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Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway.

ABSTRACT: Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.

SUBMITTER: Zhang W

PROVIDER: S-EPMC8040478 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Degenerative disc disease (DDD) is accompanied by structural changes in the intervertebral discs (IVD). Extra-cellular matrix degradation of the annulus fibrosus (AF) has been linked with degeneration of the IVD. Collagen is a vital component of the IVD. Collagen hybridizing peptide (CHP) is an engineered protein that binds to degraded collagen, which we used to quantify collagen damage in AF. This method was used to compare AF samples obtained from donors with no DDD to AF samples from patients undergoing surgery for symptomatic DDD. Fresh AF tissue was embedded in an optimal cutting temperature compound and cryosectioned at a thickness of 8 μm. Hematoxylin and Eosin staining was performed on sections for general histomorphological assessment. Serial sections were stained with Cy3-conjugated CHP and the mean fluorescence intensity and areal fraction of Cy3-positive staining were averaged for three regions of interest (ROI) on each CHP-stained section. Increases in mean fluorescence intensity (p = 0.0004) and percentage of positively stained area (p = 0.00008) with CHP were detected in DDD samples compared to the non-DDD samples. Significant correlations were observed between mean fluorescence intensity and percentage of positively stained area for both non-DDD (R = 0.98, p = 5E-8) and DDD (R = 0.79, p = 0.0012) samples. No significant differences were detected between sex and the lumbar disc level subgroups of the non-DDD and DDD groups. Only tissue pathology (non-DDD versus DDD) influenced the measured parameters. No three-way interactions between tissue pathology, sex, and lumbar disc level were observed. These findings suggest that AF collagen degradation is greater in DDD samples compared to non-DDD samples, as evidenced by the increased CHP staining. Strong positive correlations between the two measured parameters suggest that when collagen degradation occurs, it is detected by this technique and is widespread throughout the tissue. This study provides new insights into the structural alterations associated with collagen degradation in the AF that occur during DDD.

Project description: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.

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Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway.

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