Mechanical, degradation rate, and antibacterial properties of a collagen-chitosan/PVA composite nanofiber

This study synthesized collagen-chitosan/PVA nanofiber composites using the electrospinning method. Characterizations included Fourier transform infrared spectroscopy (FTIR) and surface morphology using scanning electron microscopy (SEM). Investigations were carried out on tensile strength, degradation rate, and antibacterial test. It was found that the functional groups C–H, –OH, C–O, C–N, and N–H were suitable for PVA, collagen, and chitosan materials. The SEM showed that increasing the PVA composition caused a change in fiber diameter ranging from 34.64 to 71.63 nm. The tensile strength results show that the smallest nanofiber diameter has the highest ultimate strength value of 5.6 ± 0.4 MPa. In addition, it was found that the rate of degradation was directly proportional to the increase in concentration. Antibacterial activity test was carried out using two types of bacteria, namely gram-positive bacteria S. aureus and gram-negative bacteria E. coli. The results showed that the collagen-chitosan/PVA nanofiber composite had a diameter of antibacterial inhibition for E. coli and S. aureus bacteria, respectively.


Introduction
Nanofibers are being developed for a wide variety of biomedical applications, such as drug delivery agents, tissue engineering, and regenerative medicine [1]. Nanofibers have been shown to be more efficient for biomedical applications than micro or macro-scale materials because nanofibers can mimic the extracellular matrix (ECM) [2]. Interestingly, the functional properties of nanofibers, such as mechanical strength, surface morphology, porosity, degradation rate, and antibacterial properties, can be adjusted according to the composite material used [3][4][5].
In this study, nanofiber composites were composed of three materials: Collagen, Chitosan, and Polyvinyl alcohol (PVA). Collagen is the main extracellular matrix (ECM) protein in dermal tissue and plays an active role in mediating cell adhesion [6]. The triple helix structure of collagen has a high content of the amino acid glycine, which is essential for cell attachment and proliferation [7]. In addition, collagen is biocompatible and has a porous structure and low immunogenicity [8]. However, collagen has low mechanical strength and a high degradation rate.
Meanwhile, to support its application performance, nanofiber requires good mechanical strength so that it does not suffer damage when used. In addition, the degradation rate must be suitable to provide support when applied [9]. Therefore, collagen must be combined with other materials to overcome its weaknesses.
Chitosan is an amino polysaccharide (poly-1,4D-glucosamine) widely applied in biomedical applications and has excellent mechanical properties. Thus, potentially increasing the mechanical strength of collagen [10]. Previous studies have reported that the presence of chitosan in nano-HA/collagen/poly(l-lactic acid)/chitosan increased the tensile strength from 1.42 MPa to 1.63 MPa [11]. Furthermore, it has been reported that the degradation rate of chitosan is more controlled, making it suitable to be combined with other materials [12]. In Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.
Furthermore, the presence of PVA in this study, because it is hydrophilic, is often used in biomedical applications approved by the Food and Drug Administration (FDA) [14,15]. Another advantage is that PVA can form excellent fibers through electrospinning [16]. Previous studies regarding Collagen-chitosan/PVA were still in the form of hydrogels and thin films [17,18]. However, the research on Collagen-chitosan/PVA composites in nanofiber structures has not been widely reported. This research focuses on examining the properties of collagen-chitosan/PVA nanofiber composites, including morphology, mechanical properties, rate of degradation, and antibacterial, as a basis for further studies related to their application in tissue engineering.

Materials
This study used the following ingredients: PVA (Mw 145000, hydrolyzed 98%; Merck from Germany), fish collagen, chitosan (low molecular weight; DD 75%; Aldrich), aquadest, acetic acid, and DI water. Moreover, a simulated body fluid (SBF) solution was used in the biodegradation test.
2.2. Synthesis of the electrospun nanofiber 2.2.1. Synthesis of the collagen-chitosan solution 2% collagen solution and 2% chitosan were each dissolved using 2% acetic acid with a stirrer speed of 700 rpm at room temperature for 30 min until homogeneous. Then the collagen solution and chitosan solution were mixed with a volume ratio of 7:3 at room temperature and 700 rpm for 3 h to obtain a homogeneous collagen-chitosan solution. Figure 1 shows the procedure for the synthesis of a collagen-chitosan solution.
2.3. Synthesis of the collagen-chitosan/PVA nanofiber PVA with a concentration of 10% was dissolved using distilled water at 120°C and a stirrer speed of 700 rpm for 5 h. Then the 10% PVA solution obtained was mixed with a collagen/chitosan solution with a volume ratio of 42% (CC/P 3:7), 33% (CC/P 2.5:7.5), 25% (CC/P 2:8), and 11% (CC/P 1:9), with a stirrer speed of 700 rpm at 120°C until homogeneity is reached (3 h). Then, electrospinning was carried out for 1 h on a glass slide (1 cm × 1 cm) under a voltage of 17 kV and a flow rate of 100 μl min −1 , where the distance from the needle tip to the collector was 15 cm. Figure 2 shows the synthesis procedure for the collagen-chitosan/PVA nanofiber composite.

Characterization
Characterization was performed using scanning electron microscopy (SEM, type FEI QUANTA FEG 650) to determine the surface morphology of the sample. Moreover, FTIR (type IR Prestige-21) was used to explore the properties of the molecular bonds and sample functional groups.

Mechanical test
The sample for the tensile strength test followed the ASTM D 638 type V test procedure, where it was placed in a mechanical test kit (IMADA, Titinus Olsen; universal testing machine) at room temperature. The obtained data were the initial length (L 0 ), cross-sectional area (A), length increase (ΔL), and weight of the load (F). From the obtained data, the strain (ε), stress (σ), and Young's modulus are formulated by equations (1)- (3): Meanwhile, the ultimate strength was obtained from the maximum or highest stress value from the test results. While the elongation of break is formulated by equation (4).

Degradation test
The degradation of the samples was tested in vitro using an SBF solution. Before the sample was immersed into the solution, it was weighed and recorded (initial mass: M i ). Furthermore, the sample was soaked with time variations of 1, 2, 3, and 4 weeks and stored in an incubator (B-One Model PIN 10) at a fixed temperature of 37°C . Then, the sample was removed from the incubator at the specified time and dried using an oven at 100°C. Afterward, it was weighed and recorded as dry mass M d . The weigth loss fraction is calculated using equation (5) [19]:

Antibacterial activity
An antibacterial activity test was performed using the agar diffusion method with S. aureus and E. coli. First, an agar medium was made by dissolving 8 g of nutrient agar powder into 400 ml of distilled water. It was followed by making a liquid nutrient (NC) by dissolving 0.15 g of beef extract and 0.25 g of peptone into 50 ml of distilled water as a medium for bacterial culture, where incubation was performed for 18 h. Then, NC was applied or inoculated on the entire surface of the petri dish filled with the medium so that it was evenly distributed. Next, three wells were made using a cork drill in each petri dish, and the holes were filled with the Collagen-chitosan/ PVA composite liquid. The Petri dishes were incubated at 37°C for 24 h. The sample inhibition was measured at the width of the clear zone around the well soon after the incubation.  hydroxyl band of pure alcohol, which occurred at -OH stretching without bonding, and the C-O band stretching indicated by the 1097 cm −1 peak due to the PVA spectrum [20]. The chitosan spectrum can be confirmed at 1336, 1338, 1338, and 1330 cm −1 peaks, which show amino bonds (CH bending). The amide bond at the 2943 cm −1 peak could be observed in the CC/P 42%, 25%, and 11% samples; however, in the CC/P 33% sample, it could be observed at the 2941 cm −1 peak (NH). These two groups could be obtained from the N-deacetylation bond in chitin [21]. The collagen spectrum in the successive compositions (CC/P 42%-11%) can be seen from the peak of the stretching asymmetric CH 2 functional group at 1453, 1435, 1454, and 1433 cm −1 peaks, which are associated with the stretching vibration of the carbonyl group [22]. Moreover, in the CC/P 25% sample, the stretching of the C-N bond functional group to the N-H bond combination could be confirmed, which shows the proportion of hydrogen bonds [22]. Then, the samples that showed the most significant proportion of hydrogen bonds were CC/P of 42%, 25% and 11%, , with the same peak of 1377 cm −1 , while sample 33% had the 1373.13 cm −1 peaks [23].

SEM analysis
Morphological analysis of PVA nanofiber with a concentration of 10% is presented in figure 4.
The analysis found that the fiber diameter ranged from 111-196 nm. It can also be observed that the morphology of the nanofiber has a uniform diameter without the appearance of beads covering its surface. The morphology shows that a 10% PVA concentration can produce nanofibers with good surface morphology making them suitable for matrices in collagen-chitosan/PVA nanofiber composites. The morphology of the collagen-chitosan/PVA nanofiber composite of all variations in figure 5 shows the fibers connected in random directions and without beads.
Furthermore, the analysis results in figure 5 show that variations in concentration cause changes in the size of the fiber diameter. It can be observed that the highest PVA concentration (CC/P 25% and 11%) has the widest diameter distribution, namely 203-775 nm and 218-545 nm. These results follow previous studies, where the concentration of fiber-forming polymers affects changes in diameter size [24]. The relation can happen because the nature of the solution is closely related to the flow rate and electrospinning stress, which impact the morphology of the resulting fiber [25]. Radolu et al reported that increasing polymer concentration leads to a higher increase in fiber diameter [26]. Furthermore, table 1. shows the results of the diameter range and average diameter of collagen-chitosan/PVA nanofiber.
Research related to chitosan nanofiber/PVA reported by Adeli et al showed that the diameter size changes with the increasing amount of chitosan in solution [27]. The same thing happened in the collagen-PVA nanofiber study, where the diameter size decreased from 429 nm to 283 nm with variations in increasing the weight concentration of 1% and 2%. Further analysis showed that the formed PVA-collagen fibers had different fiber yield uniformity [28]. The difference indicates that the solution's concentration plays an essential role in the stability of the electrospinning process, which leads to the quality of the nanofiber.

Mechanical properties
Nanofiber composites must possess mechanical characteristics to prevent the fiber from being damaged when applied [29]. The results of the analysis of the mechanical properties of the collagen-chitosan/PVA nanofiber composites are shown in table 2. The values of ultimate strength, young's modulus, and elongation can be identified from the mechanical properties tests.
The mechanical properties of nanofibers are closely related to their surface morphology [30]. It can be observed the illustration in figure 6 which shows that the diameter size is inversely proportional to the ultimate strength. This phenomenon is because nanofibers with small diameters have finer fibers and are connected to each other, so the bond between the fibers becomes stronger [31,32].
Furthermore, the ultimate strength and Young's modulus in this study were in the standard range of skin tissue. Each is 1-24 MPa [33], and Young's modulus range is 5-140 MPa [34], so these results can be the basis for further studies in the application of collagen-chitosan/PVA nanofiber composites for skin tissue engineering.

Degradation rate
The ability of nanofiber to be degraded is especially important to support the success of its application in biomedicals [35,36]. The degradation rate test was carried out by immersing the sample using SBF solution and measuring the reduction in the mass of the degraded sample every week for four weeks. The results of the degradation rate test are presented in figure 7.
From figure 7 it can be observed that the rate of weight loss in the range of 0-7 days (week 1) is directly proportional to the increase in concentration. Whereas in weeks 2-4 it showed a tendency for the rate of weight loss to be almost the same for all concentrations. This happens because each variation has almost the same   composition after soaking for 2-4 weeks. The collagen degradation rate is reported to be relatively fast [37]. In this study the rate of weight loss was supported by the concentration of collagen in the collagen-chitosan composite which dominated, where the ratio of collagen to chitosan was 7:3.

Antibacterial activity
The study of the antibacterial ability of nanofiber composites is needed for its application, especially in the biomedical field, where it plays a vital role in the healing process [38]. An antibacterial activity test was carried out on collagen-chitosan/PVA nanofibers using S. aureus and E. coli bacteria, which are among the 12 bacteria that are generally the most resistant to drugs [39]. Figure 8 shows the results of the collagen-chitosan/PVA nanofiber antibacterial activity test. A negative control test using distilled water was done to prove that the aquadest solvent did not play a role in the antibacterial activity. A positive control test using chloramphenicol (1%) was then compared. Chloramphenicol is an antibiotic that can inhibit gram-positive and gram-negative bacteria. The results of the measurement of the inhibition zone of antibacterial activity are shown in figure 9.  From figure 9 it appears that the decrease in concentration is directly proportional to the inhibition zone. Collagen-chitosan/PVA nanofiber composites are formed from several elements with different antibacterial abilities [40]. It is known that PVA and collagen have weak antibacterial activity [41,42], whereas chitosan is a material with strong antibacterial properties and is suitable for use in medical applications [43]. Chitosan is a suitable inhibitor for the growth of various bacteria and fungi [44]. The antibacterial properties of chitosan can be affected by the concentration of chitosan used [45]. Therefore, the highest concentration in this study had the largest inhibition zone, which is 6.49 mm and 5.08 mm for E. coli and S. aureus, respectively.

Conclusion
Based on the FTIR results, the functional group characteristics of the collagen-chitosan/PVA composite sample developed in this study showed agreement with previous studies. The SEM analysis of surface morphology showed that the nanofiber collagen-chitosan/PVA composite has differences in the size of the nanofiber diameter, which was influenced by the nature of the solution due to the variation in the concentration used. Furthermore, the smallest diameter shows the highest ultimate strength value, 5.6 ± 0.4 MPa. The rate of degradation shows the concentration of collagen-chitosan is directly proportional to the rate of weight loss. In  addition, the antibacterial activity showed a decrease in concentration directly proportional to the diameter of the inhibition zone.