Exploring hypotheses of the actions of TGF-beta 1 in epidermal wound healing using a 3D computational multiscale model of the human epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-beta 1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-beta 1 literature-derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units ( keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-beta 1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged ( by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-beta 1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-beta 1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing

Development of a Three Dimensional Multiscale Computational Model of the Human Epidermis

Transforming Growth Factor (TGF-β1) is a member of the TGF-beta superfamily ligand-receptor network. and plays a crucial role in tissue regeneration. The extensive in vitro and in vivo experimental literature describing its actions nevertheless describe an apparent paradox in that during re-epithelialisation it acts as proliferation inhibitor for keratinocytes. The majority of biological models focus on certain aspects of TGF-β1 behaviour and no one model provides a comprehensive story of this regulatory factor's action. Accordingly our aim was to develop a computational model to act as a complementary approach to improve our understanding of TGF-β1. In our previous study, an agent-based model of keratinocyte colony formation in 2D culture was developed. In this study this model was extensively developed into a three dimensional multiscale model of the human epidermis which is comprised of three interacting and integrated layers: (1) an agent-based model which captures the biological rules governing the cells in the human epidermis at the cellular level and includes the rules for injury induced emergent behaviours, (2) a COmplex PAthway SImulator (COPASI) model which simulates the expression and signalling of TGF-β1 at the sub-cellular level and (3) a mechanical layer embodied by a numerical physical solver responsible for resolving the forces exerted between cells at the multi-cellular level. The integrated model was initially validated by using it to grow a piece of virtual epidermis in 3D and comparing the in virtuo simulations of keratinocyte behaviour and of TGF-β1 signalling with the extensive research literature describing this key regulatory protein. This research reinforces the idea that computational modelling can be an effective additional tool to aid our understanding of complex systems. In the accompanying paper the model is used to explore hypotheses of the functions of TGF-β1 at the cellular and subcellular level on different keratinocyte populations during epidermal wound healing

PDGF-BB does not accelerate healing in diabetic mice with splinted skin wounds.

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used

Design and Validation of Delivery Systems for Galectin-3 for Skin Healing Applications

Chronic wounds present a significant burden to patients, causing pain, impairing limb function, and often resulting in the need for amputation. Treatment of chronic dermal wounds is challenging, with current therapies showing limited efficacy in clinical trials. As galectin-3 has been implicated in several wound healing processes, its efficacy as a therapeutic in skin healing was investigated in this study. An electrospun gelatin scaffold loaded with galectin-3 was developed as a delivery system. The influence of human recombinant galectin-3 in skin healing, when delivered topically and using an electrospun scaffold, was then investigated in wild type and diabetic mice. Electrospun gelatin/galectin-3 scaffolds were developed having an overall porosity of approximately 83% and average pore diameter of approximately 1.15 μm. The scaffolds supported the adhesion, deposition of matrix, and proliferation of human dermal fibroblasts in vitro providing evidence that they are biocompatible. In vivo treatment of wounds with topical galectin-3 and gelatin/galectin-3 scaffolds did not affect wound closure, re-epithelialization or macrophage phenotypes in the wound, casting doubt on its efficacy for these processes. Future work is required to elucidate the exact pathological contexts in which galectin-3 might modulate inflammation in skin healing

The use of Manuka honey as treatment of wounds in horses

Dissertação de Mestrado Integrado em Medicina VeterináriaAntimicrobial compounds are essentially important in reducing the global burden of infectious diseases. However, as resistant pathogens develop and spread, the effectiveness of the antibiotics is diminished. This type of bacterial resistance to the antimicrobial agents poses a very serious threat to public health, and for all kinds of antibiotics, including the major lastresort drugs, the frequencies of resistance are increasing worldwide. In addition to the generic properties of honey, Manuka honey has a non-peroxide antimicrobial activity largely attributed to methylglyoxal. Evidence that Manuka honey can also modulate the initial inflammatory response, through activation of toll-like receptor 4 on monocytes to enhance production of cytokines important in tissue repair and regeneration, have arisen. Distal limb wounds in horses usually heal by second intention because primary or delayed primary closure cannot be accomplished. In most cases these wounds have gross contamination and moderate to severe tissue loss that would make closure difficult due to a greater wound retraction, slower rates and earlier cessation of wound contraction. They must heal completely through the process of contraction, granulation, and epithelialization. The aim of applying topical medication to wounds left to heal by second intention is to manipulate the wound environment. As a topical preparation, Manuka honey has been shown to modulate the earlyperiod of second-intention wound healing in the horse, but this precise mechanism of action is still unclear, and has also been shown to control the contamination of the wound environment. The objective of this study is to show the actual evidences of the use of Manuka honey in the process of wound healing in Veterinary medicine, more specifically in horses by presenting a brief review of literature with inclusion and exclusion criteria, and by presenting three cases where Manuka honey treatment had a beneficial role and great outcomes, which provides evidence that it is an alternative treatment worth having in mind when dealing with wounds in horses.RESUMO - O uso de mel de Manuka no tratamento de feridas em cavalos - Os antibióticos são extremamente importantes na redução das doenças infeciosas que atualmente existem a nível mundial. No entanto, à medida que emergem as bactérias resistentes, a eficácia dos antibióticos torna-se mais limitada. A resistência bacteriana aos compostos antimicrobianos, representa uma ameaça muito séria à saúde pública uma vez que continua a aumentar exponencialmente em todo o Mundo. Para além das propriedades gerais do mel, o mel de Manuka tem uma atividade antimicrobiana diferente, na qual a sua atividade não surge devido a ter na sua composição o peróxido de hidrogénio mas sim devido maioritariamente à presença do metilglioxal. A qualidade e quantidade de evidência de que o mel de Manuka também tem capacidade de modular a resposta inflamatória inicial, através da ativação do TRL- 4 nos monócitos fazendo com que haja um aumento da produção de citoquinas importantes na reparação e regeneração tecidular, tem crescido. As feridas do membro distal em cavalos cicatrizam geralmente por segunda intenção, pois a oclusão por primeira intenção não é possível devido a um variado leque de fatores. Na maioria dos casos, estas feridas apresentam um nível elevado de contaminação, uma perda de tecido avaliada de moderada a grave, uma grande taxa de retração do tecido, tempos de contração da pele mais lentos e interrupção antecipada da contração da ferida. O objetivo de aplicar medicação tópica nas feridas é a manipulação do ambiente da mesma. O mel de Manuka quando utilizado topicamente demonstra que é capaz de modular as primeiras fases de cicatrização das feridas que fecham por segunda intenção em equinos, assim como é capaz de atuar no controlo da contaminação que caracteriza estas feridas. Neste trabalho, foi realizada uma revisão sistemática da literatura, com o objetivo de demonstrar as evidências que existem, assim como a sua qualidade quando se fala no uso do mel de Manuka no processo de cicatrização de feridas em medicina veterinária, mais especificamente em equinos, apresentando critérios de inclusão e exclusão. São também reportados três casos clínicos, onde o tratamento com mel de Manuka teve um papel importante e resultados benéficos de forma a validar e fornecer evidências de que é um tratamento alternativo que vale a pena ter em mente quando se lida com feridas em cavalos.N/

Investigating the Role of Epithelial-mesenchymal Crosstalk in the Pathology of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown aetiology, characterised by the progressive and irreversible scarring of parenchymal lung tissue that leads to respiratory failure and death. The disease is understood to be driven by an impaired and aberrant wound healing response, with an inappropriate reactivation of developmental signalling. The greatest risk factor for the disease is age, which is a process intimately associated with an increase in the burden of senescent cells. Such cells acquire a unique secretory phenotype and are known to have a significant impact on their local micro-environment. It was hypothesised that an alteration in epithelial-mesenchymal secretory communication, due to senescent-like changes in the fibroblast phenotype, may detrimentally contribute to lung homeostasis. An in vitro model of the lung airway was established in which primary human lung fibroblasts (HLFs) were co-cultured with human bronchial epithelial cells (HBECs). HBECs were cultured on a semi-permeable, transwell insert and co-cultured with either normal (NHLF), fibrotic (FHLF) or senescent fibroblasts. Over 72 hrs of co-culture, wound healing was assessed, via an epithelial scratch assay, and epithelial regeneration was measured, via trans-epithelial electrical resistance. Co-culture with NHLFs improves epithelial regeneration, however, FHLFs and senescent cells in co-culture show a diminished ability to promote epithelial regeneration and wound repair. The secretory repertoire of these cells contains elevated levels of IL-6, CXCL8, CXCL1 and GCSF (when assessed at both an RNA and protein level), factors strongly associated with the senescent phenotype. Targeting this secretome via treatment with the JAK 1/2 inhibitor Ruxolitinib attenuates these impairments and may point towards a new therapeutic strategy for the treatment of IPF

Self-Assembling Peptide Nanofiber Scaffolds Accelerate Wound Healing

Cutaneous wound repair regenerates skin integrity, but a chronic failure to heal results in compromised tissue function and increased morbidity. To address this, we have used an integrated approach, using nanobiotechnology to augment the rate of wound reepithelialization by combining self-assembling peptide (SAP) nanofiber scaffold and Epidermal Growth Factor (EGF). This SAP bioscaffold was tested in a bioengineered Human Skin Equivalent (HSE) tissue model that enabled wound reepithelialization to be monitored in a tissue that recapitulates molecular and cellular mechanisms of repair known to occur in human skin. We found that SAP underwent molecular self-assembly to form unique 3D structures that stably covered the surface of the wound, suggesting that this scaffold may serve as a viable wound dressing. We measured the rates of release of EGF from the SAP scaffold and determined that EGF was only released when the scaffold was in direct contact with the HSE. By measuring the length of the epithelial tongue during wound reepithelialization, we found that SAP scaffolds containing EGF accelerated the rate of wound coverage by 5 fold when compared to controls without scaffolds and by 3.5 fold when compared to the scaffold without EGF. In conclusion, our experiments demonstrated that biomaterials composed of a biofunctionalized peptidic scaffold have many properties that are well-suited for the treatment of cutaneous wounds including wound coverage, functionalization with bioactive molecules, localized growth factor release and activation of wound repair

Development Of Granulation Tissue Mimetic Scaffolds For Skin Healing

Impaired skin healing is a significant and growing clinical concern, particularly in relation to diabetes, venous insufficiency and immobility. Previously, we developed electrospun scaffolds for the delivery of periostin (POSTN) and connective tissue growth factor 2 (CCN2), matricellular proteins involved in the proliferative phase of healing. This study aimed to design and validate a novel electrosprayed coaxial microsphere for the encapsulation of fibroblast growth factor 9 (FGF9), as a component of the POSTN/CCN2 scaffold, to promote angiogenic stability during wound healing. For the first time, we observed a pro-proliferative effect of FGF9 on human dermal fibroblasts (HDF) in vitro, indicating a potential cellular mechanism of action during wound healing. POSTN/CCN2 scaffolds containing encapsulated FGF9 decreased wound diameter, with no negative or adverse effects at day 7, in a porcine model of acute cutaneous wound healing. Future work is required to investigate the effect of the POSTN/CCN2/FGF9 scaffold during impaired healing

Active regulation of the epidermal calcium profile

A distinct calcium profile is strongly implicated in regulating the multi-layered structure of the epidermis. However, the mechanisms that govern the regulation of this calcium profile are currently unclear. It clearly depends on the relatively impermeable barrier of the stratum corneum (passive regulation) but may also depend on calcium exchanges between keratinocytes and extracellular fluid (active regulation). Using a mathematical model that treats the viable sublayers of unwounded human and murine epidermis as porous media and assumes that their calcium profiles are passively regulated, we demonstrate that these profiles are also actively regulated. To obtain this result, we found that diffusion governs extracellular calcium motion in the viable epidermis and hence intracellular calcium is the main source of the epidermal calcium profile. Then, by comparison with experimental calcium profiles and combination with a hypothesised cell velocity distribution in the viable epidermis, we found that the net influx of calcium ions into keratinocytes from extracellular fluid may be constant and positive throughout the stratum basale and stratum spinosum, and that there is a net outflux of these ions in the stratum granulosum. Hence the calcium exchange between keratinocytes and extracellular fluid differs distinctly between the stratum granulosum and the underlying sublayers, and these differences actively regulate the epidermal calcium profile. Our results also indicate that plasma membrane dysfunction may be an early event during keratinocyte disintegration in the stratum granulosum