Materna Thomas, Rolf Hans J, Napp Johanna, Schulz Jutta, Gelinsky Michael, Schliephake Henning
Department of Oral and Maxillofacial Surgery, George-Augusta-University, Göttingen, Germany.
Clin Oral Implants Res. 2008 Apr;19(4):379-86. doi: 10.1111/j.1600-0501.2007.01483.x.
The aim of the present report was to evaluate current methods of in vitro analysis of three-dimensional (3D) scaffolds seeded with human bone marrow stromal cells (hBMSCs) from six bone marrow aspirates for tissue engineered growth of bone.
A series of experiments was conducted to compare methods of cell expansion and to validate analysis of proliferation and differentiation of hBMSCs in long term cultures of up to 40 days in 3D scaffolds of calcium carbonate (CaCO(3)) and mineralized collagen. Proliferation within the seeded scaffolds was monitored using cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), neutral red (NR) and DNA fluorescence assays and compared with empty controls. Differentiation was assessed by means of ELISA for osteocalcin (OC) and real time PCR for OC and collagen I (Coll I).
The results showed that the scaffold differed in seeding efficacy (CaCO(3): 53.3%, min. Coll.: 83.3%). The precise identification of the number of cells in biomaterials by MTT, NR and DNA assays was problematic, as MTT and NR assay overestimated the number of cells, whereas DNA assay grossly underestimated the number of cells on the scaffolds. Monitoring of changes over time may be biased by unspecific material-dependent background activity that has to be taken into account. Identification of osteogenic differentiation is not reliable by identifying osteogenic markers such as OC in the supernatant but has to be done on the transcriptional level.
It is concluded that monitoring of in vitro procedures for the construction of biohybrid scaffolds requires more emphasis in order to make the cell based approach a reliable treatment option in tissue engineering.
本报告旨在评估当前对三维（3D）支架进行体外分析的方法，这些支架接种了来自六份骨髓抽吸物的人骨髓基质细胞（hBMSC），用于骨组织工程生长。
进行了一系列实验，以比较细胞扩增方法，并验证在碳酸钙（CaCO₃）和矿化胶原的3D支架中长达40天的长期培养中hBMSC的增殖和分化分析。使用细胞计数、3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑（MTT）、中性红（NR）和DNA荧光测定法监测接种支架内的增殖情况，并与空白对照进行比较。通过酶联免疫吸附测定（ELISA）检测骨钙素（OC）以及通过实时聚合酶链反应（PCR）检测OC和I型胶原（Coll I）来评估分化情况。
结果表明，支架在接种效率上存在差异（CaCO₃：53.3%，矿化胶原：83.3%）。通过MTT、NR和DNA测定法精确鉴定生物材料中的细胞数量存在问题，因为MTT和NR测定法高估了细胞数量，而DNA测定法则严重低估了支架上的细胞数量。随时间变化的监测可能会受到非特异性材料依赖性背景活性的影响，这一点必须予以考虑。通过鉴定上清液中的成骨标记物如OC来确定成骨分化并不可靠，而必须在转录水平上进行。
得出的结论是，为了使基于细胞的方法成为组织工程中可靠的治疗选择，需要更加重视对生物杂交支架构建的体外程序的监测。
