Growth of Human Mesenchymal Stem Cells (MSCs)

Growth of Human Mesenchymal Stem Cells (MSCs) on Films of Enzymatically Modified Chitosan Abdulhadi Aljawish and Lionel Muniglia Universite De Lorraine, Laboratoire D’ingenierie Des Biomolecules (LIBio), TSA40602-F-54518, Vandœuvre-le`s-Nancy, France

Isabelle Chevalot Universite De Lorraine, Laboratoire Reactions Et Genie Des Procedes (LRGP-CNRS-UMR 7274), TSA40602-F-54518 Vandœuvre-le`s-Nancy, France DOI 10.1002/btpr.2216 Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com)

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco-friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA-chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EFchitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications C 2016 American Institute of Chemical Engineers Biotechespecially for tissue engineering. V nol. Prog., 000:000–000, 2016 Keywords: enzymatic modification, chitosan films, ABTS, cell adhesion, mesenchymal stem cells

Introduction Mesenchymal stem cells (MSCs) isolated from bone marrow have been used as an attractive cell source for tissue engineering and regenerative medicine.1–3 MSCs are selfrenewable adult stem cells with great therapeutic potential due to their differentiation into multiple tissue-specific lineages such as osteoblasts, chondrocytes and adipocytes.4 However, one of the main limiting steps for clinical use or biotechnological purposes is the expansion step, as MSCs are present in small amount in adult tissues.5 MSCs are classically expanded in small-scale culture systems (T-flasks). This culture system presents many drawbacks, such as poor scale-up ability, contamination risks from frequent cell passaging and a lack of pH and oxygen control.6 Tissue engineering and cell therapy require large quantities of cells that cannot be easily achieved using processes in static flasks.7 Consequently, new techniques have emerged for MSC Correspondence concerning this article should be addressed to I. Chevalot at [email protected]. C 2016 American Institute of Chemical Engineers V

expansion, especially with cells attached on microcarriers in stirred bioreactors.8–10 The main advantage of microcarrier-based culture is the high ratio of adhesion surface to medium volume, enabling the attainment of high cell densities. This culture system showed some drawbacks such as cell aggregation during expansion. To avoid cell aggregation, some studies proposed controlling operating parameters such as agitation rate, cell density, and addition of carriers.6,11 Sometimes, due to the smooth surface of microcarriers such as glass microcarriers and Cytodex, MSCs cannot attach firmly and may detach and be washed away during medium changes.12 In addition, those microcarriers cannot be digested in the human body.13 consequently, microcarriers prepared by degradable materials in the human body exhibiting heterogeneous surface such as gelatin, chitosan in the human body are recently regarded as an attractive alternative of nondegradable microcarriers with smooth surface.13–20 Among the degradable materials that can be used to prepare the microcarriers for cell growth, chitosan, the deacetylated derivative of chitin, is a copolymer of glucosamine and 1

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N-acetylglucosamine units linked by 1–4 glucosidic bonds. Because of its good biodegradability, biocompatibility, absence of toxicity, and low cost, chitosan has already been considered as an ideal biomaterial for several biomedical applications.21 Nevertheless, the inferior mechanical properties and high swelling ability causing easy deformation of chitosan, is a real limitation to use chitosan in biomedical applications.22 Moreover, the presence of positive charges on chitosan is not favorable for cell adhesion and growth on it.23–25 To overcome those drawbacks and improve cell adhesion, several studies reported chemical modification of chitosan as a way to improve physicochemical and mechanical properties such as hydrophobic character and surface topography.26–31 For instance, chitosan modification was achieved by coupling with bioactive peptides such as arginine-glycineaspartic acid (RGD) peptide32–35 or with other biopolymers such as hyaluronic acid,36 alginate,37 gelatin,38 and silk fibroin.39 Up to now, the enzymatic modification of chitosan with phenol compounds to improve the MSCs adhesion and growth has not been yet reported in the literature. Recently, enzymatic tools of chitosan modification have been demonstrated as an attractive alternative to toxic, environmentally unfriendly, and nonspecific chemical approaches.40,41 The purpose of the current work is to study cell adhesion and viability of MSCs on chitosan derivative films modified by enzymatic approach. In fact, chitosan particles were enzymatically modified with FA or EF under mild conditions in aqueous medium.40 In the present study, chitosan and its derivatives films were prepared with solution-casting technique as a function of grafted phenols quantity and film thickness in micro-plate. Some properties of these films such as thickness, phenol content, bound-water content, and antioxidant activity were evaluated. Moreover, cytotoxicity, morphology and proliferation of MSCs on chitosan and its derivatives films were investigated.

present study, enzymatic modification of chitosan was carried out for 8 or 16 h of reaction. Chitosan solution making and films casting Chitosan solution (0.5%, w/v) was prepared by dissolving chitosan and its derivatives powder in aqueous acetic acid (1%, v/v) and stirred with a magnetic stirrer (Fisher Bioblock Scientific) overnight at 700 rpm at room temperature. When chitosan was completely dissolved, the solution was filtered with cellulose nitrate membrane filters of 5 mm pore size and 47 mm in diameter (WhatmanV, 7195-004, Germany) in order to eliminate the insoluble material. Finally, a vacuum (vacuum degree was 6 3 1022 Pa) was applied for 1 h to remove air bubbles from the systems (YamatoV). Chitosan films were prepared in 24-well plates. In each assay, two 24-well plates were prepared to study the cellular adhesion as a function of three parameters: the chitosan kind, the film thickness and the phenols grafted quantity. The first 24-well plate was prepared by adding 1 mL of chitosan solution in each well, while the second 24-well plate was prepared by adding 2 mL in each well to obtain different film thicknesses. Each plate was prepared as a function of the type of chitosan kind and the quantity of grafted phenols onto the chitosan depending on the time of the functionalization reaction (8 and 16 h). Drying process was carried out at room temperature for 2 days under the flow hood to avoid dusts. Dried chitosan films were neutralized by incubation with 1 M NaOH for an hour, followed by washing with distilled water until reaching pH 7–8. Then, neutralized films were kept with PBS until use. The sterilization of films were performed by washing with 70% ethanol for 30 min and then washing two times with PBS (pH 5 7.2) and one time with fresh culture medium. R

R

Physicochemical characterisation of chitosan-based films

Materials and Methods Materials Chitosan HMW (high molecular weight, Mw 310–375 kDa), ferulic acid (FA) and ethyl ferulate (EF) (purity about 99%) were purchased from Sigma–Aldrich (France). Dulbecco’s modified eagle medium (DMEM) with high glucose (4.5 g L21), Phosphate-buffered saline without calcium (PBS), methyl thiazolyldiphenyl-tetrazolium bromide (MTT), Fibroblast Growth Factor-Basic human (FGF-2) and bmercaptoethanol were purchased from Sigma (Germany). Fetal Bovine Serum (FBS) was purchased from EuroBio (France). L-glutamine and Antibiotic Antimycotic solution (10,000 U penicillin, 10 mg streptomycin, and 25 mg amphotericin B per mL) were brought from GIBCO (USA). Acetic acid was purchased from Prolabo (France). Ethanol and isopropanol were obtained from Carlo Erba (USA). Preparation of modified chitosan Chitosan modification by oxidation products of FA or EF was enzymatically performed according to the method described by Aljawish et al.40 Reactions were carried out at 308C by the addition of 5 mL of methanol solution of 50 mM FA or EF, 45 mL of phosphate buffer (50 mM, pH 7.5), and 1 g of chitosan particles in the reactor. Reactions were started by addition of 667 UI of SuberaseV. In the R

Thickness Measurement. The measurement of film thickness was accomplished using the standard NF Q 03-016 with a manual micrometer (Mitutoyo, Japan) equipped with a head measuring 5 mm in diameter a sensitivity of 1 mm. The thickness was measured in five randomly selected points on each film and then an average value of three films was calculated (mm). Determination of Phenol Content. The phenol content of chitosan films after 8 or 16 h of reaction was determined by the method described by Singleton et al.42 with some modifications. Prior to determining the phenol content, 1 mg of each film was re-dissolved in 1 mL of aqueous acetic acid (1%, v/v). Then, 1 mL of chitosan film solution was mixed with 1 mL of 10% sodium carbonate (Na2CO3) and then this mixture was left for 10 min at 388C. Later on, 1 mL of Folin-Ciocalteau reagent (1/3) was added to the mixture and stirred for 1 h in the dark at room temperature. Absorbance was measured at 660 nm using a UV–visible spectrophotometer (Shimadzu UV-1605). Gallic acid (40 mg mL21) and water were used as standard and blank, respectively. The phenolic content was calculated by the following Eq. (1) resulted from gallic acid as a standard: y50:009 3 with ðR2 0:9997Þ

(1)

where (y) is the absorbance value at 660 nm and (x) is the concentration of phenol nongrafted (mg mL21).


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Hydrophobicity Evaluation. To evaluate the hydrophobicity of chitosan and its derivatives films, the bound water content of chitosan films prepared in micro-plate was determined. Nearly 200 mg of each dried film already prepared in 24-well plate were weighed and then dried in a vacuum oven at 1058C for 24 h. The final weight of each sample was considered as dry matter content (DMC), and then the water content (%) was calculated regarding DMC using the following Eq. (2): Water content ðhÞ ð%Þ5½ðW0 2W24 Þ=W24 3100

(2)

where W0 and W24 are the initial weight (at 0 time) and the final weight (after 24 h of drying) of the samples, respectively. Antioxidant Activity. The spectrophotometric analysis of ABTS.1 radical scavenging activity was determined according to the method of Bozic et al.43 with some modifications. The ABTS.1 cation radical was produced by the reaction between 7 mM ABTS in H2O and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h. Before use, the ABTS.1 solution was diluted with ethanol to reach an absorbance of 0.700 6 0.025 at 734 nm. Then, 1 mg of the tested chitosan film sample was mixed with 300 mL of ABTS.1 solution. The mixture was then stirred for 1 h in the dark at room temperature. The inhibition of the ABTS•1 radical was measured at 734 nm and 258C using a UV–visible spectrophotometer (Shimadzu UV- 1605). The inhibition percentage of this radical was calculated using the following Eq. (3): Inhibition or scavenging effect ð%Þ5½ðAcontrol 2Asample Þ=Acontrol 3100

(3) •1

where Acontrol is the initial concentration of the ABTS and Asample is the absorbance of the remaining concentration of ABTS•1 in the presence of the sample. All data are the averages of triplicate experiments. Biological characterizations of chitosan-based films Cells and Cell Culture. Mesenchymal stem cells (MSCs) were kindly provided by the IMOPA laboratory at the Biop^ole- medicine faculty (Nancy, France). These cells are isolated from the bone marrow of adult male aged 24-years old. MSCs were cultivated between passages 1 and 4 in DMEM and supplemented with 10% FBS, 4 mM L-glutamine, 1% Antibiotic Antimycotic solution, 5 mL FGF-2 and 3.5 mL bmercaptoethanol. The cells were usually split when 80% confluence was reached (5–7 days). The cells were then trypsinised with TrypleTM Express (Gibco-Denmark), counted by Thoma cell under an optical microscope (VI-Cell). The cells were seeded at 2 3 104 cells/cm2 in culture flasks. The cells used in these experiments were between passage number 3 (P3) and 4 (P4). Cytotoxicity Test. The cytotoxicity of chitosan films was evaluated based on a procedure adapted from the ISO109935 standard test method. The films prepared in 24-well plate were prewashed with 70% ethanol for 30 min and washed two times with phosphate buffer saline (PBS, pH 5 7.2) and one time with fresh culture medium. Then, to prepare an extraction medium, chitosan films were incubated in 1 mL of fresh culture medium at 378C for 24 h. In the other side, 1 mL of MSCs were seeded in wells of a 24-well plate at a density of 5 3 104 cells per well. After incubation for 48 h,

the culture medium (1 mL) was removed and replaced with the extraction media (1 mL) and later incubated for another 24 h. The extraction media were then removed and the cells were re-incubated for 24 h in fresh culture medium (1 mL). The number of living cells was finally quantified with MTT assay. The MTT assay is based on the reduction of the tetrazolium salt, methyl thiazolyldiphenyl-tetrazolium bromide into a crystalline blue formazan product by the cellular oxidoreductases of viable cells.44 The resultant formazan crystal formation is proportional to the number of living cells. At the end of experiment, 250 mL of MTT (2 mg/ml in PBS, pH 7.4) were added to all wells and the plate was then incubated at 378C under 5% CO2 atmosphere for 4 h. Medium with MTT was then gently removed and 1 mL of isopropanol (60%) was added to all wells for dissolving of the formazan crystals. The plates were then shaken at room temperature for 10 min and the absorbance was read at 540 nm in a microplate reader. The relative cell viability was calculated according to the following Eq. (4): Number of living cells ð%Þ5ðð12ðAbstreated

cell =Abscontrol ÞÞ3100

(4)

where Abstreated cells (with chitosan or its derivative film) and Abscontrol (polystyrene without chitosan film) are the absorbance values at 540 nm of sample with treated cells and control (polystyrene), respectively. Each test was carried out in quadruplicate and each experiment was repeated thrice. Cell Morphology and Spread. For analysis of cell morphology and spreading, images were taken after MSCs were allowed to attach onto the film scaffolds after 4 days. The cell images were taken at 1003 and 3203 magnification using an inverted optical microscope (Olympus, Tokyo, Japan) equipped with a digital camera (DXM1200F, Nikon) in the presence and the absence of chitosan films. Cell Adhesion and Viability. For cell adhesion and viability experiments, sterile 24-well plate containing chitosan films was filled with 1 mL of diluted MSCs at 53104 cells per well. Cells were allowed to attach and grow for 4 days at 378C under 5% CO2 atmosphere. The number of living cells was finally quantified with MTT assay as described above. Statistical analysis The experimental results were obtained from three biological replicates. The data were recorded as means 6 standard deviation (SD) and analyzed by SPSS (version 11.5 for Windows 2000, SPSS). One-way analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncan’s Multiple Range tests. Differences at P < 0.05 were considered significant.

Results and Discussion Chitosan as solid particles has been enzymatically modified with FA and EF under mild conditions (T 308C and pH 7.5) in aqueous medium. Thanks to catalysis by laccase, phenolic compounds could be covalently grafted onto free amino groups of chitosan at region C2 via Schiff base bond (N5C). The results demonstrated that the phenols grafting led to the increase of hydrophobicity of chitosan and to the

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Table 1. Thickness Values of Chitosan and its Derivative Films Prepared by Modified Chitosan at 8 h and 16 h of Reaction Thickness (mm) Chitosan solution (0.5% v/v) Chitosan FA-chitosan derivative at 8 h at 16 h EF-chitosan derivative at 8 h at 16 h

1 ml

2 ml a

9.7 6 0.5 10.5 6 0.6a 10.7 6 0.4a 9.9 6 0.5a 10.1 6 0.3a

20.5 6 0.8b 21.4 6 1.0b 21.8 6 0.8b 21.1 6 0.7b 21.4 6 0.6b

Each value is expressed as mean 6 standard deviation (n 5 3). Values not followed by the same letter in each column are significantly different at the 0.05% level (Duncan’s test).

Table 2. Determination of Phenolic Content onto FA or EF-chitosan Derivatives Prepared by Modified Chitosan at 8 h and 16 h of Reaction Reaction Time (h) FA-Chitosan EF-Chitosan mg of phenol/mg of chitosan

8 16

48.4 6 1.2b 71.2 6 1.8a

16.8 6 1.5b 23.2 6 1.1a

Each value represents the average 6 standard deviation of three independent experiments. Values not followed by the same letter in each column are significantly different at the 0.05% level (Duncan’s test).

decrease of the quantity of free amino groups (positive charges) of chitosan.40,41 Characterisation of chitosan-based films Thickness Measurement. The thickness of chitosan and its derivatives films prepared at 8 and 16 h of reaction time for 1 and 2 mL of chitosan solution (0.5% w/v) in each well (2 cm2) of 24-well plate was performed using a manual micrometer (Table 1). Results showed that no significant difference of thickness was observed for chitosan and its derivatives films (P < 0.05). Indeed, phenol quantity grafted onto chitosan did not influence on the film thickness. Thus, the thickness average of films prepared from chitosan solution (0.5% v/v) was almost 10 mm for 1 mL and 21 mm for 2 mL. Phenol Content. The phenol content of chitosan derivative films prepared by modified chitosan at 8 h and 16 h of reaction time was determined using gallic acid as a standard. As shown in Table 2, the quantity of FA-oxidation products grafted onto FA-chitosan films was almost three times higher than that of EF-oxidation products. Previous study indicated that the grafting capacity of FA-oxidation products onto chitosan was higher than that of EF-oxidation products.45 In fact, the presence of a free carboxyl group in FA structure led to several oxidation products46; whereas the esterification of carboxyl group in EF structure led to a single oxidation product as also already reported with peroxidise.47 This hypothesis could explain the high amount of FA-oxidation products grafted onto chitosan derivative compared to EFoxidation products. Hydrophobicity Evaluation. As shown in Table 3, the bound water content of chitosan films was higher than that of chitosan derivatives films. Moreover, the bound water content of FA-chitosan films was higher than that of EFchitosan films. The bound water content decreased with the increase of the phenol content onto chitosan derivatives. High bound water content of chitosan films is due to the

Table 3. Bound Water Content (%) and ABTS.1 Inhibition Values (%) of Chitosan and its Derivative Films Prepared by Modified Chitosan at 8 and 16 h of Reaction ABTS1 Inhibition (%) Chitosan Solution Bound Water for 1 mg of (0.5% v/v) Content (%) Chitosan Film Chitosan FA-chitosan derivative at 8 h at 16 h EF-chitosan derivative at 8 hat 16 h

6.8 6 0.3a 2.3 6 0.7c 1.2 6 0.5d 4.4 6 0.6b 2.8 6 0.5c

3.9 6 0.6e 50.3 6 1.7b 72.8 6 2.5a 23.3 6 1.1d 43.4 6 1.6c

Each value is expressed as mean 6 standard deviation (n 5 3). Values not followed by the same letter in each column are significantly different at the 0.05% level (Duncan’s test).

greater hydrophilic nature of this polymer and to the stronger interactions established between water molecules and functional groups of chitosan (AOH, ANH2) as a result of the interactions of the type of hydrogen bonds. Furthermore, the grafting of oxidation products onto chitosan derivatives reduced the bound water amount to specific polar sites of chitosan due to the presence of phenol hydrophobic site chains. These results showed that chitosan derivative films are more hydrophobic than chitosan films according to previous results.25 Moreover, the different bound water content of chitosan derivative films is related to the different quantity of phenol oxidation products. In fact, the amount of FAproducts grafted onto chitosan was higher than that of EFproducts as shown above. Consequently, FA-chitosan films are more hydrophobic than EF-chitosan films. Antioxidant Activity. ABTS•1 radical scavenging activity of chitosan and its derivative films (1 mg) at 8 h and 16 h of reaction time was shown in Table 3. In agreement with previous study,48 the results showed that the chitosan films presented almost no significant inhibitory activity towards the ABTS cation radicals mainly due to the inhibition of radical scavenging because of inter- and intra-molecular hydrogen links especially for chitosan of high molecular weight. On the contrary, chitosan derivative films exhibited much higher ABTS radical cation scavenging activities compared to chitosan films. This high antioxidant activity of chitosan derivatives increased with the increase of phenol content. In fact, the improved scavenging activity of chitosan derivatives may be partly due to the introduction of an H-atom donating group, which was produced from laccase-catalysed oxidation of FA or EF onto chitosan according to results reported in another study.40,45 This result can be attributed either to high antioxidant properties of FA49 or to high quantity of FAoxidation products grafted onto chitosan when compared with EF as presented above. Thus, these results suggested that chitosan derivatives could be considered as an efficient antioxidant polymer. Biological characterizations Cytotoxicity Test. As the chitosan used in this work is commercial, the cytotoxicity test was reported to verify the presence of different toxic molecules on chitosan such as proteins or peptides, which can have an inhibition effect towards MSCs. As the grafting of oxidation products onto chitosan can be carried out by electrostatic links, cytotoxicity test was performed to verify the washing efficacy of chitosan particles after the enzymatic modification to remove the phenols grafted by electrostatic links, which can diffuse into


Figure 1. Viability of MSCs cells cultured in the presence of extraction media from chitosan derivatives and chitosan films scaffold for a period of 24 h. Values not followed by the same letter are significantly different at the 0.05% level (Duncan’s test).

Figure 2.

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culture medium exhibiting, in turn, an inhibition effect towards MSCs. The evaluation of cytotoxicity of chitosan and its derivative films was determined using MSCs. In this test, the number of living cells reported as the percentage of the control (polystyrene wall without chitosan film), after culture of cells in extraction medium solutions for 24 h, is shown in Figure 1. The number of living cells was found to be about 95% for chitosan films, 92%, 89% for FA-chitosan films at 8 and 16 h and 90%, 88% for EF-chitosan films, respectively (P < 0.05). These results showed that no significant difference was observed and the cytotoxicity of chitosan and its derivative films was negligible in comparison with tissueculture polystyrene plate (TCPS) used as a positive control (polystyrene without chitosan film). In the literature, it was reported that the chitosan is a nontoxic polymer.50 In a previous study, the results showed that

Photographs of MSCs cells (at 3100 magnification) cultivated for 4 days either on polystyrene corresponding to the control (A) either on films of chitosan (B), of FA-chitosan (C) and of EF-chitosan (D). The 8 h and 16 h represent the enzymatic reaction time.

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Figure 3.

Morphology of MSCs cells (at 3320 magnification) cultivated for 4 days on polystyrene corresponding to the control (A) or on films of chitosan (B), of FA-chitosan (C) and of EF-chitosan (D).

Figure 4.

Percentage of MSCs living cells onto chitosan films and FA or EF chitosan derivative films with respect to film thickness and chitosan types. Values not followed by the same letter are significantly different at the 0.05% level (Duncan’s test).

laccase catalyzed oxidation products of FA or EF presented low toxicity in comparison with FA or EF initial substrates. In fact, considering the oxidation products of FA or EF at 50 mg/mL, no cytotoxicity was observed toward Human Umbilical Vein Endothelial Cells (HUVEC).45 It may be concluded that chitosan and its derivatives films were non-toxic towards MSCs cells under culture conditions. Cell Morphology and Attachment. Attachment of cells on the surface of a material is one of the prerequisites for

evaluation of its biological compatibility for possible use in biomedical applications. In this study, MSCs were cultured on chitosan and its derivative films for 4 days. Tissue-culture polystyrene plate (TCPS) was used as a positive control (polystyrene without chitosan film). As shown in Figure 2, the attachment and growth of MSCs cells on control (polystyrene without chitosan film) was better than that on chitosan and its derivative films. Moreover, chitosan derivative films showed improved attachment and growth of MSCs in comparison with chitosan films. Of note, this growth


increased with the increase of reaction time depending on the quantity of grafted phenolic compounds. The morphology of MSCs cells after culture on chitosan and its derivative films for 4 days was examined by optical microscope (Figure 3). Selected images of MSCs revealed the morphology of MSCs when they were in contact with film scaffolds. The observation of cell morphology revealed that only few MSCs could attach and spread. Most cells adhered on chitosan films remained almost round and tethered but they often did not spread according to previous studies.32,51 Results showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. Those cells displayed elongated and spread morphology. Moreover, different cell morphology types of MSCs on chitosan derivative films were observed in comparison with control (polystyrene without chitosan film). This result can be probably due to the different attachment mechanisms of cells cultured on the different surfaces. In addition, the evidence of cell-tocell interaction was indicative of non-cytotoxic response of the MSCs to the phenolic substrate grafted on chitosan derivative films. This phenomenon was reported by another study that evaluated the biocompatibility of hexanoyl chitosan films towards M929 fibroblast cells.29 Cell Proliferation. After 4 days of culture, the number of MSCs living cells was evaluated on chitosan and its derivative films by MTT test, relatively to a control (polystyrene without chitosan film) and considering the global mitochondrial activity as related to cell viability. As shown in Figure 4, chitosan derivative films showed an improved cell viability compared to chitosan films. This cell viability on films increased with the increase of oxidized phenols grafted onto chitosan derivatives. For this reason, the growth on FA-chitosan films led to high cell viability in comparison with that of EF-chitosan films. Furthermore, the MSCs cell viability also increased with the thickness of film for all films in agreement with previous results reported by the culture of MSCs on native chitosan films.52 Indeed, by increasing film thickness, the amorphous fraction and the level of vitronectin binding increased due to reduced film crystallinity.52 The cell behavior and interaction with biomaterial surfaces depend on properties such as surface topography, thickness, charge, chemistry, and surface energy.52–56 Chitosan is a typical natural polymer, positively charged, that is not favorable for cell adhesion and growth.54 Several studies already reported that chitosan film alone exhibited slight cell adhesion property due to its high hydrophilicity,51,57 its nonsuitable mechanical properties such as smooth surface,58 the absence of bioactive signals.33 Therefore, to enhance its cell adhesion, several authors reported the modification of chitosan by incorporation with other natural or synthetic polymers,36,39,59 bioactive peptides32,33 or phenolic compounds.25 In the current study, FA/EF oxidation products were enzymatically grafted onto chitosan under mild conditions (pH 7.5, 308C in aqueous medium). The presence of phenolic compounds onto chitosan derivative films could present a positive effect on MSCs growth in comparison with chitosan films. In fact, this positive effect can be due to the change of hydrophilic properties, functional groups or surface morphology as well as the introduction of antioxidant properties. Results showed that the grafting of FA-/EF-oxidation products onto chitosan could decrease hydrophilic character of chitosan obviously

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due to the presence of hydrophobic side chains of the grafted oxidized phenols as shown above. In fact, the high hydrophilic surface of materials containing high water levels within the surface is unsuitable for cell adhesion due to the decrease of protein adsorption.60,61 It was found that the moderate hydrophilic surfaces are more favorable for cell adhesion such as for HUVEC and HeLa cells due to the increase of protein adsorption.62 A previous study showed that the presence of phenolic compounds onto chitosan derivative films decreased their hydrophilic character improving the protein adsorption capacity and cell viability on chitosan films.25 Another study confirmed that N-acyl chitosan, especially N-hexanoyl chitosan, allowed best cell adhesion of fibroblast (L929) due to the decrease of hydrophilic character of chitosan film.29 Additionally, the grafting of FA/EF oxidation products onto chitosan introduced methyl and methoxyl groups (OCH3, CH3), reduced free amino groups of chitosan and increased hydroxyl and carboxyl groups as reported in previous study.40 In fact, it was shown that hydroxyl groups have a positive effect whereas free amino groups have a negative effect on osteoblast cell line function.63 Another study confirmed that free amino groups of poly (L-lysine) (PLL) have a toxic effect toward normal cells due to the interaction with cell membrane and thus these groups are not favorable for cell adhesion.64 In general, neutral polymers and polyanions showed less cytotoxicity than polycations such as protamine and poly (L-lysine) which induce cellular damage in a variety of cultured cells.65 For instance, the growth of bovine aortic endothelial cells (BAEC) increased with the increase of functional groups (AOH, ACH3).66 Another work demonstrated that the introduction of hydroxyl groups improved blood compatibility of cellulose67 and improved mouse fibroblasts cell growth on polyethylene glycol (PEG)-chitosan.68 Moreover, the grafting of FA/EF oxidation products onto chitosan increased the heterogeneity of chitosan film surface as reported in a previous study.25 In fact, among shortcomings of chitosan, the smooth surface that is considered unsuitable for cell adhesion and attachment.58 Generally, the heterogeneous surface is more favorable for cell adhesion of MSCs than homogeneous surface mainly due to improved protein adsorption on roughness surface.58 Several studies showed that the increase of surface roughness of chitosan has a positive effect on the behaviors of MSCs cells, including the increase of cell adhesion, detachment strength and proliferation.51,69,70 On the other hand, the grafting of FA/EF oxidation products onto chitosan increased antioxidant activity of chitosan derivative films as reported above. This increased character can improve MSCs growth mainly due to reduce the damage mediated by reactive oxygen species (ROS) in cell systems. In fact, normal cells especially stem cells are highly sensitive to ROS, thus the adding of antioxidant agent in culture medium is useful to reduce ROS-mediated damage in cell systems such as neuronal cell systems.71 Several studies showed that the presence of antioxidant compounds in the basal medium, such as ascorbic acid, thioctic acid, b-mercaptoethanol and sodium selenium, has positive effects on MSCs growth.12,72,73 Finally, the improvement of MSCs growth on chitosan derivative films could be due to the decrease of hydrophilic character and free amino groups as well as the increase of surface roughness, the presence of methoxyl, hydroxyl groups and the improvement of antioxidant properties.

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Conclusion The current study showed that the presence of FA-/EFoxidation products onto chitosan derivative films increased their hydrophobic and antioxidant properties. Furthermore, chitosan derivative films were not toxic toward MSCs. MSCs presented a well attachment and spread phenotype on chitosan derivative films whereas these cells often did not spread and remained round on native chitosan films. The cell viability was improved on chitosan derivative films compared to chitosan film. This cell viability increased with the increase of film thickness and with the quantity of grafted oxidized phenols. On the other hand, due to high phenol content of FA-chitosan derivative films, hydrophobic and antioxidant properties, heterogeneous character of surface and cell adhesion were higher than EF-chitosan derivative films. These results are very promising as it is shown that chitosan derivative films are compatible with the growth of MSCs for biomedical applications especially, tissue engineering. Further works are in progress to study the adhesion, growth and proliferation of MSCs onto microcarriers prepared by green enzymatically modified chitosan as attractive approach for MSCs expansion in stirred bioreactor.

Acknowledgments The authors gratefully acknowledge financial support from Forest Ecosystems, Agricultural Resources, Food and Bioprocess (EFABA) program, Lorraine University, France.

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