A Detailed Review on Wound Healing, Wound Assessment, Phases of Wound Healing, Different Evaluation Model for Wounds and Skin Disorder

Abstract

In this review there is discussion of various in vivo wound models used in research, including incision wound models, burn wound models, and dead space wound models. The incision wound model involves creating precise cuts without tissue removal, allowing researchers to study wound healing processes. This model is commonly used in animal species to test the effects of drugs, growth factors, and wound dressings on incision wound healing. Burn wound models involve creating controlled burns on living organisms to simulate human burn wounds, allowing researchers to investigate wound healing mechanisms, test therapeutic interventions, and assess treatment efficacy. However, using animals for research raises ethical concerns, and in vivo models may not fully replicate the complexity of human wound healing. Finally, the in vivo dead space wound model is used to study wound healing in living organisms by creating a space within the body where no tissue regeneration occurs. A study by Oliver et al. describes a novel small animal model used to investigate dead space management in muscle tissue, demonstrating the convenience and effectiveness of the model for assessing implant materials for dead space management. Results showed that both tested materials resolved the surgically created dead space without adverse host responses. This review will contribute to the understanding of wound healing and the development of potential treatments for various types of wounds.

Keywords

Introduction

A wound occurs when the integrity of cellular, anatomical or functional structure in living tissue is lost or damaged (1 ). A skin wound arises when the epidermal layer breaks down and loses its integrity. A wound can be defined as any anatomical disruption of tissues that are accompanied by the loss of their function. (2). Most of the time, wound healing refers to the skin’s recovery, healing of a wound starts as soon as a break occurs in the layer of cells lining the body’s surface and may continue for months or even years. This continuously changing process involves highly structured responses by different types of cells triggered by proteins in our bodies as well as other chemicals. There are three main stages- Inflammation(swelling), Proliferation (filling with new tissue) and remodeling (strengthening). If anything goes wrong at any stage along this continuum there will be an outcome such as infection or scarring (2 ). Clinicians perform wound assessment to determine the appropriate treatment. It includes determination of type of wound, whether its acute or chronic type and its cause is also determined for example, pharmacological, psychosocial etc.(1) The intricate nature of wound healing research is largely due to multifaceted nature and complexity of the healing process, which involves various cell types and repair phases such as inflammation, proliferation, re-epithelialization and remodeling. A range of preclinical models, including mice, rabbits and pigs can be utilize to mimic acute or impaired conditions, such as diabetic and nutrition related wounds (4 ). Several techniques have been developed to model human skin in vitro. Experimental methods vary in approach, including monocultures(2D) of dominant cell types(eg keratinocytes, keratinocytes derived fibroblast) Over co-cultured systems to more complex 3D tissue models (ex. Epidermis only,Reconstructed human epidermis(RHE), or dermis and epidermis based models(human skin equivalent) (5 )    

What Is Wound?

A wound occurs when the integrity of cellular,anatomical or functional structure in living tissue is lost or damaged (1 ).A skin wound arises when the epidermal layer breaks down and loses its integrity. A wound can be defined as any anatomical disruption of tissues that are accompanied by the loss of their function. (2 ).

Types of Wounds-

Acute- For every sharp wound it is important to know the duration since injury (day or hours) whether there is neurovascular supply, involvement of muscle , tendon,ligament and bony structures,and possibility of contaminants in the wound.Also important is when was patient’s last tetanus vaccine/booster dose? Clinicians should start antibiotic if the wound is very dirty or it has been more than three hours since injury.Repair all underlying tissue; irrigate the wound to remove contaminants and bacteria.

In case of traumatic /open fracture the most common classification system used is Gustilo-Anderson:

Type I- Clean wound , low energy puncture wound ,  less than 1 cm in size with minimal contamination or soft tissue damage; adequate coverage without stripping periosteum; minimal comminution.

Type II- Moderate soft tissue damage and crushing; moderate contamination; laceration, greater than 1 cm in size with good coverage over bone,without striping periosteum; minimal comminution.

Type IIIA- High energy open injury massively contaminated wounds; extensive / significant soft tissue damage and crushings; adequate coverage over bones, severly comminuted fracture and/or segmental fractures where there is complete stripping periosteum present.

Type IIIB- Wound is significantly contaminated; soft tissue is damaged extensively; it requires graft to cover bone; severely mashed and/or segmental fracture and there is periosteal stripping and bone exposure.

Type IIIC- Resemble to type A and B, it is affiliated with aerterial injuries which require repair.

Chronic - Chronic wounds are those that become stagnant during the normal stages of inflammation and healing, with a timeframe of three months for non-progression often they stagnant in the inflammatory phase. Various factors and disease conditions hinder the healing of wounds, leading to chronic , non healing wounds

The reasons for chronic wound can be categorized as follows.

Arterial- As a rule of thumb, an ABI (ankle-brachial index) below 0.9 indicates PAD (Peripheral arterial disease) and restricts blood flow to tissue while obstructing the delivery of antibiotics to infected wounds.

Venous- Due to pressure related changes in the permeability of the blood vessel wall, fibrin is able to leak into perivascular space.Fibrin accumulation has direct and adverse impact on wound healing by inhibiting collagen synthesis.

Infection- Underlying infection such as cellulitis or osteomyelitis will impede wound healing.

Pressure- Enhanced pressure to the affected area will inhibit the growth of new tissue and impede proper blood circulation to the wound area.

Radiation therapy- leads to stasis of small blood vessels and causes damage to the fibrosomes and nuclei.

Oncologic- Constantly biopsy areas of situation in non- recuperation wounds, as this can be a peculiar presentation in divers types of malignancies.

Systemic- Systemic disoreders like diabetis, obesity, immunodeficiencies, renal failure also impede wound healing.

Nutrition- Deficiency of vitamins and minerals along with protein malnutrition inhibit haling of chronic wounds. Nutrient absorption get disturbs due to glucose high level.Necessity of protein and energy may increase by 250% and 50% in chronic wound patient.

Age and hormone- A  study on health relted alterations in healing capacity showed that every stage of healing shows distinct age related alterations.Estrogen improves the age dependant impairment of healing in men and women. On the other hand , androgen negatively affect the healing of cutaneous wound.

Pharmacological- Multiple studies have shown that hydroxyurea can lead to ulceration that does not heal. Chemotherapy drugs slow down cell migration, reduce formation of wound matrix, reduce collagen production, reduce the production of fibroblast and reduce wound contraction. These drug weaken the immune system, which slows down the natural inflammatory process of wound healing and increase the risk of infection. Ingestion of steroids reduces inflammation suppresess epithelial function and reduce collagen production. The use of vitamin A an reverse delayed wound healing caused by steroids.

Psychosocial- Cell mediated immunity get disturbed due to psychological stress, inhibit the normal healing process.

Genetic- Pre-existing conditions such as hypertrophic (or keloid0 scarring , genetic predisposition and different skin types ( such as color, flexibility, thickness, sebum quality, and position)

Smoking- cigarette smoking contains over 4000 compounds- Nicotine tightens blood vessels and enhances platelet adhesion. Carbon monoxide bonds with hemoglobin and dereases oxygen delivery.Hydrogen cynide slows down oxygen transport.

Necrotizing soft tissue infection- Necrotizing soft tissue infection (NSTI) are extremely aggressive and can lead to widespread cell death. However, despite greater knowledge about the way they work, these disease often puzzle doctors because of their rarity, multiplicity of symptoms, and occasional confusing nature. A lucid method should enable diagnosis within minutes and swift antimicrobial drug prescription together with instant surgery since failure may threaten both life and limb failure to identify the cause quickly enough leads to almost certain death. (1)

Wound Healing Phases –

Wound healing is a physiological response to tissue damage. The process of healing wound requires a complex interaction between various cell types, cytokines, mediators and the vascular system. Bood vessel contraction and platelet aggregation at first are intended to prevent bleeding .A sequence of inflammatory cells, starting with neutrophils, is brought in. Various mediators and cytokines are released by inflammatory cells to promote angiogenesis, thrombosis and epidermis.Fibroblast deposit extracellular component that will act as scaffolding. Hematostasis, chemotaxis, and increased blood permeability are hallmarks of the inflammatory stage, which can restrict further damage wound closure, removal of cellular debris and bacteria, as well as promote cytological migration.The inflammatory phase typically endures for several days.Granulation tissue, re-epithelialzation and neovasularization are the three phases of the proliferative phase. This period can extend for several weeks. The wound strength is at its highest during maturation and remodeling. Upon injury, the first step is always to release lymphatic fluid and blood. At the point of complete hemostasis, both the extrinsic and intrinsic coagulation pathway are stimulated to prevent blood loss. Upon reaching the damaged endothelial linings, platelet aggregation occurs due to arterial vasoconstriction. Thrombosis is initiated by the release of adenosine 5 diphosphate (ADP) that aggregate platelets. Hemostasis and chemotaxis initiate the inflammatory phase. Both white cells and thrombocytes release more mediators and cytokines into their bodies, thus speeding up the inflammation. Other factors apart from platelet derived growth factor, promote the breakdown of collagen, the change of firoblasts, proliferation of new vessels and re-epithelialization. These process happen simultaneously in a co-ordinated manner. Platelets released mediators such as serotonin and histamine, which increase cellular permeability. The platelet derived growth factor stimulates the proliferation and division of fibroblasts, which in turn is enhanced by transforming growth factors. Collagen is produced by the fibroblasts. A fibrin scaffold is formed by platelet activation, which binds to inflammatory cells like neutrophil/monocyte and endothelial cells. Neutrophils can also phagocytoze cell debris and bacteria, thus helping to de-contaminate the wound. The  granulation or proliferation phase is not discrete and occurs continuously in the background.The fibroblast begin to secret new collagen and glycosaminoglycans by day 5 through 7. The wound’s core is composed of proteoglycans that aid in wound stabilization. Afterward, there is re-epithelialization as cells move away from the wound and surrounding areas. At first, a thin layer of epithelial cells is present, but as time passes it will be cover by progressively thicker more resilient cells. Then neovascularization occurs through angiogenesis, which involves the formation of new blood vessels from existing vessels, and vasculogenesis to form new vessels (VPCS). After the collagen fibres have been deposited on the fibrin framework, they begin to grow. The wound’s contracting is aided by the ongoing accumulation of fibroblasts and myofibro cells. The maturational or remodeling phase can last up to twelve months, beginning in week three. The loss of collagen and a peak in wound contraction occur during week three. wound contraction is much more prevent in secondary healing than in primary healers. At 11 to 14 weeks, the incision wound reaches its maximum tensile strength. The final scar will not possess all of the initial strength of this injury and only around 80% of its tensile strength. (3 )

Different Evaluation Model for Wound Healing Activity                                                                                                                       

The intricate nature of wound healing research is largely due to multifaceted nature and complexity of the healing process, which involves various cell types and repair phases such as inflammation , proliferation , re-epithelialization and remodeling. A range of preclinical models, including mice, rabbits and pigs can be utilize to mimic acute or impaired conditions, such as diabetic and nutrition related wounds (4 ). Human models are highly valued for their  ability to better reflect basic research findings and reduce the need for animal experiments during preclinical evaluation of novel therapy approaches, which aligns with the “3R” principle of humane animal research. The three tissue layer of the humane skin (epidermis,dermis,and subcutaneous fat) work together to form a dynamic and constantly evolving organ. Several techniques have been developed to model human skin in vitro. Experimental methods vary in approach, including monocultures(2D) of dominant cell types(eg keratinocytes, keratinocytes derived fibroblast) Over co-cultured systems to more complex 3D tissue models (ex. Epidermis only,Reconstructed human epidermis(RHE), or dermis and epidermis based models(human skin equivalent) (5 )  

1. In-vitro scratch assay- The scratch wound healing assay is a valuable method for evaluating cell migration during wound healing. The basic principle of the 2D healing assay involves creating a cell free region (a wound gap) in a confluent cell monolayer. This gap allows cells to migrate and repair the wound. The assay consists of three main steps:

I)Cell injury or cell wounding: The cell monolayer is disrupted to create the wound gap.

II) Monitoring of healing process: Observing how cells migrate and close the gap.

III)Data acquisition and data evaluation: Quantifying the healing process. (6)

Material use for scratch assay-

1. Cell culture medium with supplements: This includes serum and antibiotics necessary for cell growth and maintenance.
2. Phosphate buffer saline (PBS): Used for washing cells and maintaining their osmotic balance.
3. Bovine serum albumin: Often used as a blocking agent or to stabilize protein during the assay.

The procedure for a scratch assay involves the following steps:

1. Cell seeding: Cells are seeded into a 12 well culture plate at a density that allows them to reach 70-80% confluence after 24 hours
2. Scratch creation: Once the cells are confluent, a straight line scratch is made using a pipette tip or other suitable tools. The scratch should be perpendicular to the bottom of well.
3. Washing and imaging: Detached cells are washed away, and fresh medium is added. The wound area is imaged using a phase contrast microscope at regular intervals until cells migrate to close the gap (usually 24-28 hours) (7 )

Limitations of scratch assay

1. Non-unifrom cell free areas: The wound creation process in the scratch assay may result in irregular cell- free areas with non-uniform leading edges. (8).This variability can affect the accuracy of migration rate measurements.
2. Initial degree of confluence and geometry: Migration rate measurements are sensitive to the initial degree of cell confluence and the initial geometry of the wound. (9)Researchers need to consider these factors during experimental design.
3. Lack of chemical gradient: Unlike methods such as the Boyeden chamber assay, the scratch assay does not establish a chemical gradient (8 ). No chemoattractant or gradient is preset, which limits its ability to study cemotaxis.
4. Two dimensional culture: The scratch assay is performed in a 2D cell monolayer, which doesn’t fully represent the 3D architecture of tissues. In viv cells interact with ECM components and neighboring cells in a more complex environment.
5. Limited Quantifiacation: Although the assay quantifies wound closure, it doesn’t provide detailed information about cell behavior(ex. Drectionality speed).
6. Cell matrix and cell-cell interaction: The scratch assay is suitable for studying the effects ofcel-matrix and cell-cell interactions on migration (8). However , it disrupts these interactions less effectively than some other methods.
7. Time consuming: The assay usually takes several hours to overnight , not accounting for cell transfection time (8)

2. Reconstructed Human Epidermis- Reconstructed Human Epidermis (RHE) models play a crucial role in wound healing research. These in vitro models provide a human-specific context for studying wound healing processes, allowing researchers to explore cellular and molecular components without relying solely on animal experiments. Let’s delve into the significance of RHE models and their applications in wound healing studies:

What is Reconstructed Human Epidermis (RHE) Model?

An RHE model is  a three dimensional tissue construct that mimics the human epidermis.It typically consists of keratinocytes cultured on collagen-based matrix, closely resembling the native skin structure (10)

Advantages of RHE Model for wound healing research:

1)Human Relevance- RHE models recapitulate the cellular and structural components of human skin, making them more relevant for translational research.

2)Reduced Animal Experiments- By using RHE models researchers can minimize the need for animal studies during preclinical evaluations.

3)Controlled Environment- These models allow precise control over experimental conditions, facilitating reproducible wound healing studies.

Application of RHE Models in wound healing studies:

Wound closure and re-epithelialization- RHE models are used to study the re-epithelialization phase of wound healing. Researchers can monitor how keratinocytes migrate and proliferate to close wounds (11)

Chronic wounds – RHE models help to investigate chronic wounds( ex. Diabetic ulcer,venous ulcers). They allow the assessment of cellular responses and potential therapeutic interventions (10)

Hypertrophic scars and keloids- RHE model contribute to understanding the pathogenesis of hypertrophic scars and keloids. (12)

3. Human skin equivalent- Skin equivalants are valuable tool for studying skin biology,drug testing, and wound healing. The HSE model is  a 3-dimensional live cell skin model designed as an alternative to animal testing. It allows researchers to investigate the effects of products on human skin without using animals (13) HSE are generated by culturing skin keratinocyte at the air liquid interface on a dermal scaffold.

Stratified epidermis- HSE yield a fully stratified epidermis, including a functional stratum corneum.These enables the study of epidermal structure and function in a biomedical context (14)

Applications –

Rsearchers use HSE for studying skin permeation, corrosivity, irritation, compound toxicity, biochemistry, metabolism, and cellular pharmacology (15)

They have been applied in studies related to wound healing, skin barrier function and more.

Limitations-

1. Simplified complexity: HSE are made from primary human cells but lack the full complexity of native human skin. They lack hair follicles, immune cells, and other intricate features.
2. Lack of immune systems: HSE do not have a functional immune system, which is crucial for wound healing and infection control. Researchers use immune cells co-culture alongside HSEs to address this limitations
3. Static vs. Dynamic Healing: HSE provide a static environment, whereas in vivo wound healing involves dynamic processes. Real skin responds to mechanical forces, tension, and movement, which HSEs cannot fully mimic.
4. Limited vasculature and innervations: HSEs lack a fully developed vascular network and nerve endings. Blood vessels supply nutrients , oxygen and immune cells in vivo, but HSE lack this complexity.
5. Species differences :HSE are human derived , but species differences exist. Rodent skin, for example ,heals differently. Caution is needed whe translating HSE findings to clinical applications (16)(17)

4. Diabetic foot ulcer (DFU) derived chronic in-vitro models:  These models are essential for understanding wound healing processes in diabetes and evaluating potential therapies.

Complexities of DFU wound healing: DFU pose challenges due to impaired healing linked to diabetes- related complications. Cellular responses from fibroblasts, keratinocytes, and macrophages play crucial roles. Growth factors and cytokines are involved in the healing process.

Signaling pathways in DFU: Abnormalities in signaling cascades impact wound closure. Key pathway include:

PI3K/Akt: Impaired cell migration and angiogenesis due to compromised insulin signaling and oxidative stress. (19)

MAPK/ERK: Essential for inflammation and tissue remodeling

Wnt/beta-catenin: Crucial for tissue regeneration.

Factors contributing to DFU pathogenesis:

Epigenetic modifications, oxidative stress, advanced glycation end product, and more. (18)

5. Reconstructed keloid model (RKM)- The objective was to established a new reconstructed keloid model (RKM) by combining fibroblasts extracted from different areas of keloid(center, periphery, non- lesional) within a three dimensional biomaterial.This approach allows researchers to study keloid scar formation. The RKM serves a dual purpose : Understanding underlying pathology and testing potential therapeutics. (19)

Mathematical modeling of keloid- While not directly related to in vitro models, mathematical modeling can also provide insights into keloid dynamics.Excessive growth of fibrotic tissue in keloids which invades adjacent areas beyond the original wound borders. Mathematical models can help predict keloid behavior and guide therapeutic interventions (20)

6. Three dimensional hypertrophic scar model

– Researchers have developed a three dimensional model of hypertrophic scars using tissue engineering methods. This model enables the study of human fibrotic skin pathologies , including hypertrophic scars. By embedding cells within an extracellular matrix , they simulate scar tissue behavoiur in vitro. The goal is to understand scar progression and explore potential therapeutic interventions.(21)

Complex nature of scar formation- Scar formation involves intricate cellular and molecular interactions .Despiteadvances in understanding the immune system. Inflammatory responses and proteomic/genomic changes after injury, we lack a comprehensive human scar model.The timeline and mechanisms of hypertrophic scar and keloid formation remain elusive. Ideal in vitro scar model should mimic heterogenous cellular interactions and the evolving structure of human skin.

Pathophysiology and immune modulation – Hypertrophic scars and keloid may be immune-modulated or driven by inflammatory responses. Genetic predisposition alone does not fully explain scar development. (22)

In vivo wound healing model

1. In vivo Excision wound model- Wound healing is a complex process that involves various cellular and molecular events to restore tissue integrity. In the excision wound model, a defined area of skin or tissue is surgically removed , simulating a wound.The healing process is then studied by assessing parameters such as wound closure, inflammation, granulation tissue formation, and re-epithelialization. Researchers use this model to evaluate the effects of therapeutic interventions, wound dressings, and other treatments on wound healing.

Techniques and application : Excision wound models are commonly used in rodents(such as rats and mice).The wound is created by excising a circular or rectangular area of skin, often on the dorsum and flank.Healing progression is monitored over time, and various endpoints are measured, including wound contraction, histological changes, and gene expression.Researchers can assess the effects of drugs, growth factors, and biomaterials on wound closure and tissue regeneration. (23)

2. In vivo Incision wound model- Cutting of the skin or other tissue with a sharp blade results in rapid disruption of tissue integrity with minimal collateral damage.The incision wound model allows researchers to study the healing process after controlled incisions, mimicking surgical wounds. Unlike excision wounds (where tissue is removed), incision wounds involve creating a precise linear or geometric cut without tissue removal. Researchers can assess wound closure, inflammation, granulation tissue formation, and re – epithelialization in this model. (24)

Technique and application: In vivo incision wound models are commonly used in various animal species , including rats, mice, rabbits, and pigs.The wound is created by making a controlled incision using surgical instruments. Healing progression is monitored over time, and endpoints such as wound closure rate, histological changes, and gene expression are evaluated. Researchers can test the effects of drugs, growth factors, and wound dressings on incision wound healing. (25)

3. In vivo Burn wound models- In vivo burn wound models play a crucial role in understanding burn injuries and evaluating potential treatments. These models involve creating controlled burns on living organisms(typically animals) to simulate human burn wound. Researchers utilize these models to investigate wound healing mechanisms, test therapeutic interventions, and assess treatment efficacy.

Principle

Creation of Burn wounds: Researchers induce burn wounds by applying heat (ex. Hoy metal, boiling water, or flames) to specific skin areas.

Healing assessment: The healing process is monitored over time, including wound closure, tissue regeneration, inflammation, and scar formation.

Treatment Evaluation: Different treatments(such as topical agents, dressings, or medications) are tested to determine their impact on wound healing.

Types of In vivo Burn Wound models:

Excision wound models: In these models , a defined area of skin is surgically removed, simulating a deep burn injury. Researchers evaluate wound healing and treatment outcomes.

Incision wound model: Controlled incision mimic partial-thickness burns, allowing study of re-epithelialization and wound closure.

Full Thickness Burn models: These models create burns that penetrate through the entire skin layer, closely resembling severe burn injuries.

Limitations: Using animals for research raises ethical concerns. Responses to burns may differ among species, limiting direct translation to humans. In vivo model can not fully replicate the complexity of human wound healing. Conducting in vivo experiment can be expensive and time consuming. (26)

4.  In vivo Dead space wound model:  The in viv dead space wound model is a valuable tool for studying wound healing in living organisms.In this model, a space is created within the body(often subcutaneously) where no tissue regeneration occurs. Researchers can then investigate how various treatments or interventions affect wound healing in this controlled environment.A study by Oliver et al.(2015) describes a novel small animal model to investigate dead space management in muscle tissue. Here are key details

Objective: Investigate dead –space management in muscle tissue using absorbable test materials.

Materials tested : Two absorbable material were implanted in each animal: Calcium sulfate beads alone, Calcium sulfate beads loaded with vancomycin and tobramycin

METHODOLOGY: Blood samples and radiographs were  taken from each animal following implantation. Animals were sacrificed at different  time points(1,7,21,42,and 63 days post-operatively). Implant site analyzed using micro-computed tomography, histology, and immunohistochemistry.

RESULTS: Complete resorption of the materials was confirmed radiographically at 3 week post-implantation.  Histologically, the host tissue response to both materials was identical Healing at the implant sites occurred with no dead space remaining. Vancomycin was not detected in blood serum, but peak tobramycin levels were observed at 6 hours post implantation.

CONCLUSION: The model was convenient and effective for assessing implant materials for dead space management in muscle tissue. Both tested materials resolved the surgically created dead space without adverse host responses. (27)

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