Journal: Gels

Article Title: Arginine-Biofunctionalized Ternary Hydrogel Scaffolds of Carboxymethyl Cellulose–Chitosan–Polyvinyl Alcohol to Deliver Cell Therapy for Wound Healing

doi: 10.3390/gels10110679

Figure Lengend Snippet: ( A ) An illustrative representation of a cross-section of the skin, indicating the layers (epidermis and dermis) that were resembled by the 3D hybrid hydrogel, depicted with information regarding its components, chemical formula, and structure. ( B ) Scheme of the co-culture strategy of the cell therapy product simulating the skin layers, with the deposition of keratinocytes in the membrane layer (resembling basement membrane of skin) and populating the porous layer with fibroblasts to obtain a cellular biomimetic dermal–epidermal skin substitute construct (not to scale).

Article Snippet: For keratinocytes, Gold Keratinocyte Growth Basal Medium supplemented with a KGM-Gold TM Keratinocyte Single Quots TM Kit (Lonza, Morristown, NJ, USA), referred to as “complete basal keratinocyte medium”, was used.

Techniques: Co-Culture Assay, Membrane, Construct

Journal: Gels

Article Title: Arginine-Biofunctionalized Ternary Hydrogel Scaffolds of Carboxymethyl Cellulose–Chitosan–Polyvinyl Alcohol to Deliver Cell Therapy for Wound Healing

doi: 10.3390/gels10110679

Figure Lengend Snippet: ( A ) Results of cell viability (MTT cytotoxicity test) and ( B ) cell proliferation (measured using resazurin protocol) biological assays for the porous three-dimensional structures (3d = 3 days). ( C ) Calcein AM (viable cells, green fluorescence, left side images, identified as LIVE) and ethidium homodimer (dead cells, red fluorescence, right side images, identified as DEAD) staining for keratinocyte and fibroblast monoculture cells deposited onto the lower and upper faces of the L-arginine functionalized 3D constructs, respectively (scale bar = 100 µm). Details: schematic representation of cell deposition (not to scale).

Article Snippet: For keratinocytes, Gold Keratinocyte Growth Basal Medium supplemented with a KGM-Gold TM Keratinocyte Single Quots TM Kit (Lonza, Morristown, NJ, USA), referred to as “complete basal keratinocyte medium”, was used.

Techniques: Fluorescence, Staining, Construct

Journal: Gels

Article Title: Arginine-Biofunctionalized Ternary Hydrogel Scaffolds of Carboxymethyl Cellulose–Chitosan–Polyvinyl Alcohol to Deliver Cell Therapy for Wound Healing

doi: 10.3390/gels10110679

Figure Lengend Snippet: Fluorescence microscopy images showing representative images of the 3D CMC:PVA:CHI_L-Arg structure, where a co-culture of ( C ) keratinocytes (upper face—membrane) and ( D , E ) fibroblasts (lower face—porous structure) were seeded compared to controls (( A ) Keratinocyte +Control and ( B ) Fibroblast +Control). Cells were labeled with calcein AM (viable cells, green fluorescence, left column, identified as LIVE) and ethidium homodimer (dead cells, red fluorescence, right column, identified as DEAD). Scale bar = 100 µm. Detail: schematic representation of cell therapy dermal–epidermal skin substitute (not to scale).

Article Snippet: For keratinocytes, Gold Keratinocyte Growth Basal Medium supplemented with a KGM-Gold TM Keratinocyte Single Quots TM Kit (Lonza, Morristown, NJ, USA), referred to as “complete basal keratinocyte medium”, was used.

Techniques: Fluorescence, Microscopy, Co-Culture Assay, Membrane, Control, Labeling

Journal: Gels

Article Title: Arginine-Biofunctionalized Ternary Hydrogel Scaffolds of Carboxymethyl Cellulose–Chitosan–Polyvinyl Alcohol to Deliver Cell Therapy for Wound Healing

doi: 10.3390/gels10110679

Figure Lengend Snippet: Histopathological analysis of skin lesions in Hairless mice, showing representative images of ( A ) skin from the control group (no hydrogel applied) and ( B ) skin from the group treated with the 3D bilayer construct containing fibroblasts and keratinocytes (CM group). Black asterisk: intact epidermis; red asterisk: denser collagen; green asterisk: looser collagen; black arrow: reactive fibroblasts; red arrow: blood capillaries. The larger image displays the lesion area at 20× magnification, with a zoomed-in view of the highlighted area at 40× magnification.

Article Snippet: For keratinocytes, Gold Keratinocyte Growth Basal Medium supplemented with a KGM-Gold TM Keratinocyte Single Quots TM Kit (Lonza, Morristown, NJ, USA), referred to as “complete basal keratinocyte medium”, was used.

Techniques: Control, Construct

Journal: Biological Research

Article Title: H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

doi: 10.1186/s40659-024-00541-x

Figure Lengend Snippet: Downregulation of H2 antigen by FUT1 knockdown increases desmosome size and expression of desmosomal proteins. A Primary keratinocytes were transfected with NC or FUT1 (#2) siRNA cocktails. Cell sheets obtained from dispase treatment were processed for the transmission electron microscopic examination. More than 50 images per group were used to measure desmosome size using Image J software. Scale bar = 500 nm. Data were depicted as mean desmosome size (nm) ± SD. * p < 0.05, vs. NC siRNA-treated control group. B Primary keratinocytes were transfected with NC or FUT1 (#1 or #2) siRNA cocktails. The levels of desmosome assembly proteins such as plakophilin, desmoplakin, desmocollin, and desmogleins with high calcium treatment (2 mM CaCl 2 ) were investigated through Western blot. α-tubulin was used as a loading control. Relative band intensity was presented as mean fold ± SD to NC siRNA-treated control group. * p < 0.05, *** p < 0.001 vs. NC siRNA-treated control group

Article Snippet: Human primary epidermal keratinocytes were derived from foreskin tissue as described previously [ ] and cultured in a keratinocyte growth medium (calcium concentration 0.1 mM) (KGM, KBMTM GoldTM Basal Medium with KGMTM GoldTM SingleQuotsTM supplements, Lonza Bioscience, Durham, NC, USA).

Techniques: Knockdown, Expressing, Transfection, Transmission Assay, Software, Control, Western Blot

Journal: Biological Research

Article Title: H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

doi: 10.1186/s40659-024-00541-x

Figure Lengend Snippet: Downregulation of H2 antigen by FUT1 siRNA transfection increases the expression of pro-filaggrin and loricrin. A Primary keratinocytes were transfected with NC or FUT1 (#1 or #2) siRNA cocktails. The keratinocyte differentiation markers, including pro-filaggrin and loricrin, were investigated after four days of calcium treatment through Western blot ( n = 5). α-tubulin was used as a loading control. B Relative band intensity was shown as mean fold ± SD to NC siRNA-treated control group. * p < 0.05, ** p < 0.01 vs. NC siRNA-treated control group

Article Snippet: Human primary epidermal keratinocytes were derived from foreskin tissue as described previously [ ] and cultured in a keratinocyte growth medium (calcium concentration 0.1 mM) (KGM, KBMTM GoldTM Basal Medium with KGMTM GoldTM SingleQuotsTM supplements, Lonza Bioscience, Durham, NC, USA).

Techniques: Transfection, Expressing, Western Blot, Control

Journal: Biological Research

Article Title: H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

doi: 10.1186/s40659-024-00541-x

Figure Lengend Snippet: Treatment with an EGFR inhibitor, gefitinib, increases pro-filaggrin, loricrin, and desmosomal proteins. A Primary keratinocytes were treated with indicated doses of an EGFR inhibitor, gefitinib (0, 20, or 100nM). The level of proteins for keratinocyte differentiation markers and desmosome assembly following 4-day-calcium treatment were investigated through Western blot ( n = 5 ∼ 6). β-actin was used as a loading control. B Relative band intensity was demonstrated as mean fold ± standard error of the mean (SEM) to NC siRNA-treated control group. * p < 0.05, ** p < 0.01 vs. NC siRNA-treated control group

Article Snippet: Human primary epidermal keratinocytes were derived from foreskin tissue as described previously [ ] and cultured in a keratinocyte growth medium (calcium concentration 0.1 mM) (KGM, KBMTM GoldTM Basal Medium with KGMTM GoldTM SingleQuotsTM supplements, Lonza Bioscience, Durham, NC, USA).

Techniques: Western Blot, Control

Journal: Biological Research

Article Title: H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

doi: 10.1186/s40659-024-00541-x

Figure Lengend Snippet: FUT1 knockdown reduces EGFR ligands rather than directly regulating EGF receptors. A Primary keratinocytes were incubated with 2 mM CaCl 2 for 2 days, and the cell lysates were harvested for immunoprecipitation (IP). IP was performed with antibodies against epidermal growth factor receptor (EGFR), H2 antigen, or mouse immunoglobulin (IgG). The glycosylation of H2 antigen on EGFR was confirmed through Western blot analysis.​ B Primary keratinocytes were transfected with NC or FUT1 (#1) siRNA cocktails, and the phosphorylation of EGFR (Y1068) induced by treatment with EGF (0 or 20 ng/ml) was investigated through Western blot. EGF was treated in keratinocyte basal medium (KBM) for 5 min, with 24-hour pre-incubation in KBM before treatment. α-tubulin was used as a loading control. C , D Primary keratinocytes were transfected with NC or FUT1 (#1) siRNA cocktails. The levels of mRNA expression C and protein secretion into conditioned medium D of EGFR ligand proteins following high calcium treatment for the indicated days were investigated using real-time qRT-PCR and ELISA ( n = 5). The relative mRNA expression or secretion of EGFR ligands was presented as mean value (fold or %, respectively) ± SD to NC siRNA-treated control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. NC siRNA-treated control group

Article Snippet: Human primary epidermal keratinocytes were derived from foreskin tissue as described previously [ ] and cultured in a keratinocyte growth medium (calcium concentration 0.1 mM) (KGM, KBMTM GoldTM Basal Medium with KGMTM GoldTM SingleQuotsTM supplements, Lonza Bioscience, Durham, NC, USA).

Techniques: Knockdown, Incubation, Immunoprecipitation, Glycoproteomics, Western Blot, Transfection, Phospho-proteomics, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Biological Research

Article Title: H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

doi: 10.1186/s40659-024-00541-x

Figure Lengend Snippet: Enhanced expression of H2 antigen by FUT1 overexpression reverses the effects shown in FUT1 knockdown. A Primary keratinocytes were infected with 10 multiplicity of infection (MOI) of a control adenovirus vector encoding GFP gene (Ad-CNT) or recombinant adenovirus vector encoding human FUT1 gene (Ad-FUT1) in a high-calcium growth medium. The upregulation of the FUT1 protein and its product, H2 antigen, were investigated through Western blot. Changes in proteins for keratinocyte differentiation and desmosome assembly were investigated four days after the infection through Western blot. α-tubulin was used as a loading control. B Relative band intensity was shown as mean fold ± SD to the Ad-CNT-infected control group. * p < 0.05, *** p < 0.001 vs. Ad-CNT-infected control group

Article Snippet: Human primary epidermal keratinocytes were derived from foreskin tissue as described previously [ ] and cultured in a keratinocyte growth medium (calcium concentration 0.1 mM) (KGM, KBMTM GoldTM Basal Medium with KGMTM GoldTM SingleQuotsTM supplements, Lonza Bioscience, Durham, NC, USA).

Techniques: Expressing, Over Expression, Knockdown, Infection, Control, Plasmid Preparation, Recombinant, Western Blot

Journal: Biological Research

Article Title: H Antigen expression modulates epidermal Keratinocyte Integrity and differentiation

doi: 10.1186/s40659-024-00541-x

Figure Lengend Snippet: Overexpression of FUT1 increases both mRNA and protein levels of EGFR ligands. Primary keratinocytes were infected with 10 MOI of a control adenovirus vector encoding GFP gene (Ad-CNT) or recombinant adenovirus vector encoding human FUT1 gene (Ad-FUT1) in a high-calcium growth medium. The levels of mRNA expression A and protein secretion into conditioned medium B of EGFR ligand proteins with high calcium treatment for the indicated days were investigated using real-time qRT-PCR and ELISA ( n = 5). Relative mRNA expression or secretion of EGFR ligands was shown as mean value (fold or %, respectively) ± SD to the Ad-CNT-infected control group. ** p < 0.01, *** p < 0.001 vs. Ad-CNT-infected control group

Article Snippet: Human primary epidermal keratinocytes were derived from foreskin tissue as described previously [ ] and cultured in a keratinocyte growth medium (calcium concentration 0.1 mM) (KGM, KBMTM GoldTM Basal Medium with KGMTM GoldTM SingleQuotsTM supplements, Lonza Bioscience, Durham, NC, USA).

Techniques: Over Expression, Infection, Control, Plasmid Preparation, Recombinant, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay