Topographical guidance of intervertebral disc cell growth in vitro:towards the development of tissue repair strategies for the anulus fibrosus

The anulus fibrosus (AF) of the intervertebral disc consists of concentric sheets of collagenous matrix that is synthesised during embryogenesis by aligned disc cells. This highly organised structure may be severely disrupted during disc degeneration and/or herniation. Cell scaffolds that incorporate topographical cues as contact guidance have been used successfully to promote the healing of injured tendons. Therefore, we have investigated the effects of topography on disc cell growth. We show that disc cells from the AF and nucleus pulposus (NP) behaved differently in monolayer culture on micro-grooved membranes of polycaprolactone (PCL). Both cell types aligned to and migrated along the membrane's micro-grooves and ridges, but AF cells were smaller (or less spread), more bipolar and better aligned to the micro-grooves than NP cells. In addition, AF cells were markedly more immunopositive for type I collagen, but less immunopositive for chondroitin-6-sulphated proteoglycans than NP cells. There was no evidence of extracellular matrix (ECM) deposition. Disc cells cultured on non-grooved PCL did not show any preferential alignment at sub-confluence and did not differ in their pattern of immunopositivity to those on grooved PCL. We conclude that substratum topography is effective in aligning disc cell growth and may be useful in tissue engineering for the AF. However, there is a need to optimise cell sources and/or environmental conditions (e.g. mechanical influences) to promote the synthesis of an aligned ECM.

Hydroxylases regulate intestinal fibrosis through the suppression of ERK mediated TGF-β1 signaling

Fibrosis is a complication of chronic inflammatory disorders such as inflammatory bowel disease (IBD), a condition which has limited therapeutic options and often requires surgical intervention. Pharmacologic inhibition of oxygen-sensing prolyl hydroxylases (PHD), which confer oxygen-sensitivity upon the hypoxia inducible factor (HIF) pathway, has recently been shown to have therapeutic potential in colitis, although the mechanisms involved remain unclear. Here, we investigated the impact of hydroxylase inhibition on inflammation-driven fibrosis in a murine colitis model. Mice exposed to dextran sodium sulfate followed by period of recovery developed intestinal fibrosis characterized by alterations in the pattern of collagen deposition and infiltration of activated fibroblasts. Treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) ameliorated fibrosis. TGF-β1 is a key regulator of fibrosis which acts through the activation of fibroblasts. Hydroxylase inhibition reduced TGF-β1-induced expression of fibrotic markers in cultured fibroblasts suggesting a direct role for hydroxylases in TGF-β1 signalling. This was at least in part due to inhibition of non-canonical activation of extracellular signal-regulated kinase (ERK) signalling. In summary, pharmacologic hydroxylase inhibition ameliorates intestinal fibrosis, through suppression of TGF-β1-dependent ERK activation in fibroblasts. We hypothesize that in addition to previously reported immunosupressive effects, hydroxylase inhibitors independently suppress pro-fibrotic pathways