Wound Healing In Vitro Methods Experiment Name In vitro scratch assay Introduction The basic steps involve creating a ‘‘scratch’’ in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells. Compared to other methods, the in vitro scratch assay is particularly suitable for studies on the effects of cell–matrix and cell–cell interactions on cell migration, mimic cell migration during wound healing in vivo and are compatible with imaging of live cells during migration to monitor intracellular events if desired. ECIS? (Electric Cell-substrate Impedance Sensing) is a real-time, label-free, impedance-based method to study the activities of cells grown in tissue culture. These include morphological changes, cell locomotion, and other behaviors directed by the cell’s cytoskeleton. Features Convenient Inexpensive Simple No transfection Protocols References (1-5) In vitro scratch assay.pdf ECIS Assay Boyden Chamber Assay impedance-based fully intact cells The ECIS Wound Healing Assay replaces the traditional \good of disrupting the cell layer mechanically with a toothpick, needle or pipette tip and following the repeatability migration of cells to \expensive wound and monitor the healing process need special ECIS electrical wounding is only directed at the small population of cells in contact with the equipment active 250 micrometer diameter ECIS electrode, producing a well-defined 250 micrometer wound that can be verified both with the ECIS measurement and with vital staining. Unlike the traditional scrape method, with the ECIS Wound your protein coating is unaffected by the current, it remains fully intact. The Boyden chamber is a useful tool to study cell migration and cell invasion. It consists of a cylindrical cell culture insert nested inside the well of a cell culture plate. The insert contains a polycarbonate membrane at the bottom with a defined pore size. (6-10) ECIS Handbook.pdf CELL BIOLABS,INC. 2D Migration Gap Closure Assays Micro?uidics-based system
Our Gap Closure Assays (CytoSelect? Wound Healing Assay) provide a simple, convenient format to monitor cell migration with precision and accuracy. Each of these assays provides a consistently defined area across which migratory cells can move. Migration can be monitored in real time by microscopy. Microfluidic wound-healing assay Wound Healing In Vivo Methods (Experimental Animal Wound Models) A variety of normal and pathological injury paradigms can be applied in both small and large animal species.
To mimic clinical problems, rates of repair can be compromised by surgical impairment of blood supply or metabolic manipulations such as the diabetic state. Although animal wound repair is an imperfect reflection of human wound healing and its clinical challenges, these models continue to be crucial tools for the development of new strategies and approaches to rational wound therapy.
Experiment Name Acute Wound Models Excisional Incisional Primary Closure Secondary Closure Often parallels of authentic surgical procedures or traumatic injuries. Wounds closed by mechanical means heal rapidly with minimal scar tissue formation. It is less adequate for histological assessment of healing because of the limited volume/area of wound healing activity Incisions that are either deliberately or accidentally left open are said to heal by secondary intention. Incisions that are either deliberately or accidentally left open are said to heal by secondary intention. The removal of a significant volume of the target tissue, and the filling of the void created allows more ample material for determining biochemical and histological parameters. Excisional wounds can be covered with occlusive dressings, which retain the exudate (wound fluid) as a means of assessing the status of various soluble factors in the wound environment, such as nutrients, proteinases, cytokines, Introduction References and tissue degradation products Tape Stripping Blisters The simplest level of tissue excision in the skin involves partial removal of epidermis with adhesive tape. It has served as a useful model where only epidermal phenotype is to be examined. involves rupture of the epithelial basement membrane zone, forcing detachment of epidermis from underlying dermis The epidermal structures above the blister lose their nutrient supply and become necrotic, unless they are immediately reassociated with the basement membrane zone. Split-Thickness This lesion is the equivalent of the proverbial scraped knee, and the dermatome is used in clinical practice to prepare skin grafts from donor sites. This type of lesion is difficult to generate in the mouse because the skin is quite thin. This model is useful for determining rates of reepithelialization and, at least in the pig, is a good approximation of the behavior of human graft donor sites. The model has proven useful for testing many agents and devices that promote reepithelialization, including wound dressings, topical formulations, and growth factors. Full-Thickness This model involves the complete removal of epidermis and dermis to the depth of fascial planes or subcutaneous fat. The actual wound depth, as in partial-thickness wounds, is very dependent on species; the mouse has the thinnest skin, while the pig and other large domestic animals have a dermis that is as thick as or thicker than man. This model offers the advantages of significant wound volume, involvement of all dermal components, epithelialization only from the wound margins, and the ability to analyze chemistry, histology, and cell populations in the wound site. Splinting In full-thickness excision, the mechanical structure of the dermis is completely disrupted, and various forces in the surrounding dermis and the wound site can reduce the dimensions of the wound without actually filling the site with new tissue. Two distinct phenomena are associated with this type of wound closure, and they can be counteracted by physical splinting of the wound with retaining rings, biopolymer (e.g., collagen) plugs, or other mechanical devices. Contraction Contraction is a phenomenon predominantly seen in rodent and rabbit models, where the excision of loose skin results in a rapid shrinkage of the surrounding skin to reduce wound dimensions in a presumably adaptive fashion. There are clear differences in contraction rates in healing impaired animals, and this measure is often one of choice where animal numbers are limited, as in transgenic animals or targeted gene deletions in mice. Wound Contracture Wound contracture is a pathological process often associated with hypertrophic scarring, and it seems to be produced by contractile forces within wound granulation and scar tissue. The myofibroblast is often implicated as the culprit, although fibroblasts without organized bundles of a smooth muscle actin can certainly generate contractile forces. This phenomenon can be modeled in many species by administration of second- and third-degree burns. Excisional wounds in tight-skinned species, such as the pig, will also exhibit some degree of contracture, although residual scarring is minimal. Dead-Space Models All such models function by creating an artificial tissue space into which plasma infuses. Because implants have defined dimensions, they are excellent for biochemical determinations. Indeed, parameters, such as collagen content, DNA content, and quantities of various biochemicals, can be normalized on a per-implant basis or to implant weight. Limitations of the implant models include the interference of the implant with normal scar maturation, probably by the uncoupling of physical interactions among cells, the lack of epithelial components, and the likelihood of an eventual foreign body response. Wound Chambers Performed in a punch wound in the rabbit ear that was occluded on both sides by cover slips, permitting the microscopic examination of tissue reorganization. Excisional wounds may be enclosed with occlusive materials to permit the collection of wound fluid/exudate or to concentrate the administration of topical materials. Burns Immunologic Thermal Glucocorticoid Chemical, thermal, or radiation burns to the skin or other tissues produce a remarkably different healing response due to their effects on the viability of cells and tissue. This effect, in turn, induces nutrient starvation of the involved tissue. Members of this drug family are widely known for their anti-inflammatory activity with collateral effects that include thinning of the skin and diminished healing capacity. Experimentally, various species may be dosed with short-acting agents, such as hydrocortisone or dexamethasone, or, for long-term inhibition, with intramuscular methylprednisolone. The effects are demonstrable in many different wound-healing models in many species. Effects undoubtedly include the diminished capacity of monocytes to differentiate into growth factor-expressing macrophage and the reduced capacity of many fibroblasts to produce collagen and other connective tissue components. Since glucocorticoid use is clinically widespread, it is felt that these models have reasonable clinical relevance.