Engineering Superficial Zone Chondrocytes from Mesenchymal Stem Cells

Abstract

Recent cartilage engineering efforts have focused on development of zonally organized tissue. However, there remains a need for protocols that differentiate progenitor populations into chondrocytes of zonal phenotype. Here, we evaluate the potential of coculture of bovine mesenchymal stem cells (MSCs) and zonal explants of bovine cartilage tissue to drive MSC differentiation to chondrocytes with the superficial zone phenotype. Two coculture systems were set up: one between alginate encapsulated MSCs and superficial zone cartilage explants, and one between MSCs and middle/deep zone cartilage explants. Chondrogenic and superficial zone markers were monitored over a 21-day differentiation period via gene and protein expression. A control conditioned media study was used to determine the impact of communication via soluble factors between cell populations during differentiation. At day 21, results show superficial zone explant coculture without transforming-growth factor b3 supplementation induces upregulation of chondrogenic gene expression markers SOX9 and type II collagen 3.4-fold and 11.4-fold, respectively, over standard chondrogenic control media. Further, coculture of MSCs and superficial zone explants can be used to upregulate mRNA expression of the superficial zone marker proteoglycan-4 in MSCs (1.75-fold over chondrogenic control at day 21), indicating the superficial zone chondrocyte phenotype. Gene expression data show middle/deep zone explant and MSC coculture did not induce the chondrogenesis observed in superficial zone explant coculture. Likewise, poor chondrogenesis was observed in all conditioned media groups. Results highlight the importance of superficial zone cartilage and cells in guiding stem cell fate and regulating differentiation of MSCs to chondrocytes of the superficial zone type.

A developmentally inspired combined mechanical and biochemical signaling approach on zonal lineage commitment of mesenchymal stem cells in articular cartilage regeneration

“…Remarkably, the middle/deep zone explants did not induce expression of chondrogenic markers in co-cultured MSCs. 73 The results of Coates et al highlight the importance of growth factors secreted in the superficial zone on chondrogenesis whereas our results highlight the importance of BMP-7 in combination with TGF-β1 for directing differentiation of hMSCs to the superficial zone phenotype. Another factor in our experiments that may have contributed to the high SZP expression (21 fold increase) in Superficial gel was the relatively high cell density of hMSCs (60×10 6 cells/mL) consistent with that in the native cartilage tissue.…”

“…22, 72 To engineer superficial zone chondrocytes, Coates co-cultured alginate-encapsulated MSCs with explants from the superficial or middle/deep zone of bovine articular cartilage. 73 Co-cultures with the superficial zone explants without the addition of TGF-β3 to the medium resulted in up-regulation of chondrogenic markers Sox-9 and Col II and modest increase in SZP in the encapsulated MSCs after 21 days compared to standard chondrogenic medium. Remarkably, the middle/deep zone explants did not induce expression of chondrogenic markers in co-cultured MSCs.…”

Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor

“…Such uniaxial culturing system would be immensely useful in regenerative medicine applications where a co‐culture of interdependent, yet distinct cell populations is necessary. Such organizational structure is seen in the three defined zonal populations that make up cartilage (Coates and Fisher, ) or in the coupled interaction of smooth muscle cells and endothelial cells during angiogenesis and vascularization (Williams and Wick, ), where a co‐culture system of cells interact via paracrine signaling is vital. Further studies investigating the exact molecular and paracrine signals at play will allow for a complete understanding of such system.…”

Stem Cells in Skeletal Tissue Engineering: Technologies and Models

“…For example, one such study showed that co-culture of MSCs with the superficial zone cartilage explants enhanced Sox9 and Col2a1 expression along with expression of the superficial zone marker lubricin (Prg4) (235). Interestingly, building on knowledge gained from analysis of gene expression events during mesenchymal condensation and chondrogenesis during embryonic development, a recent study reported the construction of large, mechanically functional cartilage constructs from fusion of condensed mesenchymal cell bodies (236).…”