Preparation and characterization of composite scaffold of alginate and cellulose nanofiber from ramie

Abstract

Alginate scaffold with high porosity has great potential in the field of tissue engineering due to its biocompatibility and degradability. However, the poor mechanical performance of pure alginate scaffold has limited its use in many applications. Cellulose nanofibers (CNFs) have attracted attention as reinforcing agents to fabricate composite scaffolds with alginate. In this paper, CNF obtained from raw ramie fibers was incorporated with sodium alginate to make a composite scaffold by the freeze-drying method. CNF contents of 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6% were selected to study the effect of CNF on scaffold characterization. The composite scaffold exhibited fewer pores but more compact structure than the pure alginate scaffold. Fourier transform infrared spectroscopy was used to study the changes in the functional groups between the ramie fiber and its CNF, pure alginate scaffold, and the composite scaffold. X-ray diffraction indicated that the crystallinity of scaffold increased with addition of CNF. The mechanical performance of scaffold was successfully improved by adding CNF, but the porosity and swelling ratio were decreased. Hence, by combining CNF with alginate, the porous structure, mechanical properties, and swelling behaviors could be tailored, which could expand its use in the field of tissue engineering.

Keywords

cellulose nanofiber alginate composite scaffold ramie mechanical properties

Biodegradable scaffolds with open pore structures are used in tissue engineering as a template or matrix for cell adhesion, growth, and proliferation. The tissue engineering scaffold has been proved to have great potential for repairing a tissues and organs, such as skin,^1–3 bone,^4–6 vascular,⁷ and other human tissues. Every human tissue and organ has its own specific three-dimensional (3D) extracellular matrix (ECM) structure. Cells in a 3D structure typically align new ECM components according to the specific inner architecture of the bioscaffold.^8–10 The freeze-drying method is a simple and practical technique to fabricate porous scaffolds.^11,12 It is especially applied to natural polymers as there is no need for special reaction conditions or addition of organic solvent chemicals.¹³ It can be employed with hydrophilic polymers to generate pores in scaffolds by ice sublimation.^14,15

Alginate is a naturally biodegradable polymer that is typically obtained from seaweeds. It has been certificated by the US Food and Drug Administration (FDA) for tissue engineering applications.¹⁶ Sodium alginate is water-soluble at room temperature. Through the simple freeze-drying method, it could be fabricated into porous scaffolds. Alginate forms networks by crosslinking with divalent cations such as Ca²⁺, thus forming an insoluble and stable scaffold.¹⁷ With similar chemical composition to ECM, combined with its biocompatibility and biodegradability, alginate is a favorable substance for cell adhesion and growth.¹⁸ However, the use of natural polymers in the field of tissue engineering is limited due to their poor mechanical performance.¹⁹ Taking the artificial skin scaffold as an example, it has been investigated that the tensile strength of human skin was about 1500–4500 kPa, with elongation of 18.7–29.3%. The mechanical properties of the pure alginate scaffold were much weaker than this. Cellulose nanofibers (CNFs) are attractive materials as a reinforcing agent for tissue engineering scaffold due to their unique characteristics of nanoscale dimensions with high aspect ratio and high mechanical strength.^20–22 The composite scaffold with CNF was supposed to have better mechanical performance.

Cellulose is the most abundant renewable biopolymer on Earth from which CNF could be extracted.²³ Ramie, popularly known as Chinese grass, is native to China, Japan, and the Malay Peninsula. In China, the ramie yield has been recorded as 500,000 tons per year, accounting for 96–97% of world production.²⁴ The ramie fibers are among the longest and strongest of all natural bast fibers, which, combined with their natural bacterial effects and high tensile strength, mean they are widely used for fiber-reinforced composites.²⁵ CNF with high crystallinity can be extracted from ramie fibers by mechanical, steam-exploding, and oxidizing treatment.²⁶ The molecular structure of CNF is similar to that of alginate, hence it is able to form a semi-interpenetrating polymer network by creating hydrogen bonds with alginate chains and improve the mechanical properties of composite scaffolds.²⁷ In addition, CNF imparts to the composite scaffold excellent properties at the nanoscale. The huge specific surface area provides many contact points for cells, which allows more effective cell growth and tissue regeneration.²⁸

It is assumed that by blending CNF with alginate, the composite scaffold will present improved properties compared to the pure alginate scaffold. Therefore, in this paper, composite CNF–alginate scaffolds were fabricated using the freeze-drying technique, and the effect of CNF content on properties of scaffolds were investigated. Scanning electron microscopy (SEM) was used to observe the morphology of the porous structure in the scaffold. Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) were employed to study the changes in the chemical structure and the crystalline structure of scaffolds with different CNF content. The mechanical properties, porosity, and swelling behaviors are discussed. With greater understanding of the influencing mechanisms of CNF on composite scaffolds' porous structure, mechanical properties, swelling behaviors, and other properties, these scaffolds could be designed and controlled to widen the scope of application in the field of tissue engineering.

Experimental

Materials

Sodium alginate (M_w = 300,000; M_w/M_n = 1.5) was supplied by Hyzlin Biology Development Co. Ltd. (Qingdao, China). Raw ramie was obtained from Phloem Fiber Research Center (Hunan, China). Sodium chlorite (NaClO₂), acetic acid, sodium hydroxide (NaOH), sodium bromide (NaBr), TEMPO, sodium hypochlorite (NaClO), ethyl alcohol, and calcium chloride (CaCl₂) were purchased from Sinopharm Chemical Regent Co. Ltd. (Beijing, China). All reagents were of analytic grade.

CNF preparation

Raw ramie was cut into 25 cm lengths and presoaked in water at room temperature for 24 h prior to steam explosion treatment. After, the ramie was steam exploded (BGDR-4.5H; Fongs National Engineering Co. Ltd., Shenzhen, China) at 1.0 MPa (177℃) for 60 min. The steam-exploded fibers were treated with NaClO₂ solution (the mass concentration of solution, g/ml, w/v = 0.95%) at 70℃ and pH 4 by adding acetic acid each hour for 3 h to remove acid-soluble lignin. The samples were washed until neutral pH was achieved, and then treated with NaOH solution (w/v 6%) at 25℃ for 8 h and then 80℃ for 2 h. Alkaline-soluble lignin and hemicellulose were removed from samples to obtain the pretreated ramie fibers.

Then, 2 g of the oven-dried pretreated fibers were dispersed into 200 ml distilled water by velocity mixing; 0.2 g of NaBr and 0.05 g of TEMPO were added into suspension and stirred until dissolved. NaClO was added into suspension at 25℃, with magnetic stirring at 400 RPM. NaOH solution (w/v 0.2%) was added to maintain a pH value of 10 for 60 min, and 5 ml of ethyl alcohol was added to terminate the reaction. The suspension was repeatedly washed with distilled water and centrifugation till neutral pH was reached. Then, sonication (FS-450N; Sonxi Ultrasonic Instrument Co. Ltd., Shanghai, China) was conducted for 5 min to obtain a dispersed suspension of CNF.

Scaffold preparation

Sodium alginate solution with a concentration of 1.5% (w/v) was prepared by distilled water and magnetic stirring at 1500 RPM for 1 h. Then, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6% (w/v) of CNF was added into alginate solution and stirred for 1 h until uniformly dispersed. After standing at room temperature for 12 h to remove bubbles, the prepared solution was emptied into cylindrical containers (inner diameter 64 mm; height 12 mm), with the filling height of solution controlled to 5 mm. After frozen at –30℃ for 8 h in the rapid temperature change test chamber (BTKS5-150C; BellGroup, Hong Kong, China), the frozen solution was then lyophilized using a freeze drier (LGJ-IO; Beijing Songyuan Huaxing Technology Develop Co., Ltd., Beijing, China) at 0℃ for 24 h under vacuum at less than 1 Pa. The sodium alginate scaffolds were crosslinked by immersion in CaCl₂ solution (w/v 4%) for 1 h at room temperature and then flushed with distilled water. After freezing for 8 h at –30℃ and a secondary freeze-drying process (0℃, 1 Pa, 24 h), the final scaffolds were prepared. The scaffolds with different CNF contents were denoted as 0.025-CNF/alginate, 0.05-CNF/alginate, 0.1-CNF/alginate, 0.2-CNF/alginate, 0.4-CNF/alginate, 0.8-CNF/alginate, and 1.6-CNF/alginate, respectively.

Characterizations

Morphology study

CNF suspension (w/v 0.01%) was ultrasonic dispersed for 20 min and cast onto a copper grid with carbon coating, to be dried at room temperature. The sample was observed at 200 keV using transmission electron microscopy (TEM) (H-7650; Hitachi, Tokyo, Japan).

Scaffolds were cut along the vertical axial and observed using SEM (JSM-6390LV; JEOL, Tokyo, Japan) at 15–20 kV. The TEM and SEM graphs were analyzed using Image-Pro Plus.

FTIR spectroscopy analysis

The ramie, CNF, pure alginate scaffold, and 1.6-CNF/alginate scaffold were evaporated and ground for study using an FTIR spectrometer (Nicolet-5700; Thermo Fisher Scientific, MA, USA) in the range 4000 to 400 cm⁻¹.

XRD analysis

XRD (D8 Advance; Bruker, Karlsruhe, Germany) with Cu–Ka radiation (40 kV, 250 mA) was used to investigate the crystalline structure of the pure alginate scaffold, 0.1% CNF–alginate scaffold, 0.4% CNF–alginate scaffold, and 1.6% CNF–alginate scaffold. Samples were scanned at a rate of 50/min, from 2θ of 5°–60° to get the XRD pattern and the relative crystallinity, at least three samples were tested for each group.

Mechanical properties

Mechanical properties of scaffolds were examined by tensile tests with a universal strength tester (3300; Instron, Boston, USA). The scaffold was tailored into rectangular samples along the diameter of the scaffold, with length 60 mm, width 10 mm, and thickness 5 mm. Samples were stretched at a rate of 5 mm/min until broken in order to obtain the maximum strength (MPa) and elongation (%) at break. Five samples were tested for each scaffold group to get the average value.

Porosity and swelling behavior

Scaffold porosity was measured using the liquid displacement method.²⁹ The scaffold was tailored into small cubes and submerged into a known volume of alcohol (V₁). Then, evacuation was conducted to force liquid to burst into the pores and fill them until no bubbles occurred and the total volume of the alcohol–scaffold system was recorded as V₂. Then, the scaffold cubes were taken out without drips and the volume of the remaining alcohol was recorded as V₃. The porosity of the scaffold could be calculated using equation (1). Five randomly located samples were obtained for analysis.

Porosity = (V_{1} - V_{3}) / (V_{2} - V_{3}) \times 100 %

(1)

Buffer solution with neutral pH was used to test the swelling ratio of the scaffold. The scaffold was cut into cubes and dried to a constant weight (m₀). Then the sample was immersed in buffer solution for 24 h before eliminating the extra water on the surface with tissue and recording the weight (m_s). The swelling ratio was calculated using equation (2). The mean value were calculated from five samples.

Swellingratio = (m_{s} - m_{0}) / m_{0} \times 100 %

(2)

Statistical analysis

Tests values were all recorded as a mean ± standard deviation. The characterizations of scaffolds with different CNF content was compared using one-way analysis of variance (ANOVA). Results that are p < 0.05 were determined to be statistically significant.

Results and discussion

Morphology study

Figure 1(a)–(d) shows the raw ramie, pretreated ramie fiber, suspension of CNF, and TEM micrograph of CNF. After steam explosion (Figure 1(b)), the ramie fibers were dispersed and displayed whiter coloration compared to the yellow–brown strips of raw ramie (Figure 1(a)), suggesting the elimination of non-cellulose components like lignin and hemicellulose. After oxidation treatment, the suspension of CNF (Figure 1(c)) presented as a white and stable colloform. This is because the crystalline structure of cellulose was destroyed during the reaction process, and fibers were loosely arranged to absorb more water. Meanwhile, many hydrogen bonds were created between the hydroxy or carboxyl on cellulose molecular chains and water molecules, forming a colloid suspension. As shown in Figure 1(d), the CNF was about 25 nm in width and 1700 nm in length. The aspect ratio of ramie CNF was about 68, suitable for scaffold strengthening.

Figure 1.

Apparent morphology of ramie fiber: (a) raw ramie; (b) pretreated ramie fiber; (c) suspension of CNF; (d) TEM micrograph of CNF. Scale bar indicates 200 nm.

The SEM graphs of scaffold cross-section in the vertical direction are shown in Figure 2(a)–(h). The micropore structure of the scaffold was affected by addition of the CNF. The pure alginate scaffold (Figure 2(a)) showed isotropic cellular pores with high connectivity, with approximate pore size about 200 µm. Pores became smaller with addition of CNF, and the connectivity between neighboring pores decreased (Figure 2(b)–(f)). The pore size increased when CNF content rose above 0.8% (Figure 2(g)–(h)), but the number of pores decreased. It can be seen from micrographs of 1.6% CNF–alginate scaffold (Figure 2(h)) that the walls between adjacent pores are very compact, with rougher surfaces. No phase separation was observed with increase of CNF content, which indicates good integration between the alginate and CNF. By increasing the content of CNF, many hydrogen bonds were created among the –OH groups between CNF and alginate molecular chains. Meanwhile, with higher concentrations of CNF, the electrostatic interactions between these two materials were increased. A semi-interpenetrating network was set up via the interaction mechanism, with CNF diffused into alginate chains. On one hand, this restricted the formation and growth of ice crystals during the freezing process, reducing the number of micropores. On the other hand, the interaction between CNF and alginate induced more compact walls in the scaffold after the water was sublimated.

Figure 2.

Scanning electron microscopy graphs of scaffolds, scale bar indicates 100 µm: (a) pure alginate scaffold; (b) 0.0025% CNF–alginate scaffold; (c) 0.05% CNF–alginate scaffold; (d) 0.1% CNF–alginate scaffold; (e) 0.2% CNF–alginate scaffold; (f) 0.4% CNF–alginate scaffold; (g) 0.8% CNF–alginate scaffold; (h) 1.6% CNF–alginate scaffold.

FTIR spectroscopy analysis

FTIR spectroscopy was performed to analyze changes in the chemical functional groups of cellulose and alginate scaffold. The FTIR spectra of ramie fiber, CNF, pure alginate scaffold, and 1.6% CNF–alginate scaffold are shown in Figure 3(a)–(d).

Figure 3.

FTIR spectra of: (a) ramie fiber; (b) CNF; (c) pure alginate scaffold; (d) 1.6% CNF–alginate scaffold.

The spectra of ramie fiber (Figure 3(a)) showed peaks at 1739 cm⁻¹ corresponding to –C=O– stretching vibration, and 1510 cm⁻¹ assigned to aromatic ring vibration, indicating the existence of lignose and hemicellulose. However, these two peaks disappeared on spectra of CNF (Figure 3(b)), demonstrating that these two elements were completely eliminated. A new peak at 1741 cm⁻¹ on spectra of CNF indicate the carboxy group was grafted on molecular chains.

The spectra of alginate (Figure 3(c)) is similar to the spectra of CNF because of the similarity of the chemical structure between them. With addition of CNF, the FTIR spectra of the 1.6% CNF–alginate scaffold (Figure 3(d)) exhibited characteristic peaks of both calcium alginate and CNF. The peak assigned to O–H stretching vibration shifted from 3361 cm⁻¹ to 3346 cm⁻¹, the –COO– stretching vibration shifted from 1626 cm⁻¹ to 1617 cm⁻¹. These alterations proved that new hydrogen bonds were created between the alginate and CNF. Peaks at 1059 cm⁻¹ and 1033 cm⁻¹ corresponding to the vibration stretch of C–OH bonds of cellulose, illustrating the existence of CNF in the composite scaffold. The C–C stretching and C–O stretching vibration at 1085 cm⁻¹ and 1028 cm⁻¹ on the spectra in Figure 3(c) have disappeared in the spectra in Figure 3(d), suggesting hydrogen linkage between the alginate and CNF.

XRD pattern analysis

XRD was employed to investigate the crystalline structure of scaffolds with different CNF contents. The XRD patterns of the pure alginate scaffold, 0.1% CNF–alginate scaffold, 0.4% CNF–alginate scaffold, and 1.6% CNF–alginate scaffold were presented in Figure 4(a)–(d). The pure alginate scaffold (Figure 4(a)) only exhibited two broad and faint peaks around 2θ = 15° and 21°. That is because during the crosslinking process of sodium alginate scaffold by Ca²⁺, the intermolecular and intramolecular hydrogen bonds were deformed and molecular chains were more likely to be arranged in an amorphous form. With the addition of CNF, the diffractogram of the 0.1% CNF–alginate scaffold (Figure 4(b)) presented with minute peaks at around 2θ = 15° and 22°, corresponded to crystalline cellulose patterns, which was the characteristic pattern of CNF. With the increase of CNF content in the composite scaffold, the patterns of the 0.4% CNF–alginate scaffold (Figure 4(c)) and 1.6% CNF–alginate scaffold (Figure 4(d)) presented intensely elevated peaks. This result showed that the introduction of CNF could promote crystalline structure change of the alginate scaffold. Figure 5 shows the relative crystallinity of scaffolds with different CNF contents. The crystallinity of the scaffold significantly increased when the CNF content was higher than 0.0025% (p < 0.01). The crystallinity of the pure alginate scaffold was only 11.91% ± 0.72%, while the 0.4% CNF–alginate scaffold was 39.02% ± 0.71%. Then, the rate of increase slowed down as CNF increased, and the crystallinity of the 1.6% CNF–alginate scaffold was 52.1 ± 1.2% (p < 0.01). This was because the crystallinity of CNF was much higher than pure alginate, and the hydrogen bonds among the –OH groups between CNF and alginate molecular chains also make a contribution to the improvement of crystallinity.

Figure 4.

X-ray diffraction pattern: (a) pure alginate scaffold; (b) 0.1% CNF–alginate scaffold; (c) 0.4% CNF–alginate scaffold; (d) 1.6% CNF–alginate scaffold.

Figure 5.

Crystallinity of scaffolds with different CNF content.

Mechanical properties

Tensile tests were performed to investigate the reinforcement effect of CNF on alginate scaffolds. Figure 6 presents the tensile properties of scaffolds with different CNF contents. The reinforcement effect of the composite scaffold was significant (p < 0.01), with improved breaking strength and elongation at breaking with higher CNF content. The pure alginate scaffold had a breaking strength of 110.2 ± 11.9 kPa and elongation of 3.38% ± 0.13%. With CNF added, the breaking strength was 376.7 ± 15.2 kPa, 560.4 ±21.7 kPa, 732.4 ± 32.6 kPa, and 1113.2 ± 21.8 kPa, respectively, while the elongation was 5.93% ± 0.21%, 10.61% ± 0.52%, 13.94% ± 0.75%, and 22.18% ±0.66%, respectively, with CNF content of 0.05%, 0.2%, 0.4%, and 1.6% (p < 0.01). The breaking strength of the 1.6% CNF–alginate scaffold was more than 10 times that of the pure alginate scaffold, while the elongation was about seven times. The tensile strength of the composite scaffolds was still lower than the 1500–4500 kPa of human skin. However, the elongation of the scaffold with CNF content greater than 0.8% was higher than 18.7%, meeting the standard for human skin.

Figure 6.

Tensile properties of scaffolds with different CNF content.

With similar chemical structure, CNF achieved favorable compatibility and strong interaction with alginate chains. The addition of CNF created a semi-interpenetrating network with alginate molecular chains by creating hydrogen bonds among the –OH groups. Together with the electrostatic interaction between CNF and alginate, the microstructure of the scaffold pores was changed, with the connectivity between adjacent pores decreased, and the walls between pores more compact and offering higher tensile strength. Due to its high aspect ratio, CNF is able to bridge cracks in scaffolds and played the role of decelerator to retard fracturing. So the elongation of the composite scaffold was higher. The addition of CNF increased the crystallinity of the scaffold, which is shown in Figure 5. Therefore, the mechanical properties of the scaffold were significantly improved.

Porosity and swelling behavior

Porosity and swelling behavior determine the water absorption capacity of a scaffold, reflecting its porous structure. Figure 7 shows that the porosity and swelling ratio of scaffolds both declined with increased CNF content. The pure alginate scaffold had porosity of 92.76% ± 0.03% and swelling ratio of 3368.4% ±45.2%. The porosity of the composite scaffold was 92.33% ± 0.05%, 90.82% ± 0.07%, 90.14% ± 0.04%, and 88.17% ± 0.05%, respectively, while the swelling rate was 3239.7% ± 25.2%, 3041.3% ± 23.2%, 2885.9% ± 21.6% and 2491.7% ± 18.5% respectively, with CNF content of 0.05%, 0.2%, 0.4%, and 1.6% (p < 0.01). In the composite scaffold, more space was taken by CNF instead of pores as CNF content increased. It has been shown by SEM morphology (Figure 2(a)–(h)) that with higher CNF content, the number of pores in the scaffold decreases and walls between adjacent pores becomes thicker. This induces lower porosity in the scaffold, which has a negative effect on the water absorption capability. Apart from the effect on porosity, the swelling ratio of the scaffold is also impacted by the ratio of the amorphous region and free –OH groups. With higher CNF content the crystallinity of the scaffold was increased, leading to less space for the amorphous region to take water into. Meanwhile, the free –OH groups were restricted by the network of CNF and alginate chains, or created intermolecular or intramolecular hydrogen bonds. Therefore, water absorption capability of the scaffold decreased.

Figure 7.

Porosity and swelling ratio of scaffolds with different CNF content.

Conclusion

In this study, CNFs with length of 1700 nm and diameter of 25 nm were extracted from ramie by steam explosion and oxidation. The freeze-drying technique was used to produce composite scaffolds of CNF and alginate. The SEM micrograph shows that the CNF is uniformly dispersed within the scaffold. Scaffolds exhibited smaller pores with the addition of CNF, up to 0.4%. However, with the addition of more than 0.4% CNF content, pores were larger but fewer in number, with thicker and more compact walls between adjacent pores. The FTIR spectra demonstrated that the CNFs have created a semi-interpenetrating network with alginate molecular chains by creating hydrogen bonds. The XRD patterns showed that the composite scaffold has increased crystallinity with a higher CNF content. The scaffold was successfully reinforced by adding CNF, with tensile strength of the 1.6% CNF–alginate scaffold 10 times greater than the pure alginate scaffold. However, the porosity and swelling ratio of the composite scaffold decreased with higher CNF content, but was still highly porous with great water absorbency. The alginate scaffold reinforced with CNF could have wider applications in the tissue engineering field.

Footnotes

Authors' note

Yuanming Zhang and Guangting Han are also affiliated with College of Textiles, Donghua University, Shanghai, PR China.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was sponsored by the National Natural Science Foundation of China (Grant Number 51373083) and the Shandong Provincial Natural Science Foundation, China (Grant Number ZR2018BA023).

References

Wang

Shi

, et al. A biomimetic silk fibroin/sodium alginate composite scaffold for soft tissue engineering. Sci Rep 2016; 6: 39477.

Bierhalz CK and Moraes M. Tuning the properties of alginate–chitosan membranes by varying the viscosity and the proportions of polymers. J Appl Polym Sci 2016; 133.

Bahareh

Bahman

Ehsan

. Fabrication of a three dimensional spongy scaffold using human Wharton's jelly derived extra cellular matrix for wound healing. Mat. Sci. Eng. C-Mater 2017; 78: 627–638.

Chen

Zhou

, et al. Biomimetic porous collagen/hydroxyapatite scaffold for bone tissue engineering. J Appl Polym Sci 2017. 2017; 134: 45271.

Levingstone

Matsiko

Dickson

, et al. A biomimetic multi-layered collagen-based scaffold for osteochondral repair. Acta Biomater 2014; 10(5): 1996–2004.

Uchida

Sivaraman

Amoroso

, et al. Nanometer-sized extracellular matrix coating on polymer-based scaffold for tissue engineering applications. J Biomed Mater Res A 2016; 104(1): 94–103.

Chen

Kawazoe

Chen

. Biomimetic assembly of vascular endothelial cells and muscle cells in microgrooved collagen porous scaffolds. Tissue Eng C Methods 2017; 23(6): 367–376.

Pieper

van Wachem

van Luyn

MJA

, et al. Attachment of glycosaminoglycans to collagenous matrices modulates the tissue response in rats. Biomaterials 2000; 21(16): 1689–1699.

Daamen

Nillesen

STM

Hafmans

, et al. Tissue response of defined collagen–elastin scaffolds in young and adult rats with special attention to calcification. Biomaterials 2005; 26(1): 81–92.

10.

Faraj

Van

KTH

Daamen

. Construction of collagen scaffolds that mimic the three- dimensional architecture of specific tissues. Tissue Eng 2007; 13(10): 2387–2394.

11.

O'Brien

Harley

Yannas

, et al. Influence of freezing rate on pore structure in freeze-dried collagen–GAG scaffolds. Biomaterials 2004; 25(6): 1077–1086.

12.

O'Brien

Harley

Yannas

, et al. The effect of pore size on cell adhesion in collagen–GAG scaffolds. Biomaterials, 26(4): 433–441.

13.

Gong

Han

Zhang

, et al. Preparation of alginate membrane for tissue engineering. J Polym Eng 2016; 36(4): 363–370.

14.

Davidenko

Gibb

Schuster

, et al. Biomimetic collagen scaffolds with anisotropic pore architecture. Acta Biomater 2012; 8(2): 667–676.

15.

Pawelec

Husmann

Best

, et al. Understanding anisotropy and architecture in ice-templated biopolymer scaffolds. Mat Sci Eng C-Mater 2014; 37(1): 141–147.

16.

Lee

Mooney

. Alginate: properties and biomedical applications. Prog Polym Sci 2012; 37(1): 106–126.

17.

Donati

Holtan

Morch

, et al. New hypothesis on the role of alternating sequences in calcium–alginate gels. Biomacromolecules 2005; 6(2): 1031–1040.

18.

Johnston

Kumar

Choonara

, et al. Modulation of the nano-tensile mechanical properties of co-blended amphiphilic alginate fibers as oradurable biomaterials for specialized biomedical application. J Mech Behav Biomed 2013; 23: 80–102.

19.

Shapiro

Oyen

. Hydrogel composite materials for tissue engineering scaffolds. JOM 2013; 65(4): 505–516.

20.

Goh

Ching

Chuan

, et al. Individualization of microfibrillated celluloses from oil palm empty fruit bunch: comparative studies between acid hydrolysis and ammonium persulfate oxidation. Cellulose 2016; 23(3): 2245–2246.

21.

Deepa

Abraham

Pothan

, et al. Biodegradable nanocomposite films based on sodium alginate and cellulose nanofibrils. Materials 2016; 9: 50.

22.

Sampath

Ching

Chuah

, et al. Preparation and characterization of nanocellulose reinforced semi-interpenetrating polymer network of chitosan hydrogel. Cellulose 2017; 24(5): 2215–2228.

23.

Lam

Chollakup

Smitthipong

, et al. Utilizing cellulose from sugarcane bagasse mixed with poly(vinylalcohol) for tissue engineering scaffold fabrication. Ind Crop Prod 2017; 100: 183–197.

24.

Angelini

Tavarini

. Ramie [Boehmeria nivea (L) Gaud.] as a potential new fibre crop for the Mediterranean region: growth, crop yield and fibre quality in a long-term field experiment in Central Italy. Ind Crop Prod 2013; 51: 138–144.

25.

Maya

Sabu

. Biofibres and biocomposites. Carbohyd Polym 2008; 71(3): 343–363.

26.

Weng

Cao

. Morphological, thermal and mechanical properties of ramie crystallites: reinforced plasticized starch biocomposites. Carbohyd Polym, 63(2): 198–204.

27.

Aarstad

Heggset

Pedersen

, et al. Mechanical properties of composite hydrogels of alginate and cellulose nanofibrils. Polymers 2017; 9(8): 1–19.

28.

Lin

Dufresne

. Nanocellulose in biomedicine: current status and future prospect. Eur Poly J 2014; 59: 302–325.

29.

Karageorgiou

Kaplan

. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005; 26(27): 5474–5491.