. - . . SIS41 Journalof Cellular Biochemistry 67:478491 (1997) Identification of Extractable Growth Factors From Small Intestinal Submucosa Sherry L Voytik-Harbin,' Andrew 0. Brightman, Meredith R. Kraine, Beverly Waisner, and Stephen F. Badylak Hillenbrand Biomedical Engineering Center, Purdue University, West Laiayene, Indiana 47907 Abstract When implanted as a biomaterial for tissue replacement, selected submucosal layers of porcine small intestine induce site-specific tissue remodeling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix mater~al. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodeling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesize DNA and proliferate. Each o i the iour different extracts of small intestinal submucosa had measurablecell-stimulating activity when analyzed in both a whole cell proliferation assay (alamarBluedye reduction) and a DNA synthesis assay (['HI-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 fibroblast growth factor (FGF-2) in the assays. As well, the changes in cell morphology in responseto the extracted proteins mimicked the changes induced by FGF-2. Neutralization experiments with specific antibodies to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% o i the fibroblast-stimulating activity of the urea extract o i small intestinal submucosa. Western blot analvsis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence o i FGF-2. Cell stimulat~ng activity o i proteins extracted irom SIS with 4 M guanidine was neutralized by an antibody specific ior transforming growth factor P CTCFB). Changes in the morphology o i the fibroblasts exposed to this extract were nearly identical to changes induced by TGFB. Although no reactiveprotein band was detected at 25 kDa in nonreducedwestern blot analysis, several bands were reactive at higher molecular weight. The identity of this TGFf3-related component of small intestinal submucosa is unknown. Identification of FGF-2 and TGFp-related activities in SIS, two growth tactors known to significantly affect critical processes o i tissue development and difierentiation, provides the opportunity to further elucidate the mechanisms by which this extracellu- larmatrix biomaterialmodulateswound healingand tissueremodeling. J. Cell. Biochem. 67:478491,1997. Q 1997wik+~. ~nc Key words: growth iactors; biomaterial; cell proliferation; extracellular matrix; tissue repair The process of tissue regeneration, as op- injury can be enhanced by the implantation of posed to scar formation, in response to tissue various biomaterials [Bell, 1995; Langer e t al., 19951. The emerging field of tissue engineering has focused on the development of naturally Abbreviations: ECL, enhanced chemiluminescense; ECM and/or synthetic materials for tissue extraceuularmanix; EGE grodh factor,FGFd7 replacement or augmentation of wound repair basic fibroblast growth factor, GF, growth factor, GFU. factor units; m, horse~&h pemidase; NNCS, [Hubbell, 19951. The submucosa along with the neonatal calf serum: PDGF, platelet denved growth factor, adjacent connective tissue layer of 1 'an PVDF, polyvinylidene difluoride; SIS, small intestinal sub mucosa; TGF, aansforming growth factor. Contract grant sponsor: Purdue University Research Foun- dation: Conuact grant number: 'T'RASK; Contract grant sponsor: NEk Contract grant number: tID 31425 Meredith R graiae's current address is Dept. of Miaobiol- ogy and Immunology, Universiq of North Carolina, Chapel Hill NC 27599. *Correspondence to: Sherry L Voytik-Harbin. Hillenbrand Biomedical w e e m Center. Purdue UniveRity, West Lafayette, IN 47907. Received 28 January 1997; Revised 4 August 1997; Ac- cepted 8 August 1997 small intestine has proven to be an excellent natural biomaterial for wound repair and tis- sue regeneration [Badylak, 19931. Small intestinal submucosa (SIS), considing primarily of extracellular matrix material (ECM), is prepared by mechanically removing selected portions of the mucosa and the exter- nal muscle layers and then lysing resident cells with hypotonic washes. Numerous studies have shown that this biomaterial is capable o f mduc- ing host tissue proliferation, remodeling, and o 1997 wiley-Liss, Inc.