Effects of Autologous Keratinocyte Cell Spray With and Without Chitosan on Third Degree Burn Healing: An Animal Experiment

Treatment of extensive third degree burns, especially in the case of limited skin donor sites for obtaining autologous split-thickness skin grafts (STSG), has led to in vitro expansion of keratinocytes. Cultured epidermal autograft (CEA) sheets have been used for burn treatment for several years. However, time consuming processes of isolation and cultivation of keratinocytes, as well as difficult procedures of detachment of CEA from cultured flasks, led scientists to develop the technique of spraying cultured single keratinocytes (CSK) instead of using CEA. Chitosan is a well-known wound dressing biomaterial that has both biological and medical applications. In this study, the application method of CSK was used to determine whether there would be any significant difference between the treatment of burns with CSK alone in comparison to burns treated with CSK and covered with chitosan gel at a neutral pH. Materials and Methods. Thirty male Wistar rats were selected and their keratinocytes were isolated and cultured from small skin biopsy. Rats were divided randomly into 3 equal groups and 3 full-thickness round burn wounds were created on their backs. Rats were treated with either normal saline (control group), CSK (test group A), or CSK + neutral chitosan (CSK+ NCH) (test group B). The wounds were photographed on days 0, 3, 5, 7, 10, and 14, and the percentage of wound contraction was calculated with an image analyzer. Biopsy samples were taken for histological studies. Results. The results showed faster wound contraction for test groups A and B during the 14-day period than the control group (P < 0.05). Also, more contraction was found in test groups A and B in 7 days (P < 0.05). Histological observations showed significant difference in inflammation and fibrotic tissue formation between groups, but other parameters did not show any remarkable difference. Conclusion. Based on the findings of this study, the authors concluded that chitosan can prevent cells from dripping out of the wound, speed up wound contraction, and extend fibrotic tissue formation. Chitosan did not, however, have any effect on fastening the reepithelialization and granulation tissue formation during the first 14 days.

  Not only is skin the body’s first defense system against external damages and infections, it is also the largest organ of the body. Although skin is able to repair itself following minor injuries, in severe cases such as massive burns, emergency medical treatment is required to reduce scarring, as well as to avoid the dehydration and infections which are the principal causes of death in patients who have been severely burned.1 The current gold standard for treating large skin loss is the use of autologous split-thickness skin grafts (STSG). However, in massive burns, especially cases where there are limited skin donor sites for STSG, the need to rapidly dress the wound has lead to the use of other skin substitutes, such as cultured epidermal autologous (CEA) sheets.2 Cultured epidermal autologous sheets have been used in the treatment of burns and other coetaneous wounds for several years.3 Serial cultivation of keratinocytes was developed by Rheinwald and Green5 in the mid 1970s. This method can be used for producing CEA sheets that are 1,000 times larger than the initial skin specimen, up to the size of the entire body surface during a 3-4 week period.2,4,5 But CEA has some disadvantages such as unpredictable take rates, fragility, destruction, and alteration of integrins and basement membrane proteins via enzyme digestion during detachment of epithelial sheets from culture flasks.6

  Moreover, the time-consuming process of culturing keratinocytes and the difficulty of transporting them onto paraffin-impregnated gauzes, and from laboratory to hospital, led to the development of another transplantation method which involves spraying, dropping, or using a paintbrush to apply cultured single keratinocyte (CSK) suspension to the wound surface.6-8

  Wound dressings are no longer considered passive products with a minimal role in the healing process.9 An ideal wound dressing should be biocompatible, provide protection from infections, and provide a moist environment for wound beds.9 Chitosan is a biomaterial polymer that is obtained by partial N-deacetylation of chitin, a substance found in the hard shells of crustaceans and fungal mycelia. It has been proven to promote homeostasis and to have wound healing potential, bacteriostatic effects, biocompatibility, biodegrability, and immunologic activity.10 Because of these qualities, chitosan has been widely used in the form of hydrogel, fiber, membrane, and scaffold for wound healing. It also has no adverse effects on tissues after implantation.11 Since chitosan is only soluble in acidic media not suitable for cell viability, it was decided to prepare and use a viscous and neutral soluble derivative of chitosan by using azide (para-azidobenzoic acid) and lactose (lactobionic acid) that react with amino groups of chitosan. The aim of this study was to use neutral chitosan (NCH) as a wound dressing to determine if there is any significant difference between treatment of third degree burn wounds with CSK alone and CSK covered with chitosan.

  This animal experiment was approved by the Ferdowsi University of Mashhad Committee on Animals and Ethics under the project license 3998.

  Animals. Thirty male Wistar rats between 6-8 weeks of age and weighing 250 g ± 20 g were obtained from the Laboratory Animal House, School of Pharmacy, Mashhad University of Medical Sciences. The rats were housed in hygienic plastic houses and allowed at least 2 weeks to adapt to the environment, which was kept at 25°C and 12-hour light/dark cycles. Commercial food (Javaneh Khorasan, Mashhad, Iran) and tap water were available ad libitum.

  Preparation of skin biopsies. While under general anesthesia by interperitoneal injection of 5 mg/kg xylazine (Rompun, 2%, Bayer, Leverkusen, Germany) plus 60 mg/kg ketamine (Ketamine HCl 50 mg/ml, Rotexmedica, Trittuau, Germany), the body hair on the sample area of each rat was shaved and a split-thickness specimen of skin (around 2 cm × 3 cm) was taken under sterile conditions from the paravertebral region of the rats, near the cervical area. The area was sutured by 4-0 nylon to promote healing and prevent infection. Sutures were removed 6 days after biopsy harvesting. Skin biopsies were rinsed 3 times with phosphate-buffered saline (PBS) without Ca2+ or Mg2+ (Hyclone Dulbecco’s Phosphate-Buffered Saline, Thermo Fisher Scientific, Waltham, MA) containing antibiotics and antimycotics, and immediately placed into a transport medium (Dulbecco’s modified Eagle’s medium [DMEM] +10% fetal bovine serum [FBS] + 2% antibiotic and antimycotic) (PAA Laboratories, Pasching, Austria).

  Isolation and culture of keratinocytes. Rat keratinocytes were cultured according to the protocols already described by Freshney and Freshney,12 Hager et al,13 and Yano and Okochi.14Subcutaneous tissue of skin samples was eliminated with curved scissors and each biopsy was dissected into 1 cm × 0.5 cm pieces with scalpels and rinsed 2 times in PBS. The tissue samples were then immersed in cold 0.125% trypsin (Gibco, Grand Island, NY) and incubated at 4°C overnight. The epidermis was separated from the dermis using 2 fine curved forceps and collected in a 50 mL centrifuge tube containing 20 mL of complete culture medium followed by gentle pipetting to detach keratinocytes. The isolated keratinocytes were washed twice in complete culture medium by centrifugation at 500 g for 5 minutes, and finally, the total number of cells and the viable cells were counted using Trypan blue staining.

  The isolated rat keratinocytes were then seeded onto T25 flasks, one flask per rat, that had been preseeded with 3T3 cells inactivated by Mitomycin C (Kyowa Hakko Bio Co Ltd, Tokyo, Japan) as a feeder layer. The inactivated 3T3 cells were plated at 3×104 cell/cm2 density 2 days prior to keratinocyte seeding and incubated at 10% CO2 and 37°C. After 3 days, the cells were attached and the cultures were rinsed with PBS to eliminate nonattached cells. The culture medium was changed every 2 days. The cultured keratinocytes reached 80% confluence in 7-8 days. The complete culture medium was DMEM (high glucose with L-glutamine and sodium pyrovate (PAA Laboratories, Pasching, Austria) supplemented with 10% FBS, 10 ng/ml epidermal growth factor (EGF, Sigma-Aldrich, St. Louis, MO), 1 × 10-10 M. Cholera toxin (azide-free, Sigma-Aldrich, St. Louis, MO), 100 IU/ml penicillin v potassium (Jaber Ebne Hayyan Pharmaceutical Co, Tehran, Iran) and 100 µg/ml streptomycin (Rotexmedica, Trittau, Germany), 0.5 mg/ml hydrocortisone (Rotexmedica, Trittau, Germany), 5 µg/ml insulin (Exir Pharmaceutical Co, Tehran, Iran), 1.8 × 10-4 M Adenine(HiMedia, Mumbai, India), 1.4 ng/ml triiodothyronine, sodium salt (Sigma-Aldrich, St. Louis, MO), 0.5 µl/ml L-ascorbic acid (Jaber Ebne Hayyan Co, Tehran, Iran) and 5 µg/ml transferrin (Sigma-Aldrich, St. Louis, MO). Upon 80% confluency, the cells were subcultured by dispersing in 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid solution (Gibco, Grand Island, NY) and reseeded. The secondary cultures reached 80% confluence in 4-5 days.

  Preparation of neutral chitosan hydrogel. Neutral pH water-soluble chitosan was prepared as previously described by Ono et al.10 High molecular weight chitosan (800–1000 kDa) with 80% deacetylation degree (Molekula, Dorset, UK) was used. Lactobionic acid and p-azidobenzoic acid (Tokyo Chemical Industry Co Ltd, Tokyo, Japan) moieties were reacted with amino groups of chitosan molecule through a condensation reaction and, consequently, an aqueous solution of chitosan at neutral pH was achieved.

  Formation of the burn wounds. Each rat was anesthetized by an interperitoneal injection of ketamine and xylazine. The dorsal thoracic area was shaved and scrubbed using 70% ethanol. After a deep general anesthesia was reached, 3 round wounds, approximately 1 cm in diameter, were made using a 100 W electric soldering iron, heated to the point of redness (about 800° C) for 5 seconds.15 After 2 hours the damaged tissues were removed down to the panniculus carnosus muscle layer, creating third degree burn wounds.

  Experiment design. Subconfluent autologous keratinocytes (the authors used second passage cells for cell transplantation) were suspended in DMEM at a concentration of 1 x 106 cells/ml. A total of 30 rats were examined in this trial, with 10 rats per group and each rat having 3 burn wound areas including control which was sprayed by normal saline; test group A which was treated with cultured autologous keratinocytes (CAK) at a density of 5 × 105 cells/cm2 (CAK); and test group B which was treated with CAKs at a density of 5 × 105 cells/cm2 in combination with equal volume of chitosan as a cover (CAK + NCH). The authors applied NCH just once, followed by autologous keratinocyte spraying. The wound areas of all the groups were bandaged with paraffin gauze and cotton gauze dampened with normal saline. The bandages were changed every other day.

  Evaluation of wound contraction. Wounds were photographed using a digital camera (Canon IXY32S, Toyko, Japan) on days 0, 3, 5, 7, 10, and 14 post cell transplantation. The wound area was measured using image analysis software (Scion Image, Scion Corp, Frederick, MD). The percentage of wound contraction was calculated using the following formula:

    N = (N0 – Nx ) / N0 × 100
    N: percent of wound contraction
    N0: wound size ( mm2) at day 0
    Nx: wound size ( mm2) at day X

  Wound biopsies and histochemical study. Biopsy samples were taken at days 3, 7, and 14 after wound formation. Ten rats were sacrificed on each of these days to harvest histological samples. Rats were euthanized using CO2 inhalation. Whole wound biopsies with adjacent normal skin were taken and fixed in 10% buffered formalin followed by tissue processing and embedding in paraffin; 5 µm sections were prepared from the center of the wound area and stained using hematoxylin-eosin. Histological examination, including granulation tissue formation, reepithelialization, inflammatory response, and fibrous tissue formation, were evaluated and graded based on the progression of the healing process (Table 1).

  Repeated measures ANOVA, followed by Bonferroni post hoc test, were conducted to investigate the effects of treatments on wound contraction during the study period. A nonparametric Kruskal-Wallis test at P < 0.05 was used to find out whether the histopathological indices of the 3 groups differed significantly. Pairwise comparison was performed using the Mann-Whitney U test. Since there was multiple testing of the data, the significance level was adjusted using the Bonferroni correction. The 3 groups were compared and the significance level became 0.05 divided by 3 (P < 0.017). All statistical analysis was performed using SPSS statistical software version 16 (SPSS Inc, Chicago, IL).

  All rats survived the experiment and there were no obvious side effects of the chitosan and cell application. Thirty rats were used for this study and a total of 90 wounds were created. Thirty wounds were treated with CSK (test group A), 30 wounds were treated with CSK + NCH (test group B), and 30 wounds were treated with normal saline (control group). Wounds were allocated to test groups in such a way that each group had equivalent numbers of wounds in similar positions on consecutive animals. The total number of cells delivered to each wound was 5 × 105 cells/cm2 in test group A and test group B.

  Gross observation of cell application. The results showed better adherence of cells to the wound bed and less dripping out was observed in test group A compared to test group B (Figure 2).

  Evaluation of wound contraction. The average wound contraction percentage in wounds treated in test group B after 3 days (n = 30), 7 days (n = 20), and 14 days (n = 10) was greater than in the control group (P < 0.05). Also, contraction percentage in wounds treated in test group A after 7 and 14 days was greater than the control group (P < 0.05). Comparison of the average wound contraction percentage in those treated in test group A and test group B alone showed more contraction in test group B wounds during days 1, 3, and 7 (P < 0.05) (Table 2, Figure 3).

  Histological observations. In the control group, no new epithelialization was seen histologically in the center of the wound surface, just thin reepithelialization around the edge of the wounds was observed. In test groups A and B, a thin, new epithelium was observed at the center of the wound area. The degree of reepithelialization didn’t show any remarkable difference between the test groups. (P > 0.05).

  Inflammatory response on day 3 of the study and fibrous tissue formation on days 3 and 7 in rats in group B were significantly greater than those in the control group (P < 0.05). None of the other histological parameters were significantly different between the various groups (Table 3, Figure 4 and 5).

  Burn management and healing remains a complicated process. Apart from STSG, many other treatment protocols have been evaluated; however, the most effective and the fastest treatment method remains undecided.¹⁶

  Since 1998 when Fraulin et al¹⁷ described a novel method of using an epithelial cell spray on wounds in pigs, many other trials have been organized to develop this technique, lowering the time of cell culture and increasing the transplantation taking rate. Application of CSK suspension, which was used in this study, has some disadvantages such as low capacity of adhesion to the wound bed. Because of its proven effects in wound healing, chitosan was chosen as the biomaterial for the wound dressing. Chitosan is insoluble in water at neutral and alkaline pH, but dissolves in acidic pH that is not applicable for cell viability. Therefore it was decided to use neutral pH, synthesized in the author’s lab by the method described by Ono and colleagues,¹⁰ to take advantage of both the wound healing effect of chitosan and its application as a medium for coverage of the cells.

  Whereas Mori et al¹⁹ believed that chitosan doesn’t have any acceleratory effect on the production of cultured fibroblasts, Howling et al¹⁸ reported that chitosan may affect the proliferation of human fibroblast. Howling and colleagues observed more stimulation on proliferation of fibroblast with chitosan, especially with a higher degree of deacetylation.^18,19 Based on these studies, Ueno et al²⁰ suggested that chitosan can accelerate fibroblast production through indirect and direct pathways only when it is used in vivo.²⁰ The results of the present study showed that fibrotic tissue formation in test group B was greater than the control test during the first 7 days of study, which was similar to the findings of Ueno and colleagues.²⁰

  Ueno and coauthors also claimed wounds treated with chitosan showed more inflammatory response and infiltration of polymorphonuclear leukocytes in wound areas compared with the control group 3 days after treatment.²⁰ The results of the current study are in agreement with these results, showing that chitosan can promote the migration of inflammatory cells to the wound area at an early stage of wound healing; but attention should be paid to the point that the inflammatory phase is a normal and necessary reaction following injury in the first stage of healing. Determining whether the inflammatory response is part of the normal healing process or due to the effect of the material used is quite difficult.^21,22

  Previous studies also indicated that chitosan can accelerate the formation of granulation tissue and may stimulate extra cellular matrix production and fibroblast formation through promoting the production of interleukin 8 and stimulating the production of collagen type III,^20,23 but this was not found in the current study. This may be due to the small sample size. 

  In the current study, test group B was not found to have any advantage in terms of percentage of reepithelialization by itself within 14 days of wound formation when compared with test group A. Some studies reported that chitosan can interact with epidermal cells and stimulate reepithelialization in experiemental wounds on dogs and rats.^24,25 In contrast, others believe that chitosan doesn’t have any proliferative effect on human keratinocytes in vitro and even may decrease its development in some cases.¹⁸ The authors of the current study find it difficult to explain these controversial results. In contrast to the current study, previous experimental studies on rats and dogs focused only on the effect of chitosan without using autologus keratinocyte in their trial.^24,25 Also Howling et al¹⁸ tried to determine the effect of chitosan on proliferation of human keratinocytes in culture.¹⁸ When chitosan is used alone, reepithelialization of the wound edge is the aim of the study; but the main objective of the current study was to detect the difference in reepithelialization over the wound bed as an effect of cell transplantation. The authors of the current study believe that application of CSK + NCH for wound coverage in vivo may behave differently fromin vitro results or application of chitosan alone.

  Ishihara et al²⁶ examined the effect of chitosan as a wound dressing and accelerator of the healing process. Those authors reported a statistically significant difference in wound contraction between the chitosan and control groups on day 8, which is similar to the current study’s findings showing the average wound contraction during the first 7 days in test group B was greater than the other groups. In fact, the authors of the current study observed this effect as early as days 3 and 5, and speculate this could be due to the presence of CSK in test group B.

  As previously described, wound contraction percentage of the group treated with CSK was significantly greater than the control group on days 7 and 14 of this study, and CSK + NCH group data showed more contraction throughout the experiment. Comparison of the latter 2 groups demonstrated that CSK + NCH vs CSK resulted in significantly different (P < 0.05) wound contraction on days 3 (2.78% difference) and 7 (4.3% difference). It is believed that this may be because of the synergistic effect of chitosan and keratinocytes on wound contraction, although it might be a result of the moist biological dressing present over the keratinocytes, which helps better contraction of the wounds.

  The authors conclude that chitosan could act as an appropriate wound dressing and cell delivery medium that may prevent cell loss. Chitosan also can accelerate inflammatory response and fibrotic tissue formation, but in the current study it did not promote reepithelialization, which is the most important aim of wound healing. However, application of higher cell concentrations or the use of photocrosslinked chitosan containing CSK should be further explored.

  The authors would like to thank Dr. Adel Moallem for his kind support and advice during the cell culture procedure, Dr. Mohammad Azizzadeh for statistical analysis, and Dr. Ghasem Sajjadi for his advice. The authors are also grateful to Mr. Ghanbari and his colleagues at the Ghaem Hospital Laboratory of Histopathology for the preparation and staining of histological sections, and to Mr. Akbari for assisting with the animal experiments.