Sprayed Calcium phosphate substrate induced bone-like without osseoinducer supplements

摘要

Introduction: Future tissue engineering developments rely on cell-instructive scaffolds. Cell fate could be driven by topographical information coded within the biomaterial. Calcium phosphate materials (CaP) are widely studied for bone regeneration because of their biocompatibility and their chemical structure close to bone mineral phase1. Nevertheless, most studied CaP lack osseoinductive potential that could induce stem cells differentiation into mature bone cell phenotype. We report spraying technique of calcium and phosphate solutions to elaborate CaP that drives human umbilical cord Wharton's jelly stem cells (WJ-SCs) to form in vitro bone-like nodules without the use of osseoinducer supplements. Experimental Methods: An automated spraying device was used for deposition of calcium phosphate. 0.2 M NH4H2PO4 solution at pH 10 and 0.3 M Ca(NO3)2 solution at pH 4 were sprayed simultaneously 50 times then rinsed with MilliQ water and dried. The resulting substrate (CaP50) was characterized by Scanning Electron Microscopy (SEM), Fourier Transformed Infrared Spectroscopy (FTIR) and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES). The cellular adhesion process was investigated through cytoskeleton and vinculin-anchored focal adhesion labelling. At 28 days of culture, Transmission Electron Microscopy (TEM) was used for the ultrastuctural studies. Results and Discussion: SEM micrographs showed that CaP50 is constituted of almost spherical aggregates with micro-sized particles. FTIR spectras showed peaks within 500-1100 cm-1 and 3400-3600 cm-1 regions revealing octacalcium phosphate phase. CaP50 adsorbed ions from the medium within 24 h. These results highlight the bioactive properties of the sprayed CaP50. WJ-SCs on CaP50 organized themselves in small colonies and displayed a perpendicular orientation of F-actin stress fibres and a peripheral localisation of vinculin. WJ-SCs on coverslip glass substrate preserved their fibroblastic shape with F-actin running in parallel to the longitudinal cell axis and a perinuclear distribution of vinculin. Over a period of 6 days, the colonies organized themselves dense cellular structures called bone-like nodules. TEM highlighted multilayered structures containing cells that are interconnected by cell processes and bundles of mineralized type Ⅰ collagen fibrils. Conclusion: Spraying calcium and phosphate solutions allows the buildup of bioactive CaP. Without the use of ossoinduecr supplement, WJ-SCs cultivated on CaP50 form bone-like nodules, which are considered as a model of osteoblastic differentiation.