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Alpha-ketoglutaric acid based polymeric particles for cutaneous wound healing

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Abstract

Metabolites are not only involved in energy pathways but can also act as signaling molecules. Herein, we demonstrate that polyesters of alpha-ketoglutararte (paKG) can be generated by reacting aKG with aliphatic diols of different lengths, which release aKG in a sustained manner. paKG polymer-based microparticles generated via emulsion-evaporation technique lead to faster keratinocyte wound closures in a scratch assay test. Moreover, paKG microparticles also led to faster wound healing responses in an excisional wound model in live mice. Overall, this study shows that paKG MPs that release aKG in a sustained manner can be used to develop regenerative therapeutic responses.

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... When these polyesters are shaped into macroparticles (MPs), they exhibit the capacity to modulate not only the immune response of dendritic cells (DCs) but also the metabolic processes and innate immune cell phenotypes (specifically macrophages and neutrophils) through phagocytosis. [28][29][30] However, it's important to note that this phagocytosis-mediated intracellular delivery strategy may not be applicable to nonphagocytic cells (such as osteoblasts and MSCs), which have limited MPs uptake capability. Nevertheless, this implies that the AKG-based biopolymer holds promise as an innovative approach for creating biodegradable biomaterials specifically tailored for osteogenesis and bone regeneration, an area that remains largely unexplored. ...
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Dendritic cells (DCs) rely on glycolysis for their energy needs to induce pro-inflammatory antigen-specific immune responses. Therefore, inhibiting DC glycolysis, while presenting the self-antigen, may prevent pro-inflammatory antigen-specific immune responses. Previously we demonstrated that microparticles with alpha-ketoglutarate (aKG) in the polymer backbone (paKG MPs) were able to generate anti-inflammatory DCs by sustained delivery of the aKG metabolite, and by modulating energy metabolism of DCs. Herein, we demonstrate that paKG MPs-based delivery of a glycolytic inhibitor, PFK15, using paKG MPs induces anti-inflammatory DCs (CD86LoMHCII⁺) by down-regulating glycolysis, CD86, tnf and IL-6 genes, while upregulating oxidative phosphorylation (OXPHOS) and mitochondrial genes. Furthermore, paKG MPs delivering PFK15 and a self-antigen, collagen type II (bc2), in vivo, in a collagen-induced autoimmune arthritis (CIA) mouse model, normalized paw inflammation and arthritis score, by generating antigen-specific immune responses. Specifically, these formulations were able to reduce activation of DCs in draining lymph nodes and impressively generated proliferating bc2-specific anti-inflammatory regulatory T cells in joint-associated popliteal lymph nodes. These data strongly suggest that sustained glycolytic inhibition of DCs in the presence of an antigen can induce antigen-specific immunosuppressive responses, therefore, generating a technology that can be applicable for treating autoimmune diseases.
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In the present study, curcumin loaded chitosan/poly ethylene glycol nanomaterial (CUR loaded CH/PEG/AgNPs) was fabricated and characterized for wound healing efficiency after fracture surgery. The interaction of functional groups and crystal nature were recorded under FTIR and XRD spectrometer and reveals that the stabilization and purity of NPs was mediated by OH/NH2 groups in chitosan. FESEM showed the presence of spherical and well dispersed particles. The average size of the particle was 13.48 nm. The CUR loaded CH/PEG/AgNPs showed higher swelling capacity (495.6 g/g) in phosphate buffer saline compared to water (140.2 g/g). The drug loading efficiency was higher in CUR loaded CH/PEG/AgNPs compared to CH/PEG films as recorded by the absorbance peak at 460 nm corresponds to curcumin in the composite. A dose dependent cytotoxicity of CUR loaded CH/PEG/AgNPs was noticed on Vero cells. The viability of Vero cells was increased to 96.5% at 100 μg/mL. A remarkable change in Vero cells such as condensed nuclei and membrane blabbing was noticed in cells treated with CUR loaded CH/PEG/AgNPs. A greater inhibition of Staphylococcus aureus and Escherichia coli was noticed at 24 h and 48 h treated with CUR loaded CH/PEG/AgNPs. A greater healing effect by increasing the wound contraction (98% on day 12) was observed with CUR loaded CH/PEG/AgNPs compared to control. Histopathological examination demonstrated that CUR loaded CH/PEG/AgNPs showed complete tissue regeneration in wound excised rats. The results of this study conclude that CUR loaded CH/PEG/AgNPs could be promising candidate to prevent microbial infections in wound, healing wound rapidly and inhibit the proliferation of apoptotic cells. Thus, CUR loaded CH/PEG/AgNPs could be a potential therapeutic agent with broad spectrum applications in the future. • Highlights • A new approach was used to develop curcumin-loaded chitosan/poly(ethylene glycol)/AgNPs. • The CUR-loaded CH/PEG/AgNPs were confirmed to be crystals by XRD analysis. • The prepared CH/PEG/AgNPs were spherical and averaged 13.48 nm in size. • The growth of S. aureus and E. coli were inhibited mostly by CH/PEG/AgNPs treatment. • CUR loaded CH/PEG/AgNPs showed complete tissue regeneration in wound excised mice.
Article
Background: Extracellular vesicles (EVs) derived from plants have emerged as potential candidates for cosmetic and therapeutic applications. In this study, we isolated EVs from Aloe vera peels (A-EVs) and investigated the antioxidant and wound healing potential of A-EVs. Methods: A-EVs were isolated by ultracentrifugation and tangential flow filtration and were characterized using transmission electron microscopy, nanoparticle tracking analysis. The cytotoxicity and cellular uptake of A-EVs were investigated by WST-1 assay and flow cytometry. The antioxidant effect of A-EVs was evaluated by superoxide dismutase (SOD) activity assay and cellular antioxidant activity (CAA) assay. The wound healing potential was assessed by in vitro scratch assay using human keratinocytes (HaCaT) and fibroblasts (HDF). The expression of nuclear factor erythroid 2–related factor 2 (Nrf2) and their associated genes was analyzed by quantitative RT-PCR. Results: A-EVs displayed a round shape and had diameters from 50 to 200 nm. A-EVs showed good cytocompatibility on human skin cells and were internalized into HaCaT cells via clathrin-, caveolae-mediated endocytosis, and membrane fusion. The SOD activity and CAA assays exhibited that A-EVs had antioxidant activity and reduced intracellular ROS levels in H2O2-treated HaCaT cells in a dose-dependent manner. A scratch assay showed that A-EVs enhanced the migration ability of HaCaT and HDF. Moreover, A-EVs significantly upregulated the mRNA expression of Nrf2, HO-1, CAT, and SOD genes in H2O2-treated HaCaT cells. Our findings reveal that A-EVs could activate the antioxidant defense mechanisms and wound healing process via the Nrf2 activation. Conclusion: Overall results suggest that the A-EVs are promising as a potential agent for skin regeneration.
Article
An innovative approach in the functionalization of nanofibers (NFs) for wound healing relies on non-antibiotic combinational therapy to subdue microbial invasion while reducing antimicrobial resistanceand enhancing healing. Despite great potentials, wound healing efficacy of NFs embedding antimicrobial metal nanoparticles (NPs)/essential oils hasbeenscarcely documented. We developed combinational NFs using an electrospinnable hyaluronic acid/polyvinyl alcohol/polyethylene oxide blend embedding a new ZnO NPs/cinnamon essential oil(CEO) antimicrobial combination. Fourier transform infrared,X-ray diffraction and transmission electron microscopy confirmed the presence of HA and distribution of ZnO NPs and CEO within NFs.Results for mean diameter, thermal stability, hydrophilicity, tensile strength, in vitro biodegradability, and cytocompatibility of crosslinked combinational NFs were intermediatebetween those of their singly loaded counterparts. All NFs inhibited the growth of Staphylococcus aureus(S. aureus). Compared with singly loaded NFs, combinationalNFs showed the greatest healing efficacy of full thickness S. aureusinoculated incision wounds in rats in terms of bacterial inhibition following a single application, healing speed, and quality of skin structure recovery as verified by morphological, microbiological, and histopathological studies. Results highlighted thepotentials of metal NPs/essential oil functionalization of nanofibrous wound dressings as an emerging antibiotic-free combinational approach for more effective and safer wound healing.
Article
In this issue of Cell Metabolism, Asadi Shahmirzadi et al. (2020) demonstrate that late-onset dietary supplementation with calcium alpha-ketoglutarate results in increased survival, compressed morbidity, and reduced frailty in mice. The study provides further evidence for critical links between metabolism, inflammation, and aging.
Article
Metabolites affect cell growth in two different ways. First, they serve as building blocks for biomass accumulation. Second, metabolites regulate the activity of growth-relevant signaling pathways. They do so in part by covalently attaching to proteins, thereby generating post-translational modifications (PTMs) that affect protein function, the focus of this Perspective. Recent advances in mass spectrometry have revealed a wide variety of such metabolites, including lipids, amino acids, Coenzyme-A, acetate, malonate, and lactate to name a few. An active area of research is to understand which modifications affect protein function and how they do so. In many cases, the cellular levels of these metabolites affect the stoichiometry of the corresponding PTMs, providing a direct link between cell metabolism and the control of cell signaling, transcription, and cell growth.
Article
Metabolites control immune cell functions, and delivery of these metabolites in a sustained manner may be able to modulate function of the immune cells . In this study, alpha-ketoglutarate (aKG) and diol based polymeric-microparticles (termed paKG MPs) were synthesized to provide sustained release of aKG and promote an immunosuppressive cellular phenotype. Notably, after association with dendritic cells (DCs), paKG MPs modulated the intracellular metabolic-profile/pathways, and decreased glycolysis and mitochondrial respiration in vitro. These metabolic changes resulted in modulation of MHC-II, CD86 expression in DCs, and altered the frequency of regulatory T cells (Tregs), and T-helper type-1/2/17 cells in vitro. This unique strategy of intracellular delivery of key-metabolites in a sustaine manner provide a paradigm-shift in the immunometabolism field-based immunotherapy with applications in different diseases associated with immune disorders.
Article
Wound healing is one of the most complex processes in the human body. It involves the spatial and temporal synchronization of a variety of cell types with distinct roles in the phases of hemostasis, inflammation, growth, re-epithelialization, and remodeling. With the evolution of single cell technologies, it has been possible to uncover phenotypic and functional heterogeneity within several of these cell types. There have also been discoveries of rare, stem cell subsets within the skin, which are unipotent in the uninjured state, but become multipotent following skin injury. Unraveling the roles of each of these cell types and their interactions with each other is important in understanding the mechanisms of normal wound closure. Changes in the microenvironment including alterations in mechanical forces, oxygen levels, chemokines, extracellular matrix and growth factor synthesis directly impact cellular recruitment and activation, leading to impaired states of wound healing. Single cell technologies can be used to decipher these cellular alterations in diseased states such as in chronic wounds and hypertrophic scarring so that effective therapeutic solutions for healing wounds can be developed.
Article
Wound healing is a basic biological process including proliferation and migration of keratinocyte. The effects of microRNAs on skin wound healing remain largely unexplored. This study aimed to investigate the role of microRNA‐126 (miR‐126) in human skin wound healing. Relative expression of miR‐126 after injury was evaluated by qRT‐PCR. Cell viability, colony formation, cycle distribution, migration and the alternation of PI3K/AKT pathway after miR‐126 knockdown or overexpression were detected, respectively. In addition, potential target gene of miR‐126 was also explored by luciferase assay. Results showed that miR‐126 was up‐regulated during skin wound healing. Moreover, overexpression of miR‐126 promoted cell proliferation and migration, whereas inhibition of miR‐126 led to the opposite effects. Additionally, we discovered that PLK2, which inhibited cell viability, colony formation and migration of keratinocyte, was a target gene of miR‐126. The expression of PLK2 was negatively correlated with the level of miR‐126 during wound healing. Finally, we demonstrated that overexpression of miR‐126 significantly increased the expression of p‐AKT, p‐ERK2 and PI3K, indicating that overexpression of miR‐126 activated PI3K/AKT signaling pathway. In conclusion, our results demonstrated that miR‐126 acted as a critical regulator for promoting proliferation and migration in keratinocyte during skin wound healing.
Article
Staphylococcus epidermidis is a common nosocomial pathogen able to form biofilms in indwelling devices, resulting in chronic infections, which are refractory to antibiotics treatment. Staphylococcal biofilms are also associated with the delayed reepithelization and healing of chronic wounds. The human cathelicidin peptide LL‐37 has been proven active against S. epidermidis biofilms in vitro and to promote wound healing. As previous studies have demonstrated that fragments of LL‐37 could possess an equal antibacterial activity as the parent peptide, we tested whether shorter (12‐mer) synthetic fragments of LL‐37 maintained the antibiofilm and/or immune modulating activity, aiming at the identification of essential regions within the LL‐37 parent sequence. Three fragments of LL‐37 displayed improved activity against S. epidermidis in terms of biofilm inhibition and eradication, a reduced cytotoxicity to human keratinocytes and erythrocytes. In addition, KR‐12 and VQ‐12V26 enhanced wound healing potential, relative to LL37. FK‐12 and KR‐12 are truncated version of the cathelicidin, previously reported as valid antimicrobials, whereas VQ‐12V26 is a single substituted LL‐37 fragment. Remarkably, the single substitution aspartic acid to valine in position 26 caused gain of antimicrobial function in the inactive VQ‐12 fragment. The combination of antibiofilm, wound healing potential, and low cytotoxicity makes KR‐12 and VQ‐12V26 promising therapeutic agents and lead compounds for further improvement and understanding of antibiofilm and wound healing properties.
Article
Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear. Here we show that the production of α-ketoglutarate (αKG) via glutaminolysis is important for alternative (M2) activation of macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2 genes. This M2-promoting mechanism is further modulated by a high αKG/succinate ratio, whereas a low ratio strengthens the proinflammatory phenotype in classically activated (M1) macrophages. As such, αKG contributes to endotoxin tolerance after M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of macrophages through metabolic and epigenetic reprogramming.
Article
In this work, we describe the synthesis and characterization of variants of poly(diol fumarate) and poly(diol fumarate-co-succinate). Through a Fischer esterification, α,ω-diols and dicarboxylic acids were polymerized to form aliphatic polyester comacromers. Due to the carbon-carbon double bond of fumaric acid, incorporating it into the macromer backbone structure resulted in unsaturated chains. By choosing α,ω-diols of different length (1,6-hexanediol, 1,8-octanediol, and 1,10-decanediol) and controlling the amount of fumaric acid in the dicarboxylic acid monomer feed (33, 50, and 100 mol%), nine diol-based macromer variants were synthesized and characterized for molecular weight, number of unsaturated bonds per chain, and thermal properties. Degradation and in vitro cytotoxicity were also measured in a subset of macromers. As proof-of-principle, macromer networks were photocrosslinked to demonstrate the ability to perform free radical addition using the unsaturated macromer backbone. Crosslinked macromer networks were also characterized for physicochemical properties (swelling, sol fraction, compressive modulus) based on diol length and amount of unsaturated bonds. A statistical model was built using data generated from these diol-based macromers and macromer networks to evaluate the impact of monomer inputs on final macromer and macromer network properties. With the ability to be modified by free radical addition, biodegradable unsaturated polyesters serve as important macromers in the design of devices such as drug delivery vehicles and tissue scaffolds. Given the ability to extensively control final macromer properties based on monomer input parameters, poly(diol fumarate) and poly(diol fumarate-co-succinate) represent an exciting new class of macromers.
Article
As tumors employ complementary overlapping and/or independent mechanisms to evade immune surveillance, many emerging cancer immunotherapies attempt to target multiple pathways to eradicate malignant cells. Although modulation of independent pathways by simultaneous administration of multiple immune modulators (e.g., checkpoint inhibitors, cytokines, and growth factors) has shown great promise, the clinical impact remains limited due to severe toxicity associated with high systemic levels of many of these drugs. Therefore, novel platforms for efficient delivery of multi-component therapies at lower effective doses would be enabling. Here, a drug delivery platform called immunomodulatory molecule delivery system (iMods), which provides sustained extracellular delivery of a checkpoint inhibitor (anti-PD-L1) and simultaneously, targeted intracellular delivery of a tumor antigen (OVA) along with adjuvant (poly(I:C)), and the indoleamine deoxygenase inhibitor 1-MT is described. In melanoma tumor-bearing mice, combinatorial delivery of these factors with iMods leads to regression of both treated and untreated (contralateral) melanoma tumors and 100% survival. These promising therapeutic outcomes are attributed to significantly enhanced ratios of anti-tumor CD8 T-cell/tumor-protective regulatory T-cell (Treg) in tumors and tumor draining lymph nodes. Overall, the iMods delivery platform described here represents a promising advance in multi-factor cancer immunotherapy.
Article
Aged skin heals wounds poorly, increasing susceptibility to infections. Restoring homeostasis after wounding requires the coordinated actions of epidermal and immune cells. Here we find that both intrinsic defects and communication with immune cells are impaired in aged keratinocytes, diminishing their efficiency in restoring the skin barrier after wounding. At the wound-edge, aged keratinocytes display reduced proliferation and migration. They also exhibit a dampened ability to transcriptionally activate epithelial-immune crosstalk regulators, including a failure to properly activate/maintain dendritic epithelial T cells (DETCs), which promote re-epithelialization following injury. Probing mechanism, we find that aged keratinocytes near the wound edge don’t efficiently upregulate Skints or activate STAT3. Notably, when epidermal Stat3, Skints, or DETCs are silenced in young skin, re-epithelialization following wounding is perturbed. These findings underscore epithelial-immune crosstalk perturbations in general, and Skints in particular, as critical mediators in the age-related decline in wound-repair.
Article
Objective: In previous work, we demonstrated the development of a novel fusion protein containing stromal cell-derived growth factor-1 alpha juxtaposed to an elastin-like peptide (SDF1-ELP), which has similar bioactivity, but is more stable in elastase than SDF1. Herein, we compare the ability of a single topical application of SDF1-ELP to that of SDF1 in healing 1 × 1 cm excisional wounds in diabetic mice. Approach: Human Leukemia-60 cells were used to demonstrate the chemotactic potential of SDF1-ELP versus SDF1 in vitro. Human umbilical vascular endothelial cells were used to demonstrate the angiogenic potential of SDF1-ELP versus SDF1 in vitro. The bioactivity of SDF1-ELP versus SDF1 after incubation in ex-vivo diabetic wound fluid was compared. The in-vivo effectiveness of SDF1-ELP versus SDF1 was compared in diabetic mice wound model by monitoring for the number of CD31+ cells in harvested wound tissues. Results: SDF1-ELP promotes the migration of cells and induces vascularization similar to SDF1 in vitro. SDF1-ELP is more stable in wound fluids compared to SDF1. In vivo, SDF1-ELP induced a higher number of vascular endothelial cells (CD31+ cells) compared to SDF1 and other controls, suggesting increased vascularization. Innovation: While growth factors have been shown to improve wound healing, this strategy is largely ineffective in chronic wounds. In this work, we show that SDF1-ELP is a promising agent for the treatment of chronic skin wounds. Conclusion: The superior in vivo performance and stability of SDF1-ELP makes it a promising agent for the treatment of chronic skin wounds.
Article
Small molecules participate extensively in various life processes. However, specific and sensitive detection of small molecules in living system is highly challenging. Here we describe in vivo real-time dynamic monitoring of small molecules by luminescent polymer-dot oxygen transducer. The optical transducer combined with an oxygen-consuming enzyme can sensitively detect small-molecule substrates as the enzyme-catalyzed reaction depletes its internal oxygen reservoir in the presence of small molecules. We exemplify this detection strategy by using glucose-oxidase functionalized polymer dots, yielding high selectivity, large dynamic range, and reversible glucose detection in cell and tissue environments. The transducer-enzyme assembly after subcutaneous implantation provide strong luminescence signal that is transdermally detectable and continuously responsive to blood glucose fluctuations for up to 30 days. In view of a large library of oxygen-consuming enzyme, this strategy is promising for in vivo detection and quantitative determination of a variety of small molecules.
Article
Chronic skin wounds are a common complication of diabetes. When standard wound care fails to heal such wounds, a promising approach consists of using decellularized matrices and other porous scaffold materials to promote the restoration of skin. Proper revascularization is critical for the efficacy of such materials in regenerative medicine. Stromal cell-derived factor-1 (SDF-1) is a chemokine known to play a key role for angiogenesis in ischemic tissues. Herein we developed nanosized SDF-1 liposomes, which were then incorporated into decellularized dermis scaffolds used for skin wound healing applications. SDF-1 peptide associated with liposomes with an efficiency of 80%, and liposomes were easily dispersed throughout the acellular dermis. Acellular dermis spiked with SDF-1 liposomes exhibited more persistent cell proliferation in the dermis, especially in CD31(+) areas, compared to acellular dermis spiked with free SDF-1, which resulted in increased improved wound closure at day 21, and increased granulation tissue thickness at day 28. SDF-1 liposomes may increase the performance of a variety of decellularized matrices used in tissue engineering. This article is protected by copyright. All rights reserved. © 2015 by the Wound Healing Society.
Article
The cellular and molecular mechanisms underpinning tissue repair and its failure to heal are still poorly understood, and current therapies are limited. Poor wound healing after trauma, surgery, acute illness, or chronic disease conditions affects millions of people worldwide each year and is the consequence of poorly regulated elements of the healthy tissue repair response, including inflammation, angiogenesis, matrix deposition, and cell recruitment. Failure of one or several of these cellular processes is generally linked to an underlying clinical condition, such as vascular disease, diabetes, or aging, which are all frequently associated with healing pathologies. The search for clinical strategies that might improve the body's natural repair mechanisms will need to be based on a thorough understanding of the basic biology of repair and regeneration. In this review, we highlight emerging concepts in tissue regeneration and repair, and provide some perspectives on how to translate current knowledge into viable clinical approaches for treating patients with wound-healing pathologies. © 2014, American Association for the Advancement of Science. All rights reserved.
Article
Immunosuppressive therapy is increasingly being used in clinical practice and has been shown to affect wound healing to varying degrees. This article looks at the effects of the newer immunosuppressive agents on wound healing. It is shown that wound healing is impaired via different mechanisms. Some of the animal and human studies are reviewed in more detail. It is shown that some of the newer agents affect wound healing to such an extent that reduction or avoidance of these drugs until complete wound healing is achieved is advocated. More research is required for these newer agents to determine the most appropriate time to introduce them.
Article
Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.