Vuornos Kaisa, Huhtala Heini, Kääriäinen Minna, Kuismanen Kirsi, Hupa Leena, Kellomäki Minna, Miettinen Susanna
Adult Stem Cell Group, BioMediTech, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
Research, Development and Innovation Centre, Tampere University Hospital, Tampere, Finland.
J Biomed Mater Res B Appl Biomater. 2020 May;108(4):1332-1342. doi: 10.1002/jbm.b.34482. Epub 2019 Aug 31.
Lack of bone grafts appeals for bone augmentation solutions. We aimed at osteogenic differentiation of human adipose stem cells (hASCs) and microvascularization in coculture with human umbilical vein endothelial cells (HUVECs) embedded in three-dimensional (3D) gellan gum (GG) and collagen type I (COL) hydrogel mixture. We compared endothelial growth medium-2 (EGM-2) and bioactive glass extract-based endothelial and osteogenic medium (BaG EM-OM) for vascularized bone-like graft development in vitro. Cell viability, cell number, and osteogenic and endothelial gene expression were analyzed. Mineralized hydroxyapatite residues, immunocytochemical staining of endothelial marker CD31 production and late osteogenic marker osteocalcin were imaged. With both media, good cell viability was observed within 3D hydrogel. EGM-2 condition induced significantly higher cell number compared to BaG EM-OM condition at both 7 and 14 days. Interestingly, both media supported osteogenic as well as endothelial marker gene expression. Moreover, formation of reticulated cellular structures was observed in both EGM-2 and BaG EM-OM conditions. However, hydroxyapatite mineralization and strong osteocalcin staining were detected only in BaG EM-OM condition. Importantly, strong production of CD31 and elongated tube-like structures were apparent in EGM-2 culture alone. In conclusion, we demonstrated efficient hASC osteogenic differentiation and microvessel-like network formation in coculture with HUVECs.
骨移植材料的缺乏促使人们寻求骨增量解决方案。我们旨在研究人脂肪干细胞（hASCs）与嵌入三维（3D）结冷胶（GG）和I型胶原（COL）水凝胶混合物中的人脐静脉内皮细胞（HUVECs）共培养时的成骨分化和微血管化情况。我们比较了内皮生长培养基-2（EGM-2）和基于生物活性玻璃提取物的内皮和成骨培养基（BaG EM-OM）在体外构建血管化骨样移植物的效果。分析了细胞活力、细胞数量以及成骨和内皮基因表达情况。对矿化羟基磷灰石残留、内皮标志物CD31产生的免疫细胞化学染色以及晚期成骨标志物骨钙素进行了成像。在两种培养基中，3D水凝胶内均观察到良好的细胞活力。在第7天和第14天，与BaG EM-OM条件相比，EGM-2条件诱导的细胞数量显著更高。有趣的是，两种培养基均支持成骨和内皮标志物基因表达。此外，在EGM-2和BaG EM-OM条件下均观察到网状细胞结构的形成。然而，仅在BaG EM-OM条件下检测到羟基磷灰石矿化和强烈的骨钙素染色。重要的是，仅在EGM-2培养中明显观察到CD31的大量产生和细长的管状结构。总之，我们证明了hASCs与HUVECs共培养时有效的成骨分化和微血管样网络形成。
