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TGF-beta-3 Promotes Scarless Repair of Cleft Lip in Mouse Fetuses
1 Graduate School of Dental Science, Correspondence: * corresponding author, m-ohishi{at}dent.kyushu-u.ac.jp
TGF-β3 mediates epithelial-mesenchymal transformation during normal fusion of lip and palate, but how TGF-β3 functions during cleft lip repair remains unexplored. We hypothesize that TGF-β3 promotes fetal cleft lip repair and fusion by increasing the availability of mesenchymal cells. In this investigation, we demonstrated that cleft lips in mouse fetuses were repaired by fetal surgery, producing scarless fusion. At the site of the operation, we first observed an infusion of platelets expressing TGF-β3, followed by increased expression of cyclin D1 and tenascin-C, and coupled with increased mesenchymal cell proliferation. In an ex vivo serumless culture system, cleft lip explants fused in the presence of exogenous TGF-β3. Cultured lips also showed up-regulation in cyclin D1 and tenascin-C expression. These findings suggest that microsurgical repair of cleft lip in the fetus that produced scarless fusion is mediated by TGF-β3 regulation of mesenchymal cell proliferation and migration at the site of repair.
Key Words: fetal surgery • mesenchymal cells • organ culture • cyclin • tenascin
Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. The prevalence among Asian populations is particularly high, at approximately 1 in 500 live births (Mitchell and Wood, 2000). The primary treatment strategy is multidisciplinary: surgical and rehabilitative approaches. However, controversy remains regarding the optimal treatment procedures to achieve both functional and esthetic outcome (Molsted, 1999). Surgical repair of cleft lip in newborns may result in significant scar formation due to excessive tension and contraction. However, repair of cleft lip in animal fetuses in utero appears to produce relatively scarless results (Hallock, 1985; Stelnicki et al., 1999). The developmental basis for scarless repair of cleft lip in utero is unclear, although secreted growth factors have been implicated for use in augmenting repair (Chang et al., 1995). The cellular and molecular mechanisms that lead to scarless repair remain to be elucidated, and applications toward new or revised strategies for clinical treatment are yet to be formulated (Adzick and Lorenz, 1994). Gene expression studies and linkage analyses have implicated an association between the TGF-β3 gene and cleft lip with or without palate (Lidral et al., 1998; Bodo et al., 1999; Sato et al., 2001). Studies in animal models demonstrate that TGF-β3 is instrumental during normal fusion of the palate. Targeted disruption of TGF-β3 in mice resulted in cleft palate (Kaartinen et al., 1995; Proetzel et al., 1995), and exogenous TGF-β3 rescued the defect in culture (Taya et al., 1999). The mechanism of TGF-β3 action is biphasic, mediating adherence of apposing epithelia and subsequent confluence of the epithelial seam by epithelial-mesenchymal transformation (Kaartinen et al., 1997; Sun et al., 1998). A recent study shows that lip fusion progresses with a basic mechanism similar to that of palatal fusion (Sun et al., 2000). However, it is unclear whether TGF-β3 participates during the process, and whether it functions in the lip epithelium and/or mesenchymal components. Moreover, it is unclear whether TGF-β3 may augment cleft lip repair, which involves these tissue components. The present study was designed to identify the biological role of TGF-β3 during cleft lip repair in the mouse fetus. We hypothesize that TGF-β3 functions to promote cleft lip fusion by increasing the availability of mesenchymal cells. Using both in utero and ex vivo systems for the fusion of cleft lip, we demonstrated that TGF-β3 promoted fusion by increasing mesenchymal cell proliferation, and enhancing cell migration that was mediated by tenascin. We further suggest that fetal surgery is a viable option for cleft lip repair, and that our mouse model is a valuable experimental system for the study of molecular events and mechanisms associated with cleft lip repair. Finally, we also propose that TGF-β3 could be used to augment scarless repair of cleft lip.
Animal Handling and Fetal Surgery Animal handling conformed to guidelines from the Council on Animal Care at Kyushu University. Surgery was performed on timed pregnant CL/Fraser animals under the anesthesia Ethrane (Abbot, North Chicago, IL, USA). Plug day was designated as embryonic day 1 (E1). The median dorsal skin of the dam was incised to expose the uterus. This dorsal approach was taken so that the dam could not reach the operated area, thus preventing wound re-opening and possible abortion. The presence or absence of cleft lip in the fetuses was identified through the uterine wall. A microsurgical incision was made to expose the facial complex of the fetus with minimal loss of amniotic fluid. The cleft part of the upper lip and the nasal process at the center of the cleft were incised through the epithelium, positioned, and then sutured once with 11-0 nylon thread (Keisei Medical, Tokyo, Japan). The uterine wall was sutured closed. The operated fetuses were reinstated and allowed to continue development in utero until natural parturition. Mouse fetuses were divided into a control group that had not undergone surgery, and a sutured group that had undergone surgical repair of cleft lip. Only fetuses with bilateral clefts were selected for repair experiments, so that the unoperated side could serve as the contralateral control. Numerical data were subjected to an unpaired two-tailed Student’s t test, and statistical significance was taken at p < 0.05.
BrdU Labeling, Immunohistochemistry, and Cell Counting
RNA Isolation and Semi-quantitative Reverse-transcription/Polymerase Chain-reaction (RT-PCR)
Organ Culture
Fetal Surgery Performed for Repair of Cleft Lip Allowed for Scarless Fusion The CL/Fr mouse is highly susceptible to cleft lip, presenting a 25% rate of clefting (Juriloff and Fraser, 1980). In unaffected CL/Fr embryos, the upper lips begin to fuse at E12 and continue until the process is complete by E13. This represents a 24-hour delay in fusion when compared with other mouse strains without susceptibility to cleft lip. By E16, cleft lip in the fetus can be readily diagnosed through the uterine wall. We conducted fetal surgery to repair cleft lip at this stage due to a balance between viability and reproducibility. Surgery performed in E15 fetuses resulted in increased mortality, and that in E17 did not fuse properly, since the fetuses were delivered shortly after surgery.
During surgery, the left cleft was incised, positioned, and sutured (Fig. 1a
TGF-β3 was Expressed during Lip Formation and Up-regulated during Fusion of Surgically Sutured Cleft Lip To identify whether TGF-β3 participates during lip fusion and cleft lip repair, we examined the spatial distribution and expression level of TGF-β3. At E12, we detected the presence of TGF-β3 in the juxtaposed epithelia, and the merged epithelium that had just undergone fusion (Fig. 2a  ). In contrast, the oral epithelium that does not fuse did not express TGF-β3. After lip fusion, TGF-β3 disappeared. In the cleft lip, TGF-β3 was not detected in the epithelia adjacent to the cleft (Fig. 2b). Thirty min after surgical repair of cleft lip in E16, we detected an infusion of platelets into the site of operation. These platelets were positive for TGF-β3, and averaged to 12 platelets within our defined surveyed area (Figs. 2c, 2e). Some endothelial cells were also positive for TGF-β3 (inset in Fig. 2c). In controls, no platelets were observed, and the epithelium adjacent to the cleft remained negative for TGF-β3 (Figs. 2d, 2e). Using RT-PCR, we detected a significant 3.4-fold increase in TGF-β3 expression 10 min after surgery in tissues collected from the site of operation when compared with controls (Fig. 2f). Analysis of our data, taken together, suggests that TGF-β3 normally functions during lip fusion, and the absence of TGF-β3 is associated with cleft lip. The presence of TGF-β3 at the site of operation during cleft lip repair may contribute to the fusion process. Additional molecular defects may be present in the CL/Fr affected and unaffected animals that cause clefting and increase their susceptibility to clefting.
Cyclin and Tenascin Expression, and Cell Proliferation were Up-regulated during Fusion of Surgically Sutured Cleft Lip Since surgical repair of cleft lip resulted in fusion, we next examined changes in cell proliferation and extracellular matrix properties at the site of fusion. Ninety min after surgery, we detected more cyclin-positive cells at the site of the operation (Figs. 3a, 3b ). These cyclin-expressing cells were fibroblasts, since they were immunopositive for type III collagen (data not shown). Three hrs after surgery, there was an increase in the number of cells positive for BrdU label, indicative of increased cell proliferation. At the sutured site, 16.5% of the cells were BrdU-positive, vs. 11.4% in a similar area in controls (Figs. 3c, 3d). We also detected, by immunolocalization, that the extracellular matrix at the site of the operation contained more tenascin 4 hrs after surgery (Figs. 3e, 3f). Quantitative data supported these morphological observations. Within a defined surveyed area, there was a 6.2-fold increase in cells immunopositive for cyclin D1 in the sutured group vs. the control group (Fig. 3g). By 60 hrs after surgery, there was 29% more mesenchymal cells at the repaired site when compared with controls (Fig. 3g). This was accompanied by a significant 1.9-fold increase in the relative expression of cyclin D1 and a 2.5-fold elevation for tenascin-C in the sutured group (Fig. 3h). Analysis of these data suggests that the increase in cell number that was associated with successful repair could be due to an increase in cell proliferation. Further, cells could be recruited and migrate to populate the repaired area that was mediated by tenascin.
Exogenous TGF-β3 Promoted Cleft Lip Fusion in Serumless ex vivo Cultures and was Associated with Up-regulation of Cyclin and Tenascin To establish a causal relationship that TGF-β3 can promote fusion, we used an ex vivo model to support our in vivo observations. The two halves of the cleft upper lip were explanted into a serumless culture system. In this system, exogenous TGF-β3 was applied to the explants at defined concentrations and was kept free from the confounding variations introduced by serum factors. We discovered that TGF-β3 was instructive toward fusion of the lip and that the responses were dose-dependent. In the absence of exogenous TGF-β3, the lip remained clefted throughout the culture period (Figs. 4a, 4d ). In the presence of 10 ng/mL TGF-β3, 1 in 10 cleft lips (10%) achieved partial fusion. The epithelia from the 2 halves were in contact, but the halves could still be delimited (Figs. 4b, 4e). At 100 ng/mL TGF-β3, 4 out of 10 cleft lips (40%) fused by 60 hrs of culture. The area of fusion was populated with mesenchymal cells, and remnants of the epithelial seam between the 2 fused halves could be identified (Figs. 4c, 4f). Consistent with our in vivo findings, we detected a dose-dependent significant increase in the expression level and number of cells positive for cyclin D1 (Figs. 4g-4i, 4m, 4n). Similarly, the expression level as well as the amount of extracellular matrix immunopositive for tenascin were also elevated in the presence of TGF-β3 (Figs. 4j-4l, 4n). Taken together, our ex vivo findings extended our in vivo observations that TGF-β3 may direct cleft lip fusion by increasing cell proliferation and enhancing cell migration through tenascin.
In this study, we documented three distinct roles of TGF-β3: mediates epithelial adherence during developmental lip fusion, promotes lip fusion by acting on the mesenchymal component, and allows for scarless healing after surgical repair of cleft lip. The most significant findings are to support an instructive role of TGF-β3 and its mechanisms of action in promoting the fusion of cleft lip. During developmental lip fusion, TGF-β3 is transiently expressed in the epithelium, and is absent in the cleft lip. This suggests that, similar to palatal fusion, TGF-β3 may mediate lip epithelial adherence, a phase that is a prerequsite for subsequent confluence of the tissues (Kaartinen et al., 1997; Sun et al., 1998). In the absence of other serum factors in an explant system, TGF-β3 supplementation alone was able to induce the fusion of cleft lip, similar to rescue experiments performed for palatal fusion (Kaartinen et al., 1997; Sun et al., 1998; Taya et al., 1999). On the other hand, surgical repair of the cleft lip is associated with expression of TGF-β3 transcripts and the infusion of TGF-β3-positive platelets at the site of operation. However, during cleft lip repair, the epithelium is partly removed to allow for positioning and suturing. Therefore, the fact that the sutured cleft lip undergoes fusion supports an additional role for TGF-β3 on the mesenchymal component of the lip. This does not preclude the possibility that TGF-β3 is regulated by epithelial to mesenchymal interactions during developmental lip fusion. Similarly, TGF-β3 derived from the platelets could exert positive paracrine feedback onto neighboring mesenchymal and endothelial cells, resulting in further up-regulation of TGF-β3. This speculation is supported by the ability of TGF-β1 to auto-induce its expression. During fibrosis in particular, TGF-β1 from degranulating platelets auto-induces expression in neighboring monocytes and fibroblasts (Kim et al., 1990; Ashcroft et al., 1999; Piek et al., 2001). Therefore, our data support the potential of using TGF-β3 for the therapeutic augmentation of cleft lip repair. Since TGF-β3 is instrumental in promoting the fusion of cleft lip, we explored the mechanisms of its action. We discovered that TGF-β3 might promote two cellular events at the site of the operation, by enhancing cell proliferation and cell migration. The majority of the actions of TGF-βs on the cell cycle of epithelial cells is to cause growth arrest. However, in mesenchymal cells, TGF-β can enhance proliferation and induce cyclin D1 expression (Jeoung et al., 1995; Rao and Kohtz, 1995). Our findings are consistent with this literature. We detected an up-regulation of cyclin D1 transcripts in tissues at the site of the repair. This increase can be attributed to the increased expression of cyclin D1 in mesenchymal cells. An increase in cyclin D1 expression is followed by an increase in cell proliferation, as assayed by BrdU labeling. In culture, exogenous TGF-β3 results in up-regulation of cyclin D1 expression, thus further supporting a causal relationship between TGF-β3 and cell proliferation. In addition to increased cell proliferation, the total increase in mesenchymal cell number could also be the result of increased cell migration into the site of repair. This is supported by our observation that tenascin expression is up-regulated in the presence of TGF-β3. Indeed, TGF-β has been demonstrated to induce tenascin expression (Mackie et al., 1998; Chimal-Monroy and Diaz de Leon, 1999). Tenascin functions in modifying the adhesive properties of the extracellular matrix, and allows for increased cell motility. Furthermore, early tenascin expression is associated with fetal wound healing (Whitby and Ferguson, 1991). At the site of repair, we detected increased accumulation of tenascin. We speculate that this tenascin could facilitate the emigration of mesenchymal cells into the repaired site, thus in part accounting for the increase in total mesenchymal cell number and contributing toward lip fusion. In culture, we also observed up-regulation of tenascin only in the presence of TGF-β3, and conclude that TGF-β3 modulates the expression level of tenascin, directly or indirectly. In addition to promoting lip fusion, TGF-β3 also allows for scarless repair of the surgical wound after fusion. It has long been documented that wound healing in the fetus differs fundamentally from that in the adult. Fetal wounds heal without inflammation, fibrosis, and scar formation. This knowledge base thus has tremendous implications for the repair and regeneration of tissues of the fetus in utero (Molsted, 1999). One interesting point is that TGF-β3 seemingly has effects opposite those of TGF-β1 or -β2 on scar formation. TGF-β1 and -β2 are associated with adult-type wound healing, whereas TGF-β3 is associated with that in the fetus (Sullivan et al., 1995; Hsu et al., 2001). Antibodies directed against TGF-β1 and -β2, or exogenous TGF-β3, reduced scarring on cutaneous wounds (Shah et al., 1995). Although we have not compared the actions of different TGF-β family members in cleft lip repair in the fetus, our data do support a significant role of TGF-β3 in mediating such repair with scarless outcome. It is yet unclear how mechanistically the TGF-β isoforms produce different outcomes during wound healing and how specifically TGF-β3 reduces scar formation. The use of this CL/Fr mouse model coupled with fetal surgery and repair provide research opportunities for the study of the molecular events and mechanisms of TGF-β signal transduction and the developmental process of lip fusion and scarless repair of cleft lip.
We express our appreciation to Dr. Taisei Nomura, Osaka University, for his generous gift of CL/Fraser mouse breeding pairs, and to Dr. Y. Oosaki, Dr. I. Kobayashi, Dr. T. Nagata, and Dr. K. Ogata, Kyushu University, for valuable assistance and technical support for the experiments. The present study was supported in part by grants-in-aid 11470439 to M. Ohishi and 07557135 to K. Nonaka from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This paper is based on a thesis submitted to the Graduate School of Dental Science, Kyushu University, in partial fulfillment of the requirements for the PhD degree for K. Kohama. Received for publication January 29, 2002. Revision received July 9, 2002. Accepted for publication July 19, 2002.
Journal of Dental Research, Vol. 81, No. 10,
688-694 (2002) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
p < 0.01 compared with 0 ng/mL TGF-β3 group and 
p < 0.01 compared with 10 ng/mL TGF-β3 group.
