Non-Animal Models of Wound Healing in Cutaneous Repair:

In Silico, In Vitro, Ex Vivo And In Vivo Models Of Wounds And Scars In Human Skin

Sara Ud-Din, MSc1, Ardeshir Bayat, MBBS, PhD1,2

1Plastic and Reconstructive Surgery Research, Centre for Dermatology Research, University of Manchester,

Manchester, UK.

2Bioengineering Research Group, School of Materials, Faculty of Engineering & Physical Sciences, The

University of Manchester, Manchester, UK.

Corresponding Author: Dr Ardeshir Bayat (MBBS, PhD) Associate Professor Plastic and Reconstructive Surgery Research Centre for Dermatology Research University of Manchester Stopford BuildingOxford Road Manchester M13 9PT England, UK. Tel: 0161 306 0607Email: Ardeshir.bayat@manchester.ac.uk

Running title: Models of Wound Repair in Human Skin

Keywords: Models, wound repair, wound healing, tissue repair, in vivo, ex vivo, in vitro, in silico,

experimental, human

1

ABSTRACT

Tissue repair models are essential in order to explore the pathogenesis of wound healing and

scar formation, identify new drug targets/biomarkers and to test new therapeutics. However,

no animal model is an exact replicate of the clinical situation in man as in addition to

differences in the healing of animal skin; the response to novel therapeutics can be variable

when compared to human skin. The aim of this review is to evaluate currently available non-

animal wound repair models in human skin, including: in silico, in vitro, ex vivo, and in vivo.

The appropriate use of these models is extremely relevant to wound-healing research as it

enables improved understanding of the basic mechanisms present in the wound-healing

cascade and aid in discovering better means to regulate them for enhanced healing or

prevention of abnormal scarring. The advantage of in silico models is that they can be used as

a first in virtue screening tool to predict the effect of a drug/stimulus on cells/tissues and help

plan experimental research/clinical trial studies but remain theoretical until validated. In vitro

models allow direct quantitative examination of an effect on specific cell types alone without

incorporating other tissue-matrix components, which limits their utility. Ex vivo models

enable immediate and short-term evaluation of a particular effect on cells and its surrounding

tissue components compared with in vivo models that provide direct analysis of a stimulus in

the living human subject before/during/after exposure to a stimulus. Despite clear advantages,

there remains a lack of standardisation in design, evaluation and follow-up, for acute/chronic

wounds and scars in all models. In conclusion, ideal models of wound-healing research are

desirable and should mimic not only the structure plus cellular and molecular interactions, of

wound types in human skin. Future models may also include organ/skin-on-a-chip with

potential application in wound-healing research.

2

INTRODUCTION

Many types of models exist that help to investigate the processes involved in normal

cutaneous wound healing in order to optimize the ultimate results of tissue repair. 1 These

models are also essential to explore the pathogenesis of chronic wounds and abnormal scar

formation, identify new drug targets and to test new therapeutics.2 One factor in choosing the

ideal model is to determine which type of wound and phase of healing is to be investigated.3

Thus, tissue repair models can be evaluated with respect to the following distinct stages of

wound healing: wound granulation tissue formation, contraction, re-epithelialization and scar

remodelling.3

Animal models, in vitro cell culture and tissue-engineered models have been used with

varying degrees of success to represent human skin.1 However, animal models are not perfect

as they do not always accurately represent the structure of human skin or develop similar

scars, which are comparable to abnormal scars observed in humans such as keloid scars.4,5 In

addition, the potential cost, ethical, and moral issues associated with their use can be

challenging and problematic.6-8 The best current animal model in terms of dermal structure

and underlying mechanisms is considered to be the pig.9-11 Nevertheless, no animal model is

shown to be an exact replicate of the clinical wound healing scenario seen in man.

Numerous models have been developed to study wound repair in humans which aim to

identify key underlying mechanisms and better understanding of the process of healing.

These include in silico, in vitro, ex vivo and in vivo models (Figure 1). In silico models can be

useful but lack the biophysical characteristics of human skin.7 In vitro models provide

valuable information about one or few components of the skin at a time, but do not correlate

with their in vivo counterparts due to their lack of complexity.6,12 Additionally, ex vivo models

enable immediate and short-term evaluation of a particular effect on cells as well as its

surrounding tissue components although there is a lack of standardisation and the in vivo

3

environment in toto.11 Furthermore, patient and volunteer studies remain essential and pivotal

to testing of emerging novel therapeutics but the appropriate methodology should be chosen

for the type of wound or scar being studied.6 In view of this, the aim of this review is to

evaluate currently available models of wound repair in human skin and offer a detailed

overview of their clinical and scientific relevance and utility.

MATERIALS AND METHODS

The available literature was reviewed regarding non-animal models of wound repair. The

PubMed and Scopus databases were used to search the recent literature to 2016. Different

combinations of terms were used, including models, wound repair, wound healing, tissue

repair, in vivo, ex vivo, in vitro, in silico, experimental, human. Articles not written in English

and those that did not include relevant information were excluded. Relevant articles

referenced within the articles identified with the above search were also included. All

retrieved articles were reviewed for their relevance to the specific topic.

4

RESULTS

The results will now be presented under the headings in silico, in vitro, ex vivo and in vivo

models.

In silico models

The complexity of the biochemical and biophysical processes involved in tissue regeneration

highlight the need for computational models in order to understand cell growth and to design

efficient scaffolds and tissue substitutes.13 Multiphase models have been used to describe

time-dependent processes in vitro in a perfusion bioreactor, with particular focus on cell

growth, access to nutrients, and scaffold degradation.14 Other computational methods have

modelled cell spreading and tissue regeneration in vitro using porous scaffolds. They looked

at transport and nutrient intake, cell population, ECM deposition, cell attachment and

migration.13,15,16 Agent based models have been used to investigate the role of growth

factors,17 organisation of keratinocytes,18 presence of fibroblasts,19 and the importance of stem

cells in long-term skin epithelium regeneration.20 In silico models (Figure 1) may be useful

theoretically and in combination with other models but are lacking the physical

characteristics of human skin.6

A number of models have been designed using differential equations.21-24 These models have

been developed to analyse strategies for improved healing, such as wound VACs,

commercially engineered skin substitutes and hyperbaric oxygen therapy. Mathematical

models using ordinary differential equations have been developed to investigate the effects of

commercially engineered skin substitutes. Waugh and Sherratt suggest that the model can be

used to explore the effects on interpatient variability or the gradual release of treatment

5

components.22 Models using partial differential equations have also been designed to analyse

the effects of hyperbaric oxygen on the promotion of wound angiogenesis.23,24 Both models

used the ‘snail-trail’ model of angiogenesis.25

A mechanical model using a finite-element method has also been used to analyse VAC

therapy and the simulation results are consistent with the in vitro strain levels shown to

promote cell proliferation.26 This approach may be used to evaluate treatment strategies that

promote cell proliferation, growth-factor production and wound angiogenesis by applying

micromechanical forces.

Computational approaches for deterministic systems in wound healing have been tested.

There have been analyses of changes in wound morphology using either planar or

axisymmetric wounds.27 A two-dimensional epidermal wound was used to assess the wound

boundary using a level-set method. Additionally, a further study used a similar method

analysing wound contraction using the finite-element method.28 Agent-based models have

been developed to analyse different treatment strategies with wound debridement and topical

administration of growth factor.21 Many studies have used agent-based models for a multi-

scale approach to wound healing.29-32

A multi-scale approach has also been developed for deterministic systems by using a hybrid

continuous-discrete model when studying the flow through a vascular network.33,34 Recently,

a continuum hypothesis-based model has also been used for the simulation of the formation

and regression of hypertrophic scar tissue after dermal wounding.35

6

In vitro models

There are several in vitro models of tissue repair that can assist with answering specific

mechanistic questions related to wound repair.36,37 Many of these in vitro models are used to

answer basic questions related to cell signalling in response to cell stress or injury.37 In vitro

cell culture models have been used for many years to gain insight into different aspects of

scar pathogenesis.36,37 In vitro models can be categorised as single cell, co-culture and

organotypic (Figure 2, Figure S1).

Single cell models work by the relevant cells being grown in vitro after which a ‘wound’ is

made in the cell layer by scraping the dish surface with either rubber devices or the tip of a

pipette, thus removing cells from the scraped area, creating a scratch and trauma to the cells

next to the wound.37 Subsequently, the rate at which cells divide and migrate into the ‘wound’

area is determined microscopically.37 The basic physiological processes are then analysed

including proliferation, protein production and secretion, viability, migration, gene

expression and differentiation.38 Single cell models are often used with dermal fibroblasts and

keratinocytes39-41 testing various agents for the enhancement of fibroblast migration or re-

epithelialisation.

In order to overcome the limitations of single cell models, several three-dimensional in vitro

models have been utilised.42.43 Fibroblasts are seeded in a type I collagen solution which is

solidified to gel, resulting in cells being embedded in a three-dimensional collagen lattice that

is then transferred to a culture dish and covered with medium.44 This model enables the

evaluation of cell contraction and matrix compaction. Another wound healing model45

embedded fibroblasts in a 3D collagen construct and the cell migration is followed from the

denser collagen matrix into a surrounding matrix. The limitations of this model include the

use of only one type of ECM protein and one cell type.

7

A number of recent quantitative studies have analysed in vitro wounding assays to investigate

aspects of cell migration for various cell types.46-50 Human in vitro models were used in a

study to investigate human skin integrity, wound closure and scar formation.51 These methods

included, cell culture, fibroblast and endothelial cell migration scratch assay, fibroblast

chemotaxis assay, endothelial cell in vitro tube formation assay and skin equivalent. The

authors showed that these models provided a platform for testing the mode of action of novel

compounds for enhanced and scar free wound healing. Two dimensional cell culture models

do not represent the interactions and mechanisms present in whole skin, such as the effect of

the extracellular matrix (ECM) in terms of structure, as well as cell signalling mechanisms

and the processes of metabolism.52

Indirect co-cultures of keratinocytes and fibroblast monolayers using transwell systems has

also enabled the study of keratinocyte–fibroblast interactions.53,54 The transwell assay or

Boyden chamber assay has been used to analyse the chemotactic responses of leukocytes.55

The principle of this assay is based on two mediums containing chambers separated by a

porous membrane.54 The transwell migration assay can be used for many different cell types

including epithelial,56 mesenchymal57 and brain cancer cell lines58 as well as many primary

cells. However, it is difficult to obtain the 3D macroscopic fibrotic tissue structure of a scar.

The expression of biomarkers from gene and protein studies is affected by the 3D structure of

a scar.37 The use of a more 3D environment such as collagen and mechanical load can

positively influence the behaviour of fibroblasts by creating a more realistic condition.59,60

One study cultured human cells derived from patients with chronic venous ulcers using

biopsies and demonstrated that they maintain their in vivo phenotype.61 Fibroblasts derived

from different wound locations were tested for their migration capacities and another portion

of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage.

8

Another study also used chronic wounds for in vtiro assessment by taking punch biopsies

from patients with venous ulcers and evaluated their histology, biological response to

wounding (migration) and gene expression profile.62

Scratch assays are commonly used to observe the migration and proliferation of cells.63-65

Cells are placed on a culture dish and incubated, eventually forming a monolayer.66 An

artificial wound is created and images of the cell spreading by migration and proliferation are

captured over 12–24 h.67-69 Various cell types have been analysed for migration with this

assay including epithelial and mesenchymal cancer cells, keratinocytes, normal epithelial

cells, endothelial cells and fibroblasts.70-75 The majority of scratch assays falls into one of two

categories; wound assay or scrape assay. The wound assay involves the creation of a thin

wound,64,66 which produces two opposing directed cell fronts which merge.67 The scrape

assay, involves a monolayer that has been scratched further so there is only one cell type.67

Scratch assays have been used to investigate the influence of various chemical compounds

and potential drug treatments on the rates of cell motility and proliferation.63,65

When keratinocytes are cultured separately and then placed on top of the collagen gel

containing fibroblasts, an organotypic skin-like three-dimensional culture is created.76 This

model enables analysis of the interaction between these two types of cells in a three-

dimensional manner. Organotypic skin equivalents have been used to investigate scar

pathogenesis.77 3D skin equivalent models have used keloid fibroblasts in combination with

normal skin-derived keratinocytes.53,78 Using a similar method, a fully differentiated

epidermis constructed from keratinocytes isolated from hypertrophic scars on a fibroblast

populated dermal matrix showed characteristics of an abnormal scar including dermal

thickness, epidermal thickness and collagen I and illustrated the role of keratinocytes in

hypertrophic scar formation.79 Use of these models for testing therapies is limited due to the

lack of validated biomarkers and the dependence on excised scar tissue. Whilst in vitro

9

models have many benefits, they also have some limitations. The majority of cell culture

models employ serum to support and enhance the growth of cells in vitro.47,48 Serum is a

pathologic, inflammatory fluid compared to plasma and interstitial fluid that normal, resting

cells experience. Therefore, any observations made of cells in a serum environment should be

made with caution when assuming normal cellular function and behaviour.

Ex vivo models

Ex vivo human skin tissue is more appropriate for certain types of research. The use of whole

skin biopsies in culture allows the effect of individual ingredients and formulations to be

tested in an environment more closely mimicking normal skin.80 Full-thickness skin organ

culture is already an established tool used for understanding human skin pathophysiology and

evaluation of novel treatments.81-84 A number of wound healing organ culture models have

been previously reported in studying cutaneous wound healing processes.76,85,86 Therapies

including anti-inflammatory interleukin 10, anti-fibrotic streptolysin O and dermal substitutes

have been evaluated using this model.81,82,86 Despite promising results, there is a lack of

standardisation and conformity in these studies. Variations in the methodologies include the

use of different wound types such as a partial or full thickness wound, different culture

conditions, and a range of support and growth mediums.83,87 Ex vivo model types can be

divided into normal skin models and pathological skin models (Figure 3, Figure 4).

A study looking at the effects of topical formulations on human skin demonstrated that

altered expression of key gene and protein markers could be quantified in an optimised whole

tissue biopsy culture model.1 This model could be adapted to study a range of compounds or

10

disease mechanisms, negating the requirement for animal models, prior to study in a clinical

trial environment.

The ex vivo donut-shaped wound healing model has been used to investigate human

cutaneous repair.88,89 In a study by Sebastian et al, human tissue was collected and wounded

using a standardised punch biopsy (full thickness wound) and the effect of electrical

stimulation was evaluated.88 Another study also used this model for studying the effect of

dermal substitutes on human dermal fibroblasts and keratinocytes.89 Full thickness lower

human patient abdominal skin was collected and 8mm punch biopsies taken. In the centre of

these cylindrical skin biopsies, a 4mm full thickness artificial wound was created by a further

punch biopsy. Models were placed into 24-well plate inserts and prepared dermal substitutes

were inserted into the wound area. This model has been shown to be useful in studying

cutaneous wound healing.

Ex vivo models have been used when analysing stretch marks and scars including

hypertrophic and keloid scars.90-92 Multipotent keloid-derived mesenchymal-like stem cells,

found in the scar, have been implicated in keloid formation.91,92 Therefore, keloid explant

models are interesting as they allow these cells to remain in their location. Ex vivo biopsies

have been cultured at air–liquid interface, embedded in collagen gels.93,94 The functionality of

this model was confirmed by the reduced epidermal thickness and scar volume after

treatment with the dexamethasone. A further study evaluated the effect of two promising

candidate anti-fibrotic therapies: (-)-epigallocatechin-3-gallate and plasminogen activator

inhibitor-1 (PAI-1) silencing in a long-term human keloid organ culture (OC).95 This model

has several advantages; the keloid explants remain viable in culture for up to 6 weeks without

tissue deterioration, the maintenance of the keloid cellular and microenvironments enable

examination of keloid pathophysiology in situ and anti-fibrotic agents and gene silencing

using RNA interference technology can be used to investigate the cellular and molecular

11

interactions in the epidermis and dermis.94 The results showed that these therapies inhibited

growth and induced shrinkage of human keloid tissue and found this to be an effective

model.95 Another study assessed the extent to which differences relating to cell type influence

PAI-1 gene regulation with regard to growth state-associated mechanisms of expression

control using keloid fibroblasts and normal dermal fibroblasts for cell culture.96

Another ex vivo study utilised an organ culture model of dermal fibrosis to investigate the

effect of photodynamic therapy on skin scars and stretch marks in humans.90 This unique ex

vivo model showed the morphological and cellular effect of photodynamic therapy and

correlated this with the degree and severity of dermal fibrosis.

In vivo models

Human in vivo models are useful as the pathology and physiology of healing (in particular in

acute wounds) is identical to that found in the patient. There are a number of wound models

that can be inflicted upon a human patient or volunteer to provide accurate and representative

research tools (Figure 5, Figure 6). Due to the difficulty in obtaining suitable volunteers with

chronic wounds and incorporating them into a study, human acute wound models provide the

best opportunity to understand the wound healing process and the performance of different

products that aid and promote wound healing. Both patient and volunteer models have their

advantages and limitations. Patient models allow assessment in a variety of wound or scar

12

types, whilst volunteer models only enable evaluation of one type of wound/scar. This is

significant when when interpreting and analysing the results of these studies.

Patient model

Patient studies remain essential and are always necessary to validate potential novel wound

and scar therapies identified in other wound and scar models. Patient models are useful as any

wound type can be assessed using a variety of methods including incisions, excisions and

sequential punch biopsies (in non-keloid formers). However, chronic wound studies have

limitations due to the heterogeneity of the population and lack of uniformity of the wound.

Patients with scars can be used to explore the pathogenesis of scar formation, however, keloid

formers are rarely included due to concerns for recurrence and worsening of existing scars.

Incisional wound models are useful as they can be accurately reproduced and can be used for

multiple investigations. This model can evaluate the wound healing process and the influence

of different local compounds/dressings, response to energy based therapies, as well as

systemic effects of drugs. This model has been used extensively following cutaneous surgery

in order to evaluate quantitative outcome measures including rate of wound closure, quality

of healing as well as wound strength.97,98 Biomechanical analysis of soft tissue and incisional

wounds in different types of tissues have also been described.99

Acute wounds such as split-thickness donor sites have the benefit in that two wounds can be

created in a controlled fashion in the same patient. In a randomized, blinded fashion, one

wound can be treated with the therapy and the other, the control which is the optimal study

design.100,101 However, these wounds heal fast, therefore, it is difficult to demonstrate a

clinically significant increase in healing rate.

13

Volunteer models

Human volunteer models enable assessment of a homogeneous population and the volunteers

can act as their own controls. The limitations of these models are the inability to create other

wound or scar types such as chronic wounds or keloid scars or study different conditions. A

number of volunteer models have been utilised including excisional, incisional, scratch,

blister, tape stripping and burns.

Biopsy models have been used which enable a standardised approach as the subjects can act

as their own controls. These wounds are useful from an experimental perspective in that their

size and depth can be precisely controlled. As they involve all the components of the healing

process, they provide an excellent research tool and have been used to investigate

angiogenesis, wound contraction, and wound closure.102 Numerous studies have been

conducted evaluating various treatments for both wounds and scars using a sequential skin

punch biopsy model to assess various characteristics and correlated by clinical

measurements.102-107 Furthermore, studies have also monitored treatment efficacy in raised

dermal scarring including hypertrophic and keloid scars using objective non-invasive

measures.108-110 However, difficulties arise when attempting to experimentally monitor the

tissue in particular in keloid scars as we would be unable to biopsy the tissue as this would

lead to further abnormal scarring. For these in vivo studies, it is important to clarify the

definition of healing in the human wound. The endpoint should be identified (when the

wound has healed), and the determination of scar formation. The advantages of this model are

that it uses a wound with loss of tissue as found in clinical practice. These wounds enable the

evaluation of different therapies with the ability to create the same wounds each time and

measure different healing parameters quantitatively. Early healing can be monitored followed

14

by scarring and surrounding wound area can be observed. The disadvantage is that the wound

heals from the edges as well as by contraction.

A study by Dunkin et al investigated the association between scarring and depth of dermal

injury. They developed a novel device, which produced a wound that was deep dermal at one

and superficial at the other to healthy volunteer hips. They showed that this dermal scratch

model provided a standardised and reproducible model for investigating the healing response

to dermal injury of different depths.121

The blister model is used for the evaluation of epidermal regeneration and the influence of

various therapies. Blisters are produced by suction using different types of devices which

produce standardised, small, epidermal bulges.111,112 Normal human skin tends to blister after

35-55minutes and a scalpel can then remove the blister roofs, therefore, creating identical

superficial wounds of similar dimensions.113 Absoprtion effect can also be evaluated under

occlusive and semi-occlusive conditions and the trans-epidermal water loss can be measured

for the skin barrier function.114 The advantages of this model are that the wounds are identical

and can be in the same anatomical location on the body. The disadvantages are that

evaluation of such superficial wounds may have no relevance to much deeper dermal wound

types.

The tape stripping model involved successive stripping of the epidermis using adhesive tape

in order to disintegrate the skin barrier in the lowest part of the stratum corneum.115 This can

then be measure by a trans-epidermal water loss measurement device.114 This model is less

damaging to the epidermis than the blister model. A study used the tape stripping model to

assess the penetration of quantum dots.116 Adhesive tape was applied and pressed onto the

skin using a roller. The skin was stripped with 15 pieces of adhesive tape. Negative control

15

strips were obtained from a non-treated area of the upper arms. Tape strips removed from

skin were analyzed using confocal microscopy.

There have been studies that have created a burn in healthy volunteers to assess various

therapies. A randomised double-blind cross-over study in six volunteers investigated the

effects of intravenous lidocaine infusion on partial thickness skin burns.117 A thermoprobe

was used to induce a standardised thermal injury (1cm2) on the flexor side of one forearm and

was repeated on the opposite side 1 week later. Another study directly compared UVB

induced inflammation and the more established thermal burn and topical capsaicin pain

models.118 They delivered UVB to small areas of volar forearm skin in healthy volunteers and

found that the degree of inflammation and concomitant increase in sensitivity to cutaneous

stimuli were UVB dose and time dependent.

DISCUSSION

This review has evaluated currently available wound repair models in human skin, including:

in silico, in vitro, ex vivo and in vivo. The appropriate use of tissue repair models is extremely

relevant to wound healing research as it enables improved understanding of the basic

mechanisms behind the wound healing cascade and aid in discovering better means for

enhanced healing or prevention of abnormal scarring.6

16

The advantage of in silico models is that they can be used as a first in virtue screening tool to

predict the effect of a drug/stimulus on cells/tissues and help plan experimental

research/clinical trial studies but remain theoretical until validated.7 Additional limitations of

the in silico mathematical model is the lack of standardisation which is highlighted by the

arbitrary choice of specific healing parameter values use in these models.7,8 If the aim of the

model is to provide a quantitative prediction of a physiological process, the model parameters

require better quantification.13 In vitro models allow direct quantitative examination of a

particular effect on specific cell types alone without incorporating other tissue matrix

components, which limits their utility.12 Additionally, in vitro models have had a significant

role in the study of wound healing and scar formation.36,37 This had led to a better

understanding of biomarker expression, the role of keratinocytes and fibroblast activity in

wound and scar development.46-51 Although the disadvantage is that, it can be difficult to

extrapolate the findings of in vitro research to the in vivo wound scenario. Three dimensional

models including extracellular matrix, human skin constructed from keloid-derived tissue and

mesenchymal cells, have been recently made to mimic the conditions upon which these scars

form.91,92 Ex vivo models enable immediate and short-term evaluation of a particular effect on

cells and its surrounding tissue components compared with in vivo models that provide direct

analysis in the live human subject before, during and after exposure to a stimulus.12 Human in

vivo models are useful as the pathology and physiology of healing is identical to that found in

the patient.102 Both patient and volunteer models have their advantages and limitations.

Patient models allow assessment in a variety of wound or scar types, whilst volunteer models

only enable evaluation of one type of wound or similarly induced scar.102,108-110 Despite clear

advantages, there remains a lack of standardisation and conformity in design, evaluation and

follow-up, for all wound types including acute, chronic wounds and scars.

17

Many of the human wound and scar models have a limited duration of days or weeks,

whereas human scars develop over a period of months or years.52,53 Therefore, these models

enable us to look at the early stages of wound healing and scar formation but may not

accurately reflect the later stages of wound contraction or scar formation. Another limitation

is that a number of cell types that are involved in the healing process have not yet been

incorporated into the relevant human cell culture models.119,120

An emerging model is the organ-on-a-chip or skin-on-a-chip which involves engineered

tissues, which are similar to the in vivo equivalents and consist of multiple different cell types

interacting with each other under closely controlled conditions and are grown in a

microfluidic chip.121-123 These controlled conditions will make it possible to mimic the

environment of the skin (humidity, temperature, pH, oxygen levels), the elasticity of the skin,

and the complex structures and cellular interactions within and between the different cell

types.122

In summary, understanding the mechanism and pathophysiology of how scars form and

wounds heal, is essential if we are to develop optimal strategies in the future to prevent

adverse scar formation and prolonged healing. Ideal models of wound healing research

should mimic not only the structure but also the cellular and molecular interactions, of wound

types in the human skin.

Sources of funding: No sources of funding to declare.

Conflict of interest: No conflict of interest to declare.

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Figure Legends

Figure 1: Diagram summarising the four main categories for human models of wound repair

including; in silico, in vitro, ex vivo, in vivo. Within each of these domains the assays are

shown and the types of wound models.

Figure 2: In vitro models. Flowchart depicting the three types of in vitro models; single cell,

co-culture and organotypic.

Figure 3: Ex vivo models. Flowchart depicting the two categories of ex vivo models; normal

skin (full-thickness and partial thickness) and pathological skin (stretch marks, scars,

hypertrophic scars and keloid scars).

Figure 4: Ex vivo assays. A diagram to demonstrate the assays including angiogenesis,

apoptotic, proliferation, fibrotic and rate of epithelialisation.

Figure 5: In vivo models. Flowchart depicting the two categories of ex vivo models and the

types of wounds used; patients (surgical wounds, incisional wounds, excisional wounds,

donor site wounds) and human volunteers (Incisional wounds, excisional biopsy wounds,

scratch, blister tape stripping and burns).

Figure 6: In vivo assays. A diagram to demonstrate the assays including invasive (gene and

protein studies) and non-invasive devices and those used for specific healing parameters.

Supplementary Figures

Supplementary Figure S1: In vitro assays. A diagram to demonstrate the different assays for

migration, invasion proliferation and senescence.

38