Figure 5.

Collagen is sparse and disorganized in extracellular matrix deposited by fibroblasts. A: Collagen content of extracellular matrix deposited by FKBP10-null and control fibroblasts was examined. The collected matrix was extracted into three fractions: neutral salt (NS, newly incorporated collagen without cross-links), acetic acid (AA, immaturely cross-linked collagen), and pepsin digested (P, mature cross-linked collagen), and compared with collagen secreted in the media. Thirty-fold more sample was loaded for the proband to get an equivalent signal in the maturely cross-linked collagen fraction, with a loss of collagen cross-linked β-forms (n = 2). B: The extracellular matrix deposited by cultured control and FKBP10-null fibroblasts was stained with a collagen antibody (LF-68, an α1[I] C-telopeptide antibody) and DAPI, then imaged using a confocal microscope at 20× (left) or 63× (right, z-stacked images). Control matrix has abundant long fibrillar collagen strands, while collagen in proband matrix appears mesh-like, branched, thinner, and more sparse. Matrix from a patient with classical OI has abnormal fibrils, but they are as abundant as control (n = 2, representative panels shown). C: Matrix collagen to cell organics ratio in cultures measured by Raman microspectroscopy at locations where matrix and cell cytoplasm overlap. The ratios were measured in the spectral regions of amide III, CH-stretching or proline A bands. Consistent reduction in collagen of the FKBP10-null culture was also found using proline-B band, CH-bending band of all organic molecules, or amide I band of all proteins. The error bars represent the standard errors. *** P < 0.0005.