Development and characterisation of mechanical and enzymatic models of cartilage degeneration

Currently, there is a gap between pharmacological treatment and joint replacement for the
management of cartilage degradation diseases, such as osteoarthritis. It may be possible to use
cartilage substitution materials to treat small defects in cartilage tissues, delaying the need for joint
replacements, which have a limited lifetime in vivo, so are not suitable for many patients. A major
barrier to the use of cartilage substitution materials is suitable in vitro testing of cartilage materials.
Therefore mechanical and enzymatic models of cartilage degeneration were developed, which may
be used to assess novel cartilage substitution materials.

A single station pin-on-plate rig with a variable load was used to degrade the cartilage tissue
of osteochondral pins and plates to produce two mechanical models of degradation denoted “mild”
and “moderate”. A Ringer’s solution and serum based lubricant were chosen to hydrate cartilage
tissues during articulation. The lubricants used during mechanical degradation were collected and
analysed quantitatively for glycosaminoglycan (GAG) and collagen content. In addition, a method for
isolating and imaging the cartilage wear particles in the lubricant was developed. A chondroitinase
ABC enzyme was used to enzymatically degrade cartilage tissues.

The mechanical and enzymatic degradation in the models was characterised using a broad
range of mechanical and biological assessment techniques. The mechanical degradation and wear of
the tissues created using the pin-on-plate rig was evaluated using cartilage height measurements,
friction measurements, surface profilometry, histological and immunohistological staining, and
quantitative biochemical assays. The wear on the surface of the tissue was observed using
environmental scanning electron microscopy and the tissue ultrastructure was observed using
transmission electron microscopy. The tissues degraded using chondroitinase ABC were analysed
using indentation testing, histological and immunohistological staining, quantitative biochemical
assays, and transmission electron microscopy.

It was determined that an increased load used during pin-on-plate testing resulted in an
increase in tissue degradation. Mechanical degradation under the “moderate” loading condition
caused the surface of the cartilage tissue to become fibrillated and areas of tissue loss were
observed. Under the mild condition the cartilage surface remained relatively smooth however,
several small fissures were observed in some specimens. The surface of the tissue degraded under
moderate conditions was significantly rougher than that degraded under the mild condition. There
was a small loss of GAGs in the mild condition whereas a large volume of GAGs were lost from the
tissue under the moderate condition, and the aggrecan network in the tissue was heavily disrupted.

There was no significant difference between the friction measurements or the height measurements
recorded for the specimens under the two variable loading conditions. Immunohistochemical
staining for minor tissue components showed that collagen VI and cartilage oligomeric matrix
protein (COMP) were not altered by the mechanical degradation, whereas a loss in biglycan was
observed in specimens loaded under the moderate condition. It was observed that the serum
lubricant may protect the cartilage tissue from degradation during articulation. An increased number
of wear particles were observed in the lubricants recovered from the moderate loading condition
tests. Digestion of tissues with chondroitinase ABC led to an increase in tissue deformation during
indentation. The level of GAGs in the tissues was reduced and the GAGs associated with the
aggrecan network in the tissue were no longer visible. Collagen VI and COMP were not significantly
affected by chondroitinase ABC digestion, however biglycan staining was reduced at the superficial
to middle zone of the tissue.

The models produced have potential to be used in the assessment of novel cartilage
substitution materials. The parameters used in this study will also be useful in the development of in
vitro whole joint simulators.