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Characterization of Fibroblasts with Son of Sevenless-1 Mutation

¹ Human Craniofacial Genetics Section, NIDCR, National Institutes of Health, DHHS, Building 10, Room 5-2523, 10 Center Drive, Bethesda, MD 20892, USA; and
² Department of Periodontology, University of Taubate, Brazil

View larger version (92K): [in a new window]   Figure 1. Clinical appearance and histology. (A) Control gingiva. (a) Clinical aspect before the crown extension surgery; (b) histological section showing the normal tissue (hematoxylin and eosin, magnification of 5x; bar is 100 µm); (c) connective tissue section (magnification of 40x; bar is 20 µm); (d) area of connective tissue stained with sirius red F3Ba (magnification of 40x; bar is 20 µm). (B) HGF patient. (a) Clinical aspect before the surgery; (b) histological section showing the epithelial rete pegs into underlying corneum (hematoxylin and eosin, magnification of 5x; bar is 100 µm); (c) connective tissue section (magnification of 40x; bar is 20 µm); (d) area of connective tissue stained with sirius red F3Ba (magnification of 40x; bar is 20 µm).

View larger version (20K): [in a new window]   Figure 2. Cell proliferation, 5-bromo-2-deoxyuridine uptake, and cell cycle profiles in fibroblasts. Fibroblasts from normal control (N = 3 individuals) and HGF patients (N = 3 patients) were compared. At indicated time-points, total cell numbers (A) or 5-bromo-2-deoxyuridine incorporation into DNA (B) were determined from triplicate samples. The data are presented as an average with standard deviations; * indicates P < 0.05. (C–H) Fibroblast cultures were prepared under 3 different conditions as described below. Panels C, E, and G are normal control fibroblasts, and panels D, F, and H are HGF fibroblasts. (C,D) Cultures under starving media conditions for 2 days. (E,F) Cultures under starving conditions for 1 day and switched to growth media for one day. (G,H) Cultures under starving conditions for 1 day and switched to growth media for 3 days. The percentages of total cells contained in different cell cycle phases are indicated. M1 represents the "Go/G1" phase, M2 denotes the "S" phase, and M3 denotes the "G2/M" phase. Significant difference between control and HGF is marked with asterisks (P < 0.05).

View larger version (76K): [in a new window]   Figure 3. Cell attachment on different extracellular matrices. The chamber slides were coated with either collagen (c,d), fibronectin (e,f), or poly-D-lysine (g,h), and uncoated (a,b) was the control. After being plated for 30 min, cells were fixed and examined under an inverted microscope. At least 20 different fields were examined, and 1 representative image is shown. (A) Low magnification (100X). Bar is 20 µm. (B) High magnification (960X). Bar is 5 µm.

View larger version (43K): [in a new window]   Figure 4. Proliferation of fibroblasts and change of collagen matrix in three-dimensional cultures. The three-dimensional collagen matrix containing normal control fibroblasts or HGF fibroblasts was prepared at 2 different densities (low, 15,000 cells/mL; and high, 20,000 cells/mL). (A) After 2 days, fibroblasts were released from 1 set of three-dimensional cultures as described in MATERIALS & METHODS. Total cell numbers were determined, and data are presented as mean ± standard deviations from triplicate samples for controls (N = 3) and HGF patients (N = 3); * indicates P < 0.05. Another set of three-dimensional cultures (20,000 cells/mL), maintained in regular growth medium for an additional 3 days, was stained with Diff-quik to show cells (B,C). The three-dimensional cultures were fixed and stained with picrosirius red to show collagen. Panels D and F demonstrate the ordered spreading (D) and density (F) from control cells. Irregularly clumped deposition (E) and increased collagen density (G) were observed from HGF cells in three-dimensional culture. Panels D and E are low-magnification (100X) images; panels F and G are high-magnification (960X) images. Bar is 50 µm in D and E, 10 µm in F and G.