Combining nitric oxide and calcium sensing for the detection of endothelial dysfunction.

Cardiovascular diseases are the leading cause of death worldwide and are not typically diagnosed until the disease has manifested. Endothelial dysfunction is an early, reversible precursor in the irreversible development of cardiovascular diseases and is characterized by a decrease in nitric oxide production. We believe that more reliable and reproducible methods are necessary for the detection of endothelial dysfunction. Both nitric oxide and calcium play important roles in the endothelial function. Here we review different types of molecular sensors used in biological settings. Next, we review the current nitric oxide and calcium sensors available. Finally, we review methods for using both sensors for the detection of endothelial dysfunction.

Lung tumor microphysiological system with 3D endothelium to evaluate modulators of T-cell migration.

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation, and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor-on-chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a HUVEC-derived vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells migrate; and a chemoattractant/tumor (HCC0827 or NCI-H520) compartment. Here, activated T-cells extravasate and migrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose translational strategies by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration.

The vasculature system plays a critical role in inflammation processes in the body. Vascular inflammatory mechanisms are characterized by disruption of blood vessel wall permeability together with increased immune cell recruitment and migration. There is a critical need to develop models that fully recapitulate changes in vascular barrier permeability in response to inflammatory conditions. We developed a scalable platform for parallel measurements of trans epithelial electrical resistance (TEER) in 64 perfused microfluidic HUVEC tubules under inflammatory conditions. Over 250 tubules where exposed to Tumor necrosis factor alpha (TNFα) and interferon gamma (INF-γ) or human peripheral blood mononuclear cells. The inflammatory response was quantified based on changes TEER and expression of ICAM and VE-cadherin. We observed changes in barrier function in the presence of both inflammatory cytokines and human peripheral blood mononuclear cells, characterized by decreased TEER values, increase in ICAM expression as well changes in endothelial morphology. OrganoPlate 3-lane64 based HUVEC tubules provide a valuable tool for inflammatory studies in an automation compatible manner. Continuous TEER measurements enable long term, sensitive assays for barrier studies. We propose the use of our platform as a powerful tool for modelling endothelial inflammation in combination with immune cell interaction that can be used to screen targets and drugs to treat chronic vascular inflammation.

Proof-of-Concept Organ-on-Chip Study: Topical Cinnamaldehyde Exposure of Reconstructed Human Skin with Integrated Neopapillae Cultured under Dynamic Flow.

Pharmaceutical and personal care industries require human representative models for testing to ensure the safety of their products. A major route of penetration into our body after substance exposure is via the skin. Our aim was to generate robust culture conditions for a next generation human skin-on-chip model containing neopapillae and to establish proof-of-concept testing with the sensitizer, cinnamaldehyde. Reconstructed human skin consisting of a stratified and differentiated epidermis on a fibroblast populated hydrogel containing neopapillae spheroids (RhS-NP), were cultured air-exposed and under dynamic flow for 10 days. The robustness of three independent experiments, each with up to 21 intra-experiment replicates, was investigated. The epidermis was seen to invaginate into the hydrogel towards the neopapille spheroids. Daily measurements of lactate dehydrogenase (LDH) and glucose levels within the culture medium demonstrated high viability and stable metabolic activity throughout the culture period in all three independent experiments and in the replicates within an experiment. Topical cinnamaldehyde exposure to RhS-NP resulted in dose-dependent cytotoxicity (increased LDH release) and elevated cytokine secretion of contact sensitizer specific IL-18, pro-inflammatory IL-1ß, inflammatory IL-23 and IFN-γ, as well as anti-inflammatory IL-10 and IL-12p70. This study demonstrates the robustness and feasibility of complex next generation skin models for investigating skin immunotoxicity.

A Microfluidic 3D Endothelium-on-a-Chip Model to Study Transendothelial Migration of T Cells in Health and Disease.

The recruitment of T cells is a crucial component in the inflammatory cascade of the body. The process involves the transport of T cells through the vascular system and their stable arrest to vessel walls at the site of inflammation, followed by extravasation and subsequent infiltration into tissue. Here, we describe an assay to study 3D T cell dynamics under flow in real time using a high-throughput, artificial membrane-free microfluidic platform that allows unimpeded extravasation of T cells. We show that primary human T cells adhere to endothelial vessel walls upon perfusion of microvessels and can be stimulated to undergo transendothelial migration (TEM) by TNFα-mediated vascular inflammation and the presence of CXCL12 gradients or ECM-embedded melanoma cells. Notably, migratory behavior was found to differ depending on T cell activation states. The assay is unique in its comprehensiveness for modelling T cell trafficking, arrest, extravasation and migration, all in one system, combined with its throughput, quality of imaging and ease of use. We envision routine use of this assay to study immunological processes and expect it to spur research in the fields of immunological disorders, immuno-oncology and the development of novel immunotherapeutics.

TUBE Project: Transport-Derived Ultrafines and the Brain Effects.

The adverse effects of air pollutants on the respiratory and cardiovascular systems are unquestionable. However, in recent years, indications of effects beyond these organ systems have become more evident. Traffic-related air pollution has been linked with neurological diseases, exacerbated cognitive dysfunction, and Alzheimer's disease. However, the exact air pollutant compositions and exposure scenarios leading to these adverse health effects are not known. Although several components of air pollution may be at play, recent experimental studies point to a key role of ultrafine particles (UFPs). While the importance of UFPs has been recognized, almost nothing is known about the smallest fraction of UFPs, and only >23 nm emissions are regulated in the EU. Moreover, the role of the semivolatile fraction of the emissions has been neglected. The Transport-Derived Ultrafines and the Brain Effects (TUBE) project will increase knowledge on harmful ultrafine air pollutants, as well as semivolatile compounds related to adverse health effects. By including all the major current combustion and emission control technologies, the TUBE project aims to provide new information on the adverse health effects of current traffic, as well as information for decision makers to develop more effective emission legislation. Most importantly, the TUBE project will include adverse health effects beyond the respiratory system; TUBE will assess how air pollution affects the brain and how air pollution particles might be removed from the brain. The purpose of this report is to describe the TUBE project, its background, and its goals.

Reconstructed human skin shows epidermal invagination towards integrated neopapillae indicating early hair follicle formation in vitro.

Application of reconstructed human Skin (RhS) is a promising approach for the treatment of extensive wounds and for drug efficacy and safety testing. However, incorporating appendages, such as hair follicles, into RhS still remains a challenge. The hair follicle plays a critical role in thermal regulation, dispersion of sweat and sebum, sensory and tactile functions, skin regeneration, and repigmentation. The aim of this study was to determine whether human neopapilla could be incorporated into RhS (differentiated epidermis on fibroblast and endothelial cell populated dermis) and whether the neopapillae maintain their inductive follicular properties in vitro. Neopapillae spheroids, constructed from expanded and self-aggregating dermal papilla cells, synthesized extracellular matrix typically found in follicular papillae. Compared with dermal fibroblasts, neopapillae showed increased expression of multiple genes (Wnt5a, Wnt10b, and LEF1) known to regulate hair development and also increased secretion of CXCL1, which is a strong keratinocyte chemoattractant. When neopapillae were incorporated into the dermis of RhS, they stimulated epidermal down-growth resulting in engulfment of the neopapillae sphere. Similar to the native hair follicle, the differentiated invaginating epidermis inner side was keratin 10 positive and the undifferentiated outer side keratin 10 negative. The outer side was keratin 15 positive confirming the undifferentiated nature of these keratinocytes aligning a newly formed collagen IV, laminin V positive basement membrane within the hydrogel. In conclusion, we describe a RhS model containing neopapillae with hair follicle-inductive properties. Importantly, epidermal invagination occurred to engulf the neopapillae, thus demonstrating in vitro the first steps towards hair follicle morphogenesis in RhS.

Regenerative potential of adipocytes in hypertrophic scars is mediated by myofibroblast reprogramming.

Abnormal scarring is a major challenge in modern medicine. The central role of myofibroblasts and TGF-ß signaling in scarring is widely accepted, but effective treatment options are missing. Autologous fat grafting is a novel approach that has led to significant improvements in the functionality and appearance of scar tissue. While the underlying mechanism is unknown, the potential role of paracrine effects of adipocytes has been discussed. Hence, with the aim of unraveling the regenerative potential of adipocytes, their effects on in vitro differentiated myofibroblasts and on fibroblasts from hypertrophic scars were investigated. Exposure to adipocyte-conditioned medium significantly decreased the expression of the myofibroblast marker α-SMA and ECM components, indicating the occurrence of myofibroblast reprogramming. Further analysis demonstrated that myofibroblast reprogramming was triggered by BMP-4 and activation of PPARγ signaling initiating tissue remodeling. These findings may pave the way for novel therapeutic strategies for the prevention or treatment of hypertrophic scars. KEY MESSAGES: Adipocytes induce distinct regenerative effects in hypertrophic scar tissue. Adipocytes secrete several proteins which are involved in wound healing and regeneration. Adipocytes secrete BMP-4 which activates myofibroblast reprogramming. Mediators secreted by adipocytes directly and indirectly activate PPARγ which exerts distinct anti-fibrotic effects. These findings may pave the way for novel therapeutic strategies for the prevention or treatment of hypertrophic scars.

Skin substitutes are more potent than dermal or epidermal substitutes in stimulating endothelial cell sprouting.

BACKGROUND: Therapy resistant ulcers are wounds that remain open for a long time period and often arise from chronic venous disease, prolonged pressure or diabetes. For healing of chronic wounds, revitalization of the inert wound bed, which is achieved by angiogenic sprouting of new blood vessels is of great importance. An alternative treatment option to conventional therapies is the use of skin substitutes: dermal (DS), epidermal (ES) or bi-layered skin substitutes (SS). The aim of this study was to determine the mode of action of an autologous SS, ES and DS with regards to endothelial cell proliferation, migration and angiogenic sprouting into a fibrin hydrogel. RESULTS: SS consists of a fully differentiated epidermis expanding over the acellular donor dermis (AD) which has become repopulated with fibroblasts. DS is the same construct as SS but without the epidermis and ES is the same construct as SS but without the fibroblasts. As a control, AD was used throughout. It was found that the bi-layered SS was the most potent substitute in inducing migration and sprouting of endothelial cells. The cross talk between dermis and epidermis resulted in the strongest induction of sprouting via VEGF and uPAR. ES stimulated sprouting more than DS again via VEGF and uPAR. The slight induction of sprouting mediated by DS was not mediated by VEGF, but was in part stimulated through uPAR. CONCLUSION: This in vitro study supports our clinical observations that a bi-layered SS is a strong stimulator of angiogenesis and therefore has the potential to revitalize an inert wound bed.

An Organotypic Reconstructed Human Urethra to Study Chlamydia trachomatis Infection.

Organotypic models to investigate host-microbiome interactions are still a challenge for the field of tissue engineering. This is particularly the case for organs such as the urethra. Several cell line, animal, and tissue models are available to study Chlamydia trachomatis infections, but none fully reflects natural infection in native human tissue. Therefore, we developed an organotypic reconstructed human urethral model (RhU) to study invasive and noninvasive strains of C. trachomatis. Primary urethra cells were used to reconstruct epithelium on a fibroblast populated collagen-fibrin hydrogel, yielding a RhU. Immunohistochemistry was used to compare RhU with native urethral tissue and to visualize the location of C. trachomatis bacteria in RhU after 10-day exposure. RhU closely resembled native urethral tissue with respect to proliferation and differentiation markers (keratins 6, 10, 13, 17, involucrin, SKALP [skin-derived antileucoproteinase], vimentin, and CD31). Exposure of RhU to noninvasive and invasive C. trachomatis strains revealed relevant differences in infection ability because inclusions were observed (indicating active infection) in the epithelial layer after 10 days exposure only to the invasive strain. The noninvasive strain remained localized on the surface of the epithelial layer. Human primary urethral fibroblasts and keratinocytes can be used to construct RhU that closely resembles native tissue and can be used to investigate active C. trachomatis infections. RhU provides a promising model to investigate host-microbiome interactions such as, but not limited to, the human pathogenesis of C. trachomatis.

Characterization of In Vitro Reconstructed Human Normotrophic, Hypertrophic, and Keloid Scar Models.

To understand scar pathology, develop new drugs, and provide a platform for personalized medicine, physiologically relevant human scar models are required, which are characteristic of different scar pathologies. Hypertrophic scars and keloids are two types of abnormal scar resulting from unknown abnormalities in the wound healing process. While they display different clinical behavior, differentiation between the two can be difficult-which in turn means that it is difficult to develop optimal therapeutic strategies. The aim of this study was to develop in vitro reconstructed human hypertrophic and keloid scar models and compare these to normotrophic scar and normal skin models to identify distinguishing biomarkers. Keratinocytes and fibroblasts from normal skin and scar types (normotrophic, hypertrophic, keloid) were used to reconstruct skin models. All skin models showed a reconstructed differentiated epidermis on a fibroblast populated collagen-elastin matrix. Both abnormal scar types showed increased contraction, dermal thickness, and myofibroblast staining compared to normal skin and normotrophic scar. Notably, the expression of extracellular matrix associated genes showed distinguishing profiles between all scar types and normal skin (hyaluronan synthase-1, matrix-metalloprotease-3), between keloid and normal skin (collagen type IV), between normal scar and keloid (laminin α1), and between keloid and hypertrophic scar (matrix-metalloprotease-1, integrin α5). Also, inflammatory cytokine and growth factor secretion (CCL5, CXCL1, CXCL8, CCL27, IL-6, HGF) showed differential secretion between scar types. Our results strongly suggest that abnormal scars arise from different pathologies rather than simply being on different ends of the scarring spectrum. Furthermore, such normal skin and scar models together with biomarkers, which distinguish the different scar types, would provide an animal free, physiologically relevant scar diagnostic and drug testing platform for the future.

Endothelial cells enhance adipose mesenchymal stromal cell-mediated matrix contraction via ALK receptors and reduced follistatin: Potential role of endothelial cells in skin fibrosis.

Abnormal cutaneous wound healing can lead to formation of fibrotic hypertrophic scars. Although several clinical risk factors have been described, the cross-talk between different cell types resulting in hypertrophic scar formation is still poorly understood. The aim of this in vitro study was to investigate whether endothelial cells (EC) may play a role in skin fibrosis, for example, hypertrophic scar formation after full-thickness skin trauma. Using a collagen/elastin matrix, we developed an in vitro fibrosis model to study the interaction between EC and dermal fibroblasts or adipose tissue-derived mesenchymal stromal cells (ASC). Tissue equivalents containing dermal fibroblasts and EC displayed a normal phenotype. In contrast, tissue equivalents containing ASC and EC displayed a fibrotic phenotype indicated by contraction of the matrix, higher gene expression of ACTA2, COL1A, COL3A, and less secretion of follistatin. The contraction was in part mediated via the TGF-ß pathway, as both inhibition of the ALK4/5/7 receptors and the addition of recombinant follistatin resulted in decreased matrix contraction (75 ± 11% and 24 ± 8%, respectively). In conclusion, our study shows that EC may play a critical role in fibrotic events, as seen in hypertrophic scars, by stimulating ASC-mediated matrix contraction via regulation of fibrosis-related proteins.

Burn Eschar Stimulates Fibroblast and Adipose Mesenchymal Stromal Cell Proliferation and Migration but Inhibits Endothelial Cell Sprouting.

The majority of full-thickness burn wounds heal with hypertrophic scar formation. Burn eschar most probably influences early burn wound healing, since granulation tissue only forms after escharotomy. In order to investigate the effect of burn eschar on delayed granulation tissue formation, burn wound extract (BWE) was isolated from the interface between non-viable eschar and viable tissue. The influence of BWE on the activity of endothelial cells derived from dermis and adipose tissue, dermal fibroblasts and adipose tissue-derived mesenchymal stromal cells (ASC) was determined. It was found that BWE stimulated endothelial cell inflammatory cytokine (CXCL8, IL-6 and CCL2) secretion and migration. However, BWE had no effect on endothelial cell proliferation or angiogenic sprouting. Indeed, BWE inhibited basic Fibroblast Growth Factor (bFGF) induced endothelial cell proliferation and sprouting. In contrast, BWE stimulated fibroblast and ASC proliferation and migration. No difference was observed between cells isolated from dermis or adipose tissue. The inhibitory effect of BWE on bFGF-induced endothelial proliferation and sprouting would explain why excessive granulation tissue formation is prevented in full-thickness burn wounds as long as the eschar is still present. Identifying the eschar factors responsible for this might give indications for therapeutic targets aimed at reducing hypertrophic scar formation which is initiated by excessive granulation tissue formation once eschar is removed.

Progress and Future Prospectives in Skin-on-Chip Development with Emphasis on the use of Different Cell Types and Technical Challenges.

Understanding the healthy and diseased state of skin is important in many areas of basic and applied research. Although the field of skin tissue engineering has advanced greatly over the last years, current in vitro skin models still do not mimic the complexity of the human skin. Skin-on-chip and induced pluripotent stem cells (iPSC) might be key technologies to improve in vitro skin models. This review summarizes the state of the art of in vitro skin models with regard to cell sources (primary, cell line, iPSC) and microfluidic devices. It can be concluded that iPSC have the potential to be differentiated into many kinds of immunologically matched cells and skin-on-chip technology might lead to more physiologically relevant skin models due to the controlled environment, possible exchange of immune cells, and an increased barrier function. Therefore the combination of iPSC and skin-on-chip is expected to lead to superior healthy and diseased in vitro skin models.

Extensive Characterization and Comparison of Endothelial Cells Derived from Dermis and Adipose Tissue: Potential Use in Tissue Engineering.

Tissue-engineered constructs need to become quickly vascularized in order to ensure graft take. One way of achieving this is to incorporate endothelial cells (EC) into the construct. The adipose tissue stromal vascular fraction (adipose-SVF) might provide an alternative source for endothelial cells as adipose tissue can easily be obtained by liposuction. Since adipose-EC are now gaining more interest in tissue engineering, we aimed to extensively characterize endothelial cells from adipose tissue (adipose-EC) and compare them with endothelial cells from dermis (dermal-EC). The amount of endothelial cells before purification varied between 4-16% of the total stromal population. After MACS selection for CD31 positive cells, a >99% pure population of endothelial cells was obtained within two weeks of culture. Adipose- and dermal-EC expressed the typical endothelial markers PECAM-1, ICAM-1, Endoglin, VE-cadherin and VEGFR2 to a similar extent, with 80-99% of the cell population staining positive. With the exception of CXCR4, which was expressed on 29% of endothelial cells, all other chemokine receptors (CXCR1, 2, 3, and CCR2) were expressed on less than 5% of the endothelial cell populations. Adipose-EC proliferated similar to dermal-EC, but responded less to the mitogens bFGF and VEGF. A similar migration rate was found for both adipose-EC and dermal-EC in response to bFGF. Sprouting of adipose-EC and dermal-EC was induced by bFGF and VEGF in a 3D fibrin matrix. After stimulation of adipose-EC and dermal-EC with TNF-α an increased secretion was seen for PDGF-BB, but not uPA, PAI-1 or Angiopoietin-2. Furthermore, secretion of cytokines and chemokines (IL-6, CCL2, CCL5, CCL20, CXCL1, CXCL8 and CXCL10) was also upregulated by both adipose- and dermal-EC. The similar characteristics of adipose-EC compared to their dermal-derived counterpart make them particularly interesting for skin tissue engineering. In conclusion, we show here that adipose tissue provides for an excellent source of endothelial cells for tissue engineering purposes, since they are readily available, and easily isolated and amplified.

Inhibited early immunologic response is associated with hypertrophic scarring.

This study aimed to examine changes in the inflammatory response in early hypertrophic compared to normal wound healing. The immune system is thought to be involved in hypertrophic scar formation. However, the exact mechanism and time of onset of the derailment remain unknown. In a prospective observational study, skin biopsies were taken directly postwounding and 3 hours later from patients who had elective cardiothoracic surgery. The skin biopsies were analysed for mRNA, proteins and cells involved in the early inflammatory phase of wound healing. The endpoint was scar outcome (hypertrophic (HTS) or normal (NTS)) at one year after surgery. There were significant differences between the NTS and HTS groups regarding the fold changes of mRNA expression of P-selectin during surgery. Postoperative skin concentrations of inflammatory proteins IL-6, IL-8 and CCL2 were significantly lower in the HTS compared to the NTS group. Also, a trend of higher pre-operative M2 macrophage numbers was observed in the HTS group. Neutrophil numbers increased equally during surgery in both groups. The increase of P-selectin mRNA in hypertrophic wound healing could affect leucocyte migration. The decreased concentrations of inflammatory proteins in hypertrophic wound healing indicate a reduced inflammatory response, which has consequences for the treatment of hypertrophic scarring during the early inflammatory phase. In a conclusion, alterations of wound healing associated with hypertrophic scarring are visible as early as 3 hours postwounding and include a reduced rather than increased inflammatory protein response.

Immune-competent human skin disease models.

All skin diseases have an underlying immune component. Owing to differences in animal and human immunology, the majority of drugs fail in the preclinical or clinical testing phases. Therefore animal alternative methods that incorporate human immunology into in vitro skin disease models are required to move the field forward. This review summarizes the progress, using examples from fibrosis, autoimmune diseases, psoriasis, cancer and contact allergy. The emphasis is on co-cultures and 3D organotypic models. Our conclusion is that current models are inadequate and future developments with immune-competent skin-on-chip models based on induced pluripotent stem cells could provide a next generation of skin models for drug discovery and testing.

Methods to study differences in cell mobility during skin wound healing in vitro.

Wound healing events which occur in humans are difficult to study in animals due to differences in skin physiology. Furthermore there are increasing restrictions in Europe for using animals for testing the therapeutic properties of new compounds. Therefore, in line with the 3Rs (reduction, refinement and replacement of test animals), a number of human in vitro models of different levels of complexity have been developed to investigate cell mobility during wound healing. Keratinocyte, melanocyte, fibroblast and endothelial cell mobility are described, since these are the residential cells which are responsible for restoring the main structural features of the skin. A monolayer scratch assay is used to study random fibroblast and endothelial cell migration in response to EGF and bFGF respectively and a chemotactic assay is used to study directional fibroblast migration towards CCL5. In order to study endothelial sprouting in response to bFGF or VEGF, which involves continuous degradation and resynthesis of a 3D matrix, a fibrin gel is used. Human physiologically relevant tissue-engineered skin models are used to investigate expansion of the stratified, differentiated epidermis (keratinocytes and melanocytes) over a fibroblast populated dermis and also to study migration and distribution of fibroblasts into the dermis. Together these skin models provide a platform for testing the mode of action of novel compounds for enhanced and scar free wound healing.

Different wound healing properties of dermis, adipose, and gingiva mesenchymal stromal cells.

Oral wounds heal faster and with better scar quality than skin wounds. Deep skin wounds where adipose tissue is exposed, have a greater risk of forming hypertrophic scars. Differences in wound healing and final scar quality might be related to differences in mesenchymal stromal cells (MSC) and their ability to respond to intrinsic (autocrine) and extrinsic signals, such as human salivary histatin, epidermal growth factor, and transforming growth factor beta1. Dermis-, adipose-, and gingiva-derived MSC were compared for their regenerative potential with regards to proliferation, migration, and matrix contraction. Proliferation was assessed by cell counting and migration using a scratch wound assay. Matrix contraction and alpha smooth muscle actin was assessed in MSC populated collagen gels, and also in skin and gingival full thickness tissue engineered equivalents (reconstructed epithelium on MSC populated matrix). Compared to skin-derived MSC, gingiva MSC showed greater proliferation and migration capacity, and less matrix contraction in full thickness tissue equivalents, which may partly explain the superior oral wound healing. Epidermal keratinocytes were required for enhanced adipose MSC matrix contraction and alpha smooth muscle actin expression, and may therefore contribute to adverse scarring in deep cutaneous wounds. Histatin enhanced migration without influencing proliferation or matrix contraction in all three MSC, indicating that salivary peptides may have a beneficial effect on wound closure in general. Transforming growth factor beta1 enhanced contraction and alpha smooth muscle actin expression in all three MSC types when incorporated into collagen gels. Understanding the mechanisms responsible for the superior oral wound healing will aid us to develop advanced strategies for optimal skin regeneration, wound healing and scar formation.

Suppressed inflammatory gene expression during human hypertrophic scar compared to normotrophic scar formation.

Hypertrophic scar formation is a result of adverse cutaneous wound healing. The pathogenesis of hypertrophic scar formation is still poorly understood. A problem next to the lack of suitable animal models is that often normal skin is compared to hypertrophic scar (HTscar) and not to normotrophic scar (NTscar) tissue. Another drawback is that often only one time period after wounding is studied, while scar formation is a dynamic process over a period of several months. In this study, we compared the expression of genes involved in inflammation, angiogenesis and extracellular matrix (ECM) formation and also macrophage infiltration in biopsies obtained before and up to 52 weeks after standard surgery in five patients who developed HTscar and six patients who developed NTscar. It was found that HTscar formation coincided with a prolonged decreased expression of inflammatory genes (TNFα, IL-1α, IL-1RN, CCL2, CCL3, CXCL2, CXCR2, C3 and IL-10) and an extended increased expression of ECM-related genes (PLAU, Col3A1, TGFß3). This coincided with a delayed but prolonged infiltration of macrophages (type 2) in HTscar tissue compared to NTscar tissue. These findings were supported by immunohistochemical localization of proteins coding for select genes named above. Our study emphasizes that human cutaneous wound healing is a dynamic process that is needed to be studied over a period of time rather than a single point of time. Taken together, our results suggest innate immune stimulatory therapies may be a better option for improving scar quality than the currently used anti-inflammatory scar therapies.