Odontogenic cell culture in PEGDA hydrogel scaffolds for use in tooth regeneration protocols

ABSTRACT

In order to obtain a tooth-like structure, embryonic oral ectoderm cells (EOE) and bone marrow-derived stem cells (BMSC) were stratified within a synthetic hydrogel matrix (PEGDA) and implanted in the ileal mesentery of adult male Lewis rats. Wholemount in situ hybridization was used to evaluate the expression of Pitx2, Shh and Wnt10a signals indicative of tooth initiation. In rats, expression of the three markers was present in the oral ectoderm starting at embryonic stage E12.5, which was therefore selected for cell harvesting. Embryos were obtained by controlled service of young female Lewis rats in which estrus was detected by impedance reading. At E12.5, pregnant rats were humanely euthanized and embryos were collected. The mandibular segment of the first branchial arch was dissected and the mesenchyme separated from the ectoderm by enzymatic digestion with pancreatin trypsin solution. BMSCs were collected by flushing the marrow of tibiae and femurs of adult Lewis rats with a-MEM and cultured in a-MEM in 25 cm2 flasks. Second passage BMSC's were recombined with competent oral ectoderm (E12.5-E13) stratifying them within a 3D PEGDA scaffold polymerized by exposure to UV (365nm) inside a pyramidal polypropylene mold. Constructs were incubated from 24 to 48 hrs in a-MEM and then implanted for four to six weeks in the mesentery of adult male (3- 6 month old) Lewis rats. 76 constructs were implanted (37 experimental, 27 negative controls and 12 positive controls). Upon maturation, constructs were harvested, fixed in buffered formalin, processed and stained with hematoxylin eosin (HE). Histological evaluation of the experimental and negative constructs showed that BMSCs underwent an apoptotic process due to lack of matrix interactions, known as anoikis, and were thus incapable of interacting with the competent ectoderm. In contrast, embryonic oral ectoderm was able to proliferate during the mesenteric implantation. In conclusion, PEGDA scaffolds are incompatible with BMSCs, therefore it is essential to continue the search for an ideal scaffold that allows proper tissue interactions.

RESUMEN

Para lograr la formacion de una estructura similar a un diente se estratificaron celulas de ectodermo oral embrionario (EOE) con celulas troncales de medula osea (BMSC) dentro de una matriz de diacrilato de polietilenglicol (PEGDA) que se implanto en el mesenterio ileal de machos adultos de ratas Lewis. Mediante hibridizacion in situ de bloque completo se evaluo la expresion de tres genes iniciadores putativos de la formacion dental (Pitx2, Shh y Wnt10a), estableciendo que en ratas las senales iniciadoras de dentogenesis aparecen entre E12.5 y E13. El tejido ectodermico embrionario se obtuvo haciendo cruces controlados de hembras a las que se les detecto el estro mediante impedanciometria. En E12.5 las hembras se sacrificaron y se extrajeron los embriones. Se diseco la porcion mandibular del primer arco branquial y el ectodermo se separo del mesenquima mediante disociacion enzimatica con una solucion de pancreatina tripsina. Las BMSC se extrajeron de los huesos largos de las extremidades inferiores de ratas mediante lavado con a-MEM y se cultivaron en cajas de 25cm2 hasta un segundo pasaje. Las BMSC fueron recombinadas con ectodermo embrionario competente (E12.5 - E13) estratificandolas en un soporte tridimensional de PEGDA, polimerizado con luz ultravioleta (365nm) dentro de un molde piramidal de polipropileno (PP). Los constructos se cultivaron entre 48 y 72 horas en a-MEM y posteriormente fueron implantados en el mesenterio ileal de machos adultos (3 a 6 meses de edad) por un periodo de cuatro a seis semanas. Se implantaron 76 constructos (37 experimentales, 27 controles negativos y 12 controles positivos). En la fecha determinada los animales se sacrificaron mediante asfixia con una mezcla de CO2 y aire recuperando los constructos que se fijaron en formalina tamponada para luego procesarlos y tenirlos con hematoxilina eosina (HE). La evaluacion histologica de los constructos experimentales, positivos y negativos mostro que las BMSC incluidas en el hidrogel sufrieron un proceso de apoptosis conocido como anoikis que impidio su interaccion con las celulas ectodermicas. En contraste el EOE prolifero durante el periodo de implantacion. A futuro se debe buscar la matriz portadora ideal que permita el confinamiento de los dos grupos celulares y que brinde el soporte estructural necesario para la proliferacion de las BMSC facilitando su interaccion con las celulas inductoras de origen ectodermico.