Bioactive chitosan scaffold co cultured with keratinocyte and fibroblast cells.
Abstract: Polymeric cell seeded sponge scaffolds play a prominent role in supporting the regeneration of skin for burn patients. The three-dimensional scaffolds provide physical support and acts as an excellent matrix regulating cell growth, adhesion and differentiation. Natural macromolecule polymeric scaffolds from chitosan have advantages over synthetic polymers in that they encourage cell attachment and maintain differentiation of cells. Chitosan is the most used natural, biodegradable polymer as tissue scaffolds. It is known in the wound management area for its haemostatic properties. It possesses other biological activities and affects macrophage functions that assist in faster wound healing. It provides a non-protein matrix for three-dimensional tissue growth. Chitosan seems to generate desirable migrant and stimulatory effects on the stromal cells of the surrounding cells. Patients suffering from extensive skin loss like burns are in danger of succumbing to either massive infection or excessive fluid loss. Chitosan scaffold with the optimum combination of fibroblast cells and keratinocyte cells is guided to produce the desired tissues, which ultimately helps in the complete regeneration of functional skin. The present study demonstrates the importance of chitosan as a tissue engineered scaffold for burn wounds.
Article Type: Report
Subject: Keratinocytes (Properties)
Keratinocytes (Research)
Fibroblasts (Research)
Fibroblasts (Properties)
Biopolymers (Research)
Biopolymers (Properties)
Cell culture (Research)
Biomedical materials (Research)
Authors: Paul, Willi
Rekha, M.R
Mohanan, P.V.
Sharma, Chandra P.
Pub Date: 01/01/2012
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: Jan, 2012 Source Volume: 26 Source Issue: 1
Topic: Event Code: 310 Science & research
Product: SIC Code: 2836 Biological products exc. diagnostic
Geographic: Geographic Scope: India Geographic Code: 9INDI India
Accession Number: 304842707
Full Text: Introduction

Chitosan is a common biopolymer that is derived from chitin a key component of crustacean outer skeletons. It is the second most abundant natural polymer after cellulose and is known in the wound management area for its haemostatic properties. It has wide application in waste management, food processing, medicine and biotechnology. It possesses other biological activities that affects macrophage function and helps in faster wound healing [1]. It also has an capability to stimulate cell proliferation and histoarchitectural tissue organisation [2]. The biological properties including bacteriostatic and fungistatic properties are particularly useful for wound treatment. Various studies have been reported on the beneficial effects of chitosan accelerated wound closure and healing [3-6]. Chitosan facilitated rapid wound re-epithelialization and the regeneration of nerves within the vascular dermis and early returns to normal skin color at chitosan-treated areas [7]. Treatment with chitin and chitosan demonstrated a significant reduction in treatment time with minimal scar formation on various animals [8]. Biochemistry and histology of chitosan in wound healing has been reviewed by Muzzarelli et al. [9] and Feofilova et al. [10]. The silver sulfadiazine incorporated bilayer chitosan wound dressing showed excellent oxygen permeability, controlled water vapor transmission rate, and water-uptake capability along with excellent antibacterial activity [11]. Antibacterial, antifungal and antiviral properties found chitosan particularly useful for biomedical applications such as wound dressings, surgical sutures, as aids in cataract surgery, periodontal disease treatment, etc. [12]. Research has shown that chitin and chitosan are nontoxic and non-allergic so that the body will not reject these compounds. Both chitin and chitosan possess many properties that are beneficial for wound healing like biocompatibility, biodegradability [13], hemostatic activity [14], healing acceleration, non-toxicity, adsorption properties and anti infection properties [15-17]. An effective wound dressing not only protects the wound from its surroundings but also promotes the wound healing by providing an ideal microenvironment for healing, removing any excess wound exudates and allowing continuous tissue reconstruction [12]. Chitosan has been widely studied as a wound dressing material [18,19]; however, a wound-dressing product based on chitosan is yet to be commercialized.

The use of temporary skin substitutes such as Biobrane, TransCyte, Integra and Terudermis have become more widely used in the treatment of mild to deep dermal burn injury. However, they are extremely expensive. Also, they contain collagen which is not recommended for third degree burns where dermis, epidermis and hypodermis are totally destroyed [20]. Cell based artificial skin substitute Dermagraft derived from fibroblast cells was the first one to be approved by FDA for use in severe burns in 1997. However, is used only for diabetic foot ulcers now due to complications in burn cases. Apligraft a bovine collagen matrix cultured with fibroblast cells and keratinocyte cells is approved for venous stasis and diabetic foot ulcers only by the FDA. Epicel is a layer of autologous keratinocytes (skin cells) used to replace the epidermal or top layer of skin on severely burned patients. This is approved by the FDA, however, is allergic to some patients and is expensive.

These cell based skin substitutes had significant wound healing and scar reducing effect on patients. They were prepared by primary cultures of fibroblast and keratinocyte cells on hydrated collagen sponges. However, because of its sheer high cost and complication of utilizing collagen in burn patients there is still scope for the development of sponge dressing with co cultured fibroblast and keratinocyte cells. This can essentially replace collagen and is highly compatible to human tissue for use in severe burn wounds. Optimization of fibroblast cells and keratinocyte cells play a pivotal role in controlling collagen production with reduced scar formation. Globally there are over 40-45 million surgical procedures performed every year. Out of which 8-10 million are leg ulcers and 7-8 million procedures are for burn wounds. The predicted sales of wound dressing and wound care products for the year 2012 is US$ 12500 million [21]. However, chitosan's share is negligible. Chitosan is a natural hemostat which interacts with RBC and aggregates [22] which also helps in wound healing. An attempt has been made here to construct a perfect scaffold material for the co culture of fibroblasts and keratinocyte cells from chitosan. Optimization of fibroblast cells and keratinocyte cells have been attempted to reduce excess collagen production which may produce scar formation. This cell loaded scaffolds were tested on full thickness rabbit wounds for the possible use in burn wounds.

Materials and Methods

Chitosan (MW = 354kD, DDAc = 86%) was supplied by India Sea Foods, Kochi as gift. Polyethylene glycol (MW6000 was from E Merck, Germany. Sodium alginate (medium viscosity) and keratinocyte growth factor were from Sigma Chemical Co. All other chemicals and solvents were of analytical reagent grade.

Chitosan scaffold construction

Wound healing scaffold were prepared by freeze drying method by optimizing the content of chitosan, sodium alginate and polyethylene glycol in solution (Patent pending) at -40[degrees]C. The freeze drying cycle was optimized to achieve the desired chitosan sponge. The keratinicyte growth factor was loaded onto the sponge matrix by diffusion filling [23]. Briefly, 1[cm.sup.2] of chitosan sponge was incubated with 50ng/ml of growth factor solution in Tris buffer for one hour and freeze dried.

Degradation studies

The chitosan scaffold was incubated in 1mg/ml of lysozyme solution in phosphate buffered saline (pH 7.4) for 1 week. The enzyme solution was replaced every 24 hours with fresh solution. At specified intervals, the samples were taken out of the enzyme solution, washed with distilled water and dried. The weights of the samples were noted, and the weight losses were calculated.

Cell culture

Fibroblast cells and Keratinocyte cells were isolated from one healthy rabbit skin by the reported method [24]. Cultures were established by a single-cell suspension technique following enzymatic digestion of the skin samples with collagenase Type IV overnight. Cultures were maintained in the medium containing fetal calf serum (20mL), L-glutamine (1.5mL, 200mM) penicillin/ streptomycin (1mL, 10,000 units/mL penicillin, 10 mg/ mL streptomycin). This was incubated in an atmosphere consisting of 5% C[O.sup.2] at 37[degrees]C for the specified time. Upon reaching confluence, the cultures were split into half, to make almost 1 x [10.sup.5] cells per 35mm diameter tissue culture dish. Keratinocyte cells were first seeded onto the sponges for 6, 12 and 24 hours, and then fibroblast cells were also seeded for the extent of 12 hours. Cell proliferation and collagen production were evaluated using Laser Scanning Confocal Microscopy. These samples with proliferated keratinocytes and fibroblast cells were studied for wound healing on rabbits.

Wound healing studies on rabbits

Normal, healthy adult albino rabbits with either sex with body weight above 2 Kg were selected for the study. Animals were anesthetized by giving ketamine (80mg/ Kg) and Xylazine (5mg/Kg) i.m. The fur on the back of the animals was clipped, and the areas to be cut from the body were marked. Under aseptic conditions, six open wounds were made on each animal by cutting a full thickness of skin along the marking (three on the left and three on the right). Oozing blood was blotted with sterile cotton. Immediately following the excision of the skin (1 x 1 [cm.sup.2]), the cell seeded sponges with the same dimensions were placed on the wound bed and covered by gauze and bandaged. Animals were kept in individual cages. The grafts were applied to animals in the following pattern. 9 rabbits were divided into four groups of three animals each. First group of three animals received chitosan sponge alone on the right side wounds. Second group of three animals received co culture of fibroblast and keratinocyte cell cultured chitosan/alginate sponge on the right side wounds. Third group of three animals received no grafts on the right side and were treated as a negative control. All the above 9 rabbits received three grafts each on the left wounds with a commercial sample (Surgicel, Johnson and Jognson) as control. Each animal from each group (four animals each) was observed for 7 days, 14 days and 30 days for the quality of healing. After the respective observation, periods animals were sacrificed, and the newly grown skin along with the adjacent normal skin was taken for histopathological studies.

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Results and Discussion

Ideal tissue engineering scaffold should be highly biocompatible. They should have an appropriate microstructure with interconnected pores of 100-200 microns with porosity above 90% [25]. It should also have controlled biodegradability with suitable mechanical support for use at the wound site. The chitosan sponge scaffolds prepared have a pore size distribution of 100-200 microns with greater than 90% open pore volume. It can absorb water or exudate 20 times its weight. The SEM micrograph of the sponge is shown in figure 1. It has been shown that the degradation of chitosan sponge was 20% during six days duration in enzymatic solution as shown in figure 2. It has been reported that 7% of natural chitosan degrades in a period of 6 days and 20% in 15 days. Further degradation after 15 days seems to be slower [26].

A significant fibroblast proliferation was observed for fibroblast growth factor loaded chitosan sponge as shown in figure 3. Similarly, collagen production on growth factor loaded sponges were high (figure 4) with remarkably reduced collagen production on chitosan sponge without growth factor. In in vivo condition, the glucosamine availability (by degradation of chitosan) seems to be rate limiting for hyaluronic acid synthesis. Increased hyaluronic acid stimulates the migration and mitosis of mesenchymal and epithelial cells, leading to prolific collagen fibril structure [27]. The significant collagen production may lead to scar formation. It has been reported that the relative changes of the collagen al(I) messenger RNA expression in fibroblasts is significantly less in keratinocyte-fibroblast co culture compared to fibroblast monoculture [28]. The chitosan sponge was loaded with keratinocyte growth factor. The amount of growth factor loaded in the sample was 5ng/ [cm.sup.2]. The release of growth factor was monitored by HPLC, and the cumulative release profile is shown in figure 5. Keratinocyte cells were seeded onto the growth factor loaded sponges for the extent of 6 hours, 12 hours and 24 hours. Onto these cell loaded sponges, fibroblast cells were seeded for the extent of 12 hours. The co culture of keratinocyte and fibroblast on keratinocyte growth factor loaded chitosan sponge was optimized as shown in figure 6. Corresponding topography of collagen production is shown in figure 7. Cell loaded chitosan sponge with 12 hours of keratinocyte culture and further 12 hours of fibroblast culture seems to be suitable for optimal collagen production.

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The optimal cell loaded chitosan scaffold was applied on full thickness wound in rabbits. This was compared with chitosan sponge and a commercial wound dressing (Surgicel, Johnson & Johnson). Extend of healing was similar in case of cell loaded chitosan sponges, and other samples and re-epithelialisation was completed in 21 days. However, sham did not heal perfectly even at 21 days. Figure 8 and figure 9 reveal the healing of the wounds in 14 days and 21 days respectively. The quality of healing was better with cell seeded bioactive chitosan sponge. Collagen deposition was more on chitosan scaffold than on cell-loaded chitosan scaffolds. This may effectively limit the uncontrolled collagen production and scar formation with effective healing in the case of burn wounds. The specific histopathological studies are on going.

Various kinds of dressings are available for chronic wounds, including venous ulcers, diabetic ulcers and pressure sores, in wound-care management that helps to improve patient mobility, quality of life, and well-being. However, in most cases these dressings are used for prevention of infection or to make a moist environment helping in normal wound healing. Chronic wounds like ulcers and burn wounds disrupt normal process of healing and are often not sufficient in itself to effect repair. A burn injury results in either the loss or disruption of some or all of the functions of the normal skin. Impairment of blood flow in the zone of stasis can occur from shortly after the burn injury up to 48 hours post-burn [29]. If blood flow is compromised, this may lead to the eventual necrosis of cells in this zone.

Although bioactive wound dressing sponges based on chitosan containing sodium alginate and poly ethylene glycol is excellent in wound healing, with beneficial effect in the control of infection, incorporation of growth hormones helps in faster wound healing and will especially be useful in cases of burn wounds. Growth factors are endogenous substances that promote wound healing, including platelet-derived growth factor, epidermal growth factor, fibroblast growth factors, transforming growth factors, and insulin-like growth factors. In other words, it is proteins that communicate activities to cells. Fibroblasts, keratinocyte cells, epithelial cells, endothelial cells and growth factors all work together to achieve wound repair. Each of these factors is essential as cell migration, proliferation, and extra cellular matrix deposition are accomplished through the activity of these and other growth factors [30]. It has been established that the possible cause of wounds not healing in a timely manner (like chronic ulcers), is the deficiency in growth factors, or the growth factors cannot perform to their full potential or growth factors are entrapped in wound components like fibrin cuffs. Supplementing the keratinocyte growth factor and co culturing a combination of fibroblast and keratinocyte cells on chitosan scaffold may help in faster wound healing in cases of burn cases with no scar formation.

Conclusion

Tissue engineered scaffold for skin tissue engineering is an expanding area of research. With its unique combination of biological, physical, and chemical properties, chitosan is widely used in both the industrial and medical fields. These properties present a novel, versatile biopolymer that can be tailor-made to suit a particular application with the required modes of function. The development and commercial applications for chitosan have expanded in recent years. There is considerable potential for exploitation of chitosan in the field of biomedical, pharmaceutical, food and beverage, cosmetic, agriculture, industry, and water treatment. With a wide range of potential applications in medicine, pharmaceutics, and industry, there is still considerable scope for future research on chitosan towards skin tissue engineering. Wound healing is a complex process that can be compromised by a number of factors. Although, with proper care, some wounds fail to heal in an appropriate manner and may become chronic. From the individual studies reported in literature and the present study based on cell seeded chitosan sponge seems to be an excellent candidate material for the wound healing applications in chronic wounds. The chitosan based bioactive wound dressing sponges containing keratinocyte growth factor, which was seeded with keratinocyte and fibroblast cells in an optimized ratio exhibited significantly wound healing for full thickness wounds in rabbits. Controlled collagen production by culture of keratinocyte cells initially and then fibroblast cells, with optimized percentage and duration that helps in controlling the overgrowth of tissue and reduces scar production with faster wound healing. This is particularly useful for chronic ulcer and burn wounds where there is a total loss of tissue and cells.

Acknowledgement

We are grateful to the Director and the Head BMT Wing of SCTIMST for providing facilities for the completion of this work. This work was supported by the Department of Science & Technology, Govt. of India through the project 'Facility for nano/microparticle based biomaterials--advanced drug delivery systems' #8013, under the Drugs & Pharmaceuticals Research Programme.

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Willi Paul (1), Rekha M.R (1)., Mohanan P.V (2)., Chandra P. Sharma (1) *

(1) Division of Biosurface Technology, (2) Toxicology Division

Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology Poojappura, Thiruvananthapuram 695012, India

* Corresponding author: sharmacp@sctimst.ac.in
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