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Objective To prepare biphasic calcium phosphate/polyvinyl alcohol scaffolds by 3D printing at room temperature and explore the effect of 3D scaffolds on in vitro osteogenic differentiation of the bone marrow mesenchymal stem cells (BMSCs).Methods After biphasic calcium phosphate and polyvinyl alcohol solutions were mixed,the biphasic calcium phosphate/polyvinyl alcohol composite scaffolds were prepared by room temperature 3D printing combined with freeze drying technique.Non-printing scaffolds were prepared by injection molding.The surface microstructure,porosity,elastic modulus and hydrophilicity of the 2 sorts of scaffolds were measured.The cytological experiments were carried out in 3 groups (n =3):printed scaffold group,non-printed scaffold group and blank control group (no scaffold).After the BMSCs were seeded onto the scaffolds for 7 and 14 days,the 3 groups were compared in terms of cellular proliferation,alkaline phosphatase activity and expression levels of osteogenesis-related genes.Results 3D composite scaffolds with controllable pore size and porosity were prepared successfully,with an average porosity of 59.6％ ± 3.6％ and an average elastic modulus of 429.3 ± 54.3 kPa.After culture for 7 and 14 days,the cellular absorbance values in the printed scaffold group (0.987 ± 0.047 and 1.497 ± 0.076) were significantly higher than those in the non-printed scaffold group (0.767 ±0.063 and 1.181 ±0.098) (P ＜ 0.05) which were in turn significantly higher than those in the blank control group (0.532 ±0.046 and 0.895 ± 0.062) (P ＜ 0.05).After culture for 7 and 14 days,the ALP activity and expression levels of osteogenesis-related genes in the printed and non-printed scaffold groups showed no significant between-group differences (P ＞ 0.05),but were significantly higher than those in the blank control group (P ＜ 0.05).Conclusions Tissue-engineered composite biphasic calcium phosphate/polyvinyl alcohol scaffolds with controllable pore size and good connectivity can be prepared by freeze-drying and room temperature 3D printing techniques.Co-culture of the scaffolds and BMSCs in vitro promotes adhesion,proliferation and osteogenic differentiation of the cells.
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Resumen Introducción y objetivo: Los tejidos dentales contienen células madre mesenquimales con diversas capacidades de diferenciación. El objetivo de este estudio fue comparar la capacidad de osteodiferenciación de células madre de pulpa dental y ligamento periodontal cultivadas con diferentes medios de osteodiferenciación. Materiales y métodos: Se obtuvieron 24 muestras de tejido pulpar y 30 de ligamento periodontal de terceros molares, se aislaron y se cultivaron con 5 condiciones diferentes de osteoinducción. La osteodiferenciación fue evaluada mediante RT-PCR y Rojo Alizarin. Resultados: Las células aisladas fueron caracterizadas mediante citometría de flujo. Se observó una mayor expresión de Colágeno tipo I para células de pulpa dental, mientras que las células de ligamento periodontal expresaron mayores niveles de RUNX2. Las células Madre de ligamento periodontal presentaron mejor perfil de osteodiferenciación cuando fueron cultivadas en el medio 5, mientras que las células madre extraídas de la pulpa dental presentaron mejores características de osteodiferenciación en el medio de cultivo 4. La única diferencia entre estos dos medios fue la concentración de 50uM de Ácido Ascórbico para el medio 5 y 200uM de Ácido Ascórbico para el medio 4. Conclusiones: Las células obtenidas de pulpa dental y ligamento periodontal mostraron características de células madre. La capacidad de osteodiferenciación de estas células fue dependiente de las concentraciones de los sustratos aplicados al cultivo.
Introduction and objective: Dental tissues have mesenchymal stem cells with capacities to differentiate in several tissues. The aim of this research was to compare the ability of osteodifferentiation of stem cells isolated of dental pulp and periodontal ligament cultured with different media. Materials and methods: 24 samples of pulp tissue and 30 samples of periodontal ligament were obtained of third molars. Stem cells isolated by the explant method were subjected to 5 osteodifferentiation conditions. Osteodifferentiation was evaluated by RT-PCR and Alizarin Red. Results: The isolated cells were characterized as stem cells by flow cytometer. Collagen type I showed the highest expression in dental pulp Stem Cells while Runx2 was increased in periodontal ligament Stem Cells. Medium 5 showed the best conditions for osteodifferentiation of periodontal ligament Stem cells while Dental pulp Stem Cells showed better osteogenic profile in medium 4. The unique difference between mediums 4 and 5 was the concentration of Ascorbic Acid 50um in medium 5 and 200um in medium 4). Conclusions: The cells obtained both pulp and ligament showed features of Stem Cells. The osteogenic capacity of cells was depending of the concentrations of the substrates applied.