A Macro, Nano Level Study to Evaluate the Impact of Trp2 Allele in Theα 2 Chain of Collagen IX on the Biomechanics of Human Intervertebraldiscs

A Macro, Nano Level Study to Evaluate the Impact of Trp2 Allele in the α2 Chain of Collagen IX on the Biomechanics of Human Intervertebral Discs and Disc Collagens
Submitted by Darwesh Mohideen ALADIN KADERBATCHA for the Degree of Master of Philosophy at the University of Hong Kong in March 2007

Intervertebral disc degeneration is a common problem in young individuals causing sciatica and low back pain. Recently, a heterozygous substitution of tryptophan in COL9A2 coding for the α2 chain of collagen IX was found to be a predisposing genetic factor for disc degeneration. The precise function of collagen IX and how this alteration in coding sequence results in disc degeneration are not known. With collagen IX's physical location on collagen II fibrils and their multiple cross-links, collagen IX may play an important role in the physical integrity of the extracellular matrix. This study hypothesizes that collagen IX molecules containing product of the Trp2 allele are incorporated into the disc and subsequently predisposes it to degeneration by altering the disc mechanics.
The aim of this study was to compare the mechanical properties of human nucleus pulposus from those that carry the Trp2 allele with those that do not. Moreover, since collagen fibrils are the basic structural elements of the i discs, this study also aimed at comparing the morphology and nano mechanics of the individual collagen fibrils to study the influence of the Trp2 allele.
Blood and whole-disc samples were retrieved from adolescents and young adults during scoliosis surgery, degenerated discs were retrieved from patients with back pain during spinal fusion. DNA were extracted from blood and Trp2 status was determined by sequencing. Non-degenerated (Trp2-), non- degenerated (Trp2+), degenerated (Trp2-) and degenerated (Trp2+) nucleus pulposus samples were tested in confined compression. Swelling pressure and compressive modulus were measured and compared between groups.
At the nano level, collagen II fibrils were extracted from the nucleus pulposus of non-degenerated (Trp2-) and non-degenerated (Trp2+) discs and were studied under the Atomic Force Microscope (AFM). The morphology and tensile mechanical properties of individual collagen II fibrils were evaluated and compared.
Confined compression test results showed that the mean swelling pressure and compressive modulus of 6 non-degenerated (Trp2+) samples were significantly lower (P 0.8). The compressive modulus of the degenerated (Trp2+) group was also significantly lower than the degenerated (Trp2-) group (P=0.001).
ii In the AFM study the length of the collagen II fibrils ranged between 1- 13μm. The mean diameter of collagen II fibrils from Trp2+ group was marginally larger than of those from the Trp2- discs. The 67nm characteristic D-periodic banding of fibril forming collagens was observed throughout the length of the fibrils from the Trp2+ discs. Interestingly, this banding was significantly wider in the fibrils extracted from the Trp2+ discs (P = 0.