Coculture between mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) represents a promising strategy for cartilage regeneration. This study aimed at elaborating how ACs were regulated by MSCs. Rabbit ACs (rACs) and rabbit MSCs (rMSCs) were seeded separately in a Transwell system to initiate non-contact coculture in growth medium without chondrogenic factors. Cell morphology, cell proliferation, production of extracellular matrix (ECM), and gene expression of rACs were characterized. Upon coculture, rACs underwent a morphological transition from a rounded or polygonal shape into a fibroblast-like one and proliferation was provoked simultaneously. Such effects were dependent on the amount of rMSCs. Along with these changes, ECM production and gene expression of rACs were also perturbed. Importantly, when a ROCK inhibitor (Y27632) was supplemented to coculture, the effects except that on cell proliferation were inhibited, suggesting the involvement of RhoA/ROCK signaling. By applying an inhibitor (BIBF1120) of VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β in coculture, or supplementing FGF-1, VEGF-A and PDGFbb in monoculture, it was confirmed that the paracrine factors by rMSCs mediated the compounding effects on rACs. These findings shed light on MSCs-ACs interactions and might confer an insight view on cell-based cartilage regeneration.
Proliferation stimulation of rACs by rMSCs was noted in coculture and the number of rACs within 6 days at the presence of the highest density of rMSCs was almost twice as that in monoculture. MSCs are known to secrete a plethora of trophic factors including FGF, VEGF and PDGF36,37,38. In the present study, by adding an inhibitor of VEGFR1/2/3, FGFR1/2/3 and PDGFRα/β, BIBF1120, proliferation of rACs was drastically inhibited in coculture. At the same time, cell morphology, GAG production and gene expression in coculture were all reverted to those as in monoculture, implicating the critical roles of these growth factors in mediating interactions between rMSCs and rACs. This observation was in contrast to a study by Wu et al., wherein MSCs stimulated both proliferation and GAG production of chondrocytes in a pellet coculture with cell-cell contacts21,39. However, in our previous study, while MSCs had been shown to downregulate both cartilaginous ECM production and gene expression of ACs encapsulated in alginate gel beads in non-contact coculture, stimulation of proliferation was not observed26. Moreover, Lee et al. reported that adipose-derived MSCs could downregulate the differentiated phenotype of costochondral chondrocytes by secreting VEGF-A and FGF-2 in a similar coculture setting and a reduction in chondrocyte proliferation in coculture was noted25. This was possibly due to variations in the secretome of MSCs derived from different tissue origins (bone marrow versus adipose tissue). To further confirm our finding, FGF-1, VEGF-A and PDGFbb were supplemented in individual or in combination in monoculture of rACs. Growth factors including TGF-β1, FGF-2 and PDGFbb had previously been applied to stimulate proliferation of ACs during monolayer expansion40. It should be pointed out that the three tested growth factors (FGF-1, VEGF-A and PDGFbb) exerted differential effects on rACs. FGF-1, PDGFbb and FGF-1&VEGF-A&PDGFbb, but not VEGF-A or FGF-1&VEGF-A, promoted proliferation of rACs. These growth factors also displayed distinct effects on morphology, gene expression and GAG production of rACs. In fact, Lee et al. suggested the opposite effects of VEGF-A and FGF-2 on proliferation of costochondral chondrocytes, with VEGF-A showing the inhibitory effect25. Previously, Wu et al. reported that MSCs provoked proliferation of chondrocytes in a pellet coculture possibly through FGF-1 signaling41. In the present study, when a specific FGFR-1 inhibitor (PD173046) was added to coculture, changes in both proliferation and morphology of rACs induced by rMSCs were inhibited, confirming the significant role of FGF signaling. However, taken into consideration of all aspects including the morphology, GAG production, gene expression as well as proliferation of rACs, only a combination of FGF-1, VEGF-A and PDGFbb (FGF-1&VEGF-A&PDGFbb) could essentially recapitulate the compounding effects of rMSCs on rACs.
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One of the challenges in applying ACs for cartilage regeneration is dedifferentiation and coculture between MSCs and ACs has been expected to overcome this issue5. Dedifferentiated chondrocytes display a fibroblast-like morphology, reduced ECM synthesis, downregulated chondrocytic gene expression (e.g. Col2a1) and upregulated fibroblastic gene expression (e.g. Col1a1) as well as an altered cell surface antigen profile2,50. In coculture with rMSCs, by displaying a fibroblast-like shape, downregulated chondrocytic gene expression (Acan, Col2a1 and Sox9), upregulated fibroblastic gene expression (Vcan and Col1a1), and reduced cartilaginous ECM production (GAG and type II collagen), rACs seemed to undergo an expedited dedifferentiation process compared to those in control monoculture. In literature, to evaluate the differentiated phenotype of chondrocytes, gene expression ratios of Col2a1/Col1a1 and Acan/Vcan are considered as convenient quantitative indices48,51 and recently, a high ratio of CD14/CD90 (i.e., Thy1) has also been correlated with human chondrocyte differentiation50,52. We had previously confirmed that dedifferentiation of rACs was associated with upregulation of CD90 and a steady level of CD14 expression26. Strikingly, upon coculture with rMSCs, upregulation of CD14 and downregulation of CD90 were consistently observed, suggesting that coculturing rMSCs with rACs might have not provoked a bona fide chondrocyte dedifferentiation process. Monolayer culture of ACs has been speculated to be a selective expansion of progenitor cells52. However, it seemed not to be the case in coculture in the present study, since gene expression of CD90, a well-known surface molecular marker for MSCs, was downregulated in rACs upon coculture. Nevertheless, considering that dedifferentiated chondrocytes might share a similar pattern of surface markers with those OA chondrocytes52, changes of rACs in coculture with rMSCs distinct from dedifferentiation might be considered beneficial when applying these cells in cartilage repair. As a matter of fact, ACs expanded in monolayer at the presence of growth factors have been demonstrated to have an improved redifferentiation potential during subsequent chondrogenic induction40. In this sense, chondrocytes can be conditioned in coculture with MSCs and possibly rejuvenated with a better chondrogenic potential for subsequent cartilage regeneration applications.
We carried out three different experiments to investigate the parasitism hypothesis. First, we used commercial McPhail traps to test for the potential attraction effect of nestling feces alone on flying insects. We found that traps with fecal sacs attracted significantly more flies (Order Diptera), but not ectoparasites, than the two control situations. Second, we used artificial blackbird (Turdus merula) nests to investigate the combined attraction effect of feces and nest materials on arthropods (not only flying insects). Flies, again, were the only group of arthropods significantly attracted by fecal sacs. We did not detect an effect on ectoparasites. Third, we used active blackbird nests to investigate the potential effect of nestling feces in ecto- and endoparasite loads in real nestlings. The presence of fecal sacs near blackbird nestlings did not increase the number of louse flies or chewing lice, and unexpectedly reduced the number of nests infested with mites. The endoparasite prevalence was also not affected. In contrast, feces provoked an activation of the immune system as the H/L ratio of nestlings living near excrements was significantly higher than those kept under the two control treatments.
Includes unlimited streaming via the free Bandcamp app, plus high-quality downloads of Snowflake Supernova, Appraisal, Blind (lead the blind), Self-Control, Provoked by Motions, Legacy of K, From The Past, Constant Loop, and 3 more. , and , . Purchasable with gift card Buy Digital Discography $19.30 USD or more (40% OFF) Send as Gift about Second single from MORE, taken from the forthcoming album Sentimental Sounds. $(".tralbum-about").last().bcTruncate(TruncateProfile.get("tralbum_about"), "more", "less"); credits released May 22, 2020 license all rights reserved tags Tags electronic synth pop electro pop retrosynth synth pop synthpop Sweden Shopping cart total USD Check out about MORE Sweden
The publishing of the Prajateerpu report of a workshop in southern India provoked a huge debate amongst people involved in participatory practices throughout the world. This article reports some contributor's opinions on the use of citizens' juries in participatory research and problems with representation within the juries, as expressed in an e-forum after the publication of the report. 2ff7e9595c
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