In vitro manipulation of cleft palate connective tissue: setting the bases of a proposed new treatment.
BACKGROUND
Palatoplasty has the undesired side effect of impaired mid-facial growth. To avoid this problem, we propose an alternative to palatoplasty. We hypothesize that if BMP-2 is injected together with a carrier into the periosteum of the cleft palate borders, border volume will increase and connective tissue cells will be activated to produce extra bone. Once these borders supported by bone reach the midline, extraction of their covering epithelia with trypsin will permit adhesion of the underlying tissues. We investigated in vitro the ability of cleft palate connective tissue cells to produce extra bone in the presence of BMP-2 and the possibility of using trypsin to remove the epithelium covering the cleft palate borders without impairing the underlying tissues' ability to adhere.


METHODS
We used the cleft palate presented by tgf-beta(3) null mice and small fragments of human cleft palate mucoperiosteum as models. Immunolabeling BMP-2-treated or untreated cultures with TUNEL and anti-osteocalcin or PCNA antibodies was performed. The epithelium of the cleft palate borders was removed with a trypsin solution, and the de-epithelialized tissues were cultured in apposition.


RESULTS
BMP-2 induces differentiation toward bone on cleft palate connective tissue cells without producing cell death or proliferation. Trypsin removal of the cleft palate margins' epithelium does not impair the underlying tissues' adhesion.


CONCLUSIONS
It is possible to generate extra bone at the cleft palate margins and to chemically eliminate their covering epithelia without damaging the underlying tissues, which allows further investigation in vivo of this new approach for cleft palate closure.
Resel
Eva
E
Departamento de Anatomía y Embriología Humana I, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
Martínez-Sanz
Elena
E
González
Ignacio
I
Trinidad
Eva
E
Garcillán
Beatriz
B
Amorós
María
M
Alonso-Bañuelos
Carmen
C
González-Meli
Beatriz
B
Lagarón
Emilio
E
Murillo
Jorge
J
Del Río
Aurora
A
Barrio
Carmen
C
López
María
M
Martínez-Alvarez
Concepción
C
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology* / Q000502:physiology; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002972:Cleft Palate / Q000188:drug therapy* / Q000473:pathology; D003238:Connective Tissue / Q000187:drug effects / Q000502:physiology; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000166:cytology; D010159:Palate / Q000187:drug effects* / Q000473:pathology* / Q000502:physiology; D011247:Pregnancy; D046509:Tissue Culture Techniques; D016212:Transforming Growth Factor beta / Q000494:pharmacology* / Q000502:physiology; D014357:Trypsin / Q000494:pharmacology
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta; D014357:Trypsin