The Spondyloepiphyseal Dysplasia Congenita homozygous Mutant: A Model for the Mechanism of Osteoarthritis

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David Macdonald and Dr. Robert Seegmiller, Department of Physiology and Developmental Biology

Articular cartilage is a hard-wearing and specialized form of hyaline cartilage which allows for an almost frictionless surface during joint movement (Ofek et al. 2008). These unique characteristics are primarily due to chondrocyte secretions, and the composition and properties of the extracellular matrix (ECM) (Buckwalter and Mankin 1998). Chondrocytes play a crucial role in cartilage durability because of their extracellular secretions (Knudson and Knudson 2001). Being immotile, chondrocytes depend greatly upon the equilibrium of the ECM for protection, integrity, and regulation (Aszodi et al. 2003). The ECM is composed primarily of type II collagen, glycoproteins, water and aggrecan proteoglycan (Hollander et al.1994).

The COL2A1 gene codes for type II collagen. After the chains are translated and secreted into the rough endoplasmic reticulum (RER), the fibrils associate to stabilize the collagen’s helical shape (Seegmiller et al. 2008). These are secreted into the ECM and incorporated into the three-dimensional, net-like collagen meshwork (Eyre 2002). Proteoglycans, while similarly produced, are comprised of a core protein covalently covered by sulfated glycosaminoglycan (GAG) chains that attract sodium ions and water molecules via osmosis. These inter-fibrillar proteoglycans ‘swell’ causing the tangled meshwork of collagen fibrils to ‘inflate’ allowing for resistance to mechanical forces and elasticity (Scott 1975). Compromises to the integrity of any of these components result in early-onset osteoarthritis (OA) (Holt et al. 2012).

Spondeloepipheseal dysplasia congenital (sedc) is a mutation of the COL2A1 gene: causing errors in type II collagen formation (Donahue et al. 2003). Therefore, the sedc mutant displays ECM disgenesis and collagen alterations. Further analysis of the homozygous sedc model provides a causative approach at further understanding OA and the severity of chondrocyte and ECM malformations in humans. This was interesting since it was not something that I was originally trying to show. Initially, when I approached this project I thought that I would see something similar to what had been published by Holt et al.: a mechanism of OA. However, as we did more testing, we noticed that the homozygous mutant displayed unique characteristics and degradation patterns. This shifted our focus to what was happening in the ECM, particularly with the collagen fibrils. It also changed the research from mechanistic to causative in nature.

We encountered several problems that were significant time deterrents: people graduating/leaving, and breeding problems. At the end of the Winter 2012 semester many members of my team graduated, and my mentor, Dr. Seegmiller, retired. This caused a great lack of communication, guidance and assistance. To resolve this, I enlisted the help of new lab members in the Seegmiller and Kooyman research labs. I also began emailing/calling Dr. Seegmiller every couple days so that we could keep each other informed. Another problem was acquiring enough homozygous mutants to make the study significant. The only way to fix this was to breed as many heterozygotes as possible.

The change in focus provided us with the results we expected to observe. Histological stains in the mutant showed increased proteoglycan staining in the area immediately surrounding the cell, increased cartilage thickness, and cartilage fissuring as compared to the wildtype. Mankin scoring demonstrated significantly increased OA present in the homozygous mutant mice at all ages studied. Immunohistochemical staining visualized type II collagen in both the wildtype and mutant. Electron microscopy showed the swollen nature of the space immediately outside of the chondrocyte of the mutant, as well as smaller type II collagen fibrils.

The irregularities that we have noticed in the homozygous mutant affect ECM components and chondrocyte characteristics, both of which induce early OA. We suggest that proteoglycan is unable to leave the space around the cell, causing the area to swell due to osmotic properties. Furthermore, a lack of viable type II collagen will not form a competent meshwork to provide structure and resistance. Thus, the cartilage will swell uncontrollably and eventually tear: OA. Further research would be needed in identifying which types of aggrecan proteoglycans are present in the homozygous mutant ECM. This information would help in the comparisons to the wildtype, as well as allowing researchers to come closer to finding possible preventative therapies. We are now in the final stages of editing the manuscript with the end goal of submitting it to the Journal of Histochemistry and Cytochemistry in early January 2013. I am very grateful to my mentor, Dr. Robert Seegmiller, as well as the suppliers of the ORCA money that helped make this possible.

References

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