Nano-Structured Injectable Cell Carriers for Dental Regeneration

Objectives: The combination of Human dental pulp stem cells (DPSCs) with novel nano-structured injectable microspheres as cell carriers offer a promising strategy to regenerate mineralized dental tissues. In this work, we have evaluated the histomorphometric, viability and physiologic profiles of DPSCs in both nanofibrous microspheres (NF-MS) and conventional microspheres with smooth surfaces (C-MS) developed from poly (L-lactic acid) (Liu et al.,Nat Mater 2011) to establish the optimal conditions for the use of dentin-like tissue regeneration. 

Methods: Human DPSCs (1x10⁵) were seeded on 0.25 g microspheres (NF-MS or C-MS) and cultured for 24h, 3 and 7 days by using 1 ml of supplemented Dulbecco’s modified Eagle’s medium. To determine the cell viability, adhesion and proliferation, a LIVE/DEAD®, DNA quantification, PCNA detection and PKH67 assay were performed. Subsequently, cell differentiation was determined by flow cytometry using CD105, CD146, CD90, and CD45 markers.

Results: Our results revealed an efficient cell attachment and proliferation of DPSCs to NF-MS at 24h, 3 and 7 days. In contrast, we observed a progressive release of DNA to the culture medium in DPSCs cultured with C-MS, and a reduction of the cell viability was detected in C-MS along 7 days. The cell proliferation study by PCNA detection demonstrated higher levels at 24 h and 3 days in both, NF-MS and C-MS. Finally, flow cytometry analysis showed a negative expression of CD45 and positive expression of CD90, CD105 and CD146 markers in DPSCs cultured with NF-MS at 24h and 7days. However, a lack of CD105 and CD146 expression was observed in C-MS.

Conclusion: Our results show that DPSCs on NF-MS had better cell viability, adhesion and proliferation capabilities than those on C-MS, suggesting that NF-MS might be a promising micro-carrier of DPSCs for dental regeneration.