Influence of the three‐dimensional culture of human bone marrow mesenchymal stromal cells within a macroporous polysaccharides scaffold on Pannexin 1 and Pannexin 3

Abstract

Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three-dimensional (3D) microenvironment provided by a polysaccharide-based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large-pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two-dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell-cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast-specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi-scid/IL-2Rγ ) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.

Mechanisms of pore formation in hydrogel scaffolds textured by freeze-drying

“…A homogenous seeding of MC3T3-E1 cells within the pore network was achieved by the simple addition of a cell suspension on freeze-dried scaffolds. Isolated cells of different origins (species, tissues), two or more cell types, or cell clusters can be accommodated in these 3D porous scaffolds [17][18][19]21]. Such scaffolds are suitable for cell culture in 3D (especially when cell clustering and spheroid growth is necessary for studying cell development and differentiation), for tissue engineering or for pharmacological assays in 3D.…”

“…In this work, we address the mechanisms of pore formation by freeze-drying in a polysaccharide-based hydrogel scaffold designed for bone tissue engineering [16]. This biomaterial is under research and industrial developments [17][18][19][20][21]. The scaffolds are manufactured according to Route 1 with a low cooling rate.…”

Pannexin biology and emerging linkages to cancer

“…Peptides that mimic regulatory sequences of pannexins have been widely considered as a selective means to block PANX1 channels [69]. The best known of these is 10 PANX1, which is considered a specific blocker of PANX1 and is widely used in both cell and organoid cultures [70]. With the recent arrival of high-resolution structural knowledge of PANX1 channels in different activation states, it should now be possible to creatively design and synthesize strategic peptides that best regulate PANX1 channels [71][72][73][74].…”

Development of a cell-free and growth factor-free hydrogel capable of inducing angiogenesis and innervation after subcutaneous implantation

“…The upregulation of some osteoblastic genes in VKAIV-containing hydrogels can be explained by the cellular organization as spheroids. Our group has already shown that spheroid organization can provide numerous cellular contacts, stimulating junctional proteins such as connexin 43 or pannexins that could drive the osteogenic differentiation of MSCs [57,58].…”