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Intervertebral disc (IVD) herniation is a leading cause of disability and lower back pain, causing enormous socioeconomic burdens. The standard of care for disc herniation is nucleotomy, which alleviates pain but does not repair the annulus fibrosus (AF) defect nor recover the biomechanical function of the disc. Existing bioadhesives for AF repair are limited by insufficient adhesion and significant mechanical and geometrical mismatch with the AF tissue, resulting in the recurrence of protrusion or detachment of bioadhesives. Here, we report a composite hydrogel sealant constructed from a composite of a three-dimensional (3D)-printed thermoplastic polyurethane (TPU) mesh and tough hydrogel. We tailored the fiber angle and volume fraction of the TPU mesh design to match the angle-ply structure and mechanical properties of native AF. Also, we proposed and tested three types of geometrical design of the composite hydrogel sealant to match the defect shape and size. Our results show that the sealant could mimic native AF in terms of the elastic modulus, flexural modulus, and fracture toughness and form strong adhesion with the human AF tissue. The bovine IVD tests show the effectiveness of the composite hydrogel sealant for AF repair and biomechanics recovery and for preventing herniation with its heightened stiffness and superior adhesion. By harnessing the combined capabilities of 3D printing and bioadhesives, these composite hydrogel sealants demonstrate promising potential for diverse applications in tissue repair and regeneration.

Avascular dense connective tissues (e.g., the annulus fibrosus (AF) rupture, the meniscus tear, and tendons and ligaments injury) repair remains a challenge due to the "biological barrier" that hinders traditional drug permeation and limits self-healing of the injured tissue. Here, accurate delivery of nitric oxide (NO) to penetrate the "AF biological barrier" is achieved thereby enabling programmable AF repair. NO-loaded BioMOFs are synthesized and mixed in a modified polyvinyl alcohol and PCL-composited electrospun fiber membrane with excellent reactive oxygen species-responsive capability (LN@PM). The results show that LN@PM could respond to the high oxidative stress environment at the injured tissue and realize continuous and substantial NO release. Based on low molecular weight and lipophilicity, NO could penetrate through the "biological barrier" for accurate AF drug delivery. Moreover, the dynamic characteristics of the LN@PM reaction can be matched with the pathological microenvironment to initiate programmable tissue repair including sequential remodeling microenvironment, reprogramming the immune environment, and finally promoting tissue regeneration. This tailored programmable treatment strategy that matches the pathological repair process significantly repairs AF, ultimately alleviating intervertebral disc degeneration. This study highlights a promising approach for avascular dense connective tissue treatment through intelligent NO release, effectively overcoming "AF biological barriers" and programmable treatment., (© 2024 Wiley‐VCH GmbH.)

Oxidative stress and apoptosis play important roles in the pathogenesis of various degenerative diseases. Previous studies have shown that naringin can exert therapeutic effects in multiple degenerative diseases by resisting oxidative stress and inhibiting apoptosis. Although naringin is effective in treating degenerative disc disease, the underlying mechanism remains unclear. This study is aimed at investigating the effects of naringin on oxidative stress, apoptosis, and intervertebral disc degeneration (IVDD) induced by cyclic stretch and the underlying mechanisms in vitro and in vivo . Abnormal cyclic stretch was applied to rat annulus fibrosus cells, which were then treated with naringin, to observe the effects of naringin on apoptosis, oxidative stress, mitochondrial function, and the nuclear factor- (NF-) κ B signaling pathway. Subsequently, a rat model of IVDD induced by dynamic and static imbalance was established to evaluate the effects of naringin on the degree of degeneration (using imaging and histology), apoptosis, and oxidative stress in the serum and the intervertebral disc. Naringin inhibited the cyclic stretch-induced apoptosis of annulus fibrosus cells, reduced oxidative stress, improved mitochondrial function, enhanced the antioxidant capacity, and suppressed the activation of the NF- κ B signaling pathway. Additionally, it reduced the degree of IVDD (evaluated using magnetic resonance imaging) and the level of oxidative stress and inhibited apoptosis and p-P65 expression in the intervertebral discs of rats. Thus, naringin can inhibit cyclic stretch-induced apoptosis and delay IVDD, and the underlying mechanism may be related to the inhibition of oxidative stress and activation of the NF- κ B signaling pathway. Naringin may be an effective drug for treating degenerative disc disease., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2022 Yue-Hui Zhang et al.)

Background: Intervertebral disc is responsible for absorbing and transmitting mechanical compression. Under physiological conditions, the peripheral annulus fibrosus (AF) cells are subjected to different magnitudes of transverse mechanical stretch depending on the swelling of the central nucleus pulposus tissue. However, the biological behavior of AF cells under mechanical stretch is not well studied., Objective: This study was performed to study the effects of mechanical tension on AF cell senescence and the potential signaling transduction pathway., Methods: Rat AF cells were made to experience different magnitudes of mechanical stretch (2% elongation and 20% elongation for 4 hours every day at 1 Hz) in a 10-day experiment period. The inhibitor RKI-1447 of the Rho-associated coiled-coil-containing protein kinases (ROCK) was added along with culture medium to investigate its role. Cell proliferation, cell cycle, telomerase activity, and expression of senescence markers (p16 and p53) were analyzed., Results: We found that 20% elongation significantly decreased cell proliferation, promoted G0/G1 cell cycle arrest, decreased telomerase activity, and upregulated mRNA/protein expression of p16 and p53. Moreover, the inhibitor RKI-1447 partly resisted effects of 20% elongation on these parameters of cell senescence., Conclusion: High mechanical stretch obviously induces AF cell senescence through the RhoA/ROCK pathway. This study provides us a deeper understanding on the AF cell's behavior under mechanical stretch., Competing Interests: The authors declared no conflicts of interest., (Copyright © 2021 Li Ning et al.)

During disc degeneration, the increase of inflammatory cytokines and decrease of disc cell density are two prominent features. Enhanced inflammatory reaction contributes to disc annulus fibrosus (AF) cell apoptosis. In this study, we investigated whether resveratrol can suppress AF cell apoptosis in an inflammatory environment. Rat disc AF cells were cultured in medium with or without tumor necrosis factor- α (TNF- α ). Resveratrol was added along with the culture medium supplemented with TNF- α . Caspase-3 activity, cell apoptosis ratio, expression of apoptosis-associated molecules (Bcl-2, Bax, caspase-3, cleaved PARP, and cleaved caspase-3), reactive oxygen species (ROS) content, and the total superoxide dismutase (SOD) activity were measured. Our results showed that TNF- α significantly increased caspase-3 activity and AF cell apoptosis ratio and upregulated gene/protein expression of Bax, caspase-3, cleaved caspase-3, and cleaved PARP, whereas it downregulated the expression of Bcl-2. Moreover, TNF- α significantly increased ROS content but decreased the total SOD activity. Further analysis demonstrated that resveratrol partly attenuated the effects of TNF- α on AF cell apoptosis-associated parameters, decreased ROS content, and increased the total SOD activity in the AF cells treated with TNF- α . In conclusion, resveratrol attenuates inflammatory cytokine TNF- α -induced AF cell apoptosis through regulating oxidative stress reaction in vitro. This study sheds a new light on the protective role of resveratrol in alleviating disc degeneration., Competing Interests: No conflicts of interest exist in this work., (Copyright © 2021 Qunqun Shan et al.)

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16 INK4a , p19 ARF , and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration., (© 2021. The Author(s).)

Inflammation response is an important reason for disc cell senescence during disc degeneration. Recently, melatonin is suggested to protect against disc degeneration. However, the effects of melatonin on annulus fibrosus (AF) cell senescence are not fully studied. The main purpose of this study was to investigate the effects of melatonin on AF cell senescence in an inflammatory environment and the underlying mechanism. Rat disc AF cells were cultured in a medium with tumor necrosis factor- α (TNF- α ). Melatonin was added along with the medium to observe its protective effects. Compared with the control AF cells, TNF- α significantly declined cell proliferation potency and telomerase activity, elevated senescence-associated β -galactosidase (SA- β -Gal) activity, upregulated protein expression of senescence markers (p16 and p53), and increased reactive oxygen species (ROS) content and activity of the NF- κ B pathway. However, when the TNF- α -treated AF cells were incubated with melatonin, ROS content and activity of the NF- κ B pathway were decreased, and those parameters reflecting cell senescence indicated that AF cell senescence was also partly alleviated. Together, melatonin suppresses AF cell senescence through regulating the ROS/NF- κ B pathway in an inflammatory environment. This study sheds a new light that melatonin may be promising to retard inflammation-caused disc degeneration., Competing Interests: There are no conflicts of interest in this work., (Copyright © 2021 Jing Li et al.)

Background: Collagen cross-links contribute to the mechanical resilience of the intervertebral disc (IVD). UVA-light-activated riboflavin-induced collagen crosslinking (UVA-CXL) is a well-established and effective ophthalmological intervention that increases the mechanical rigidity of the collagen-rich corneal matrix in Keratoconus. This study explores the feasibility, safety and efficacy of translating this intervention in reinforcing the IVD., Methods: Annulus fibrosus (AF) cells were isolated from bovine IVDs and treated with different combinations of riboflavin (RF) concentrations (0.05-8 mM) and UVA light intensities (0.3-4 mW/cm2). Metabolic activity (resazurin assay), cell viability (TUNEL assay), and gene expression of apoptosis regulators C-FOS and PT5 were assessed immediately and 24 hours after treatment. Biomechanical effects of UVA-CXL on IVDs were measured by indentation analysis of changes in the instantaneous modulus and by peel-force delamination strength analysis of the AF prior and after treatment., Results: Different intensities of UVA did not impair the metabolic activity of AF cells. However, RF affected metabolic activity (p < 0.001). PT53 expression was similar in all RF conditions tested while C-FOS expression decreased 24 hours after treatment. Twenty-four hours after treatment, no apoptotic cells were observed in any condition tested. Biomechanical characterizations showed a significant increase in the annular peel strength of the UVA-CXL group, when compared to controls of UVA and RF alone (p < 0.05). UVA-CXL treated IVDs showed up to 152% higher (p < 0.001) instantaneous modulus values compared to the untreated control., Conclusion: This is the first study on UVA-CXL treatment of IVD. It induced significantly increased delamination strength and instantaneous modulus indentation values in intact IVD samples in a structure-function relationship. RF concentrations and UVA intensities utilized in ophthalmological clinical protocols were well tolerated by the AF cells. Our findings suggest that UVA-CXL may be a promising tool to reinforce the IVD matrix., Competing Interests: The authors have declared that no competing interests exist.

Ferroptosis is a necrotic form of regulated cell death that was associated with lipid peroxidation and free iron-mediated Fenton reactions. It has been reported that iron deficiency had been implicated in the pathogenesis of intervertebral disc degeneration (IVDD) by activating apoptosis. However, the role of ferroptosis in the process of IVDD has not been illuminated. Here, we demonstrate the involvement of ferroptosis in IVDD pathogenesis. Our in vitro models show the changes in protein levels of ferroptosis marker and enhanced lipid peroxidation level during oxidative stress. Safranin O staining, hematoxylin-eosin staining, and immunohistochemical were used to assess the IVDD after 8 weeks of surgical procedure in vivo. Treatment with ferrostatin-1, deferoxamine, and RSL3 demonstrate the role of ferroptosis in tert-butyl hydroperoxide (TBHP)-treated annulus fibrosus cells (AFCs) and nucleus pulposus cells (NPCs). Ferritinophagy, nuclear receptor coactivator 4 (NCOA4)-mediated ferritin selective autophagy, is originated during the process of ferroptosis in response to TBHP treatment. Knockdown and overexpression NCOA4 further prove TBHP may induce ferroptosis of AFCs and NPCs in an autophagy-dependent way. These findings support a role for oxidative stress-induced ferroptosis in the pathogenesis of IVDD., (© 2020 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Deformity or fissure within the annulus fibrosus (AF) lamellar structure often results in disc herniation leading to the extrusion of nucleus pulposus (NP), which pushes the adjacent nerve, causing low back pain. Low back pain, frequently associated with the degeneration of the intervertebral disc (IVD), affects around 80% of the population worldwide. The difficulty in mimicking the unique structural characteristics of the native AF tissue presents several challenges to the tissue engineering field for the development of the long-term effective therapeutic strategy for AF tissue regeneration. The AF cell niche possesses less reparative capacity for regeneration and thus compels to develop a strategy to recapitulate damaged AF tissues. We have fabricated a polycaprolactone-supported electrocompacted type-I collagen patch (A-PCL-NH 2 +Col-I) using surface-modified electrospun-aligned polycaprolactone (A-PCL) nanofibers cross-linked with an electro-compacted type-I collagen patch (Col-I) using EDAC-NHS (1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride and N -hydroxy succinimide). This subtle approach offered a 3D biodegradable scaffold with dense aggregates of anisotropic collagen-I nanofibrils coupled with electrospun-aligned PCL nanofibers, which provide high tensile strength (4.21 ± 1.07 MPa), moduli (24.496 ± 4.85 MPa), low subsidence to failure, and high-water absorption ability. The systemic organization of both the polymers within the scaffold, evident from attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, revealed a uniform degree of fiber alignment assessed by differential interference contrast (DIC) microscopy, field-emission scanning electron microscopy (FE-SEM), and cryo-SEM. The aminolysis of A-PCL nanofibers was established by energy-dispersive X-ray analysis (EDX), while circular dichroic spectra showed that the electro-compacted Col-I patch displayed a triple helical structure, characteristic of collagens. Moreover, the scaffold revealed more hydrophilic, rough nano-features, which provided ample ligands for cell attachment supporting adequate proliferation of primary goat annulus fibrosus (AF) cells, oriented along the fiber direction, and also favored sufficient production of collagen type-I (+32-fold change) and a glycosaminoglycan extracellular matrix (+2.3-fold change) as compared to cell control, respectively. This study thus demonstrates for the first time the practicability of creating an aligned polycaprolactone-supported electrocompacted type-I collagen hydrogel (A-PCL-NH 2 +Col-I) with significant biomechanical properties, which can be used as an alternative to repair and regenerate AF fissures in degenerated IVD.

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.

Back pain is a leading cause of global disability associated with intervertebral disc (IVD) pathologies. Discectomy alleviates disabling pain caused by IVD herniation without repairing annulus fibrosus (AF) defects, which can cause accelerated degeneration and recurrent pain. Biological therapies show promise for IVD repair but developing high-modulus biomaterials capable of providing biomechanical stabilisation and delivering biologics remains an unmet challenge. The present study identified critical factors and developed an optimal formulation to enhance the delivery of AF cells and transforming growth beta-3 (TGFβ-3) in genipin-crosslinked fibrin (FibGen) hydrogels. Part 1 showed that AF cells encapsulated in TGFβ-3-supplemented high-modulus FibGen synthesised little extracellular matrix (ECM) but could release TGFβ-3 at physiologically relevant levels. Part 2 showed that AF cells underwent apoptosis when encapsulated in FibGen, even after reducing fibrin concentration from 70 to 5 mg/mL. Mechanistic experiments, modifying genipin concentration and integrin binding site presence demonstrated that genipin crosslinking caused AF cell apoptosis by inhibiting cell-biomaterial binding. Adding integrin binding sites with fibronectin partially rescued apoptosis, indicating genipin also caused acute cytotoxicity. Part 3 showed that FibGen formulations with 1 mg/mL genipin had enhanced ECM synthesis when supplemented with fibronectin and TGFβ-3. In conclusion, FibGen could be used for delivering biologically active compounds and AF cells, provided that formulations supplied additional sites for cell-biomaterial binding and genipin concentrations were low. Results also highlighted a need for developing strategies that protect cells against acute crosslinker cytotoxicity to overcome challenges of engineering high-modulus cell carriers for musculoskeletal tissues that experience high mechanical demands.

Objective: To explore if chemokine (C-C motif) ligand 5 (CCL5) delivery could recruit annulus fibrosus (AF) cells to the injury sites and facilitate the repair of ruptured AF., Design: The effects of CCL5 on bovine AF cells in vitro were tested by transwell assay and quantitative real-time polymerase chain reaction. Fibrin gel containing CCL5 was used to treat annulotomized bovine caudal discs cultured under dynamic loading conditions. After 14 days of loading, the samples were collected for histological examination. A pilot animal study was performed using sheep cervical discs to investigate the effect of fibrin gel encapsulated with CCL5 for the treatment of ruptured AF. After 14 weeks, the animals were sacrificed, and the discs were scanned with magnetic resonance imaging before histopathological examination., Results: CCL5 showed a chemotactic effect on AF cells in a dose-dependent manner. AF cells cultured with CCL5 in vitro did not show any change of the gene expression of CCL5 receptors, catabolic and proinflammatory markers. In vitro release study showed that CCL5 exhibited sustained release from the fibrin gel into the culture media; however, in the organ culture study CCL5 did not stimulate homing of AF cells toward the defect sites. The pilot animal study did not show any repair effect of CCL5., Conclusions: CCL5 has a chemotactic effect on AF cells in vitro , but no ex vivo or in vivo regenerative effect when delivered within fibrin gel. Further study with a stronger chemotactic agent and/or an alternate biomaterial that is more conductive of cell migration is warranted.

Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor β1 (TGF-β1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-β1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-β1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-β1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-β1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-β1-containing collagen-PU constructs can induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

Aim: To investigate the effects of metformin, a drug used widely for the treatment of type 2 diabetes mellitus, on human primary cell cultures prepared from uninjured segment of disc material intervertebral disk tissues., Material and Methods: Primary cell cultures were prepared using the tissues of six patients (three males and three females) who had undergone lumbar microdiscectomy and sequestrectomy. Untreated samples served as the control group, and metformintreated samples served as the experimental group. All the samples were evaluated using an inverted light microscope, acridine orange/propidium iodide staining (AO/PI), and a fluorescence microscope. The cytostatic and cytotoxic effects of metformin, which was administered to the samples using a commercial MTT assay kit, were also evaluated. The data obtained were statistically assessed, and the alpha significance value was accepted as less than 0.05. In addition, for the groups’ changes in the expressions of chondroadherin (CHAD), cartilage oligomeric matrix protein (COMP), interleukin-1β (IL-1β) matrix metalloproteinase 7 (MMP-7), and matrix metalloproteinase 19 (MMP-19), genes related to the extracellular matrix synthesis and degradation were determined using gene-specific TaqMan Gene Expression Assays., Results: The administration of the drug adversely affected nucleus pulposus (NP)/annulus fibrosus (AF) cells and extracellular matrixâ€"like structures. This was statistically significant (p < 0.05)., Conclusion: Clinicians should not disregard the adverse effects of metformin, which is used widely in clinical practice, on the components of intervertebral disk tissues.

Low back pain caused by degenerative disc disease affects many people worldwide and brings huge economical burden. Thus, attentions have focused on annulus fibrosus (AF) tissue engineering for treatment of intervertebral disc degeneration. To engineer a functional replacement for the AF, it is important to fabricate scaffolds that mimic the structural and mechanical properties of native tissue. AF-derived stem cells are promising seed cells for AF tissue engineering due to their tissue specificity. In the present study, decellularized AF matrix (DAFM)/chitosan hybrid hydrogels were fabricated using genipin as a crosslinker. AF stem cells were cultured on hydrogel scaffolds with or without basic fibroblast growth factor (bFGF), and cell proliferation, morphology, gene expression, and AF tissue synthesis were examined. Overall, more collagen-I , collagen-II , and aggrecan were secreted by AF stem cells grown on hydrogels with bFGF compared to those without. These results support the application of DAFM/chitosan hybrid hydrogels as an appropriate candidate for AF tissue engineering. Furthermore, incorporation of bFGF into hydrogels promoted AF-related tissue synthesis. Impact Statement The investigation of annulus fibrosus (AF)-related tissue secretion and gene expression in extracellular matrix (ECM) of AF-derived stem cells (AFSCs) provided theoretical and practical basis for the choice of scaffold materials and growth factors for AF tissue engineering. The innovations of the present work are obvious. First, AFSCs were used because they are more easily differentiated into AF cells, thereby producing more AF-related ECM. Second, the decellularized AF matrix (DAFM) was derived from native AF tissue, but had reduced immunogenicity after decellularization. Furthermore, the DAFM structure mimicked the fibrous network of actual AF tissue, which was advantageous to AFSC adhesion and growth. Third, basic fibroblast growth factor was successfully incorporated into the DAFM, showed gradual sustained release, and effectively promoted production of AF tissue ECM factors collagen-I, collagen-II, aggrecan, and glycosaminoglycan.

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.

Background: The aim of this study is to explore the effects of TGF-β1 on autophagy and apoptosis induced by exogenous hydrogen peroxide (H 2 O 2 ) in annulus fibrosus (AF) cells and possible signal pathways involved in this process., Methods: AF cells were isolated from rat lumbar discs and subjected to different concentrations of exogenous H 2 O 2 (50, 100, 200 μmol/L) for different time periods (0.5, 1, 2, and 4 h). Cell viability was determined by CCK-8 assay, and the levels of autophagy and apoptosis were evaluated by Western blotting and caspase 3, 8, 9 activity assay. By administration with different concentrations of TGF-β1 (5, 10, 20 ng/mL), the effects of TGF-β1 on autophagy and apoptosis induced by H 2 O 2 were observed, and the possible signaling pathways were also investigated by using various apoptosis inhibitors or an autophagy inhibitor Bafilomycin A (Baf A) in AF cells., Results: H 2 O 2 significantly impaired cell viability in a dose- and time-dependent manner. H 2 O 2 also induced a sudden and the highest level of autophagy at 1 h, and gradually increased apoptosis through ERK pathway. The mitochondrial pathway was involved in H 2 O 2 -induced apoptosis in AF cells. TGF-β1 reduced the expression of p-ERK and downregulated the expressions of Beclin-1, LC3 II/I, and mitochondrial-related apoptotic proteins (Bax/Bcl-2, caspase-9). Meanwhile, TGF-β1 downregulated the level of intracellular H 2 O 2 through upregulating the expression level of glutathione peroxidase-1 (GPx-1)., Conclusions: TGF-β1 reduced autophagy and apoptosis induced by exogenous H 2 O 2 through downregulating the expression of ERK in AF cells. TGF-β1 could downregulate the level of ERK and intracellular H 2 O 2 by upregulating GPx-1.

Focal defects in the annulus fibrosus (AF) of the intervertebral disc (IVD) arising from herniation have detrimental impacts on the IVD's mechanical function. Thus, biomimetic-based repair strategies must restore the mechanical integrity of the AF to help support and restore native spinal loading and motion. Accordingly, an annulus fibrosus repair patch (AFRP); a collagen-based multi-laminate scaffold with an angle-ply architecture has been previously developed, which demonstrates similar mechanical properties to native outer AF (oAF). To further enhance the mimetic nature of the AFRP, interlamellar (ILM) glycosaminoglycan (GAG) was incorporated into the scaffolds. The ability of the scaffolds to withstand simulated impact loading and resist herniation of native IVD tissue while contributing to the restoration of spinal kinematics were assessed separately. The results demonstrate that incorporation of a GAG-based ILM significantly increased (p < 0.001) the impact strength of the AFRP (2.57 ± 0.04 MPa) compared to scaffolds without (1.51 ± 0.13 MPa). Additionally, repair of injured functional spinal units (FSUs) with an AFRP in combination with sequestering native NP tissue and a full-thickness AF tissue plug enabled the restoration of creep displacement (p = 0.134), short-term viscous damping coefficient (p = 0.538), the long-term viscous (p = 0.058) and elastic (p = 0.751) damping coefficients, axial neutral zone (p = 0.908), and axial range of motion (p = 0.476) to an intact state. Lastly, the AFRP scaffolds were able to prevent native IVD tissue herniation upon application of supraphysiologic loads (5.28 ± 1.24 MPa). Together, these results suggest that the AFRP has the strength to sequester native NP and AF tissue and/or implants, and thus, can be used in a composite repair strategy for IVDs with focal annular defects thereby assisting in the restoration of spinal kinematics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis.

Study Design: Experimental study., Objective: 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., Summary of Background Data: 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., Methods: 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., Results: 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., Conclusion: 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., Level of Evidence: N/A.

Background: Disc degeneration (DD) is one of the common diseases worldwide, which deeply influences normal life and leads to excruciating pain. However, an effective treatment for DD is still not identified., Method: The present study systemically examined the effect of melatonin on annulus fibrosus (AF) cells of patients with DD., Results: Melatonin had the effect of promoting proliferation, inducing autophagy, and suppressing apoptosis on AF cells of patients with DD. Moreover, melatonin contributed to the translation and transcription of autophagy-related protein ATG7 and inhibited the function of miR-106a-5p in AF cells. In addition, the results suggested that miR-106a-5p mediated the expression of ATG7 by directly binding to its 3'UTR in AF cells., Conclusion: This research not only gained a deep insight of melatonin mode of action, but also indicated its potential target signaling pathway in AF cells., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: There is an increasing local application of methylene blue (MB) in the treatment of discogenic low back pain (LBP) and percutaneous transforaminal endoscopic discectomy (PTED) procedures. MB could generate DNA damage and induce apoptosis in different cell types; however, the effects of MB on intervertebral disc (IVD) annulus fibrosus (AF) cells are not clearly understood., Objective: The objective of this study was to investigate the effects of different concentrations of MB on rat AF cells in vitro., Study Design: This study used an experimental design., Setting: This research was conducted at the Orthopaedic Institute of the Clinical Medical College of Yangzhou University., Methods: AF cells were isolated and cultured with different concentrations of MB (0, 2, 20, and 200 mu-g/mL) and assessed to determine the possible cytotoxic effects of MB. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. The inverted phase-contrast microscopy was used to perform morphological observation of apoptotic cells, and flow cytometry was used to measure the incidence of cell apoptosis. The mRNA and protein expression levels of apoptosis-associated genes (caspase-3, Bcl-2, and Bax) and other related genes (collagen type I, transforming growth factor beta1 [TGF-beta1], fibroblast growth factor [bFGF], and tissue inhibitor of metalloproteinase-1 [TIMP-1]) were analyzed by quantitative real-time PCR (RT-PCR) and Western blotting., Results: Our results indicated that MB reduced cell viability in a concentration- and time-dependent manner. MB also induced marked AF cell apoptosis in a concentration-dependent manner observed by inverted phase-contrast microscopy, flow cytometry, and indicated by the increased expression of caspase-3. Both RT-PCR and Western blotting revealed significant up-regulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl-2 in a concentration-dependent manner. Moreover, collagen type I, TGF-beta1, bFGF, and TIMP-1 mRNA and protein levels were also found to be decreased by MB in a concentration-dependent manner., Limitations: Limitations of this study were the in vitro study design and lack of in vivo validation of the observed effects of MB on human IVD cells., Conclusions: Our results indicate that a high concentration of MB can not only inhibit proliferation and paracrine function of AF cells, but can also induce cell apoptosis in a concentration-dependent manner, suggesting that it is necessary to choose low concentrations of MB in practical application and limit the use of MB in the treatment of discogenic LBP to research protocols., Key Words: Methylene blue, annulus fibrosus cell, proliferation, apoptosis, paracrine.

The surgical standard of care for lumbar discectomy leaves the annulus fibrosus (AF) defect unrepaired, despite considerable risk for a recurrent herniation. Identification of a viable defect repair strategy has until now been elusive. The scope of this ex vivo biomechanical study was to evaluate crosslinking hydrogels as potentially promising AF defect sealants, and provide a baseline for their use in combination with collagen scaffolds that restore disc volume. This study directly compared genipin crosslinked fibrin hydrogel (FibGen) as a promising preclinical candidate against a clinically available adhesive composed of glutaraldehyde and albumin (BioGlue). Forty-two bovine coccygeal functional spine units (FSU) were randomly allocated into four groups, namely untreated (control, n = 12), repaired with either one of the tested hydrogels (BioGlue, n = 12; FibGen, n = 12), or FibGen used in combination with a collagen hydrogel scaffold (FibGen+Scaffold, n = 6). All specimens underwent a moderate mechanical testing protocol in intact, injured and repaired states. After completion of the moderate testing protocol, the samples underwent a ramp-to-failure test. Lumbar discectomy destabilized the FSU as quantified by increased torsional range of motion (28.0° (19.1, 45.1) vs. 41.39° (27.3, 84.9), p<0.001), torsional neutral zone (3.1° (1.2, 7.7) vs. 4.8° (2.1, 12.1), Z = -3.49, p < 0.001), hysteresis(24.4 J (12.8, 76.0) vs. 27.6 J (16.4, 54.4), Z = -2.61, p = 0.009), with loss of both disc height (7.0 mm (5.0, 10.5) vs 6.1 mm (4.0, 9.3), Z = -5.16, p < 0.001) and torsional stiffness (0.76 Nmdeg-1 (0.38, 1.07) vs. 0.66 Nmdeg-1 (0.38, 0.97), Z = -3.98, p < 0.001). Most FibGen repaired AF endured the entire testing procedure whereas only a minority of BioGlue repaired AF and all FibGen+Scaffold repaired AF failed (6/10 vs. 3/12 vs. 0/6 respectively, p = 0.041). Both BioGlue and FibGen+Scaffold repaired AF partially restored disc height (0.47 mm (0.07, 2.41), p = 0.048 and 1.52 mm (0.41, 2.57), p = 0.021 respectively) compared to sham treatment (0.08 mm (-0.63, 0.88)) whereas FibGen-only repaired AF had no such effect (0.04 mm (-0.73, 1.13), U = 48.0, p = 1). The AF injury model demonstrated considerable change of FSU mechanics that could be partially restored by use of an AF sealant. While inclusion of a volumetric collagen scaffold led to repair failure, use of FibGen alone demonstrated clinically relevant promise for prevention of mechanical reherniation, outperforming an FDA approved sealant in this ex vivo test series., Competing Interests: The authors have declared that no competing interests exist.

Objective: The aim is assessing the in vivo efficacy of annulus fibrosus (AF) cells seeded into collagen by enhancing the reparative process around annular defects and preventing further degeneration in a rat-tail model., Summary of Background Data: Treating disc herniation with discectomy may relieve the related symptoms but does not address the underlying pathology. The persistent annular defect may lead to re-herniation and further degeneration. We recently demonstrated that riboflavin crosslinked high-density collagen gels (HDC) can facilitate annular repair in vivo., Methods: 42 rats, tail disc punctured with an 18-gauge needle, were divided into 3 groups: untreated (n = 6), injected with crosslinked HDC (n = 18), and injected with AF cell-laden crosslinked HDC (n = 18). Ovine AF cells were mixed with HDC gels prior to injection. X-rays and MRIs were conducted over 5 weeks, determining disc height index (DHI), nucleus pulposus (NP) size, and hydration. Histological assessments evaluated the viability of implanted cells and degree of annular repair., Results: Although average DHIs of both HDC gel groups were higher than those of the puncture control group at 5 weeks, the retention of disc height, NP size and hydration at 1 and 5 weeks was significant for the cellular group compared to the punctured, and at 5 weeks to the acellular group. Histological assessment indicated that AF cell-laden HDC gels have accelerated reparative sealing compared to acellular HDC gels., Conclusions: AF cell-laden HDC gels have the ability of better repairing annular defects than acellular gels after needle puncture., Statement of Significance: This project addresses the compelling demand of a sufficient treatment strategy for degenerative disc disease (DDD) perpetuated by annulus fibrosus (AF) injury, a major cause of morbidity and burden to health care systems. Our study is designed to answer the question of whether injectable, photo-crosslinked, high density collagen gels can seal defects in the annulus fibrosus of rats and prevent disc degeneration. Furthermore, we investigated whether the healing of AF defects will be enhanced by the delivery of AF cells (fibrochondrocytes) to these defects. The use of cell-laden collagen gels in spine surgery holds promise for a wide array of applications, from current discectomy procedures to future nucleus pulposus reparative therapies, and our group is excited about this potential., (Copyright © 2018. Published by Elsevier Ltd.)

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.

Study Design: Institutional review board-approved research using human annulus cells cocultured with F11 nerve cells., Objective: To perform functional, kinetic assays of neurite dynamics and media neurotrophin measurements to test whether proinflammatory cytokines influence annulus cells' signaling cues for neurite growth/repulsion., Summary of Background Data: Nerves grow in response to signaling molecules called neurotrophins, which disc cells produce (e.g., brain-derived neurotrophic factor [BDNF], glial cell line-derived neurotrophic factor [GDNF], and neurotrophin 3 [NT3]) and which influence neuron survival, differentiation, and migration. How proinflammatory cytokines influence disc signaling cues for neurite growth/repulsion is poorly understood., Methods: Studies used our previous model of 4-day human annulus cell-F11 nerve cell coculture to assess effects of added proinflammatory cytokines interleukin 1 beta (IL-1β; 10 pmol/L) or tumor necrosis factor alpha (TNF-α) (10 pmol/L). Annulus cells were cultured from 6 Thompson grade I, 9 grade II, 8 grade III, 11 grade IV, and 7 grade V discs. Neurite lengths were measured following control conditions or with added IL-1β or TNF-α, and conditioned media assayed with RayBiotech Growth Factor Arrays. Standard statistical methods used analysis of variance and Spearman correlation coefficient testing associations of neurite length with neurotrophin levels., Results: IL-1-β or TNF-α significantly increased neurite lengths (P < 0.001) and BDNF, NT3, and GDNF media levels (P ≤ 0.01) versus controls. Significant positive correlations were present between media neurotrophin levels for BDNF, NT3, and GDNF and neurite lengths under control conditions, following addition of IL-1β, and following addition of TNF-α. Novel data showed production of the neurotrophin amphiregulin., Conclusion: In vitro data supported the hypothesis that nerve-disc cell interactions may be influenced by the heightened proinflammatory milieu present in degenerating discs, leading to increased nerve migration. Data may have direct clinical relevance/implications for nerve ingrowth and pain in the outer annulus (where disc cell numbers are high), and in regions where nerves penetrate into the disc via annular tears., Level of Evidence: N/A.

Annulus fibrosus (AF) defects from intervertebral disk (IVD) herniation and degeneration are commonly associated with back pain. Genipin-crosslinked fibrin hydrogel (FibGen) is an injectable, space-filling AF sealant that was optimized to match AF shear properties and partially restored IVD biomechanics. This study aimed to enhance mechanical behaviors of FibGen to more closely match AF compressive, tensile, and shear properties by adjusting genipin crosslink density and by creating a composite formulation by adding Poly(D,L-lactide-co-glycolide) (PDLGA). This study also evaluated effects of thrombin concentration and injection technique on gelation kinetics and adhesive strength. Increasing FibGen genipin concentration from 1 to 36 mg/mL significantly increased adhesive strength (∼5 to 35 kPa), shear moduli (∼10 to 110 kPa), and compressive moduli (∼25 to 150 kPa) with concentration-dependent effects, and spanning native AF properties. Adding PDLGA to FibGen altered the material microstructure on electron microscopy and nearly tripled adhesive strength, but did not increase tensile moduli, which remained nearly 5× below native AF, and had a small increase in shear moduli and significantly decreased compressive moduli. Increased thrombin concentration decreased gelation rate to < 5 min and injection methods providing a structural FibGen cap increased pushout strength by ∼40%. We conclude that FibGen is highly modifiable with tunable mechanical properties that can be formulated to be compatible with human AF compressive and shear properties and gelation kinetics and injection techniques compatible with clinical discectomy procedures. However, further innovations, perhaps with more efficient fiber reinforcement, will be required to enable FibGen to match AF tensile properties.

Study Design: Laboratory study., Objective: To elucidate the potential involvement of the interleukin-6 (IL-6)/Janus kinase (JAK)/signal transducers and activator of transcription (STAT3) pathway in the development of intervertebral disc (IVD) degeneration., Summary of Background Data: IL-6 plays a crucial role in IVD degeneration; however, the downstream intracellular signaling of IL-6 in the IVD is not fully understood., Methods: The expression levels of IL-6 and suppressors of cytokine signaling 3 (SOCS3), a target gene of the IL-6/JAK/STAT3 pathway, were evaluated in rat and human degenerated IVD samples. The effects of IL-6 on primary rat annulus fibrosus (AF) cells were analyzed using quantitative PCR, immunocytochemistry, and Western blotting. The potential efficacy of a JAK inhibitor, CP690,550, in neutralizing the effect of IL-6 was evaluated in vitro., Results: A high expression of IL-6 and SOCS3 was observed in both rat and human degenerated IVD samples. In rat AF cells, IL-6 markedly induced the phosphorylation of STAT3 and the expression of cyclooxygenase-2 and matrix metalloprotease-13. CP690,550 significantly suppressed the phosphorylation of STAT3 and offset the catabolic effect of IL-6 in rat AF cells., Conclusion: Our results suggest that the IL-6/JAK/STAT3 pathway is involved in the pathogenesis of IVD degeneration and that CP690,550 suppresses the catabolic effect of the IL-6 in the IVD., Level of Evidence: N/A.

Low back pain which is a major cause of disability for people aged between 20 and 50years imposes a serious socio-economic burden. The current focus of regenerative medicine is on identifying molecular markers to facilitate the design of targeted therapeutics. Previously, we have demonstrated that expression of the anti-proliferative interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and pro-apoptotic insulin-like growth factor-binding protein-3 (IGFBP3), are up-regulated as downstream targets of the inflammatory cytokine interferon α (IFNα) signaling pathway in the human annulus fibrosus (AF). Here, we hypothesised that injection of hyaluronic acid (HA) would have an anti-inflammatory and matrix modulatory effect on injured and IFNα 2 β inflamed bovine intervertebral discs (IVD). Discs with an AF defect and challenged with IFNα 2 β were used in a bovine IVD organ culture model to test the effect of HA on the IFNα 2 β pathway, as well as the matrix proteins aggrecan and collagen I. qRT-PCR was used to assess the gene expression of IFNα 2 β signaling molecules. Additionally, immunostaining was used to measure protein expression. Our results show that HA treatment significantly down-regulates IFNAR1, IFNAR2, STAT1/2, JAK1, IFIT3 and IGFBP3 mRNA expression in the inflamed groups. Protein analysis confirmed the PCR results. In the extracellular matrix, aggrecan and collagen I were up-regulated while ADAMTS4 was down-regulated upon treatment of the injured and inflamed discs with HA. Hence, HA demonstrates both an anti-inflammatory role, resulting in the down-regulation of IFIT3 and IGFBP3 in the AF, and a matrix modulatory effect by up-regulating aggrecan and collagen I expression., Statement of Significance: The pro-inflammatory environment of the degenerated IVD represents a challenge for regenerative therapies. The study demonstrates that hyaluronan acts as an anti-inflammatory molecule by down-regulating IFNAR1 and IFNAR2, the signaling molecules STAT1, STAT2, JAK1 and the downstream apoptotic targets IGFBP3 and IFIT3. We also demonstrated that hyaluronan modulates the disc matrix environment by increasing aggrecan and collagen I synthesis and down-regulating ADAMTS4 that degrades the matrix under inflammatory conditions. The significance of this work lies in the fact that hyaluronan acts as an anti-inflammatory molecule by shifting the disc environment towards a more anabolic state and by promoting native IVD matrix production., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Tissue engineering provides a promising approach to treat degenerative disc disease, which usually requires a large quantity of seed cells. A simple and reliable in vitro culture system to expand seed cells in a timely fashion is necessary to implement the application clinically. Here, we sought to establish a cost-effective culture system for expanding human annulus fibrosus cells using extracellular matrix (ECM) proteins as culture substrates. Cells were cultured onto a plastic surface coated with various types of ECMs, including fibronectin, vitronectin, collagen type I, gelatin and cell-free matrix deposited by human nucleus pulposus cells. AF cell morphology, growth, adhesion and phenotype (anabolic and catabolic markers) were assessed by microscopy, real-time RT-PCR, western blotting, zymography, immunofluorescence staining and biochemical assays. Fibronectin, collagen and gelatin promoted cell proliferation and adhesion in a dose-dependent manner. Fibronectin elevated mRNA expression of proteoglycan and enhanced glycosaminoglycan production. Both collagen and gelatin increased protein expression of type II collagen. Consistent with increased cell adhesion, collagen and fibronectin promoted formation of focal adhesion complexes in the cell-matrix junction, suggesting enhanced binding of the actin network with both ECM substrates. On the other hand, fibronectin, collagen and gelatin decreased expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 in media. Finally, a mixture of fibronectin (1.7 μg/mL) and collagen (1.3 μg/mL) was identified as the most promising in vitro culture substrate system in promoting proliferation and maintaining anabolic-catabolic balance. Our method provides a simple and cost-effective platform for tissue engineering applications in intervertebral disc research.

Background: Different biologic approaches to treat disc regeneration, including growth factors (GFs) application, are currently under investigation. Human annulus fibrosus (hAF) repair or regeneration is one of the key elements for maintenance and restoration of nucleus pulposus function. However, so far there is no effective treatment for this purpose. The aim of the present study was to investigate the response of hAF cells to different combinations of GFs, and develop a protocol for efficient culture expansion., Methods: hAF cells were harvested from degenerated disc tissues during surgical intervertebral disc removal, and hAF cells were expanded in a monolayer. The experiments were categorized based on different protocols with transforming growth factor (TGF-β1) and fibroblast growth factor (FGF-2) culture for 14 days: group 1 had no GFs (control group); group 2 received TGF-β1; group 3 received FGF-2; group 4 received both GFs; and group 5 (two-step) received both GFs for the first 10 days and TGF-β1 only for the next 4 days. Cell proliferation, collagen, and noncollagen extracellular matrix (ECM) production and genes expression were compared among these groups., Results: At days 3, 7 and 10 of cultivation, groups 4 and 5 had significantly more cell numbers and faster cell proliferation rates than groups 1, 2, and 3. At 14 days of cultivation, significantly more cell numbers were observed in groups 3 and 4 than in group 5. The group 4 had the most cell numbers and the fastest proliferation rate at 14 days of cultivation. After normalization for cell numbers, group 5 (two-step) produced the most collagen and noncollagen ECM at 10 and 14 days of cultivation among the five groups. In group 5, ECM gene expression was significantly upregulated. High expression of matrix metalloproteinase-1 was upregulated with FGF-2 on the different days as compared to the other groups. Annulus fibrosus cell phenotypes were only marginally retained under the different protocols based on quantitative polymerase chain reaction results., Conclusion: Taken together, the two-step protocol was the most efficient among these different protocols with the most abundant ECM production after normalization for cell numbers for culture expansion of hAF cells. The protocol may be useful in further cell therapy and tissue engineering approaches for disc regeneration.

Study Design: Establishment of immortalized cell lines derived from rat intervertebral disc cells by Rho-associated kinase (ROCK) inhibitor, Y-27632., Objective: To determine whether rat nucleus pulposus (NP) and annulus fibrosus (AF) cells could be immortalized, retain their phenotype, and used as cell lines for in vitro cell biology., Summary of Background Data: Intervertebral disc degeneration is a major factor for most low-back pain. However, the mechanism of the disease is not well understood by the limitation to obtain sufficient amounts of primary disc cells. Therefore, the establishment of disc cell lines will help in vitro molecular signaling studies to understand the mechanism of degenerative disc disease., Methods: Cells were isolated from the NP and AF tissues of lumbar discs of adult Sprague Dawley rat. Tissues were digested and cultured with DMEM/Ham's F-12 (1:1) and 20% FBS and antibiotics. From day 3, cells were grown in the presence of 10  μM Y-27632, a well-characterized inhibitor of the ROCK, and subcultured by trypsinization, passaging them 1:3 onto 100  mm culture dishes. Morphologic and genetic analyses were performed on the different passaged cells., Results: ROCK inhibitor successfully immortalized rat NP and AF cells. They passaged for over 50 generations with sustained expression levels of several NP and AF cell markers. In addition, they retained phenotypic features similar to the primary parental NP and AF cells when the cells were challenged with different cytokines and growth factors., Conclusion: We established immortalized rat NP and AF cell lines using a method of treating cells with ROCK inhibitor Y-27632 and demonstrated that these immortalized cells retain the properties of primary cells and could serve as useful tools for in vitro signaling studies or drug screening studies to develop novel therapeutic strategies., Level of Evidence: N/A.