|
Sign In to gain access to subscriptions and/or personal tools.
|
TGF-β3 Decreases Type I Collagen and Scarring after Labioplasty
R. Hosokawa1,*,
K. Nonaka2,*,
M. Morifuji3,
L. Shum4 and
M. Ohishi3,**
1 Graduate School of Dental Science,
2 Pediatric Dentistry, Division of Oral Health, Growth & Development, and
3 Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan; and
4 Cartilage Biology and Orthopaedics Branch, National Institutes of Health, Bethesda, MD, USA;
View larger version (94K):
[in this window]
[in a new window]
|
Figure 1. TGF-β3 reduced scar formation in vivo and was normally expressed in sutured cleft lip. The upper lip of the neonatal mouse was incised, sutured, and allowed to recover until post-natal day 24. Control animals that had not been operated upon showed smooth epidermis and dermal tissues (A,E). Animals that had been operated upon showed conspicuous macroscopic scar formation (B; between white arrowheads). Histological analysis revealed deeply creviced scarring, with abundant fibrous connective tissues in the dermis and loss of salivary and vibrissae follicles (F). Animals that had been operated upon and treated with exogenous TGF-β3 showed a significant reduction in scar formation. The site of the surgery was identified by the presence of the suture thread (C; between white arrowheads). Histological evaluation showed smooth epidermis, and dermis with abundant mesenchymal cells and capillaries (G). Animals treated with PBS instead of TGF-β3 (D,H) were similar to untreated controls (B,F). Dotted lines delineate site of surgery.
Cleft lip of the CL/Fr neonatal mouse (I) was repaired by labioplasty (L). Black-and-white arrowheads indicate non-repaired and repaired cleft lips, respectively. Immunohistochemistry for TGF-β3, 30 min after surgery, showed positive reaction (brown deposits) at the site of the surgery (M). Higher magnification of the boxed area in (M) represented in (N) identified TGF-β3 expression in mesenchymal cells (white arrowheads). TGF-β3 immunoreactivity was not detectable in unoperated controls (J, boxed area represented in K). Scale bars in A-D are 3 mm, in E-H are 340 µm, in I and L are 2 mm, in J and M are 350 µm, and in K and N are 140 µm.
The number of mesenchymal cells positive for TGF-β3 was significantly higher in the sutured group (7.2 ± 0.5, N = 5) compared with the non-sutured group (0.2 ± 0.2, N = 5) (O). Ten min following labioplasty, there was a significant increase in TGF-β3 expression in the sutured cleft lip (10.8 ± 0.3, N = 5) as compared with the non-sutured cleft lip (8.4 ± 0.3, N = 5) (P). Numerical data are expressed as mean ± standard error of the mean.  p < 0.05.
|
|
View larger version (130K):
[in this window]
[in a new window]
|
Figure 2. TGF-β3 reduced type I collagen accumulation in repaired cleft lip. CL/Fr mouse cleft lip was sutured, placed in serum-free organ culture, and assayed for type I collagen expression. Suturing of cleft lip (B) resulted in a significant increase in the accumulation of type I collagen (brown deposit indicates immunoreactivity) 3 days after explant culture, as compared with non-sutured cleft lip (A,E). Dotted lines and asterisks delineate site of surgery. Scale bars in A-D are 140 µm. In the presence of exogenous TGF-β3, type I collagen immunoreactivity was reduced, and this decrease was dose-dependent on the concentration of TGF-β3 (38.2 ± 0.5 for control, 95.2 ± 0.7 for 0 ng/mL, 66.1 ± 1.4 for 10 ng/mL, and 51.2 ± 1.4 for 100 ng/mL TGF-β3, N = 5 for all groups) (C,D,E). Ten hrs after explant culture, a dose-dependent decrease in type I collagen message level was detected by RT-PCR (5.4 ± 0.6 for control, 8.6 ± 0.5 for 0 ng/mL, 5.2 ± 0.2 for 10 ng/mL, and 3.1 ± 0.4 for 100 ng/mL TGF-β3, N = 5 for all groups) (F). Numerical data are expressed as mean ± standard error of the mean. p < 0.05 compared with non-sutured group;  p < 0.05 compared with group with no TGF-β3;  p < 0.05 compared with group with 10 ng/mL TGF-β3.
|
|
View larger version (112K):
[in this window]
[in a new window]
|
Figure 3. TGF-β3 inhibited alpha-smooth-muscle actin expression. CL/Fr mouse cleft lip was sutured, and placed in serum-free organ culture, and assayed for alpha-smooth-muscle actin expression. Suturing of cleft lip (B) resulted in a significant increase in alpha-smooth-muscle actin (brown deposit indicates immunoreactivity) 3 days after explant culture, as compared with non-sutured cleft lip (A,E). Dotted lines and asterisks delineate site of surgery. Scale bars in A-D are 140 µm. In the presence of exogenous TGF-β3, the number of mesenchymal cells immunopositive for alpha-smooth-muscle actin was reduced, and this decrease was dose-dependent on the concentration of TGF-β3 (2.4 ± 0.2 for control, 8.8 ± 0.4 for 0 ng/mL, 7.4 ± 0.2 for 10 ng/mL, and 5.8 ± 0.4 for 100 ng/mL TGF-β3, N = 5 for all groups) (C,D,E). Eight hrs after explant culture, a dose-dependent decrease in alpha-smooth-muscle actin message level was detected by RT-PCR (2.9 ± 0.3 for control, 11.9 ± 1.3 for 0 ng/mL, 7.1 ± 0.4 for 10 ng/mL, and 3.3 ± 0.5 for 100 ng/mL TGF-β3, N = 5 for all groups) (F). Numerical data are expressed as mean ± standard error of the mean. p < 0.05 compared with non-sutured group; p < 0.05 compared with group with no TGF-β3; p < 0.05 compared with group with 10 ng/mL TGF-β3.
|
|
View larger version (62K):
[in this window]
[in a new window]
|
Figure 4. TGF-β3 promoted MMP-9 expression and activity. CL/Fr mouse cleft lip was placed in serum-free organ culture and assayed for MMP-9. Suturing of cleft lip (B) resulted in a significant increase in MMP-9 (brown deposit indicates immunoreactivity) 10 hrs after explant culture, as compared with non-sutured cleft lip (A,E). Dotted lines and asterisks delineate site of surgery. Scale bars in A-D are 140 µm. In the presence of exogenous TGF-β3, the number of mesenchymal cells immunopositive for MMP-9 was elevated, and this increase was dose-dependent on the concentration of TGF-β3 (0.2 ± 0.2 for control, 7.4 ± 0.5 for 0 ng/mL, 14.6 ± 0.7 for 10 ng/mL, and 27 ± 0.8 for 100 ng/mL TGF-β3, N = 5 for all groups) (C,D,E). Eight hrs after explant culture, a dose-dependent increase in MMP-9 message level was detected by RT-PCR (7.3 ± 0.1 for control, 8.4 ± 0.5 for 0 ng/mL, 11.1 ± 0.4 for 10 ng/mL, and 13.1 ± 0.9 for 100 ng/mL TGF-β3, N = 5 for all groups) (F). In contrast, MMP-1 message level remained unchanged in the presence of TGF-β3 (G). Numerical data are expressed as mean ± standard error of the mean. p < 0.05 compared with non-sutured group; p < 0.05 compared with group with no TGF-β3; p < 0.05 compared with group with 10 ng/mL TGF-β3.
In situ zymography showed that TGF-β3-soaked beads implanted into the upper lip resulted in a cleared gelatinolytic zone surrounding (asterisks) the bead in neonatal (H) and fetal (I) tissues. Control PBS beads had no effect (J). Scale bars in H-J are 140 µm. The upper lip was dissociated into primary cell culture, and the conditioned media were assayed for MMP expression and activity by gelatinolytic zymography (K). In the absence of TGF-β3, latent pro-MMP2 (72 kDa) and active MMP-2 (62 kDa) were detected. No MMP-9 was present. In the presence of 100 ng/mL TGF-β3, MMP-2 remained unchanged. However, the latent pro-MMP-9 (92 kDa) as well as the active MMP-9 (82 kDa) were detected.
We performed Western analysis to quantitate the levels of protein expression for TGF-β3, MMP-9, and type I collagen (L). Sutured cleft lip had a 3.3-fold increase in TGF-β3 protein level (p < 0.05). Similarly, MMP-9 levels also increased by 3.5-fold and 5.8-fold at 10 and 100 ng/mL exogenous TGF-β3, respectively (p < 0.05). Type I collagen levels decreased by 0.8-fold and 0.1-fold at 10 and 100 ng/mL exogenous TGF-β3, respectively (p < 0.05).
|
|
Journal of Dental Research, Vol. 82, No. 7,
558-564 (2003)
DOI: 10.1177/154405910308200714
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati  Twitter What's this?
|
|
