An-Najah National University 

Faculty of Graduate Studies 

 

 

DEVELOPMENT OF AN OPTIMIZED SCAFFOLD 

FOR TISSUE ENGINEERING BASED ON THE 

NON-COVALENT FUNCTIONALIZATION OF 

CARBON NANOMATERIALS  

 

 

By 

Salsabeel Mamon Odeh 

 

Supervisors 

Dr. Mohyeddin Assali 

Dr. Naim Kittana 

 

 

This Thesis is Submitted in Partial Fulfillment of the Requirements for the Degree 

of Master of Pharmaceutical Sciences, Faculty of Graduate Studies, An-Najah 

National University, Nablus - Palestine. 

 
 

2022 



 II 

 

 

 

 

 

 

 

 

 

 

DEVELOPMENT OF AN OPTIMIZED SCAFFOLD 

FOR TISSUE ENGINEERING BASED ON THE 

NON-COVALENT FUNCTIONALIZATION OF 

CARBON NANOMATERIALS 
 

 
By  

Salsabeel Mamon Odeh 

 

This Thesis was Defended Successfully on 92/29/2022 and approved by 

Dr. Mohyeddin Assali 

Supervisor 

 

Signature 

Dr. Naim Kittana 

Co-Supervisor 

 

Signature  

Dr. Hani Naseef 

External Examiner 

 

Signature 

Dr. Ramzi Shawahna 

Internal Examiner 

 

Signature 



 III 

Dedication 

This is dedicated to: 

The greatest dad and mum in the whole world for their unconditional love, prayers, 

endless support, and for raising me to believe that anything was possible  

My beloved brothers and sister for being the pillows and cheerleading squad I have 

needed. I love you to the moon and back 

To a very special, my lovely husband Rashid for his continued and unfailing love, 

supporting all my dreams, and making everything possible. You were always around at 

times I thought that it is impossible to continue, you helped me to keep things in 

perspective. I greatly value his contribution and deeply appreciate his belief in me. You 

make me the happiest wife; I love you endlessly  

I consider myself the luckiest in the world to have such a lovely and caring family, 

standing beside me with their love and unconditional support. 

All the people in my life who touch my soul somehow. 

 

  



 IV 

Acknowledgment 

I want to start by thanking Allah for giving me the strength, willpower, and guidance I 

needed to complete this research despite all difficulties.  

I am greatly indebted to my research guide, Dr. Mohyeddin Assali, who accepted me as 

his master student and offered me his mentorship. My deepest thanks to Dr. Naim 

Kittana for his good advice and scientific support. This work would not have been 

possible without their guidance and involvement. 

I would like to thank the rest of the examination committee, for reading my thesis, their 

insightful comments and suggestions and invaluable criticisms 

Thanks to Dr. Muna Hajj Yahya, Department of Physics, Faculty of Science, at An-

Najah National University for her collaboration in conductivity measurements. 

I’m thankful to the staff of the laboratories of the College of Pharmacy, Science and 

Medicine for supplying help and assistance. Especially, the supervisor of the 

laboratories of the College of Pharmacy Mr. Mohammad Arar and the lab workers 

Tahreer Shtayeh, Linda arar and thankful to the supervisor of laboratories of the College 

of Medicine Mr. Ahmed Musa and the lab worker mohammad nabeel and abed zarour. 

 

 

 

 

 

 

 

 

 

 

 



 V 

Declaration 

 

I, the undersigned, declare that I submitted the thesis entitled: 

 

DEVELOPMENT OF AN OPTIMIZED SCAFFOLD FOR TISSUE 

ENGINEERING BASED ON THE NON-COVALENT FUNCTIONALIZATION 

OF CARBON NANOMATERIALS  

 

I declare that the work provided in this thesis, unless otherwise referenced, is the 

researcher’s own work, and has not been submitted elsewhere for any other degree or 

qualification. 

 

_____________________________________ Student's Name:  

_____________________________________ Signature: 

_____________________________________ Date: 

 

 

 

 

 

 

 

 

 

 

 



 VI 

List of Contents 
 

 

Dedication ....................................................................................................................... III 

Acknowledgment ............................................................................................................ IV 

Declaration ....................................................................................................................... V 

List of Contents ............................................................................................................... VI 

List of Tables ............................................................................................................... VIII 

List of Figures ................................................................................................................. IX 

List of Schemes ................................................................................................................ X 

ABSTRACT ................................................................................................................... XII 

Chapter one: Introduction ................................................................................................. 1 

1.1 Regenerative medicine ................................................................................................ 1 

1.2 Tissue engineering ...................................................................................................... 1 

1.4 Carbon Nanotubes (CNTs) ......................................................................................... 6 

1.4.1 Different Techniques For Carbon Nanotubes Synthesis .......................................... 7 

1.4.2 Properties of CNTs ................................................................................................ 11 

1.4.3 Applications of CNTs ............................................................................................ 13 

1.4.5 Functionalization of CNTs ..................................................................................... 16 

1.4.5.1 Covalent functionalization of CNTs ................................................................... 17 

1.4.5.2 Non-covalent functionalization of CNTs ............................................................ 18 

1.6 Chitosan .................................................................................................................... 20 

1.7 Literature Review ..................................................................................................... 21 

1.8 Aims of the study ...................................................................................................... 25 

1.9 Objectives ................................................................................................................. 25 

Chapter Two: Methodology ............................................................................................ 26 

2.1 Reagents and instrumentation ................................................................................... 26 

2.2 Synthesis of pyrene derivatives ................................................................................ 27 

2.2.1 Tosyl-TEG-Tosyl Synthesis (1) ............................................................................. 27 

2.2.2  N3-TEG-N3 Synthesis (2)...................................................................................... 28 

2.2.3 OH-TEG-alkyne Synthesis (3) ............................................................................... 28 

2.2.4 Pyrene-TEG-alkyne Synthesis (4) ......................................................................... 29 

2.2.5 Pyrene-TEG-triazole-TEG-azide Synthesis (5) ..................................................... 30 

2.2.6 pyrene-TEG-triazole-TEG-amine Synthesis (6) .................................................... 30 

2.2.7 OH-TEG-Tosyl ...................................................................................................... 31 

2.2.8 OH-TEG-N3 Synthesis (8) ..................................................................................... 31 

2.2.9 COOH-TEG-N3 synthesis  (9) ............................................................................... 31 

2.2.10 Pyrene-TEG-triazole-TEG-COOH Synthesis (10) .............................................. 32 



 VII 

2.4 Functionalization of MWCNTs with Py-COOH and Py-NH2 ................................. 32 

2.5 Atomic force microscope characterization ............................................................... 33 

2.6 Primary dermal fibroblast culture and maintenance ................................................. 33 

2.7 Generation of Engineered Connective Tissue (ECT) with/without functionalized 

MWCNT ................................................................................................................ 34 

2.7.1 Double-strength cell culture media (2x DMEM) preparation ............................... 34 

2.7.2 Preparation of 0.1% NaOH .................................................................................... 34 

2.7.3 Preparation of 1.5 % (W/V) chitosan ..................................................................... 34 

2.7.4 Primary cell suspension preparation ...................................................................... 34 

2.7.5 Preparation of MWCNTs-NH2 and MWCNTs-COOH Stock solutions ............... 35 

2.7.6  Preparation of engineered connective tissues (ECTs) .......................................... 35 

2.7.6.1 Preparation of f-MWCNTs containing ECT ....................................................... 36 

2.7.6.2 preparation of chitosan and f-MWCNTs containing ECT .................................. 37 

2.8 Characterization of Engineered Connective tissues .................................................. 38 

2.8.1 Electrical conductivity of ECTs ............................................................................. 38 

2.8.2 Cell viability test (MTS assay) .............................................................................. 38 

2.8.2.1  Preparation of the treatment solution for MTS assay ........................................ 38 

2.8.2.2 Cell culture .......................................................................................................... 39 

2.8.2.3  MTS assay .......................................................................................................... 39 

Chapter Three: Results and Discussion .......................................................................... 40 

3.1 Synthesis and functionalization of carbon nanomaterials ......................................... 40 

3.2.2 Electrical conductivity test of ECTs ...................................................................... 44 

3.2.3 Cell viability test (MTS assay) .............................................................................. 47 

Chapter Four: Discussions and Conclusions .................................................................. 49 

4.1 Conclusion ................................................................................................................ 49 

List of abbreviations ....................................................................................................... 51 

References ....................................................................................................................... 52 

Appendices ...................................................................................................................... 61 

 ب  ............................................................................................................................... الملخص

 

 

 

 

 

 

 



 VIII 

List of Tables 
 

Table 1: ECT general composition ................................................................................. 35 

Table 2: ECT contains 0.025% f-MWCNT general composition .................................. 36 

Table 3: ECT contains 0.05% f-MWCNT general composition .................................... 36 

Table 4: ECT contains 0.1% f-MWCNT general composition ...................................... 37 

Table 5: General composition of chitosan-containing ECT ........................................... 37 

Table 6: chitosan and 0.025%MWCNT-COOH containing ECT .................................. 38 

 

 

 

 

 
 

  



 IX 

List of Figures 
 

Figure 1.1: types of carbon nanomaterials (24). ............................................................... 4 

Figure 1.2: carbon nanotubes structures: A) SWCNTs; B) MWCNTs (34, 35). .............. 7 

Figure 1.3: graphene sheet folded in three different patterns: a) chair, b ) zigzag, c) 

chiral (49). .................................................................................................... 12 

Figure 1.4: Multilayer nanotubes' transverse structure is represented schematically: a) 

Russian doll, b) parchment (49). .................................................................. 13 

Figure 1.5: structures of pyrene-functional polyesters. b) pyrene-functional polyesters 

interact non-covalently with the surface of MWCNTs via π-π stacking (70)

 ...................................................................................................................... 20 

Figure 1.6: MWCNT dispersion on the surface of PLGA-MWNT scaffolds as shown by 

SEM. The agglomeration size distribution within the unmodified MWCNTs 

is shown in red circle (77). ........................................................................... 22 

Figure 3.1: Image of dispersion of A) pristine MWCNTs; B) MWCNTs-Py-COOH and 

(C) MWCNTs-Py-NH2. ............................................................................... 43 

Figure 3.2: A) pristine MWCNTs, B) f-MWCNTs with py-COOH, C) f-MWCNTs with 

py-NH2. ........................................................................................................ 43 

Figure 3.3: primary fibroblast-containing ECT with varying MWCNTs loading: A) 

Control; B) MWCNTs-Py-COOH (0.025%); C) MWCNTs-Py-COOH 

(0.05%); D) MWCNTs-Py-COOH (0.10%); E) MWCNTs-Py-NH2 

(0.025%); F) MWCNTs-Py-NH2 (0.05%) and G) MWCNTs-Py-NH2 

(0.10%). ........................................................................................................ 44 

Figure 3.4: Bright-field images at 100× for primary dermal fibroblast cells treated with 

A) Control (cells without treatment); B cells treated with Chitosan, C) 

0.025%MWCNT-COOH with Chitosan, D-F) different concentrations of 

MWCNT-COOH (0.025%, 0.050%, and 0.100%) respectively. ................. 47 

Figure A.1: Noncovalent functionalization of MWCNTs with Py-COOH and Py-NH2.

 ...................................................................................................................... 61 

Figure A.2: 48-well plate with stretchers. ....................................................................... 61 

 

 

 

 
 

 

 

 

 

 

 

 

 



 X 

List of Schemes 
 

Scheme 1: Tissue engineering approach for tissue regeneration (2). ............................... 2 

Scheme 2: Figure showing the different graphene rolling directions that can produce 

single-wall carbon nanotubes with differing chiralities (50) ....................... 12 

Scheme 3: Covalent functionalization of CNTs (43). ..................................................... 18 

Scheme 4: Noncovalent adsorption of SWCNTs with 1-pyrenebutanoic acid, 

succinimidyl ester via π–π stacking (68) ..................................................... 19 

Scheme 5: chemical structure of chitosan (72) ............................................................... 20 

Scheme 6: The synthesized OH-TEG-alkyne linker (1) was reacted with pyrene butyric 

acid to obtain Py-TEG-alkyne (2). ............................................................... 41 

Scheme 7: Synthesis of Py-TEG-triazole-TEG-COOH (8) and Py-TEG-triazole-TEG-

NH2 (9). ....................................................................................................... 42 

Scheme 8: Average electrical conductivity of ECT structures of primary dermal 

fibroblast cells with different A)MWCNT-COOH loading and B)MWCNT-

NH2 loading. The symbol (*) indicates significance (P ≤ 0.05) compared to 

the control (0.000%). The two-way ANOVA was used to determine the 

statistical significance. ................................................................................. 45 

Scheme 9: Average electrical conductivity of ECT structures of primary dermal 

fibroblast cells with different Chitosan and 0.025% MWCNT-COOH 

loading. The symbol (*) indicates significance (P ≤ 0.05) compared to the 

control (0.000%). The two-way ANOVA was used to determine the 

statistical significance. ................................................................................. 46 

Scheme 10: Concentration-dependent effect of MWCNTs and Chitosan on the viability 

of primary dermal fibroblast cells over 24 hours. The symbol (*) indicates 

significance (P ≤ 0.05) compared to the control (0.000%). The two-way 

ANOVA was used to determine the statistical significance. ....................... 48 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 XI 

List of Appendices 
 

Appendix  A: Figures of Study ....................................................................................... 61 

 

 

 
 

 

 

 

 

 

 

 

  



 XII 

DEVELOPMENT OF AN OPTIMIZED SCAFFOLD FOR TISSUE 

ENGINEERING BASED ON THE NON-COVALENT FUNCTIONALIZATION 

OF CARBON NANOMATERIALS 

By 

Salsabeel Mamoon Odeh 

Supervisors 

Dr. Mohyeddin Assali 

Dr. Naim Kittana 

 

ABSTRACT 

One of the main approaches for tissue engineering for therapeutic purposes involves the 

utilization of primary cells that are cultured on a biocompatible scaffold with 

appropriate characteristics. Such tissues aim to maintain and/or restore normal tissue 

functions. Over the past few years, significant attention has been given to carbon 

nanomaterials, like carbon nanotubes (CNTs), and their potential applications in 

generating tissue scaffolds as they tune some of the tissue's physical characteristics such 

as flexibility, elasticity, and porosity.  

A major obstacle to the employment of CNTs in biological applications was the poor 

limited water dispersibility and cytotoxicity, however, it was found that the 

functionalization of CNTs adequately with polar functional groups can solve this 

problem and improve their biocompatibility. Similar benefits could be achieved by 

coating the CNTs with chitosan, which is biodegradable, biocompatible, and can 

form porous structures that are appropriate for cell growth.  

Therefore, in our project, we aimed to investigate the characteristics of an engineered 

connective tissue (ECTs) that is composed of primary dermal fibroblasts and collagen 

hydrogel that is enriched by varying concentrations of noncovalently functionalization 

multiwall CNTs (MWCNTs) with pyrene moiety and coated with chitosan. The tested 

concentrations were 0.025%, 0.05%, and 0.1%.  

 



 XIII 

Our data demonstrated that the enrichment of the ECTs with the functionalized 

MWCNTs significantly increased the electrical conductivity of the tissues in a kind and 

concentration-dependent manner. Furthermore, the conductivity enhancement was 

greater with MWCNTs- COOH compared to MWCNTs-NH2, the concentration of 

0.025% of MWCNT-COOH was enough to sufficiently enhance the electrical 

conductivity of the tissue compared to the control tissue. however, this concentration 

was still associated with some cytotoxicity where it reduces the cell viability by around 

(20-30) % relative to the control, while there was no significant reduction in viability 

when adding chitosan to 0.025%MWCNT-COOH, which indicates that chitosan might 

have a cytoprotective effect against CNT-induced toxicity, in addition to electrical 

conductivity enhancement.  

Keywords: carbon nanomaterials, tissue engineering, chitosan, MWCNT, primary 

fibroblast. 

 

 

 

 

 

 

 

  



 

 1 

Chapter one 

Introduction 

1.1 Regenerative medicine 

Regenerative medicine is a recent multidisciplinary science that aims to improve, 

maintain, or repair the function of tissues and organs. combining living cells, which 

provide biological functions, with substances that serve as scaffolds for cell 

proliferation, can be used to regenerate tissues (1). 

Regenerative medicine has raised expectations for a variety of current human disorders 

around the world. Parkinson, Alzheimer's , spine injuries, osteoporosis, and cancer may 

all be treated in the near future with treatments aimed at regenerating damaged or 

diseased tissues (2). The development of cell biology, biological chemistry, and 

biomaterial science has resulted in new tissue and organ engineering choices (3). 

1.2 Tissue engineering 

Transplanted organs or cells are used to treat patients who have damaged or diseased 

organs. However, because of the aging population, There is a severe lack of donor 

organs and tissues. A new area of medicine called "regenerative medicine" treats 

diseased or damaged organs and tissues. In 1987, at a meeting of the National Science 

Foundation, the term "tissue engineering" was first used. 

Tissue engineering is one of the most important components of regenerative medicine, 

which builds on the principles of materials engineering and cell transplantation to create 

biological alternatives that can maintain and/or restore normal tissue function. Tissue 

engineering is divided into two categories: the first, depends on the body's natural 

ability for regeneration when using acellular matrices to promote the optimal orientation 

and direction of new tissue growth, which are typically created by making up artificial 

scaffolds or extracting cellular components from tissues through mechanical and 

chemical manipulation to create collagen-rich matrices (4, 5). The second category is 

the use of matrices with cells, in vitro production, or in vivo regeneration using 

scaffolds built of synthetic or natural materials and filled with living cells. In both in 

vitro and in vivo procedures, cells are loaded onto a porous scaffold. On the scaffold 

surface, the nanometer-scale topographical structure can be produced. To improve cell 



 

 2 

functions, the scaffold can be functionalized with various chemicals. Components like 

medications, peptide sequences like arginine–glycine–aspartic acid (RGD), genes, or 

proteins can be encapsulated in nanoparticles and then released in a controlled manner 

to reach a specific target like cells, bacteria, or viruses (sheme1) (2, 6). 

Scheme 1 

Tissue engineering approach for tissue regeneration (2) 

 

All tissues contain the extracellular matrix (ECM), a non-cellular component., is one of 

the most essential components in tissue engineering. (7). This environment, which 

surrounds all cells, is important for normal tissue’s function and is also important for 

tissue regeneration following pathology or damage because it allows cells to grow, 

move, adhere, proliferate, and differentiate. To enable tissue engineering, the suitable 

synthetic ECM should identify as closely as possible to the natural ECM . Collagen is a 

typical biological material used to create a synthetic ECM because it is an abundant 

protein present in the ECM of body tissues and can provide binding sites to help cells 

adhere, migrate, proliferate, and differentiate (8, 9).  

Epithelial, muscle, nerve and connective tissue are the four main types of body tissues, 

which are related but not identical cells that are linked and act together to achieve 

specific objectives. Epithelial tissue is a protective tissue that makes up the body's outer 

layer and lines many of its cavities. Smooth, skeletal, and cardiac all are types of muscle 

tissue that can contract and generate distinct voluntary or involuntary movements. 

Nerve tissue is divided into central and peripheral types and is responsible for 

transporting chemical and electrical impulses from the central nervous system and brain 



 

 3 

to the periphery and back. The final tissue is connective tissue, which is a biological 

tissue found in practically every organ and creates a larger part of the skin, muscles, 

blood vessels, joints, tendons, and ligaments (10, 11). Connective tissue connects and 

supports other tissues as the term indicates, the characteristics of connective tissue 

unlike the characteristics of other tissues depend on their cellular elements, the 

connective tissue is vital for cell migration, mechanical support, directing metabolic 

processes in other tissues, and wound healing. Connective tissues are mainly composed 

of fibroblasts, collagen fibers, and extracellular matrix (ECM), however, the kind of 

intracellular matrix changes depending on the type of connective tissue (11). Because of 

the low inherent healing ability of connective tissue, certain injuries are a typical 

therapeutic challenge, requiring the development of a scaffold that preserves normal 

tissue function (12). One of the challenges in developing this scaffold is mimicking the 

qualities of real tissues, which was overcome by integrating customized nanoparticles 

into these scaffolds (13, 14). 

Carbon nanomaterials are one of the most often used nanoparticles in scaffolds because 

they share characteristics with a natural extracellular matrix such as flexibility, 

elasticity, and porosity with similar sizes (15-17). Electrical conductivity is another 

remarkable attribute of carbon nanomaterials. This characteristic is responsible for 

improving the electrical connection between cells, resulting in a better connective 

cardiac tissue engineering (15, 18). 

Finally, the functionalized carbon nanomaterials may be coupled with polymeric 

hydrogels, such as collagen, to create new scaffolds that match the composition and 

characteristics of the ECM as closely as possible, thus enhancing cell proliferation in 

injured tissue (19). Therefore functionalized carbon nanotubes can be used as 

nanomaterials for tissue regeneration (20, 21). Both collagen and functionalized carbon 

nanomaterials are used to create these scaffolds, which have several advantages. They 

are bioresorbable, biodegradable, and biocompatible, as well as having the requisite 

mechanical stiffness and electrical conductivity while preserving a porous three-

dimensional nanostructure (20).  

 



 

 4 

Chitosan is also biocompatible, biodegradable, and may be molded into a variety of 

geometries and has antibacterial activity, chitosan also forms porous structures that are 

appropriate for cell growth. Because of its biodegradability and biocompatibility, 

chitosan is a good choice for a number of tissue engineering applications. To improve 

the mechanical strength of these composites, recently combined chitosan with carbon 

nanotubes (22). The combination of these characteristics enables the target cells to 

adhere to the scaffold surface, proliferate and differentiate. These one-of-a-kind 

scaffolds can help regenerate several tissue types, including skin and myocardium (23). 

1.3 Carbon nanomaterials (CNMs) 

Carbon nanomaterials are one of the most extensively researched and utilized 

nanomaterials with diameters between 1 to 100 nm. These include nano-diamond, 

graphene nanosheets, fullerene (or Buckyball), graphene quantum dots (GQDs), 

graphene oxide (GO), carbon nanotubes (single-walled and multi-walled), amorphous 

nanocarbon, and carbon foam are some examples of CNMs (figure1.1). CNM families 

have special features that can be used in a variety of biological applications (24). CNTs 

are one of the highly searched carbon nanomaterials in the scientific world due to their 

unique thermal, mechanical and electrical characteristics (25). 

Figure 1.1 

types of carbon nanomaterials (24) 

 



 

 5 

1.3.1 Types of carbon nano allotropes 

 Fullerenes 

The third carbon allotrope, known as buckminsterfullerene (C60), was identified in 

1985. Fullerene molecules are hollow spheres or tubes made of carbon atoms. 

Buckyballs are another name for the bucky forms of spherical fullerenes. It is 

considered that their three-dimensionality, length, hydrophobicity, and electrical 

configurations make them an interesting topic in medicinal chemistry (26). 

 Nanodiamonds 

Carbon nanoparticles called nanodiamonds (NDs) have a curved octahedral architecture 

and are typically 2 to 8 nm in diameter. The surface of nanodiamonds is often covered 

with a layer of functional groups that reduces dangling bonds and stabilizes the particle. 

The particles become more stable as a result of the conversion of sp3 hybridization 

carbon with sp2. The bulk of surface atoms are terminated by groups containing 

oxygen. While most nanodiamonds are spherical in shape, some of them contain facets 

(27). 

 Carbon Quantum Dots 

Carbon nanoparticles with a size of fewer than 10 nanometers are known as carbon 

quantum dots (CQDs). Due to its qualities of being water-dispersible, non-toxic, and 

fluorescence, CQDs have recently been the topic of research. Graphene, cellulose, and 

other materials can be used to create CQDs at high temperatures and pressures. CQDs 

have drawn a lot of interest because of their unique features (28). 

 Graphene 

An allotrope of carbon called graphene is composed of a single sheet of atoms arranged 

in a two-dimensional hexagonal arrangement. The term, which is a mix of "graphite" 

and the suffix -ene, relates to the carbon allotrope made up of stacked layers of 

graphene known as graphite. A hexagonal lattice of sp2-hybridized carbon atoms forms 

the two-dimensional sheet known as graphene. Due to its mechanical, electrical, and 

thermal properties, graphene has become extremely popular in both the scientific and 

technical fields (29). 



 

 6 

 Carbon Nanotubes 

Carbon nanotubes are members of the CNM family. The use of carbon nanoparticles in 

pharmaceuticals has attracted a lot of attention. This is a result of their highly flexible 

characteristics plus a few good attributes that they inherently have (24). 

1.4 Carbon Nanotubes (CNTs) 

In the periodic table, carbon is one of the basic elements. that is necessary for life on 

Earth to exist. It's also critical for a wide range of technical uses, from pharmaceuticals 

to synthetic materials (30). Carbon nanotubes (CNTs) are synthetic carbon allotropes 

(30, 31), also called buckytubes, are nanoscale hollow carbon tubules. They are 

described as sheets of graphene formed of a hexagonal lattice of condensed benzene 

rings that have been rolled into a seamless tubule and concentric cylinder with 

diameters in the range of 5–350 nm and lengths in the micrometer range (32). These 

cylindrical carbon molecules typically have aspect ratios (length-to-diameter values) 

greater than 103 (24).  

several techniques have been documented for the synthesis of CNTs but the most 

popular techniques are arc-discharge, laser ablation, and chemical vapor deposition 

(CVD). The two main types of CNTs are single-walled carbon nanotubes (SWCNTs) 

and multi-walled carbon nanotubes (MWCNTs) (31, 32). In 1991, Graphitic carbon 

microtubules with outer diameters of 4 to 30 nm and lengths of up to 1 mm were found. 

These tubules were made up of two or more concentrically arranged seamless graphene 

cylinders. These tubes are called MWCNTs (multi-wall carbon nanotubes). While 

single-wall carbon nanotubes (SWCNTs) are first described in 1993. Which are made of 

only one seamless cylinder of the graphene sheet (Fig. 1.2). Their lengths are typically 

in the micrometer range, and their diameters range from 0.4 to 2-3 nm (33). 

  



 

 7 

Figure 1.2 

carbon nanotubes structures: A) SWCNTs; B) MWCNTs (34, 35) 

 

1.4.1 DIFFERENT TECHNIQUES FOR CARBON NANOTUBES SYNTHESIS 

To create MWNTs or SWNTs, a variety of procedures are used. Widely used processes 

for producing CNTs include electric arc discharge, laser vaporization, and chemical 

vapor deposition. In the sections below, these techniques are described. 

1.4.1.1 Electric-Arc Discharge  

Often, carbon nanotubes (CNTs) are made by creating an arc between graphite 

electrodes in an inert environment (argon or helium), a procedure that also results in 

fullerene-containing carbon soot (36). The carbon arc offers a traditional and practical 

method for producing the high temperatures (>3000oC) required to evaporate carbon 

atoms into plasma. The stability of the plasma produced between the electrodes, the 

current density, the pressure of the inert gas, and the cooling of the electrodes and 

chamber all affect the yield of the CNTs.  

Helium (He) produces the greatest results when compared to the other inert gases, likely 

because of its high ionization potential. In the arc growth procedure, the well-cooled 

electrodes and arc chamber serve to enhance the yield of nanotubes. For making 

MWNTs, the parameters are set up to reduce soot creation during arc evaporation and to 

produce 75% of the evaporated carbon from pure anodes to deposit onto the graphite 

cathode surface.  



 

 8 

The arc deposit is composed of an internal soft black powder formed of around two-

thirds CNTs and one-third graphitic nanoparticles, and an exterior hard gray shell built 

of pyrolitic graphite. The ideal synthesis conditions were 20-25V, 50-100Amp direct 

current, and 500 torr of helium pressure. The arc discharge is a quick and easy 

procedure that produces high-quality CNTs with excellent structural properties. 

Conventional arc discharge, on the other hand, is a discontinuous and unstable process 

and cannot produce a significant amount of CNTs. When the CNTs are made on the 

cathode surface and the electrode spacing varies, neither the current flow nor the electric 

fields are homogeneous. As a result, carbon nanoparticles and contaminants always 

coexist with nanotubes and the density of carbon vapor and temperature distribution are 

not uniform. Many efforts have been made to create a steady and highly effective 

discharge, and numerous studies have been conducted to understand the nanotube 

development mechanism. 

The arc method typically uses high-purity graphite electrodes, metal powders (only for 

creating SWNTs), and high-purity He and Ar gases; as a result, the cost of producing 

SWNTs and MWNTs is expensive. Although the material has a high degree of 

crystallinity, the length and diameter of the tubes cannot be controlled. Unfortunately, 

other by-products are also produced, including amorphous carbon, polyhedral graphite 

particles (in MWNTs), and encapsulated metal particles (in SWNTs) (37). 

1.4.1.2 Laser Vaporization  

The laser evaporation method is a productive way to create bundles of SWNTs with a 

limited distribution. With this technique, a graphite target is heated to a high 

temperature and exposed to an argon atmosphere before vaporizing under laser 

irradiation. When a pure graphite target was used, MWNTs were discovered. The yield 

and quality of these products have been proven to be temperature-dependent. At a 

reaction temperature of 1200oC, the greatest quality is produced. Lower temperatures 

cause the structural quality to decrease and the CNTs to begin showing numerous 

defects. When small amounts (a few percent or less) of transition metals (Ni, Co) acting 

as catalysts are added to the graphite pellet, the results undergo dramatic changes, and 

SWNTs rather than MWNTs are produced.  

The type of metal catalyst employed and furnace temperature are two important 

variables that have a significant impact on the yield of SWNTs. In the condensing vapor 



 

 9 

in a heated flow tube, a high yield with around 50% conversion of transition-

metal/graphite composite rods to SWNTs was recorded (operating at 1200oC). 

Due to the utilization of high-purity graphite rods, the high laser strengths required (in 

some circumstances, two laser beams are needed), and the lower daily production of 

CNTs compared to the arc discharge approach, the laser technique is not an 

economically viable (37, 38). 

1.4.1.3 Chemical Vapor Deposition  

One of the most used techniques for depositing thin films is chemical vapor deposition 

(CVD). The two other popular techniques for making CNTs, electric arc discharge, and 

laser vaporization, are very different from CVD. Catalytic CVD is a medium temp (700-

1473K) and long-time reactions (usually minutes to hours) method, whereas arc 

discharge and laser vaporization can be categorized as high temperature (>3000K) and 

short time reaction (s-ms) approaches. Arc discharge and laser vaporization both make 

CNTs stand-alone materials, which is one of their main technological limitations. CNTs 

do not develop on a typical or designed substrate. 

Although the majority of CVD-grown CNTs were initially "spaghetti-like" and 

defective, the technique's ability to meet technical needs was understood. Since 1998, 

significant and quick improvement has been made in CVD, establishing it as a highly 

regulated process for the creation of CNTs. High quality single-walled carbon 

nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) can now be 

produced in bulk as raw material or directly onto substrates. 

A significant advantage of CVD is that the CNTs can be employed immediately without 

additional purification, except for the need to remove the catalyst particle. CNTs are 

produced using the CVD technique by decomposing an organic gas over a substrate 

coated in metal catalyst particles. Many CVD techniques are reported, including 

catalytic pyrolysis of hydrocarbons, thermal CVD, and plasma assisted CVD (37). 

 Thermal Chemical Vapor Deposition (Hydrocarbon Gas Decomposition on 

Metal Catalysts)  

A quartz tube surrounded by a furnace makes up a thermal CVD reactor, which is easy 

to build and relatively inexpensive. Si, alumina, silica quartz, and mica are just a few 

possible substrate materials. By adjusting many parameters, including the hydrocarbon 



 

 10 

sources, the type of catalytic metals and their supports, the gas flows, the reaction time, 

the reaction temperature, etc., the reaction's nature and yield may be regulated. The 

physical (e.g., length, shape, diameter) and chemical (e.g., flaws, graphitization) 

features of CNTs can be planned in advance by choosing the appropriate conditions. 

Ethylene (C2H4) or acetylene (C2H2) gas is typically employed as the carbon feedstock 

while Co, Ni, or Fe nanoparticles are used as the catalyst in thermal CVD procedures to 

generate MWNTs (37). 

 Plasma Enhanced Chemical Vapor Deposition  

The selective positioning and vertical alignment of CNTs can be achieved using the 

promising upcoming growth technology known as plasma-enhanced chemical vapor 

deposition (PECVD). Applications that use vertical alignment are crucial. This is 

especially helpful for field emitters, that are currently being evaluated for use in flat 

panel displays. 

According to conventional wisdom, plasma processing is superior to thermal CVD 

because the precursor is dissolved by extremely energetic electrons, allowing the 

substrate temperature to be significantly lower. These techniques make it abundantly 

clear that PECVD is a high yield and controllable method of creating CNTs with 

vertical alignment. 

 Catalytic Pyrolysis of Hydrocarbon  

This process is frequently used to produce CNTs in bulk or in large quantities using 

CVD. The main benefit of utilizing this method is that CNTs can be recovered from the 

substrate without the need for purification. The easiest way is to directly introduce 

catalyst nanoparticles into the CVD chamber, for example, in the form of a 

colloidal/particle suspension or organometallic precursors with a carbon feedstock. 

Precursors for the catalyst are frequently composed of organometallic compounds, such 

as iron pentacarbonyl, metallocenes, and iron (II) phthalocyanine. When heated, these 

precursors often sublime and generate catalyst nanoparticles in the process when the 

chemical is broken down or reduced by heat or hydrogen. Due to the different 

temperatures required for nanotube development and organometallic sublimation, a 

double stage furnace is frequently required. 



 

 11 

 In general, the structural properties of the nanotubes, like length and diameter, can be 

only moderately controlled through the sublimation of metallocenes. However, it has 

been demonstrated that the average diameter of the structures can be altered by altering 

the reactive concentration of the metallocene to carbon in the gas phase. 

The nanotubes are nearly always produced on quartz (SiO2), either as the reactor wall 

or a particular substrate. This technology has the advantage of producing aligned CNT 

bundles in a single step at a low cost without the need to prepare substrates before (37). 

1.4.1.4 Other Synthesis Techniques for Carbon Nanotubes  

Diffusion flame synthesis (39), electrolysis with graphite electrodes submerged in a 

liquid ionic salts (40, 41), graphite ball milling (42), and heat treating a polymer (37) 

are further methods for producing CNTs. 

1.4.2 Properties of CNTs 

One of the most searched carbon nanomaterials by scientists are carbon nanotubes 

(CNTs), because of their unique features (excellent mechanical characteristics plus 

thermal and electrical conductivities) (25, 43). CNTs have a nanoscale size and 

micrometer length, forming a structure like a needle with a large surface area (35), they 

are lightweight with highly porous materials (44). CNTs have exceptional physical 

characteristics, MWCNTs have a tensile strength that can reach 63 GPa, about 50 times 

that of steel . CNTs and their elastic modulus might range from 1.0 to 1.8 terapascals 

(TPa) (45), and have a mean fracture strength of more than 100 gigapascals (GPa). At 

room temperature, MWCNTs' thermal conductivity is measured to be more than 3000 

WK-1m-1 (46).  

Carbon nanotubes possess special electrical characteristics (47),  depending on chirality 

and how the hexagon rings are arranged along the tubular surface, they can either be 

metallic or semiconductors (48). In SWCNT, the graphene layer can be rolled up in a 

variety of ways: perpendicular to the face (zigzag tubes), along the face of the hexagon 

(armchair tubes), and in all intermediate layers (chiral) (figure 1.3) (49).  

 

 

 



 

 12 

Figure 1.3 

graphene sheet folded in three different patterns: a) chair, b ) zigzag, c) chiral (49) 

 

The chirality of nanotubes determines their characteristics including their electronic 

conductivity. An SWCNT's chirality is determined by the chiral vector Ch, Ch = na1 + 

ma2, where a1 and a2 are the lattice vectors of graphene while n and m are the 

graphene's geometrical parameters (scheme 2). for example, When n= m (n, n), 

SWCNT with an armchair structure will have no band gap and always be metallic. 

SWCNT adopts a zigzag chirality with either metallic or semiconductive properties 

when m = 0. If n > m > 0  and n = 3q (q is an integer) metallic chiral-structured 

SWCNT  is also possible (50). 

Scheme 2 

Figure showing the different graphene rolling directions that can produce single-wall carbon 

nanotubes with differing chiralities (50) 

 



 

 13 

In  MWCNTs cylindrical graphene sheets can be described by two structural models: 

the parchment model (as rolled paper, rolled around itself)  and the Russian doll model 

(single-layer nanotubes coaxially nested into one another) (figure 1.4). When any sheet 

has metallic chirality, MWCNT is metallic. Because the majority of the current 

traveling through is restricted to the outermost layer, it has also been shown that the 

electron transport in MWCNTs is comparable to that of the SWCNTs (50). 

Figure 1.4 

Multilayer nanotubes' transverse structure is represented schematically: a) Russian doll, b) 

parchment (49) 

 

1.4.3 Applications of CNTs 

Due to the special characteristics of CNTs, they are extensively studied in a variety of 

fields, including physics, biology, chemistry, medicine, and engineering. For 

electrochemical transduction in biosensors, CNTs have been identified as one of the 

most crucial materials, due to the great surface area that enables signal amplification 

and chemical stability (51). Additionally, CNTs used for food safety, environmental 

monitoring, clinical diagnostics, and drug targeting. They are crucial in identifying 

different pathogenic bacteria and also aid in the treatment of cancer. CNTs even exhibit 

a variety of antibacterial properties (52). Due to the ability to give the scaffold unique 

features and steering cell growth, CNTs are an important component in the tissue 

engineering (53). 

 

 

 



 

 14 

 CNT Use for Diagnostic  

Due to their strong biocompatibility, CNT is being used frequently in biomedical 

applications, whether for disease diagnosis or treatment.  

The development of detection technologies is particularly important because early 

diagnosis is a necessity for effective therapy. Several teams have examined employing 

CNTs as the primary component of electrochemical sensors due to their electrical 

properties, and numerous label-free CNT-based biosensors have been produced as a 

result. CNT can also act as a contrast agent in several bio-imaging methods. When 

functionalized and coupled with different biomarkers, they can effectively and 

accurately identify the presence and location of targeted cells (54). 

 Biosensors Based on CNT  

In the field of biosensors, CNT has been suggested as a sensing component to identify 

and detect a variety of diseases, mainly diabetes and also bacterial infections. In general, 

the presence of CNT at the electrode surface accelerates the transport of electrons and 

increases the sensitivity of the electrochemical detection (55). Recently, resistive 

sensors, and more specifically differential resistive pulse sensors (RPS) based on 

MWCNT, demonstrated their remarkable efficacy in passing the single molecule 

detection threshold (56). 

 Imaging Methods Based on CNT  

There are numerous CNT-based technologies available for use in imaging. In order to 

penetrate further, photoluminescent imaging, for instance, uses excited SWCNT's 

fluorescence in the Near InfraRed-I (700–900 nm) and NIR-II (1100–1400 nm) ranges, 

where tissues and water are practically transparent. This characteristic was employed by 

Welsher et al. to optically monitor in real time the biodistribution of SCWNTs injected 

into deep tissues and arteries of mice (57). 

 Tips for scanning probe 

The usage of scanning probe tips is crucial for obtaining images with higher resolutions. 

When a MWNT-bound scanning probe is employed in place of a standard probe, picture 

resolution can be improved compared to past results. These probes are now available 



 

 15 

commercially by Daiken Chemical Company and Seiko Instruments. Nanotube tips that 

have been chemically modified can be utilized as sensors to identify particular chemical 

and/or biological groups. These sensors are crucial for identifying additional or even 

illicit compounds (58). 

 Targeted Treatments using CNT 

In the medical industry, effective drug administration is a serious challenge. Moreover, 

low selectivity and short half-lives might result in multiple administrations, which can 

have side effects and even be toxic. CNT are extensively researched as nanocarriers for 

drug and gene delivery as well as a cancer treatment because of their expected 

biocompatibility and acceptable size (54). 

 CNTs as fillers  

One of the most advanced fields in nanotechnology is the use of CNTs as fillers in 

various materials to create nano-composites. CNTs have been used as a filler by 

numerous scientists studying nanocomposites. The primary goal of incorporating CNTs 

into various polymeric and other materials is to enhance their properties. The 

mechanical, electrical, and thermal properties are raised to their most optimum level as 

a result.  

According to Soichia et al, adding SWNT loading to a SWNTs/polyimide 

nanocomposite enhanced the material's tensile modulus and yield strength. They 

demonstrated that the mechanical properties of polyimide were improved with a good 

dispersion (59). 

 CNT’s  medical applications  

The science of nanomedicine has developed into the most quickly expanding field of 

study as a result of the rising profits connected with medical technology related to gene 

therapy, cancer treatments, and innovative new therapies for life-threatening diseases. 

Scientists can create new subfields of nanomedicine due to the special 

characteristics and properties of CNTs. SWNTs and MWNTs have already proved their 

potential to act as safer and more effective alternatives to earlier drug delivery systems.  

 



 

 16 

They have the ability to cross membranes and deliver vaccinations, nucleic acids, and 

therapeutic drugs deep inside the cell to their intended substrate sites. They act as the 

best nontoxic carriers, which in some situations increase the drug's solubility for 

improved effectiveness and safety. Overall, CNTs have a very promising future in 

medicine, according to recent studies (58). 

1.4.4 Toxicity of CNTs 

The use of CNTs in the industry has been considered thoroughly because of their 

exceptional physical and chemical capabilities, but there are some drawbacks, 

particularly those linked to their toxicity. To reduce the toxicity of CNTs, specific 

actions must be taken. Incorporating CNTs into polymeric materials is expected to 

change their surface characteristics, which may change their toxicity without 

compromising their unique properties or potential for application in the future. A 

strategy based on the development of inherently safer nanomaterials, however, appears 

to have much more potential. Such methods are important for the further improvement 

and safe application of CNTs, both for professionals and everyday users. It is evident 

from numerous research on the toxicity of CNTs that surface modifications are essential 

for decreasing CNT toxicity. And also coating allows for the alteration of the CNTs' 

external surface environment while preserving their intrinsic structure and essential 

properties, it may be a useful tool (58). 

biological applications have been hampered due to the limited water dispersibility, 

unfunctionalized CNTs (CNTs free of any polar functional groups) tend to agglomerate 

together and precipitate in aqueous solutions rather than disperse in water  (60, 61). 

adding the polar functional groups to CNTs can dramatically increase their dispersibility 

in aqueous conditions while also greatly reducing their toxicity because pristine CNTs 

may induce a number of toxic reactions (62, 63). 

1.4.5 Functionalization of CNTs  

CNTs' poor solubility in most organic solvents and aqueous solutions makes them 

unsuitable for biological applications. As a result, appropriate surface functionalization 

on CNTs can increase their biocompatibility and ability to disperse in water and thus 

reduce their toxicity (64). Carbon nanotube cytotoxicity is primarily determined by their 



 

 17 

surface functionalization, nanotubes with well-functionalized, serum-stable have the 

least harmful and toxic effects. 

For the functionalization of CNTs, there are two basic protocols: covalent and 

noncovalent functionalizations (65). 

1.4.5.1 Covalent functionalization of CNTs 

During chemical reactions, the functional groups are generated, and covalent reactions 

occur between these groups on the π- conjugated carbon nanomaterial skeleton (65) 

(scheme 3). The goal of this functionalization approach is to prevent connected 

biomolecules from being released before they reach the target location, reducing their 

side effects. The high stability of covalently functionalized carbon nanotubes makes 

them ideal for drug delivery . covalent functionalization is providing beneficial 

functional groups on the surface of CNT while maintaining high dispersion stability. 

Conversely, covalent techniques can result in a huge number of defects on the CNT’s 

sidewall as well as the fragmentation of CNTs with missing inherent properties in the 

worst situation layers . the covalent method has been observed to disrupt the π-network, 

resulting in the loss of mechanical and electrical characteristics (66). 

 There have been numerous reports of successful covalent surface functionalization. The 

two general strategies of covalent functionalization that result in biomedical 

applications are nonselective attack of nanotube sidewalls utilising highly reactive 

species like nitrenes; and the formation of amide bonds at ends of nanotubes using 

carboxyl group, which is typically generated on CNTs by oxidation in strong acid(67). 

 

 

 

 

 

 



 

 18 

Scheme 3 

Covalent functionalization of CNTs (43) 

 

 

1.4.5.2 Non-covalent functionalization of CNTs 

The ability to retain the intrinsic features of CNT while giving additional functionality 

makes non-covalent functionalization attractive. Non-covalent methods may aid to 

preserve the conformational structure following immobilization on carbon nanotubes, 

but covalent methods may alter the carbon nanotubes’ sp2 structures, affecting their 

mechanical and electrical properties. In general, the best method for non-covalent CNT 

functionalization should include biocompatible functionalization agents which are 

stable on the CNT surface without detachment in a variety of conditions and have 

appropriate functional moieties for attaching to various biomolecules (50, 68). 

The non-covalent functionalization of CNTs can be done through CH- interaction, van 

der Waals force (π–π stacking), and s electrostatic interaction between CNTs and 

biomolecules (65, 69). Dai and coworkers announced the first general and 

uncomplicated strategy for functionalizing SWCNTs with proteins via π–π stacking 

(scheme 4) (68). π–π stacking occurs between the sidewall of the SWCNTs and the 

pyrenyl group of the bifunctional 1-pyrenebutanoic acid, succinimidyl ester. Then, 

proteins formed an amide bond through the amino group, proteins were immobilized by 

anucleophilic substitution of N-hydroxysuccinimide (36). 



 

 19 

Scheme 4 

Noncovalent adsorption of SWCNTs with 1-pyrenebutanoic acid, succinimidyl ester via π–π 

stacking (68) 

 

In another study, to increase the dispersion of multi-walled carbon nanotubes 

(MWCNTs), utilized functional polyesters, which have a number of functional groups (-

OH, -COOH, and -CCH), to modify MWCNTs by stacking the pyrene functional 

polymer scaffold on top of the aromatic carbon nanotubes via π-π interaction. First, 

create a number of polyesters with alkyne moieties that lack electrons that enable 

Huisgen 1,3-cycloaddition processes to effectively introduce a variety of terminal 

functional groups, including pyrene, hydroxyl, carboxylic, and alkyne. Then, use 

pyrene-functional polyesters to interact non-covalently with the surface of MWCNTs 

via π-π stacking (figure 1.5). Because of the hydrophilic properties of the tetraethylene 

glycol and the terminal hydroxyl groups in the polyester's backbone, functionalized 

MWCNT well disperses in water (70). 

 

 

 

 



 

 20 

Figure 1.5 

structures of pyrene-functional polyesters. b) pyrene-functional polyesters interact non-

covalently with the surface of MWCNTs via π-π stacking (70) 

 

1.6 Chitosan 

chitosan is a natural polyglucosamine (scheme 5), it has the benefits of being not toxic 

and biocompatible. Chitosan has been used in a variety of topical formulations, 

including hydrogels, powders, and dressings, which have all been studied in the wounds 

and burn animal models. Chitosan-containing products can enhance the re-

epithelialization of wounds, collagen deposition, the proliferation of fibroblasts, and in 

general the healing wound's tensile strength (71). 

Scheme 5 

chemical structure of chitosan (72) 

 

When administered chitosan to fresh wounds, it suppresses or reduces the number of 

fibroplasia. Chitosan has been shown to minimize exophytic callus in bone regeneration 

and allow vascular grafts to develop. It allows normal tissue elements to regenerate in 

skin lesions with minimum scar formation (73). Chitosan preparations increased the 



 

 21 

histoarchitectural order and vascularization of the dermis layer, according to clinical 

investigations (74). SWCNTs and MWCNTs complexed with chitosan, via van der 

Waals interactions, so that the hydrophilic parts of the chitosan polymer would be 

exposed to the aqueous environment and wrap around the CNT, a hydrogel with a CNT 

scaffold was created. The addition of polar functional groups to CNTs may dramatically 

improve their dispersibility in aquatic settings and lower their toxicity, as a result, the 

hydrogel enhanced wound healing and re-epithelialization (75). 

1.7 Literature Review 

Several efforts were made to create suitable tissue engineering scaffolds using carbon 

nanomaterials, especially multi-walled carbon nanotubes. There are several reported 

approaches for utilizing MWCNTs to advance tissue engineering, which are presented 

in this literature review. 

In 2011, Eri Hirata et al. prepared composite materials containing a rat primary 

osteoblast (ROBs), which were cultured on an MWCNT-coated collagen sponge 

(MWCNT-coated sponge) for bone tissue engineering, a surface coating treatment using 

MWCNTs was applied to a 3D collagen scaffold. MWCNT-coated sponges had 

considerably greater calcium and osteopontin concentrations than untreated sponges 

after 7 days. On the MWCNT-coated sponge, ROBs began to discriminate before they 

did on the uncoated sponge. Additionally, the tissue response to the MWCNT-coated 

sponge was evaluated. After implantation in subcutaneous tissue at 7 and 28 days, mild 

inflammation was seen surrounding MWCNTs. At 28- and 56-days following 

implantation in the femur, there was much more bone growth around the MWCNT-

coated sponges than around the uncoated sponges, as well as new bone connected to 

MWCNTs directly. Furthermore, at 4 weeks after implantation bone tissues were 

successfully generated in the pores corresponding to the honeycomb structure of the 

MWCNT-coated sponge. As a result, MWCNT coating looks to be useful in the bone 

tissue engineering (76). 

Mikael et al. in 2014 enhanced the mechanical strength of microsphere scaffolds of 

poly-lactic-co-glycolic acid (PLGA) by structuring them with carbon nanotubes,  

allowing them to be used for regeneration and load-bearing repair. They molded pristine 

and modified MWCNTs (with carboxylic acid (COOH) and hydroxyl (OH)) into 3-D 



 

 22 

porous scaffolds to create PLGA microsphere scaffolds. The modulus and strength 

properties of pure PLGA scaffolds were dramatically boosted when only 3% MWCNTs 

were added. In the SEM picture, there was a significant difference in the distribution of 

MWCNTs on the surface of PLGA-MWCNTs scaffolds. While agglomeration was 

observed in all cases, modified MWCNTs had lower size agglomerates than non-

modified MWCNTs, demonstrating the functionalized CNTs' dispersible nature (figure 

1.6). As well cell adhesion and proliferation appeared to be perfect in SEM pictures. 

While tests on osteoblast-like MC3T3-E1 cells in vitro showed high cellular viability, 

proliferation, and mineralization. The in vivo investigation on 28 male rats showed 

differences in inflammatory response over the course of 12 weeks, with OH-MWCNTs 

having the lowest reaction and pristine MWCNTs and COOH-MWCNTs having a more 

prominent response. Overall, the results indicated that PLGA scaffolds including water-

dispersed functionalized MWCNTs were mechanically stronger and had good tissue 

compatibility, indicating that they might be used in the bone tissue engineering (77).   

Figure 1.6 

MWCNT dispersion on the surface of PLGA-MWNT scaffolds as shown by SEM. The 

agglomeration size distribution within the unmodified MWCNTs is shown in red circle (77) 

  

In another paper published a year later, researchers from Stony Brook University's 

Department of Biomedical Engineering reported on the fabrication and 

cytocompatibility of 3-D chemically cross-linked macro-sized porous CNTs scaffolds. 

The radical initiated thermal crosslinking of both SWCNTs and MWCNTs were used to 

create these scaffolds, and after 5 days, MC3T3 cells on SWCNTs and MWCNTs 

scaffolds had cell viability very well comparable to PLGA scaffolds because at day 3 



 

 23 

and 5 there was no significant difference in total LDH release between the PLGA, 

MWCNT, and SWCNT scaffold groups. MC3T3 cells could connect and multiply 

SWCNTs and MWCNT scaffolds, as shown by immunofluorescence imaging and 

confocal live cell imaging. As a result, MC3T3 cells became round on low-surface-

roughness scaffolds (SWCNTs) but elongated on high-surface-roughness scaffolds 

(MWCNTs). These findings showed that crosslinked SWCNTs and MWCNTs scaffolds 

are cytocompatible, opening the way for their development in tissue engineering 

applications (78) 

in 2017, Shayan Gholizadeh et al. used chitosan and -Glycerophosphate with 

functionalized multi-walled carbon nanotubes, and the novel scaffold was prepared and 

characterized. according to in vitro experiments, increased f-MWCNT content improves 

electrical conductivity, but in the case of mechanical properties, The compressive 

strength of the samples was increased by increasing CNT content up to 0.1%, while 

compressive mechanical strength was slightly diminished by increasing CNT content up 

to 0.5%, and significantly degraded in mechanical strength by raising this value to 1%. 

Higher concentrations of CNTs can result in a significant increase in water uptake and 

porosity because they can increase the distances between chitosan chains and possibly 

break the chains' crosslinking (79). One year later, Valentina Martinelli et al. created an 

elastomeric scaffold made of microporous and self-standing polydimethylsiloxane 

(PDMS) material with micrometric voids, and they included multiwall carbon 

nanotubes (MWCNTs) in it. They used microscopy, cell biology, and calcium imaging 

to see if 3D-PDMS+MWCNT cultured neonatal rat ventricular myocytes (NRVMs) 

develop a more viable and mature phenotype than 3D-PDMS control. When compared 

to control PDMS, 3D-PDMS+MWCNT increased good cell proliferation while 

inhibiting cardiac fibroblast proliferation. 3D-PDMS+MWCNT can increase cardiac 

myocyte viability, proliferation, and functional maturation. These characteristics are 

critical in cardiac tissue engineering (80). 

In 2021, Human foreskin fibroblasts (HFF-1) in collagen type I were used to make 

engineered connective tissues (ECTs), which were enriched with 3 different percentages 

of chitosan-coated MWCNTs (C-MWCNT); 0.025, 0.05, and 0.1 %. At whatever 

concentration, the used C-MWCNTs did not interfere with the embedding cells' 

alignment, and C-MWCNTs were distributed uniformly throughout the tissues. In 



 

 24 

comparison to tissues without C-MWCNT, supplementation with 0.025% C-MWCNT 

increased tissue stiffness moderately but significantly. While adding 0.1% C-MWCNT 

to ECTs reduced tissue contraction and improved extensibility, elasticity, and 

biomechanical properties, This could be used for future in vivo connective tissue repair 

applications, such as the dermis layer in the skin, ligaments, and tendons, which demand 

strong tolerance capacities against external stressors. Regardless of the condition, C-

MWCNT supplementation could improve the biophysical properties of ECTs, which 

could be beneficial for connective tissue repair applications (81).  

In another study, the electrospinning method was used to create polyurethane nanofiber 

with varying concentrations of gelatin and SWNT. Gelatin and SWNTs were combined 

to reduce the mean diameter of nanofibrous scaffolds from 210 to 140 nm, which had an 

effect on the initial behavior of the cells. It was discovered that nanofibrous scaffolds' 

hydrophilicity significantly increased after gelatin and SWNTs were added. Gelatin was 

used to modify the scaffold degradation profile. The scaffolds' electrical conductivity 

was significantly improved by the addition of SWNTs. After 7 days of culture, 

biological evaluation by SEM and MTT assay showed that the nanofibrous surface was 

covered by a dense layer of confluent endothelial cells and myocardial myoblast, which 

is essential for cardiovascular tissue engineering. The results demonstrated the potential 

of the manufactured scaffolds for the cardiovascular tissue engineering (82). 

Previous literature has shown that conductive materials, such as carbon nanomaterials 

(83), stimulate the proliferation and differentiation of electrical stimulus-responsive 

cells such as muscle cells (84, 85), neuron cells (86-88), bone cells (89, 90). Carbon 

nanotubes, a conductive material, are used to achieve the functional differentiation of 

transplanted cells in tissue engineering by mimicking the properties of the 

microenvironment, they stimulate stem cells to differentiate into electroactive lineages, 

emphasizing the potential involvement of stem cells in electroactive tissue regeneration 

(e.g. nerve and heart) (91-93).  

Our previous works, started by functionalizing the CNTs non-covalently based on 

pyrene derivatives and studying their suitability as a scaffold for the tissue engineering 

(94). In this work, we will construct a connective tissue based on primary cells extracted 

from mice skin and continue to test collagen hydrogel that is fortified by MWCNTs, 



 

 25 

which are noncovalently functionalization with pyrene moiety and coated with chitosan 

as a scaffold for connective engineering.  

1.8 Aims of the study 

The main aim of this project is to utilize the previously functionalized MWCNTs 

terminated with COOH or amine group in our research group to develop a scaffold for 

connective tissue based on primary cells extracted from mice skin, and decorted with 

chitosan to achieve enhanced electrical conductivity and acceptable biocompatibility as 

shown in figure A.1 (in appendix A). 

1.9 Objectives  

1. Synthesize the pyrene derivatives terminated with carboxyl or amine groups that have been 

previously developed in our group for the effective functionalization of the MWCNTs. 

2. Non-covalently functionalization of the MWCNTs and also decort them with chitosan. 

3. Characterization of the developed nanocomposites 

4. Assess the electrical properties of the formed engineered tissue 

5. Assess the biocompatibility of the engineered tissue. 

  



 

 26 

Chapter Two 

Methodology 

2.1 Reagents and instrumentation 

tetraethylene glycol (TEG) (Catalog # B23990), propargyl bromide (Catalog # 

A16580), EDC (catalog # A10807), 1-Pyrenebutyric acid (Catalog # A17760) and L-

ascorbic acid sodium salt (Catalog # A17759) were obtained from (Alfa Aesar company 

in England). MWCNTs were obtained from (Nanostructured and Amorphous Materials, 

USA). We obtained Sodium azide (NaN) (Catalog # 0E30428) from Germany 

(RiedeldeHaёn Company). Methanol (MeOH), Aceton, dichloromethane (DCM), 

ethanol (EtOH), and isopropyl alcohol were obtained from (Haifa C.S. Company,). 

Diethyl ether (catalog # 38132) and triethylamine (Et3N) (Catalog # 40502L05) were 

obtained from (Merck Millipore). 4-(dimethylamino) pyridine (DMAP) (Catalog # 

1122583)  and anhydrous copper sulfate (Catalog # 451657) were obtained from 

(Sigma-Aldrich, USA). n-hexane (Hex) (Catalog # 2355544800024) and ethyl acetate 

(EtOAc) (Catalog # 2355516100024) solvents were obtained from (Frutarom Company, 

Haifa). While Tetrahydrofuran (THF) solvent (Catalog # 487308) was obtained from 

(Carlo Erba Company, Italy).  

Purification of the products using column chromatography with silica gel (pore size 60 ) 

from the Sigma Aldrich Company. Water bath sonicators were used in the dispersion 

and preparation of functionalized MWCNTs (MRC DC-200H Digital Ultrasonic 

Cleaner) and centrifuges (UNIVERSAL 320, Hettich Zentrifugen, Germany). A rotary 

evaporator (MRC, ROVA-100, laboratory equipment company) was employed to dry 

the solvents. TLC was used to monitor the reactions (DC-Fertigfolien 

ALUGERAM®SIL G/UV254, MACHEREY NAGEL Company, Germany). 

For biological tests utilized Penicillin/streptomycin solution (pen/strep) (REF #03-031-

1B), phosphate-buffered saline (PBS, Ca+2 free), and high glucose Dulbecco's Modified 

Eagle Medium (DMEM) from (Biological Industries, Jerusalem). Fetal Bovine Serum 

(catalog # F7524), DMEM powder (Catalog # 56436C-10L), Trypsin-EDTA solution 

1X (Catalog # 59417C), L-Glutamine solutions (Catalog #G7513), Chitosan powder 

(CAS 912_76_4 ) and bovine skin collagen solution (Catalog # C4243-20ML) were 

obtained from Sigma-Aldrich, USA.  



 

 27 

Pipetting was done using variable Accumax Micropipette (made in the UK), incubating 

the cell line was done in an Esco cell culture CO2 incubator. In the cell viability test, To 

scan the plate, a Unilab Microplate Reader 6000 was employed. Vector Network 

Analyzer (made in Switzerland) was used to measure electrical conductivity, Digital 

microscope, and Leica ICC50 HD used to obtain digital images. 

2.2 Synthesis of pyrene derivatives 

The pyrene-COOH was synthesized according to our previously published work (94). 

Regarding the pyrene-NH2 has been synthesized as follows: 

2.2.1 Tosyl-TEG-Tosyl Synthesis (1) 

 

Tetra ethylene glycol (TEG) (4g, 20.59 mmol), triethylamine (Et3N) ( 9.23ml, 51.25 

mmol) were dissolved in 40 ml Tetrahydrofuran (THF) and stirred for five minutes, the 

reaction then cooled in an ice bath, and tosyl chloride (10 g, 51.25 mmol) was added 

gradually to the mixture. At room temperature and overnight the reaction was stirred. 

The product was diluted by dichloromethane (DCM) (100ml) and 1 M Hydrochloride 

(HCl) (50 ml). 100 ml of DCM was used to wash the aqueous layer. After that, sodium 

sulfate (Na2SO4) was used to dry the collected organic layers, and then evaporated the 

excess solvent under vacuum and obtained a mixture product of (11.3 g). the product 

was purified using silica column chromatography in a 1:1 Hexane/Ethyl acetate to get a 

product that is oily yellow and has a yield of (5.92 g, 24.24 mmol, 52.4%) 

Rf : 0.42 (Hexane/Ethyl acetate (1:1)). 

1
H NMR: (500 MHz, CDCl3): δ7.76 (d, 4H, J = 8.2 Hz, Ts), 7.30 (d, 4H, J = 7.8 Hz, 

Ts), 4.11 (t, 4H, J = 4.6 Hz, 2CH2OTs), 3.63 (t, 4H, J = 4.6 Hz, 2OCH2CH2OTs),  3.54-

3.50 (m, 8H, 4CH2O), 2.40 (s, 6H, 2CH3).  

13
C NMR (125.7 MHz, CDCl3): δ 144.9, 133.0, 129.9, 128.0, 70.7, 70.5, 69.3, 68.7, 

21.6.  

 



 

 28 

2.2.2  N3-TEG-N3 Synthesis (2) 

 

Compound (1) (1.2g , 4.912 mmol) was added to a 100 ml round bottom flask (RBF) 

and was dissolved by 10 ml Ethanol. Then sodium azide (798.5mg, 12.28mmol) was 

added to the solution, the reaction was stirred in reflux at 70 ºC overnight. The product 

was diluted using diethyl ether (100 ml) and saturated sodium chloride (NaCl) (50 ml). 

100 ml of diethyl ether was used to wash the aqueous layer. After that, sodium sulfate 

was used to dry the obtained organic layers (Na2SO4), and the excess solvent was 

evaporated under vacuum. The obtained yield was (0.6195 g, 2.538 mmol). 

Rf: 0.625 (Ethyl acetate/Hexane (2:1)) 

1
H NMR: (500 MHz, CDCl3): δ 3.62-3.60 (m, 8H, 4CH2O), 3.33 (t, 4H, J = 5.0 Hz, 

2OCH2CH2N3), 2.56 (t, 4H, J = 4.5 Hz, 2CH2N3).  

13
C NMR (125.7 MHz, CDCl3): δ70.7, 70.0, 50.7, 40.9. 

2.2.3 OH-TEG-alkyne Synthesis (3) 

 

In 100 ml Round bottom flask, 3 g of TEG (15.4 mmol) was added and dried under 

vacuum and then dissolved in 20 ml of anhydrous THF under argon. A dried solution of 

sodium hydride (NaH) (741 mg, 30.9 mmol) and anhydrous THF (10 ml) were added 

drop by drop to the TEG solution until the release of Hydrogen gas (H2). After 30 min, 

Anhydrous THF (10 ml) was mixed in a dry solution of propargyl bromide (1.9 ml, 21.6 

mmol) and added to the reaction. The reaction was continuously and vigorously stirred 

at room temperature overnight. The following day, drop by drop added of 5 ml of water 

to the reaction, and THF was evaporated. DCM (100 ml) and HCl (50 ml) were used to 

dilute the crude product then it was dried with Na2SO4., filtered and evaporated, the 

crude product was (2.3462g). Silica column chromatography in ethyl acetate was used 

to purify the product to produce a product that is oily yellow and has a yield of (1.2742 

g, 5.49 mmol, 54.31%). 



 

 29 

Rf: 0.3714 (Ethyl acetate). 

1
H NMR(500 MHz, CDCl3): δ 4.24 (d, 2H, J = 2.9 Hz, CH2C), 3.65–3.58 (m, 14H, 

7CH2O), 3.57–3.51 (m, 2H), 2.95 (s, 1H, CH), 1.1 (s, 1H, OH). 
13

C NMR (125.7 MHz, 

CDCl3): δ 92.6, 78.8, 74.5, 72.3, 70.8, 70.7, 70.7, 70.6, 70.3, 69.1, 61.5, 58.4. 

2.2.4 Pyrene-TEG-alkyne Synthesis (4) 

 

Compound (3) (1 g, 4.3 mmol), 620.7 mg of pyrenebutyric acid ( 2.2 mmol), 1.2 g  of  

EDC (6.5 mmol) and 396.2 mg of  4-Dimethylaminopyridine (DMAP) (3.2 mmol) were 

dissolved in 20 ml  DCM and were reacted under argon. For 24 hours, the reaction was 

stirred continuously at room temperature. The resulting product was dried with Na2SO 

after being collected using DCM (170 mL) and 1 M HCL (50 mL), filtered and 

evaporated. the product was purified using silica column chromatography in a 3:2 

mixture of ethyl acetate/n-hexane to produce a product that is oily yellow and has a 

yield of (916 mg, 1.7 mmol, 41.3%). 

Rf: 0.3 (Ethyl acetate/n-Hexane (2:3)). 

1
H NMR: (500 MHz, CDCl3): δ8.29-7.83 (m, 9H,Py), 4.23 (s, 2H, CH2OCO), 4.15 (s, 

2H, OCH2C≡CH), 3.67-3.53 (m, 14H, 7CH2O), 3.37 (t, 2H, J = 7.2 Hz, Py-CH2), 2.47 

(t, 2H, J = 7.2 Hz, CH2COO), 2.39 (s, 1H, C≡CH), 2.20-2.15 (quint, 2H, Py-CH2CH2). 

13
C NMR (125.7 MHz, CDCl3): δ173.5, 135.8,131.4, 130.9, 130.0, 128.7, 127.5, 

127.4, 126.7, 125.9, 125.1, 125.0, 124.9. 124.8, 123.4, 72.5, 70.5, 70.4, 70.2, 69.7, 69.2, 

69.1, 64.6, 63.5, 61.6, 61.5, 61.0, 33.8, 32.7, 29.7, 26.8.  

 

 

 

 



 

 30 

2.2.5 Pyrene-TEG-triazole-TEG-azide Synthesis (5) 

 

compound (4) (350 mg, 0.69mmol) and Compound (2) (200 mg, 0.82 mmol) were 

dissolved in DCM (3ml), and a solution of sodium ascorbate (62.1mg, 0.313mmol), and 

anhydrous copper sulfate (22.23mg, 1.4mmol) in H2O (3ml) was added. The reaction 

was stirred for twenty-four hours at room temperature. The product was first diluted 

with DCM (100 ml) and water (50 ml), dried with Na2SO4, filtered, and evaporated. 

Then the product was purified using silica column chromatography in 20:1 mixture of 

DCM/MeOH, to produce a product that is oily yellow and has a yield of (192mg, 

0.25mmol. 54%). 

Rf: 0.33 (DCM/MeOH (20:1)). 

1
H NMR: (500 MHz, CDCl3): δ 8.27 (d, 1H, J = 9.2 Hz, Py), 8.13 (dd, 2H, J = 6.9 Hz, 

J = 5.5 Hz, Py), 8.08 (dd, 2H, J = 3.7 Hz, J = 2.3 Hz, Py), 8.00-7.93 (m, 3H. Py), 7.83 

(d, 1H, J = 7.8 Hz, Py), 7.74 (bs, 1H, CH triazole),  4.62 (s, 2H, CH2-triazole),4.46 (t, 

2H, J = 5.0 Hz, CH2OCO), 4.21 (t, 2H, J = 4.8 Hz, CH2N-triazole), 3.79 (t, 2H, J = 5.0 

Hz, CH2CH2N-triazole), 3.65-3.53 (m,26H, 13CH2O),3.36-3.30(m, 4H, CH2CO, 

CH2N3), 2.45 (t, 2H, J = 7.3 Hz, Py-CH2), 2.15 (quint, 2H, Py-CH2CH2). 

13
C NMR (125.7 MHz, CDCl3): δ173.5, 135.8, 131.4, 130.9, 130.0, 128.7, 127.5, 

127.4, 126.7, 125.9, 125.0, 124.9, 124.8, 123.4, 70.7, 70.6, 70.5, 70.0, 69.7, 69.4, 69.2, 

64.5, 63.5, 50.7, 33.8, 32.7, 26.8.  

2.2.6 pyrene-TEG-triazole-TEG-amine Synthesis (6) 

 

Compound (5)(90 mg, 0.118 mmol) and palladium on carbon (9 mg) (as a catalyst) 

were dissolved in 5 ml methanol under hydrogen. For twenty-four hours and at room 

temperature, the reaction was stirred. The product was extracted by 100 ml of DCM, 



 

 31 

3ml of methanol, and evaporated, before being filtered and rewashed with Mmethanol 

then re-evaporated. The obtained yield was 93 mg. 

1
H NMR: (500 MHz, CDCl3): δ8.36 (d, 1H, J = 11.0 Hz, Py), 8.24-7.96 (m, 8H, Py), 

7.90 (s, 1H, CH triazole),4.47-4.44 (m, 4H, CH2-triazole, NH2),4.31 (t, 2H, J = 4.6 Hz, 

CH2OCO), 4.11 (t, 2H, J = 5.0 Hz, CH2N-triazole), 3.78-3.73 (m, 2H, CH2CH2N-

triazole), 3.55-3.35 (m, 30H, 13CH2O, CH2CO, CH2NH2),  2.44-2.42 (m, 2H, Py-CH2), 

2.12 (quint, 2H, Py-CH2CH2). 

13
C NMR (125.7 MHz, CDCl3): δ173.3, 144.3, 136.7, 131.4, 130.9, 129.9, 128.6, 

127.9, 127.8, 127.0, 126.6, 125.4, 125.3, 124.7, 124.63, 123.9, 72.8, 70.2, 70.1, 70.0, 

69.4, 69.2, 68.8, 64.0, 63.6, 60.7, 56.5, 49.8, 33.6, 33.4, 32.3, 29.5, 27.3, 27.2. 

2.2.7 OH-TEG-Tosyl synthesis  (7) 

 

The compound was synthesized and identified as in the literature (95) . 

2.2.8 OH-TEG-N3 Synthesis (8) 

 

The compound was synthesized and identified as in the literature (96). 

2.2.9 COOH-TEG-N3 synthesis  (9) 

 

The compound was synthesized and identified as in the literature (96). 

 

 

 



 

 32 

2.2.10 Pyrene-TEG-triazole-TEG-COOH Synthesis (10) 

 

The compound was synthesized and identified as in the literature (97). 

2.3 Optimizing the needed amount of compound (6) or (10) to functionalize  

MWCNTs in water 

Compounds (6) or (10) in varying concentrations (0.25, 0.5, and 1 mg) were dissolved 

in 1 mL of water before being mixed with 1 mL of MWCNTs and sonicated for 30 

minutes. The various dispersions were then kept under observation so that the best 

concentration could be determined for dispersing carbon nanomaterials in water without 

precipitation or an excess of micellar structures visible in TEM images. 

2.4 Functionalization of MWCNTs with Py-COOH and Py-NH2 

 



 

 33 

Optimum amount of Py-COOH and Py-NH2 were dissolved in H2O (1 mL) then 1 mL 

of MWCNTs were added up to the solution and the mixture was sonicated for 30 

minutes. The products were then dried by a freeze-dryer. 

2.5 Atomic force microscope characterization  

The morphology and diameters of the unfunctionalized and functionalized multiwalled 

carbon nanotubes were determined using an atomic force microscope (CoreAFM, 

nanosurf, Switzerland) using the tape mode and the cantilever (Dyn190Al with 

Resonant frequency = 190 kHz). 

2.6 Primary dermal fibroblast culture and maintenance 

 Preparation of fibroblast growth medium with 20% FBS 

A bottle of 500 ml of high glucose DMEM was supplied with 100 ml fetal bovine serum 

(FBS), 5ml penicillin/streptomycin, and 5ml L- glutamine. 

 Isolation and culturing of primary dermal neonatal mouse fibroblasts 

Neonatal mice were euthanized by cervical dislocation. After being sterilized with 70% 

ethyl alcohol followed by a povidone-iodine solution, the limbs and the tails of the mice 

were cut off before the skin was taken off. The skin was then washed twice with PBS 

under a sterile laminar flow. After that, the skin was transferred to a 100 mm petri dish 

and by using surgical scissors it was cut into small pieces of about 2-3 mm
2
. Then, the 

tissue pieces were transferred to a flacon tube containing 5ml 0.25%trypsin solution and 

were incubated at 37
o
C for 15 minutes. Every 5 minutes the falcon tubes were shaken 

gently and 5 ml of 0.25% fresh trypsin solution was added to each one. After that, a 

sufficient fresh culture medium containing 20% FCS was added to each tube to 

inactivate the trypsin. Then the tissue pieces were transferred to 6-well plates with the 

dermis layer facing downwards. The tissues were left to partially dry under the laminar 

flow until they stuck to the plastic surface, then approximately 700 microliters medium 

were added carefully.  



 

 34 

The tissue culture plates were incubator at 37°C with 5% CO2 to allow the outgrowth of 

the cells from the tissue pieces. Every other day the medium was changed until the cells 

were about 90% confluent. 

2.7 Generation of Engineered Connective Tissue (ECT) with/without 

functionalized MWCNT  

2.7.1 Double-strength cell culture media (2x DMEM) preparation 

To create a medium with double strength, 267. 2 mg DMEM powder (13.36g/L), 74 mg 

of NaHCO3 (3.7g/L), and 2 ml of FBS were added to 18 ml of the ready-to-use1x 

DMEM. The mixture was stirred for 30 minutes before sterile filtering with a 0.22-

micrometer syringe filter. 

2.7.2 Preparation of 0.1% NaOH 

Forty mg of sodium hydroxide (NaOH) was dissolved in 10 mL of distilled water  and 

was then filtered through a 0.22-micrometer syringe filter. 

2.7.3 Preparation of 1.5 % (W/V) chitosan 

By combining 20 ml of 1% (v/v) acetic acid with 300 mg of chitosan. The mixture was 

sonicated for 1 hour to completely dissolve chitosan. 

2.7.4 Primary cell suspension preparation 

First, the cells were washed twice with Ca
+2

-free PBS after the old medium was 

removed. After that, 0.05 percent trypsin was added to the cells just enough to cover 

them, and the plate was left in the cell culture incubator at 37 °C and 5% CO2 for up to 

5 minutes. The cells were examined under the microscope continually until most cells 

detached from the flask. A sufficient amount of DMEM growth medium (containing 

20% FCS) was added to inactivate the trypsin. The cell suspension was collected in a 50 

ml falcon tube, and the cell concentration was determined by a cell counting chamber. 

The cells were then centrifuged at 200 RCF for 7 minutes at 4
o
C. The supernatant was 

removed and the cell pellet was resuspended in a fresh growth DMEM medium to 

obtain the desired cell concentration (7.510
6
 cell/ml). 



 

 35 

2.7.5 Preparation of MWCNTs-NH2 and MWCNTs-COOH Stock solutions 

Fifty mg of each f-MWCNTs species was mixed with 1200 microliters of PBS to obtain 

a 4% (w/v) of f-MWCNTs suspension. The mixture was sonicated for at least 120 min 

to enhance the dispersion of f-MWCNTs. After that, the suspension was autoclaved in a 

humidified environment. 

2.7.6  Preparation of engineered connective tissues (ECTs) 

The ECTs were formulated according to a method described by Kittana et al., 2021(81). 

Table 1 presents the formula of a single ECT (about 255 μL volume) without the 

addition of any type of f-MWCNTs.  

All of the used materials in this experiment were maintained at 4°C and kept cold 

during the experiments. Here the ECTs were prepared by mixing collagen (3 mg/mL), 

2x DMEM, and PBS. The pH was then titrated to neutralization with 0.1 N NaOH until 

the liquid became pink in color. The cell suspension (containing the required cell 

number per ECT) was then added to the Eppendorf, and the mixture was pipetted 

immediately in the form of a ring around a pair of flexible poles fitted in a special 

model of 48-well mold plate (Myriamed, Göttingen, Germany) (figure A.2 in appendix 

A). The plate was then transferred to the incubator (37°C, 5% CO2) for 45 minutes. A 

fresh DMEM growth medium was added at this step, and the plate was placed in the 

incubator for 5 days, with the medium being changed every other day. The formula was 

scaled up based on the number of ECTs to be prepared. 

Table 1  

ECT general composition 

Constituent Volume (μL) 

Collagen  100 

2x DMEM 100 

PBS 6.4 

NaOH (0.1 N) ~ 5 

Cell suspension (7.5 x 10
6 

cell/mL). 

44 

Total ~ 255.4 

 

 

 



 

 36 

2.7.6.1 Preparation of f-MWCNTs containing ECT 

 ECT containing F-MWCNT  

The various ECTs containing different types of f-MWCNTs were prepared using the 

same methodology as before, but instead of PBS, we used an equivalent volume of a 

stock solution containing enough CNTs to obtain the target total concentration in the 

ECT master mix. Tables 2, 3, and 4 contain the formula for a single ECT containing 

0.025%,0.05%, and 0.1% f-MWCNT (either MWCNT-NH2 or MWCNT-COOH), And 

the formula was scaled up based on the number of ECTs to be prepared. 

Table 2  

ECT contains 0.025% f-MWCNT general composition 

Constituent Volume (μL) 

Collagen  100 

2x DMEM 100 

4% f-MWCNT 1.6 

PBS 4.8 

NaOH (0.1 N) ~ 5 

Cell suspension (7.5 x 10
6 
cell/mL). 44 

Total ~ 255.4 

Table 3 

ECT contains 0.05% f-MWCNT general composition 

Constituent Volume (μL) 

Collagen  100 

2x DMEM 100 

4% f-MWCNT 3.06 

PBS 3.34 

NaOH (0.1 N) ~ 5 

Cell suspension (7.5 x 10
6 
cell/mL). 44 

Total ~ 255.4 

 

 

 

 



 

 37 

Table 4 

ECT contains 0.1% f-MWCNT general composition 

Constituent Volume (μL) 

Collagen  100 

2x DMEM 100 

4% f-MWCNT 6.4 

NaOH (0.1 N) ~ 5 

Cell suspension (7.5 x 10
6 

cell/mL). 

44 

Total ~ 255.4 
 

2.7.6.2 preparation of chitosan and f-MWCNTs containing ECT 

 Chitosan containing ECT (control) 

The ECTs were prepared as described earlier, except that chitosan solution was included 

in the formula at the expense of the collagen proportion (Table 5).  

Table 5  

General composition of chitosan-containing ECT 

Constituent Volume (μL) 

Collagen  50 

Chitosan 50 

2x DMEM 100 

NaOH (0.1 N) ~ 50 

Cell suspension (7.5 x 10
6 

cell/mL). 

33 

Total ~ 283 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

 38 

 Chitosan and 0.025% MWCNT-COOH containing ECT  

Table 6  

chitosan and 0.025%MWCNT-COOH containing ECT 

Constituent Volume (μL) 

Collagen  50 

Chitosan 50 

2x DMEM 100 

4% MWCNT-COOH 1.83 

NaOH (0.1 N) ~ 50 

Cell suspension (7.5 x 10
6 

cell/mL). 

33 

Total ~ 284 

 

 

2.8 Characterization of Engineered Connective tissues 

2.8.1 Electrical conductivity of ECTs 

After DMEM growth medium removal, PBS was used to wash the tissue samples, then 

the electrical conductivity was measured by Vector Network Analyzer which provides 

high-precision measurements of dielectric parameters (conductivity, permittivity, loss 

tangent) over an extremely broad frequency range from 200 MHz to 14 GHz, the probe 

is coupled to a vector network analyzer (VNA) to measure the complex reflection 

coefficient at the probe end. 

2.8.2 Cell viability test (MTS assay) 

2.8.2.1  Preparation of the treatment solution for MTS assay 

Medium samples containing different concentrations of the tested compounds were 

prepared to use for MTS assay, preparation of medium solutions containing 0.025%, 

0.05%, and 0.1% MWCNT-COOH, Chitosan solution (add 300 μL chitosan to 1100 μL 

medium and neutralized the mixture with approximately 360 μL NaOH), 0.025% 

MWCNTs with Chitosan solutions, and 0.025%,0.05% and 0.1% Pyrene-COOH 

solutions.  

 

 



 

 39 

2.8.2.2 Cell culture 

Primary fibroblasts were cultured in DMEM growth media and incubated at 37˚c with 

5% CO2. The cells were collected by trypsinization as mentioned above when they were 

around 80-90 percent confluent. The concentration of the cell suspension was adjusted 

to 100,000 cells/mL. A 96-well plate was then filled with the cell suspension, as 100 

microliters per well, so that each well receives 10,000 cells. Some wells did not receive 

cells to serve as blank samples. The cells were left to adhere and adapt for 48 hours 

before starting the treatment. 

2.8.2.3  MTS assay 

After primary fibroblast cells were sub-cultured and left for 48 hours to adhere and 

accommodate in a 96-well plate, the DMEM growth medium was replaced by 100 uL of 

a treatment solution. Control wells and blank wells received fresh DMEM growth 

medium. The cells were incubated for 24 hours with the treatment conditions. After 

that, the treatment conditions were replaced by a 100 uL fresh DMEM growth medium 

containing 10% MTS reagent, and the plate was put in the incubator at 37 °C and 5% 

CO2. After one hour, a microplate reader was used to measure the absorbance at 492 

nm. 

  



 

 40 

Chapter Three 

Results and Discussion 

3.1 Synthesis and functionalization of carbon nanomaterials 

This project aimed to non-covalently functionalize carbon nanotubes such that the 

electrical properties of the carbon nanostructure can be preserved to generate an 

electrically conductive scaffold for tissue engineering. We achieved this by using the 

pyrene moiety to interact with the MWCNT surface via π-π stacking. In addition, the 

pyrene moiety was linked to a hydrophilic linker to provide an appropriate 

hydrophobic/hydrophilic ratio capable of dispersing carbon nanotubes in water. 

We synthesized two hydrophilic linkers COOH-TEG-N3 and NH2-TEG-N3 which were 

reacted with Py-TEG-alkyne through click reaction. Forming Py-TEG-triazole-TEG-

COOH or Py-TEG-triazole-TEG-NH2. We choose two hydrophilic linkers that 

terminated with a carboxyl or amine group to study if this terminated group has an 

effect on the electrical property or in the formation of the constructed tissue. The 

synthesis began by reacting the tetraethylene glycol with propargyl bromide to form the 

terminal alkyne linker which reacted with pyrene butyric acid to from pyrene terminal 

alkyne as shown in Scheme 6. 

  



 

 41 

Scheme 6 

The synthesized OH-TEG-alkyne linker (1) was reacted with pyrene butyric acid to obtain     

Py-TEG-alkyne (2) 

 

After that, two hydrophilic derivatives of tetraethylene glycol were synthesized to react 

with the pyrene moiety to have a terminal cabroxyl or amine group. In order to 

synthesize the TEG-COOH, The tetraethylene glycol was reacted with 1 equivalent of 

tosyl chloride followed by the conversion to azide by reacting with sodium azide. Then 

a final oxidation step using Jones reagent to form compound 5 (COOH-TEG-N3). 

Finally, compound 5 was reacted with pyrene-alkyne 2 using click reaction to obtain the 

final product compound 8 as shown in scheme 6. 

To synthesize the terminal amine group, the tetraethylene glycol was reacted with two 

equivalents of tosyl chloride to obtain the ditosylated linker which was converted to 

diazide using two equiavlents of sodium azide to synthesize compound 7. After that 

compound 7 was reacted with pyrene-alkyne 2 using a click reaction followed by a 

reduction reaction to obtain the final compound 9 as shown in Scheme 7.  

 

 

 

 



 

 42 

Scheme 7 

Synthesis of Py-TEG-triazole-TEG-COOH (8) and Py-TEG-triazole-TEG-NH2 (9) 

 

After the successful synthesis of the two pyrene derivatives, they were utilized to 

functionalize the MWCNTs non-covalently. It has been found that the needed amount 

from compound 8 or 9 to disperse the MWCNTs is 0.5 mg/ml where the pristine 

MWCNTs form aggregates and precipitate immediately whereas the functionalized 

MWCNTs with py-COOH or Py-NH2 showed good water dispersibility and stability as 

shown in figure 3.1.  

After the dispersion and the functionalization of the MWCNTs, an atomic force 

microscope (AFM) was conducted to evaluate the morphology and dispersibility of the 

MWCNTs. As can be observed in figure 3.2, the pristine MWCNTs showed the 

formation of a huge mesh of carbon nanotubes due to the hydrophobic interaction 

between the walls of the nanotubes. However, upon the non-covalent functionalization 

with the pyrene derivatives the carbon nanotubes become dispersed in water due to the 



 

 43 

introduction of the hydrophilic groups as can observe in Figure 3.2 B and C, we noticed 

the separation of the nanotubes and we can see single multiwalled carbon nanotubes 

with diameter ranges between 20 and 30 nm. These images confirm the successful 

functionalization of the carbon nanotubes with the successful separation of the 

nanotubes.  

Figure 3.1 

Image of dispersion of A) pristine MWCNTs; B) MWCNTs-Py-COOH and (C) MWCNTs-Py-

NH2 

 

 

Figure 3.2 

A) pristine MWCNTs, B) f-MWCNTs with py-COOH, C) f-MWCNTs with py-NH2 

 

 

 

 



 

 44 

3.2 Casting and characterization of connective tissues 

3.2.1 ECTs Generation with primary dermal fibroblast 

With varying kinds and concentrations of f-MWCNTs, ECTs including primary dermal 

fibroblast cells were created (figure 3.3). The control tissues were whitish, opaque, and 

cohesive ring around stretchers, As expected, the addition of f-MWCNTs changed the 

tissue’s color into dark gray-black, depending on the concentration of f-MWCNTs. 

Over 5 days of incubation, the tissues condensed in the form of a ring and contracted the 

stretchers. At this point, the tissues were mature enough to undergo the conductivity 

test. 

Figure 3.3 

primary fibroblast-containing ECT with varying MWCNTs loading: A) Control; B) MWCNTs-

Py-COOH (0.025%); C) MWCNTs-Py-COOH (0.05%); D) MWCNTs-Py-COOH (0.10%); E) 

MWCNTs-Py-NH2 (0.025%); F) MWCNTs-Py-NH2 (0.05%) and G) MWCNTs-Py-NH2 

(0.10%) 

 

 
 

3.2.2 Electrical conductivity test of ECTs 

Our group previously reported that the development of ECT using chitosan-

functionalized CNTs improves wound healing and re-epithelization(75). Another study 

by our group showed that adding CNTs to ECTs with collagen matrix could improve 

several of their biomechanical characteristics, including tissue stiffness, elasticity, 

toughness, and extensibility(81). However, the electrical conductivity of ECT is very 



 

 45 

important for effective application in tissue engineering, electrical transfer through the 

tissue improves tissue repair and stimulates growth and cellular activity by enhancing 

cell differentiations (91-93).  

The extracellular matrix in natural tissues serves as a 3D environment for tissue 

development, and human tissues are more complex in structures than monolayers of 

cells. It is now appreciated that the 3D environment demonstrates unique effects on cell 

behavior as compared to 2D environments, including cell migration, proliferation, and 

differentiation. 3D biomaterials were required to simulate the native extracellular 

matrix. Hydrogels and porous scaffolds are now the most studied 3D biomaterials forms 

(83). In this study, prepared 3D chemically cross-linked macro-sized porous f-

MWCNTs with chitosan scaffolds. the formation of a three-dimensional network of f-

MWCNTs within the collagen matrix was linked to a significant increase in tissue 

electrical conductivity that was kind and concentration-dependent, as in MWCNT-

COOH and MWCNT-NH2 the conductivity was increased when increasing the 

concentration of f-MWCNTs, (scheme 8). 

Scheme 8 

Average electrical conductivity of ECT structures of primary dermal fibroblast cells with 

different A)MWCNT-COOH loading and B)MWCNT-NH2 loading. The symbol (*) indicates 

significance (P ≤ 0.05) compared to the control (0.000%). The two-way ANOVA was used to 

determine the statistical significance 

 

 



 

 46 

As expected, data showed that enriching tissues with f-MWCNTs significantly raised 

the electrical conductivity of the ECTs as compared to the control (collagen-only ECTs) 

which is consistent with a previous report by our lab (98). Conductivity enhancement 

was greater with MWCNTs- COOH compared to MWCNTs-NH2, as the conductivity 

of ECT with 0.1% MWCNT-COOH loading is approximately 9 s/m, but the 

conductivity with 0.1% MWCNT-NH2 loading is between 2-3s/m, therefore we opt to 

proceed with the MWCNT-COOH for the next experiments and to use the lowest tested 

concentration (0.025%). 

The concentration of 0.025% of MWCNT-COOH was enough to sufficiently enhance 

the electrical conductivity of the tissue compared to the control tissue as reported in 

previous literature, however, this concentration was still associated with some 

cytotoxicity (98),  which could arise from the uptake of CNTs by the cells and the 

associated oxidative stress reactions (99), therefore we decided to incorporate a 

biocompatible polymer that can coat the CNTs, enhance their distribution and sticking 

in the ECM, and for that we selected chitosan and tested its effect first of the tissue 

conductivity.   

Scheme 9 

Average electrical conductivity of ECT structures of primary dermal fibroblast cells with 

different Chitosan and 0.025% MWCNT-COOH loading. The symbol (*) indicates significance 

(P ≤ 0.05) compared to the control (0.000%). The two-way ANOVA was used to determine the 

statistical significance 

 



 

 47 

Interestingly, the data demonstrated that chitosan not only did not decrease but instead 

enhanced the conductivity of 0.025% MWCNTs-COOH (Scheme 9). Therefore, we 

tested its effect on cell viability in the next step.  

3.2.3 Cell viability test (MTS assay) 

Primary dermal fibroblast cells were incubated in monolayer and cultures for 24 hours 

in culture media containing several treatment conditions at doses equivalent to those 

used in ECT. The viability of primary dermal fibroblast cells was investigated through 

subjective visual microscopic evaluation concerning cell morphology and by MTS 

assay. The control cells and those treated with 0.025% of MWCNT-COOH or 0.025% 

MWCNTs with Chitosan loading appeared normal, spread, and well-adhered to the 

plastic surface, as shown in (figure 3.4). However, higher MWCNT-COOH 

concentrations, caused some of the cells' morphologies to be abnormal and the cells to 

appear less spread out over the plastic surface. Such variations appeared to depend on 

concentration. 

Figure 3.4 

Bright-field images at 100× for primary dermal fibroblast cells treated with A) Control (cells 

without treatment); B cells treated with Chitosan, C) 0.025%MWCNT-COOH with Chitosan,  

D-F) different concentrations of MWCNT-COOH (0.025%, 0.050%, and 0.100%) respectively 

 
 

 

 

 



 

 48 

Scheme 10 

Concentration-dependent effect of MWCNTs and Chitosan on the viability of primary dermal 

fibroblast cells over 24 hours. The symbol (*) indicates significance (P ≤ 0.05) compared to the 

control (0.000%). The two-way ANOVA was used to determine the statistical significance 

 

The results of the MTS analysis (Scheme 10) showed that there was a statistically 

significant concentration-dependent reduction in the viability of primary dermal 

fibroblast cells with MWCNT-COOH compared to the control cells, the concentrations 

of 0.025% of MWCNTs reduced the cell viability by around (20-30)%. While there was 

no significant reduction in viability when adding Chitosan to 0.025%MWCNT-COOH, 

which indicates that chitosan might have a cytoprotective effect against CNT-induced 

toxicity. Therefore, it can be concluded that the addition of chitosan to the matrix of the 

ECTs can provide two advantages; a cytoprotective effect and enhancement of electrical 

conductivity. 

  



 

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Chapter Four 

Discussions and Conclusions 

4.1 Conclusion 

MWCNTs have been successfully functionalized noncovalently, and a good 

dispersibility of functionalization was demonstrated. The pristine MWCNTs form 

aggregates and precipitate immediately whereas the functionalized MWCNTs with py-

COOH or Py-NH2 showed good water dispersibility and stability. The successful π-π 

stacking between the carbon nanotubes and the pyrene moieties was confirmed by 

atomic force microscope (AFM). The successful functionalization of the carbon 

nanotubes with the successful separation of the nanotubes.  

Primary dermal fibroblast cells extracted from mice skin and ECTs including these 

primary cells were created, collagen hydrogel that is fortified by MWCNTs which are 

noncovalently functionalization with pyrene moiety and coated with chitosan was tested 

as a