Collagen VII Expression Is Required in Both Keratinocytes and Fibroblasts for Anchoring Fibril Formation in Bilayer Engineered Skin Substitutes

The blistering disease recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding collagen VII (COL7), which forms anchoring fibrils that attach the epidermis to the dermis. Cutaneous gene therapy to restore COL7 expression in RDEB patient cells has been proposed, and cultured epithelial autograft containing COL7-modified keratinocytes was previously tested in clinical trials. Because COL7 in normal skin is expressed in both fibroblasts and keratinocytes, cutaneous gene therapy using a bilayer skin substitute may enable faster restoration of anchoring fibrils. Hypothetically, COL7 expression in either dermal fibroblasts or epidermal keratinocytes might be sufficient for functional anchoring fibril formation in a bilayer skin substitute. To test this, engineered skin substitutes (ESS) were prepared using four combinations of normal + RDEB cells: (1) RDEB fibroblasts + RDEB keratinocytes; (2) RDEB fibroblasts + normal keratinocytes; (3) normal fibroblasts + RDEB keratinocytes; and (4) normal fibroblasts + normal keratinocytes. ESS were incubated in vitro for 2 weeks prior to grafting to full-thickness wounds in immunodeficient mice. Biopsies were analyzed in vitro and at 1, 2, or 3 weeks after grafting. COL7 was undetectable in ESS prepared using all RDEB cells (group 1), and macroscopic blistering was observed by 2 weeks after grafting in ESS containing RDEB cells. COL7 was expressed, in vitro and in vivo, in ESS prepared using combinations of normal + RDEB cells (groups 2 and 3) or all normal cells (group 4). However, transmission electron microscopy revealed structurally normal anchoring fibrils, in vitro and by week 2 in vivo, only in ESS prepared using all normal cells (group 4). The results suggest that although COL7 protein is produced in engineered skin when cells in only one layer express the COL7 gene, formation of structurally normal anchoring fibrils appears to require expression of COL7 in both dermal fibroblasts and epidermal keratinocytes.

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A gelatin/collagen/polycaprolactone scaffold for skin regeneration

PeerJ ◽

10.7717/peerj.6358 ◽

2019 ◽

Vol 7 ◽

pp. e6358 ◽

Cited By ~ 3

Author(s):

Lin-Gwei Wei ◽

Hsin-I Chang ◽

Yiwei Wang ◽

Shan-hui Hsu ◽

Lien-Guo Dai ◽

...

Keyword(s):

Stem Cells ◽

Wound Healing ◽

Dermal Fibroblasts ◽

Skin Regeneration ◽

Collagen Content ◽

Wound Dressings ◽

Skin Substitute ◽

Epidermal Keratinocytes ◽

Adipose Derived Stem Cells

Background A tissue-engineered skin substitute, based on gelatin (“G”), collagen (“C”), and poly(ε-caprolactone) (PCL; “P”), was developed. Method G/C/P biocomposites were fabricated by impregnation of lyophilized gelatin/collagen (GC) mats with PCL solutions, followed by solvent evaporation. Two different GC:PCL ratios (1:8 and 1:20) were used. Results Differential scanning calorimetry revealed that all G/C/P biocomposites had characteristic melting point of PCL at around 60 °C. Scanning electron microscopy showed that all biocomposites had similar fibrous structures. Good cytocompatibility was present in all G/C/P biocomposites when incubated with primary human epidermal keratinocytes (PHEK), human dermal fibroblasts (PHDF) and human adipose-derived stem cells (ASCs) in vitro. All G/C/P biocomposites exhibited similar cell growth and mechanical characteristics in comparison with C/P biocomposites. G/C/P biocomposites with a lower collagen content showed better cell proliferation than those with a higher collagen content in vitro. Due to reasonable mechanical strength and biocompatibility in vitro, G/C/P with a lower content of collagen and a higher content of PCL (GCLPH) was selected for animal wound healing studies. According to our data, a significant promotion in wound healing and skin regeneration could be observed in GCLPH seeded with adipose-derived stem cells by Gomori’s trichrome staining. Conclusion This study may provide an effective and low-cost wound dressings to assist skin regeneration for clinical use.

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Adipose Tissue-Derived Mesenchymal Cells Support Skin Reepithelialization through Secretion of KGF-1 and PDGF-BB: Comparison with Dermal Fibroblasts

Cell Transplantation ◽

10.3727/096368912x637064 ◽

2012 ◽

Vol 21 (11) ◽

pp. 2441-2454 ◽

Cited By ~ 17

Author(s):

Vassilia-Ismini Alexaki ◽

Despoina Simantiraki ◽

Marianna Panayiotopoulou ◽

Olga Rasouli ◽

Maria Venihaki ◽

...

Keyword(s):

Wound Healing ◽

Adipose Tissue ◽

Normal Skin ◽

Human Adipose Tissue ◽

Mesenchymal Cells ◽

Dermal Fibroblasts ◽

Skin Substitutes ◽

Keratinocyte Proliferation

Epidermal organization and homeostasis are regulated by mesenchymal influences through paracrine actions. Until today, dermal fibroblasts (DFs) are used in the “dermal” layer to support keratinocyte growth in vitro in dermal and skin substitutes. In the present work, we used human adipose tissue-derived mesenchymal cells (ADMCs) as a support of keratinocyte growth in vitro (in monolayer culture and in 3D skin cell culture models) and in vivo (mouse wound healing models) and compared our findings with those obtained using dermal fibroblasts. ADMCs induce reepithelialization during wound healing more efficiently than DFs, by enhancing keratinocyte proliferation through cell cycle progression, and migration. This effect is mediated (at least partially) by a paracrine action of KGF-1 and PDGF-BB, which are more prominently expressed in ADMCs than in DFs. Furthermore, replacement of DFs by ADMCs in the dermal compartment of organotypic skin cultures leads to an artificial epidermis resembling to that of normal skin, concerning the general histology, although with a higher expression of cytokeratins 5 and 19. In Rag1 knockout mice, ADMCs induced a more rapid reepithelialization and a more effective wound healing, compared to dermal fibroblasts. In conclusion, we provide evidence that ADMCs can serve as supportive cells for primary keratinocyte cultures. In addition, because of their abundance and the great cell yield achieved during ADMC isolation, they represent an interesting cell source, with potential aspects for clinical use.

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PREPARATION AND EVALUATION OF GAG-INCORPORATED SKIN SUBSTITUTE: AN IN VITRO STUDY

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s1016237206000257 ◽

2006 ◽

Vol 18 (03) ◽

pp. 153-157 ◽

Cited By ~ 1

Author(s):

TZU-WEI WANG ◽

HSI-CHIN WU ◽

YI-CHAU HUANG ◽

JUI-SHENG SUN ◽

FENG-HUEI LIN

Keyword(s):

Lower Layer ◽

In Vitro Study ◽

Dermal Fibroblasts ◽

Skin Equivalent ◽

Skin Substitute ◽

Epidermal Keratinocytes ◽

Different Temperatures ◽

Air Liquid Interface ◽

Epidermis Structure

A bi-layered gelatin-C6S-HA membrane with different pore sizes was prepared by freeze-drying at different temperatures - 20°C and -196°C, respectively Glycosaminoglycans (GAGs) were incorporated within the gelatin matrices to mimic the dermal composition and to create an appropriate environment for cell growth. The gelatin-C6S-HA membrane was cross-linked by 1-ethyl-3(3-dimethylaminopropryl) carbodiimide (EDC) to resist rapidly biodegradation by matrix enzymes. In this study, the lower layer of the sponge was inoculated with dermal fibroblasts for dermis development and as the feeder layer for epidermal keratinocytes. The upper layer was seeded with keratinocytes for epidermalization. After cultured for a period of time in air-liquid interface, the upper layer was developed into an epidermis structure with stratified epidermal layers. The lower part was developed into dermis-like structure synthesized by dermal fibroblasts surrounding with its own secreted extracellular matrix. In brief, the bi-layered skin equivalent with biological dermal analog and epidermal analog would be a suitable tool for autologous skin equivalent tissue engineering.

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A Novel Substance P-Based Hydrogel for Increased Wound Healing Efficiency

Molecules ◽

10.3390/molecules23092215 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2215 ◽

Cited By ~ 4

Author(s):

Da Kim ◽

Ji Jang ◽

Song Jang ◽

Jungsun Lee

Keyword(s):

Wound Healing ◽

Substance P ◽

Dermal Fibroblasts ◽

Epidermal Keratinocytes ◽

Human Epidermal Keratinocytes ◽

In Vivo Experiments ◽

And Migration ◽

Proliferation And Migration

The neuropeptide substance P (SP) is known to stimulate wound healing by regulating the production of relevant cytokines as well as cell proliferation and migration. However, the therapeutic application of SP is limited by its low stability under biological conditions and oxidation during purification, formulation, and storage. To address this problem, we developed a novel formulation of SP as an SP gel, and investigated its wound healing activity both in vitro and in vivo. SP in SP gel was stable at various temperatures for up to 4 weeks. In vitro, SP gel exhibited more potential as a candidate wound-healing agent than SP alone, as evidenced by the observed increases in the proliferation and migration of human epidermal keratinocytes and human dermal fibroblasts. In vivo experiments showed that SP gel treatment enhanced the healing of full-thickness wounds in mice as compared to SP alone. These results demonstrate the benefits of SP gel as a promising topical agent for wound treatment.

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Development of the Mechanical Properties of Engineered Skin Substitutes After Grafting to Full-Thickness Wounds

Journal of Biomechanical Engineering ◽

10.1115/1.4026290 ◽

2014 ◽

Vol 136 (5) ◽

Cited By ~ 19

Author(s):

Edward A. Sander ◽

Kaari A. Lynch ◽

Steven T. Boyce

Keyword(s):

Mechanical Properties ◽

Dermal Fibroblasts ◽

Epidermal Keratinocytes ◽

Type I ◽

Full Thickness ◽

Skin Substitutes ◽

Maximum Extension ◽

Engineered Skin Substitutes ◽

Native Skin

Engineered skin substitutes (ESSs) have been reported to close full-thickness burn wounds but are subject to loss from mechanical shear due to their deficiencies in tensile strength and elasticity. Hypothetically, if the mechanical properties of ESS matched those of native skin, losses due to shear or fracture could be reduced. To consider modifications of the composition of ESS to improve homology with native skin, biomechanical analyses of the current composition of ESS were performed. ESSs consist of a degradable biopolymer scaffold of type I collagen and chondroitin-sulfate (CGS) that is populated sequentially with cultured human dermal fibroblasts (hF) and epidermal keratinocytes (hK). In the current study, the hydrated biopolymer scaffold (CGS), the scaffold populated with hF dermal skin substitute (DSS), or the complete ESS were evaluated mechanically for linear stiffness (N/mm), ultimate tensile load at failure (N), maximum extension at failure (mm), and energy absorbed up to the point of failure (N-mm). These biomechanical end points were also used to evaluate ESS at six weeks after grafting to full-thickness skin wounds in athymic mice and compared to murine autograft or excised murine skin. The data showed statistically significant differences (p <0.05) between ESS in vitro and after grafting for all four structural properties. Grafted ESS differed statistically from murine autograft with respect to maximum extension at failure, and from intact murine skin with respect to linear stiffness and maximum extension. These results demonstrate rapid changes in mechanical properties of ESS after grafting that are comparable to murine autograft. These values provide instruction for improvement of the biomechanical properties of ESS in vitro that may reduce clinical morbidity from graft loss.

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Transient Hyperproliferation of a Transgenic Human Epidermis Expressing Hepatocyte Growth Factor

Cell Transplantation ◽

10.3727/000000002783985819 ◽

2002 ◽

Vol 11 (4) ◽

pp. 385-395 ◽

Cited By ~ 20

Author(s):

Karen E. Hamoen ◽

Jeffrey R. Morgan

Keyword(s):

Growth Factor ◽

Hepatocyte Growth Factor ◽

Human Keratinocytes ◽

Epidermal Keratinocytes ◽

Skin Substitutes ◽

Proliferating Cells ◽

Ki 67 ◽

Hepatocyte Growth

Hepatocyte growth factor (HGF) is a fibroblast-derived protein that affects the growth, motility, and differentiation of epithelial cells including epidermal keratinocytes. To investigate the role of HGF in cutaneous biology and to explore the possibility of using it in a tissue engineering approach, we used retroviral-mediated gene transfer to introduce the gene encoding human HGF into diploid human keratinocytes. Modified cells synthesized and secreted significant levels of HGF in vitro and the proliferation of keratinocytes expressing HGF was enhanced compared with control unmodified cells. To investigate the effects of HGF in vivo, we grafted modified keratinocytes expressing HGF onto athymic mice using acellular dermis as a substrate. When compared with controls, HGF-expressing keratinocytes formed a hyperproliferative epidermis. The epidermis was thicker, had more cells per length of basement membrane, and had increased numbers of Ki-67-positive proliferating cells, many of which were suprabasal in location. Hyperproliferation subsided and the epidermis was equivalent to controls by 2 weeks, a time frame that coincides with healing of the graft. Transient hyperproliferation may be linked to the loss of factors present in the wound that activate HGF. These data suggest that genetically modified skin substitutes secreting HGF may have applications in wound closure and the promotion of wound healing.

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Needleless electrospinning of PVP/PGS fibrous scaffolds for skin tissue engineering applications

10.31224/osf.io/xs64k ◽

2018 ◽

Cited By ~ 1

Author(s):

Antonios Keirouz ◽

Giuseppino Fortunato ◽

Anthony Callanan ◽

Norbert Radacsi

Keyword(s):

Tissue Engineering ◽

Tensile Modulus ◽

Dermal Fibroblasts ◽

Skin Tissue ◽

Skin Substitute ◽

Electrospinning Technique ◽

Skin Tissue Engineering ◽

Needleless Electrospinning ◽

High Concentrations

Scaffolds and implants used for tissue engineering need to be adapted for their mechanical properties with respect to their environment within the human body. Therefore, a novel composite for skin tissue engineering is presented by use of blends of Poly(vinylpyrrolidone) (PVP) and Poly(glycerol sebacate) (PGS) were fabricated via the needleless electrospinning technique. The formed PGS/PVP blends were morphologically, thermochemically and mechanically characterized. The morphology of the developed fibers related to the concentration of PGS, with high concentrations of PGS merging the fibers together plasticizing the scaffold. The tensile modulus appeared to be affected by the concentration of PGS within the blends, with an apparent decrease in the elastic modulus of the electrospun mats and an exponential increase of the elongation at break. Ultraviolet (UV) crosslinking of PGS/PVP significantly decreased and stabilized the wettability of the formed fiber mats, as indicated by contact angle measurements. In vitro examination showed good viability and proliferation of human dermal fibroblasts over the period of a week. The present findings provide important insights for tuning the elastic properties of electrospun material by incorporating this unique elastomer, as a promising future candidate for skin substitute constructs.

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Cytotoxicity Studies of Curcumin Loaded-Cockle Shell-Derived Calcium Carbonate Nanoparticles

Nanoscience &Nanotechnology-Asia ◽

10.2174/2210681209666191128155819 ◽

2019 ◽

Vol 09 ◽

Cited By ~ 1

Author(s):

Maryam Muhammad Mailafiya ◽

Mohamad Aris Mohd Moklas ◽

Kabeer Abubakar ◽

Abubakar Danmaigoro ◽

Samaila Musa Chiroma ◽

...

Keyword(s):

Calcium Carbonate ◽

Safety Margin ◽

Bone Diseases ◽

In Vivo Studies ◽

Normal Cells ◽

Cytotoxicity Studies ◽

Standard Techniques ◽

Cockle Shell

Background: Cockle shell-derived calcium carbonate nanoparticles (CSCaCO3NP) are natural biogenic inorganic material that is used in drug delivery mainly as a bone-remodeling agent as well as a delivery agent for various therapeutics against bone diseases. Curcumin possess wide safety margin and yet puzzled with the problem of poor bioavailability due to insolubility. Propounding in vitro and in vivo studies on toxicity assessments of newly synthesized nanoparticles are ongoing to overcome some crucial challenges regarding their safety administration. Nanotoxicology has paved ways for concise test protocols to monitor sequential events with regards to possible toxicity of newly synthesized nanomaterials. The development of nanoparticle with no or less toxic effect has gained tremendous attentions. Objective: This study aimed at evaluating the in vitro cytotoxic effect of curcumin-loaded cockle shell-derived calcium carbonate nanoparticles (Cur-CSCaCO3NP) and assessing its biocompatibility on normal cells using standard techniques of WST’s assay. Method: Standard techniques of WST’s assay was used for the evaluation of the biocompatibility and cytotoxicity. Result: The result showed that CSCaCO3NP and Cur-CSCaCO3NP possess minimal toxicity and high biocompatibility on normal cells even at higher dose of 500 µg/ml and 40 µg/ml respectively. Conclusion: CSCaCO3NP can be termed an excellent non-toxic nanocarrier for curcumin delivery. Hence, curcumin loaded cockle shell derived calcium carbonate nanoparticles (Cur-CSCaCO3NP) could further be assessed for various in vivo and in vitro therapeutic applications against various bone related ailments.

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Design, Synthesis, Molecular Modeling and Antitumor Evaluation of Novel Indolyl-Pyrimidine Derivatives with EGFR Inhibitory Activity

Molecules ◽

10.3390/molecules26071838 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1838

Author(s):

Naglaa M. Ahmed ◽

Mahmoud M. Youns ◽

Moustafa K. Soltan ◽

Ahmed M. Said

Keyword(s):

Molecular Modeling ◽

Antitumor Activity ◽

Cell Lines ◽

Cancer Cell ◽

Antiproliferative Activity ◽

Antitumor Agents ◽

Cancer Cell Lines ◽

Normal Cells

Scaffolds hybridization is a well-known drug design strategy for antitumor agents. Herein, series of novel indolyl-pyrimidine hybrids were synthesized and evaluated in vitro and in vivo for their antitumor activity. The in vitro antiproliferative activity of all compounds was obtained against MCF-7, HepG2, and HCT-116 cancer cell lines, as well as against WI38 normal cells using the resazurin assay. Compounds 1–4 showed broad spectrum cytotoxic activity against all these cancer cell lines compared to normal cells. Compound 4g showed potent antiproliferative activity against these cell lines (IC50 = 5.1, 5.02, and 6.6 μM, respectively) comparable to the standard treatment (5-FU and erlotinib). In addition, the most promising group of compounds was further evaluated for their in vivo antitumor efficacy against EAC tumor bearing mice. Notably, compound 4g showed the most potent in vivo antitumor activity. The most active compounds were evaluated for their EGFR inhibitory (range 53–79 %) activity. Compound 4g was found to be the most active compound against EGFR (IC50 = 0.25 µM) showing equipotency as the reference treatment (erlotinib). Molecular modeling study was performed on compound 4g revealed a proper binding of this compound inside the EGFR active site comparable to erlotinib. The data suggest that compound 4g could be used as a potential anticancer agent.

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Regulatory Mechanism for Exfoliative Toxin Production inStaphylococcus aureus

Infection and Immunity ◽

10.1128/iai.00872-10 ◽

2011 ◽

Vol 79 (4) ◽

pp. 1660-1670 ◽

Cited By ~ 24

Author(s):

Fuminori Kato ◽

Noriko Kadomoto ◽

Yuko Iwamoto ◽

Katsuaki Bunai ◽

Hitoshi Komatsuzawa ◽

...

Keyword(s):

Mouse Model ◽

Regulatory Mechanism ◽

Toxin Production ◽

Neonatal Mouse ◽

Epidermal Keratinocytes ◽

Global Regulators ◽

Exfoliative Toxin ◽

Blistering Disease

ABSTRACTThe exfoliative toxin (ET) is a major virulence factor ofStaphylococcus aureusthat causes bullous impetigo and its disseminated form, staphylococcal scalded-skin syndrome (SSSS). ET selectively digests one of the intracellular adhesion molecules, desmoglein 1, of epidermal keratinocytes and causes blisters due to intraepidermal cell-cell dissociation. MostS. aureusstrains that cause blistering disease produce either ETA or ETB. They are serologically distinct molecules, where ETA is encoded on a phage genome and ETB is enocded on a large plasmid. ETA-producingS. aureusstrains are frequently isolated from impetigo patients, and ETB-producingS. aureusstrains are isolated from SSSS. ET-induced blister formation can be reproduced with the neonatal mouse. To determine the regulatory mechanism of ET production, we investigated the role of the two-component systems and global regulators foretaoretbexpressionin vitroandin vivowith the mouse model. Western blot and transcription analyses using a series of mutants demonstrate ETA production was downregulated bysigB,sarS, andsarA, while ETB production was downregulated bysigBandsarAbut not bysarS. Production of both toxins is upregulated bysaeRS,arlRS, andagrCA. Furthermore, by thein vivoneonatal mouse model,sigBandsarSbut notsarAnegatively regulate the exfoliation activity of the ETA-producing strain, whilesarAnegatively regulates the ETB-producing strain. In both strains,saeRS,arlRS, andagrCApositively regulate the exfoliation activityin vivo. The data illustrate similar but distinct regulatory mechanisms for ETA and ETB productionin S. aureus in vitroas well asin vivo.

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