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TGF-beta-3 Promotes Scarless Repair of Cleft Lip in Mouse Fetuses
K. Kohama1,
K. Nonaka2,
R. Hosokawa1,
L. Shum3 and
M. Ohishi4,*
1 Graduate School of Dental Science,
2 Pediatric Dentistry, Division of Oral Health, Growth & Development, and
4 Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan; and
3 Cartilage Biology and Orthopaedics Branch, National Institutes of Health, Bethesda, MD, USA;
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Figure 1. Fetal surgery performed for repair of cleft lip allowed for scarless fusion. E16 mouse embryo with cleft upper lip (black arrowheads) was identified through the uterine wall, and exposed for fetal surgery (a). Bilateral clefts were identified, and the left cleft was repaired by suturing; the contralateral side was allowed to develop without repair as control. Sixty hrs following surgery (b), the repaired left cleft was fused (white arrowheads), and the control side remained clefted (black arrowheads). Newborn animal with repaired left cleft (white arrowheads) displayed smooth, scarless surface (d), as compared with control unoperated side (black arrowheads), and control animal that did not receive surgery (c). Serial cross-sections were obtained from the lower face region (e) for further histological and molecular analyses of cleft lip repair. Histological section of fetus with left cleft lip that was repaired and fused (g) showed continuity of epithelial and mesenchymal components (between open arrowheads), although an apparent decrease in the number of vibrissae follicles was observed as compared with the similar region in the unaffected fetus (f). The unoperated right cleft remained open (between black arrowheads). Higher magnification of the repaired region (h and box in g) showed no inflammation, fibrotic changes, or histological evidence of scarring. This region was filled with loose mesenchymal cells. Scale bars in (a) for (a-d) and in (f) and (g) are 1 mm. Scale bar in (h) is 100 µm.
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Figure 2. TGF-β3 was expressed during lip formation and up-regulated during fusion of surgically sutured cleft lip. The upper lip of the unaffected CL/Fr mouse embryo begins to fuse at E12. Immunohistochemistry showed juxtaposing surface epithelia of the lips immunopositive for TGF-β3 (a) as detected by the russet color positive reaction. TGF-β3 was not detected in the surface epithelium of the cleft lip (b). In the E16 fetus with cleft lip that was surgically repaired, a large number of platelets (black arrowheads) was observed populating the site of operation 30 min after surgery (c). These platelets were immunopositive for TGF-β3. Some endothelial cells were also positive for TGF-β3 (inset in c). In control unoperated clefts, tissues remained immunonegative for TGF-β3 (d). Sections were counterstained with hematoxylin. Asterisks indicate oral epithelium. Scale bars in (a-d) are 100 µm. The average number of TGF-β3-positive platelets was 12 + 1.86 (N = 5), whereas none was observed in the control group (N = 5) (e), *p < 0.01. Samples were also collected 10 min after surgery and assayed for TGF-β3 expression level (f). In the sutured group (1.86 + 0.21, N = 5), the expression of TGF-β3, normalized for β-actin expression level, was 3.4-fold higher than that of the control group (0.55 + 0.13, N = 5), *p < 0.01.
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Figure 3. Cyclin expression, cell proliferation, and tenascin expression were up-regulated during fusion of surgically sutured cleft lip. Following surgery to repair cleft lip in the fetuses, the expression of cyclin (a,b,h) and tenascin (e,f,h) was examined. An apparent increase in the number of cyclin-positive cells (b, arrowheads) when compared with control (a) was observed at the site of the operation 1.5 hrs after surgery. Furthermore, a 1.9-fold increase in the relative expression level of cyclin D1 was detected in the sutured group (1.81 + 0.16, N = 5) compared with controls (0.95 + 0.08, N = 5) (h), *p < 0.01. Similarly, 4 hrs after surgery, an increase in tenascin-positive extracellular matrix was observed at the site of the operation in the sutured group (f, arrowheads) when compared with the control (e). A 2.5-fold elevation in the relative expression level of tenascin-C was also detected; control was 5.54 + 1.05 and sutured group was 13.84 + 2.41, N = 5, for both groups (h). We performed BrdU labeling to examine cell proliferation. Sixteen and a half percent of the mesenchymal cells at the site of the operation was positive for BrdU label (d), compared with only 11.4% of the cells in the control group (c). Asterisks indicate oral epithelium. Increased cell proliferation was associated with a significant increase in total mesenchymal cell number (g), with more of these cells positive for cyclin (2.5%; 23.79 + 2.41 cyclin-positive cells compared with 919.19 + 76.77 total cells, N = 5) in the sutured group when compared with the control group (0.5%; 4.07 + 2.34 cyclin-positive cells compared with 727.27 + 30.30 total cells, N = 5), *p < 0.01. Scale bar in (a) for (a-f) is 50 µm.
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Figure 4. Cyclin and tenascin expression was up-regulated in upper lips cultured in the absence of serum and supplemented only with exogenous TGF-β3. Cleft upper lips were isolated and explanted into serumless cultures in the absence (a,d,g,j) or presence of 10 (b,e,h,k) or 100 ng/mL (c,f,i,l) of TGF-β3. Explants cultured for 60 hrs in the absence of TGF-β3 exhibited a significant cleft (between arrowheads) remaining between the 2 halves of the cleft lip (a,d). In the presence of 10 ng/mL TGF-β3, 1 in 10 of the cleft lips achieved partial fusion (b,e) in which an epithelial seam (asterisks) demarcated the 2 halves of the cleft lip. In the presence of 100 ng/mL TGF-β3, 4 in 10 of the cleft lips fused (c,f), with small remnants of the epithelium remaining (asterisk) and continuity of the mesenchyme throughout the fusion area. In the presence of TGF-β3 (h,i,k,l), the number of cells positive for cyclin (g-i) or extracellular matrix positive for tenascin (j-l) was apparently increased when compared with those in controls (g,j). The total number of mesenchymal cells at the fusion area in lips treated with 100 ng/mL TGF-β3 was increased slightly but significantly. However, the percentage of cyclin-positive mesenchymal cells was substantially increased: 4.5% (33.75 + 8.75 cyclin-positive cells compared with 753.68 + 98.95 total cells, N = 5), 6.8% (55.42 + 7.92 cyclin-positive cells compared with 818.95 + 143.16 total cells, N = 5), and 16.5% (157.5 + 14.58 cyclin-positive cells compared with 953.68 + 77.89 total cells, N = 5) for 0, 10, and 100 ng/mL TGF-β3, respectively (m). TGF-β3 inceased cyclin expression dose-dependently (0.47 + 0.28 for control, 2.63 + 0.45 at 10 ng/mL and 4.32 + 0.58 at 100 ng/mL TGF-β3, N = 5, for all groups) (n). Similarly, TGF-β3 promoted tenascin-C expression (5.06 + 1.03 for control, 6.92 + 1.24 at 10 ng/mL, and 9.51 + 1.32 at 100 ng/mL TGF-β3, N = 5, for all groups) (n). Scale bar in (a) for (a-c) is 1 mm; those in (d) for (d-f) and in (g) for (g-l) are 100 µm.  p < 0.01 compared with 0 ng/mL TGF-β3 group and  p < 0.01 compared with 10 ng/mL TGF-β3 group.
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Journal of Dental Research, Vol. 81, No. 10,
688-694 (2002)
DOI: 10.1177/154405910208101007
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