Type of Document	Master's Thesis
Author	Cissell, James Michael
URN	etd-08172009-170542
Title	In Vitro Equine Flexor Tendonitis: New Model Development and Therapeutic Investigation
Degree	Master of Science
Department	Veterinary Medical Sciences
Advisory Committee	Advisor Name Title Linda Dahlgren Committee Chair Jennifer Barrett Committee Member John Robertson Committee Member
Keywords	in vitro horse prostaglandin E2 model Tendonitis
Date of Defense	2009-07-08
Availability	unrestricted
Abstract Flexor tendonitis is a common cause of lameness and wastage in the equine athlete. Current techniques for tendonitis therapy provide limited success, and horses that do recover tend to return at a decreased level of performance. Current treatment techniques have begun to focus on regenerative medicine to improve tissue healing. Investigations of new treatments are made difficult by the lack of reliable in vitro models that allow for accurate comparison of treatment protocols. New techniques are often implemented into the clinical setting prior to thorough investigation for safety and efficacy. In vitro testing is an important step in the development of new therapeutic agents. However, results of in vitro tests should only be deemed as useful if the model used is one that is reliable and mimics the clinical situation that the reseachers are attempting to investigate. Equine flexor tendonitis is believed to be the result of microdamage caused by cyclic loading of tendons. Cyclic loading of fibroblasts results in increased production of the inflammatory cytokine prostaglandin E2 (PGE2). Thus the exposure of tendon fibroblasts to exogenous PGE2 may induce metabolic changes in the cells similar to what is seen in clinically affected animals making this a useful model for the investigation of therapeutic techniques. Currently a variety of techniques exist for treatment of flexor tendonitis; however, no single treatment has separated itself as superior. A new technique using autogenous conditioned serum (ACS) in humans for treatment of muscle injury has been shown to speed tissue regeneration. ACS produced from human blood has been shown to contain significantly increased levels of III growth factors that may improve tendon fibril formation and strength. We propose to investigate the effect of ACS on cellular metabolism in equine tendon fibroblast monolayers. This will involve cell culture, PGE2-induced cellular injury, and analysis of the cellular response to injury when treated with ACS. Controls will include fetal bovine serum, normal equine serum, and ACS without PGE2-induced cellular injury. The cellular response will be investigated biochemically by quantification of DNA, glycosaminoglycan, and soluble collagen levels and by real time PCR to assess gene expression for matrix metalloproteinases (MMP)-1, MMP-3, and MMP-13, collagen types I and III, and the non-collagenous proteins cartilage oligomeric matrix protein (COMP) and decorin. Data will be analyzed by analysis of variance and post-hoc comparisons. Significance will be set at p<0.05. We hypothesize that the addition of exogenous PGE2 to culture media for monolayers of equine tendon fibroblasts will insight alterations in cellular metabolism that will generate a suitable model for the in vitro study of fibroblast response to novel therapies. We then hypothesize that the addition of ACS to PGE2-treated fibroblasts will result in increased gene expression for collagen types I and III, cartilage oligomeric matrix protein, and decorin. ACS will also stimulate increased protein production of collagen and glycosaminoglycans, and stimulate increased cell proliferation. The use of ACS will decrease gene expression of inflammatory molecules important in tendon degradation, namely matrix metalloproteinases -1, -3, and -13.
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