IDENTIFICATION OF EXTRACTABLE GROWTH-FACTORS FROM SMALL-INTESTINAL SUBMUCOSA

When implanted as a biomaterial for tissue replacement, selected submu cosal layers of porcine small intestine induce site-specific tissue re modelling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix material. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodelling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesi ze DNA and proliferate. Each of the four different extracts of small i ntestinal submucosa had measurable cell-stimulating activity when anal ysed in both a whole cell proliferation assay (alamarBlue dye reductio n) and a DNA synthesis assay ([H-3]-thymidine incorporation). Proteins extracted from SIS with 2 M urea induced activity profiles in the two assays which were very similar to the activity profiles of basic fibr oblast growth factor (FGF-2) in the assays. As well, the changes in ce ll morphology in response to the extracted proteins mimicked the chang es induced by FGF-2. Neutralization experiments with specific antibodi es to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% of the fibroblast-stimulating activit y of the urea extract of small intestinal submucosa. Western blot anal ysis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence of FGF-2. Cell stimulating activity of proteins extracted from SIS with 4 M guanidine was neutralized by an antibody specific for transforming growth factor beta (TGF beta). Changes in the morphology of the fibro blasts exposed to this extract were nearly identical to changes induce d by TGF beta. Although no reactive protein band was detected at 25 kD a in nonreduced western blot analysis, several bands were reactive at higher molecular weight. The identity of this TGF beta-related compone nt of small intestinal submucosa is unknown. Identification of FGF-2 a nd TGF beta-related activities in SIS, two growth factors known to sig nificantly affect critical processes of tissue development and differe ntiation, provides the opportunity to further elucidate the mechanisms by which this extracellular matrix biomaterial modulates wound healin g and tissue remodelling. J. Cell. Biochem. 67:478-491, 1997. (C) 1997 Wiley-Liss, Inc.