Creation of functional skin substitutes within a clinically acceptable time window is essential for timely repair and management of large wounds such as extensive burns. The aim of this study was to investigate the possibility of fabricating skin substitutes via a bottom-up nanofiber-enabled cell assembly approach and using such substitutes for full-thickness wound repair in nude mice. Following a layer-by-layer (L-b-L) manner, human primary skin cells (fibroblasts and keratinocytes) were rapidly assembled together with electrospun polycaprolactone (PCL)/collagen (3:1 w/w, 8% w/v) nanofibers into 3D constructs, in which fibroblasts and keratinocytes were located in the bottom and upper portion respectively. Following culture, the constructs developed into a skin-like structure with expression of basal keratinocyte markers and deposition of new matrix while exhibiting good mechanical strength (as high as 4.0 MPa by 14 days). Treatment of the full-thickness wounds created on the back of nude mice with various grafts (acellular nanofiber meshes, dermal substitutes, skin substitutes and autografts) revealed that 14-day-cultured skin substitutes facilitated a rapid wound closure with complete epithelialization comparable to autografts. Taken together, skin-like substitutes can be formed by L-b-L assembling human skin cells and biomimetic nanofibers and they are effective to heal acute full-thickness wounds in nude mice.

The repair and management of full-thickness skin defects resulting from burns and chronic wounds remain a significant unmet clinical challenge. For those skin defects exceeding 50%-60% of total body surface area, it is impractical to treat with autologous skin transplants because of the shortage of donor sites. The possibility of using tissue-engineered skin grafts for full-thickness wound repair is a promising approach. The primary goal of tissue-engineered skin grafts is to restore lost barrier function, but regeneration of appendages, such as hair follicles, has to be yet achieved. The successful regeneration of hair follicles in immunodeficient mice suggests that creating human hair follicles in tissue-engineered skin grafts is feasible. However, many limitations still need to be explored, particularly enriching isolated cells with trichogenic capacity, maintaining this ability during processing, and providing the cells with proper environmental cues. Current advances in hair follicle regeneration, in vitro and in vivo, are concisely summarized in this report, and key requirements to bioengineer a hair follicle are proposed, with emphasis on a three-dimensional approach.

While achieving the spatial organization of cells within 3D assembled nanofiber/cell constructs via nanofiber-enabled cell layering, the small sizes of inter-fiber pores of the electrospun nanofiber mats could significantly limit cell penetration across the layers for rapid formation of an integrated tissue construct. To address this challenge, efforts were made to improve cellinfiltration of electrospun nanofiber mats by modulating the density distribution and spatial organization of the fibers during electrospinning. Collection of collagen-containing electrospun nanofibers (300-600 nm in diameter) onto the surface of a stainless steel metal mesh (1 mm × 1 mm in mesh size) led to the periodic alternation of fiber density from densely packed to loosely arranged distribution within the same mat, in which the densely packed fibers maintained the structural integrity while the region of loose fibers allowed for cell penetration. Along with improved cell infiltration, the distinct fiber organization between dense and loose fiber regions also induced different morphology of fibroblasts (stellate vs. elongated spindle-like). Assembly of cell-seeded nanofiber sheets into 3D constructs with such periodically organized nanofiber mats further demonstrated their advantages in improving cell penetration across layers in comparison to either random or aligned nanofiber mats. Taken together, modulation of nanofiber density to enlarge the pore size is effective to improve cell infiltration through electrospun mats for better tissue formation.

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To investigate the seroprevalence of Herpes Simplex Viruses (HSV1 and HSV2), Ebstein-Barr Virus (EBV) and Hepatitis B Virus (HBV) in children with acute lymphoblastic leukemia (ALL) in southern Iran. 90 patients with ALL and 90 age-sex matched healthy participants were enrolled in this study. Antibodies (IgG) against HBsAg, HSV1, HSV2, EBV different antigens including Epstein-Barr nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA) and early antigen (EA) were measured by enzyme-linked immunosorbent assay (ELISA). There were 54 (60%) male and 36 (40%) female in both study groups with mean age of 8.47 ± 3.61 and 8.61 ± 2.84 years in case and control group respectively (P = 0.812). The prevalence of antibodies against HBsAg (P = 0.002), HSV1 (P < 0.0001), VCA (P = 0.021) and EA (P < 0.0001) antigens of EBV were significantly higher in ALL patients. With the results of this study, we could not exclude a connection between these viral infections and later leukemogenesis in childhood ALL, although nor latent infection nor congenital infection cannot be excluded by this method.