www.ietdl.org Published in IET Nanobiotechnology Received on 24th December 2012 Revised on 19th June 2013 Accepted on 2nd July 2013 doi: 10.1049/iet-nbt.2012.0050

ISSN 1751-8741

Tissue engineered poly(caprolactone)-chitosan-poly (vinyl alcohol) nanofibrous scaffolds for burn and cutting wound healing Adeleh Gholipour-Kanani1, S. Hajir Bahrami1, Mohammad Taghi Joghataie2, Ali Samadikuchaksaraei2,3, Hossein Ahmadi-Taftie4, Shahram Rabbani4, Alireza Kororian2, Elham Erfani2 1

Textile Engineering Department, Amirkabir University of Technology, No. 529, 424 Hafez Avenue, Tehran, Iran Cellular and Molecular Research Center, Iran University of Medical Science, Tehran, Iran 3 Faculty of Allied Medicine, Department of Medical Biotechnology, University of Medical Sciences, Tehran, Iran 4 Tehran Heart Center, Tehran University of Medical Science, Tehran, Iran E-mail: [email protected] 2

Abstract: Natural-synthetic blend nanofibres have recently attracted more interest because of the ability of achieving desirable properties. Poly(ε-caprolactone) (PCL)-chitosan (Cs)-poly(vinyl alcohol) (PVA) blend nanofibrous scaffolds were electrospun in 2:1:1.33 mass ratio of PCL:Cs:PVA. The presence of PCL in the blend leads to improvement in web hydrophobicity and helped the web to retain its integrity in aqueous media. The scaffolds were used in two forms of acellular and with mesenchymal stem cells. They were applied on burn (n = 12) and excisional cutting (n = 12) wounds on dorsum skin of rats. Macroscopic investigations were carried out to measure the wounds areas. It was found that the area of wounds that were treated with cell-seeded nanofibrous scaffolds were smaller compared to other samples. Pathological results showed much better healing performance for cell-seeded scaffolds followed by acellular scaffolds compared with control samples. All these results indicate that PCL:Cs:PVA nanofibrous web would be a proper material for burn and cutting wound healing.

1

Introduction

Electrospun nanofibrous scaffolds offer numerous advantages in comparison with other tissue engineered scaffolds because of their innate properties, that is high surface area and micro-porous structure which mimic the nanofibrils structure of native extracellular matrix (ECM) [1]. These properties afford quickly start signalling pathway and attract fibroblasts to the derma layer, which can excrete important ECM components, such as collagen and several cytokines (e.g. growth factors and angiogenic factors) to repair the damaged tissue. The electrospun membrane is also important for cell attachment and proliferation in wound healing [2]. Apart from nanofibrous structure, polymer type is also very important. Natural polymers such as chitosan, chitin, collagen and so on are used in biomedical applications because of their high biological properties, that is, biocompatibility, cell adhering, antimicrobial ability and so on. Although their low electrospinability and low mechanical properties restrict their applications in some cases, but these polymers are used with other polymers which impart better mechanical properties [3–5]. Since high hydrophilicity of natural polymers, produced scaffolds have not shown enough physical properties in aqueous biological environments. So they need to be modified or blend with

other synthetic polymers. On the other hand, most of the synthetic biopolymers have low biological properties in comparison with natural polymers, but they often have high mechanical properties with high electrospinability. In recent years, polyblend nanofibres have been the area of interest in biomedical applications. The electrospun nanofibres fabricated from blending synthetic and natural polymers have demonstrated desired and unique combinations of biochemical, structural and mechanical properties that cannot be achieved by any single polymer. Polyblend nanofibres represent an emerging class of biomimetic nanostructures which has found many applications in a wide range of tissue engineering, wound dressings and drug delivery systems [6–8]. This provides nanofibrous scaffolds with proper mechanical properties whereas maintaining a very low density. These fibrous mats are commonly made with pore and fibre size distributions that mimic the structure of the ECM of the body, the fibrous protein and glycosaminoglycan (GAG) network that surrounds and supports cellular activity. Natural–synthetic polymer blend nanofibres have attracted more interest than natural–natural and synthetic–synthetic polyblend nanofibres. The primary reason is the flexibility in nanofibres engineering to achieve both mechanical strength of a synthetic component and biological

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www.ietdl.org functionality of a natural polymer. On the other hand, in many cases synthetic polymers can facilitate the electrospinning of natural polymers. For instance, casein, silk fibroin and chitosan cannot readily be electrospun, because of their strong intermolecular forces or three-dimensional structures of the native polymers [9]. One of the considerable natural–synthetic nanofibrous web is chitosan-poly(caprolactone) blend nanofibrous scaffold. Poly(ε-caprolactone) (PCL) is a synthetic semi-crystalline biocompatible poly(α-hydroxyl ester). Its hydrophobicity leads to low cell loading and reduces cell adhesion, proliferation and differentiation compared with natural polymers [10, 11]. To enhance these biological properties, PCL has been blended with other hydrophilic polymer(s) to improve the hydrophilicity [12]. Although the hydrophilic natural polymers such as chitosan have great biological properties (biocompatibility, biodegradability, non-toxicity and antimicrobial ability); the resulted scaffold from these polymers alone cannot provide sufficient mechanical property. Therefore apart from structural and chemical functionality, biochemical and mechanical functionality is also critical in tissue-engineered structures. The scaffold must necessarily possess adequate mechanical properties depending on the tissue [1]. In this regard, Shalumon et al. [13, 14] fabricated a fibrous scaffold comprising of chitosan (Cs) (0.5–2%) and PCL (6%) using electrospinning from a novel solvent mixture consisting of formic acid and acetone. In another work, they examined the cyto-compatibility of the CS/PCL scaffold using human osteoscarcoma cells (MG63) and found to be non-toxic. The results showed that CS/PCL scaffold supported the attachment and proliferation of various cell lines such as mouse embryo fibroblasts (NIH3T3), murine aneuploid fibro sarcoma (L929) and MG63 cells. The authors concluded that the results indicate that CS/PCL nanofibrous scaffold would be an excellent system for bone and skin tissue engineering. Wound healing requires a coordinated interplay among cells, growth factors and ECM proteins. In this regards, cell-seeded scaffolds have recently attracted more interest in biomedical field, because of their higher performance in tissue regeneration. The scaffolds provide the ECM structure and the cells could have important role in wound healing. Central to this process is the endogenous mesenchymal stem cell (MSC), which coordinates the repair response by recruiting other host cells and secreting growth factors and matrix proteins [15]. The Wharton’s jelly of the umbilical cord contains smucoid connective tissue and fibroblast-like cells. Thus, human umbilical cord mesenchymal cells can be expanded in culture and induced to form several different types of cells. In addition to multilineage differentiation capacity, MSCs regulate immune response and inflammation and possess powerful tissue protective and reparative mechanisms, making these cells attractive for treatment of different diseases [15, 16]. In this study, PCL:Cs:PVA blend nanofibrous webs were electrospun from PCL:Cs:PVA solution in 2:1:1.33 mass ratio. The solution preparation and electrospinning conditions are reported elsewhere [17]. PCL:Cs:PVA nanofibrous webs were taken and used as wound dressing in two forms of acellular scaffolds and stem cell-seeded scaffolds. MSC were seeded on half of scaffolds. In vivo studies were carried out on the dorsal skin of rats and two different round cutting wounds and burn wounds were created on them. Round cutting wounds were created with scalpel. Dorsum skin of rats were burnt using a hot brass

cylinder in 1 cm diameter. The resulted scaffolds were applied on the round cutting and burn wounds for in vivo studies. Macroscopic (measurement of the wound’s areas) and microscopic (histological assay) studies were performed to investigate the healing process.

2

Materials and methods

Poly(caprolactone) (Mw 80 kDa) was purchased from Sigma-Aldrich. Chitosan was obtained from Chitotech Co., PVA, acetic acid and other chemical materials were purchased from Merck Company. PCL:Cs:PVA blend nanofibrous webs were prepared by electrospinning technique and the results were reported elsewhere [17]. Briefly, 5% chitosan solution was prepared in 80% acetic acid as solvent. PVA was dissolved in distilled water to obtain 10% concentration. Ninety percent acetic acid was used as solvent to prepare 10% PCL solution. The different solutions were blended slowly in 2:1:1.33 PCL:Cs:PVA mass ratio, under stirrer at 40°C temperature conditions. The blend solution was electrospun with 15 kV applied voltage, 15 cm nozzle to collector distance, and 1 ml/h extrusion rate. Morphological investigations were carried out using SEM micrographs taken by scanning electron microscopy (SEM, XL30-SFEG, FEI Philips).

3 3.1

Experimental Cell-seeded scaffolds

Half of the scaffolds were selected for applying mesenchyme stem cells. Institutional review board approval was obtained for all procedures. With the consent of the parents, fresh human umbilical cords were obtained after birth and stored in Hanks’ balanced salt solution for 1–24 h before tissue processing to obtain mesenchymal cells. After removal of blood vessels, the mesenchymal tissue was scraped off from the Wharton’s jelly with a scalpel and centrifuged at 250 g for 5 min at room temperature and the pellet was washed with serum-free Dulbecco’s modified Eagle’s medium (DMEM). Next, the cells were centrifuged at 250 g for 5 min at room temperature and then treated with collagenase (2 mg/ml) for 16 h at 37°C, washed and treated with 2.5% trypsin for 30 min at 37°C with agitation. Finally, the cells were washed and cultured in DMEM supplemented with 10% fetal bovine serum and glucose (4.5 g/l) in 5% CO2 in a 37°C incubator [16]. MSC of 4 × 104 were seeded on Cs: PVA nanofibrous scaffolds and maintained at 37°C in a humidified CO2 incubator for 3 days before applying on the wounds. 3.2

In vivo study

For in vivo study, all experiments were approved by the ethical committee of Tehran University Heart Center. All animals received human care in accordance with the ‘Guide for the care and use of laboratory animals’ published by the US National Institute of Health (publication no. 85–23 revised 1996). Twenty four adult male Sprague Dawley rats weighing 250 ± 50 g were used for this study. All animals were housed separately in plastic cages with free access to water. They were kept in animal house for adaptation one week prior to surgery. The temperature of the animal house was 22°C ± 2°C and the humidity was 50–60%. Day/night cycle was 12/12 [18]. All animals were divided into two groups: group A with full thickness

124 IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 123–131 This is an open access article published by the IET under the Creative Commons doi: 10.1049/iet-nbt.2012.0050 Attribution License (http://creativecommons.org/licenses/by/3.0/)

www.ietdl.org excisional round cutting wounds [19–21] and group B with full thickness round burn wound [22–26]. Both groups were divided into two sub-groups. Acellular scaffolds and cell-seeded scaffolds were applied on the test wounds in group 1 (n = 6) and group 2 (n = 6), respectively. Three time points were selected to sacrifice the animals, that is, on days 5, 10 and 15. In group A, for each animal two round cutting wounds were created (one as control and the other as test), in group B, for each animal, four burn wounds were created (two as control and two as test ones). The animals were anesthetised with single intra-muscular injections of 50 mg/kg ketamine + 5 mg/kg xylazine. The dorsum [27–29] of the animals were shaved, sterilised and then draped. All procedures were done on a heating pad for a better recovery of animals. Codes given to the groups are summarised as follows in Table 1. 3.3

Surgery

3.3.1 Excisional cutting injury: Briefly, rats were anesthetised (n = 12) and the shaved area prepared with 10% antiseptic povidone–iodine solution (Kim-Pa, Poviiodeks, 10% povidone–iodine). Circular wounds of 1 cm in diameter were removed from the skin on the dorsolateral flank through the panniculus carnosus layer. Two wounds created on each rat, one of them (left) as test and other (right) as control. PCL:Cs:PVA of 15 × 15 mm2 blend nanofibrous scaffolds were applied on test wounds in two forms. For group A1, acellular nanofibrous PCL:Cs: PVA scaffolds were applied on test wound in each rat. For group A2, stem cell-seeded scaffolds were placed on test wound in each rat. For each time point two rats were operated. All wounds were left un-sutured and then dressed with sterile gauze. 3.3.2 Thermal injury: Twelve rats were anesthetised and pre-operation treatments were performed. Animals were subjected to full-thickness second to third-degree skin burns

Table 1 Divided groups in different types of wounds Wound type

Group code

Acellular scaffolds (group 1)

Cell-seeded scaffolds (group 2)

cutting wounds burn wounds

A

A1 (n = 6)

A2 (n = 6)

B

B1 (n = 6)

B2 (n = 6)

with 1 cm surface area diameter by brass probes. The brass probe was set on a soldering iron to reach 150°C. The probe was then placed on the back of the rats for 60 s without applying pressure. All wounds were debrided with pistol. Because of the extra heat of the device, the real diameter of the wounds was 1.1 cm. For both group B1 and B2, four wounds were made on each rat, equidistance from the midline. Up-left and down-right wounds were selected for tests and up-right and down-left wounds were selected for controls. All wounds were left un-sutured. For group B1, acellular nanofibrous PCL:Cs:PVA scaffolds (15 × 15 mm2) were applied on two test wounds in each rat. For group B2, stem cell-seeded scaffolds were placed on two test wounds in each rat. For each time point two rats were operated. After that, all wounds were dressed with sterile gauze to avoid rats accessing to the wounds. Animals were then recovered and kept in their cages. At each time point, four animals were sacrificed by intra-peritoneal injection of 100 mg/kg sodium thiopental. Five, ten and 15 days post operations were selected as time points. Full thickness of the skin was excised and fixed in 10% formaldehyde for pathologic evaluation. All specimens were stained with hematoxylin and eosin for epithelialisation, collagen regeneration and pattern, skin appendage and total wound healing factor studies. In this study, there was no mortality because of infection and other complications.

4 4.1

Results Morphological study

The SEM micrograph of resulted nanofibrous webs is shown in Fig. 1a [17]. The presence of PCL in the blend leads to reduction of hydrophilicity of the blend and preserves webs integrity in aqueous media. From the results obtained in our previous work [17], by adding PVA to PCL:Cs in 2:1:1.33 (PCL:Cs:PVA) blend ratio (Fig. 1a), uniform nanofibrous web with narrower distribution of fibre diameter can be obtained. Nanofibrous web with bead-less morphology is electrospun from PCL:Cs:PVA (2:1:1.33) blend solution. Figs. 1b and c show the morphology of stem cells on the resulted scaffolds in SEM micrographs and under optical microscopy, respectively. Cells were attached and spread well on nanofibrous scaffold (Fig. 1b). In Fig. 1c, cells good growth shows the viability and cell-compatibility on the PCL:Cs:PVA nanofibrous scaffold. This effect could be

Fig. 1 Morphological study a SEM micrographs of PCL:Cs:PVA (2:1:1.33) nanofibres (10 000 × ); b SEM micrographs of cell morphology on the scaffold (1000 × ); c Stem cells under optical microscopy (100 × ) IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 123–131 125 doi: 10.1049/iet-nbt.2012.0050 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/)

www.ietdl.org because of high biological properties of chitosan in blend. However, higher physical properties of blend webs are because of the presence of PCL which result in better cell attachment.

4.2

In vivo macroscopic results

Wounds areas were measured in all groups (A1, A2, B1 and B2) for primary investigations. In this regard, wound area was considered as a criterion for investigating wound healing visually. At the same time points, smaller wound area shows a better wound healing. The areas of wounds in each group were measured using Image J software and the wounds digital images. The reported area in each time point is the average of four measurements. The results of acellular scaffolds (A1) and cell-seeded scaffolds (A2) which treated cutting-wounds are shown in Table 2. The results show that wounds areas in test specimens are smaller than the control wounds after the same time points. On the other hand, cell-seeded scaffold treated wounds show smaller wound areas compared with acellular scaffold treated and control wounds. From Table 2, cutting wounds that were treated with cell-seeded PCL:Cs:PVA nanofibrous scaffolds were healed completely on day 15 and no scar was found on the skin of the rats. Fig. 2 shows wound healing processes by applying acellular scaffolds for 5, 10 and 15 days post operating. As it was found, the wounds that were treated with acellular PCL:Cs:PVA nanofibrous webs are smaller in area, compared with the control wounds. Table 3 also shows the measured wounds areas for burn wounds that were treated with acellular scaffolds (B1) and cell-seeded scaffolds (B2). In addition Table 3 shows the average area of burn wounds that were treated with Table 2 Round cutting wounds measured areas in different time point Sample

control acellular scaffolds (A1) cell-seeded scaffolds (A2)

Primary area of wound, mm2

Wound area by 5-day, mm2

Wound area by 10-day, mm2

Wound area by 15-day, mm2

78.5 ± 0.01 78.5 ± 0.01

68.2 ± 0.22 50.4 ± 0.17

52.18 ± 0.4 29.5 ± 0.27

25.9 ± 0.35 14.1 ± 0.12

78.5 ± 0.01

44.0 ± 0.24

21.11 ± 0.23

0

cell-seeded PCL:Cs:PVA scaffold was about half the area of primary wound on day 15. Also, from Fig. 3c, the difference between the average area of test and control is clear. Scaffold-treated specimens show very small wounds; however, they show good healing at the surface. From Tables 2 and 3, and Figs. 2 and 3, the effects of acellular and cell-seeded scaffolds on healing of cutting wounds were faster than burn wounds in the same time. This fact could be because of collapsing blood vessels and damaging nerve tissues in the burn area reducing the signal pathways and speed of fibroblasts attraction to the derma layer till 10-day post operation. By day 15, the effects of acellular and cell-seeded scaffolds were more visible in burn wound healing. It seems that the burn wounds needed more time to heal, but the presence of PCL:Cs:PVA nanofibrous scaffolds accelerated the healing process compared with the control specimens. 4.3

In vivo microscopic results

The rats were sacrificed and the wounded skin tissue samples were collected for pathological examinations on 5, 10 and 15 days post surgery. Standard hematoxylin and eosin staining of wounds were applied on fixed removed wounds. The pathological images for cutting (A) and burn (B) wounds that were treated with both acellular and cell-seeded scaffolds are shown in Figs. 4 and 5, respectively. From bio-pathological studies (Figs. 4 and 5) and based on [29, 30], four criteria were considered to evaluate pathological scores of wound healing (Table 4). 4.3.1 Cutting wounds pathological data: As it is shown in histological images (Fig. 4), in day 5 only blood scabs, fibrins and a lot of inflammatory agents were found in control specimens. Fibrin layer is directly in contact with the wound area and causes slower wound healing process (Fig. 4a). There was no evidence of collagen regeneration and epithelialisation. Figs. 4b and c show that in test specimens, porous nanofibrous webs absorb fibrin layers on the wounds and improve the healing process. In day 5 post-surgeries, from Fig. 4b and c, wounds that were treated with acellular and cell-seeded PCL:Cs:PVA nanofibrous scaffolds showed better collagenous regeneration compared with controls. In acellular scaffold treated wounds epithelial layer had partially formed; however, the total wound healing factor was weak. Collagen with much fibrils and moderate epithelial layer was formed in cutting wounds that were treated with cell-seeded scaffolds and the total healing factor score was about 2.

Fig. 2 Cutting wounded rats treated with acellular PCL:Cs:PVA nanofibrous scaffolds in group A1: test (left wound) and control (right wound) in three time points: a Day 5 b Day 10 c Day 15 post surgery 126 IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 123–131 This is an open access article published by the IET under the Creative Commons doi: 10.1049/iet-nbt.2012.0050 Attribution License (http://creativecommons.org/licenses/by/3.0/)

www.ietdl.org Table 3 Burn wounds measured areas in different time point Sample

control acellular scaffolds (B1) cell-seeded scaffolds (B2)

Primary area of wound, mm2

Wound area by 5-day, mm2

Wound area by 10-day, mm2

Wound area by 15-day, mm2

95 ± 0.01 95 ± 0.01

94.1 ± 0.32 82.3 ± 0.24

86.12 ± 0.21 68.27 ± 0.21

60.5 ± 0.2 53.2 ± 0.35

95 ± 0.01

74.31 ± 0.04

64.21 ± 0.3

48.18 ± 0.12

By day 10 post operating, control wounds showed a moderate collagen generation, no skin appendage and a moderate epithelialisation (Fig. 4d). On the other hand, wound treated with acellular scaffold showed a complete epidermal layer, collagen was formed in bundle shape with an irregular pattern, skin appendages were not found and the healing score was developing to 2–3 (Fig. 4e). In this time point, there were some attractive points in healing process of wound treated with cell-seeded scaffold. Epidermis layer reached a complete thickness and collagen regeneration had complete bundle form with regular pattern in some points. Good granulation tissues with some skin appendages such as hair follicles on the edge of wounds were established (Fig. 4f ). These considerable improvements could be due to stem cells ability for collagen regeneration and their active chemical signalling to absorb fibroblast in dermis layer. Figs. 4h and i indicated that in day 15 post surgery, wounds that were treated with scaffolds (with cells or without cells) showed higher grade of healing compared with control specimens (Fig. 4g). Cutting wound treated with acellular scaffold showed good collagen regeneration with complete epidermal layer. Some skin appendages such as hair follicles spread in all areas under the wound which is also visible in Fig. 2c. In addition, tissue engineered scaffolds with stem cells result in better and faster healing compared with other samples. Cutting wounds that had been treated with cell-seeded scaffold healed completely and was explored as normal tissue (Fig. 4i). Epithelialisation, collagen regeneration and pattern, granulation tissue and skin appendages are four considered

criteria in histological studies were graded according to Table 4, and the resulted grades are presented in Table 5. 4.3.2 Burn wound pathological data: Comparing all specimens, control wounds displayed a greater degree of inflammation (heat, redness and swelling) whereas they reduced in wounds that were treated with acellular and cell-seeded PCL:Cs:PVA nanofibrous scaffolds. As it is shown in histological image, in day 5 for control specimens (Fig. 5a), there were a lot of infections on the wound surface and fibrin layer was directly in contact with the wound which caused the slower wound healing process. Control specimen showed no epidermal regeneration and no considerable collagen fibrils. So the wound healing process is very slow. At the same time point, however, wounds that were treated with acellular scaffolds and cell-seeded scaffolds showed better collagenous regeneration and epithelial layer had partially formed, but the total wound healing factor was weak (Figs. 5b and c). By day 10 post operating, Fig. 5e shows weak to moderate collagen regeneration, no skin appendages and partial epidermal regeneration on the edge of acellular scaffold treated wounds, whereas control wounds showed no obvious development on healing because of high amount of inflammatory cell infiltration (Fig. 5d ). Wounds that were treated with stem cell-seeded scaffolds showed higher range of healing process. Collagen fibrils were formed in bundle shape with irregular pattern, granulation tissue got thick and epidermis layer reached good thickness on the edge of wounds. Therefore the healing process has developed very well because of stem cells ability for collagen regeneration and their active chemical signalling to absorb fibroblast in dermis layer. By day 15 post surgery, control wound still showed no development in healing process. Moderate collagen regeneration in bundle shape with irregular pattern, complete epidermal on the edge of wound and no skin appendages were indicated in acellular scaffold treated wounds (Fig. 5h). In the same time point, wound treated with cell-seeded scaffold showed high amount of collagen bundle with irregular pattern and good epidermal layer on the edge of wounds (Fig. 5i). Generally, wounds that were treated with scaffolds (with cells or without cells) showed higher grade of healing compared with control samples.

Fig. 3 Burn wounded rats treated with acellular PCL:Cs:PVA nanofibrous scaffolds in group B1: test (up-left and down-right) and control (up-right and down-left) in three time points a Day 5 b Day 10 c Day 15 post surgery IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 123–131 127 doi: 10.1049/iet-nbt.2012.0050 This is an open access article published by the IET under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/)

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Fig. 4 Haematoxylin and eosin histology of round cutting wound tissue samples removed from (a, d, g) control wounds; (b, e, h) acellular scaffold treated wound and (c, f, i) cell-seeded scaffold treated wounds; in different time points a–c 5 days d–f 10 days g–i 15 days (10 × )

The considered criteria in burn wound healing were graded according to Table 4, and the resulted grades for each group are presented in Table 6.

5

Discussions

Cells, ECM and signals are three basic components which would be considered in tissue engineering. Scaffolds fabricated from suitable biopolymers and with proper structures often can play ECM role for tissue replacement or regeneration. In this study, PCL:Cs:PVA nanofibrous scaffolds were applied to cutting and burn wounds as tissue matrix. The main aim of this study was the addition of nanofibres special properties (such as 3-D and high porous structure) to high biological properties of chitosan and high physical–mechanical properties of PCL to fabricate a unique scaffold. The resulted electrospun nanofibres from blended synthetic and natural polymers have demonstrated desired and unique combinations of biochemical, structural and mechanical properties that cannot be achieved by any

single polymer. Addition of PCL to Cs:PVA reduced web hydrophilicity and maintained physical integrity of the resulted web when it was applied to the wounds. It was found from pathological results that the nanofibrous scaffolds absorbed the liquid (fibrin or blood) from the wounds because of the nanoporous structure and hydrophilic nature of the polymers. The scaffolds could also help oxygen ventilation for faster and better repairing. Owing to antimicrobial property of chitosan, the numbers of inflammatory cells in the wound were reduced on first day post operating. Pathological examination demonstrated advanced granulation tissue formation and collagen regeneration in wounds that had been treated with cell-seeded scaffolds. These results could be due to two different reasons. First, because of nanofibres innate properties that can offer quick signalling pathway and attract fibroblasts to the derma layer. Moreover, the second is the incorporation of cells in nanofibrous scaffolds that often provides the signals needed for tissue building. The involvement of MSCs in the wound-healing process is

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Fig. 5 Haematoxylin and eosin histology of burn wound tissue samples removed from (a, d, g) control wounds; (b, e, h) acellular scaffold treated wound and (c, f, i) cell-seeded scaffold treated wounds; in different time points a–c 5 days d–f 10 days g–i 15 days (10 × )

Table 4 Healing process criteria and their scores [28] Criteria

1

2

3

4

Score epithelialisation collagen regeneration and pattern granulation tissue (GT) and skin appendage (SA) wound healing

no or very little very little and thin fibril thin GT, no SA

little little and thicker fibril

weak with inflammation

moderate with inflammation

moderate GT , no SA

critical, in particular for difficult non-healing wounds resulting from burn, trauma, diabetes, vascular insufficiency and numerous other conditions. MSCs have a role in the inflammatory, proliferative and remodelling phases of wound healing, and their presence supports healthy physiologic functioning towards successful healing. As such, therapeutic

moderate moderate bundles irregular pattern moderate GT, with SA

complete complete bundles regular pattern complete GT, with SA

good

complete

application of MSCs has been shown to enhance and improve wound healing in clinical settings [15, 16]. From macroscopic results (Tables 2 and 3) and microscopic results (Figs. 4 and 5), the healing effects of acellular and cell-seeded scaffolds on cutting wounds were faster than burn wounds in the same time. This fact could be because

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www.ietdl.org Table 5 Scored criteria for each round cutting wound in different time point Sample

5-day

10-day

15-day

Criteria

control acellular web cell-seeded web control acellular web cell-seeded web control acellular web cell-seeded web

Epithelialisation

Collagen regeneration and pattern

Granulation tissue (GT), skin appendage (SA)

Wound healing

1 1 2

1 1–2 2

1 2 2

1 1–2 2

2 4 4

2 2 3

2 2 3

2 2–3 3

4 4 4

2 3 4

2 3 4

2–3 3–4 4

Table 6 Scored criteria for each burn wound in different time point Sample

5-day

10-day

15-day

Criteria

control acellular web cell-seeded web control acellular web cell-seeded web control acellular web cell-seeded web

Epithelialisation

Collagen regeneration and pattern

Granulation tissue (GT), skin appendage (SA)

Wound healing

1 1 1

1 1 1

1 1 1

1 1 1

1–2 2 2

1 2–3 3

1 1 1–2

1 1–2 2

2 2 2–3

1–2 3 3

1 2 2–3

1–2 2 2–3

of blood vessels collapsing and nerve tissues damaging in the burn area, which reduced the signal pathways and caused lower speed of fibroblasts attraction to the derma layer till 10-day post operation. By the day 15, the effects of acellular and cell-seeded scaffolds were more visible in burn wound healing. It seems the burn wounds need more time to heal, but the presence of PCL:Cs:PVA nanofibrous scaffolds accelerate the healing process. According to Tables 5 and 6, epithelialisation, granulation tissue thickness and collagen regeneration were growing up by day 15 for all groups. It was found that wound healing process in test specimens (wound treated with acellular and cell-seeded scaffolds) began earlier than the controls and these high levels were maintained in all time points both for cutting and burn wounds. From the above discussion, it is clear that the wound treated with PCL:Cs:PVA nanofibrous webs showed faster wound healing. Although, the healing performance of cell-seeded scaffolds showed better rate compared with acellular ones. During the inflammatory phase, MSCs coordinate the effects of inflammatory cells and support wound clearance from infection via direct secretion of antimicrobial factors and by stimulating phagocytosis by immune cells. The ability of MSCs to promote the transition from the inflammatory to the proliferative phase is particularly critical for treating chronic wounds where high levels of inflammation prevent healing. MSCs also contribute to the proliferative phase by expressing growth factors to promote

granulation and epithelialisation. Lastly, MSCs regulate remodelling of the healed wound by promoting organised ECM deposition [15]. Thus, multiple mechanisms are involved in MSC-mediated wound healing, including antiinflammatory and antimicrobial, immunomodulative and tissue reparative activities.

6

Conclusions

PCL:Cs:PVA nanofibrous scaffolds were electrospun successfully in 2:1:1.33 blend ratio, respectively. The presence of PCL in the blend reduces the hydrophilicity of the blend and preserves the webs integrity when it is applied to wound aqueous media. Tissue engineered scaffolds were established by seeding MSC on the scaffolds. The scaffolds were used in two forms of acellular and cell-seeded. They were applied on burn (n = 12) and excisional cutting (n = 12) wounds on the dorsum rats skins. Macroscopic investigations were carried out to measure the wounds areas. Wounds which were treated with cell-seeded scaffolds showed smaller scabs areas in comparison with ones treated with acellular scaffolds. However, all treated wounds (with cells or without cells) demonstrated better wound healing compared with control specimens visually. To study the effect of scaffolds on wound healing process, pathological results were conducted. PCL:Cs:PVA nanofibrous scaffolds showed positive effects on healing process of both cutting and burn wounds. The resulted

130 IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 123–131 This is an open access article published by the IET under the Creative Commons doi: 10.1049/iet-nbt.2012.0050 Attribution License (http://creativecommons.org/licenses/by/3.0/)

www.ietdl.org effect might be related to nanofibrous structure of the scaffolds that mimic the natural ECM, high biological properties of chitosan (anti-inflammatory, antioxidant and antibacterial effect) and high physical–mechanical properties of PCL that causes to maintain the integrity of webs next to blood and fibrin. Histo-chemical results showed much better healing performance for scaffolds stem cells followed by acellular scaffolds compared with control samples because of stem cells ability of collagen regeneration and providing the signals needed for tissue building. In conclusion, application of blend PCL:Cs:PVA nanofibrous scaffolds are effective in healing of cutting and burning related to skin wounds in the rat models.

7

Acknowledgments

This study was supported by the Iran National Elites Foundation. The authors wish to acknowledge the support extended by the Tehran Heart Center.

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References

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