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.
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.
The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains of Escherichia coli use this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in the periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 -strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. IMPORTANCEPili are critical virulence factors for many bacterial pathogens. Uropathogenic E. coli relies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points for therapeutic intervention. Pilus biogenesis is a multistep process. This work investigates the oligomeric state of the pilus chaperone in the periplasm, which is important for understanding early assembly events. Our work unambiguously demonstrates that both PapD and FimC chaperones are monomeric in solution. We further demonstrate that the solution behavior of the FimH and PapGII adhesins differ, which may be related to functional differences between the two pilus systems.A n important step in the pathogenesis of uropathogenic Escherichia coli is the binding to host receptors within the urinary tract. This is accomplished by the recognition of sugar moieties on the surface of urothelial cells by lectins at the tip of elongated organelles, termed pili or fimbriae (1, 2). Specifically, uropathogenic E. coli expresses type 1 and P pili, which mediate binding to the bladder and kidney, respectively (3, 4). Type 1 and P pili are assembled by the chaperone/usher pathway, which is a conserved secretion system used to assemble a wide variety of pilus-type struct...
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