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The effect of oxygen tension on response of equine cartilage/subchondral bone explants to lipopolysaccharide stimulation

James E. Johnson, BS and Deborah T. Kochevar, DVM, PhD, ACVCP

Veterinary Pysiology and Pharmacology, Texas A&M University, College of Veterinary Medicine, College Station, TX 77843-4467


Degenerative joint disease afflicts a large number of individuals in both the human and animal populations. In vivo studies of joint disease in patients or in animal models are difficult and potentially painful to the subject. In contrast, in vitro systems provide a more humane approach and greater control over variables but at the expense of true physiologic conditions. While there have been many studies that utilize in vitro models for studying articular cartilage, there remains no comprehensive in vitro model that adequately simulates in vivo conditions of a cartilaginous joint. Furthermore, cells or explanted tissue are routinely grown under culture conditions that approximate 20% oxygen tension. Cartilage in vivo, being an avascular tissue, is typically exposed to oxygen levels ranging from 10% at the articular surface down to less than 1% in the very deepest tissue layers.

Objective- To 1) establish a well-controlled, physiological culture environment for cartilage/subchondral bone (C/SCB) explants and 2) to use this system to compare the effects of oxygen tension on C/SCB explant responses to stimulation.

Animals - Tissue from 2 adult horses, euthanized for reasons unrelated to joint disease

Experimental Design- C/SCB explants were taken from the weight bearing regions of the antebrachiocarpal and middle carpal joints of horses euthanized for reasons unrelated to joint disease. Explants were placed in custom holders designed to allow media to contact only the articular surface and the layer of underlying bone. Explants were cultured for 3 days in a sealed incubator at 37C at either 20% or 10% oxygen and then stimulated by incubation with lipopolysaccharide (LPS) for 24 hours. Control explants in each oxygen condition were not simulated. Media samples were collected daily for 6 days. Tissue samples were taken on days 2-6. Explant health and response to stimulation will be assessed by determining changes in selected biomarkers (in either media or explant tissue) including metabolic parameters (lactate, lactate dehydrogenase (LDH)), glycosaminoglycans (chondroitin sulfate (CS), hyaluronic acid (HA)), matrix metalloproteinase activity (MMP-3, MMP-13) and expression of selected gene products (hyaluronic acid synthase 2, aggrecanase, MMP-3, MMP-13, and interleukin-1beta (IL-1beta))

Results - Pending analysis of collected samples. Media samples from days 1-6 currently stored at -80 degrees, tissue samples from days 2-6 stored under liquid nitrogen. 

Clinical Relevance - Should the proposed hypothesis prove correct and the explants respond differently depending on oxygen, the implications for culturing articular cartilage could be significant. In vitro systems offer a number of advantages over in vivo conditions, but only if the results obtained from such systems provide reasonable approximations of physiologic events. This is particularly the case for studies considering the effects of pharmaceuticals or other experiments in which a physiologically accurate response is essential.  By using full thickness cartilage explants, including a layer of subchondral bone, in a 10% oxygen environment, this study seeks to provide a culture system that more accurately simulates conditions that would be found in an in vivo joint, and thereby increase the usefulness and applicability of data derived using such a system.