The zonal organization of articular cartilage is crucial in providing the tissue with mechanical properties to withstand compression and shearing force. Current treatments available for articular cartilage injury are not able to restore the hierarchically organized architecture of the tissue. Implantation of zonal chondrocyte as a multilayer tissue construct could overcome the limitation of current treatments. However, it is impeded by the lack of efficient zonal chondrocyte isolation protocol and dedifferentiation of chondrocytes during expansion on tissue culture plate (TCP). This study aims to develop a protocol to produce an adequate number of high-quality zonal chondrocytes for clinical application via size-based zonal chondrocyte separation using inertial spiral microchannel device and expansion under dynamic microcarrier culture.

Full thickness (FT) chondrocytes isolated from porcine femoral condyle cartilage were subjected to two serial of size-based sorting into three subpopulations of different cell sizes, namely small (S1), medium (S2), and large (S3) chondrocytes. Zonal phenotype of the three subpopulations was characterised. To verify the benefit of stratified zonal chondrocyte implantation in the articular cartilage regeneration, a bilayer hydrogel construct composed of S1 chondrocytes overlaying a mixture of S2 and S3 (S2S3) chondrocytes was delivered to the rat osteochondral defect model. For chondrocyte expansion, two dynamic microcarrier cultures, sort-before-expansion and sort-after-expansion, which involved expansion after or before zonal cells sorting, were studied to identify the best sort-expansion strategy.

Size-sorted zonal chondrocytes showed zone-specific characteristics in qRT-PCR with a high level of PRG4 expression in S1 and high level of aggrecan, Type II and IX collagen expression in S2 and S3. Cartilage reformation capability of sorted zonal chondrocytes in three-dimensional fibrin hydrogel showed a similar trend in qRT-PCR, histology, extracellular matrix protein quantification and mechanical compression test, indicating the zonal characteristics of S1, S2 and S3 as superficial (SZ), middle (MZ) and deep (DZ) zone chondrocytes, respectively. Implantation of bilayered zonal chondrocytes resulted in better cartilage tissue regeneration in a rat osteochondral defect model than FT control group, with predominantly Type II hyaline cartilage tissue and significantly lower Type I collagen. Dynamic microcarrier expansion of sorted zonal chondrocytes was able to retain the zonal cell size difference that correlate to zonal phenotype, while maintaining the rounded chondrocyte morphology and F-actin distribution similar to that in mature articular cartilage. With the better retention of zonal cell size and zonal phenotype relation on microcarrier, zonal cells separation was achievable in the sort-after-expansion strategy with cells expanded on microcarrier, in comparison to cells expanded on TCP.

Inertial spiral microchannel device provides a label-free and high throughput method to separate zonal chondrocytes based on cell size. Stratified implantation of zonal chondrocytes has the potential to improve articular cartilage regeneration. Dynamic microcarrier culture allows for size-based zonal chondrocyte separation to be performed on expanded chondrocytes, thus overcoming the challenge of limited tissue availability from the patients. Our novel zonal chondrocyte isolation and expansion protocol provide a translatable strategy for stratified zonal chondrocyte implantation that could improve articular cartilage regeneration of critical size defects.