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

  • Vaishnavi Dhanvantri Department of Regenerative Medicine and Department of oral Pathology
  • R Ramya Department of oral Pathology
  • Kotturathu Mammen Cherian Department of Regenerative Medicine
  • Balasundari Ramesh Department of Regenerative Medicine https://orcid.org/0000-0002-0090-2827
Keywords: Dental Pulp Stem Cells, Nanofibers, Conditioned Media, Nanotopography, Poly Lactic Acid

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 biodegradable
poly-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.

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References

Lanza R, Gearhart J, Hogan B, Melton D, Pedersen R, Thomas ED, Thomson J, editors. Essentials of Stem Cell Biology. Cambridge: Academic Press; 2006.

Weissman IL, Anderson DJ, Gage F. Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. Annu Rev Cell Dev Biol. 2001;17:387-403. doi: https://www.doi.org/10.1146/annurev.cellbio.17.1.387

Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002;81:531-5. doi: https://www.doi.org/10.1177/154405910208100806

Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97(25):13625-30. doi: https://www.doi.org/10.1073/pnas.240309797

Maher A, Núñez-Toldrà R, Carrio N, Ferres-Padro E, Ali H, Montori S, Al Madhoun A. The Effect of Commercially Available Endodontic Cements and Biomaterials on Osteogenic Differentiation of Dental Pulp Pluripotent-Like Stem Cells. Dent J (Basel). 2018;6(4). doi: https://www.doi.org/10.3390/dj6040048

Karaöz E, Doğan BN, Aksoy A, Gacar G, Akyüz S, Ayhan S, et al. Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol. 2010;133(1):95-112. doi: https://www.doi.org/10.1007/s00418-009-0646-5

Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100(10):5807-12. doi: https://www.doi.org/10.1073/pnas.0937635100

Friedlander LT, Cullinan MP, Love RM. Dental stem cells and their potential role in apexogenesis and apexification. Int Endod J. 2009;42(11):955-62. doi: https://www.doi.org/10.1111/j.1365-2591.2009.01622.x

Mitsiadis TA, Woloszyk A, Jimenez-Rojo L. Nanodentistry: combining nanostructured materials and stem cells for dental tissue regeneration. Nanomedicine (Lond). 2012;7(11):1743-53. doi: https://www.doi.org/10.2217/nnm.12.146

Nishikawa SI, Osawa M. Generating quiescent stem cells. Pigment Cell Res. 2007;20(4):263-70. doi: https://www.doi.org/10.1111/j.1600-0749.2007.00388.x

Wang J, Ma H, Jin X, Hu J, Liu X, Li L, Ma PX. The effect of scaffold architecture on odontogenic differentiation of human dental pulp stem cells. Biomaterials. 2011;32(31):7822-30. doi: https://www.doi.org/10.1016/j.biomaterials.2011.04.034

Brafman DA. Constructing stem cell microenvironments using bioengineering approaches. Physiol Genomics. 2013;45(23):1123-35. doi: https://www.doi.org/10.1152/physiolgenomics.00099.2013

Gupte MJ, Ma PX. Nanofibrous Scaffolds for Dental and Craniofacial Applications. J Dent Res. 2012;91(3):227-34. doi: https://www.doi.org/10.1177/0022034511417441

Khanna-Jain R, Vanhatupa S, Vuorinen A, Sandor GKB, Suuronen R, et al. Growth and Differentiation of Human Dental Pulp Stem Cells Maintained in Fetal Bovine Serum, Human Serum and Serum-free/Xeno-free Culture Media. J Stem Cell Res Ther. 2012; 2(4). doi: https://www.doi.org/10.4172/2157-7633.1000126

Scadden DT. The stem-cell niche as an entity of action. Nature. 2006;441(7097):1075-9. doi: https://www.doi.org/10.1038/nature04957.

Estrela C, Alencar AH, Kitten GT, Vencio EF, Gava E. Mesenchymal stem cells in the dental tissues: perspectives for tissue regeneration. Braz Dent J. 2011;22(2):91-8.

Fitzgerald M, Chiego JD Jr, Heys DR. Autoradiographic analysis of odontoblast replacement following pulp exposure in primate teeth. Arch Oral Biol. 1990;35(9):707-15. doi: https://www.doi.org/10.1016/0003-9969(90)90093-p.

Kim DH, Provenzano PP, Smith CL, Levchenko A. Matrix nanotopography as a regulator of cell function. J Cell Biol. 2012;197(3):351-60. doi: https://www.doi.org/10.1083/jcb.201108062

Turner LA, Matthew JD. Nanotopography – potential relevance in the stem cell niche. Biomater Sci. 2014;2:1574-94. doi: https://www.doi.org/10.1039/C4BM00155A

Sloan AJ, Waddington RJ. Dental pulp stem cells: what, where, how? Int J Paediatr Dent. 2009;19(1):61-70. doi: https://www.doi.org/10.1111/j.1365-263X.2008.00964.x

Soares DG, Zhang Z, Mohamed F, Eyster TW, Souza Costa CA, Ma PX. Simvastatin and nanofibrous poly (l-lactic acid) scaffolds to promote the odontogenic potential of dental pulp cells in an inflammatory environment. Acta Biomater. 2018;68:190-203.

Alleman M, Low E, Truong K, Huang E, Hill CK, Chen TY, et al. Dental pulp-derived stem cells (DPSC) differentiation in vitro into odontoblast and neuronal progenitors during cell passaging is associated with alterations in cell survival and viability. Int J Med Biomed Res. 2013;2(2):133-41.

Bonnamain V, Thinard R, Sergent-Tanguy S, Huet P, Bienvenu G, Naveilhan P, et al. Human dental pulp stem cells cultured in serum-free supplemented medium. Front Physiol. 2013;4:357.

Deng XL, Xu MM, Li D, Sui G, Hu XY, Yang XP. Electrospun PLLA/ MWNTs/HA hybrid nanofiber scaffolds and their potential in dental tissue engineering. Key Eng Mater. 2007;330-332:393-6. doi: https://www.doi.org/10.4028/www.scientific.net/KEM.330-332.393

Matsuoka F, Takeuchi I, Agata H, Kagami H, Shiono H, Kiyota Y, et al. Morphology-Based Prediction of Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells. PLoS One. 2013;8(2):e55082. doi: https://www.doi.org/10.1371/journal.pone.0055082

Woo KM, Chen VJ, Ma PX. Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment. J Biomed Mater Res A. 2003;67(2):531-7.

Zainal Ariffin SH, Kermani S, Zainol Abidin IZ, Megat Abdul Wahab RM, Yamamoto Z, Senafi S, et al. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium. Stem Cells Int. 2013;2013(250740). doi: https://www.doi.org/10.1155/2013/250740

Kichenbrand C, Velot E, Menu P, Moby V. Dental pulp stem cell-derived conditioned medium: an attractive alternative for regenerative therapy. Tissue Eng Part B Rev. 2019;25(1):78-88. doi: https://www.doi.org/10.1089/ten.TEB.2018.0168

Ramesh B, Bishi DK, Rallapalli S, Arumugam S, Cherian KM, Guhathakurta S. Ischemic cardiac tissue conditioned media induced differentiation of human mesenchymal stem cells into early stage cardiomyocytes. Cytotechnology. 2012;64(5):563-75. doi: https://www.doi.org/10.1007/s10616-012-9440-7

Shi S, Bartold PM, Miura M, Seo BM, Robey PG, Gronthos S. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res. 2005;8(3):191-9. doi: https://www.doi.org/10.1111/j.1601-6343.2005.00331.x

Aanismaa R, Hautala J, Vuorinen A, Miettinen S, Narkilahti S. Human dental pulp stem cells differentiate into neural precursors but not into mature functional neurons. Stem Cell Discovery. 2012;2(3):85-91. doi: https://www.doi.org/10.4236/scd.2012.23013

Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials. 2005;26(15):2603-10. doi: https://www.doi.org/10.1016/j.biomaterials.2004.06.051

Abe S, Hamada K, Miura M, Yamaguchi S. Neural crest stem cell property of apical pulp cells derived from human developing tooth. Cell Biol Int. 2012;36(10):927-36.

Published
2019-12-27
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Section
Original Research