Evaluation of human dental stem cell growth characteristics and cellular morphological changes in response to extracellular matrix nanotopography

Abstract

Objective: Nanotopography and soluble extracellular factors are present in the dental stem cell niche in the pulp. Their effect on dental stem cell survival and differentiation is yet to be established. We aimed to analyze the individual and combined roles of extracellular matrix (ECM) nanotopography and serum (soluble factors) on the growth, differentiation potential, and morphological characteristics of the human dental pulp stem cells (hDPSC). This study aimed to evaluate and compare the hDPSC response to different environmental cues – nanofibers, serum, and conditioned media. Materials and methods: In this study, fabricated PLLA nanofibers were used as the in vitro structural biomimetic of the native nanotopography found in the in vivo ECM/stem cell niche. Serum and conditioned media were used as the in vitro mimic of the soluble factors to which stem cells get exposed in vivo. hDPSC were grown in the presence and absence of biodegradablepoly-L-lactic-acid nanofibers and serum. The growth characteristics of hDPSC were assessed in terms of cell viability and doubling time at the interval of every passage. Cellular morphological changes were studied using inverted microscopy and H&E. As the second part of the study, hDPSC in all culture conditions were exposed to Dental Pulp Conditioned Media (DPCM) for a short duration of 3 days. After transient exposure to DPCM, the growth characteristics and the morphological changes of hDPSC were assessed. In addition, scanning electron microscopy was used for the morphological study of hDPSC on nanofibers, exposed to conditioned media. The differentiated cells were analyzed by qRT-PCR for neurogenic and odontogenic expression of RUNX2, osteopontin, and β-tubulin III genes. Results: hDPSC showed better survival and proliferation in the presence of nanofibers and serum. Absence of nanofibers or serum greatly altered stem cell survival and proliferation and also indicated differentiation. In addition, it was observed that after transient exposure to DPCM, the presence of both PLLA nanofiber and serum favoured higher odontogenic and neurogenic differentiation potential, without characteristic morphological changes of terminal differentiation. Conclusion: hDPSC has the ability to sense nanoscale geometric cues from their microenvironment. Nanotopography and soluble factors of the extracellular matrix both affect hDPSC. Further studies are essential to identify the key pathways that play a vital role in such interactions. The hDPSC demonstrated better survival and proliferation in the presence of nanofibers and serum. Absence of nanofibers or serum greatly altered stem cell survival and proliferation and also showed changes indicative of differentiation. The results were compared and analyzed using GraphPad Prism 5 Software. hDPSC possess the ability to sense nanoscale geometric cues from their microenvironment. Nanotopography and soluble factors of the Extracellular matrix together influence the fate of hDPSC. Further studies are essential to identify the key pathways that play a vital role in such interactions.