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Abstract

This investigation encompassed three projects. First was the examination of growth factor induction of[³H]-thymidine uptake as a measure of cell division in porcine periodontal ligament epithelial (PLE) cells cultured on tissue culture plastic. Experimental parameters affecting[³H]-thymidine uptake of PLE cells were established (culture medium, plating density, substrate effect and onset of[³H]-thymidine uptake) and then the effects of EGF, KGF, PDGF and IGF-1 were assayed. EGF induced an increase in[³H]-thymidine uptake (220%), while KGF, IGF-1 and PDGF in the presence of 1% FBS did not. Under serum free conditions PDGF induced a large concentration-dependent decrease in[³H]-thymidine uptake suggesting under specific conditions PDGF may inhibit PLE cell proliferation. The second aspect of this project examined growth factor regulation of matrix metalloproteinase activity with emphasis on the role of KGF. The culture conditions had a significant effect on cellular responses to the growth factor. In histiotypic-culture on porous-polycarbonate membranes, porcine ligament epithelial cells responded to KGF with increased 92 kDa gelatinase (MMP-9) activity. No such response was observed in cells maintained on plastic plates. EGF and PDGF also increased MMP-9 activity in the histiotypic cultures of epithelial cells. Addition of heparin with KGF produced a further increase in MMP-9 activity with heparin alone having Iittle effect. Precoating of polycarbonate membranes with matrix components showed that fibronectin or an engineered poly RGD molecule substrate were required for KGF plus heparin to increase MMP-9 activity. Precoating plastic culture plates with the same proteins did not generate the same response. Concomitant with gelatinase activity KGF also increased urokinase-type plasminogen activator in the epithelial cells which suggests active gelatinase may be induced with KGF stimulation. The last aspect of this investigation examined the role of KGF, heparin and heparan sulfate in the control of epithelial collagenase activity and synthesis utilizing the histiotypic culture model. Both heparin and heparan sulfate induced the activity of a 58 kDa (nonreduced) gelatin degrading enzyme which was subsequently identified as collagenase (MMP-1). For each concentration tested heparin was more effective than heparan sulfate at increasing enzyme activity. The increase in collagenase activity by heparin was further increased by the addition of KGF. KGF alone did not increase collagenase activity. Analysis of secreted radiolabelled proteins showed that the increase in collagenase activity was not due to a general increase in protein synthesis. Synthesis of collagenase protein was specifically increased by heparin and further increased by KGF plus heparin. Further investigation identified the induction of MMP-1 activity by KGF plus heparin was not dependant on fibronectin precoating of polycarbonate membranes as was MMP-9 activity. Therefore, heparin in combination with KGF may have important roles in the regulation of MMP-9 and -1 enzyme activity in epithelial cells during inflammation and wound healing.