Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA.
Acta Biomater. 2013 Jan;9(1):4688-97. doi: 10.1016/j.actbio.2012.08.009. Epub 2012 Aug 16.
There has been little research on the seeding of human umbilical cord mesenchymal stem cells (hUCMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of this study were: (i) to seed hUCMSCs in a fibrin hydrogel containing fast-degradable microbeads (dMBs) to create macropores to enhance cell viability; and (ii) to investigate the encapsulated cell proliferation and myogenic differentiation for muscle tissue engineering. Mass fractions of 0-80% of dMBs were tested, and 35% of dMBs in fibrin was shown to avoid fibrin shrinkage while creating macropores and promoting cell viability. This construct was referred to as "dMB35". Fibrin without dMBs was termed "dMB0". Microbead degradation created macropores in fibrin and improved cell viability. The percentage of live cells in dMB35 reached 91% at 16 days, higher than the 81% in dMB0 (p<0.05). Live cell density in dMB35 was 1.6-fold that of dMB0 (p<0.05). The encapsulated hUCMSCs proliferated, increasing the cell density by 2.6 times in dMB35 from 1 to 16 days. MTT activity for dMB35 was substantially higher than that for dMB0 at 16 days (p<0.05). hUCMSCs in dMB35 had high gene expressions of myotube markers of myosin heavy chain 1 (MYH1) and alpha-actinin 3 (ACTN3). Elongated, multinucleated cells were formed with positive staining of myogenic specific proteins including myogenin, MYH, ACTN and actin alpha 1. Moreover, a significant increase in cell fusion was detected with myogenic induction. In conclusion, hUCMSCs were encapsulated in fibrin with degradable microbeads for the first time, achieving greatly enhanced cell viability and successful myogenic differentiation with formation of multinucleated myotubes. The injectable and macroporous fibrin-dMB-hUCMSC construct may be promising for muscle tissue engineering applications.
对于人脐带间充质干细胞（hUCMSCs）在三维支架中用于肌肉组织工程的接种，研究甚少。本研究的目的是：（i）将 hUCMSCs 接种到含有快速降解微珠（dMBs）的纤维蛋白水凝胶中，以形成大孔来提高细胞活力；（ii）研究包封细胞的增殖和肌源性分化，用于肌肉组织工程。测试了 dMBs 质量分数为 0-80%，结果表明 35%的 dMBs 在纤维蛋白中既避免了纤维蛋白收缩，又形成了大孔并促进了细胞活力。这种构建体被称为“dMB35”。不含 dMBs 的纤维蛋白被称为“dMB0”。微珠降解在纤维蛋白中形成大孔并提高了细胞活力。第 16 天，dMB35 中的活细胞百分比达到 91%，高于 dMB0 的 81%（p<0.05）。dMB35 中的活细胞密度是 dMB0 的 1.6 倍（p<0.05）。包封的 hUCMSCs 增殖，使 dMB35 中的细胞密度在 1 至 16 天增加了 2.6 倍。与 dMB0 相比，第 16 天 dMB35 的 MTT 活性显著更高（p<0.05）。hUCMSCs 在 dMB35 中的肌球蛋白重链 1（MYH1）和α-辅肌动蛋白 3（ACTN3）等肌管标记物的基因表达较高。用肌生成特异性蛋白（包括肌生成素、MYH、ACTN 和肌动蛋白α 1）的阳性染色形成了伸长的多核细胞。此外，在肌生成诱导时检测到细胞融合显著增加。总之，hUCMSCs 首次被包封在具有可降解微珠的纤维蛋白中，实现了大大提高的细胞活力和成功的肌源性分化，形成了多核肌管。可注射的多孔纤维蛋白-dMB-hUCMSC 构建体可能在肌肉组织工程应用中具有广阔的前景。
