Wound Repair and Regeneration: Literature Review
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The skin is the largest organ and one of its functions is as a protective barrier of human body. As it is the largest organ, hence it is prone to have injuries. Once injury occurs, this barrier will be disrupted and thus it needs to regain its function to prevent invasion of pathogens and blood loss. Wound healing involves multiple steps which includes blood clotting, re-epithelialization, granulation tissue formation, inflammation and scarring (Richardson et al., 2013). Many different cell lineages play roles during this overlapping wound repair phases such as the innate response, the inflammatory response, the proliferation, the migration of cells, the contraction phase and the resolution phase. The resolution phase is very important by ensuring the dispersion of leukocytes at the site of injury or their apoptosis to prevent chronic inflammation which is the root causes of many diseases and slowing the healing process (Sonnemann & Bement, 2011; Thomas, 2007). These crucial phases in wound healing are being studied progressively in model organisms by understanding them through identification of the most conserved fundamental players in those steps and process and this helps in initial steps of wound healing study. Besides that, the simpler organisms are better healers than complex organisms even though the general mechanisms and molecules used in both organisms are the same. Hence it is good to study wound healing in simpler organisms such as in invertebrates, fish and amphibians so that rapid and complete healing can be recapitulated in humans (Sonnemann & Bement, 2011). Wound healing in embryo is associated with efficient repair mechanism and scar free compared to adult wound healing which is imperfect and producing fibrosis and scar. Hence, by studying Zebrafish (Danio rerio) embryo hopefully it can give insights in establishing new therapeutic ways to improve adult wound healing (Redd et al., 2004) .
Many technical advances recently have facilitated the emerging knowledge of wound healing. Life is dynamic, thus to observe living tissue over time is needed. Visualization techniques through the use of fluorescence microscopy has revolutionized with the introduction of genetically encoded fluorophores such as Green Fluorescent Protein (GFP) to localize protein in living cells. GFP has helped in biological research as GFP gene can be expressed in any possible genetically-engineered species. They encoded for fluorescent protein that can act as a spatial reporter for gene expression and the movement of these fusion proteins can be followed in live imaging (Plautz et al., 1996; Yuste, 2005). For example, a transgenic model organism is produced by using Green Fluorescent Protein (GFP) that will enhance the ability to observe live imaging in vivo of targeted gene products that fused with GFP (Redd et al., 2004).
A few of animal models can be used to study wound healing such as chick embryo, mouse, Drosophila and zebrafish. Every model has its own advantages in understanding wound healing and normally Drosophila and zebrafish are helpful for genetics. They are being used extensively as they are genetically tractable. Zebrafish larvae are relatively transparent thus feasible to follow GFP-targeted gene fusion in them and they are bred largely in numbers for genetic screening (Denis et al., 2013). Zebrafish is a good vertebrate model to study human genetic diseases by knowing gene function through knocking down or overexpression of the gene (Howe et al., 2013). Mouse is the best animal model to study wound healing as it is a mammal like humans however it demands for high cost of housing and feeding (Denis et al., 2013). In addition transgenic animal can be produced through reverse genetic technique such as knock out which can help in understanding the effect of sequence on phenotype. Thus, by inactivating suspected genes that probably involve in wound healing, we can suggest its role in this mechanism.
Wound healing is an essential physiological process to maintain tissue homeostasis. Thus, in this case it is necessary to understand the development process as a guideline in understanding the wound healing so that we can compare what is missing or what have been reactivated again to restore its normal state. Drosophila has been studied at various stages of development including embryo, larvae and larval imaginal disc, pupa and adult to model mammalian tissue repair (Razzell et al., 2011). Wound healing repair mechanism and epithelial fusion resemble morphogenetic movement in embryogenesis during dorsal closure in Drosophila larva at its late stage of development (Cordeiro & Jacinto, 2013). Once injury happens, epidermis will disintegrate and it must be repaired to close this ruptured area and as a consequence it is a necessary for re-epithelialization. Re-epithelialization might happen through cell division or the epidermal cells might stretch themselves until wound edges meet and fuse. From the study of mammalian skin healing, it has been proven that cell division happened in the advanced wound epithelium (Werner et al., 2014).
However, through live confocal imaging of Drosophila closure wounded by means of a laser beam, it has been found that wound-edge epithelia are stretching themselves between two opposing epithelial sheets and zipping them together instead of undergoing cell division. This changing in shape of wound-edge epithelia was observed by using life confocal time-lapse analysis. The e22c-GAL4 driver was used to express a GFP-actin fusion protein in all epithelial cells for visualizing dynamic actin in wound closure. Time-lapse confocal movie showed that actin machineries that responsible to drive wound re-epithelialisation. It has been found that this wound closure is mediated by contractile actomyosin cable and filopodia. The interdigitation of filopodial protrusions from opposing wound-edge epithelial cells was also illustrated by using transmission electron micrograph and this was similar protrusions happened in the dorsal closure in Drosophila (Redd et al., 2004). Thus, it can be said that cell division per se is not necessary for wound healing as Drosophila’s embryo can fill the gap by changing the shape of cells to increase their surface area. In conclusion, embryos are poor model to investigate the role of cell division in re-epithelialization. However a study done in wounded larval imaginal disc showed cell proliferation at the wound margin and might have role in regeneration process and also in zebrafish larvae epidermis showed regenerative capacity once wounded (Dale & Bownes, 1981; Richardson et al., 2013). This can be a good insight for further studies to dissect the genetics that might involve in vertebrate wound response.
In order to test the function of genes that involves in actin machineries in fruit flies, transgenic ActinGFP- expressing Drosophila embryos will be used. Rho mutant embryos were able to seal their wounds even though they were unable to assemble an actin cables. One of Rho GTPases, the dominant negative CDC42 had been made to see the effect of blocking the activity of CDC42. The rate of wound closure was not affected until the adhesion step failed to seal the wound as this mutant blocked assembly of filopodia that knitting together the wound edge fronts (Wood et al., 2002).
In wound healing, inflammatory response is vital to prevent intrusion of pathogens at wound sites. It develops in mid-embryogenesis onwards but early embryos do not have it. A range of chemotactic signals are released at the wound site and will attract leukocytes to migrate to that area from circulating bloodstream. Drosophila and zebrafish can be modelled to observe recruitment of innate immune cells to wounds. Haemocyte which is an innate immune cell lineage is very amenable to live imaging and able to respond to tissue damage cues such as hydrogen peroxide (H2O2). Haemocyte recruitment and resolution can be seen based on time course series in the in vivo healing wound (Razzell et al., 2011). Zebrafish has become the new model to study inflammatory response through fin inflammation assay as mice are not feasible to observe this dynamic process of leukocytes recruitment. The zebrafish larva is translucent thus neutrophils migration to the wound can be observed in situ by using differential interference contrast (DIC) optics and there are several lines with fluorescently tagged neutrophils (Mathias et al., 2006; Redd et al., 2004; Renshaw et al., 2006). It has small number of neutrophils about 20 to 30 cells drawn to a wound and each of them can be tracked with time and location precision (Martin & Feng, 2009).
In Zebrafish, the potential genes that might involve in inflammatory response can be knocked down by using morpholinos to know their functions by applying loss of function. They block targeted messenger mRNA translation and as a result no respective protein will be synthesized (Heasman, 2002). A combination of genetic and pharmacological studies has been used to identify potential sources of the H2O2 signal. It is believed that H2O2 is produced through the activation of Duox enzyme. Duox is NADPH oxidase that generates ROS during metabolic process. Knocking down Duox with the use of morpholinos which prevents the specific mRNAs into proteins mimics the drug block that prevents the establishment of H2O2 gradient and ultimately inhibit the migration of neutrophils to the wound (Heasman, 2002; Niethammer et al., 2010). In order to monitor neutrophils dispersion from inflammatory site, a photoconvertable protein, named Kaede has been used in the reverse migration assays in transgenic zebrafish larvae. Leukocytes labelled with the Kaede protein can be photoconverted from green to red fluorescence at the wound site (Henry et al., 2013). In order to achieve anti-inflammatory wound healing therapies, zebrafish has been used for anti-neutrophil drug discovery. High-throughput chemical screening can be executed by using zebrafish embryos in multiwell plates that contain compound libraries to see the effect of the drugs. These compound screens can identify hit compounds interfering with wound healing or alleviating inflammation in wound healing pathologies (Henry et al., 2013; Richardson et al., 2013).
A finding of new model organisms that able to undergo scarless wound healing or having regenerative capacity is very fascinating. This is possible as inflammatory response is dispensable during wound healing proven by PU.I null mouse which is ‘macrophageless’ but heals rapidly without fibrosis (Cooper et al., 2005). This is because of healing by fibrosis has caused public health and economic burden. Congestive heart failure is believed as a result of myocardial scar tissue resulting from myocardial infarction which causes insufficient blood supply to the body’s tissue. This disease accounts for 100, 000 deaths each year in the United State and lifelong disability caused by fibrotic healing has a great cost impact (Gurtner et al., 2008). A new model organism named Axolotl has been proposed to study scarless wound healing. It has been found that there was almost no neutrophils at the wound at early time after postwounding (Lévesqu et al., 2010). It is a vertebrate that has tight-skinned like humans and long-lived to monitor the impact of treatments. Even though it is unfavourable for genetic screening like zebrafish, however this model can be used to study mechanistic nature in wound healing. There are available tools that can be used to fulfil this investigation. There are available of transgenic Axolots to track cell fate and inducible promoters (Sobkow et al., 2006; Whited et al., 2012). In addition there is available of specific microarrays for salamanders to assess the expression of thousand of genes (Monaghan et al., 2009). Fortunately, as Axolots is an aquatic life thus, the treatment can be bathed around it to see the therapeutic effects in wound healing and regeneration.
All in all we are getting to improve the wound healing therapies by studying wound healing in several model organisms that complement the knowledge of wound healing coupled with technical advances which provide dynamic imaging. Inflammation is not necessary for wound healing and embryos can do so as they have immature immune system to produce immune cells that triggers inflammation. It suggests that efficient repair is possible and offers therapies to improve clinical adult healing. Thus, we need to understand cell biology and genetics in inflammation to identify target genes for modulating them in inflamed diseases as we cannot totally shut down the immune system. The antibiotic treatment can be monitored together in the patient to reduce the bacterial infection.
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