Botulinum Toxin Type a Iontophoresis for Postburn Hypertrophic Scar
Phase: NA
Conditions: Hypertrophic Scar
Sponsor: Cairo University

Approved date: 17/5/2022 
 
 
 
 
BOTULINUM TOXIN TYPE A IONTOPHORESIS 
FOR POSTBURN HYPERTROPHIC SCAR 
 
 
 
 
 
 
By
 
HAYAM MAMOUD ABOULMAATI ALNAWAGY
 
 
 
 
 
 
 
 
 
Submitted in a Partial Fulfillment of the Requirements of Doctoral 
Degree in Department of Physical Therapy for Surgery
 
Faculty of Physical Therapy
 
Cairo University
 
2022
 

Approved date: 17/5/2022 
 
 
 
 
Supervisors 
 
 
Prof. Dr. Amal Mohamed Abd El Baky 
Professor of Physical Therapy for Surgery and Vice 
Dean for Post Graduate Studies and Research
 
Faculty of Physical Therapy
 
Cairo University
 
 
 
 
Prof. Dr. Ashraf Elsebaie Mohamed 
Professor of Plastic and General Surgery
 
Faculty of Medicine
 
Cairo University
 
 
 
 
Ass. Prof. Dr. Ereny Sobhy Wahba Khalil
 
Assistant Professor of Physical Therapy for Surgery
 
Faculty of Physical Therapy
 
Cairo University

Approved date: 17/5/2022 
 
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CHAPTER Ι 
INTRODUCTION  
A hypertrophic scar (HS) is a skin condition characterized by 
excessive fibrosis with disordered collagens from skin fibroblasts. HS 
usually develops during the wound healing process after deep-thickness 
trauma or burn injury and hinders normal function, resulting in physical, 
psychological and aesthetic problems for patients. Major risk factors for 
HS formation include gender, age, genetic predisposition, immunological 
responses of the patient, type of injury, wound size and depth, anatomical 
site and mechanical tension on the wound. HS proliferate exponentially 
for 2–18 months after injury (Shirakami et al., 2020). 
Hypertrophic scars are more common in burn injuries and occur in 
30–90% of patients with burn injuries. Delay (>3 weeks) in wound 
healing increases the risk of hypertrophic scarring, which is typically 
restricted to the confines of the initial injury and does not recur after 
excision. Decreased collagenase activity in hypertrophic scars leads to 
perturbed collagen production and degradation, resulting in bundles of 
crosslinked collagen oriented parallel to the epidermal surface. In 
particular, mature type I collagen expression is reduced and type III 
collagen is over-synthesized (Jeschke et al., 2020). 
Botulinum toxin A is produced by anaerobic spore-forming 
bacteria and is used for various therapeutic and cosmetic purposes 
(Samizadeh and De Boulle., 2018). 
It is a potent neurotoxin that blocks neuromuscular transmission. 
Some authors have reported that botulinum toxin type A can minimize 
scar formation by reducing muscle tension during wound healing, 
causing the fibroblast cell cycle to be paused in a non-proliferative state 
and influencing TGF-β1 expression (Lee and Janget., 2018). 

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Statement of the problem: 
Does Botulinum toxin type A iontophoresis help in minimizing 
postburn hypertrophic scars?  
Purpose of the study: 
The main objective of the study is to evaluate the therapeutic effect 
of Botulinum toxin type A iontophoresis in improving postburn 
hypertrophic scars.  
Significance of the study: 
Hypertrophic scars result from fibroblast proliferation which is 
responsible for the enhanced collagen production that leads to excessive 
scarring. By decreasing the tension in the scar, botulinum toxin causes 
local fibroblasts to gradually change their functional status to proliferate 
slower, secrete less biologically active mediators and synthesize less 
extracellular matrix and collagen resulting in the improvement of 
hypertrophic scars (Elhefnawy., 2016). 
One of the most significant drawbacks of BTX-A injection is pain. 
Iontophoresis, as an alternative modality for drug delivery, has several 
advantages. It is a painless modality with a reduction of systemic adverse 
effects due to localized drug administration and its convenience to the 
patient (Montaser-Kouhsari et al., 2014). 
 
Delimitations: 
This study was delimited in the following aspects: 
1. Subjects: 
Seventy-six patients with postburn hypertrophic scar will be 
randomly divided into two equal groups each one has 38 patients. 
 
 
 

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2. Equipment and tools: 
2.1. Measurement equipment: 
− Sonography (Reinholz et al., 2016). 
− Patient and Observer Scar Assessment Scale (POSAS) (Reinholz 
et al., 2016). 
2.2. Therapeutic equipment: 
• Iontophoretic device. 
Hypothesis: 
It will be hypothesized that: 
Botulinum toxin type A iontophoresis may help in minimizing 
postburn hypertrophic scars. 
Basic Assumptions: 
It will be assumed that: 
• All patients will receive the same kinds of medication and the 
same nursing care. 
• All subjects are free from any old previous hypertrophic scars. 
• All subjects will continue in the study. 
• All patients will follow the instructions during the treatment 
 
 
 
 
 
 
 
 
 

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Definitions of terms:      
The following terms are defined for clear understanding of the 
terminology used in the present study: 
Botulinum toxin type A (BTA): 
Botulinum toxin type A (BTA) is an exotoxin of the anaerobic 
spore-forming bacterium, Clostridium botulinum. It had been extensively 
used in medicine owing to its ability to inhibit the release of 
acetylcholine which is a helpful tool for treating the hyperactive muscles 
(Elsaie., 2021). 
Hypertrophic scar: 
A hypertrophic scar is a wide, thickened, and often raised scar that 
develops at the site of skin injury that can be itchy and typically does not 
extend beyond the boundary of the original wound. A leading cause of 
hypertrophic scar formation is the mechanical tension on the wound 
(Zhang et al., 2020). 
Iontophoresis: 
It is a method for transdermal drug delivery based on the transfer 
of charged molecules using a low-intensity electric current. This original 
route of drug delivery is non-invasive and presents several advantages in 
comparison to the usual passive transdermal administration, such as 
faster release of the drug into the skin, the capacity to deliver 
macromolecules and better control of the delivered dose. Depending on 
the properties of the molecule, systemic administration can also be 
achieved without first-pass metabolism (Roustit et al., 2014). 
 
 
 

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CHAPTER ΙΙ 
REVIEW OF RELATED LITERATURE 
 The review of the related studies and literature of the main 
concept of this study has been presented under the following headings: 
1.1- Wound healing process: 
The classical model of wound healing involves three distinct, but 
overlapping phases that follow a time sequence: the inflammatory phase, 
the proliferative phase and the remodelling phase. 
The first phase of wound healing is the inflammatory phase which 
starts immediately after tissue injury and lasts for approximately 2–3 days 
after injury. Coagulation cascades, complement activation and platelet 
degranulation prevent further fluid and blood losses by creating platelet 
plugs and a fibrin matrix. The immune system and inflammatory reactions 
are activated to prevent infection and remove devitalized tissues. 
Neutrophils are recruited by chemotactic factors produced by platelet and 
bacterial degranulations, and monocytes are recruited and differentiated 
into macrophages 2–to 3 days after injury (Lee and Janget., 2018). 
The second phase of wound healing is the proliferative phase. This 
phase of new tissue formation occurs approximately 2–3 days after tissue 
damage and may last for 3–6 weeks. Active cellular proliferation and 
migration characterize this phase. Keratinocytes migrate to the damaged 
dermis; new blood vessels grow inward within the damaged tissue; and 
new capillaries replace the fibrin matrix with granulation tissue via the 
actions of macrophages and fibroblasts. Granulation tissue forms a new 
substrate for keratinocyte migration. Keratinocytes proliferate and mature 
within granulation tissue along the wound margin, restoring the protective 
function of the epithelium. In the late proliferative phase, a portion of the 

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fibroblasts differentiates into myofibroblasts in association with 
macrophages. Fibroblasts and myofibroblasts produce extracellular matrix 
(ECM), mainly in the form of collagen; this accumulated collagen forms 
most of the eventual scar. Other constituents of ECM include elastin, 
hyaluronic acids and proteoglycans. Myofibroblasts, which contain actin 
filaments, have contractile properties and help bring the edges of the 
wound together over time (Lee and Janget., 2018). 
Once wound closure is accomplished, the final remodelling phase 
commences. This phase is characterized by the degradation of excessive 
tissue, transforming immature healing products into a mature form. 
Remodelling may last for a year or more. Excessive ECM is degraded and 
remodelled from type III collagen, the main component of ECM present 
during the early wound healing process, to mature type I collagen (Lee 
and Janget., 2018). 
1.2- Hypertrophic scar formation: 
An imbalance between the proliferation and degradation of 
extracellular matrix components leads to excessive scar formation. Risk 
factors for this include delayed epithelialization, wound infection, specific 
anatomic locations, or genetic predispositions (Jeschke et al., 2020). 
On a molecular level, increased activity regarding scar proliferation 
is commonly mediated through inflammatory cells that secrete TGF-β1 
and β2 which induce a fibrogenic response within the scar tissue. At the 
same time, scar remodeling stimulated through matrix metalloproteases 
and TGF-β3 is decreased. Excessive scar formation is dependent on both 
the severity of the inflammation and the type of the immune response 
(Jeschke et al., 2020). 
2.1: Botulinum toxins: 
Botulinum neurotoxin (BoNT) is produced naturally by Clostridium 
botulinum, an anaerobic, Gram-positive, spore-forming bacillus. Seven 

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serotypes of BoNT have been discovered (A to G), with only types A and 
B available for therapeutic usage. BoNTs have been used for spasticity, 
depression, hyperhidrosis, migraines, ageing of the neck, face, and 
decolletage on and off label in the medical and cosmetic areas since their 
commercial availability (Naik et al., 2021). 
2.2: BoNT-A formulations: 
BoNT-A is synthesized as macromolecular protein complexes in 
nature. The BoNT-A protein has a molecular weight of 150 kDa. 
The three botulinum toxin formulations that have been approved by 
the US Food and Drug Administration and are well known in the Western 
hemisphere are as follows: 
• Onabotulinum toxin A (ONA; Botox®/Vistabel®; Allergan Inc., 
Dublin, Ireland)  
• Abobotulinum toxin A (ABO; Dysport®/Azzalure®; Ipsen, Paris, 
France/Galderma, Lausanne, Switzerland) 
• Incobotulinum toxin A (INCO; Xeomin®/Bocouture®, NT 201; 
Merz Pharmaceuticals GmbH, Frankfurt, Germany) (Samizadeh 
and De Boulle., 2018). 
2.2: Mechanism of action of BONTs: 
The most recognized mechanism of action of BoNTs is the 
inhibition of neurotransmitters (acetylcholine, norepinephrine, substance 
P, calcitonin gene-related peptide [CGRP], and glutamate) release at the 
presynaptic neuromuscular junction. However, BoNTs can affect both 
sympathetic and parasympathetic functionality since acetylcholine is also 
a neurotransmitter of the autonomic nervous system. Furthermore, recent 
evidence shows the effect of BoNTs on different human cell types, both 
neuronal and non-neuronal cells. These latter include epidermal 
keratinocytes, mesenchymal stem cells from subcutaneous adipose, 
neutrophils and macrophages, dermal fibroblasts, mast cells, sebocytes 

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and vascular endothelial cells. Thus, several clinical applications are 
emerging in dermatology (Guida et al., 2018). 
2.3: Effect of BoNT A in hypertrophic scars: 
Hypertrophic scars occur mainly as a result of the tension exerted 
on wound edges. Tension can, directly and indirectly, lead to hypertrophic 
scars. BoNT A works by inhibiting the release of acetylcholine in the 
neuromuscular 
junction 
which 
indirectly 
causes 
neuromuscular 
transmission, leading to muscle paralysis and thus reducing wound 
tension. As a result of this and other mechanisms, BoNT A can be used 
not only as treatment but also as prevention of hypertrophic scars. BoNT 
A may directly modulate the activity of fibroblasts by altering apoptotic, 
migratory, and fibrotic pathways on pathological scars and thus improve 
their appearance (Kasyanju Carrero et al., 2019). 
3.1: Transdermal iontophoresis: 
Iontophoresis is a method of introducing ionized and neutral drugs 
into the skin by using an electric current. As shown in (Fig. 1), during 
iontophoresis, electromigration and electroosmosis are the principal 
mechanisms of enhancing drug transport across the skin into the systemic 
circulation. Electromigration refers to the ordered movement of ionized 
drug molecules in the presence of the applied electric field. Therefore, 
charged drugs are forced across the skin by electronic repulsion of similar 
charges. Cationic drugs can permeate through the skin by using a 
positively charged working electrode. Similarly, anionic drugs can cross 
skin by negatively charged electrodes. Electroosmosis is the fluid moving 
in a direction in the presence of electric field, forming an electroosmotic 
flow and driving the movement of hydrated ions. At physiological pH, 
skin has a slight negative charge. Thus, electroosmotic flow occurs from 
anode to cathode (Wang et al., 2021). 
  

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Fig. (1): A schematic diagram of iontophoretic technique (Adapted from 
Wang et al., 2021). 
3.2: Factors influencing iontophoretic process:  
There are various factors affecting iontophoresis as: The drug used 
should be water-soluble, of low dosage and susceptible to ionization. 
Smaller particles are more mobile. Increasing drug concentration results 
in greater drug delivery, but only to some extent. If buffer ions are 
present, they compete with the drug, reducing its delivery. The effect of 
iontophoresis also depends on the tissue on which the electrodes are 
applied (e.g., thickness, permeability, the presence of pores). In the skin, 
sweat glands are the most significant way the charges are carried 
(Karpiński., 2018). 
3.3: Iontophoretic device: 
 An iontophoretic device consists of four parts: power source, 
control circuit, electrodes, and reservoirs. The power source can be 
connected to the skin through a control circuit and two electrodes (the 
anode and the cathode). Two electrodes are attached to two reservoirs 

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(one reservoir containing drug ions and the other containing 
physiologically compatible salts such as NaCl). Generally, Ag/AgCl is 
selected as the electrode material in iontophoretic systems (Wang et al., 
2021). 
3.4: Iontophoretic delivery of peptides and proteins: 
Iontophoresis transiently increases the skin permeability of 
peptides and proteins. Peptides and proteins play a determining role in 
modern therapy. Their potency and specificity make them excellent 
therapeutic agents; however, their physicochemical properties and 
stability requirements almost invariably necessitate their administration 
by subcutaneous, intramuscular or intravenous injection. These act as 
effector agents that regulate and/or mediate physiological processes, 
serving as hormones, neurotransmitters and signal transducing factors. It 
has long been known that iontophoresis can administer therapeutic 
amounts of biologically active peptides into the body. More recent studies 
have shown that it is also capable of delivering structurally intact, 
functional proteins non-invasively into and across intact human skin and 
becomes the most popular choice of drug delivery (Dhote et al., 2012). 
 
 
 
 
 
 
 
 
 
 
 

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CHAPTER ΙII 
SUBJECTS, MATERIALS AND METHODS
 
In this part of the study, the materials and methods will be 
presented under the following headings: subjects, equipment, procedures 
of the study and statistical procedures.  
1-Subjects (Sample size): 
Sample size calculation is performed using G*POWER statistical 
software (version 3.1.9.2; Franz Faul, Universitat Kiel, Germany) based 
on data of VSS from the previous study (Elshahed et al., 2020) who 
reported a significant effect of botulinum toxin compared with control in 
treating hypertrophic scars. The required sample size for this study was 
38 subjects per group. Calculations were made using α=0.05, power 90% 
and effect size = 0.76 and allocation ratio N2/N1 =1.  
Seventy-six patients who have postburn hypertrophic scars will 
participate in this study. Their ages will be ranged from 20 to 40 years. 
The participants will be selected from the outpatient clinic of the faculty 
of physical therapy, at Cairo University and randomly distributed into 
two equal groups. 
1.1 
Design of the study: 
In this study, the patients will be randomly assigned into two equal 
groups (38 patients for each group). 
1.1(a) Group A (Study group): 
This group includes 38 postburn hypertrophic scar patients who 
will receive Botox iontophoresis once monthly for 3 months; in addition 
to their physical therapy program (Positioning, Stretching exercises, 

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Pressure Therapy and Massage) 2 sessions per week for 3 months 
(Mowafy et al., 2021). 
1.1(b) Group B (Control group): 
This group includes 38 patients who will receive traditional 
physical therapy (Positioning, Stretching exercises, Pressure Therapy and 
Massage) 2 sessions per week for 3 months (Mowafy et al., 2021). 
1.2 Criteria for the patient selection: 
1.2. a. Inclusion Criteria: 
The subject selection will be according to the following criteria: 
• Patient with hypertrophic scar (3-6) months  after burn healing. 
• Age range between 20-40 years. 
• Male and female patients will participate in the study. 
• All patients have a postburn hypertrophic scar at different body 
sites.    
• All patients enrolled on the study will have their informed consent. 
1.2.b Exclusion Criteria: 
Exclusion Criteria related to iontophoresis (Sheikh and Dua., 2020): 
• Patients with prior medical histories of cardiac arrhythmias. 
• Patients with cardiac pacemakers. 
• Patients with orthopaedic implants. 
• Areas of skin with lesions and impaired sensation. 
• During pregnancy and breastfeeding. 
• Patient with diabetes mellitus. 
 

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Exclusion Criteria related to the delivered drug itself (Elshahed et al., 
2020): 
• Patients with a history of hypersensitivity or adverse reactions 
associated with (BTX_A).  
• Recent BTA administration 6 months before the study. 
•  Any subject complaining of psychiatric disorders or neurological 
disorders such as myasthenia gravis. 
2-Equipment: 
2.1-Measurement equipment: 
2.1. (a)Sonography 
A high-resolution B-image sonogram of every patient will be taken 
before and after treatment using an 11-Mhz receiving transducer of Logiq 
P6 Pro (GE Healthcare, Solingen, Germany). This method allows a good 
penetration depth of up to 40 mm into the skin and a resolution of around 
158 micro m. It has been successfully utilized for objectively measuring 
pathological scars (Reinholz et al., 2016). 
2.1. (b) Patient and Observer Scar Assessment Scale (POSAS) 
 POSAS is an adequate tool to objectively measure scar quality and 
the therapeutic success of the applied scar treatment. It consists of two 
parts; one for the patient (Patient scale; POSAS Patient) and one for the 
physician (Observer scale; POSAS Observer). Both contain six items on a 
10-point rating scale and an extra category “Overall Opinion”. All 
characteristic features of the pathological scars are covered by the 
questionnaire: 
vascularity, 
pigmentation 
disorders, 
relief/texture, 
thickness, pliability, surface area, pain, and itching/pruritus. The latter 
items, in particular, concern the well-being of the patients (Reinholz et 
al., 2016). 

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2.2-Therapeutic equipments:  
2.2. (a) Iontophoretic device: 
 
 
Fig. (2): The iontophoresis device (Adapted from Ibrahim et al., 2021). 
 
2.2. (b) Botulinum toxin type A: 
• Botox Allergan ®(Irvine, CA, USA) will be used .  
It is 100 U vacuum-dried powder in a single-use vial for reconstitution 
diluted in 2 mL of sterile, preservative-free 0.9% saline to constitute a 
solution at a concentration of 4 U/0.1 mL) (Elhefnawy et al., 2016). 
 
3-Procedures of the study: 
3.1-Measurement procedures:   
Both sonography and POSAS will be used for assessment of hypertrophic 
scar pre treatment and post treatment ( after 3 months then after 6 months 
as follow up). 
3.1. (a)Sonography 

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High frequency ultrasound is the most common used technique for 
scar assessment. Its inferior resolution compared to optical equivalents is 
mitigated by its superior penetration depth allowing thickness analysis, 
even in severe scar thickening. The working mechanism is based on 
refection of sound waves of structures with different acoustic impedances 
and the analysis of the refection time to determine the depth of the 
structure. The penetration depth ranges from the upper dermal layers to 
full-thickness skin and subcutaneous structures, depending on the 
employed frequency (Téot et al., 2020). 
3.1. (b) Patient and Observer scar Assessment Scale (POSAS): 
The scar will be rated numerically on a ten-step scale by both the 
patient and doctor on six items: vascularity, pigmentation, thickness, 
relief, pliability, and surface area on the Observer Scale. The Patient 
Scale consists of pain, itchiness, color, stiffness, thickness, and 
irregularity of the scar. One of the reasons POSAS was chosen for scar 
evaluation is because it is the only scar assessment tool to include a 
component for patients to fill in. Furthermore, its distinctive feature of 
reflecting subjective symptoms like pain and pruritus and because of its 
appropriateness for everyday practice (Kant et al., 2018). 
 
Also, The Arabic version of the (POSAS) is an easy-to-administer, 
simple, reliable and valid tool for assessment of burn scar and for use on 
Egyptian population. It is advised to be used in clinical practice as well as 
scientific researches (Nossier et al., 2018). 
 

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(Adapted from Draaijers et al., 2004). 
 
3.2-Therapeutic procedures: 
3.2. (a) Procedures of iontophoretic device (Ibrahim et al., 2021): 
Iontophoretic drug delivery system (Phoresor IIAuto,Model 
PM850, IOMED). The active electrode is placed directly over the marked 
area. The dispersive electrode was applied to the skin 6 inches distal from 

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the active electrode (Figure 3). The dose required is selected on the 
device; depending on the subject’s tolerance, the current intensity is 
gradually increased, ranging from 2 to 4 mA. 
3.2. (b) -Procedures of Botox iontophoresis (Elhefnawy et al., 2016)An 
intralesional injection of botulinum toxin type A (Botox Allergan ®, 
Irvine, CA, USA).  
1. 100 U vacuum-dried powder in a single-use vial for reconstitution 
diluted in 2 mL of sterile, preservative-free 0.9% saline to 
constitute a solution at a concentration of 4 U/0.1 mL) will be 
administered once a month for a total period of three months. 
2. The dose will be adjusted to 2.5 U/cm2 of the lesion.   
3. The dose shouldn't exceed 100 units per session. 
3.2. (c) Physical therapy program for both groups: 
Positioning: 
Positioning should begin immediately post injury and maintain 
during the entire process. Positioning should be carried out together with 
proper ROM training, otherwise, a prolonged fixed position will also 
result in reduced ROM and contracture. Positioning could be achieved 
through various modalities including pads, pillows, headboard, foam 
pads, splints and restraint belts (Cen et al., 2015). 
Stretching and early mobilization: 
Patients should be encouraged to get out of bed and exercise as 
soon as they are fit enough to do so. Therapeutic exercise encompasses 
ambulation of joints, consideration of neurovascular integrity, improving 
cardiovascular and respiratory capacity, coordination, balance, muscle 
strength and endurance, exercise performance and functional capacity. 

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Exercise also helps the patient to experience a general feeling of 
wellbeing and a sense of confidence and achievement (Procter., 2010). 
Massage therapy: 
Massage therapy, manual or mechanical (i.e., compressed air, 
threadlike showers, vacuotherapy, etc.), is standard therapy in 
rehabilitation centers specializing in the treatment of scars and burns. 
Although there is no scientific evidence, it has been shown that massage 
therapy not only reduces scar-related pain and itching, but also increases 
range of motion, reveals patients’ anxiety and improves their mood and 
mental status. Mechanical compression is shown to reduce collagen 
synthesis by several mechanisms. These include decreased blood, oxygen 
and nutrients delivery to the scar, MMP-28 (matrix metalloprotease-28) 
downregulation and increase of PGE2 (prostaglandin-E2), which 
activates collagenase (Arno et al., 2014). 
Pressure therapy: 
pressure therapy is believed to work by reducing perfusion and 
oxygen concentrations reaching the scar surface resulting in reduced 
collagen synthesis. Moreover, pressure on scars reduces hydration, 
decreases angiogenesis, and induces apoptosis. A variety of materials can 
be used to provide pressure such as custom-fitted splints and adhesive 
plaster fittings while the efficacy is mostly dependent on the scar location 
with limbs and trunks being most appropriate for pressure therapy. 
Pressure garments can help reduce pain and itching associated with scars 
(Elsaie., 2021). 
4-Statistical procedures: 
Descriptive statistics: 

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In this study, the descriptive statistics (the mean and the standard 
deviation) will be calculated for all subjects in all groups of the study to 
determine the homogeneity of the groups. 
Analytical statistics: 
Comparison will be made by student's t-test to compare the 
variables between all groups of the study. Paired t-test was used to 
compare before and after treatment in the same group. A value of p<0.05 
will be considered statistically significant (Maronna et al., 2006). 
 
 
 
Sample size:  
Sample size calculation  is performed using G*POWER statistical 
software (version 3.1.9.2; Franz Faul, Universitat Kiel, Germany) based 
on data of VSS from the previous study (Elshahed et al., 2020) who 
reported a significant effect of botulinum toxin compared with control in 
treating hypertrophic scars. The required sample size for this study was 
38 subjects per group. Calculations were made using α=0.05, power 90% 
and effect size = 0.76 and allocation ratio N2/N1 =1.  
 

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REFERENCES 
 
1. Arno AI, Gauglitz GG, Barret JP and Jeschke MG, (2014):  "Up-
to-date approach to manage keloids and hypertrophic scars: A useful 
guide." Burns; 40(7): 1255-1266. 
2. Cen Y, Chai J, Chen H, Chen J, Guo G and Han C, (2015): 
"Guidelines for burn rehabilitation in China." Burns & trauma, 3. 
3. Dhote V, Bhatnagar P, Mishra PK, Mahajan SC and Mishra DK, 
(2012): "Iontophoresis: A potential emergence of a transdermal drug 
delivery system." Scientia pharmaceutica; 80(1): 1-28. 
4. Draaijers LJ, Tempelman FR, Botman YA, Tuinebreijer WE, 
Middelkoop E, Kreis RW and Van Zuijlen PP, (2004): "The 
patient and observer scar assessment scale: A reliable and feasible 
tool for scar evaluation." Plastic and reconstructive Surgery; 113(7): 
1960-1965. 
5. Elhefnawy A, (2020):  "Assessment of intralesional injection of 
botulinum toxin type A injection for hypertrophic scars." Indian 
journal of dermatology, venereology and leprology, 82(3). 
6. Elsaie ML, (2021): "Update on management of keloid and 
hypertrophic scars: A systemic review." Journal of cosmetic 
dermatology; 20(9): 2729-2738. 
7. Elshahed AR, Elmanzalawy KS, Shehata H and ElSaie ML, 
(2020):  "Effect of botulinum toxin type A for treating hypertrophic 
scars: A split‐scar, double‐blind randomized controlled trial." Journal 
of Cosmetic Dermatology; 19(9): 2252-2258. 
8.  Ibrahim NA, Abdel Raoof NA, Mosaad DM and Abu el kasem 
ST, (2021): “Effect of magnesium sulfate iontophoresis on 
myofascial trigger points in the upper fibres of the trapezius.” J 
Taibah Univ Med Sc 2021;16(3):369e378 

Approved date: 17/5/2022 
 
22 
 
9. Guida S, Farnetani F, Nisticò SP, Mariarosaria CG, Babino G, 
Pellacani G and Fulgione E, (2018):  "New trends in botulinum toxin 
use in dermatology." Dermatology practical & conceptual; 8(4): 277. 
10. Jeschke 
MG, 
Kamolz 
LP, 
Sjöberg 
F 
and 
Wolf 
SE, 
(2020):  "Handbook of Burns Volume 1." 2nd (ed). Springer 
International Publishing; 475-488. 
11. Jeschke MG, van Baar ME, Choudhry MA, Chung KK, Gibran 
NS and Logsetty S, (2020): "Burn injury." Nature Reviews Disease 
Primers; 6(1): 1-25. 
12. Kant SB, Van den Kerckhove E, Colla C, Tuinder S, Van der 
Hulst R and de Grzymala AP, (2018):  "A new treatment of 
hypertrophic and keloid scars with combined triamcinolone and 
verapamil: A retrospective study." European journal of plastic 
surgery; 41(1): 69-80. 
13. Karpiński 
TM, 
(2018): 
"Selected 
medicines 
used 
in 
iontophoresis." Pharmaceutics; 10(4): 204. 
14. Kasyanju Carrero LM, Ma WW, Liu HF, Yin XF and Zhou BR, 
(2019): "Botulinum toxin type A for the treatment and prevention of 
hypertrophic scars and keloids: Updated review." Journal of cosmetic 
dermatology; 18(1): 10-15. 
15. Lee HJ and Jang YJ, (2018): "Recent understandings of biology, 
prophylaxis and treatment strategies for hypertrophic scars and 
keloids." International journal of molecular sciences; 19(3): 711. 
16. Maronna R, Martin R and Yohai V, (2006):"Robust statistics 
theory and methods:" Journal of the American statistical association; 
90: 330-341. 
17. Montaser-Kouhsari L, Zartab H, Fanian F, Noorian N, Sadr B, 
Nassiri-Kashani M and Firooz A, (2014): "Comparison of 
intradermal injection with iontophoresis of abobotulinum toxin A for 

Approved date: 17/5/2022 
 
23 
 
the treatment of primary axillary hyperhidrosis: A randomized, 
controlled trial." Journal of dermatological treatment; 25(4): 337-341. 
18. Mowafy Z, Elgohary HM, Hassan KM and Mohamed KE, (2021): 
"Effect of cryotherapy on hypertrophic scar." European Journal of 
Molecular & Clinical Medicine; 8(3): 1184-1191. 
19. Naik PP, (2021):  "Utilities of botulinum toxins in dermatology and 
cosmetology." 
Clinical, 
 Cosmetic 
and 
Investigational 
Dermatology; 14: 1319. 
20. Nossier AA, Abdelmotaleb EA and Saied SM, (2018):  "Validity 
and Reliability of Arabic Version of the patient and observer scar 
assessment scale with burned patients." The Medical Journal of Cairo 
University; 86: 2311-2316. 
21. Procter F, (2010):  "Rehabilitation of the burn patient." Indian journal 
of plastic surgery: official publication of the Association of Plastic 
Surgeons of India; 43(Suppl), S101. 
22. Reinholz M, Schwaiger H, Poetschke J, Epple A, Ruzicka T, Von 
Braunmühl T and Gauglitz GG, (2016): "Objective and subjective 
treatment evaluation of scars using optical coherence tomography, 
sonography, photography, and standardised questionnaires." European 
Journal of Dermatology; 26(6): 599-608. 
23. Roustit M, Blaise S and Cracowski JL, (2014):  "Trials and 
tribulations of skin iontophoresis in therapeutics." British journal of 
clinical pharmacology;77(1): 63-71. 
24. Samizadeh S and De Boulle K, (2018): "Botulinum neurotoxin 
formulations: Overcoming the confusion." Clinical, cosmetic and 
investigational dermatology; 11: 273. 
25. Sheikh 
NK 
and 
Dua 
A, 
(2020):"Iontophoresis 
Analgesic 
Medications. " 

Approved date: 17/5/2022 
 
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26. Shirakami E, Yamakawa S and Hayashida K, (2020): "Strategies 
to prevent hypertrophic scar formation: A review of therapeutic 
interventions based on molecular evidence." Burns & trauma; 8. 
27. Téot L, Mustoe TA, Middelkoop E and Gauglitz GG, (2020): 
"Textbook on Scar Management: State of the art management and 
emerging technologies. " 
28. Wang Y, Zeng L, Song W and Liu J, (2021): "Influencing factors 
and drug application of iontophoresis in transdermal drug delivery: 
An overview of recent progress." Drug Delivery and Translational 
Research; 1-12. 
29. Zhang T, Wang XF, Wang ZC, Lou D, Fang QQ, Hu YY and Tan 
WQ, (2020):  "Current potential therapeutic strategies targeting the 
TGF-β/Smad signaling pathway to attenuate keloid and hypertrophic 
scar formation." Biomedicine & Pharmacotherapy; 129: 110287.