Identification of Intraoral Mucosal Wound Hyaluronidase

Bertolami, Charles

Abstract

The purpose of the proposed study will be to identify, purify, and characterize hyaluronidase of intraoral mucosal wound origin and to define associated changes in wound hyaluronate content. In developmental systems, a regulatory role has been envisaged for glycosamine-glycans (GAG) which correlates the synthesis and enzymatic removal of hyaluronate with the proliferation, migration, and differentiation of mesenchymal cells. Historically, parallels have been recognized between development and wound healing, but only recently have skin wounds been examined systematically for hyaluronidase activity; intraoral mucosal wounds have not yet been studied in the manner proposed. The importance of identifying an intraoral wound hyaluronidase is based upon previous laboratory and clinical studies and upon ongoing work which suggest that those wounds which experience a discrete, time-related increase in hyaluronidase activity and which demonstrate a predictable decline in hyaluronate content during healing may be characteristically susceptible to experimental manipulation of tissue GAG composition. The ability to alter GAG levels in healing tissues may be of value for studying hyaluronate-mediated effects on cellular behavior in vivo and for favorably influencing rates of wound reepithelialization, contraction, and connective tissue repair. Buccal mucosal wounds will be studied in 42 New Zealand white rabbits (Oryctolagus cuniculus) to identify and enzyme with hyaluronidase activity; appropriate precautions will be taken to assure that the wound-associated enzyme is not of oral bacterial or leukocytic origin. Changes in the enzyme's specific activity as a function of postwound interval will be determined and correlated to alterations in wound hyaluronate content. The mucosal wound enzyme will be purified by anion-exchange and molecular sieve column chromatography. Enzyme characterization will include determining rate limiting substrate concentrations, pH optimum, linearity of enzyme activity as a function of incubation time, molecular weight, endoglycosidic/exoglycosidic mode of action, and cleavage products.