Engineering an Adhesive and Antimicrobial Nanocomposite

Hydrogel

A Thesis Presented By

Brijesh Hirani

To

The Department of Chemical Engineering

In partial fulfilment of the requirements

For the degree of

Master of Science

In the field of

Chemical Engineering

Northeastern University

Boston, Massachusetts

(May 16, 2018)

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Acknowledgements

I would like to thank Northeastern University for funding. I also thank every faculty member

and staff in the Department of Chemical Engineering. I am grateful to everybody who has

supported my research during this year. Professor Nasim Annabi is the first one I would like to

thank. She gave me this great opportunity to work in her Biomaterials Innovation and Tissue

Engineering, and always her professional, innovative ideas to my project. Also, I would also

like to thank Ebrahim Mostafavi, my mentor who trained me on various experimental skills,

technical writing, and presentation skills. In addition, I would like to thank every lab member

in the Annabi Lab. I appreciate the kindness of Prof. Sidi Bencherif and Prof. Ambika Bajpayee

for being my committee members for my masters’ thesis. Assistance from faculty staff was

also momentous during my research. Especially, I need to thank Willian Fowle who helped me

with Scanning electron microscope images. I would also like to thank my parents for their

support when I was pursuing my master’s degree. I know they stand by me because they love

me. It was their love that gave me power to fight against difficulties during my masters’

education period. Finally, I would like to thank all again who helped me with this MS thesis.

You guided me with the right research direction, helped me to overcome challenges, led me to

face problems bravely. Without your help, it is hard to imagine whether my MS thesis could

be completed successfully or not. I really appreciate it!

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Abstract

Hydrogel-based adhesives become very popular nowadays for various surgical and tissue

engineering applications due to their biocompatibility and biomimetic properties. However,

there are always major challenges persist in fabricating hydrogels which possesses adequate

tissue adhesiveness, strong antimicrobial properties, and excellent mechanical properties. An

estimated 700,000 people die yearly around the world because they have an infection that has

become resistance to the antibiotics and drugs used to treat it. Therefore, designing adhesive

hydrogels with desired antibacterial activity, sufficient adhesion and mechanical properties is

of importance for many tissue engineering applications. To address these limitations, we

engineered an antimicrobial nanocomposite adhesive by incorporating Zinc-Oxide tetrapods

(ZnO-T) in a photocrosslinkable hydrogel prepolymer solution which can be topically applied

on wound site by spraying and easily crosslinked to form an adhesive hydrogel by UV

irradiation in a few seconds. These nanocomposite hydrogels were engineered by dispersing

various concentrations of ZnO-T in the range of 0 to 2 %(w/v) in gelatin methacryloyl (GelMA)

prepolymer solution with different concentrations ranging from 5 to 20%(w/v). Then, the

physical properties of the nanocomposite hydrogels were evaluated by measuring the elastic

and compressive modulus, elasticity, swellability and degradation. Moreover, we determined

various tissue adhesive properties of hydrogel based on various standard adhesion tests such as

wound closure, lap shear and burst pressure. We also compared these properties with the

hydrogels loaded with commercially available ZnO spherical (ZnO-C) nanoparticles. In

addition, we exposed the engineered nanocomposites to (Escherichia coli (E.Coli) and

Pseudomonas aeruginosa (P.a) bacteria) to evaluate their antimicrobial properties. We

demonstrated that the nanocomposite hydrogels loaded with ZnO-T had significantly higher

tensile and compressive strength as compared to hydrogel containing ZnO-C. This could be

due to the aggregation of ZnO-C, particularly at higher concentration which can adversely

affect mechanical properties whereas in case of ZnO-T, the distance between tetrapod arms can

prevent aggregation of nanoparticles in solution and resulting in improved mechanical

properties. In addition, adhesion tests revealed significantly higher tissue adhesion for ZnO-T

loaded hydrogel as compared to ZnO-C incorporated nanocomposite. This can be due to the

shape of ZnO-T, which may facilitate mechanical interlocking with the native tissues.

Antimicrobial results showed that not only the zone of inhibition increased by increasing the

concentration of the both types of ZnO nanoparticles from 0 to 2%(w/w), hydrogels loaded

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with ZnO-T also offered higher antibacterial activity as compared to Kanamycin (commercial

antibiotic) as well as control samples containing ZnO-C. Our results demonstrate that the

engineered nanocomposite adhesive hydrogels have potential to be used for various surgical

procedures that prone to risk of infection.

Key words: gelatin methacryloyl, tissue adhesion, Zinc Oxide tetrapod, Antimicrobial, tissue

engineering applications tensile and compression modulus, zinc oxide spherical, Kanamycin

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TABLE OF CONTENTS

List of figures ....................................................................................................... vi

List of Tables ...................................................................................................... vii

1.0 Introduction ................................................................................................... 1

1.1 Motivation and Background ................................................................................... 1

1.2 Objective and Scope ............................................................................................. 1

2.0 Critical literature review .............................................................................. 3

2.1 Introduction ......................................................................................................... 3

2.2 Hydrogel-based Adhesives and sealants ................................................................... 3

2.3 Nanocomposite Hydrogel ...................................................................................... 5

2.3.1 Carbon based nanomaterials............................................................................. 5

2.3.2 Inorganic nanoparticles .................................................................................... 7

2.3.3 Metal and Metal oxide nanoparticles ............................................................... 9

2.4 Zinc Oxide Tetrapods(ZnO-T) ............................................................................... 9

2.4.1 Polymer nanocomposite materials ................................................................... 14

2.4.2 ZnO-T Biomedical applications ....................................................................... 14

2.5 Antibacterial activity of ZnO ................................................................................ 17

2.6 Summary .......................................................................................................... 19

3.0 Materials and methods ...................................................................................... 20

3.1.1 GelMA preparation ........................................................................................ 20

3.1.2 ZnO tetrapod synthesis ................................................................................... 20

3.1.3 GelMA-ZnO nanocomposite hydrogel ............................................................. 20

3.2 Scanning Electron Microscopy (SEM) ................................................................... 20

3.3 Tissue Adhesive properties .................................................................................. 21

3.3.1 Wound closure test ......................................................................................... 21

3.3.2 Lap shear test ................................................................................................ 22

3.3.3 Burst pressure test ......................................................................................... 24

3.4 In vitro swelling ratio and degradation .................................................................. 24

3.5 Characterization of mechanical properties .............................................................. 25

v

3.6 Scanning electron microscopy of hydrogel/ porcine skin interface ............................. 26

3.7 Antibacterial properties (Zone of Inhibition) ........................................................... 26

4.0 Results and Discussion ................................................................................ 28

4.1 Synthesis and structural characterization of nanocomposite hydrogel ......................... 28

4.2 Mechanical properties of nanocomposite hydrogel .................................................. 29

4.3 Tissue Adhesive properties of nanocomposite hydrogel ............................................ 30

4.4 In vitro swelling and degradation .......................................................................... 33

4.5 In vitro Antimicrobial properties of nanocomposite hydrogel .................................... 35

5.0 Conclusion .................................................................................................... 37

6.0 Recommendations ....................................................................................... 38

6.1 Alternatives of UV irradiation for crosslinking of prepolymer solution ....................... 38

6.2 Mechanism of enhancing mechanical strength of ZnO-T incorporated ....................... 38

nanocomposite hydrogel

6.3 ZnO antibacterial property mechanisms ................................................................. 39

6.4 In vitro cytocompability study ............................................................................. 40

6.5 NMR study ........................................................................................................ 42

7.0 Nomenclature .............................................................................................. 44

8.0 References .................................................................................................... 45

vi

List of figures

Figure 1. Engineered nanocomposite hydrogels ........................................................................ 5

Figure 2. Nanocomposite hydrogels from CNTs and GelMA ................................................... 7

Figure 3. Highly elastomeric hydrogel network from PEG-Silicate.......................................... 8

Figure 4. Overview of different research areas ....................................................................... 11

Figure 5. ZnO tetrapods for advanced self-reporting/healing .................................................. 14

Composites

Figure 6. Biomedical applications of ZnO tetradpods ............................................................. 15

Figure 7. Schematic of synthesis of Nanocomposite hydrogel ............................................... 21

Figure 8. Schematic of Wound closure test ............................................................................ 22

Figure 9. Schematic of Lap shear test ..................................................................................... 23

Figure 10. Schematic of Burst pressure test ............................................................................ 24

Figure 11. Schematic of mechanical test ................................................................................ 25

Figure 12. Morphology of hydrogel nanocomposite by scanning electron microscope .......... 28

Figure 13. Comparison of mechanical properties between nanocomposite hydrogels ........... 30

Figure 14. Comparison of in vitro adhesion properties of nanocomposite hydrogels ............ 32

Figure 15. Scanning electron microscope images of nanocomposite hydrogel/porcine ......... 33

skin Interface

Figure 16. In vitro swelling and degradation properties of nanocomposite hydrogels ........... 35

Figure 17. Antimicrobial properties of nanocomposite hydrogels against .............................. 36

E. coli and P. aeruginosa

Figure 18. SEM images reveal size and shape of different fillers used in the polymer .......... 38

composite

Figure 19. Antibacterial different mechanisms ....................................................................... 39

Figure 20. ZnO nanoparticles antibacterial mechanisms ........................................................ 39

Figure 21. In vitro 3D encapsulation of 3T3 fibroblasts in ZnO-T & ZnO-C loaded ............ 41

GelMA hydrogels

Figure 22. 1H NMR analysis of nanocomposite hydrogel ...................................................... 42

Figure 23. Comparison of crosslinking degree between nanocomposite hydrogels ................ 43

vii

List of Tables

Table 1. ZnO tetrapod material: application in various fields ................................................. 13

viii

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1.0 Introduction

1.1 Motivation and background

Adhesive hydrogels with high water content, superior mechanical properties and a structure

similarity to native soft tissue are one of the most salient properties of biomaterials. They

exhibit excellent wound closure and tissue regeneration properties[1] instead of current invasive

methods used for surgical closures such as stapling and suturing. Suturing is a tedious method

that needs technical and professional skill and can also induce inflammation, scar formation,

pathogenic infections and wound edema[2]. Although major development in health care

standards and medical technology, infectious diseases caused by pathogenic microorganisms

such as viruses, bacteria, fungi, parasites, remain a major menace that may convert into

substantial socio-economic problems. Surprisingly, according to a World Health Organization

(WHO) report, infectious diseases are the second leading cause of global mortality[3]. Thus,

there is an imperative need to develop antimicrobial tissue adhesives that will allow surgeons

to replace conventional suturing with flexible and safe techniques which also diminish chances

of bacterial infection in various surgical and tissue engineering applications. Several classes of

unique materials, such as antimicrobial peptides (AMPs)[4], synthetic cationic polymers[5] and

antimicrobial nanoparticles[6-7], have emerged as potential substitutes for conventional

antibiotics.

1.2 Objective and Scope

The main objective of this research was to synthesize nanocomposite polymer hydrogel that

represents superior mechanical properties, sufficient tissue adhesion, controlled degradation

and swelling, excellent antimicrobial property, and supporting growth, spreading, and low

cytotoxicity to fibroblast cells. Therefore, particular aims of this thesis include following:

1. Synthesis of GelMA/ZnO-T and ZnO-C polymer nanocomposite hydrogel with varying

concentration of ZnO including 0.01, 0.05 and 2 %(w/v).

2. Characterization of both nanoparticles, morphology of nanocomposite hydrogel by

scanning electron microscope(SEM) images

3. Comparison of mechanical properties of hydrogels such as elastic and compression

modulus, ultimate tensile strength, elongation

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4. To determine native tissue adhesion property of nanocomposites by various standard

adhesion tests such as wound closure, lap shear and burst pressure test

5. To verify mechanical interlocking of tetrapod arms into porcine skin by SEM images of

hydrogel-porcine skin cross-section

6. To analyse swelling and in vitro degradation behaviour of hydrogels in PBS for different

time intervals

7. To identify UV irradiation crosslinking time effect (4, 6, and 10 min) on compression and

burst pressure results

8. To evaluate antibacterial property of samples against bacteria (Escherichia coli (E. Coli)

and Pseudomonas aeruginosa (P.a) bacteria) by Zone of inhibition

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2.0 Critical Literature Review

2.1 Introduction

This critical literature review depicts on basic motivation behind this project and is divided into

four sections:

1. Hydrogel-based Adhesives and sealants: This section introduce various tissue

adhesive property of hydrogels and how it supports sutureless and injection free

surgery in several tissue engineering applications, finally how it overcomes various

limitations and drawbacks in conventional techniques of staples and sutures.

2. Nanocomposite hydrogel: This section talks briefly about different

nanocomposite hydrogels based on variety of nanoparticles used such as carbon-

based nanomaterials, inorganic particles, polymeric nanoparticles and finally

discussion on several biomedical applications.

3. Zinc Oxide Tetrapods(ZnO-T): This section reviews about various shape of zinc

oxide nanoparticles and its antibacterial activity, briefly possible mechanism about

reactive oxygen species. Then finally, discussion on how Zinc Oxide tetrapod

shape advantageous as compared to other shapes on various properties such as

native tissue adhesion, antibacterial, cell cytotoxicity etc.

4. Antimicrobial property: This section goes first into brief discussion on

burgeoning bacterial resistance against traditional antibiotics and how this can be

diminished by using Zinc Oxide nanoparticles (ZnO). At the end, explanation on

possible mechanism for antibacterial property by introducing reactive oxygen

species generation.

2.2 Hydrogel-based Adhesives and sealants

Hydrogels have a similar structure to biological soft tissues and can be engineered to resemble

an extracellular matrix, and therefore have great potential for tissue engineering applications[8].

Hydrogels for tissue repair must be designed with excellent tissue adhesive property so that

they can accelerate tissue regeneration after implantation[9]. However, tough hydrogels usually

lack sufficient tissue adhesion properties and therefore cannot be fixed with surrounding tissues

during surgical operation[10]. In general, ideal hydrogels for tissue engineering application

should possess several specific properties. They need an adequate adhesive strength that is

compatible with the fragile skin tissue of patients, that means that the adhesives should be

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easily released from the fragile surface. However, most of the conventional adhesive products,

such as adhesive medical or surgical dressings and bandages, normally have excessive adhesion

strength that leads to cause localized trauma and pain to patient, particularly for patients with

fragile skin[11].

Depending on applications several important properties are needed, for example, in defective

vascularized tissues needs adhesives with hemostatic properties, liquid or air leakages demands

effective sealants that can not only withstand high pressures, but also maintain their

functionality. This is necessary to control the adhesion and physical properties of tissue

adhesives based on desired applications. Several types of adhesives synthesized from natural,

synthetic polymers have been previously already developed[12]. Generally, fibrin and collagen-

based natural adhesives widely applied due to their known biocompatibility, but still they don’t

have sufficient tissue adhesion strength as well as possess very low mechanical strength[13]. On

the contrary, synthetic-based adhesives have sufficient adhesion strength but exhibits poor

biocompatibility and biodegradability, sometimes also become toxic to cells. Moreover, most

of the tissue adhesives restricted to dry surfaces only, however, synthesis polymeric hydrogels

can be crosslinked even in wet conditions[14]. Additionally, because of low mechanical and

adhesive characteristics of commercially available hydrogel-based adhesives, their clinical

indications are mostly focused on the additional sealing of sutures, not suture-free

techniques[15]. Although plethora of hydrogel-based sealants developed for sealing and closure

of elastic tissues, most of them lack appropriate mechanical properties, adhesion strength and

burst pressure for sealing of lung tissue leakages[16]. Sometimes, air leakage after lung surgery

leads to risk of infections and consequently longer stay in hospitals associated with higher

healthcare costs[17]. To avoid these obstacles, variety of natural and synthetic-based polymers

have been examined for use, including fibrin, collagen-based sealants and synthetic glues[18].

2.3 Nanocomposite Hydrogel

A plenty of innovations in chemistry, micro- and nanofabrication technologies, biotechnology

alter the designing of composite hydrogel networks with controlled and desired

functionality[19]. Recent trends also indicate growing interest in developing nanocomposite

hydrogels for several biomedical applications. Nanocomposite hydrogels can be defined as

hydrated polymer networks, either physically or covalently crosslinked with each other and

with nanostructures[20]. A range of nanoparticles such as carbon-based nanomaterials (carbon

nanotubes (CNTs), graphene), polymeric nanoparticles, inorganic

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nanoparticles(hydroxyapatite, silica, calcium phosphate), metal/metal-oxide nanoparticles

(gold, silver, iron-oxide, zinc) are incorporated with the polymer chains to obtain

nanocomposite hydrogels (Fig.1).

Figure 1. Engineered nanocomposite hydrogels : A range of nanoparticles such as carbon-based

nanomaterials, polymeric nanoparticles, inorganic nanoparticles, and metal/metal-oxide nanoparticles are

combined with the synthetic or natural polymers to obtain nanocomposite hydrogels with desired property

combinations. These nanocomposite networks are either physically or chemically crosslinked. By controlling the

polymer-polymer or polymer-nanoparticles interactions, the physical, chemical, and biological properties of the

nanocomposite hydrogels can be tailored (adopted from [20]).

2.3.1 Carbon-based nanomaterials

Carbon-based nanomaterials such as CNTs, graphene, nanodiamonds are being explored for

various potential biomedical applications[21]. Generally, CNTs and graphene are widely

incorporated for broad multifunctional properties such as high mechanical, electrical

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conductivity, and optical properties to natural or synthetic polymers[22], for example, both

CNTs- or graphene-based utilised for application such as actuators, biosensors, tissue

engineering scaffolds, drug delivery systems and biomedical devices[23]. However, one of the

problems associated with CNTs is that their hydrophobic nature that cause limited interaction

with hydrophilic polymers which eventually leads to aggregation problems. To circumvent this

drawback, several techniques are developed to enhance the dispersion of the CNTs by

modifying the surface with various polar groups, surfactants to modify the surface

properties[24]. In a preliminary study, GelMA was reinforced with COOH-functionalized CNTs

to fabricate hybrid nanocomposite hydrogels[25]. GelMA coating with multi-wall CNTs to

generate a fibrous structure in which CNTs were embedded within an interconnected and

porous hydrogel network(Fig. 2). There was an observation in increasing tensile modulus

almost three times due to the addition of 0.5% CNTs to GelMA hydrogels. It was also showed

that cardiomyocytes seeded onto CNT-GelMA nanocomposite hydrogel had a threefold

increase in their beating frequency compared with cells that were seeded on GelMA-alone

hydrogels. Although CNT-based hydrogels are able to demonstrate the lab-grown tissues can

mimic some of the function of native tissue , their application in human body as tissue

replacements still need to be further investigated due to cytotoxicity concerns.

Graphene is also carbon-based nanomaterial with high mechanical strength and is an excellent

conductor of electricity. In one study, genetically engineered elastin-like polypeptides (ELPs)

non-covalently reinforced with GO to obtain hydrogel actuators that can be mechanically

actuated to bend, stretch and twist when subjected to different radiation intensities. The

covalent crosslinking between polymer and nanoparticles enables the transfer of mechanical

force within polymeric network and thus leads to enhanced mechanical strength and toughness.

For example, mechanically stiff and highly resilient nanocomposite hydrogels were fabricated

by covalently conjugating GO sheets to polyacrylamide[26]. In this study, GO sheets were

functionalized via radiation-induced peroxidation to obtain graphene peroxide(GPO) and this

GPO was covalently crosslinked with polyacrylamide to obtain nanocomposite hydrogels.

Here, more than 900% and 500% increases in tensile strength and elongation, respectively,

were showed due to addition of the GPO (3 mg/mL) compared with conventional polymeric

hydrogels. In summary, significant efforts have been focused on designing and developing

hybrid hydrogels containing carbon-based nanoparticles for biomedical applications, however,

deeper mechanistic studies are required to identify biological properties of such hydrogels

under in vitro and in vivo conditions.

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Figure 2. Nanocomposite hydrogels from CNTs and GelMA. (a) Schematic showing synthesis of

nanocomposite network. First, CNTs are coated with GelMA and then the composite is subjected to UV radiation

to obtain photocrosslinked network. (b) Due to photo crosslinking ability of the nanocomposite network,

microfabrication technologies can be used to control cellular interactions. (c) Cardiac cells that were seeded on

CNTs-GelMA nanocomposites retained their phenotype as determined by the expression of sarcomerica-actinin

and troponin I. (d) The engineered cardiac patch obtained by seeding cardiac cells on CNTs-GelMA surface

showed macroscopic mechanical displacement due to continuous contraction and relaxation of the patch (adopted

from [20]).

2.3.2 Inorganic Nanoparticles

Recently, number of bioactive nanoparticles have been reported including hydroxyapatite,

synthetic silicate nanoparticles, bioactive glasses, silica, calcium phosphate and glass ceramic

for biomedical applications[27]. For example, nHA was incorporated within polyethylene

glycol(PEG) matrix to obtain highly elastomeric nanocomposite hydrogel which showed

enhanced mechanical strength and improved physiological stability. Moreover, the addition of

nHA resulted in enhanced cell adhesion characteristics when compared with PEG hydrogels.

Due to enhanced bioactivity and high mechanical strength, these nanocomposite

networks(PEG-nHA and PEG-silica) can be used as injectable fillers for various orthopedic

applications[28].

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Figure 3. Highly elastomeric hydrogel network was obtained from PEG-Silicate

nanocomposites. (a) Precursor solution containing silicate nanoplatelets and acrylated PEG when subjected

to UV radiation results in the formation of a covalently crosslinked nanocomposite network. The nanocomposite

hydrogels stick to soft tissues (b) and undergoes high deformation (c) and (d). (e) The addition of synthetic silicate

results in significant increase in mechanical stiffness and elongation of the nanocomposite network compared to

polymeric hydrogels. (f) Moreover, the addition of silicate also promotes cells adhesion on the nanocomposite

hydrogels(adopted from [20])

Synthetic silicates nanoparticles, also known as nanoclays, have been observed to enhance the

physical and mechanical properties of polymeric hydrogels[29]. Although their unique physical

and chemical properties, only a few studies available for biomedical applications such as

controlled cell adhesion, injectable drug delivery matrix, and antimicrobial films[30]. Synthetic

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silicate nanoplatelets, when mixed with linear and branched polymers, resulting into

mechanically strong and tissue adhesive nanocomposite hydrogels[31]. Other types of ceramic

nanoparticles, such as calcium phosphate, bioglasses are also incorporated in different synthetic

and natural polymers to obtain bioactive nanocomposite hydrogels[32]. The degradation or

dissolution products of these ceramic nanoparticles can result in favourable biological response

and thus providing opportunities for use in various biomedical applications.

2.3.3 Metal and Metal-Oxide Nanoparticles

Several types of metallic nanoparticles used to fabricate nanocomposite hydrogels for

biomedical applications include gold (Au), Silver (Ag), Zinc (Zn), whereas metal-oxide

nanoparticles include iron oxide, titanium, zinc oxide. Metal and metal-oxide nanoparticles

have been shown to possess desired physical properties such as magnetic properties( iron and

zinc oxides), and antimicrobial properties (Ag and Zn nanoparticles). Therefore,

nanocomposite hydrogels containing metal or metal-oxide nanoparticles are extensively used

as imaging agents, drug delivery systems, conductive scaffolds, actuators and sensors[33]. In

one study, Au nanoparticles entrapped within the polymeric network consisting of hyaluronic

acid (HA) and gelatin do not improve the mechanical properties[34]. However, when thiol-

functionalized Au nanoparticles can crosslink with the polymeric network, significant increase

in the stiffness are observed.

Other types of metal-oxide nanoparticles can be used to enhance the bioactivity of hydrogels.

For example, nanophase alumina and titanium, after incorporating in polymer matrix such as

poly-(L-lactic-co-glycolic acid) (PLGA), result in enhanced osteoblast adhesion and

proliferation[35]. Recently, the surface of titanium was functionalized with amine groups to

make interactions between the nanoparticles and carboxymethylcellulose[36]. These hybrid

nanocomposite hydrogels can be used to encapsulate cells for tissue engineering applications.

2.4 Zinc Oxide Tetrapods(ZnO-T)

Zinc is one of the most important nutrients for the human body that can control more than 300

metabolic functions in body[37]. Interestingly, a large variety of ZnO nano and microstructures

with any complex shape can be easily found in literatures[38]. That basically depends on the

kinetics and the method of synthesis for several different ZnO structures. The broad nano and

micro structuring abilities of ZnO could play a very crucial role in material science engineering

and technology. Typically, ZnO tetrapods is made of four arms interconnected together via a

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central core at angles ranging from ~105º to 110º with respect to each other and this unique 3D

morphology offers easy accessibility of nanoscale features[39]. Consequently, this 3D

morphology offers a greater advantage over spherical nanoparticles in terms of porosity that

even if a large amount of tetrapods accumulated together, it will produce highly stable

macroporous structure. Additionally, because of ZnO tetrapod is built from 1D ZnO nanorods,

it includes all physical and chemical features of 1D nanorod shape as well.

Tetrapods represents a potential versatile technological applications and glimpse of various

possible opportunities demonstrated in Figure 4. This tetrapodal shape is unique in the sense

that irrespective of how they are placed, one arm is pointing upwards, hence it offers various

1D nanoscale mechanical, electrical, optical, luminescent and other interesting qualities.

Because of complex 3D shape, the ZnO tetrapods are better linker/filler candidates toward

fabricating advanced multifunctional composites in contrast to conventional spherical and 1D

nanostructures that suffering agglomeration issues[40]. Moreover, the developed ZnO tetrapods

have already shown potentials against viral infections and several other fatal diseases[41-43].

Also, these tetrapods could be electrospinned together with polymers to synthesize rose-spike

like biocompatible fibers with unique biocompatible features for advanced biomedical

applications[44]. The sacrificial nature of ZnO tetrapods is very advantageous that they can also

be used to design other new materials in very efficient ways. The sacrificial template-based

strategy offers plenty of nanostructuring opportunities in terms of combining various

inorganics (metals, oxides, nitrides, phosphides, carbides, etc.), carbon (carbon nanodots,

fullerenes, graphenes, single/ multi-walled carbon nanotubes, etc.), organic, polymers, zeolites,

etc. materials together in the form of hybrid 3D nanomaterials for multifunctional applications.

Although its difficult to list all possible application, herein we tried to represent some of them

in Table 1 that will generally provide idea about the various fields where these several million

tones ZnO materials are consumed and how important tetrapods could be for future

technologies.

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Figure 4. Overview of different research areas: ZnO nano and micro tetrapods could play a very

important role. They can be efficiently utilized in a lot of innovative applications as well as toward growth of new

and hybrid multifunctional 3D micro- and nanostructured materials(Adopted from [39])

2.4.1 Polymer Nanocomposite materials

Polymer-based composites become the most utilized material, which has become possible

mainly by the availability of various kinds of filler nanoparticles (Table 1). Therefore, the role

of filler particles is rather very important, and their various nano structural forms have been

developed. In this respect, tetrapods with 3D shape exhibit a special role toward engineering

the properties of polymer-based composite materials in contrast to conventional spherical

nanoparticles or one-dimensional nanostructures such as nanofibers[45]. It is very well known

that filling the polymers with appropriate fillers in the form of composite leads to a noticing

12

improvement in their overall properties, and they are being utilized in many applications.

However, the conventional fillers (0D and 1D structures) have strong agglomeration tendencies

in contrast to tetrapod- shaped nanostructures that do not agglomerate irrespective of how they

are put together. Moreover, a lesser amount of tetrapod- shaped fillers than conventional fillers,

is required to achieve similar improvement properties of the resultant polymer composite. With

spherical fillers, getting a very uniform distribution is an issue, at the same time 1D long fibers

result in unidirectional improvement in properties (along their length). In contrast the tetrapod-

shaped fillers improve the overall properties of the composite in all three dimensions because

of their unique spatial complex shape. Such a concept using tetrapod- shaped fillers is thus very

helpful for designing rough and tough composite materials with engineered properties for

advanced technologies. Not only this, some aspects like joining two un-joinable polymers (e.g.,

silicone and Teflon) which was a challenge, can be easily achieved by following this purely

interlocking strategy with tetrapod-shaped fillers[46]. A typical concept for using the ZnO

tetrapods as linking elements (zippers) is shown as inset in Figure 5a, in which they are

embedded at the interface between the two polymer layers. After integration, the adhesion

strength between the two polymer layers (silicone and Teflon) was increased significantly. The

overall peeling strength from tetrapodal ZnO-T structures was compared with 1D ZnO rods

(Figure 5b) and tetrapodal shape indeed exhibits an important role in the improving adhesion

between two polymers. The adhesion is mainly due to shape-induced mechanical interlocking

(Figure 5c) at the interface because no chemical interactions between ZnO surface and

polymers were observed[46]. Such tetrapod-based polymer composites have thus the ability to

report about any damage occurring internally and could lead to designing the new class of self-

reporting and smart composites. A typical concept for designing the self-reported composites

using ZnO tetrapods is shown in Figure 5d– f in which the applied stress on the composite is

reflected as a change in luminescence (Figure 5f). With an increase in the T-ZnO filling fraction

(wt%), the Young’s modulus of the nanocomposite also improved (Figure 4e) leading toward

better mechanical properties. The mechano-luminescence response of the tetrapodal tetrapodal

ZnO polymer composites can be further enhanced (up to several mechanical cycles) by

controlling the distribution of tetrapods within the PDMS polymer as can be seen in Figure 4g

and h[48]. Spiropyran molecules have got unique features in terms of conformational

transformation (reflected as color change) when subjected to some energy in the form of stress,

heat, light, etc., and they can be nicely utilized for self-reporting, healing, and advanced

composites (Figure 4i). The required energy for conformational transformation is in the visible

range of the electromagnetic (EM) spectrum, which is difficult to provide locally. However, if

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ZnO tetrapods are also embedded along with spiropyran molecules in the polymer, they can

easily provide the necessary energy for conformational transformation because of their broad

luminescent emission in the visible region and can also improve the overall mechanical

properties of the composite. A large variety of advanced polymer composites using ZnO

tetrapods have been fabricated and utilized for various applications, especially robust coatings

for underwater appliances (ships, marines, etc.)[50]. and many more must be further realized. It

is worth to mention that embedding the ZnO tetrapods in a polymer improves its wettability

features, too, hence the ZnO- T-based polymer hydrophobic coatings/paints can be easily

developed and mounted on all those areas that need to be water proofed. Improving the

properties of rubber-based composites, e.g. tire and related industries, has been a big issue, and

because of their unique shape, ZnO-T could add wings to this field. Therefore, in milieu of

composites, ZnO-T have got a lot of scope toward fabricating various interesting composites[51-

53], electronic-skin[54], and smart-textiles[55] and, therefore, will introduce several novel and

broad applications.

Table 1. ZnO tetrapod material: application in various field(adopted from [39])

ZnO tetrapod materials: application in various fields

Electronics Nanodevices, Photo detection, Gas sensing, Biosensor

Photonics Luminescent devices, light scattering elements, Imaging technologies

Composites Linkers-joining polymers, Advanced polymer composites, Antifouling

coatings, Rubber engineering technologies

Chemistry &

environment

Paints, pigments, inks, catalyst, water purification, water treatment,

Advanced membranes

Biomedical ROS generation, Dental implants & paints, Antiviral, Antibacterial,

Antifungal, Antitumor, Antioxidant

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Figure 5. ZnO tetrapods for advanced self-reporting/healing polymer composites: (a–c)

Linker element for joining the unjoinable polymers. (d–f) Intelligent filler candidates for self-reporting polymer

composites [85]. (g and h) Regular arrangement of tetrapods in polymer-enhanced mechanical, luminescent, and

self-reporting behaviour. (i) ZnO tetrapods improve the self-recovery response of spiropyran-based polymer

composite(Adopted from [39]).

2.4.2 ZnO-T biomedical applications

15

Figure 6. Biomedical applications of ZnO tetrapods: (a–c) Plasmid DNA delivery. (d) Cell viability

study demonstrating lower cytotoxicity of nano- and microscale ZnO tetrapods as compared to spherical ZnO

nanoparticles and ZnCl2 powders. (e and f) Capturing of HSV-1 viruses by ZnO tetrapods and virus-binding

mechanism. (g–l) In vivo animal studies for role of ZnO tetrapods against genital herpes virus, (m–p)

anticancerous potential of ZnO structures against glioblastoma cancer (Adopted from [39]).

16

Due to the exceptional physical and chemical properties, nanomaterials have got special

perspectives toward biomedical engineering, and that is why, nanobio-based interdisciplinary

research has been trending since past two decades. Plethora of strategies have been followed

to develop efficient drugs, the so-called ‘nanodrugs’, and up to some extent, a few approaches

have delivered successful results; still, this field of nanobio must witness a lot more to reach

on certain conclusion. The requirement for an effective drug is the least cytotoxicity at dosage

targeted for the desired therapeutic effects. Despite their promising therapeutic effects,

nanoscopic structures from several materials are out of scope because of high cytotoxicity

issues. Only a few materials, e.g., gold, silver, titanium etc. overcome, but apart from

cytotoxicity, they need to overcome several other challenges, like cost effective synthesis, etc.

ZnO material is very advantageous in the sense that its nanostructures (with adequate sizes and

shapes) exhibit very low toxicity as found by many studies[56]; even nanowires were found to

be completely biocompatible and biosafe[57]. A low cell cytotoxicity is observed from micro-

and nanoscale ZnO tetrapods on healthy cells[58], which are prepared using the solvent-free

FTS approach[59]. Adverse effects of ZnO seem to be observed only at unrealistic high doses

like 1 g/kg[60]. This is important as micro- and nanoparticles, especially the spiky ones, are

always suspicious to cause cancer or asbestos-like effects. Probably, the good biocompatibility

of ZnO, including ZnO tetrapods, results from the fact that Zn is an essential trace element for

humans (15–30 mg/day recommended by world health organization ‘WHO’ for a male adult[61]

) and it can be easily metabolized by human body.

Today ZnO nanomaterials are an everyday ingredient in many creams, powders, and

ointments[62]. Most biomedical effects are believed to be based on hydrophilic, antiseptic

effects and the solution of zinc. For example, according to one study, ZnO-T can be

functionalized for plasmid DNA delivery (Figure 6a–c)[63]. Plasmid DNA easily binds to

functionalized tetrapod surfaces due to electrostatic interactions between positively charged

amino groups and negatively charged phosphate groups from DNA. The ratio of

functionalizing agents (silica and amino groups) is of much importance since it ensures binding

of enough DNAs because only above a certain ratio, the DNA binding could be confirmed

(Figure 6b). Cells (A375) can also easily bind to functionalized tetrapods with amino groups[64].

The unique binding capability of functionalized ZnO-T with respect to DNA, as well as cells

(A375), could facilitate unique plasmid-DNA delivery. Inspired by the biomedical potential

and extremely low cytotoxicity (Figure 6d) of ZnO-T, we targeted a unique mechanism for

capturing different viruses (Figure 6f) with oxygen vacancies on our ZnO-T. This can be easily

17

equipped with oxygen vacancies to create polar surfaces with positively charged defects, which

we intended to trap the virus with its functional glycoprotein groups on the surface like a fly

with a flypaper (Figure 6e). This binding mechanism could be confirmed by illuminating the

T-ZnO crystals with UV light in ambient air to create more oxygen vacancies (more viruses

are bound with the surface) by the known auto photocatalytic process (Figure 6f)[65]. There is

also one study about the immune system picks up viruses from the T-ZnO and performs a tri-

functional vaccination (Figure 6g–l)[67]. Furthermore, the wound healing was much faster in

animals treated with ZnO-T (Figure 6g). The use of ZnO-T reduced the development of chronic

infection (Figure 6h and i) and the ZnO-treated animals survived longer (Figure 6l). Viral

particle bound with ZnO tetrapods is easily taken up by dendritic cells (Figure 6j and k). After

successful animal studies, first observations on human patients were also performed, which

again confirmed the results in similar with the animal studies. Thus, it is very important to

perform systematic studies and to widen them for further similar viruses and other species. The

ongoing experiments with human papillomavirus (HPV), ZIKA, m-AIDS, Dengue also showed

promising results. This is not limited to viruses; the recent work[68] about the response of ZnO

nanostructures against glioblastoma cancer cells (Figure 6m–p) opens further a broad the

perspective about use of ZnO tetrapods in biomedical engineering.

2.5 Antibacterial Activity of ZnO

Nanotechnology is a research hot trend in modern materials science. This distinct property of

nano-size allows their possible applications in diverse fields such as biosensors,

nanomedicines, and biotechnology[69]. The intrinsic properties of metal NPs such as zinc oxide

(ZnO), TiO2, and silver are mostly characterized by their size, composition, crystallinity and

morphology. Reducing the size to nanoscale can modify their chemical, mechanical, electrical,

morphological properties. Nano-sized ZnO demonstrates varying morphologies and shows

significant antibacterial activity over a wide spectrum of bacterial species explored by many

researchers[70-74]. ZnO exhibits significant antimicrobial activities when particle size reduced,

then this nano-sized ZnO can interact with bacterial surface and/or with the bacterial core where

it enters inside the cell, and consequently represents distinct bactericidal mechanisms[75].

Increased outbreaks and infections of pathogenic strains, bacterial antibiotic resistance, lack of

suitable vaccine in underdeveloped countries, hospital-associated infections, are global health

hazard to human. For example, infections by Shigella flexneri cause 1.5 million deaths

annually[76]. Thus, developing novel antibacterial agents against bacteria strains, such as

Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa has become utmost

18

demand. Antibacterial activity is known according to The American Heritage Medical

Dictionary 2007, as the action by which bacterial growth is destroyed or inhibited.

Various methods have been employed for the assessment and investigation of antibacterial

activity in vitro. These methods include disk diffusion, broth dilution, agar dilution, and the

microtiter plate-based method[77]. The most commonly used method is the broth dilution

method, followed by colony count, through plating serial culture broths dilutions which

contained ZnO-NPs and the targeted bacteria in appropriate agar medium and incubated. Many

researchers[78] have examined the antibacterial activity of ZnO-NPs to determine bacterial

growth through the culture turbidity and the viable cells percentage by the colony counting test,

while others considered that the antibacterial activity rate was much improved by decreasing

the initial number of bacterial cells from 102 to 106 colony forming unit (CFU). The MIC of an

antimicrobial agent and MBC can be measured by using the susceptibility test methods.

However, there are some variations in the established laboratory methods and protocols in the

assessment of the bactericidal activity[79]. The agar diffusion method is the most frequently

used method and has been standardized as an official method for detecting bacteriostatic

activity by the (ATCC). The microdilution method is a modification of the broth macrodilution

test, which utilizes the advances in miniaturization to allow multiple tests to be performed on

a 96-well plate. Modified procedures along with the standard methods are also used. In all of

the aforementioned methods, the culture media [trypticase soy broth (TSB), Luria–Bertani

broth (LB), nutrient agar (NA), tryptic soy agar (TSA), and blood agar (BA; see

Abbreviations)] were accordingly selected to autoclave and stored at 4–5 ºC. The stocks of

ZnO-NPs suspensions are also usually prepared, and serially diluted to different concentrations,

and then characterized using techniques [X-ray diffraction (XRD), field emission scanning

electron microscope (FESEM), transmission electron microscope (TEM), energy dispersive X-

ray spectroscopy (EDX), electron spectroscopy imaging (ESI), etc. to correlate the antibacterial

response with ZnO properties. Growth curves were typically obtained via monitoring the

optical density (OD), at wavelength of 600 nm, a typical wavelength for cells. The density of

bacterial isolates must be adjusted to an optimal density of 0.5 McFarland standards. The OD

should serially be monitored hourly up to 12 h of incubation, and finally after 24 h of overnight

incubation for the determination of the percentage of growth inhibition[80]. The inhibition rate

varies with the tested organisms and the utilized NP-oxide[81].

2.6 Summary

19

This critical literature review was aimed to develop justification behind choosing this project.

Brief overview of literature study is as follows:

1. To synthesize biocompatible hydrogel that possesses sufficient native tissue

adhesion property, excellent mechanical property, controlled degradation and high

swelling ratio that can be utilized for several tissue engineering applications.

2. To incorporate nanoparticles into hydrogel to produce nanocomposite hydrogel

for controlling various physiochemical properties such as tensile and compression

modulus, elongation etc., subsequently that can be applied for desired biomedical

applications.

3. Micro-nano size ZnO-T that has rose-spike like special shape that can facilitate

mechanical interlocking with tissues, therefore its broad variety of applications in

tissue engineering fields, particularly antimicrobial resistance and wound healing.

4. Antimicrobial properties exhibited by ZnO nanoparticles against gram-positive

and gram-negative bacteria and possible mechanisms that can be applied in

infectious diseases for several surgical applications by incorporating into adhesive

hydrogel .

20

3.0 Materials and methods

3.1.1 GelMA preparation

GelMA was synthesized using a method previously described in the literature[87]. Briefly, 10%

(w/v) gelatin solution was reacted with 8 mL of methacrylic anhydride for 3 h. The solution

was then dialyzed for 5 days to remove any unreacted methacrylic anhydride, and then placed

in a −80 °C freezer for 24 h. The frozen acrylated polymer was then freeze-dried for 7 days.

3.1.2 ZnO tetrapod synthesis

To produce branched ZnO nanoparticles, a flame transport synthesis technique was used. This

method offered direct conversion from metallic Zn microparticles into complex shaped ZnO

nano- and microstructures in a single step conversion within the flame in the presence of normal

air environment. The mixture (2:1 weight ratio) of sacrificial polyvinylbutyral polymer and /

or ethanol and Zn microparticles from Goodfellow, UK was burned in a simple muffle type

oven where Zn particles were directly converted into branched ZnO nanostructures via solid-

vapor solid growth in a further modified flame transport synthesis process, as described in a

previous work.

3.1.3 GelMA-ZnO nanocomposite hydrogel

ZnO-T and ZnO-C stack solution of 5% (w/v) was prepared in PBS followed by vortexing of

solution for 10 min to make uniform distribution in solvent. To break up aggregates of micron-

sized particles, solution was put in sonication bath for 30 min. Meanwhile, freeze-dried GelMA

prepolymer was dissolved in PBS at concentration of 20 % (w/v) at 37 ℃ in heat-block.

Additionally, we added 0.5 % (w/v) 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2 methyl-1

propane-1-one (Irgacure 2959, BASF, Florham Park, NJ, USA) as a photo-initiator. Then,

series of different concentrations of ZnO-T and ZnO-C solutions such as 0.01, 0.05 and 2 %

(w/v) were prepared by diluting stack solution in 20 % (w/v) GelMA to prepare Nano-

composite pre-polymer solution. Afterwards, this precursor solution was photo-crosslinked

through UV light irradiation with intensity 6.9 w/cm2 (Omnicure S2000, 320-500 nm filter,

EXFO Photonic Solutions Inc., Quebec, Canada) to form hydrogels.

3.2 Scanning Electron Microscopy (SEM)

SEM imaging and analysis were conducted to evaluate morphology of the crosslinked

composite hydrogels. Lyophilized hydrogel samples were prepared using the compressive

molds. SEM images were obtained using a Hitachi S-4800 Scanning Electron Microscope

(SEM).

21

Fig. 7 Schematic of synthesis of nanocomposite hydrogel: (a) represents various steps of synthesis

of polymer nanocomposite. (b) shows crosslinking structure of hydrogel formed by UV light irradiation for 4

min followed by various mechanics such as Tensile and Compression Tests to determine mechanical strength of

polymer.

3.3 Tissue Adhesive Properties

3.3.1 Wound closure test

The adhesion strength of composite hydrogels was demonstrated by using the standard test

method for wound closure strength of tissue adhesives and sealants ASTM F2458-05.

22

Fig. 8 Schematic of wound closure test: (a) Krazy glue is applied on glass slides for attachment of

porcine skin (b) skin sample was cut using Razor blade from middle (c) sample was placed in between gap (d)

UV irradiation of to form hydrogel (e) sample was put on an Instron machine for tensile test (adopted from [89]).

According to test, fresh porcine skin from local slaughterhouse brought and pieces of (10 mm

x 15 mm) dimension were prepared by removing extraneous layers of fats from tissues. Pieces

were soaked into phosphate buffer saline(PBS) to prevent from local environment. Then, pieces

were blotted dry before fixing onto two polymethyl methacrylate glass sheets (25 mm x 60

mm) using Krazy Glue. Gap between two sheets was kept 6 mm followed by cutting tissue

from centre by using razor blade in straight, thereafter filling the space and surrounding area

with 60 µl of sample, followed by crosslinking by UV light (6.9 w/cm2) for 4 min, turned out

to be crosslinked hydrogel which was then placed for mechanical test and maximum adhesion

strength was obtained at failure at strain rate of 1 mm/min.

23

3.3.2 Lap shear test

Fig. 9 Schematic of lap shear test: (a) coating of glass slides with fish gelatin (b) applying adhesive

sample on gelatin film (c) UV irradiation for 4 min through glass slide to form hydrogel (d) shear strength of

sample using Instron machine (adopted from [89])

The shear strength was determined by Standard Test Method for strength properties of tissue

adhesives in Lap shear by Tension Loading ASTM F2255-05. Briefly, glass sheets of

polymethyl methacrylate (25 mm x 10 mm) were taken for test. The part of sheets (10 mm x

10 mm) was coated by 20 % Fish Gelatin, which was then incubated for around 30 min at 37

℃ Thereafter, 20 µl of sample was applied on top portion of one glass sheet (10 mm x 10 mm),

and the other sheet was placed on top of solution followed by irradiation by UV light for 4 min.

These prepared hydrogels, then, placed in Instron mechanical testing machine for shear testing

by tensile loading at strain rate of 1 mm/min. Shear strength was determined at failure of

sample.

24

3.3.3 Burst pressure test

Fig. 10 Schematic of Burst pressure test: (a) Collagen sheet is placed in between burst pressure

assembly with 2.5 mm diameter hole in middle of sheet (b) Adhesive sample was placed above hole (c) UV

crosslinking for 4 min to form hydrogel (d) collagen film is ready for burst pressure test (adopted from [89])

The burst pressure of hydrogels was determined by using a standard test method, ASTM

F2392-04 for burst strength of surgical sealants. Briefly, collagen sheets (40 mm x 40 mm)

were soaked in PBS for around 10 min. Then, circular hole of 3 mm diameter was made in

centre of sheet which later placed on Burst pressure experiment set-up consisting of a pressure

detection and recorder unit and syringe pump. Thereafter, this circular hole was covered with

70 µl of precursor solution of composite hydrogel followed by UV irradiation for 4 min. This

hydrogel, then, pressurized by air syringe pump at a rate of 5 ml/min until it burst, and this

maximum pressure was recorded as a burst strength.

3.4 In vitro swelling ratio and degradation

To determine swelling ability, crosslinked samples were prepared in compression molds and

then incubating samples in PBS at 37 ℃ for 24 h. Samples were taken out at different time

intervals such as 2 h, 4 h, 8 h, 14 h and 24 h and drying with kimwipes before weighing.

Changes in mass were noted each time intervals and swelling ratio was calculated as describe

d below formula:

25

Swelling ratio = Ws − Wd

Wd

Here, Ws = weight of swollen polymer, Wd = weight of dry polymer

Degradation percentage of nano-composite hydrogels determined by incubating samples in

PBS at 37 ℃ and then measuring weight after freeze-dried of samples overnight. Four

replicates were performed for both swelling and degradation Weights were measured at

different days 1, 2, 4, 8, 15 and 21 and following equation used to check degradation

percentage:

Degradation percentage = 𝐶ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑜𝑙𝑦𝑚𝑒𝑟

𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 * 100

3.5 Characterization of Mechanical Properties

Mechanical testing of engineered Nano-composites was performed by using an Instron 5542

mechanical testing machine. For Tensile strength, the samples were first prepared in

Rectangular shaped tensile molds (length: 13 mm, width: 5 mm, depth: 1.5 mm) by pouring

approximately 70 µl of precursor solution followed by UV crosslinking for 4 min.

Fig. 11 Schematic of mechanical test: (a) schematic showing assembly of tensile machine including

sample holder, grips and load cell assembly (b) figure showing hydrogel sample attached to tensile taps for tensile

test

26

Mechanical testing of engineered Nano-composites was performed by using an Instron 5542

mechanical testing machine. For Tensile strength, the samples were first prepared in

Rectangular shaped tensile molds (length: 13 mm, width: 5 mm, depth: 1.5 mm) by pouring

approximately 70 µl of precursor solution followed by UV crosslinking for 4 min. Then, this

hydrogel was placed on tensile taps and both ends were fixed with Krazy glue to prevent slip

of samples during experiment. Thereafter, sample was fixed in Instron machine with the help

of tensile grips and stretched from both ends at a rate of 1 mm/min until failure. Extensibility

and Ultimate tensile strength were determined from the failure point of stress-strain curve,

whereas Elastic modulus was calculated from the slope of straight line.

For compression strength, the same method of sample preparation used as described for Tensile

strength. Here, compression molds (diameter: 6.5 mm, height: 4.5 mm) used to make

cylindrical shape to facilitate test. Then, sample was placed on compression plate which already

immersed in PBS water-bath and compression applied at a rate of 1 mm/min until deformation

of sample. The compression modulus then calculated from initial linear part of slope of stress-

strain curve.

3.6 Scanning electron microscopy of hydrogel/ porcine skin interface

To verify the mechanical interlocking of ZnO-T arms on tissue, we tried to take SEM images

of hydrogel-porcine skin interface using a Hitachi S-4800 Scanning Electron Microscope

(SEM) by using critical point drying (CPT) method.

3.7 Antibacterial properties (Zone of Inhibition)

Hydrogels with ZnO-T and ZnO-C of 0.01, 0.05 and 2 %(w/v) were prepared as described

previously using a compressive mold. Next, these nanocomposite hydrogels were deposited

into a 24 well plate, in separate wells, and sterilized under UV light. Pseudomonas aeruginosa

and Escherichia coli were used to evaluate the antimicrobial properties of the composite

hydrogels. A single colony of each strain of bacteria was mixed in tryptic soy broth (TSB;

Sigma-Aldrich; 5 mL) and incubated overnight in a bacterial shaker incubator (200 rpm at 37

ºC). The optical density (OD) of the resulting bacterial suspension was adjusted to an OD of

0.52 (109 CFU/ml) at a wavelength of 570 nm using a spectrophotometer. This suspension was

then serially diluted to a density of 106 CFU/ml. Using a sterile swab, a suspension of the pure

culture is spread evenly over the face of a sterile agar plate. The antimicrobial agent is applied

to the centre of the agar plate by creating hole using punch. Then, this agar plate was incubated

27

for 18-24 hr in incubator at 37 ºC. The antimicrobial activity then can be determined by

measuring size of zone of inhibition-larger zone of inhibition generally means that the

antimicrobial is more potent.

28

4.0 Results and Discussion

4.1 Synthesis and structural characterization of nanocomposite hydrogel

Herein, we synthesized nanocomposite hydrogel by using different shape of Zinc Oxide

nanoparticles such as tetrapod and spherical by incorporating them into 20 %(w/v) GelMA

prepolymer followed by UV irradiation for 4 min. Then, we characterized these samples

including control, ZnO-T incorporated hydrogel and ZnO-C incorporated hydrogel by SEM

images. As shown in Fig. 15, ZnO-T incorporated hydrogel has spikes on hydrogel surface

similar to rose spikes that protrude from surface which facilitate several enhancing

physiochemical properties such as enhancing mechanical strength, mechanical interlocking etc.

Likewise, ZnO-C incorporated hydrogel has spherical particles on hydrogel surface, but it

aggregates as depicts from figure which retards several mechanical and adhesion properties of

nanocomposites.

Fig. 12 Morphology of hydrogels by Scanning electron microscope: (a) shows GelMA hydrogel

(b) represents hydrogel with ZnO-C as aggregated nanoparticles at few places (c) shows ZnO-T arms pointing

outward from hydrogel surface

29

4.2 Mechanical Properties of nanocomposite hydrogels

The mechanical properties of composite hydrogels were assessed through tensile and

compression test by incorporating various concentration of ZnO-T and ZnO-C (0.01,0.05 and

2 %(w/v)) in 20 %(w/v) GelMA to analyse increasing nanoparticle effect on mechanics. Our

results demonstrated that incorporating nanoparticles in GelMA significantly alter mechanical

properties. Elastic modulus increased from 61.8 ± 8.5 kPa to 98.06 ± 12.16 kPa (0.01 %(w/v)

ZnO-T) but was almost same as 66.45 ± 16.15 kPa (0.01 %(w/v ZnO-C)). Then, we

hypothesized that higher particle concentration should show improved results, therefore

comparison with 2 %(w/v) represented significant increase in modulus to 244.45 ± 9.15 kPa

for ZnO-T while it decreased to 29.77 ± 16.83 kPa for ZnO-C (Fig.16). We also tested samples

to find compressive modulus and results showed same trend for varying concentrations as

observed in elastic modulus for both ZnO (Fig.17). Ultimate strength also revealed drastic

increase from 21.22 ± 8.07 kPa to 78.66 ± 15.47 kPa for 2 %(w/v) ZnO-T (Fig.18). This

composite hydrogel also able to retain extensibility even after incorporating higher amount of

ZnO-T (28.89 ± 12.4) close to control sample (34.66 ± 11.6) while it showed enhanced

extensibility (67.41 ± 1.16) for 2 %(w/v) ZnO-C (Fig.19). This is likely due to the very low

strength of material at higher concentration for ZnO-C. In addition, the phenomena of lower

extensibility at higher concentration in case of ZnO-T was proposed by Xin Jin et al. that due

to the mechanical interlocking of tetrapods with each other and with polymer chains reform

polymer matrix in stronger shape which further retard the movement of particles and thus

resulted into lower elongation.

100 µm

30

Fig.13 Comparison of mechanical properties between nanocomposite hydrogels. (a) and (b)

represent elastic and compressive modulus respectively for varying concentration of both ZnO loaded in GelMA.

(c) and (d) describes UTS and Extensibility respectively. Here, each graph compares between ZnO-T and ZnO-C

incorporated in 20 %(w/v) GelMA. (All Hydrogel Nano-composite were prepared by 4 min UV crosslinking using

0.5 %(w/v) Irgacure as a Photo-initiator agent). Data is represented as mean ± SD (*p < 0.05, **p < 0.01, ***p <

0.001, ****p < 0.0001 and n =3)

4.3 Tissue Adhesive properties of nanocomposite hydrogels

Properties that are important for effective sealants, including adhesion strength, shear strength,

and burst pressure, were examined in vitro according to ASTM standard tests. In these tests,

the sealing capability and adhesion strength of nanocomposite hydrogels, produced by using

different concentrations and photo-crosslinking times were compared. The adhesion strength

of the engineered sealants was measured by using a modified wound closure test based on

ASTM F2458-05 (Fig. 20). Results represented significant increase in adhesion strength from

50 ± 3 kPa to 150 ± 5 kPa (three times) for 0.01 %(w/v) ZnO-T whereas there was no

improvement observed in case of ZnO-C incorporated hydrogels. It might be due to mechanical

interlocking of ZnO-T arms on tissue surface that lead to higher adhesion strength. Moreover,

we observed reducing adhesion strength at higher concentration (40 ± 5 kPa for ZnO-T) for

31

both hydrogels because of highly crosslinking and interaction of tetrapod arms with polymer

chains turned into no free arms available to attach to tissue surface. Similarly, adhesion strength

is almost same(50 ± 5 kPa) as control sample for ZnO-C 2 %(w/v) due to aggregation of

particles at higher concentration. The shear strength of the engineered nanocomposite was also

characterized by using a modified lap shear test based on ASTM F2255-05 (Fig. 21). Similar

to the wound closure test, the highest shear strength was obtained for the 0.01 % (w/v) ZnO-T

hydrogel (290 ± 10 kPa) which was also higher than ZnO-C 0.01 %(w/v) (130 ± 10 kPa). This

test also demonstrated similar trend observed in wound closure in a way that at higher

concentrations of both nanocomposite hydrogels (2 %(w/v)) lead to drastic decrease in shear

strength. In order to test the burst pressure of the engineered nanocomposite, continuously

increasing air pressure was exerted on hydrogels covering a standardized defect in a collagen

sheet based on ASTM F2392-04 (Fig. 22). The burst pressure of 0.01 %(w/v) ZnO-T

significantly enhanced from 10.0 ± 0.6 kPa to 22.0 ± 3.0 kPa as compared to 20 %(w/v)

GelMA, but there were no changes observed for 0.01 %(w/v) ZnO-C (9 ± 1.1 kPa ) hydrogel.

In addition, it also showed decrease in burst pressure strength to 7 ± 2 kPa for 2 %(w/v) ZnO-

T and 8 ± 2 kPa for 2 %(w/v) ZnO-C. Moreover, the burst pressure value for 0.01 %(w/v) ZnO-

T nanocomposite hydrogel decrease from 22.0 ± 3.0 kPa to 12.0 ± 2.0 kPa by increasing UV

exposure time from 4 min to 10 min, although it was not statistically significant (Fig.23).

Taken together, the mechanical testing and ASTM standard tests for adhesives showed

excellent mechanical and adhesive properties for GelMA sealants produced by using 0.01

%(w/v) ZnO-T nanocomposite hydrogel. The wound closure strength, the shear resistance and

especially the burst pressure were significantly higher for a 0.01 %(w/v) ZnO-T incorporated

in 20 %(w/v) GelMA as compared to all other concentrations of ZnO-C loaded in 20 %(w/v)

GelMA. Additionally, to verify the mechanism of mechanical interlocking of tetrapod arms on

native tissue surface, we took SEM images of nanocomposite hydrogel/ porcine skin interface.

SEM images obviously reveal mechanical interlocking of those spikes onto tissue surface,

whereas it doesn’t show any attachment for ZnO-C loaded samples. Therefore, this could be

the reason for increasing tissue adhesiveness which apparent in all three standard adhesion

tests.

32

Fig.14 Comparison of in vitro adhesion properties of Nanocomposite hydrogels. Comparison

of in vitro wound closure adhesion test in (a) between ZnO-T and ZnO-C with varying concentrations in 20

%(w/v) GelMA. (b) Lap shear test to evaluate shear strength of composite for both type of particles on 20% fish

gelatin coated glass slides (c) Air burst pressure of samples on collagen sheets and comparison between ZnO-T

and ZnO-C. In all these three standard adhesion tests, UV light was used as source of photo-polymerization for 4

min (6.9 W/cm2). (d) Comparison of Burst pressure for different time of UV crosslinking (4, 6 and 10 min) for

0.01 %(w/v) ZnO-T to verify increasing crosslinking density effect on tissue adhesive property. Data is

represented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and n = 3)

33

Fig. 15 Scanning electron microscope images of nanocomposite hydrogel/ porcine skin

interface: Control represents the interface of porcine skin/ hydrogel sample, Spherical shows interface between

nanocomposite and porcine skin where there was no attachment of nanoparticles observed on porcine skin,

Tetrapod exhibit the mechanical interlocking of spikes inside the porcine skin at interface that facilitate higher

adhesion strength compared to spherical particles

4.4 In vitro swelling and degradation

Swelling ratios of the nanocomposite hydrogels were also determined at various time points,

throughout 24 h of incubation in phosphate buffer saline at 37 ºC. Our results demonstrated

that both ZnO-T and ZnO-C incorporated nanocomposite hydrogels exhibit almost same

swelling behaviour ranging from 3.0 to 3.8 even after incorporating higher concentration of

34

nanoparticles(Fig.25 and 26). However, 2 %(w/v) ZnO-C loaded hydrogels, due to extremely

low mechanical strength, deformed immediately in PBS and therefore it was not possible to

measure swelling ratio for this concentration. The wide range of swelling ratios obtained for

GelMA/ZnO hydrogels is advantageous for tissue engineering applications since they could be

finely tuned based on their final application This is mainly due to their enhanced structural

stability in physiological environments, long lasting tissue interactions, and sustained

mechanical performance. This further regulates elimination of exudates and production of

ECM components that promote cellular function and tissue regeneration[87].

Another advantage of hydrogels used for sutureless wound closure is their controlled

degradation in wet environments. Therefore, we aimed to investigate the in vitro degradation

of both nanocomposite hydrogels various concentrations in PBS (Fig. 27 and 28) Results

demonstrated that the in vitro degradation rate of the composite hydrogels was dependent on

type and concentration of ZnO. Overall, in vitro degradability was consistently higher for 2

%(w/v) ZnO-C loaded hydrogel samples. Rest all samples represent almost similar controlled

degradation behaviour as control 20 %(w/v) GelMA. In contrast, previous studies have shown

that the incorporation of antimicrobial agents such as ZnO in protein-based biopolymers, could

significantly alter the degradation rate of the resulting biomaterials[88].

35

Fig.16 In vitro swelling and degradation properties of the nanocomposite hydrogels. (a)

represents swelling ratio in PBS for ZnO-T with varying concentrations (0, 0.01, 0.05 and 2 %(w/v)) in 20 %(w/v)

GelMA at different time intervals (2,4, 8, 14 and 24 h). (b) shows swelling ratio for ZnO-C (0, 0.01, 0.05 %(w/v)

in PBS. (c) and (d) represent nanocomposite degradation percentage in PBS for various concentration of ZnO-T

and ZnO-C (0, 0.01, 0.05 and 2 %(w/v)) respectively in 20 %(w/v) GelMA at fixed time intervals (1, 2, 3, 7, 15

and 21 day). Data is represented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and n = 3)

4.5 In vitro Antimicrobial properties of nanocomposite hydrogel

Many findings suggest that inadequate selection and abuse of antimicrobials may lead to

resistance in various bacteria and make the treatment of bacterial infections more difficult.

Antimicrobial resistance in E. coli has been reported worldwide. Treatment for E. coli infection

has been increasingly complicated by the emergence of resistance to most first-line

antimicrobial agents. Therefore, the antibacterial activity of the engineered nanocomposite was

tested against (Escherichia coli (E.Coli) and Pseudomonas aeruginosa (P.a) bacteria) to

evaluate their antimicrobial properties by Zone of Inhibition method as they are widely

prevalent as Gram-negative pathogens in human. Herein, we compared our results with

commercially available antibiotic kanamycin. Our results demonstrated that 2 %(w/v) ZnO-T

and ZnO-C loaded hydrogels shows almost similar behaviour as Kanamycin against E.Coli

bacteria(Fig.28). In addition, 0.01 %(w/v) ZnO-T and ZnO-C loaded hydrogels showed better

36

antibacterial property compared to control sample without nanoparticles. Similarly, both ZnO

nanoparticles of 0.01 %(w/v) showed excellent antimicrobial property against P. aeruginosa

bacteria compared to other concentrations.

Fig.17 Antimicrobial property nanocomposite hydrogels against E.coli and P.aeruginosa:

(a) and (b) represents zone of inhibition against E.coli for ZnO-C and ZnO-T nanocomposite hydrogels

respectively, (c) and (d) shows zone of inhibition for ZnO-T and ZnO-C nanocomposite hydrogels respectively

against P.aeruginosa bacteria

37

5.0 Conclusion

An adhesive and antimicrobial nanocomposite hydrogel was investigated in this thesis by

incorporating novel ZnO-T micro-nano tetrapods into GelMA. Herein, we utilized

biocompatibility, biodegradability of GelMA and special shape of tetrapods that facilitate

mechanical interlocking, superior mechanical as well as antimicrobial properties in

synthesizing composite which has potential for various tissue engineering applications. This

precursor solution can be crosslinked by UV irradiation in just 4 min to prepare crosslinked

polymer structure. Various physical properties can be controlled easily by varying the

concentrations of ZnO-T in GelMA solution such as elastic and compression modulus,

elongation etc.

Additionally, this novel material can be applied for various surgical applications such as wound

healing where this material can be sprayed topically on infected site. Future studies are needed

to evaluate several important properties such as cell cytotoxicity of nanocomposite with

different concentrations, elucidate mechanism behind antibacterial activity by measuring ion

release concentration, in vivo biocompatibility and biodegradation.

38

6.0 Recommendations

6.1 Alternatives of UV irradiation for crosslinking of prepolymer solution

Although UV light is widely applied to crosslink prepolymer solution by using crosslinker to

form crosslinked structure, its detrimental and toxic effects on human skin circumscribe its

various applications. Instead of this, this could also be crosslinked by using other available

various methods such as visible light, by dispersing various chemical substances that induce

reaction to form crosslinked structure without applying light sources, etc.

6.2 Mechanism of higher mechanical strength of ZnO-T incorporated nanocomposite

hydrogel

The reason for higher mechanical strength of tetrapod at higher concentration might be special

geometry of tetrapod that exhibits mechanical interlocking in the polymer and with each other.

Moreover, tetrapod represent a concave shape that four legs pointing into different directions

which facilitate strong locking in polymer matrix. At a critical strain, the ZnO-T network starts

to break up by bending of the legs of tetrapod, which later move away from each other and lose

the interlocking effect. Unlike other shapes particles which prone to aligned with direction of

stress and lose their restrictive effects, the tetrapod always has legs that are not parallel to

direction of stress and therefore could interlock at higher filling factors[40].

Figure 18. SEM images reveal the size and shape of different fillers used in the polymer

composite. (a) ZnO microfibers and particulate produced by grinding tetrapodal ZnO particles (G-ZnO). (b)

Agglomerated ZnO nano spherical particles (S-ZnO), upper corner shows the magnified image. (c) Tetrapodal

ZnO microparticles (T-ZnO)(Adopted from [40])

39

6.3 ZnO antibacterial property mechanism

Fig.19 Antibacterial different mechanisms Correlation between (a) influence of essential ZnO-NPs

parameters on the antibacterial response and the (b) different possible mechanisms of ZnO-NPs antibacterial

activity, including: ROS formation, Zn2+ release, internalization of ZnO-NPs into bacteria, and electrostatic

interactions (Adopted from [88])

Fig. 20 ZnO Nanoparticles antibacterial mechanism (a) NPs internalization into the cell and

translocation. NPs penetrate through holes, pits or protrusions in the cell wall. (b) Schematic representation of

collapsed cell showing disruption of cell wall and extrusion of cytoplasmic contents. (c) Bacterial cell showing

important variations in envelope composition (slight invaginations and thickening of cell wall) and extrusion of

cytoplasm. (d) Probable mechanisms involve the following: metal ions uptake into cells, intracellular depletion,

40

and disruption of DNA replication, releasing metallic ions and ROS generation and accumulation and dissolution

of NPs in the bacterial membrane(Adopted from [88]).

There is possible mechanism underlying the interaction of NPs with bacteria : (1) excessive

ROS generation, mostly hydroxyl radicals (HO*) and singlet oxygen (1O2 )[82] . Its already

demonstrated that effect to a direct interaction between NPs and the membrane as well as to

ROS generation nearby bacteria membrane. Moreover, it was also demonstrated that

electrostatic interaction between NPs and bacterial cell surface as a cause of growth inhibition,

and that the total bacterial charge is negative, because of the excessive formation of separated

carboxyl groups. Thus, the cell surface is negatively charged, and ZnO-NPs contain a positive

charge in a water suspension[83]. Such reverse charges enhance the total effect by creating

electrostatic forces, which serve as a powerful bond between NPs and bacterial surface.

Therefore, the cell membrane is damaged. Although the detailed mechanism of ZnO

antibacterial activity is under discussion, a three most widely accepted, and reported

hypothetical mechanisms in the literature[84] are: (i) metal ions uptake (translocation and

particle internalization) into cells followed by depletion of intracellular ATP production and

disruption of DNA replication[85] (ii) ROS generation from NPs metal oxides and ions with

subsequent oxidative damage to cellular structures[86], and (iii) changes in bacterial membrane

permeability (progressive release of lipopolysaccharides, membrane proteins, and intracellular

factors) and dissipation of the proton motive force as a result of accumulation and dissolution

of NPs in the membrane.

6.4 In viro cytocompabilty study

The in vitro cytocompatibility of hydrogels sample were assessed by measuring metabolic

activity of the 3T3 fibroblasts cells cultured in hydrogels using PrestoBlue assay after day 1 ,

3. 5 and 7. Our results showed that ZnO-T did not offer any cytotoxicity on cells incorporating

with GelMA hydrogels. Whereas ZnO-C showed slight inhibitory effect on cells. Therefore,

we can say that ZnO-T incorporated hydrogels offered excellent cell affinity compared to ZnO-

C Hydrogels.

41

Fig. 21 In vitro 3D encapsulation of 3T3 fibroblasts in ZnO-T and ZnO-C loaded GelMA

hydrogels. 3T3 cells were encapsulated inside UV light irradiated with control(20% GelMA) and 0.01 %(w/v)

ZnO-T and ZnO-C incorporated GelMA. Representative live/ dead images from control as well as ZnO-T and

ZnO-C incorporated GelMA in Figure (a) for day 1 and day 5 post encapsulation. (b) represents Quantification

of metabolic activity, relative fluorescence units (RFU), using PrestoBlue assay at days 1, 3, 5 and 7 post

encapsulation.

(a)

(b)

42

6.5 NMR study

(a)

(b)

(c)

Fig. 22 1H NMR analysis of nanocomposite hydrogel. 1H NMR (500 MHz; D2O) spectra of (a) 20

%(w/v) GelMA precursor (b) 20 %(w/v) GelMA 2 % ZnO-C precursor and (c) 20 %(w/v) GelMA 2 % ZnO-C

hydrogels.

43

To determine degree of crosslinking within 2 %(w/v) ZnO-C incorporated GelMA hydrogels,

1H NMR (500 MHz) spectra were taken from GelMA prepolymer(Fig a) and ZnO-C

incorporated GelMA hydrogels. The extent of crosslinking was determined by the change in

the integrated areas of the peaks from the methacrylated groups after exposure with UV light.

Using this method, the degree of crosslinking was found 13.2 ± 0.1%.

Fig. 23 Comparison of crosslinking degree between nanocomposite hydrogels

As we can see from Fig.23 that 2 %(w/v) ZnO-C incorporated hydrogels was not fully

crosslinked as compared to GelMA and ZnO-T incorporated hydrogels. It shows only 13.2 %

degree of crosslinking for ZnO-C. Therefore, it shows very low mechanical strength as well as

and deforms immediately totally during swelling.

44

7.0 Nomenclature

SEM Scanning electron microscopy

GelMA Gelatin Methacryloyl

ZnO-T Zinc oxide tetrapods

ZnO-C Zinc Oxide sphericals

DI Water Deionized Water

DLS Dynamic Light Scattering

ECM Extracellular Matrix

E. coli Escherichia coli

NPs Nanoparticles

OD Optical density of absorbance

PBS Phosphate buffer saline

SA Staphylococcus aureus

TSB Tryptic Soy Broth

UV Ultra Violet

ZnO Zinc Oxide

45

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