Bionic, porous, functionalized hybrid scaffolds with vascular endothelial growth factor promote rapid wound healing in Wistar albino rats

Abstract

Bionic collagen-poly(dialdehyde) locust bean gum based hybrid scaffolds synergistically combined with vascular endothelial growth factor were prepared to regenerate tissue formation for wound healing applications. The dialdehyde functionalities introduced in the locust bean gum were responsible for the improved collagen stability, biostability and immobilization of vascular endothelial growth factor in the hybrid scaffolds. In vitro Swiss 3T6 mouse fibroblast cell culture studies reveal that the prepared hybrid scaffolds have enhanced cell viability and infiltration. An in vivo wound healing study demonstrates that the collagen-poly(dialdehyde) locust bean gum-vascular endothelial growth factor hybrid scaffolds boost the level of fibroblast and neovascular content as well as collagen deposition; complete epithelialization occurs within 16 ± 0.9 days. The results show that the vascular endothelial growth factor immobilized hybrid scaffold induces chemotactic effects to promote rapid tissue regeneration and wound repair, thereby demonstrating its potential for burn wounds, chronic wounds and diabetic foot ulcer treatments.

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1. Introduction

Tissue bionics is a technique to design and engineer human tissues/organs by employing biomimetic compounds.¹ It performs a significant role in repairing and regenerating the defective tissues in tissue engineering and wound healing applications.¹ Wound healing is a basic response to tissue damage that involves a multifaceted regenerative process and remodelling by collagenous tissue formation.² Wounds associated with diabetics and burn injuries face challenges such as improper collagen deposition, undesirable wound contraction and non-functional neotissue formation.^3–5 This is primarily due to ineffective cellularization and vascularization development in the wound region.^5,6 The formation of blood vessels promotes tissue regeneration through the necessary transport of nutrients, oxygen, and immunocompetent cells and the clearance of metabolic wastes.^5,6 A deficiency of blood vessels impairs the delivery, leading to inadequate cell proliferation at the wound site.^5,6 Therefore, the preparation of growth factor incorporated tissue engineering constructs to stimulate cellularization and vascularization is a new approach to address this problem. Growth factors are large polypeptides that play crucial role in stimulating cellular growth, proliferation, differentiation, migration, adhesion and gene expression. Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor that stimulates vasculogenesis and angiogenesis.^7,8 It is produced by endothelial, keratinocyte, fibroblast smooth muscle, platelet, neutrophil and macrophage cells. It functions as an endothelial cell mitogen, chemotactic agent and inducer of vascular permeability.⁷ VEGF plays an important role in wound healing because it promotes early events in angiogenesis, particularly endothelial cell migration and proliferation.^7,9–11 Several studies have shown that the direct administration of VEGF improves re-epithelialization of diabetic wounds associated with enhanced vessel formation.^12,13 Direct administration of VEGF also restores the impaired angiogenesis in diabetic ischemic limbs.¹⁴ However, the half-life of VEGF is extremely short, which necessitates controlled release systems to stimulate biological activities in the wound healing environment.^10,11

Collagen (C) is a well-known structural protein abundantly found in the extracellular matrix and widely used in biomedical applications due to its biomimicking architecture with weak antigenicity and excellent biocompatibility.¹⁵ It also plays an important role in the induction of clotting, cell proliferation, migration and regeneration of new tissues in wound healing.^15,16 However, its instability requires crosslinking, which is typically achieved through the addition of synthetic polymers or crosslinking agents, resulting in incompatibility and toxicity.¹⁷ Biocompatible and crosslinkable biopolymer is an ideal choice for the stabilization of collagen and also for the immobilization of bioactive factors in tissue engineering constructs.¹⁸ Locust bean gum (LB) is a water-soluble galactomannan polysaccharide extracted from the seeds of Ceratonia siliqua. It has been widely used as a thickening and gelling agent in the food industry. Several studies have been carried out to modify the structure of polysaccharides and create crosslinkable or reactive functional groups.^18–20 Sodium periodate oxidation is a popular method to cleave the carbon–carbon bonds of adjacent diols in polysaccharides and form dialdehyde groups without significant side reactions. These dialdehyde groups in polysaccharides support the immobilization of drugs as well as the stabilization of biopolymers through covalent imine bond formation.^18–20 Herein, we have synthesized poly(dialdehyde) locust bean gum (PDALB) for stabilizing collagen and immobilizing VEGF and prepared hybrid scaffolds. We have also investigated the effectiveness of the prepared scaffolds for application on an open excision wound model in Wistar albino rats. The crosslinking efficiency, structural morphology, biostability, and drug release of the prepared hybrid scaffolds were characterized; in vitro cell culture analysis and in vivo open excision wound healing studies were also performed.

2. Materials and methods

2.1. Materials

Human recombinant vascular endothelial growth factor-A was procured from Prospec Bio, Isreal. The human VEGF enzyme-linked immunosorbent assay development kit was purchased from Peprotech Ltd. Locust bean gum from Ceratonia siliqua seeds, 2,4,6-trinitrobenzene sulfonic acid (TNBS, 5% w/v in H₂O), protease from Aspergillus saitoi, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Dulbecco's modified Eagle's medium (DMEM) and the supplementary antibiotics for tissue culture were purchased from Sigma-Aldrich, India. Fetal bovine serum (FBS) was procured from Gibco Life Technologies. Sodium periodate was purchased from M/s Sisco Research Lab, India. Ethylene glycol was purchased from Himedia, India. Swiss 3T6 mouse fibroblast cells were obtained from the National Centre for Cell Science (NCCS), Pune, India. Thiopentone sodium (Thiosol®) was procured from Neon Laboratories, Mumbai, India. All other reagents were of analytical grade.

2.2. Preparation of collagen solution

Raw cowhide trimmed wastes were collected from the pilot tannery at the Central Leather Research Institute (CLRI), Chennai. The collected hide trimmings were processed into hide powder according to our previously reported method.²¹ Briefly, the collected rawhide trimming pieces were treated as per conventional leather processing procedures, namely, soaking, liming, dehairing, reliming, fleshing and deliming.^22,23 The delimed hide trimming pieces were dehydrated using 35% and 70% acetone for 3 h followed by 100% methanol five times, each of 3 h duration. Finally, the dehydrated hide pieces were vacuum dried and made into a fine powder using a Willy mill of mesh size 2 mm. About 2 g of hide powder was weighed and blended with 100 mL of 0.5 N glacial acetic acid at 4 °C for 2 h. After complete solubilisation, the collagen solution was stored in an amber bottle under refrigerated conditions. The concentration of collagen in the hide powder was analyzed using a hydroxyproline assay²⁴ and was found to be 90% ± 2%.

2.3. Synthesis of poly(dialdehyde) locust bean gum

The synthesis of poly(dialdehyde) locust bean gum was carried out by oxidizing the OH groups to CHO groups in the monosaccharide units of LB using a sodium periodate based oxidation method according to our previous protocol.¹⁸ The schematic of PDALB synthesis is shown in Fig. 1a. Briefly, 1 g of LB was dissolved in 200 mL of distilled water in a dark bottle and stirred for 30 min to obtain a homogenous solution. Subsequently, 1.5 g sodium periodate dissolved in 25 mL distilled water was added and maintained with stirring at 35 °C for 24 h. Then, the reaction was quenched by the addition of 1 mL of ethylene glycol and stirred for 1 h at 35 °C. The reaction solution was centrifuged at 10 000 rpm for 10 min and the supernatant solution was mixed with acetone in a 1 : 4 ratio to precipitate the PDALB biopolymer. The collected PDALB biopolymer was dissolved in distilled water and re-precipitated by the addition of acetone. This process was repeated three times. Finally, the PDALB biopolymer was freeze dried and then powdered. The yield of synthesized PDALB biopolymer was found to be 60%.

Fig. 1 Schematic showing (a) the modification of LB to PDALB by the periodate oxidation reaction and (b) the covalent immobilization of VEGF in the 100/100 wt% C/PDALB hybrid scaffold.

2.4. Degree of oxidation

The degree of LB oxidation reaction was determined based on the potassium iodine-soluble starch indicator method.²⁵ Briefly, the indicator solution was freshly prepared by mixing 10 mL of 20% (w/v) potassium iodine and 10 mL of 1% (w/v) soluble starch using phosphate buffer solution (pH 7). Before adding the quencher to the oxidation reaction, 1 mL samples were drawn at different time intervals and diluted to 250 mL with distilled water. Then, 3 mL of diluted sample was mixed with 1.5 mL indicator solution in a clean test tube and the total volume was made up to 5 mL with distilled water. The blank solution was prepared using 3.5 mL of distilled water and 1.5 mL of indicator solution. The absorbance of the triiodide–starch complex was measured using an UV-visible spectrophotometer (UV-1800, Shimadzu) at 486 nm. The degree of oxidation was calculated using the equation as follows:

2.5. Preparation of hybrid scaffolds

1.5 g of PDALB was weighed and dissolved in 50 mL of distilled water with continuous stirring at 80 °C for 2 h. In a clean vial, 10 mL of collagen solution was stirred with different concentrations of PDALB solution from 25 to 100 wt% for 30 min at 35 °C. The blending of collagen and PDALB biopolymer induced the crosslinking through a Schiff base reaction. The blended viscous solutions of 100/0, 100/25, 100/50, 100/75 and 100/100 wt% C/PDALB were poured into Petri plates (75 × 12 mm²) and lyophilized to obtain hybrid scaffolds. Finally, the prepared C/PDALB hybrid scaffolds were stored under refrigerated conditions.

2.6. Characterization of hybrid scaffolds

2.6.1. Fourier transform infrared (FTIR) spectroscopic analysis. The collagen, LB, PDALB and hybrid scaffolds were ground with KBr and compressed to form pellets. The formed pellets were analyzed using a Perkin Elmer FTIR spectrometer in single beam mode in the range of 400–4000 cm⁻¹ with an average of 4 scans and 2 cm⁻¹ resolution.

2.6.2. TNBS assay. The crosslinking efficiency of PDALB during the stabilization of collagen was determined using a TNBS assay.²⁶ Briefly, 500 μL of un-lyophilized solution was taken into a clean test tube and mixed with 1 mL of 4% (w/v) sodium bicarbonate solution. Subsequently, 1 mL of freshly prepared 0.5% (v/v) TNBS solution was added and the mixture was maintained at 40 °C for 2 h. The samples were treated with 3 mL of 6 M HCl solution at 60 °C for 1.5 h. Then, the reaction mixture was diluted with 5 mL of distilled water. The maximum absorbance of the formed trinitrophenyl complex was measured using a UV-visible spectrophotometer (UV-1800, Schimadzu) at 420 nm. The native collagen solution was used as a control and treated with TNBS in a similar manner. The crosslinking efficiency and free amino groups were determined using the equations as follows:

2.6.3. Differential scanning calorimetric (DSC) analysis. The hydrothermal stability of the select hybrid wet 100/0 and 100/100 wt% C/PDALB scaffolds was determined using a DSC analyzer (DSC Q200, TA Instruments) at the rate of 1 °C min⁻¹ from 30 to 100 °C in a nitrogen flow of 50 mL min⁻¹.

2.6.4. Mechanical and thickness analysis. The mechanical properties of the prepared hybrid scaffolds were characterized using an Instron 4501 universal testing machine. The hybrid scaffolds were cut into dumbbell shapes with dimensions of 40 × 5 mm before the analysis. The samples were analyzed using a 100 N load cell at a crosshead speed of 10 mm min⁻¹. The thickness of the hybrid scaffolds was measured using a graduated scale and was expressed in millimeters (mm).

2.6.5. Porosity studies. The porosity of the prepared hybrid scaffolds was measured by immersing known weights of the scaffolds into 5 mL ethanol for 24 h at 30 °C.^27,28 After the incubation, the samples were weighed after removing the excess ethanol from the surface of the scaffolds. The porosity of the hybrid scaffolds was calculated using the equation as follows:

2.6.6. Scanning electron microscopy (SEM) analysis. Samples from the 100/0 and 100/100 wt% C/PDALB hybrid scaffolds were mounted on aluminium stubs and coated with gold using a Tescan sputter coater. Then, the surface morphologies of the gold coated hybrid scaffold samples were analyzed using a scanning electron microscope (Tescan Vega 3 SB) at an accelerating voltage of 3 kV.

2.6.7. Swelling studies. The in vitro swelling behavior of the 100/0, 100/25, 100/50, 100/75 and 100/100 wt% C/PDALB hybrid scaffolds was studied using 0.05 M phosphate buffered saline (PBS) solution (pH 7.4). Known weights of the scaffolds were immersed in 25 mL of PBS solution. At different time intervals, the swollen scaffolds were weighed after removing the surface water using filter paper. The extent of swelling of the hybrid scaffolds was calculated using the equation as follows:

2.6.8. In vitro biodegradation studies. In vitro biodegradation of the 100/0, 100/25, 100/50, 100/75 and 100/100 wt% C/PDALB hybrid scaffolds was carried out using bacterial protease from Aspergillus saitoi. A known weight of scaffold was incubated in 10 mg of protease dissolved in 25 mL of PBS (pH 7.4) solution at 37 °C for 24 h. Subsequently, the samples were centrifuged, freeze dried and then weighed. This step was repeated 4 times using fresh protease solution. The in vitro biodegradation of the hybrid scaffolds was calculated using the equation as follows:

2.7. Immobilization of VEGF in hybrid scaffolds

The immobilization of VEGF in hybrid scaffolds was carried out by blending 5 mL of collagen solution with 3.35 mL of PDALB and 1 mL of 1 μg mL⁻¹ VEGF solution for 30 min at 30 °C, as shown in Fig. 1b. Then, the blended solution of 100/100 wt% C/PDALB loaded with VEGF was poured into Petri plates (50 × 12 mm²) and freeze dried. The prepared 100/100 wt% C/PDALB loaded with VEGF hybrid scaffolds were stored under refrigerated conditions.

2.8. In vitro VEGF release studies

The prepared 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold was incubated in 10 mL of PBS solution (pH 7.4) at 37 °C. At different time intervals, 100 μL of the medium was replaced by fresh PBS for measuring the concentration of VEGF. A human VEGF enzyme-linked immunosorbent assay (ELISA) development kit was used and measurements were made at 405 nm with wavelength correction set at 650 nm employing a microplate reader (Epoch, BioTek). The in vitro VEGF release was calculated using the equation given as follows:

2.9. In vitro cell culture studies

The biocompatibility of the select 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffolds was investigated using Swiss 3T6 mouse fibroblast cell lines. Circular pieces of scaffolds with a size of 0.5 cm × 0.6 mm were sterilized using UV irradiation and placed in 48 well culture plates. 250 μL of Swiss 3T6 mouse fibroblast cell suspension containing 2 × 10⁴ cells was seeded into each well and maintained using DMEM high glucose with 10% FBS supplemented with penicillin (100 units per mL), streptomycin (100 μg mL⁻¹), gentamicin (50 μg mL⁻¹) and amphotericin B (2.5 μg mL⁻¹) at 37 °C, humidified with 5% CO₂. Swiss 3T6 mouse fibroblast cells were incubated for 24 and 72 h and the medium was changed every day. The number of viable cells in each well was determined by MTT assay at 570 nm using a Bio-Rad ELISA plate reader. The percentage of cell viability was calculated using the equation as follows:

For the cell morphology and attachment studies, hybrid scaffolds with sizes of 1 cm × 0.3 mm were seeded with Swiss 3T6 mouse fibroblast cells in 48 well culture plates. After 24 and 72 h incubation, the cell morphology and attachment was detected by staining the Swiss 3T6 mouse fibroblast cells with fluorescein diacetate staining agent. The stained cells were photomicrographed using a fluorescence phase contrast microscope (Leica Systems) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

2.10. In vivo animal studies

Healthy male Wistar albino rats (190 ± 10 g body weight) were used to form full-thickness skin wound models. All animal experiments were carried out after obtaining the approval of the Institutional Animal Ethical Committee (Reg. no. 466/01a/CPCSEA) of CSIR-Central Leather Research Institute, Chennai, India in compliance with institutional guidelines. A total of 20 animals were divided into four groups, each consisting of 5 rats, corresponding to control, 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffolds treated groups. After intraperitoneal injection of thiopentone sodium (dose 50 mg kg⁻¹ body weight), the dorsal hair of each rat in the experimental groups was shaved and disinfected with 70% ethanol. A 2 × 2 cm² sized full thickness excision wound was created by excising the disinfected dorsal skin using sterile surgical tools. On the surface of the excised skin wound, 2.5 × 2.5 cm² hybrid scaffolds were applied and tied with sterile absorbent gauze to hold the material on the wound area. A 2.5 × 2.5 cm² sized 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold contains 300 ng of VEGF drug concentration. The surface wounds on the control group were covered with the sterile absorbent gauze material alone. At regular intervals of 4, 8, 12 and 16 days post wounding, the granulation tissues were removed and redressed with the respective materials after cleaning the wound surface with sterile distilled water. The collected granulation tissues from the wound surface were evaluated by biochemical and histological analyses.

2.11. Biochemical and wound assessment

The total collagen content in the defatted and dried granulation tissues was analyzed biochemically using a procedure reported by Woessner.²⁴ The time taken for the re-epithelialization was observed in the experimental groups until the wounds completely healed. The rate of wound closure was determined using a standard planimetric method by tracing the wound area in a transparent graph sheet on the 0^th, 4^th, 8^th, 12^th and 16^th days. The percentage of wound closure was calculated using the formula as follows:
where n = number of days after treatment (4^th, 8^th, 12^th and 16^th day).

2.12. Histopathology

Histopathology of the collected granulation tissues was performed by fixing in 10% formalin-saline, dehydration using a graded alcohol series, clearing in xylene and embedding in paraffin wax. The samples were sliced into 5 μm thick sections and separately stained with haematoxylin and eosin stain as well as Masson's trichrome stain. The stained sections were examined and photomicrographed using a TCM400 Labomed microscope. The number of fibroblast, inflammatory cells and blood vessels formation in the haematoxylin and eosin stained photomicrographs was quantified and expressed in percentage.²⁹

2.13. Statistical analysis

Data were expressed as mean ± standard deviation (SD). Statistical analyses were evaluated using the unpaired Student's t-test by GraphPad Prism software. The probability value of <0.05 was considered as statistically significant.

3. Results and discussion

3.1. Characterization of PDALB

Sodium periodate oxidation leads to the cleavage of diol groups into dialdehyde groups in the monosaccharide units of LB polysaccharide backbones. The degree of oxidation directly indicates the formation of aldehyde groups in polysaccharides.²⁵ Degree of oxidation in LB at different time intervals during the course of reaction is shown in Fig. 2a. As can be observed, the degree of oxidation increases up to 98% at the end of the 24^th hour of reaction. It is also interesting to note that more than 50% of LB was oxidized within the first hour of reaction, after which it attained a steady state. After the completion of the reaction, up to 2% residual periodate was observed, which indicates an unutilized quantity at the end of the reaction. The unutilized periodate was inactivated by adding ethylene glycol to the reaction solution after the completion of the reaction in 24 h.

Fig. 2 Synthesis of PDALB and its crosslinking efficiency with collagen. (a) Degree of oxidation during the modification of LB using sodium periodate; (b) FTIR spectra of pure LB and PDALB; (c) FTIR spectra of C/PDALB hybrid scaffolds; (d) crosslinking efficiency of PDALB with collagen and free amino groups in collagen with varying amounts of PDALB; and (e) DSC traces showing the hydrothermal stability of the wet 100/0 and 100/100 wt% C/PDALB hybrid scaffolds.

Fig. 2b shows the FTIR spectra of pure LB and PDALB. The pure LB spectrum displays the broad characteristic peak at 3229 cm⁻¹, representing a significant quantity of OH groups in the polysaccharide units. Moreover, the PDALB spectrum shows a slight reduction in that region and the peak is shifted to 3389 cm⁻¹ due to the oxidation of the OH groups. The PDALB spectrum also shows characteristic peaks at 1724, 1027 and 735 cm⁻¹, which are assigned to C=O, C–O–C stretching and C–H out of plane bending. The observed low intensity in the peak at 1724 cm⁻¹ is due to the fact that most carbonyl groups are in hemiacetal form in the molecular structure. The intense peaks at 1027 and 735 cm⁻¹ are because of the formation of hemiacetal and hydrated bonds between the aldehyde and hydroxyl groups. It should be noted that the transformation of aldehyde functionalities into hemiacetal groups in the molecular structure is an unstable and reversible reaction. The results indicate that the adjacent hydroxyl groups have been oxidized and confirm the formation of aldehyde groups in LB, thereby producing PDALB.^25,30

3.2. Crosslinking efficiency

The crosslinking efficiency of collagen with PDALB was investigated using FTIR, TNBS and DSC analyses. Fig. 2c shows the FTIR spectra of pure collagen and C/PDALB hybrid scaffolds. The characteristic peaks of collagen are observed at 1642, 1554 and 1240 cm⁻¹, which correspond to the amide I, amide II and amide III groups, respectively. The distinctive aldehyde peak of PDALB at 1724 cm⁻¹ is not detectable in the prepared hybrid scaffolds. This may be due to the crosslinking of the aldehyde groups of PDALB with the amino groups of collagen, resulting in the formation of stable covalent imine bonds through Schiff base reactions. The distinctive peak of the imine bond associated with the C=N linkage is generally shown as a strong stretching absorption around 1620 cm⁻¹.^31,32 However, the presence of amide I in the collagen is also observed as a sharp peak in the 1642 cm⁻¹ region. Nevertheless, the peak at 1620 cm⁻¹ became sharper and more intense as the concentration of PDALB increased in the hybrid scaffolds, which signifies the formation of imine bonds, as shown by the arrows in Fig. 2c. It can also be observed that the characteristic peaks of PDALB at 1027 and 735 cm⁻¹ became sharper and more intense as the concentration of PDALB increased in the C/PDALB hybrid scaffolds. Enlarged FTIR spectra of the hybrid scaffolds are given in Fig. S1† for better comparison and visualization. These results confirm the imine bond formation and homogenous nature of the prepared hybrid scaffolds.

The percentage of crosslinking efficiency of PDALB with collagen was determined by estimating the free amino groups using the TNBS assay; this is shown in Fig. 2d. It can be observed that the crosslinking efficiency of PDALB with collagen gradually increased and attained a maximum of 60% for the 100/100 wt% C/PDALB hybrid scaffolds. This indicates that the increased crosslinking is due to the increased availability of aldehyde groups in PDALB. As also can be observed, the percentage of free amino groups in the prepared 100/100 wt% C/PDALB hybrid scaffolds is about 40%. This value is slightly higher than that obtained using modified guar gum.¹⁸

The shrinkage temperature or hydrothermal stability of collagen is another key indicator of its stabilization or crosslinking efficiency. DSC traces of wet pristine collagen and 100/100 wt% C/PDALB hybrid scaffolds are shown in Fig. 2e. It can be observed that the shrinkage temperature of wet pristine collagen scaffold is around 58 °C. Moreover, the crosslinked 100/100 wt% C/PDALB hybrid scaffolds exhibit a significantly increased value of 68 °C. The increase in the hydrothermal stability of the 100/100 wt% C/PDALB hybrid scaffolds may be due to the interaction of PDALB and collagen. Therefore, the FTIR, TNBS and DSC results confirm the covalent crosslinking of PDALB with collagen, which provides good stability and prevents the need for external toxic crosslinking agents for the stabilization of collagen.

3.3. Physical properties of hybrid scaffolds

The mechanical, thickness and porosity properties of the prepared hybrid scaffolds are shown in Table 1. As can be observed, the tensile properties gradually decrease as the amount of PDALB increases in collagen, whereas the thickness and porosity gradually increase in the hybrid scaffolds. The thickness of the scaffolds is not even, as observed from the large standard deviation. This may be because the base of the Petri plates is not uniform (convex edges) and the rate of evaporation of solvent during lyophilisation is also not uniform. Therefore, scaffolds with uneven thickness are generally obtained. The prepared hybrid scaffolds exhibit decreasing tensile strength from 4.6 to 0.3 MPa and increasing porosity from 93% to 95.4% as the content of PDALB increases. This indicates that the decrease in tensile properties is mainly due to the increased porosity of the hybrid scaffolds.^33,34 The increased porosity also demonstrates the 3-dimensional structure of the prepared hybrid scaffolds. The observed mechanical and porosity results are highly comparable to those of biodegradable porous polyurethane scaffolds.³³

Table 1 Mechanical, thickness and porosity properties of the hybrid scaffolds

Composition of hybrid scaffolds	Stress (MPa)	Strain (%)	Thickness (mm)	Porosity (%)
100/0 wt% C/PDALB	4.6 ± 0.4	6.1 ± 1.9	1.7 ± 0.9	93.0 ± 0.6
100/25 wt% C/PDALB	1.6 ± 0.7	5.7 ± 0.6	2.4 ± 1.1	93.9 ± 0.6
100/50 wt% C/PDALB	1.2 ± 0.3	5.1 ± 1.2	3.2 ± 1.3	94.4 ± 0.5
100/75 wt% C/PDALB	0.6 ± 0.3	4.3 ± 1.5	3.6 ± 1.3	94.9 ± 0.1
100/100 wt% C/PDALB	0.3 ± 0.1	7.0 ± 4.2	4.0 ± 1.8	95.4 ± 0.1

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3.4. Structural morphology of hybrid scaffolds

The structural morphology of 100/0 and 100/100 wt% C/PDALB hybrid scaffolds is shown in Fig. 3a–d. A scanning electron microscopy image showing the surface morphology of the un-crosslinked 100/0 wt% C/PDALB scaffold exhibits a fairly smooth surface with fewer pores (Fig. 3a). However, the hybrid 100/100 wt% C/PDALB scaffold displays a rough surface with numerous pores spread over several layers (Fig. 3c). Additional SEM images showing the surface morphology are presented in Fig. S2.† The digital images of as-prepared freeze dried hybrid scaffolds are shown in Fig. 3b, d and S3.† It is observed that the thickness and compactness of the scaffolds gradually increase as the concentration of PDALB increases in the hybrid scaffolds (Fig. S3†). The cross sectional images clearly show that the pores are interconnected in several layers. These results are in agreement with the results obtained from the thickness and porosity of the hybrid scaffolds. Therefore, these results suggest that the prepared C/PDALB hybrid scaffolds are highly porous and are expected to greatly promote drug release, cell adhesion and proliferation.

Fig. 3 Structural morphology and biostability of the prepared C/PDALB hybrid scaffolds. Scanning electron microscopy and digital images showing the morphology of the as-prepared freeze dried (a and b) 100/0 and (c and d) 100/100 wt% C/PDALB hybrid scaffolds, respectively; (e) in vitro swelling and (f) in vitro biodegradation patterns of the 100/0, 100/25, 100/50, 100/75 and 100/100 wt% C/PDALB hybrid scaffolds. The insets in Fig. 3b and d show cross sectional digital images of the respective hybrid scaffolds.

3.5. In vitro biostability of hybrid scaffolds

The in vitro swelling and biodegradation studies demonstrate the biostability of the developed hybrid scaffolds. The in vitro swelling behavior of the prepared C/PDALB hybrid scaffolds is shown in Fig. 3e. It can be observed that the increase in PDALB concentration reduces the swelling behavior in the hybrid scaffolds. The maximum swelling for pure collagen scaffold is found to be 1490% at the end of the 12^th h of incubation. Moreover, 100/100 wt% C/PDALB hybrid scaffolds exhibit a maximum swelling of 545% after 12 h. The results also illustrate that the pure collagen scaffold reaches equilibrium stage after 8 h, whereas the C/PDALB hybrid scaffolds attain equilibrium within the first hour of incubation. Fig. 3f shows the enzymatic degradation patterns of pure collagen and different C/PDALB hybrid scaffolds. The enzymatic degradation of the un-crosslinked collagen scaffold is as high as 43% on the 1^st day and complete digestion is observed at the end of the 5^th day of incubation. Moreover, the enzymatic degradation in C/PDALB hybrid scaffolds is gradually decreased and attains the lowest degradation of 19% for the 100/100 wt% C/PDALB hybrid scaffolds at the end of the 5^th day of incubation. It indicates that the crosslinking of PDALB with collagen may protect the active sites of collagen recognized by the protease enzymes.³⁵ These results show that the prepared C/PDALB hybrid scaffolds possess improved biostability against swelling and enzymatic degradation due to the crosslinking of PDALB with the collagen.

3.6. Drug delivery studies

As can be observed, the 100/100 wt% C/PDALB hybrid scaffolds exhibited enhanced crosslinking, porosity and biostability in comparison to other C/PDALB compositions. Therefore, we selected the 100/100 wt% C/PDALB composition for VEGF loading and further in vitro and in vivo studies. The release profile of VEGF from 100/100 wt% C/PDALB loaded with VEGF hybrid scaffolds is shown in Fig. 4a. It can be observed that the VEGF is released rapidly up to 65% at the end of 1^st day of incubation. This may be due to the quick biodegradation tendency of C/PDALB hybrid scaffold during the initial period of incubation, as observed in Fig. 3f. Subsequently, sustained VEGF release is seen up to the 6^th day of incubation, which may be due to the interaction between the amine groups of VEGF and the aldehyde groups of PDALB. The results suggest that the sustained release of VEGF from the C/PDALB hybrid scaffolds can facilitate cell proliferation and blood vessel formation in tissue engineering and wound healing applications.¹⁰

Fig. 4 (a) In vitro drug release pattern of covalently immobilized 100/100 wt% C/PDALB loaded with VEGF hybrid scaffolds; (b) in vitro Swiss 3T6 mouse fibroblast cell proliferation patterns of 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffolds; (c) fluorescence microscopy images of Swiss 3T6 mouse fibroblast cells on 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold. The inset of (a) shows the digital image of 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold.

3.7. In vitro cell culture studies

The biocompatibility of the hybrid scaffolds plays a vital role in wound healing and tissue engineering applications. The in vitro Swiss 3T6 mouse fibroblast cell proliferation and viability on 100/0 wt% C/PDALB, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF scaffolds are shown in Fig. 4b. The MTT assay results show that the cell viability is above 90% in all the treated scaffolds at the end of 24 and 72 h culture periods. This means that the scaffolds did not show significant toxicity against Swiss 3T6 mouse fibroblast cells. Fig. 4c shows the fluorescence microscopy images of the hybrid scaffolds after 24 and 72 h culture periods. It can be observed that the Swiss 3T6 mouse fibroblast cells proliferated and adhered on the surface of the pristine and hybrid scaffolds. These results demonstrate that the prepared hybrid scaffolds are not cytotoxic and promote cell proliferation and adhesion in a three dimensional fashion, presumably due to the 3D porous structure of the scaffolds. Enlarged individual images of fluorescein diacetate stained Swiss 3T6 mouse fibroblast cells on the hybrid scaffolds are shown in Fig. S4 and S5† for greater clarity.

3.8. In vivo wound healing studies

The collagen content in the granulation tissues of the control and experimental groups is shown in Fig. 5a. The total collagen content in the granulation tissues on the 4^th day is 2.7 ± 0.3, 3.1 ± 0.3, 3.7 ± 0.3 and 4.9 ± 0.4 mg/100 mg of dry weight for the control (untreated wound), 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated groups, respectively. Moreover, on the 8^th day, the collagen content of the granulation tissues is 5.8 ± 0.2, 6.4 ± 0.3, 6.5 ± 0.3 and 7.2 ± 0.2 mg/100 mg of dry weight for the control, 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated groups, respectively. It can be observed that the collagen content is significantly higher in the treated groups until the 8^th day. The increase of collagen concentration in the granulation tissues indicates the level of cellular proliferation and protein synthesis on the wound surface. The synthesis of collagen in the wound region plays an essential role in the extracellular matrix, homeostasis and epithelialization at the later phase of wound healing.^2,29

Fig. 5 (a) Levels of collagen content in the granulation tissue for the control and experimental groups on the 4^th and 8^th days; (b) Masson's trichrome staining of the granulation tissue of the control and experimental animal groups on the 4^th, 8^th and 12^th days. The blue stained region in the images indicates collagen formation. (c) Wound closure rates of the control and experimental animal groups. (d) Epithelialization period of the control and experimental animal groups. Values are expressed as mean ± SD and the level of significance is expressed as *, ** and ***, corresponding to p < 0.05, p < 0.01 and p < 0.001 compared with the corresponding control group.

Fig. 5b shows the photomicrographs of Masson's trichrome staining sections and displays the varying degree of collagen formation in the granulation tissues of the control and experimental treated groups. The control, 100/0 and 100/100 wt% C/PDALB hybrid scaffold treated groups show moderate levels of collagen formation. Moreover, the 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated group exhibits a high amount of collagen formation compared to the other experimental groups. The Masson's trichrome staining data correlate well with the quantified data of collagen content in the granulation tissues. Individual Masson's trichrome staining images of the control and experimental groups are shown in Fig. S6–S9† for better visualization.

Fig. 5c shows the wound closure rates of the control and experimental groups. As can be observed, the wound closure rates are 70%, 82%, 86% and 100% for the control, 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated groups, respectively, at the end of the 16^th day of treatment. The 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated group contracted the wound rapidly when compared to other groups. A similar trend has been observed in the epithelialization period analysis. The epithelialization periods of the control, 100/0, 100/100 wt% C/PDALB and 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated groups were 22 ± 0.7, 20 ± 1.1, 19 ± 1.3 and 16 ± 0.9 days, respectively (Fig. 5d). These results suggests that the 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated group experienced rapid and significant wound contraction compared to the other groups. The planimetric results of wound closure corroborate well with the photographic wound closure patterns (Fig. 6). These results are comparable to those of platelet derived growth factor incorporated hybrid scaffolds.²⁹

Fig. 6 Photographic representations of wound closure patterns on different days of the control and experimental groups.

3.9. Histopathology and quantification of cells

Fig. 7 shows the photomicrographs of haematoxylin and eosin staining sections and the quantified data of fibroblast, inflammatory cell and blood vessel formation in the granulation tissues. The untreated control group shows a lower number of fibroblast and a higher number of inflammatory cells compared to the other groups. Moreover, the 100/0 and 100/100 wt% C/PDALB hybrid scaffold treated groups show moderate levels of fibroblast and inflammatory cells. Amongst all the groups, the 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated group shows a high number of fibroblast cells and blood capillaries with fewer inflammatory cells up to the 12^th day of treatment; this is quantitatively shown in Fig. 7b–d. Inflammatory cells are a necessary prerequisite for normal wound healing, which involves phagocytosis activity to kill bacteria and generates several growth factors.³⁶ However, a high infection of bacteria in the wound region leads to a prolonged inflammatory phase and the development of chronic wounds.³⁶ Fibroblast proliferation and blood vessel formation in the wound region facilitates the formation of extracellular matrix, collagen synthesis and the transfer of oxygen and essential nutrients required for re-epithelialization.^5,6 The 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated group promotes less inflammation, more extracellular matrix production and the formation of numerous blood vessels, which may be due to the release of VEGF from the hybrid scaffolds. Individual haematoxylin and eosin staining images of the control and experimental groups are shown in Fig. S10–S13† for better comparison and visualization.

Fig. 7 (a) Haematoxylin and eosin staining of the granulation tissue of the control and experimental animal groups on the 4^th, 8^th and 12^th days. F, I and BV refer to fibroblasts, inflammatory cells and blood vessels, respectively. The schematic shows the structural morphology of the fibroblasts, inflammatory cells and blood vessels. Quantification of fibroblast, inflammatory cell and blood vessel formation in the granulation tissues on the (b) 4^th, (c) 8^th and (d) 12^th days. Values are expressed as mean ± SD of five photomicrographs in each group, and the level of significance is expressed as *, ** and ***, corresponding to p < 0.05, p < 0.01 and p < 0.001 compared with the corresponding control group.

These results indicate that the 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold treated group enhances the early phases of wound healing through proper cellularization and vascularization development. This may be due to the fact that VEGF acts as an endothelial cell mitogen and chemotactic agent and promotes angiogenesis at the early stage of wound healing.^7,8,13 The formation of angiogenesis restores tissue perfusion, re-establishes microcirculation and increases oxygen tension at the wound site.⁷ It is also interesting to note that the usage of 100/0 wt% C/PDALB and 100/100 wt% C/PDALB hybrid scaffolds moderately enhances the wound healing process, which may be due to the porosity, biostability and biocompatibility of pristine collagen as well as crosslinked collagen.

VEGF is a potent proangiogenic factor and stimulates healing through multiple mechanisms such as collagen deposition, epithelization and angiogenesis.^7,10 The development of new blood vessel formation in the earlier stage of wound repair provides a conduit for nutrients and other mediators of the healing response as well as the removal of metabolites.^7,10 Fibroblasts in the wound region depend on oxygen to proliferate and lay down the new extracellular matrix. The release of VEGF in the wound region involves vasodilation, basement membrane degradation, endothelial cell migration and proliferation.⁷ It induces the procoagulant factors in endothelial cells, which mediates the platelet adhesion and aggregation. It also increases endothelial cell secretion of matrix metalloproteinases to enhance the tissue remodeling. The proliferation of endothelial cells in the wound region promotes blood vessel formation.⁷ Wounds associated with diabetics and burn injuries show ischemia or hypoxia problems because of nerve damage and vascular lesions.⁷ To overcome this problem, an adequate blood supply and a desirable local environment are required. Therefore, VEGF incorporated C/PDALB hybrid scaffolds (100/100 wt% C/PDALB loaded with VEGF) protect the wound as well as deliver a local concentration of VEGF to the wound region.

The observed results of 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold in wound healing are highly comparable with other VEGF incorporated biomaterials. VEGF loaded pullulan–dextran–fucoidan electrospun fibers promote endothelial cell migration and angiogenesis at the 14^th day of post implantation in mice.¹⁰ The pullulan–dextran–fucoidan scaffolds potentiate the VEGF bioactivity on endothelial cells to trigger an angiogenic response and cellular infiltration.¹⁰ Calcium alginate microspheres have also been used as vehicles to deliver VEGF at the subcutaneous site in a rat model.¹¹ This treatment shows a considerable level of capillary network formation at the subcutaneous site after the first week of implantation.¹¹ It has also been shown that the collagen binding domain-VEGF in the collagen scaffold exhibits a higher wound healing rate and better vascularization in a diabetic rat model.³⁷ Therefore, the results lead us to conclude that topical 100/100 wt% C/PDALB loaded with VEGF hybrid scaffold dressings provides good biocompatibility, cellularization, and vascularization as well as rapid wound healing.

4. Conclusions

Biomimetic and covalently immobilized collagen–PDALB–VEGF hybrid porous scaffolds were prepared to promote early in vivo wound repair and tissue regeneration in wound healing applications. FTIR, crosslinking efficiency and DSC studies reveal that the inclusion of PDALB with collagen forms imine bonds through the crosslinking of the amino groups of collagen and the aldehyde groups of PDALB. An increase of PDALB concentration in collagen significantly improves the thermal, 3D porous morphology, swelling and biodegradation properties of the hybrid scaffolds. In vitro cell culture studies show the excellent proliferation and attachment of Swiss 3T6 mouse fibroblast cells on the surface of the hybrid scaffolds. In vivo wound healing evaluation reveals that the collagen–PDALB–VEGF hybrid scaffolds stimulate biological activities in the wound region and promote collagen deposition, wound closure, re-epithelialization and blood vessel formation. Therefore, these results suggest that the functionalized porous collagen–PDALB–VEGF hybrid scaffolds provide rapid tissue regeneration and wound repair and therefore are promising as an effective system for the treatment of chronic wounds.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

R. M. thank the Indian Council of Medical Research (ICMR), New Delhi, for the award of Senior Research Fellowship. IRIS ID: 2014-21000. CSIR-CLRI Communication No. 1191.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Enlarged FTIR spectra of the C/PDALB hybrid scaffolds, additional SEM images of the 100/0 and 100/100 wt% C/PDALB hybrid scaffolds, digital images of as-prepared freeze dried C/PDALB hybrid scaffolds showing the surface and cross section morphology, individual fluorescence cell culture microscopy images, individual photomicrographs of haematoxylin and eosin staining and Masson's trichrome staining of granulation tissues. See DOI: 10.1039/c5ra27571g

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