Structure-properties relationship of chitosan/collagen films with potential for biomedical applications

1 Structure-properties relationship of chitosan/collagen films with potential for biomedical applications Mireia Andonegia, Kevin Las Herasb, Edorta Santos-Vizcaínob,c, Manoli Igartuab,c, Rosa Maria Hernandezb,c, Koro de la Cabaa*, Pedro Guerreroa* aBIOMAT Research Group, University of the Basque Country (UPV/EHU), Escuela de Ingeniería de Gipuzkoa, Plaza de Europa 1, 20018 Donostia-San Sebastián, Spain. bNanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain. cBiomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN). Vitoria-Gasteiz, Spain. *Corresponding authors Koro de la Caba BIOMAT research group University of the Basque Country (UPV/EHU) Escuela de Ingeniería de Gipuzkoa Plaza de Europa 1, 20018 Donostia-San Sebastián (Spain) E-mail: [email protected] Pedro Guerrero BIOMAT research group University of the Basque Country (UPV/EHU) Escuela de Ingeniería de Gipuzkoa Plaza de Europa 1, 20018 Donostia-San Sebastián (Spain) E-mail: [email protected] This is the accepted manuscript of the article that appeared in final form in Carbohydrate Polymers 237 : (2020) // Article ID 116159, which has been published in final form at https://doi.org/10.1016/ j.carbpol.2020.116159. © 2020 Elsevier under CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/) 2 Abstract 1 Chitosan/collagen films were developed and characterized in order to assess 2 the suitability of these films for biomedical applications. Hence, 3 physicochemical, thermal, barrier and mechanical properties were analyzed and 4 related to the film structure, which showed the prevalence of the triple helix of 5 native collagen after the addition of chitosan. Furthermore, collagen fiber 6 diameter changed from 3.9 ± 0.6 μm, for collagen films without chitosan, to 1.8 7 ± 0.5 μm, for collagen films with low molecular weight chitosan. These results 8 suggested interactions between collagen and chitosan molecules, as observed 9 by Fourier transform infrared (FTIR) analysis. Regarding film barrier properties, 10 chitosan/collagen films showed a water vapor transmission rate around 1174 11 g∙m-2∙day-1, suitable for biomedical applications such as wound healing. 12 Additionally, biological tests confirmed that the chitosan/collagen films 13 developed are suitable for biomedical applications. 14 Keywords: chitosan, collagen, film, triple helix, biomaterial. 15 3 1. Introduction 16 Skin is the largest organ of human body and it is the first line of protection 17 against environmental exposure, preventing unregulated loss of body fluid. 18 Annually, millions of people suffered cutaneous wounds caused by physical and 19 chemical factors or diseases (Proksch, Brandner, & Jensen, 2008). Those 20 wounds might be exposed to infection by pathogenic bacteria so, wound healing 21 is an essential process for remodeling injured tissues (Boateng, Matthews, 22 Stevens, & Eccleston, 2008). In fact, cutaneous wound healing can be divided 23 into four overlapping phases, haemostasis, inflammation, proliferation and 24 remodeling, which involve diverse types of cells (Chen et al., 2019; Dabiri, 25 Damstetter, & Phillips, 2016). Any disturbance in these phases causes 26 prolonged healing, increasing the risk of infection (Li et al., 2019). Therefore, 27 different wound dressings, such as cotton gauze dressings (Frykberg & Bank, 28 2015), nanofibrous dressings (Garcia-Orue et al., 2019), human amniotic 29 membrane (Zelen, Serena, Denoziere, G., & Fetterolf, 2013), and 30 polysaccharide-based dressings (Ding, Deng, Du, Shi, & Wang, 2014; Khalid, 31 Khan, UI-Islam, Khan, & Wahid, 2017; Qin, 2008), have been investigated with 32 the aim to assist and promote the cutaneous wound healing process, 33 maintaining optimum conditions for the reestablishment of the damaged tissue. 34 In this context, the use of biopolymers such as collagen and chitosan has been 35 suggested as an alternative treatment of wounds, taking into account that an 36 ideal wound dressing should be non-allergenic and non-toxic, preserve moist 37 environment, promote effective oxygen exchange, protect the wound against 38 microbial organisms and absorb wound exudates (Perez-Puyana, Jiménez-39 Rosado, & Guerrero, 2019) 40 4 In this regard, collagen is a fibrous protein that plays an important role in tissue 41 healing, providing the biological microenvironment for cell growth and 42 supporting cell attachment, migration, and proliferation (Sorkio et al., 2015). At 43 the same time, it is biocompatible, biodegradable and low immunogenic 44 (Gopinath et al., 2004). For these reasons, collagen has become a promising 45 biopolymer in regenerative medicine (Pawelec, Best, & Cameron, 2016). 46 However, modification or cross-linking, using aldehydes (Tonndorf, Aibibu, & 47 Cherif, 2020) or carbodiimides (Beghetto, Gatto, Conca, Bardella, & Scrivanti, 48 2019), is essential for tissue engineering, since the degradation rate and 49 mechanical stability of single collagen are insufficient (Li et al., 2019). 50 Nevertheless, the crosslinking agents used frequently cause cytotoxicity 51 problems (Bae, Wang, & Kurisawa, 2013; Perez-Puyana, Jiménez-Rosado, & 52 Guerrero, 2019) and, thus, new crosslinking strategies must be assessed. 53 Regarding chitosan, this is a semi-crystalline polysaccharide obtained by 54 deacetylation of chitin and it is widely used as a biomaterial due to its 55 biocompatibility, biodegradability, non-toxicity, antimicrobial and antifungal 56 activity (Ahmed & Ikram, 2016). Chitosan also has a beneficial effect as a 57 wound healing promoter, improving the functions of fibroblasts, macrophages 58 and inflammatory cells (Rezaii, Oryan, & Javeri, 2019). In the last decades, 59 chitosan has also been studied for cutaneous wound healing in the form of 60 membranes, colloidal solutions or sponges (Bui, Park, & Lee, 2017; Elsabee & 61 Abdou, 2013). However, the effect of pure chitosan on promoting cell 62 proliferation is limited, and its biodegradability depends on its deacetylation 63 degree (Gamiz-Gonzalez et al., 2017). 64 5 Considering the functional properties of collagen and chitosan, their 65 combination could lead to composites with enhanced mechanical properties, 66 positive effects on cell proliferation, and controlled degradation velocity. 67 Although collagen-chitosan composites have been previously investigated for 68 different tissue engineering applications (Raftery et al., 2016; Si, Yang, Xing, 69 Yang, & Shan, 2019; Yan et al., 2019), there are few reports related to collagen-70 chitosan composites for cutaneous wound healing and those works referred to 71 gels (Li et al., 2019) and scaffolds (Rezaii, Oryan, Javeri, 2019). In this context, 72 the aim of this work is focused on the development of films based on native 73 collagen, assessing the effect of the incorporation of chitosan with high and low 74 molecular weight on physicochemical, thermal, barrier, mechanical and 75 biological properties of the resulting composite films and relating these 76 properties to the film structure. It is worth noting that, in contrast to other works 77 on collagen films, in which crosslinkers such as 1-ethyl-3-(3-dimethyl 78 aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added 79 (Socrates et al., 2016), in this work there was no need to incorporate any 80 crosslinker into the film forming formulation due to the prevalence of the triple 81 helix structure of native collagen for control and chitosan-incorporated films. 82 2. Materials and methods 83 2.1. Materials 84 Bovine collagen from trimmings and the splitting-derived residues, with 33% 85 glycine, 22% imino acids, 12% proline, 11% alanine and 10% hydroxyproline, 86 was supplied by Tenerias Omega (Spain). Low molecular weight (LMW, 190 87 kDa) chitosan (batch MKBB9037) and high molecular weight (HMW, 375 kDa) 88 chitosan (batch MKBC0059) with a degree of deacetylation higher than 75% 89 6 were provided by Sigma-Aldrich (Spain). Glycerol with a purity of 99.01% and 90 analytical grade, used as plasticizer, was supplied by OPPAC Products S.A 91 (Spain). 92 2.2. Preparation of films 93 The treatments of bovine skin to obtain native collagen were carried out 94 according to the method of Andonegi et al. (2020). Collagen films with 30 wt. % 95 (on collagen dry basis) LMW chitosan and HMW chitosan were prepared by 96 solution casting. Firstly, collagen skins were treated with NaOH 1 M at room 97 temperature for 12 h. These samples were neutralized with phosphate buffer 98 saline (PBS). Collagen was grinded and freeze-dried in order to facilitate the 99 subsequent processing. Afterwards, 1.5 g chitosan were dissolved in 100 mL of 100 0.5 M acetic acid under continuous stirring for 30 min and then, 5.0 g collagen 101 and 20 wt. % glycerol (on collagen dry basis) were added. The blend was 102 maintained under mechanical stirring for 3 h and then, poured into Petri dishes 103 and left drying at room temperature to obtain the films. Control samples were 104 prepared without chitosan. 105 2.3. Moisture content (MC) and mass loss (ML) 106 To determine the MC of films, samples were weighed (w0) and then freeze-107 dried. After that, samples were reweighed (w1) and MC was calculated as: 108 MC ሺ%ሻ=w0-w1 w0 x 100 109 Mass loss values were calculated using dried specimens. Rectangular film 110 pieces (w1) were immersed into PBS (pH 7.4) for 5 days and then, the samples 111 were reweighed (w2). The mass loss for three specimens of each sample was 112 calculated as: 113 7 Mass loss ሺ%ሻ=w1-w2 w1 x 100 114 2.4. Swelling 115 Rectangular pieces were weighed (wi) and then, immersed into PBS in order to 116 maintain the solution pH constant. Samples were weighed after immersion into 117 PBS for specific times (wf), until constant values were achieved. The swelling 118 was calculated as follows: 119  %1 00 fi f ww Swelling x w   120 2.5. Fourier transform infrared (FTIR) spectroscopy 121 Fourier transform infrared (FTIR) spectra were recorded on a Nicolet 380 FTIR 122 spectrometer equipped with horizontal attenuated total reflectance (ATR) crystal 123 (ZnSe). A total of 32 scans were made at 4 cm-1 resolution. All spectra were 124 smoothed using the Savitzky–Golay function. Second-derivative spectra of the 125 amide region were used at peak position guides for the curve fitting procedure, 126 using OriginPro 2019b software. 127 2.6. Thermo-gravimetric analysis (TGA) 128 The thermal stability of samples was measured by using the Mettler Toledo 129 TGA/SDTA 851 thermo-balance. Dynamic scans from 25 to 750 ºC were carried 130 out at a constant rate of 10 ºC/min under nitrogen atmosphere (10 mL N2/min) 131 to avoid thermo-oxidative reactions. 132 2.7. Differential Scanning Calorimetry (DSC) 133 DSC measurements were performed on a Mettler Toledo DSC 822. Samples of 134 around 3 mg were subjected to a heating ramp from 25 to 250 ºC at a rate of 10 135 8 ºC/min under nitrogen atmosphere to avoid oxidative reactions. Sealed 136 aluminum pans were used to prevent mass loss during the experiment. 137 2.8. X-ray diffraction (XRD) 138 XRD was performed with a PANalytical Xpert PRO diffraction unit, operating at 139 40 kV and 40 mA. The radiation was generated from a Cu-Kα (λ= 1.5418 Å) 140 source. The diffraction data were collected from 2θ values from 2 to 50 º, where 141 θ is the angle of incidence of the X-ray bean on the sample. 142 2.9. Scanning electron microscopy (SEM) 143 A Hitachi S-4800 scanning electron microscope was used. Prior to observation, 144 samples were mounted on a metal stub with double-side adhesive tape and 145 coated under vacuum with gold, using a JEOL fine-coat ion sputter JFC-1100, 146 in an argon atmosphere. All samples were examined using an accelerating 147 voltage of 15 kV. For the collagen fiber diameter evaluation (ᴓ) SEM images 148 were analyzed by ImageJ software. 149 2.10. Ultraviolet-visible (UV-vis) spectroscopy 150 The light-barrier properties of films were determined by measuring their light 151 absorption at wavelengths ranging from 200 nm to 800 nm, using a UV-Jasco 152 V-630 spectrophotometer. 153 2.11. Water vapor transmission rate (WVTR) 154 WVTR was determined in a controlled humidity environment chamber, using a 155 Labthink PERME™ W3/0120 water vapor transmission rate tester. Circles of 156 7.40 cm diameter, with a test area of 33 cm2, were cut. The test was carried out 157 according to ASTM E96-00 (ASTM, 2000). The setup was subjected to a 158 9 temperature of 38 °C and a relative humidity of 90%. WVTR values were 159 calculated by the following expression: 160 WVTR ൬g day cmଶ൰=G t x A 161 where G is the change in weight (g), t is time (day), and A is the test area (cm2). 162 2.12. Water contact angle (WCA) 163 Water contact angle measurements were performed using a DataPhysics OCA 164 20 contact angle system. A 3 μL droplet of distilled water was placed on film 165 surface to estimate its hydrophobic or hydrophilic character. The image of the 166 drop was captured using SCA20 software. 167 2.13. Mechanical properties 168 Tensile strength (TS) and elongation at break (EB) were measured at room 169 temperature, using a MTS Insight 10 electromechanical testing system (MTS, 170 Spain), for dry and wet specimens. Wet samples were hydrated for 2 h into 200 171 mL of distilled water. Tensile tests were performed according to ASTM D 638-172 03 standard (ASTM, 2003). Specimens for each sample were cut into bone-173 shaped samples of 4.75 mm × 22.25 mm and the crosshead rate was 174 1 mm/min. 175 2.14. Degradation studies 176 For the determination of the weight loss due to hydrolytic and enzymatic 177 processes, films (n=3) with a diameter of 8 mm and an area of 110 m2 were 178 weighed and subsequently immersed into different solutions. 8 mm diameter 179 disks were used due to the ease of handling and the suitability for testing in 24-180 well plates, as shown in previous in vivo studies (Garcia-Orue et al., 2019). PBS 181 16 Concerning DSC measurements, all samples exhibited three endothermic 314 peaks, as shown in Figure 2B. The peak around 90 ºC was related to the free 315 and bound water release (Kaczmarek, Sionkowska, & Skopinska-Wisniewska, 316 2018). The denaturation temperature was associated to the endothermic peak 317 around 150 ºC (Chakrapani, Gnanamani, Giridev, Madhusoothanan, & Sekaran, 318 2012). The temperature at which the maximum of this peak appeared increased 319 with the addition of chitosan, and the highest value was found for the films with 320 LMW chitosan. This fact would confirm that collagen-chitosan interactions would 321 be favored for the chitosan with lower molecular weight, as previously shown by 322 swelling results. Finally, the third endothermic peak appeared between 210 and 323 250 ºC, and it was associated to conformational changes from the triple helix 324 structure of collagen to random coil (Bozec & Odlyha, 2011). The temperature 325 corresponding to the maximum of this peak increased with the addition of 326 chitosan due to the existence of interactions between collagen and chitosan, as 327 observed by FTIR results. In particular, DSC revealed the characteristic 328 collagen-collagen and collagen-water interactions for control films. In the films 329 with chitosan, these interactions are partially replaced by interactions between 330 collagen and chitosan, indicating that the formation of hydrogen bonds between 331 collagen and chitosan competed with hydrogen bonding between collagen 332 chains. Similar results have been found for collagen-fucoidan films, showing the 333 increase of thermal stability by the incorporation of polysaccharides into 334 collagen film forming formulations (Perumal et al., 2018). 335 3.3. Morphological properties 336 The changes showed above require information about the structure of 337 chitosan/collagen films, thus XRD and SEM analyses were carried out. All the 338 17 films exhibited XRD patterns of amorphous materials (Figure 3A). The small 339 reflection peak at 10° is attributed to the chitosan crystal form (Leceta, Peñalba, 340 Arana, Guerrero, & de la Caba, 2015) and  the peak around 7º is related to the 341 triple helix structure of collagen (Valencia, Luciano, Lourenço, Bittante, & 342 Sobral, 2019). Collagen molecules generally assemble into fibrils and then form 343 collagen fibers by intermolecular cross-linking, leading to the characteristic 344 hierarchical interwoven structure of collagen. According to XRD results, the 345 addition of chitosan did not cause any relevant change, indicating that the 346 helical structure of collagen was maintained after chitosan addition, in 347 consistence with FTIR results. Additionally, a broad band appeared around 20º, 348 associated to the diffuse scattering of collagen fibers, indicating the amorphous 349 structure of the films (Zou et al., 2017). As can be seen, all samples showed 350 similar XRD patterns with a slight increase in the intensity of the band at 20º for 351 the films with HMW chitosan, indicative of the increase in the structural order. 352 353 Figure 3. Morphological properties of chitosan/collagen films: A) XRD patterns 354 and SEM images of surfaces for B) control films, C) collagen films with LMW 355 chitosan, and D) collagen films with HMW chitosan. 356 To further asses the changes caused by the addition of chitosan in collagen 357 films, SEM analysis was carried out and the images of film surface are shown in 358 Figure 3B, C and D. SEM micrographs exhibited the fibril structure of collagen 359 for all the films, showing the collagen periodicity corresponding to native 360 18 collagen (Socrates et al., 2019). With the addition of chitosan, the surface was 361 rougher and more fibers could be observed, especially for collagen films with 362 HMW chitosan. Furthermore, a significant (P < 0.05) decrease of collagen fiber 363 diameter was observed from 3.9 ± 0.6 μm for control films to 1.8 ± 0.5 μm and 364 1.9 ± 0.5 μm for the films with LMW and HMW chitosan. 365 3.4. Barrier and mechanical properties 366 Light barrier properties of chitosan/collagen films were analyzed and UV-vis 367 spectra are shown in Figure 4. As can be seen, collagen films provided a 368 maximum UV light barrier from 200 to 250 nm, associated with carbonyl, 369 carboxyl, and amide groups in the polypeptide chains of collagen (Pal, 370 Nidheesh, & Suresh, 2015; Veeruraj, Arumugam, & Balasubramanian, 2013), 371 and a small absorption peak at 250-280 nm, associated to tyrosine and 372 phenylalanine amino acid residues in collagen (Duan, Zhang, Du, Yao, & 373 Konno, 2009; Huang, Shiau, Chen, & Huang, 2011). The addition of chitosan 374 showed a strong UV blocking capacity, increasing the absorbance at 250-280 375 nm due to the hydroxyl auxochrome groups of chitosan (Uranga et al., 2019). 376 200 300 400 500 600 700 800 Absorbance Wavelength (nm) Control LMW HMW 377 Figure 4. UV-vis spectra of chitosan/collagen films. 378 Biomaterials for wound dressing need to maintain moisture loss from the wound 379 at an optimum rate and, thus, favor evaporation and inhibit excess fluid 380 19 absorption (Singh, Gupta, & Gupta, 2018). Taking the above into consideration, 381 water vapor transmission rate was measured. For normal skin, the inherent 382 water vapor evaporation rate is about 204 g∙m-2∙day-1, while the evaporation 383 rate increases to 279 g∙m-2∙day-1 and 5138 g∙m-2∙day-1 for injured skin and burn 384 skin, respectively (Lamke, Nilsson, & Reithner, 1977). As can be seen in Table 385 3, there was no significant (P > 0.05) difference in WVTR values with the 386 addition of chitosan and all samples showed a high occlusive character, 387 appropriate for biomedical applications such as wound healing. Additionally, 388 WCA values were measured to determine the film hydrophilic character, which 389 is known to have a great effect on the interaction between the biomaterial and 390 the tissue. As shown in Table 3, WCA values significantly (P < 0.05) decreased 391 with the incorporation of chitosan, which would favor the adhesion of fibroblasts 392 and endothelial cells during wound healing. This decrease of WCA values has 393 also been reported for collagen-fucoidan films (Perumal et al., 2018). 394 Table 3. Water vapor transmission rate (WVTR), water contact angle (WCA), 395 tensile strength (TS), and elongation at break (EB) of dry and wet 396 chitosan/collagen films. 397 Film WVTR (g∙m-2∙day-1) WCA (º) TSdry (MPa) EABdry (%) TSwet (MPa) EABwet (%) Control 1172a 103 ± 7c 7.7 ± 0.6a 13.2 ± 0.5a 2.0 ± 0.1a 50.6 ± 1.9a LMW 1176a 90 ± 7a 12.4 ± 0.8b 24.1 ± 0.8b 2.8 ± 0.1b 34.0 ± 0.4b HMW 1174a 93 ± 7b 13.0 ± 0.6b 21.9 ± 0.7c 2.3 ± 0.1c 39.3 ± 1.1c a-cTwo means followed by the same letter in the same column are not 398 significantly (P > 0.05) different through the Turkey’s multiple range test. N = 5 399 was the minimum number of replications. 400 Mechanical properties of films are largely associated with distribution and 401 density of intermolecular and intramolecular interactions in the network, so the 402 effect of chitosan on mechanical properties is shown in Table 3. For the dry 403 20 films, TS and EAB significantly (P < 0.05) increased when chitosan was added, 404 induced by the interactions between collagen and chitosan, as found by FTIR 405 analysis. As previously shown in Table 2, collagen is mainly composed of α406 helix chains with intense hydrogen bonds and Van der Waals forces among 407 these chains. Due to the triple helix structure, collagen shows appropriate 408 tensile strength, but the intense interactions among chains can prevent 409 stretching. However, in the wet state, water can weaken these interactions 410 among collagen chains, facilitating stretching and providing the films with a 411 greater flexibility. Nevertheless, this effect was less noticeable for the films with 412 chitosan due to the additional interactions among collagen and chitosan, 413 especially for the films with LMW chitosan, since the interactions with smaller 414 size-molecules are favored. This fact is in accordance with the swelling 415 behavior shown in Figure 1, where water uptake was higher for control films, 416 followed by the films with HMW chitosan and finally, by the films with LMW 417 chitosan. It is worth noting that all films showed a fibril structure in the fracture 418 surface after mechanical testing, as shown in Figure 5. 419 420 21 Figure 5. SEM images of the fracture surface in the break zone after 421 mechanical analysis for a) control films, b) collagen films with LMW chitosan, 422 and c) collagen films with HMW chitosan. 423 3.5 Biodegradation and cytotoxicity assessment 424 On the one hand, degradation studies showed that there was no progressive 425 weight decrease due to the hydrolytic action after 21 days in any of the 426 chitosan/collagen films. The initial loss, approximately 15% for the films with 427 chitosan and 23% for the control films (Figure 6A), was likely due to the 428 dissolution of glycerol. These findings also suggested that chitosan decreased 429 the dissolved glycerol since the films with chitosan showed a lower percentage 430 of degraded weight through the entire assay and non-significant differences 431 were observed in any time-point between the films with LMW or HMW chitosan. 432 The insoluble character of collagen in water explains the observed film 433 resistance to hydrolysis. This property was improved with the addition of 434 chitosan that formed hydrogen bonds with collagen molecules, as previously 435 shown by FTIR analysis. 436 On the other hand, enzymatic degradation studies showed a complete 437 degradation of the control films in less than 12 h; however, for collagen films 438 with chitosan, only 27-28% of the weight was lost at 12 h (Figure 6B). These 439 values are in accordance with those found by Perumal et al. (2018), who 440 reported 27% of degradation after 12 h for collagen films with 40% of fucoidan. 441 After 96 h, approximately 70% loss of the initial weight was reached, with no 442 significant differences between the films with LMW and HMW chitosan. The 443 30% of the remaining weight in the films with chitosan corresponded to 444 chitosan, which was not affected by the enzymatic action. Thus, it is 445 22 demonstrated that chitosan increased the resistance of the collagen to the 446 action of collagenase. These findings may be explained due to the fact that the 447 enzymatic degradation depends on the number of cleavage sites in the forming 448 polymer and on the concentration of available enzymes in the scaffold 449 environment (Drury & Mooney, 2003). In that sense, the addition of chitosan 450 hindered the access of the enzyme to the cleavage sites of collagen, reducing 451 the degradation caused by the collagenase. As previously reported (Etxabide et 452 al., 2017), films of these characteristics can be loaded with growth factors such 453 as epidermal growth factor (EGF). The slow degradation profile allows a 454 controlled release of bioactives, thus protecting them from the harsh 455 microenvironment with increased metalloprotease activity present in 456 inflammatory diseases such as chronic wounds (Barrientos, Brem, Stojadinovic, 457 & Tomic-Canic, 2014). 458 459 Figure 6. Degradation and cytotoxicity assessment: (A) hydrolytic degradation 460 with *** indicating P < 0.001 for control vs both LMW and HMW groups and N.S. 461 indicating non-significant differences between any time-point and day 1 for each 462 group; (B) enzymatic degradation with *** indicating P < 0.001 for control vs 463 both LMW and HMW groups, # indicating P < 0.05, and ### indicating P < 0.001 464 between any time-point and 2 h for each group; (C) cytotoxicity assay with N.S. 465 indicating non-significant differences vs blank group and dashed line marking 466 the 70% of cell viability. 467 23 Since films for biomedical applications must fulfill additional requirements such 468 as biocompatibility, in vitro cytotoxicity assay was performed following an 469 adapted protocol from the ISO 10993-5:2009 guidelines for biological evaluation 470 of biomedical devices. As can be seen in Figure 6C, all the films showed more 471 than 70% of cell viability after 48 h of direct contact with the film. These results 472 are in agreement with the biocompatibility of each component of the film forming 473 formulation. Taking these findings into account, chitosan/collagen films are in 474 compliance with the ISO 10993-5:2009 and may be considered as non-cytotoxic 475 biomaterials. 476 Conclusions 477 Chitosan/collagen films with enhanced functional properties for biomedical 478 applications such as wound healing were developed. The addition of chitosan 479 reduced the swelling degree of collagen films, which could allow a controlled 480 release of epidermal growth factors, substances of interest if films were used as 481 wound dressings. This behavior was explained by the interactions between 482 collagen and chitosan, as found by FTIR analysis, which showed the 483 prevalence of the triple helix structure of native collagen, as confirmed by XRD 484 results. No significant differences were found in most of functional properties as 485 a function of the molecular weight of chitosan, including mechanical resistance 486 in wet state and a cell viability higher than 70% for all the films assessed. 487 Acknowledgments 488 Authors thank the Basque Government (KK-2019/00006) for financial support. 489 M. Andonegi (PRE_2017_1_0025) and K. Las Heras (PRE_2018_1_0412) 490 thank the Basque Government for their PhD grants. Thanks also Advanced 491 Research Facilities (SGIker) from the UPV/EHU. Authors also wish to thank the 492 24 intellectual and technical assistance from the ICTS “NANBIOSIS”, more 493 specifically by the Drug Formulation Unit (U10) of the CIBER-BBN at the 494 UPV/EHU. 495 References 496 Ahmed, S. & Ikram, S. (2016). Chitosan Based Scaffolds and Their Applications 497 in Wound Healing. Achievements in the Life Sciences, 10, 27-37. 498 https://doi.org/10.1016/j.als.2016.04.001 499 Andonegi, M., Peñalba, M., de la Caba, K. & Guerrero, P. (2020). ZnO 500 nanoparticle-incorporated native collagen films with electro-conductive 501 properties. Materials Science and Engineering C, 108, 110394. 502 http://doi.org/10.1016/j.msec.2019.110394 503 ASTM D 638-03 (2003). Standard test method for tensile properties of plastics. 504 In Annual book of ASTM standards. Philadelphia, PA: American Society of 505 Testing and Materials. 506 https://www.astm.org/DATABASE.CART/HISTORICAL/D638-03.htm 507 ASTM E 96-00 (2000). Standard Test Methods for Water Vapor Transmission of 508 Materials. In Annual book of ASTM standards. Philadelphia, PA: American 509 Society of Testing and Materials. 510 https://www.astm.org/DATABASE.CART/HISTORICAL/E96-00.htm 511 Bae, K.H., Wang, L.S., & Kurisawa, M. (2013). Injectable biodegradable 512 hydrogels: progress and challenges. Journal of Materials Chemistry B, 1, 5371-513 5388. https://doi.org/10.1039/C3TB20940G 514 Bam, P., Bhatta, A., & Krishnamoorthy, G. (2019). Design of biostable scaffold 515 based on collagen crosslinked by dialdehyde chitosan with presence of gallic 516 25 acid. International Journal of Biological Macromolecules, 130, 836-844. 517 https://doi.org/10.1016/j.ijbiomac.2019.03.017 518 Barrientos, S., Brem, H., Stojadinovic, O., & Tomic-Canic, M. (2014). Clinical 519 application of growth factors and cytokines in wound healing. Wound Repair 520 and Regeneration, 22, 569-578. https://doi.org/10.1111/wrr.12205 521 Beghetto, V., Gatto, V., Conca, S., Bardella, N., & Scrivanti, A. (2019). 522 Polyamidoamide dendrimers and cross-linking agents for stabilized 523 bioenzymatic resistant metal-free bovine collagen. Molecules, 24, 3611. 524 https://doi.org/10.3390/molecules24193611 525 Boateng, J.S., Matthews, K.H., Stevens, H.N., & Eccleston, G.M. (2008). 526 Wound healing dressings and drug delivery systems: A review. Journal of 527 Pharmaceutical Sciences, 97, 2892-2923. https://doi.org/10.1002/jps.21210 528 Bozec, L., & Odlyha, M. (2011). Thermal Denaturation Studies of Collagen by 529 Microthermal Analysis and Atomic Force Microscopy. Biophysical Journal, 101, 530 228-236. http://doi.org/10.1016/j.bpj.2011.04.033 531 Bui, V., Park, D., & Lee, Y.C. (2017). Chitosan combined with ZnO, TiO2, and 532 Ag nanoparticles for antimicrobial wound healing applications: a mini review of 533 the research trends. Polymers, 9, 21. https://doi.org/10.3390/polym9010021 534 Chakrapani, V.I., Gnanamani, A., Giridev, V.R., Madhusoothanan, M., & 535 Sekaran, G. (2012). Electrospinning of type I collagen and PCL nanofibers 536 using acetic acid. Journal of Applied Polymer Science, 125, 3221-3227. 537 https://doi.org/10.1002/app.36504 538 Chen, Q., Wu, J., Liu, Y., Li, Y., Zhang, C., Qi, W., Yeung, K. W.K., Wong, T.M., 539 Zhao, X. & Pan, H (2019). Electrospun chitosan/PVA/bioglass Nanofibrous 540 32 nanoparticles for orthopedic application. Materials Letters, 161, 759-762. 680 https://doi.org/10.1016/j.matlet.2015.09.089 681 Sorkio, A.E., Vuorimaa-Laukkanen, E.P., Hakola, H.M., Liang, H., Ujula, T.A., 682 Valle-Delgado, J.J., Österberg, M., Yliperttula M.L., & Skottman, H. (2015). 683 Biomimetic collagen I and IV double layer Langmuir-Schaefer films as 684 microenvironment for human pluripotent stem cell derived retinal pigment 685 epithelial cells. Biomaterials, 51, 257-269. 686 https://doi.org/10.1016/j.biomaterials.2015.02.005 687 Tonndorf, R., Aibibu, D., & Cherif, C. (2020). Collagen multifilament spinning. 688 Materials Science and Engineering C, 106, 110105. 689 https://doi.org/10.1016/j.msec.2019.110105 690 Uranga, J., Puertas, A.I., Etxabide, A. Dueñas, M.T., Guerrero, P. & de la Caba, 691 K. (2019). Citric acid-incorporated fish gelatin/chitosan composite films. Food 692 Hydrocolloids, 86, 95-103. https://doi.org/10.1016/j.foodhyd.2018.02.018. 693 Valencia, G.A, Luciano, C.G., Lourenço, R.V., Bittante, A.M.Q.B, & Sobral, P.J. 694 A. (2019). Morphological and physical properties of nano-biocomposite films 695 base on collagen loaded with laponite®. Food Packaging and Shelf Life, 19, 24-696 30. https://doi.org/10.1016/j.fpsl.2018.11.013. 697 Veeruraj, A., Arumugam, M., & Balasubramanian, T. (2013). Isolation and 698 characterization of thermostable collagen from marine eel-fish (Evenchelys 699 macrura). Process Biochemistry, 48, 1592-1602. 700 https://doi.org/10.1016/j.procbio.2013.07.011 701 Wu, X., Liu, Y., Liu, A., & Wang, W. (2017). Improved thermal-stability and 702 mechanical properties of type I collagen by crosslinking with casein, keratin and 703 33 soy protein isolate using transglutaminase. International Journal of Biological 704 Macromolecules, 98, 292-301. https://doi.org/10.1016/j.ijbiomac.2017.01.127 705 Yan, F., Li, M., Zhang, HQ., Li, G.L., Hua, Y., Shen, Y., Ji, X.M., Wu, C.J., An, 706 H., & Ren, M. (2019). Collagen-chitosan scaffold impregnated with bone 707 marrow mesenchymal stem cells for treatment of traumatic brain injury. Neural 708 Regeneration Research, 14, 1780-1786. https://doi.org/10.4103/1673-709 5374.257533 710 Zelen, C.M., Serena, T.E., Denoziere, G., & Fetterolf, D.E. (2013). A 711 prospective randomised comparative parallel study of amniotic membrane 712 wound graft in the management of diabetic foot ulcers. International Wound 713 Journal, 10, 502-507. https://doi.org/10.1111/iwj.12097 714 Zou, Y., Wang, L., Cai, P., Li, P., Zhang, M., Sun, Z., Sun, C., Xu, W., & Wang, 715 D. (2017). Effect of ultrasound assisted extraction on the physicochemical and 716 functional properties of collagen from soft-shelled turtle calipash. International 717 Journal of Biological Macromolecules, 105, 1602-1610. 718 https://doi.org/10.1016/j.ijbiomac.2017.03.011 719