Rabbit Model Of Disc Degeneration Research Articles

Proteoglycan-depleted regions of annular injury promote nerve ingrowth in a rabbit disc degeneration model.

BackgroundTo assess the effects of proteoglycan-depleted regions of annular disruptions on nerve ingrowth in the injury site in vivo.MethodsNew Zealand white rabbits (n = 18) received annular injuries at L3/4, L4/5, and L5/6. The experimental discs were randomly assigned to four groups: (a) an annular defect was created; (b) an annular defect implanted with a poly lactic-co-glycolic acid (PLGA)/fibrin/PBS plug; (c) an annular defect implanted with a PLGA/fibrin/chondroitinase ABC (chABC) plug; and (d) an uninjured L2/3 disc (control). Disc degeneration was evaluated by radiography, MRI, histology, and analysis of the proteoglycan (PG) content. Immunohistochemical detection of nerve fibers and chondroitin sulfate (CS) was performed.ResultsThe injured discs produced progressive and reliable disc degeneration. In the defective discs, the lamellated appearance of AF (Annulus fibrosus) was replaced by extensive fibrocartilaginous-like tissue formation outside the injured sites. In contrast, newly formed tissue was distributed along small fissures, and small blood vessels appeared in the outer part of the disrupted area in the PLGA/fibrin/PBS discs. More sprouting nerve fibers grew further into the depleted annulus regions in the PLGA/fibrin/chABC discs than in the control discs and those receiving PLGA/fibrin/PBS. In addition, the innervation scores of the PLGA/fibrin/chABC discs were significantly increased compared with those of the PLGA/fibrin/PBS discs and defected discs.ConclusionChABC-based PLGA/fibrin gel showed promising results by achieving biointegration with native annulus tissue and providing a local source for the sustained release of active chABC. Disc-derived PG-mediated inhibition of nerve and blood vessel ingrowth was abrogated by chABC enzymatic deglycosylation in an annular-injured rabbit disc degeneration model.

Transition from static culture to stirred tank bioreactor for the allogeneic production of therapeutic discogenic cell spheres

BackgroundCulturing cells as cell spheres results in a tissue-like environment that drives unique cell phenotypes, making it useful for generating cell populations intended for therapeutic use. Unfortunately, common methods that utilize static suspension culture have limited scalability, making commercialization of such cell therapies challenging. Our team is developing an allogeneic cell therapy for the treatment of lumbar disc degeneration comprised of discogenic cells, which are progenitor cells expanded from human nucleus pulposus cells that are grown in a sphere configuration.MethodsWe evaluate sphere production in Erlenmeyer, horizontal axis wheel, stirred tank bioreactor, and rocking bag format. We then explore the use of ramped agitation profiles and computational fluid dynamics to overcome obstacles related to cell settling and the undesired impact of mechanical forces on cell characteristics. Finally, we grow discogenic cells in stirred tank reactors (STRs) and test outcomes in vitro (potency via aggrecan production and identity) and in vivo (rabbit model of disc degeneration).ResultsComputation fluid dynamics were used to model hydrodynamic conditions in STR systems and develop statistically significant correlations to cell attributes including potency (measured by aggrecan production), cell doublings, cell settling, and sphere size. Subsequent model-based optimization and testing resulted in growth of cells with comparable attributes to the original static process, as measured using both in vitro and in vivo models. Maximum shear rate (1/s) was maintained between scales to demonstrate feasibility in a 50 L STR (200-fold scale-up).ConclusionsTransition of discogenic cell production from static culture to a stirred-tank bioreactor enables cell sphere production in a scalable format. This work shows significant progress towards establishing a large-scale bioprocess methodology for this novel cell therapy that can be used for other, similar cell therapies.

Efficacy of matrilin-3-primed adipose-derived mesenchymal stem cell spheroids in a rabbit model of disc degeneration

BackgroundChronic low back pain is a prevalent disability, often caused by intervertebral disc (IVD) degeneration. Mesenchymal stem cell (MSC) therapy could be a safe and feasible option for repairing the degenerated disc. However, for successful translation to the clinic, various challenges need to be overcome including unwanted adverse effects due to acidic pH, hypoxia, and limited nutrition. Matrilin-3 is an essential extracellular matrix (ECM) component during cartilage development and ossification and exerts chondrocyte protective effects.MethodsThis study evaluated the effects of matrilin-3-primed adipose-derived MSCs (Ad-MSCs) on the repair of the degenerated disc in vitro and in vivo. We determined the optimal priming concentration and duration and developed an optimal protocol for Ad-MSC spheroid generation.ResultsPriming with 10 ng/ml matrilin-3 for 5 days resulted in the highest mRNA expression of type 2 collagen and aggrecan in vitro. Furthermore, Ad-MSC spheroids with a density of 250 cells/microwell showed the increased secretion of favorable growth factors such as transforming growth factor beta (TGF-β1), TGF-β2, interleukin-10 (IL-10), granulocyte colony-stimulating factor (G-CSF), and matrix metalloproteinase 1 (MMP1) and decreased secretion of hypertrophic ECM components. In addition, matrilin-3-primed Ad-MSC spheroid implantation was associated with optimal repair in a rabbit model.ConclusionOur results suggest that priming MSCs with matrilin-3 and spheroid formation could be an effective strategy to overcome the challenges associated with the use of MSCs for the treatment of IVD degeneration.

A novel magnetic resonance imaging postprocessing technique for the assessment of intervertebral disc degeneration-Correlation with histological grading in a rabbit disc degeneration model.

IntroductionEstimation of intervertebral disc degeneration on magnetic resonance imaging (MRI) is challenging. Qualitative schemes used in clinical practice correlate poorly with pain and quantitative techniques have not entered widespread clinical use.MethodsAs part of a prior study, 25 New Zealand white rabbits underwent annular puncture to induce disc degeneration in 50 noncontiguous lumbar discs. At 16 weeks, the animals underwent multi‐echo T2 MRI scanning and were euthanized. The discs were stained and examined histologically. Quantitative T2 relaxation maps were prepared using the nonlinear least squares method. Decay Variance maps were created using a novel technique of aggregating the deviation in the intensity of each echo signal from the expected intensity based on the previous rate of decay.ResultsDecay Variance maps showed a clear and well demarcated nucleus pulposus with a consistent rate of decay (low Decay Variance) in healthy discs that showed progressively more variable decay (higher Decay Variance) with increasing degeneration. Decay Variance maps required significantly less time to generate (1.0 ± 0.0 second) compared with traditional T2 relaxometry maps (5 (±0.9) to 1788.9 (±116) seconds). Histology scores correlated strongly with Decay Variance scores (r = 0.82, P < .01) and weakly with T2 signal intensity (r = 0.32, P < .01) and quantitative T2 relaxometry (r = 0.39, P < .01). Decay Variance had superior sensitivity and specificity for the detection of degenerate discs when compared to T2 signal intensity or Quantitative T2 mapping.ConclusionOur results show that using a multi‐echo T2 MRI sequence, Decay Variance can quantitatively assess disc degeneration more accurately and with less image‐processing time than quantitative T2 relaxometry in a rabbit disc puncture model. The technique is a viable candidate for quantitative assessment of disc degeneration on MRI scans. Further validation on human subjects is needed.

Elevated expression of hypoxia-inducible factor-2alpha regulated catabolic factors during intervertebral disc degeneration

Headings aimsThe present study determined whether nucleus pulposus (NP) cells express hypoxia-inducible factor-2alpha (HIF-2α) and assessed its role in regulating the expression of catabolic factors during intervertebral disc degeneration. Materials and methodsHuman degenerated NP tissues were acquired to examine the HIF-2α expression levels using immunohistochemistry, western blotting, and Real-time PCR. Human NP cells were cultivated under normoxic or hypoxic conditions, and the HIF-2α expression was determined. Then, human NP cells were treated with HIF-2α plasmids, HIF-2α siRNA, and tumor necrosis factor-alpha (TNF-α) to evaluate the role of HIF-2α in regulating matrix metalloproteinase (MMP) and aggrecanase expression. An in vivo rabbit disc degeneration model was established to demonstrate that HIF-2α plays a critical role in disc degeneration. Key findingsWe found that HIF-2α had a markedly elevated expression in human degenerated discs in the Grade III stage. HIF-2α protein and gene transcript levels in vitro were relatively higher under hypoxic conditions. The expression of MMP-13, ADAMTS-4 was decreased significantly in HIF-2α silencing condition, while the over-expression resulted in significantly increased levels of MMP-13 and ADAMTS-4. When cytokine TNF-α was added, HIF-2α was induced by nuclear factor-κB (NF-κB). The in vivo experiments showed that the HIF-2α controlled the catabolic factors MMP-13 and ADAMTS-4 that regulated the collagen II and aggrecan metabolism in disc degeneration. SignificanceHIF-2α is a catabolic regulator in disc degeneration and directly controls the catabolic genes. The suppression of HIF-2α expression leads to decelerates extracellular matrix degradation that might represent a therapeutic target for the degenerative disc.

Roles of SDF-1/CXCR4 axis in cartilage endplate stem cells mediated promotion of nucleus pulposus cells proliferation

Stem cells transplantation has shown considerable promise in intervertebral disc repair and low-back pain release. Cartilage endplate stem cells (CESCs) also showed potential for nucleus pulposus (NP) regeneration in a rabbit disc degeneration model, the precise mechanism was unclear. Here we investigated the effects of CESCs on NP cells (NPCs) proliferation and the mechanism in vitro. CESCs and NPCs were isolated from surgical specimens of degenerative human lumbar disc. NPCs were co-cultured with CESCs at a 1:1 ratio or cultured in CESCs conditioned medium (CESCs-CM). NPCs proliferation was evaluated by Ki-67 staining, CCK-8 assay and cell cycle analysis. Gene expressions were detected by qRT-PCR and activation of Akt and ERK1/2 was detected by western blot. CXCR4 antagonist AMD3100 was used to block SDF-1/CXCR4 axis. ERK1/2 and Akt inhibitors were used to block Akt and ERK1/2 activation. Results showed that NPCs proliferation was promoted by direct-contact co-culturing with CESCs as well as culturing in CESCs-CM. SDF-1 expression level in CESCs was significantly higher than that in NPCs, while CXCR4 was the opposite. Promotion of NPCs proliferation mediated by CESCs-CM could be partially attenuated by AMD3100. CESCs-CM activated both Akt and ERK1/2 in NPCs, while rhSDF-1 scarcely activated Akt but obviously activated ERK1/2. Akt and ERK1/2 inhibitors could partially inhibited CESCs-CM mediated promotion of NPCs proliferation and showed cumulative effect, while ERK1/2 inhibitor and AMD3100 could significantly abrogate SDF-1 mediated promotion of NPCs proliferation. Our results suggested that CESCs might promote NPCs proliferation in a paracrine pathway, which was partially mediated by SDF-1/CXCR4 axis via ERK1/2 signaling transduction pathway.

Short Link N promotes disc repair in a rabbit model of disc degeneration

BackgroundThe degeneration of the intervertebral disc (IVD) is characterized by proteolytic degradation of the extracellular matrix, and its repair requires the production of an extracellular matrix with a high proteoglycan-to-collagen ratio characteristic of a nucleus pulposus (NP)-like phenotype in vivo. At the moment, there is no medical treatment to reverse or even retard disc degeneration. The purpose of the present study was to determine if a low dose of short link N (sLN), a recently discovered fragment of the link N peptide, could behave in a manner similar to that of link N in restoring the proteoglycan content and proteoglycan-to-collagen ratio of the disc in a rabbit model of IVD degeneration, as an indication of its potential therapeutic benefit in reversing disc degeneration.MethodsAdolescent New Zealand white rabbits received an annular puncture with an 18-gauge needle into two noncontiguous discs to induce disc degeneration. Two weeks later, either saline (10 μL) or sLN (25 μg in 10 μL saline) was injected into the center of the NP. The sLN concentration was empirically chosen at a lower molar concentration equivalent to half that of link N (100 μg in 10 μL). The effect on radiographic, biochemical and histologic changes were evaluated.ResultsFollowing needle puncture, disc height decreased by about 25–30% within 2 weeks and maintained this loss for the duration of the 12-week study; a single 25-μg sLN injection at 2 weeks partially restored this loss in disc height. sLN injection led to an increase in glycosaminoglycans (GAG) content 12 weeks post-injection in both the NP and annulus fibrosus (AF). There was a trend towards maintaining control disc collagen-content with sLN supplementation and the GAG-to-collagen ratio in the NP was increased when compared to the saline group.ConclusionsWhen administered to the degenerative disc in vivo, sLN injection leads to an increase in proteoglycan content and a trend towards maintaining control disc collagen content in both the NP and AF. This is similar to link N when it is administered to the degenerative disc. Thus, pharmacologically, sLN supplementation could be a novel therapeutic approach for treating disc degeneration.

Therapeutic effects of cell therapy with neonatal human dermal fibroblasts and rabbit dermal fibroblasts on disc degeneration and inflammation

BACKGROUND CONTEXTIncreasing evidence suggests transplanting viable cells into the degenerating intervertebral disc (IVD) may be effective in treating disc degeneration and back pain. Clinical studies utilizing autologous or allogeneic mesenchymal stem cells to treat patients with back pain have reported some encouraging results. Animal studies have shown that cells injected into the disc can survive for months and have regenerative effects. Studies to determine the advantages and disadvantages of cell types and sources for therapy are needed. PURPOSEThe objective of this study is to determine the impact of donor source on the therapeutic effects of dermal fibroblast treatment on disc degeneration and inflammation. STUDY DESIGNUsing the rabbit disc degeneration model, we compared transplantation of neonatal human dermal fibroblasts (nHDFs) and rabbit dermal fibroblasts (RDFs) into rabbit degenerated discs on host immune response, disc height, and IVD composition. METHODSNew Zealand white rabbits received an annular puncture using an 18-guage needle to induce disc degeneration. Four weeks after injury, rabbit IVDs were treated with 5 × 106 nHDFs, RDFs, or saline. At eight weeks post-treatment, animals were sacrificed. X-ray images were obtained. IVDs were isolated for inflammatory and collagen gene expression analysis using real-time polymerase chain reaction and biochemical analysis of proteoglycan contents using dimethylmethylene blue assay. These studies were funded by a research grant from SpinalCyte, LLC ($414,431). RESULTSEight weeks after treatment, disc height indexes of discs treated with nHDF increased significantly by 7.8% (p<.01), whereas those treated with saline or RDF increased by 1.5% and 2.0%, respectively. Gene expression analysis showed that discs transplanted with nHDFs and RDFs displayed similar inflammatory responses (p=.2 to .8). Compared to intact discs, expression of both collagen types I and II increased significantly in nHDF-treated discs (p<.05), trending to significant in RDF-treated discs, and not significantly in saline treated discs. The ratio of collagen type II/collagen type I was higher in the IVDs treated with nHDFs (1.26) than those treated with RDFs (0.81) or saline (0.59) and intact discs (1.00). Last, proteoglycan contents increased significantly in discs treated with nHDF (p<.05) and were trending toward significance in the RDF-treated discs compared to those treated with saline. CONCLUSIONSThis study showed that cell transplantation with nHDF into degenerated IVDs can significantly increase markers of disc regeneration (disc height, collagen type I and II gene expression, and proteoglycan contents). Transplantation with RDFs showed similar regenerative trends, but these trends were not significant. This study also showed that the human cells transplanted into the rabbit discs did not induce a higher immune response than the rabbit cells. These results support that the IVD is immune privileged and would tolerate allogeneic or xenogeneic grafts.

An injectable nucleus pulposus cell-modified decellularized scaffold: biocompatible material for prevention of disc degeneration.

We developed a nucleus pulposus cell (NPC)-modulated decellularized small intestinal submucosa (SIS) scaffold, and assessed the ability of this material to prevent Intervertebral disc degeneration (IVD) degeneration. Decellularized porcine SIS was squashed into particles and the biological safety and efficiency of these particles were evaluated. Next, SIS particles were seeded with rabbit NPCs, cultured for two months in vitro, decellularized again and suspended for intervertebral injection. We demonstrated that use of the decellularization protocol resulted in the removal of cellular components with maximal retention of extracellular matrix. The xenogeneic decellularized SIS did not display cytotoxicity in vitro and its application prevented NPC degradation. Furthermore, the xenogeneic SIS microparticles were effective in preventing IVD progression in vivo in a rabbit disc degeneration model. In conclusion, our study describes an optimized method for decellularized SIS preparation and demonstrated that the material is safe and effective for treating IVD degeneration.

Repair of the Degenerated Disc: A Perspective Derived from Cell-Based Studies

Like articular joints, intervertebral discs (IVD) suffer from degeneration which is thought to be irreversible after initiation. Disc degeneration is associated with low back pain, and the degenerative IVD can be a source of irritation. While the cause of IVD degeneration is not entirely understood to date, both genetic and environmental factors have shown contribution to its etiology. Various studies investigated disc repair or regeneration via cell-based approaches. These findings help us to derive new insights or principles about the degeneration, and the use of related therapies in future. Nucleus pulposus (NP) has a role in disc height maintenance and supporting motion of spinal segment. The loss of NP integrity is one of the earliest events of disc degeneration, implying a critical function of NP in IVD homeostasis. Recovery of the NP function is therefore an important goal in treating disc degeneration. Transplantation of chondrocytes or NP cells has shown some extent of reparative capacity. Nevertheless, the use of mesenchymal stromal/stem cells (MSCs) in treating IVD degeneration has drawn much attention in the last decade. Studies have attempted to engineer MSCs into NP-like cells directly for de novo disc engineering or disc implantation, aiming to restore the matrix and hence recover the native NP function. This includes the use of growth factors such as TGF-beta, GDFs, and BMPs to induce MSC differentiation in vitro. MSCs may also be delivered directly into the disc where the engrafted MSCs acquire a differentiated phenotype to benefit NP function. Results from pilot clinical trials support that intradiscal MSC implantation may alleviate symptoms and possibly delay IVD degeneration progression. While the use of models and regime of MSC introduction may vary, the results from the in vivo studies have laid important foundation to our understanding in the capacity and mechanism of MSC-based therapies. One school of thoughts is that MSCs can attain a collage II and proteoglycan expressing phenotype in the degenerated discs. However, other reports have also suggested that MSCs and NP cells can mutually exchange biological information and promote anabolic activities through cell-cell contact or paracrine action. Therefore MSCs may contribute to disc repair via pathways other than direct differentiation. MSCs may have other capacities in alleviating disc degeneration. Autopsy and surgical specimens show evidence of fibrosis in the majority of degenerated discs. We recently show that human IVD degeneration exhibits features of fibrosis and that a rabbit disc degeneration model shows similar features. Implantation of bone marrow-derived MSCs can inhibit fibrosis-related events in the degenerative NP and effectively preserves its mechanical characteristics and the overall motion segment function. MSCs may achieve this by repressing the profibrotic mediators that are implicated in mediating collagen anomalies in fibrotic diseases. Altogether, these support a model where MSCs may have a capacity in actively modifying the local microenvironment to potentiate resident progenitor function for tissue repair. Disclosures: None

BMP7 enhances the effect of BMSCs on extracellular matrix remodeling in a rabbit model of intervertebral disc degeneration

Intervertebral discs (IVDs) provide stability and flexibility to the spinal column; however, IVDs, and in particular the nucleus pulposus (NP), undergo a degenerative process characterized by changes in the disc extracellular matrix (ECM), decreased cell viability, and reduced synthesis of proteoglycan and type II collagen. Here, we investigated the efficacy and feasibility of stem cell therapy using bone marrow mesenchymal stem cells (BMSCs) over-expressing bone morphogenetic protein 7 (BMP7) to promote ECM remodeling of degenerated IVDs. Lentivirus-mediated BMP7 over-expression induced differentiation of BMSCs into an NP phenotype, as indicated by expression of the NP markers collagen type II, aggrecan, SOX9 and keratins 8 and 19, increased the content of glycosaminoglycan, and up-regulated β-1,3-glucuronosyl transferase 1, a regulator of chondroitin sulfate synthesis in NP cells. These effects were suppressed by Smad1 silencing, indicating that the effect of BMP7 on ECM remodeling was mediated by the Smad pathway. In vivo analysis in a rabbit model of disc degeneration showed that implantation of BMSCs over-expressing BMP7 promoted cell differentiation and proliferation in the NP, as well as their own survival, and these effects were mediated by the Smad pathway. The results of the present study indicate the beneficial effects of BMP7 on restoring ECM homeostasis in NP cells, and suggest potential strategies for improving cell therapy for the treatment of disc diseases.

Transplantation of human Wharton's jelly-derived mesenchymal stem cells highly expressing TGFβ receptors in a rabbit model of disc degeneration.

IntroductionMesenchymal stem cells (MSCs) are widely considered to hold promise for the treatment of intervertebral disc (IVD) degeneration. However, variation in the therapeutic efficacy of MSCs is a major problem and the derivation of MSCs for use in IVD regeneration has not been optimized. Additionally, no data are available on the efficacy of Wharton’s Jelly-derived MSC (WJ-MSC) transplantation in an animal model of IVD degeneration.MethodsThis study evaluated the effectiveness of a cross-linked hyaluronic acid (XHA) scaffold loaded with human WJ-MSCs, according to their expression levels of transforming growth factor-β receptor I/activin-like kinase receptor 5 (TβRI/ALK5) and TβRII, for IVD regeneration in a rabbit model. We compared the degree of IVD regeneration between rabbits transplanted with a XHA scaffold loaded with WJ-MSCs highly and lowly expressing TβRI/ALK5 and TβRII (MSC-highTR and MSC-lowTR, respectively) using magnetic resonance imaging (MRI) and histological analysis.ResultsAt 12 weeks after transplantation, T2-weighted MRI analysis showed significant restoration of the disc water content in rabbits treated with a MSC-highTR-loaded XHA scaffold in comparison to rabbits treated with the scaffold alone or a MSC-lowTR-loaded XHA scaffold. In addition, morphological and histological analyses revealed that IVD regeneration was highest in rabbits transplanted with a MSC-highTR-loaded XHA scaffold.ConclusionTaken together, our results suggest that a MSC-highTR-loaded XHA scaffold supports IVD regeneration more effectively than a MSC-lowTR-loaded XHA scaffold. This study supports the potential clinical use of MSC-highTR-loaded XHA scaffolds to halt IVD degeneration or to enhance IVD regeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0183-1) contains supplementary material, which is available to authorized users.

Electroacupuncture stimulates remodeling of extracellular matrix by inhibiting apoptosis in a rabbit model of disc degeneration.

The present study was designed to determine whether EA stimulates remodeling of extracellular matrix by inhibiting apoptosis in degenerated disc. 40 rabbits were randomly assigned to one of the four groups. Animal model was established by a loading device. Magnetic resonance imaging and Pfirrmann's classification were obtained to evaluate both the model and the EA treatment on disc degeneration. The ultrastructure of discs was observed by TEM. Apoptosis involvement was determined with TUNEL staining and western blot for the protein expression of Bax and Bcl-2. The results indicated that EA intervention decreased the MRI grades. TEM analysis showed an apparent remodeling and rearrangement of disc ECM after EA intervention for 28 days. The number of TUNEL-positive cells in the EA group was significantly lower than that in the compression group. The protein expression demonstrated an antiapoptosis effect mediated by EA. Increased expression of Bcl-2 proteins and reduced Bax protein expression were detected after 28 days treatment. It was concluded that antiapoptosis pathway probably participates in the mechanism of EA stimulating the remodeling of ECM in disc degeneration.

Link-N Exerts Its Growth Factor-Like Effects through Activation of Smad 1/5 Signaling in Human IVD Cells

Introduction Back pain is a fairly common problem, which affects a large portion of the population and has an impact on quality of life. Intervertebral disc (IVD) degeneration is one of the most common causes of back pain. Link-N peptide represents the 16 amino acid sequence (DHLSDNYTLDHDRAIH) from the N-terminus of the link protein that stabilizes the proteoglycan aggregates present in cartilage and IVDs. We have previously shown that Link-N can stimulate collagen and proteoglycan synthesis in IVD cells in vitro1 and in intact human IVDs ex vivo,2 as well as increase disc height in a rabbit model of disc degeneration.3 Recently, BMPII receptor has been shown to interact with Link-N in rabbit IVD cells.4 The purpose of this study was to determine the mechanism by which Link-N exerts its beneficial effects by promoting matrix production in human IVD cells. Materials and Methods Human lumbar spines were retrieved through the Transplant Quebec organ donation program. IVD cells were isolated from nucleus pulposus (NP) and annulus fibrosus (AF) regions. The cells were cultured to 90% confluence in NP Cell Medium (NPCM, Cat. No.4801, ScienCell). Then the cells were incubated overnight in serum-free DMEM followed by treatment with Link-N (1 μg/mL), TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) for 10 minutes to 4 hours. TGF-β (10 ng/mL) or BMP-2 (25 ng/mL) was used as positive controls. Cell extracts were prepared using NP40 lysis buffer. Total protein in the cell extracts was determined by the bicinchoninic acid assay (BCA). Protein expression was analyzed by immunoblotting using specific antibodies. Western blot images were quantified using ImageJ (NIH) software. For gene expression studies, cells were incubated for 24 hours with Link-N (1 μg/mL) in the presence or absence of human recombinant noggin (100 ng/mL) and total RNA was isolated using Trizol reagent and relative gene expression was determined by RT PCR. For receptor-binding studies, AF and NP cells were treated with biotinylated Link-N (1 μg/mL) with or without untagged Link-N (10 μg/mL) for competitive displacement. Scrambled biotinylated Link-N (1 μg/mL) was used as a specificity control. After treatments, cells were fixed in 2% paraformaldehyde. Fixed cells were treated with fluorescent-dye conjugated streptavidin (Alexa Fluor 700 conjugate, streptavidin). We assessed whether noggin directly binds with biotinylated Link-N by dot-blot technique. Results Link-N addition to human IVD cells did not promote the phosphorylation of Smads 2 and 3. This suggested that Link-N signaling does not function in a fashion similar to TGF-β, which stimulates the phosphorylation/activation of Smads 2 and 3. Further, similar to BMP-2, Link-N promoted the phosphorylation of Smad ? (Fig. 1). These results suggest that Link-N exerts its growth factor-like effects via a BMP-signaling mechanism rather than TGF-β. Noggin is a known physiological negative modulator of BMP signaling and noggin levels are elevated in disease conditions. As we observed that Link-N signaling is similar to BMP, it is essential to assess if noggin can interfere with the function of Link-N. Dot-blot results showed that noggin does not directly bind with Link-N. Therefore, noggin inhibition of Link-N signaling is unlikely because of noggin binding with Link-N. This suggests that Link-N may have an added advantage as a therapeutic agent over BMPs. In addition, Link-N increased BMP 4 expression significantly in both AF and NP cells, with the increase in AF cells being significantly higher than that in NP cells. Specific binding (also internalization) of biotinylated Link-N was found both in AF and NP cells, as this binding was competitively displaced by nonbiotinylated Link-N. No binding was seen with scrambled biotinylated Link-N. Conclusion Link-N represents a potential economic growth factor with beneficial effects on disc repair and the identification of the intracellular signaling pathways that it transduces is a step further toward clinical trials on NP repair in the degenerate human IVD. Disclosure of Interest None declared References Mwale F, Demers CN, Petit A, et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. J Cell Biochem 2003;88(6):1202–1213 Gawri R, Antoniou J, Ouellet J, et al. Best paper NASS 2013: link-N can stimulate proteoglycan synthesis in the degenerated human intervertebral discs. Eur Cell Mater 2013;26:107-119, discussion 119 Mwale F, Masuda K, Pichika R, et al. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011;13(4):R120 Wang Z, Weitzmann MN, Sangadala S, Hutton WC, Yoon ST. Link protein N-terminal peptide binds to bone morphogenetic protein (BMP) type II receptor and drives matrix protein expression in rabbit intervertebral disc cells. J Biol Chem 2013;288(39):28243–28253

LINK between Degeneration and Regeneration of Human Intervertebral Discs

Back pain affects a large portion of the population across all ages and intervertebral disc (IVD) degeneration is its most implicated common cause. Presently, there are no established treatments to prevent, stop, or even retard disc degeneration, and surgery is often the offered option. Previous studies have shown that Link N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens in bovine IVD in vitro and in intact human IVDs ex vivo, as well as increase disc height in a rabbit model of disc degeneration. However, the sequences in Link N involved in modulating cellular activity are not well understood. To determine if disc cells can proteolytically process Link N, human disc cells in monolayer were exposed to native Link N over a 48-hour period and mass spectrometric analysis revealed that a peptide spanning residues 1 to 8 was generated in the presence of AF cells but not NP cells. Link N 1-8 significantly induced proteoglycan production in the presence of IL-1β in both NP and AF cells, confirming that the biological effect is maintained in the first eight amino acids of the peptide and indicating that the effect is sustained in an inflammatory environment. Thus Link N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration. Acknowledgments The authors thank Ms. Areej Khabut for her assistance with mass spectrometry. This work would not have been possible without funding from the Faculty of Medicine McGill University, AO Spine, North American Spine Society and Canadian Institutes of Health Research. We also thank Transplant Quebec for coordinating the retrieval of human lumbar spines through their organ donation program. Lastly, we thank The Crafoord Foundation and the Inga-Britt &amp; Arne Lundberg Foundation for funding the mass spectrometers used in this study. Disclosure of Interest None declared

Link-N is Cleaved by Human Annulus Fibrosus Cells Generating a Fragment with Retained Biological Activity

Introduction Back pain affects a large portion of the population across all ages, and intervertebral disc (IVD) degeneration is its most common cause. Presently, there are no established treatments to prevent, stop, or even retard disc degeneration, and surgery is often the offered option. Previous studies have shown that Link-N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens in articular cartilage, in bovine IVD in vitro, and in intact human IVDs ex vivo, as well as increase disc height in a rabbit model of disc degeneration. However, the sequences in Link-N involved in modulating cellular activity and the stability of Link-N in the presence of IVD cells are not well understood. Materials and Methods Human lumbar spines were obtained through the Transplant Quebec's organ donation program. Six spinal segments were retrieved (mean age 30 years) and IVD cells were isolated from the nucleus pulposus (NP) and inner annulus fibrosus (iAF) regions by sequential enzymatic digestion. IVD cells were cultured in monolayer in the presence of Link-N to assess its stability in culture. Media was collected at designated time points from the cultures and was analyzed by MALDI-TOF and MS-MS mass spectrophotometry to evaluate the stability of Link-N peptide. IVD cells embedded in 1.2% alginate were exposed to the identified Link-N fragments and to the native scrambled or reversed Link-N peptides in the absence and presence of IL-1β to assess the biological activity of the peptides. SO4 incorporation was used to assess proteoglycan synthesis in response to the peptides. All peptides were used at equimolar concentrations. Results Mass spectrometric analysis revealed that a peptide spanning residues 1 to 8 was generated in the presence of human AF, while the original 16 amino acids peptide remained intact in the presence of NP cells. Link-N 1-8 significantly induced proteoglycan production in the absence and presence of IL-1β in both human NP and AF cells, confirming that the biological effect is maintained within the first 8 amino acids of the peptide. A reversed or scrambled Link-N peptide, as well as residues 9-16 of Link-N, had no biological effect. Conclusion In conclusion, Link-N 1-8 peptide is a promising therapeutic alternative for treating early disc degeneration before major collagen degradation has occurred. One advantage in using this shorter 8 amino acid peptide rather than the original 16 amino acid Link-N in therapy is the production cost. More important, this small size should be amenable to medicinal chemists allowing them to optimize the structure for maximal biological activity and stability. Thus Link-N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration. Disclosure of Interest None declared

Roles of Biomaterials in Cell-based Therapies for Disc Degeneration

Degenerative disc disease poses an increasing threat to our quality of life as we age. Existing treatments have limitations. New treatment modalities using biological approaches are appealing. While cells including mesenchymal stem cells (MSC) are proving themselves potential candidates for treating disc degeneration, biomaterials are demonstrating their important roles in supporting and potentiating such therapies. During intradiscal injection, the vast majority of cells leaked out via back flow from the injection portal, reducing the efficacy of treatment. Recent evidence even suggests that annulus puncture is associated with cell leakage and contributes to osteophyte formation, leading to an undesirable side-effect of MSC-based therapy. This suggests the significance of developing appropriate cell carriers and effective approaches to prevent leakage and associated osteophyte formation before MSC-based therapies can be applied in a clinical setting. Using a collagen microencapsulation platform, we fabricated collagen microspheres entrapping MSCs, supporting their survival, proliferation, differentiation and matrix remodeling. Using a rabbit disc degeneration model, we showed that, comparing with delivering MSC in saline, delivering MSC in collagen microspheres significantly reduced the risk of osteophyte formation. Using a photochemical crosslinking technology, we further fabricated an injectable collagen-based annulus plug to block the injection portal during intradiscal delivery. Ex vivo studies showed that the plug survived physiologically relevant loadings and significantly reduced leakage and enhanced retention of the injected materials. In vivo rabbit disc degeneration model showed that the plug significantly reduced osteophyte formation, suggesting the potential of the annulus plug as an adjunct or annulus closure device for intradisc delivery of cells and materials. Apart from facilitating cell delivery as shown in the above mentioned example, biomaterials play important roles in immobilizing soluble signals such as growth factors, providing structural and mechanical support to cells and tissues, presenting microenvironmental clues or niches, and acting as the template for functional remodeling of cells and tissues. Detailed discussion and examples on these functions of biomaterials will be given in this study. Disclosure of Interest None declared

Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue Engineering

Introduction In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus, nucleus pulposus (NP), and cartilage endplate. Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. Materials and Methods In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, and so on. Results We found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs ( p&lt; 0.05); in addition, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray ( p&gt;0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. Conclusion Our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases. Disclosure of Interest None declared

The effect of electroacupuncture on the extracellular matrix synthesis and degradation in a rabbit model of disc degeneration.

The present study was aimed at determining if the electroacupuncture (EA) is able to protect degenerated disc in vivo. New Zealand white rabbits (n = 40) were used for the study. The rabbits were randomly assigned to four groups. EA intervention was applied to one of the four groups. Magnetic resonance imaging and Pfirrmann's classification were obtained for each group to evaluate EA treatment on the intervertebral disc degeneration. Discs were analyzed using immunofluorescence for the labeling of collagens 1 and 2, bone morphogenetic protein-2 (BMP-2), matrix metalloproteinase-13 (MMP-13), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). For protein expression analysis, western blot was used for biglycan and decorin. Outcomes indicated that EA intervention decreased the grades compared with the compressed disc. Immunofluorescence analysis showed a significant increase of collagens 1 and 2, TIMP-1, and BMP-2 positive cells, in contrast to MMP-13 after EA treatment for 28 days. The protein expression showed a sign of regeneration that decorin and biglycan were upregulated. It was concluded that EA contributed to the extracellular matrix (ECM) anabolic processes and increased the ECM components. MMPs and their inhibitors involved in the mechanism of EA intervention on ECM decreased disc. It kept a dynamic balance between ECM synthesis and degradation.

Utilization of Stem Cells in Alginate for Nucleus Pulposus Tissue Engineering

In a general view of anatomy, intervertebral disc is composed of three parts: annulus fibrosus (AF), nucleus pulposus (NP), and cartilage endplate (CEP). Recently, several types of stem cells were successfully isolated from these corresponding regions, but up to now, no research was performed about which kind of stem cells is the most efficient candidate for NP tissue engineering or for stem cell-based disc regeneration therapy. In this study, we compared the regenerative potentials of the above-mentioned three kinds of disc-derived stem cells with that of the classic bone marrow (BM)-mesenchymal stem cells (MSCs) in a rabbit disc degeneration model. By magnetic resonance imaging (MRI), X-ray, histology, etc. evaluations, we found that cartilage endplate-derived stem cells (CESCs) showed superior capacity compared with the annulus fibrosus-derived stem cells (AFSCs), nucleus pulposus-derived stem cells (NPSCs), and BM-MSCs (p<0.05); additionally, when comparing the CESC group with the normal control group, there existed no statistical difference in X-ray (p>0.05). Those results demonstrated that the CESC-seeded alginate construct performed the most powerful ability for NP regeneration, while AFSCs showed the most inferior potency, NPSCs and BM-MSCs had similar regenerative capacity and located in the middle. All in all, our study showed that CESCs might act as an efficient seed cell source for NP tissue engineering, which paved a new way for the biological solution of disc degeneration diseases.