Regional Variations in the Cellular, Biochemical, and Biomechanical Characteristics of Rabbit Annulus Fibrosus

<div><p>Tissue engineering of annulus fibrosus (AF), the essential load-bearing disc component, remains challenging due to the intrinsic heterogeneity of AF tissue. In order to provide a set of characterization data of AF tissue, which serve as the benchmark for constructing tissue engineered AF, we analyzed tissues and cells from various radial zones of AF, i.e., inner AF (iAF), middle AF (mAF), and outer AF (oAF), using a rabbit model. We found that a radial gradient in the cellular, biochemical, and biomechanical characteristics of rabbit AF existed. Specifically, the iAF cells (iAFCs) had the highest expression of collagen-II and aggrecan genes, while oAF cells (oAFCs) had the highest collagen-I gene expression. The contents of DNA, total collagen and collagen-I sequentially increased from iAF, mAF to oAF, while glycosaminoglycan (GAG) and collagen-II levels decreased. The cell traction forces of primary AFCs gradually decreased from iAFCs, mAFCs to oAFCs, being 336.6±155.3, 199.0±158.8, and 123.8±76.1 Pa, respectively. The storage moduli of iAF, mAF, and oAF were 0.032±0.002, 2.121±0.656, and 4.130±0.159 MPa, respectively. These measurements have established a set of reference data for functional evaluation of the efficacy of AF tissue engineering strategies using a convenient and cost-effective rabbit model, the findings of which may be further translated to human research.</p></div

Mechanical tests of AF tissues.

<p>(A–B) The picture of a paraffin-embedded AF sample and a schematic showing how the testing regions and points were selected. Note NP was removed from the IVD. (C) The pre-test and post-test images of a sample under nanoindentation test, from which an imprint is clearly seen (shown by the arrowhead). (D) The storage moduli of oAF, mAF and iAF measured using nanoindentation at different frequencies. (E) The Young’ moduli of oAF, mAF and iAF measured using tensile test. All data are presented as mean±SD. Asterisk (*) indicates significant difference between groups (<i>p</i><0.01, n≥10).</p

CTFM measurement of AF cells.

<p>(A) CTFM for measuring CTFs of iAFC, mAFC and oAFC. <i>a–c</i>, primary cells from each region; <i>d–f</i>, the substrate displacement fields; <i>g–i</i>, the CTF maps. (B–C) CTFs and spread areas of iAFC, mAFC and oAFC, respectively. All data are presented as mean±SD. Asterisk (*) indicates significant difference between groups (<i>p</i><0.05, n≥30).</p

Sequences of primers for RT-PCR.

<p>Sequences of primers for RT-PCR.</p

Histological and immunohistochemical analysis of AF tissue.

<p>(A–C) H&E stain of iAF, mAF and oAF, respectively. (D) H&E stain of a whole AF tissue section. (E) Safranin Orange–Fast Green stain of a whole AF tissue section. (F–G) Immunohistochemical stain for collagen-I and collagen-II expression of whole AF tissue sections.</p

Morphology, proliferation, and gene expression of AF cells.

<p>(A–C) Phase contrast images of primary iAFCs, mAFCs, and oAFCs. (D) Proliferation of primary AFCs. (E–G) Real time quantitative PCR analysis of collagen-I, collagen-II, and aggrecan genes, respectively, for primary AFCs of various regions. Gene expression was normalized to GAPDH expression, and fold differences were calculated using the △△Ct method by comparing to gene expression of oAFCs. All data are presented as mean±SD. Asterisk (*) indicates significant difference between groups (<i>p</i><0.01, n = 3).</p

Schematic illustration of the sample preparation for nanoindentation test.

<p>Schematic illustration of the sample preparation for nanoindentation test.</p

Recherches archéologiques au Luristan (Iran)

info:eu-repo/semantics/publishe

Identification of Rabbit Annulus Fibrosus-Derived Stem Cells

<div><p>Annulus fibrosus (AF) injuries can lead to substantial deterioration of intervertebral disc (IVD) which characterizes degenerative disc disease (DDD). However, treatments for AF repair/regeneration remain challenging due to the intrinsic heterogeneity of AF tissue at cellular, biochemical, and biomechanical levels. In this study, we isolated and characterized a sub-population of cells from rabbit AF tissue which formed colonies <i>in vitro</i> and could self-renew. These cells showed gene expression of typical surface antigen molecules characterizing mesenchymal stem cells (MSCs), including CD29, CD44, and CD166. Meanwhile, they did not express negative markers of MSCs such as CD4, CD8, and CD14. They also expressed Oct-4, nucleostemin, and SSEA-4 proteins. Upon induced differentiation they showed typical osteogenesis, chondrogenesis, and adipogenesis potential. Together, these AF-derived colony-forming cells possessed clonogenicity, self-renewal, and multi-potential differentiation capability, the three criteria characterizing MSCs. Such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using cell therapies or tissue engineering approaches.</p></div

Number of patients with adjacent disc degeneration in two groups.

<p><i>P</i> = 0.033.</p