Monitoring the Process of Healing by Means of Bioimpedance ...



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 Group |Laser Stimulation Group |Monitorization of healing by means of |

| |(630-650nm red light) |Bioimpedance Spectroscopy and Histology |

| | |Analysis |

|No external stimulation |Dosimetry and application protocol studies|Bio-electrical impedance measurements |

|- |Laser stimulation on pre-defined days of |Histology (H&E and Trichrome Staining) |

| |healing | |

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.

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.

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 |24 | |Experiments |C |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.

REFERENCES

1. Ruthinéia Diógenes Alves Uchôa Lins, et.al. “Biostimulation effects of low-power laser in the repair process" An Bras Dermatol, 2010 Dec; 85(6):849-55. Review. English, Portuguese.

2. Hüseyin Demir, Halil Balay, Mehmet Kirnap, “A comparative study of the effects of electrical stimulation and laser treatment on experimental wound healing in rats” Journal of Rehabilitation and Research and Development, Volume 41, Number 2, Pages 147–154 May/April 2004.

3. Bisht D, Gupta SC, Misra V, Mital VP, Sharma P. “Effect of low intensity laser radiation on healing of open skin wounds in rats” Indian Journal of Medical Research. 1994;100:43–46.

4. Abergel P, Lyons RF, Castel JC, Dwyer RM, Uitto J. “Biostimulation of wound healing by lasers: experimental approaches in animal models and in fibroblast cultures” Journal of Dermatological Surgery and Oncology. 1997;13:127–33.

5. Lyons RF, Abergel P, White R. “Biostimulation of wound healing in vivo by a helium-neon laser” Annals of Plastic Surgery. 1987;18:47–51.

6. Conlan M, Rapley JW, Cobb CM. “Biostimulation of wound healing by low-energy laser irradiation: a review” Journal of Clinical Periodontology. 1996;23:492–96.

7. Bravermen B, McCarthy RJ, Ivankovich DE, Forde DE, Overfield M, Bapna MS. “Effect of helium-neon and infrared laser irradiation on wound healing in rabbits” Lasers in Surgery and Medicine. 1989;9:50–58.

8. Ghamsari SM, Taguchi K, Abe N, Acorda JA, Sato M, Yamada H. “Evaluation of low level laser therapy on primary healing of experimentally induced full thickness teat wounds in dairy cattle” Veterinary Surgery1997;26:114–20.

9. Karu Tiina I. “Photobiological Fundamentals of Low-Power Laser Therapy” IEEE Journal of Quantum Electronics, Vol. QE-23, NO. 10, October 1987.

10. Pereira AN, Eduardo Cde P, Matson E, Marques MM. “Effect of low-power laser irradiation on cell growth and procollagen synthesis of cultured fibroblasts” Lasers in Surgery and Medicine 2002;31:263–7.

11. Medrado AR, Pugliese LS, Reis SR, Andrade ZA. “Influence of low level laser therapy on wound healing and its biological action upon myofibroblasts” Lasers in Surgery and Medicine 2003;32:239–44.

12. Almeida-Lopes L, Rigau J, Zangaro RA, et al. “Comparison of the low level laser therapy effects on cultured human gingival fibroblasts proliferation using different irradiance and same fluence” Lasers in Surgery and Medicine 2001;29:179–84.

13. Kana JS, Hutschenreiter G, Haina D, Waidelich W. “Effect of low-power density laser radiation on healing of open skin wounds in rats” Archives of Surgery 1981;116:293–6.

14. Bisht D, Gupta SC, Misra V, et al. “Effect of low intensity laser radiation on healing of open skin wounds in rats” Indian Journal of Medical Research 1994;100:43–6.

15. Anneroth G, Hall G, Ryden H, Zetterqvist L. “The effect of lowenergy infra-red laser radiation on wound healing in rats” British Journal of Oral and Maxillofacial Surgery 1988;26:12–7.

16. Schlager A, Oehler K, Huebner KU, et al. “Healing of burns after treatment with 670-nanometer low-power laser light” Plastic and Reconstructive Surgery 2000;105:1635–9.

17. Schlager A, Kronberger P, Petschke F, Ulmer H. “Low-power laser light in the healing of burns: a comparison between two different wavelengths (635 nm and 690 nm) and a placebo group” Lasers in Surgery and Medicine 2000;27:39–42.

18. Cambier DC, Vanderstraeten GG, Mussen MJ, van der Spank JT. “Low-power laser and healing of burns: a preliminary assay Plastic and Reconstructive Surgery 1996;97:555–8; discussion 559.

19. Mester E, Korenyi-Both A, Spiry T, Tisza S. “The effect of laser irradiation on the regeneration of muscle fibers (preliminary report)” Z Exp Chirurg 1975;8:258–62.

20. Schindl A, Schindl M, Schindl L. “Successful treatment of a persistent radiation ulcer by low power laser therapy” Journal of the American Academy of Dermatology 1997;37:646–8.

21. D. Warren Spence and Bruce Pomeranz, “Surgical wound healing monitored repeatedly in vivo using electrical resistance of the epidermis” Physiological Measurements 1996, 57–69.

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Xylene

5 min

96% alcohol

10 dip

96% alcohol

10 dip

Haematoxylin

2 min

Lithium

Carbonate

96% alcohol

10 dip

Eosine

2 min

96% alcohol

10 dip

96% alcohol

10 dip

96% alcohol

10 dip

Acetone

10 dip

Xylene

3 dip

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

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