NOVEL THERAPEUTICS IN THE PREVENTION OF FLEXOR TENDON ADHESION FORMATION

Benjamin Robert Klass

A thesis submitted to University College London for the degree of Doctor of Medicine (Research) MD (Res)

The RAFT Institute of Plastic Surgery, Mount Vernon Hospital, Middlesex

The Royal Free Hospital, Hampstead, London

2011

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Declaration I, Benjamin Robert Klass, confirm that the research forming the basis of this thesis is original and the ideas were developed in conjunction with my supervisors. I performed the majority of experiments myself with guidance and technical assistance from the scientific and laboratory staff in each of the institutes where the work was undertaken. Histological processing was performed by the RAFT histopathologist, Liz Clayton, and adhesion analysis was performed in a blinded fashion by a mixed group of scientists and surgeons from the RAFT institute. The institutes include The RAFT Institute, Mount Vernon Hospital and the Biological Services Department, The Royal Free Hospital. Where I have drawn on the work, ideas and results of others this has been appropriately acknowledged in the thesis.

This work has been partly funded by Britannia Pharmaceuticals Ltd. (suppliers of Pumactant) however the research undertaken has been performed independently without any personal financial interest.

Ben Klass

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Abstract Tendon injuries of the hand are common with nearly one-third of a million digital flexor tendon injuries per year in the United States. Injuries in zone II of the flexor tendon are notoriously difficult to repair and the main complications are either tendon rupture or adhesion formation. Adhesions remain a problem despite many attempts at prevention using various chemicals and physical barrier techniques. The overall aim of this thesis was to further understand the biology of tendon adhesion formation and to develop novel treatments targeting this process.

Uninjured flexor tendons were obtained from New Zealand White rabbits. Tenocytes derived from different parts of the flexor tendon-sheath complex were grown using standard tissue culture techniques. Each of the three different cell types (endotenon, epitenon and tendon sheath) was subjected to various assays (proliferation/toxicology, cell adhesion, and mRNA expression,) using TGF-β1 and our proposed treatments; epigallocatechin-3-gallate (EGCG), Resveratrol and Pumactant.

A further study then compared the three treatments in vivo. New Zealand White rabbits (n=8 per group; 32 in total) were anaesthetised and the flexor digitorum profundus (FDP) of digits 2 and 4 of the forepaw was subjected to a partial tenotomy. The three treatments (compared with control groups) were infiltrated into the flexor sheath of immobilised tendons and the wound was then sutured closed. After two weeks the tendons were harvested and randomised to either mechanical or histological assessment of adhesion formation.

The major findings from the in vitro study were as follows: TGF-β1 showed a statistically significant increase in collagen type I gene expression in epitenon cells at 24 and 48 hours and an increase in collagen type III in sheath cells between 6 and 24 hours. There was a statistically significant down-regulation of collagen type III in endotenon and epitenon cells at various time points. Resveratrol showed a statistically significant increase in collagen type I gene expression in epitenon cells with a corresponding down-regulation of fibronectin and PAI-1 in both epitenon and sheath cells. Resveratrol also up-regulated collagen type III at late time points in tendon

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sheath cells. Pumactant also showed some therapeutic advantages at the gene expression level with a statistically significant increase in collagen type III in endotenon cells at late time points, corresponding with an overall down-regulation of PAI expression in the same cell type and sheath cells.

The results from the in vivo study were that all three treatments showed a statistically significant reduction of tendon adhesion formation when compared to operated controls in both mechanical and histological assessments (p