Design and validation of an ex vivo, whole organ joint model using post mortem specimens

Abstract

Arthritis is a disease associated with high morbidity, affecting the quality of life of both human and veterinary patients. Therapeutic advances are aided by the use of in vivo rodent models however their translational value for human disease has been questioned. Therefore alternative models using large animals have come to the forefront of translational research. However despite the potentially greater applicability of experimental observations, the associated loss of animal life remains a concern. Availability of an ex vivo whole organ joint model has the potential to promote therapeutic advances without the drawbacks of in vitro and in vivo models.

Isolated limb perfusion (ILP), a technique in which appendices are separated from the body’s blood circulation but maintained under physiologic conditions using an extracorporeal circuit, is well established in transplantation surgery and selected research applications. Extending this principle to the maintenance of joint viability through the use of porcine cadaver limbs offers a significant opportunity to study post interventional short-term events relevant to arthritis in a relatively physiological environment. The body of work described in the thesis focused on (a) the establishment, validation and attempted optimisation of this novel approach and (b) the potential applicability of the model to arthritis research.

After dissection of porcine distal hind limb specimens, two arteries (A. dorsalis pedis and Ramus caudalis of the saphenous artery) were selected to link the specimen to a non-circulating ILP set-up. The model was perfused (low flow) with oxygenated adapted Tyrode solution warmed to body temperature. An intra-arterial pressure monitoring system allowed continuous perfusate pressure assessment, while scales recorded weight gain of the perfused limb. For cellular migration experiments, a second controlled fluid channel, formed by a syringe pump, was introduced into the circuit. Studies on the porcine cadaver limb model revealed good overall specimen viability over a period of six hours, as evidenced by oxygen consumption and glucose metabolism and further supported by multiphoton laser scanning microscopy (MLSM) of joint specific tissue stained for live and dead cells. Constant lactate, potassium, and LDH levels further substantiated functionality of the model. Weight gain as an indirect measure of oedema formation was in line with results reported in the literature using comparable models. Inflammatory cell migration towards an intra-articular stimulus was assessed by MSLM of joint capsule samples following isolation and fluorescent labelling of porcine neutrophils and their incorporation into the perfusate; this revealed very limited neutrophil migration. Quantitative PCR analysis of synovium for inflammatory gene (TNF-α, IL-1β, IL-6, IFN-γ, and COX-1) expression revealed a trend towards an upregulation of some genes in response to joint injection; perfusion itself did not seem to induce inflammatory gene expression. Results of the presented work suggest that the model is applicable to arthritis research as a pharmaceutical tool for testing new drugs and delivery systems; however, further refinements are required to ensure its full potential value is achieved.