Rabbit Annulus Fibrosus Research Articles

Lactate oxidative phosphorylation by annulus fibrosus cells: evidence for lactate-dependent metabolic symbiosis in intervertebral discs

BackgroundIntervertebral disc degeneration contributes to low back pain. The avascular intervertebral disc consists of a central hypoxic nucleus pulpous (NP) surrounded by the more oxygenated annulus fibrosus (AF). Lactic acid, an abundant end-product of NP glycolysis, has long been viewed as a harmful waste that acidifies disc tissue and decreases cell viability and function. As lactic acid is readily converted into lactate in disc tissue, the objective of this study was to determine whether lactate could be used by AF cells as a carbon source rather than being removed from disc tissue as a waste byproduct.MethodsImport and conversion of lactate to tricarboxylic acid (TCA) cycle intermediates and amino acids in rabbit AF cells were measured by heavy-isotope (13C-lactate) tracing experiments using mass spectrometry. Levels of protein expression of lactate converting enzymes, lactate importer and exporter in NP and AF tissues were quantified by Western blots. Effects of lactate on proteoglycan (35S-sulfate) and collagen (3H-proline) matrix protein synthesis and oxidative phosphorylation (Seahorse XFe96 Extracellular Flux Analyzer) in AF cells were assessed.ResultsHeavy-isotope tracing experiments revealed that AF cells imported and converted lactate into TCA cycle intermediates and amino acids using in vitro cell culture and in vivo models. Addition of exogenous lactate (4 mM) in culture media induced expression of the lactate importer MCT1 and increased oxygen consumption rate by 50%, mitochondrial ATP-linked respiration by 30%, and collagen synthesis by 50% in AF cell cultures grown under physiologic oxygen (2-5% O2) and glucose concentration (1-5 mM). AF tissue highly expresses MCT1, LDH-H, an enzyme that preferentially converts lactate to pyruvate, and PDH, an enzyme that converts pyruvate to acetyl-coA. In contrast, NP tissue highly expresses MCT4, a lactate exporter, and LDH-M, an enzyme that preferentially converts pyruvate to lactate.ConclusionsThese findings support disc lactate-dependent metabolic symbiosis in which lactate produced by the hypoxic, glycolytic NP cells is utilized by the more oxygenated AF cells via oxidative phosphorylation for energy and matrix production, thus shifting the current research paradigm of viewing disc lactate as a waste product to considering it as an important biofuel. These scientifically impactful results suggest novel therapeutic targets in disc metabolism and degeneration.

Biomimetic angle-ply multi-lamellar scaffold for annulus fibrosus tissue engineering.

Constructing a biomimetic scaffold that replicates the complex architecture of intervertebral disc annulus fibrosus (AF) remains a major goal in AF tissue engineering. In this study, a biomimetic angle-ply multi-lamellar polycaprolactone/silk fibroin (PCL/SF) AF scaffold was fabricated. Wet-spinning was used to obtain aligned PCL/SF microfiber sheets, and these were excised into strips with microfibers aligned at +30° or -30° relative to the strip long axis. This was followed by stacking two strips with opposing fiber alignment and wrapping them concentrically around a mandrel. Our results demonstrated that the scaffold possessed spatial structure and mechanical properties comparable to natural AF. The scaffold supported rabbit AF cells adhesion, proliferation, infiltration and guided oriented growth and extracellular matrix deposition. In conclusion, our angle-ply multi-lamellar scaffold offers a potential solution for AF replacement therapy and warrants further attention in future investigations.

Rabbit Annulus Fibrosus Cells Express Neuropeptide Y, Which Is Influenced by Mechanical and Inflammatory Stress.

ObjectiveRabbit annulus fibrosus (AF) cells were exposed to isolated or combined mechanical and inflammatory stress to examine the expression of neuropeptide Y (NPY). This study aims to explore the ability of AF cells to produce NPY in response to mechanical and inflammatory stress. MethodsLumbar AF cells of 6- to 8-month-old female New Zealand white rabbits were harvested and exposed to combinations of inflammatory (interleukin-1β) and mechanical (6% or 18%) tensile stress using the Flexcell System. NPY concentrations were measured in the media via enzyme-linked immunosorbent assay. The presence of NPY receptor-type 1 (NPY-1R) in AF cells of rabbit intervertebral discs was also analyzed via immunohistochemistry and immunofluorescence. ResultsExposure to inflammatory stimuli showed a significant increase in the amount of NPY expression compared to control AF cells. Mechanical strain alone did not result in a significant difference in NPY expression. While combined inflammatory and mechanical stress did not demonstrate an increase in NPY expression at low (6%) levels of strain, at 18% strain, there was a large—though not statistically significant—increase in NPY expression under conditions of inflammatory stress. Lastly, immunofluorescence and immunohistochemistry of AF cells and tissue, respectively, demonstrated the presence of NPY-1R. ConclusionThese findings demonstrate that rabbit AF cells are capable of expressing NPY, and expression is enhanced in response to inflammatory and mechanical stress. Because both inflammatory and mechanical stress contribute to intervertebral disc degeneration (IDD), this observation raises the potential of a mechanistic link between low back pain and IDD.

Metformin decreases LPS-induced inflammatory response in rabbit annulus fibrosus stem/progenitor cells by blocking HMGB1 release.

The present study aimed to investigate the mechanism of intervertebral disc degeneration (IVDD) and identify an efficient treatment for low back pain. Rabbit annulus fibrosus stem cells (AFSCs) were treated with metformin and lipopolysaccharide (LPS). The results indicated that LPS induced HMGB1 release from the nuclei of AFSCs and caused cell senescence in a concentration-dependent manner. The production of PGE2 and HMGB1 was increased in the medium of the LPS-treated AFSCs. Certain inflammation-associated genes (IL-β1, IL-6, COX-2 and TNF-α) and proteins (IL-β1, COX-2 and TNF-α) and specific catabolic genes (MMP-3 and MMP-13) exhibited increased expression in LPS-treated AFSCs. However, the expression levels of other anabolic genes, such as collagen I and collagen II were decreased in LPS-treated AFSCs. Following addition of metformin to LPS-containing medium, HMGB1 was retained in the nuclei of AFSCs and the production of PGE2 and HMGB1 was reduced. The expression levels of the catabolic genes and proteins were decreased and those of the anabolic genes were increased. The findings indicated that metformin exerted an anti-inflammatory effect by blocking the HMGB1 translocation and by inhibiting catabolic production and cell senescence in AFSCs. Therefore, metformin may be used as an efficient treatment for the disc degenerative disease.

Mitochondrial Pathway Is Involved in Advanced Glycation End Products-Induced Apoptosis of Rabbit Annulus Fibrosus Cells.

Experimental study. The purposes of this study were to evaluate whether advanced glycation end-products (AGEs) induce annulus fibrosus (AF) cell apoptosis and further to explore the mechanism by which this process occurs. Recent studies revealed that AGEs accumulation is considered an important factor in diabetic intervertebral disc (IVD) degeneration. However, the effect of AGEs on intervertebral disc remains unclear. AF cells were treated with various concentrations of AGEs for 3 days. Cell viability and cell proliferation were measured by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays, respectively. Cell apoptosis was examined by Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3, and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by real-time PCR (RT-PCR). Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by 5,5',6,6' -Tetrachloro-1,1',3,3'- tetraethyl-imidacarbocyanine iodide (JC-1) staining and 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probes, respectively. Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine (NAC) significantly reversed AGE-induced MMP decrease and AF cell apoptosis. These results suggested that AGEs induce rabbit AF cell apoptosis and mitochondrial pathway may be involved in AGEs-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration. N/A.

Rapamycin prevents the intervertebral disc degeneration via inhibiting differentiation and senescence of annulus fibrosus cells.

The effects of bleomycin and rapamycin on cellular senescence and differentiation of rabbit annulus fibrosus stem cells (AFSCs) were investigated using a cell culture model. The results showed that bleomycin induced cellular senescence in AFSCs as evidenced by senescence-associated secretory phenotype. The morphology of AFSCs was changed from cobblestone-like cells to pancake-like cells. The senescence-associated β-galactosidase activity, the protein expression of P16 and P21, and inflammatory-related marker gene levels IL-1β, IL-6, and TNF-α were increased in bleomycin-treated AFSCs in a dose-dependent manner. Rapamycin treatment decreased the gene expression of MMP-3, MMP-13, IL-1β, IL-6, TNF-α, and protein levels of P16 and P21 in bleomycin-treated AFSCs. Furthermore, neither bleomycin nor rapamycin changed the ribosomal S6 protein level in AFSCs. However, the phosphorylation of the ribosomal S6 protein was increased in bleomycin-treated AFSCs and decreased in rapamycin-treated AFSCs. AFSCs differentiated into adipocytes, osteocytes, and chondrocytes when they were cultured with respective differentiation media. Rapamycin inhibited multi-differentiation potential of AFSCs in a concentration-dependent manner. Our findings demonstrated that mammalian target of rapamycin (mTOR) signaling affects cellular senescence, catabolic and inflammatory responses, and multi-differentiation potential, suggesting that potential treatment value of rapamycin for disc degenerative diseases, especially lower back pain.

Annulus fibrosus cells express and utilize C-C chemokine receptor 5 (CCR5) for migration

Background ContextDisc degeneration is associated with the progressive loss of the proteoglycan content of the intervertebral disc, decreased matrix synthesis, higher concentrations of proteolytic enzymes, and increased levels of proinflammatory cytokines. In previous studies, we have shown that C-C chemokine ligand (CCL)2, CCL3, and CCL5 are highly expressed by cultured nucleus pulposus (NP) and annulus fibrosus (AF) cells that have been treated by interleukin-1. The major function of these chemokines is to recruit immune cells into the disc. It is unclear if disc cells can respond to these chemokines. Recent studies by Phillips et al. (2015) showed that NP cells express a number of cytokines and chemokine receptors. PurposeThe purpose of this study is to determine the gene and protein expression of C-C chemokine receptor (CCR)1, CCR2, and CCR5 in NP and AF cells, and to test if these receptors can respond to their ligands in these cells by cell signaling and migration. Study Design/SettingThis is an in vitro study. MethodsFor RNA, surface expression, and cell signaling studies, human cells were isolated from the NP and AF tissues collected after spine surgery or from donated spine segments (Gift of Hope Human Donor & Tissue Network of Illinois) and cultured in monolayer. The gene expression of human CCR1, CCR2, and CCR5 was analyzed using real-time polymerase chain reaction. The surface expression of CCR1, CCR2, and CCR5 was analyzed using flow cytometry and fluorescently tagged antibodies specific for these proteins. Extracellular signal-regulated kinase (ERK) phosphorylation was analyzed from the cell lysates of NP and AF cells treated with CCL2 and CCL5 for 1 hour using enzyme-linked immunosorbent assay. Migration of primary rabbit AF cells was assayed using 8-µm Corning Transwell inserts in the presence or absence of CCL5. This study was partially funded by a North American Spine Society 2014 Basic Research Grant Award ($50,000). ResultsRNA analysis showed that gene expression of CCR1, CCR2, and CCR5 was evident in human NP and AF cells (n=6). Only a small population of NP and AF cells expressed CCR1 (1.9% and 1.2%, respectively) and CCR2 (0.8% and 1.4%, respectively) on the cell surface, whereas a larger percentage expressed CCR5 (12.7% and 11.6%, respectively). Significantly higher levels of ERK phosphorylation were detected in AF cells after treatment with CCL5 and not CCL2. Treatment with either chemokine did not cause significantly higher ERK phosphorylation in NP cells. There was an increase in average AF cell migration in the presence of CCL5. The increase was significant when the migration was induced with CCL5 (500 ng/mL) at both 2- and 6-hour time points. ConclusionsCCR5 is expressed at the RNA level and on the cell surface of NP and AF cells. In the presence of CCL5, we detected increased levels of ERK phosphorylation and AF cell migration, suggesting that the CCR5 receptors in AF cells are functional. These data suggest that AF cells may have the ability to migrate in response to disc damage or inflammation.

Preliminary construction of a novel tissue-engineered BMG/PBST biphasic scaffold for annulus fibrosus in vitro

Objective To construct a novel tissue-engineered bone matrix gelatin (BMG)/poly[butylene succinate-co-terephthalate] (PBST) biphasic scaffold for annulus fibrosus. Methods The PBST spinning fibers were prepared by electrospinning and the porosity and water absorption rate were tested. Rabbit annulus fibrosus cells were isolated, cultured and identified through Safranin O staining, and collagen Ⅱ immunohistochemical staining in vitro. And then annulus fibrosus cells were implanted on the PBST fiber, whose growth situation was observed by scanning electron microscope (SEM). Then the BMG/PBST biphasic scaffold was constructed by BMG as the outer annular fibrosus and PBST fiber as the inner annular fibrosus. The annulus fibrosus cells were implanted on the biphasic scafflod and cultured for 3, 7 and 21 days in vitro. The biomechanical and biological property was observed at the predetermined time point. Results The porosity of the fiber was 61.83%±7.33% and its water absorption rate was 297.34%±57.13%. The identified result of annulus fibrosus cells were positive, suggesting that the cells have still kept their annulus fibrosus cells characteristics. The cells growth could be observed through SEM at 3rd and 7th day after implanted on the fibers. After cultured on the BMG/PBST scaffold, HE staining proved that the cells could ingress into the inner of fiber with time. Safranin O staining and collagen Ⅱ immunohistochemical staining proved that the cells can secreted abundant proteoglycan and collagen Ⅱ, the special annulus fibrosus cell extracellular matrix. Compared with the BMG/PBST scaffold without cells, the elastic modulus of biphasic scaffold was increased from 14.83±1.02 MPa to 17.56±1.47 MPa after cultured with cells for 21 days in vitro. Conclusion The novel tissue-engineered biphasic scaffold for annulus fibrosus constructed with BMG and PBST fiber spinning has good cytocompatibility and biomechanical characteristics, which provide a basis for the complete tissue engineered intervertebral disc. Key words: Tissue engineering; Tissue saffolds; Gelatin

Ropivacaine- and bupivacaine-induced death of rabbit annulus fibrosus cells in vitro: involvement of the mitochondrial apoptotic pathway

The purposes of this study were to assess whether local anesthetics (LAs), such as ropivacaine and bupivacaine, could induce apoptosis of rabbit annulus fibrosus (AF) cells in vitro and further to explore the possible underlying mechanism. Rabbit AF cells at second passage were treated with saline solution and various concentrations of LAs. Apoptosis of AF cells were examined by cell counting kit-8 (CCK-8), Annexin V assays, Hoechst 33342 staining, and Caspase-3, -9 activity assays. The expression of apoptosis-related markers was detected by real-time PCR (RT-PCR) and Western Blot. The JC-1 staining was used to evaluate the change of mitochondrial membrane potential (MMP). Moreover, the levels of reactive oxygen species (ROS) were determined with fluorescent probe DCFH-DA. The results of flow cytometry indicated that LAs could induce apoptosis of rabbit AF cells in a dose-dependent manner. Apoptosis was confirmed by cell morphology, condensed nuclei and activation of Caspase-3 and -9. In addition, the molecular data showed that LAs could significantly up-regulate the expression of Bax, accompanied by a significant down-regulation of Bcl-2 expression. Furthermore, we also observed that LAs resulted in alteration of MMP and accumulation of intracellular ROS in AF cells. Blockade of ROS production by N-acetyl-l-cysteine (NAC) inhibited LAs-induced apoptosis. These findings suggest that LAs in clinically relevant concentrations could induce apoptosis of rabbit AF cells in vitro, and the mitochondrial pathway was, at least in part, involved in the LAs-mediated apoptosis. Further investigations focusing on the potential cytotoxicity of LAs on IVD cells are needed.

616. Disc Gene Therapy: Development of a Novel Inducible System To Regulate Expression of the Therapeutic Transgene TIMP1

Introduction: Gene therapy using the anti-catabolic factor Tissue Inhibitor of Metalloproteinase-1 (hTIMP1) has shown promising efficacy in treating disc degeneration. However, a major limitation of the current gene therapy technique is the use of a constitutive promoter to drive transgene expression that results in uncontrolled and excessive expression of therapeutic gene product which could result in undesirable side effects. To overcome this important limitation, we successfully developed a new gene therapy recombinant adeno-associated viral (rAAV)vector, rAAV-NFkB-hTIMP1, in which the therapeutic recombinant human TIMP1 gene is placed under the control of a promoter containing the NF-kB element. The rAAV-NFkB-hTIMP1 construct is designed to produce the therapeutic gene product TIMP1 only when the cells experience stress which activates the NF-kB pathway. Hence we expect using this strategy that hTIMP1 will be selectively expressed in cells under inflammatory conditions typically found in active degenerating discs and not under normal conditions, in which the therapeutic gene was not needed. In this study we tested our hypothesis that rabbit annulus fibrosus cells transfected with aserotype 2 of rAAV vector carrying a hTIMP1 gene will not express high level of hTIMP1 unless stimulated with the IL-1b.Methods: Cultured rabbit annulus fibrosus (rAF) cells were divided into six groups; “Control” (cell only); “rAAV-CMV-hTIMP1(cells transfected with AAV-CMV-hTIMP1 plasmid DNA)” and “rAAV-NFkB-hTIMP1(cells transfected with AAV-NFkB-hTIMP1 plasmid DNA)” treated with and without IL-1b (n=4 for each group). Production of hTIMP1 was determined by RT-PCR. To determine if MMP activity was inhibited by the produced TIMP1, MMP activity assay was measured by following cleavage of a fluorogenic substrate.Results: RT-PCR analysis demonstrated that the level of hTIMP1 transcription from cells and culture media transfected with rAAV-NFkB-hTIMP1 construct was greater than in the IL-1b stimulated condition compared to unstimulated control. The result shows that the level of MMP activity was decreased compared to baseline levels or cells exposed to IL-1.Discussion: The RT-PCR result showed cells transfected with AAV-NFkB-hTIMP1 plasmid DNA had significant increase in the hTIMP-1 mRNA expression after stimulation with IL-1b. These results demonstrate the novelty of this strategy in which the NF-kB element containing promoter ensures that the transgene hTIMP1 is expressed highly only under conditions of inflammation typically found in injured and degenerating discs which would act to block MMP-mediated matrix proteolytic destruction. Low levels of hTIMP1 were also observed under unstimulated conditions and the bioenergetic consequences of the cell are unclear.

Co-culture of annulus fibrosus cells and bone marrow mesenchymal stem cells.

To investigate the effect on cell proliferation and extracellular matrix expression of annulus fibrosus (AF) cells when co-cultured with bone marrow mesenchymal stem cells (BMSCs). Primary isolated rabbit BMSCs and AF cells were cultured and harvested cells were placed into a 15-mL centrifugal tube co-culture system in a ratio of 2:1 (A group), 1:1 (B group), and 1:2 (C group). Cell proliferation was evaluated using cell counting kit-8, and mRNA of collagen II and mucopolysaccharide was quantified using real-time polymerase chain reaction (PCR) on Days 7, 14, and 21. The cell count, synthesized collagen II and mucopolysaccharide increased in a time-dependent manner, with a peak at Day 21. Cells in Group B proliferated faster and synthesized more collagen II and mucopolysaccharide than groups A and C, where the difference was significant. AF cells and BMSCs in the ratio of 1:1, cultured using the centrifugal tube three-dimensional co-culture system showed that BMSCs can promote AF cell proliferation and extracellular matrix synthesis.

Synergistic effect of decellularized annulus fibrosus (AF) matrix and substrate stiffness on the gene expression of rabbit AF stem cells

Synergistic effect of decellularized annulus fibrosus (AF) matrix and substrate stiffness on the gene expression of rabbit AF stem cells

Identification of rabbit annulus fibrosus-derived stem cells.

Annulus fibrosus (AF) injuries can lead to substantial deterioration of intervertebral disc (IVD) which characterizes degenerative disc disease (DDD). However, treatments for AF repair/regeneration remain challenging due to the intrinsic heterogeneity of AF tissue at cellular, biochemical, and biomechanical levels. In this study, we isolated and characterized a sub-population of cells from rabbit AF tissue which formed colonies in vitro and could self-renew. These cells showed gene expression of typical surface antigen molecules characterizing mesenchymal stem cells (MSCs), including CD29, CD44, and CD166. Meanwhile, they did not express negative markers of MSCs such as CD4, CD8, and CD14. They also expressed Oct-4, nucleostemin, and SSEA-4 proteins. Upon induced differentiation they showed typical osteogenesis, chondrogenesis, and adipogenesis potential. Together, these AF-derived colony-forming cells possessed clonogenicity, self-renewal, and multi-potential differentiation capability, the three criteria characterizing MSCs. Such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using cell therapies or tissue engineering approaches.

Annulus fibrosus cell characteristics are a potential source of intervertebral disc pathogenesis.

In the end stage of intervertebral disc degeneration, cartilage, bone, endothelial cells, and neurons appear in association with the worsening condition. The origin of the abnormal cells is not clear. This study investigated the properties of progenitor cells in the annulus fibrosus (AF) using one in vitro and two in vivo models. Cultivation of rabbit AF cells with chondrogenic media significantly increased expressions of collagen and aggrecan. Upon exposure to osteogenic conditions, the cultures showed increased mineralization and expression of osteopontin, runx2, and bmp2 genes. Two models were used in the in vivo subcutaneous implantation experiments: 1) rabbit AF tissue in a demineralized bone matrix (DBM) cylinder (DBM/AF), and, 2) rat intact and needle punctured lumbar discs. Bone formation in the AF tissue was detected and hypertrophic chondrocytes and osteoblasts were present 1 month after implantation of the DBM/AF to nude mice. In addition to collagen I and II, immunostaining shows collagen X and osteocalcin expression in DBM/AF specimens 4 months after implantation. Similar changes were detected in the injured discs. Almost the entire needle punctured disc had ossified at 6 months. The results suggest that AF cells have characteristics of progenitor cells and, under appropriate stimuli, are capable of differentiating into chondrocytes and osteoblasts in vitro as well as in vivo. Importantly, these cells may be a target for biological treatment of disc degeneration.

Regional Variations in the Cellular, Biochemical, and Biomechanical Characteristics of Rabbit Annulus Fibrosus

Tissue engineering of annulus fibrosus (AF), the essential load-bearing disc component, remains challenging due to the intrinsic heterogeneity of AF tissue. In order to provide a set of characterization data of AF tissue, which serve as the benchmark for constructing tissue engineered AF, we analyzed tissues and cells from various radial zones of AF, i.e., inner AF (iAF), middle AF (mAF), and outer AF (oAF), using a rabbit model. We found that a radial gradient in the cellular, biochemical, and biomechanical characteristics of rabbit AF existed. Specifically, the iAF cells (iAFCs) had the highest expression of collagen-II and aggrecan genes, while oAF cells (oAFCs) had the highest collagen-I gene expression. The contents of DNA, total collagen and collagen-I sequentially increased from iAF, mAF to oAF, while glycosaminoglycan (GAG) and collagen-II levels decreased. The cell traction forces of primary AFCs gradually decreased from iAFCs, mAFCs to oAFCs, being 336.6±155.3, 199.0±158.8, and 123.8±76.1 Pa, respectively. The storage moduli of iAF, mAF, and oAF were 0.032±0.002, 2.121±0.656, and 4.130±0.159 MPa, respectively. These measurements have established a set of reference data for functional evaluation of the efficacy of AF tissue engineering strategies using a convenient and cost-effective rabbit model, the findings of which may be further translated to human research.

The effects of glucosamine sulfate on intervertebral disc annulus fibrosus cells in vitro

Background contextGlucosamine has gained widespread use among patients, despite inconclusive efficacy data. Inconsistency in the clinical literature may be related to lack of understanding of the effects of glucosamine on the intervertebral disc, and therefore, improper patient selection. PurposeThe goal of our study was to investigate the effects of glucosamine on intervertebral disc cells in vitro under the physiological conditions of inflammation and mechanical loading. Study designControlled in vitro laboratory setting. MethodsIntervertebral disc cells isolated from the rabbit annulus fibrosus were exposed to glucosamine sulfate in the presence and absence of interleukin-1β and tensile strain. Outcome measures included gene expression, measurement of total glycosaminoglycans, new proteoglycan synthesis, prostaglandin E2 production, and matrix metalloproteinase activity. The study was funded by NIH/NCCAM, and the authors have no conflicts of interest. ResultsUnder conditions of inflammatory stimulation alone, glucosamine demonstrated a dose-dependent effect in decreasing inflammatory and catabolic mediators and increasing anabolic genes. However, under conditions of mechanical stimulation, although inflammatory gene expression was decreased, PGE2 was not. In addition, matrix metalloproteinase-3 gene expression was increased and aggrecan expression decreased, both of which would have a detrimental effect on matrix homeostasis. Consistent with this, measurement of total glycosaminoglycans and new proteoglycan synthesis demonstrated detrimental effects of glucosamine under all conditions tested. ConclusionsThese results may in part help to explain the conflicting reports of efficacy, as there is biological plausibility for a therapeutic effect under conditions of predominate inflammation but not under conditions where mechanical loading is present or in which matrix synthesis is needed.

A novel integrated biphasic silk fibroin scaffold for intervertebral disc tissue engineering

The intervertebral disc (IVD) tissue engineering construct provides a promising approach for the treatment of symptomatic degenerative disc diseases. In this study, a novel 3D silk fibroin biphasic scaffold for integrated IVD tissue engineering was fabricated by sequentially using the paraffin sphere-leaching method for annulus fibrosus (AF) phase and the phase separation method for nucleus pulposus (NP) phase. Both phases were perfectly integrated and possessed a highly interconnected porous structure with pore size of 220±23.1μm for AF and 90±17.8μm for NP phase. Furthermore, the scaffolds were found to have a high porosity of 91% and 93% for AF and NP phases, respectively. In addition, this silk biphasic scaffold had a relative high compressive modulus under wet conditions (150.7±6.8kPa). Rabbit AF and NP cells could attach, grow and further penetrate into the scaffold and distribute uniformly. This silk fibroin biphasic IVD scaffold emerges as a potential candidate for IVD tissue engineering.

Link Protein N-terminal Peptide Binds to Bone Morphogenetic Protein (BMP) Type II Receptor and Drives Matrix Protein Expression in Rabbit Intervertebral Disc Cells

Intervertebral disc (IVD) degeneration and associated spinal disorders are leading sources of morbidity, and they can be responsible for chronic low back pain. Treatments for degenerative disc diseases continue to be a challenge. Intensive research is now focusing on promoting regeneration of degenerated discs by stimulating production of the disc matrix. Link protein N-terminal peptide (LPP) is a proteolytic fragment of link protein, an important cross-linker and stabilizer of the major structural components of cartilage, aggrecan and hyaluronan. In this study we investigated LPP action in rabbit primary intervertebral disc cells cultured ex vivo in a three-dimensional alginate matrix. Our data reveal that LPP promotes disc matrix production, which was evidenced by increased expression of the chondrocyte-specific transcription factor SOX9 and the extracellular matrix macromolecules aggrecan and collagen II. Using colocalization and pulldown studies we further document a noggin-insensitive direct peptide-protein association between LPP and BMP-RII. This association mediated Smad signaling that converges on BMP genes leading to expression of BMP-4 and BMP-7. Furthermore, through a cell-autonomous loop BMP-4 and BMP-7 intensified Smad1/5 signaling though a feedforward circuit involving BMP-RI, ultimately promoting expression of SOX9 and downstream aggrecan and collagen II genes. Our data define a complex regulatory signaling cascade initiated by LPP and suggest that LPP may be a useful therapeutic substitute for direct BMP administration to treat IVD degeneration and to ameliorate IVD-associated chronic low back pain.

Demineralized Bone Particle Impregnated Poly(l-Lactide-co-Glycolide) Scaffold for Application in Tissue-Engineered Intervertebral Discs

Demineralized bone particle (DBP) contains powerful bioactive molecules that facilitate new bone or cartilage growth. We developed hybrid scaffolds of poly(l-lactide-co-glycolide) (PLGA) with various concentrations of DBP (DBP/PLGA), of which phenotypes on intervertebral disc (IVD) cells were investigated. The hybrid scaffold has a cylindrical donut shape with two distinct parts; the inner is for the nucleus pulposus (NP) and the outer is for annulus fibrosus (AF). Rabbit NP and AF cells were seeded into the inner and outer regions of the DBP/PLGA scaffolds separately. Disc cell viability in DBP/PLGA scaffolds was superior to pure PLGA scaffold and increased with increasing DBP concentration. In vitro- and in vivo-formed tissues were characterized by RT-PCR, Safranin-O, Masson’s trichrome staining and immunohistochemi- cal staining for type-I and type-II collagen. DBP/PLGA hybrid scaffolds revealed more active expression of disc phenotypes, as characterized by protein and mRNA expression, than the PLGA control. This study provides valuable information for potential disc replacement using DBP and PLGA.

Determination of annulus fibrosus cell response to tensile strain as a function of duration, magnitude, and frequency

As clinical evidence suggests that mechanical forces can have both reparative and traumatic effects on the spine, we investigated responses to different magnitudes, frequencies, and durations of applied tensile strain in an in vitro system. We examined the interactions of inflammatory and mechanical stimuli on cells isolated from the annulus fibrosus. Rabbit annulus fibrosus fibrochondrocytes were cultured in the presence or absence of an inflammatory stimulus. Cells were exposed to various magnitudes and frequencies of tensile strain for 4 or 24 h, and mRNA expression of catabolic mediators of inflammation and matrix degradation was measured by quantitative real time PCR and compared to control cells. Conditioned media were analyzed for matrix metalloprotease activity and production of prostaglandin E(2) . Application of low magnitudes and frequencies of tensile strain resulted in down-regulation of catabolic mediators, particularly under inflammatory stress. However, loss of this protective effect was observed at higher frequency and magnitude, and after prolonged duration. These in vitro data confirm the existence of magnitude, frequency, and duration based effects, which determine biochemical response of disc tissue resulting in either anti- or pro-catabolic outcomes. This may help to explain the beneficial effects of motion-based therapies as well as the destructive effect of traumatic levels of applied strain.