Pig palate stem cells and smart scaffold as a challenge for cleft palate

摘要

Since the concept of tissue engineering was first introduced, researchers have been seeking a solution for bone repair and regeneration, by combining stem cells, growth factors and the use of a porous scaffold. The design and composition of the scaffold itself, is critical for its ability to promote and allow the cells to adhere, so that they may proliferate and differentiate. Orally derived mesenchymal stem cells (MSCs) for use in tissue engineering approaches, has great potential for solving clinical and surgical problems related to craniofacial wound healing. The dynamics of bone healing are controlled by growth factors, such as biological cues to promote these requirements; several strategies for bone and tissue regeneration have been developed. Yang F et al., ported that strontium could enhance the osteogenic differentiation of the MSCs. Strontium folate (SrFO) is a recently developed bone promoting agent of keen interest in the medical and pharmaceutical fields. In that sense, our group has synthesized and characterized a smart scaffold (SrFO-chitosan-polyethylene glycol dimethacrylate and beta tri calcium) In our present work, we have evaluated the capacity of the smart scaffold to improve the proliferation and differentiation of stem cells, in the ongoing challenge of cleft palate repair. Methods: The palatal stem cells from mini pigs were isolated using enzymatic digestion method with collagenase (Clostridium histolyticum, Sigma Aldrich C-8051, Saint Louis, Missouri, USA) and cultured in T25 flasks, for 14 days, with DMEM (Dulbecco's Modified Eagle's Medium) (Low-glucose Biowest-L0060- Riverside -MO, United States). The medium was replaced twice a week and the cells were grown until a confluence of 70-80%, with the third passages used for assay. Scaffolds were created as previously described Pig palate stem cells were seeded onto scaffolds, at a density of 4 x 10~4 cells. The scaffold/cells constructs were cultured as described above for in vitro studies, such a cell proliferation was assessed by MTT assay (Roche Life Science). Cell induction or differentiation to osteogenic was evaluated by Von Kossa (abl50687) and alkaline phosphatase activity (QuantiChrom™ Alkaline Phosphatase Assay Kit (DALP-250) (Hayward, CA, USA). Results: MSCs phenotype was confirmed by the presence of surface markers CD44, CD90, CD105 and the absence of CD34 and CD45, a good proliferation index was detected in the absorbance and osteogenic differentiation can be achieved with the detection of calcium deposits evaluated by Von Kossa assay as well as alkaline phosphatase activity. Conclusions: Therefore, we can conclude that the scaffold increased cell proliferation at different periods of time and improve differentiation as observed in the osteogenesis tests, palate stem cells and a smart scaffold, loaded with biological cues, appear to be a good challenge for use in pre-clinical therapy in in vivo cleft palate or craniofacial defect.