pathogenesis by impairing nucleus pulposus cell function, decreasing matrix deposition, and accelerating fibrosis.PURPOSEThis study was conducted to evaluate the effects of lorecivivint (LOR; SM04690), a small-molecule Wnt pathway inhibitor, on IVD cells and in an animal model of DDD.STUDY DESIGNWe used in vitro assays and a rat model of DDD to test the effects of LOR on nucleus pulposus cell senescence and viability, annulus fibrosus (AF) cell fibrosis, and cartilage regeneration and protection.METHODSWnt pathway gene expression was measured in human NP and AF cell cultures treated with LOR or DMSO (vehicle). Chondrocyte-like differentiation of rat and human NP cells, NP cell senescence and protection, and AF cell fibrosis were assessed using gene expression and immunocytochemistry. Disc and plasma pharmacokinetics were analyzed following intradiscal LOR injection in rats. In vivo effects of LOR and vehicle on AF integrity, AF/NP junction, NP cellularity and matrix, and disc height were compared using histopathology and radiography in a rat coccygeal IVD needle-puncture model of DDD.RESULTSIn NP and AF cell cultures, LOR-inhibited Wnt pathway gene expression compared with vehicle. In NP cells, LOR inhibited senescence, decreased catabolism, and induced differentiation into chondrocyte-like cells; in AF cells, LOR decreased catabolism and inhibited fibrosis. A single intradiscal LOR injection in rats resulted in therapeutic disc concentrations (~30 nM) for >180 days and minimal systemic exposure. DDD-model rats receiving LOR qualitatively demonstrated increased cartilage matrix and reduced AF lamellar disorganization and fragmentation with significantly (p<.05) improved histology scores and increased disc height compared with vehicle.CONCLUSIONSLOR showed beneficial effects on IVD cells in vitro and reduced disease progression in a rat model of DDD compared with vehicle, suggesting that LOR may have disease-modifying therapeutic potential.CLINICAL SIGNIFICANCEThe current therapeutic options for DDD are pain management and surgical intervention; there are no approved therapies that alter the progression of DDD. Our data support advancing LOR into clinical development as an injectable, small-molecule, potential disease-modifying treatment for DDD in humans.     

Effect of cell number on mesenchymal stem cell transplantation in a canine disc degeneration model

Transplantation of mesenchymal stem cells (MSCs) inhibits the progression of disc degeneration in animal models. We know of no study to determine the optimal number of cells to transplant into the degenerated intervertebral disc (IVD). To determine the optimal donor cell number for maximum benefit, we conducted an in vivo study using a canine disc degeneration model. Autologous MSCs were transplanted into degenerative discs at 10⁵, 10⁶, or 10⁷ cells per disc. The MSC‐transplanted discs were evaluated for 12 weeks using plain radiography, magnetic resonance imaging, and gross and microscopic evaluation. Preservation of the disc height, annular structure was seen in MSC‐transplantation groups compared to the operated control group with no MSC transplantation. Result of the number of remaining transplanted MSCs, the survival rate of NP cells, and apoptosis of NP cells in transplanted discs showed both structural microenvironment and abundant extracellular matrix maintained in 10⁶ MSCs transplanted disc, while less viable cells were detected in 10⁵ MSCs transplanted and more apoptotic cells in 10⁷ MSCs transplanted discs. The results of this study demonstrate that the number of cells transplanted affects the regenerative capability of MSC transplants in experimentally induced degenerating canine discs. It is suggested that maintenance of extracellular matrix by its production from transplanted cells and/or resident cells is important for checking the progression of structural disruption that leads to disc degeneration. Published by Wiley Periodicals, Inc. J Orthop Res 28:1267–1275, 2010     

The intervertebral disc from embryonic development to disc degeneration: insights into spatial cellular organization

Background contextLow back pain is commonly attributed to intervertebral disc (IVD) degeneration. IVD resembles articular cartilage in its biochemical and cellular composition in many ways. For articular cartilage, degeneration stage-specific characteristic spatial chondrocyte patterns have recently been described.PurposeThis study addresses how spatial chondrocyte organization in the IVD changes from early embryonic development to end stage degeneration.Study designEx vivo immunohistochemical analysis.MethodsWe immunohistochemically investigated bovine IVD-tissue (n=72) from early embryonic development to early disc degeneration and human adult IVD-tissue (n=25) operated for trauma or degeneration for cellular density and chondrocyte spatial organization. IVD samples were sectioned along the main collagen fiber orientation. Nuclei were stained with DAPI and their number and spatial patterns were analyzed in an area of 250,000 µm² for each tissue category.ResultsThe initially very high cellular density in the early embryonic bovine disc (11,431 cells/mm²) steadily decreases during gestation, growth and maturation to about 71 cell/mm² in the fully grown cattle. Interestingly, in human degenerative discs, a new increase in this figure could be noted (184 cells/mm). The IVD chondrocytes appear to be predominantly present as single cells. Especially in the time after birth, string-formations represent up to 32% of all cells in the anulus fibrosus, although single cells are the predominant spatial pattern (>50%) over the entire time. With increasing degeneration, the relative proportion of single cells in human IVDs continuously decreases (12%). At the same time, the share of cells organized in clusters increases (70%).ConclusionSimilar to articular cartilage, spatial chondrocyte organization appears to be a strong indicator for local tissue degeneration in the IVD.Clinical SignificanceIn the future these findings may be important for the detection and therapy of IVD degeneration in early stages.     

Exogenous and autocrine growth factors stimulate human intervertebral disc cell proliferation via the ERK and Akt pathways

Intervertebral disc (IVD) degeneration is accompanied by growth factor-overexpression and increased cell proliferation, probably representing a tissue repair process. Accordingly, we studied the effect of exogenous and autocrine growth factors on the proliferation of human IVD cells. We observed that Platelet-Derived Growth Factor (PDGF), basic Fibroblast Growth Factor (bFGF), and Insulin-like Growth Factor-I (IGF-I) stimulate DNA synthesis of human IVD cells, through the activation of the MEK/ERK and the PI-3K/Akt signal transduction pathways. Furthermore, medium conditioned (CM) by IVD cells induced DNA synthesis in the same cells, indicating the secretion of autocrine growth factors. The MEK/ERK and PI-3K/Akt pathways were also induced by CM, while their inhibition reversed in large part the DNA synthesis induction by CM. These responses to the exogenous and autocrine growth factors were qualitatively similar in both nucleus pulposus (NP) and annulus fibrosus (AF) cell cultures. Immunohistochemical studies in human biopsies showed significant activation of both signaling pathways, which was most prominent in the clusters of proliferating cells. These in vitro and in vivo data indicate that the proliferation of human IVD cells is regulated by exogenous and autocrine growth factors mainly via the MEK/ERK and PI-3K/Akt pathways; this may contribute to the design of future interventional approaches.     

Differential expression of galectin‐1 and its interactions with cells and laminins in the intervertebral disc

Galectin‐1 (Gal‐1), an endogenous β‐galactoside‐binding protein, binds to laminins, which are highly expressed in the nucleus pulposus (NP) of the intervertebral disc (IVD). The objective of this study is to evaluate the expression of Gal‐1 protein in IVD tissues during aging and the effect of Gal‐1 on IVD cell adhesion to laminins. Tissues from rat, porcine, and human (scoliosis or disc degeneration) IVDs were used to evaluate Gal‐1 expression via immunostaining, RT‐PCR, and Western blot analysis. Attachment of isolated IVD cells (porcine and human) on select laminin isoforms (LM‐111 and LM‐511) was compared with/without pre‐incubation with exogenous Gal‐1. A biotinylated Gal‐1(B‐Gal‐1) was used to evaluate for binding to IVD cells and to select for IVD cells by magnetic activated cell sorting (MACS). NP cells expressed high levels of Gal‐1 protein as compared to anulus fibrosus (AF) cells in immature tissues, while exogenous Gal‐1 increased both NP and AF cell attachment to laminins and exhibited a similar binding to both cell types in vitro. With aging, Gal‐1 levels in NP tissue appeared to decrease. In addition, incubation with B‐Gal‐1 was able to promote the retention of more than 50% of IVD cells via MACS. Our results provide new findings for the presence and functional role of Gal‐1 within IVDs. Similar staining patterns for Gal‐1 and LM‐511 in IVD tissue suggest that Gal‐1 may serve as an adhesion molecule to interact with both cells and laminins. This MACS protocol may be useful for selecting pure IVD cells from mixed cells of pathological tissue. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1923–1931, 2012