Health and emotional benefits are linked to participation in exercise, however
single-impact load (e.g. trauma) and altered joint loading can cause bone
fracture(s) resulting in permanent cartilage damage and increased risk of
osteoarthritis (OA).
This study investigated the role of single mechanical load and the effects of
chondroprotective agents over short periods of time (2-30 min) post impact.
Mechanical load (force 1.14 N) induced by a drop tower device caused cell
death from as early as 2 min. Pre-incubation in hypertonic media protected
chondrocytes from cell death, whereby at 30 min, death was decreased from
9.22 % to 3.42 % (p<0.01), thus implicating volume regulatory changes as a
potential key mechanism for chondroprotection, with in situ chondrocytes
altering their cell volume in response to hypertonicity by 20 %. Investigation
of the cell cytoskeleton, showed that hypertonicity increased cortical actin by
29 % within the superficial zone (SZ) only (p<0.01).
Volume and actin polymerisation regulation are governed by intracellular
Ca2+ and the regulatory volume decrease (RVD) inhibitor REV5901 has been
linked to both and was therefore tested for its potential chondroprotective
properties. Impact led to cell death, which was significantly reduced by
REV5901 from 10.92 % to 5.44 % (p<0.001) and initial cell volume was
reduced by 25%. Cortical actin staining was increased by 20 % within the SZ
of articular cartilage only (p<0.01). GdCl3 (blocker for stretch-activated
calcium channels), uridine-5’-triphosphate (uridine; calcium mobilizing
mediator) and the phosphoinositide 3-kinases (PI3K) inhibitor wortmannin
were used to determine its mechanism of action. Wortmannin alone
increased cell death and inhibited REV5901 chondroprotective effects with
no alteration in cell volume compared to REV5901 alone. Whilst uridine
reduced cell volume with 20 % (p<0.05) it did not reduce cell death
significantly from 10.92 % to 8.10 % (p>0.05) compared to control. GdCl3
inhibited REV5901 chondroprotective effects by increasing cell death by ~ 5
% compared to control (p<0.05), F-actin staining appeared reduced 72.84 AU
and not significantly different from control (p>0.05).
The role of the cell cytoskeleton is important for cell mechanotransduction
and for maintaining integrity as actin microfilaments are recognized to bear
tension therefore, alterations of the actin-binding proteins responsible for
actin treadmilling cofilin, profilin and gelsolin mRNA was compared to
untreated chondrocytes. REV5901 was observed to reduce cofilin (known
actin depolymerizing factor) with 30 % (p<0.05) and increase profilin
significantly by 75 % (p<0.001) respectively. Western blot analysis showed
that only cofilin and gelsolin were expressed in all samples with no detection
of profilin. REV5901 was observed to significantly reduce cofilin by 28 %
(p<0.05).
This data highlights that REV5901 exhibits chondroprotective properties in
part due to the polymerisation of the actin cytoskeleton via PI3Ks pathway.
This could offer a novel therapeutic opportunity for perention of irreversible
cartilagedamage following acute impact trauma
