Effects of TGFbeta2 on collagen synthesis in cultured normal and wounded fetal mouse palates.

OBJECTIVE

It has been demonstrated in a number of models that fetal wounds heal with little or no scar. Since collagen is an integral part of the extracellular matrix in adult scar formation, we studied the synthesis and localization of collagen in an in vitro mouse palate model for fetal wound healing.

METHODS

Palates, dissected from fetal mice at 15, 16, and 17 days of gestation and from newborn mice, were cultured in medium containing serum (for 8 hours); this was followed by culture in serum-free medium (for 12 hours). One-half of the samples from each age group were wounded in the midline. All samples were placed in serum-free medium containing 20 microCi/mL 3H-proline for 8 hours. In addition, palates from 15-day gestation and from newborn mice were also incubated with transforming growth factor TGF-beta2 (10 ng/mL). Palates were washed with saline, homogenized, and radioactivity was counted. Proline uptake was calculated for each sample as counts per milligram of protein and was subjected to statistical analysis (three-way analysis of variance). Samples of the homogenate were subjected to sodium dodecyl sulfate-gel electrophoresis and Western blotting in order to determine the types of collagen that were synthesized. Immunohistochemical localization of collagen types I, III, and VI was carried out on paraffin-embedded samples from each group.

RESULTS

There were no significant differences in proline uptake between wounded mouse palates and nonwounded mouse palates at any age, and there was no histological evidence of regeneration of the palate at the site of the wound. Proline uptake was significantly greater in untreated wounded palates at 15 days' gestation than it was in newborns. After treatment with TGF-beta2, proline uptake was significantly greater in both wounded and nonwounded palates in the newborn group and had no effect on collagen synthesis in palates from 15-day gestation animals. Collagen types I and III were localized in histological specimens using immunohistochemistry and on nitrocellulose using Western blotting. No type VI collagen was demonstrated by Western blotting, but it was localized around blood vessels and on basement membranes using immunohistochemistry.

CONCLUSION

Treatment with TGF-beta2 significantly increased collagen synthesis, as assessed by 3H-proline uptake, in cultured palates from newborn mice as compared with palates from untreated newborn mice and from both treated and untreated palates of 15-day gestation mice. These data suggest a differential response to TGF-beta2 by mouse palates as a function of fetal development.