Viability and osteo/odontogenic differentiation of dental pulp stem cells in crosslinked chitosan-gelatin scaffolds

Abstract

Event Abstract Back to Event Viability and osteo/odontogenic differentiation of dental pulp stem cells in crosslinked chitosan-gelatin scaffolds Maria Chatzinikolaidou1, 2, Athina Bakopoulou3 and Anthie Georgopoulou1 1 University of Crete, Materials Science and Technology, Greece 2 FORTH, IESL, Greece 3 Aristotle University of Thessaloniki, Department of Fixed Prosthesis and Implant Prosthodontics, Greece Introduction: Chitosan-gelatin scaffolds have been proposed due to their suitable mechanical, biodegradation and biocompatibility properties for different biomedical applications, including cartilage, skin and peripheral nerve regeneration [1][2]. Aim of this study was to investigate the potential of glutaraldehyde (GTA)-crosslinked chitosan-gelatin scaffolds to promote attachment and osteo/odontogenic differentiation of Dental Pulp Stem Cells (DPSCs) towards targeted dental tissue regeneration. Methods: Porous scaffolds were prepared by dissolving 2% (w/v) chitosan in a 1% (v/v) acetic acid solution and 2% (w/v) gelatin in ultrapure water at 50 oC. The two solutions were poured together to reach a composition of 40%-60% chitosan-gelatin and stirred for 2 h at 50 oC. GTA was then added to the mixture at 0.1% (w/v) under agitation at 50 oC until gelation. The gel mixture was then casted into 24-well plates and lyophilized for 24 h at -20 oC. Scaffolds were neutralized using 0.1 M NaOH and rinsed thoroughly with ultrapure water. DPSC cultures were established from third molars of young healthy donors and characterized for several stem cell (SC) markers with flow cytometry [3]. For in vitro cell culture, scaffolds were washed with PBS (3 times, 30 minutes each), followed by culture medium and again PBS (3 times, 30 minutes each). Finally, scaffolds were incubated with culture medium for 24 h at 37 oC. Each scaffold was then spotted with 100 μl complete culture medium (a-MEM containing 15% Fetal Bovine Serum-FBS, 100 mM L-ascorbic acid phosphate and antibiotics/antimycotics) containing 106 DPSCs (from passages 2-6). Spotted scaffolds were incubated for 2 h at 37 oC and 5% CO2 and then fully covered with 1.2 ml culture medium/well. Medium change was performed every 2 days. After 3, 7 and 14 days, live/dead fluorescent staining (Calcein M/PI staining) was used to assess cell viability by means of confocal microscopy. Moreover, total RNA was isolated after 7 and 14 days and real time PCR was performed to evaluate expression of osteo/odontogenic markers (BSP, DSPP, BMP-2, ALP, osterix, Runx2). The results were adjusted by amplification efficiency (LinRegPCR) and were normalized to the two most stable housekeeping genes evaluated by geNorm (SDHA and B2M). Results and Discussion: We prepared stable 40%-60% chitosan-gelatin scaffolds with interconnective pores and pore size of 100 μm. The established DPSC cultures were positive (>95% of the population) for SC markers, like CD90, CD73, CD29, CD81, CD49f, CD51, CD166, Nestin, Nanog, and partly for CD105, CD146, STRO-1, CD24, CD271, Oct3/4, SSEA-4. The 40%-60% chitosan-gelatin scaffolds could support high levels of cell attachment and viability over time. Real time PCR showed that cells grown inside the scaffolds presented a significant upregulation of DSPP [up to16-fold], BSP [up to 5.4-fold], BMP-2 [up to 2.6-fold], osteocalcin [up to 4.7-fold], osterix [up to 8.8-fold] and ALP [up to 1.5-fold]. Conclusion: These results indicate that the proposed 40%-60% chitosan-gelatin scaffolds support osteo/odontogenic differentiation of DPSCs even in absence of inductive media and therefore can be further considered for other properties, such as their ability to induce dentin or bone-like mineralized tissue formation to be used for targeted dental and periodontal tissue regeneration. This study was financially supported by the Hellenic General Secretariat for Research and Technology grant Aristeia II ‘Osteobiomimesis 3438’ and funded by the European Union (EU) and National Resources