Summary: Wound healing studies are intricate, mainly because of the multifaceted nature of the wound environment and the complexity of the healing process, which integrates a variety of cells and repair phases, including inflammation, proliferation, reepithelialization and remodelling. There are a variety of possible preclinical models, such as in mice, rabbits and pigs, which can be used to mimic acute or impaired for example, diabetic and nutrition‐related wounds. These can be induced by many different techniques, with excision or incision being the most common. After determining a suitable model for a study, investigators need to select appropriate and reproducible methods that will allow the monitoring of the wound progression over time. The assessment can be performed by non‐invasive protocols such as wound tracing, photographic documentation (including image analysis), biophysical techniques and/or by invasive protocols that will require wound biopsies. In this article, we provide an overview of some of the most often needed and used: (a) preclinical/animal models including incisional, excisional, burn and impaired wounds; (b) methods to evaluate the healing progression such as wound healing rate, wound analysis by image, biophysical assessment, histopathological, immunological and biochemical assays. The aim is to help researchers during the design and execution of their wound healing studies.

Context: The quantification of total collagen is of major importance in a wide range of research areas, including the study of cutaneous wound healing and new drugs trials. Objective: The total collagen content in skin biopsies was compared by biochemical hydroxyproline assay and by two computer-aided histomorphometric analyses of histological sections. Materials and methods: Two methods were used to evaluate collagen formation: the hydroxyproline assay, as the gold standard and histomorphometric image analysis of the filled areas by corresponding stained collagen fibres, using picrosirius and Gomori’s trichrome staining. The image analyses were determined by digital densitometry recognition using computer-aided ImageJ software. One-way ANOVA, simple linear regression and ANCOVA were applied for the statistical analysis and correlation. Results: In a simple linear regression analysis carried out on the 14th day period after the induction of skin injury, three techniques, picrosirius red (F = 33.57, p = 0.00), Gomori’s trichrome (F = 81.61, p = 0.00) and hydroxyproline content (F = 16.85, p = 0.00) were able to detect collagen production. After scale adjustment, there were no significant differences among either the slopes (F = 1.17, p = 0.32) or the intercepts (F = 0.69, p = 0.51) of the estimated regression lines. It seems that a highly significant correlation exists between the histomorphometrical analysis and hydroxyproline assay. Discussion and conclusion: The morphometric analysis proved to be adequate and can be used as a simple, rapid, low-cost technology for evaluating total collagen in cutaneous wound specimens, compared with the gold standard hydroxyproline assay.

Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of fibroblasts in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2^nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to 19% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, growth factor and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

Lipoxin A₄ (LXA₄) is involved in the resolution of inflammation and wound healing; however, it is extremely unstable. Thus, to preserve its biological activities and confer stability, we encapsulated LXA₄ in poly-lactic-co-glycolic acid (PLGA) microparticles (LXA₄-MS) and assessed its application in treating dorsal rat skin lesions. Ulcers were sealed with fibrin adhesive and treated with either LXA₄-MS, unloaded microparticles (Un-MS), soluble LXA₄, or PBS/glue (vehicle). All groups were compared at 0, 2, 7, and 14 days post-lesions. Our results revealed that LXA₄-MS accelerated wound healing from day 7 and reduced initial ulcer diameters by 80%. Soluble LXA₄, Un-MS, or PBS closed wounds by 60%, 45%, and 39%, respectively. LXA₄-MS reduced IL-1β and TNF-α, but increased TGF-β, collagen deposition, and the number of blood vessels. Compared to other treatments, LXA₄-MS reduced inflammatory cell numbers, myeloperoxidase (MPO) concentration, and metalloproteinase-8 (MMP8) mRNA in scar tissue, indicating decreased neutrophil chemotaxis. In addition, LXA₄-MS treatment increased macrophages and IL-4, suggesting a positive impact on wound healing. Finally, we demonstrated that WRW4, a selective LXA₄ receptor (ALX) antagonist, reversed healing by 50%, indicating that LXA₄ must interact with ALX to induce wound healing. Our results show that LXA₄-MS could be used as a pharmaceutical formulation for the treatment of skin ulcers.