Human Annulus Research Articles

An annulus fibrosus closure device based on a biodegradable shape-memory polymer network

Injuries to the intervertebral disc caused by degeneration or trauma often lead to tearing of the annulus fibrosus (AF) and extrusion of the nucleus pulposus (NP). This can compress nerves and cause lower back pain. In this study, the characteristics of poly(d,l-lactide-co-trimethylene carbonate) networks with shape-memory properties have been evaluated in order to prepare biodegradable AF closure devices that can be implanted minimally invasively. Four different macromers with (d,l-lactide) to trimethylene carbonate (DLLA:TMC) molar ratios of 80:20, 70:30, 60:40 and 40:60 with terminal methacrylate groups and molecular weights of approximately 30 kg mol−1 were used to prepare the networks by photo-crosslinking. The mechanical properties of the samples and their shape-memory properties were determined at temperatures of 0 °C and 40 °C by tensile tests- and cyclic, thermo-mechanical measurements. At 40 °C all networks showed rubber-like behavior and were flexible with elastic modulus values of 1.7–2.5 MPa, which is in the range of the modulus values of human annulus fibrosus tissue. The shape-memory characteristics of the networks were excellent with values of the shape-fixity and the shape-recovery ratio higher than 98 and 95%, respectively. The switching temperatures were between 10 and 39 °C. In vitro culture and qualitative immunocytochemistry of human annulus fibrosus cells on shape-memory films with DLLA:TMC molar ratios of 60:40 showed very good ability of the networks to support the adhesion and growth of human AF cells. When the polymer network films were coated by adsorption of fibronectin, cell attachment, cell spreading, and extracellular matrix production was further improved. Annulus fibrosus closure devices were prepared from these AF cell-compatible materials by photo-polymerizing the reactive precursors in a mold. Insertion of the multifunctional implant in the disc of a cadaveric canine spine showed that these shape-memory devices could be implanted through a small slit and to some extent deploy self-sufficiently within the disc cavity.

Mechanical properties of the extra‐fibrillar matrix of human annulus fibrosus are location and age dependent

The mechanical behavior of the annulus fibrosus (AF) of the intervertebral disc can be modeled as a mixture of fibers, extra-fibrillar matrix (EFM), ions, and fluid. However, the properties of the EFM have not been measured directly. We measured mechanical properties of the human EFM at several locations, determined the effect of age and degeneration, and evaluated whether changes in EFM properties correspond to AF compositional changes. EFM mechanical properties were measured using a method that combines osmotic loading and confined compression. AF samples were dissected from several locations, and mechanical properties were correlated with age, degeneration, and composition. EFM modulus was found to range between 10 and 50 kPa, increasing nonlinearly with compression magnitude and being highest in the AF outer-anterior region. EFM properties were not correlated with composition or degeneration. However, the EFM modulus, its relative contribution to tissue modulus, and model parameters were correlated with age. These measurements will result in more accurate predictions of deformations in the intervertebral disc. Additionally, parameters such as permeability and diffusivity used for biotransport analysis of glucose and other solutes depend on EFM deformation. Consequently, the accuracy of biotransport simulations will be greatly improved.

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.

Insights into the Hallmarks of Human Nucleus Pulposus Cells with Particular Reference to Cell Viability, Phagocytic Potential and Long Process Formation

Objective: As a main cellular component within the disc, nucleus pulposus (NP) cells play important roles in disc physiology. However, little is known on the biologic hallmarks of human NP cells. Therefore, the present study aimed to address the features of human NP cells.Methods: Human NP samples were collected from normal cadavers, patients with scoliosis and disc degeneration as normal, disease control and degenerative NP, respectively. The NP samples were studied using transmission electron microscopy and TUNEL assay. Pre-digested NP samples were studied using flow cytometry with PI/Annexin V staining.Results: Both control and degenerative human NP consisted of mainly viable cells with a variety of morphology. Both necrosis and apoptosis were noted in human NP as forms of cell death with increased apoptosis in degenerative NP, which was further confirmed by the TUNEL assay. Phagocytic NP cells had the hallmarks of both stationary macrophages with lysosomes and NP cells with the endoplasmic reticulum. Annulus fibrosus cells have similar morphologic characteristics with NP cells in terms of cell nest, phagocytosis and intracellular organs. Moreover, NP cells with long processes existed in degenerative and scoliotic NP rather than normal NP. When cultured in glucose-free medium, NP cells developed long and thin processes.Conclusion: Human degenerative NP consists of primarily viable cells. We present direct and in vivo evidence that both human annulus fibrosus and NP cells have phagocytic potential. Moreover, NP cells with long processes exist in both scoliotic and degenerative NP with lack of glucose as one of the possible underlying mechanisms.

Rabbit Notochordal Cells Modulate the Expression of Inflammatory Mediators by Human Annulus Fibrosus Cells Cocultured With Activated Macrophage-Like THP-1 Cells

We evaluated the influence of rabbit notochordal cells on the expression of inflammatory mediators by human annulus fibrosus (AF) cells cocultured with macrophage-like cells. To identify the protective effect of rabbit notochordal cells on AF during in vitro inflammation. Discogenic pain, which is an important cause of intractable lower back pain, is associated with macrophage-mediated inflammation in the AF. Although rabbit notochordal cells prevent intervertebral disc degeneration, their effects on human AF inflammation remain unknown. Human AF pellets were cocultured for 48 hours with notochordal cell clusters from adult New Zealand White rabbits and phorbol myristate acetate (PMA)-stimulated human macrophage-like THP-1 cells. Conditioned media (CM) from the cocultures were assayed by enzyme-linked immunosorbent assay. The expression of inflammatory mediators in the AF pellets was evaluated by real-time reverse-transcription polymerase chain reaction. The levels of mRNA for interleukin (IL)-6, IL-8, and inducible nitric oxide synthase (iNOS) in the AF pellets cocultured with notochordal cells and macrophages (hAF[rNC-M]) were significantly lower than those in the AF pellets cultured with macrophages alone (hAF[M]) (P < 0.05). The levels of IL-6 and IL-8 proteins in the CM of hAF(rNC-M) were significantly lower than those in the CM of hAF(M) (P < 0.05). Coculturing with notochordal cells significantly decreased the levels of mRNA for IL-6, IL-8, and iNOS in the macrophage-exposed AF pellets (P < 0.05). After 1 ng/mL IL-1β stimulation, the levels of IL-6 and IL-8 mRNA and the level of IL-8 protein production were significantly decreased in the AF pellets with notochordal cells compared with naïve AF pellets (P < 0.05). In an in vitro coculture system, rabbit notochordal cells reduced the levels of main inflammatory mediators and gene expression in the human AF during inflammation. Therefore, rabbit notochordal cells may constitute an important protective tool against symptomatic disc development.

Rabbit Notochordal Cells Modulate the Expression of Inflammatory Mediators by Human Annulus Fibrosus Cells Cocultured with Activated Macrophage-Like THP-1 Cells

Discogenic pain, which is an important cause of intractable lower back pain, is associated with macrophage-mediated inflammation in the AF. Although rabbit notochordal cells prevent intervertebral disc degeneration, their effects on human AF inflammation remain unknown.

Nutrient Transport in Human Annulus Fibrosus is Affected by Compressive Strain and Anisotropy

The avascular intervertebral disc (IVD) receives nutrition via transport from surrounding vasculature; poor nutrition is believed to be a main cause of disc degeneration. In this study, we investigated the effects of mechanical deformation and anisotropy on the transport of two important nutrients--oxygen and glucose--in human annulus fibrosus (AF). The diffusivities of oxygen and glucose were measured under three levels of uniaxial confined compression--0, 10, and 20%--and in three directions--axial, circumferential, and radial. The glucose partition coefficient was also measured at three compression levels. Results for glucose and oxygen diffusivity in AF ranged from 4.46 × 10(-7) to 9.77 × 10(-6) cm(2)/s and were comparable to previous studies; the glucose partition coefficient ranged from 0.71 to 0.82 and was also similar to previous results. Transport properties were found to decrease with increasing deformation, likely caused by fluid exudation during tissue compression and reduction in pore size. Furthermore, diffusivity in the radial direction was lower than in the axial or circumferential directions, indicating that nutrient transport in human AF is anisotropic. This behavior is likely a consequence of the layered structure and unique collagen architecture of AF tissue. These findings are important for better understanding nutritional supply in IVD and related disc degeneration.

Commentary: Does needle injection cause disc degeneration? News in the continuing debate regarding pathophysiology associated with intradiscal injections

COMMENTARY ON Gruber HE, Rhyne AL, Hansen KJ, et al. Deleterious effects of discography radiocontrast solution on human annulus cell in vitro: changes in cell viability, proliferation, and apoptosis in exposed cells. Spine J 2012;12:329–35 (in this issue). Gruber HE, Rhyne AL, Hansen KJ, et al. Deleterious effects of discography radiocontrast solution on human annulus cell in vitro: changes in cell viability, proliferation, and apoptosis in exposed cells. Spine J 2012;12:329–35 (in this issue). Deleterious effects of discography radiocontrast solution on human annulus cell in vitro: changes in cell viability, proliferation, and apoptosis in exposed cellsThe Spine JournalVol. 12Issue 4PreviewCarragee et al. have recently shown that modern discography injections are associated with subsequent acceleration of disc degeneration, herniation, and loss of disc height. Although needle puncture and pressurization are known trauma events that can create disc degeneration in animal models, another likely culprit in clinical discography–associated degeneration is a direct effect of the contrast agent itself on disc cells. Full-Text PDF

Deleterious effects of discography radiocontrast solution on human annulus cell in vitro: changes in cell viability, proliferation, and apoptosis in exposed cells

Carragee etal. have recently shown that modern discography injections are associated with subsequent acceleration of disc degeneration, herniation, and loss of disc height. Although needle puncture and pressurization are known trauma events that can create disc degeneration in animal models, another likely culprit in clinical discography-associated degeneration is a direct effect of the contrast agent itself on disc cells. To test the hypothesis that discography contrast solution would have a deleterious effect on human annulus cells invitro. An invitro study using cultured human annulus cells to assay cell death, cell proliferation, and apoptosis. Annulus cells from eight surgical disc specimens were evaluated (two Thompson Grade III discs and six Grade IV discs) for cell death and proliferation, and an additional five cultures were tested for apoptosis. The proportion of dead and live cells, cell proliferation, and the proportion of apoptotic cells in control and experimental groups. After internal review board approval, experimental design used two sets of controls: untreated cells under our normal culture conditions (control) and a set with added glucose to adjust the osmolality to match respective Isovue radiocontrast solution treatments (glucose controls) using a freezing point osmometer. Treated cells received Isovue 200 (iopamidol, Isovue-M 200; Bracco Diagnostics, Inc., Princeton, NJ, USA) at 12.5, 25, 50, or 100 mg/mL. Twenty thousand cells/well were seeded in triplicate in 24 well plates, control or test media added, and incubated for 24 hours. At termination, dead cells were identified with trypan blue staining and percentage dead cells determined. Cells were also tested to determine the percentage of apoptotic cells after 50 or 100 mg/mL Isovue exposures. Proliferation assays used standard plate reader methods. Statistical analysis used repeated measures analysis of variance with SAS software (version 9.2; SAS Institute, Inc., Cary, NC, USA). Analysis of cell death showed a significant increase in the percentage of dead cells with increasing Isovue concentrations compared with control cells (p=.018-.0008). Cell proliferation analyses showed significantly reduced division in Isovue-treated cells (p=.004), and apoptosis assays revealed a significantly higher proportion of apoptotic cells in cells exposed to 50 and 100 mg/mL Isovue (p=.016 and .0003, respectively). Discography is used extensively in the evaluation of low back pain. Because the lifetime prevalence of disc degeneration and low back pain is high (80% in the general population), many patients may undergo this procedure. Data presented here show that cells exposed invitro to a radiocontrast agent with adjustments for osmolality have significantly reduced proliferation, increased cell death, and increased programmed cell death (apoptosis). In light of the well-recognized age- and degeneration-related decrease in disc cell numbers, it is possible that radiocontrast exposure may be contributing significantly to disc cell loss with subsequent progression of disc degeneration. Findings presented here provide a plausible cell-based explanation for the previously reported disc degeneration in patients receiving discography contrast solutions.

Genome-Wide Analysis of Pain-, Nerve- and Neurotrophin -Related Gene Expression in the Degenerating Human Annulus

BackgroundIn spite of its high clinical relevance, the relationship between disc degeneration and low back pain is still not well understood. Recent studies have shown that genome-wide gene expression studies utilizing ontology searches provide an efficient and valuable methodology for identification of clinically relevant genes. Here we use this approach in analysis of pain-, nerve-, and neurotrophin-related gene expression patterns in specimens of human disc tissue. Control, non-herniated clinical, and herniated clinical specimens of human annulus tissue were studied following Institutional Review Board approval.ResultsAnalyses were performed on more generated (Thompson grade IV and V) discs vs. less degenerated discs (grades I-III), on surgically operated discs vs. control discs, and on herniated vs. control discs. Analyses of more degenerated vs. less degenerated discs identified significant upregulation of well-recognized pain-related genes (bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase). Nerve growth factor was significantly upregulated in surgical vs. control and in herniated vs. control discs. All three analyses also found significant changes in numerous proinflammatory cytokine- and chemokine-related genes. Nerve, neurotrophin and pain-ontology searches identified many matrix, signaling and functional genes which have known importance in the disc. Immunohistochemistry was utilized to confirm the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus.ConclusionsFindings point to the utility of microarray analyses in identification of pain-, neurotrophin and nerve-related genes in the disc, and point to the importance of future work exploring functional interactions between nerve and disc cells in vitro and in vivo. Nerve, pain and neurotrophin ontology searches identified numerous changes in proinflammatory cytokines and chemokines which also have significant relevance to disc biology. Since the degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines, these substances are not cleared from the tissue and remain there over time. We hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production.

The Chemokines CXCL10 and XCL1 Recruit Human Annulus Fibrosus Cells

Human annulus fibrosus tissue and cells were analyzed for the presence of chemokine receptors and the migratory effect of selected chemokines. To investigate spontaneous repair mechanisms and underlying cell recruitment in response to annular tears and degenerative defects. Resorption of herniated disc tissue and the attempt to close annulus tears with repair tissue occur spontaneously. Although chemokines are suggested to play a role in resorption of herniated disc tissue, the role of chemokines in annulus fibrosus homeostasis and repair remains unclear. Cells were isolated from annulus fibrosus tissue and expanded in the presence of human serum. Multiwell chemotaxis assays were used to analyze the migratory effect of human serum and 0 to 1000 nM concentrations of the chemokines CXCL7, CXCL10, CXCL12, CCL25, and XCL1 on annulus fibrosus cells (AFCs) (n = 9 per chemokine and dose). Presence of corresponding chemokine receptors in AFCs was determined by real-time polymerase chain reaction analysis and immunohistochemistry. Serum (0.1%-10%) significantly (P < 0.01) stimulates the migration of AFCs. Compared with untreated cells, the migration of cells was significantly (P < 0.01) enhanced upon stimulation with 100 to 1000 nM CXCL10 and 1000 nM XCL1. Chemokine receptors showed low expression levels in expanded AFCs as assessed by polymerase chain reaction. Immunohistochemical staining of the CXCL10 receptor CXCR3 and the XCL1 receptor XCR1 showed that the presence of the particular receptors in AFCs expanded under conventional cell culture conditions. In native annulus fibrosus tissue, CXCR3 was evident, whereas XCR1 could not be detected. The findings suggest that chemokines, in particular CXCL10, effectively recruit isolated AFCs. This suggests that chemokines are involved in annulus fibrosus homeostasis and potentially in spontaneous annulus repair attempts. This might have important implications for biological annulus-sealing strategies.

Type IX Collagen Neo-Deposition in Degenerative Discs of Surgical Patients Whether Genotyped Plus or Minus for COL9 Risk Alleles

Immunohistochemical analysis of type IX collagen in disc tissue from spinal fusion patients. To determine if collagen IX can be detected in adult disc tissue removed at spinal fusion surgery from patients either with or without degeneration-associated tryptophan single nucleotide polymorphisms (SNPs) and whether the distribution is associated either with severity of degeneration or incidence of a collagen IX SNP genotype. Genetic factors are strongly associated with risk of development and/or progression of disc degeneration. Two SNPs that introduce tryptophan polymorphisms in COL9A2 and COL9A3 are independently linked to an increased risk of lumbar disc disease. Although tryptophan variants are associated with accelerated degeneration, it is not known if collagen IX can be detected in adult disc tissue. We selected age-matched disc samples from five clinical groups: fracture with Trp(-) (six cases), herniation (six cases), degeneration (five cases), spondylolisthesis with Trp(-) (eight cases), and spondylolisthesis/herniation/fracture with Trp(+) (six cases of Trp3 allele and one case of Trp2 allele). Using hematoxylin and eosin staining and immunohistochemical staining (collagens IX and IIA), 78 sections from 32 patients were analyzed. Selected disc tissues were assayed biochemically for collagen IX. Focal deposition of collagen IX was observed in regions of adult human disc tissue from spines showing degenerative changes in patients whether or not they were positive for a tryptophan SNP. However, in nondegenerative control disc tissue from fracture cases, little or no collagen IX was detected. The latter finding was confirmed by direct biochemical analyses for collagen IX in pooled samples of normal adult human annulus fibrosus or nucleus pulposus. During growth and maturation of the disc, collagen IX is presumably removed completely during matrix remodeling so that the protein is absent from normal adult annulus and nucleus but can reappear at sites of degeneration presumably as part of a repair response to mechanical injury.

Interleukin 1-beta (IL-1) Exposure Significantly Upregulates Neurotrophin Gene Expression for Brain-Derived Neurotrophic Factor, Neurotrophin 3 and Neuropilin 2 During 3D Culture of Human Annulus Cells

Interleukin 1-beta (IL-1) Exposure Significantly Upregulates Neurotrophin Gene Expression for Brain-Derived Neurotrophic Factor, Neurotrophin 3 and Neuropilin 2 During 3D Culture of Human Annulus Cells

Deleterious Effects of Discography Radiocontrast Solution on Human Annulus Cells in Vitro: Changes in Cell Viability, Proliferation and Apoptosis in Exposed Cells

Deleterious Effects of Discography Radiocontrast Solution on Human Annulus Cells in Vitro: Changes in Cell Viability, Proliferation and Apoptosis in Exposed Cells

Matrix metalloproteinase-26, a novel MMP, is constitutively expressed in the human intervertebral disc in vivo and in vitro

Matrix metalloproteinase (MMP) regulation and expression is important in the aging/degenerating human intervertebral disc. MMP-26 (also known as matrilysin-2 or endometase) is a newly discovered MMP which degrades type IV collagen, fibronectin, fibrinogen, vitronectin, denatured collagen types I–IV, insulin-like growth factor binding protein 1, and activated pro-MMP-9. Our objective here was to determine if it is present in human disc tissue and cultured disc cells. Immunohistochemistry and microarray gene expression analyses were used to evaluate the presence of MMP-26 in human disc tissue from healthy and degenerated discs. Immunohistochemistry was also applied to human annulus cells cultured in a collagen sponge. Cellular and matrix localization of MMP-26 was identified in the outer and inner annulus and in the nucleus pulposus. Fewer cells showed localization in the inner vs. outer annulus, and localization was sparse in the nucleus. During in vitro culture of annulus cells, MMP-26 was also expressed. Molecular analyses showed significant downregulation of expression of MMP-26 (p=0.03), and significant 9.8-fold upregulation of TGF-beta (p=0.01) in more degenerated discs vs. healthier discs. Findings document the first identification of MMP-26 in the disc at the molecular and protein levels. Results point to the potentially important role of MMP-26 in matrix modulation during disc health and degeneration.

Fibre–matrix interaction in the human annulus fibrosus

Although the mechanical behaviour of the human annulus fibrosus has been extensively studied, the interaction between the collagen fibres and the ground matrix has not been well understood and is therefore ignored by most constitutive models. The objective of this study is to identify the significance of the fibre–matrix interaction in the human annulus fibrosus by careful investigation of the experimental data, the theoretical constitutive models, and the numerical simulation results in the literature. Based on the experimental results from biaxial and uniaxial tests, it is shown that the mechanical behaviour of the matrix can be well simulated by an incompressible neo-Hookean type model, but the effective stiffness of the matrix depends on fibre stretch ratio, which can only be explained by fibre–matrix interaction. Furthermore, we find that this interaction takes place anisotropically between the matrix and the fibres distributed in different proportions in different directions. The dependence of the tangent stiffness of the matrix on the first invariant of the deformation tensor can also be explained by this fibre orientation dispersion.

Mitochondrial gene expression in the human annulus: in vivo data from annulus cells and selectively harvested senescent annulus cells

Mitochondrial dysfunction is recognized during cell senescence and apoptosis, two important components of human disc aging/degeneration. We hypothesize that mitochondrial dysfunction is present in the degenerating and senescent annulus cells. The objective of the present study was to analyze gene expression profiles related to mitochondrial function invivo. This study had two objectives in the analysis of gene expression patterns related to mitochondria in the human annulus: First, to assess human annulus cells in a genome-wide microarray analysis approach to evaluate mitochondrial gene expression in annulus tissue from degenerated compared with healthier discs. Second, to use laser capture microdissection (LCM) to selectively isolate senescent versus nonsenescent annulus cells to evaluate their mitochondrial gene expression patterns. Following approval by our Human Subjects Institutional Review Board, annulus cells from 20 human lumbar discs were analyzed for gene groups related to mitochondrial function; a subset was also analyzed, which focused on senescent versus nonsenescent annulus cells in a study of annulus cells from 10 lumbar discs. Human annulus tissue was used in molecular studies following institutional review board approval. Gene expression levels identified with microarray analyses were statistically evaluated using GeneSifter Web-based software (VizX Labs, Seattle, WA, USA). Human annulus specimens were assessed for gene expression related to mitochondrial function. Approaches used whole annulus tissue and senescent or nonsenescent annulus cells selectively harvested using LCM. Microarray data were analyzed using gene ontology searches and GeneSifter Web-based software. Analysis of annulus cells compared mitochondrial gene expression patterns in annulus cells from more degenerated discs with patterns in annulus cells derived from healthier discs. Important findings included significant upregulation of p53 and several proapoptotic genes (including apoptosis-inducing factor, mitochondrion-associated 1, BCL2-like 11 [an apoptosis facilitator]; caspase 7 apoptosis-related cysteine peptidase; proteasome 26S subunit nonadenosine triphosphatase 10, programmed cell death 6, and reticulon 3). Methionine sulfoxide reductase (Msr), a repair enzyme that reduces methionine sulfoxide residues in proteins damaged by oxidation, was also significantly upregulated (2.02-fold increase). The gene "membrane-associated ring finger (C3HC4) 5" was significantly upregulated and relevant because it is believed to play a role in preventing cell senescence acting to regulate mitochondrial quality control. Nitric oxide synthase 3 (endothelial nitric oxide synthase [eNOS]) showed a 5.9-fold downregulation in more degenerated versus healthier annulus cells. In LCM-harvested senescent cells, Msr was significantly downregulated in senescent versus nonsenescent cells, a finding previously recognized in other types of senescent cells. Novel data showed that significant gene expression patterns are present in the human annulus related to mitochondrial dysfunction; changes were identified in important genes involving apoptosis, eNOS and Msr expressions, and solute carrier genes. Because current research efforts are focusing on bioactive compounds for mitochondria, we suggest that future biologic cell-based therapies for annulus degeneration should also consider mitochondrial-focused therapies.

Human annulus fibrosus material properties from biaxial testing and constitutive modeling are altered with degeneration

The annulus fibrosus (AF) of the intervertebral disk undergoes large and multidirectional stresses and strains. Uniaxial tensile tests are limited for measuring AF material properties, because freely contracting edges can prevent fiber stretch and are not representative of in situ boundary conditions. The objectives of this study were to measure human AF biaxial tensile mechanics and to apply and validate a constitutive model to determine material properties. Biaxial tensile tests were performed on samples oriented along the circumferential-axial and the radial-axial directions. Data were fit to a structurally motivated anisotropic hyperelastic model composed of isotropic extra-fibrillar matrix, nonlinear fibers, and fiber-matrix interactions (FMI) normal to the fibers. The validated model was used to simulate shear and uniaxial tensile behavior, to investigate AF structure-function, and to quantify the effect of degeneration. The biaxial stress-strain response was described well by the model (R (2)>0.9). The model showed that the parameters for fiber nonlinearity and the normal FMI correlated with degeneration, resulting in an elongated toe-region and lower stiffness with degeneration. The model simulations in shear and uniaxial tension successfully matched previously published circumferential direction Young's modulus, provided an explanation for the low values in previously published axial direction Young's modulus, and was able to simulate shear mechanics. The normal FMI were important contributors to stress and changed with degeneration, therefore, their microstructural and compositional source should be investigated. Finally, the biaxial mechanical data and constitutive model can be incorporated into a disk finite element model to provide improved quantification of disk mechanics.

Enhancement of Matrix Production and Cell Proliferation in Human Annulus Cells Under Bioreactor Culture

Tissue engineering is a promising approach for treatment of disc degeneration. Herein, we evaluated effects of rotating bioreactor culture on the extracellular matrix production and proliferation of human annulus fibrosus (AF) cells. AF cells were embedded into alginate beads, and then cultured up to 3 weeks in a rotating wall vessel bioreactor or a static vessel. By real-time reverse transcription-polymerase chain reaction, expression of aggrecan, collagen type I and type II, and collagen prolyl 4-hydroxylase II was remarkably elevated, whereas expression of matrix metalloproteinase 3 and a disintegrin and metalloproteinase with thrombospondin motifs 5 was significantly decreased under bioreactor. Biochemical analysis revealed that the levels of the whole cell-associated proteoglycan and collagen were approximately five- and twofolds in rotating bioreactor, respectively, compared to those in static culture. Moreover, AF cell proliferation was augmented in rotating bioreactor. DNA contents were threefolds higher in rotating bioreactor than that in static culture. Expression of the proliferating cell nuclear antigen was robustly enhanced in rotating bioreactor as early as 1 week. Our findings suggested that rotating bioreactor culture would be an effective technique for expansion of human annulus cells for tissue engineering driven treatment of disc degeneration.

Differential effects of nicotine and tobacco smoke condensate on human annulus fibrosus cell metabolism

Tobacco smoking increases the risk of intervertebral disc degeneration (IDD) and back pain, but the mechanisms underlying the adverse effects of smoking are largely unknown. Current hypotheses predict that smoking contributes to IDD indirectly through nicotine-mediated vasoconstriction which limits the exchange of nutrients between the discs and their surroundings. We alternatively hypothesize that direct contact of disc cells, that is, cells in the outermost annulus and those present along fissures in degenerating discs, with the vascular system containing soluble tobacco smoking constituents could perturb normal metabolic activities resulting in IDD. In this study, we tested our hypothesis by comparing the effects of direct exposure of human disc cells to tobacco smoke condensate and nicotine on cell viability and metabolic activity. We showed that smoke condensate, which contains all of the water-soluble compounds inhaled by smokers, exerts greater detrimental effects on human disc cell viability and metabolism than nicotine. Smoke condensate greatly induced an inflammatory response and gene expression of metalloproteinases while reduced active matrix synthesis and expression of matrix structural genes. Therefore, we have demonstrated that disc cell exposure to the constituents of tobacco smoke has negative consequences which have the potential to alter disc matrix homeostasis.