Isolation and characterisation of two epithelial-like cell lines from the gills of Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon) and their use in aquatic toxicology.

In vitro gill models are becoming increasingly important in aquatic toxicology, yet the fish gill invitrome is underrepresented, encompassing approximately 0.1% of extant species. Here, we describe the establishment and characterisation of two gill-derived, epithelial-like cell lines isolated from fish species of significant importance to New Zealand: Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon). Designated CAgill1PFR (Chrysophrys auratus, gill 1, Plant & Food Research) and OTgill1PFR (Oncorhynchus tshawytscha, gill 1, Plant & Food Research), these cell lines have each been passaged greater than each 70 times over several years and are considered spontaneously immortalised. Both cell lines required serum for growth and exhibited differential responses to basal media formulations. CAgill1PFR was sensitive to low temperatures (4 °C) but replicated at high temperatures (30 °C), whereas OTgill1PFR was sensitive to high temperatures but remained viable at low temperatures, mirroring the natural environment of their host species. Immunostaining revealed expression of epithelial cell markers cytokeratin and E-cadherin, alongside positivity for the mesenchymal cell marker, vimentin. CAgill1PFR was more sensitive to the environmental toxin 3,4 dichloroaniline than OTgill1PFR through measurements of metabolic activity, membrane integrity, and lysosomal function. Furthermore, CAgill1PFR produced less CYP1A activity, indicative of ongoing biotransformation processes, in response to beta-naphthoflavone than OTgill1PFR. These cell lines expand the toolbox of resources and emphasise the need for species-specific aquatic toxicology research.

Macrocystis pyrifera Lipids Reduce Cytokine-Induced Pro-Inflammatory Signalling and Barrier Dysfunction in Human Keratinocyte Models.

Atopic dermatitis is a chronic condition where epidermal barrier dysfunction and cytokine production by infiltrating immune cells exacerbate skin inflammation and damage. A total lipid extract from Macrocystis pyrifera, a brown seaweed, was previously reported to suppress inflammatory responses in monocytes. Here, treatment of human HaCaT keratinocytes with M. pyrifera lipids inhibited tumour necrosis factor (TNF)-α induced TNF receptor-associated factor 2 and monocyte chemoattractant protein (MCP)-1 protein production. HaCaT cells stimulated with TNF-α, interleukin (IL)-4, and IL-13 showed loss of claudin-1 tight junctions, but little improvement was observed following lipid pre-treatment. Three-dimensional cultures of HaCaT cells differentiated at the air-liquid interface showed increased MCP-1 production, loss of claudin-1 tight junctions, and trans-epidermal leakage with TNF-α, IL-4, and IL-13 stimulation, with all parameters reduced by lipid pre-treatment. These findings suggest that M. pyrifera lipids have anti-inflammatory and barrier-protective effects on keratinocytes, which may be beneficial for the treatment of atopic dermatitis or other skin conditions.

ICTV Virus Taxonomy Profile: Poxviridae 2023.

Poxviridae is a family of enveloped, brick-shaped or ovoid viruses. The genome is a linear molecule of dsDNA (128-375 kbp) with covalently closed ends. The family includes the sub-families Entomopoxvirinae, whose members have been found in four orders of insects, and Chordopoxvirinae, whose members are found in mammals, birds, reptiles and fish. Poxviruses are important pathogens in various animals, including humans, and typically result in the formation of lesions, skin nodules, or disseminated rash. Infections can be fatal. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Poxviridae, which is available at ictv.global/report/poxviridae.

Methods to Assess Chemokine Binding and Anti-chemotactic Activity of Virus Proteins.

Chemokines are key instigators of inflammatory and immune responses. Viruses can suppress these responses by secreting proteins that interfere with chemokine action. These proteins bind to chemokines and block the host's ability to recruit immune cells to sites of infection, thus facilitating virus replication and spread. When produced recombinantly, chemokine binding proteins provide a formidable resource to deploy against human disease. Here, we describe an enzyme-linked immunosorbent inhibition assay and a chemotaxis inhibition assay that are employed to assess the chemokine binding strength and anti-chemotactic activity of viral proteins. These assays are quick and reproducible, and are thus ideal for screening putative or modified chemokine binding proteins as the first step in their development as therapeutics.

Parapoxvirus Interleukin-10 Homologues Vary in Their Receptor Binding, Anti-Inflammatory, and Stimulatory Activities.

Homologues of interleukin (IL)-10, a pleiotropic immunomodulatory cytokine, have been identified in the Parapoxvirus genus. The first identified, Orf virus (ORFV) IL-10, greatly enhanced infection of its host, exhibiting immune modulatory effects equivalent to human IL-10. IL-10-like genes were then identified in Bovine papular stomatitis virus (BPSV), Pseudocowpox virus (PCPV), Red deerpox virus (RDPV) and Grey sealpox virus (GSPV). This study aimed to produce and characterise recombinant parapoxvirus IL-10s, then quantitatively compare their receptor binding and immunomodulatory activities. Recombinant IL-10s were expressed, purified, then characterised using bioinformatic, biochemical and enzymatic analyses. Anti-inflammatory effects were assessed in lipoteichoic acid-activated THP-1 monocytes, and stimulatory effects in MC/9 mast cells. IL-10 receptor (IL-10R)1 binding was detected in a competitive displacement assay. BPSV IL-10 inhibited production of monocyte chemoattractant protein (MCP)-1, IL-8 and IL-1ß, induced mast cell proliferation, and bound IL-10R1 similarly to ORFV IL-10. PCPV IL-10 showed reduced MCP-1 inhibition, mast cell proliferation, and IL-10R1 binding. RDPV IL-10 displayed reduced inhibition of IL-8 and MCP-1 production. GSPV IL-10 showed limited inhibition of IL-1ß production and stimulation of mast cell proliferation. These findings provide valuable insight into IL-10 receptor interactions, and suggest that the parapoxvirus IL-10s play similar pathogenic roles during infection of their hosts.

Oestrogen deprivation induces chemokine production and immune cell recruitment in in vitro and in vivo models of oestrogen receptor-positive breast cancer.

BACKGROUND: Oestrogen receptor-positive (ER+) breast cancer is commonly treated using endocrine therapies such as aromatase inhibitors which block synthesis of oestradiol, but the influence of this therapy on the immune composition of breast tumours has not been fully explored. Previous findings suggest that tumour infiltrating lymphocytes and immune-related gene expression may be altered by treatment with aromatase inhibitors. However, whether these changes are a direct result of impacts on the host immune system or mediated through tumour cells is not known. We aimed to investigate the effect of oestrogen deprivation on the expression of chemokines and immune infiltration in vitro and in an ER+ immunocompetent mouse model. METHODS: RT-qPCR and a bead-based Bioplex system were used to investigate the expression of chemokines in MCF-7 breast cancer cells deprived of oestrogen. A migration assay and flow cytometry were used to measure the migration of human peripheral blood mononuclear cells (PBMCs) to MCF-7 cells grown without the main biologically active oestrogen, oestradiol. Using flow cytometry and immunohistochemistry, we examined the immune cell infiltrate into tumours created by injecting SSM3 ER+ breast cancer cells into wild-type, immunocompetent 129/SvEv mice. RESULTS: This study demonstrates that oestrogen deprivation increases breast cancer secretion of TNF, CCL5, IL-6, IL-8, and CCL22 and alters total human peripheral blood mononuclear cell migration in an in vitro assay. Oestrogen deprivation of breast cancer cells increases migration of CD4+ T cells and decreases migration of CD11c+ and CD14+ PBMC towards cancer cells. PBMC migration towards breast cancer cells can be reduced by treatment with the non-steroidal anti-inflammatory drugs, aspirin and celecoxib. Treatment with endocrine therapy using the aromatase inhibitor letrozole increases CD4+ T cell infiltration into ER+ breast cancer tumours in immune competent mice. CONCLUSIONS: These results suggest that anti-oestrogen treatment of ER+ breast cancer cells can alter cytokine production and immune cells in the area surrounding the cancer cells. These findings may have implications for the combination and timing of anti-oestrogen therapies with other therapies.

Strategies for inclusion of growth factors into 3D printed bone grafts.

There remains a critical need to develop new technologies and materials that can meet the demands of treating large bone defects. The advancement of 3-dimensional (3D) printing technologies has allowed the creation of personalized and customized bone grafts, with specific control in both macro- and micro-architecture, and desired mechanical properties. Nevertheless, the biomaterials used for the production of these bone grafts often possess poor biological properties. The incorporation of growth factors (GFs), which are the natural orchestrators of the physiological healing process, into 3D printed bone grafts, represents a promising strategy to achieve the bioactivity required to enhance bone regeneration. In this review, the possible strategies used to incorporate GFs to 3D printed constructs are presented with a specific focus on bone regeneration. In particular, the strengths and limitations of different methods, such as physical and chemical cross-linking, which are currently used to incorporate GFs to the engineered constructs are critically reviewed. Different strategies used to present one or more GFs to achieve simultaneous angiogenesis and vasculogenesis for enhanced bone regeneration are also covered in this review. In addition, the possibility of combining several manufacturing approaches to fabricate hybrid constructs, which better mimic the complexity of biological niches, is presented. Finally, the clinical relevance of these approaches and the future steps that should be taken are discussed.

Skin antigen-presenting cells and wound healing: New knowledge gained and challenges encountered using mouse depletion models.

The role of antigen-presenting cells in the skin immune system, in particular Langerhans cells and dendritic cells, has not been well defined. We recently published a study in 'Immunology' where we reported that the loss of langerin-positive cells in the skin accelerated wound repair in the Lang-DTR mouse. The study published here by Li, et al. reports delayed wound closure following depletion of CD11c-positive cells in the CD11c-DTR mouse. In this commentary, we attribute the differences between these results to several factors that differ between the studies including the depletion of different cell populations; differences in the age and the sex of mice; differences in antibiotic use between the studies; and differences in the location of the biopsies that were taken. Here, we describe the impact of these differences on wound healing and conclude that further standardization of the wound model, and further characterization of the specific cells that are depleted in these mice, is necessary to better understand how antigen-presenting cells contribute to wound healing.

Advancements in the Delivery of Growth Factors and Cytokines for the Treatment of Cutaneous Wound Indications.

Significance: Wound healing involves the phasic production of growth factors (GFs) and cytokines to progress an acute wound to a resolved scar. Dysregulation of these proteins contributes to both wound chronicity and excessive scarring. Direct supplementation of GFs and cytokines for treatment of healing and scarring complications has, however, been disappointing. Failings likely relate to an inability to deliver recombinant proteins at physiologically relevant levels to an environment conducive to healing. Recent Advances: Inspired by the extracellular matrix, natural biomaterials have been developed that resemble human skin, and are capable of delivering bioactives. Hybrid biomaterials made using multiple polymers, fabrication methods, and proteins are proving efficacious in animal models of acute and impaired wound healing. Critical Issues: For clinical translation, these delivery systems must be tailored for specific wound indications and the correct phase of healing. GFs and cytokines must be delivered in a controlled manner that will target specific healing or scarring impairments. Preclinical assessment in clinically relevant animal models of impaired or excessive healing is critical. Future Directions: Clinical success will likely depend on the GF or cytokine selected, their compatibility with the chosen biomaterial(s), degradation rate of the fabricated system, and the degree of control over release kinetics. Further testing is essential to assess which wound indications are most suited to specific delivery systems and to prove whether they provide superior efficacy over direct protein therapies.

Murine Model of Thermal Burn Injury for Evaluating Protein Therapeutics Derived from Viruses.

In vivo wound healing models are predictive preclinical tests for therapeutics that enhance skin repair or limit scarring. Large animals, such as swine, heal in a manner similar to humans, but testing is impractical and expensive. Experiments in mice are more economic, but may be less translatable as this species heals primarily through contraction, not by the processes of epithelialization and granulation tissue formation as seen in human wounds. Here, we describe a murine model of thermal burn injury that closely mimics human healing, resulting in a large, hypertrophic-like scar. This practical, reproducible model is ideal for testing promising wound-healing therapies, such as virus-derived growth factors and immune-modulatory proteins.

Anti-fibrotic Actions of Equine Interleukin-10 on Transforming Growth Factor-Beta1-Stimulated Dermal Fibroblasts Isolated From Limbs of Horses.

Fibroproliferative disorders occur in both humans and horses following skin injury. In horses, wound healing on the limb is often complicated by the formation of fibroproliferative exuberant granulation tissue, characterized by persistent expression of pro-fibrotic transforming growth factor-beta1 (TGF-ß1) and deficient expression of anti-inflammatory interleukin-10 (IL-10). IL-10 has been shown to directly modulate fibrotic gene expression in human fibroblasts, so we hypothesized that equine IL-10 (eIL-10) may exert similar anti-fibrotic effects on equine dermal fibroblasts. Cell-lines were created from the limb skin of six individual horses. Recombinant eIL-10 was produced and purified, and its effects on the cells investigated in the presence and absence of equine TGF-ß1 (eTGF-ß1). Myofibroblast differentiation and collagen production were examined using immunofluorescent cytometry, cell contractility in a collagen gel assay, and fibrotic gene expression using quantitative PCR. In response to eTGF-ß1, fibroblasts increased in contractility and expression of alpha-smooth muscle actin, collagen types 1 and 3, and matrix metalloproteinase 1, 2, and 9. Equine IL-10 limited cell contractility and production of alpha-smooth muscle actin and type 3 collagen, and decreased mRNA levels of eCol3a1 and eMMP9, while increasing that of eMMP1. Opposing effects on eTGF-ßR3 and eIL-10R1 gene expression were also observed, with mRNA levels decreasing following eTGF-ß1 treatment, and increasing with eIL-10 treatment. These findings indicate that eIL-10 limits the pro-fibrotic effects of eTGF-ß1, potentially through the modulation of fibrotic and receptor gene expression. Further investigations are warranted to assess the therapeutic utility of eIL-10 in the treatment of exuberant granulation tissue.

Vascular endothelial growth factor encoded by Parapoxviruses can regulate metabolism and survival of triple negative breast cancer cells.

Dysbiotic microbiomes are linked to many pathological outcomes including different metabolic disorders like diabetes, atherosclerosis and even cancer. Breast cancer is the second leading cause of cancer associated death in women, and triple negative breast cancer (TNBC) is the most aggressive type with major challenges for intervention. Previous reports suggested that Parapoxvirus signatures are one of the predominant dysbiotic viral signatures in TNBC. These viruses encode several genes that are homologs of human genes. In this study, we show that the VEGF homolog encoded by Parapoxviruses, can induce cell proliferation, and alter metabolism of breast cancer and normal breast cells, through alteration of MAPK-ERK and PI3K-AKT signaling. In addition, the activity of the transcription factor FoxO1 was altered by viral-encoded VEGF through activation of the PI3K-AKT pathway, leading to reprogramming of cellular metabolic gene expression. Therefore, this study provides new insights into the function of viral-encoded VEGFs, which promoted the growth of the breast cancer cells and imparted proliferative phenotype with altered metabolism in normal breast cells.

Visible light mediated PVA-tyramine hydrogels for covalent incorporation and tailorable release of functional growth factors.

The translation of growth factors (GFs) into clinical applications is limited by their low stability in physiological environments. Controlled GF delivery through biomaterial vehicles provides protection from proteases, targeted delivery, and longer term release profiles. However, current methods used to incorporate GFs into biomaterials still present limitations. While direct adsorption and encapsulation result in burst release, covalent incorporation provides a tailorable release profile but generally requires more complicated processes and chemical modification of the GFs. Bioaffinity methods provide long-term release profiles but fail in their specificity, resulting in GF-dependent applicability and release profiles. In the present study, we introduce tyraminated poly-vinyl-alcohol (PVA-Tyr) as a GF-delivery vehicle that can covalently incorporate native GFs through a photo-initiated cross-linking process via formation of bi-phenol bonds. Mass loss and release studies revealed that protein-loaded PVA-Tyr hydrogels had highly tailorable degradation times from 7 to 92 days, during which the covalently incorporated proteins were released in a linear fashion. The incorporation of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), or brain-derived growth factor (BDNF) resulted in similar incorporation efficiencies and release profiles, demonstrating the low specificity and versatility of the system. Furthermore, functional studies demonstrated that VEGF, bFGF and BDNF released from the PVA-Tyr hydrogels retained the ability to increase the metabolic activity, migration, and 3D vessel formation of endothelial cells and mesenchymal stem cells. Taken together, this demonstrates that PVA-Tyr shows high potential as a highly tailorable GF delivery tool for a range of different regenerative medicine applications.

Orf Virus IL-10 and VEGF-E Act Synergistically to Enhance Healing of Cutaneous Wounds in Mice.

Orf virus (OV) is a zoonotic parapoxvirus that causes highly proliferative skin lesions which resolve with minimal inflammation and scarring. OV encodes two immunomodulators, vascular endothelial growth factor (VEGF)-E and interleukin-10 (ovIL-10), which individually modulate skin repair and inflammation. This study examined the effects of the VEGF-E and ovIL-10 combination on healing processes in a murine wound model. Treatments with viral proteins, individually and in combination, were compared to a mammalian VEGF-A and IL-10 combination. Wound biopsies were harvested to measure re-epithelialisation and scarring (histology), inflammation, fibrosis and angiogenesis (immunofluorescence), and gene expression (quantitative polymerase chain reaction). VEGF-E and ovIL-10 showed additive effects on wound closure and re-epithelialisation, and suppressed M1 macrophage and myofibroblast infiltration, while allowing M2 macrophage recruitment. The viral combination also increased endothelial cell density and pericyte coverage, and improved collagen deposition while reducing the scar area. The mammalian combination showed equivalent effects on wound closure, re-epithelialisation and fibrosis, but did not promote blood vessel stabilisation or collagen remodeling. The combination treatments also differentially altered the expression of transforming growth factor beta isoforms, Tgfß1 and Tgfß3. These findings show that the OV proteins synergistically enhance skin repair, and act in a complimentary fashion to improve scar quality.

Deriving Immune Modulating Drugs from Viruses-A New Class of Biologics.

Viruses are widely used as a platform for the production of therapeutics. Vaccines containing live, dead and components of viruses, gene therapy vectors and oncolytic viruses are key examples of clinically-approved therapeutic uses for viruses. Despite this, the use of virus-derived proteins as natural sources for immune modulators remains in the early stages of development. Viruses have evolved complex, highly effective approaches for immune evasion. Originally developed for protection against host immune responses, viral immune-modulating proteins are extraordinarily potent, often functioning at picomolar concentrations. These complex viral intracellular parasites have "performed the R&D", developing highly effective immune evasive strategies over millions of years. These proteins provide a new and natural source for immune-modulating therapeutics, similar in many ways to penicillin being developed from mold or streptokinase from bacteria. Virus-derived serine proteinase inhibitors (serpins), chemokine modulating proteins, complement control, inflammasome inhibition, growth factors (e.g., viral vascular endothelial growth factor) and cytokine mimics (e.g., viral interleukin 10) and/or inhibitors (e.g., tumor necrosis factor) have now been identified that target central immunological response pathways. We review here current development of virus-derived immune-modulating biologics with efficacy demonstrated in pre-clinical or clinical studies, focusing on pox and herpesviruses-derived immune-modulating therapeutics.

Depletion of langerin+ cells enhances cutaneous wound healing.

Langerin is a C-type lectin receptor that is expressed on Langerhans cells and langerin-positive dermal dendritic cells in the skin. Little is known about the function of langerin+ cells in wound healing. In this study, the effects of ablation of langerin+ cells on healing of a full-thickness excision wound were investigated using the langerin-DTR depletable mouse. Strikingly, depletion of langerin+ cells resulted in more rapid reduction in wound area. Accelerated wound healing in the langerin+ -cell-depleted group was characterized by enhanced neo-epidermis and granulation tissue formation, and increased cellular proliferation within the newly formed tissues. Accelerated healing in the absence of langerin+ cells was associated with increased levels of granulocyte-macrophage colony-stimulating factor, F4/80+ cells and blood vessels within the granulation tissue. These data support an inhibitory role for langerin+ cells during wound healing. Therapies that suppress langerin+ cells or their function may therefore have utility in progressing the healing of wounds in humans.

Human Papillomavirus E6/E7 Expression in Preeclampsia-Affected Placentae.

Whether HPV is causative of pregnancy complications is uncertain. E6 and E7 affect functions underling preeclampsia (PET) in cultured trophoblasts, but whether E6 and E7 is produced in the placenta is uncertain. Here, we investigated whether E6/E7 was expressed in the placentae from pregnancies with PET, other pregnancy complications (fetal growth restriction (FGR) and diabetes mellitus), and uncomplicated pregnancies. Placental tissues collected from two geographical locations were subjected to RNAscope analyses of high- and low- risk E6/E7, and individual HPV types identified using an HPV array. High-risk E6/E7 expression was increased in both PET cohorts, (81% and 86% of patients positive for high-risk HPV DNA compared to 13% of control patients). Various HPV types were identified. Although HPV 18 was the most frequent in all cohorts, the majority of individuals had multiple HPV types (55% of the PET compared to 25% of the control cohort). Further evidence that E6 and E7 is present early when placental pathology underlying preeclampsia is established, is provided with the finding of high-risk E6/E7 in the first-trimester placenta anchoring trophoblast. In conclusion, E6/E7 expression and multiple HPV types were frequent in placentae from preeclampsia-complicated pregnancies.

Chemokine-Binding Proteins Encoded by Parapoxvirus of Red Deer of New Zealand Display Evidence of Gene Duplication and Divergence of Ligand Specificity.

Parapoxvirus of red deer in New Zealand (PVNZ) is a species of the Parapoxvirus genus that causes pustular dermatitis. We identified a cluster of genes in PVNZ that encode three unique chemokine-binding proteins (CBPs) namely ORF112.0, ORF112.3 and ORF112.6. Chemokines are a large family of molecules that direct cell trafficking to sites of inflammation and through lymphatic organs. The PVNZ-CBPs were analyzed by surface plasmon resonance against a broad spectrum of CXC, CC, XC and CX3C chemokines and were found to differ in their specificity and binding affinity. ORF112.0 interacted with chemokines from the CXC, CC and XC classes of chemokines with nM affinities. The ORF112.3 showed a preference for CXC chemokines, while ORF112.6 showed pM affinity binding for CC chemokines. Structural modeling analysis showed alterations in the chemokine binding sites of the CBPs, although the core structure containing two ß-sheets and three α-helices being conserved with the other parapoxvirus CBPs. Chemotaxis assays using neutrophils and monocytes revealed inhibitory impact of the CBPs on cell migration. Our results suggest that the PVNZ-CBPs are likely to have evolved through a process of gene duplication and divergence, and may have a role in suppressing inflammation and the anti-viral immune response.

The role of Langerhans cells in pathologies of the skin.

Langerhans cells (LCs) are epidermal immune cells of myeloid origin. Although these cells were primarily thought to play a defensive role in the skin, evidence now indicates a diverse range of LC-mediated effects including the relay of viral antigens in herpes simplex infection, recruitment of eosinophils in atopic dermatitis and promotion of a Th17 response in Candida infection. LCs may have a protective or suppressive function in pathologies of the skin, with differing functions being driven by the skin milieu. Understanding LC function will help guide the development of interventions that modulate these cells for therapeutic benefit.

The Cutaneous Inflammatory Response to Thermal Burn Injury in a Murine Model.

Many burn interventions aim to target the inflammatory response as a means of enhancing healing or limiting hypertrophic scarring. Murine models of human burns have been developed, but the inflammatory response to injury in these models has not been well defined. The aim of this study was to profile inflammatory cell populations and gene expression relative to healing and scarring in a murine model of thermal burns. Cutaneous injuries were created on the dorsal region of C57Bl/6 mice using a heated metal rod. Animals were euthanized at selected time points over ten weeks, with the lesions evaluated using macroscopic measurements, histology, immunofluorescent histochemistry and quantitative PCR. The burn method generated a reproducible, partial-thickness injury that healed within two weeks through both contraction and re-epithelialization, in a manner similar to human burns. The injury caused an immediate increase in pro-inflammatory cytokine and chemokine expression, coinciding with an influx of neutrophils, and the disappearance of Langerhans cells and mast cells. This preceded an influx of dendritic cells and macrophages, a quarter of which displayed an inflammatory (M1) phenotype, with both populations peaking at closure. As with human burns, the residual scar increased in size, epidermal and dermal thickness, and mast cell numbers over 10 weeks, but abnormal collagen I-collagen III ratios, fibre organization and macrophage populations resolved 3⁻4 weeks after closure. Characterisation of the inflammatory response in this promising murine burn model will assist future studies of burn complications and aid in the preclinical testing of new anti-inflammatory and anti-scarring therapies.