Long bone lengthening occurs at the growth plate (GP) by well-regulated
chondrocyte proliferation, hypertrophy and terminal matrix deposition. GP
chondrocyte (GPC) hypertrophy has been implicated to be the main determinant of
bone growth rate; however the mechanism is poorly understood. The work of this
thesis examined some of the cellular process that drives the chondrocyte swelling
or hypertrophy particularly in a mammalian post natal GPs using living in situ GPC
and fixed GP tissues.
Confocal scanning microscopy (CLSM) was used to determine living in situ
GPC volume and dimension changes in proliferative zone (PZ) through to
hypertrophic zone (HZ) chondrocytes of different GPs of various bones. While PZ
cells showed similar volumes and dimensions, HZ cells varied in different GPs,
even within the same long bone but at opposite ends. However, the hypertrophic
cell volume measured at a single post natal age (day 7) was independent of the
corresponding bone length. This could reflect a complex interplay between local
and systemic factors in different GPs, which occurs throughout the active phase of
bone growth.
Maintaining GPC morphology was critical in studying GPC hypertrophy
using fixed tissues. This work highlighted a problem caused by conventional
fixative solutions, which caused up to 44% hypertrophic GPC shrinkage following
GP fixation. This artifact appeared to be associated with the hyperosmotic nature of
the fixatives used and could be abolished by adjusting the fixative osmolarity close
to physiological level (280 mOsm), or could be significantly reduced by bisecting
bone tissues prior to tissue fixation.
This thesis proposed roles for plasma membrane transporter(s) in mediating
GPC hypertrophy. This hypothesis was tested by examining roles of sodium-hydrogen exchanger (NHE) and anion exchanger (AE) in GPC hypertrophy using
an ex vivo bone growth inhibition model. Inhibition of bone growth by inhibitors of
NHE (EIPA) and AE (DIDS) respectively was shown to be dose-dependent. The
histology of bones demonstrated that the late HZ width was significantly reduced
in GPs treated with EIPA or DIDS. Although in situ GPC volumes in the PZ and
HZ were not notably different in DIDS-treated GP, the cell volumes in both zones
were significantly reduced by EIPA treatment. Fluorescence
immunohistochemistry revealed distinctive cellular localisations of NHE1 and AE2
in the PZ and early HZ. These results suggest a possible role of AE in mediating
GPC volume increase in PZ chondrocytes and those in the early stages of cell
hypertrophy, whereas NHE could possibly maintain intracellular pH of GPC
throughout all GP zones.
This thesis has characterized various changes in volume and dimensions of
living in situ GPC from PZ through to HZ of different GPs of postnatal rats. This
work emphasized the importance of fixative osmolarity in order to accurately
preserve the normal volume/morphology of cells within tissues. Most importantly,
this thesis confirmed a potential role of the plasma membrane transporters, AE and
NHE in GPC hypertrophy of growing bones