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 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.

This study investigated the coculture of chondrocytes and mesenchymal stem cells (MSCs) on electrospun fibrous polymer scaffolds to produce polymer/extracellular matrix (ECM) hybrid constructs with the objective of reducing the number of chondrocytes necessary to produce ample cartilage-like ECM within the scaffolds. To generate these hybrid constructs, electrospun poly(e-caprolactone) fibrous scaffolds were seeded at both high and low initial densities with five different ratios of chondrocytes to MSCs: 1:0, 1:1, 1:3, 1:5, and 0:1, and cultured for 7, 14, and 21 days. Glycosaminoglycan production and distribution within the three coculture groups was similar to quantities generated by chondrocyte-only controls. Conversely, as the concentration of chondrocytes was increased, the collagen content of the constructs also increased at each time point, with a 1:1 chondrocyte to MSC ratio approximating the collagen production of chondrocytes alone. Histological staining suggested that cocultured constructs mimicked the well-distributed ECM patterns of chondrocyte generated constructs, while improving greatly over the restricted distribution of matrix within MSC-only constructs. These results support the capacity of cocultures of chondrocytes and MSCs to generate cartilaginous matrix within a polymeric scaffold. Further, the inclusion of MSCs in these cocultures enables the reduction of chondrocytes needed to produce cell-generated ECM.

Recently, scaffolds for tissue regeneration purposes have looked 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 discreet materials, specifically fibrin and poly(ε-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 in 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.

This work examined the chondrogenic potential of chondrocyte and mesenchymal stem cell (MSC) coculture generated poly(ɛ-caprolactone) (PCL)/extracellular matrix (ECM) hybrid scaffolds. Five different ratios of chondrocytes and MSCs were cocultured to generate cartilage-like ECM within electrospun fibrous scaffolds for 7, 14, and 21 days. These constructs were then devitalized to isolate the chondrogenic effects of the ECM alone. Devitalization was successful at removing cellular matter from the scaffolds, yet did reduce the amount of matrix present in the scaffolds. Following devitalization, the PCL/ECM scaffolds were then cultured with MSCs in serum-free conditions with or without TGF-β3 treatment for 21 days. TGF-β3 supplemented culture caused an induction of chondrogenesis in each scaffold type, but also somewhat masked the subtle differences of the different ECM coatings. Without TGF-β3, the cartilaginous matrix generated by 1:1 cocultures of chondrocytes to MSCs for 14 days supported similar chondrogenic gene expression patterns of MSCs cultured on scaffolds generated by chondrocytes alone. These scaffold formulations had a positive chondrogenic effect on aggrecan, collagen type II, and collagen II/I expression when compared to PCL controls. This study demonstrates that it is possible to utilize cocultures of chondrocytes and MSCs to coat a polymer scaffold with cartilage-like ECM capable of supporting chondrogenic differentiation of MSCs.

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