Characterization of Human Endplate Biopsies: Preliminary Results on Cellular Composition, Metabolic Activity and Interactions with Disk Cells

Abstract

Introduction Tissue alterations that result in an impaired endplate (EP) permeability are assumed to play a crucial role in the pathogenesis of intervertebral disk (IVD) degeneration. Therefore, disturbance of endplate permeability is one of the main problems that need to be solved for successful disk tissue engineering. However, the biological mechanisms underlying these alterations are poorly understood. To shed light on this aspect, a standardized analysis of EP structure and cellular composition is essential to understand the degeneration processes. Purpose of this study was the development of standardized protocols for a descriptive characterization of EP samples by histology, transmission electron microscopy (TEM), analysis of differentiation and metabolic activity of EP cells. In first preliminary experiments we also aimed to detect possible interactions of the EP with disk cells of the same donor patients in a bioassay with EP-conditioned media. The suitability of these methods for EP characterization and possible correlations with degenerative alterations is discussed. Materials and Methods Cartilaginous endplates obtained during spine surgery of total 27 human donors underwent the following procedures: (1) isolation of endplate cells, in vitro expansion and characterization of chondrogenic differentiation in 3D pellet cultures ( n = 6), (2) preparation of standardized EP-sections for histological and ultrastructural analyses ( n = 8), and (3) preparation of standardized endplate punches that were incubated for 24 hours in serum-free culture medium followed by determination of metabolic activity of endplate cells by MTT-test ( n = 13) and further analysis of conditioned media (CM) in a bioassay with NP cells of the same donor ( n = 2). Pellet cultures of EP cells were characterized for matrix protein expression by Alcian blue staining. Influences of CM on gene expression of matrix proteins, matrix degrading enzymes, and inflammation factors by NP cells were determined. Results Differentiation of endplate cells in pellet cultures varied strongly not only between the six individual donors, but also in different disk levels of the same patient. Part of the samples exhibited a very low proteoglycan production whereas others were strongly positive in the Alcian blue staining intensity suggesting high variations in the differentiation capacity of EP cells. Histological examination of the endplate samples allowed a characterization of both the endplates and an observation of the adjacent structures as the very thin endplate layer could not be clearly separated from adjacent tissue structures. High variations between tissue samples from different donors could be found. TEM images showed signs of degeneration both in the matrix and the EP cells whereas higher numbers of apoptotic cells and matrix degradation products could be found in stronger degenerated tissue samples. Standardized EP punches that were cultured for 24 hours in DMEM and incubated for 4 hours with MTT showed different staining intensities suggesting donor-specific variations in metabolic activity of EP cells. In first two experiments with CM of EP samples, a dose-dependent upregulation of inflammation-factors and matrix metalloproteinases (Interleukins IL-1β, IL-6, IL-8, MMP-3, and MMP13) by NP-cells of the same donors could be detected. The extent of this effect differed strongly between the two patients with different degeneration degree. Conclusion The present results suggest that a combination of different methods allows the characterization of EP tissue and possible interactions with the adjacent disk tissue. As the cartilaginous EP itself is very thin and with low cellularity, isolation of an almost homogeneous EP population is very difficult and the cell yield is very poor. Therefore, methods that directly analyze the cells in situ for example by laser capture micro-dissection might be more suitable to determine the gene expression profile of EP cells. Analysis of metabolic activity of standardized EP punches by MTT test is a promising approach provided that the samples are standardized with regard to diameter and height with no adjacent other tissue regions. TEM investigation is appropriate to investigate cellular details, but also with this method, the poor cellularity of EP tissue is a challenge. A very promising approach is the analysis of CM in a bioassay with NP cells of the same donor that allows the detection of possible interactions between both tissue areas. In ongoing investigations, we analyze higher numbers of EP samples with different degrees of degeneration to enable a correlation between the biological and clinical data of the patients. For this purpose, analyses of nondegenerated control samples are essential. In summary, all these methods together allow a holistic view on the mechanisms that contribute to the morphological and pathophysiological changes of the EP and potential interaction with the adjacent disk tissue. Acknowledgement Part of this research work was supported by the EU framework 7 project Geneodisc (FP7, 2007–2013) under grant agreement no. HEALTH-F2-2008-201626. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared