Efecto de medios condicionados derivados de membranas de fibrina rica en plaquetas y leucocitos de fumadores y no fumadores en la diferenciación epitelial de un modelo de mucosa oral 3D in vitro

Abstract

La fibrina rica en plaquetas y leucocitos (L-PRF) es un concentrado plaquetario que tiene un efecto benéfico en la cicatrización debido a su la capacidad de liberar biomoléculas y factores de crecimiento. Las membranas de L-PRF de no fumadores pueden aumentar el grosor de la mucosa queratinizada. Sin embargo, poco se sabe del efecto de L-PRF de no fumadores en el epitelio oral y no hay evidencia del efecto de L-PRF de fumadores en el epitelio oral. El objetivo de este estudio es evaluar el efecto de medios condicionados derivados de membranas de fibrina rica en plaquetas y leucocitos (CM-LPRF) de fumadores y no fumadores en la estratificación epitelial en un modelo de mucosa oral 3D (tridimensional) in vitro. Se estableció un modelo de mucosa oral 3D in vitro, utilizando colágeno tipo I, fibroblastos gingivales primarios humanos y queratinocitos de la línea celular TR146 incubados en interfaz aire-líquido y se suplementaron con distintas concentraciones de CM-LPRF de no fumador a 12,5%, 25%, 50% y 100%. Los modelos de mucosa se suplementaron con CM-PRF de fumadores o no fumadores a una concentración de 50%. También se realizaron controles con DMEM/F12+ SFB (suero fetal bovino) 10%+ Glutamina 1%. Imágenes del epitelio se analizaron mediante tinción H&E (hematoxilina & eosina), PAS (ácido peryódico de Schiff) e inmunofluorescencia para pancitoqueratinas. El grosor epitelial se midió con el programa ImageJ. Se estableció un modelo de mucosa oral con características similares a las nativas. Los CM-LPRF de fumadores y no fumadores a una concentración de 50% fueron capaces de aumentar la estratificación epitelial en comparación con el control. Se evidenciaron similitudes en el grosor epitelial al suplementar con CM-LPRF de fumadores y no fumadores. Nuestros resultados sugieren el uso de CM-LPRF de fumadores y no fumadores como suplemento para inducir la estratificación epitelial en un modelo de mucosa oral 3D in vitro. No obstante, son necesarios más estudios para analizar los efectos de CM-LPRF en la diferenciación del tejido epitelial.

Leukocyte and platelet-rich fibrin (L-PRF) is a second-generation platelet concentrate used for stimulating tissue wound healing due to the presence of platelets and leukocytes entrapped in a fibrin matrix and enabled biomolecules and growth factors release. L-PRF membranes derived from non-smokers have been reported to promote wound healing in many clinical situations as periodontal defects and as coadjuvant in surgical procedures for root coverage and gingival gain. However, L-PRF effects in the oral epithelium are scarce, and there is limited evidence about smoking effects on the L-PRF functional effects. This study aimed to evaluate the effects of L-PRF conditioned media (CM-LPRF) derived from smokers and nonsmokers in the epithelial stratification on an in vitro oral mucosa 3D cell culture model. Oral mucosa 3D cell culture was established, mixing human primary gingival fibroblasts inside of collagen type I gel and the keratinocyte cell line TR146. Both were harvested at an air-liquid interface for 14 days. To establish a better concentration to promote epithelial stratification, oral mucosa 3D cell cultures were fed with nonsmoker CM-LPRF at different concentrations of 12,5%, 25%, 50%, and 100%. Later, the oral mucosa 3D cell cultures were fed with smokers or nonsmokers CM-LPRF at a concentration of 50%. Positive controls using the conventional cell culture medium composed by DMEM/F12 supplemented with FBS (fetal bovine serum) 10%, and Glutamine 1% were used. Oral mucosa 3D cell cultures were fixed and stained with H&E. Besides, immunofluorescence analyzed pan-cytokeratin. The epithelial size was quantified using ImageJ software. In this study, an oral mucosa 3D cell culture model was established with similar characteristics as native oral mucosa. CM-LPRF derived from smokers and nonsmokers at 50% was able to induce a significant epithelial stratification in comparison with control. However, the epithelial width was similar between 3D oral mucosa cell cultures treated with CM-LPRF recovered from smokers or nonsmokers. Our results suggest that CM-LPRF derived from smokers and nonsmokers is a source of growth factors to induce epithelial growth and differentiation. However, more studies are required to analyze the effects of CM-LPRF on epithelial tissue differentiation.