Wound Medicine 20 (2018) 43–53

Contents lists available at ScienceDirect

Wound Medicine
journal homepage: www.elsevier.com/locate/wndm

In vivo models for assesment of wound healing potential: A systematic T

review
⁎
Alankar Shrivastava, , Arun Kumar Mishraa, Syed Salman Alia, Aftab Ahmadb,
Mohammed F. Abuzinadahc, Najam Ali Khana
a
School of Pharmaceutical Sciences, IFTM University, Moradabad, Uttar Pradesh, India
b
Department of Health Information Technology, Jeddah Community College, King Abdulaziz University, Jeddah, Saudi Arabia
c
Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Keywords: Introduction: The wound is defined as an injury of a living tissue, arose due to either an accidental injury like cut
Wound healing of the cells or skin or sometime, maybe broken the tissue, which may cause the anomalies in the cellular ability
Incision to exhibit the protective mechanism. Wound healing is process of regaining the integrity of cell structure and
Excision layers of the skin. The normal healing process of wounds are categorized in 4 phases which includes hemostatic,
Burn
inflammatory, fibrolastic and maturation.
Dead space
Purpose: The present manuscript embarks on up to date information on different methods being used for wound
healing assessment including incision, excision, burn wound and dead space models. The biochemical parameter
which acts as wound healing markers are collagen estimation (hydroxyproline), hexosamine, DNA estimation,
total protein estimation, wound Index, contraction area, epithelization period and tensile strength etc. Histologic
evaluations is also an integral part of wound healing evaluation which, help in evaluating the cellular and matrix
detail within the wound.
Conclusion: At present, molecular approaches are important to heal the wound. For assessment of potential of
any herbal drug/synthetic compound, incision wound model, excision wound model, burn wound model and
dead space model and their parameter are used.

1. Introduction
A wound may be defined as an injury of living tissue or break in the
epithelial integrity of the upper layer of skin. According to WHS
(wound healing society), wound or physical injury which may lead to
opening or break down the skin system, may further lead to disturbance
in skin anatomy, physiology and their function [1]. Wound Healing is a
natural phenomenon, occurs by replacing devitalized and missing cel-
lular structure and tissue layers of the skin.
hematoma (blood tumor) and crush injury etc. Acute wound is a normal
tissue injury and timely operative process, results in restoration of
anatomic and physiologic integrity of skin. Cuts or surgical incisions are
the example of acute wounds, which may be restored within the spe-
cified time frame. Chronic wound generally fails to restore the healing
process. Most of the local infections, lack of blood supply, foreign
particles and systemic problems like diabetes, immuno deficiency and
others biological mediators are the most dangerous causes of chronic
wound.

1.1. Classification of wound 1.2. Mechanism of wound healing

The wound can be divided into two main categories which include
open wounds and closed wounds (Fig. 1). Open wounds are defined, in
which blood escapes the body and bleeding is clearly observed. e.g.
incised wound, puncture wound, penetration wound and tears wounds
(laceration) [2]. Closed wound, covers the system, in which, blood es-
capes in the circulatory system but remains under the skin e.g.
Wound healing occurs in four processes which includes hemostasis,
inflammatory phase, proliferative or fibroblasts phase (this phase in-
cludes collagen synthesis, neovasculature formation and re-epitheliali-
zation) and wound contraction (Fig. 2). The process is called as re-
modeling of tissue [3].
Summary of work done so far on wound healing is presented in

⁎
Corresponding author.
E-mail address: alankar1994.ss@gmail.com (A. Shrivastav).

https://doi.org/10.1016/j.wndm.2018.01.003
Received 12 December 2017; Accepted 19 January 2018
2213-9095/ © 2018 Elsevier GmbH. All rights reserved.
A. Shrivastav et al.
Fig. 2. Mechanism of wound healing.
Table 1.
2. Formulation better for wound healing
For a good wound healing formulation, it must contain active in-
gredient which posses good aqueous solubility, better tissue absorption,
delayed metabolism and long plasma half-life. Few of the active agents
as curcumin is considered unsuitable for systemic administration but
contemporary researchers have worked to solve the problems and for-
mulations of curcumin such as films, fibers, emulsion, hydrogels and
different nanoformulations have been developed for targeted delivery
at wounded sites [43].
Eupatorium glandulosum m. extract contains number of active con-
stituents. Wound healing activity of Eupatorium glandulosum michx was
44
Wound Medicine 20 (2018) 43–53
Fig. 1. Open and Closed Wounds.
investigated by formulating it into gel formulations. Initial investiga-
tion revealed that formulations exhibited promising results for pH,
viscosity, spreadability and anti-microbial studies. In vivo studies of gel
formulations were studied by applying the formulations on shaved skin
of wistar rats. It was observed that formulation showed a shorter period
of epithelization with greater rate of wound contraction [35].
Polyherbal formulation prepared from the plant extracts accelerates
the wound healing process by proliferation and mobilization of fibro-
blast and keratinocytes and angiogenesis at the site of injury. It ex-
hibited quick wound closure with beneficial improvement in tissue
biochemical and antioxidant parameters [36].
One of natural polymer, Chitosan causes lethal pneumonia in dogs if
given a high dose of chitosan. The intratumor injection of chitosan on
mice bearing tumor increases the rate of metastasis and tumor growth.
 Table 1
A. Shrivastav et al.

Work Done on Wound Healing so far-.

S. No. Model used Plant Remark Reference

1. Excision Aegle marmelos The leaves of Aegle marmelos ethanolic extract also showed faster rate of healing when compared with standard topical application. [4]
2. Excision and Incision Tamarindus indica T. indica cork and seed ash has significant wound healing potential and comparatively T. indica cork ash has significant wound [5]
healing activity than T. indica seed.
3. Excision Azadirachta indica The presence of various phytoconstituents which were found to be present in the stem bark of Azadirachta indica (Neem) [6]
4. Excision Artemisia indica the formulated dendri-creams showed better wound healing activity [7]
5. Excision Malva sylvestrisand Solanum nigrum and Rosa Polyherbal cream experimentally and histopathologically revealed a burn wound healing activity due to the antioxidant, anti- [8]
damascena inflammatory and antimicrobial activities of its phytochemical contents. Therefore, this study confirms the use of M. sylvestris, S.
nigrumand R. damascenain burn prescriptions.
6. Excision Anredera cordifolia The research shows the, significance of the A. cardifloia leaf extract has a potential for wound healing in guinea pig. [9]
7. Burn Areca catechu Areca catechu has polyphenols and some alkaloids which enhances the healing of incision and excision wounds by increasing the [10]
breaking strength of granulation tissue.
8. Excision Acorus calamus The extracts could effectively inhibit the mRNA expressions of inflammatory mediators induced by lipopolysaccharide in RAW [11]
264.7 cells. These wound-healing activity of aqueous extracts in the animal model of excise wound healing, and anti- inflammatory
activity in vitro was observed as positive.
9. Excision and burn Curcumin (Curcum longa) Comparative acceleration in wound healing was due to early implementation of fibroblasts and its differentiation (increased level of [12,34]
α- smooth muscle actin). Western blotting and semiquantitative PCR analysis clearly indicated that COP bandage can efficiently
quench free radicals leading to reduced antioxidative enzyme activity.
10. Excision Solanum xanthocarpum The ethanol extract has remarkable wound healing potential and appeared to justify the traditional use of Solanum xanthocarpum in [13]
wound healing.
11. Excision, Incision and Dead space Buteamono sperma At present work made scientific base to the ethno medicinal use of Buteamono sperma, which is largely responsible for additive or [14]
synergistic effect of their constituents.

45
12. Excision, Incision and Dead space Mimuso pselengi In this study, collagen maturation by increased cross-linking of collagen fibers. The increased weight of the granulation tissue also [15]
indicated the presence of higher protein content.
13. Excision and incision Hippo phaerhamnoides Aqueous leaf extract exhibited significant healing potential in burn wounds and has a positive influence on the different phases of [16]
wound repair.
14. Excision and Burn Mimosa pudica The results of present study further suggested the Mimosa pudica facilitates healing by increasing the rate and extent of wound [17]
closure. The topical formulations containing herbal extract possess good wound healing activity.
15. Excision and Incision Acorus calamus A. calamus extract ointment-treated animals showed remarkable effect on collagen level. [18]
16. Burn Carica papaya Papaya latex formulated in the Carbopol gel is effective in the treatment of burns and thus supports its traditional use [19]
17. Incision, Excision, Burn and Dead Sesamum indicum In this study, a significant increase in the breaking strength, dry weight and hydroxyproline content of the granulation tissue was [20]
space observed. The results suggested that S. indicum seeds and oil applied topically or administered orally possesses wound healing
activity.
18. Excision, Incision and Dead space Cordia dichotoma In this study, hydroxyl groups of flavonoids make the radical inactive by combining with radical, flavonoids [OH] + R → Flavonoids [21]
[O] + RH.
19. Excision Cassia հstula Ethanolic extract of leaves exhibited potent wound healing action. [22]
20. Excision, incision and dead space Allium sativum Aqueous and ethanolic extract of bulbs are useful in the treatment of wounds [23]
21. Excision Ziziphus nummularia Ethanolic extract of leaves are benifical for quick wound healing. [24]
22. Excision, incision and dead space Weddelia chinensis Leaves were used to treat microbial attack and help to heal the wound [25]
23. Excision, incision Sida acuta Methanolic extract whole plant increase tensile strength of wound [26]
24. Excision, incision Rubia cordifolia Root are used in the treatment of wound [27]
25. Excision, incision and dead space Quercus infectoria Leaves or juice are applied directly to the wound and quick relief is observed due to collagen production. [28]
26. Excision, incision and dead space Piper betle Tubers are used in the infections ROS are also disabled. [29]
27. Excision Mirabilis jalapa Hydromethanolic extract of flower are used to treat wound. [30]
28. Excision, incision Michelia champaca/Champaca Methanolic extract of the stem bark increase wound healing. [31]
29. Excision, incision and dead space Lycopodium serratum Whole plant are used in the treatment of wound [32]
30. Excision, incision and dead space Kaempferia galangal Ethanolic extract of Rhizomes are used to treat wounds with increase of collagen level. [33]
Wound Medicine 20 (2018) 43–53
A. Shrivastav et al.
Therefore, it is important to consider these effects of chitosan, prior to
drug delivery [37]. It indicates that chitosan should be modified before
its application for wound healing purpose.
In another study, Water-soluble chitin (WSC) was prepared by
deacetylating chitins to about 50% of N-acetyl content and topical
formulations were prepared to evaluate wound healing on rabbit ear
model. The application of WSC ointments significantly accelerated
wound healing and wound contraction. The grown granulation tissues
including dense fibroblast deposition were observed under the thick-
ened epithelium of the wound treated with WSC ointments. The in-
flammatory cells in WSC ointment treated animals were significantly
decreased. Neovascularization was the most prominent in WSC oint-
ment group. The result demonstrates that the topical formulation based
on WSC is excellent dressing for wound healing process [38].
In another study topical gel formulations of lawsone, prepared by
dispersion technique using natural and synthetic polymers like tara
gum and HPMC K15 M. The results of the animal studies showed better
wound closure when applied topically. Significant decrease in time of
epithelization was observed in groups treated with lawsone [39].
3. Molecular approaches involved in wound healing
Wound healing remains a challenging clinical problem therefore
correct and efficient wound management is essential. Much effort has
been focused on wound care with an emphasis on new therapeutic
approaches and the development of technologies for acute and chronic
wound management. Wound healing involves multiple cell populations,
the extracellular matrix and the action of soluble mediators such as
growth factors and cytokines. The process of wound healing involves
several molecular steps as epithelial-mesenchymal interaction,
Hedgehog signaling pathway etc.
3.1. Epithelial-mesenchymal interaction in healing
The epithelial cells undergo an epithelial-mesenchymal transition
(EMT) and thereafter migrate to the organs to differentiate themselves
in their mesenchymal components, including fibroblasts, smooth
muscle cells of blood vessels. Even more likely, pericytes [40]. The skin,
and other organ as intestines, liver and glandular tissues, contains
epithelial and mesenchymal cells. The epithelial cells adhere one to
another strongly and makes layers. The mesenchymal cells are non-
polarized [41]. The biological cascade occurring in the epithelial-me-
senchymal transition make it feasible for a polarized epithelial cell
leading to molecular alteration, acquiring a mesenchymal phenotype,
with migratory capacity through the extracellular matrix and increase
the production of the matrix [42].
3.2. Hedgehog signaling pathway
Hedgehog (Hh) is a family of secreted signaling molecules which
associated are in many steps, including the action as key agents in the
standardization of numerous tissues types [43]. Hh comprehends the
process of proteins that regulate the diverse biological processes, in-
ccluding embryological development, homeostasis, and cell repair.
Keeping in view the fact that the Hh ligand can regulate angiogenesis,
its signaling can also influence tissue remodeling.
The Hh gene was first identified in genetic works about the seg-
mentation of the body in Drosophila melanogaster, which is the fruit fly.
The German researchers, Nüsslein and Wieschaus, in 1980, studying the
cytogenetics of the embryos of Drosophila melanogaster, identified that
the loss of a gene caused projections within these [44].
3.3. Pivotal genes in wound healing: hub nodes of clusters as the key
elements in regulation
In view of wound healing, cluster hubs are pivotal nodes in highly
46
Wound Medicine 20 (2018) 43–53
connected modules (i.e., clusters). Which are intrinsically important to
understand the underlying genetic mechanisms. In order to select the
hub nodes, partitioning of the network with cluster is don [45]. Hub
nodes were ordered according to their degree and used as gene set.
3.4. Pivotal genes in wound healing: bridge nodes
Bridge nodes are measures of the betweenness of clusters in wound.
These nodes are connected among clusters. Identifying bridge proteins
is generally done to unveil viable targets for pharmaceuticals [46,47].
using the bridging procedure, 45 bridge nodes were identified and stot
score was calculated. Next, we calculated the Stot score of each bridge
node by considering the wound healing-related KEGG pathways and/or
GO terms; it represents the estimated likelyness of a bridge node being
pivotal in wound healing. Tnf, Cxcl2, Ccl12, and Fosb gene regulations
also play vital role in wound healing [48,49].
3.5. Angiogenesis and granulation tissue formation
Modelling and establishment of new blood vessels is essential in
wound healing and takes place concurrently during all phases of the
wound healing process. Along with attracting neutrophils and macro-
phages, numerous angiogenic factors secreted during the haemostatic
phase promote angiogenesis [50,51]. Resident endothelial cells respond
to number of angiogenic factors, including FGF, vascular endothelial
growth factor (VEGF), PDGF, angiogenin, TGF-α and TGF-β. A fine
balance is kept by the action of inhibitory factors, such as angiostatin
and steroids. [52,53] Inhibitory and stimulatory agents act on pro-
liferating endothelial cells directly as well as indirectly, by activating
mitosis, promoting locomotion and by stimulating the host cells to re-
lease endothelial growth factors. [54,55] Under hypoxic conditions,
molecules are secreted from the surrounding tissue, promote pro-
liferation and growth of endothelial cells.
3.6. Protrusion
In view of interconnected filaments in wound healing process, it is
said that the cytoskeleton is anchored at cell–cell junctions and cel-
l–extracellular matrix adhesions, which provide mechanical strength
for the cell [56]. The network is well known for its dynamic re-
organization and is important for coordinating cell migration. During
the wound healing, actin polymerization occur at the leading edge,
which pushes the plasma membrane outside. A protruding structure is
formed, are known as filopodia, which is filled with filamentous actin.
[57] Unidirectional movement of the cell is maintained through the
action of a cyclic assembly and disassembly of actin filaments in front of
and well behind the leading edge [58]. Multiple signalling pathways
and regulatory proteins control actin dynamics and the changes of cell
morphology, therefore wound healing process is regulated [59,60].
3.7. Adhesion
During wound healing adhesion to a solid substratum is a particu-
larly important step in cell migration step [61,62]. It is mediated by
integrins, which act as the primary receptors for extracellular matrix
proteins and are consequently required for cell motility [63]. Integrins
plays vital role in signal transduction, and in regulating and stimulating
migration [64,65].
4. Pharmacological models
4.1. Excision wound model
Excision wound model used to evaluate the wound healing potential
cover epithilezation, area of wound contraction area, wound index and
also helpful for the estimation of collagen formation like hexosamine
A. Shrivastav et al.
and hydroxyproline estimation [66].
In this model, the rats or animals were anesthetized with the help of
ketamine HCl at a dose of 80 mg/kg of body weight in i.p route and the
hair of the back region are removed with the help of shaving cream.
After that an impression is made on the dorsal thoracic region of the
rats and a 300 mm2 circularareais removed with the help of surgical
blade and scissor. Wound of depth not more than 2 mm is created [67].
4.1.1. Evaluation parameters of excision wound model
4.1.1.1. Measurement of wound area. The progressive change in the
wound area is monitored on a full HD camera on pre-determined days
such as, 2, 4, 8, 12, 16, and 20 and so on until the wound is completely
healed. Take the trace on a trace paper and calculatethe areawith the
help of mm scale graph paper [68].
4.1.1.2. Determination of period of epithelization. The dropping of the
scab from the wound is considered as indicator of wound healed, or as
the end point of complete epithilization. The number of days is required
for this is known as epithilization period [69].
4.1.1.3. Determination of wound index. Wound index [70] is estimated
by an arbitrary scoring system as presented in Table 2.
4.1.1.4. Estimation of collagen. In collagen, more than 30%
hydroxyproline exists. Therefore, estimation of collagen is main
marker for wound healing. For the same, 50 mg of the tissue is taken
with 1.0 ml of 6.0N Hydrochloric acid in the glass tube and the tube is
closed by a cotton plug carefully. The tubes are autoclaved; further the
prepared hydrolysate sample is neutralized to pH 7.0. The tubes are
marked as sample, blank and standard. 1.0 ml of test sample is added to
test tubes marked as sample, 1.0 ml double distilled water is taken in
test tube marked as blank, and 1.0 ml of a standard solution of
hydroxyproline to test tube marked as standard.
Then freshly prepared 1.0 ml of 0.01 M copper sulphate solution is
added in all the test tubes, and then 1.0 ml of 2.5N sodium hydroxide
and 1.0 ml of 6% hydrogen peroxide are added. The resulting solution is
mixed well and heated for 5 min on a water bath at 80 °C. Then after the
tube chilled immediately on ice bath and then 4.0 ml of 3N sulphuric
acid is added in all the tubes with agitation. Then after 2.0 ml of Ehrlich
reagent is added in all the test tubes and the solution again heated on a
water bath for 15 min at 70 °C temperature [71].
The optical density is measured at 540 nm with the help of the
colorimeter.
OD of the sample
concentration of the sample =
OD of the standard
× concentration of standard
4.1.1.5. Estimation of protein. In order to estimate total protein, 0.2 ml
serum or tissue extract, 1.8 ml NaOH (O.1N) and 5 ml alkaline copper
reagent (prepared by mixing 0.5 ml 0.5% Copper sulphate, 0.5 ml 1%
sodium potassium tartarate and 50 ml 2% sodium carbonate) are added
and kept for 15 min. Then 0.5 ml diluted Folin's reagent (diluted 1: 1
with distilled water) is added. Mixed and kept for 30 min and read at
Table 2
Wound Index.
S.No. Gross change
1. Formation of pus-evidence of necrosis
2. Healing yet not be start but environment is healthy
3. Delayed, but healthy healing
4. Incomplete but healthy healing
5. Complete healing
Total
Wound Medicine 20 (2018) 43–53
675 nm. In the blank, 0.2 ml water and to the standard (100 mg%
bovine serum albumin in 0.1 N NaOH), 0.2 ml working standard
(100 pg/ml- diluted stock standard 1 in 10 using 0.1 N NaOH) is
added instead of tissue extract and treated as above [72].
4.1.1.6. Estimation of hexosamine content. For the preparation of the
sample (protein hydrolysate), 50 mg of the tissue is taken with 1.0 ml of
6.0N hydrochloric acid in the glass tube and the tube is closed by a
cotton plug carefully. The tubes are autoclaved, further the hydrolysate
sample is neutralized to pH 7.0.
For estimation of hexosamine content from granulation tissue, the
acetyl acetone is mixed with the diluting solution and then the mixture
is heated for 40 min at 96 °C temperature. Finally, the resulting mixture
is kept in an ice-bath for sudden cooling and a fraction of 96% is added,
followed by adding Ehrlich’s reagent.
The resulting solution is kept a side for 1 h at room temperature and
absorbance is measured at 530 nm. The amount of hexosaime is esti-
mated by comparing with standard curve of D (+) Glucosaime HCl. The
content of hexosamine is expressed as mg/g dry tissue weight [73].
4.1.2. Histopathology
The rats are sacrificed with the high dose of ketamine HCl and tissue
is excised from the wound site of each animal of the group. These tissue
samples are separately stored in the formalin solution, for microscopic
examination [74].
4.2. Incision wound model
Incision wound model is used for evaluation of the effectiveness or
healing potential. In this, tensile strength (the force required to open
the healing skin) is estimated [75].
For this, the rats or animals areanesthetized with the help of keta-
mine HCl at a dose of 80 mg/kg of body weight in i.p route and the hair
of the back region are removed with the help of shaving cream. Incision
wound is created on the shaved back of the rat after 30 m.a 6 cm long
and 2 mm depth incision is made on the rat skin with the help of sur-
gical blade (No. 9), stitched out the skin with the help of the surgical
needle (No. 32) and surgical thread (No. 000) at a distance of 5 cm
intervals. Finally, the different groups of rats are treated with the to-
pical ointment/oral administration of drugsfor the period of 10 days.
Considering the day 0th as wounding day, for this activity, the su-
tures are removed on the 8thday and the tensile strength is evaluated on
10th post wounding day with the help of tensiometer [76].
4.2.1. Measurement of tensile strength
On the 10th day of the model, the animals are sacrificed with the
help of high dose of ketamine HCL and the tensile strength is measured.
After sacrificing the rats, a wound stripe of equal size (length and
width) is excised out carefully from the rats and fixed at a fix distance
on the tensiometer. Then both ends of the skin strip are fixed by using a
pair of stainless clips. One clip allowshangingon the stand and another
clip for holding polyethylene bottle to fill with water gradually till the
wound strip is to be ruptured. The amount of water is noted and ex-
pressed as the tensile strength of the wound in grams (g) [77,78].
4.3. Burn wound model
In this model, the rats or animals are anaesthetized with the help of
ketamine HCL at a dose of 80 mg/kg of body weight in i.p route and the
Score
hairs of the back region are removed. The shaved area is disinfected
4 with the help of 70% v/v ethanol. In this model the wound created by
3 the help of a cylindrical metal rod about 10 mm in diameter, being
2 heated for 30 s. in open flame firstly and immediately pressed to the
1
shaved skin of rats under the anesthesia.
0
10 The treatment is applied as per your dose selection, followed by
continuous treatment of skin/tissue integrity. The percentage wound
47
A. Shrivastav et al.
contraction and the epithelization times are evaluated by using this
model [19].
4.3.1. Parameters for burn wound healing activity
For the evaluation of burn wound activity, the two main parameters
are employed which includes Wound contraction, their percentage and
the period of epithelization [10].
4.3.1.1. Wound contraction rate. The progressive change in the wound
area should be monitoring of a full HD camera on predetermined days
such as, 0, 4, 8, 12, 16, and 20 and so on until the wound is completely
healed. A trace is taken on a trace paper and the area is calculated with
the help of a mm scale graph paper.
4.3.1.2. Period of epitheliazation. The dropping of the scab from the
wound is considered as the wound healed, or as the end point of
complete epithelization. The number of days, required for this is known
as epithelization period.
4.4. Dead space wound model
Dead space model is used to evaluate the wound healing potential
and collagen strength of the tissue. In this model, Hydroxyproline,
Hexosamine, Hexuronic, Protein estimation and DNA estimated.In this
model, the rats or animals are anaesthetized with the help of ketamine
HCL at a dose of 80 mg/kg of body weight in i.p route. The dead space
wound is created by making a small transverse incision on the dorsal
paravertebral lumbar skin region. a polypropylene tube (2.5 × 0.5 cm)
implanted in the lumbar region.
After 10 days treatment and under the anesthesia, the granulation
tissue formed on the tube is harvested carefully on the 10th day and
breaking strength of the granulation tissue is measured [79].
4.4.1. Evaluation parameters
For tissue hydrolysate, following procedure is adopted in which
about 150 mg of wet dried tissue is triturated in a 3 ml of 6N HCl in a
glass air tight container and the tube is kept in an autoclave for
15–20 m at 121 °C. The hydrolysate is cooled at room temperature,and
excess acid is neutralized by 10N NaOH solution.
The final hydrolysate is used to determinate the Hydroxyproline,
Hexosamine, Hexuronic Acid, Protein estimation and concentration of
DNA estimation.
4.4.1.1. Hydroxyproline. Hydroxyproline content is estimated from the
wound tissue, which is a basic component of collagen.For the prepared
protein hydrolysate sample, the tube is marked as sample, blank and
standard. 1.0 ml of test sample is added to test tubes marked as sample,
1.0 ml double distilled water is taken in test tube marked as blank, and
1.0 ml of a standard solution of hydroxyproline to test tube marked as
standard.
Then freshly prepared 1.0 ml of 0.01 M copper sulphate solution is
added in all the test tubes. 1.0 ml of 2.5N sodium hydroxide and 1.0 ml
of 6% hydrogen peroxide are added in each tube. The solution is mixed
well and heated for 5 m on a water bath at 80 °C. Further the tube is
chilled and 4.0 ml of 3N sulphuric acid is added in all the tubes with
agitation. Afterward 2.0 ml of Ehrlich reagent is added in all the test
tubes and the solution again is heated on a water bath for 15 m at 70 °C
temperature [80].
The optical density is measured at 540 nm with the help of the
colorimeter.
OD of the sample
concentration of the sample =
OD of the standard
× concentration of standard
48
Wound Medicine 20 (2018) 43–53
4.4.1.2. Hexosamine (HXA). 0.05 ml of hydrolyzed fraction is diluted
to 0.5 ml with distilled water.The acetyl acetone mixed with the
diluting solution and the mixture is heated for 40 m at 96 °c
temperature. The resulting mixture is suddenly kept in an ice bath for
sudden cooling and a fraction of 96% is added, followed by adding
Ehrlich’s reagent.
After mixing, each tube is kept a side 1 h at room temperature and
the absorbance is measured at 530 nm. The amount of hexosamine is
determined upon comparison with a standard curve of D (+) glucosa-
mine hydrochloride. Hexosamine content is presented as mg/g dry
tissue weight [81].
4.4.1.3. Hexuronic acid (HUA). 2.5 ml of 0.025 M Borax in
concentrated sulphuric acid is placed in stoppered tubes fixed in a
rack and cooled to 4 °C. Further 0.125 ml of hydrolysate is diluted
0.5 ml by adding distilled water. 0.5 ml of hydrolysate is layered
carefully on Borax-sulphuric acid mixture, kept in rack at 4 °C. The
tubes are closed with glass stoppers. The chilled tubes are then heated
for 10 m in a boiling water bath and cooled to room temperature.
Thereafter, 0.1 ml of 0.125% carbazole reagent in absolute ethanol is
added to each tube, heated in the boiling water bath for further 10 m,
and then set a side for 1hr. Color intensity is measured at 530 nm
against the blank.
The Hexuronic acid content of the samples is calculated by the
standard curve prepared with D (+) Glucurono-6, 3- lactone [82].
4.4.1.4. Estimation of DNA. In order to extract the DNA sample from
the tissue, 0.5 ml of tissue hydrolysate sample or standard solution in a
glass tube then add 0.5 ml 1N perchloric acid and hydrolyze at 70 °C for
7m. The resulting chilled solution is centrifuged at 1500 rpm for 5 min.
In supernatant, 1 ml 0.5N perchloric acid is incorporated and the
process done in triplet. Diphenylamine (LR) and supernatants in the
ratio of 2:1 are mixed and incubate at 30 °C for 18 h. The absorbance is
measured at 595 and 650 nm by using 0 μg/ml standards as a blank
[83].
4.4.1.5. Tensile strength. For determining the tensile strength of the
dead space model, the granulomas is dissected out from the
surrounding tissue on 10th day post wounding under the light
anesthesia. The strength of tissue is measured by taking a piece of
tissue about 15 × 8 mm on the 10th day adopting continuous water
flow techniques [21].
5. Recent advancement in wound healing assessment
Apart from methods incision, excision, burn and dead space models
are used nowadays to evaluate the wound healing. Assessment of en-
zymatic debridement (Collagenase), high pressure water irrigation, bio
debridement by medicated Larvae, topical insulin, pressure therapy
(negative and positive) and use of growth factors are some new tech-
niques being involved in wound healing at contemporary time.
5.1. Enzymatic debridement
Debridement is one of the essential tools of wound management
[84]. It is defined as the removal of nonviable material, foreign bodies,
and poorly healing tissue from a wound and it facilitates the processes
of granulation, contraction, epithelialization, and healing. The most
direct formof debridement is surgical excision, although other reason-
able options exist for patients who are poor surgical candidates or who
have wounds in need of less aggressive debridement. These alternative
debridement options include the following: mechanical debridement,
which is exemplified by wet to dry dressings or pressure irrigation;
autolytic debridement, in which occlusive dressings allow wound pro-
teases to liquefy necrotic tissue; biologic debridement, which utilizes
maggot therapy; and enzymatic debridement, which utilizes agents
A. Shrivastav et al.
such as collagenase or papain-urea. Collagenase has been shown to be
useful for degredation of collagen and elastin but not fibrin. Papain-
urea’s main action is to solubilize fibrin [85].
Collagenase, which are available as ointments used for over a
quarter of a century for wound healing purpose. They assist in digesting
necrotic tissue without damaging healthy tissue. Collagenase is a pro-
teolytic enzyme which specifically attacks and breaks down native
collagen and is gentle on viable cells. It is therefore useful in main-
tenance phase of wound debridement, i.e., gradual breakdown of tissue
[86].
Debridement is an integral component for wounds of all etiologies
that contain necrotic tissue, high bacterial burden, or other compli-
cating unwanted elements [84]. Surgical (sharp) debridement is the
most rapid, direct and effective methods of debridement. Enzymatic
debriding agents have long been used in burn wound treatment due to
their ease of use [87]. Enzymatic debridement in the face of tissue
bacterial loads greater than 10 [88] may predispose to worsening in-
vasive infection, leading to sepsis. Additional antimicrobial topical
formuation is given to avoid complication [89,90].
Wound care regimens that are simple and effective have greater
compliance and success. Simplification of wound care by safely redu-
cing the number of preparations used for care may improve wound
healing process. Commercially available collagenase and papain-urea
enzymatic debriding agents are commonly used for wound care and
show effectiveness in wound conditions. Both collagenase and papai-
nurea appear to reduce bacterial load to a level of 10 in process of
wound healing. Wounds treated with collagenase or papain-urea close
at an accelerated rate than with wounds treated with isotonic sodium
chloride solution. Although this effect to reduction of bacterial burden.
Some rapid wound healing may be due to other wound healing prop-
erties of these specific enzymatic preparations. It appears that because
of their antimicrobial properties, collagenase and papainurea may be
safely used without concomitant topical antimicrobials in chronic
wounds [89].
5.2. High pressure water irrigation
Hydrocision or pressurized irrigation has now entered the foray of
tools in surgical debridement and cleaning of the wound with high or
low pressure, razor thin stream of water, saline or antibiotic solution
and acts on good option when compared to the above methods [91].
High pressure irrigation is effective in removing bacteria, particu-
late matter and necrotic debris from the wounds, thus lowering the rate
of infection compared to low pressure irrigation.
Wound irrigation is important in of wound management, which
endeavours to remove devitalised tissue debris, dirt and bacteria,
bearing in mind that all traumatic wounds can be categorised as dirty
wounds [92,93]. Irrigation of particles such as fat detritus increases the
rate of bacterial clearance, which are engulfed and eradicated by leu-
cocytes. Healing get accelerated by prior removal of bacteria and hae-
matoma from the acute wound [94]. The concept of irrigation in the
treatment of wounds (traumatic and elective) is divided into irrigation
technique (pressure related) and the irrigant used.
The traumatic wounds are acute wounds that is healed by hae-
mostasis, inflammation, new-tissue formation and remodeling process.
Irrigation has been defined as ‘the process of washing a wound’ with a
‘solution’. In recent years, researches showed that irrigation with
normal saline solution causes decreased level of wound infection and
the effectiveness was proportional to the volume of solution used.
Irrigation provides benefit to wound healing and therefore patient
outcome [95].
Different lavage irrigating tips (single orifice, multijet and radiant)
and varying irrigating pressures were reviewed recently [96]. This in-
volved radiographic and histological analysis to assess dispersion of
radiopaque material and to detect fibre separation, respectively. The
wounds were 5 mm deep except for those irrigated with the catheter tip.
49
Wound Medicine 20 (2018) 43–53
It was observed that using the multijet tip, no spread of liquid beyond
the debris originally present in the wound was observed.
The single jet tip and radiant catheter tip produced dispersion and
fibre separation at increase pressures. It concluded that the multijet tip
is less traumatic to the tissues. The reduce debris could be substantially
more using the single jet tip and radiant catheter tip, which could then
provide benefit. The finding suggested that acute human wounds get
cured by reduction in tissue trauma with less dispersion of debris ma-
terial if multijet tip is used, but the quantification of debris material is
still under investigation [97].
5.3. Bio debridement by medicated larvae (myiasis or maggot therapy)
The usage maggots for wound debridement is in practice since
centuries. The process is preferred because of the ability of maggot to
breed sterile flies commercially [98]. The secretions from maggot
contain proteolytic enzymes, which digests necrotic debris. The maggot
enzymes are having bactericidal effective against MRSA and b- hae-
molytic streptococcus [99].
Mechanical debridement is caused by the mouth hooks of the
maggots and their cause rough surface of body which both scratch the
necrotic tissue [100]. Apart from this, maggots excretions and secretion
(ES) that contains proteolytic enzymes, which may dissolve the dead
and/or infected matrix on the wound bed [101,102].
Maggots excretions and secretions contain, sulfhydryl radicals, cal-
cium, glutathione, growth stimulating factors for fibroblasts, carbox-
ypeptidases A and B, leucine aminopeptidase, collagenase and serine
proteases (trypsin-like and chymotrypsin-like enzymes, metalloprotei-
nase and aspartyl proteinase) [103].
Larval therapy with free-range maggots and maggots in Biobags
were compared with hydrogel. Result showed faster debridement with
the maggots [104]. The maggots in Biobags required 28 days to debride
and free range maggots required 14 days, both therapies were very ef-
fective in debridement. Maggot can prevent, inhibit, and break down
biofilms of various bacteria on prosthetic materials, and therefore in the
future they may provide treatment of biofilm-associated infections of
orthopedic biomaterials [105,106].
5.4. Negative pressure wound therapy (NPWT)
NPWT or vacuum-assisted closure (VAC) is important as it play
major role as a bridge to reconstruction. NWPT is advanced wounf
healing therapy in which process promotes active wound healing at the
cellular level by negative pressure. Pressure employed is sub atmo-
spheric pressure (100–125 mm Hg) in view of NPWT [107].
The VAC system possess 4 major components: (1) a sponge is kept in
to the wound; (2)a dressing is placed to isolate the wound environment
and allow the vacuum system to transmit sub atmospheric pressures to
the wound surface; (3) a connecting tube;(4)and a vacuum system. A
fluid collection canister is also incorporate with the device [108]. The
structure of the packing material in to the wound may be important in
the efficacy of NPWT. Although different materials may be used with
various NPWT devices, reticulated, open-pore foam is the most
common. This foam is made of bubbles of highly interconnected net-
work. This structure, as the name suggests, resembles 3-dimensional
net. The increased density and smaller pores of the White Foam help to
reduce the growth of granulation tissue, thereby diminishing pain as-
sociated with wound dressing [108,109]. NPWT therapies are most
commonly employed to an open wound, speeding the wound healing
phenomenon and bringing the wound margins closer together. The
wound healing of both types acute and chronic is improved through the
combination of components that include the wound-foam interface and
semi occlusive micro environment.
A. Shrivastav et al.
5.5. Growth factors
Growth factors are naturally available proteins in the body system
which regulate many key cellular activities during tissue repair process.
The macromolecules of wound fluid and bed trap growth factors within
fibrin bind to the extracellular matrix [110].
The growth factors are produced wound healing. Blood platelets
release platelet-derived growth factor (PDGF) and transforming growth
factor (TGF)-b along with insulin like growth factor (IGF), epidermal
growth factor (EGF), and TGF-a. During the process of wound healing,
Blood-borne monocytes reach the wound and differentiate into mac-
rophages, which are activated and become the producers of growth
factors and cytokines. Macrophages may produce 100 growth proteins
that are active in stimulating cell migration, proliferation and matrix
synthesis. Keratinocytes, endothelial cells and plasma help to release
growth factors white wound healing.
5.6. Collagen
Collagen produced by fibroblasts is the most important constituent
of connective tissue and play role in wound healing. Out of the many
subtypes of collagen, type I collagen is mostly seen in healing tissues.
Chronic wounds are now treated with topical collagen products which
causes improved wound healing by laying down a matrix favoring the
deposition of new tissue and attracts cells required for wound healing.
The collagen is thought to provide a temporary scaffold which allow in
growth of tissue [111].
There are a number of different collagen dressings, which uses
variety of combining agents as gels, pastes, polymers, oxidized re-
generated cellulose (ORC), and ethylene diamine tetraacetic acid
(EDTA). The collagen within these products tends to be derived from
bovine, porcine, equine, or avian sources, which is purified in order to
act as nonantigenic [112]. The collagen in a dressing can vary in con-
centration. Certain collagen dressings are comprised of Type I (native)
collagen; whereas, other collagen dressings contain denatured collagen.
A collagen dressing may contain ingredients, as alginates and cellulose
derivatives that can enhance absorbency, flexibility and comfort. Col-
lagen dressings have a variety of pore sizes and surface areas. All of
these properties are meant to enhance the wound management aspects
of the dressings. Many collagen dressings contain an antimicrobial
agent to control pathogens within the wound [113]. Some collagen-
based dressings causes a significant increase in the fibroblast produc-
tion; which may be important in encouraging fibroblast permeation
[114]. The mechanism of action of collagen dressings includes the in-
hibition excess MMPs because increased MMPs are a key contributor to
wound chronicity.
5.7. Topical insulin
Topical insulin in combination with zinc in animal studies heals the
wounds faster [115]. It control wound inflammatory response by pro-
liferation and migration of macrophages and keratinocytes in nearby
tissues. However, method for routine administration of insulin is still
under investigation [116].
Liu et al. [117] stated that insulin acts through its receptors and
helps in the migration of keratinocytes in wounds without interaction
with the epidermal growth factor receptors. The activation of peroxi-
some-proliferator activated receptor-gamma (PPArϒ) by insulin is also
considered as one of the factors for angiogenesis [118]. Insulin through
insulin like growth factor-1 has also been shown to inhibit apoptotic
pathway, attenuates the production of anti-inflammatory cytokines and
stimulates production of extracellular matrix component. Ghahary et al.
[119] stated that insulin like growth factor 1 promotes conversion of
growth factor- beta in dermal fibroblasts for wound healing. Apart from
cutaneous cells, to heal faster with topical insulin, corneal ulcer in ro-
dents has shown to rapidly heal via mechanism involving production of
50
Wound Medicine 20 (2018) 43–53
insulin-like factor 3 [120]. The efficacy of topical insulin in patients
with traumatic tympanic membrane perforations have been proved
[121]. Dhall et al. [122] reported that alginate sponge dressings that
release insulin promote regeneration of the burn wounds in rats. To-
pical insulin has also been reported to increase bacterial clearance rate
in the wounds [123].
6. Wound healing in different organ system
The process of wound healing involved hemostasis, inflammation,
repair, and remodeling. For non-degradable smooth-surfaced implants,
repair and remodeling causes isolation of the implant by tissue en-
capsulation. The nature of the encapsulation tissue and the cellular
participants in the immune reaction varies depending on the site of
implantation and the type of tissue of hosts.
6.1. Partial-thickness skin repair
Skin layers basically, the epidermis and the dermis play important
role in wound healing. In epidermal wounds, only the epidermis is
damaged, but the basement membrane is intact along with hair follicles
and sebaceous glands. As during wound healing, the epidermal surface
require to be replaced and epithelial progenitor cells stays intact below
to wound, the deposition of collagen is not needed. For repairing of
surface wound, the site of repair should be re-epithelialized by migra-
tion of keratinocytes from the wound and at the wound edge. The de-
gree of re-epithelialization depends on the amount of tissue loss and the
depth and width of the wound.
Re-epithelialization starts within 24 h as uninjured keratinocytes
separates from the basal layer to underneath the fibrin clot, across the
wound. This process is called as lamellopoidial crawling [124]. The
keratinocytes migrates at the wound edge and upregulate their pro-
duction of matrix metalloproteinases (MMPs), releasing the cells from
to the surface of basal lamina [125,126]. Keratinocytes resting on the
basal lamina migrate across the wound site and get attached to fi-
bronectin and vitronectin contained within the clot by upregulating
their expression of α β and α β integrins [127,128].
6.2. Stabilized bone repair
When movement at the bone fracture site is prevented and bone
fractured ends are held in place immediately after injury, primary mi-
neralized tissue repair occurs. In process of primary bone repair,
structural support not required because of stabilized tissue [129].
6.3. Gap repair for wound healing
In gap repair, healing begins in parallel as blood vessels and con-
nective tissue fill the wound. After 14 days mesenchymal cells of the
bone marrow reaches at the wound surface and start dividing into bone-
producing cells. These are called as osteoblasts. Osteoblasts fill gaps in
the tissue by the layers of unmineralized bone matrix, termed as osteoid
[130]. As osteoid becomes mineralized whereas some osteoblasts re-
main embedded in the matrix and become entrapped as the tissue is
mineralized. These osteoblasts become osteocytes and become re-
sponsible for mechanical support of tissue. Osteoblasts are activated by
proteins belonging to the TGF-β class such as bone morphogenic pro-
teins (BMPs) [131,132]. Therefore osteocytes plays important role in
gap repair and leading to wound healing.
6.4. Peripheral nervous system (PNS) repair
Several neurons and their supporting cells are termed as Schwann
cells. Which works as supporting cells, surrounding the signaling pro-
cesses of neurons and produce myelin, which increases signal propa-
gation velocity? These Schwann cells are encircled by a connective
A. Shrivastav et al.
tissue matrix called the endoneurium, subsequently grouped into bun-
dles, and bounded by another thin connective tissue matrix called the
perineurium. A number of these bundles along with vasculature make
up a nerve with an outer sheath, termed epineurium [133,134]. In-
crease amount of Schwann cells to PNS repair leads to wound healing.
6.5. Full-thickness cutaneous tissue repair
If the basement membrane is damaged in a full thickness skin
wound and substantial dermis is lost, the wound heal by re-
epithelialization is tough. The dermal fibroblasts available at wound
site are proliferated resulted into migrating, leading to wound repair
[125].
6.6. Bone repair
For bone repair, secondary healing is important than primary frac-
ture healing bone because most bones are not rigidly supported after
injury [133]. When injury results in the disruption of the bone’s ex-
ternal vascular covering (periosteum). The unstabilized mineralized
tissue will undergo secondary repair where the first step is creating an
ECM-rich bridge to support the fracture.
6.7. CNS repair (Gliosis)
The repair of a wound in the CNS is not followed by neuronal re-
generation. Unlike the response seen in the PNS, where degenerated
axons can regenerate, damaged axons of the CNS initially sprout, but
regeneration is impeded as the growth cones collapse within a day.
Gliosis starts just after injury as astrocytes outside the lesion core and
the surrounding area get activated. This marked glial response, i.e.
gliosis, is made up of a multilayered sheet of activated astrocytes re-
sulting a boundary around areas of tissue damage. The glial scar is
believed to protect neuronal function following injury by repairing the
blood–brain barrier and to subsequently limit inflammatory response to
cells of the CNS [136,138].
Like the response seen in unmineralized tissue repair, astrocytes
alter their integrin expression, migrating toward the lesion while also
secreting MMPs for degradation. MMP expression by astrocytes and
neurons 1 to 2 weeks after injury promotes wound healing by growth
factors production to initiate angiogenesis [135]. Once astrocytes
reaches at the site of injury, their processes encircle the lesion and
become tightly intertwined to give the glial barrier a highly disordered
appearance, forming glial scar and result in wound healing [135,137].
7. Conclusion
The present models, used for wound healing evaluation includes
incision wound model, excision wound model, burn wound model, and
dead space model. Researchers are using growth factors, negative and
positive pressure therapy and biochemical parameters including col-
lagenase. The findings of literature study concluded that more sophis-
ticated models as digital tensiometer, PCR based molecular finger-
printing etc may be used for further accurate evaluation of wound.
Conflict to interest
All authors declare no conflict of interest.
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