Current clinical approaches for articular cartilage repair have not been able to restore the tissue with zonal architecture, and its biomechanical and functional properties. Mimicking the zonal organization of articular cartilage in neo-tissue by implanting zonal chondrocyte subpopulations in multilayer construct could enhance the functionality of the graft, engineering of stratified tissue has not yet been realized due to lack of efficient and specific zonal chondrocyte isolation protocol. We show that by using a spiral microchannel device, the superficial, middle and deep zone chondrocytes can be separated based on cell size, and enriched from full thickness porcine cartilage in a high-throughput, label-free manner. The size-sorted cells show zone-specific characteristics in RT-PCR analysis of zonal cartilage markers. Both freshly sorted and two-passage expanded zonal chondrocytes formed cartilage tissue in 3D hydrogel, bearing respective zonal characteristics, indicated by RT-PCR, histology, extracellular matrix proteins, and mechanical compression test. In the proof-of-concept in vivo study using a rodent cartilage defect model, the size-sorted zonal chondrocytes when delivered in bi-layered hydrogel construct, facilitated better cartilage repair with mechanically enhanced cartilage tissue, in comparison to conventional chondrocytes implantation. This study provides an effective approach to obtain large numbers of zonal chondrocytes, and demonstrates the translational potential of stratified zonal chondrocyte implantation for clinical repair of critical size cartilage defects.

当前用于关节软骨修复的临床方法尚不能通过带状结构及其生物力学和功能特性来恢复组织。通过在多层构建体中植入带状软骨细胞亚群来模仿新组织中的关节软骨的带状组织可以增强移植物的功能，但由于缺乏有效和特异性的带状软骨细胞分离方案，因此尚未实现分层组织的工程化。我们显示，通过使用螺旋微通道设备，可以根据细胞大小分离浅层，中层和深层软骨细胞，并以高通量，无标签的方式从全厚度猪软骨中富集。大小分类的细胞在区域软骨标志物的RT-PCR分析中显示区域特有的特征。新鲜分选的和经两次通道扩增的带状软骨细胞均在3D水凝胶中形成了软骨组织，并具有各自的带状特征，这由RT-PCR，组织学，细胞外基质蛋白和机械压缩试验表明。在概念证明中使用啮齿动物软骨缺损模型进行的体内研究表明，与传统的软骨细胞植入相比，大小不同的带状软骨细胞在双层水凝胶构建物中递送时，通过机械增强的软骨组织促进了更好的软骨修复。这项研究提供了一种获得大量带状软骨细胞的有效方法，并证明了分层带状软骨细胞植入的翻译潜力可用于临床修复关键尺寸的软骨缺损。