Effect of DMSO-free cryopreservation on MSCS osteogenesis in 3D porous scaffolds.

Successful clinical application of bone tissue engineered constructs depends on efficiency of osteogenic differentiation of cells within 3D scaffolds. Dimethyl sulfoxide (DMSO)-free cryopreservation can have a direct effect on osteogenesis. Therefore, the aim of this research was to evaluate the ability of cryopreserved mesenchymal stromal cells (MSCs) to differentiate into osteogenic direction within 3D scaffolds. Human adipose tissue-derived MSCs were cryopreserved without DMSO and serum using sucrose-based pretreatment protocol. After thawing non-cryopreserved and cryopreserved MSCs were seeded into gelatin, collagen and collagen / hydroxyapatite scaffolds, afterwards cell attachment, viability, metabolic and proliferation activity were tested. Efficiency of induced osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and calcium content. After seeding into all investigated scaffolds cryopreserved and non-cryopreserved cells attached to their surface and were able to proliferate. Cryopreservation resulted in an increased ALP expression in all investigated groups; however had no effect on calcium contents. The maximum levels of ALP activity and calcium deposition were obtained for MSCs grown within collagen-based scaffolds, while cells within gelatin scaffolds demonstrated less efficiency of osteogenic differentiation. To sum up, the developed DMSO- and fetal serum (FS) - free cryopreservation protocol preserves the potential of MSCs to differentiate towards osteogenic direction in 3D environment.

骨组织工程构建体的临床成功应用，取决于3D支架内细胞成骨分化的效率。无二甲基亚砜（Dimethyl sulfoxide, DMSO）的冷冻保存技术，可对成骨过程产生直接影响。因此，本研究旨在评估经冷冻保存的间充质基质细胞（Mesenchymal Stromal Cells, MSCs）在3D支架内向成骨方向分化的能力。本研究采用基于蔗糖的预处理方案，在不含DMSO与血清的条件下，对人脂肪来源间充质基质细胞进行冷冻保存。复苏后，将未冷冻保存与经冷冻保存的细胞接种至明胶、胶原以及胶原/羟磷灰石支架中，随后检测细胞的黏附能力、存活率、代谢活性与增殖活性。通过碱性磷酸酶（Alkaline Phosphatase, ALP）活性与钙含量，评估诱导成骨分化的效率。将细胞接种至所有受试支架后，经冷冻保存与未冷冻保存的细胞均可黏附于支架表面并实现增殖。冷冻保存可使所有受试组的ALP表达水平升高，但对钙含量无显著影响。在胶原基支架中培养的间充质基质细胞，其ALP活性与钙沉积水平达到最高；而明胶支架中的细胞成骨分化效率则相对较低。综上，本研究开发的无DMSO与胎牛血清（Fetal Serum, FS）冷冻保存方案，可保留间充质基质细胞在3D环境内向成骨方向分化的潜能。