Hybrid scaffolding strategy for dermal tissue reconstruction: a bioactive glass/chitosan/silk fibroin composite

Abstract

Regeneration of deep burn wounds is a very complex process that strongly relies on the tissue response between the dermal substitute and the newly-formed dermis. In this research, we introduced a multifunctional integrated strategy for dermal tissue reconstruction. A porous BG/CHI/SF composite scaffold was prepared by freeze-drying a silk fibroin (SF) solution containing bioactive glass (BG) and chitosan (CHI). Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and thermogravimetry (TG) confirmed the chemical structure of the composite scaffold and its three-dimensional (3D) architecture. In vitro and in vivo investigations indicated that this composite scaffold presented excellent biocompatibility and tissue-repairing capability. Most importantly, a vascularisation evaluation demonstrated that the BG/CHI/SF composite scaffold possessed an outstanding ability to promote newly-formed and maturated blood vessels, which may accelerate the wound regeneration process. We therefore believe that this BG/CHI/SF multifunctional integrated scaffold has great potential in dermal tissue reconstruction.

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1. Introduction

Skin loss because of trauma, burns or chronic diseases is a very common problem in the clinic, and thus dermal tissue regeneration is one of the most common orthopaedic procedures. Millions of patients worldwide require skin repair because of burn injuries.^1,2 Various skin substitutes have been used to promote dermal tissue generation during surgical procedures.³ Of the traditional treatments, the split-thickness autograft remains the “gold standard” for implanting. Although autografts have good fusion rates and present no risk of rejection, they are limited by the availability of donor sites and timing.⁴ Hence, the design of an adequate bioscaffold to substitute tissue defects remains a significant challenge.

Within the context of rapid development of modern regenerative medicine, dermal tissue engineering has a promising future for dermal tissue regeneration. The development of dermal tissue engineering partly yet strongly depends upon the implanted scaffolds. A scaffold can be considered as a temporary platform that provides mechanical support for the regenerating region and provides sufficient space for cell proliferation.^5–7 Various bioscaffold materials are currently used for dermal tissue engineering, including protein fibres, chitosan, silk fibroin and hyaluronic acid, as well as their hybrid scaffolds.^8–11

Chitosan (CHI) is a positively-charged alkaline amylose with good biocompatibility and a fast in vivo degradation rate. Chitosan has also antibacterial activity. However, its poor cellular affinity makes it unsuitable as a scaffold for skin tissue engineering alone.¹² Silk fibroin (SF) from the Bombyx mori silkworm is a structural polymer possessing unique physical properties including good biocompatibility and water solubility. It has been demonstrated that SF has great potential for applications in clinical repair, tissue engineering and modified materials because of several outstanding characteristics such as having the same cellular affinity as collagen, good mechanical toughness and causing minimal inflammatory reaction. It degrades relatively slow although the degradation products may provide nutrient for restoring function of skin tissue. In the previously study, Luangbudnark et al. prepared chitosan/silk fibroin (CHI/SF) blend films and demonstrated cell proliferation on CHI/SF films. In their experiments, β-sheet conformation of silk fibroin was enhanced by the chitosan content, and flexibility, swelling index and enzyme degradation of the blend films were also increased. Furthermore, these results showed possibility of using the CHI/SF films as a supporting material for further study on skin tissue engineering.¹³

The lack of bioactive components, especially the vascularisation promotion factor, limits these scaffolds in terms of practical applications. Recently, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were introduced into dermal tissue engineering scaffolds because they could accelerate the formation of blood vessels.^2,14,15 However, the high costs, safety concerns and short half-lives of these growth factors restrict extensive applications. Faced with these issues, a bioactive glass (Bioglass 58S) was introduced to improve the vascularisation of the scaffolds in tissue regeneration;^16,17 it has been very successful in dermal tissue repairing.

We therefore hypothesised that hybrid scaffolds would be more beneficial for dermal tissue reconstruction in vivo. Herein, we used the BG/CHI/SF composite scaffold as an example to present the multifunctional integrated strategy for dermal tissue reconstruction. Our integrated system has three main components, each having quite specific roles: BG mainly serves as a bioactive component to promote vascularisation,¹⁸ CHI acts as an assembler to adsorb and enrich the growth factors during tissue reconstruction⁸ and SF acts as a template for tissue formation to provide a three-dimensional (3D) porous structure and mechanical support.¹⁹ The integrated components, providing a novel and multifunctional composite dermal tissue scaffold with improved repairing capability, illustrates the potential of this multifunctional integrated strategy for dermal tissue reconstruction.

2. Materials and methods

2.1 Preparation of the BG/CHI/SF composite scaffold

Micron-sized Bioglass 58S (BG) particles were composed of 58 wt% SiO₂, 33 wt% CaO and 9 wt% P₂O₅. Water-soluble chitosan (CHI, DAC ≥ 85%) was purchased from Jinan Haidebei Marine Bioengineering Co. Ltd. and was purified before use. Silk fibres were purchased from Zhejiang Second Silk Co. Ltd.

Preparation of the SF solution was previously described.²⁰ Raw silk fibres were firstly degummed five times with 0.05 wt% Na₂CO₃ solution at 100 °C for 1 h, washed with deionised water and oven-dried. The extracted silk fibroin was then dissolved in aqueous 9.3 M LiBr solution at about 60 °C for 5 h. A 3.5 wt% silk fibroin solution was obtained after dialysis for 3 days. The silk fibroin was isolated by filtration and stored at 4 °C until needed.

Bioactive glass powders (10 wt%) were mixed with silk fibroin and chitosan under vigorous stirring. The mixture solution was then poured into a 24-well plate and vacuum freeze-dried for 24 h at −80 °C. The percentage of the bioactive glass was selected according to the Chinese Pharmacopoeia (2010 edition). Scaffolds containing only one or two components were also fabricated for comparison. All of the SF-based scaffolds were immersed in aqueous methanol (90% v/v) for 30 min to induce a structural transition that generated the water-insoluble SF scaffolds.^21,22

2.2 Materials characterisation

The molecular structures of the as-prepared composite scaffolds were confirmed by Fourier transform infrared (FTIR) spectroscopy over the range of 4000–600 cm⁻¹ using a Nicolet Magna 750 spectrophotometer at 2 cm⁻¹ resolution in absorbance mode. The porous microstructures of the single-component and composite scaffolds were examined by a Zeiss scanning electron microscope (SEM) at an accelerating voltage of 3.0 kV. The porosity of the scaffolds was measured using pycnometer with ethanol according to a previously published method.²³ The thermal behaviour of all scaffolds was evaluated with a Netzsch STA 409 C/CD instrument from room temperature (ca. 25 °C) to 800 °C at a rate of 10 °C min⁻¹.

2.3 Swelling property

Water absorption property of the SF-based scaffolds was determined by immersion in PBS (pH 7.4) at 37 °C for 24 h. The dry weight (W_d) and wet weight (W_s, swollen weight) were measured. Then, the swelling index was calculated as shown:

2.4 In vitro cell viability assay

The proliferation of NIH 3T3 fibroblast cells was analysed to evaluate the cytotoxicity of the scaffolds using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Sigma-Aldrich). Generally, the cells were seeded on the composite scaffolds and incubated at 37 °C with 5% CO₂ for periods of up to 7 days. Culture medium was replenished every 2 days. After 1, 3, 5 and 7 days of cultivation, the cell-seed scaffolds were incubated in MTT solution for 4 h, and then replaced by DMSO. The optical density (OD) at 490 nm was measured using a microplate reader (Model 550, Bio-Rad Corp.). All data are presented as the mean ± SD of five tests.

2.5 In vivo dermis regeneration in a rat model

All procedures and handling of the animals were carried out in accordance with the guidelines of Jinan University and the US National Institutes of Health. Five groups with five Sprague-Dawley (SD) rats per group weighing ca. 180–200 g were anesthetised with pentobarbital sodium. For each rat, hairs of the back surface were totally depilated by Na₂S (8.0% w/v). Burn wounds (15 mm in diameter) were made on the back of a rat using a hot copper cylinder (90 °C) for 20 s. After 24 h, the necroses of the burned skin were removed to simulate the clinical treatment of a burn injury. Four full-thickness wounds (15 mm in diameter) were made on the upper back of each rat. Then, BG/CHI, BG/SF, CHI/SF and BG/CHI/SF composite scaffolds were implanted into the defect sites. All wounds were closed after the implantations. Each rat was caged individually after surgery and provided with sufficient water and food.

2.6 Histology

All scaffolds with surrounding tissue were excised at different time points and fixed in 4% formaldehyde/PBS solution. Then, the harvested samples were dehydrated with a gradient series of ethanol and embedded in paraffin. The embedded tissue blocks were then sectioned and stained with hematoxylin and eosin (H&E) and Masson trichrome, and studied by optical microscopy (BX41, Olympus).

2.7 Immunohistochemistry

Newly-formed and matured blood vessels were evaluated by immunohistochemical staining of CD31 and alpha smooth muscle actin (α-SMA), which are the critical factors related to the process of angiogenesis.

The detailed pre-treatment for immunohistochemical staining is described elsewhere.^4,24 Generally, paraffin-embedded tissue was washed three times in PBS for 5 min, and then blocked with 5% serum for about 30 min. It was then sectioned and incubated to rabbit anti-CD31 primary antibody (Abcam) and rabbit anti-α-SMA primary antibody (Abcam) at 4 °C overnight. After that, the slides were washed three times in PBS and exposed to goat-anti-rabbit secondary antibody (Dako) at 37 °C for 30 min, and then developed and counterstained with 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution and hematoxylin. The amounts of newly-formed and matured blood vessels were counted by CD31 and α-SMA positive staining per area, respectively.

2.8 Statistical analysis

Statistical analysis of all data in this paper was performed using SPSS for Windows software (ver 16.0; SPSS, Inc.). All experimental data are presented as means ± standard deviation (SD). Differences between groups were set as P < 0.05.

3. Results and discussion

3.1 Physical characterisation

FTIR spectroscopy was used to probe the structural conformation of scaffolds made with the different components (Fig. 1). Pure SF showed three characteristic absorption bands at 1641, 1515 and 1237 cm⁻¹ corresponding to the amide I (C=O stretching), amide II (N–H deformation) and amide III (O–C–N bending) features.^25,26 All of the samples with the SF component, i.e. SF/CHI, BG/SF and BG/CHI/SF, showed these three bands, which indicated that SF was successfully doped into the composite scaffolds. Similarly, absorption bands at 1640 (C=O stretching) and 1511 cm⁻¹ (N–H deformation) were also found for pure CHI,²⁷ although the absorptions were much less intense than for SF. The band at 1152 cm⁻¹ was attributed to C–O–C anti-symmetric stretching, and the absorption bands at 1070 and 1022 cm⁻¹ were associated with C–O stretching.²⁷ The SF/CHI, BG/CHI and BG/CHI/SF samples also displayed these bands. Pure BG absorption bands at 1044 and 791 cm⁻¹ were attributed to the Si–O (s) and Si–O (b) vibrations, respectively.²⁸

Fig. 1 Fourier transform infrared (FTIR) spectra of pure bioactive glass (BG), chitosan (CHI) and silk fibroin (SF) scaffolds, SF/CHI, BG/SF and BG/CHI two-component scaffolds and BG/CHI/SF three-component scaffold.

The surface morphology of each sample was investigated by SEM (Fig. 2). The BG particles were spherical with diameters of ca. 5–10 μm (Fig. 2a). The microstructures of CHI and SF were very similar to each other, presenting highly porous skeleton structures with diameters of ca. 100 μm for the CHI scaffold (Fig. 2b) and ca. 120 μm for the SF scaffold (Fig. 2c). When composited with each other, the SF/CHI sample also had a porous microstructure, but its porosity was denser than that of the CHI or SF scaffold (Fig. 2d). The micron-sized BG particles were homogeneously distributed throughout the CHI and SF scaffolds when BG was doped into the CHI and SF samples. Noticeably, the CHI structure collapsed when it was composited with BG (Fig. 2f), yet BG/SF presented a porous skeleton structure (Fig. 2e). The BG/CHI/SF three-component composite scaffold had a similar microstructure to BG/SF, exhibiting spherical BG particles that were uniformly distributed and integrated throughout the SF structure and fixed firmly to the walls or intersections of the scaffolds (Fig. 2g). As the addition of BG, the surfaces of BG/SF, BG/CHI and BG/CHI/SF became rough (partly exposed BG). According to previous reports, the surface roughness can affect cell adhesion and morphology. As the CS content decreased, the pore size increased. When SF content was high, the internal structure of the scaffold material had laminar pore walls. Furthermore, the high porosity (90%) and interconnectivity of the pores were well maintained and not affected by the BG. The 3D structure of the BG/CHI/SF composite scaffold is better suited to skin tissue regeneration applications.

Fig. 2 Scanning electron microscopy (SEM) images of (a) pure BG, (b) CHI and (c) SF scaffolds, (d) SF/CHI, (e) BG/SF and (f) BG/CHI two-component scaffolds and (g) BG/CHI/SF three-component scaffold.

The pore diameters and porosity of the CHI/SF samples with different composition ratios were also evaluated (Table 1). The CHI/SF (4 : 6) composite had the largest pore diameter of 120 ± 9.4 μm while that of CHI/SF (5 : 5) was only 74 ± 3.5 μm. The porosity increased with increasing SF content, probably because increasing SF content in the system lead to more interaction between fibroin molecules and subsequently larger volume shrinkage effect during the freeze drying process. And the 91.3 ± 5.4% porosity measured for CHI/SF (2 : 8) was the largest of all four samples. Scaffolds with porosities greater than 90% may be more beneficial to the dermal regeneration process.²⁹ The CHI/SF (2 : 8) composite had the best overall balance of pore diameter and porosity, and so was chosen for further evaluation.

Table 1 Pore diameters and porosities of chitosan (CHI)/silk fibroin (SF) scaffolds with different composition ratios

Sample	Pore diameter (μm) (mean ± SD)	Porosity (%) (mean ± SD)
CHI/SF (5 : 5)	74 ± 3.5	74.3 ± 4.7
CHI/SF (4 : 6)	120 ± 9.4	83.3 ± 5.6
CHI/SF (3 : 7)	100 ± 7.3	90.2 ± 6.2
CHI/SF (2 : 8)	112 ± 7.2	91.3 ± 5.4

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A 3D structure, with pore size and porosity characteristics, is one of the most important requirements for a skin tissue regeneration scaffold.^30,31 Pore sizes ranging from 20 to 125 μm support the regeneration of adult mammalian skin.³² Higher porosity can benefit cell attachment and proliferation and the flow transport of nutrients and metabolic waste.³³ The pore diameter and porosity data presented in Fig. 1 and Table 1 indicate that our experimental samples had appropriate pore sizes and high porosities, which may lead to good in vivo performance. The porosity increased with increasing SF content while decreasing CHI content. Increasing content of SF in the system would lead to the more interaction between fibroin molecules. Hydrophobic domain, which is large proportion in SF and bound to fibroin with short-chain amino acids (mainly Ala and Gly), would be packed and arranged more closely with the increasing SF.^13,34,35 Then the gap between pore boundaries would get narrow and the pore would get large. Even the small pore would get enough large to visible. More pore volume would be occupied by water. Thus after freeze drying, the porosity of the CHI/SF structure is increased when the silk fibroin proportion is increased.

The thermograms of the series of samples are presented in Fig. 3. Thermogravimetric analysis found that all of the scaffolds presented a similar trend of decreasing mass as the temperature increased from room temperature (ca. 30 °C) to 800 °C (Fig. 3a). Differential thermogravimetry (DTG) was used to evaluate the thermal behaviour in detail. As an example, Fig. 3b shows the loss in mass of CHI with increasing temperature. The first stage of mass loss that occurred from 30–135 °C is associated with water evaporation. Then, the mass loss rate gradually increased and reached a maximum at 305 °C. This prolonged loss of mass is ascribed to a complex thermal degradation process, such as the dehydration of the saccharide rings, and depolymerisation and decomposition of acetylated and deacetylated polymer units.²⁵

Fig. 3 Thermal behaviour of the different scaffolds. (a) Thermogravimetry (TG) curves of pure BG, CHI and SF scaffolds, SF/CHI, BG/SF and BG/CHI two-component scaffolds and BG/CHI/SF three-component scaffold. (b) Differential thermogravimetry (DTG) curve of the CHI scaffold.

Interestingly, there was more residue when BG was doped into a scaffold. This is because the SiO₂, CaO and P₂O₅ components of BG are difficult to degrade. For example, a residue of 56.1% was observed for the BG/CHI composite scaffold, while only 31.6% was left behind with pure CHI. The presence of residue after heating to 800 °C in the DTG instrument also indicated a successful preparation of composite scaffolds. This result is also in accordance with those reported by other groups.^36,37

The water absorption ability reflects capability of the scaffold in holding aqueous medium which is necessary for the cell growth. In addition, the fluid-absorbing capacity of the scaffold is an important criterion for maintaining a moist environment over the wound bed. The water absorption ability of SF and CHI/SF (2 : 8) scaffold was shown in Table 2. The swelling index of SF and CHI/SF (2 : 8) scaffold were 1.12 and 4.89, respectively. The water absorption ability of CHI/SF (2 : 8) scaffold is higher than SF scaffold. Due to SF is hydrophobic material, the water absorption ability of composite SF scaffolds were dependent on the hydrophilicity of the materials. CHI is a hydrophilic material because of its chemical and physical structure. The results confirmed that CHI improved the water absorption ability of composite SF scaffolds.

Table 2 The water absorption ability of SF and CHI/SF (2 : 8) scaffold

	Dry weight (g)	Wet weight (g)	Swelling index
SF scaffold	0.005	0.0097	1.12 ± 0.104
	0.0051	0.0109
	0.005	0.0115
CHI/SF (2 : 8) scaffold	0.0049	0.0263	4.89 ± 0.338
	0.0051	0.0295
	0.0042	0.0274

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3.2 Biocompatibility in vitro

The proliferation of NIH 3T3 model cells was used to evaluate the in vitro biocompatibility of the composite scaffolds. After cultivating with the BG/SF, BG/CHI, CHI/SF and BG/CHI/SF scaffolds for 1, 3, 5 and 7 days, the viability and proliferation of the NIH 3T3 cells were tested by an MTT assay (Fig. 4). All of the samples showed increasing MTT values with longer cultivation times. During the initial 3 days' incubation, the viability and proliferation of NIH 3T3 cells on the CHI/SF scaffold were always superior to those on the other three scaffolds (P < 0.05). This phenomenon may result from the better 3D connectivity of its architecture, which is indicated by the SEM image of Fig. 2d. However, the BG/CHI/SF three-component composite scaffold exhibited a significantly higher proliferation rate after 5 and 7 days of culture (P < 0.05). This long-term beneficial effect in cell proliferation suggests that the BG/CHI/SF composite scaffold would be more suitable for skin tissue regeneration.

Fig. 4 NIH 3T3 cell viability on different composites scaffolds as a function of culture time. Asterisk (*) denotes a statistically significant difference (P < 0.05, n = 5).

The biocompatibility of scaffolds, which comprises in vitro biocompatibility and in vivo biocompatibility, is one of the absolutely critical parameters for a skin tissue regeneration scaffold. Compared with the complex evaluation process of in vivo biocompatibility, that for in vitro biocompatibility is simpler yet effective.^38,39 In vitro biocompatibility of scaffolds is defined as the ability of these biomaterials to be used for a specific application without having toxic or injurious effects on biological function.^33,40 Fig. 4 shows that the composite scaffolds could significantly promote the migration of NIH 3T3 cells, suggesting that they could possibly be used for skin tissue regeneration.

3.3 In vivo study

(1) Macroscopic observations. To investigate their wound healing responses, composite scaffolds were implanted into rats as dermal substitutes. First, the hairs of the back surface of each anaesthetic rat were totally depilated by Na₂S (8.0% w/v) (Fig. 5a). Then, a hot copper cylinder (90 °C) of 15 mm diameter was pressed onto the back of the rat for 20 s to artificially create four burn wounds (Fig. 5b). After 24 h, the necroses of the burned skin were removed to simulate the clinical treatment of a burn injury, and four full-thickness wounds (15 mm in diameter) were made (Fig. 5c). Finally, corresponding counterparts of BG/CHI, BG/SF, CHI/SF or BG/CHI/SF composite scaffolds were implanted into the defect sites (Fig. 5d).

Fig. 5 Surgical procedure protocol: (a) depilate the hairs of the rat back surface, (b) create burn wounds, (c) debride the wounds and (d) implant the composite scaffold in the defect sites.

Fig. 6 shows photographs of the four composite scaffolds implanted as dermal substitutes after 3, 7, 14, 21 and 28 days. The wound areas were all significantly reduced over 28 days, suggesting that all of the full-thickness wounds were well-repaired. At day 3, white scaffolds were observed on the wounds in all cases. Meanwhile, some infection or necrosis between the implanted scaffolds and the surrounding tissue were detected for the BG/SF and BG/CHI scaffolds, indicating that infection clearly occurred, whereas the wounds repaired with the CHI/SF and BG/CHI/SF scaffolds were clean without any empyema. From day 7 to day 14, the BG/CHI/SF scaffold presented a significantly greater reduction in wound area compared with the other three scaffolds. In contrast, wound healing was low and the pus was still visible for the BG/SF and BG/CHI cases. At day 21, the wounds were almost completely healed and nearly sealed for the BG/CHI/SF case, while with other three scaffolds, there were still small wounds at the defect sites and the wounds gradually disappeared over 28 days. These results indicated that the wound healing process was accelerated by the implanted BG/CHI/SF scaffold compared with the other three scaffolds.

Fig. 6 Direct observation of burn wounds implanted with SF/CHI, BG/SF, BG/CHI and BG/CHI/SF composite scaffolds for 3, 7, 14, 21 and 28 days.

Wound healing is a complex process that may involve network interactions of many kinds of cells, cytokines and ECMs.^41,42 During the healing process, an implanted scaffold plays a crucial role in treating full-thickness skin defects.⁵ We conclude from our results that the porous structure of the BG/CHI/SF scaffold can provide a favourable moist microenvironment for cell proliferation. Simultaneously, the presence of each component of the BG/CHI/SF composite scaffold could promote the antibacterial property and skin tissue regeneration, which may explain the better performance of the BG/CHI/SF scaffold in wound healing (Fig. 6).

(2) Histology evaluation. Regenerated tissues were induced by the various composite scaffolds. Fig. 7 presents the H&E stained images of repaired dorsal skin lesions. Generally, newly-formed tissue was found surrounding the composite scaffolds at different implantation intervals for all of the scaffolds. At day 3, the implanted composite scaffolds could still be clearly seen, while neither oedema nor severe inflammation was exhibited for the BG/CHI/SF scaffold. However, the burn wound treated with the other three scaffolds presented severe inflammatory tissue response. At day 7, the BG/CHI and BG/SF cases still displayed some inflammation and oedema, while wounds treated with CHI/SF and BG/CHI/SF showed much less occurrence of infiltrated inflammatory cells in granulation tissues. The burn wounds in all cases gradually formed regenerated tissues from day 14 to day 21, but especially with the BG/CHI/SF scaffold. With the BG/CHI/SF scaffold after 14 days of recovery, blood vessels were also observed in the interior regions, accompanied by moderate infiltration of fibroblasts. These results suggested that the degradation of the BG/CHI/SF scaffold could significantly induce endothelial cell formation. The wound showed almost complete vascularisation with the BG/CHI/SF scaffold after 21 days. Moreover, many fibroblasts were observed in the entire scaffold, and extracellular matrix was found in the pores of the marginal parts of the scaffold; they were almost entirely organised and replaced by newly-formed connective tissue at day 28. These results indicated that the BG/CHI/SF scaffold was more beneficial for wound healing at all time points.

Fig. 7 Hematoxylin and eosin (H&E) staining of sections of burn wounds treated with SF/CHI, BG/SF, BG/CHI and BG/CHI/SF composite scaffolds for 3, 7, 14, 21 and 28 days.

To further evaluate the wound healing responses, we used Masson's trichrome stain to demonstrate the deposition of collagen in regenerated tissue for the different composite scaffolds at various time points (Fig. 8). At day 3, a little deposition of collagen was observed with the BG/CHI/SF scaffold, while much less collagen fibre was discovered and accompanied by more neotissue formation in the other three cases. At day 14, more collagen fibres were deposited in the wound area in all cases, especially with the CHI/SF and BG/CHI/SF composite scaffolds. Uniform distribution of collagen fibres was found with the BG/CHI/SF case. After 4 weeks, all groups exhibited significant collagen deposition in regenerated tissue. Furthermore, the alignment of collagen fibres was regular and uniform in the BG/CHI/SF case, which resembles the morphology of normal dermal tissue. These results demonstrated that the three-composite scaffold had more potential to promote wound healing.

Fig. 8 Masson's trichrome staining of sections of burn wounds treated with SF/CHI, BG/SF, BG/CHI and BG/CHI/SF composite scaffolds for 3, 7, 14, 21 and 28 days.

(3) Vascularisation. It is widely accepted that adequate vascularisation is a prerequisite for formation of regenerated tissue.^43,44 We therefore used the expression of CD31 to evaluate the effect on neovessel formation. CD31 is a highly specific marker for vascular and endothelial cells, and would express much less for normal tissue than newly-formed tissue.^4,24 Fig. 9 shows that CD31 expression was induced in the initial repair process (day 3) for all groups, which suggested that these composite scaffolds could significantly promote the angiogenic process at the wound site. For the BG/CHI/SF case, more neovessels were formed in the granulation tissue, which indicated good expression of CD31 in this group. At longer time points, the amount of new blood vessels increased in all groups, especially in the BG/CHI/SF group. Additionally, large luminal vessels were formed in the BG/CHI/SF case, but were scarcely found in the other three groups.

Fig. 9 CD31 immunohistochemical staining of sections of burn wounds treated with SF/CHI, BG/SF, BG/CHI and BG/CHI/SF composite scaffolds for 3, 7, 14, 21 and 28 days.

To quantitatively determine the amount of newly-formed blood vessels, we statistically analysed CD31 expression for the various implantation periods (Fig. 10). The amount of vessels in the BG/CHI/SF group was larger than that in the other three groups for all time points (P < 0.05). The amount of new blood vessels in the granulation tissue was detected in the first 3 days after implantation. The amount continuously increased from day 7 over the next 3 weeks, with the peak observed at 21 days after scaffold transplantation. At day 28, the amount showed an obvious reduction in all groups. This behaviour can be explained as follows: newly-formed blood vessels would aid the transportation of nutrients and oxygen in the initial part of the regeneration segment, and then the amount of vessels would reduce upon the formation of normal skin tissue in the remodelling process.^8,45

Fig. 10 Number of newly-formed blood vessels in areas treated with SF/CHI, BG/SF, BG/CHI and BG/CHI/SF composite scaffolds for 3, 7, 14, 21 and 28 days. Asterisk (*) denotes a statistically significant difference (P < 0.05, n = 5).

Next, we used α-SMA staining to investigate the maturated blood vessels because it is a specific marker for myofibroblast.^4,24 Fig. 11 shows that at day 3, the maturated blood vessels were found in all scaffolds, and especially in the BG/CHI/SF one. The amount of maturated blood vessels always increased with longer times. The maturated blood vessels area for the various implantation periods were statistically analysed (Fig. 12); the area increased over the first 21 days, then decreased. The amount of maturated blood vessels in the BG/CHI/SF group was significantly larger than for other three groups at all time points (P < 0.05).

Fig. 11 α-SMA immunohistochemical staining of sections of burn wounds treated with SF/CHI, BG/SF, BG/CHI and BG/CHI/SF composite scaffolds for 3, 7, 14, 21 and 28 days.

Fig. 12 Number of mature blood vessels in areas treated with SF/CHI, BG/SF, BG/CHI and BG/CHI/SF composite scaffolds for 3, 7, 14, 21 and 28 days. Asterisk (*) denotes a statistically significant difference (P < 0.05, n = 5).

As noted above, CD31 can be used to detect newly-formed blood vessels because it is a transmembrane protein expressed in the initial regeneration process, while α-SMA can be used to indicate the smooth muscle cells, which surround endothelial cells to form mature blood vessels. The results presented in Fig. 9–12 confirmed that the BG/CHI/SF composite scaffold significantly promoted newly-formed and mature blood vessels, revealing its great potential for dermal regeneration.

4. Conclusion

In this research, a multifunctional integrated BG/CHI/SF composite scaffold was successfully prepared and evaluated for dermal tissue reconstruction. Compared with the BG/CHI, BG/SF and CHI/SF scaffolds, the three-component composite scaffold had a better porous 3D structure and biocompatibility in vitro. The BG/CHI/SF composite scaffold also displayed excellent dermis regeneration and perfect promotion in vascularisation. We believe that the BG/CHI/SF multifunctional integrated scaffold, with its excellent capacity for burn regeneration, could lead to the development of novel multifunctional biomaterials for dermal tissue reconstruction and many other biomedical applications.

Acknowledgements

This study was supported financially by National Basic Research Program of China (2012CB619100), the 111 Project (B13039), Key grant of Chinese Ministry of Education (313022), Program for Changjiang Scholars and Innovative Research Team in University (IRT 0919).

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