Novel wound-healing models developed in rodents

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An important biological reaction, wound healing comprises different phases including inflammation, proliferation, and remodelling. Humans and animals have distinct wound-healing outcomes, which could be due to variances in skin structure, hair density, and skin tightness. When simulating wound healing in humans, it’s therefore critical to use animal models that closely resemble the human wound-healing process. Dr Shiro Jimi and fellow researchers from Fukuoka University in Japan have developed two different wound models in rodents – splint and scar. Their research has provided a breakthrough in the development of models for wound healing and opened doors for future investigations.
Animals have been used in numerous wound-healing experiments during the last half-century. The relevance of these research studies to human wound healing has been a key concern due to structural variations between human and animal skin. Indeed, animal wound-healing research may lead to erroneous conclusions about human wound healing because humans and animals have distinct wound-healing outcomes, which could be due to variances in skin structure, hair density, and skin tightness. As a result, it’s critical to use animal models that closely resemble the human wound-healing process.

Understanding the mechanism of wound healing

Haemostasis, inflammation, proliferation, and remodelling are the four overlapping stages of wound healing in humans, which are complicated and well-orchestrated responses to tissue injury. Damaged cells release damage-associated molecular patterns and their intracellular contents during tissue injury, which activate the inflammasome and cause the secretion of pro-inflammatory cytokines (proteins secreted by the cells of the immune system that are involved in cell signalling). Platelets get activated when they come into contact with the extracellular matrix, releasing clotting factors and encouraging matrix formation for moving inflammatory cells and different growth factors involved in wound healing.

The human wound-healing process consists of four overlapping stages: haemostatis (forming a blood clot to stop the bleeding), inflammation, proliferation, and remodelling. Designua/Shutterstock.com

Pro-inflammatory cytokines attract neutrophils (a type of white blood cell) to the injury site, where they produce reactive oxygen species and, if present, phagocytose cell debris and pathogens. Phagocytosis is the process of ingesting and eliminating particles larger than 0.5μm in diameter. Furthermore, recruited neutrophils release chemokines and other chemicals that drive immune system cell migration. Monocytes are recruited and develop into either pro-inflammatory or reparative macrophages. The macrophages are a type of white blood cells that surround and kill microorganisms, remove dead cells, and stimulate the action of other immune-system cells. Macrophages also encourage fibroblast infiltration by phagocytosing the residual debris and dead neutrophils.

Migrated fibroblasts produce matrix metalloproteinases, which help remove disordered extracellular matrix and make room for newly formed extracellular matrix components. A proliferation and differentiation factor closes the area of injury. In addition, other molecules lead to the proliferation of endothelial cells and thus angiogenesis. Angiogenesis is a necessary and vital process that involves the formation of new blood vessels. This permits oxygen and nutrients to be transported to the site of injury, which can help the wound heal faster.

“Plenty of research is yet to be conducted to develop novel models mimicking human skin and to yield better results in wound healing.”

The need for novel wound-healing models

Wound-healing delays not only lengthen hospital stays and raise healthcare expenses but also lower patients’ quality of life. Wound healing is a basic tissue reaction in which wounds heal after the creation of granulation tissue (new connective tissue and microscopic blood vessels that form on the surfaces of a wound during the healing process) and tissue regeneration at the sites of tissue defects. Treatment becomes difficult if such reactions do not progress adequately, especially in patients with metabolic problems such as diabetes, nutritional insufficiency, and blood-flow disturbance. The micrograft technique for wound healing was first reported by Reverdin in 1869, and it has since been used in a variety of applications with various graft sizes. The Rigenera protocol, an advanced and unique micrograft procedure, was recently established.

Neutrophils are an essential part of our immune system. ALIOUI MA/Shutterstock.com

An animal study using gingival (gum) connective tissue was recently described in a wound-healing study (Noda et al, 2018). Clinical trials on dehiscent surgical wounds, complex wounds, chronic ulcers, and chronic scars have also been conducted using the approach. The conclusions of all trials were positive. However, the pathophysiological processes by which this method accelerates wound healing are still unknown. Plenty of research is yet to be conducted to develop novel models mimicking human skin and to yield better results in wound healing.

Dr Shiro Jimi and fellow researchers from Fukuoka University, Japan, have conducted a tremendous amount of research over the years in the field of wound healing. They have created two wound models in mice to stimulate wound healing in humans. One is the splint model to avoid wound contraction, which is specialised for properly predicting epithelial wound healing, and the other is the abdominal wall wound model, which develops scar tissues.

The splint wound model

To simulate human wound healing, the mouse excisional dorsal full-thickness wound model with a silicone splint adhered to the skin has been widely employed. In this model, mice were anesthetised with pentobarbital and the dorsal hair was removed. A circular tattoo was made on the centre of the lumbar area, and this section of skin was excised with scissors. A round splint was inserted beneath the skin near the wound defect and attached to the fascia with six-stitch ligations. The splint was then fixed to the skin with surgical silk thread. To prevent thread removal by the mice, they were dressed and fixed with a tight silicone vest.

Histopathology of granulation tissue at 11 days after injury, showing fibroblasts, bleeding, and lymphocytes.

However, because the initial point of epithelialisation (the process of covering defects on the epithelial surface after injury) on the wound surface is unclear, this approach cannot precisely assess dermal remodelling. To circumvent this constraint, the researchers from Fukuoka University created a new mouse excisional wound model that uses a plastic ring-shaped splint anchored beneath the surrounding epidermal tissue to measure the degree of epithelial extension and regeneration. This method, in comparison to earlier splint models, provides a more realistic assessment of epidermal processes in wound healing and can be used to test the impact of various wound-healing factors.

A micrograft technology has recently been created that minces tissue into micro-fragments >50μm. Its pathophysiological mechanisms in wound healing, however, are still unknown. As a result, Jimi’s group used normal mice to conduct a wound-healing experiment. The researchers employed a humanised mouse model of a skin wound with a splint. Following total skin excision, the Rigenera technique was used to obtain tissue micro-fragments, which were then infused into the incisions. The changes observed in their experimental investigations proved to be beneficial for epithelial regeneration on the wound.

“Jimi’s splint model provides a more realistic assessment of epidermal processes in wound healing and can be used to test the impact of various wound-healing factors.”

In another study (2020), Jimi and his team used a novel chitosan-based cryogel. The cryogel was applied to the muscular fasciae after total skin excision for the experimental splint wound-healing model. According to the results obtained, the dissolution of cryogel fragments may help to prevent chronic inflammation and severe scar formation by preventing continuous inflammation. Through their study, they showed that chitosan-based hydrogels are effective in wound healing.

In an alternative study (2020), the researchers focused on five major tissue reactions in wound healing: regeneration, migration, granulation, neovascularisation, and contraction. Their findings indicated that micrograft accelerated a series of wound-healing reactions and could be useful for treating intractable wounds in clinical situations.

After excising the abdominal muscle wall, granulation tissue developed in the wound. Phosphorylated SMAD (pSMAD) expression was examined in the granulation tissue, muscle wall adjusting to the wound (MA), and muscle wall distancing to the wound (MD) on days 7, 14, and 21. Expression levels of pSMAD were increased in all abdominal portions over time and were greatest in the granulation tissue. The result indicates tensile force may load significantly in the granulation tissue during the study and activate the intracellular SMAD pathway that leads to fibrosis.

The scar-based wound-healing models

Scar lesions rarely occur in mice, which has been known for a long time. Under an in situ tensile stress on the abdomen, Jimi and his group of researchers generated an abdominal muscle-wall excision model that forms fibrosis-prone scar lesions. They studied the morphological changes in fibrous scar lesions and muscle differentiation in the granulation tissue of mice treated with Pro-Hyp (Prolyl-hydroxyproline) and discovered that Pro-Hyp can help reduce scarring and increase muscle regeneration in the granulation tissue of the abdominal muscle wall.

Experimental animals rarely acquire hypertrophic scars similar to those found in humans, probably due to their looser skin structure. This makes understanding the origins of scar lesions difficult. As a result, suitable animal models are urgently required. In recent work (2021), Jimi and fellow researchers created a unique experimental model of a scar-forming wound in mice by resecting a tiny part of the abdominal muscle wall on the lower centre of the belly and exposing the surrounding muscle tissue to contractive pressures. The animal model established in this work is unique in that it allows researchers to examine the pathophysiology and preclinical therapy of scar lesions by allowing fibrous scar tissues to form under physiological conditions without the use of artificial stimuli.

Wound healing is a complex process and requires a complete understanding of the physiological mechanism along with better therapeutic techniques. Jimi and his fellow scientists have contributed immensely to this area. Their discoveries have provided a breakthrough in the development of models for wound healing and opened doors for future investigations.

What other aspects of wound healing would you like to investigate in the future?
After wounding, our tissues may produce tissue factors from injured cells and matrices for accelerating wound healings, including scarless healings. We have to determine the effects of those factors acting in the microenvironment using the animal models established and further expand our knowledge about wound healing. Our goal leads to future regenerative medicine, which challenges the treatments of unhealed ulcers and unregenerative organs such as the heart and the central nervous system in humans.

 

References

  • Jimi, S, Koizumi, S, Sato, K, Miyazaki, M, Saparov, A, (2021) Collagen-derived dipeptide Pro-Hyp administration accelerates muscle regenerative healing accompanied by less scarring after wounding on the abdominal wall in mice. Scientific Reports, 11(1), 18750. doi.org/10.1038/s41598-021-98407-9
  • Jimi, S, Saparov, A, Koizumi, S, Miyazaki, M, Takagi, S, (2021) A novel mouse wound model for scar tissue formation in abdominal muscle wall. The Journal of Veterinary Medical Science, 83(12), 1933–1942. doi.org/10.1292/jvms.21-0464
  • Jimi, S, Jaguparov, A, Nurkesh, A, Sultankulov, B, Saparov, A, (2020) Sequential Delivery of Cryogel Released Growth Factors and Cytokines Accelerates Wound Healing and Improves Tissue Regeneration. Frontiers in Bioengineering and Biotechnology, 8, 345. doi.org/10.3389/fbioe.2020.00345
  • Jimi, S, Takagi, S, De Francesco, F, Miyazaki, M, Saparov, A, (2020) Acceleration of Skin Wound-Healing Reactions by Autologous Micrograft Tissue Suspension. Medicina (Kaunas, Lithuania), 56(7), 321. doi.org/10.3390/medicina56070321
  • Sato, K, Asai, TT, Jimi, S, (2020) Collagen-Derived Di-Peptide, Prolylhydroxyproline (Pro-Hyp): A New Low Molecular Weight Growth-Initiating Factor for Specific Fibroblasts Associated With Wound Healing. Frontiers in Cell and Developmental Biology, 8, 548975. doi.org/10.3389/fcell.2020.548975
  • Noda, S, Sumita, Y, Ohba, S, Yamamoto, H, Asahina, I, (2018) Soft tissue engineering with micronized-gingival connective tissues. Journal of Cellular Physiology, 233(1), 249–258. doi.org/10.1002/jcp.25871
  • Jimi, S, Kimura, M, De Francesco, F, et al, (2017) Acceleration Mechanisms of Skin Wound Healing by Autologous Micrograft in Mice. International Journal of Molecular Sciences, 18(8), 1675. doi.org/10.3390/ijms18081675
  • Jimi, S, De Francesco, F, Ferraro, GA, Riccio, M, Hara, S, (2017) A Novel Skin Splint for Accurately Mapping Dermal Remodeling and Epithelialization During Wound Healing. Journal of Cellular Physiology, 232(6), 1225–1232. doi.org/10.1002/jcp.25595
DOI
10.26904/RF-140-2302910766

Research Objectives

Dr Shiro Jimi developed two rodent wound models, splint and scar, that simulate wound healing in humans.

Funding

  • R&D Center, Nitta Gelatin Inc (Osaka, Japan)
  • TMSC Co Ltd (Tokyo, Japan)

Collaborators

  • Prof Kenji Sato, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
  • Dr Francesco De Francesco, Department of Reconstructive Surgery and Hand Surgery, University Hospital (AOU Ospedali Riuniti di Ancona), Ancona, Italy
  • Prof Satoshi Takagi, Department of Plastic Reconstructive and Aesthetic Surgery, Faculty of Medicine, Fukuoka University, Fukuoka, Japan

Bio

Dr Shiro Jimi is a Designated Research Professor at Fukuoka University in Japan. Currently, his main research subjects are wound healing and bacterial biofilm infections using animal models.

Dr Shiro Jimi

Contact

E: sjimi@fukuoka-u.ac.jp
T: +81 92 801 1011

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