We are currently studying the physiology of in situ chondrocytes exhibiting normal or abnormal morphology, to try to determine what causes the changes in shape and hence matrix metabolism that characterise osteoarthritis.
Osteoarthritis (OA, or more correctly osteoarthrosis) is a very common and debilitating syndrome of human articular cartilage.
It used to be thought that it arose from cartilage ‘wear-and-tear’, but there is increasing evidence that changes to the normal physiology of cartilage cells (chondrocytes) plays a key role, although the details are largely unclear.
Human chondrocytes are normally ellipsoidal/spheroidal, but using confocal microscopy and our state-of-the-art imaging facility (IMPACT) which enables the visualisation of living cells within their native environment, we have observed cells with remarkable ‘processes’ extending for significant distances from the cell body (Figure 1; Bush & Hall 2003).
These are present even in non-degenerate cartilage, and preliminary work suggests their incidence increases markedly with the degree of OA.
Changes to chondrocyte morphology alters the type and mechanical properties of the extracellular matrix which the cells produce. Are these changes to morphology an early and preventable step in the development of OA?
Changes to cell shape may arise from alterations to chondrocyte biology (such as control of cell volume, cytoskeletal arrangement).
We are currently studying the physiology of in situ chondrocytes exhibiting normal or abnormal morphology, to try to determine what causes the changes in shape and hence matrix metabolism that characterise OA.
We are also developing a three-dimensional culture system to study the factors involved in the regulation of chondrocyte morphology under more defined conditions.
Figure 1 shows the abnormal morphology of an in situ chondrocyte within the tibial plateau of osteoarthritic cartilage. Articular chondrocytes are normally elliptical/spheroidal in shape, however a surprising proportion of cells demonstrated abnormal morphology (Figure courtesy of Dr Peter G Bush; also see Bush & Hall 2003 for further details).
The regulated swelling of chondrocytes in the growth plate (termed hypertrophy) is an essential step, crucial for the orderly and rapid lengthening of bones during skeletal development.
The failure of hypertrophy is directly implicated in growth and skeletal disorders and also childhood injuries of the growth plate.
However the process(es) by which chondrocytes swell is unknown. It is highly probable that the accumulation of osmolytes (especially ions) by chondrocyte membrane transporters coupled with modulation of normal volume regulatory pathways are involved.
This project utilises confocal laser scanning microscopy (CLSM) to study the role of the various volume-sensitive membrane transporters of living in situ and isolated growth plate chondrocytes.
By loading chondrocytes with appropriate fluorescent indicators and subjecting them to various experimental manoeuvres, we are characterising the osmolyte transporters and determining the gradients of intracellular ions (with concentrations where possible) as a function of hypertrophy.
We also culture bone rudiments under conditions which will increase/decrease chondrocyte swelling in an attempt to accelerate/retard bone development.
The aim of the project is to identify the osmolyte transporters involved in chondrocyte hypertrophy in the growth plate.
Figure 2 shows the increase in chondrocyte volume in the mammalian growth plate. On the left of the figure is the cartilage surface. On moving to the right, the columns of cells of increasing volume in the growth plate can clearly be seen.
Finally the cells die and are replaced by the advancing bone (right of figure). The increase in chondrocyte volume is large (~10 times) and the major driving force for bone lengthening, although the mechanisms responsible are poorly understood. (Also see Huntley et al 2003 for further details.)
Articular cartilage is routinely exposed to mechanical stresses both during normal in vivo activity, and in surgical procedures e.g. during cutting with a scalpel.
Rather surprisingly, cartilage cells (chondrocytes) can be very sensitive to mechanical stresses. For example the cutting of cartilage with even an extremely sharp scalpel blade causes the rapid death of a band of cells either side of the wound.
Chondrocyte death in cartilage is highly significant because the cells are not replaced, leaving the remaining cells with an increased burden for the maintenance of the extracellular matrix.
We are interested in the mechanisms which cause chondrocyte injury and death in an attempt to identify procedures that might protect the cells. Explants of articular cartilage are subjected to defined impact loads using a simple drop tower or controlled cutting.
The fluorescently-labelled cells within cartilage and their intracellular constituents are visualised by CLSM, and the response of cells either near to, or removed from the injury studied (Figure 3).
Our preliminary data suggest a complex response following impact/cartilage cutting with marked changes to chondrocyte physiology occurring before cell death. An understanding of these processes might identify targets for therapeutic intervention to protect cells from mechanical trauma, and thereby limiting or inhibiting the development of OA resulting from mechanical injury to the joints.
Figure 3 shows the effect of a single injurious impact on articular cartilage. The cartilage surface was uppermost, and following impact, some of the chondrocytes survived (labelled green) whereas many died quickly (within ~20 minutes, red).
Note the complex pattern of sensitive cells. (Also see Bush et al 2005 for further details.)
Click an image to view the full size version.
Amin, A.K., Huntley, J.S., Patton, J.T., Brenkel, I.J., Simpson, A.H.W.R. & Hall, A.C. (2011). Hyperosmolarity protects chondrocytes from mechanical injury in human articular cartilage: An experimental report. J. Bone Jt. Surg. (Br). 93(2), 277-284.
Murray, D.H., Bush, P.G., Brenkel, I.J. & Hall, A.C. (2010). Abnormal human chondrocyte morphology is related to increased levels of cell-associated IL-1? and disruption to pericellular collagen type VI. J. Orthop. Res.28(11), 1507-1514.
Amin, A.K., Huntley, J.S., Simpson, A.H.R.W. & Hall, A.C. (2010). Increasing the osmolarity of joint irrigation solutions may avoid injury to cartilage: A pilot study. Clin. Orthop. Related Res. 468, 875-884
Amin, A.K., Huntley, J.S., Simpson, A.H.W. & Hall, A.C. (2009). Chondrocyte survival in articular cartilage: the influence of subchondral bone in a bovine model. J. Bone Jt. Surg. (Br).91 B:691-699.
Bush, P.G., Hodkinson, P.D., Hamilton, G.L., & Hall, A.C. (2005). Viability and volume of in situ bovine articular chondrocytes - changes following a single impact and effects of medium osmolarity. Osteoarth. & Cart. 13, 54-65.
This article was published on Sep 14, 2011