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Journal of Biomaterials Science, Polymer Edition

ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsp20

Mechanical enhancement and in vitrobiocompatibility of nanofibrous collagen-chitosanscaffolds for tissue engineering

Fengjuan Zou, Runrun Li, Jianjun Jiang, Xiumei Mo, Guofeng Gu, ZhongwuGuo & Zonggang Chen

To cite this article: Fengjuan Zou, Runrun Li, Jianjun Jiang, Xiumei Mo, Guofeng Gu, ZhongwuGuo & Zonggang Chen (2017) Mechanical enhancement and in vitro biocompatibility of nanofibrouscollagen-chitosan scaffolds for tissue engineering, Journal of Biomaterials Science, PolymerEdition, 28:18, 2255-2270, DOI: 10.1080/09205063.2017.1392672

To link to this article: http://dx.doi.org/10.1080/09205063.2017.1392672

Accepted author version posted online: 16Oct 2017.Published online: 19 Oct 2017.

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SHORT COMMUNICATION

Mechanical enhancement and in vitro biocompatibility of nanofibrous collagen-chitosan scaffolds for tissue engineering

Fengjuan Zoua#, Runrun Lia#, Jianjun Jiangb, Xiumei Moc, Guofeng Gua, Zhongwu Guoa and Zonggang Chena

aNational Glycoengineering Research Center, and Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, People’s Republic of China; bDepartment of Vascular Surgery, Qilu Hospital, Shandong University, Jinan, People’s Republic of China; cCollege of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, China

ABSTRACTThe collagen–chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen–chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen–chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen–chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young’s modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen–chitosan nanofiber reinforced by TPU, both the break strength and the Young’s modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen–chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.

KEYWORDSCollagen; chitosan; poly (vinyl alcohol); thermoplastic polyurethane; tissue engineering scaffold; mechanical property

ARTICLE HISTORYReceived 9 September 2017 Accepted 12 October 2017

© 2017 Informa UK Limited, trading as Taylor & Francis Group

CONTACT Zonggang Chen chenzg@sdu.edu.cn#These authors contributed equally to this work.

JOURNAL OF BIOmATERIALS SCIENCE, POLymER EDITION, 2017VOL. 28, NO. 18, 2255–2270https://doi.org/10.1080/09205063.2017.1392672

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1. Introduction

Native extracellular matrix (ECM) is a dynamic fibrillar network of self-assembled proteins and polysaccharides [1,2] such as collagen, elastin, proteoglycan, glycosaminoglycan. They create a highly defined and specialized cell microenvironment that is full of information [3]. In addition to its conventional role in providing a support scaffold for developing and repairing tissues, the ECMs direct cellular growth and provide instructional information for regulating cell behavior and cell fate. The quest to understand both structural and functional attributes of ECM has motivated researchers to design materials with objec-tive of mimicking matrices’ architecture, physicochemical properties, and biomolecular composition. The ultimate desired for these bioinspired templates is to induce cellular responses as natural ECM, with special emphasis on matrix’s cell adhesion, proliferation and differentiation potential [2].

In natural ECM, proteoglycans, and glycosaminoglycans are usually coupled with col-lagen to provide a microenvironment [4], which plays a critical role in guiding local cell behavior. Collagen is the major structural and functional protein of ECM in mammals. It has been reported that collagen has been utilized as a biomaterial for a breadth of medical devices and tissue engineered scaffolds [5]. But using just collagen for tissue engineering applications is restricted due to its fast biodegradation, poor mechanical properties and issues related to in vivo contraction [2,6]. Chitosan derived from chitin is an abundant polysaccharide, which has also been widely used as biomaterial in the pharmaceutical and medical fields because of its abundant production in nature, excellent biocompatibility, appropriate biodegradability, excellent physicochemical properties and commercial avail-ability at relatively low cost [7,8]. Furthermore, as a glycosaminoglycan-like biodegradable polymer, it may be used to replace glycosaminoglycan in the biomimetic ECM. Therefore, incorporation of chitosan into collagen-based scaffolds may help in overcoming the potential limitations of collagen matrices [2].

The native ECM comprises a three-dimensional hierarchical fibrous structure of nano-meter-scale dimensions [2,9]. Multiscale fibrillar networks have also been developed in recent years so as to mimic the ECM architecture and elucidate better cellular behavior [2]. Electrospinning is a versatile technique, which can generate fibers with diameter ranging from nanometer to microns [10,11]. The electrospun fibrous structures possess a large surface area to volume ratio, high porosity, and potential for mimicking the structure and function of natural ECM [12]. It has been shown that electrospun scaffolds can promote cell attachment, spreading and proliferation, which could potentially enhance tissue regen-eration [9,12]. Because of the good biocompatibility and biodegradability, the collagen–chitosan complex have been fabricated into a three-dimensional nanofiber structure to mimic both components and structure of native ECM for tissue repair and biomedical applications in our previous researches [13]. Although previous studies have shown that the collagen–chitosan nanofibers demonstrate good cell viability [12,13], and thus have the potential as tissue engineering scaffolding for vascular graft [12], skin repair [2] and bone regeneration [14], the insufficient mechanical properties are not conducive for use as a scaffold for tissue engineering or biomedical applications due to the mechanical limitation of natural materials [12]. The mechanical properties need to closely match those of the real tissue in order to provide support during the initial stages of tissue growth. The mechanical

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properties of several tissues are listed in Table 1, indicating a large mechanical property range among a variety of human tissues [15].

In this study, we tailored and enhanced the mechanical properties of collagen–chitosan nanofiber scaffold by the introduction of PVA or TPU into the collagen–chitosan nanofiber, which would be suitable for a wide variety of tissue engineering scaffold applications (e.g. for healing and regeneration of skin and blood vessel).

2. Materials and methods

2.1. Materials

Collagen (molecular weight 0.8–1  ×  105  Da) was purchased from Sichuan Minrang Biotechnology Co., Ltd (China) and chitosan (85% deacetylated, molecular weight ~106 Da) was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd (China). TPU pol-ymer (Tecoflex EG-80A) was purchased from Noveon, Inc. (USA) and PVA (molecular weight 1.1–1.3 × 105 Da) was obtained from Sinopharm Chemical Reagent Co., Ltd (China). 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) from Fluorochem Co., Ltd. (United Kingdom) and 2,2,2-trifluoroacetic acid (TFA) from Sinopharm Chemical Reagent (China) were used to dissolve the collagen, chitosan, PVA, PU or their blends.

2.2. Electrospinning

Collagen (6  wt%) and TPU (6  wt%) were, respectively dissolved in HFP solvent while chitosan (6 wt%) and PVA (1 wt%) were dissolved in HFP/TFA mixture (v/v, 90/10) with sufficient stirring. After these solutions were blended at various weight ratio of collagen/chitosan (2/8, 5/5 and 8/2) and collagen/chitosan/PVA (8/2/1, 5/5/1 and 2/8/1) with suf-ficient stirring at room temperature for 1 h, they were placed into a 5-ml plastic syringe with 21-gauge blunt-end needle, respectively. The syringe was located on a syringe pump (Model 100，KD Scientific Inc., US) and dispensed at a feedrate of 1.0 ml·h−1. A voltage of 20 kV was applied from a high voltage power supply (DW-P503-1ACDF, Tianjin Dongwen High Voltage Power Supply Co. Ltd., China) across the needle and grounded aluminum foil collector at a distance of 15 cm. The blended fiber scaffolds of collagen/chitosan or collagen/chitosan/PVA were collected on a flat aluminum foil collector. A sandwich structure (col-lagen/chitosan-TPU-collagen/chitosan) of nanofibrous scaffold was also fabricated using the same parameters as mentioned above. The weight ratio of collagen/chitosan-TPU-col-lagen/chitosan in the fiber scaffolds corresponds to 4–2–4 based on each weight ratio of collagen/chitosan (2/8, 5/5 or 8/2, respectively). The collected fibers were stored and dried in a vacuum oven at room temperature for several days to removal the remained solvents.

Table 1. mechanical properties of several human tissues [15].

Tensile strength (MPa) Compressive strength (MPa) Young’s modulus (MPa)Cancellous bone 8 4–12 50–100Cortical bone 60–160 130–180  3–30 × 103

Cartilage 3.7–10.5 N/A 0.7–15.3Ligament 13–46 N/A 65–541Tendon 24–112 N/A 143–2310

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2.3. Morphologic analysis

After the fibers were coated with gold sputter, the morphology of the spun fibers was observed with a scanning electronic microscope (SEM) (JSM-5600, JEOL, Japan).

2.4. Tensile test

According to the method described in our previous works [16], the tensile test of samples of fiber membrane (30 mm × 10 mm) was performed using a universal materials tester (H5 K-S, Hounsfield, UK) with a 50 N load cell at room temperature. A tensile speed of 10 mm min-1 was used for all the tested specimens. The machine-recorded data were used to plot the tensile stress–strain curves of the specimens. The ultimate tensile stress and the ultimate elongation were also determined, and the Young’s modulus was been calculated by dividing the tensile stress by the extensional strain in the elastic (initial, linear) portion of the stress–strain curve. Six samples were tested for each condition.

2.5. Materials biocompatibility

Porcine iliac artery endothelial cells (ECs) were cultured on the collagen –chitosan nano-fibrous scaffold in order to evaluate materials biocompatibility. The fibrous scaffolds spun onto some round coverslips with a diameter of 14 mm were placed into a 24-well plates. The materials were sterilized with 75% ethanol for 4 h, soaked and rinsed five times with phosphate buffer solution (PBS). Then they were soaked in culture medium for 30 min in order to facilitate protein adsorption and cell attachment onto the materials. The cells were seeded onto the sterilized scaffolds at a density of 1 × 104 cells cm−2, and 400 μl Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 50 U ml−1 pencillin and 50 μg ml−1 streptomycin were also added into each well. The cultures were then maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air incubator, and the medium was refreshed every 2 days. In order to evaluate materials bicompatibility, cell behavior on the materials was monitored for 2, 4 and 6 days (n = 3 for each time point per group) by methylthiazol tetrazolium (MTT) assay. Briefly, the culture medium was removed, and the cultures were washed three times with PBS. About 200  μl serum-free DMEM medium and 20 μl MTT solution were added into each sample, followed by incubation at 37 °C for 4 h to allow the formation of MTT formazan. Then the medium and MTT were replaced by 400 μl dimethylsulfoxide to dissolve the formazan crystals. After 30 min, the solution was put into 96-well plates, and the samples were read using a microplate reader (Bio-Rad, Model 680, USA) at 490 nm. SEM analyses were also performed after cells were cultured for 3 days. Briefly, the samples were washed with PBS to remove non-adherent cells, fixed with 3% glutaraldehyde solution for 2 h at room temperature. Then the substrates were washed twice with PBS, dehydrated through a graded series of ethanol (50, 70, 80, 90, 95, and 100%), 10 min for each process. The samples were sputter coated with gold and observed by SEM after they were freeze-dried in vacuum.

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2.6. Statistical analysis

Data are presented as mean  ±  standard deviation (SD). The experimental results were compared by one-way analysis of variance (ANOVA) using Origin 8.0 software (OriginLab, USA), and p < 0.05 was considered statistically significant.

3. Results

3.1. Morphologies of collagen–chitosan fiber scaffold

The collagen–chitosan fiber scaffold with multiple levels of hierarchical organization has been fabricated by electrospinning. As shown in Figure 1, the lowest level of this hierarchy is the organization of single collagen–chitosan fiber (Figure 1(A)). The second level of the hierarchy is the organization of intertwined assembly of collagen–chitosan fiber network (Figure 1(B)). And the third level of the hierarchy is the organization of membrane consist-ing of collagen–chitosan fiber network and single fiber (Figure 1(C)). There was no obvious difference in the morphology of fiber after the fiber was reinforced by PVA or TPU.

Figure 1. Representative morphological assessment of electrospun collagen-chitosan fiber scaffold with microscopic methods. (A) SEm micrographs of a single fiber, (B) SEm micrographs of fiber network, (C) Photograph of fibrous membrane (white).

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3.2. Mechanical properties of electrospun collagen–chitosan fiber scaffold

Figure 2 gives the typical tensile stress–strain curves of electrospun collagen–chitosan fiber membrane with different collagen content. The mechanical properties of the fibrous mem-brane were improved with the increase of collagen content in spun fibers, which had also been demonstrated in our previous works [16]. But the resulting mechanical properties of materials are insufficient to meet the requirements of materials as tissue engineering scaffold, especially, no elastic deformation besides the plastic deformation occurs when the electrospun collagen–chitosan fiber member with 80% content of collagen are tensed. Great efforts should be made to enhance the mechanical properties of materials.

3.3. Mechanical properties of scaffold reinforced by PVA

PVA was introduced into collagen–chitosan fiber scaffolds to enhance their mechanical properties. Figures 3 and 4 show the tensile stress–strain curves of electrospun collagen–chitosan fiber membrane reinforced by PVA. It was found that both ultimate tensile strength and ultimate tensile elongation of fibrous membrane also increased with the increase of collagen content in spun fibers. Figure 5 gives the comparison of tensile properties of elec-trospun collagen–chitosan fiber membrane before and after reinforced by PVA. The average break strength of materials was heightened after enhanced by PVA with the increase of colla-gen content in the fibers. The average break elongation of materials was also improved after reinforced by PVA when collagen content is equal to 50% in the fibers, but it was decreased when collagen content is equal to 80% in the fibers. This is mainly attributed to the tensile plastic deformation of unreinforced materials with 80% content of collagen in the fibers (Figure 2). The introduction of PVA did not have significant effect on the average Young’s modulus of materials besides the improved plastic behavior of the materials with 80% content of collagen. Figure 6 shows the comparison of typical tensile stress–strain curves of collagen–chitosan nanofiber membrane before and after enhanced by PVA. Obviously, the tensile properties of materials were heightened.

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Figure 2.  Typical tensile stress–strain curves of collagen–chitosan superfine fiber membrane with thicknesses of about 0.060–0.070 mm and different collagen content: (a) 20%; (b) 50%; (c) 80%.

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3.4. Mechanical properties of scaffold reinforced by TPU

We try to enhance the mechanical properties of materials by fabricating a sandwich structure

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Figure 3. Tensile stress–strain curves of collagen–chitosan superfine fibre membrane reinforced by PVA with various collagen contents. (A) 20%; (B) 50%; (C) 80%.Note: The legends in the plots represent different thicknesses of the membranes in mm.

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Figure 4. Typical tensile stress–strain curves of electrospun collagen–chitosan fiber membrane reinforced by PVA with thicknesses of about 0.085–0.090 mm and different collagen content: (a) 20%; (b) 50%; (c) 80%.

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(collagen/chitosan – TPU – collagen/chitosan) of fibrous membrane. Figure 7 gives typical tensile stress–strain curves of collagen–chitosan nanofiber membrane enhanced by TPU with different collagen content. Owing to the significant difference of mechanical proper-ties between the complex collagen/chitosan fiber and TPU fiber, it experienced two-time sharp drop of stresses in the course of tensile test (Figure 7(A)), which implied two-time

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Figure 5. Comparison of tensile properties of electrospun collagen–chitosan fiber membrane before and after reinforced by PVA.Notes: No young’s modulus for the unreinforced membrane with 80% content of collagen due to no elastic deformation besides the plastic deformation.

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Figure 6.  Comparison of typical tensile stress–strain curves of electrospun collagen–chitosan fiber membrane before and after reinforced by PVA.Notes: The legends in the plots represent different composition of nanofibrous scaffolds, ch-co-x-y represents the ratio of chitosan content to collagen content being x–y.

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breaks of fiber membrane, the first one for that of collagen/chitosan fibers, and the second one for that of TPU fibers. Here, only the tensile properties of fiber membrane before the first fracture were discussed as shown in Figure 7(B) because the fiber membrane had been broken after the first fracture. Figure 8 shows the differences of the tensile behavior of colla-gen–chitosan nanofiber membrane enhanced by TPU with the variety of collagen contents

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Figure 7. Typical tensile stress–strain curves of electrospun collagen–chitosan fiber membrane reinforced by TPU with thicknesses of about 0.070–0.090 mm and different collagen content: (a) 20%; (b) 50%; (c) 80%.

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Figure 8. Tensile stress–strain curves of electrospun collagen–chitosan fiber membrane reinforced by TPU with various collagen contents. (A) 20%; (B) 50%; (C) 80%.Notes: The legends in the plots represent different thicknesses of the membranes in mm.

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and the thicknesses of the fiber membrane. It was found that the mechanical properties of the fibrous membrane were enhanced to some extent. Compared with the mechanical properties of unreinforced fibers, the tensile properties of materials were also enhanced as a whole. From Figure 9, both the average break strength and the average Young’s modulus of materials were enhanced in various degrees with the variety of collagen content in the fibers, which had also been shown by the comparison of typical tensile stress–strain curves of collagen–chitosan fiber membrane between before and after enhanced by TPU as shown Figure 10. The decrease of average break elongation of materials is mainly attributed to the tensile plastic deformation of unreinforced materials.

3.5. Materials biocompatibility

Materials biocompatibility in vitro was evaluated by culturing ECs on the scaffolds. The results showed that not only did the cells attach and spread well on both the fibrous scaf-folds reinforced by PVA and TPU, but also they interacted with the surface of materials well when they were cultured for 3 days (Figure 11). Moreover, interactions also occured among the cells on the materials, and this even resulted in the overlap of cells of multilayer cells (Figure 11). MTT assay indicated that ECs grew well on all the different fibrous scaffolds, no obvious difference was found between the proliferation of cell on the unreinforced and reinforced fibrous scaffolds (Figure 12). The All these indicate that the introduction of PVA or TPU has no obvious effect on the biocompatibility of collagen–chitosan fibrous scaffolds.

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Figure 10.  Comparison of typical tensile stress–strain curves of electrospun collagen–chitosan fiber membrane between before and after reinforced by TPU.The legends in the plots represent different composition of nanofibrous scaffolds, ch-co-x-y represents the ratio of chitosan content to collagen content being x–y.

Figure 11. SEm micrographs of 3-day cultured cells on the fibrous scaffolds reinforced by PVA (A) and TPU (B).

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4. Discussion

Tissue engineering evolved from the field of biomaterials development and refers to the practice of combining engineering scaffolds, cells, and suitable biochemical factors into functional tissues. The goal of tissue engineering is to assemble functional constructs that restore, maintain, or improve damaged tissues or whole organs. Its typical method is to incorporate patients’ own isolated living cells into 3-dimensional (3D) scaffolds and to create conditions for cells to proliferate in vitro, then transplant them back to the patient by surgical implantation or in a minimally invasive manner to develop into the desired tissues or organs. A second strategy is in situ tissue regeneration, where an absorbable scaffold is implanted directly into a defect site and the body is employed as its own bioreactor [17–20]. Obviously, tissue engineering requires 3D scaffold materials that have been engineered to cause desirable cellular interactions to contribute to the formation of new functional tissues for medical purposes. The scaffolds can provide the appropriate mechanical, structural and biological environments for tissue repair and regeneration. The mechanical proper-ties of scaffold affect not only cellular response but also the success of a tissue engineered construction [21–26]. Therefore, the mechanical properties of any scaffold are important in order to facilitate applications such as cell culture in vitro, in vivo implantation and mechanical functionality once implanted [27,28]. Mechanical properties are also important for maintaining 3D architecture for cell attachment and cell migration, applying biophysical stimuli to cells within the scaffold and mediating adaptive cell stiffening due to cell focal adhesions attachment [29–32]. it was also shown that mesenchymal stem cells differentiate toward different phenotypes depending on the stiffness of the substrate upon which they are seeded in seminal work by Engler and colleagues [30]. Therefore, mechanical strength is an important factor for the clinical applications of tissue engineering scaffold. It should match the mechanical property of natural tissue located in the implanted or repaired site. Moreover, the scaffold should provide initial biomechanical support before new tissues form. Therefore, many efforts should be made to improve the mechanical properties of tissue engineering scaffolds.

Natural biomaterials such as collagen show promise in tissue engineering applications because collagen is the major structural and functional protein of ECM in mammals. Chitosan is a derivative of chitin and is similar in structure to glycosaminoglycans, which are a common ECM element. Therefore, the collagen–chitosan complex has been fabricated into a three-dimensional nanofiber structure to mimic both components and structure of native ECM for tissue repair and biomedical applications in our previous researches [13]. The scaffold also showed acceptable cell adhesion and proliferation over 7 and 14 days using porcine iliac artery endothelial cells and myocardial artery smooth muscle cells of mouse [13]. The versatile scaffolds incorporating collagen and chitosan would have shown great potential as appropriate platforms for promoting orthopaedic tissue repair if it is not the limit of the poor mechanical strength of natural materials, which has stopped their further development as a scaffold for tissue engineering or biomedical applications.

Recently, polymer blending has received increasing attention from both the scientific and industrial communities as it is widely accepted as an efficient method to offer an attractive low-cost substitute to the development of entirely new materials with excellent performance.

PVA has a carbon chain backbone with hydroxyl groups that can act as a source of hydrogen bonding to enhance the formation of polymer complexes [33]. Many researchers

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have dispersed the fibers in the polymers of PVA, resulting in strong reinforcement effects [34]. In fact, PVA is also contemplated as the most attractive biomedical polymers due to a combination of qualities, such as biocompatibility, excellent mechanical property, and absence of toxicity [35]. Its mechanical behavior is similar to rubber-like materials, such as time dependent viscoelastic behavior. Thus, considering the advantages of biocompatibility and good mechanical properties of the PVA materials, it can be used in the pharmaceutics and biomaterial areas [36], e.g. tissue mimicking, vascular cell culturing, vascular implant-ing [37].

Synthetic polymers such as poly lactic acid (PLA), poly glycolic acid (PGA) and poly lac-tic-co-glycolic acid (PLGA) are widely used for polymer scaffold to enhance the properties of natural materials, such as mechanical properties. However, some drawbacks still exist upon using polymers including: formation of acidic species, this leads to a decrease in pH and tissue inflammation [38,39]. Among several choices of polymers, PVA, a hydrophilic semicrystalline polymer, has been frequently explored as an implant material in wide array of biomedical applications such as drug delivery systems, wound dressings, membranes, arti-ficial skin and the replacement or repair of the wounded tissue like liver, kidney or damaged articular cartilage [40], mainly due to its excellent mechanical strength, biocompatibility, and nontoxicity [39]. In this study, the introduction of PVA into the collagen–chitosan complex fibers had enhanced the mechanical properties of the materials. Both the tensile strength and the break elongation of nanofibrous scaffold were reinforced, moreover, the tensile plastic deformation of materials was also improved.

TPU is a linear block copolymer composed of alternating soft and rigid segments. The hard segments are formed by adduct of diisocyanate and small glycols. The soft segments consist of flexible polyether or polyester chains (polyols) connecting two hard segments. The soft and hard segments are incompatible at room temperature and aggregate into soft and hard domains, respectively, resulting in a polymeric system characterized schematically by hard domains formed amid the rubbery soft domains [41]. It is generally recognized that the existence of the microphase separation is responsible for the unique properties of TPUs, such as the high tensile strength, high elongation at break, good wear and tear resistance, low temperature elasticity, etc. [42]. Based on the above listed advantages, TPUs as a variety of polymeric materials are now widely used. A high-performance engineering materials might be produced by blending TPU with the other polymer.

Bioresorbable polyurethane (PU) scaffolds have attracted considerable attention and their great potential in tissue engineering has been reported [15]. TPU as a class of PU has been widely employed in medical applications due to its biocompatibility and flexibility, and has been used in medical devices such as catheters, pacemaker leads, skin, and vascular grafts. TPUs offer high elongation, moderate tensile strength and Young’s modulus, and excellent abrasion and tear resistance [15]. Therefore, it is a potential mechanical strengthening agent. In this study, TPU were introducing into the collagen–chitosan complex fibers by fabricating a sandwich structure (collagen/chitosan – TPU – collagen/chitosan) of fibrous membrane to enhance the mechanical properties of the collagen–chitosan nanofibers for potential use as tissue engineering scaffolds. It was found that the mechanical properties of the fibrous membrane were heightened after enhanced by TPU. Especially, both the average break strength and the average Young’s modulus of materials were enhanced in various degrees. Besides the enhanced mechanical properties, the sandwich structure of fiber scaffold m be better for cell adhesion, proliferation, migration and hemocompatibility

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because cell grows directly on the fiber composed of natural collagen/chitosan instead of the intermediate layer of synthetic TPU [43–45].

Cell culture on the materials offers an effective way to evaluate the biocompatibility of scaffolds. The results demonstrated that the reinforced collagen–chitosan complex with a three-dimensional nanofiber structure could provide adequate environment for ECs adhe-sion and proliferation, which indicated that the materials could be potential scaffolds for cardiovascular tissue engineering.

5. Conclusions

Though the collagen–chitosan nanofiber network has the potential as tissue engineering scaffold because they can offer satisfactory biological environment to facilitate cell adhe-sion, migrate and proliferation, the insufficient mechanical properties of natural materials has stopped their further development as a scaffold for tissue engineering or biomedical applications. In order to overcome this problem, two kinds of biocompatible synthetic polymers including PVA and TPU were introduced into the collagen–chitosan fibrous scaf-folds to enhance their mechanical properties. The results showed that the materials were reinforced. Both the break strength and the break elongation of materials reinforced by PVA were heightened. The materials reinforced by TPU had better break strength and Young’s modulus. Both the collagen–chitosan nanofibers reinforced by PVA and TPU can provide adequate environment for cell adhesion and proliferation. This would help in a wide vari-ety of tissue engineering scaffold applications according to different clinical requirements. Compared with the mechanical properties of several human tissues shown in Table 1. The reinforced collagen–chitosan complex fibers may be considered to develop tissue engineer-ing scaffold for soft tissue repair and regeneration. Further investigations will have to focus on large animal models or clinical applications.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the National Natural Science Foundation of China [grant num-ber 21472114], the Natural Science Foundation of Shandong Province of China [grant number ZR2016EMM15]; the Science and Technology Development Project of Shandong Province of China [grant number 2014GSF118113], [grant number 2017GSF18119]; the Fundamental Research Funds of Shandong University [grant number 2015JC004].

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