Monitoring the Process of Wound Healing by Means of

Bioimpedance Spectroscopy

Hakan Solmaz

Boğaziçi University Institute of Biomedical Engineering

Thesis Proposal Committee:

Prof. Yekta Ülgen....................................................(Thesis Supervisor)

Assoc. Prof. Murat Gülsoy.......................................(Thesis Co-Advisor)

Assoc. Prof. Can Yücesoy........................................

DATE: 13.01.2012

ABSTRACT

Wound healing has always been an important subject for researchers aiming to

increase the knowledge and understanding of physiology of chronic wounds and the

complex process of healing. There are economic and medical approaches for the

efficiency and effectiveness of wound care. The medical approach consists of using a

wide variety of topical dressings, oral and systemic medications, with the ultimate goal of

secondary healing. The surgical approach is based on surgical intervention to prepare and

heal the wound.

Wound healing can be simply defined as the process in which the skin or other

tissues and organs start repairing itself immediately after an injury. The entire wound

healing process is a complex series of events that begins at the moment of injury and can

continue for months to years. The physiological and chemical events happening

throughout this complex process is generally discussed in three subsections which are

inflammation, proliferation and maturation phases. These phases are discussed in detail in

the next sections of my proposal.

Monitorization of wound healing has been an important subject for understanding

the physiology of the healing process. The conventional method used for monitoring the

wound tissue during healing is the histological analysis based on examining specific types

of cells by means of tissue staining procedures. However, histological examinations are

complex and time consuming in vitro procedures that have to be performed by a

professional researcher.

The aim of this project is to develop a new in-vivo method for monitoring the

process of healing by means of bioimpedance spectroscopy and evaluating the affects of

laser biostimulation on wound healing process by examining the results using the

proposed bioimpedance spectroscopy method and histological analysis. The electrical

impedance of the tissues during the process of healing with and without the application of

laser stimulation will be measured and compared with the results of histological analysis

in order to make a relationship between the changes of electrical properties of wound

tissue and the phases of healing process.

I. INTRODUCTION (LITERATURE SURVEY)

In now days, many researchers have been working on the investigation of new

methods for understanding the complex physiology of wound healing and reduce the time

of healing as much as possible. One of the most important reasons of studying on the

wound healing process is that, it enables to increase the knowledge and understanding the

physiology of chronic wounds (venous ulcers, diabetic and pressure ulcers) that may

provide development in the treatment of these wounds resulting with increased patient

comfort and satisfaction.

Although there are various approaches that have been studied in the intent of wound

healing, the method of laser-biostimulation has been considered to be quite effective on

progression of healing process. There are various studies represented by many researchers

in the literature using this method in wound healing because of its positive affects

obtained from the experiment results.

The use of laser in medical purposes can be classified into two, high-power surgical

lasers with cutting, vaporization and hemostasis properties and low-power therapeutic

lasers with analgesic, anti-inflammatory and biostimulation properties. It is shown in

literature that radiation emitted by low-power lasers has shown analgesic, anti-

inflammatory and healing properties. It is emphasized that therapeutic lasers instead of

having a direct healing effect, act as an important pain-relieving agent providing the body

with a better inflammatory response, as they help to reduce edema and minimize pain, in

addition to promoting tissue repair of the injured region quite effectively through cellular

biostimulation [1]. Because of the biostimulation effect at those wavelengths, 632.8nm

HeNe and 904nm GaAs lasers are the mostly used types of lasers in wound healing studies

[1-9].

Low-level laser therapy studies on cell cultures show that the amount of connective

tissue, endothelial and epithelial tissues with the granulation tissue have increased while

the number of cells increased locally [3-6]. Experiments performed on human subjects

using 904nm GaAs lasers indicate that fibroblast production after laser stimulation

increased while no positive effect was observed in collagen synthesis when compared to

control groups. Some similar studies using 670, 692, 780 and 786nm laser represent

similar results which indicate that the phases of healing gained acceleration with the laser

irradiation [10-12]. Although the importance of cell studies for understanding the process

of wound healing is agreed by many researchers, it is obviously not enough to recognize

the phases of healing separately and prepare a substructure for human skin studies. For

that purpose, researchers emphasize the necessity of animal studies. In some studies

examining the affect of energy densities of laser irradiation using 632.8nm HeNe and

514.5nm Argon lasers, it is found that lower energy stimulations were much more

effective in wound healing for those specific wavelengths [13, 14]. However, some other

studies in the literature state the opposite results obtained with low-level laser application.

904nm GaAs laser is reported as having neither positive nor negative affect on the

production of granulation tissue [15]. It is stated in the same article that stimulation of

wound tissue with this wavelength did not shorten the phases of healing. Moreover,

researchers report in their studies that 635, 670 and 690nm diode lasers also did not have

any therapeutic affect on burn injuries [16-18].

Besides the contribution of results obtained from researches including cell culture

and animal studies on the background information of wound healing, it has been agreed

that human studies should have also been examined for better understanding of healing of

human skin despite the difficulties of standardization of procedures and patient monitoring

in human experiments. In literature it is possible to find such examinations reporting the

beneficial affect of low-level laser therapy on soft tissue healing [19, 20].

Monitoring a tissue during the complex process of healing is one another important

subject at which the number of studies on wound monitorization has been increasing day

by day. Bioimpedance spectroscopy, which has been thought to improve the

monitorization of healing process and understanding the physiology of the complex

healing, is a relatively new method that has not been used very often until now days [21].

Thus, it might provide basis for future studies of wound treatment monitorization

investigating the process of healing especially on human subjects. As a matter of fact

some of the advantages of electrical resistance measurements of tissues by means of

impedance spectroscopy are:

- it makes possible the observation of healing process in vivo without damaging the

tissues,

- large number of measurements may be done on the same tissue for repeatability and

reliability examinations with no need to increase the number of samples,

- some previous studies used to prefer visual investigation and planimetry methods for

their measurements to be non-invasive. However, these methods required large wound

surface areas and are no longer trustable, since their results were subject dependent.

Thus, it may be very elucidative to make further analysis on electrical resistance

measurements of wound tissues during healing process, which were stimulated with either

laser or electrical current.

II. THESIS STATEMENT

II-1 Motivation

The wound healing process has always been an excellent subject for researchers.

Treatment of chronic wounds (venous ulcers, diabetic and pressure ulcers) has particular

importance for patients suffering from non-healing wounds. As I have already discussed

in previous parts of my proposal, monitorization of treated wounds is one very important

subject that must be considered for understanding the complex process of healing.

Monitorization of healing wounds by means of bioimpedance spectroscopy, which is a

new in-vivo method, may prepare a framework for feature studies with human subjects.

II-2 Aim

The aim of this proposed study is to develop a new in-vivo method for

monitorization of healing wounds by means of bioimpedance spectroscopy and examine

the effects of laser biostimulation on the healing process by comparing the results of

conventional histology and the new method of impedance spectroscopy.

II-3 Originality of Project

- Monitorization of a healing wound is one of the most important issues of

understanding the physiology of healing process. However, recent methods are

generally based on in vitro examinations performed on experiment animals and

require removal of wound tissue followed by sacrificing the animal. This necessity

undesirably increases the number of experiment animal used. In this project, the

new in vivo method based on impedance spectroscopy for monitorization of

wound treatment may be used as a conventional method of wound monitorization

in the future. This may lead to decrease the usage of experiment animals and

prepare a basis for human studies.

III. APPROACH AND METHODS

The project will include two groups of rats, which are the control group and laser

stimulation group.

1. Control Group (G1): Animals in this group will not be exposed to external stimulation.

Samples taken from this group for histological examinations will be used as a reference.

2. Laser Stimulation Group (G2): This group will be irradiated with laser of certain

wavelength, power and frequency for a pre-defined amount of time for the first ten days of

treatment following the day of wound excision. This group will also be divided into two

for two different energy densities of irradiation.

The project will include the following main parts;

Control GroupLaser Stimulation Group

(630-650nm red light)

Monitorization of healing

by means of Bioimpedance

Spectroscopy and

Histology Analysis

No external stimulationDosimetry and application

protocol studies

Bio-electrical impedance

measurements

-Laser stimulation on pre-

defined days of healing

Histology (H&E and

Trichrome Staining)

III-1 Monitorization of Healing: Bioimpedance Spectroscopy and Histology Analysis

The aim of monitoring the progression of a healing wound is important for

understanding the physical and chemical changes occurring during the healing process.

The conventional method of examining those changes is making histological

examinations. The detailed information of how tissues are going to be prepared for

histological analysis will be given in the oncoming sections of my proposal.

In this project, the method of monitorization based on bioimpedance spectroscopy is an

alternative to the current histology examinations. The aim is to investigate the relationship

between the changes of electrical properties of tissues and the physical and chemical

alternations occurring during the process of healing. The in vivo non-invasive

measurement of bio-electrical impedance of wound tissue by means of an impedance

measurement device may be an evidence of the success of the treatment modality during

the progression of healing.

III-2 Laser Stimulation Group

According to papers in literature, the most distinctive biostimulation affects on

wound treatment are obtained in studies using laser irradiations of 630-650nm

wavelengths. Therefore, group (G2) will be illuminated with a red laser of 630-635nm

wavelength. Laser irradiation procedure for a certain amount of time will be repeated for

the first ten days following the day of excision. In order to examine the affect of the

application, samples taken from control group and laser group are going to be compared

with histological examinations. These examinations are going to be performed on days 3,

7, 14 and 21 following the day of wound excision. The reason of selecting those days for

histological examinations is that, these days correspond to times at which one phase of

healing will have completed. Thus, this will allow us to investigate all the phases of

healing process.

Laser group is going to be divided into two, which will be exposed to two different

energy densities of irradiation. The two groups are also going to be compared with each

other in order to see the affect of dose on the same wounds.

III-4 Surgical Procedures

Throughout the experiments, healthy, randomly selected, 5–6 months old, male

Wistar rats, weighing 250-300 g, will be used. The animals will be obtained from

Psychobiology Laboratory of Bogazici University. All of the experiment protocols will be

conducted under a protocol approved by the Institutional Animal Research and Care Ethic

Committee at Bogazici University. Rats are housed in plastic cages and maintained on a

12-h-light/12-h-dark cycle in a temperature-controlled vivarium (22±2°C). Food and

water are available ad libitum. Rats will be anesthetized with ketamine (90mg/kg) and

10mg/kg xylazine by intraperitoneal injection (1.65 ml/kg). Hair at the site of application

of each subject will be shaved. Then, all of the rats will undergo en block excision of the

skin at the median region of the back measuring 8mm in diameter by use of a punch. The

wound model will be as shown in the following figure.

Wound Model

Figure 1: Wound excision of 8 mm in diameter.

III-5 Histology and Tissue Preparation Procedures

III-5.1 Tissue Preparation

The samples will be fixed in 10% formalin and processed by dehydration.

Dehydrated tissues will be embedded into paraffin, 3 or 5-micrometer-thick tissue sections

will be obtained via microtome. Tissue sections will be obtained from removal of paraffin

over tissues by inserting into 40ºC water-bath. Tissues will be aligned on top of glass

slides. These slides will be kept in incubator overnight, in order to remove remaining

paraffin.

III-5.2 Hematoxylin&Eosine Staining

Hematoxylin and eosin (H&E) stains have been used for at least a century and are

still essential for recognizing various tissue types and the morphologic changes. This stain

gives idea about the general structure of tissue. Hematoxylin, which appears blue, is a

basic stain that binds with acidic cell components. These components are therefore termed

basophilic. Nucleic 'acids' such as the nucleus of the cell and the endoplasmic reticulum

stain with this dye, due to their high affinity for Hematoxylin. In contrast, Eosin is an

acidic dye which binds structures that are basic. Eosin binds basic components of the cell

and extracellular matrix, such as proteins. Thus eosin colors those eosinophilic structures

bright pink. Hematoxylin and eosin staining will enable to examine poymorphonuclear

leukocytes, macrophages and fibroblasts. Hematoxylin and eosin staining protocol is

described in the following table.

III-5.3 Trichrome Staining

Trichrome staining will be used to differentiate between collagen matrix and smooth

muscle structure. As the name implies, three dyes are employed selectively staining

muscle, collagen fibers, fibrin, and erythrocytes.

III-5.4 Toluidine Blue Staining

Toluidine Blue will be used for staining mast cells. Mast cells are found in the

connective tissue. Toluidine blue stains mast cells red-purple and the background blue.

Xylene5 min

96% alcohol10 dip

96% alcohol10 dip

Haematoxylin2 min

Lithium Carbonate

96% alcohol10 dip

Eosine2 min

96% alcohol10 dip

96% alcohol10 dip

96% alcohol10 dip

Acetone10 dip

Xylene3 dip

IV. FACILITIES

IV-1 Histology Equipments

Paraffin Embedding System: Leica EG 1150 H

Rotary Microtome: Leica RM2255

Cold Plate: Leica EG1150 C

Etuve: Nüve EN 025

Digital Biological Microscope: DMWB1-223, Motic China Group Co.

IV-2 Bioimpedance Measurement Equipments

LCR meter: HP 4284 A.

Slide Staining Set: Bio-Optica Strumentazioni Scientifiche Slide Staining Set

V. TIME SCHEDULE

Months 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24E

xper

imen

tsC x x x x

L1 x x x x x

L2 x x x x x

SA x x x x x x

TW x x x x x x x x x

C: Control Group,L1: Laser Stimulation Group 1,L2: Laser Stimulation Group 2,SA: Data Examinations and Statistical Analysis, TW: Thesis Writing.

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