The Effects of Dried Amniotic Membrane on Epithelialization in Secondary Wound Healing : An Experimental Study

Aims: To investigate the efficacy of dried gamma-irradiated amniotic membrane dressing for the healing of full-thickness skin wounds, using a rabbit model. Materials and Methods : This study was performed in the Oral and Maxillofacial sugery Department, College of Dentistry, University of Mosul, Iraq. Twenty, locally bred, six to eight months old male rabbits were planned to conduct the study. Bilateral full-thickness circular segments approximately 10 mm in diameter and nearly (3-4)mm in thickness were excised from the the back of each rabbit ( near to the mid-dorsum area) with a surgical blade; one dorsal skin wounding was performed lateral to the midline on right side, the other wound was performed lateral to the midline on left side. The right wound was used as an experimental wound and covered with dried amniotic membrane whereas the left wound was considered as a control wound. All wounds were covered with non adherent moist occlusive gauze. Animals were divided into four groups according to sacrificing date ( 3, 7, 14, 30) days. Skin specimens were collected after sacrificing the rabbits. The specimens were placed in formalde-hyde and sent for histopathological examination and scoring for wound healing. Results : The histopathological findings of this study revealed that there was a significant difference of reepithelialization between the two groups at two weeks period, at p-value < 0.05, while there was no significant difference at 3 days, one week, and one month. Conclusions : This study concluded that amniotic membrane can be used as a safe dressing, promoting epithelialization on secondary wounds.


INTRODUCTION
Wound healing is the body's natural biological process for regenerating dermal and epidermal tissue. This process involves time and a balanced activity of inflammatory, vascular, connective tissue, and epithelial cells leading to the restoration of skin integrity (1). Although many efforts have been made, wound healing has become an increasingly difficult clinical, scientific and economic problem, especially taking into account the current population development (2). In clinical practice, the best way to heal a wound is to close it according to surgical standards as quickly as possible after injury. However, this procedure is limited to those wounds located in anatomical regions that permit adaptation of wound borders allowing wound closure by primary intention. This means closing it by suturing and restoring of the skin continuity (3) .In large-surface and deep wounds in which primary wound closure is neither possible nor practicable, the most important issue is to dress the wound with appropriate materials in order to keep it free from infection, to reduce or eliminate pain and all potential factors inhibiting natural healing, and to replace or substitute the missing tissue as much as possible (4).
Current strategies point out the acceleration of the wound repair by systematically designed dressing materials. By this direction, most efforts have experimentally and clinically utilized the biologically derived, which are capable of accelerating the healing processes at molecular, cellular, and systemic levels, as materials to produce wound dressings (5,6,7). Amniotic membrane is a tissue of interest as a biological dressing due to its biological properties and immunologic characteristics. It has low immunogenicity and beneficial re -epithelialization effects, with anti-inflammatory, antifibrotic, anti-microbial, and nontumorigenic properties (4). The amniotic membrane is the innermost layer of the fetal membrane, which first appears at about days 7-8 following conception. It results from a separation from the inner cell mass of the germ disk at the periphery of the ectodermal layer (8). As shown in Figure  The amniotic membrane(AM) consists of five layers from within outward: epithelium, basement membrane, compact layer, fibroblast layer and spongy layer (9). The epithelium consists of a single layer of epithelial cells which are polygonal in shape (10). The basement membrane of the AM which contains types І, Ш, IV, V collagen, laminin-1, laminin-5 and fibronectin, plays an important role in the other cells prolifera-tion and differentiation (9,11). The compact layer is a dense layer almost totally devoid of cells and consists mainly of a complex reticular network. The fibroblastic layer is the thickest layer of the amnion and consists of mesenchymal cells. The spongy layer forms the interface between the AM and chorion and consists of wavy bundles of reticulum bathed in mucin. This layer is normally separated from fibroblast layer when the AM is peeled from the underlying chorion. The AM varies in thickness from 0.02 mm to 0.5 mm, so it can easily be used as a biological dressing in ischemic areas (10).
The AM has low immunogenicity, and well-documented re-epithelialization effects, anti-inflammatory, anti-fibrotic, antimicrobial, and non-tumorigenic properties. The AM has low immunogenicity, and welldocumented re-epithelialization effects, anti-inflammatory, anti-fibrotic, antimicrobial, and non-tumorigenic properties. These pleiotropic functions are related in part to its capacity to synthesize and release biological active substances including cytokines and signaling molecules such as the tumor necrosis factor, interferon, transforming growth factor (TGF)-a, TGF-b, basic fibroblastic growth factor, epidermal growth factor, keratinocyte growth factor, hepatic growth factor, interleukin-4 (IL-4), IL-6, IL-8, natural inhibitors of metalloproteases,b-defensins,prostaglandins etc (12-14,).
In wounds healing by secondary intention reepithelialization occurs after the wound defect is filled with granulation tissue. As the cells migrate, they increase in number by mitosis. Once the wound is covered with cells, cellular differentiation occurs, restoring normal epithelial layers. Epithelialization proceeds at approximately 1 mm/day (23,24).
The mechanism by which amniotic membrane enhance epithelialization could be attributed to the production of growth factors, and the basement membrane of amniotic membrane facilitates migration of epithelial cells which reinforces the adhesion of basal epithelial cells, and may promote epithelial differentiation (19,20). Amniotic membrane exerts anti-scaring effect through secreting anti-inflammatory cytokines and suppressing TGF-ß signaling at the transcriptional level, leading to downregulation of several downstream genes that are responsible for scar formation (25).

MATERIALS AND METHODS
This study was performed in the Oral and Maxillofacial Surgery Department, College of Dentistry, University of Mosul, Iraq, from 28/2/2013 to 21/6/2013.Twenty, locally bred, 6-8 months old male rabbits were planned to conduct the study. The weight of each rabbit was about 1.5 + 0.5 kg. The animals were housed in an animal house prepared for that purpose, given free access to water and food, and maintained on a 12-h light-dark cycle.
According to sacrificing time, animals could be divided into four groups: G1: Animals sacrificed after 3 days. G2: Animals sacrificed after 1 week. G3: Animals sacrificed after 2 weeks. G4: Animals sacrificed after 1 month. Each one of these groups included two sub groups which are: Subgroup C: Which included the control group.
Subgroup AM: Which included the study group. For achieving general anesthesia each animal was given intramuscular dose of xylazine base (sedative and muscle relaxant) (5 mg/kg) and ketamine hydrochloride (anesthetic and analgesic) (50 mg/kg) injected into rabbit's thigh muscle (16) . The animal was laid on its ventral side on the surgical board ,the hair was shaved using lotion hair remover, the area was washed with a tap water. The animal was covered with sterile towel exposing dorsal side only. The surgical area was then disinfected by scrapping using piece of gauze with povidone-iodine solution (10 %) before performing the wound. A metal template measuring 10 mm in diameter was placed on the skin and an outline of the template was traced on the skin using a pen marker. The wound was made by excising the skin,within the border of the template. Bilateral full-thickness circular segments approximately 10 mm in diameter and nearly (3)(4) mm in thickness were excised from the back of each rabbit ( near to the mid-dorsum area) with a surgical blade (No. 11), one dorsal skin wounding was performed lateral to the midline on right side, another was performed lateral to the midline on left side, (with at least 1 cm the distance between two wounds). The right wound was used as experimental wound and covered with dried amniotic membrane, the left wound was considered as a control wound. All wounds were covered with non adherent moist occlusive gauze (17) Figure (2).

Figure (2).Surgical Procedure
A:The hair was shaved using lotion hair remover. B: Bilateral full-thickness circular segments approximately 10 mm in diameter were excised from the back of each rabbit C: The Amniotic Membrane was cut into appropriate size. D: The material was put on gauze and moistened with normal saline. E: The material was applied to the study wound. F: Both control and study wounds were covered with moist occlusive gauze. G: Both control and study wounds were covered with strips H: A bandage was wrapped around the trunk of animals At day of sacrifice ,samples were taken and placed in 10% formalin and sent for histopathological study. Histopathological Examination used to assess scoring for reepthelialization wich include (18) .
Score 0: Reepithelialization at the edge of the wound. Score 1: Reepithelialization covering less than half of the wound. Score 2: Reepithelialization covering more than half of the wound. Score 3: Reepithelialization covering the entire wound, irregular thickness. Score 4: Reepithelialization covering the entire wound, normal thickness.
Statistical analysis was made by using spss 19 computer soft ware program. Data were analyzed by using Mann -Whitney NPar Test was used to compare means between groups .

RESULTS AND DISCUSSION
In this study the effects of gammairradiated amniotic membrane on the epithelialization of the secondary wound sites were assessed. Since the use of gauze dressing is cheaper as compared to other commercial available dressings and is the normal practice for coverage of wound, healing of secondary wound with gamma irradiated amniotic membrane dressing and standard gauze dressing was compared. The results included reepithelialization examination in the four periods of the study as shown in Table (1),Chart (1). In the three days period, there is a minimal reepithelialization at the wound edge in both groups Figure (3).

Amniotic Membrane Induced Epithelialization In Secondary
Al -Rafidain Dent J Vol. 16 No statistically significant variation in the reepithelialization process between groups at this time. This result agreed with comparative study of Nordback etal., they concluded that the difference in epitheliazation was not statistically significant between the dispase de-epithelialized, glycerol cryopreserved amniotic membrane group and sodium chloride-moistened gauze group at 3 day period (17). A study conducted by Waikakul S, etal,(1990) used freeze-dried amniotic membrane applied in the form of splitthickness skin grafts over an area from which skin had been surgically removed. they found that covering the wound with the amniotic membrane had good results in terms of pain reduction, but the promotion of healing were not demonstrated (26). In contrast ,Woob etal.,(2001) investigated the effects of amniotic membrane on epithelial wound healing and stromal remodelling on eye of the rabbits by creating a wound 6.0 mm in diameter using an excimer laser, one eye was covered by the AM while the other eye served as a control they found that the course of healing was started with a slower initial phase, a faster middle phase, and increasing healing latency towards complete epithelial closure and most of the eyes in the AM group were entirely reepithelialised by 72 hours after surgery (27). Corneal and dermal epithelialisation cannot be stated as equal because corneal wound healing is thought to be a simpler process than when the healing process occurs in the skin. For instance, cornea lacks blood vessels in the cornea (28) .
In one week period ,there was no statistically significant variation in reepithelialization between groups but the degree of reepithelialization of wounds cover by amniotic membrane were better Figure (  This result agreed with comparative study of Nordback etal., they concluded that the epithelialization was somewhat larger in the AM group on days 7 but the difference was not statistically significant (17). In two weeks period, There was a statistically significant enhancement of epithelialization of amniotic membrane group compared to control group as in Figure (  The amniotic membrane has beneficial reepithelialization effects, the AM contains many growth factors and the basement membrane facilitates migration of epithelial cells which reinforces the adhesion of basal epithelial cells, and may promote epithelial differentiation ,the AM provides a suitable substrate, such as laminins for rapid attachment of epithelial cells. AM might exert its wound healing effect by accelerating keratinocyte migration from the wound edge and inducing its differentiation, thereby contributing to the generation of intact epithelium (4,(19)(20)(21) .
Mermet etal., used AM on the treatment of chronic venous leg ulcers, they found that the greatest effect of AM was observed at the 14th day but no significant effect was seen at the 7th day (22) .Insausti et al. concluded, although the process was slow, AM application in human patients with post-traumatic wounds inducing the formation of a well-structured epidermis with all relevant layers and restoring skin integrity (4) .
In the one month period ,both groups showed complete epithelialization and there was no statistically significant variation in reepithelialization between groups. Both groups showed well-recovered epidermal and dermal layers. The epidermis showed mature differentiation, and the dermal layer showed thick collagen bundle deposition and well-recovered skin appendages .Usually the healing at this period is by far completed and inflammation susbsided .
Nordback etal., found that the effect of AM decreases in later stages. they concluded that the difference in epitheliazation was not statistically significant between the dispase de-epithelialized, glycerol cryopreserved amniotic membrane group and sodium chloride-moistened gauze group at 21 day period (17) .

CONCLUSIONS
Dried Amniotic membrane has properties that are helpful in wound healing. According to this study, the use of amniotic membrane as a dressing in secondary wound to enhance epithelialization is beneficial.