Figure 1.
The schematic representation showing fabrication of sericin matrices from “Sericin hope” silkworm cocoons and co-culture system (A).
Keratinocytes-fibroblast coculture was done by seeding fibroblasts on sericin matrices pre-soaked in DMEM medium at day 0. On day 2, keratinocytes were directly seeded on the surface of sericin matrices containing fibroblasts. The cells attached to the surface of matrices and proliferated over time to cover the surface of the entire sericin matrices. Optical microscope images (B); of (a) human dermal fibroblasts and, (b) human keratinocytes cells used in the experiments. Scale bars = 100 micrometer.
Figure 2.
Scanning electron micrograph of surface of 2% (a) sericin, (b) chitosan matrices and (c) average pore size of sericin and chitosan matrices.
The magnification bar represents 100 micrometer.
Figure 3.
FTIR spectra of sericin porous matrices (A).
Non-crosslinked (a) and genipin (b) crosslinked sericin matrices; Mechanical properties (B); and swelling ratio (C) of sericin and chitosan porous matrices in water at 37 °C. Error bars represent the standard error of the mean (n=3).
Figure 4.
Percentage degradation in terms of weight loss of sericin crosslinked and uncrosslinked matrices in phosphate buffer saline pH 7.4 solution and in lysozyme solution at 37 °C (n= 3, Mean ±standard deviation, *p< 0.05).
Figure 5.
Alamar blue assay of co-cultured cells on sericin and chitosan porous matrices (n= 3, Mean ±standard deviation).
Figure 6.
The viability of co cultured cells on sericin and chitosan porous matrices using Live/Dead assay with confocal microscopy of day 7, 14, 21 and 28 culture.
Live cells produced green fluorescence and dead cells showed red fluorescence. Scale bars are 50 micrometer.
Figure 7.
Growth and proliferation of human keratinocytes and fibroblasts on sericin and chitosan matrices observed under confocal micrographs for day 1, 7, 14, 21 and 28 days.
Scale bars = 100 micrometer. Chitosan and sericin matrices show red and green auto fluorescence respectively in lasers used in confocal microscope.
Figure 8.
Three dimensional confocal images of fibroblasts and keratinocytes co-cultured on (a) sericin and (b) chitosan matrices showing two different cell types distribution over the matrices at day 14.
Figure 9.
Figure represents morphology of seeded cells observed by confocal microscope and SEM on sericin matrices (a; c) keratinocytes, (b; d) fibroblasts.
Figure 10.
The in vitro release profile of the growth factors TGF beta, b-FGF and cytokine IL-8 by cocultured cells, fibroblasts and keratinocytes monoculture cells seeded on sericin matrices for 72 h, essential for epidermal morphogenesis.
Figure 11.
H & E stained cross-sections of co-culture constructs composed of epidermal and dermal layers formed by culturing fibroblasts and keratinocytes on the constructs for 7, 14, 21 and 28 days.
White broken line outlines the border between epidermis and dermis (scale bar =100 micrometer).
Figure 12.
Expression of fibroblast surface protein, involucrin and collagen type IV in co-cultured sericin matrices.
Immunohistochemical staining performed on deparaffinized sections using the HRP-conjugated secondary antibody and visualized using DAB as substrate. The antibodies used were anti-type IV collagen, mouse monoclonal anti-involucrin and mouse monoclonal anti fibroblast surface protein. Pictures were taken after 14, 21 and 28 days of culture at the air–liquid interface (scale bar = 100 µm).
Figure 13.
Production of TNF- α (A), IL-1β (B) and of nitric oxide (C) by RAW 264.7 murine macrophages on sericin and chitosan matrices with tissue culture plates (TCP as negative control) for day 1 and day 3.
The TNF α, IL-1beta and nitric oxide production by the cells estimated from a standard graph.