Dataset Information

Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

ABSTRACT: The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus fibrosus harvested from intervertebral disc of seven patients undergoing surgery. Samples were loaded to 6% tensile strain in 1% steps. Elastic modulus was calculated from loading curves, and micromechanical strains were calculated from the images using custom software. The same protocol was applied to twenty strips of annulus harvested from cow tail discs. Significant morphological differences were found between human and cow tail samples, the most striking being the lack of collagen fibre crimp in the former. Fibres were also observed bending and running from one lamella to the other, forming a strong flexible interface. Interdigitation of fibre bundles was also present at this interface. Quantitative results show complex patterns of inter-bundle and inter-lamellar behaviour, with inter-bundle sliding being the main strain mechanism. Elastic modulus was similar between species, and it was not affected by the degree of degeneration. This work gives an insight into the complex structure and mechanical function of the annulus fibrosus, which should be accounted for in disc numerical modelling.

SUBMITTER: Vergari C

PROVIDER: S-EPMC5511600 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

Vergari Claudio C Chan Daniel D Clarke Andrew A Mansfield Jessica C JC Meakin Judith R JR Winlove Peter C PC

Biomechanics and modeling in mechanobiology 20170404 4

The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus f ...[more]

PMID: 28378119

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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.

Project description:INTRODUCTION:Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors. METHODS:Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor ? (TGF?). RESULTS:The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGF? induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections. CONCLUSION:We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.

Project description:BACKGROUND:The lifetime prevalence of degenerative disc disease is dramatically high. Numerous investigations on disc degeneration have been performed on cells from annulus fibrosus (AF) and nucleus pulposus (NP) of the intervertebral disc (IVD) in cell culture experiments utilising a broad variety of basal culture media. Although the basal media differ in nutrient formulation, it is not known whether the choice of the basal media itself has an impact on the cell's behaviour in vitro. In this study, we evaluated the most common media used for monolayer expansion of AF and NP cells to set standards for disc cell culture. METHODS:Human AF and NP cells were isolated from cervical discs. Cells were expanded in monolayer until passage P2 using six different common culture media containing alpha-Minimal Essential Medium (alpha-MEM), Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F-12 medium (Ham's F-12) as single medium or in a mixture of two media (alpha/F-12, DMEM/alpha, DMEM/F-12). Cell morphology, cell growth, glycosaminoglycan production and quantitative gene expression of cartilage- and IVD-related markers aggrecan, collagen type II, forkhead box F1 and keratin 18 were analysed. Statistical analysis was performed with two-way ANOVA testing and Bonferroni compensation. RESULTS:AF and NP cells were expandable in all tested media. Both cell types showed similar cell morphology and characteristics of dedifferentiation known for cultured disc cells independently from the media. However, proceeding culture in Ham's F-12 impeded cell growth of both AF and NP cells. Furthermore, the keratin 18 gene expression profile of NP cells was changed in alpha-MEM and Ham's F-12. CONCLUSION:The impact of the different media itself on disc cell's behaviour in vitro was low. However, AF and NP cells were only robust, when DMEM was used as single medium or in a mixture (DMEM/alpha, DMEM/F-12). Therefore, we recommend using these media as standard medium for disc cell culture. Our findings are valuable for the harmonisation of preclinical study results and thereby push the development of cell therapies for clinical treatment of disc degeneration.

Dataset Information

Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

Publications

Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

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