During 1996-1998, I was a member of Alan Grodzinky's lab at MIT, where I completed the following dissertation for my master of engineering degree.



Injurious Compression Induced Apoptosis in Articular Cartilage
by
Andreas Markus Loening

Submitted to the Department of Electrical Engineering and Computer Science
in partial fulfillment of the requirements for the degree of
Master of Engineering in Electrical Engineering and Computer Science
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 1999
© Massachusetts Institute of Technology 1999. All rights reserved.

Abstract

Increased numbers of apoptotic cells have been identified in OA cartilage, and it has been suggested that apoptosis may play an important role in the pathogenesis of OA. Autoradiography studies of injuriously compressed normal cartilage tissue have shown subpopulations of cells exhibiting little or no matrix turnover and condensed nuclei suggestive of apoptotic cell death. The objectives of this thesis were to determine if mechanical loading could induce apoptosis in non-arthritic cartilage in vitro, and to compare any mechanically induced apoptosis with other markers of tissue injury. Bovine cartilage was subjected to unconfined compression-release cycles producing peak stresses of 0-20 MPa. Tissue was stained via TUNEL for apoptotic nuclei and via ethidium bromide/fluorescein diacetate for viability. Additionally, wet weight changes, sulfated glycosaminoglycan (sGAG) loss, and nitric oxide (NO) release were measured. Compression of explants to just 4.5 MPa peak applied stress produced a significant increase in the number of nuclei staining positive for apoptosis via TUNEL. A dose dependent response was seen, with ~50% of chondrocytes staining positive for apoptosis following a 20 MPa compression. Changes in cell viability corresponded closely with those of the TUNEL assay. Wet weight changes and sGAG loss also increased in a dose-dependent manner and were indicative of collagen network damage from the injury. NO release increased for only the most severely compressed condition (20 MPa). In summary, mechanical injury of articular cartilage tissue was found to induce chondrocytic apoptosis. Additionally, this effect was observable at injury levels (4.5 MPa) below what was needed to induce detectable effects in wet weight (13 MPa), sGAG release(6 MPa), and viability (10 MPa). This suggests that programmed cell death may be one of the earliest events in response to injury of articular cartilage and could hypothetically lead to subsequent tissue degeneration due to tissue hypocellularity. An alternative interpretation is that the in vitro mechanical injury could be more representative of fibrillated tissue and be interpreted as evidence of apoptosis as a secondary effect of fibrillation, perhaps relevant to the OA process.

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