Tendinopathy is a tendon pathology often resulting from a failed healing response to tendon injury. Activated protein C (APC) is a natural anti-coagulant with anti-inflammatory and wound healing promoting functions, which are mainly mediated by its receptors, endothelial protein C receptor (EPCR) and protease activated receptors (PARs). This study aimed to determine whether APC stimulates tenocyte healing and if so, to assess the involvement of the receptors.

Mouse-tail tenocytes were isolated from 3-week-old wild type (WT), PAR- 1 knockout (KO) and PAR-2 KO mice. The expression of EPCR, PAR-1 and −2 and the effect of APC on tenocytes tendon healing and the underlying mechanisms were investigated by Reverse transcription real time PCR, western blot, 3- (4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, zymography, and scratch wound healing/ migration assay.

When compared to WT cells, PAR-1 KO tenocytes showed increased cell proliferation (3.3-fold, p<0.0001), migration (2.7-fold, p<0.0001) and wound healing (3-fold, p<0.0001), whereas PAR-2 KO cells displayed decreased cell proliferation (0.6-fold, p<0.05) and no change in cell migration or wound healing. APC at 1 μg/ml stimulated WT and PAR-1 KO tenocyte proliferation (~1.3, respectively, p<0.05) and wound healing (~1.3-fold, respectively, p<0.05), and additionally promoted PAR1-KO cell migration (1.4-fold, p<0.0001). APC only increased the migration (2-fold, p<0.05) of PAR-2 KO tenocytes. The activation of AKT, extracellular signal-regulated kinase (ERK)-2, and glycogen synthase kinase (GSK)-β3, the intracellular molecules that are associated with cell survival/growth, and matrix metalloproteinase (MMP)-2 that is related to cell migration and wound healing, were increased in all three cell lines in response to APC treatment.

These findings show that PAR-1 and PAR-2 act differentially in tenocyte proliferation/migration/wound healing. APC likely promotes tenocyte proliferation/ wound healing via PAR-2, not PAR-1.

Rotator cuff repair is performed to treat shoulder pain and disability. Failure of the tendon repair site is common; one strategy to improve healing is to enforce a period of post-operative immobilisation. Immobilisation may have unintended effects on tendon healing. Tenocytes under uniaxial strain form more organised collagen and up regulate expression of proliferative genes. Vitamin C (ascorbic acid), an anti-oxidant that is a co-factor for collagen synthesis, has also been reported to enhance collagen deposition and organisation. The purpose of this study was to compare human tenocyte cultures exposed to uniaxial cyclical strain with or without slow-release ascorbic acid (ascorbyl-2 phosphate) to determine their individual and combined effects on tissue remodelling and expression of tissue repair genes. Rotator cuff tissues were collected from degenerative supraspinatus tears from eight patients. Tenocytes were incorporated into 3D type I collagen culture matrices. Cultures were divided into four groups: 1) ascorbic acid (0.6mMol/L) + strain (1%–20% uniaxial cyclic strain at 0.1 Hz), 2) ascorbic acid unstrained, 3) strain + vehicle 4) unstrained + vehicle. Samples were fixed in paraffin, stained with picrosirius red and analysed with circular polarising light. A second set of cultures were divided into three groups: 1) 0.5mM ascorbic acid, 2) 1mM ascorbic acid, 3) vehicle cultured for 24, 72, 120 and 168 hours. Cell-free collagen matrix was used as a control. Tenocyte proliferation was assessed using the water soluble tetrazolium-1 (WST1) assay and f tissue repair gene expression (TGFB1, COL1A1, FN1, COLIII, IGF2, MMP1, and MMP13), were analysed by qPCR. The data were analysed using a Split model ANOVA with contrast and bonferroni correction and a one-way ANOVAs and Tukey's test (p<0.05 was significant). Our results indicated that unstrained cultures with or without exposure to slow release ascorbic acid exhibited greater picrosirius red birifringency and an increase in collagen fiber deposition in a longitudinal orientation compared to strained tenocytes. We found that slow release ascorbic acid promoted significant dose and culture-time dependent increases in tenocyte proliferation (p<0.05) but no obvious enhancement in collagen deposition was evident over cultures without ascorbic acid supplementation. Based on these data, applying strain to tenocytes may result in less organised formation of collagen fibers, suggestive of fibrotic tissue, rather than tendon remodelling. This may indicate that a short period of immobilisation post-rotator cuff repair is beneficial for the healing of tendons. Exposure to slow release ascorbic acid enhanced tenocyte proliferation, suggesting that supplementation with Vitamin C may improve tendon repair post-injury or repair. Future studies will assess levels of tissue repair-associated proteins as well as comparing traumatic and degenerative rotator cuff tears to healthy uninjured rotator cuff tissue