Background/Purpose: Basic calcium phosphate (BCP) crystals frequently deposit in
tendons and may cause an acute inflammatory syndrome of calcific tendinitis. Tenocytes
are the stromal cells of the tendon and are responsible for maintenance of
tendon extracellular matrix.  The aim of this study was to determine the
effects of BCP crystals on tenocyte function, using an in vitro model of
calcific tendinitis. 

Methods : BCP
crystals were synthesized by alkaline hydrolysis of brushite.  Primary human
tenocytes were prepared by collagenase/dispase digestion from human tendon
(biceps, supraspinatus, hamstring) obtained from patients undergoing
orthopaedic surgery. BCP crystals (0-50μg/mL) were added to the tenocyte
cultures. After 24 hours, cell viability was assessed using the alamarBlue
assay and changes in the relative mRNA expression levels of tendon-related
genes were analysed using real-time PCR.  The effects of cyclo-oxygenase (COX)-1
and COX-2 inhibition on gene expression were analysed by adding 1 µM SC-560 or SC-236
respectively for 45 minutes prior to addition of BCP crystals. 

Results: BCP
crystals did not alter tenocyte viability or gene expression of tendon
extracellular matrix proteins (type 1 collagen, type 10 collagen, and tenomodulin)
and inflammatory cytokines (IL-1, IL-6 and TNF-α).  In contrast, BCP
crystals induced gene expression of matrix metalloprotease (MMP)-1, MMP-3, and a
disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 in a
dose dependent manner (Figure).  Gene expression of tissue inhibitor of
metalloproteinases (TIMP)-1 was also induced (for 50µg/ml BCP, mean (SD)
relative expression 2.2 (0.7), ANOVA p for linear trend =0.002), but not TIMP-2
or TIMP-3.  COX-1 gene expression was induced (for 50µg/ml BCP, mean (SD)
relative expression 2.3 (1.1), ANOVA p for linear trend =0.008), with a similar
trend for COX-2 (for 50µg/ml BCP, mean (SD) relative expression 2.6 (1.1), ANOVA
p for linear trend =0.08).  The COX-2 inhibitor SC-236 suppressed BCP
crystal-induced gene expression of MMP-1 (mean change -83%), MMP-3 (-25%),
ADAMTS-4 (-51%) and TIMP-1 (-47%).  Similar, but generally less pronounced
effects were observed with the COX-1 inhibitor SC-560 (mean change MMP-1 -63%,
MMP-3 -56%, ADAMTS-4 -31%, TIMP-1 -39%).

Conclusion :
BCP crystals induce tenocyte catabolic and anabolic gene expression in a cyclo-oxygenase (1 and 2) dependent manner.  These data suggest
that BCP crystals interact with tenocytes to influence tendon extracellular
matrix integrity.

Figure: Effect of BCP crystals on MMP-1,
MMP-3 and ADAMTS-4 gene expression in tenocytes. Data
were pooled from four biological repeats and analysed using one-way ANOVA with
post hoc Dunnett’s tests compared to control (no treatment). *p<0.05,
**p<0.01, ***p<0.001.