nanodiamond-polymer composites for regenerative medicinezceeg99/project/1.pdfd ire ctio n al e q u...

Nanodiamond-polymer composites for regenerative medicine

This report proves that advances in materials technology now enable a new gen-

eration of biodegradable nerve conduits with high electrical conductivity to be de-

veloped to vastly improve treatments in neuronal injuries. Peripheral nerve trauma

is a common injury to which existing surgical techniques, harvesting donor tissue,

is susceptible to problems such as sensory loss. Building on the previous use of

UCL-NanoBio as a strong base biomaterial, a layer of nanodiamond coating would

mimic a protein coating. The conduit could be made to conduct by inserting small

amounts of nano-onions, a form of carbon. The biodegradable polymer would be

complemented by using bioprocessable species of carbon leaving only the recovered

tissue in situ. Detailing how these materials will be produced, combined, and ana-

lytically studied shows this project is viable. This new device will be of important

consequence and may provide a standard for future bioengineered treatments.

Contents

I. Introduction 2

II. Background 2

III. Aims and Objectives 5

IV. Impact 6

V. Approach 6

VI. Conclusions 7

References 7

Glossary of analytical terms 11

Nanodiamond-polymer composites for regenerative medicine Joe Smith

I. INTRODUCTION

This document has been written to examine the validity and impact of building a new

tubular device for neuronal repair, termed a conduit. This will differ from current con-

duits by harnessing the latest developments in nanotechnology to add biodegradability and

conductivity to a strong polymer. First the basic biology of the neuron shall be reviewed,

problems with current medical techniques shown, and evidence given as to why these forms

of carbon should be effective in a composite nerve conduit.

II. BACKGROUND

A neuron is a cell that transmits electrical signals. It can be thought of as an analogue to

digital converter. The cell takes in multiple inputs from branched connections or dendrites

attached to it and sends a pulse response along its long tail or axon (see Fig 1). Neurons

form a network through the body termed the peripheral nervous system, distinguished from

the central nervous system of the brain and spine. Damage to the peripheral nerve system,

caused when the tissue is crushed, stretched, or lacerated due to sharp objects [1], accounts

for 3% of all trauma injuries [2].

After trauma occurs, the nerve attempts reparation. First, the stump of the axon furthest

from the injury degenerates [3]. The near stump sends axonal sprouts. Schwann cells

are generated and align to form columns, termed Bands of Bungner, which direct axonal

regeneration towards the target nerve end as shown in Fig 2 [4, 5].

Injuries beyond the regenerative capabilities of the tissue must be surgically operated on.

The established technique is to harvest donor nerve tissue from another part of the body to

bridge between two nerve ends [1, 6–8]. Several difficulties can occur here. Donor tissue may

be the wrong diameter or of insufficient length. Surgery from harvesting tissue introduces

disease termed donor site morbidity [1, 6, 7] resulting in permanent loss of feeling in almost

all cases [9].

Using a synthetic conduit is a viable alternative. A conduit approximates nerve stumps

by constraining regeneration along its cylindrical shape. Bioengineers design conduits that

are as architecturally similar to the injured nerve as possible [1]. This project explores novel

ways of achieving this in the development of a new conduit.

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The base polymer for the conduit to has been developed at UCL and is trademarked

UCL-NanoBio [10]. It retains mechanical strength in biological surroundings [11] yet is

biodegradable hence removal surgery to prevent scar tissue forming, compressing the nerve,

is not required [12].

Nanodiamond (ND) will be added to the conduit. These are nanometre size pieces of

diamond. Research shows ND provides an excellent growth medium when added as a coating

of individual particles [13] suitably replacing the Extra Cellular Matrix proteins found in

real neural tissue (see Fig 3). ND is biocompatible [14] and bioprocessable. Nanoparticles

with a diameter of less than 6nm leave the body quickly through the kidneys [15].

Concentric rings of C60 (see Fig 5), termed the nano-onion, can be made from ND [16]

and other notable methods [17, 18]. Adding small quantities of this molecule to UCL-

NanoBio should vastly improve conductivity through percolation [19](see [20] on percolation

theory). The function of neurons as biological electronic components evidently suggests

optimal generation in an electronically conductive environment. This has been verified

experimentally by [21]. Nano-onions are nontoxic in a cellular environment [22] and crucially,

their sub 5nm size makes them processable like ND.

(Figure 3.16b), bone, cartilage, and adipose tissue. Muscle tissue provides movement for thebody through its specialized cells that can shorten in response to stimulation and then returnto their uncontracted state. Figure 3.16c shows the three types of muscle tissue: skeletal(attached to bones), smooth (found in the walls of blood vessels), and cardiac (found onlyin the heart). Nervous tissue consists of neurons (Figure 3.16d) that conduct electricalimpulses and glial cells that protect, support, and nourish neurons.

3.4 MAJOR ORGAN SYSTEMS

Combinations of tissues that perform complex tasks are called organs, and organs thatfunction together form organ systems. The human body has 11 major organ systems: integ-umentary, endocrine, lymphatic, digestive, urinary, reproductive, circulatory, respiratory,nervous, skeletal, and muscular. The integumentary system (skin, hair, nails, and various

CARDIAC

SKELETAL

SMOOTH

DENDRITES

AXON

NODE OFRANVIER

CELL BODY

NUCLEUS

PRESYNAPTICTERMINALS

(c) (d)

(a)(b)

HAIR SHAFT

SWEATGLAND

FAT

HAIRFOLLICLE

EPIDERMIS

DERMIS

SEBACEOUSGLAND

ARRECTORPILI MUSCLE

REDBLOODCELLS

WHITEBLOODCELLS

FIGURE 3.16 Four tissue types. Skin (a) is a type of epithelial tissue that helps protect the body. Blood (b) is aspecialized connective tissue. The three types of muscle tissue (c) are cardiac, skeletal, and smooth. Motor neurons(d) are a type of nervous tissue that conducts electrical impulses from the central nervous system to effector organssuch as muscles.

94 3. ANATOMY AND PHYSIOLOGY

FIG. 1: The neuron, taken from [23]

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Injury

Schwann cells Macrophage Monocycle

Nerve cellbody

Attachedmuscle

FIG. 2: Schwann cells forming Bands of Bungner to repair a damaged peripheral nerve, adapted

from [12]

FIG. 3: Neurons grown show similar results with ND as a protein coating (LN/p-ORN) compared

to no coating. Taken from[13]

FIG. 4: Schwann cells grown on UCL-NanoBio taken from [6]

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invented the geodesic dome; each C60 is simply a molecular replica of such a dome,which is often referred to as ‘‘buckyball’’ for short. The term fullerene is used todenote the class of materials that are composed of this type of molecule.

Diamond and graphite are what may be termed network solids, in that all ofthe carbon atoms form primary bonds with adjacent atoms throughout the entiretyof the solid. By way of contrast, the carbon atoms in buckminsterfullerene bondtogether so as to form these spherical molecules. In the solid state, the C60 unitsform a crystalline structure and pack together in a face-centered cubic array.

As a pure crystalline solid, this material is electrically insulating. However, withproper impurity additions, it can be made highly conductive and semiconductive.As a final note, molecular shapes other than the ball clusters recently have beendiscovered; these include nanoscale tubular and polyhedral structures. It is antici-pated that, with further developments, the fullerenes will become technologicallyimportant materials.

3.14 LINEAR AND PLANAR ATOMIC DENSITIES

The two previous sections discussed the equivalency of nonparallel crystallographicdirections and planes. Directional equivalency is related to the atomic linear densityin the sense that equivalent directions have identical linear densities. The directionvector is positioned so as to pass through atom centers, and the fraction of linelength intersected by these atoms is equal to the linear density.

Correspondingly, crystallographic planes that are equivalent have the sameatomic planar density. The plane of interest is positioned so as to pass through atomcenters. And planar density is simply the fraction of total crystallographic planearea that is occupied by atoms (represented as circles). It should be noted that theconcepts of linear and planar densities are one- and two-dimensional analogs ofthe atomic packing factor; their determinations are illustrated in the following twoexample problems.

EXAMPLE PROBLEM 3.11

Calculate the linear density of the [100] direction for BCC.

S OLUT I ON

A BCC unit cell (reduced sphere) and the [100] direction therein are shownin Figure 3.26a; represented in Figure 3.26b is the linear packing in this direction.

S-4 ! Chapter 3 / Structures of Metals and Ceramics

FIGURE 3.18 The structure of a C60 molecule.

FIG. 5: A C60 molecule forms a geodesic dome, adapted from [24]. Several of these molecules in

concentric shells display a cross-section like an onion as seen in [25]

III. AIMS AND OBJECTIVES

The objective of this project is to research optimal synthesis for the addition of detonation

nanodiamonds and nanoonions to UCL-NanoBio. From this, a conduit could be synthesised

to provide biocompatibility and conductivity for axonal regeneration. Product must be

carefully analysed at each stage using a mixture of spectroscopic and microscopic techniques.

The aim is to achieve this through the stages listed below.

1. Produce Nanodiamonds

(a) form stable colloids

(b) determine surface terminations and ζ−potential

(c) obtain ND functional groups

(d) physical and electrical characterisation

2. Produce Carbon Nano-onions

(a) by transformation of nanodiamond

(b) through CuCl2 hydrate reaction with calcium carbide

(c) using methane decomposed over Ni-Fe catalyst

(d) physical and electrical characterisation

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3. Effect of nanodiamonds in UCL-NanoBio

(a) synthesise polymer

(b) investigate chemical linkage

(c) assess physical and electrical influence

(d) evaluate conduction mechanisms

4. Effect of nanoonions in UCL-Nanobio

(a) synthesise polymer

(b) investigate chemical linkage

(c) assess physical and electrical influence

(d) evaluate conduction mechanism

IV. IMPACT

This research should produce a material that provides suitably quick and efficient axonal

regrowth, making the harvest of donor tissue unnecessary. Aside from the economic, time

saving and risk-reducing effects of simplifying surgical procedures, recovery avoids permanent

sensory loss (researched in the background section [9]). Should the device be adopted, this

research will be of great importance given that peripheral nerve trauma occurs in 3% of all

injuries [2]. The nanodiamond-polymer conduit may provide a standard for the future use

of bioengineered components in medical treatment.

V. APPROACH

The initial task is to obtain detonation nanodiamonds with appropriate surface termina-

tions. Single digit mono-dispersed colloids will be formed through sonication and centrifugal

treatments. Particle dispersion and ζ−potential can then be determined using DLS. High

temperature annealing and photolytic treatments will produce different functional groups.

The samples should then be characterised by Raman, FTIR and UV-visible spectroscopy

with electrical properties characterised using IS, Hall effect and AFM.

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The second task is to produce carbon nano-onions using three separate methods. First,

nanodiamonds will be transformed at 1000-1200◦C in a vacuum (see [16]). Also, CuCl2

hydrate will be reacted with calcium carbide under high temperature and pressure (see

[17]). Finally, CH4 will be decomposed over a Ni-Fe catalyst at 850◦C (see [18]). The

synthesised molecules will be characterised as before with the addition of TEM, XRD and

SEM analysis.

Next, the addition of the nanodiamond to the UCL-NanoBio polymer will be explored.

Synthesis of the polymer is fully detailed in [10]. Nanodiamonds will be added at the pre-

polymer stage with different concentrations and functional groups. Physical and electrical

changes in the composite will be observed using AFM, nano-indentation, measurement of

ζ−potential, IS and Hall effect. Conduction mechanisms will be evaluated.

Finally, nano-onions will be added to UCL-NanoBio. The polymer must be synthesised

as before and molecules produced from all three methods added. Physical and electrical

characterisation will be performed similar to the previous stage.

An approximate time scale for the procedures is outlined in Fig 6. With no prior experi-

ence with most of these experimental techniques, this Gantt chart will require refining over

the duration of the project. With limited lab time, it is likely that only a fraction of the

tasks can be satisfied.

VI. CONCLUSIONS

A detailed literature survey has been produced looking at a thorough selection of papers

from medical, chemical, biotechnology and nanotechnology journals. This has enabled a wide

ranging understanding of the task in hand and its implications. Clear steps to undertake

this project has been set out and a Gantt chart provided. This report is sufficiently detailed

to allow work to begin in an educated and structured manner.

[1] B. J. Pfister, T. Gordon, J. R. Loverde, A. S. Kochar, S. E. Mackinnon, and D. K. Cullen,

“Biomedical engineering strategies for peripheral nerve repair: surgical applications, state of

the art, and future challenges.,” Critical reviews in biomedical engineering, vol. 39, pp. 81–124,

Jan. 2011.

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FIG. 6: A Gantt chart of the work ahead. Week numbers align with UCL Common Timetable

[2] J. Noble and C. Munro, “Analysis of upper and lower extremity peripheral nerve injuries in a

population of patients with multiple injuries,” The Journal of trauma, 1998.

[3] A. Waller, “section of the glossopharyngeal and hypoglossal nerves of the frog, and observations

of the alterations produced thereby in the structure of their primitive fibres,” Philosophical

Transactions of the Royal Society of . . . , vol. 140, no. 1850, pp. 423–429, 1850.

[4] R. B. Donoff, “Nerve regeneration: basic and applied aspects.,” Critical reviews in oral biology

and medicine : an official publication of the American Association of Oral Biologists, vol. 6,

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pp. 18–24, Jan. 1995.

[5] S. Y. Fu and T. Gordon, “The cellular and molecular basis of peripheral nerve regeneration.,”

Molecular neurobiology, vol. 14, no. 1-2, pp. 67–116, 1997.

[6] A. Pabari, S. Y. Yang, A. M. Seifalian, and A. Mosahebi, “Modern surgical management of

peripheral nerve gap.,” Journal of plastic, reconstructive & aesthetic surgery : JPRAS, vol. 63,

pp. 1941–8, Dec. 2010.

[7] T. Sedaghati, S. Y. Yang, A. Mosahebi, M. S. Alavijeh, and A. M. Seifalian, “Nerve re-

generation with aid of nanotechnology and cellular engineering.,” Biotechnology and applied

biochemistry, vol. 58, no. 5, pp. 288–300.

[8] J. S. Belkas, M. S. Shoichet, and R. Midha, “Axonal guidance channels in peripheral nerve

regeneration,” Operative Techniques in Orthopaedics, vol. 14, pp. 190–198, July 2004.

[9] F. F. a. IJpma, J.-P. a. Nicolai, and M. F. Meek, “Sural nerve donor-site morbidity: thirty-four

years of follow-up.,” Annals of plastic surgery, vol. 57, pp. 391–5, Oct. 2006.

[10] A. SEIFALIAN, H. SALACINSKI, and S. HANCOCK, “Polymer for use in conduits and

medical devices,” WO Patent WO/2005/070,998, 2005.

[11] J. Raghunath, G. Georgiou, D. Armitage, S. N. Nazhat, K. M. Sales, P. E. Butler, and

A. M. Seifalian, “Degradation studies on biodegradable nanocomposite based on polycapro-

lactone/polycarbonate (80:20%) polyhedral oligomeric silsesquioxane.,” Journal of biomedical

materials research. Part A, vol. 91, pp. 834–44, Dec. 2009.

[12] C. E. Schmidt and J. B. Leach, “Neural tissue engineering: strategies for repair and regener-

ation.,” Annual review of biomedical engineering, vol. 5, pp. 293–347, Jan. 2003.

[13] A. Thalhammer, R. J. Edgington, L. a. Cingolani, R. Schoepfer, and R. B. Jackman, “The

use of nanodiamond monolayer coatings to promote the formation of functional neuronal

networks.,” Biomaterials, vol. 31, pp. 2097–104, Mar. 2010.

[14] K.-K. Liu, C.-L. Cheng, C.-C. Chang, and J.-I. Chao, “Biocompatible and detectable car-

boxylated nanodiamond on human cell,” Nanotechnology, vol. 18, p. 325102, Aug. 2007.

[15] H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. Itty Ipe, M. G. Bawendi, and J. V.

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diffraction and molecular dynamics,” Diamond & Related Materials, vol. 20, no. 10, pp. 1333–

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[17] F.-D. Han, B. Yao, and Y.-J. Bai, “Preparation of Carbon Nano-Onions and Their Appli-

cation as Anode Materials for Rechargeable Lithium-Ion Batteries,” The Journal of Physical

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[18] C. Zhang, J. Li, C. Shi, E. Liu, X. Du, W. Feng, and N. Zhao, “The efficient synthesis of

carbon nano-onions using chemical vapor deposition on an unsupported NiFe alloy catalyst,”

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[20] A. Bunde and J. Kantelhardt, “Diffusion and Conduction in Percolation SystemsTheory and

Applications,” uni-giessen.de, pp. 1–19.

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Glossary of analytical terms

ζ−potential is a name for the electrokinetic potential in a colloid system between the

dispersed particle and its medium. It is used as a measurement for the system stability.

AFM Atomic Force Microscopy (AFM) uses a cantilever to probe a sample. Deflections of

the cantilever are mapped to image the sample surface at a very high resolution.

annealing is a heat treatment of materials in which the energy added allows redistribution

of atoms before cooling slowly.

colloids are particles microscopically dispersed in a medium such as the butterfat suspended

in water that constitutes milk.

DLS Dynamic Light Scattering (DLS) determines the size and distribution of particles in

a solution by measuring fluctuations in scattered light intensity due to diffusion.

FTIR Fourier Transform Infrared spectroscopy (FTIR) collects data in a wide spectral

range. A Fourier transform obtains the actual spectrum which is compared with that

of known molecules to help identify a sampl.

functional groups are recognised collections of atoms responsible for characteristic chem-

ical reactions such as alkanes and amides.

Hall effect can be used to measure electrical properties, such as mobility and carrier con-

centration in semiconductors.

IS Impedance Spectroscopy (IS) measures the sample impedance over a range of frequencies.

mono-dispersed is where dispersed particles are of approximately the same size.

photolytic treatments breakdown material through the introduction of light energy, re-

distributing atoms.

Raman spectroscopy uses laser light to interact with chemical bonds. This is compared

with the spectrum of a known molecule. This spectroscopy is highly sensitive to the

morphology of carbon hence is useful in identifying allotropes [26].

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SEM Scanning Electron Microscopy (SEM) uses a beam of electrons to image a sample.

This provides a higher resolution than traditional optical microscopy.

sonication is the agitation of particles with sound energy in order to affect their dispersion.

surface terminations are the atomic configuration at the surface of a material which give

rise to important electronic states at the interface.

TEM Transmission Emission Microscopy (TEM) sends a beam of electrons through an

extremely thin specimen. The interactions with this beam are captured by a sensor

on the other side.

UV-visible spectroscopy gathers information in the ultraviolet and visible light range.

XRD X-ray Diffraction (XRD) is used to find the chemical and physical properties of a

material by sending a beam of X-rays and observing the scattering intensity.

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