Introduction: Tissue engineering approaches appear highly promising for the regeneration of injured bone tissues. This strategy combine three essential components such as: scaffolds, mesenchymal stem cells (MSCs) and growth factors and is based on the culture of stem or progenitor cells on scaffolds in order to generate new bone by osteoinductive cue. Recently bioengineering has been focused on electrospinning technique use to fabricate a nanofibrous mat with appropriate pore size and internal/external scaffold geometry suitable for stem cells growth. Natural and synthetic polymers electrospun scaffolds with seeded stem cells have drawn great interest in tissue engineering. The aim of this study was to investigate the impact of an hydrolytically modified hybrid electrospun nonwoven poly(-L-lactide) and co-caprolactone (PLCL) scaffold on biological behaviour of human dental pulp stem cells (hDPSCs) growing on the nanofiber scaffold.
Methodology: Hybrid hydrolytically modified nanofibrous scaffold was fabricated by blending different weight ratio of poly(-L-lactide) and co-caprolactone. Stem cells were isolated from human dental pulp of extracted the third molars taken from four healthy donors. hDPSCs were seeded on PLCL for evaluation of cell viability, adhesion, proliferation, migration, and osteogenic differentiation. After one, three and seven days of hDPSCs seeding on PLCL scaffold the viability and proliferation of hDPSC was measured using the MTT assay and fluorescence intensity after cells staining with PKH26. hDPSC adhesion to PLCL scaffold was determined through cells counting, and migration was assessed through the hematoxylin-eosin staining of hDPSCs-PLCL samples. hDPSCs osteogenic potential was confirmed by mineralization status detected by Alizarin Red staining and bone-related gene expression evaluated by qRT-PCR analysis. As a control for all experiments, the hDPSCs were cultured as a monolayer.
Results: The results showed that the PLCL scaffold supported hDPSC viability and proliferation. The hDPSCs adhesion rate on PLCL increased with time of culture and was significantly higher compared to control group (p < 0.001). hDPSCs were able to migrate inside the PLCL electrospun scaffold after 7 days of seeding. No differences in morphology and immunophenotype of hDPSCs grown on PLCL and in flasks was observed. The mRNA levels of bone-related genes (OCN, OPN, BSP, DSPP) were significantly higher in hDPSCs after osteogenic differentiation on PLCL compared with undifferentiated hDPSCs on PLCL (p < 0.005). The ability of osteogenic differentiation of hDPSCs was also confirmed by the presence of mineral deposits on PLCL by Alizarin Red staining.
Conclusions: This study confirmed that the mechanical properties of a modified PLCL mat support hDPSC attachment and viability/proliferation. The biological features such as: adhesion, proliferation and migration of hDPSCs growing on PLCL scaffold, and good PLCL biocompatibility with stem cells indicate that this mat may be applied in designing of a bioactive hDPSCs-PLCL construct for tissue engineering. Moreover, high osteogenic potential of hDPSCs growing on PLCL suggest that this mat provide appropriate environment for dental stem cells differentiation into osteoblasts and might be used in bone tissue engineering.