Cellularity of tendons.

<p>Both (A) cellularity scores and (B) DNA content demonstrated reduced cellularity in MSC-treated tendons. The values for normal horses are shown as mean (thick dashed line) with two standard deviations (thin dashed lines). (A) * p=0.007; ** p=0.001; (B) * p=0.037; ** p<0.001.</p

Morphological parameters of tendons.

<p>(A) Organisation and (B) crimp as assessed by blinded scores were taken from two separate histological sections for each tendon. Representative micrographs of haematoxylin and eosin stained histological SDFT sections from the treated and remote (less injured) sites in both MSC-treated and saline-treated groups are shown in the panels on the right. Normal tendon has a score of zero and the higher the score the more disrupted the tendon matrix and less crimp pattern, respectively. (A) * p = 0.003; ** p < 0.002; (B) * p = 0.046; ** p < 0.0001 (scale bars 50 µm).</p

Trilineage differentiation of putative bone marrow MSCs.

<p>Representative microscopic images of von Kossa and Oil-Red-O staining to confirm osteogenic and adipogenic differentiation of plastic adherent bone marrow derived cell populations (scale bars 100 µm). A higher magnification inset is shown in the adipogenesis image to demonstrate the presence of intracellular lipid droplets. Chondrogenic differentiation was confirmed by the formation of dense cell pellets (haematoxylin and eosin staining) that were positive for Safranin O (orange-red stain) (scale bars 1 mm). All respective controls (non-induced cultures) are shown in the upper row panels.</p

Typical microscopic appearance of normal and injured equine flexor tendons.

<p>Longitudinal histology sections stained with Haematoxylin and Eosin showing: (A) normal superficial digital flexor tendon (SDFT) from a 6 year old horse showing regular arrangement of parallel collagen fibrils. Scale bar = 100 µm. (B) Sub-acutely injured SDFT 3 weeks post injury from a 4 year old horse showing marked cellular infiltration (black arrows). Scale bar = 100 µm. (C) Chronic injured SDFT >3 months post injury from a 12 year old horse showing increased cellularity and poor organisation of collagen fibrils compared to (A). Scale bar = 100 µm.</p

Collagen re-modelling in injured and stem cells treated tendons.

<p>(A) Tissue-linked fluorescence and (B) MMP-13 activity. The increased MMP-13 activity seen in the saline-treated group matched the lower tissue-linked fluorescence suggesting greater remodelling of the collagen matrix in the saline-treated compared to the MSC-treated tendons. (A) * p = 0.039; ** p = 0.003; (B) * p = 0.045; ** p = 0.015.</p

Cross-sectional area of the superficial digital flexor tendon.

<p>(A) Cross-sectional areas were measured ultrasonographically at the time of implantation (Stem Cell 1, Saline 1 and Contra 1) and at 6 months (Stem Cell 6, Saline 6 and Contra 6). (B) Relative change in cross-sectional area between groups. The Wilcoxon Signed-Rank test showed that there was no difference in cross-sectional area between 1 and 6 months for either the stem cell treated group or the saline group, or between the contralateral limbs in each group. Contralateral limbs were therefore combined from both groups for statistical analysis. The values for normal horses are shown as mean (thick dashed line) with two standard deviations (thin dashed lines). * p = 0.006, ** p= 0.003, ***p<0.045.</p

Biomechanical properties of tendons.

<p>SDFTs were subjected to mechanical testing within 2 h of post euthanasia to determine (A) structural stiffness and (B) material stiffness (modulus), which was calculated by expressing the ratio of the structural stiffness to the ultrasonographically-determined cross-sectional area. Structural stiffness was significantly reduced in stem cell treated tendons and closer to that of the uninjured (contralateral) tendons. * p=0.015.</p

FPR2/ALX protein expression in natural tendon injury.

<p>The relationship between FPR2/ALX levels with age is shown in injured flexor tendons (n = 10). Horse age ranged between 4 and 16 years (mean 11±4 years). FPR2/ALX expression was significantly reduced with increasing age (<i>P</i> = 0.0008, r² = 0.77). Overlapping points are present for tendons derived from more than one 15 and 16 year old horses.</p

Levels of prostaglandins and lipoxin A₄ in extracts of normal and injured tendons.

<p>(A) Mean PGE₂ and (B) mean PGF_2α levels in normal (n = 19), sub-acute (3–6 weeks post injury, n = 6) and chronic injured (>3 months post injury, n = 9) equine superficial digital flexor tendons. PGE₂ levels are significantly reduced in sub-acute injury compared to normal and chronic injuries. In contrast PGF_2α levels are 3 fold lower than PGE₂ and do not change with injury. (C) Mean LXA₄ levels in normal (n = 8), sub-acute (n = 7) and chronic (n = 6) injured tendons, showing significantly increased levels in sub-acute injury compared to normal and chronic injuries. Error bars denote standard deviation. * <i>P</i><0.05, **<i>P</i><0.01, *** <i>P</i><0.001.</p

mPGES-1 and PGDH expression in normal and injured tendons.

<p>mPGES-1 and PGDH mRNA expression were normalised to GAPDH or 18S ribosomal RNA and are shown expressed as mPGES-1: PDGH ratio in each case. (A) Median values for GAPDH normalized ratio of mPGES-1: PGDH gene expression in normal (n = 6), sub-acute (n = 8) and chronic injured flexor tendons (n = 6). (B) Median values for 18S normalized ratio of mPGES-1: PGDH gene expression in normal (n = 6), sub-acute (n = 6) and chronic injured flexor tendons (n = 5), showing elevated mPGES-1:PGDH expression in sub-acute injury compared to normal and chronic injured tendons.</p