Plant-derived Nanocellulose as Structural and Mechanical

Subscriber access provided by Nottingham Trent University

Article

Plant-derived Nanocellulose as Structural and Mechanical Reinforcement of Freeze-Cast Chitosan Scaffolds for Biomedical Applications Kaiyang Yin, Prajan Divakar, and Ulrike Gesa K WEGST Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00784 • Publication Date (Web): 27 Aug 2019 Downloaded from pubs.acs.org on August 28, 2019

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 57 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

Biomacromolecules

Plant-derived Nanocellulose as Structural and Mechanical Reinforcement of Freeze-Cast Chitosan Scaffolds for Biomedical Applications Kaiyang Yin, Prajan Divakar, Ulrike G.K. Wegst Thayer School of Engineering, Dartmouth College, Hanover, NH, USA KEYWORDS: structure-property-processing correlation, cooling rate, anisotropy, confocal, compression.

ABSTRACT

Despite considerable recent interest in micro- and nanofibrillated cellulose as constituents of lightweight structures and scaffolds for applications that range from thermal insulation to filtration, few systematic studies have been reported to date on structure-property-processing correlations in freeze-cast chitosan-nanocellulose composite scaffolds, in general, and their application in tissue regeneration, in particular. Reported in this study are the effects of the addition of plant-derived nanocellulose fibrils (CNF), crystals (CNC) or a blend of the two (CNB) to the biopolymer chitosan on structure and properties of the resulting composites. Chitosan-nanocellulose composite scaffolds were freeze cast at 10°C/min and 1°C/min, and their microstructures were quantified in both the dry and fully-hydrated states using scanning electron and confocal microscopy,

ACS Paragon Plus Environment

Biomacromolecules 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 57

respectively. The modulus, yield strength, toughness (work to 60% strain) were determined in compression parallel and the modulus also perpendicular to the freezing direction to quantify anisotropy. Observed were the preferential alignment of nanocellulose crystals and/or fibrils parallel to the freezing direction. Additionally, observed was the self-assembly of the nanocellulose into micro-struts and micro-bridges between adjacent cell walls (lamellae), features that affected the mechanical properties of the scaffolds. When freeze cast at 1°C/min, chitosannanocellulose fibril scaffolds had the highest modulus, yield strength, toughness, and smallest anisotropy ratio, followed by chitosan and the composites made with the nanocellulose blend, and that with crystalline cellulose. These results illustrate that the nanocellulose additions homogenize the mechanical properties of the scaffold through cell-wall material self-assembly, on the one hand, and add architectural features such as bridges and pillars, on the other. The latter transfer loads and enable the scaffolds to resist deformation also perpendicular to the freezing direction. The observed property profile and the materials’ proven biocompatibility highlight the promise of chitosan-nanocellulose composites for a large range of applications, including those for biomedical implants and devices.


2

Page 3 of 57 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

Biomacromolecules

1.

Introduction Surprisingly few systematic studies, to date, explore the promise of chitosan-nanocellulose

composite scaffolds for biomedical applications; this is in contrast to a considerable body of literature on chitosan, and bacterial and plant-based cellulose scaffolds1–8 and hydrogels 9–15. The nanocellulose-chitosan composite scaffolds assessed in the framework of this study, are an attractive advancement that takes the concept of chitosan-16–20 and chitosan-nanocellulose cellular solids21–29 to the next level. Such porous materials emulate many attractive features that provide natural composites30–32 and hybrid materials26,27,33–35 with exceptional structural, mechanical, and functional properties.32,36–38 With the biocompatibility of pure chitosan,39–43 pure cellulose,44,45 and chitosan-46–49 and cellulose-containing composites9,12,50–54 already established, we focus in this systematic study of structure-property-processing correlations on highly porous materials made from the two most abundant polysaccharides chitosan (Chi) and plant-based nanocellulose, the latter in the form of nanocellulose crystals (CNC), nanocellulose fibrils (CNF), or a blend of the two (CNB) embedded in the chitosan matrix. The development of the chitosan-nanocellulose composite scaffolds was motivated by our studies of chitosan-based scaffolds for peripheral nerve repair,17,18,20,55–62 which provide with their chitosan matrix a regenerative environment for biomedical applications and, through the addition of nanocellulose, not only attractive additional nano- and microtopographical structures, but also mechanical properties that allow them to be handled with ease during surgery. Chitosan, the deacetylated form of the polysaccharide chitin, was chosen as the composite matrix, because of its well established biocompatibility and attractive structural, mechanical, and chemical properties, which are primarily determined by the polymers’ molecular weight and degree of deacetylation63,64, processing conditions, and moisture content.65,66


3


Page 4 of 57

Freeze-cast chitosan and its composites with other biopolymers, such as nanocellulose, are attractive for biomedical applications in general, and peripheral nerve repair, in particular, due to their highly aligned porosity and additional micro-and nanostructural features on the cell wall surfaces.17,18,20,55–62 Since no enzyme exists in the human body to digest cellulose, a chitosannanocellulose scaffold will degrade slowly and thus be attractive for the regeneration of large nerve gaps (>50 mm).23 Freeze casting, the directional solidification of typically water-based solutions and slurries, was chosen as the process for the manufacture of the chitosan-nanocellulose composite scaffolds, because of its versatility for the custom-design and production of hierarchically-structured materials with complex architectures and well-defined pore morphologies (Figure 1A).32,67,68 Factors such as chitosan properties and concentration, nanocellulose type used, viscosity, and pH of the solution and slurry combined with the applied cooling direction and rate define ice crystal growth, velocity, and instability formation parallel to the crystal growth direction, and through these the ice-templated pore morphology of the freeze-cast scaffold at both the micro- and nanoscopic length scales.18 During solidification, diffusion-controlled and shear-flow-driven selfassembly processes occur that determine the preferential alignment of molecules,

69,70

fibers and

crystals, bridge and pillar formation, and through these both the cell wall and overall scaffold structure and properties.71,6,72 Important for tissue regeneration, but not yet established for chitosan-nanocellulose composites, are structure-property-processing correlations that will enable the custom-design of scaffolds with the desired pore morphology and cell wall structure, and mechanical performance for a given application. Of great interest is also, how the freezing conditions, particularly the applied cooling


4

Page 5 of 57 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

Biomacromolecules

rate, affect the nanocellulose self-assembly in the chitosan matrix, depending on particle morphology (crystal, fibril, blend).6,73,74 Presented in this study, are the structural and mechanical properties of pure chitosan (Chi) and Chi-CNC, Chi-CNB, and Chi-CNF composites freeze cast with applied cooling rates of 10°C/min and 1°C/min. The resulting scaffolds were tested in compression both parallel and perpendicular to the freezing direction. New structure-property-processing correlations were discovered.

Figure 1. (A) Schematic of the freeze casting process used to create aligned porosities parallel to the axis of the cylindrical mold; (B) Fibrillation formation at the freezing front; (C) Ridged structure ice-templated by instabilities during ice crystal growth, adapted from Donius et al.75 (D) Pore structure of a Chi-CNF scaffold (scale bar:100 µm).


5


2.

Materials and Methods

2.1

Materials

Page 6 of 57

Chitosan (Chi, 95% degree of deacetylation, 150-300 kDa; Heppe Medical Chitosan GmbH, Germany) was used as received; three different types of nanocellulose suspensions (BioPlus® hydrophilic cellulose, American Process, Atlanta, GA): nanofibrils (CNF, 5-20 nm diameter, 500 nm length, 3% w/w), nanocrystals (CNC, 4-5 nm diameter, 50-500 nm length, 6% w/w), and nanoblend (CNB: CNF and CNC mixed in 2:3 ratio, 3% w/w) were used as received76; and sodium hydroxide (anhydrous, reagent grade, Sigma Aldrich, St. Louis, MO, USA), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC, Sigma Aldrich, St. Louis, MO, USA), acetic acid (Glacial, ACS grade, EMD Millipore, Burlington, MA, USA), phosphate buffered saline (PBS, biotechnology grade; VWR, Radnor, PA, USA), and 200 proof ethanol (VWR, Radnor, PA, USA) were used as received.

2.2

Slurry Preparation

The CNF, CNB, and CNC suspensions were diluted to 1% w/v in deionized water. Chitosan was dissolved in 1.5% v/v acetic acid (0.265 M) in deionized water to prepare a 2.4% w/v chitosan solution. The chitosan solution was mixed with the diluted CNF, CNB or CNC suspension in a 50:50 volume ratio, respectively, by sonicating at high power for 20 minutes (1500A-MT, VWR, Radnor, PA, USA), and homogenizing in a Speed Mixer (FlackTek, Landrum, SC, USA) at 2000 rpm for 2 minutes. To obtain pure chitosan scaffolds, the chitosan solution was diluted to 1.7% w/v. The overall polymer mass percentage in the composite slurries was 1.7% w/v. The pH values of the chitosan solution and chitosan-nanocellulose slurries were determined at 23°C using


6

Page 7 of 57

a pH meter (Orion Star A111, ThermoFisher, Waltham, MA, USA). The viscosities of the chitosan solution and chitosan-nanocellulose slurries were determined with a Brookfield DVE viscometer (Brookfield, Middleboro, MA, USA) with spindle 63 at 100 rpm at 4°C. The Brookfield viscosities of the slurries were determined to be h = 846 cP for Chitosan (pH = 4.19), in good agreement with the manufacturer’s specifications, h = 1122 cP for Chi-CNF (pH = 4.23), h = 445 for Chi-CNB (pH = 4.31), and h = 311 cP for Chi-CNC (pH = 4.25). The hydrodynamic radius of the stored chitosan was determined by dynamic light scattering (DLS, DynaPro Nanostar, Wyatt Technology, Santa Barbara, CA, USA) after first diluting the 1.7% w/v chitosan solution to 0.1% w/v with 1% acetic acid, averaging ten acquisitions of 60s at 20°C. The hydrodynamic radius of the chitosan was found to be 580 nm (Figure 2), a value that agrees well with previous observations;77,78 two weaker peaks presented around 70 nm and 5 nm, represent the degraded portion during one week of storage.

Chitosan

70 60

Intensity %

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

Biomacromolecules

50 40 30 20 10 0

1

10

100

1000

10000

RH (nm)

Figure 2. Hydrodynamic radius of chitosan measured by DLS (DynaPro Nanostar, Wyatt Technology, Santa Barbara, CA, USA) of ten acquisitions at 20°C.


7


Page 8 of 57

All slurries were stored at 4°C for the one week after preparation, and sonicated and homogenized, again, directly before freeze casting.

2.3

Freeze Casting

The scaffolds were freeze cast in a system detailed in Wegst et al. (2010, 2015) (Figure 1A).32,68 Briefly, a polytetrafluoroethylene (PTFE) tube (25.4 mm outer, 20 mm inner diameter, 50 mm height) was sealed with a copper mold bottom and filled with 12.5 mL of the solution or slurry using a syringe. The mold was then placed with its copper bottom on a liquid nitrogen cooled copper cold finger whose temperature is PID controlled. The molds were equilibrated to 4°C for 10 min before a cooling rate of either 10°C/min or 1°C/min was applied until the mold reached a temperature of -150°C. The frozen slurries were then demolded with an Arbor press and lyophilized (FreeZone 6 Plus, Labconco, Kansas City, MO, USA) for 72 hours at 0.008 mbar and a coil temperature of -85°C.

2.4

Scanning Electron Microscopy

Transverse and longitudinal scaffolds cross sections were prepared with a razor blade (Astra Superior Platinum Double Edge) at a height of 28 mm measured from the scaffold bottom. Scanning electron microscopy (SEM) was performed on non-neutralized samples without prior application of a conductive coating (Vega 3, Tescan, Brno-Kohoutovice, Czech Republic). High resolution micrographs were obtained, again, on non-neutralized, uncoated cell wall membranes removed from the scaffolds by peeling under a stereomicroscope (M205C, Leica Microsystems Inc., Buffalo Grove, IL, USA).


8

Page 9 of 57 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

Biomacromolecules

2.5

Laser Scanning Confocal Microscopy

For imaging by confocal microscopy, the scaffolds were first neutralized in 0.4% w/v NaOH in 95% EtOH for 15 minutes, followed by three 15-minute washes in deionized water. In a second step, the samples were stained following a previously reported method, which does not affect the scaffold structure.56 Briefly, samples were for 6 hours soaked in 0.05M EDC and 0.05 mg/mL fluorescein sodium salt in PBS solution, followed by three washes with sample palpitation in deionized water. All liquids were filtered through a 0.2 µm PTFE filter before use, and all steps were performed in the dark. Transverse cross-sections were imaged using a Nikon A1R laser scanning confocal microscope (Nikon Instruments Inc., Melville, NY, USA) under a 488 nm excitation laser using a 10´ objective. No z-stacking was used. Cell walls were peeled off and imaged using a 488 nm excitation laser using a 20´ objective lens; z-stacking was used to image the entire depth of the membrane.

2.6

Mechanical Testing of Scaffold Material in Compression

For the mechanical testing of the freeze-cast scaffold material in compression, cubes of 5 mm side length were cut using a diamond wire saw (Model 4240, Well Diamond Wire Saws, Norcross, GA, USA) operated with 0.22 mm diamond-studded steel wire and a cutting speed of 0.7 m/s, at three different heights in the scaffold, with cube-center positions at 7 mm (bottom), 17.5 mm (middle) and 28.00 mm (top) measured from the sample bottom, respectively. To determine the density of the samples, their mass was determined using a precision balance (XP105, Mettler Toledo, Columbus, OH, USA) and their dimensions measured along the three cube axes, averaging three measurements each using a stereomicroscope M205C, Leica Microsystems Inc., Buffalo Grove, IL, USA. All cubic specimens were acclimatized for 48 hours at 55% relative humidity


9


Page 10 of 57

and 20°C, then compression tested under the same environmental conditions in a BioPuls Bath chamber (Instron 5498, Norwood, MA, USA). A strain rate of 0.1 min-1 was applied up to 80% strain both parallel and perpendicular to the freezing direction with forces measured by a 50 N load cell. Four specimens were tested for each composition, applied cooling rate, sample height, and compression direction. The modulus was calculated from the initial linear region of the stressstrain curve. The yield strength was determined from the upper yield point, when it existed, or calculated as the intersection of the extensions of the initial linear section and the plateau section, when it did not.67 Toughness was defined as the area underneath the stress-strain curve up to 60% strain. Linear regression and integration were performed in Origin 9.1 (Origin Lab, Northampton, MA, USA). Property charts were created using the CES Constructor & Selector 2018 software.79

2.7

Mechanical Testing of Film in Tension

To determine benchmark properties for the cell wall material of the freeze-cast scaffolds, thin films were prepared, oven-dried for 24 hours and acclimatized to 55% r.h., and then tested in tension. Briefly, the slurries were slip cast onto an air plasma treated acrylic sheet equipped with 1 mm thick glass slides on four edges to confine the slurries (a method adapted from Abba et al. 2016).66 To determine the density of the films, disks of 3 mm in diameter were punched out from the film using a 3mm biopsy punch (Robbins Instruments, Chatham, NJ). The thickness of the disks was measured with a micrometer screw gauge (IP65, Mitutoyo, Kawasaki, Japan), their mass was determined using a precision balance (XP105, Mettler Toledo, Columbus, OH, USA); the density was calculated as mass per volume averaging n = 24-36 disks. For mechanical testing, the films were cut into a dumbbell shape (ASTM D1708-06) with a narrow section 17 mm long and 5 mm wide, and taped into paper-frames with a gauge length of 22 mm for testing. Three samples


10

Page 11 of 57 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

Biomacromolecules

were tested for each composition with an Instron with a 50 N load cell and a crosshead speed of 1 mm/min. The modulus of the films was calculated from the slope of the stress-strain curve. The yield strength was determined as the intersection of the extensions of the initial linear section and the secondary linear section, when it existed, or the stress at break, when it did not.

2.8

Sample Moisture Content

To determine the moisture content of the different samples, 2 mm thick sections with a sample center at a height of 24 mm measured from the bottom were weighed and then heated in an oven at 110°C, which is a temperature below the decomposition temperature of chitosan and nanocellulose. The weight was monitored every 20 minutes until no further weight change was observed in two consecutive measurements. The moisture percentage was calculated as the percentage reduction in weight before and after sample heating.

3.

Results and Discussion

3.1

Scaffold Structure

The structural characterization of all scaffolds revealed their highly aligned, honeycomb-like porosity parallel to the freezing direction, with elongated pore cross sections and regularly-spaced cell walls of uniform thickness transverse to it (Figure 3). Decreasing the applied cooling rate from 10°C/min to 1°C/min resulted in a pore size increase, frequently defined also as the lamellar spacing or short axis of the pore, and measured in freeze-dried biopolymer samples as the distance between the center of neighboring cell walls (Table 1). The correlation between lamellar spacing, S, and applied cooling rate, 𝐶̇ , can conveniently be described as:71


11


𝑆 = 𝑘𝐶̇ &'

Page 12 of 57

(1)

where k and n are material and mold specific quantities. The k and n values calculated for the results obtained in this study for chitosan and chitosan-nanocellulose composites are comparable to those observed and reported before for collagen and frozen foods (Table 1).71,80 The trends reported here can be extrapolated to higher and lower cooling rates, as reported in an earlier publication of ours (Divakar et al. 2019). We find, however, that in the case of our freeze casting system, limitations in, for example, heat flow and thermal insulation become noticeable through deviations from the trends, for applied cooling rates below 0.1°C/min and higher than 30°C/min. The cell walls of the pure chitosan scaffolds are straight and exhibit regularly spaced ridges parallel to the ice growth direction. These ridges are typically observed only on one face of the cell wall (Figure 3), as reported18,75. Both the spacing and height of the ridges increase with decreasing cooling rate, from 23 µm and 5 µm to 44 µm and 21 µm, respectively, with values agreeing well with previously reported observations.18,75,81 In contrast to the cell walls in pure chitosan scaffolds, the cell walls of the Chi-CNF scaffolds do not exhibit ridges, but are similarly straight. The cell wall structure reveals that the CNFs are homogeneously distributed with a preferential alignment parallel to the freezing direction (Figure 4). Regularly spaced fibrillar pillars and bridges connect neighboring cell walls with one another (Figures 3, 4). As is the case for the chitosan ridges, the fibrillar bridges increase in spacing and thickness, when the applied cooling rate decreases. In contrast to Chi-CNF, the Chi-CNB cell walls are undulated parallel to the direction of ice crystal growth. Confocal microscopy reveals that finger-like protrusions emerge from the blunt ridges on the cell walls in both Chi-CNB and Chi-CNC scaffolds frozen at 10°C/min (Figures 4C1, 4D1). Again, in contrast to the nanocellulose fibrils, the nanocellulose crystals of the Chi-CNC show


12

Page 13 of 57 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

Biomacromolecules

agglomeration in the cell walls (Figures 5B3, 5D3); the Chi-CNC cell walls are also more porous than those in the other scaffold compositions (Figures 3, 5). Much of the structure formation during freezing and solidification is due to phenomena that occur in parallel to the phase separation into pure water ice crystals and an increasingly acidic and upconcentrated chitosan solution, in which the nanocellulose is suspended. The polymer phase solidifies when, finally, its glass transition temperature is reached; this glass transition temperature depends on the cooling rate, which also determines the amount of moisture retained in the cell wall material.68,75,82 Table 1. Values for lamellar spacing (mean ± standard deviation, SD) and the respective quantities for kc, nc, and ks, ns obtained applying equation (1) to measurement performed on confocal (Sc) and SEM (Ss) micrographs of the top sections in Chitosan, Chi-CNF, Chi-CNB and Chi-CNC scaffolds frozen at 10°C/min and 1°C/min applied cooling rates (𝐶̇ ).

Composition Chitosan Chi-CNF Chi-CNB Chi-CNC

𝑪̇ [°C/min]

Sc [µm]

10

39.4 ± 6.4

1

77.5 ± 18.5

10

25.5 ± 1.6

1

64.6 ± 13.0

10

47.4 ± 5.5

1

60.9 ± 5.4

10

40.6 ± 7.8

1

52.1 ± 17.4

kc

nc

77.54

0.29

64.59

0.40

60.89

0.11

52.11

0.11

Ss [µm] 25.9 ± 6.4 53.9 ± 9.4 20.8 ± 0.8 33.1 ± 2.1 17.9 ± 1.4 33.8 ± 9.6 23.0 ± 4.3 33.3 ± 4.1

ks

ns

53.89

0.32

33.11

0.20

33.80

0.28

33.34

0.16

As the dependence on both the freezing rate and slurry composition indicates, ridges are favored, when the solutes/particles are well dispersed and no fibrillation can occur in the material system. Bridges result from fibril and fiber trapping in the increasingly viscous polymer solution during


13


Page 14 of 57

the solidification process,28,71,83–85 while the majority of CNFs remain homogeneously dispersed to form slightly textured film-like cell walls, as earlier observed in freeze-cast scaffolds composed only of CNFs (Figure 1B).86 The fibrillar bridges formed bend or buckle, when the overall scaffold relaxes and shrinks during lyophilization, which effectively is a second step of drying (Figures 3B2, 3B4). The spacing and dimensions of the ridges and fibrillar bridges increase, when the freezing rates decreases due to the resulting longer timespan available for both larger ice crystal growth and polymer assembly. Interesting features in the cell wall microstructure are observed in both the Chi-CNB and ChiCNC scaffolds. It appears that the presence of nanocellulose fibrils in the Chi-CNB results in a more homogeneous distribution of nanocellulose crystals, likely due to crystals decorating the fibrils. This means that fewer crystals remain distributed in the chitosan solution without association to fibrils in comparison to Chi-CNC composition, which is reflected in the amount of pore formation in the cell wall material. The pores are formed, when CNCs aggregate while the chitosan solution changes, first into a hydrogel then into a polymer film, during the two-step drying process of freezing and lyophilization. The shrinkage associated with the drying of the chitosan phase “rips” the cell walls open and accumulates the CNC clusters along the perimeter of the newly formed pore. Such a process leads to a smaller number of pores in the Chi-CNB scaffolds compared to that in the Chi-CNC scaffolds, just as we observe.


14

Page 15 of 57 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

Biomacromolecules

Figure 3. SEM micrographs of longitudinal cross-sections of (A) Chitosan, (B) Chi-CNF, (C) ChiCNB, and (D) Chi-CNC scaffolds frozen at the applied cooling rate of 10°C/min (Row 1), 1°C/min (Row 2). Transverse cross-sections of the scaffolds of different compositions (same column ordering) frozen at the applied cooling rate of 10°C/min (Row 3), 1°C/min (Row 4). Scale bar of A1 applies to all micrographs shown.


15


Page 16 of 57

Figure 4. Confocal micrographs of transverse cross-sections of (A) Chitosan, (B) Chi-CNF, (C) Chi-CNB, and (D) Chi-CNC scaffolds frozen at the applied cooling rate of 10°C/min (Row 1), 1°C/min (Row 2). Scale bar of A1 applies to all micrographs shown.


16

Page 17 of 57 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

Biomacromolecules

Figure 5. SEM micrographs (Row 1 and 2) and confocal micrographs (Row 3 and 4) of peeledoff cell walls of (A) Chitosan, (B) Chi-CNF, (C) Chi-CNB, and (D) Chi-CNC scaffolds frozen at the applied cooling rate of 10°C/min (Row 1 and 3), 1°C/min (Row 2 and 4). Scale bars of A1 and A3 applies to all SEM and Confocal micrographs shown, respectively.


17


3.2

Page 18 of 57

Density and Mechanical Properties and Performance

Both the relative density and moisture content are known to significantly affect the mechanical properties of cellular solids; their modulus, E, and yield strength, 𝜎+ , increase with the relative density, (𝜌 ∗ /𝜌1 ), of the scaffold as:87 𝐸 𝜌 ∗ '8 = C5 6 7 𝐸1 𝜌1

(2)

𝜎+ 𝜌 ∗ ': = C9 6 7 𝜎+,1 𝜌1

(3)

where 𝜌 ∗ is the density of the cellular material, and 𝐸1 , 𝜎+,1 and 𝜌1 are those of the solid, of which the cell walls are composed. C1 and C2 are material specific constants, and n1 and n2 depend on the architecture of the porosity, e.g. foam (n1 = 2, n2 = 1.5) versus honeycomb (n1 = n2 = 1), the structure of the latter is the one to which the freeze-cast scaffolds of this study are structurally most akin. The densities of nanocellulose-containing scaffolds (Chi-CNF, Chi-CNB and Chi-CNC) frozen at 1°C/min were overall slightly lower than those frozen at 10°C/min (Figure 7D). Since the 10°C/min and 1°C/min scaffold manufacture started with identical slurry compositions and identical processing steps, apart from the applied cooling rate, the difference in scaffold densities can be attributed to two key factors: the slurry composition, on the one hand, and the applied cooling rate, on the other. A lower freezing rate affects the properties 𝜌1 , 𝐸1 , and 𝜎+,1 of the solid of the cell wall material as it results in its more effective drying, due to a longer time for water molecules to diffuse out of the slurry and onto the growing ice crystal.75,82 Material composition affects the outcome, because the different nanocellulose types (CNC, CNF, CNB) offer different amounts of surface area for


18

Page 19 of 57 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

Biomacromolecules

the formation of shells of hydration. At 55% relative humidity, the moisture content of the chitosan-nanocellulose composite scaffolds was with 17.0-20.6% about 50% higher than the 12.214.1% of pure chitosan (Table 2). Based on our findings, the amount of water tightly bound to the composite cell wall materials is expected to be higher in the nanocellulose-containing scaffolds. Comparing our composite films, which were made with AVAP processed nanocellulose, with pure chitosan films, with TEMPO-oxidized nanocellulose films, and with films made from blends of these two, we find that at 55% r.h. they have comparable moisture contents of about 15-19%,88 about 15%,89,90 and about 13%

91,92

. Moisture is a plasticizer in biopolymers, which causes

modulus and strength to decrease with increasing moisture content; in parallel, it can lead to an increase in toughness through increased ductility.66

Table 2. Moisture content of Chitosan, Chi-CNF, Chi-CNB and Chi-CNC scaffolds frozen with applied cooling rates, 𝐶̇ , of 10°C/min and 1°C/min.

Composition Chitosan Chi-CNF Chi-CNB Chi-CNC

𝑪̇ [°C/min]

Moisture Content [%]

10

12.2 ± 2.2

1

14.1 ± 0.1

10

20.6 ± 0.9

1

18.1 ± 0.1

10

19.9 ± 0.1

1

17.0 ± 3.9

10

19.8 ± 2.2

1

17.6 ± 2.4

In addition to relative density and moisture content, the architecture that results from the freezing conditions affects the mechanical properties. The applied cooling rate determines the pore size and cell wall thickness; the higher the applied cooling rate, the smaller the pore size, which we define


19


Page 20 of 57

as the short pore axis. Additional structural features of mechanical importance that form and whose size and number depend on the applied cooling rate, are ridges, pillars, and bridges between the cell walls; the higher the applied cooling rate for a given composition, the higher the number of ridges, pillars, and bridges (Figures 3, 5). These features are observed, because at a higher applied cooling rate the timespan is shorter for fibrils to align and be drawn out by the shear flow that develops between the ice dendrites, while the slurry upconcentrates and becomes increasingly viscous (Figure 1B). Typical stress-strain curves for the different scaffold compositions are shown in Figure 6. For all compositions, an increase in modulus, strength, and toughness was observed with increasing freezing rate, decreasing short pore axis, decreasing cell wall thickness, and increasing number of ridges, pillars, and bridges, all of which enhance the mechanical properties of the scaffolds (Figures 7A-C, Table 1).87 Reported in Table 3 are property predictions for foams and honeycombs calculated with equations 2 and 3, for samples from the middle section freeze cast with an applied cooling rate of 10°C/min, assuming that the values for modulus, Es, σ (y,s), and ρ s obtained on fully dense chitosan and chitosan-nanocellulose composite films are similar to the properties of the solid of which the respective cell walls are composed. A comparison of the calculated scaffold properties with the experimentally determined ones reported in Table 4 reveal that the scaffolds more closely resemble in their mechanical performance foams than honeycombs, which is typical for freezecast scaffolds. This more foam-like behavior is due to imperfect alignment of the cell wall with the loading direction, undulations of the cell walls, defects in the cell wall material, and irregularities in cell wall thickness, all of which result in bending rather than stretch-dominated deformation and corresponding lower property values.


20

Page 21 of 57 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

Biomacromolecules

Table 3. Young’s modulus (mean ± SD) of Chitosan, Chi-CNF, Chi-CNB and Chi-CNC films. Predicted modulus, E, and yield strength,σy, for foams and honeycombs, respectively, calculated using C1 = C2 = 1, and n1 = 2, n2 = 1.5 for foams and n1 = n2 = 1 for honeycombs. Film

Density

Modulus

Es

Yield Strength

𝐸

Plant-derived Nanocellulose as Structural and Mechanical

Recommend Documents