Skin wound healing is a complex physiological event, involving many cellular and molecular components. The event of
wound healing is the coordinated overlap of a number of distinct phases, namely haemostasis, inflammatory, proliferative
and remodelling. The molecular events surrounding wound healing, particularly the reepithelialisation, has been reported
to be similar to the epithelial to mesenchymal transition (EMT). In this review, the mechanism between epithelialisation
and EMT were compared. Both are characterised by the loss of epithelial integrity and increased motility. In terms of
the signalling kinases, Smad and mitogen-activated protein kinase (MAPK) has been reported to be involved in both
reepithelialisation and EMT. At the transcriptional level, SLUG transcription factor has been reported to be important for
both reepithelialisation and EMT. Extracellular matrix proteins that have been associated with both events are collagen
and laminin. Lastly, both events required the interplay between matrix metalloproteinases (MMPs) and its inhibitor. As a
conclusion, both reepithelialisation and EMT shares similar signaling cascade and transcriptional regulation to exhibit
decreased epithelial traits and increased motility in keratinocytes.
Galectin-3 (Gal-3) is a carbohydrate-binding protein, that promotes angiogenesis through mediating angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). There is strong evidence confirming FGF involvement in tumor growth and progression by disrupting cell proliferation and angiogenesis. In this study, we investigated the effect of β-cyclodextrin:everolimus:FGF-7 inclusion complex (Complex) on Caco-2 cell migration, cell motility and colony formation. In addition, we examined the inhibitory effect of the Complex on the circulating proteins; Gal-3 and FGF-7. Swiss Target Prediction concluded that Gal-3 and FGF are possible targets for β-CD. Results of the chemotaxis cell migration assay on Caco-2 cell line revealed that the Complex has higher reduction in cell migration (78.3%) compared to everolimus (EV) alone (58.4%) which is possibly due to the synergistic effect of these molecules when used as a combined treatment. Moreover, the Complex significantly decreased the cell motility in cell scratch assay, less than 10% recovery compared to the control which has ~ 45% recovery. The Complex inhibited colony formation by ~ 75% compared to the control. Moreover, the Complex has the ability to inhibit Gal-3 with minimum inhibitory concentration of 33.46 and 41 for β-CD and EV, respectively. Additionally, β-CD and β-CD:EV were able to bind to FGF-7 and decreased the level of FGF-7 more than 80% in cell supernatant. This confirms Swiss Target Prediction result that predicted β-CD could target FGF. These findings advance the understanding of the biological effects of the Complex which reduced cell migration, cell motility and colony formation and it is possibly due to inhibiting circulating proteins such as; Gal-3 and FGF-7.
Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion.
Bacterial cellulose (BC)/acrylic acid (AA) hydrogel has successfully been investigated as a wound dressing for partial-thickness burn wound. It is also a promising biomaterial cell carrier because it bears some resemblance to the natural soft tissue. This study assessed its ability to deliver human epidermal keratinocytes (EK) and dermal fibroblasts (DF) for the treatment of full-thickness skin lesions. In vitro studies demonstrated that BC/AA hydrogel had excellent cell attachment, maintained cell viability with limited migration, and allowed cell transfer. In vivo wound closure, histological, immunohistochemistry, and transmission electron microscopy evaluation revealed that hydrogel alone (HA) and hydrogel with cells (HC) accelerated wound healing compared to the untreated controls. Gross appearance and Masson's trichrome staining indicated that HC was better than HA. This study suggests the potential application of BC/AA hydrogel with dual functions, as a cell carrier and wound dressing, to promote full-thickness wound healing.
Diabetes patients are at risk of having chronic wounds, which would take months to years to resolve naturally. Chronic wounds can be countered using the electrical stimulation technique (EST) by dielectrophoresis (DEP), which is label-free, highly sensitive, and selective for particle trajectory. In this study, we focus on the validation of polystyrene particles of 3.2 and 4.8 μm to predict the behavior of keratinocytes to estimate their crossover frequency (fXO) using the DEP force (FDEP) for particle manipulation. MyDEP is a piece of java-based stand-alone software used to consider the dielectric particle response to AC electric fields and analyzes the electrical properties of biological cells. The prototypic 3.2 and 4.8 μm polystyrene particles have fXO values from MyDEP of 425.02 and 275.37 kHz, respectively. Fibroblast cells were also subjected to numerical analysis because the interaction of keratinocytes and fibroblast cells is essential for wound healing. Consequently, the predicted fXO from the MyDEP plot for keratinocyte and fibroblast cells are 510.53 and 28.10 MHz, respectively. The finite element method (FEM) is utilized to compute the electric field intensity and particle trajectory based on DEP and drag forces. Moreover, the particle trajectories are quantified in a high and low conductive medium. To justify the simulation, further DEP experiments are carried out by applying a non-uniform electric field to a mixture of different sizes of polystyrene particles and keratinocyte cells, and these results are well agreed. The alive keratinocyte cells exhibit NDEP force in a highly conductive medium from 100 kHz to 25 MHz. 2D/3D motion analysis software (DIPP-MotionV) can also perform image analysis of keratinocyte cells and evaluate the average speed, acceleration, and trajectory position. The resultant NDEP force can align the keratinocyte cells in the wound site upon suitable applied frequency. Thus, MyDEP estimates the Clausius-Mossotti factors (CMF), FEM computes the cell trajectory, and the experimental results of prototypic polystyrene particles are well correlated and provide an optimistic response towards keratinocyte cells for rapid wound healing applications.
Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting AP1S3, a disease gene previously identified by our group and validated here in a newly ascertained patient resource. We first showed that AP1S3 expression is distinctively elevated in keratinocytes. Because AP1S3 encodes a protein implicated in autophagosome formation, we next investigated the effects of gene silencing on this pathway. We found that AP1S3 knockout disrupts keratinocyte autophagy, causing abnormal accumulation of p62, an adaptor protein mediating NF-κB activation. We showed that as a consequence, AP1S3-deficient cells up-regulate IL-1 signaling and overexpress IL-36α, a cytokine that is emerging as an important mediator of skin inflammation. These abnormal immune profiles were recapitulated by pharmacological inhibition of autophagy and verified in patient keratinocytes, where they were reversed by IL-36 blockade. These findings show that keratinocytes play a key role in skin autoinflammation and identify autophagy modulation of IL-36 signaling as a therapeutic target.
The angiogenic potential of native skin (NS), keratinocytes single skin equivalent (SSE-K), fibroblasts single skin equivalent (SSE-F) and bilayered skin equivalent secreting angiogenic growth factors such as transforming growth factor beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) in the in vitro systems at 24, 48, 72 hours and 7 days was compared using Enzyme-Linked Immunosorbent Assay (ELISA). Bilayered skin equivalent exhibit highest release of growth factors within 24 hours to 7 days of culture compared to NS, SSE-K and SSE-F. This proved the potential of bilayered skin equivalent in producing and sustaining growth factors release to enhance angiogenesis, fibroblasts proliferation, matrix deposition, migration and growth of keratinocytes.
A major factor limiting survival following extensive thermal injury is insufficient availability of donor sites to provide enough skin for the required grafting procedures. Limitation of autologous grafting promotes the usage of allograft skin substitutes to promote wound healing. Here, we investigated the wound healing potential of allograft single layered tissue engineered skin which comprises of either keratinocytes (SLTES-K) or fibroblast (SLTES-F) with fibrin as the delivery system. Results from gross and microscopic evaluation showed our single layered tissue engineered skin constructed with keratinocytes or fibroblast after gamma radiation with the dosage of 2Gy could serve as allograft for the treatment of skin loss.
The treatment of major burn injuries are a formidable challenge to the burn surgeon. Early aggressive surgery for deep to full thickness burn injuries is vital in the prevention of infection. The ultimate goal in major burn injuries is to prevent the onset of multi-resistant organisms and achieve early wound cover. The field of tissue engineering can help to expedite the healing of these burn wounds. The development of keratinocyte culture delivery system can be used clinically to fasten the healing process and save many lives.
Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
The ability to regenerate new bone for skeletal use is a major clinical need. In this study, two novel porous calcium phosphate materials pure HA and biphasic HA/beta-Tricalcium phosphate (HA/beta -TCP) were evaluated as potential scaffolds for cell-seeded bone substitutes using human osteoblast-like cells (HOS) and primary human mesenchymal stem cells (hMSCs). A high rate of proliferation was observed on both scaffolds. A greater increase in alkaline phosphatase (ALP- an indicator of osteoblast differentiation) was observed on HA/beta -TCP compared to HA. This observation indicates that HA/TCP may play a role in inducing osteoblastic differentiation. Although further evaluation is required both materials show potential as innovative synthetic substitutes for tissue engineered scaffolds.
Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.
Single-walled carbon nanotubes (SWCNTs) are carbon-based nanomaterials that possess immense industrial potential. Despite accumulating evidence that exposure to SWCNTs might be toxic to humans, our understanding of the mechanisms for cellular toxicity of SWCNTs remain limited. Here, we demonstrated that acute exposure of short (1-3μm) and regular-length (5-30μm) pristine, carboxylated or hydroxylated SWCNTs inhibited cell proliferation in human somatic and human stem cells in a cell type-dependent manner. The toxicity of regular-length pristine SWCNT was most evidenced in NP69>CYT00086>MCF-10A>MRC-5>HaCaT > HEK-293T>HepG2. In contrast, the short pristine SWCNTs were relatively less toxic in most of the cells being tested, except for NP69 which is more sensitive to short pristine SWCNTs as compared to regular-length pristine SWCNTs. Interestingly, carboxylation and hydroxylation of regular-length SWCNTs, but not the short SWCNTs, significantly reduced the cytotoxicity. Exposure of SWCNTs also induced caspase 3 and 9 activities, mitochondrial membrane depolarization, and significant apoptosis and necrosis in MRC-5 embryonic lung fibroblasts. In contrast, SWCNTs inhibited the proliferation of HaCaT human keratinocytes without inducing cell death. Further analyses by gene expression profiling and Connectivity Map analysis showed that SWCNTs induced a gene expression signature characteristic of heat shock protein 90 (HSP90) inhibition in MRC-5 cells, suggesting that SWCNTs may inhibit the HSP90 signaling pathway. Indeed, exposure of MRC-5 cells to SWCNTs results in a dose-dependent decrease in HSP90 client proteins (AKT, CDK4 and BCL2) and a concomitant increase in HSP70 expression. In addition, SWCNTs also significantly inhibited HSP90-dependent protein refolding. Finally, we showed that ectopic expression of HSP90, but not HSP40 or HSP70, completely abrogated the cytotoxic effects of SWCNTs, suggesting that SWCNT-induced cellular toxicity is HSP90 dependent. In summary, our findings suggest that the toxic effects of SWCNTs are mediated through inhibition of HSP90 in human lung fibroblasts and keratinocytes.
Biopolymer chitosan (beta-1,4-d-glucosamine) comprises the copolymer mixture of N-acetylglucosamine and glucosamine. The natural biocompatibility and biodegradability of chitosan have recently highlighted its potential use for applications in wound management. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability, but it is unknown as to what degree. Hence, the biocompatibility of the chitosan porous skin regenerating templates (PSRT 82, 87 and 108) was determined using an in vitro toxicology model at the cellular and molecular level on primary normal human epidermal keratinocytes (pNHEK). Cytocompatibility was accessed by using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay from 24 to 72h. To assess the genotoxicity of the PSRTs, DNA damage to the pNHEK was evaluated by using the Comet assay following direct contact with the various PSRTs. Furthermore, the skin pro-inflammatory cytokines TNF-alpha and IL-8 were examined to evaluate the tendency of the PSRTs to provoke inflammatory responses. All PSRTs were found to be cytocompatible, but only PSRT 108 was capable of stimulating cell proliferation. While all of the PSRTs showed some DNA damage, PSRT 108 showed the least DNA damage followed by PSRT 87 and 82. PSRT 87 and 82 induced a higher secretion of TNF-alpha and IL-8 in the pNHEK cultures than did PSRT 108. Hence, based on our experiments, PSRT 108 is the most biocompatible wound dressing of the three tested.
Ticks are important ectoparasites which transmit many disease pathogen to animals; these are labelled tick borne diseases (TBD). Tick induced damage to skin and hides has not received attention. Skin and hides are important for the leather product industry, particularly in Pakistan. Due to economic importance and financial loss by ticks in leather industry, the present study was designed to investigate skin and hides damage due to ticks at microscopic level. Naturally tick infested tissue samples of hides and skin were collected from slaughter houses. Primary lesions at tick feeding sites showed epidermal edema with adjacent dermal edema. Histopathological examination revealed degeneration of epidermal layer down to the basal layer. Epidermal and sub dermal layers often displayed focal necrosis infiltrated with neutrophils and mononuclear cells at tick bite sites. Hyperplasia of keratinocytes was also seen at sites of ruptured epidermis. Quality of leather depends upon the grain (Outer) surface skin/hides. Ticks infestation damages the outer surface, due to bites, inflammatory responses, and secondary bacterial infections that often become established at feeding sites. Control of ticks should be given consideration to reduce infestation induced losses in the leather industry in Pakistan.