Fig. 4: Collagen fiber morphometry and organization in laser-ablated corneas undergoing cell therapies.

The corneas were harvested 21 days after cell injections. a Synchrotron small-angle X-ray scattering (SAXS) was employed to elucidate the interfibrillar distance (IFD), fibrillar diameter (FD), and degree of collagen fibril organization (matrix order) in the corneas centrally. The IFD (b) and FD (c) were only marginally different between all groups, namely the naïve, quiescent corneal stromal keratocytes (qCSKs)-treated, stromal fibroblasts (SFs)-treated, and non-treated corneas, with naïve corneas having the smallest IFD and FD. d The matrix order in the qCSK-injected corneas was akin to the naïve corneas and was significantly higher than the SFs-injected and non-treated samples. e Low-magnification transmission electron microscopy (TEM) images of the anterior stroma region revealed a more regular alternation of darker (collagen fibers that appeared in longitudinal orientation) and lighter (fibers that appeared in cross-sectional orientation) bands of lamellae in the naïve and qCSKs groups. At high magnification, especially within the darker band, the fibers were typically long and continuous in both naïve and qCSK-treated rats but appeared relatively shorter due to the interruption by the cross-sectionally appearing fibers in the lamellar layer. f Further collagen fiber profiles were characterized with a Histoindex Genesis 200 multiphoton microscope. The built-in analytical system then measured the tissue area ratio (TAR) (g), collagen area ratio in tissue (CART) (h), collagen fiber density (CFD) (i), collagen fiber count (CFC) (j), collagen fiber length (CFL) (k), and collagen area reticulation density (CARD) (l), revealing the restoration of collagen fiber profile by qCSKs to the naïve corneal state (m). In contrast, the collagen fiber profile of SF-injected corneas was closer to that of the non-treated corneas. Data are presented as mean ± SEM (n = 9 in each group for SAXS and n = 3 in each group for multiphoton microscopy). TEM was repeated on 3 independent samples. Statistical significance was analyzed with one-way ANOVA, followed by post hoc Tukey test. Naïve, qCSKs, SFs, and non-treated groups are represented by pink, blue, yellow, and gray, respectively, in the bar graphs and radar chart. Source data are provided as a Source Data file.