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Rescue of Cleft Lip Ex Utero by Exogenous Reactivation of Craniofacial Developmental Programs
Wilmina N. Landford, B.A., James Hart, D.V.M., Elisabetta Ferretti, Ph.D., Ope A. Asanbe, M.D., Xue Dong, B.S., Kerri A. Morrison, B.S., Licia Selleri, M.D., Ph.D, Jason A. Spector, M.D., FACS.
Weill Cornell Medical College, New York, NY, USA.

BACKGROUND:Cleft lip/palate (CLP) affects approximately 1 in 500-700 live births, representing the most common congenital craniofacial anomaly. Presently, no single gene has been identified as a universal culprit for nonsyndromic cases but several accounts of interplay between genetic and environmental factors have shown a close relationship to the etiology. Previously, we have demonstrated that Pbx-Wnt signaling at the embryonic lambdoidal (λ) junction has been shown to mediate midfacial morphogenesis via localized apoptotic programs. We not only developed a unique compound Pbx-deficient murine model with fully penetrant CLP, but also demonstrated genetic rescue strategies to reconstitute Wnt signaling and correct midface clefting. We now seek to identify novel avenues for tissue repair of this mutilating facial abnormality by using Wnt-soaked collagen microspheres to restore craniofacial developmental programs in our Pbx-deficient embryonic murine model ex utero.
METHODS:A modified Whole Embryo Culture (WEC) system was established to enable normal development of murine embryos. Wildtype and Pbx-deficient viable murine embryos were dissected from the uterus and embryos placed in a 37°C incubator. In vitro studies were done to determine optimal formulations of collagen microspheres. At gestation day E11.5, collagen microspheres soaked in murine purified Wnt9b protein were microsurgically implanted at the midface λ junction. Correction of CLP was assessed by gross morphology, histology, and evaluation of the restoration of apoptotic programs. In addition, titration assays were conducted to optimize the dose of Wnt by assessing protein content and release kinetics as well as its range of action in the embryonic face with regard to space and time.
RESULTS:Prelimary results demonstrate that embryos continue to develop normally in this "artificial uterus" for about 24 hours, evidenced by normal facial development in the cultured embryos when compared to embryos fixed at immediate removal from the uterus. Type I collagen microspheres, approximately 50-70 microns, were fabricated and demonstrated appropriate release kinetics. Targeted delivery of Wnt9b was demonstrated by implantation of Wnt9b-soaked microspheres at the lambdoidal junction, resulting in augmented expression of Wnt in the area surrounding the collagen microsphere when compared to the normal endogenous Wnt signaling pattern evaluated under X-gal staining. The implanted microspheres themselves did not affect normal development of the lambdoidal junction in wild type embryos or alter cleft development in Pbx knockouts.
CONCLUSIONS:We have successfully demonstrated the viability of our ex vivo whole embryo culture system as well as the feasibility of implanting collagen microspheres as targeted drug delivery vehicles. Wnt9b-soaked collagen microspheres are currently being implanted into Pbx-deficient embryos to determine if such local delivery can correct the clefting associated with that genotype. We believe that our research will open new avenues towards unconventional and innovative prenatal interventions for patients with CL/P as well as provide future applicable repair approaches for congenital head and neck disorders.

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