Rat Annulus Fibrosus Research Articles

Repair of annulus fibrosus defects using decellularized annulus fibrosus matrix/chitosan hybrid hydrogels

Degenerative disc disease is the leading cause of lower back and leg pain, considerably impacting daily life and incurring substantial medical expenses for those affected. The development of annulus fibrosus tissue engineering offers hope for treating this condition. However, the current annulus fibrosus tissue engineering scaffolds fail to accurately mimic the natural biological environment of the annulus fibrosus, resulting in limited secretion of extracellular matrix produced by the seeded cells and poor biomechanical properties of the constructed biomimetic annulus fibrosus tissue. This inability to match the biomechanical performance of the natural annulus fibrosus hinders the successful treatment of annulus fibrosus defects. In this study, we fabricated decellularized annulus fibrosus matrix (DAFM)/chitosan hydrogel-1 (DAFM: Chitosan 6:2) and DAFM/chitosan hydrogel-2 (DAFM: Chitosan 4:4) by varying the ratio of DAFM to chitosan. Rat annulus fibrosus (AF)-derived stem cells were cultured on these hydrogel scaffolds, and the cell morphology, AF-related gene expression, and Interleukin-6 (IL-6) levels were investigated. Additionally, magnetic resonance imaging, Hematoxylin and eosin staining, and Safranine and Fast Green staining were performed to evaluate the repair effect of the DAFM/chitosan hydrogels in vivo. The gene expression results showed that the expression of Collagen type I (Col-I), Collagen type I (Col-II), and aggrecan by annulus fibrosus stem cells (AFSCs) cultured on the DAFM/chitosan-1 hydrogel was higher compared with the DAFM/chitosan-2 hydrogel. Conversely, the expression of metalloproteinase-9 (MMP-9) and IL-6 was lower on the DAFM/chitosan-1 hydrogel compared with the DAFM/chitosan-2 hydrogel. In vivo, both the DAFM/chitosan-1 and DAFM/chitosan-2 hydrogels could partially repair large defects of the annulus fibrosus in rat tail vertebrae. In conclusion, the DAFM/chitosan-1 hydrogel could be regarded as a candidate scaffold material for the repair of annulus fibrosus defects, offering the potential for improved treatment outcomes.

Annulus Fibrosus Injury Induces Acute Neuroinflammation and Chronic Glial Response in Dorsal Root Ganglion and Spinal Cord-An In Vivo Rat Discogenic Pain Model.

Chronic painful intervertebral disc (IVD) degeneration (i.e., discogenic pain) is a major source of global disability needing improved knowledge on multiple-tissue interactions and how they progress in order improve treatment strategies. This study used an in vivo rat annulus fibrosus (AF) injury-driven discogenic pain model to investigate the acute and chronic changes in IVD degeneration and spinal inflammation, as well as sensitization, inflammation, and remodeling in dorsal root ganglion (DRG) and spinal cord (SC) dorsal horn. AF injury induced moderate IVD degeneration with acute and broad spinal inflammation that progressed to DRG to SC changes within days and weeks, respectively. Specifically, AF injury elevated macrophages in the spine (CD68) and DRGs (Iba1) that peaked at 3 days post-injury, and increased microglia (Iba1) in SC that peaked at 2 weeks post-injury. AF injury also triggered glial responses with elevated GFAP in DRGs and SC at least 8 weeks post-injury. Spinal CD68 and SC neuropeptide Substance P both remained elevated at 8 weeks, suggesting that slow and incomplete IVD healing provides a chronic source of inflammation with continued SC sensitization. We conclude that AF injury-driven IVD degeneration induces acute spinal, DRG, and SC inflammatory crosstalk with sustained glial responses in both DRGs and SC, leading to chronic SC sensitization and neural plasticity. The known association of these markers with neuropathic pain suggests that therapeutic strategies for discogenic pain need to target both spinal and nervous systems, with early strategies managing acute inflammatory processes, and late strategies targeting chronic IVD inflammation, SC sensitization, and remodeling.

A novel rat model of annulus fibrosus injury for intervertebral disc degeneration

BACKGROUND CONTEXTIn clinical practice, acute trauma and chronic degeneration of the annulus fibrosus (AF) can promote further degeneration of the intervertebral disc (IVD). Therefore, it is critical to understand the AF repair process and its consequences on IVD. However, the lack of cost-effective and reproducible in vivo animal models of AF injury has limited research development in this field. PURPOSESThe purpose of this study was to establish and evaluate the utility of a novel animal model for full-thickness AF injury. Three foci were proposed: (1) whether this new modeling method can cause full-layer AF damage; (2) the repair processes and pathological changes in the damaged area after AF injury, and (3) the morphological and histological changes in the IVD are after AF injury. STUDY DESIGN/SETTINGIn vivo rat AF injury model with characterization of AF damage repair, IVD degeneration. METHODSA total of 72,300 g male rats were randomly assigned to one of the two groups: experimental or sham. Annulus fibrosus was separated layer by layer under the microscope with a #11 blade up to the AF- nucleus pulpous (NP) junction. The repair process of the horizontal AF and morphological changes in the sagittal IVD were evaluated with HE staining. Sirius red staining under polarized light. Immunofluorescence was conducted to analyze changes in the expression of COL1 and COL3 in the AF injury area and 8-OHdg, IL-6, MMP13, FSP1, and ACAN in the IVD. The disc height and structural changes after AF injury were measured using X-ray and contrast-enhanced micro-CT. Additionally, the resistance of the AF to stretching was analyzed using three-point bending. RESULTSAnnulus fibrosus-nucleus pulpous border was identified to stably induce the full-thickness AF injury without causing immediate NP injury. The AF repair process after injury was slow and expressed inflammation factors continuously, with abundant amounts of type III collagen appearing in the inner part of the AF. The scar at the AF lesion had decreased resistance to small molecule penetration and weakened tensile strength. Full-thickness AF injury induced disc degeneration with loss of disc height, progressive unilateral vertebral collapse, and ossification of the subchondral bone. Inflammatory-induced degeneration and extracellular matrix catabolism gradually appeared in the NP and cartilage endplate (CEP). CONCLUSIONSWe established a low-cost and reproducible small animal model of AF injury which accurately replicated the pathological state of the limited AF self-repair ability and demonstrated that injury to the AF alone could cause further degeneration of the IVD. CLINICAL RELEVANCEThis in vivo rat model can be used to study the repair process of the AF defect and pathological changes in the gradual degeneration of IVD after AF damage. In addition, the model provides an experimental platform for in vivo experimental research of potential clinical therapeutics.

Inhibition of PP2A ameliorates intervertebral disc degeneration by reducing annulus fibrosus cells apoptosis via p38/MAPK signal pathway

BackgroundIntervertebral disc degeneration (IVDD) is considered one of the main reasons for low back pain (LBP). To date, the specific pathology of IVDD remains unclear. The annulus fibrosus (AF) is an important part of the intervertebral disc, and AF cell oxidative stress, apoptosis plays a vital role in disc degeneration. Protein phosphatase 2 A (PP2A), a serine/threonine phosphatase, has regulatory functions in various processes, including apoptosis and autophagy. However, thus far, the effect of PP2A on IVDD is not clear. MethodsAF cells derived from caudal intervertebral discs in SD rats were used to analyze the levels of oxidative stress, apoptosis and degeneration as well as PP2A expression. A PP2A agonist (FTY720), inhibitor (microcystin-LR) and siRNA (si-PPP2CA) were employed in IVDD induced by H2O2 to investigate the levels of apoptosis and degeneration. The p38/MAPK signal pathways were evaluated, and a p38 inhibitor (SB203580) and ERK inhibitor (U0126) were added for verification. Finally, FTY720 and microcystin-LR were administered to IVDD rats to assess the effects on levels of apoptosis and degeneration and the relief of IVDD. ResultsThe expression of PP2A was increased in rat AF cells after H2O2 intervention.The levels of apoptosis and degeneration were higher with upregulation of PP2A but were significantly reduced after inhibition of PP2A. The PP2A inhibitor relieved cell apoptosis and degeneration by downregulating the p38/MAPK pathway. In vivo, the knockdown of PP2A resulted in a more complete morphology of discs and less apoptotic and degenerative expression. ConclusionsThis study suggests that the downregulation of PP2 A could reduce AF cell apoptosis and degeneration via the p38/MAPK pathway. It also revealed that the inhibition of PP2 A is expected to be a therapeutic target for IVDD.

Single-cell RNA sequencing reveals resident progenitor and vascularization-associated cell subpopulations in rat annulus fibrosus.

One of the main causes of low back pain is intervertebral disc degeneration (IDD). Annulus fibrosus (AF) is important for the integrity and functions of the intervertebral disc (IVD). However, the resident functional cell components such as progenitors and vascularization-associated cells in AF are yet to be fully identified. Identification of functional AF cell subpopulations including resident progenitors and vascularization-associated cells. In this study, the single-cell RNA sequencing data of rat IVDs from a public database were analyzed using Seurat for cell clustering, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analysis, StemID for stem cell identification, Monocle and RNA velocity for pseudotime differentiation trajectory validation, single-cell regulatory network inference and clustering (SCENIC) for gene regulatory network (GRN) analysis, and CellChat for cell-cell interaction analysis. Immunostaining on normal and degenerated rat IVDs, as well as human AF, was used for validations. From the data analysis, seven AF cell clusters were identified, including two newly discovered functional clusters, the Grem1 + subpopulation and the Lum +​subpopulation. The Grem1 + subpopulation had progenitor characteristics, while the Lum +​subpopulation was associated with vascularization during IDD. The GRN analysis showed that Sox9 and Id1 were among the key regulators in the Grem1 + subpopulation, and Nr2f2 and Creb5 could be responsible for the vascularization function in the Lum +​subpopulation. Cell-cell interaction analysis revealed highly regulated cellular communications between these cells, and multiple signaling networks including PDGF and MIF signaling pathways were involved in the interactions. Our results revealed two new functional AF cell subpopulations, with stemness and vascularization induction potential, respectively. These findings complement our knowledge about IVDs, especially the AF, and in return provide potential cell source and regulation targets for IDD treatment and tissue repair. The existence of the cell subpopulations was also validated in human AF, which strengthen the clinical relevance of the findings.

Smad7 Is Highly Expressed in Human Degenerative Discs and Participates in IL-1β-Induced Apoptosis of Rat AF Cells via the Mitochondria Pathway.

Background Abnormal Smad7 expression can lead to apoptosis in different cell types. Previously, we found high expression of Smad7 in rat degenerative discs. However, the exact role of Smad7 in the apoptosis of disc cells and the possible underlying mechanism remain unclear. Methods Degenerative and nondegenerative human lumbar intervertebral discs were collected from patients during operation. The expressions of SMAD7 mRNA and protein in the different components of these discs were measured with real-time PCR and Western blotting, respectively. Annulus fibrosus (AF) cells were isolated and cultivated from the discs of young healthy rats. Smad7 in the AF cells was overexpressed with adenovirus and knocked down with siRNA. IL-1β was used to induce apoptosis in the AF cells. Loss-and-gain cell function experiments were performed to show the effect of Smad7 on the apoptosis of AF cells. The function recovery experiments were performed to verify whether Smad7 regulates the apoptosis of AF cells through the mitochondria-mediated pathway. Results Both the mRNA and protein expressions of Smad7 were significantly higher in the different components of human degenerative discs than in those of the nondegenerative discs. IL-1β stimulated apoptosis while upregulating the Smad7 expression in the AF cells in vitro. Overexpression of Smad7 in AF cells exaggerated the IL-1β-induced apoptosis in the cells while knockdown of Smad7 expression suppressed this apoptosis. With the exaggerated apoptosis in the AF cells with Smad7 overexpression, both active cleaved caspase-3 and cleaved caspase-9, the ratio of Bax/Bcl-2, and Cyt-c increased significantly. However, the inhibitor of caspase-9, Z-LEHD-FMK, significantly diminished the apoptosis in these cells. Conclusion Smad7 is highly expressed in human degenerative discs and participates in IL-1β-induced apoptosis of rat AF cells via the mitochondria pathway. Smad7 may be a potential target for the prevention and treatment of degenerative disc disease.

TGF-β1-supplemented decellularized annulus fibrosus matrix hydrogels promote annulus fibrosus repair.

Annulus fibrosus (AF) repair remains a challenge because of its limited self-healing ability. Endogenous repair strategies combining scaffolds and growth factors show great promise in AF repair. Although the unique and beneficial characteristics of decellularized extracellular matrix (ECM) in tissue repair have been demonstrated, the poor mechanical property of ECM hydrogels largely hinders their applications in tissue regeneration. In the present study, we combined polyethylene glycol diacrylate (PEGDA) and decellularized annulus fibrosus matrix (DAFM) to develop an injectable, photocurable hydrogel for AF repair. We found that the addition of PEGDA markedly improved the mechanical strength of DAFM hydrogels while maintaining their porous structure. Transforming growth factor-β1 (TGF-β1) was further incorporated into PEGDA/DAFM hydrogels, and it could be continuously released from the hydrogel. The in vitro experiments showed that TGF-β1 facilitated the migration of AF cells. Furthermore, PEGDA/DAFM/TGF-β1 hydrogels supported the adhesion, proliferation, and increased ECM production of AF cells. In vivo repair performance of the hydrogels was assessed using a rat AF defect model. The results showed that the implantation of PEGDA/DAFM/TGF-β1 hydrogels effectively sealed the AF defect, prevented nucleus pulposus atrophy, retained disc height, and partially restored the biomechanical properties of disc. In addition, the implanted hydrogel was infiltrated by cells resembling AF cells and well integrated with adjacent AF tissue. In summary, findings from this study indicate that TGF-β1-supplemented DAFM hydrogels hold promise for AF repair.

Investigation into the anti-inflammatory properties of metformin in intervertebral disc cells.

IntroductionIntervertebral disc degeneration (IDD) is closely related to heightened inflammation in the annulus fibrosis (AF) and nucleus pulposus (NP) cells in the intervertebral disc. An imbalanced matrix homeostasis has been shown to contribute to disc degeneration and associated discogenic low back pain. Metformin, a diabetes medication, has been noted to exhibit anti‐inflammatory properties through upregulation of the AMPK pathway, leading to various anti‐inflammatory‐related responses in hepatocytes. However, it is still unclear how metformin influences disc cellular response to inflammatory stress and the corresponding mechanism. Hence, the objective of this study is to elucidate the effects of metformin on expression of key pro‐inflammatory, catabolic, and anabolic factors within rat AF cells in response to inflammatory stimulation and mechanical tensile stress.MethodsFive Fischer 344 rats were sacrificed and their spines isolated. AF cells were cultured and plated in flexible silicone membrane‐based six‐well plates. Wells were split into eight groups and subjected to metformin, IL‐1β, mechanical stretch, and combined treatments. Relative gene expressions of MMP‐13, COX‐2, iNOS, AGC, and Col1 were assessed with quantitative real‐time polymerase chain reaction (qRT‐PCR), and downstream prostaglandin E2 (PGE2) production was quantified with enzyme‐linked immunosorbent assay (ELISA). NF‐kB nuclear translocation was also quantified.ResultsMetformin in the presence of the combined stress treatments (M + IL/S) significantly increased Col1, COX‐2, and MMP‐13 gene expression, decreased PGE2 production compared to IL/S conditions alone. Metformin treatment of cultured rat annulus fibrosus cells significantly reduced the nuclear translocation of NF‐κB after 4 h of IL‐1β treatment from 43.1% in case of IL‐1β treatment down to 26.2% in the case of metformin + IL‐1β treatment.DiscussionThe lack of metformin‐mediated suppression of inflammatory response in the nonstretch groups indicates that metformin may be enacting its effects through a stretch‐dependent manner. These results suggest a foundation for pursuing further research into metformin's potential role as an anti‐inflammatory agent for curtailing intervertebral disc degeneration.

Naringin Inhibits Apoptosis Induced by Cyclic Stretch in Rat Annular Cells and Partially Attenuates Disc Degeneration by Inhibiting the ROS/NF-κB Pathway.

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-) κB 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-κB 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-κB signaling pathway. Naringin may be an effective drug for treating degenerative disc disease.

Mechanical Stretch Induces Annulus Fibrosus Cell Senescence through Activation of the RhoA/ROCK Pathway.

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.

Icariin Attenuates Serum Deprived-Induced Premature Senescence in Rat Annulus Fibrosus Cells and Ameliorates Disc Degeneration In Vivo

Icariin Attenuates Serum Deprived-Induced Premature Senescence in Rat Annulus Fibrosus Cells and Ameliorates Disc Degeneration In Vivo

Inner-heating acupuncture of lumbar degenerative lesions based on β-catenin signaling pathway

By observing the effect of inner-heating acupuncture on the expression of Wnt1, Axin and β-catenin in the intervertebral disc of rats with lumbar degenerative disease and to explore the regulatory mechanism of inner-heating acupuncture on the apoptosis of annulus fibrosus cells in rats with lumbar degenerative disease. Forty SD rats were randomly divided into normal group, model group, inner heating acupuncture group and acupuncture group with 10 rats in each group. Except for normal group, all rats were modeled. Bilateral Da Chang Shu were selected for inner-heating acupuncture and acupuncture treatment. Western-bolt assay was used to detect the expression of Wnt1, Axin and β-catenin in rat intervertebral discs. Compared with the model group, Wnt1 and β-catenin expression in annulus fibrosus cells of rats in the inner heating acupuncture group and the acupuncture group were significantly decreased(P<0.05);the expression of Axin in annulus fibrosus of rats in the inner heating acupuncture group was significantly increased(P<0.05). There was no significant difference in the expression of Axin in annulus fibrosus of rats between acupuncture group and model group(P>0.05). Inner heating acupuncture can down regulate the expression of Wnt1 and β-catenin and up regulate the expression of Axin in annulus fibrosus of rats with lumbar degenerative desease. It is suggested that the mechanism may be to regulate the expression of related factors in the β-catenin signaling pathway, so as to achieve the goal of treating lumbar degenerative diseases.

Biological activity and regulatory effect of extracellular matrix expression of bone marrow mesenchymal stem cellsconditioned medium on the degenerated nucleus pulposus cells

Objective To observe the biological activities and expression of extracellular matrix of degenerative nucleus pulposus cells which were cultured in the bone marrow mesenchymal stem cells conditioned medium. Methods Bone marrow mesenchymal stem cells were extracted from the femur and tibia of adult SD rats. After subculture, the conditioned medium of bone marrow mesenchymal stem cells was collected. We destroy the annulus fibrosus of the adult rats, and the intervertebral disc was obtained 2 weeks later. Degenerative rat model was used to extract rat nucleus pulposus cells. The nucleus pulposus cells were cultured in the bone marrow mesenchymal stem cells conditioned medium for 24 hours. Results Compared with normal nucleus pulposus cells, the activity of degenerative nucleus pulposus cells [(59.43±12.33)%, t=11.647, P 0.05) and type Ⅱ collagen in cells (1.52±0.18, t=0.299, P>0.05, 1.21±0.31, t=1.543, P>0.05), proteoglycan (1.81±0.32, t=0.486, P>0.05, 0.99±0.16, t=0.094, P>0.05) and matrix metalloproteinase 3 (0.24±0.14, t=0.486, P>0.05, 0.55±0.11, t=0.593, P>0.05) Protein and mRNA expression levels were not significantly affected. Conclusion The bone marrow mesenchymal stem cell conditioned medium promoted the proliferation of extramedullary nucleus cells and the expression of extracellular matrix proteins. Key words: Intervertebral disc herniation; Nucleus pulposus cells; Extracellular matrix protein; Bone marrow mesenchymal stem cell conditioned medium; Type Ⅱ collagen; Aggrecan; Matrix metalloproteinase 3

TGF-\u03b21 reduces the oxidative stress-induced autophagy and apoptosis in rat annulus fibrosus cells through the ERK signaling pathway

BackgroundThe aim of this study is to explore the effects of TGF-β1 on autophagy and apoptosis induced by exogenous hydrogen peroxide (H2O2) in annulus fibrosus (AF) cells and possible signal pathways involved in this process.MethodsAF cells were isolated from rat lumbar discs and subjected to different concentrations of exogenous H2O2 (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 H2O2 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.ResultsH2O2 significantly impaired cell viability in a dose- and time-dependent manner. H2O2 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 H2O2-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 H2O2 through upregulating the expression level of glutathione peroxidase-1 (GPx-1).ConclusionsTGF-β1 reduced autophagy and apoptosis induced by exogenous H2O2 through downregulating the expression of ERK in AF cells. TGF-β1 could downregulate the level of ERK and intracellular H2O2 by upregulating GPx-1.

High glucose promotes annulus fibrosus cell apoptosis through activating the JNK and p38 MAPK pathways.

Diabetes mellitus (DM) is an important risk factor of intervertebral disc degeneration. A high glucose niche-mediated disc cell apoptosis is an implicate causative factor for the spine degenerative diseases related with DM. However, the effects of a high glucose niche on disc annulus fibrosus (AF) cell apoptosis and the potential signaling transduction pathway is unclear. The present study is to investigate the effects of high glucose on disc AF cell apoptosis and the role of two potential signaling pathways in this process. Rat AF cells were cultured in baseline medium or medium with different concentrations (0.1 and 0.2 M) of glucose for 3 days. Flow cytometry was used to assess the degree of apoptosis. Activity of caspase 3/9 was evaluated by chemical kit. Expression of pro-apoptotic and anti-apoptotic molecules was analyzed by real-time polymerase chain reaction and Western blot. In addition, activity of the C-Jun NH2-terminal kinases (JNK) pathway and p38 mitogen-activated protein kinase (MAPK) pathway was evaluated by Western blot. Compared with the control group, high glucose culture increased cell apoptosis ratio and caspase-3/9 activity, up-regulated expression of bax, caspase-3, cleaved caspase-3 and cleaved PARP, and down-regulated expression of bcl-2 in a glucose concentration-dependent manner. Additionally, high glucose culture increased expression of the p-JNK and p-p38 MAPK in a concentration-dependent manner. Further results showed that inhibition of the JNK or p38 MAPK pathway attenuated the effects of high glucose on AF cell apoptosis. Together, high glucose promoted disc AF cell apoptosis through regulating the JNK pathway and p38 MAPK pathway in a glucose concentration-dependent manner.

Secreted Factors From Intervertebral Disc Cells and Infiltrating Macrophages Promote Degenerated Intervertebral Disc Catabolism.

Rat nucleus pulposus (NP) cells or annulus fibrosus (AF) cells were stimulated with conditioned media of RAW 264.7 macrophages and vice versa under healthy culture conditions and in the presence of pro-inflammatory mediators. The gene expression of pro-inflammatory mediators, extracellular matrix (ECM)-modifying enzymes, and chemokines, which play important roles in intervertebral disc degeneration (IDD), was determined. To test whether the interaction between native disc cells and infiltrating macrophages accelerates inflammation state, disrupts matrix homeostasis, and promotes inflammatory cells infiltration. With macrophages infiltration, the disc resident cells would be inevitably exposed to macrophages. Macrophages have been shown to play pro-inflammatory role in the cellular interactions with disc cells under healthy culture conditions. However, the biologic interactions between macrophages and disc cells under degenerated disc inflammatory environment remain unknown. Murine Macrophages RAW 264.7 were cultured in the conditioned media of Rat AF or NP cells culture in the presence or absence of IL-1β stimulation. Similarly, Rat AF or NP cells were also cultured in the conditioned media of Murine Macrophages RAW 264.7 culture in the presence or absence of IFN-γ stimulation. The mRNA levels difference of pro-inflammatory genes, catabolic genes and chemokines genes for AF cells, NP cells and Macrophages RAW 264.7 were analyzed by qRT-PCR, respectively. Compared with serum-free media exposure, RAW 264.7 macrophages exposed to AF or NP cells conditioned media selectively modestly upregulated mRNA levels of the aforementioned cytokines. Exposure of RAW 264.7 macrophages to conditioned media from AF or NP cells with IL-1β stimulation dramatically increased mRNA levels of all the investigated cytokines. Similarly, compared with serum-free media exposure, AF or NP cells exposed to RAW 264.7 macrophages conditioned media selectively modestly upregulated mRNA levels of the aforementioned cytokines. Exposure of AF or NP cells to conditioned media from RAW 264.7 macrophages with IFN-γ stimulation dramatically increased mRNA levels of all the investigated cytokines. The biologic interactions between infiltrating macrophages and native disc cells under degenerated disc inflammatory environment lead to an increasingly severe inflammatory conditions, which may be a self-stimulated process from the macrophages infiltration occurrenceLevel of Evidence: 5.

Extensive mechanical tension promotes annulus fibrosus cell senescence through suppressing cellular autophagy.

Background: Mechanical load contributes a lot to the initiation and progression of disc degeneration. Annulus fibrosus (AF) cell biology under mechanical tension remains largely unclear.Objective: The present study was aimed to investigate AF cell senescence under mechanical tension and the potential role of autophagy.Methods: Rat AF cells were cultured and experienced different magnitudes (5% elongation and 20% elongation) of mechanical tension for 12 days. Control AF cells were kept static. Cell proliferation, telomerase activity, cell cycle fraction, and expression of senescence-related molecules (p16 and p53) and matrix macromolecules (aggrecan and collagen I) were analyzed to evaluate cell senescence. In addition, expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I were analyzed to investigate cell autophagy.Results: Compared with the control group and 5% tension group, 20% tension group significantly decreased cell proliferation potency and telomerase activity, increased G1/G0 phase fraction, and up-regulated gene/protein expression of p16 and p53, whereas down-regulated gene/protein expression of aggrecan and collagen I. In addition, autophagy-related parameters such as gene/protein expression of Beclin-1 and LC3, and the ratio of LC3-II to LC3-I, were obviously suppressed in the 20% tension group.Conclusion: High mechanical tension promotes AF cell senescence though suppressing cellular autophagy. The present study will help us to better understand AF cell biology under mechanical tension and mechanical load-related disc degeneration.

Toxicity Effects of Methylene Blue on Rat Intervertebral Disc Annulus Fibrosus Cells

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 μg/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 β1 [TGF-β1], 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 timedependent 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 upregulation of Bax and caspase-3 expression levels accompanied by decreased expression of Bcl2 in a concentration-dependent manner. Moreover, collagen type I, TGF-β1, 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

Potential involvement of semaphorin 3A in maintaining intervertebral disc tissue homeostasis.

Intervertebral discs (IVDs) are avascular; however, ingrowth of blood vessels into their outer regions has been noted during the progression of degeneration. The mechanisms underlying vascularization in IVD degeneration are not completely understood. Semaphorin 3A (Sema3A), originally characterized as a chemorepulsive factor for growing axons in the developing nervous system, inhibits angiogenesis. This study aimed to elucidate the potential involvement of Sema3A in maintaining tissue homeostasis within the avascular IVD. We demonstrated that the mRNA expression of Sema3A was higher in rat annulus fibrosus (AF) than in nucleus pulposus (NP) and that its expression level decreased with age. Both mRNA and protein expression level of Sema3A was also markedly suppressed in AF tissues of a rat IVD degeneration model. Both real-time RT-PCR and Western blot clearly indicated that Sema3A expression significantly reduced by treating inflammatory cytokines in rat AF cells. In a gain- and loss-of-function study, we observed that Sema3A reduced the catabolic shift in rat AF cells. In addition, our results indicated that Sema3A potentially inhibited the IL-6/JAK/STAT pathway. Finally, BrdU assay and tube formation assay revealed that treatment of recombinant Sema3A significantly blocks both proliferation and tube formation of HUVEC. Our results indicate that Sema3A may help maintain IVD tissue homeostasis. Thus, although further studies are needed, Sema3A may be a potential molecular target for suppressing IVD degeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Responses of apoptosis and matrix metabolism of annulus fibrosus cells to different magnitudes of mechanical tension in vitro.

Background: Annulus fibrosus (AF) is important to confine disc nucleus pulposus (NP) tissue during mechanical load experience. However, the knowledge on AF cell biology under mechanical load is much limited compared with disc NP. Objective: The present study aimed to investigate responses of apoptosis and matrix metabolism of AF cells to different magnitudes of mechanical tension in vitro. Methods: Rat AF cells were subjected to different magnitudes (5, 10, and 20% elongations at a frequency of 1.0 Hz for 6 h per day) of mechanical tension for 7 days. Control AF cells were cultured without mechanical tension. Cell apoptosis ratio, caspase-3 activity, gene/protein expression of apoptosis-related molecules (Bcl-2, Bax, caspase-3/cleaved caspase-3 and cleaved PARP), matrix macromolecules (aggrecan and collagen I) and matrix metabolism-related enzymes (TIMP-1, TIMP-3, MMP-3, and ADAMTS-4) were analyzed. Results: Compared with 5% tension group and control group, 10 and 20% tension groups significantly increased apoptosis ratio, caspase-3 activity, up-regulated gene/protein expression of Bax, caspase-3/cleaved caspase-3, cleaved PARP, MMP-3, and ADAMTS-4, whereas down-regulated gene/protein expression of Bcl-2, aggrecan, collagen I, TIMP-1, and TIMP-3. No significant difference was found in these parameters apart from Bcl-2 expression between the control group and 5% tension group. Conclusion: High mechanical tension promotes AF cell apoptosis and suppresses AF matrix synthesis compared with low mechanical tension. The present study indirectly indicates how mechanical overload induces disc degeneration through affecting AF biology.