Pasquinelli G, Orrico C, Foroni L, Bonafè F, Carboni M, Guarnieri C, Raimondo S, Penna C, Geuna S, Pagliaro P, Freyrie A, Stella A, Caldarera C M, Muscari C
Clinical Pathology, Clinical Department of Radiological and Histocytopathological Sciences, University of Bologna, Bologna, Italy.
J Anat. 2008 Nov;213(5):520-30. doi: 10.1111/j.1469-7580.2008.00974.x.
The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.
制备富含多能干细胞的可生物降解三维支架似乎是修复不可逆损伤组织的一种有前景的策略。研究了大鼠间充质干细胞（rMSCs）与基于透明质酸的支架（即HYAFF(R)11）相互作用的精细机制，以评估这种工程构建体的潜在临床应用。将rMSCs接种（2×10(6)个细胞/cm(-2)）到支架上，培养21天，并使用适当技术进行分析。未经处理的支架样品的光学显微镜（LM）、扫描电子显微镜（SEM）和透射电子显微镜（TEM）显示，支架具有高度多孔的结构，由表面粗糙的15微米厚的微纤维组成。经台盼蓝染色检测，第1天细胞黏附率高。CFDA检测显示rMSCs存活长达14天，MTT检测显示其在支架上稳定增殖。光学显微镜显示第3天时rMSCs位于支架的最内部。扫描电子显微镜显示第21天时亚汇合的细胞单层覆盖了支架表面的40±10%。早期培养的透射电子显微镜显示rMSCs以规则间隔的粘着斑包裹单个纤维，而共聚焦显微镜显示CD44透明质酸受体的极化表达；14天培养的透射电子显微镜证明了纤维连接的形成。21天培养物的免疫组织化学显示，纤连蛋白是分泌到细胞外空间的主要基质蛋白；核心蛋白聚糖和多功能蛋白聚糖仅见于细胞质中，IV型胶原表达极少。在细胞培养结束时，发现间充质干性标志物CD90的表达未受影响。我们的结果表明，在无任何化学诱导的静态培养条件下，HYAFF(R)11支架支持rMSCs的黏附、迁移和增殖，以及细胞外基质成分的合成和分泌。接种细胞的高保留率和活力以及它们与底物相互作用的良好方式表明，在修复如软骨修复、伤口愈合和大血管置换等广泛组织缺损时，这种支架可能具有潜在用途。
