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Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: tissue engineering; biosynthesis; chondrocyte. Includes bibliographical references (p. 57-62).

Plant tissue culture regeneration is chiefly regulated by exogenous phytohormones. To stop regeneration and induce undifferentiated callus growth auxins are used. Unfortunately auxins influence many plant responses, most unrelated to development. Using the thymidine analogue 5-bromodeoxyuridine (BrdU) a phytohormone independent means for differentiation inhibition has been developed. Studies were focused on the target site and mechanism of BrdU action. The BrdU inhibited step in development is indicative of a plant response necessary for normal differentiation. BrdU (5-30 uM) interrupts callus growth in all tested plants. Exogenous cytokinin does not restore growth while thymidine and deoxycytidine rescue plant growth and differentiation in the presence of BrdU. Endogenous cytokinin levels are not greatly affected by subtoxic BrdU levels and indicate that cytokinin and BrdU act upon independent sites. Domestic carrot cells were used in further studies. Normally carrot cells are undifferentiated in medium with the auxin 2,4D. When 2,4D is removed, somatic embryogenesis takes place. By including 5 uM BrdU in the hormoneless medium, the cells fail to differentiate. The growth of carrots in 2,4D is not affected by 5 uM BrdU. Thus, BrdU influences growth during differentiation to a greater extent than the growth of callus cells. BrdU is effective in halting development when applied 0-24 hours after differentiation induction. An event required for differentiation (the first and second replications) must take place at this time. BrdU action begins with DNA incorporation. The consequent cellular replication becomes slowed and DNA repair results. At the same time RNA and protein levels are similar in BrdU treated and untreated cultures. BrdU thymidine substitution into DNA increases from 28% (2 days) to 68% (3 days) after embryogenic induction. A second BrdU effect follows DNA incorporation. Factors (MIFs) in the medium of BrdU treated cells arrest differentiation. After BrdU is repaired from the DNA, the cells are only able to differentiate after a medium change. Understanding MIF production could explain why some plants differentiate more readily than others. BrdU provides the means for further study of MIF's in the auxin-free inhibition of development.

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Effects of homologous extracts prepared from mature avian skeletal muscle on the development of isolated myoblasts from the thigh muscle of 11-12 day old chick embryoswere studied in vitro under previously unpublished culture conditions. Results of these experiments indicate that:1) fusion of myoblasts can occur in a predictable manner under the culture conditions described in this report2) some factor(s) present in extracts of homologous adult organ is able to partially inhibit this fusion3) such inhibition occurs maximally between 12 and 24 hours after myoblasts are placed in an in vitro environment, and is only partially reversible by the re-establishment of optimal culture conditions4) the inhibitory factor(s) is apparently long lived, non-dialyzablQ, heat labile, and subject to inactivation by proteolytic enzymes.