Collagen Fibril Formation in a Wound Healing Model Jacinta F. White,* Jerome A. Werkmeister,* Ian A. Darby,† Teresa Bisucci,† David E. Birk,‡ and John A. M. Ramshaw* ,1 *CSIRO Molecular Science, 343 Royal Parade, Parkville, Australia; †Department of Human Biology & Movement Science, RMIT University, Bundoora, Australia; and ‡Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania Received November 29, 2001, and in revised form February 25, 2002 Control of tissue composition and organization will be a key feature in the development of success- ful products through tissue engineering. However, the mechanism of collagen fibril formation, growth, and organization is not yet fully understood. In this study we have examined collagen fibril formation in a wound healing model in which the newly formed fibrils were kept distinct from preexisting tissue through use of a porous tubular biomaterial im- plant. Samples were examined after 4, 6, 14, and 28 days by light microscopy, in situ hybridization, and immunofluorescence microscopy. These showed a normal wound healing response, with significant collagen formation at 14 and 28 days. Individual collagen fibrils were isolated from these samples by gentle extraction in a gentamicin-containing buffer which allowed extraction of a large proportion of intact fibrils. Examination by transmission electron microscopy showed that 80% of the intact fibrils showed a single polarity reversal, with both ends of each fibril comprising collagen amino-terminal do- mains; the remaining fibrils had no polarity rever- sal. All fibrils had similar diameters at both time points. Immunoelectron microscopy showed that all labeled fibrils contained both type I and III collag- ens. These data indicate that this wound healing model provides a system in which collagen fibril formation can be readily followed. © 2002 Elsevier Science (USA) Key Words: collagen; fibril formation; gentamicin; heterotypic fibrils; histology; in situ hybridization; immunoelectron microscopy; immunofluorescence; wound healing. INTRODUCTION The rapidly emerging field of tissue engineering provides a unique opportunity to provide homolo- gous tissue for regeneration of diseased or damaged tissues (Langer and Vacanti, 1999). Research has shown that if appropriate cells can be taken from a patient and grown on a designed bioresorbable mo- lecular scaffold in a bioreactor, then replacement tissue for the patient can be produced. It is impor- tant that the organization and composition of the collagenous tissue match that of the natural tissue that is being replaced so that the durability and complex mechanical properties of the natural tissue are accurately reproduced. For example, in liga- ments the collagen fibrils are highly oriented along the length of the tissue, and their diameters can be correlated with the load requirements for the tissue (Parry and Craig, 1988). These characteristics are quite distinct from the collagen composition and or- ganization in a general wound healing response, where a different collagen organization and compo- sition are found (Bailey et al., 1975). This wound response could emerge during the production of a tissue-engineered replacement. Thus, in order to match the appropriate tissue characteristics it will be necessary to control the organization and compo- sition of the new collagen that is deposited in the tissue-engineered construct. Although there is extensive information on the biosynthesis of collagen and on the properties of collagenous tissues, the processes by which procol- lagen forms into functional fibrils in tissues are less well understood. Evidence suggests that the nucle- ation steps (Silver et al., 1992) that initiate genera- tion of fibrils may occur within cell surface crypts (Birk and Trelstad, 1984), and that nonstaggered SLS aggregates may be involved in this process (Bruns et al., 1979), although it is possible that nucleation may occur in the matrix further from the cell. There have been various in vitro studies (see Veis and Payne, 1988) on fibril formation, many utilizing pepsin or acid-soluble collagen extracted from tissue. Of most interest, however, have been 1 To whom correspondence should be addressed. Fax: +61 3 9662 7218. E-mail: John.Ramshaw@csiro.au. Journal of Structural Biology 137, 23–30 (2002) doi:10.1006/jsbi.2002.4460 23 1047-8477/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.