Electrospun Nanofibers for Tissue Engineering: Desired Properties
RESEARCH ARTICLE

Electrospun Nanofibers for Tissue Engineering: Desired Properties

Luis Jesús Villarreal Gómez1 , * Open Modal
Authors Info & Affiliations
The Open Biomaterials Science Journal 19 Oct 2022 RESEARCH ARTICLE DOI: 10.2174/26659956-v1-e2209030

Abstract

Electrospun nanofibers have gained great attention in the biomedical industry, especially in tissue engineering, because of their interesting properties that promote cell growth and tissue cicatrization or regeneration, where any biological tissue can be beneficiated by choosing the proper biomaterials. Hence, the objective of this perspective article is to give an insight into the desired properties of the electrospun nanofibers dedicated to the tissue engineering approach. A high tensile strength, flexibility, reduced permeability of water, high surface area, biocompatibility, and biodegradability are some of the properties recognized and discussed to be more important for tissue engineering applications. The purpose of these properties is to mimic the surrounding tissue or create the optimal condition for the targeted cell growth. Despite all the reported literature, it still is missing a complete screening of the above mentioned properties specific to their respective target tissues.

Keywords: Tissue engineering, Electrospinning technique, Nanofibers, Electrospun nanofibers, Bone tissue, 3D tissue formation.

1. INTRODUCTION

The value of the electrospinning technique has been reported over the last 10 years [1-13]. This technique leads to the production of versatile nanofibers that possess a diverse set of properties [3] and can be used in several applications such as tissue engineering [1, 2, 4, 12], drug delivery systems [4, 5, 7, 8, 10, 11], and biotechnology [6, 13], amongst others.

Among the interesting properties of electrospun nanofibers, the high tensile strength, flexibility, reduced permeability of water [14], high surface area [15], biocompatibility [2], and biodegradability [16], are a few mentioned.

The above properties can always be designed through the choice of a specific polymer [4] that is used as a matrix and can be functionalized with a great variety of biomaterials such as metals [9, 12], ceramics [1, 2] or other polymers [6, 13], these above biomaterials can be used to functionalize the nanofibers with extra properties.

Such is the case of the integration of an antimicrobial effect on the nanofibers thanks to some bioactive properties, for example, silver nanoparticles [12] and curcumin [13], or conductive properties by adding graphene [17], polyaniline [18] or having therapeutics effect loading pharmaceutical drugs in/on the surface of the nanofibers such as dexamethasone [10], sildenafil citrate [11], just to mention some examples.

Tissue engineering is one of the approaches beneficiated by the electrospun technology [1-4], because nanofibers create a tridimensional structure that simulates the extracellular matrix made by tissues (Fig. 1). These structures can be prepared with biomaterials that resemble the chemical composition of tissues, using natural biomaterials, for example, hydroxyapatite [1, 2] which promotes the regeneration of bone tissue. By the same time, the nanofibers are reabsorbed and become part of the tissue [19]. The main objective of tissue engineering is to avoid tissue and organ transplantation. Hence, natural and synthetic biomaterials are provided through the electrospinning technique of 3D tissue formation, which is regularly enhanced by the seeding of the cells into the material structure.

In Fig. (1), it can be observed that electrospun fibers with different diameters (yellow arrows) create a tridimensional scaffold that resembles the extracellular matrix of tissues.

Despite all tissues can be susceptible to the use of electrospun nanofibers for regeneration/cicatrization improvement, just a few of them have been extensively studied, such as bone [1, 2, 4, 14, 16, 17, 19], cartilage [20, 21], and skin [4, 12, 18], this can be due to the accessibility of the tissue and less complicated applicative experiments in universities or research institutes [7].

Fig. (1). Tridimensional matrix made by electrospun nanofibers.

For tissue regeneration, bioactivity, biocompatibility, biodegradability, and adequate mechanical properties are the main factors to resolve in the design of an excellent system [12].

Bioactivity can be incorporated on the surface of the fibers in case the polymeric blend has no bioactivity by itself (such as collagen or hydroxyapatite [1, 2] whose sole presence increases the stimulation of bone formation), for example, antimicrobial agents [12, 13], proliferation enhancers such as growth factors [22], pharmaceutical drugs for tissue stress relief [23], being all these additions promoter of enhancers of the function of the 3D structure of the scaffolds.

Conversely, biodegradability offers a lifetime of the scaffolds to prevent overstaying its presence in the tissue and avoiding additional surgery or treatments, and this degradation time is dependent on the severity of the lesion and the rate at which the tissue is proliferating [24]. Moreover, degradation metabolites have to be carefully considered in designing an implantable scaffold because this bioproduct will lead to the success or failure of the membrane [25].

Mechanical properties of the scaffolds depend on the tissue, the zone of implantation, and handling requirements [12]. These properties vary and always are desired that the mechanical properties of the scaffolds assemble in the tissue. The adequate characteristics of the fibers are crucial to the successful use of the system due to their structural support for the newly formed tissue [26].

Finally, biocompatibility is the most important feature of the electrospun scaffolds for tissue engineering since the implanted materials have to realize a specific function with an adequate response from the surrounding tissue and not over stress [1, 2, 12]. With this property, cytotoxicity is avoided and tissue response is evaluated. The equilibrium between bioactivity, biodegradability, and mechanical properties is desired depending on the specific tissue and injury to treat [4, 22, 23].

Research groups that are dedicated to tissue engineering and proposed electrospun nanofibers need to carefully study all these features and demonstrate the feasibility of their systems. Biomedical systems always need not to be toxic and do not provoke chronic alteration of the tissue.

2. BIODEGRADABILITY

The biodegradability of the electrospun fibers depends on the chosen polymer used to fabricate the fibers. Biodegradable polymers propose significant advantages for disposable or fast consuming products in medical applications [27].

The poly (hydroxyalkanoates) (PHA), a class of naturally occurring poly (esters) that are secondary metabolites of microorganisms in excess of carbon source conditions, are among the most significant biodegradable polymers and are highly recommended for biomedical applications due to their biodegradability [27].

Moreover, biodegradable aliphatic polyesters (poly (lactide) (PLA), poly (glycolide) (PGA), and poly (lactide-co-glycolide) (PLGA) are the most characteristic synthetic polymers for tissue engineering and regenerative medicine. Despite that, aseptic inflammation is one of their disadvantages due to their acidic degradation creating conditions for further implantation [28].

The desired degradation time of the electrospun fibres varies depending on the application; some, like poly (vinyl pyrrolidone) (PVP) [29], are designed to disintegrate in seconds, while others, like poly (caprolactone) (PCL) [30], are intended to degrade over the years.

In Table 1 are enlisted some examples of biodegradable polymers that can be used in the electrospinning technique and have been used for tissue engineering.

Table 1.
Some examples of biodegradable electrospun fibers for tissue engineering.
Electrospun Fibers Biodegradable Polymers Degradation Time Application Refs.
PHBV PHBV 420 days Osteoblast and fibroblast regeneration [27]
CTS/PLGA PLGA Several weeks to several months (55 days) Amelioration inflammatory responses [28]
GT/PCL PCL 2 or 3 years Postoperative Cardiac Adhesion [30]
PLCL PLCL 72 days at 50 °C, 177 days at 37 °C Orthopedic shoulder surgery [31]
PPC PPC 8 months Peripheral nerve regeneration [32]
CTS/PU PU 20-23 years Soft tissue engineering [33]
PLA/PEG PEG 10-12 hrs Endothelial cell regeneration [34]
CTS/PLGA: Chitosan/ poly(lactide-co-glycolide; GT/ PCL: Gelatin/ polycaprolactone; PHBV: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); PLA/PEG: Poly(D,L-lactide)-b-poly(ethylene glycol); PLCL: Poly(L, L-lactide-co-ε-caprolactone); PPC: Poly (propylene carbonate); CTS/PU: Chitosan based-poly(urethane urea).

3. MECHANICAL PROPERTIES

One of the main problems of the electrospun fibrous scaffolds is the lack of evaluation of the impact of solvent retention in fibers on the scaffold’s mechanical properties [35].

D'Amato et al. 2018, discussed that the retained solvent can act as a plasticizer, modifying the mechanical properties of the fibrous scaffolds such as brittleness and stiffness. In that sense, that study evaluated the retention of solvent and its effect in PGA, PLCL, and PET electrospun fibers through themogravimentric analyses. The obtained results showed that polymers that were electrospun below their glass transition temperature (Tg) held solvent and polymers that were electrospun above their Tg did not, which affect mechanical properties such as Young’s moduli, toughness and failure strain as the solvent evaporates [35, 36].

One of the most intriguing polymers utilised in the electrospinning process is poly (caprolactone), which has promising mechanical qualities, is less expensive than other polymers, is biocompatible, and has a slow rate of degradation, making it perfect for tissue engineering applications [36]. It is important to highlight that besides the polymers' properties, the fibers' thickness confer the mechanical properties. Hence, PCL possesses great mechanical properties for tissue engineering in trabecular bone, skin, blood vessels, liver, lungs, heart and kidney because its tensile module is about 62±26 MPa in PCL fibers of 440-1040 nm, which is higher than the tensile module of all the above tissues Table 2 [36].

Table 2.
Tensile module of some human tissues.
Human Tissue Tensile Module References
Trabecular bone 10-3,000 MPa [37]
Skin 27.2±9.3MPa [38]
Blood vessels 8.3 ± 1.7 MPa [39]
Liver 12.16 ± 1.20 KPa [40]
Lungs 142 ± 8.84 kPa [41]
Heart 2.15 ± 0.15 MPa [42]
Kidney 180.32 ± 11.11 kPa [43]

4. BIOCOMPATIBILITY

Electrospun nanofibrous scaffolds possess morphological parallels with the extracellular matrix in tissues, which give them thanks to cell adhesion, proliferation, and cell function properties making them ideal for tissue engineering applications (Table 3) [44]. Biocompatibility is the most important feature in electrospun nanofibers intended for tissue engineering [1, 2] because the successful role of the electrospun fibers implanted in tissue is the main and final objective of any biomedical biodevices. It is important to remark that in order to achieve biocompatibility, the low cytotoxicity of the nanofibers is crucial [1-4].

Çakmakçı et al. 2012, combined UV curing and electrospinning technologies to produce methacrylatd cellulose acetate butyrate (CABIEM) electrospun nanofibers. The cytotoxicity of these nanofibers was evaluated using the MTT assay in human umbilical vein endothelial (ECV304) and mouse embryonic fibroblasts (3T3) cells. Also, a modified collagen presence in the CABIEM fibers was proposed in order to enhance cell adhesion and proliferation. Regarding their results, the CABIEM fibers appear to be non-toxic; authors discussed that cell viability was related to collagen proportion. The study encountered that cell adhesion and proliferation were improved as the collagen concentration increased [45].

In another study, conductive electrospun fibrous scaffolds of poly (pyrrole) (PPy) were proposed to rush the healing of damaged tissues. Authors tested different proportions of PPy in polymeric fibers and concluded that the presence of PPy enhanced conductivity. It is well known that conductivity in the electrospun fibers promotes cell adhesion, growth, and cell proliferation. Hence, conductive fibers can be proposed for tissue regeneration of the heart, nerve, skin, and other tissues where electrical signals are required for cell communication [46].

Table 3.
Some examples of biocompatibility studies performed in electrospun fibers.
Electrospun Fibers Cell Line Biocompatibility Study References
PCL/GP Rat stem cells Cell attachment and differentiation assay [44]
CABIEM ECV304 and 3T3 cells MTT assay [45]
PPy/CTS/COL Fibroblast cells MTT assay [46]
TPU/PDMS Human skin fibroblast cells MTT assay [47]
PHBV/PVA HUVECs, SMCs and MSCs cells Flow cytometry and immunocytochemistry [48]
CABIEM: methacrylated cellulose acetate butyrate; PPy/CTS/COL: polypyrrole/ chitosan/ collagen; PCL/GP: polycaprolactone-cyclopentanone/ graphene; TPU/PDMS: Thermoplastic polyurethanes/ Polydimethylsiloxane. PHBV/PVA: poly(hydroxy butyrate-co-hydroxy valerate)/poly(vinyl alcohol).

Ginestra P. 2019, prepared different proportions of graphene loaded in poly (caprolactone)/cyclo pentanone electrospun fibers, presenting interesting mechanical behaviors. Statistically significant differences in mechanical and biological properties were related to graphene concentration. In the study, rat stem cells were exposed to these fibers that found a great relationship between the graphene presence, taking into account that graphene confers conductivity on the polymeric fibers. Also, a higher proportion of dopaminergic neurons were identified in the study related to higher percentages of graphene [44].

Finally, Drupitha et al. 2019, evaluated the biocompatibility of thermoplastic polyurethanes/ polydimethylsiloxane (TPU/PDMS) electrospun fibers using the MTT and cell proliferation assay with human skin fibroblast cells. It was found that fiber morphology, porosity, surface wettability, and biological and mechanical properties were influenced by the presence of the PDMS fraction [47].

CONCLUSION

Electrospun nanofibers have demonstrated their capacity to improve cell proliferation in different cell lines in vitro and in vivo. For the success of the fibrous scaffolds, it is necessary to test and design proper formulations to confer specific properties that will help the fibers interact with cells and the surrounding tissue. The high tensile strength, flexibility, reduced permeability of water, high surface area, biocompatibility, and biodegradability are the most interested properties for tissue engineering. However, not all the reported literature completely screen these parameters.

LIST OF ABBREVIATIONS

PHA = Poly (Hydroxyalkanoates)
PLA = (poly lactide)
PLGA = (lactide-coglycolide)

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

Luis Jesús Villarreal-Gómez is the Editorial Advisory Board of The Open Biomaterials Science Journal.

ACKNOWLEDGEMENTS

To Dr. Daniel Grande, East Paris Institute of Chemistry and Materials – CNRS, Paris, France, for taking the micrograph figure.

REFERENCES

1
Villarreal-Gómez LJ, Vera-Graziano R, Vega-Rios MR, Pineda-Camacho JL, Mier-Maldonado PA, Almanza-Reyes H, et al. In vivo biocompatibility of dental scaffolds for tissue regeneration. Advanced Materials Research Trans Tech Publications Ltd 2014.https://www.scientific.net/AMR.976.191
2
Cornejo-Bravo JM, Villarreal-Gómez LJ, Vera-Graziano R, et al. Biocompatibility evaluation of electrospun scaffolds of poly (L-lactide) with pure and grafted hydroxyapatite. J Mexi Chem Soc 50(4): 435-.
3
Villareal-Gómez LJ, Álvarez Suárez AS, Villarreal Gómez LJ, Paz González JA, Iglesias AL, Vera Graziano R. Designing a low-cost electrospinning device for practical learning in a bioengineering biomaterials course. Rev Mex Ing Biomed 2015; 37: 27-36.
4
Villarreal-Gómez LJ, Cornejo-Bravo JM, Vera-Graziano R, Grande D. Electrospinning as a powerful technique for biomedical applications: A critically selected survey. J Biomater Sci Polym Ed 2016; 27(2): 157-76.
5
Torres-Martinez EJ, Cornejo Bravo JM, Serrano Medina A, Pérez González GL, Villarreal Gómez LJ. A Summary of Electrospun Nanofibers as Drug Delivery System: Drugs Loaded and Biopolymers Used as Matrices. Curr Drug Deliv 2018; 15(10): 1360-74.
6
Velasco-Barraza RD, Vera-Graziano R, López-Maldonado EA, et al. Study of nanofiber scaffolds of PAA, PAA/CS, and PAA/ALG for its potential use in biotechnological applications. Int J Polym Mater 2018; 67(13): 800-7.
7
Pérez-González GL, Villarreal-Gómez LJ, Serrano-Medina A, Torres-Martínez EJ, Cornejo-Bravo JM. Mucoadhesive electrospun nanofibers for drug delivery systems: Applications of polymers and the parameters’ roles. Int J Nanomedicine 2019; 14: 5271-85.
8
Torres-Martinez EJ, Pérez-González GL, Serrano-Medina A, et al. Drugs loaded into electrospun polymeric nanofibers for delivery. J Pharm Pharm Sci 2019; 22(1): 313-31.
9
López-Covarrubias JG, Soto-Muñoz L, Iglesias AL, Villarreal-Gómez LJ. Electrospun nanofibers applied to dye solar sensitive cells: A review. Materials (Basel) 2019; 12(19): 3190.
10
Pérez-González GL, Villarreal-Gómez LJ, Olivas-Sarabia A, Valdez R, Cornejo-Bravo JM. Development, characterization, and in vitro assessment of multilayer mucoadhesive system containing dexamethasone sodium phosphate. Int J Polym Mate Polym Biomat 2020; 1: 1-13.
11
Torres-Martínez EJ, Vera-Graziano R, Cervantes-Uc JM, et al. Preparation and characterization of electrospun fibrous scaffolds of either PVA or PVP for fast release of sildenafil citrate. e-Polymers 2020; 20: 746-58.
12
Álvarez-Suárez AS, Dastager SG, Bogdanchikova N, et al. Electrospun fibers and sorbents as a possible basis for effective composite wound dressings. Micromachines (Basel) 2020; 11(4): 441.
13
Pompa-Monroy DA, Figueroa-Marchant PG, Dastager SG, et al. Bacterial biofilm formation using PCL/Curcumin electrospun fibers and its potential use for biotechnological applications. Materials (Basel) 2020; 13(23): 5556.
14
Conte AA, Sun K, Hu X, Beachley VZ. Effects of fiber density and strain rate on the mechanical properties of electrospun polycaprolactone nanofiber mats. Front Chem 2020; 8: 610.
15
Shah M, Yang Z, Li Y, Jiang L, Ling J. Properties of electrospun nanofibers of multi-block copolymers of [Poly-ε-caprolactone-b-poly(tetrahydrofuran-co-ε-caprolactone)]m synthesized by janus polymerization. Polymers (Basel) 2017; 9(11): 559.
16
Blachowicz T, Ehrmann A. Production and application of biodegradable nanofibers using electrospinning techniques. Electrospun Nanofibers: Fabrication, Functionalization and Applications 2021; 559-24.
17
Bateni F, Hashemi Motlagh G. Electrospun polyamide/graphene oxide nanofibers as fillers for polyethylene: Preparation and characterization. J Appl Polym Sci 2022; 139(3): 51506.
18
Abd Razak SI, Wahab IF, Fadil F, Dahli FN, Md Khudzari AZ, Adeli H. A review of electrospun conductive polyaniline based nanofiber composites and blends: processing features, applications, and future directions. Adv Mater Sci Eng 2015; 2015: 1-19.
19
Raja IS, Preeth DR, Vedhanayagam M, et al. Polyphenols-loaded electrospun nanofibers in bone tissue engineering and regeneration. Biomater Res 2021; 25(1): 29.
20
Li G, Shi S, Lin S, et al. Electrospun fibers for cartilage tissue regeneration. Curr Stem Cell Res Ther 2018; 13(7): 591-9.
21
Yilmaz EN, Zeugolis DI. Electrospun polymers in cartilage engineering—state of play. Front Bioeng Biotechnol 2020; 8: 77.
22
Sahoo S, Ang LT, Goh JCH, Toh SL. Growth factor delivery through electrospun nanofibers in scaffolds for tissue engineering applications. J Biomed Mater Res A 2009; 93(4): 1539-50.
23
Ye K, Kuang H, You Z, Morsi Y, Mo X. Electrospun nanofibers for tissue engineering with drug loading and release. Pharmaceutics 2019; 11(4): 182.
24
Silva M, Ferreira FN, Alves NM, Paiva MC. Biodegradable polymer nanocomposites for ligament/tendon tissue engineering. J Nanobiotechnology 2020; 18(1): 23.
25
Stojanov S, Berlec A. Electrospun nanofibers as carriers of microorganisms, stem cells, proteins, and nucleic acids in therapeutic and other applications. Front Bioeng Biotechnol 2020; 8: 130.
26
Rashid TU, Gorga RE, Krause WE. Mechanical properties of electrospun fibers—a critical review. Adv Eng Mater 2021; 23(9): 2100153.
27
Kaniuk Ł, Stachewicz U. Development and advantages of biodegradable pha polymers based on electrospun phbv fibers for tissue engineering and other biomedical applications. ACS Biomater Sci Eng 2021; 7(12): 5339-62.
28
Shen Y, Tu T, Yi B, et al. Electrospun acid-neutralizing fibers for the amelioration of inflammatory response. Acta Biomater 2019; 97: 200-15.
29
Samprasit W, Akkaramongkolporn P, Kaomongkolgit R, Opanasopit P. Cyclodextrin-based oral dissolving films formulation of taste-masked meloxicam. Pharm Dev Technol 2018; 23(5): 530-9.
30
Wang X, Xiang L, Peng Y, et al. Gelatin/Polycaprolactone electrospun nanofibrous membranes: The effect of composition and physicochemical properties on postoperative cardiac adhesion. Front Bioeng Biotechnol 2021; 9: 792893.
31
Yue B, Ye P, Liu C, Chang Z. Application of poly( L, L -lactide-co-ε-caprolactone) copolymer medical implants to the treatment of massive rotator cuff tears in orthopedic shoulder surgery. AIP Adv 2020; 10(11): 115212.
32
Wang Y, Zhao Z, Zhao B, et al. Biocompatibility evaluation of electrospun aligned poly (propylene carbonate) nanofibrous scaffolds with peripheral nerve tissues and cells in vitro. Chin Med J (Engl) 2011; 124(15): 2361-6.
33
Vieira T, Carvalho Silva J, Botelho do Rego AM, Borges JP, Henriques C. Electrospun biodegradable chitosan based-poly(urethane urea) scaffolds for soft tissue engineering. Mater Sci Eng C 2019; 103: 109819.
34
Kruse M, Greuel M, Kreimendahl F, et al. Electro-spun PLA-PEG-yarns for tissue engineering applications. Biomed Eng 2018; 63(3): 231-43.
35
D’Amato AR, Bramson MTK, Corr DT, Puhl DL, Gilbert RJ, Johnson J. Solvent retention in electrospun fibers affects scaffold mechanical properties. Electrospinning 2018; 2(1): 15-28.
36
Baker SR, Banerjee S, Bonin K, Guthold M. Determining the mechanical properties of electrospun poly-ε-caprolactone (PCL) nanofibers using AFM and a novel fiber anchoring technique. Mater Sci Eng C 2016; 59: 203-12.
37
Morgan EF, Unnikrisnan GU, Hussein AI. Bone mechanical properties in healthy and diseased states. Annu Rev Biomed Eng 2018; 20(1): 119-43.
38
Trotta A, Ní Annaidh A. Mechanical characterisation of human and porcine scalp tissue at dynamic strain rates. J Mech Behav Biomed Mater 2019; 100: 103381.
39
Camasão DB, Mantovani D. The mechanical characterization of blood vessels and their substitutes in the continuous quest for physiological-relevant performances. A critical review. Mater Today Bio 2021; 10: 100106.
40
Karimi A, Shojaei A. An experimental study to measure the mechanical properties of the human liver. Dig Dis 2018; 36(2): 150-5.
41
Karimi A, Razaghi R. The role of smoking on the mechanical properties of the human lung. Technol Health Care 2018; 26(6): 963-72.
42
Kwon J, Ock J, Kim N. Mimicking the mechanical properties of aortic tissue with pattern-embedded 3d printing for a realistic phantom. Materials (Basel) 2020; 13(21): 5042.
43
Simmons WN, Cocks FH, Zhong P, Preminger G. A composite kidney stone phantom with mechanical properties controllable over the range of human kidney stones. J Mech Behav Biomed Mater 2010; 3(1): 130-3.
44
Ginestra P. Manufacturing of polycaprolactone - Graphene fibers for nerve tissue engineering. J Mech Behav Biomed Mater 2019; 100: 103387.
45
Çakmakçı E, Güngör A, Kayaman-Apohan N, Kuruca SE, Çetin MB, Dar KA. Cell growth on in situ photo-cross-linked electrospun acrylated cellulose acetate butyrate. J Biomater Sci Polym Ed 2012; 23(7): 887-99.
46
Zarei M, Samimi A, Khorram M, Abdi MM, Golestaneh SI. Fabrication and characterization of conductive polypyrrole/chitosan/collagen electrospun nanofiber scaffold for tissue engineering application. Int J Biol Macromol 2021; 168: 175-86.
47
Drupitha MP, Bankoti K, Pal P, et al. Morphology-induced physico-mechanical and biological characteristics of TPU–PDMS blend scaffolds for skin tissue engineering applications. J Biomed Mater Res B Appl Biomater 2019; 107(5): 1634-44.
48
Deepthi S, Nivedhitha Sundaram M, Vijayan P, Nair SV, Jayakumar R. Engineering poly(hydroxy butyrate-co-hydroxy valerate) based vascular scaffolds to mimic native artery. Int J Biol Macromol 2018; 109: 85-98.