Department of Bioengineering, Schools of Engineering and Medicine, Stanford University, Stanford, California.
Department of Biology, School of Humanities and Sciences, Stanford University, Stanford, California.
Tissue Eng Part A. 2019 Oct;25(19-20):1404-1412. doi: 10.1089/ten.TEA.2018.0289. Epub 2019 May 2.
While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, most tissue engineering strategies for cartilage regeneration encapsulate MSCs as single cells, partially due to the technical challenge to homogeneously encapsulate cell pellets in three-dimensional (3D) hydrogels. It remains unclear whether encapsulating MSCs as single cell suspension or cell aggregates in 3D hydrogels would enhance MSC-based cartilage formation. In this study, we determined that the optimal size of MSC micropellets (μPellets) that can be homogeneously encapsulated in hydrogels with high cell viability is 100 cells/pellet. Using optimized μPellet size, MSCs were encapsulated either as single cell suspension or μPellets in four soft hydrogel formulations with stiffness ranging 3-6 kPa. Regardless of hydrogel formulations, single cell encapsulation resulted in more neocartilage deposition with improved mechanical functions over μPellet encapsulation. For single cell encapsulation, polyethylene glycol (PEG) hydrogels containing chondroitin sulfate led to the most cartilage matrix deposition, with compressive modulus reaching 211 kPa after only 21 days, a range approaching the stiffness of native cartilage. The findings from this study offer valuable insights on guiding optimal method design for MSCs and hydrogel-based cartilage regeneration. The optimized μPellet encapsulation method may be broadly applicable to encapsulate other stem cell types or cancer cells as aggregates in hydrogels. Impact Statement While the gold standard for inducing mesenchymal stem cell (MSC) chondrogenesis utilizes pellet culture, it remains unclear whether encapsulating MSCs as cell pellets in three-dimensional hydrogels would enhance MSC-based cartilage formation. In this study, we determined the optimal size of MSC micropellet (μPellet) that can be homogeneously encapsulated in hydrogels with high cell viability. Unexpectedly, single cell encapsulation resulted in more robust new cartilage formation than μPellet encapsulation. Furthermore, tuning hydrogel formulation led to rapid cartilage regeneration with stiffness approaching that of native cartilage. The findings from this study would facilitate clinical translation of MSCs and hydrogel-based therapies for cartilage regeneration with optimized parameters.
虽然诱导间充质干细胞（MSC）软骨生成的金标准是使用微球培养，但大多数软骨再生的组织工程策略都将 MSC 包裹为单个细胞，部分原因是在三维（3D）水凝胶中均匀包裹细胞微球存在技术挑战。目前尚不清楚将 MSC 包裹为单细胞悬液或 3D 水凝胶中的细胞聚集体是否会增强基于 MSC 的软骨形成。在这项研究中，我们确定了可以均匀包裹在高细胞活力水凝胶中的 MSC 微球（μPellets）的最佳尺寸是 100 个细胞/微球。使用优化的 μPellet 尺寸，将 MSC 包裹为单细胞悬液或 μPellets 分别封装在 4 种具有 3-6kPa 硬度的软质水凝胶制剂中。无论水凝胶制剂如何，单细胞包封均导致更多的新生软骨沉积，并改善了机械功能，优于 μPellet 包封。对于单细胞包封，含有硫酸软骨素的聚乙二醇（PEG）水凝胶导致软骨基质沉积最多，仅 21 天后压缩模量达到 211kPa，接近天然软骨的刚度。这项研究的结果为指导 MSC 和基于水凝胶的软骨再生的最佳方法设计提供了有价值的见解。优化的 μPellet 包封方法可能广泛适用于将其他干细胞类型或癌细胞包裹为水凝胶中的聚集体。
