Direct and indirect co-culture of chondrocytes and mesenchymal stem cells for the generation of polymer/extracellular matrix hybrid constructs

In this work, the influence of direct cell–cell contact in co-cultures of mesenchymal stem cells (MSCs) and chondrocytes for the improved deposition of cartilage-like extracellular matrix (ECM) within nonwoven fibrous poly(∊-caprolactone) (PCL) scaffolds was examined. To this end, chondrocytes and MSCs were either co-cultured in direct contact by mixing on a single PCL scaffold or produced via indirect co-culture, whereby the two cell types were seeded on separate scaffolds which were then cultured together in the same system either statically or under media perfusion in a bioreactor. In static cultures, the chondrocyte scaffold of an indirectly co-cultured group generated significantly greater amounts of glycosaminoglycan and collagen than the direct co-culture group initially seeded with the same number of chondrocytes. Furthermore, improved ECM production was linked to greater cellular proliferation and distribution throughout the scaffold in static culture. In perfusion cultures, flow had a significant effect on the proliferation of the chondrocytes. The ECM contents within the chondrocyte-containing scaffolds of the indirect co-culture groups either approximated or surpassed the amounts generated within the direct co-culture group. Additionally, within bioreactor culture there were indications that chondrocytes had an influence on the chondrogenesis of MSCs as evidenced by increases in cartilaginous ECM synthetic capacity. This work demonstrates that it is possible to generate PCL/ECM hybrid scaffolds for cartilage regeneration by utilizing the factors secreted by two different cell types, chondrocytes and MSCs, even in the absence of juxtacrine signaling

Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration

Recently, scaffolds for tissue regeneration purposes have been observed to utilize nanoscale features in an effort to reap the cellular benefits of scaffold features resembling extracellular matrix (ECM) components. However, one complication surrounding electrospun nanofibers is limited cellular infiltration. One method to ameliorate this negative effect is by incorporating nanofibers into microfibrous scaffolds. This study shows that it is feasible to fabricate electrospun scaffolds containing two differently scaled fibers interspersed evenly throughout the entire construct as well as scaffolds containing fibers composed of two discrete materials, specifically fibrin and poly(?-caprolactone). In order to accomplish this, multiscale fibrous scaffolds of different compositions were generated using a dual extrusion electrospinning setup with a rotating mandrel. These scaffolds were then characterized for fiber diameter, porosity and pore size and seeded with human mesenchymal stem cells to assess the influence of scaffold architecture and composition on cellular responses as determined by cellularity, histology and glycosaminoglycan (GAG) content. Analysis revealed that nanofibers within a microfiber mesh function to maintain scaffold cellularity under serum-free conditions as well as aid the deposition of GAGs. This supports the hypothesis that scaffolds with constituents more closely resembling native ECM components may be beneficial for cartilage regeneration