Adult Human Intervertebral Disc Research Articles

Immunohistochemical analysis of protein expression in formalin fixed paraffin embedded human intervertebral disc tissues.

Immunohistochemistry (IHC) is a useful technique for the localization and semiquantification of protein expression within tissues. Adult human intervertebral disc (IVD) tissues contain a large amount of auto‐fluorescence which often makes immunofluorescence techniques inappropriate on tissue samples but can be applied to isolated cell samples. Thus, IHC remains one of, if not the most common application for protein detection within IVD tissue. Immunostaining localizes antigen expression through specific epitope‐antibody interactions. Within the field of IVD research, IHC is commonly used on fresh frozen and paraffin embedded tissues to elucidate the expression of antigens. Here, we discuss the principles of IHC applied to formalin fixed paraffin embedded IVD tissue and supply optimized protocols for antibodies used within our group to guide research within the IVD field.

The non-aggregated aggrecan in the human intervertebral disc can arise by a non-proteolytic mechanism.

Analysis of both the aggregated and non-aggregated fractions of aggrecan isolated from adult human intervertebral disc using immunoblotting with antibodies specific for the different domains constituting the aggrecan core protein or atomic force microscopy revealed that many components contained the G1 domain. However, little of the disc aggrecan was able to reform aggregates with hyaluronan, as determined by gel filtration chromatography, suggesting that the G1 domains had been rendered non-functional. Since previous studies have shown that disc aggrecan undergoes non-enzymatic glycation with age, the functional effect of such modification was investigated in vitro using bovine aggrecan isolated from young animals. Incubation of monomeric aggrecan with ribose to induce glycation rendered it unable to form complexes with hyaluronan stable to agarose gel electrophoresis or gel filtration chromatography. Similarly, extended treatment of intact proteoglycan aggregate with ribose resulted in destabilisation of the complex with separation of the aggrecan from the hyaluronan. Although it is clear that proteolysis occurs in the intervertebral disc and gives rise to some non-aggregating molecules, a different mechanism is required to explain the presence of many non-aggregating molecules bearing the G1 domain. The products of non-enzymatic glycation of the globular domains of aggrecan would account for this phenomenon and explain why some of the non-aggregating molecules are still large proteoglycans. While such molecules may be retained in the nucleus pulposus, they may be able to diffuse within it, reducing the ability of the tissue to resist compression under asymmetric loading such as bending and ultimately contributing to disc degeneration.

The Fuzzy Logic of Degenerative Disc Disease: From a Lorenz Attractor to a Percolation Threshold Model

Apart from a small number of capillaries that penetrate only a few millimeters into the outermost annulus, the normal adult human intervertebral disc is virtually avascular (9). Therefore, the nutrients necessary for the survival of the cells inside the disc (especially glucose and oxygen) are derived mainly from diffusion from vascular channels (which have been called “vascular buds”) present in the limit between the intervertebral disc and the vertebral cartilagenous endplate (3). In a very elegant study, a research group from the Biomedical Engineering Department of the University of Miami investigated the effects of mechanical deformation and anisotropy on the transport of oxygen and glucose in the annulus fibrosus of human intervertebral discs obtained from a tissue bank (7). Using custom-designed chambers (Figure 1A and B), the diffusivities of oxygen and the partition coefficient of glucose were measured under 3 levels of confined uniaxial compression (0%, 10%, and 20%) and in 3 directions (axial, circumferential, and radial) (Figure 1C). The results demonstrated that glucose

Development of an Organ Culture System for Long-Term Survival of the Intact Human Intervertebral Disc

Human intervertebral discs were used to develop an intact whole disc organ culture system with long-term cell viability. To develop and validate a long-term organ culture system for intact human intervertebral discs, in which the potential for biologic repair of disc degeneration can be studied. Intervertebral disc degeneration is a common cause of back pain, which can be costly to the health care system and have a negative impact on the quality of life of the patient. Once injured the adult human intervertebral disc seems incapable of intrinsic repair, but the early stages of disc degeneration can potentially be retarded or even reversed by the administration of growth factors to promote new extracellular matrix synthesis. Intervertebral discs were prepared by three isolation techniques and placed in free swelling organ culture. Cell viability, disc swelling, glycosaminoglycan content, and extracellular matrix degradation were assessed under a variety of culture conditions. Human intervertebral discs isolated with intact cartilage end plates retained cell viability and did not undergo matrix degradation when cultured for 4 weeks with both a high and low nutrient level. This contrasted with the excessive cell death that was observed if the cartilage end plates were removed before culture or if vertebral bone was retained. Retention of the cartilage end plates limits tissue swelling and permits efficient nutrient supply, thus allowing viable long-term organ culture. The availability of such a system will permit the repair potential of therapeutic candidates to be studied in human discs with naturally occurring degeneration. Furthermore, the system is simple and economical, as no apparatus is needed to limit the detrimental effects of excessive tissue swelling.

The influence of nutrient supply and cell density on the growth and survival of intervertebral disc cells in 3D culture

The adult human intervertebral disc (IVD) is normally avascular. Changes to the extracellular matrix in degenerative disc disease may promote vascularisation and subsequently alter cell nutrition and disc homeostasis. This study examines the influence of cell density and the presence of glucose and serum on the proliferation and survival of IVD cells in 3D culture. Bovine nucleus pulposus (NP) cells were seeded at a range of cell densities (1.25 × 10(5)-10(6) cells/mL) and cultured in alginate beads under standard culture conditions (with 3.15 g/L glucose and 10 % serum), or without glucose and/or 20% serum. Cell proliferation, apoptosis and cell senescence were examined after 8 days in culture. Under standard culture conditions, NP cell proliferation and cluster formation was inversely related to cell seeding density, whilst the number of apoptotic cells and enucleated "ghost" cells was positively correlated to cell seeding density. Increasing serum levels from 10% to 20% was associated with increased cluster size and also an increased prevalence of apoptotic cells within clusters. Omitting glucose produced even larger clusters and also more apoptotic and senescent cells. These studies demonstrate that NP cell growth and survival are influenced both by cell density and the availability of serum or nutrients, such as glucose. The observation of clustered, senescent, apoptotic or "ghost" cells in vitro suggests that environmental factors may influence the formation of these phenotypes that have been previously reported in vivo. Hence this study has implications for both our understanding of degenerative disc disease and also cell-based therapy using cells cultured in vitro.

The presence and absence of lymphatic vessels in the adult human intervertebral disc: relation to disc pathology

Although the normal adult human intervertebral disc is considered to be avascular, vascularised cellular fibrous tissue can be found in pathological conditions involving the disc such as disc herniation. Whether lymphatics vessels form a component of this reparative tissue is not known as the presence or absence of lymphatics in herniated and normal disc tissue is not known. We examined spinal tissues and discectomy specimens for the presence of lymphatics. The examination used immunohistochemistry to identify the specific lymphatic endothelial cell markers,podoplanin and LYVE1. Lymphatic vessels were not found in the nucleus pulposus or annulus fibrosus of intact, non-herniated lumbar and thoracic discs but were present in the surrounding ligaments. Ingrowth of fibrous tissue was seen in 73% of herniated disc specimens of which 36% contained LYVE1+/podoplanin + lymphatic vessels. Lymphatic vessels were not seen in the sacrum and coccyx or biopsies of four sacrococcygeal chordomas, but they were noted in surrounding extra-osseous fat and fibrous tissue at the edge of the infiltrating tumour. Our findings indicate that lymphatic vessels are not present in the normal adult intervertebral disc but that, when there is extrusion of disc material into surrounding soft tissue, there is ingrowth of reparative fibrous tissue containing lymphatic vessels. Our findings also indicate that chordoma, a tumour of notochordal origin, spreads to regional lymph nodes via lymphatics in para-spinal soft tissues.