Cellulose Nanofiber Blend Scaffolds Prepared at Various pH

Subscriber access provided by UNIV TEXAS SW MEDICAL CENTER

Article

Collagen/Cellulose Nanofiber Blend Scaffolds Prepared at Various pH Conditions Chun-Yen Liu, Daisuke Goto, Chizuru Hongo, Takuya Matsumoto, and Takashi Nishino ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00302 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

Collagen/Cellulose Nanofiber Blend Scaffolds Prepared at Various pH Conditions

Chun-Yen Liu, Daisuke Goto, Chizuru Hongo, Takuya Matsumoto, Takashi Nishino*

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Rokko, Nada, Kobe, 657-8501, Japan. E-mail: [email protected]

KEYWORDS Cellulose Nanofiber, Collagen, Cell culture, Scaffold, pH

1

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

ABSTRACT Living tissues modules consist of highly organized cells and extracellular matrices (ECMs) in a hierarchical manner. Among these ECMs, type I collagen (COL), which is the most abundant protein, is widely accepted in tissue engineering field due to its biocompatibility. However, improvement of mechanical properties of COL scaffolds still remains challenge. We prepared the scaffold sheets with blends of COL and 2,2,6,6-tetramethylpiperidine-1-oxil(TEMPO)-oxidized cellulose nanofiber (TOCN). TOCNs with high mechanical properties reinforced COL scaffolds. Moreover, we prepared their blends under various pH conditions and investigated their mechanical properties and biocompatibilities. Sheets prepared at higher pH possess better mechanical performance. From Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy measurements, it is proved that the higher mechanical properties were attributed to COL triple inter helix structure, hydrogen interaction and electro static interaction with TOCN. The biocompatibilities of COL/TOCN prepared at higher pH were increased. We successfully demonstrated COL/TOCN blend materials with high mechanical strength and high biocompatibility for scaffold materials.

2


Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Regenerative medicine is based on the combination of cells and scaffolds to construct artificial tissues or organs to replace, repair, or enhance biological functions of injured patients.1,2 For construction of vascularized 3D-tissues, several bottom-up approaches have been reported recently; for example, cell sheets,3–6 hydrogels composed of extracellular matrices (ECMs),7–9 micro fluidic channel models,10–12 and angiogenic factors embedded scaffolds.13,14 However, these techniques have crucial limitations because of low biocompatibility or insufficient of mechanical properties of scaffold, which are significant factors to establish functionalized three-dimensional (3D) tissues in the field of tissue engineering.15–17 From the perspective of biocompatibility, which is an important factor for tissue engineered materials, native ECMs originated from living things such as our human beings body are promising materials and are widely accepted as frameworks of 3D-tissues. The native ECMs including collagen, gelatin, elastin, and fibronectin are important for cell adhesion, proliferation, and differentiation in native tissues. Among these ECNs, type I collagen (COL) is one of the most abundant proteins which are

3



Page 4 of 36

widely employed in biomedical or tissue engineering fields18–20 and are sensitively denatured by environmental factors.21 COL molecules have rod-like triple-helical structures composed of three polypeptide chains. The conventional amino acid sequences of COL are glycine-proline-X or glycine-X-hydroxyproline, where X indicates any amino acid other than glycine, proline and hydroxyproline.22 COL shows higher adhesion abilities against specific sensitive cells on culture dishes, or enhances differentiation of cells.23,24 However, in most research reports, COL scaffolds have been investigated under wet gel conditions and they showed low mechanical strength.25 Accordingly, development of biocompatible materials with high mechanical strength still remains a challenge in the tissue engineering fields. Cellulose is a structural component of the primary cell wall of plants, and is the most abundant organic polysaccharide with a linear chain. Cellulose nanofibers have recently attracted attentions as high performance materials because of their lightweights, robust mechanical properties, low linear thermal expansion coefficients and high biocompatibilities. In particular, their mechanical properties are attributed to larger surface areas, entanglements with nanofibers and the decrease of structural defects. The elastic modulus of the crystalline region of cellulose I in the direction

4


Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


parallel to the chain axis is 138 GPa. This value is twice of the modulus of aluminum, glass and comparable with those of high performance polymers such as aramid and liquid

crystalline

polyesters.

2,2,6,6-tetramethylpiperidine-1-oxil

Recently,

many

(TEMPO)-oxidized

research cellulose

reports

on

nanofibers

(TOCNs) for advanced applications have been published.26–28 Hydroxyl groups of 6-positions of cellulose molecules are oxidized to carboxyl groups by TEMPO. Through oxidation by TEMPO, TOCNs with 3 nm in diameter are obtained and possess high flexibility, strength as well as proper biocompatible properties. Their mechanical properties and biocompatibilities suggest that TOCN would employ as reinforcement materials in 3D-tissue engineering.29–32 Herein, we reinforced COL by blending of TOCN and investigated the mechanical properties and biocompatibilities of COL/TOCN blends. COL/TOCN composites were prepared under various pH conditions, then characterized by a series of analyses. The carboxyl groups on the surface of TOCN would form electrostatic interactions with amino acids of collagen molecules. It is suggested that higher pH resulted higher mechanical strength even in wet states and well cell adhesion ability, which can be considered as a suitable scaffold material for tissue engineering.

5



Page 6 of 36

MATERIALS & METHODS Materials Type I collagen from pork skin was purchased from Nitta Gelatin Inc. (Osaka, Japan). Bast fibers of kenaf (Hibiscus cannabinus, Indonesia, 2011) were provided by Toyota Boshoku Co. (Aichi, Japan), which are accepted as “green composite materials” in the motor vehicle industry such as world-leading company TOYOTA Co. Ltd. Human normal dermal fibroblast cells (NHDFs) were purchased from Lonza (NJ, USA). Alpha modified minimum essential medium (α-MEM), trypsin-EDTA, penicillin-streptomycin mixed solution, toluene, sodium chlorite, sodium hypochlorite, acetic acid, potassium hydroxide, sodium bromide, sodium hydroxide, hydrochloric acid, oxalic acid dihydrate, and benzene were purchased from Nakalai Tesque (Kyoto, Japan). Ethanol and carbon tetrachloride were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Aldrich-Sigma (MO, USA). Polystyrene was purchased from Asahi Kasei (Tokyo, Japan). TEMPO, Live/Dead cell viability kit, anti-rabbit IgG Alexa 488, Phalloidin-Rhodamine and DAPI were purchased from Thermo Fisher Scientific (MA, USA). All reagents were used without further purification.

6


Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Purification of Cellulose from kenaf According to reported literature,33 cellulose was purified from rough kenaf bast fibers by removing grease, lignin with Wise methods and hemicellulose using alkaline treatment as shown in Figure S1 in Supporting Information. Preparation of TEMPO-Mediated Cellulose Nanofibers Purified kenaf fibers were oxidized by TEMPO as shown in Figure S1 in Supporting Information.26 Firstly, 1.0 g of sodium bromide (NaBr) and 0.16 g of TEMPO was added to 1 L of distilled water and dissolved. 10 g of purified kenaf fibers were added into the prepared solutions, following addition of 25 mL of 1M sodium hypochlorite (NaClO) solution. The pH of the solution was maintained at 10 by titrating 0.5 M sodium hydroxide (NaOH) for 10 h with an automatic titration apparatus (AUT-501, DKK-TOA Co., Tokyo, Japan). After the titration, treated cellulose was washed and filtered with 10 times volume of distilled water to remove exceed reactants and impurities. TEMPO-oxidized cellulose hydrogels were obtained after washing steps and were dispersed in 800 mL of acetate buffer (pH 4.8). 9.1 g of sodium chlorite was added in order to oxidize remaining aldehyde groups to carboxyl groups. After washing the resulted oxidized cellulose fibers with excess of distilled

7



Page 8 of 36

water, and adjust the concentration to 0.4 wt% for fibrillation treatment. Briefly, 0.4 wt% oxidized cellulose in aqueous solutions were fibrillated using high speed mixer (MX1200XT, Xtreme Hi-Power Blender, Waring, TX, USA) at 22000 rpm for 10 min and TEMPO oxidized cellulose nanofibers (TOCNs) were obtained. Preparation of COL, TOCN and COL/TOCN blends We prepared COL, TOCN, and COL/TOCN (1:1 weight ratio) sheets at different pH conditions by casting method. Before casting, pH of aqueous dispersion was adjusted by adding 0.02 M hydrochloric acid solution and 0.02 M ammonium solution. The dispersion were controlled at various pH (3, 5, 7, 9, and 11). The dispersion at various pH were poured into polytetrafluoroethene (PTFE) dishes and dried at 10 ºC, then sample sheets were obtained. Characterizations Fourier transform infrared spectrophotometer (Perkin Elmer, Ltd., Spectrum GX FT-IR System I-KS) was used for the investigation of functional groups of each sample. The dried (under vacuum oven at 40 ºC for 48 h) samples were dispersed into KBr pellets including COL, TOCN and COL/TOCN. The prepared samples were measured at a range between 4000-400 cm-1 with 2 cm-1 resolutions for 10 times

8


Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


accumulation. Conductometric titration was performed and the conversion ratio of carboxyl groups on the surface of TOCNs were evaluated. Frozen TOCN hydrogels were dried under vacuum for 1 day to obtain cryogel. Cryogel (0.1 g) was then dispersed in distilled water with addition of sodium chloride (NaCl) solution under stirring. The density of carboxyl group in prepared TOCNs was then calculated from the amount of sodium hydroxide consumed during the coordination of sodium ions to carbonyl groups by following equation (1). Carboxyl group density (mmol/g)=

(1)

Atomic force microscope (Nano Navi E-sweep, Hitachi, Tokyo, Japan) was used for the observation of TOCN. The nanofibers on the mica substrates were observed with a dynamic mode AFM in topological images and phase images of TOCNs. X-ray diffraction profiles were obtained using RINT-Ultima + 2200 (Rigaku Co. Ltd.) with a symmetrical reflection geometry. CuKα beam (λ = 1.5418 Å) was generated at 40 kV and 20 mA. The scanning speed was 2º/ min, and the step sampling was 0.02º. The X-ray beam was also irradiated on the sample from the edge direction to

9



Page 10 of 36

obtain out-of-plane orientation images on an imaging plate (camera distance: 64 mm). The degree of the crystallite orientation of COL, TOCN, and COL/TOCN samples were calculated from intensity distribution curve along the Debye-Scherrer ring using the following equation (2). π = (180° − H°)/180°

(2)

where H° is the half-width of the intensity distribution curve for the diffraction. UV-vis absorbance measurements were carried out with a Jasco V-750 UV/Vis spectrometer. The scan speed was 400 nm/min. Density were measured by floatation method with benzene (d= 0.88 g/cm3), tetrachloromethane (d= 1.60 g/cm3), and tetrabromoethane (d= 2.97 g/cm3) at 30 ºC. The morphology of COL, TOCN, and COL/TOCN blend sheets were observed using a scanning electron microscope (SEM, JSM-7500F; JEOL) at 7 kV. All prepared sheets were vacuum dried (40 ºC) for 24 h, then coated with osmium prior to the observation. Mechanical properties All the sheets were cut into rectangles with 30 mm length and 5 mm width, then vacuum dried (40 ºC) for 6 h. Tensile tests were performed with a tensile tester

10


Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Autograph, AGS-1kND, Shimadzu Co.) equipped with a 500 N load cell. Specimens were tested at a cross-head speed of 20 mm/ min, which corresponds to an initial strain rate of 0.1 min-1. The relative humidity was kept at 50% and the temperature at 23 °C. The numbers of tested specimens were 5 for each cast sample. The average values and standard deviations of Young’s modulus and tensile strength σmax were evaluated. Wet samples of the kinds of cast sheet were also tested. The wet samples were prepared by immersing the dried samples into deionized water for 1 min. In Vitro Cell Adhesion Test The sheets were sterilized by UV irradiation for about 3 h. After that, the sterilized sheets were washed 3 times with PBS, followed by rinsing with culture medium (α-MEM) for 30 min. Arround 4 x 103 cells were seeded onto each 10 mm × 10 mm square sheet and cultured in the incubator under humidified condition with 5% CO2 at 37 ºC for an hour. After all seeded sheets were washed with PBS for removing of dead cells, cells on the cast sheets were incubated for 1, 5, 10, and 15 days. To count the numbers and densities of cells on COL, TOCN, and COL/TOCN sheets, paraformaldehyde

(PFA)

Phalloidin-Rhodamine

was

and

cell

applied

and

nuclei

with

actin

was

stained

with

4',6-diamidino-2-phenylindole,

11



Page 12 of 36

dihydrochloride (DAPI), respectively.

12


Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS & DISCUSSION We prepared TOCNs from bast fibers of kenaf through TEMPO-mediated oxidation and nanofibrillization. The obtained TOCNs were characterized by FT-IR, conductometric titration and AFM as shown in Figure 1. Form IR spectra, the increase of carboxyl absorption indicated that the hydroxyl groups at 6-position of cellulose were translated to carboxyl groups. The concentration of carboxyl groups was 1.41 mmol/g vs glucopyranose unit. This indicated that, in the 6×6 cellulose fibril model, 82% hydroxyl groups at 6-positions on the cellulose surface were oxidized. The degree of oxidation per hydroglucose unit of cellulose (DO) was 0.23. From AFM images, the diameters of TOCNs were about 4-5 nm and those length were several micrometers.

Figure 1. Characterizations of TOCNs by (a) FT-IR, (b) electric conductometance by 13



Page 14 of 36

conductometric curve methodology and (c) AFM image. We prepared COL, TOCN and COL/TOCN blends at various pH (pH 3, 5, 7, 9, and 11) by casting at 10 ºC. Figure 2 shows the transmittance of TOCN and COL/TOCN blend sheets. For TOCN cast sheets, the transmittance were decreased at pH 3. Under acidic condition, the sodium carboxylate groups were protonated to carboxyl groups and TOCNs were agglomerated, which brought light scattering in the TOCN sheets prepared at pH 3. Under higher pH conditions, the transparency of TOCN cast sheets were kept high. However, all the COL/TOCN blends cast under lower pH conditions were more turbid relative to both COL and TOCN sheets. These results suggested that COL and TOCN in the blends were separated with each component.

Figure 2. UV-vis transmittance spectra of COL, TOCN and COL/TOCN blend sheets cast at pH 3-11. (thickness 100 µm)

14


Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. FT-IR spectra of (a) COL, TOCN, and COL/TOCN sheets cast at pH 3-11. (b) Stretching of O-H and N-H at 3200-3600 cm-1 was magnified to examine each COL/TOCN cast sheets at different pH conditions.

For the investigation of the pH effect on the structure of COL/TOCN blends, FT-IR spectra of COL, TOCN and COL/TOCN blends prepared under various pH conditions were measured. Figure 3 shows FT-IR spectra of COL/TOCN blends and Figure S2 in Supporting Information shows those of COL and TOCN prepared under various conditions, respectively. In the FT-IR spectra of all the COL/TOCN blends, the bands originated from O–H and N–H stretching, C–H stretching, C=O stretching and amide stretching vibration were observed at around 3400 cm−1, 2950 cm−1, 1650 cm−1 and 1550 cm−1, respectively. All the functional groups of COL and TOCN components were remained even in COL/TOCN blends. The O–H and N–H stretching bands of COL/TOCN blends under higher pH conditions were shifted to the side of

15



Page 16 of 36

lower wavenumber. These shifts were also observed in the spectra of COL sheets under higher pH conditions, as shown in Figure S2a in Supporting Information. In particular, that of COL/TOCN prepared at pH 7 was observed at 3370 cm−1, which was the lowest relative to others. In addition, the C=O bands of COL/TOCN blends prepared at pH 7 were shifted to 1665 cm−1 (that of TOCN at 1615 cm−1). These results suggested that the carboxyl groups of TOCN would interact with amine groups of COL.

Figure 4. X-ray diffraction profiles of COL/TOCN sheets cast at pH 3-11 conditions.

In order to further confirm pH effects on structures of COL, TOCN and COL/TOCN blends, their X-ray diffraction measurements were performed. Figure 4 shows X-ray diffraction profiles of COL/TOCN blend samples. COL sheets prepared 16


Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


at various pH showed two diffraction peaks at 2θ = 8° and 16°, as shown in Figure S3a of Supporting Information. Their peaks were originated from inter and intra triple helical structures of COL, respectively. No shift and change of relative intensity of their peaks were observed. Regardless of isoelectric points of COL at pH 7, the structure of COL had no dependence on pH condition. Figure S3b of Supporting Information shows X-ray diffraction profiles of TOCN sheets prepared at various pH conditions. In X-ray diffraction profiles of all the TOCN sheets, the diffraction peaks at 15° and 22° were observed. They were indexed as (110)/(1−10) and (200) plane of cellulose I, respectively. There was no apparent difference between TOCN sheets at various pH. This indicates that cellulose I was stable at any pH conditions. In Figure 4, the X-ray diffraction profiles of COL/TOCN blends prepared at pH 3-11 are shown. The results also revealed no definitive difference of cellulose peak between each sample, suggesting that crystalline structures of cellulose I were remained at different pH. In contrast, the intensities of COL peaks increased with the increasing of pH (pH ≥9), and at lower pH (pH < 7), the relative intensities of diffraction peaks were drastically decreased. This means that, under acidic condition at

17



Page 18 of 36

lower pH than isoelectric point of collagen, the triple helix structure of collagen might be dstroyed by the interaction with TOCN. In this study, all the sheets were prepared in the casting method. In their sheets, the crystallites were aligned in the direction parallel to sheet surface. In order to investigate the orientation of their crystallites, we took X-ray diffraction photograph from the edge direction of sample sheets, as shown in Figure 5. The oriented diffractions from inter and intra helix of COL, and those from (110)/(1−10) plane of cellulose I were observed in the COL/TOCN sheets. Degree of the crystallite orientation of COL and TOCN were summarized in Table 1. The COL and TOCN showed relatively lower orientation at pH 3, whereas higher orientation were observed for the COL/TOCN (>pH 5). Under acidic condition, the dispersity of TOCN in water was decreased and partially gelation was occurred. Therefore, in COL/TOCN blends prepared under acidic condition, the blend sheets were fabricated before completely alignment of COL and TOCN. These led to lower crystallite orientation of COL and TOCN.

Figure 5. X-ray diffraction photographs at cross-section sliced COL/TOCN blends at various pH conditions 18


Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Orientation of COL and TOCN crystallites in COL/TOCN blends cast at pH 3-11.

COL Orientation

TOCN Orientation

%

%

COL/TOCN pH3

81

82

COL/TOCN pH5

88

89

COL/TOCN pH7

89

90

COL/TOCN pH9

90

91

COL/TOCN pH11

90

89

Figure 6 shows scanning electron microscopic (SEM) images to observe cross-section sliced COL/TOCN blend sheets Morphologies of blend sheets were dramatically changed depends on pH conditions. Sheets prepared at pH 3 showed random structures at the sliced surfaces, which may result from the aggregation of TOCN as mentioned above part. In the case at other pH conditions, it was observed that the layered structures were aligned along their surface planes in their sheets. These results were coincident with those of X-ray diffraction profiles.

19



Page 20 of 36

Figure 6. Cross-sectional SEM images of COL/TOCN blends cast at (a) pH3, (b) pH5, (c) pH7, (d) pH9, and (e) pH11.

Figure 7. Stress–strain curves of COL, TOCN, and COL/TOCN blend sheets prepared at pH 3-11 in dry state.

Stress-stain curves of COL, TOCN, and COL/TOCN blend sheets at different pH in dry states are shown in Figure 7. Under all the pH conditions, COL/TOCN blends 20


Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


showed higher Young’s modulus than COL and lower modulus than TOCN. On the other hand, the elongation at break of COL/TOCN blends were improved and the tensile strength was keeping constants, compared to the reference TOCN. The reason is for the in-plane orientation of triple helix collagen and crystalline structure of cellulose I in TOCN. As the pH increased, both tensile strength and elongation at break of COL/TOCN sheets increased. This is considered to be that the amine groups of COL interact with carboxylate groups of TOCN under basic condition, remaining the triple helix structure of COL and crystalline structure of TOCN. The obtained values of tensile strength was superior to PLLA (around 60 MPa), a conventional biocompatible polymer.

Figure 8. Stress–strain curves in wet state of COL, TOCN and COL/TOCN blends cast at pH3-11.

21



Page 22 of 36

In order to evaluate the utility as a suitable scaffold material for cell culture, mechanical properties of COL, TOCN, and COL/TOCN blends were investigated under wet condition. The tensile tests were performed for the sheets after immersing in deionized water for 1 min. Figure 8 shows stress–strain curves of COL, TOCN and COL/TOCN blends in wet state. For COL sheets, Young’s modulus dropped from 3.5 GPa to 4.0 MPa ,and tensile strength decreased from 142 to 3.5 MPa after immersing in water. These abrupt decreases brought COL difficult to be used as a cell culture material. The mechanical properties of TOCN sheets also decreased in wet states from 9.2 GPa to 0.11 GPa (Young’s modulus) and 211 MPa to 27 MPa (tensile strength), respectively, which would be attributed to the weakening of intermolecular hydrogen bonding. Regarding COL/TOCN blends sheets at various pH conditions, all the sheets possessed higher tensile strength than COL sheets. In particular, blend sheets prepared at pH 7 showed the highest Young’s modulus and tensile strength. Actually, their tensile strength were much larger than those of gel scaffolds (< 1 MPa).

22


Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. NHDFs cultured on COL, TOCN and COL/TOCN blends cast at pH 3-11 (a) at day 15 were stained by DAPI to confirm growth condition and cell density. (b) Cell densities of all samples for consecutive 15-day incubation were calculated to compare the difference between each sample.

Important factor as a scaffold material for cell growth is not only mechanical properties of sheets, but also biocompatibility of prepared sheets. Normal human dermal fibroblasts (NHDFs) were seeded on COL, TOCN, and COL/TOCN blend sheets for 15 days to evaluate their toxicity as shown in Figure S9 of Supporting Information. Cells on sheets were fixed and stained with fluoresce for counting the number of cells. Figure S10 in Supporting Information shows cell densities 1, 5, 10 and 15 days after NHDFs were cultured on COL sheets. There was no obvious difference after 10 and 15 days incubation. In addition, no dependence of pH condition in preparation of COL sheets were observed. In contrast, NHDFs were

23



Page 24 of 36

cultured slightly on TOCN sheets. The cultivation on COL/TOCN blends were also carried out for 15 days. Figure 9a shows NHDFs cultured on COL, TOCN, and COL/TOCN blends at day 15. COL/TOCN blends prepared at higher pH showed higher affinity for cell incubation. Cell densities of all samples with different pH conditions for 0, 5, 10 and 15 days are shown in Figure 9b. For the first week of culture, there was no significant difference between samples. COL sheets showed the higher cell densities after 10-day incubation, compared with those of TOCN and COL/TOCN blends. Cell densities of NHDFs on COL/TOCN blends were increased at high pH condition, which corresponded to native extracellular matrix in our body. On COL/TOCN prepared at pH 7, cell proliferation was most rapidly progressed.

24


Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. Cell viabilities of NHDFs on (a) COL, (b) TOCN and (c-g) COL/TOCN blends cast at pH3-11 for 15 days.

Cell viability of NHDFs on all prepared sheets were evaluated by LIVE/DEAD assay. Living cells can be labelled as green fluorescence, and dead cells are labelled into red fluorescence. Figure 10 shows the results after 15-day incubation on COL, TOCN and COL/TOCN blends. All the COL/TOCN blends possessed higher biocompatibility relative to TOCN sheets. Cells seeded on COL/TOCN blends at pH 3, 5, and 7 blend sheets showed relatively red fluorescence, whereas, in COL/TOCN

25



Page 26 of 36

blends at pH 9 and 11, their surface were covered with living cells, as shown in Table S6 in the Supporting Information. The cell viabilities in COL/TOCN blends at pH 9 and 11 were comparable with that of collagen as a conventional ECM. These viabilities would be achieved by the proper electrostatic interaction between collagen and cellulose nanofibers under basic condition and high swelling ratio of the COL/TOCN. Therefore, the COL/TOCN blends prepared under basic condition possessed high biocompatibilities as well as high mechanical properties. It is concluded that the COL/TOCN blends were promising materials as scaffolds in 3D tissue engineering.

26


Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSIONS COL, TOCN, and COL/TOCN blends prepared at various pH were characterized with FTIR, X-ray diffraction, and UV—vis spectra as well as SEM observations, tensile tests. UV-vis spectra indicated higher dispersion of nanofibers in the sheets under alkaline condition. From the X-ray diffraction measurements, fibrillation of collagen and the crystallites of cellulose were clarified. X-ray diffraction and SEM results also proved that the orientation of TOCN induced COL fibril orientation. In addition, from the results of tensile tests and cell incubation, COL/TOCN blend sheets prepared at pH 11 possessed the high biocompatibility and high mechanical strength. In this study, we achieved COL/TOCN blend sheets as scaffold materials with high biocompatibility, low cell toxicity, and proper mechanical performance.

27



Page 28 of 36

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXXXXX. The Supporting Information included Purification and TEMPO oxidation procedures, FT-IR spectra of COL and TOCN, X-ray diffraction profiles of COL and TOCN, X-ray diffraction photographs of COL and TOCN, Strain–stress curves of COL and TOCN in dry and wet states, Mechanical parameters of COL and TOCN in dry and wet states, Incubation methods of cells, Fibroblast cell densities cultured on COL, Cell viabilities cultured on COL, TOCN, and COL/TOCN blends.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing financial interest.

28


Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGMENTS This study was supported by the Core Research for Evolutional Science and Technology (CREST, JPMJCR13B2), and Japan Science and Technology Agency (JST).

29



Page 30 of 36

REFERENCE (1)

Badylak, S. F. Regenerative Medicine and Developmental Biology: The Role of the Extracellular Matrix. Anat. Rec. - Part B New Anat. 2005, 287 (1), 36– 41.

(2)

Upadhyay, R. K. Role of Regeneration in Tissue Repairing and Therapies. J.

Regen. Med. Tissue Eng. 2015, 4 (1), 1. (3)

Sasagawa, T.; Shimizu, T.; Sekiya, S.; Haraguchi, Y.; Yamato, M.; Sawa, Y.; Okano, T. Design of Prevascularized Three-Dimensional Cell-Dense Tissues Using a Cell Sheet Stacking Manipulation Technology. Biomaterials 2010, 31 (7), 1646–1654.

(4)

Asakawa, N.; Shimizu, T.; Tsuda, Y.; Sekiya, S.; Sasagawa, T.; Yamato, M.; Fukai, F.; Okano, T. Pre-Vascularization of in Vitro Three-Dimensional Tissues Created by Cell Sheet Engineering. Biomaterials 2010, 31 (14), 3903– 3909.

(5)

Sakaguchi, K.; Shimizu, T.; Okano, T. Construction of Three-Dimensional Vascularized Cardiac Tissue with Cell Sheet Engineering. J. Control. Release 2015, 205, 83–88.

(6)

Yang, J.; Yamato, M.; Kohno, C.; Nishimoto, A.; Sekine, H.; Fukai, F.; Okano, T. Cell Sheet Engineering: Recreating Tissues without Biodegradable Scaffolds.

Biomaterials 2005, 26 (33), 6415–6422. (7)

Papavasiliou, G.; Turturro, M. V.; Christenson, M. Synthetic PEG Hydrogel Extracellular Matrix Mimics for the Vascularization of Engineered Tissues.

Cardiovasc. Pathol. 2013, 22 (3), e31–e32. (8)

DeQuach, J. A.; Lin, J. E.; Cam, C.; Hu, D.; Salvatore, M. A.; Sheikh, F.; Christman, K. L. Injectable Skeletal Muscle Matrix Hydrogel Promotes Neovascularization and Muscle Cell Infiltration in a Hindlimb Ischemia Model.

Eur. Cell. Mater. 2012, 23, 400–412. (9)

Moon, J. J.; Saik, J. E.; Poché, R. A.; Leslie-Barbick, J. E.; Lee, S.-H.; Smith, A. A.; Dickinson, M. E.; West, J. L. Biomimetic Hydrogels with Pro-Angiogenic Properties. Biomaterials 2010, 31 (14), 3840–3847.

(10)

Sung, K. E.; Su, G.; Pehlke, C.; Trier, S. M.; Eliceiri, K. W.; Keely, P. J.; Friedl, A.; Beebe, D. J. Control of 3-Dimensional Collagen Matrix Polymerization for Reproducible Human Mammary Fibroblast Cell Culture in Microfluidic Devices. Biomaterials 2009, 30 (27), 4833–4841.

(11)

Tien, J. Microfluidic Approaches for Engineering Vasculature. Curr. Opin. 30


Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chem. Eng. 2014, 3, 36–41. (12)

Choi, N. W.; Cabodi, M.; Held, B.; Gleghorn, J. P.; Bonassar, L. J.; Stroock, A. D. Microfluidic Scaffolds for Tissue Engineering. Nat. Mater. 2007, 6 (11), 908–915.

(13)

Akiyama, H.; Ito, A.; Kawabe, Y.; Kamihira, M. Genetically Engineered Angiogenic Cell Sheets Using Magnetic Force-Based Gene Delivery and Tissue Fabrication Techniques. Biomaterials 2010, 31 (6), 1251–1259.

(14)

Zheng, Y.; Chen, J.; Craven, M.; Choi, N. W.; Totorica, S.; Diaz-Santana, a.; Kermani, P.; Hempstead, B.; Fischbach-Teschl, C.; Lopez, J. a.; et al. From the Cover: In Vitro Microvessels for the Study of Angiogenesis and Thrombosis.

Proc. Natl. Acad. Sci. 2012, 109 (24), 9342–9347. (15)

Hollister, S. J. Porous Scaffold Design for Tissue Engineering. Nat. Mater. 2005, 4 (July), 518–524.

(16)

Chen, G.; Ushida, T.; Tateishi, T. Scaffold Design for Tissue Engineering.

Macromol. Biosci. 2002, 2 (2), 67–77. (17)

O’Brien, F. J. Biomaterials & Scaffolds for Tissue Engineering. Mater. Today 2011, 14 (3), 88–95.

(18)

Nishiguchi, A.; Yoshida, H.; Matsusaki, M.; Akashi, M. Rapid Construction of Three-Dimensional Multilayered Tissues with Endothelial Tube Networks by the Cell-Accumulation Technique. Adv. Mater. 2011, 23 (31), 3506–3510.

(19)

Liu, C.-Y.; Matsusaki, M.; Akashi, M. Control of Vascular Network Location in Millimeter-Sized 3D-Tissues by Micrometer-Sized Collagen Coated Cells.

Biochem. Biophys. Res. Commun. 2016, 472 (1), 131–136. (20)

Liu, C.-Y.; Matsusaki, M.; Akashi, M. The Construction of Cell-Density Controlled Three-Dimensional Tissues by Coating Micrometer-Sized Collagen Fiber Matrices on Single Cell Surfaces. RSC Adv. 2014, 4 (86), 46141–46144.

(21)

Sherman, V. R.; Yang, W.; Meyers, M. A. The Materials Science of Collagen.

J. Mech. Behav. Biomed. Mater. 2015, 52, 22–50. (22)

Shoulders, M. D.; Raines, R. T. Collagen Structure and Stability. Annu Rev

Biochem 2010, 78, 929–958. (23)

Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de. Endogenous Collagen Influences Differentiation of Human Multipotent Mesenchymal Stromal Cells. Tissue Eng Part A. 2010, 16 (5), 1693–1702.

(24)

Fernandes, H.; Dechering, K.; Someren, E. Van; Steeghs, I.; Apotheker, M.; Leusink, A.; Bank, R.; Janeczek, K.; Blitterswijk, C. Van; Boer, J. de. The Role 31



Page 32 of 36

of Collagen Crosslinking in Differentiation of Human Mesenchymal Stem Cells and MC3T3-E1 Cells. Tissue Eng Part A. 2009, 15 (12), 3857–3867. (25)

Roeder, B. A.; Kokini, K.; Sturgis, J. E.; Robinson, J. P.; Voytik-Harbin, S. L. Tensile Mechanical Properties of Three-Dimensional Type I Collagen Extracellular Matrices With Varied Microstructure. J. Biomech. Eng. 2002, 124 (2), 214–222.

(26)

Isogai, A.; Saito, T.; Fukuzumi, H. TEMPO-Oxidized Cellulose Nanofibers.

Nanoscale 2011, 3 (1), 71–85. (27)

Saito, T.; Isogai, A. TEMPO-Mediated Oxidation of Native Cellulose. The Effect of Oxidation Conditions on Chemical and Crystal Structures of the Water-Insoluble Fractions. Biomacromolecules 2004, 5 (5), 1983–1989.

(28)

Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Cellulose Nanofibers Prepared by TEMPO-Mediated Oxidation of Native Cellulose. Biomacromolecules 2007,

8 (8), 2485–2491. (29)

Takano, Y.; Turner, C. H.; Owan, I.; Martin, R. B.; Lau, S. T.; Forwood, M. R.; Burr, D. B. Elastic Anisotropy and Collagen Orientation of Osteonal Bone Are Dependent on the Mechanical Strain Distribution. J. Orthop. Res. 1999, 17 (1), 59–66.

(30)

Riggs, C. M.; Vaughan, L. C.; Evans, G. P.; Lanyon, L. E.; Boyde, A. Mechanical Implications of Collagen Fibre Orientation in Cortical Bone of the Equine Radius. Anat. Embryol. (Berl). 1993, 187 (3), 239–248.

(31)

Martin, R. B.; Lau, S. T.; Mathews, P. V.; Gibson, V. A.; Stover, S. M. Collagen Fiber Organization Is Related to Mechanical Properties and Remodeling in Equine Bone. A Comparsion of Two Methods. J. Biomech. 1996, 29 (12), 1515–1521.

(32)

Portigliatti Barbos, M.; Bianco, P.; Ascenzi, A.; Boyde, A. Collagen Orientation in Compact Bone: II. Distribution of Lamellae in the Whole of the Human Femoral Shaft with Reference to Its Mechanical Properties. Metab.

Bone Dis. Relat. Res. 1984, 5 (6), 309–315. (33)

Nobuta, K.; Teramura, H.; Ito, H.; Hongo, C.; Kawaguchi, H.; Ogino, C.; Kondo, A.; Nishino, T. Characterization of Cellulose Nanofiber Sheets from Different Refining Processes. Cellulose 2016, 23 (1), 403–414.

32


Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract

33



338x316mm (96 x 96 DPI)


Page 34 of 36

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


338x316mm (96 x 96 DPI)



338x316mm (96 x 96 DPI)


Page 36 of 36

Cellulose Nanofiber Blend Scaffolds Prepared at Various pH

Recommend Documents