Full-thickness skin wounds are common and could be a heavy physical and economic burden. With the development of three dimensional (3D) printing technology, skin-like constructs have been fabricated for skin wound healing and regeneration. Although the 3D printed skin has great potential and enormous advantages before vascular networks can be well-constructed, living cells are not recommended for 3D skin printing for in vivo applications. Herein, we designed and printed a bilayer membrane (BLM) scaffold consisting of an outer poly (lactic-co-glycolic acid) (PLGA) membrane and a lower alginate hydrogel layer, which respectively mimicked the skin epidermis and dermis. The multi-porous alginate hydrogel of the BLM scaffolds promoted cell adhesion and proliferation in vitro, while the PLGA membrane prevented bacterial invasion and maintained the moisture content of the hydrogel. Skin regeneration using the bilayer scaffold was compared with that of PLGA, alginate hydrogel and the untreated defect in vivo. Tissue samples were analyzed using histopathological and immunohistochemical staining of CD31. In addition, mRNA expression levels of collagen markers [collagen type 1 alpha 1 (COL1a1) and collagen type 3 alpha 1 (COL3a1)] and inflammatory markers [interleukin-1β (IL-1β), as well as tumor necrosis factor (TNF-α)] were measured. Conclusively, the application of BLM scaffold resulted in highest levels of best skin regeneration by increasing neovascularization and boosting collagen I/III deposition. Taken together, the 3D-printed BLM scaffolds can promote wound healing, and are highly suitable for a wide range of applications as wound dressings or skin substitutes.
Peripheral naive CD4(+) T cells selectively differentiate to type 1 T(h), type 2 T(h) and IL-17-producing T(h) (T(h)17) cells, depending on the priming conditions. Since these subsets develop antagonistically to each other to elicit subset-specific adaptive immune responses, balance between these subsets can regulate the susceptibility to diverse immune diseases. The present study was undertaken to determine whether poly-gamma-glutamic acid (gamma-PGA), an edible and safe exopolymer that is generated by microorganisms such as Bacillus subtilis, could modulate the development pathways of T(h) subsets. The presence of gamma-PGA during priming promoted the development of T(h)1 and T(h)17 cells but inhibited development of T(h)2 cells. gamma-PGA up-regulated the expression of T-bet and ROR-gammat, the master genes of T(h)1 and T(h)17 cells, respectively, whereas down-regulating the level of GATA-3, the master gene of T(h)2 cells. gamma-PGA induced the expression of IL-12p40, CD80 and CD86 in dendritic cells (DC) and macrophages in a Toll-like receptor-4-dependent manner, and the effect of gamma-PGA on T(h)1/T(h)2 development was dependent on the presence of antigen-presenting cells (APC). Furthermore, gamma-PGA-stimulated DC favored the polarization of naive CD4(+) T cells toward T(h)1 cells rather than T(h)2 cells. In contrast, gamma-PGA affected T(h)17 cell development, regardless of the presence or absence of APC. Thus, these data demonstrate that gamma-PGA has the potential to regulate the development pathways of naive CD4(+) T cells through APC-dependent and -independent mechanisms and to be applicable to treating T(h)2-dominated diseases.
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