Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin-eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely from human cell lines, provides an excellent opportunity to study in vitro skin biology and can also be used for drug targeting and testing new therapeutics, and ultimately, for incorporating into skin-on-a chip in the future

Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa produces a number of proteases that are associated with virulence and disease progression. A substrate able to detect P. aeruginosa-specific proteolytic activity could help to rapidly alert clinicians to the virulence potential of individual P. aeruginosa strains. For this purpose we designed a set of P. aeruginosa-specific fluorogenic substrates, comprising fluorescence resonance energy transfer (FRET)-labeled peptides, and evaluated their applicability to P. aeruginosa virulence in a range of clinical isolates. A FRET-peptide comprising three glycines (3xGly) was found to be specific for the detection of P. aeruginosa proteases. Further screening of 97 P. aeruginosa clinical isolates showed a wide variation in 3xGly cleavage activity. The absence of 3xGly degradation by a lasI knock out strain indicated that 3xGly cleavage by P. aeruginosa could be quorum sensing (QS)-related, a hypothesis strengthened by the observation of a strong correlation between 3xGly cleavage, LasA staphylolytic activity and pyocyanin production. Additionally, isolates able to cleave 3xGly were more susceptible to the QS inhibiting antibiotic azithromycin (AZM). In conclusion, we designed and evaluated a 3xGly substrate possibly useful as a simple tool to predict virulence and AZM susceptibility

Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1

Evaluation of a novel oral mucosa in vitro implantation model for analysis of molecular interactions with dental abutment surfaces

Background: Abutment surfaces are being designed to promote gingival soft tissue attachment and integration. This forms a seal around prosthetics and consequently ensures long-term implant survival. New scalable and reproducible models are necessary to evaluate and quantify the performance of these surfaces. Purpose: To evaluate a novel implantation model by histomorphometric and immunohistochemical characterization of the interactions between human oral gingival tissue and titanium abutments with either novel anodized or conventional machined surface. Materials and Methods: Abutments were inserted into an organotypic reconstructed human gingiva (RHG) model consisting of differentiated gingival epithelium cells on a fibroblast populated lamina propria hydrogel following a tissue punch. Epithelial attachment, down-growth along the abutment surface, and phenotype were assessed via histomorphology, scanning electron microscopy, and immunohistochemistry 10 days after implantation. Results: The down-growing epithelium transitioned from a gingival margin to a sulcular and junctional epithelium. The sulcus depth and junctional epithelial length were similar to previously reported pre-clinical and clinical lengths. A collagen IV/laminin 5 basement membrane formed between the epithelium and the underlying connective tissue. The RHG expanded in thickness approximately 2-fold at the abutment surface. The model allowed the evaluation of protein expression of adhering soft tissue cells for both tested abutments. Conclusions: The RHG model is the first in vitro 3D model to enable the assessment of not only human epithelial tissue attachment to dental abutments but also the expression of protein markers involved in soft tissue attachment and integration. The two abutments showed no noticeable difference in epithelial attachment

Monocytes co-cultured with reconstructed keloid and normal skin models skew towards M2 macrophage phenotype

Several abnormalities have been reported in the peripheral blood mononuclear cells of keloid-forming patients and particularly in the monocyte cell fraction. The goal of this in vitro study was to determine whether monocytes from keloid-prone patients contribute to the keloid phenotype in early developing keloids, and whether monocyte differentiation is affected by the keloid microenvironment. Therefore, keloid-derived keratinocytes and fibroblasts were used to reconstruct a full thickness, human, in vitro keloid scar model. The reconstructed keloid was co-cultured with monocytes from keloid-forming patients and compared to reconstructed normal skin co-cultured with monocytes from non-keloid-formers. The reconstructed keloid showed increased contraction, dermal thickness (trend) and α-SMA+ staining, but co-culture with monocytes did not further enhance the keloid phenotype. After 2-week culture, all monocytes switched from a CD11chigh/CD14high/CD68low to a CD11chigh/CD14low/CD68high phenotype. However, only monocytes co-cultured with either reconstructed keloid scar or normal skin models skewed towards the more fibrotic M2-macrophage phenotype. There was negligible fibroblast and fibrocyte differentiation in mono- and co-cultured monocytes. These results indicate that monocytes differentiate into M2 macrophages when in the vicinity of early regenerating and repairing tissue, independent of whether the individual is prone to normal or keloid scar formation

Stable reconstructed human gingiva-microbe interaction model: Differential response to commensals and pathogens

BACKGROUND: To investigate human oral health and disease, models are required which represent the interactions between the oral mucosa and microbiome. Our aim was to develop an organotypic model which maintains viability of both host and microbes for an extended period of time. METHODS: Reconstructed Human Gingiva (RHG) were cultured air-lifted with or without penicillin-streptomycin (PS) and topically exposed to Streptococcus gordonii (commensal) or Aggregatibacter actinomycetemcomitans (pathogen) for 72 hours in agar. RHG histology, viability and cytokines (ELISA), and bacterial viability (colony forming units) and location (FISH) were assessed. RESULTS: The low concentration of topically applied agar did not influence RHG viability. Topically applied bacteria in agar remained localized and viable for 72 hours and did not spill over to infect RHG culture medium. PS in RHG culture medium killed topically applied bacteria. Co-culture with living bacteria did not influence RHG viability (Ki67 expression, MTT assay) or histology (epithelium differentiation, Keratin10 expression). RHG exposed to S. gordonii (with or without PS) did not influence low level of IL-6, IL-8, CCL2, CCL5, CCL20 or CXCL1 secretion. However, all cytokines increased (except CCL2) when RHG were co-cultured with A. actinomycetemcomitans. The effect was significantly more in the presence of living, rather than dead, A. actinomycetemcomitans. Both bacteria resulted in increased expression of RHG antimicrobial peptides (AMPs) Elafin and HBD-2, with S. gordonii exposure resulting in the most Elafin secretion. CONCLUSION: This technical advance enables living human oral host-microbe interactions to be investigated during a 72-hour period and shows differences in innate immunology triggered by S. gordonii and A. actinomycetemcomitans

Non–heat inactivated autologous serum increases accuracy of in vitro CFSE lymphocyte proliferation test (LPT) for nickel

Background: Skin patch testing is still seen as the gold standard for the diagnosis of allergic hypersensitivity. For several metals and for patients with a suspected adverse reaction to their medical device implant material, patch testing can be unreliable. The current alternative to metal allergy patch testing is the in vitro lymphocyte proliferation test (LPT) using tritiated thymidine. This method is well-established but requires handling of radioactive material, often uses heat-inactivated allogenic human pooled serum and cannot determine T cell subsets. Objective: To develop a radioactive free LPT by using carboxyfluorescein succinimidyl ester (CFSE) and to evaluate the influence of serum source (heat-inactivated human pooled serum [HI HPS] vs autologous serum) on the sensitivity and specificity of the nickel-specific LPT. Methods: Peripheral blood mononuclear cells derived from nickel-allergic patients and healthy controls were collected, labelled with CFSE and cultured in medium containing 10% HI HPS or 10% autologous serum with or without additional T cell skewing cytokine cocktails (Th1: IL-7/IL-12, Th2: IL-7/IL-4 or Th17: IL-7/IL-23/IL-1β) in the absence or presence of NiSO4. The stimulation index (SI) was calculated as the ratio of divided cells, that is the percentage of CFSElow/neg CD3+CD4+ T-lymphocytes upon nickel stimulation compared to the percentage of CFSElow/neg CD3+CD4+ T-lymphocytes without antigen. These results were compared with the history of Ni allergy, patch test results and the MELISA test. Results: Autologous serum positively influenced Ni-specific proliferation while HI HPS negatively influenced Ni-specific proliferation. The test protocol analysing CD4+ cells and autologous serum without skewing cytokines scored the best diagnostic values (sensitivity 95%; specificity 93%; and overall accuracy 94%) compared to the parallel test using HI HPS (accuracy 60%). Cytokine supplements did not further improve the test protocol which used autologous serum. The protocol using HI HPS could be further improved by addition of the cytokine skewing cocktails. Conclusions: Here, we describe an optimized and highly accurate flow cytometric LPT which comprises of CFSE-labelled cells cultured in autologous serum (not heat inactivated) and without the presence of T cell skewing cytokines. Clinical relevance: The sensitivity and specificity of LPT is enhanced, compared to HI HPS, when autologous serum without skewing cytokines is used

Development of a Full-Thickness Human Skin Equivalent In Vitro

Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin-eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely from human cell lines, provides an excellent opportunity to study in vitro skin biology and can also be used for drug targeting and testing new therapeutics, and ultimately, for incorporating into skin-on-a chip in the future