Small Angle Light Scattering for Dermal Wound Collagen Structural Analysis

Abstract

MTRODUCTION. The process of demal wound heaiing is a very complex one, taking place in several consecutive stages. Cytokines, in particular growth factors, are important in rhe regulation of these stages. Transforming Growth Factor p (TGFP) is known to play a crucial role during inflammation and extracellular ma^ deposition. Chronic exposure to large amounts of TGFP may promote excessive fibrosis. Attempts to prevent fibrosis and scarring have been aimed at lowering the levels of TGFP in the wound site. Local application of neutralizing antibodies to TGFP (Ab-TGFP) has been shown to signpiticantly decrease the amount of scarring during wound h e a h g . Small angle light scattering (SALS) offers a means to quantitatively determine differences in collagen fiber orientation and directional uniformity during the would healing process in the presence of TGFP or Ab-TGFP. In this study, we demonstrate that SALS can be used to quantify differences in the collagen fiber architecture of histological sections taken from dermal wounds that exposed to TGFP and Ab-TGFP. METHODS. Recombinant human TGFP2 and Ab-TGFP;, of the IgG subtype were utilized with a methylcellulose 2% gel as the delivery vehicle. Two white specific pathogen pigs (15-20Kg) were wounded according to established procedures following AAALAC standards. Biopsies were performed on days 7, 14, and 46 post-wounding, with normal unwounded s!un also taken as the conuol. Tissue specimens were fixed and standard histological sections prepared using H&E staining. A detailed description of the SALS device and its accuracy in tissue structural analysis has been previously presented (Sacks, et al. 1997). Briefly, a 4 mW HeNe (X = 632.8 nm) continuous unpolarized wave laser is passed Wough the tlssue specimen, which scatters light according to the internal fiber structure within the light beam envelope (Fig. 1). The information obtainable from thc scattered light distribution I (0) includes the preferred fiber . drrection, defined as the cennoid of I(O), and the degree of orientation defined as the angle containmg half of the total area under the I(cD), representing 50% of the fibers (Sacks, et al. 1997). After SALS scanning, the data was analyzed and the degree of orientation and collagen preferred directions mapped (Fig. 2). SALS data from the wounded section was extracted and the means for degree of orientation computed. RESULTS A N D DISCUSSION. Normal skin revealed the lowest degree of orientation (54.6"), while the TGFP treated wounds had a significantly greater (p < 0.0001) degree of orientation at days 7 and 14. The greater degree of orientation is consistent the presence of fibrosis in the wound site. Specimens treated with Ab-TGFPL3 displayed a degree of orientation intermediate between normal and TGFP treated wounds. By day 46, wound architectures were indistinguishable. These results suggest that SALS can be used to detect a d quanti@ the differences in collagen fiber onentation in normal ealing skin wounds. t Scattering I