Physiological tissues adapt their structure and composition to functional demands. Since a major function of connective tissues is mechanical support, physical forces are believed to play a significant role in connective tissue growth and development. This thesis focuses on the response of epiphyseal plate chondrocytes to mechanical loading. A basic system theory approach is used as a framework for examining the complex interactions which exist between the applied signal, tissue matrix, cells, and measurable response. Under physiological loading conditions, many events occur simultaneously within the tissue. These include deformation, streaming currents and potentials; fluid flow, changes in hydrostatic pressure, and physicochemical changes associated with consolidation. Any one or
more of these events may act as a modulating signal to the cell. Experimental configurations which decouple these events and which provide a spatially uniform signal were used to probe and characterize the cellular response.
Reserve zone epiphyseal plate cartilage was harvested from newborn calves immediately after slaughter and maintained in organ culture for 2 days. For the next 12 hours tissue was subjected to one of three exposure conditions: (1) sinusoidal currents up to 1 mA/cm2 at frequencies ranging from 0.1 to 100 Hz, (2) static compressive loads up to 3 MPa, and (3) physicochemical alterations of [SO4-] (0.8 mM to 1.6 mM), [K+] (5.4 mM to 10.4 mM) or pH (5.5 to 7.9). During the exposure period, tissue was bathed in media containing 35S-sulfate and 3H-proline to assess glycosaminoglycan and protein synthesis. In a separate series of experiments the kinetics of the response to mechanical loading was examined for step loading and step unloading. The applied currents, which were similar to those expected to occur under in vivo loading conditions, did not significantly alter the incorporation of proline and sulfate over the 12 hour period. Under static loading conditions there was a dose-dependent depression in proline and sulfate incorporation. This depression was strongly dependent on compression for compressions greater than 35%.
Proline incorporation was found to decrease under load in less than 1/2 hour, while sulfate incorporation decreases in 2 to 6 hours. The response to unloading following a 12 hour preload was not simply the inverse of the response to loading; proline incorporation exceeded control levels for 4 hours, while sulfate incorporation remained depressed for over 4 hours. Because of the high negative fixed charge density of cartilage, - 4 - compression leads to increases in interstitial cation concentration (e.g. [K+], [H+]) and decreases in anion concentration (e.g. [S02-]) consistent with Donnan equilibrium. Increasing [S0O-] did not alter the incorporation of sulfate under free swelling or loading conditions. When the potassium concentration was increased under unloaded conditions to levels expected to occur at 60% consolidation there was no detectable effect on either sulfate or proline incorporation. In contrast, adjustment of bath pH with bicarbonate led to changes in incorporation consistent with those seen under equivalent loading conditions. The insensitivity of chondrocytes in organ culture to electric fields (and/or associated fluid flow) suggests that such fields either have a minimal effect on the total biosynthetic behavior of chondrocytes, or result in a very slow response by the chondrocytes. The dose-dependent response to static loads suggests that longitudinal growth rate is modulated, in part, by the time-average load. This response may be accounted for by the decreased interstitial pH which occurs with consolidation. Compression induced changes in [K+] and [SO4-] do not appear to influence the response to compressive loads. The nonlinear response seen when comparing step increases in load to step decreases suggests that the response to dynamic loads may not simply reflect the response to the time-average load. -5- ACKNOWLEDGEMENTS To appropriately acknowledge and thank all those who have helped and encouraged me over the years would require a document in itself. This dissertation truly represents the work and ideas of many people; in
all honesty, it should not be called mine. I hope that all involved will accept my most sincere thanks. The members of my committee spent considerable time and effort guiding the research and reading the document. For 7 years Alan
Grodzinsky has been my research advisor, patiently encouraging and teaching me, all the while treating me as a colleague rather than as the naive young graduate student that I was. The support and guidance provided by Raphael Lee were instrumental to this project. He helped me to maintain a broad perspective by introducing me to many of the
relevant clinical and research issues. David Swann taught me the necessary cell culture and biochemical techniques and was always helpful in analyzing and interpreting the results. Steve Trippel's enthusiasm for the work was a major source of encouragement. He and his co-workers were very helpful in teaching me practical procedures such as harvesting the epiphyseal plate. Bill Siebert has always been very supportive as was demonstrated by his willingness to serve on my committee, despite a very busy schedule. The success of this project is due in large part to the wonderful people in the Continuum Electromechanics Group. Chapter 6 is dedicated to my dear friend Angelina Pizzanelli, for without her those experiments would not have been possible. In addition, she is responsible for the
careful and tedious biochemical analyses. Caroline Wang and Pauline Liu performed most of the electrical experiments. Caroline has been extremely helpful in running control experiments and harvesting the tissue. Lori Tsuruda faithfully hunted down numerous references, patiently calibrated thermistors and load cells, and willingly assisted in preparing samples for counting. I am particularly indebted to Eliot Frank both for his friendship and assistance. In a calm, type B way he revolutionized the production of theses and he provided regular expert consultation on topics ranging from cartilage electrokinetics to circuit design. Linda Bragman was always ready to help out in every way. It would be impossible to list all she has done for me but to name a few: she typed some of these chapters; labeled sample tubes; took data; brought me lunch; and prevented many crises. TOATEOH will be remembered always for the support and friendship and for introducing me to the lore of Continuum Electromechanics. Susan arrived on the scene with a helping hand just as desperation had begun to sink in; no thesis is complete
without a figure from Kevin; Laura, Bob and Debbie spent inordinate amounts of time reading and rereading the document. Professor Sol taught me a tremendous amount about cartilage and research and was always ready and willing to listen and offer suggestions. As a colleague and friend, KJ has had a tremendous influence in my life. Working with him was always fun (even though it usually meant getting drenched in the rain) and I am very much looking forward to working together in the coming -6 - years. Finally, it seems appropriate here to express my appreciation to the faculty and students in HST. Roger Mark convinced me that MIT and MEMP would provide a wonderful and exciting graduate education. As usual, he was right. Ernie Cravalho has been a continuing source of encouragement and friendship. He was instrumental in making MEMP a very special experience in the midst of this institution. The MEMP students comprise a truly remarkable group. The friendship provided by Dave and Linda, Debbie, Jose, and many others deserves my most sincere appreciation.
Xanya Sofra Weiss
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