Fabrication of Amyloid Curli FibersâAlginate Nanocomposite

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Characterization, Synthesis, and Modifications

Fabrication of amyloid curli fibers–alginate nanocomposite hydrogels with enhanced stiffness Eneko Axpe, Anna Duraj-Thatte, Yin Chang, Domna-Maria Kaimaki, Ana SanchezSanchez, H. Burak Caliskan, Noémie-Manuelle Dorval Courchesne, and Neel S. Joshi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00364 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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ACS Biomaterials Science & Engineering

Fabrication of amyloid curli fibers–alginate nanocomposite hydrogels with enhanced stiffness Eneko Axpe* †‡§▲○, Anna Duraj-Thatte‡§, Yin Chang†, Domna-Maria Kaimaki†, Ana SanchezSanchez†⊥, H. Burak Caliskan†, Noémie-Manuelle Dorval Courchesne‡§ and Neel S. Joshi‡§

†

Nanoscience Centre, Department of Engineering, Cambridge University, 11 JJ Thomson Ave,

Cambridge CB3 0FF, United Kingdom; ‡ Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Cir, Boston, MA 02115, United States; § School of Engineering and Applied Sciences, Harvard University, 29 Oxford St, Cambridge, MA 02138, United States; ⊥

Electrical Engineering Division, Department of Engineering, Cambridge University,

Trumpington St, Cambridge CB2 1PZ, United Kingdom Email: [email protected], Phone: 415-244-2844 KEYWORDS. Curli fibers, Alginate, Nanocomposite hydrogels, Mechanical properties

ABSTRACT

Alginate hydrogels are biocompatible, biodegradable, low-cost and widely used as bioinks, cell encapsulates, three-dimensional culture matrices, drug delivery systems and scaffolds for tissue engineering. Nevertheless, their limited stiffness hinders their use for certain

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biomedical applications. Many research groups have tried to address this problem by reinforcing alginate hydrogels with graphene, carbon nanotubes or silver nanoparticles. However, these materials present nanotoxicity issues, limiting their use for biomedical applications. Other studies show that electrospinning or wet spinning can be used to fabricate biocompatible, microand nanofibers to reinforce hydrogels. As a relatively simple and cheap alternative, in this study we used bioengineered bacteria to fabricate amyloid curli fibers to enhance the stiffness of alginate hydrogels. We have fabricated for the first time bioengineered amyloid curli fibers– hydrogel composites, and characterise them by a combination of (i) Atomic Force Microscopy (AFM) to measure the Young´s modulus of the bioengineered amyloid curli fibers and study their topography, (ii) nanoindentation to measure the Young´s modulus of the amyloid curli fibers–alginate nanocomposite hydrogels and (iii) Fourier-Transform Infrared Spectroscopy (FTIR) to analyse their composition. The fabricated nanocomposites resulted in a highly improved Young´s Modulus (up to 4 fold) and showed very similar physical and chemical properties, opening the window for their use in applications where the properties alginate hydrogels are convenient but do not match the stiffness needed.

INTRODUCTION Alginate hydrogels are widely used as bioinks1, cell encapsulates2, three-dimensional culture matrices3, drug delivery systems4 and scaffolds for tissue engineering5, among other biomedical applications6. Alginate hydrogels are biocompatible7, relatively cheap to fabricate and can easily be modified to promote cell adhesion8. Despite their desirable properties and widespread use, one major weakness is their limited stiffness, which hinders their use for certain


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biomedical applications9–13. A variety of nanocomposite alginate hydrogels made with graphene14, carbon nanotubes15–17 or silver nanoparticles18,19 have been fabricated to try to address this issue. However, many of the filler materials lack the biocompatibility that would be required for in vivo implantation. Previous studies have shown the potential of the use of biocompatible fibers to reinforce hydrogels. Electrospinning and wet spinning are widely employed techniques to fabricate micro- and nanofibers that are then mixed with a crosslinking gel to form reinforced composites. Here, we use a recently developed technique that can be used as a simple and inexpensive alternative to other approaches to obtain nanofibers. As an alternative to the approaches discussed above we sought to use amyloid fibers as a versatile nanofiller material in alginate hydrogels. Amyloids are among the stiffest known biopolymeric materials20,21, and they have the potential to be more biocompatible than other fillers (e.g. carbon nanotubes). Indeed, several microorganisms (e.g. Escherichia coli22, Pseudomonas spp.23, Salmonella spp.24 and Bacillus subtilis25) exploit the remarkable properties of amyloid fibrils to promote the mechanical rigidity and strength on their respective biofilms26. In addition to their mechanical properties, amyloids are often highly resistant to degradation by proteases and denaturation, and can be customized to display functional domains and mediate interaction will cells27. Amyloid fibrils often exhibit elastic modulus values on the order of 3-20 GPa28, making them attractive for the reinforcement of hydrogels. Despite numerous studies on both amyloid fibrils and alginates as building blocks for functional materials26, there is a surprising lack of studies that use them together in a single biomaterial. In this study, our goal was to enhance the stiffness of alginate hydrogels by adding low quantities of amyloid curli fibers, without altering other physical or chemical properties


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dramatically. We selected curli fibers, a type of bacterial functional amyloid, because of their programmability and the existence of straightforward protocols for their purification in large enough quantities for hydrogel fabrication29. We used Atomic Force Microscopy (AFM) to characterize the morphology and Young´s modulus of dried amyloid curli fibers. We studied the concentration dependent reinforcement effects of amyloids in alginate hydrogels by nanoindentation. We monitored swelling ratio and sol fraction of the bioinspired nanocomposites. We also used Fourier-Transform Infrared Spectroscopy (FTIR) to confirm the chemical composition of the nanocomposite hydrogels. To our knowledge, this is the first example of a composite alginate/amyloid material and the first demonstration that amyloids can serve as mechanical reinforcement in an alginate hydrogel.

MATERIALS AND METHODS Materials: Calcium chloride dihydrate (CaCl2*2H20), alginic acid sodium salt (100-200 kDa), guanidinium chloride (GdmCl) and benzonase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium dodecyl sulfate (SDS) was purchased from TEKNOVA INC (USA). Fabrication of the amyloid curli fibers: To express curli nanofibers, PQN4 cells were transformed with pET21d plasmids encoding CsgA variant, CsgA-TFF2 (TFF2 - trefoil factor 2). Transformed cells were streaked onto fresh lysogeny broth (LB) agar plates supplemented with 100 µg/ml carbenicillin and 0.5 w/v% glucose and were grown at 37 °C. A single colony was picked, inoculated in LB medium containing 100 µg/ml carbenicillin and 2 w/v% glucose and incubated overnight at 37 °C. The overnight culture was diluted 1:100 in fresh LB medium with


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100 µg/ml carbenicillin and the protein expression was carried out at 37 °C for 24-48h. Next, a semi-purification protocol adapted from a previously described method29 was applied to the expressed curli fibers. Culture expressing curli fibers was incubated with guanidinium chloride (GdmCl) (to a final concentration 0.8M) at 4°C for 1 h. Next, the culture was concentrated onto a 47 mm polycarbonate filter membrane with 10 µm pores (EMD Millipore) using vacuum filtration. Curli fibers deposited on a filter membrane were incubated with 5 ml of 8 M GdmCl for 5 min, followed by vacuum filtration of the liquid and 3 rinses with 25 ml of sterile DI water. Next, to remove DNA/RNA bound to curli fibers, the curli-TFF fibers were treated with 5 ml of an aqueous solution (2 µM MgCl2) of nuclease (Benzonase, 1.5 U/ml) for 10 min. Finally, the filtered fibers were incubated with 5 ml of 5 w/v% SDS in water for 5 min followed by vacuum filtration of the liquid and 3 rinses with 25 ml of DI water. Semi-purified curli nanofibers were removed from the filter membrane by gently scraping the filter with a flat spatula. Purified curli nanofibers were lyophilized and stored at 4 °C. Fabrication of the alginate hydrogels: Alginic acid sodium salt was slowly added while stirring to dH20 at 50 ºC and stirred to make a solution of 2 w/v%. Subsequently, 0.5 ml of this solution was mixed with 0.5 ml of a 200 mM CaCl2 solution after pouring into a 1 cm3 mould. The process was repeated three times, making three identical samples. The moulds were covered by aluminium foil and allowed to crosslink at room temperature overnight. The fully cross-linked hydrogels were washed by dH2O. The samples were immersed in dH2O for 24h and kept at 4 ºC to allowing them to swell. Fabrication of the amyloid curli fibers–alginate hydrogel nanocomposites: Suspensions of lyophilized amyloid curli fibers in dH2O were prepared at different concentrations: 0.5, 1.0, 2.0, 4.0, and 8.0 mg/ml. The suspensions were stirred for 3 h at room temperature, followed by


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warming to 50 ºC in a water bath. Alginic acid sodium salt (100-200 kDa) was slowly stirred into these suspensions at 50 ºC to a 2 w/v% concentration of alginate and sonicated in an ultrasonic bath. 0.5 ml of this solution was mixed with 0.5 ml of a 200 mM CaCl2 solution after pouring into a 1 cm3 mould. Three identical samples were made for each of the concentrations. The moulds were covered by aluminium foil and allowed to crosslink at room temperature overnight. The fully cross-linked hydrogels were washed by dH2O. The samples were immersed in dH2O for 24h and kept at 4 ºC to allow them to swell. Swelling ratio and sol fraction of the hydrogels: The following equations30 were used to calculate swelling ratio and sol fraction of the hydrogel nanocomposites: Swelling ratio = ୛౩ ି୛ౚ ୛ౚ

, and, Sol fraction (%) =

୛౟ ି୛ౚ ୛౟

x 100, where Ws is the weight of hydrogel composites

after swelling in dH20 for 24 h, Wd is the weight of dried hydrogels, and Wi is the weight of hydrogels after crosslinking and before swelling in dH20. Thus, the swelling ratio was defined as the fractional increase in the weight of the hydrogel due to water absorption. The sol fraction was defined as the polymer fraction following a crosslinking reaction that is not part of a crosslinked network. Atomic Force Microscopy (AFM) imaging and force spectroscopy: A Solver Pro AFM by NT-MDT was used to conduct morphological and mechanical property studies. The semicontact modality of the instrument was used to obtain topography and phase images of the samples and the HybriD modality was used to map mechanical properties. This was done by acquiring a force-distance curve at each point and fitting it with the appropriate contact mechanics model so as to obtain Young’s modulus31. The tip was a Multi75Al-G with a resonance frequency of 75 kHz and a nominal stiffness of 3 N/m. It was calibrated using both the thermal and the Sader


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method, giving a stiffness of 3.53 N/m and a sensitivity of 39.5 nm/nA. Images ranging from a scan size of 10 by 10 µm2 to 500 by 500 nm2 were acquired. All measurements were conducted in air, at 20 °C and 52% relative humidity. Scanning electron microscopy (SEM): samples were prepared by fixing purified amyloid curli fibers with 2 w/v% glutaraldehyde and 2 w/v% paraformaldehyde for 2 hours at room temperature. The fibers were rinsed with water, and the solvent was gradually exchanged to ethanol with an increasing ethanol step gradient (25, 50, 75 and 100 v/v% ethanol). The sample was dried in a critical point dryer, placed onto SEM sample holders using silver adhesive (Electron Microscopy Sciences, PA, USA), and sputtered until they were coated in a 5 nm layer of Pt/Pd. Imaging was performed using a Zeiss Ultra 55 Field Emission SEM (Germany). Nanoindentation: Young’s modulus measurements of the hydrogels were performed using a Piuma nanoindenter (Optics11, Netherlands). A probe from the same manufacturer with a stiffness of 0.041 N/m and a tip radius of 40.0 µm was used. Calibration was conducted as on glass under wet conditions as per the manufacturer's instructions. Each sample was immersed in dH20 for 24 h before the nanoindentation experiments. Force vs. distance measurements were conducted under wet conditions (dH20). The indentation depth was fixed to be < 7 µm in order to avoid bottom effects. For each hydrogel type, 3 replicates from 2 different batches were tested to ensure the reproducibility of the experiments. At least 19 force curves were used to determine the local Young’s modulus of each sample, using the Optics11 Nanoindenter V2.1.13 sotware. The results are shown as mean ± standard error of the mean. A Hertzian contact model parameter was used with a 20% of Pmax for the fit of the curves and assuming that the Poisson's ratio of the samples is νsample = 0.5.


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Fourier-Transform Infrared Spectroscopy (FTIR): A Perkin-Elmer Spotlight 400 FTIR (PerkinElmer, Waltham, MA, USA) was used in the attenuated total internal reflection (ATR) mode for spectroscopic analyses of the dried hydrogel samples. The wavelength resolution was set to 4 cm−1. The average of 8 scans was recorded from 4000 cm-1 to 650 cm-1.

RESULTS AND DISCUSSION Purified recombinant curli amyloid nanofibers were isolated in hydrogel form, dried, and subjected to AFM analysis to determine their morphology and stiffness. The fibers exhibited a uniform thickness of ~7 nm, which is close to the expected diameter for a single fiber composed of the CsgA protein. Their lateral dimension, measured via a topography cross section, was more variable and of the order of 35 nm, indicating that the fibers were laterally aggregated (see phase imagining, Figure 1 and Supplementary Figure S1). This type of aggregation is very common in amyloids, as previously reported32. Nanomechanical (Hybrid mode) AFM was also performed to determine the mechanical properties of the bioengineered amyloids contained in the film. Five different areas of 1 by 1 µm2 scan size were mapped and the resulting force-distance curves were fitted using the DerjaguinMueller-Topolov (DMT) model33,34. This analysis produced a Young’s modulus of 1508 ± 882 MPa (see the histogram in Supplementary Figure S2). This value is on the lower end of the range of Young’s modulus values reported in the literature for amyloids28.


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Figure 1. AFM topography (top) and phase (bottom) images of the film containing amyloid fibers and their respective cross sections.

Nanoindentation analysis of the amyloid–alginate composite hydrogels under wet conditions revealed that their Young´s moduli are higher than alginate hydrogels, and depend on the amyloid concentration (Figure 2 and Figure S3). The reinforcement is statistically significant (P < 0.01) for amyloid curli fibers/alginate mass ratios of 1:10, 1:5 and 1:2.5. More specifically, when the mass ratio is 1:10, the mean value of the Young´s modulus (‫ )ܧ‬increases by 275% compared to that of alginate alone. When the mass ratio is 1:5, the ‫ ܧ‬is 341% higher. Finally, when the mass ratio is 1:2.5, the nanocomposite hydrogels showed that ‫ ܧ‬is 410% higher than alginate hydrogels.


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Figure 2. Young´s moduli of amyloid–alginate nanocomposite hydrogels as a function of amyloid concentration. Alginate only (grey) and amyloid–alginate (red) at concentrations of 0.5, 1.0, 2.0, 4.0, and 8.0 mg/ml are represented (n=3 hydrogel replicates). All data are represented as mean ± s.e.m. with n = 33, 19, 21, 30, 27 and 23 technical replicates, respectively. NS = not significant, **P < 0.01 by one-way ANOVA followed by post-hoc Tukey HSD multiple comparisons test.

Figure 3 shows the measured swelling ratio (A) and sol fraction (B) of the alginate and the nanocomposites hydrogels. No significant difference was observed in either the swelling ratio or the sol fraction when comparing the amyloid–alginate nanocomposites with the alginate hydrogels. In addition, there was no significant difference in swelling between nanocomposites


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with different concentrations of amyloid fibers. The physiognomy of the nanocomposite hydrogels at the macroscale was almost exactly as the alginate hydrogel alone: transparent, stable and easy to handle (see Supplementary Figure S4).

(A)

(B)

Figure 3. (A) Swelling ratio (a.u.) and (B) sol fraction (%) of alginate hydrogels (grey) and amyloid curli fibers– alginate composite hydrogels (red). Data represent mean ± s.d. of triplicates (NS, not significant, by one-way ANOVA followed by post-hoc Tukey HSD multiple comparisons test).

We performed compositional analysis using FTIR for composite samples that showed a significant increase in the Young´s modulus, compared to pure alginate gels. Pure alginate gels showed a chemical “fingerprint” between 500-1700 cm-1. Pure amyloid gels showed distinguishable peaks near 2750 cm-1 that also appeared in the composite hydrogels in proportion to the amyloid concentration (Figure 3). On the other hand, a very sharp peak appears in the amyloid curli fibers at about 1200 cm-1, which is not present in the nanocomposites.


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Figure 4. Compositional analyses of dried amyloid curli fibers–alginate (2 w/v%) composite hydrogels with 2.0, 4.0 and 8.0 mg/ml amyloid compared to amyloid fibers and alginate alone. Spectra from Attenuated total internal reflection Fourier Transform Infrared (ATR-FTIR) spectra were normalized and shifted vertically for clarity.

Thus, we fabricated the first example of nanocomposite hydrogels of alginate and genetically engineered amyloid curli fibers. We found that even relatively low concentrations of amyloids (2 mg/ml) significantly increase the stiffness of alginate hydrogels, as measured by nanoindentation tests on hydrated samples. The highest alginate/amyloid ratio of 1:2.5 led to a 4fold increase in Young´s modulus. AFM analysis of dried films composed of pure amyloid fibers revealed their nanoscale features, indicating their high surface to volume ratio. As is the case in other composite hydrogels35, it is likely that favourable interactions between curli amyloid fibers


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and the alginate matrix polymer (Supplementary Figure S5), combined with the characteristically high rigidity of amyloids, lead to the observed increase in Young’s modulus for the composite materials. Importantly, addition of curli amyloid fibers to the alginate gels did not significantly affect their swelling ratio or sol fraction, indicating that amyloids do not interfere with the water absorption capability or crosslinking reaction of alginate. Together with FTIR-based compositional analysis, these results indicate that curli amyloid fibers could serve as reinforcing agents that could be mixed with alginate polymers without altering any other physico/chemical properties that may be desired. Both components of the composites have shown good biocompatibility in previous studies. Amyloid curli fibers have been used as cell growth scaffolds36 and in the presence of mammalian cells, showing no toxicity37. Alginate hydrogels have been used in numerous biomedical applications12. However, the biocompatibility in vivo of the composites should be addressed in future studies. The limitations of this study also include the fact that we are yet to establish cell–nanocomposite hydrogel interactions, (i.e., cell spreading within the nanocomposite hydrogels). In this regard, it is logical to think that the amyloid curli fibers are filling some of the hydrogel pores, and this could have effects on the diffusion properties in the hydrogel and cell spreading within the matrix; both would be important features to consider for future in vitro applications of the hydrogels reinforced with amyloid curli fibers. Taken together, our findings suggest a practical use for curli amyloid fibers in enhancing and modulating the mechanical performance of alginate hydrogels. Alginate hydrogels are already used for a range of biomedical applications9, including tissue engineering38 and drug delivery39, some of which depend integrally on precise tuning of gel mechanical properties. Curli amyloid fibers are relatively cheap to produce and have the added benefit of being highly


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customizable through straightforward genetic engineering37, which could be leveraged to influence cell-matrix interactions by modulating the stiffness of hydrogels.

CONCLUSION In conclusion, our findings demonstrate that our goal to enhance the stiffness of alginate hydrogels by amyloid curli fibers was achieved, opening the window for its use in applications where alginate hydrogels are convenient but do not match the stiffness needed. Inspired by nature, we have fabricated for the first time bioengineered amyloid curli fibers–alginate nanocomposite hydrogels. These nanocomposites resulted in a highly improved Young Modulus (up to 4 fold). At the same time, these bioinspired nanocomposites showed very similar physical and chemical properties –reflected by the swelling ratio, sol fraction and FTIR tests– to alginate hydrogels. This study shows that amyloid fiber reinforced composites can capture the benefits of hydrogels (including water content and transparency) and overcome their mechanical limitations. The up to 4-fold increase in the Young´s modulus could address the limitation of alginate in resembling the stiffness of biological soft tissues like arteries, skin or cornea in potential applications such as 3D cell cultures or tissue repair applications. Future research could continue to explore the incorporation of bioengineered amyloid curli fibers as an effective, cheap and simple strategy to modulate the stiffness of other hydrogels. Looking forward, we hope this study will inspire researchers to fabricate new hydrogels with enhanced mechanical properties for 3D culture matrices, bioinks, drug delivery systems, or scaffolds for tissue engineering by employing amyloid curli fibers.


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SUPPORTING INFORMATION The following files are available free of charge: Supplementary Figure S1. Scanning Electron Microscopy (SEM) image of the amyloid curli fibers (PDF) Supplementary Figure S2. Histogram of Young´s modulus of the amyloid fibers (PDF) Supplementary Figure S3. Force versus indentation curves obtained by nanoindentation (PDF) Supplementary Figure S4. Picture of an amyloid curli fibers–alginate hydrogel nanocomposite sample (PDF) Supplementary Figure S5. AFM topography and phase images of 8 mg/ml amyloid curli fibers– alginate hydrogel nanocomposites (PDF) AUTHOR INFORMATION Corresponding Author *Dr. Eneko Axpe Department of Materials Science and Engineering Stanford University, Stanford California 94305, United States Email: [email protected] Phone: +1 415-244-2844


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Present Addresses ▲

Department of Materials Science and Engineering, Stanford University, 496 Lomita Mall

Stanford, CA 94305, United States;

○

Space Biosciences Division, NASA-Ames Research

Center, Moffett Blvd, Mountain View, CA 94035, United States Author Contributions Eneko Axpe and Neel S. Joshi conceived and coordinated the study. Anna Duraj-Thatte and Eneko Axpe fabricated the amyloid curli fibers. Eneko Axpe and Ana Sanchez-Sanchez designed the protocols for the fabrication of the nanocomposites. Ana Sanchez-Sanchez fabricated the alginate and nanocomposite hydrogels. Yin Chang carried out the nanoindentation measurements. Domna-Maria Kaimaki carried out the AFM studies. Noémie-Manuelle Dorval Courchesne did the SEM experiment. H. Burak Caliskan tested the samples by FTIR. All authors participated in the writing of the article. Conflict of interest The authors declare no conflict of interest. ACKNOWLEDGMENT We would like to acknowledge the discussion and valuable comments by Dr. M. B. Avinash and Doreen Chan, and the help with the design of the graphical abstract by Margherita Gallieni. DM. Kaimaki would like to acknowledge the Durkan lab for the use of the AFM. Dr. E. Axpe is thankful for the Postdoctoral Fellowship of the Basque Government and Dr. A. Sanchez-Sanchez for the Marie Curie IF BIKE Project No. 743865.


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ABBREVIATIONS AFM, Atomic Force Microscopy; FTIR, Fourier-Transform Infrared Spectroscopy; CaCl2*2H20, Calcium chloride dehydrate; GdmCl, Guanidinium Chloride; SDS, Sodium Dodecyl Sulfate; TFF2, Trefoil Factor 2; LB, Lysogeny Broth, E, Young´s modulus. REFERENCES (1)

Jia, J.; Richards, D. J.; Pollard, S.; Tan, Y.; Rodriguez, J.; Visconti, R. P.; Trusk, T. C.; Yost, M. J.; Yao, H.; Markwald, R. R.; et al. Engineering Alginate as Bioink for Bioprinting.

Acta

Biomater.

10

2014,

(10),

4323–4331,

DOI:

10.1016/J.ACTBIO.2014.06.034 (2)

Orive, G.; Hernández, R. M.; Gascón, A. R.; Calafiore, R.; Chang, T. M. S.; Vos, P. De; Hortelano, G.; Hunkeler, D.; Lacík, I.; Shapiro, A. M. J.; et al. Cell Encapsulation: Promise and Progress. Nat. Med. 2003, 9 (1), 104–107, DOI: 10.1038/nm0103-104

(3)

Caliari, S. R.; Burdick, J. A. A Practical Guide to Hydrogels for Cell Culture. Nat. Methods 2016, 13 (5), 405–414, DOI: 10.1038/nmeth.3839

(4)

Li, J.; Mooney, D. J.; Buffle, J.; DeRouchey, J. E.; Langer, R. Designing Hydrogels for Controlled

Drug

Delivery.

Nat.

Rev.

Mater.

2016,

1

(12),

16071,

DOI:

10.1038/natrevmats.2016.71 (5)

Venkatesan, J.; Bhatnagar, I.; Manivasagan, P.; Kang, K.-H.; Kim, S.-K. Alginate Composites for Bone Tissue Engineering: A Review. Int. J. Biol. Macromol. 2015, 72, 269–281, DOI: 10.1016/j.ijbiomac.2014.07.008

(6)

Bidarra, S. J.; Barrias, C. C.; Granja, P. L. Injectable Alginate Hydrogels for Cell Delivery in

Tissue

Engineering.

Acta

Biomater.

2014,

10

(4),

1646–1662,

DOI:

10.1016/J.ACTBIO.2013.12.006 (7)

Shapiro, L.; Cohen, S. Novel Alginate Sponges for Cell Culture and Transplantation. Biomaterials 1997, 18 (8), 583–590, DOI: 10.1016/S0142-9612(96)00181-0


17


(8)

Page 18 of 22

Pawar, S. N.; Edgar, K. J. Alginate Derivatization: A Review of Chemistry, Properties and Applications.

Biomaterials

2012,

33

(11),

3279–3305,

DOI:

10.1016/J.BIOMATERIALS.2012.01.007 (9)

Axpe, E.; Oyen, M. L. Applications of Alginate-Based Bioinks in 3D Bioprinting. Int. J. Mol. Sci. 2016, 17 (12), DOI: 10.3390/ijms17121976

(10)

Zhao, Z.; Fang, R.; Rong, Q.; Liu, M. Bioinspired Nanocomposite Hydrogels with Highly Ordered Structures. Adv. Mater. 2017, 29 (45), 1703045, DOI: 10.1002/adma.201703045

(11)

Sun, G.; Li, Z.; Liang, R.; Weng, L.-T.; Zhang, L. Super Stretchable Hydrogel Achieved by Non-Aggregated Spherulites with Diameters

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