Two-photon laser scanning microscopy of epithelial cell-modulated collagendensity in engineered human lung tissue

Tissue remodeling is a complex process that can occur in response to a woun d or injury. In lung tissue, abnormal remodeling can lead to permanent stru ctural changes that are characteristic of important lung diseases such as i nterstitial pulmonary fibrosis and bronchial asthma. Fibroblast-mediated co ntraction of three-dimensional collagen gels is considered an in vitro mode l of tissue contraction and remodeling, and the epithelium is one factor th ought to modulate this process. We studied the effects of epithelium on col lagen density and contraction using two-photon laser scanning microscopy (T PLSM). TPLSM was used to image autofluorescence of collagen fibers in an en gineered tissue model of the human respiratory mucosa-a three-dimensional c o-culture of human lung fibroblasts (CCD-18 lu), denatured type I collagen, and a monolayer of human alveolar epithelial cell line (A549) or human bro nchial epithelial cell line (16HBE14o(-)). Tissues were imaged at days 1, 8 , and 15 at 10 depths within the tissue. Gel contraction was measured concu rrently with TPLSM imaging. Image analysis shows that gels without an epith elium had the fastest rate of decay of fluorescent signal, corresponding to highest collagen density. Results of the gel contraction assay show that g els without an epithelium also had the highest degree of contraction (19.8% +/- 4.0%). We conclude that epithelial cells modulate collagen density and contraction of engineered human lung tissue, and TPLSM is an effective too l to investigate this phenomenon.