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

机译：交联的壳聚糖-明胶支架中牙髓干细胞的活力和成骨/成牙本质分化

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Introduction: Chilosan-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. 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 add 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 . 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 pi 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 denlin or bone-like mineralized tissue formation to be used for targeted dental and periodontal tissue regeneration.
机译：简介：由于具有适合各种生物医学应用（包括软骨，皮肤和周围神经再生）的合适的机械，生物降解和生物相容性，已经提出了Chilosan-明胶支架。这项研究的目的是调查戊二醛（GTA）交联的壳聚糖-明胶支架在促进牙髓干细胞（DPSC）附着和骨/牙源性分化方面朝着目标牙齿组织再生的潜能。方法：将50％的超纯水中的2％（w / v）壳聚糖溶解在1％（v / v）的乙酸添加溶液和2％（w / v）的明胶中，制备多孔支架。将两种溶液倒入一起以达到40％-60％壳聚糖-明胶的组成，并在50 oC下搅拌2小时。然后在50°C搅拌下以0.1％（w / v）将GTA添加到混合物中直至凝胶化。然后将凝胶混合物倒入24孔板中，并在-20 oC冻干24 h。使用0.1 M NaOH中和支架，并用超纯水彻底冲洗。从年轻的健康供体的第三磨牙建立DPSC培养物，并用流式细胞仪对几种干细胞（SC）标记物进行表征。对于体外细胞培养，将支架用PBS洗涤（3次，每次30分钟），然后用培养基洗涤，再用PBS洗涤（3次，每次30分钟）。最后，将支架与培养基在37 oC下孵育24小时。然后在每个支架上点样包含100个pi完全培养基（包含15％胎牛血清FBS，100 mM L-抗坏血酸磷酸酯和抗生素/抗真菌药的a-MEM），该培养基包含106个DPSC（来自第2-6代）。将斑点的支架在37 oC和5％CO2下孵育2小时，然后用1.2 ml培养基/孔完全覆盖。每2天进行一次培养基更换。 3,7和14天后，通过共聚焦显微镜，使用活/死荧光染色（Calcein M / PI染色）评估细胞活力。此外，在7和14天后分离出总RNA，并进行实时PCR评估骨/牙源性标记（BSP，DSPP，BMP-2，ALP，osterix，Runx2）的表达。通过扩增效率（LinRegPCR）调整结果，并通过geNorm评估的两个最稳定的管家基因（SDHA和B2M）进行标准化。结果与讨论：我们制备了稳定的40％-60％壳聚糖-明胶支架，该支架具有连通的孔和100μm的孔径。建立的DPSC培养物对SC标记（例如CD90，CD73，CD29，CD81，CD49f，CD51，CD166，Nestin，Nanog）呈阳性（占总人口的95％），部分对CD105，CD146，STRO-1，CD24， CD271，Oct3 / 4，SSEA-4。 40％-60％的壳聚糖-明胶支架可以支持高水平的细胞附着和存活力。实时PCR显示，在支架内生长的细胞显着上调了DSPP [高达16倍]，BSP [高达5.4倍]，BMP-2 [高达2.6倍]，骨钙蛋白[高达4.7倍]。 ]，osterix [最高8.8倍]和ALP [最高1.5倍]。结论：这些结果表明，拟议的40％-60％壳聚糖-明胶支架即使在没有诱导性培养基的情况下也支持DPSC的骨/牙源性分化，因此可以进一步考虑其其他性质，例如它们诱导牙本质或骨形成的能力。例如矿化的组织形成，可用于有针对性的牙齿和牙周组织再生。

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