Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Summary

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

Abstract

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

Introduction

The intervertebral disc (IVD) is a cartilage-based structure that biochemically and with respect to cellular architecture, at first sight, resembles in many ways the articular cartilage¹. Indeed, both IVD degeneration and osteoarthritis (OA) of articular cartilage are characterized by joint space narrowing due to cartilage wear, subchondral cyst and osteophyte formation, and subchondral sclerosis^2,3. Despite these seeming similarities architecture and functional role of both tissues differ. While the matrix of articular cartilage is mainly formed of an arcade-forming collagen type II ne....

Protocol

For the analysis of embryonic development and maturation, bovine discs were used. To evaluate degeneration of the IVD, human samples were analyzed.

Human IVD tissue was obtained from patients undergoing surgery for lumbar disc degeneration, disc prolapse, or spinal trauma in the Department of Orthopaedic Surgery, University Hospital of Tübingen and the BG Trauma Centre Tübingen. Full ethical committee approval was obtained before the commencement of the study (project number 244/2013BO2). Written informed consent was received from all patients before participation. The methods were carried out in accordance with the approved guidelines.

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Results

Using mosaic images, the architecture of the IVD with its dense collagen fiber network in the anulus and the softer nucleus can clearly be recognized (Figure 4). A continuous decrease in cellular density can be observed during embryonic development (Figure 5). While in the early stages of IVD development a cell density of 11,435 cells/mm² in the bovine anulus fibrosus and 17,426 cells/mm² in the bovine nucleus pulposus can be found, these numbers decrease rapidl.......

Discussion

Using fluorescence microscopy augmented by mosaic imaging and optical sectioning, we evaluated the spatial arrangement of chondrocytes in the anulus of the lumbar IVD throughout development, maturation, and degeneration. While degenerative tissue could be harvested from patients receiving spine surgery for disc degeneration, analysis of the embryonic period and maturation phase required the use of a model organism (bovine). High cellular densities were noted in the anulus during early embryonic development. In the furthe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amphotericin B	Merck KGaA,  Germany	A2942
Adhesion Microscope Slides SuperFrost Plus	R. Langenbrinck, Germany	03-0060
ApoTome	Carl Zeiss MicroImaging GmbH, Germany	462000115
AxioVision Rel. 4.8 with Modul MosaiX	Carl Zeiss MicroImaging GmbH, Germany
CellMask Actin Tracking Stain	Thermo Fischer Scientific, US	A57249
Cryostat	Leica Biosystems, US	CM3050S
DAPI	Thermo Fischer Scientific, US	D1306
Dulbecco's modified Eagle's medium (DMEM)	Gibco, Life Technologies, Germany	41966052
Ethylenediaminetetraacetic acid	Sigma-Aldrich, US	60004
Fluorescence Miscoscope - Axio Observer Z1 with Axio Cam MR3 and Colibri	Carl Zeiss MicroImaging GmbH, Germany	3834000604
Formaldehyde	Merck KGaA,  Germany	104002
Image J 1.53a, with Cell counter plugin	National Insittute of Health (NIH), US
Invitrogen Alexa Fluor 568 Phalloidin	Thermo Fischer Scientific, US	A12380
Microscopic Cover Glasses	R. Langenbrinck, Germany	01-1818/1
PAP Pen Liquid Blocker	Science Sevices  GmbH, Germany	N71310
Penicillin-Streptomycin	Sigma-Aldrich, US	P4333
Phosphate buffered saline	Sigma-Aldrich,US	P5119
Scalpel	pf medical AG, Germany	2023-01
Tissue-tek O.C.T. Compound	Sakura Finetek, Netherlands	SA6255012