Application of Allogeneic Fibroblast Cultured on Acellular Amniotic Membrane for Full-thickness Wound Healing in Rats

Objective. Utilization of the autologous and allogeneic skin substitutes seems to be a promising treatment option. In this study, the authors used amniotic membrane covered with cultured allogenic fibroblast as a skin substitute in the treatment of acute wounds.Materials and Methods. Full-thickness wounds were created on rats’ dorsum regions and treated with cultured allogenic fibroblast on an acellular amniotic membrane (AAM+F), an acellular amniotic membrane (AAM) alone, an allogenic fibroblast suspension (AFS), or normal saline as a control (C). Specimen biopsies were obtained 7  days after wounding. Quantitative wound healing parameters including the epidermal thickness, the mean number of keratinocytes, fibroblasts, and lymphocytes were assessed. Results. All transplanted wounds exhibited significantly further contraction compared with the nontransplanted wounds. Wounds transplanted with AAM+F and AAM showed a significant increase in epidermal thickness compared to nontransplanted wounds. Wounds transplanted with AAM+F or AAM showed improved epidermal healing compared to nongrafted wounds. Furthermore, granulation of tissue formation in the AAM+F group was more organized when compared to AFS and the normal saline groups. Conclusion. Quantitative assessment of the full-thickness wounds showed transplantation of AAM+F and AAM better improve wound healing parameters when compared to treatment with AFS and the normal saline groups.

In deep and extensive wounds, it is not possible to use a bandage for intial treatment, as the quick healing of acute wounds can lead to more inflammation, proliferation, and a prolonged remodeling phase. Therefore, it is suggested the wound be covered with special materials to avoid infection.^1,2 Various biomaterials are used as a dermal matrix to accelerate the fibroblast and keratinocyte growth and proliferation,^3-7 and skin substitutes made of dermal matrices and fibroblasts facilitate reepithelialization via dynamic interaction.^8-10

The human acellular amniotic membrane (AAM) is proposed as a suitable scaffold for fibroblasts and keratinocytes. The amniotic membrane (AM) is biodegradable, antimicrobial, antifibrosis, antiscarring, antitumorigenic, and promotes angiogenesis.^11,12 It is cost-effective, easy to process, and can be stored by freezing. The viability and adherence properties of cultured fibroblast on the AAM have been shown in previous studies,^13,14 and the extracellular matrix constituents of AM are suitable for skin substitute preparation.^15,16

The purpose of this study was to evaluate the potential of AAM as a supportive factor for full-thickness wound healing in the experimental rats. Therefore, the authors used the AAM as a scaffold and studied the role of allogenic fibroblasts cultured on AAM stroma in wound healing, histologically.

Animals. Prior to the start of the study, approval was obtained from the Mazandaran University of Medical Sciences Ethic Committee for the animal experiments. Twenty wistar male rats (5 rats per group) weighing 170g-200g were selected and kept in individual cages at 21ºC ± 1ºC for 1 week in a 12-hour alternating light and dark cycle to acquaint the animals with their new environment.

The subjects under study were randomly divided into 4 groups treated as follows: 1) 3×10⁵ allogenic fibroblasts cultured on acelluar amniotic membrane  (AAM+F); 2) AAM alone;  3) 3×10⁵ allogenic fibroblast suspension (AFS); and 4) 0.9% normal saline (C).

Isolation and cultivation of fibroblasts. Fibroblasts were isolated from the dermis as previously described.¹⁷ Briefly, the full-thickness skin sample was removed from the abdominal region. Samples digested by trypsin-ethylenediaminetetraacetic acid and epidermis were separated from the dermis. The dermis samples were minced and explanted in tissue culture dishes, incubated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin at 37°C with 5% CO₂. Cultivated cells were fed every 3 days. When they reached 85%-90% confluence, the cells were passaged 5 times.

Wound model and surgical procedure. The full-thickness skin wounds were created in the dorsum region.¹⁷ The rats were anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The skin was shaved, disinfected and 1.5 cm² × 1.5 cm² full-thickness wound was created on the dorsal neck.

Acellular amniotic membrane preparation. The placentas were obtained from healthy mothers following elective cesarean section deliveries in Shohadaye Tajrish Educational Hospital, Tehran, Iran. Written consent was obtained from the mothers to harvest the AM, which was detached from the chorion and washed in 1000 ml physiological saline solution containing penicillin/streptomycin 3 times under sterile conditions. The amnion was washed with phosphate buffered saline twice to separate blood clots, dead cells, debris, and mucous. Then the amnion was mechanically decellularized using a scraper after 3 freeze-and-thaw cycles and, finally, followed by overnight trypsinization at 4ºC. The AAM was cut into 6 cm × 6 cm pieces and stored at -80ºC. This scaffold could be preserved for several months in the sterile vial containing freezing solution at -80ºC. Before fibroblast culturing, the samples were incubated at 37ºC for 1 hour in the culture medium.

Cellular skin substitute preparation. The fifth passage of fibroblast (3 × 10⁵ cells/cm²) was seeded on the center of AAMs in 6-well plates. After 2 hours of incubation at 37ºC to attach the cells, 3 mL complete DMEM with final concentrations of 10% FCS and 1x penicillin/streptomycin was added to the wells and incubated at 37ºC for 24 hours in 5% CO₂. The prepared skin substitutes were transplanted onto the full-thickness wound in the rat models. The rats under experiment were kept in separate cages.

Histologic analysis. Skin biopsies from the healed wounds and normal tissue were studied for pathological changes. Samples were fixed in 10% paraformaldehyde and processed for routine hematoxylin and eosin (H&E) staining. In this study, the paraffin-embedded sections with 5 µm thickness were serially prepared with 15 µm intervals and stained with H&E. The criteria of tissue repair evaluated by a pathologist who was blinded to the details of the experiment were as follows: the epidermal thickness, the mean number of keratinocytes, fibroblasts, and lymphocytes. The epidermal thickness was measured by light microscopy and using analysis software (OLYSIA BioReport version 3.2, Soft Imaging System Corp, Lakewood, CO) at least in 5 consecutively x100 magnified fields from the edge towards center of wounds. The number of keratinocytes in the reepithelialized region and the fibroblasts and lymphocytes in granulation tissue bed were counted at least in 5 consecutively x400 magnified fields from the edge towards the center of the wounds, and then the means of the cell numbers in wounds were calculated.

The qualitatively obtained data in the experiment and control groups were evaluated using SPSS version 15 software (IBM, New York, NY). All of the data were expressed as ANOVA, one-way ANOVA, and Tukey tests. Significant differences between the experiment and the control group were analyzed statistically. The significant differences between the groups were determined, and P < 0.05 was accepted as significant.

Macroscopic findings showed contraction and dryness of the wounds in experimental groups AAM+F and AAM on the second day, while the same results in the AFS group and the control group were observed on the third day. During the biopsy harvesting (on day 7), a high thickness of fibrous tissue and ulcer was observed clearly in the control group (Figure 1). 

Seven days postoperation, biopsies were collected from the wounds. Figure 2 shows the quantitative indexes of wound healing in the 4 groups. The histopathological investigation of the wound biopsies has shown wound reepithelialization in the experimental groups occurred completely without any clinical symptoms, inflammation, or epithelial defects (Figure 2). 

Microscopic observation showed epidermal thickness, the fibroblast, and keratinocyte cells numbers in the experimental groups were more than the control group (P < 0.05). In addition, the granulated tissue was denser, more fibrotic, and less inflamed in groups receiving the amniotic membrane with and without fibroblast (Figures 2A, 2B). The wounds that only received AFS formed less-organized epithelium layers compared to the transplanted groups (Figure 2C). In treated wounds with normal saline, the epithelialization showed a significant delay compared to the experimental groups (Figure 2D). All of the grafted wounds showed a dominant increase in the number of fibroblasts and keratinocytes and a decrease in the lymphocyte number in comparison to the nongrafted wounds. Also in the control group, the inflammation severity was more in comparison with the experimental groups (Figure 3). 

Transplantation with AAM to large and deep traumatic wounds has been shown to accelerate the reepithelialization.¹⁸ The previous studies showed AAM can support fibroblast adherence and infiltration.^13,14 In the current study, the skin substitute was constructed by cultured allogenic fibroblast on AAM and investigated in vivo. It was found the stroma of amnion is appropriate biomaterial for fibroblast cell delivery onto the wound bed. Additionally, transplanting of AAM+F showed better results than using AAM treatment alone.

The beneficial effects of transplantation of fibroblast and keratinocyte cells suspension were previously demonstrated in wound healing.^17,19 In the present study, the wounds treated with allogeneic fibroblasts showed significant improvement in epidermal repair compared to normal saline. Here, the authors investigated the effect of AAM as a wound dressing on wound healing parameters including epidermal thickness and the number of fibroblast, keratinocyte, and lymphocyte cells. They found transplantation of allogeneic fibroblast cells cultured on AAM scaffold results were better than nontransplanted wounds. Both AAM and AAM+F demonstrated enhanced healing outcomes compared to the control group.

An increase of epidermal thickness is one of the indicators of improvement in wound repair. In this study, epidermal thickness was measured 7 days after injury. Increased epidermal thickness was observed in wounds grafted with AAM+F and AAM. Further criteria should be taken into account to better evaluate the outcome of wound healing. Treated wounds with AAM+F and AAM exhibited significantly higher fibroblast and keratinocyte numbers when compared to the control group. In addition, AAM+F and AAM transplantation leads to faster wound healing than repairing wounds with AFS and normal saline. Recent studies suggested that amniotic membrane as a skin substitute can facilitate the reepithelialization.^18,20,21

As a biomaterial, the AAM is able to reduce inflammation, facilitate healing, and promote reepithelialization.¹⁵ In this study, the severity of inflammation in the healed wounds that received AAM with and without fibroblasts was significantly lower than the treated wounds with the cell suspension and normal saline.

Worth mentioning, the results show significant improvement in the wound healing process when grafted with AM with and without cells. This finding agrees with the authors’ previous finding, which indicates AM is suitable for transferring cells onto the wounds.¹³

There are several reasons explaining why transplantation with AAM or AAM+F improves wound healing parameters to a greater extent than the cultured fibroblast. It has been demonstrated that wound healing with skin substitutes containing living cells occurs more quickly, more effectively, and with less fibrosis.^19,22 In AAM+F, the fibroblasts are surrounded by a supportive microenvironment, increasing the regenerative capacity of the cells when transplanted. One can assume the presence of these supporting structures facilitates keratinocyte and fibroblast migration and promotes wound healing.

Although the design of the present study is not sufficient to reach a conclusion on the efficiency of AAM, the authors demonstrated that AAM+F and AAM transplantation could be useful and safe to promote acute full-thickness wound healing.

Acellular amniotic membrane seems to act as a safe skin substitute. As previously observed with other allogeneic skin substitutes,²² AAM and AAM+F transplantation on the acute wounds markedly and rapidly reduces fibrous and granulation tissue formation in comparison to AFS and normal saline. Therefore, AAM transplantation could be considered an alternative method for treating acute wounds. This study was a preliminary report and showed AAM can be used as a safe skin substitute for human wound healing. The authors recommend clinical trial studies are needed for further understanding. Likewise, due to the many similarities between porcine and human skins,²³ the authors also recommend the research be conducted using a  porcine animal model.

The authors wish to thank the Vice of Research of Mazandaran University of Medical Sciences, Sari, Iran and the Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran for their financial support. They would also like to thank Ms. Niki Mahmoudi Rad for her assistance during the development of this study.

From the Skin Research Centre, Shahid Beheshti University of Medical Sciences, Tehran, Iran; Anatomy Department, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Amol Faculty of Nursing and Midwifery, Mazandaran University of Medical Sciences, Amol, Iran; Pathology Department, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; and Immunology Department, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Address correspondence to:
Fereshteh Talebpour Amiri, PhD 
Anatomy Department, Faculty of Medicine
Mazandaran University of Medical Sciences
Sari, Iran
ftaleb2001@yahoo.co.uk

Disclosure: This research was supported by research grants from Molecular and Cell biology Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran (Grant code; 91.51, date; 2012.8.17), Sari, Iran and Skin Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.