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Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc.

ABSTRACT: Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col Iα 1, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

SUBMITTER: Bhunia BK

PROVIDER: S-EPMC5776988 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Intervertebral disc degeneration impairs the quality of patients lives. Even though there has been development of many therapeutic strategies, most of them remain unsatisfactory due to the limited understanding of the mechanisms that underlie the intervertebral disc degeneration. Questions/purposes: This study is meant to identify the key modules and hub genes related to the annulus fibrosus in intervertebral disc degeneration (IDD) through: (1) constructing a weighted gene co-expression network; (2) identifying key modules and hub genes; (3) verifying the relationships of key modules and hub genes with IDD; and (4) confirming the expression pattern of hub genes in clinical samples. Methods: The Gene Expression Omnibus provided 24 sets of annulus fibrosus microarray data. Differentially expressed genes between the annulus fibrosus of degenerative and non-degenerative intervertebral disc samples have gone through the Gene Ontology (GO) and pathway analysis. The construction of a gene network and classification of genes into different modules were conducted through performing Weighted Gene Co-expression Network Analysis. The identification of modules and hub genes that were most related to intervertebral disc degeneration was proceeded. In order to verify the relationships of the module and hub genes with intervertebral disc degeneration, Ingenuity Pathway Analysis was operated. Clinical samples were adopted to help verify the hub gene expression profile. Results: One thousand one hundred ninety differentially expressed genes were identified. Terms and pathways associated with intervertebral disc degeneration were presented by GO and pathway analysis. The construction of a Weighted Gene Coexpression Network was completed and clustering differentially expressed genes into four modules was also achieved. The module with the lowest P-value and the highest absolute correlation coefficient was selected and its relationship with intervertebral disc degeneration was confirmed by Ingenuity Pathway Analysis. The identification of hub genes and the confirmation of their expression profile were also realized. Conclusions: This study generated a comprehensive overview of the gene networks underlying annulus fibrosus in intervertebral disc degeneration. Clinical Relevance: Modules and hub genes identified in this study are highly associated with intervertebral disc degeneration, and may serve as potential therapeutic targets for intervertebral disc degeneration.

Project description:Annulus fibrosus (AF) damage commonly occurs due to intervertebral disc (IVD) degeneration/herniation. The dynamic mechanical role of the AF is essential for proper IVD function and thus it is imperative that biomaterials developed to repair the AF withstand the mechanical rigors of the native tissue. Furthermore, these biomaterials must resist accelerated degradation within the proteolytic environment of degenerate IVDs while supporting integration with host tissue. We have previously reported a novel approach for developing collagen-based, multi-laminate AF repair patches (AFRPs) that mimic the angle-ply architecture and basic tensile properties of the human AF. Herein, we further evaluate AFRPs for their: tensile fatigue and impact burst strength, IVD attachment strength, and contribution to functional spinal unit (FSU) kinematics following IVD repair. Additionally, AFRP resistance to collagenase degradation and cytocompatibility were assessed following chemical crosslinking. In summary, AFRPs demonstrated enhanced durability at high applied stress amplitudes compared to human AF and withstood radially-directed biaxial stresses commonly borne by the native tissue prior to failure/detachment from IVDs. Moreover, FSUs repaired with AFRPs and nucleus pulposus (NP) surrogates had their axial kinematic parameters restored to intact levels. Finally, carbodiimide crosslinked AFRPs resisted accelerated collagenase digestion without detrimentally effecting AFRP tensile properties or cytocompatibility. Taken together, AFRPs demonstrate the mechanical robustness and enzymatic stability required for implantation into the damaged/degenerate IVD while supporting AF cell infiltration and viability. STATEMENT OF SIGNIFICANCE:The quality of life for millions of individuals globally is detrimentally impacted by IVD degeneration and herniation. These pathologies often result in the structural demise of IVD tissue, particularly the annulus fibrosus (AF). Biomaterials developed for AF repair have yet to demonstrate the mechanical strength and durability required for utilization in the spine. Herein, we demonstrate the development of an angle-ply AF repair patch (AFRP) that can resist the application of physiologically relevant stresses without failure and which contributes to the restoration of functional spinal unit axial kinematics following repair. Furthermore, we show that this biomaterial can resist accelerated degradation in a simulated degenerate environment and supports AF cell viability.

Project description:Degeneration of the intervertebral disc (IVD) is characterized by marked degradation and restructuring of the annulus fibrosus (AF). Although several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF and to identify the manner by which AF cells use MMP-2.Two established animal models of disc degeneration, static compression and transannular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. With lentiviral transduction of an shRNA expression cassette, we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both noninfected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels.Both static compression and 18-g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, whereas 22-g and 26-g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knockdown of transcript levels by as much as 88%, and protein levels by as much as 95% over a 10-day period. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is used locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices.Our study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally induced degenerative changes in rodent animal models. AF cells appear to use MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease and could be a potential therapeutic target.

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Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc.

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