Background

Proteoglycans (PGs) have long been known to be important to the functioning of the intervertebral disc. The most common PG is aggrecan, but there are also small leucine-rich proteoglycans (SLRPs) which constitute only a small percentage of the total PGs. However, they have many important functions, including organising the collagen, protecting it from degradation and attracting growth factors to the disc. We have examined how the core proteins of these molecules vary in intervertebral discs from patients with different pathologies.

Purpose

Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response.

Purpose: It has been shown that ischemic/hypoxic stress as well as shear stress and mechanical injury are linked to the pathogenesis of osteoarthritis. The aim of this study was to determine whether Egr-1 (Early Growth Response protein-1), a transcription factor induced by stress or injury, affects articular cartilage and intervertebral discs (IVDs).

Method: Experimental studies used 6- to 7-month-old adult female wild-type C57Bl/6 or Egr-1-deficient (knockout) mice. All animals were sacrificed at the same age interval (8- to 9-months) and stored at −20°C. Prior to dissection, posterior-anterior and lateral x-rays of whole mice were done. Right knee joint and cervical to lumbar spine were stained with hematoxylineosin (H& E), Safranin-O/Fast green, and Weigert’s hematoxylin/alcian blue/picrosirius red for histological analysis. Bone mineral density (BMD) was measured using a PIXImus Bone Densitometer System. Micro computed tomography (CT) data were acquired on a SkyScan T1072 X-ray Microscope-Microtomograph.

Results: Results showed that the articular cartilage of knee joints of Egr-1 knockout mice was more irregular and degenerative than in the wild type mice. Furthermore, a lower concentration of proteoglycans (predominantly aggrecan) was observed in articular cartilage of knockout mice. The nucleus pulposus (NP) of the knockout mice IVD also showed signs of degeneration and a loss of notochordal cells. The overall disc height was also reduced compared to wild type mice. The number of cells in the endplate was higher in the knockout mice than in the wild type animals. Furthermore, there was a trend for increased lumbar vertebrae BMD as well a significant increase in BMD in the femur of the Egr-1 knockout mice. The relative bone volume (BV/TV) was significantly smaller in Egr-1 knockout mice as was trabecular number and trabecular separation, while there was increased bone surface to bone volume.

Conclusion: Our findings showed signs of degeneration in both articular cartilage and IVD in Egr-1 knockout mice. Moreover, the loss of height in IVD and cellular components of the NP, as well as the increased cell numbers in cartilage of the endplate showed some resemblance to those of human degenerative disc diseases. However, further studies are needed to determine the mechanism by which Egr-1 leads to articular cartilage and IVD degeneration.

Purpose: Hormone replacement therapy for the menopause seems to be associated with a decrease in the prevalence of symptoms and radiological alterations related to hip and knee osteoarthritis. However, little is known on the effects of estrogen in articular cartilage and intervertebral disc (IVD). The aim of this study was to evaluate the developmental changes in mouse articular cartilage and intervertebral discs under estrogen deficiency.

Method: Experimental studies used 6- to 7-month-old adult female wild-type or bilaterally ovariectomized (OVX) C57Bl/6 mice. All animals were sacrificed at the same age interval (8- to 9-months) and stored at −20°C. Prior to dissection, posterior-anterior and lateral x-rays of whole mice were done. Right knee joint and cervical to lumbar spine were stained with hematoxylineosin (H& E), Safranin-O/Fast green, and Weigert’s hematoxylin/alcian blue/picrosirius red for histological analysis. Bone mineral density (BMD) was measured using a PIXImus Bone Densitometer System. Micro computed tomography (CT) data were acquired on a SkyScan T1072 X-ray Microscope-Microtomograph.

Results: Degeneration, including the loss of notochordal cells, was observed in the nucleus pulposus (NP) of the IVD of OVX mice. The annulus fibrosus (AF) showed marked thinning as compared to the wild type. Furthermore, the OVX group showed decreased IVD heights and trend of endplate ossification. Knee joints of OVX mice showed a trend towards having more gross degenerative changes, like areas of cartilage erosion. A decrease in articular cartilage thickness was also observed. Certain layers of cartilage were more affected than others, suggesting a specific role of estrogens in the developing cartilage. Also, the BMD was reduced in both the femur and lumbar vertebrae of the OVX group. Finally, MicroCT results showed a decrease in percent bone volume, trabecular thickness, trabecular number, and an increase in trabecular separation.

Conclusion: The present study showed AF thinning, decreased IVD height, NP degeneration, and loss of cellular components in the NP in ovariectomized mice. Likewise, the articular cartilage revealed more degenerative changes, including a decrease in articular thickness. Results suggest that estrogens play a role in maintaining healthy cartilage and IVD.

Hyaline cartilage and immature nucleus pulposus possess similar macromolecules in their extracellular matrix, and there is no unique molecular marker to distinguish the two tissues. We show that in normal disc (fifteen to twenty-five years old), the GAG to hydroxyproline ratio (proteoglycan to collagen ratio) within the nucleus pulposus is approximately 28:1. However, the GAG to hydroxyproline ratio within hyaline cartilage of the same group is 2.5:1. This information is important in identifying stem cell conversion to a nucleus pulposus cell phenotype rather than a chondrocyte phenotype for tissue engineering of intervertebral disc.

Tissue engineering of intervertebral discs (IVDs) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. Since there is no unique marker for disc tissue, and because cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. In this study, we compare the proteoglycan to collagen ratio in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals.

To distinguish between a normal NP and hyaline cartilage phenotype for tissue engineering of IVDs.

Human lumbar spine specimens were harvested from fresh cadavers, aged twelve week to seventy-nine year. Discs and endplates were examined for total collagen using the hydroxyproline assay and glycosaminoglycan (GAG) content using a standard assay.

In a mature disc with no degeneration (fifteen to twentyfive years), the GAG to hydroxyproline ratio within the NP is approximately 28:1. However, the ratio within the hyaline cartilage endplate of the same group is 2.5:1.

A high proteoglycan to collagen ratio can be used to distinguish NP cells from chondrocytes. The lower NP collagen content is probably responsible for its gelatinous nature rather than the firm texture of hyaline cartilage, and this is essential for normal disc function. This information is crucial in identifying a NP-like phenotype when MSCs are induced to differentiate into a disc cell as opposed to a chondrocyte, for tissue engineering of IVDs.

Purpose: In vivo, intertervertebral disc cells exist in a low oxygen environment ranging from 5% O2 for the annulus fibrosus (AF) cells to 1% O2 for the nucleus pulpous (NP) cells. Various conditions have been used for in vitro cell culture and seem that AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply, O2 levels and biomechanical loading. The aim of this study was to determine how AF and NP cells respond to different O2 concentrations when cultured in a 3 dimensional system consisting of an alginate scaffold.

Methods: Bovine AF and NP cells were embedded in alginate beads and incubated in airtight polypropylene containers at different O2 concentration of 1%, 5% or 21%. Culture medium was changed every third day and the culture was carried out for 21 days. The pro-teoglycan content of the medium was analyzed using the DMMB assay. Cells were recovered from the alginate beads at two time points, day 8 and day 21 and RT-PCR was performed to amplify gene expression of GAPDH and aggrecan.

Results: In both cell types, the cumulative production of GAG increased with time in culture up to day 9, and then tended to plateau in the AF cells but continue to increase in the NP cells. At all time points, the level of GAG synthesis by NP cells was greater than by AF cells. All GAG synthesis trends were the similar at all O2 levels (1%, 5% and 21%).

Conclusions: In the alginate scaffold NP cells continue to exhibit their in situ behaviour by producing more proteoglycan than AF cells. Perhaps surprisingly, both cell types showed little change in GAG production with variations of O2 levels from 1–21%. This contrasts with other studies where GAG production is dependent on O2 concentrations. In the culture system used in this work, both cell types metabolize easily well at low oxygen as they do in normal conditions. Funding: Other Education Grant