Characterisation of Resin-Composite Structure and Collagen Assembly at Nanometre to Micrometre Length Scales

Characterisation of resin-composite structure and collagen assembly at nanometre to micrometre length scales

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2018

Alvaro E. Munoz Schiemann

School of Dentistry

TABLE OF CONTENTS

Abstract ...... 12

Declaration ...... 13

Chapter I. Introduction and aims ...... 15

I.1. Introduction ...... 16 I.2. Tooth structure ...... 16 I.2.1. Enamel ...... 17 I.2.2. Dentine ...... 18 I.2.3. Pulp ...... 20 I.3. Dentine matrix ...... 20 I.3.1. Collagen ...... 20 I.3.1.1. Fibrillogenesis ...... 24 I.3.1.2. Mineralisation ...... 29 I.3.2. Non-collagenous proteins ...... 31 I.3.2.1. Enzymes ...... 34 I.3.2.1.1. Matrix metalloproteinases (MMPs) ...... 34 I.3.2.1.2. Cysteine Cathepsins...... 36 I.4. Dental Caries ...... 38 I.4.1. Caries treatment ...... 39 I.4.1.1. Non-invasive ...... 39 I.4.1.2. Invasive treatment ...... 39 I.4.1.2.1. Etch and rinse approach ...... 40 I.4.1.2.2. Self-etch approach ...... 42 I.5. Tooth-resin interface ...... 43 I.5.1. Adhesion/micromechanical interlocking ...... 43 I.5.1.1. Adhesion to Enamel...... 44 I.5.1.2. Adhesion to Dentine ...... 45 I.5.2. Failure of the adhesion ...... 46 I.5.2.1. Hydrolysis of the resin adhesive ...... 46 I.5.2.2. Collagen degradation ...... 47 I.5.3. Longevity of resin restorations ...... 49 I.5.4. Current strategies to prevent degradation of the hybrid layer ...... 50 I.5.4.1. Adhesive-related strategies to prevent hybrid layer degradation ...... 51 I.5.4.2. Enzyme-related strategies to prevent hybrid layer degradation ...... 52 I.6. Resin-composites roughness and measurement techniques ...... 53 I.7. Clinical implications ...... 56 I.8. Atomic Force Microscopy ...... 57

I.8.1 Background ...... 57 I.8.2 Configuration of the microscope ...... 59 I.8.3 Imaging modes ...... 62 I.8.3.1 Contact mode ...... 62 I.8.3.2 AC modes ...... 63 I.8.3.3 Kelvin Probe Force microscopy (KPFM) ...... 64 I.9. Rationale ...... 66 I.10. Aims and Objectives ...... 67

Chapter II. “Optimisation of a collagen fibril model system” ...... 68

II.1. Overview ...... 69 II.2. Materials and Methods ...... 69 II.2.1. Comparison of collagen models ...... 69 II.2.1.1. Model-1 Reconstituted Collagen ...... 69 II.2.1.2. Model-2 Extracted collagen ...... 70 II.2.2. Optimisation of AFM experimental conditions ...... 71 II.3. Results ...... 72 II.3.1. Comparison of models ...... 72 II.3.1.1. Reconstituted collagen ...... 72 II.3.1.2. Extracted collagen ...... 75 II.3.2. Optimisation of AFM experimental conditions ...... 76 II.4. Discussion ...... 79 II.5. Conclusion ...... 83

Chapter III. “Influence of dental bonding procedures on collagen fibril nano- structure and nano-charge distribution” ...... 84

III.1. Overview ...... 85 III.2. Materials and Methods ...... 85 III.2.1 Sample preparation ...... 85 III.2.2 AFM imaging ...... 86 III.2.3 Image post-processing and analysis ...... 87 III.2.4 Statistical analysis ...... 88 III.3. Results ...... 88 III.3.1 Height ...... 91 III.3.2 Width ...... 95 III.3.3 D-Period ...... 97 III.3.4 Surface Potential (KPFM)...... 99 III.3.5 Height difference between Gap and Overlap regions ...... 101 III.3.6 KFM Profiles ...... 102 III.4. Discussion ...... 104 III.5. Conclusion ...... 107

Chapter IV. “The surface roughness of resin-composites is dependent on the measurement area: an AFM study” ...... 108

IV.1 Overview ...... 109 IV.2 Materials and Methods ...... 109 IV.2.1 Surface Roughness Measurement ...... 110 IV.2.2 Statistical analysis ...... 112 IV.3 Results ...... 112 IV.3.1 Comparison of Roughness Average (Sa) ...... 116 IV.3.2 Comparison of Root Mean Square Roughness (Sq) ...... 118 IV.3.3 Comparison of the Maximum height (Sz) ...... 119 IV.4 Discussion ...... 121 IV.5 Conclusion ...... 123

Chapter V. Discussion ...... 124

Future work ...... 127

References ...... 128

Appendix 1 ...... 156

LIST OF FIGURES

Figure I-1. Molar tooth diagram. Note the difference between inner and outer dentine,

density of tubules: 6.5 x 106 tubules/cm2 and 1.9 x 106 tubules/cm2; tubule

dimension: 3 µm and 0.8 µm, respectively (12)...... 17

Figure I-2. Dentine structure, note size and density of tubules regarding their

proximity to the dentine-enamel junction (DEJ). Taken from Katz et al. (31) ...... 19

Figure I-3. Hierarchical structure of fibrillar type I collagen. Adapted from Orgel et al.

(2011) ...... 24

Figure I-4. Electron micrograph of collagen fibril. A, positive staining pattern of a

double stained fibril, first with phosphotungstic acid and then with uranyl

acetate; showing different bands. B, labelling of the bands and representation of

the stain intensities. C, negative staining pattern with sodium phosphotungstate,

showing gap (0.54D) and overlap (0.46D) regions in a D-period of 67nm.

Adapted from Chapman et al. (70) ...... 29

Figure I-5. Annual failure rate (AFR) of different adhesive systems. SEs: Self-etch

strong acidity, SEm: Self-etch mild acidity, E&R: etch and rinse, GI: Glass ionomer.

Adapted from Peumans et al. (205) ...... 50

Figure I-6. Diagram of an AFM control system showing the main components. Here

can be observed how a sample mounted on a piezo is raster scanned by a flexible

cantilever. The bending of the cantilever due to the interaction with the sample,

produce a deflection of the laser beam reflecting in the photodetector. The signal

obtained in the photodetector allows reconstruction of the topography and give

feed-back to the piezo in order to avoid crashing the tip on the sample. In KPFM

mode an electric bias is used on the tip to detect variation of the surface potential

of the sample. Adapted from Morris (254)...... 65

Figure II-1. AFM images of collagen fibrils at different concentration. Images A and B

are dry samples (12µg/ml) and their successive images following re-hydration

are captioned from 1 to 4 (at 9 minutes intervals approx). Image C is the dry

sample at 24µg/ml and C.1 depicts the mica substrate lacking collagen after re-

hydration. Insets depict fast Fourier transform (FFT) used to determine D-period.

...... 74

Figure II-2. AFM images of representative collagen type I extracted from rat tail

tendon. Fibril A, presented an average height of 143.8nm and fibril B of 164.6 nm.

Amplitude channel of images A and B are presented in C and D, respectively. Scale

bar 500nm. Calibration bar in nm...... 75

Figure II-3. AFM image of the same section of collagen type I extracted from rat tail

tendon before (A) and after (B) acid treatment. The configuration used was 10%

phosphoric acid during 5 sec. In image B can be seen almost complete

disintegration of the fibril. Same calibration bar on both images, in nm...... 76

Figure II-4. AFM images of the same section of a type I collagen fibril extracted from

rat tail tendon before (A) and after (B) acid treatment (10% Citric acid, 5s).

Images C and D correspond to the amplitude channel of fibril presented in A and

B, respectively. Here the D-period is observable despite the reduction of height.

Same calibration bar on both height images, in nm...... 77

Figure II-5. AFM topographic (A and B) and KPFM (C and D) map of the same section

of a type I collagen fibril before and after acid treatment (10% Citric acid, 5s). It

can be seen height and potential changes on the same area of the same fibril.

Topographic bar in nm, KPFM bar in milivolts...... 78

Figure III-1. AFM images of the same 2 x 2 µm area of a collagen fibril submitted

sequentially to deionised water (A-B), phosphoric acid- (C-D), chlorhexidine- (E-

F) and primer-treatment (G-H). Left images correspond to topographic maps

(calibration bar in nm), right images to KPFM surface potential maps (calibration

bar in mV)...... 90

Figure III-2. Tri-dimensional reconstruction of the same collagen fibril section

subjected to consecutives phosphoric acid (PA), chlorhexidine (Chx) and primer

interventions. Dimensions expressed in nanometre ...... 91

Figure III-3. Height variation across the interventions. Note effect of acid etching on

colllagen. Mean and SD are plotted for each group...... 92

Figure III-4. Individual variation of the collagen height after different interventions

and statistical differences of height are presented by groups treated with citric

acid ...... 93

Figure III-5. Individual variation of collagen height after different interventions within

groups and statistical differences in groups treated with phosphoric acid and

PA/Chx solution...... 94

Figure III-6. Effects on the FWHM of collagen fibrils submitted to a dental adhesion

procedure. In general, width had a slight increase after acid treatment (p>0.05).

Some reductions are also detected especially in group Ca+P, where one fibrils

reduced its width in more than 100nm. Individual variations per group and mean

(SD) results are presented...... 96

Figure III-7. AFM image of the same section of a collagen fibril initially and after

treatment with a solution of phosphoric acid and chlorhexidine (Acid/Chx) and

primer. Note the preservation of the D-banding (66±1 nm) throughout

interventions...... 97

Figure III-8. D-period variation across interventions. Individual variations and means

(SD) per groups are presented...... 98

Figure III-9. Tri-dimensional reconstruction of the same section of a fibril topography

(left) and surface potential (right). A: Initial, B: citric acid, C: chlorhexidine D:

Primer. Left: An appreciable reduction in height without major width variation is

observed. Right: initially is measure a mean surface potential of 22.5 mV,

increasing to 37.6mV after acid treatment, after chlorhexidine decreased to 26.6

mV and primer application reduced the value to 21.7 mV (values correspond to

an average of the top area of the fibrils). Calibration bar in mV...... 99

Figure III-10. Surface potential variation of individual fibrils and average per groups

are presented. It can be observed a great initial dispersion in values but in

general an increased of surface potential after acid treatment and primer

aplication...... 100

Figure III-11. Height profiles of the same section of a collagen fibril from group

PA+Chx+P. Here, acid induced a slight reduction of step-height and primer

increased some and reduced other areas of gap-overlap...... 101

Figure III-12. Topography (blue) and potential (red) profiles of the same section of an

individual fibril across interventions. It can be seen gap areas had a higher

surface potential compared to overlap...... 103

Figure IV-1. Tri-dimensional reconstruction of resin-composite surfaces obtained

after AFM scanning an area of 5x5 µm. Irregularities produced by brushing

procedure are observed especially in CeramX sample...... 112

Figure IV-2. Tri-dimensional reconstruction of resin-composites surfaces after AFM

scanning. At a higher area of 20x20 µm, CeramX and VDiamond can be seen with

greater irregularities on its surface...... 113

Figure IV-3. Tri-dimensional reconstruction of dental resin-composites surfaces after

AFM scanning. At an area of 100x100 µm, the irregularities produced by brushing

are again seen but the amount of events has also increased...... 113

Figure IV-4. Average roughness (Sa) determined for area in the range 0.01 to 10,000

µm2. It can be seen an increasing roughness average at incremental dimensions.

Brushed samples are seen to vary more than polished surfaces. Note abruptly

increase of Sa at just over 25 µm2 area of observation for Tetric and VDiamond

brushed samples...... 114

Figure IV-5. RMS (Sq) roughness determined for area in the range 0.01 to 10000 µm2.

Increase value RMS is observed at higher areas. Major changes for Tetric and

VDiamond brushed values at just over 25 µm2 observations...... 115

Figure IV-6. Maximum height determined (Sz) for area in the range 0.01 to 10000 µm2.

Incremental Sz values are found at higher areas of observation...... 115

LIST OF TABLES

Table I-1. Vertebrate collagens. Classification according to domain structure homology

and supra-structural assembly. FACIT, fibril-associated collagens with

interrupted triple helices; MACIT, membrane-associated collagen with

interrupted triple helices; MULTIPLEXIN, multiple triple-helix domains and

interruptions...... 23

Table I-2. Non collagenous proteins in dentine matrix...... 33

Table I-3. Methods for measuring roughness (†) ...... 55

Table I-4. Comparison of Atomic Force Microscopy, Scanning electron microscopy and

Transmission electron microscopy main characteristics...... 59

Table III-1. Groups and treatment sequence ...... 86

Table IV-1. Name and product details used in this study ...... 110

Table IV-2. Multiple comparison analysis (Tukey test) of mean Sa results of resin-

composites at three different dimensions...... 117

Table IV-3. Roughness Average Ratio (Brushed:Polished) at different scales of

measurement ...... 117

Table IV-4. Multiple comparison analysis (Tukey test) of mean of Sq results of resin-

composites at three different dimensions ...... 118

Table IV-5. RMS roughness Ratio (brushed:polished) at different scales of

measurement ...... 119

Table IV-6. Multiple comparison analysis (Tukey test) of mean Sz results of resin-

composites at three different dimensions ...... 120

Table IV-7. Maximum height ratios (brushed:polished) at different scales of

measurement ...... 120

Abstract

Despite significant improvements of resin-composite in dentistry, when used as a restorative material, the main clinical reasons for failure are fracture and secondary caries. The quality of the dentine collagen in adhesive procedures is a critical aspect and affects the restorations longevity. Scarce evidence of the bonding agents effects on collagen at a nanometre scale and a lack of standardisation on the evaluation of surface roughness applied to dental materials are found in the scientific literature. Therefore, the aims of this study were i) to identify the most appropriate model for the study of collagen interactions with bonding agents, ii) to use this model to evaluate nano-scale morphological and electrical changes as consequence of bonding procedures, and iii) to determine the optimal area to assess roughness of resin- composite surfaces. Topographic and surface potential maps were obtained from collagen samples seeded on silicon wafer using advanced imaging techniques: Atomic Force Microscopy (AFM) and Kelvin Probe Force microscopy (KPFM). The samples were then divided in 5 groups of 8 specimens and submitted to the effects of dental chemical agents (citric acid, phosphoric acid, phosphoric acid/chlorhexidine, chlorhexidine, HEMA/TEGDMA). After each agent, the same area of the fibrils was reassessed. Important reductions in height and electrical surface potential were detected. Therefore, it was concluded that chemical agents induce changes in the structure of collagen type I. Moreover, topographic maps at different dimensions were obtained from polished and brushed samples from three different resin-composites using AFM. Using Matlab routines, arithmetical mean height (Sa), root mean square height (Sq) and maximum height (Sz) were obtained at incremental size areas. It was observed a non-linear increase of surface roughness at higher dimensions. These observations suggest that to successfully characterise surface roughness of dental materials is essential measuring and report Sa, Sq and Sz at both, small and large areas and to standardise procedures.

Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.library.manchester.ac.uk/about/regulations/) and in The University’s policy on Presentation of Theses

Chapter I. Introduction and aims

I.1. Introduction

Resin-composites are widely used nowadays where minimally invasive dentistry is one of top priorities of dentists (1, 2). Undoubtedly, patients are demanding more aesthetic results (3). Evidence shows significant improvements in terms of function and mimetic ability during the last two decades (4). However, the interface of tooth- resin restorations continues to be the downside for durable restorations (5, 6).

Independently of the primary factor causing failure, it has been reported an annual failure rate of 1-3% of resin restoration placed in posterior permanent teeth (7-9).

Thus, time and resources are limited; therefore the increasing demand for replacing resin restoration (7, 10) should be tackled.

This literature review describes briefly, the tooth, its structure and composition as a general knowledge, then the caries disease is presented and described, before discussing resin restorations, how the adhesive restorative treatment is time-affected, what is the role of collagen and others proteins in affecting this treatment and the current strategies to delay degradation.

I.2. Tooth structure

Teeth are divided in two sections, crown and root (Figure I-1). The crown is the visible portion of the tooth and the root is embedded in the alveolus, which is part of the maxillary bones (11).

Histologically, three tissues are recognizable: enamel, dentine and pulp (12).

Figure I-1. Molar tooth diagram. Note the difference between inner and outer dentine, density of tubules: 6.5 x 106 tubules/cm2 and 1.9 x 106 tubules/cm2; tubule dimension: 3 µm and 0.8 µm, respectively (12).

I.2.1. Enamel

Enamel is the hardest mineralised tissue of the body, which covers the crown of the tooth (Figure I-1). This tissue contains hydroxyapatite (HA) 96 wt%, water 3wt% and organic materials 1wt% (11). The scarce organic content comprises proteins and lipids; proteins are the main fraction and are divided in two groups: amelogenins and non-amelogenins (such as enamelin and tufteline) (13). Although enamel is the

hardest tissue, it can be lost due to caries, attrition, erosion and abrasion. Moreover, once lost it cannot be repaired as it is an acellular tissue (11).

The enamel structure is an ordered arrangement (prisms) of tightly packed HA crystals (14). The enamel prism, also known as rod, is oriented from the dentine- enamel junction to the surface (Figure I-1). The length is variable, having the longest extension at the cusps or incisal edges of about 2.5mm with a diameter of 5-6 µm (11).

The inter-crystalline space or space between prisms is filled with water and organic materials.

I.2.2. Dentine

Dentine is the mineralised tissue that surrounds the pulp and it is covered by enamel

(crown) or cementum (root) (Figure I-1), also defined as a bio-composite of a collagen matrix infiltrated with nano-hydroxyapatite (15). Its composition has been estimated at 50wt% inorganic, 40wt% organic, and 10wt% water, similar to bone (16). The main component of the organic part (30 vol%) is collagen type I (90%) and the other

10% is composed by several non-collagenous components including proteoglycans, phospholipids and enzymes (17, 18). More detailed description of the organic matrix is found in section I.3

From the morphological point of view, the dentine contains tubules where the odontoblast process is located. This cytoplasmic prolongation is part of the cell responsible for dentine development (19, 20). The tubules run from the pulp to the dentine-enamel junction (Figure I-2), decreasing its diameter from 2.5µm to 0.8µm

and consequently their number per unit area from 45000-65000/mm2 at the pulp to

15000-20000/mm2 at the dentine-enamel junction (21). The tubules are responsible of the dentine permeability (19). Surrounding the tubules a highly mineralised zone called peritubular dentine is observed and between them the intertubular dentine

(22). A particular difference between intertubular and peritubular dentine is the absence of collagen type I in the latter (23, 24), where its organic content is constituted by phosphorylated proteins (25, 26), proteoglycans and glycosaminoglycans (23). This particular scenario yield to dentine having different micro-mechanical properties (27) such as crack bridging and crack deflection as toughening mechanisms (28-30).

Figure I-2. Dentine structure, note size and density of tubules regarding their proximity to the dentine-enamel junction (DEJ). Taken from Katz et al. (31)

I.2.3. Pulp

The pulp is a connective tissue located in the inner part of the tooth (pulp chamber and root canal) (Figure I-1). It is responsible for maintenance of dentine and hosts blood vessels, nerves and cells, i.e. fibroblast, stem cells, defensive cells and unlike other soft tissues, contains odontoblasts (11).

The pulp is composed by water 75%w and 25%w organic material. It slowly produces secondary dentine but also responds to strong stimulus such as caries, trauma or tooth movement producing tertiary dentine (11). The extracellular matrix content of this connective tissue includes: collagens (type I, III, V, VI), non-collagenous proteins

(Bone sialoprotein, osteopontin, fibronectin, osteonectin, and others), glycoproteins, enzymes (e.g. MMPs, cathepsins, TIMPs), growth factors and some plasma-derived components (32).

The pulp is organised in a semifluid gel due to the complex scaffold formed by collagen (type I and III), proteoglycans and glycoproteins. This loose connective tissue allows cell migration, proliferation, adhesion, differentiation and function (11).

I.3. Dentine matrix

Due to the complexity of the organised protein assembly, i.e. dentine matrix, this section is dedicated to collagen and non-collagenous component of dentine.

I.3.1. Collagen

The collagens are a large family of structurally related proteins. In humans over 30% of the total proteins are collagens (33). To date, 28 different types of collagen have

been identified (33) (see Table I-1). All collagen molecules are formed by three polypeptide chains or trimers, which are chemically and genetically distinct macromolecular species that can aggregate forming visible fibres, and basement and pericellular membranes of connective tissues (34). Some of these structural proteins are homotrimers and other heterotrimers, conformed either by 2 or 3 different chain types (35). Despite the variety described, a common characteristic of the collagen family is a structural motif, a right-handed twisted triple helix, in which each of the three polypeptide chains (α-chains) are left-handed coiled (in a polyproline II-type conformation) and staggered about each other by one residue as proposed in several models (33, 35-38).

The most widely accepted model has been proposed by Ramachandran and Kartha

(36, 39); and posteriorly corrected by Rich and Crick (37, 38) and by Cowan et al. (40).

The structure is composed of three polypeptide chains of triplets where due to steric constrains of the close packed helix (35), Glycine (Gly) is required at every third position (X-Y-Gly) and the X and Y are frequently proline and 4-hydroxyproline (41).

Thus, the triple-helix formed is stabilised by only one inter-stranded hydrogen bond per tripeptide (33). Here, the bond is formed between the amino group of glycine and the C=O group of the amino/imino acid located in the X position (33, 38, 42).

According to Bella et al. (43), the length of this bond was not long enough to be established directly. As a result of their experimental data, a water molecule was confirmed to be in these intra and intermolecular bonds (defining thereafter the inherent presence of cylinders of hydration surrounding the collagen triple helix) (44,

45).

It is noteworthy that the collagen structure has two non-helical portions located at each extreme of the triple helix (N-terminal and C-terminal) (Figure I3). Despite their size, these non-collagen segments are important on the structure (46). For example, the C-propeptide could have an important role in the early stages of the triple helix formation (46). In addition, N-propeptide has been related to the control of the fibril diameter (46). Nevertheless, after being processed the remaining C- and N-terminal portions of the collagen molecule are involved in the molecular cross-linking.

Additionally, these telopeptide domains have the ability to bind other structures or molecules (46).

The fibrillar collagen I is one of the most abundant proteins in animals (33, 47), it has not imperfections (36-40). However, as a result of post-translational changes or splicing during collagen assembly (34), the triple helix can be disrupted in some portions, originating non-fibrillar collagens (48). Thus, collagen types have been classified as fibrillar and network-forming collagens, fibril-associated collagens with interrupted triple helices (FACITs), membrane-associated collagen with interrupted triple helices (MACITs); and multiple triple-helix domains and interruptions

(MULTIPLEXINs) (Table I1) (33).

Table I-1. Vertebrate collagens. Classification according to domain structure homology and supra-structural assembly. FACIT, fibril-associated collagens with interrupted triple helices; MACIT, membrane-associated collagen with interrupted triple helices; MULTIPLEXIN, multiple triple-helix domains and interruptions. Type Classification Composition Distribution I Fibrillar α1(I)2α2(I) Dermis, bone, tendon, ligaments II Fibrillar α1(II)3 Cartilage, vitreous III Fibrillar α1(III)3 Skin, blood vessels, intestine α1(IV)2α2(IV) IV Network α3(IV)α4(IV)α5(IV) Basement membranes α5(IV)2α6(IV) α1(V)3 V Fibrillar α1(V)2α2(V) Bone, dermis, cornea, placenta α1(V)α2(V)α3(V) α1(VI) α2(VI) α3(VI) VI Network Bone, cartilage, cornea, dermis α1(VI) α2(VI) α4(VI) VII Anchoring fibrils α1(VII)2 α2(VII) Dermis, bladder α1(VIII)3 VIII Network α2(VIII)3 Dermis, brain, heart, kidney α1(VIII)2 α2(VIII) IX FACIT α1(IX) α2(IX) α3(IX) Cartilage, cornea vitreous X Network α1(X)3 Cartilage XI Fibrillar α1(XI) α2(XI) α3(XI) Cartilage, intervertebral disc XII FACIT α1(XII)3 Dermis, tendon XIII MACIT - Endothelial cells, dermis, eye, heart XIV FACIT α1(XIV)3 Bone, dermis, cartilage XV MULTIPLEXIN - Capillaries, testis, kidney, heart XVI FACIT - Dermis, kidney XVII MACIT α1(XVII)3 Hemidesmosomes in epithelia XVIII MULTIPLEXIN - Basement membrane, liver XIX FACIT - Basement membrane XX FACIT - Cornea(chick) XXI FACIT - Stomach, kidney XXII FACIT - Tissue junctions XXIII MACIT - Heart, retina XXIV Fibrillar - Bone, cornea XXV MACIT - Brain, heart, testis XXVI FACIT - Testis, ovary XXVII Fibrillar - Cartilage XXVIII - - Dermis, sciatic nerve Adapted from Shoulders and Raines (2009)

It is widely accepted that 90 wt% of the organic content of dentine is a supramolecular assembly of several hierarchically organised substructures in a fibrillar arrangement (collagen I) (49, 50), secreted by odontoblasts into the 23

extracellular space as tropocollagen during dentinogenesis (16). Thus, the following paragraphs are primarily oriented to describe the fibrillogenesis and mineralisation of this class of collagens.

Figure I-3. Hierarchical structure of fibrillar type I collagen. Adapted from Orgel et al. (2011)

I.3.1.1. Fibrillogenesis

The current knowledge about the complex fibril formation process is mainly based on in vitro studies (46). The collagen fibril has a hierarchical structure where individual tropocollagen molecules are self-assembled outside cells, i.e. fibrillogenesis (33)

(Figure I-3). The first step of this complex process begins with the cleavage of the terminal globular portions by specific proteins, leaving only two small non-helical

portions that fold tightly along the triple helix (41). These telopeptide regions (N- terminal and C-terminal) account for 2% of the structure are critical for fibril formation, and do not form triple helix (51).

Fibril-forming collagen molecules (i.e. collagen types I, II, III, V, XI, XXIV and XXVII) have their characteristic triple helical domain with a length of 300nm and a diameter of 1.4 nm, approximately (33, 34). These insoluble molecules start to aggregate even at nanomolar concentrations (41).

According to the model proposed by Hodges and Petruska (52), 5 molecules (in width) are arranged in a staggered fashion (Figure I-3) to provide the most characteristic feature of the collagen fibril, the D-periodic banding of near to 67nm in average (33, 41). This banding pattern can be observed by electron microscopy (33,

34, 53) and atomic force microscopy (AFM) in both dehydrated and hydrated samples

(16, 54). This characteristic banding is formed due to the length of the collagen molecule, which is not an exact multiple of D (it is 4.46D); giving rise to overlapping and gapping zones (0.46D and 0.54D respectively) (Figure I-3). The dimension of the molecule along with its staggered arrangement seems to be critical for the mechanical properties of the fibril, avoiding brittle fracture and maximizing dissipation of energy under large deformation (55). In addition, the staggered organisation with a determined D-period depends on the state of hydration of the fibril (44) and could vary slightly across the techniques (41). Using AFM, Habelitz et al. (16) reported an axial repeat distribution of dentine-derived collagen ranging from 54 to 75nm in hydrated samples; although the majority was measured around 67-68 nm. Similar results, but in rat tail tendon showed a distribution of 63 to 74 nm (56) and 60 to 70

nm (57). In addition, the height difference of distance between gap and overlap has been measured to be between 4 and 6 nm in fully demineralised collagen (16, 54).

The fibrils are assembled in segments, where each of these sections are asymmetric

(58). The extension and organisation of the fibril assembly determines the mechanical and physical characteristics of the tissue. For example, narrow fibril formation occur in the cornea, here the perfect orthogonal arrangement provides optical transparency required for its purpose. In contrast, when tensile strength is required, such as in tendon, the fibril formation is organised in parallel bundles of thicker fibrils (51).

A discrepancy with the model of Hodge and Petruska is that the axis of the alpha- helices is not parallel to the fibrillar axis, having 5 degrees in respect to each other

(34), as seen in Figure I-3 (tri-dimensional arrangement). Thus, the model seems too simple and is unable to explain for example, why the D-banding is independent of the fibril diameter or why the surface is corrugated (59). Recent findings have led to new proposals to explain such controversy (33). Orgel et al. (60) demonstrated an orthohexagonal lattice of the collagen microfibril, which continues even throughout the gap region and it is maintained by four other collagen molecules forming a right- handed supertwisted arrangement (33, 60). This network structure was considered to be as a rope (60). In addition, Bozec et al. using AFM of tendon collagen, modelled a similar rope-like structure of the collagen fibril (59). Observations on AFM confirm that collagen microfibrils (composed by 5 molecules) in dentine possess a diameter of

4nm (16), similar to those previously found in native collagen in several X-Ray diffraction (XRD) studies (60-63).

Another remarkable characteristic of the collagen fibril is observed as a tilted terminal region of the collagen molecule through the overlap region (60). In addition, it is possible these regions of the collagen molecule interact with other chains within the microfibril or neighbouring microfibrils via covalent lysine and hydroxy-lysine cross- linking (60). Orgel et al. described the presence of several clusters of these peptides

(within the telopeptide region) having an adequate bonding distance to make possible those interactions (60). This continuous and uninterrupted arrangement within the microfibril could explain why these microfibrils despite being identifiable entities are still not possible to extract and isolate from tissue samples (33). Moreover, the mechanical properties could also be explained by these interactions (60).

Based on in vitro observations, it has been established that collagen fibrils increase their diameter in multiples of 8nm (64), ranging from 30 to 500nm depending on tissue, age or genetic defects. According to Scott (65), larger fibrils are arranged in 10-

25nm bundles of microfibrils, which is consistent with the multiple embrace of four or five collagen microfibrils by proteoglycan such as decorin (66). Although studies of demineralised dentine describe a wide range of collagen diameters, the dimension commonly described is around 100nm (16, 67).

The fibrillar organization of the collagen is vital in the formation of bone and teeth

(68). This has been associated to the molecular architecture of the fibril, where the amino acid residues generate electrically charged areas (69). Within the fibrillar structure, groups of charged residues of amino acids tend to occur in groups (70). The predicted charged distribution has been compared with staining patterns using heavy metal salts in electronic microscopy (70). Figure I-4 shows the different dark bands

occurring within a D-period (C) due to uptake of the heavy metal staining by the charged lateral residues (A) with the notation introduced by Hodge and Schmidt (71)

(B). The bands are distributed within the D-period and marked as a3, a2, a1, e2, e1, d, and c3 (located in the gap area) and c2, c1, b2, b1 and a4 (overlap area). These bands not only have electrical charges, but also provide flexibility to the microfibril (72).

However due to the amphoteric nature of collagen, the surface potential could be affected by the pH. Thus, Uquillas and Akkus (73) developed a model to predict the charge profile of collagen type I at different pH. According to their prediction, collagen presents positive net charge in acidic solution, it is equally positive and negatively charged in a slightly basic medium, and it is negatively charged at pH 13.

Using a novel approach, Stone and Mesquida (74) reported that it is possible to detect the inhomogeneous distribution of ionisable residues of collagen type I after been immersed in different solutions using Kelvin probe force microscopy (KPFM). This approach allowed them to correlate potential- and topographic profiles obtained for the collagen surface (74).

Figure I-4. Electron micrograph of collagen fibril. A, positive staining pattern of a double stained fibril, first with phosphotungstic acid and then with uranyl acetate; showing different bands. B, labelling of the bands and representation of the stain intensities. C, negative staining pattern with sodium phosphotungstate, showing gap (0.54D) and overlap (0.46D) regions in a D-period of 67nm. Adapted from Chapman et al. (70)

I.3.1.2. Mineralisation

Collagen mineralization is a complex process (67). The composition and configuration of the collagen matrix provides in some way the adequate microenvironment for the hydroxyapatite formation (67). Depending on the type of collagen present within the matrix, it is possible to differentiate the mineralization phase of the calcifying tissue.

Lane et al. (75) examined bone tibial healing process to characterise the expression profile and localisation of collagen types at different stages. The authors described the expression of collagen type III at early stages of callous formation, with no visible detection in pre-existing bone; posteriorly replaced by type I in zones where lamellar bone was found; type II collagen was found only in zones of chondroid differentiation(75). Moreover, collagen type I, undergoes differential post-

translational modification (for example glycosylation and cross-linking) in hard and soft tissues (67).

The configuration of fibrils is important in the process of mineralization. Channels of

2nm are present within bundles of microfibrils. These nanochannels seem to facilitate the movement of calcium and phosphate ions, and even allowing the tissue to remain almost structurally unchanged after nucleation (76). The entry gaps for these channels are located throughout the D-period (77), and may serve as entry point for the mineral. However, the infiltration of mineral has been observed in positively charged areas within the gap region, more specifically within the a-band (Figure I-4) of collagen (69). This site was described, based on an analysis of net charge, as having the lowest electrostatic potential energy, thus enhancing interaction with negative charges(69).

Additionally, functional modifications such as those occurring under stress have confirmed the relationship between form and orientation of the inorganic crystal with the macromolecular arrangement of the collagen fibre (78).

The tooth mineralization apparently is commanded by non-collagenous proteins (16).

Glycoproteins and proteoglycans are among these non-collagenous proteins located in dentine, which are covering collagen fibrils and it is believed they have association with the hydroxyapatite (HA) (16). Thus, phosphoproteins seem to be critical in binding calcium phosphates and facilitating the nucleation of HA (79-81).

The shape of the mineral in dentine varies from needle-like near the pulp to plate-like at the dentine-enamel junction (82). The reason for this is not clear. However, a possible explanation is likely to be a different maturation degree of the crystal

throughout the dentine (82). In addition, the thickness of the crystal has been measured using X-ray techniques between 4 to 5nm (82, 83).

In contrast to enamel, the apatite content of dentine is organised with respect to collagen fibrils in extrafibrillar (between fibrils) (84, 85) and intrafibrillar mineral especially in the gap zone of fibrils but also extending between molecules (54, 86).

Some evidence shows that after continuous demineralisation of dentine, the extrafibrillar mineral more accessible (ca. 65-70%) was easily removed and the 30%-

35% remaining corresponding to intrafibrillar was more resistant to dissolution (87).

Interestingly, the proportion of intrafibrillar mineral seems to influence the mechanical properties of the tissue especially concerning to elasticity and hardness

(54). Dry dentine lacking intrafibrillar mineralization was tested for hardness and modulus, obtaining values similar to normal dentine (E= 20.4±1.8 GPa vs 23.9±1.1

GPa and Hardness= 0.65±0.04 GPa vs 0.83±0.08 GPa respectively). However in wet conditions the results were clearly dissimilar (E= 5.7±1.4 GPa vs 20.0±1.0 GPa and

Hardness= 0.2±0.03 GPa vs 0.85±0.1 GPa, respectively) (88).

I.3.2. Non-collagenous proteins

The non-collagenous proteins (NCPs) are proteins and proteoglycans, which during dentinogenesis play an active role in promoting, controlling and regulating fibrillogenesis, crystal growth and mineralisation (89). They constitute the remaining

10% of the organic content in dentine (90). Similar to collagen, NCPs are synthesised and secreted by odontoblasts (Table I-2).

The proteoglycans have structural, metabolic, and functional roles and are key during mineralisation of dentine (89). Decorin and biglycan, part of the small leucine-rich proteoglycan (SLRP) family, are important in directing crystal growth

(mineralisation) and regulating fibril growth during dentinogenesis by binding type I pro-collagen and collagen (89, 91). Although the exact role of all NCPs is not completely elucidated, apparently the lack of some NCPs could result in abnormal development of teeth such as dentinogenesis imperfect Shields type II which has been observed as a product of mutation of the dentine sialophosphoprotein (DSPP) gen

(89, 92).

Table I-2. Non collagenous proteins in dentine matrix. Decorin Small leucine-rich Biglycan Proteoglycans proteoglycan (SLRP) Fibromodulin (PG) family Lumican Osteoadherin Large aggregating PGs Versican Vitamin K-dependent Osteocalcin Glycoproteins Secretory calcium- binding phosphoprotein Osteonectin (SPARC) (SCPP) family Glycoproteins Osteopontin Dentine matrix protein 1 Bone sialoprotein SIBLING proteins Dentine sialophosphoprotein Matrix extracellular phosphoglycoprotein Albumin

IgG Serum proteins Transferin

Fetuin-A

MMP-8 (collagenase-2) Matrix MMP-2 (gelatinase-A) metalloproteinases MMP-9 (gelatinase-B) Enzymes (MMPs) MMP-20 (enamelysine) Cysteine Cathepsins Cathepsin B Insulin-like growth factor-I (IGF-I)

Skeletal growth factor/Insulin-like growth factor II (SGF/IGF-II) Transforming growth factor-beta 1 (TGF-β1) Platelet-derived growth factor Growth (PDGF) Factors Vascular Endothelial grwoth factor (VEGF) Placental growth factor (PIGF)

Fibroblast growth factor-2 (FGF-2)

Epidermal growth factor (EGF)

Adrenomodullin (AM)

Adapted from Orsini et al. (88)

I.3.2.1. Enzymes

Physiological and pathological dentine formation conditions have been extensively investigated (93). The organic matrix consisting mainly of type I collagen is highly resistant to general proteolysis and for its degradation requires the action of specific enzymes families (i.e. matrix metalloproteinases and Cathepsins) (94).

I.3.2.1.1. Matrix metalloproteinases (MMPs)

MMPs are a family of 24 mammalian Zn+2 and Ca+2-dependent enzymes also known as matrixins, which are involved in normal tissue remodelling and in pathologies where the turnover of extracellular matrix is affected (93, 95). MMPs have been classified in six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMP

(MT-MMPs) and other MMPs (96). Nonetheless, most of them are able to degrade different substrates at different rates (or with variable specificity) (93). These enzymes are detected during remodelling or reparation, in diseased or inflamed tissues (97). They are kept in an inactive state (ProMMPs) within the tissue and are activated by proenzymes (97) or by ectopic perturbation of the cysteine-zinc interaction (96). Once activated, they could be inhibited by natural inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), synthetics inhibitors such as tetracycline, chlorhexidine; or by internalization (97). Similarly, α2-macroglobulins are able to inactivate MMPs in body fluids. However, this type of inactivation is irreversible by forming a MMP-macroglobulin complex, which is endocytosed and eliminated posteriorly (96).

Interestingly, in vitro studies have shown that MMPs have low substrate specificity

(able to degrade different proteins), thus, several strategies such as proteomics, substrate-binding motifs and deduction have been used to identify their potential role or substrate (97). For example, MMP-14 has been identified as key factor in the turnover of the extracellular matrix (98-100). Nevertheless, only three groups are directly related in degrading fibrillar collagen: collagenases, gelatinases and MT-

MMPs (93). In fact, in highly packed collagen (triple helix) the only enzymes able to cleave this structure are collagenases and cathepsin K (96, 101).

The ability of a MMP to degrade collagen is depending on the structure of each part

(catalytic site, C-terminal hemopexin-like domain and the hinge region) (102, 103).

Although the mechanism is not clearly understood, some alternatives have been proposed. Possibly binding the catalytic and hemopexin-like domains simultaneously to the triple helix would cause the collagen structure to bend and distort, facilitating cleavage (95). Thus, the collagen molecule would be sliced in ¼ and ¾ segments (95).

I.3.2.1.1.1. MMPs and tooth

MMPs have been identified as part of the tooth development, organising components and compartments of the pulp-dentine complex (93). In early stages, the expression of

MMP-1, -2, -3, -9, -14 and -20 and TIMP-1, -2 and -3 have been detected(104-110); in later stages such as dentinogenesis, MMPs-2, -9 and -20 have been identified (111,

112). In addition, it has been suggested that MMP-3 and TIMP-1 participate actively in the formation of intratubular dentine (113-115). Moreover, MMP-2 could regulate the

differentiation of mesenchymatic cells to odontoblasts through its action over the dentine matrix acidic phosphoprotein-1 (DMP-1) (116).

In adult teeth, odontoblasts have the potential to produce almost the entire family of

MMPs (117). However in human, only gelatinases MMP-2 and -9, collagenase MMP-8 and -20 have been described as synthesised by odontoblasts (111, 112, 117, 118).

I.3.2.1.1.2. MMPs in mineralised dentine

In addition to gelatinases (MMP-2 and -9) and collagenases (MMP-8 and -20), MMP-3 has also been reported in mature human dentine (119), but its expression is reduced in odontoblasts (117).

Although MMPs are important in physiological processes in dentine, they also are involved in pathologies affecting the pulp-dentine complex. For example, the organic matrix composed almost exclusively by collagen type I is degraded by MMPs during dentine caries (120-122) and during the process of degradation of the collagen-resin interface or hybrid layer (123-126).

MMPs are also able to activate growth factors such as transforming growth factor beta

(TGF-β) (127) and therefore regulating the cellular defensive response (128) and tertiary dentine formation (129). Same process has been described for cysteine cathepsins (130).

I.3.2.1.2. Cysteine Cathepsins

In humans, these enzymes are a group of 11 proteases, which are active and stable in slightly acidic conditions (131). Their functions involve tissue remodelling and

turnover of the extracellular matrix, regulation of the immune system, and modulation and alteration of cell function (93). They are also involved in pathological processes where amplification of the degradative cascade could lead to tissue damage, facilitating cancer cells invasion (93). Cysteine cathepsins are synthesised in an inactive form and later activated either via endosomic acidification or by other proteases (132). In addition, cysteine cathepsins can be inactivated at neutral pH.

Exceptionally cathepsin S is active at neutral or slightly basic pH and cathepsin B needs a pH of 7.4 for its endopeptidic action (93, 133-135). The most relevant cysteine cathepsin associated to mineralised tissue resorption is cathepsin K, which is highly expressed in osteoclasts and odontoclasts (136, 137). In bone resorption, osteoclasts reduce the pH in the lacunae. This reduction enables the cysteine cathepsin collagenolytic activity, especially cathepsin K. With the increasing calcium and phosphate available due to demineralization, the pH increases, this inactivates the cathepsin K but creates an ideal environment for MMPs to continue the collagen degradation (138).

The activity of cysteine cathepsins has been detected in dentine under normal conditions and in carious lesions (94, 130). Their expression has been demonstrated in odontoblasts and pulp tissue (94). Their activity is higher in carious lesions compared to sound dentine, as well as in deeper dentine (closer to the pulp) especially in younger patients (130). Therefore, they could have a great impact in the success of dental restorations, which are principally indicated as treatment of caries lesions with some degree of pulpal inflammation, and probably higher expression and activity of cysteine cathepsins (131).

I.4. Dental Caries

The dental caries is a pathological process of gradual demineralisation of the tooth structure due to disequilibrium between physiological process of remineralisation and demineralisation (139). The disease rarely is self-limiting (only when dental plaque is removed) and it also involves the destruction of the organic component of the tooth (17, 139). The destruction of the tooth structure is often referred as caries lesion (139). However, the range of lesions vary from ultrastructural or nanoscale level (still reversible) to total destruction of the tooth (irreversible) (139).

Several factors are associated in the development of caries; amongst them is the presence of bacteria in an organised biofilm or dental plaque (139). In early stages of the process, bacterial acids diffuse into the enamel (or cementum) and consequently the hydroxyapatite is solubilised (17). If the local pH is maintained below 5.5 remineralisation is avoided and then further destruction could take place (17).

Once dentine is affected, the process is irreversible and bacterial invasion is found in the dentine-enamel junction (17). In addition, following the dissolution of the mineral content of dentine, the collagenous organic matrix is exposed (18). Evidence shows that host-derived enzymes are responsible for the collagen degradation, i.e. matrix metalloproteinases (MMPs) and cysteine cathepsins (17, 120).

Although in most industrialised countries, including the United Kingdom there is evidence of a reduction in the incidence of caries related to an improvement of the economic wealth since the 1980s (140), the caries disease or mainly its consequences continues to be one of the main tasks of the dental workforce (141). In fact, nowadays

dentists must frequently direct their efforts towards repairing or replacing defective restoration rather than treating new lesions or preventing them (141).

I.4.1. Caries treatment

I.4.1.1. Non-invasive

The current philosophy to treat caries is to prevent and detect the disease before restorative treatment is required (2). For this, it is essential early detection of the disease and clinicians need to perform a risk-assessment of each individual patient to propose an adequate plan to avoid disease or intervene as minimally as possible when lesions are detected (142).

Clinically, early stages of caries are detected as a sub-surface enamel demineralisation, often referred as a white spot. At this level, the lesion can be arrested by several strategies; all of them include the use of fluoride because it can enhance the uptake of calcium and phosphate ions forming fluorapatite (2). This fluorapatite is more resistant than hydroxyapatite, demineralising at pH 4.5 or lower (143). However, implementing a prevention strategy and follow-up schedule after this non-invasive treatment are crucial steps that require patient commitment and involvement (142).

I.4.1.2. Invasive treatment

When early detection of the disease is not possible and partial destruction of the tooth has happened, removal of the damaged tissue and posterior restoration are needed.

Currently, restorative treatments are commonly driven by aesthetic requirements of

patients. The use of resin-composite materials is widely spread as an alternative to resolve those requirements allowing at the same time the adoption of minimal intervention techniques (144). This technology relies in the adhesion or micromechanical retention of the resin-composite to the tooth structure, i.e. enamel and dentine (145). Early attempts to adhere resin-composites to the tooth structure were successful after enamel acid etching, reported by Buonocore (146). However, adhesion to untreated dentine was still challenging to achieve due to its inherent structure and moisture. It was not until the 1980s, when Nakabayashi (147) started to etch dentine and enamel (total-etch technique) that adhesive treatments began to be more reliable. From that point to present time, several adhesives systems have been launched onto the market. Thus, nowadays the union between the hydrophobic resin- composite and a hydrophilic substrate (dentine) is achieved through bonding systems.

Prior to application of bonding agents, the calcium-phosphate mineral contained in the hydroxyapatite is partially dissolved by acid etching (etch-and-rinse approach) or acidic monomers (self-etch approach) in order to expose a porous dentine collagen matrix where posterior monomer penetration and formation of the hybrid layer occurs (16).

I.4.1.2.1. Etch and rinse approach

The use of etch-and-rinse system demineralises the dentine surface exposing the collagen network, which is then infiltrated with primers. In other words, when dentine is demineralised, extrafibrillar mineral is rapidly removed and the collagen fibril and non-collagenous proteins remain exposed and swell in water (54). Although

it is not clear if extrafibrillar demineralization occurs faster than intrafibrillar, it has been observed that intrafibrillar apatite requires longer times to be completely removed from the collagen gap zones, up to 3600s in 10% citric acid (54), which suggest the mineral is protected from extracellular fluids. Thus, the acid treatment provides spaces where monomer infiltration is possible. Once primer and bonding (or both together) are applied into this collagen web, the new matrix is called hybrid layer. Therefore, the hybrid layer is composed by collagen fibrils and adhesive resin

(148).

It has been described that application of 37% phosphoric acid for 15s is able to remove the smear layer, as well as tubular plugs and peritubular dentine (149). Using

AFM no denaturation of the collagen fibril, based on 67nm banding preservation, was found after acid treatment (149). Moreover, spectroscopic analysis using Attenuated

Total Reflectance technique of Fourier Transform Infrared spectroscopy (ATR-FTIR) has demonstrated chemical variations of amides, proline and hydroxyproline in the collagen type I after acid application (150). In addition, the depth of demineralisation has been measured between 3 and 7 microns (linear) (148). However, it has been described a decreasing infiltration of resin monomers through this demineralised dentine (151). As a result of this incomplete diffusion, the bottom of the hybrid layer contains denuded fibrils (152, 153). Interestingly, the use of acid etching increase the bond strength but the wetness degree of dentine before priming greatly influences bond strength achieved in vitro as well as in clinical situations (148).

The collapse of the collagen matrix after acid treatment can be effectively reduced by the presence of water (149, 154). In fact, it has been established that the collapsed

collagen mesh might be raised up again after water treatment (155-157). The water- filled porosity of the un-collapsed collagen is vital to adhesive infiltration (19).

I.4.1.2.2. Self-etch approach

The essential difference between etch-and-rinse and self-etch systems is based in the application of conditioner and primer at the same time, as self-etching systems contains acidic monomers performing both actions (158). Due to the elimination of the water-rinsing step, this simplified system has been more attractive to clinicians as it claims to be user-friendlier (reducing application time and steps) and less sensitive

(no wetness evaluation of dentine is required) than etch-and-rinse (148, 158). In addition and as a consequence of the technique, the smear layer (left after the cavity preparation) is included within the hybrid layer (148). The preservation of the smear layer has been associated with lower incidence of postoperative sensitivity (159, 160), due to the resistance of fluid movement caused by the smear plugs obliteration of the tubules (161).

Furthermore, self-etching adhesives require to be ionised in order to demineralise the tooth structure. The ionisation of the acidic groups is usually achieved by incorporation of water in the compounds (148). Therefore, these products are hydrophilic by essence, which could cause water uptake and posterior plasticisation of the resin (162). The water sorption is markedly higher in one-bottle adhesives

(163).

Similar to etch and rinse, an incomplete resin infiltration of the dentine resulting in nanoleakage has been found with this approach (164). In this case, the cause has been

attributed to incomplete removal of water, which remains bonded to the hydrophilic resin monomer (123). It might be important to highlight at this point that the effects of demineralising agents (used either in etch and rinse or self-etch approach) on the collagen fibrils has received less attention. Most of the knowledge has been described limited to changes on the collagen banded pattern (fibrillar-scale) (19, 149, 157).

However, no information of their effects at a molecular or microfibrillar scale is found in the literature (15). These changes, if any, could expose active sites where MMPs could bind and start their collagenolytic activity (15).

I.5. Tooth-resin interface

I.5.1. Adhesion/micromechanical interlocking

The adhesion of resin-composites to the tooth structure relies on exchanging inorganic tooth material (HA) for synthetic materials (resin-composites) (165). For this, calcium and phosphates are superficially removed, leaving micro-porosities exposed in enamel and dentine. Hence, resins are able to infiltrate these substrates; to be subsequently polymerised in situ in a process called hybridization. As a result, a micromechanical interlocking based mostly on diffusion mechanisms is achieved

(166). Although micromechanical interlocking is considered as a prerequisite to achieve good bonding, there is scarce information on how monomers interact with collagen. For example, Spencer et al. (167) reported no evidence of covalent bond formation between HEMA and collagen; however the chances of other types of interactions such as hydrogen bonds or van der Waals or even ionic bond formation

cannot be ruled out. In fact, some authors using FT Raman spectroscopy have suggested Hydrogen bonds between monomer (hydroxyethylmethacrylate) and collagen (168). Similarly, H-bonds formation have been observed between an experimental primer (N-methacryloyl glycine) and dentine collagen (169). Moreover, computer modelling techniques have supported the interactions through hydrogen bonds or van de Waals forces between some ligands (for example HEMA and GLUMA) and the collagen molecule (170). However, the authors established these interactions could be limited by kinetic constrains (170). Another evidence of HEMA binding to collagen was observed as a reduction in the T1 value (spin-lattice relaxation time) in the ester carbonyl carbon of HEMA using 13C NMR (Nuclear Magnetic Resonance), which suggest hydrogen bonding formation between monomer and collagen (171).

It is important to establish that the diffusion mechanism will be different between enamel and dentine due to higher collagen content in dentine.

I.5.1.1. Adhesion to Enamel

The adhesion to enamel is effectively achieved with the use of etch-and-rinse bonding systems. Here, a selective dissolution of the HA caused commonly by 30-40% phosphoric acid etching is followed by application and posterior in situ polymerisation of resin monomers. Once applied on the surface, the monomers are readily attracted to infiltrate by the capillary effect created on the etched enamel surface. Finally, the polymerised resin-composite is mechanically interlocked to the enamel by macro and micro tags formation (result of a combination of infiltration/polymerisation within prismatic spaces) (166).

I.5.1.2. Adhesion to Dentine

At dentine, most systems rely on demineralization treatments of its surface (172-

174). For this, the mineral content on the surface is removed, similarly to enamel, totally or partially by acid treatment or by using acidic primers, respectively (175). As a result, the dentine collagen matrix is exposed; and subsequently it could be infiltrated with adhesive monomers, filling the space left by the inorganic content.

Interestingly, most of the research regarding the consequence of acid solutions on dentine, overlook the effect on collagen (176). In fact, the dentine matrix is considered to be relatively inert (177). Often, it is concluded that acid has not had an effect on collagen due to conservation of the D-period (178, 179).

Based on the observation of hierarchical assemblies of the collagen fibril (65), it could be assumed that 100nm diameter collagen fibres described in dentine (180, 181) are also disassembled in 10-20nm micro-bundles due to the conditioning treatment, similar to subfibrillar structures described by Scott (65).

However, the process of infiltration is far from perfect. Here, the encapsulation of collagen is not complete leaving zones of denuded fibrils especially at the bottom of the hybrid layer (148, 175), maybe due to the complexity and structural barriers formed after conditioning (15). In addition, and based on the model of the collagen structure proposed more recently by Orgel (60), the spaces left as a product of the lateral packing within a collagen microfibril fluctuate between 1.26 to 1.33nm. These spaces should be theoretically filled by resin monomers, however these monomeric molecules such as TEGDMA have a size of at least 2nm (15). Therefore, these spaces

filled with water (182), are where a time-dependant degradation of the hybrid layer begin (175). Although self-etch approach was designed to demineralise and infiltrate at the same depth, incomplete infiltration leading to nanoleakage of the hybrid layer has also been described (182, 183). Sano et al. (164, 183) described the nanoleakage phenomena using SEM and TEM, which was referred to the porosities found within or underneath the hybrid layer. In conclusion, it is still not elucidated how the structure of dentine at a nanometer scale interacts with resin monomers and how the dentine morphology may be altered by acid etchants, which could have implications on that interaction, leading to nanoleakeage and ulterior adhesive failure.

I.5.2. Failure of the adhesion

In general, it is said that hydrophilic dentine adhesives produce a resin-dentine bond that deteriorates over time (123, 152, 184-186). Thus, the degradation of the hybrid layer involves hydrolytic dissolution of collagen and hydrophilic resins, ending in reduction or total loss of the bond strength between its components. In fact, several studies have found evidence of elution and/or collagen degradation in aged hybrid layers (187-190). Thus, the two major mechanisms by which a time-dependent deterioration of the hybrid layer occurs are: hydrolysis of the adhesive and degradation of water-rich, resin-sparse collagen (123, 191).

I.5.2.1. Hydrolysis of the resin adhesive

Water present within the hybrid layer due to a lack of infiltration or trapped after polymerisation added to a loosely cross-linked resin, or the presence of hydrophilic monomers is responsible to promote hydrolysis of the ester bonds in the resin. This 46

slow reaction allow monomer leaching, but it also permit the access of esterases, which are able to accelerate the process of hydrolysis (192). In addition, the continuous elution of resins could expose previously encapsulated collagen to the attack of enzymes (123) .

I.5.2.2. Collagen degradation

In dentine, the degradation of collagen fibrils is a consequence of the incomplete infiltration of resin. Evidence of dentine collagen degradation in resin-restored tooth has been found in lateral branches of dentinal tubules. Hashimoto et al. (187, 193) described widening of these zones after a period of ageing. The hydrolysis of collagen within the hybrid layer suggest a lack of monomer infiltration and therefore encapsulation of the fibres within a matrix zone previously demineralised (148).

Similarly, in vitro studies have demonstrated degradation using morphological analysis in samples aged in water (188, 193). These non-encapsulated collagen fibrils are a subject to enzymatic attack. Two major groups of enzymes (MMPs and cysteine cathepsins) are thought to be responsible of the complete degradation of some segments of the hybrid layer (186, 188). Accordingly, the evidence indicates that

MMPs trapped within the dentine matrix are exposed after acid demineralization (18,

120, 194). Although most of the MMPs remain bound to the collagen after acid treatment (194), they are kept activated, and consequently they slowly degrade the collagen matrix (123).

The model of collagen degradation in dentine establishes that although MMPs are released and activated during dentine bonding (123, 195, 196), the molecular

arrangement of collagen is governing the exposure of the binding sites where MMPs could be attached and thus start their collagenolytic attack (15). In general, it was assumed that collagen-degrading MMPs could easily digest collagen monomers as the latter would freely rearrange to accommodate the MMP binding site (197, 198).

However, the mechanism seems to be much more complex (15). In the molecular model based on the interaction of MMP1-collagen type I, MMPs have a 0.5nm wide active site (199) unable to allocate the intact triple helix (1.4nm) and therefore it would require to bind and then unwind the triple helix before the active site is located in one α-chain of the collagen molecule and hydrolysis could begin (199). In addition,

Chung et al. (199) demonstrated that the region where cleavage can take place is entirely protected by the C-telopeptide and thus, intact collagen molecules are difficult to degrade.

The current hypothesis of collagen degradation in dentine establishes a molecular rearrangement of the fibrils induced by acid attack (i.e. in the caries process or during dentine conditioning), which could be able to break cross-links at the C-terminal region, and thus exposing the binding site for MMP cleavage (15).

Nevertheless, the mechanism by which MMPs and cysteine cathepsins are interrelated in degrading the hybrid layer is still unclear (131). Most of the knowledge is based in association occurring in other tissues, consequently it has to be taken as hypothetical until dentine-based research had been done (131). For example, cathepsin B (from cartilage) is able to degrade TIMPs (-1 and -2), consequently MMPs activity is maintained due to loss of the regulatory role of these inhibitors (200). In turn, active

MMPs (from peritoneal macrophages) are able to cleave procathepsin B (201). Once

active, cathepsin B was found to be able to cleave MMP-1 in gingival fibroblast cultures (202).

Another important association between MMPs and proteoglycan/glycosaminoglycans has been established, which could affect the MMPs activation and activity (203).

Similarly, GAGs also could have participation in the cysteine cathepsins activity, allowing them even be stabilised and functional at neutral pH (204). Therefore, it is also possible that GAGs regulate the action of cathepsins in dentine (131).

Undoubtedly, further research is required to achieve a complete understanding of the failure mechanisms of the adhesion and hence increase the durability of restorations.

I.5.3. Longevity of resin restorations

An important predictor of bonding effectiveness of adhesives is related to the clinical failure of the restorative treatment (205). Accordingly, better results of the adhesive interface are translated to longer life or longevity of resin-composites. The evaluation of restorations in non-carious cervical lesions to compare the annual failure rate of different bonding systems, namely etch-and-rinse (E&R) and self-etch (SE) adhesives is commonly used as an indirect measure of longevity (205). Six groups of adhesives have been compared and contrasted to glass ionomer in an extensive systematic review (205). The groups analysed were: 3-step E&R, 2-step E&R, 2-step SE mild and intermediary strong (pH≥1.5), 2-step SE strong (pH<1.5), 1-step SE mild and intermediary strong, and 1-step strong. From this research, Peumans et al. (205) established three groups with the lowest annual failure rates namely 2SEm, 3E&R and

1SEm; the intermedium performance was described to 1SEs and 2E&R; and 2SEs obtained the worst results (Figure I-5).

Annual Failure Rate 9 8

7 6 5

4 AFR (%)AFR 3 2 1 0 1SEs 1SEm 2E&R 2SEs 2SEm 3E&R GI Adhesive systems

Figure I-5. Annual failure rate (AFR) of different adhesive systems. SEs: Self-etch strong acidity, SEm: Self-etch mild acidity, E&R: etch and rinse, GI: Glass ionomer. Adapted from Peumans et al. (205)

I.5.4. Current strategies to prevent degradation of the hybrid layer

Despite huge improvements of resin-composites in terms of sealing and bonding capabilities, the interface tooth-restoration is the weakest point of this restorative system, especially in dentine (206). As a result, several strategies to increase the stability of the hybrid layer are continually investigated. In general, these strategies can be divided in adhesive-related and enzyme-related.

I.5.4.1. Adhesive-related strategies to prevent hybrid layer degradation

Although most of the adhesives systems used nowadays have demonstrated positive results after short term in vitro test, mostly by counteracting polymerisation shrinkage (206), there are still problems in long term results due to the afore mentioned hybrid layer degradation. Several attempts or strategies to increase the bond stability have been and continue to be explored. Amongst them the use of an additional hydrophobic layer (207-210), multiple layers (211), enhancing evaporation of the adhesive carrier (212), extension of photo-curing times (213), or the use of electrical devices able to induce deepest monomer infiltration (214, 215) seem to achieve the goal of retarding degradation.

The rationale for the use of hydrophobic layer and multiples layers, and even enhancing solvent evaporation is based in the inherent permeability of the hybrid layer. Thus, additional coatings reduce nanoleakage (207-211), and phase separation is reduced by enhancing evaporation (212). Also considering the problems related to the resin-based problems such as elution and permeability, Cadenaro et al. (213) proposed increasing the curing-time period to avoid sub-optimally polymerised resin, which was associated with these two problems (elution and permeability).

The use of an electric potential difference between a device and the tooth preparation to encourage infiltration showed to reduce the nanoleakage and improved bond strength results compared to controls (214). In addition, Toledano et al. (216) demonstrated an improvement of wettability of the dentine substrate with the use of this device (Electrobond).

I.5.4.2. Enzyme-related strategies to prevent hybrid layer degradation

Due to the increasing amount of evidence of host-derived proteases degrading the collagen within the hybrid layer (17, 119, 195, 217, 218) several investigations are focussed in identifying inhibitors of MMPs and cysteine cathepsins, most of them involving chlorhexidine (123, 125, 219). In addition, chlorhexidine has been used largely in treatment of periodontal diseases as antimicrobial agent (220) and as a disinfectant before restorations (221). One of the most known MMPs-inhibitors used in the oral environment is tetracycline and its derivatives (93). This family exerts its inhibitory action by chelating cations and by scavenging reactive oxygen species that allow activation of pro-MMPs (222, 223). In addition, tetracyclines cause down regulation of MMP transcription levels (224-227). Other potential inhibitors of MMPs found in the literature are galardin, epigallocatechin-3-gallate, quaternary ammonium salts and ethylenediaminetetraacetic acid (EDTA) (221). In addition, recent studies related to bisphosphonates which are pyrophosphate analogues used to treat conditions where abnormal bone resorption occurs such as in Paget’s disease or osteoporosis (228), have shown down-regulation and inhibition of MMPs (229-232).

I.6. Resin-composites roughness and measurement techniques

Dental resin-composites have been used increasingly since their introduction more than 50 years ago (233). The success of these restorations rely not only their ability to adhere to the tooth but also on the resilience of the surface to mechanical wear (233,

234). These materials are in general terms, a combination of a polymeric matrix, inorganic fillers, a coupling agent to allow interaction between them, and initiators of the polymerization (233). During the evolution of these materials, one of the most important alterations was the introduction of filler particles to reinforce the organic matrix (233). The reduction in size of the reinforcing filler and modification in the organic matrices led to improved aesthetics (by facilitating polish) and also a higher wear resistance (233, 235). The surface texture of resin-composites has also been described as critical on plaque accumulation, gingival inflammation and wear behaviour (236). Roughness is defined as fluctuations of high frequency and short wavelengths of a measured surface (237). According to Quirynen and Bollen (238,

239) and Teughels (240) there is a clinically acceptable roughness threshold located at Ra 0.2µm (Roughness average). Thus, rougher surfaces increase the risk of periodontal disease and secondary caries (by promoting bacterial adhesion) as well as they could cause excessive wear of the opposing tooth (240-243). In contrast, smoother surfaces retain aesthetics and improve longevity of restorations (241).

Roughness is characterised by peaks (local maxima) and valleys (local minima). Due to the impossibility of measuring the height and location of every peak, only a representative section of the sample is used to calculate roughness. There are different techniques to study surface topography (Table I-3), such as surface 53

profilometer, optical microscopy, scanning electron microscopy, transmission electron microscopy, scanning tunnelling electron microscopy and atomic force microscopy (237). The description of each technique goes beyond the scope of this thesis. However, all these methods register together roughness and form error or waviness (irregularities of longer wavelengths), which can be then decoupled.

According to Kakaboura et al. (244), atomic force microscopy is able to discriminate more accurately than profilometer methods and provide superior surface texture definition compared to SEM.

Measures of surface roughness are commonly used to compare different composites, polishing procedures or the effect of brushing (241, 245-249) with a view to selecting the most suitable materials or polishing systems for clinical practice. However, not only surface roughness can be characterised using different parameters but the relative roughness may depend on the size of the measure area and the resolution of the instrument used (237, 241). Interestingly, in dental research investigating roughness surface, the dimensions used to determine roughness were either different or not mentioned (240, 241, 245-250).

Table I-3. Methods for measuring roughness (†) Method Quantitative 3-D Resolution at Limitations/Comments data data maximum magnification (nm) Horizontal Vertical

Operates along linear Stylus track, contact type can Yes Yes 15-100 0.1-1 profilometer damage sample, slow speed in 3-D mapping Light Qualitative Limited Yes 500 0.1-1 sectioning * Taper Destructive, tedious Yes No 500 25 sectioning * speciment preparation Specular Semiquantitative No No 105-106 0.1-1 reflection * Diffuse Smooth surfaces Limited Yes 105-106 0.1-1 reflection * (<100nm) Speckle Smooth surfaces Limited Yes pattern * (<100nm) Optical Yes Yes 500-1000 0.1-1 interference * Operates in vacuo, limits SEM Limited Yes 10 0.1 on specimen size Operates in vacuo, TEM Limited Yes 0.5 0.02 requires replication of surface Requires a conducting STM Yes Yes 0.2 0.02 surface, scans small areas AFM Yes Yes 0.2-1 0.02 Scans small areas (†) Adapted from Sahoo, P (237) (*) Optical Methods

I.7. Clinical implications

Resin-composites are increasingly used as the preferred choice when a direct restoration is required (3, 144). However, biodegradation occurring at the tooth- restoration interface is considered as the main culprit of secondary loss of adhesion, nanoleakage, and together an increased roughness and subsequent plaque accumulation, results in secondary caries (250). Failure of restorations leads to their replacement, which requires additional clinical time and resources and loss of tooth structure as a product of this re-intervention. In fact, according to the National

Institute of Dental and Craniofacial Research (10) of the USA, 60% of the average dentist’s practice time is consumed replacing restorations.

Certainly, further research involving the hybrid layer is essential. Previous research has been centred in the relationship of monomers with the hydroxyapatite but is scarce regarding the effects on collagen after acid treatments or its interaction with monomers. In addition, it is clear that a consensus to evaluate roughness needs to be reached in order to allow comparison between different publications.

I.8. Atomic Force Microscopy

I.8.1 Background

The advance in sciences is related closely with our ability to interact with the environment. The study of physical processes has advance from macro- to micro- and increasingly into nanoscale aided by the creation of new tools and techniques (251).

Thus, in dentistry, it is possible to recognise that the nano-structure and the chemistry of collagen are likely to play a key role in mediating the resin-composite restoration longevity whilst at the micro scale the influence of mechanical wear is also important.

Atomic force microscopy (AFM) is an ideal technique to characterise these key aspects due to its superior resolution and because allows the observation in real time and with little or no sample preparation (244, 252).

AFM is a form scanning probe microscopy (SPM), which gather information of specimens by touching or feeling their surfaces rather than looking at them (253,

254). A sharp probe is use to scan the surface, allowing resolutions on the order of nanometres. Thus, molecules or even sub-molecular particles can be imaged. A big advantage of this technique compared to electron microscopy is that samples can be scanned under natural conditions (255). These characteristics have attracted researchers from biology and biophysics, as AFM potentially can evaluate biological systems with great resolution, under natural conditions and in real-time (254).

As said before, SPM cannot be described exactly as microscopes since images are generated by sensing the surface with a probe. In conventional microscopy, the image is generated by collecting the radiation transmitted or reflected from a specimen. The

highest resolution will depends on the wavelength from the source of radiation and it is limited by the diffraction. Thus, traditional light microscopy has a limited resolution of approximately 200 nm, which in practical terms is a magnification of 1500x (254).

In order to achieve higher resolutions, high energy electrons with shorter wavelengths are used in the electron microscopy. However, this approach requires vacuum or partial vacuum, therefore the specimens need to be prepared in order to maintain their native structure (254). In contrast, AFM allows good resolution of samples with little or no preparation (255). Table I-4 presents main characteristics

AFM compared to SEM and TEM.

AFM images are obtained by registering changes on the probe due to the interacting surface while scanning. The deflection of the probe is perceived by a laser beam and a photo detector (256). Therefore, the resolution is given by the sharpness of the probe and how precise can be tracked the sample surface beneath the probe. So, atomic resolution can be obtained when the tip of the probe is in intimate contact with the surface of a flat sample, either in air or liquid-environment (253).

Table I-4. Comparison of Atomic Force Microscopy, Scanning electron microscopy and Transmission electron microscopy main characteristics. AFM SEM TEM Sample type Conductive Conductive Conductive / non Conductive Sample easy or easy to easy to preparation none difficult difficult Sample Vacuum or Any Vacuum environment Gas Horizontal 0.1 nm 5 nm 0.1 nm resolution Vertical 0.05 nm NA NA Resolution Depth of Poor Good Poor field Imaging 2 - 5 min 0.1 - 1 min 0.1 - 1 min time Maximum 100 um 1 mm 100 nm field of view Maximum sample size Unlimited 30 mm 2 mm Cost Low Medium High Adapted from Eaton and West (252)

I.8.2 Configuration of the microscope

In order to obtain an image using AFM, the surface of a specimen is tracked accurately using a probe that is touching or sensing the surface. The magnitudes of the changes due to the topology and the nature of the sample are registered and the image is built.

To sense the surface on AFM a probe consisting on a cantilever with a spike or tip fix at its end is used. The length and sharpness of this tip influence the resolution of the instrument. In addition, the cantilever helps tracking the surface by allowing the tip to move up and down. Moreover, to control the force of the interaction between tip and sample by the AFM, cantilevers have usually very low spring constant. For this, probes

are usually fabricated of silicon or silicon-nitride due to the wear resistance, hardness and its suitability to be micro-fabricated (254).

To scan the surface of a specimen, the sample or the tip should be moved in relation to the other. The movement must be controlled and registered accurately in x, y and z directions. For this purpose is used a piezoelectric transducer. When mechanical stress is applied to a piezoelectric material, this material accumulates an electric charge producing a potential difference. As this phenomenon is reversible, the application of a potential difference will result in an expansion of the material. These changes are very sensitive and reproducible. The precise and controllable expansion of piezoelectric ceramics is used in AFM to accurately scan tri-dimensionally a sample.

In fact, each channel (x, y and z) can be controlled individually, allowing the x, y raster-movement without losing the close relation between tip and surface (z- channel) (254).

To monitor the movement of the probe during the scanning-movement is commonly used an optical lever system. Here, commonly a laser beam is directed on the back of the cantilever and a photodiode detector receives the reflected beam and turns that input into an electric signal that varies according to the intensity of the laser beam received. The detector is divided in four quadrants. As the cantilever moves in response to the sample surface, it also changes the angle of the reflected beam and consequently its position on the photodiode detector. The positional changes create a variation on the intensity captured by each quadrant allowing differentiation of up- down (top against bottom quadrants) as well as twisting (left against right quadrants) movements of the tip. The signal gathered on the photodiode detector quadrants is

then collected by a differential amplifier, which is able to discriminate atomic scale changes (253, 254).

An important part of the AFM microscope is the feedback mechanism. During the scanning process the cantilever deflects as the interacting force tip-surface changes.

Without a feedback mechanism, there is a risk of crashing the tip on the surface or losing contact with the surface. For example during contact mode or DC mode, the cantilever is maintained at a specific deflection while the scanning is performed. The information gather by the photodiode is sent to the amplifier to generate an image and then sent to the feedback system to control the z-piezo to maintain or correct the force tip-surface (254).

The entire information gather so far is analogue (i.e. voltage); therefore to be interpreted by a computer is needed a signal conversion to digital (binary) using an analogue to digital converter (ADC), which is part of the AFM electronic system (also called controller box). The now digital signal is sent to the computer’s central processing unit (CPU), which performs all the processing and calculations regarding the operation of the AFM in real-time. All the corrections required to keep the tip- surface relationship are calculated and sent back in a digital form back to the controller box where are transformed in analogue signal by the digital to analogue converter (DAC). This signal feeds another part of the electronic system known as high voltage amplifier. Thus, with this amplified voltage the piezoelectric ceramics are controlled tri-dimensionally, as described earlier (254).

The last piece of electronics inside a common AFM is the laser driver which is well isolated along with the differential amplifier from the other components that generate

electrical noise. This device provides the variable power to maintain the laser intensity stable using its own feedback loop (254).

I.8.3 Imaging modes

There are several imaging techniques in atomic force microscopy. They differ on the interacting force probe-sample aiming to characterise materials. Thus, along with the topography it is possible to describe mechanical properties (e.g. adhesion or friction), electrical properties (e.g. electric potential difference, electrostatic forces), magnetic properties, and optical spectroscopic properties (257). Due to the large amount of techniques, the following paragraphs are dedicated only to the most common imaging modes and to Kelvin Probe Force Microscopy mode (KPFM).

I.8.3.1 Contact mode

Contact DC mode is the most direct mode. Here, the tip is continually in contact with the surface during the scanning of a sample at a determined pre-set force, where the cantilever is deflected by a fixed amount and maintained by the feedback loop; consequently another name for this mode is constant force or deflection feedback mode. The force applied on the surface affects the image contrast and/ or reduce damage to the sample. Softer cantilevers are used for more friable samples, especially considering that shear forces can be generated during the raster scan, thus causing sample damage. Additionally to the risk of damaging the specimen due to shear forces, capillary forces as a result of a thin water layer condensation (scan in air at normal relative humidity-RH) is able to attract even further the tip onto the surface. This force is difficult to compensation by the operator. In order to minimise capillary forces, the

scanning may be performed in low RH or eliminated completely in scanning in liquid.

The latter option is been preferred to study biological samples, as a wide range of buffers can be used (254).

I.8.3.2 AC modes

Another possible alternative to overcome capillary forces caused by the permanent contact tip-sample is to drive the cantilever at a certain vibration frequency. Two modes, tapping (in air or in liquid) and non-contact are the most known (254).

a) Tapping mode (also known as Amplitude modulation). In this technique, an

electrical oscillator vibrates the cantilever at a user-defined amplitude, so when

the tip interacts with the surface of the specimen a dampening on the oscillation

amplitude can be detected and registered. Here, capillary forces can be supressed

but also is possible to ease lateral forces and therefore avoiding distortion or

damage of the sample.

b) Non-contact mode (also known as Frequency modulation). Although in this

technique the cantilever is also vibrated at a pre-set amplitude (here, only a few

nanometres), the tip is kept hovering above the sample (without touching it). In

this case the cantilever oscillating frequency shifts, caused by the van der Waals

attractive forces between tip and sample. Two major weaknesses of this mode are

that it cannot be performed in liquid and when performed in air there is a high

risk of touching and becoming stuck to the sample surface due to the low energy

used to vibrate the cantilever. Nevertheless, due to the ability of sensing a force

gradient rather than force (as in taping), this mode has a great spatial resolution

able to achieve real atomic resolution.

I.8.3.3 Kelvin Probe Force microscopy (KPFM)

KPFM is a technique that measures a contact potential difference (CPD) between the tip (conductive probe) and the sample. Here, the same line is scanned twice; the topography is acquired first (using Tapping mode) and consecutively the surface potentials are measured at a pre-determined height from the surface (258). During the second pass, the mechanical drive is replaced by an AC bias to produce the cantilever oscillation. The electric bias will produce an electric force between the tip and the surface. This force can be nullified by a controller-derived DC-voltage (second feedback) and therefore the local potential difference between the tip and the surface can be known (259, 260). Thus, it is possible to measure the surface potential with nanoscopic resolution (261).

Figure I-6. Diagram of an AFM control system showing the main components. Here can be observed how a sample mounted on a piezo is raster scanned by a flexible cantilever. The bending of the cantilever due to the interaction with the sample, produce a deflection of the laser beam reflecting in the photodetector. The signal obtained in the photodetector allows reconstruction of the topography and give feedback to the piezo in order to avoid crashing the tip on the sample. In KPFM mode an electric bias is used on the tip to detect variation of the surface potential of the sample. Adapted from Morris (254).

I.9. Rationale

This brief review has identified several implications for the design of the current study.

 Failure of the composite resin restorations are related to collagen degradation

and secondary caries.

 Scarce evidence is found regarding the effects of bonding procedures on

collagen type I. These studies are usually limited to analysing the D-Period to

report structural changes.

 Only a pilot study has evaluated the action of different pHs on the surface

potential of collagen type I

 Despite of multiple reports of roughness in the literature, they fail to use same

dimensions of measurement or simply are unreported.

 AFM can non-destructively detect structural changes as well as ionization state

changes in native fibrils without needing sample preparation.

Therefore, the following hypothesis emerge from these findings

1. Bonding procedures induce structural and surface potential changes on collagen

type I

2. The surface roughness of resin-composites are different depending on the

observation size-area

I.10. Aims and Objectives

In order to test the hypothesis “Bonding procedures induce structural and surface potential changes on collagen type I”, this study aimed to evaluate morphological and electrical changes of type I collagen as a consequence of chemical agents commonly used during the dentine bonding treatment. Key objectives in achieving this were i) the comparison reconstituted collagen versus extracted collagen, ii) the evaluation of changes in height, width and D-periodicity on type I collagen before and after application of acid agents, chlorhexidine and a solution of primers; and iii) the detection of electrical potential changes on the surface of type I collagen before and after application of acid agents, chlorhexidine and a solution of primers.

To test the hypothesis “The surface roughness of resin-composites are different depending on the observation area”, it was aimed to determine an optimal area for roughness measurement. Key objectives to achieving this were i) the comparison of the surface roughness of different resin-composites and ii) the identification of the effect of surface area on the AFM analysis of resin-composites

Chapter II. “Optimisation of a collagen fibril model system”

II.1. Overview

This experiment consisted in the use of AFM to characterise the structure of two models: 1) Reconstituted and 2) Extracted collagen fibrils before and after exposure to dental environments (i.e. acid etching at different concentrations) in order to establish which model is more suitable to carry out sequential measurements.

II.2. Materials and Methods

II.2.1. Comparison of collagen models

II.2.1.1. Model-1 Reconstituted Collagen

Two solutions at different concentrations of bovine collagen type I (Nippi, Japan) were prepared for this comparison, 12 and 24µg/ml, in a buffer containing 50mM glycine,

200mM KCl at pH9.2 following the protocol proposed by Cisneros et al. (262). The samples were incubated at 27 °C on mica and rinsed/dried after completing the time determined for reaction in buffer (60min and 30min, respectively). The samples were stored at room temperature until AFM observation. 5x5 µm observations were performed using a Bioscope Catalyst AFM mounted with a Bruker ScanAsyst Fluid triangular silicon nitride cantilever (nominal spring constant of 0.7N/m and a pyramidal tip of nominal radius of 20nm) with a sampling frequence of 512 x 512 (for a lateral spacing of 9.8nm). One image per sample was captured in dry state and at least 4 consecutives captures of the same area were promptly captured after being rehydrated with a droplet of purified water (18MΩcm). The images obtained were

evaluated using WSxM software (263) with Fast Fourier Transform (FFT) and roughness analysis of the height images to determine periodicity, alignment and to determine average height.

II.2.1.2. Model-2 Extracted collagen

Tails from rat donors from tissue bank kept at -20 °C were deskinned and their tendons harvested. Small sections of tendon ca. 5 mm were frozen and crushed in liquid nitrogen following the protocol described by Holmes and Kadler (264). The powder resulting from this process was dispersed in buffer (50mM Tris-HCl (pH 7.4),

100 mM sucrose, 150mM NaCl) using a hand-held Dounce homogeniser. From this suspension, 10 microliter was seeded on freshly cleaved mica and left overnight to allow collagen attach to the substrate. The following day the samples were rinsed using ultrapure water (18MΩcm) to remove any residual solution and stored. An area of 2 x 2 µm were imaged using tapping mode or AC mode performed on a MFP-3D

AFM microscope (Asylum Research) using Silicon nitride probe (Scanasyst-air,

Bruker) with a nominal tip radius of 2 nm at a resonance frequency near 70 kHz, with a sampling frequence of 512 x 512 (for a lateral spacing of 3.9 nm). The images were exported to ascii files (.txt) without performing any modification. Axial dimension were determined using routines written in Microsoft Visual Basic (265). Further post- processing was carried out using ImageJ software (National Institutes of Health: available on the internet al. https://imagej.nih.gov/ij/). Height and periodicity were determined.

II.2.2. Optimisation of AFM experimental conditions

Reconstituted collagen and extracted collagen were prepared as described previously and used to determine acid solutions and time.

Acid solutions

a. Phosphoric Acid: Sequential solutions of 37%, 10% and 1% were prepared by

diluting a stock solution 85% (Sigma Aldrich)

b. Citric Acid: a solution of 10% citric acid was prepared by adding ultrapure water

into citric acid anhydrous 99.7% (AnalaR, BDH Chemicals)

Experimental procedure

1. The samples prepared on mica were initially imaged using tapping mode or AC

mode performed on a MFP-3D AFM microscope (Asylum Research) using Silicon

nitride probe (Scanasyst-air, Bruker) with a nominal tip radius of 2 nm at a

resonance frequency near 70 kHz. Secondly, the samples were acid-treated using

phosphoric acid 37% and citric acid 10% during and initial time of 15 seconds.

After this, the samples were re-scanned; if no image was achieved, new samples

were etched with a reduced time of 10 and 5 seconds. When after these time

reductions still an image was not captured, new acid dilutions were used.

2. Once an optimal acid concentration and time was reached, new samples seeded

on silicon wafer were scanned using tapping mode or AC mode performed on a

MFP-3D AFM microscope (Asylum Research) using a Silicon-Pt coated probe (AC-

240TM, Olympus Corporation) with a nominal tip radius of 15 nm at a resonance

frequency near 70 kHz. During the scanning, a region was selected (2 x 2 µm)

where height and potential channel were registered (scan rate 1 Hz, resolution 71

512 lines; lateral step 3.9 nm). Prior to scan the chosen area, the KPFM signal was

optimised using the fine tune KPFM feedback procedure using an external voltage

source following the procedure described by Jacobs et al. (266), at a lift height of

10-15 nm. Once the initial scan was finished, the sample was acid treated either

with 1% phosphoric acid or 10% citric acid during 5 seconds and rinsed with

ultrapure water and blown-dried. Finally, a new scan was performed after

localising the same region and executing a new fine tune of the KPFM feedback.

II.3. Results

II.3.1. Comparison of models

II.3.1.1. Reconstituted collagen

The collagen molecules assembled into aligned fibrils and establishing a D-band periodicity of 66.19±2.88nm (n=16). The D-period measurement on the dry samples at 12 µg/ml (n=2) was 67.8±0.72nm. After rehydration, 4 images were captured continuously with an interval of ca. 9 minutes; the D-period was 71.59±0.06nm,

64.43±0.22nm, 67.16±1.85nm and 62.48±0.11nm successively. In contrast, only 1 sample at 24µg/ml was successfully scanned and its D-period was 64.07nm. However, after rehydration no image could be taken. Interestingly, the width of the fibrils formed with a concentration of 12µg/ml collagen (Figure II-1, A and B) apparently was more regular than those observed at 24µg/ml (Figure II-1 C). In addition, the average height was variable ranging from 2.1nm (Figure II-1 B.4) to 7.9nm (Figure

II-1 A.2). When clearer images were obtained (from the second sample at 12µg/ml

collagen), the D-period average (D) and average height (H) of the rehydrated images

(Figure II-1 B.1 to B.4) remained close to the values obtained from the dry image

(Figure II-1B) with values D=66.9nm and H=2.38nm (of rehydrated) compared to

D=67.07nm and H=2.34nm of the dry sample.

Figure II-1. AFM images of collagen fibrils at different concentration. Images A and B are dry samples (12µg/ml) and their successive images following re-hydration are captioned from 1 to 4 (at 9 minutes intervals approx). Image C is the dry sample at 24µg/ml and C.1 depicts the mica substrate lacking collagen after re-hydration. Insets depict fast Fourier transform (FFT) used to determine D-period.

II.3.1.2. Extracted collagen

In general, the sample preparation following the protocol proposed by Holmes and

Kadler (264) was able to deliver a good yield of collagen type I. The previously rehydrated specimens were explored using optic microscopy aiming to select an area where an isolated fibril could be scanned using AFM microscopy (Figure II-2). The D- period was estimated in 65.4±2 nm (n=4) and the average height was 154.2±9.3 nm.

Figure II-2. AFM images of representative collagen type I extracted from rat tail tendon. Fibril A, presented an average height of 143.8nm and fibril B of 164.6 nm. Amplitude channel of images A and B are presented in C and D, respectively. Scale bar 500nm. Calibration bar in nm.

II.3.2. Optimisation of AFM experimental conditions

As none of the configuration acid/time produced observable fibrils of the reconstituted fibrils, this section will be focussed on the extracted collagen.

Once the selected areas of collagen samples were successfully scanned, the head of the microscope was removed and the sample was acid treated and rinsed. The effect of phosphoric acid on the collagen samples could not be explored at higher concentrations due to the complete disintegration or removal of the collagen from the surface of the substrate, despite reducing the action time to 5 seconds. Figure II-3B shows a sample treated with 10% phosphoric acid during 5s, where it is not possible to recognise any feature of fibrillar collagen. Once a concentration of 1% phosphoric acid during 5 seconds and subsequent rinse and dry were employed, it was possible to scan again the same fibril in a predictable manner.

Figure II-3. AFM image of the same section of collagen type I extracted from rat tail tendon before (A) and after (B) acid treatment. The configuration used was 10% phosphoric acid during 5 sec. In image B can be seen almost complete disintegration of the fibril. Same calibration bar on both images, in nm.

The procedure was repeated using this time citric acid. The starting concentration of

10% citric acid was applied for 15 seconds with no results. As phosphoric acid was applied successfully for 5s, the second attempt with citric acid was using the same concentration for 5s. This configuration allowed fibrils to be scanned once more

(Figure II-4).

Figure II-4. AFM images of the same section of a type I collagen fibril extracted from rat tail tendon before (A) and after (B) acid treatment (10% Citric acid, 5s). Images C and D correspond to the amplitude channel of fibril presented in A and B, respectively. Here the D-period is observable despite the reduction of height. Same calibration bar on both height images, in nm.

The last part of the experiment was the optimisation of the KPFM signal. For this, the samples were seeded on a conductive silicon wafer, to allow the application of an external AC bias following the KPFM feedback fine tuning procedure described by

Jacobs et al. (266). Height and KPFM images were successfully registered before and after acid treatment.

Figure II-5. AFM topographic (A and B) and KPFM (C and D) map of the same section of a type I collagen fibril before and after acid treatment (10% Citric acid, 5s). It can be seen height and potential changes on the same area of the same fibril. Topographic bar in nm, KPFM bar in milivolts.

II.4. Discussion

The study of collagen type I during bonding procedures used in dentistry is difficult because dentine is a mineralised tissue. Extraction of collagen from dentine would require some chemical substances to remove the hydroxyapatite allowing collagen fibrils to be exposed (179); however during this extraction, alteration of these fibrils is highly likely. Reconstituted collagen and rat tail tendon are common sources of collagen type I and are widely used to investigate these fibrils.

The collagen assembly obtained resulted in a fibrillar structure with a regular orientation and D-period, which is similar to the structure described by Cisneros et al. and Jiang et al. (262, 267), which proposed the protocol to obtain assembly of collagen into microribbons. Particularly, the 12µg/ml collagen samples (Figure II-1, A and B) exhibited more regular fibrillar patterns compared to the higher concentration sample

(24µg/ml collagen, Figure II-1 C). Thus, the dehydrated images at lower collagen concentration suggest independent fibril growth agreeing with the results of Cisneros et al. (262), where the authors established that lateral growth of single fibrils occurs independently and they fused with adjacent fibrils when they came into contact (262).

In contrast, the sample at 24µg/ml showed fibrils interconnected with each other through smaller fibrils forming a film-like structure. A possible explanation of this difference is related to the isoelectric point (pI) of collagen I. This pI has been reported at pH 9.3 (268) and according to Jiang et al. (267), increased microfibrillar spaces occur when the pH of the solution is above 8.9. Assuming that less concentrated samples in this study have higher pH, the proposed explanation of higher repulsion of microfibrils at pH close the pI cannot be discarded. In addition, the 79

fibrillar appearance in samples at 12µg/ml (Figure II-1, A and B) is surrounded by a halo or sheath, not visible in sample C (24µg/ml), which could indicate the lateral apposition of collagen molecules allowing the fibril to grow independently rather than as a network.

Another similarity of the less concentrated samples with the proposed protocol was the average height of the collagen fibrils, which in dried samples was 2.36nm compared to 3nm measured by Cisneros et al. and Jiang et al. (262, 267). This height

(around 4nm) has been described for single fibrils (60, 269), and therefore a monolayer of variable width is formed following the present procedure

(microribbon). In contrast, the sample at collagen concentration of 24µg/ml resulted in two times the height at 12µg/ml. Nevertheless, when this dimension was measured after re-hydration the values were not constant, increasing only in sample A (sample C was lost after rehydration) to almost 7.9nm (Figure II-1 A.2). Sample B remained unchanged probably due the inability of water of re-entering into the structure of collapsed collagen.

Several techniques such as electronic microscopy, X-ray diffraction and AFM support the model where the fibrillar collagen has a characteristic D-period of 67nm (57, 270,

271). According to Jiang et al. (267) and Cisneros et al. (262), the presence of potasssium ion in the buffer solution allows the formation of this periodicity. The fibril formation obtained in this study proved formation of a D-period, which could be measured by FFT at 67.8±0.72nm in the samples A and B and 64nm in sample C. After re-hydration, the samples A and B (considering only samples depicted in Figure II-1

A.2, B.2 and A.4 and B.4, which are similar to A and B for more accurate comparison)

maintained the morphology but reduced their D-period to 64.43±0.22nm and

62.48±0.11nm after 18 and 36 minutes, respectively. These measurements could be explained by swelling of the collagen structure affecting the definition of the probe during the scanning procedure.

The extraction procedure used here and proposed by Holmes and Kadler (264) is an easy way to obtain individual collagen fibrils . The suspension obtained from this procedure was seeded on mica to determine if the sample could be resolved by AFM microscopy after acid etching. An initial scan was obtained for each isolated fibril prior to acid treatment to allow comparison. The dental procedure labelled as “etch and rinse” consists in the use of an acid, frequently phosphoric acid 37%, during a suggested time frame (often 15 seconds) on the surface of dentine to expose collagen by removing the hydroxyapatite. Thus, the starting values of this experiment (acid concentration and time frame) should be the ones used clinically. When the samples already treated were re-examined, only remnants of collagen were left on the surface.

Therefore, protocol modifications were made by decreasing time and acid concentration. Several configurations were tested unsuccessfully, until 1% phosphoric acid and 10% citric acid applied on the extracted samples during 5 seconds allowed to resolve consistently the fibrils. In contrast to reconstituted samples, the extracted- collagen samples treated using these values were regularly observed by AFM. This inferior resistance of the reconstituted collagen has been associated with modification of original features after the complex chemical process involved in its production (16).

Kelvin probe force microscopy allows obtaining a surface potential map of the samples (258). Therefore, it was interesting to investigate if the local electric potential

of the collagen was affected by bonding procedures. However, the technique requires a conductive substrate to detect potential differences between tip and sample. For this, the samples were deposited on silicon wafer. As reported by Stone and Mesquida

(74), the surface potential of type I collagen could reach values of 150 mV when examined after immersion in deionized water, and drop to 50 mV after immersion in a diluted hydrochloric solution, pH 2. Furthermore, these authors reported that a surface potential variation between gap and overlap potential could be resolved by

KPFM. For this, it was necessary to calibrate the instrument using an external source, a wave generator following the fine tune of the feedback procedure proposed by

Jacobs et al. (266). Considering this, the second part consisted on checking the feasibility of a similar study under different conditions. As a result, the collagen fibrils were successfully imaged.

II.5. Conclusion

The protocol suggested by Cisneros et al. (262) was reproduced in this study rendering fibrillar collagen at lower concentration of collagen solutions. The morphology was assessed successfully by AFM and therefore, allowed comparison between dried and re-hydrated samples. However, it was not a good candidate for studies regarding structural changes as a consequence of acid treatment. In contrast, the model developed by Holmes and Kadler (264) rendered individual fibrils that were successfully scanned after the action of acid solution. Thus, the study of collagen type I changes as a consequence of acid treatment, as part of the bonding procedure used in dentistry, requires the modification of clinical protocols. Using diluted phosphoric acid and citric acid during a few seconds allows under these experimental conditions, improved the AFM and KPFM resolution of collagen fibrils before and after treatment.

Chapter III. “Influence of dental bonding procedures on collagen fibril nano-structure and nano-charge distribution”

III.1. Overview

In this research, possible changes occurring on collagen type I with etching procedures that could affect the interaction with bonding agents were explored.

However, a clear disadvantage of using dentine is that as a mineralised tissue, the hydroxyapatite must be removed totally or partially to expose collagen. Any method used could have potential effects on the structure or the properties measured. Hence, it was decided to use a more readily available source of collagen type I, i.e. rat tail tendon, which according to Wallace et al. (272) possess similar morphological characteristic of collagen at a nanoscale.

Thus, in order to test the hypothesis “Bonding procedures induce structural and surface potential changes on collagen type I”, this study aimed to evaluate morphological changes of type I collagen as a consequence of chemical agents commonly used during the dentine bonding treatment.

III.2. Materials and Methods

III.2.1 Sample preparation

Tendon samples were harvested from rat tails (stored at -20°C). After removing and discarding the skin, the tendons were taken from the tail and cut in small sections of

0.5 cm approximately. Each section was frozen and crushed in liquid nitrogen following the protocol proposed by Holmes and Kadler (264). The powder resulting from this process was dispersed in buffer [50mM Tris-HCl (pH 7.4), 100 mM sucrose,

150mM NaCl] using a hand-held Dounce homogeniser. From this suspension, 10 microliter was seeded on silicon wafer (Sigma-Aldrich) and left overnight to allow collagen attach to the substrate. The following day the samples were rinsed using ultrapure water to remove any residual solution and stored.

Previous to AFM imaging, the samples were rehydrated using ultrapure water during

5 minutes, then the excess was blown dry using a rubber blower and imaged immediately (Control/initial).

Each sample was sequentially treated according to the assigned group (Table III-1):

Table III-1. Groups and treatment sequence Rins Rins Primer Rins Chlorhexi AFM Acid e / AFM e / AFM (HEM e / AFM dine 2% Group Scan Solution Air- Scann Air- Scann A / Air- Scann pH 7.4 ning (5sec) blo ing blo ing TEGD blo ing (1 min) wn wn MA) wn Ca+ Citric 10% ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ Chx+P pH 1.52 Citric 10% Ca+P ✔ ✔ ✔ - - - ✔ ✔ ✔ pH 1.52 Pa+ Phosphoric ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ ✔ Chx+P 1% pH 1.4 Phosphoric Pa+P ✔ ✔ ✔ - - - ✔ ✔ ✔ 1% pH 1.4 Phosphoric PaChx 1%/Chlorh ✔ ✔ ✔ - - - ✔ ✔ ✔ +P exidine 2% pH 1.95 Ca: Citric Acid; Pa: Phosphoric Acid; PaChx: solution Phosphoric Acid and Chlorhexidine; Chx: Chlorhexidine; P: solution HEMA/TEGDMA

III.2.2 AFM imaging

The samples were imaged using tapping mode or AC mode performed on a MFP-3D

AFM microscope (Asylum Research) using a Silicon-Pt coated probe (AC-240TM,

Olympus Corporation) with a nominal tip radius of 15 nm at a resonance frequency 86

near 70 kHz. Prior to scanning the already selected area (2 x 2 micron at a resolution of 512 lines; lateral step 3.9nm), the KPFM signal was optimised using the fine tune

KPFM feedback procedure using an external voltage source (following (266) at lift height of 10nm. Height and Potential channels were recorded simultaneously at a scanning rate of 1 Hz.

III.2.3 Image post-processing and analysis

All images were exported to ascii files (.txt) without performing any modification.

Axial dimension were determined using routines written in Microsoft Visual Basic

(265). Further post-processing was carried out using ImageJ software (National

Institutes of Health: available on the internet al. https://imagej.nih.gov/ij/). For this, both channels were stacked together and using the height image as reference, the full width of the fibril was selected; straightening the fibril if required. Each channel was calibrated using the conversion value registered previously. The next step was the alignment of each image based on the height images to obtain the same area at each interval of treatment. Once alignment was achieved, the images were cropped (full width x 1.007 micron).

Having the same area of the fibril before and after each intervention allowed comparison with high reliability of changes in height, width (full width at half maximum), D-period and surface potential. The profiles of each individual were extracted from the central portion of fibrils (41 pixel wide x 257 pixels long). From these profiles an average height and potential was calculated. In addition, from the same area selected for profiles, the D-period was calculated using a two dimensional

fast Fourier transform (2D FFT) analysis (272). Furthermore, full width at half maximum (FWHM) was used to measure the collagen fibril width. Lastly, using the roughness tool in Gwyddion, height and potential profiles previously selected were levelled and plotted together to investigate relative differences between gap and overlap.

III.2.4 Statistical analysis

The results were analysed using repeated measures one-way ANOVA, with

Greenhouse-Geisser correction, followed by Tukey’s multiple comparisons test, with individual variances computed for each comparison using GraphPad Prism version

7.05 for Windows (GraphPad Software, USA).

III.3. Results

The effects of different agents commonly used during dentine bonding procedures on collagen fibrils were investigated by AFM imaging and KPFM methods. For this, type I collagen fibrils from rat tail tendon were dispersed following the aforementioned procedure and left to adsorb on Si-wafer. This procedure to obtain isolated fibril of collagen type I was successful. Figure III-1 shows an example of the initial and subsequent images of a fibril treated according to the method described above. The tapping-mode topography map (left images) of an isolated fibril showing the classical

D-period was resolved throughout irrespective of the treatment. On the right hand side, images are shown KPFM surface potential maps (color scale range in mV) of the same fibril. Striking differences were observed across the experiment. In Figure III-2

is presented a tri-dimensional reconstruction of the same fibril as in Figure III-1 allowing a better visual comparison of the topography during the different stages of the bonding procedure.

Figure III-1. AFM images of the same 2 x 2 µm area of a collagen fibril submitted sequentially to deionised water (A-B), phosphoric acid- (C-D), chlorhexidine- (E-F) and primer-treatment (G-H). Left images correspond to topographic maps (calibration bar in nm), right images to KPFM surface potential maps (calibration bar in mV).

Primer

Chx

PA Control

Figure III-2. Tri-dimensional reconstruction of the same collagen fibril section subjected to consecutives phosphoric acid (PA), chlorhexidine (Chx) and primer interventions. Dimensions expressed in nanometre

III.3.1 Height

The collagen fibrils analysed in this study had an initial height ranging between 99 and 255 nm (mean 154±21 nm). The average height per group was 174±31 nm,

144±26 nm, 150±30, 176±53 nm and 128±22 nm (Figure III-3). These heights were significantly affected, regardless the type of acid treatment (Citric acid, Phosphoric acid or acid solution consisting of Phosphoric acid and Chlorhexidine) (Figure III-4

and Figure III-5). Subsequent treatments (Chlorhexidine and/or Primer treatment) apparently have no impact on the fibril height. It should be noted that after primer application the fibrils height increased slightly. In those samples treated with phosphoric acid followed by chlorhexidine (Pa+Chx+P group), the difference between control and primer height was not significant (p=0.08) (Figure III-5 top).

A v e ra g e H e ig h t V a ria tio n C a + C h x + P 2 5 0 C a + P P a + C h x + P 2 0 0 P a + P P a C h x + P

1 5 0

m n 1 0 0

5 0

0 In itia l A c id C h x P rim e r

Figure III-3. Height variation across the interventions. Note effect of acid etching on colllagen. Mean and SD are plotted for each group.

C a + C h x + P 9 5 % C o n fid e n c e In te rv a ls (T u k e y )

2 5 0 C h x - P rim e r

2 0 0 A c id - P rim e r )

A c id - C h x m

n 1 5 0 (

In itia l - P rim e r

h g

i 1 0 0 In itia l - C h x e

h In itia l - A c id 5 0

-5 0 0 5 0 1 0 0 1 5 0 0 D iffe r e n c e b e tw e e n g ro u p m e a n s

l d x r ia i h e it c A C im In r P

C a + P 9 5 % C o n fid e n c e In te rv a ls (T u k e y )

2 5 0

A c id - P rim e r

2 0 0 )

m In itia l - P rim e r

n 1 5 0

(

h g

i 1 0 0

e In itia l - A c id h 5 0 -2 0 0 2 0 4 0 6 0 0 D iffe r e n c e b e tw e e n g ro u p m e a n s

l d r ia i e it c A im In r P

Figure III-4. Individual variation of the collagen height after different interventions and statistical differences of height are presented by groups treated with citric acid

P a + C h x + P 9 5 % C o n fid e n c e In te rv a ls (T u k e y )

2 5 0 C h x - P rim e r

2 0 0 A c id - P rim e r

) A c id - C h x m

n 1 5 0

( In itia l - P rim e r

t h

g In itia l - C h x

i 1 0 0 e

h In itia l - A c id 5 0 -5 0 0 5 0 1 0 0 D iffe r e n c e b e tw e e n g ro u p m e a n s 0

l d x r ia i h e it c A C im In r P

P a + P 9 5 % C o n fid e n c e In te rv a ls (T u k e y )

3 0 0

A c id - P rim e r )

2 0 0 m

n In itia l - P rim e r

(

g i

e 1 0 0 In itia l - A c id h

-5 0 0 5 0 1 0 0 1 5 0 0 D iffe r e n c e b e tw e e n g ro u p m e a n s

l d r ia i e it c A im In r P

P a C h x + P 9 5 % C o n fid e n c e In te rv a ls (T u k e y )

2 0 0

A c id /C h x - P rim e r

1 5 0 )

m In itia l - P rim e r

(

t 1 0 0

h g

i In itia l - A c id /C h x

e h 5 0 -2 0 0 2 0 4 0 6 0 D iffe r e n c e b e tw e e n g ro u p m e a n s 0

l x r a e ti h i C m n / i I d r i P c A

Figure III-5. Individual variation of collagen height after different interventions within groups and statistical differences in groups treated with phosphoric acid and PA/Chx solution. 94

III.3.2 Width

In order to measure accurately the width of fibrils before and after treatments, the full width at half maximum was calculated. The fibril ranged from 271 nm up to 661 nm wide (mean 420±41 nm). Per group the calculated average before treatments were

393±27 nm, 412±64 nm, 472±96 nm, 373±91 nm and 450±93 nm (Figure III-6). The statistical analysis showed that despite of an average increase of width after acid treatment, this difference was no significant for all groups (p>0.05). Another remarkable observation was that some fibrils reduced their width (one of them decreased more than 100 nm its width), most remained generally unchanged but some increase in more than 150 nm their width regardless the acid used (Figure

III-6). Subsequent treatments with Chlorhexidine and/or Primer did not vary significantly except when citric acid treatment was followed by chlorhexidine, showing a reduction of their width (p=0.014).

C a + C h x + P - W id th C a + P - W id th

8 0 0 8 0 0

6 0 0 6 0 0 m

4 0 0 m 4 0 0

n n

2 0 0 2 0 0

0 0

l d x r l r a i e a id x ti c h i h e i C m it c n A i A C im I r In r P P

P a + C h x + P - W id th P a + P - W id th

1 0 0 0 6 0 0

8 0 0

4 0 0

6 0 0

n n 4 0 0 2 0 0

2 0 0

0 0

l d x r l d x r ia i h e ia i h e it c it c A C im A C im In r In r P P

P a C h x + P - W id th A v e ra g e F W H M C a + C h x + P C a + P 8 0 0 8 0 0 P a + C h x + P P a + P P a C h x + P 6 0 0

6 0 0

m n

m 4 0 0 n 4 0 0

2 0 0

l d x r 0 ia i h e it c A C im l In r d x r P ia i h e it c A C im In r P

Figure III-6. Effects on the FWHM of collagen fibrils submitted to a dental adhesion procedure. In general, width had a slight increase after acid treatment (p>0.05). Some reductions are also detected especially in group Ca+P, where one fibrils reduced its width in more than 100nm. Individual variations per group and mean (SD) results are presented.

III.3.3 D-Period

From the 40 fibrils examined using fast Fourier transform analysis was determined an average D-period of 67±1.4 nm. This average measurement remained mainly unchanged after acid (66.7±0.6 nm), chlorhexidine (66.5±0.1 nm) and primer

(67.4±2.4 nm) interventions (Figure III-8). A visual example of a fibril from group

Pa/Chx+P is presented in Figure III-7.

Figure III-7. AFM image of the same section of a collagen fibril initially and after treatment with a solution of phosphoric acid and chlorhexidine (Acid/Chx) and primer. Note the preservation of the D-banding (66±1 nm) throughout interventions.

C a + C h x + P - D p e rio d C a + P - D p e rio d

7 5 7 5

7 0 7 0 m

6 5 m 6 5

n n

6 0 6 0

5 5 5 5

l d x r l r a i e a id x ti c h i h e i C m it c n A i A C im I r In r P P

P a + C h x + P - D p e rio d P a + P - D p e rio d

1 2 0 7 5

1 0 0

7 0 m

8 0 m

n n

6 5 6 0

4 0 6 0

l d x r l d x r ia i h e ia i h e it c it c A C im A C im In r In r P P

P a C h x + P - D p e rio d A v e ra g e D -P e rio d 9 0 C a + C h x + P 9 0 C a + P P a + C h x + P P a + P 8 0 8 0 P a C h x + P

m 7 0 n

6 0 6 0

5 0

l d x r 5 0 ia i h e it c A C im l In r d x r P ia i h e it c A C im In r P

Figure III-8. D-period variation across interventions. Individual variations and means (SD) per groups are presented.

III.3.4 Surface Potential (KPFM)

The bonding procedures induced changes in the surface potential of type I collagen fibrils. An example of the variation is presented in Figure III-9.

The absolute values of fibril surface potentials before interventions were in average

34±9.1 mV, once acid solutions took effect, this potential increased to 44.9± 6.5 mV, after Chlorhexidine was 25±3.3 mV, and finally reaching 41±11.5 mV after primer application.

When analysed by acid solution, those samples treated with citric acid had the highest potential increase (from 30±18 mV to 46±10 mV). The solution phosphoric acid/chlorhexidine had a variation of ca. +12mV (from 35±14 mV to 47±18 mV).

Phosphoric acid affected the least, although the difference was also positive (from

36±28 mV to 41±15 mV) (Figure III-10).

Figure III-9. Tri-dimensional reconstruction of the same section of a fibril topography (left) and surface potential (right). A: Initial, B: citric acid, C: chlorhexidine D: Primer. Left: An appreciable reduction in height without major width variation is observed. Right: initially is measure a mean surface potential of 22.5 mV, increasing to 37.6mV after acid treatment, after chlorhexidine decreased to 26.6 mV and primer application reduced the value to 21.7 mV (values correspond to an average of the top area of the fibrils). Calibration bar in mV.

C a + C h x + P - P o te n tia l C a + P - P o te n tia l

6 0 1 0 0

8 0

4 0

6 0

m m 4 0 2 0

2 0

0 0 l d x r l d x r o i h e o i e tr c r c h C m t m n A i n A C i o r r P o P C C

P a + C h x + P - P o te n tia l P a + P - P o te n tia l

5 0 1 5 0

4 0

1 0 0

3 0

m m 2 0 5 0

1 0

0 0

l d x r l d x r o i h e o i h e tr c tr c A C im A C im n r n r o P o P C C

P a C h x + P - P o te n tia l A v e ra g e P o te n tia l C a + C h x + P C a + P 1 0 0 1 0 0 P a + C h x + P P a + P 8 0 P a C h x + P

6 0 V m

V 5 0 m 4 0

2 0

l d x r 0 o i h e tr c A C im l n r d x r o P o i h e C tr c A C im n r o P C

Figure III-10. Surface potential variation of individual fibrils and average per groups are presented. It can be observed a great initial dispersion in values but in general an increased of surface potential after acid treatment and primer aplication.

100

III.3.5 Height difference between Gap and Overlap regions

The differences found between Gap and Overlap regions in the fibrils without any treatment ranged from 1 to 6 nm. These differences were generally reduced irrespective the acid agent used (Figure III-11). Interestingly, after applying chlorhexidine 2% the gap-overlap difference remained, improving the shape of the overlap area, with a reduction or an increase of their difference. In addition, the use of primer on acid- or acid- and chlorhexidine-treated fibrils caused random effects, where within the same fibril some D-periods were kept unaltered, others increased or decreased that difference between gap and overlap. The difference was affected either by filling the gap region, depositing over the overlap or by reducing the overlap height

(Figure III-11).

In itia l A c id

2 2 )

1 ) 1

(

t h

0 h 0

H  -1  -1

-2 -2 0 5 0 0 1 0 0 0 0 5 0 0 1 0 0 0 nm

C h x P r im e r

2 2 )

) 1 1

m m

n n

( (

t t h

h 0 0

g g

i i

e e

H H   -1 -1

-2 -2 0 5 0 0 1 0 0 0 0 5 0 0 1 0 0 0

Figure III-11. Height profiles of the same section of a collagen fibril from group PA+Chx+P. Here, acid induced a slight reduction of step-height and primer increased some and reduced other areas of gap-overlap.

101

III.3.6 KFM Profiles

The effects of acid agents, chlorhexidine and primer on the KFM profile of collagen fibrils were also investigated. For this, the same areas considered for the topographic evaluation were used.

In general, KPFM analysis was unable to find differences between gap and overlap regions or between control and the different agents used. However, when alterations were detected (Figure III-12), the gap region presented a higher surface potential than the overlap; this difference remained or slightly increased by phosphoric acid or the solution phosphoric acid/chlorhexidine. Chlorhexidine application after phosphoric acid caused a reduction of the potential difference. Finally, the application of the primer blend had a more random effect, where most of the differences increased markedly reaching up to a maximum of 8-10mV, but in other sectors the difference became lower or even negligible.

102

In itia l A c id

1 0 1 0 1 0 H e ig h t 1 0 H e ig h t P o te n tia l P o te n tia l

5 5 5 5

 

) )

P P

o o

m m

t t

n n

e e

( (

n n

t t

h h 0 0 i 0 0 i

a a

g g

i i

l l

e e

( (

m m

h h

V V

 

) )

-5 -5 -5 -5

-1 0 -1 0 -1 0 -1 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 n m n m

C h x P r im e r

1 0 1 0 1 0 H e ig h t 1 0 H e ig h t P o te n tia l P o te n tia l

5 5 5 5

 

) )

P P

o o

m m

t t

n n

e e

( (

n n

t t

h h 0 0 i 0 0 i

a a

g g

i i

l l

e e

( (

m m

h h

V V

 

) )

-5 -5 -5 -5

-1 0 -1 0 -1 0 -1 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 n m n m

Figure III-12. Topography (blue) and potential (red) profiles of the same section of an individual fibril across interventions. It can be seen gap areas had a higher surface potential compared to overlap.

103

III.4. Discussion

Many restorative procedures in dentistry rely on the adhesion of synthetic materials to the tooth. Due to its heterogeneity, the main challenge of adhesive techniques is dentine. This tissue contains roughly 50 vol% of minerals (hydroxyapatite), 30 vol% organic (mainly collagen type I) and 20 vol% water (273). The adhesive procedure replaces the loss tissue with a polymer material (274). This requires dentine demineralisation with acids to increase the surface area and to enhance infiltration of hydrophobic monomers into the exposed collagen network (6, 274). Here, interaction forces are developed between the two parts. These forces, i.e. Van der Waals, capillary, electrostatic and steric forces are massively affected by the medium in which the process occur (274). However, morphological changes of collagen due to the effect of acids may be significant. Evidence using KPFM (74), suggests that a change in the ionisation state of collagen type I after been immersed in an acid solution HCl (pH 2) occurs. Despite several studies investigating the collagen organisation, only a few have focussed on the changes occurring after acid treatment and are mainly centred at a fibrillar level no further than the D-period.

The study of changes on collagen type I as a consequence of the procedures to achieved adhesion to dentine remains a challenge. One of the main reasons lies on the mineralised nature of the tissue. Therefore, in order to gain access to collagen, the mineral part must be removed using different chemical techniques. Thus, any intervention irrespective of the method to remove hydroxyapatite could induce changes on the collagen structure and properties measured (272). Therefore, rat tail tendon which is composed by 90 wt% collagen type I (275), was used as an initial step 104

to determine if the acids used commonly in dental adhesion procedures cause changes on its structure and surface potential. The main goal of this adhesive interaction is the complete imbibition of collagen network by the monomer (126, 206). The detection and understanding of these changes, which in our opinion has been underestimated so far, could potentially be addressed in the future and thus, improve the interaction of restorative materials with dentine.

Initially, fibrils collagen were analysed in order to provide a reference point for posterior comparison. Three acid solutions were used, namely 1% phosphoric acid,

10% citric acid and a solution of 1% phosphoric acid / 2% chlorhexidine. The effect on height was significant, regardless of the solution. All samples showed a reduction of ca. 30%. As this modification could have been due to alteration of the shape (or interaction with the substrate), the width was measured before and after treatments.

Although most of the fibrils became slightly wider than the control, these differences were not statistically significant, confirming an alteration of the structure

(dissolution) rather than shape alteration. According to Venturoni et al. (276), in solutions with values below pH 3, collagen fibrils are dissolved, losing completely their structure. However, this outcome was prevented by immersing the samples overnight at low pH in a solution containing high concentrations of NaCl or CaCl2.

After this, the classical D-period was observed. Interestingly in the current experiment, even though fibrils were dissolved, the classic D-period remained at ca.

67nm throughout the experiment, observation in agreement with those described in dentine (16, 149, 179). When the analysis was focussed on the height difference between gap and overlap, acid treatments produced a slight reduction of height,

105

suggesting that the overlap is more susceptible to attack compared to the gap region.

A possible explanation for this is the presence of decorin in the gap region. This proteoglycan has been described at the bottom of the gap region from rat tail tendon using techniques such as histochemical (277), immunohistochemical (278) and by

AFM (279). Hitherto, the superficial reduction of the overlap in dentine has not been described; on the contrary, a progressive increase in gap-overlap height difference was found during citric acid treatment on sound dentine (179), but it is possible that the continual acid dissolution of the intrafibrillar apatites could be masking the effect of the acid on the overlap region. Another evidence of alteration seen on these fibrils was the increase of surface potential after acid treatment. This rise indicates a positive net charge on the surface of collagen caused by acid, which is in agreement with the prediction model of collagen in low pH presented by Uquillas and Akkus (73).

Interestingly, this potential surge remained even after rinsing with water. In contrast to our findings, Stone and Mesquida (74) found that KPFM was able to find differences between gap and overlap when analysed after immersion in water but this difference was insignificant after immersing the collagen in an acid solution. Here, only few samples exhibited differences within the D-period and no remarkable differences were found after acid treatment. The lack of agreement could be related with the size of the probe-tip, the lift height and the instrument noise. Bigger tips receive influence of a wider area. A similar situation could occur at lower lift heights, where the cantilever might also be stimulated by the sample’s potential masking the interaction taking place at the tip (280).

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III.5. Conclusion

This study confirms that AFM is an excellent tool to evaluate morphological and electrical surface potential changes of collagen samples. Thus, under the conditions described, it can be declared that type I collagen fibrils are affected by dental chemical agents, especially during acid etching.

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Chapter IV. “The surface roughness of resin-composites is dependent on the measurement area: an AFM study”

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IV.1 Overview

The aim of this study was to use topography maps, generated by atomic force microscopy (AFM), of three dental restorative materials subjected to either brushing or polishing to compare the surface roughness of different resin-composites and to identify the effect of surface area on the AFM analysis of resin-composites.

IV.2 Materials and Methods

Two disc-shape (12 x 2mm) specimens per sample were prepared for each resin- composite (CeramX duo, Tetric EvoCeram and Venus Diamond) as described in Table

IV-1. Each disc was polymerised using a QTH light curing unit (Optilux 501) emitting

550mW/cm2 for 40s. Subsequently, the discs were finished and polished with an

OptiDisc system (Kerr, KerrHawe, Switzerland). All specimens were polished, thus, for each specimen a finish disc, a fine polish disc and a high gloss disc were used following a 30s cycle per disc to standardise the time for all samples. Once polished, the samples were placed in an ultrasonic water bath (Elma ultrasonic T310, Germany) for 5min.

One disc per sample was used as a control (polished) and the other was mounted and fixed in a custom made toothbrush simulating machine. The experimental specimens

(brushed) were subjected to 16,000 cycles (equal to 32,000 strokes to simulate 3 years of toothbrushing) (281), using Oral B Plus P-40 toothbrush heads (P&G,

Cincinnati, OH, USA) attached to a holder moving back and forth under a vertical load of 2.5N. A slurry of toothpaste was prepared by mixing Colgate Total Advance

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toothpaste (Colgate-Palmolive, Manchester, UK) of RDA value 70 and water in a ratio of 1:1.

Table IV-1. Name and product details used in this study

Material Type Manufacturer Composition Filler type, size and content (%) Ceram X Nanoceramic Dentsply, Methacrylate Barium – Duo / Nanohybrid Germany modified aluminium – polysiloxane, borosilicate glass, dimethacrylate 1.1-1.5µm, 76 wt% resin / 57 vol% Tetric Nanohybrid Ivoclar Dimethacrylates Barium glass 1µm, EvoCeram Vivadent, Ba-Al-Silicate glass Liechtenstein (0.4-0.7µm), Ytterbium trifluoride, mixed oxide, prepolymer, 82.5 wt% / 68 vol% Venus Nanohybrid Heraeus TCD-DI-HEA, Ba-Al-F- Diamond Kulzer, UDMA Borosilicate glass, Germany silica nanofiller (5nm - 20µm), 81.2 wt% / 64.3 vol%

IV.2.1 Surface Roughness Measurement

Surface roughness parameters Sa, Sq and Sz; defined previously (282) as the arithmetic mean of the absolute ordinate values within a defined area; the root mean square value if the ordinate values within a definition area; and the sum of the maximum peak height and the maximum pit height value within a definition area, respectively, were determined for all the specimens (polished and brushed). Each specimen was fixed to a 15mm metallic disc and their nanoscale topography was

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scanned and registered using a MultiMode 8 atomic force microscope (AFM) and a

Nanoscope IIIa controller (Bruker, Coventry, UK) in ScanAsyst mode and using Bruker

Scanasyst Air triangular silicon nitride cantilever (nominal spring constant of 0.4N/m and a pyramidal tip of nominal radius of 2nm). Scans were performed for areas of

25µm2, 400µm2 and 10000µm2 (i.e. 5x5, 20x20 and 100x100 µm, respectively) and a sampling frequency of 512 x 512 (for a lateral sampling frequencies of 9.8nm, 39nm and 195nm, respectively). Specimens were scanned in air at room temperature at 3 different locations (per scan size) and their images were saved and analysed afterwards. Following a similar methodology as described by Sherratt et al. (265), roughness analyses to determine Sa (roughness average), Sq (root mean square roughness) and Sz (peak to peak) were carried out with MatLab R2014a routines written by Dr M. Ozols that uses the same equations employed by the Nanoscope software (Eq. 1, 2 and 3) where Zave is the average Z-value within a given area, Zi is the current Z-value, N is the number of points within the given area, zmax is the maximum peak height value within a given area and zmin is the maximum pit height value. The

MatLab code calculates each parameter for multiples areas per scan, set al. 10pixels incremental area sizes, until the area is exceeded. All the measures are averaged, giving the measure for the specific box size.

1 푆푎 = ∑ |푍i − 푍ave | (Eq.1) 푁

2 푆푞 = √∑(푍i − 푍ave) /푁 (Eq.2)

푆푧 = 푧max − 푧min (Eq.3)

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IV.2.2 Statistical analysis

The data obtained at dimension of 5x5, 20x20 and 100x100 µm were analysed using

ANOVA and Tukey’s post-hoc test at 5% significance level to compare Sa, Sq and Sz among experimental and control groups at different scan sizes.

IV.3 Results

Representative AFM images of the resin-composites polished and brushed at three areas of observation are presented in Figure IV-1, Figure IV-2 and Figure IV-3.

Significant differences in the topography induced by the brushing procedure were observed.

Figure IV-1. Tri-dimensional reconstruction of resin-composite surfaces obtained after AFM scanning an area of 5x5 µm. Irregularities produced by brushing procedure are observed especially in CeramX sample.

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Figure IV-2. Tri-dimensional reconstruction of resin-composites surfaces after AFM scanning. At a higher area of 20x20 µm, CeramX and VDiamond can be seen with greater irregularities on its surface.

Figure IV-3. Tri-dimensional reconstruction of dental resin-composites surfaces after AFM scanning. At an area of 100x100 µm, the irregularities produced by brushing are again seen but the amount of events has also increased.

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Without exception, it was observed an increase of roughness when increasing the area of analysis, regardless of the parameter analysed. In specific, the roughness surface increased more markedly on the brushed samples. The samples where the roughness was least affected by area Tetric Evo Ceram and Venus Diamond, both polished.

S a

) 4 0 0

m C e ra m X b ru s h e d

n ( C e ra m X p o lis h e d

e 3 0 0

g a

r T e tric b ru s h e d e v T e tric p o lis h e d

A 2 0 0

s V d ia m o n d b ru s h e d e

n 1 0 0 V d ia m o n d p o lis h e d

u o

R 0 1 4 5 4 1 0 0 5 8 6 8 0 .0 .3 .1 .4 .2 .0 .1 .8 .5 .5 .2 .0 0 0 1 2 4 5 4 3 1 6 2 0 2 4 7 6 7 8 0 4 9 6 0 1 0 1

2 A re a (µ m )

Figure IV-4. Average roughness (Sa) determined for area in the range 0.01 to 10,000 µm2. It can be seen an increasing roughness average at incremental dimensions. Brushed samples are seen to vary more than polished surfaces. Note abruptly increase of Sa at just over 25 µm2 area of observation for Tetric and VDiamond brushed samples.

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S q

5 0 0 C e ra m X b ru s h e d 4 0 0

) C e ra m X p o lis h e d

m 3 0 0 n

( T e tric b ru s h e d

S 2 0 0

M T e tric p o lis h e d R 1 0 0 V d ia m o n d b ru s h e d

0 V d ia m o n d p o lis h e d

1 4 5 4 1 0 0 5 8 6 8 0 0 .0 .3 .1 .4 .2 .0 .1 .8 .5 .5 .2 . 0 0 1 2 4 5 4 3 1 6 2 0 2 4 7 6 7 8 0 4 9 6 0 1 0 1

2 A re a (µ m )

Figure IV-5. RMS (Sq) roughness determined for area in the range 0.01 to 10000 µm2. Increase value RMS is observed at higher areas. Major changes for Tetric and VDiamond brushed values at just over 25 µm2 observations.

S z

) 4 0 0 0

C e ra m X b ru s h e d

n (

C e ra m X p o lis h e d t 3 0 0 0

h T e tric b ru s h e d

g i

e T e tric p o lis h e d

h 2 0 0 0

m V d ia m o n d b ru s h e d u V d ia m o n d p o lis h e d

m 1 0 0 0

x a

M 0

1 4 5 4 1 0 0 5 8 6 8 0 0 .0 .3 .1 .4 .2 .0 .1 .8 .5 .5 .2 . 0 0 1 2 4 5 4 3 1 6 2 0 2 4 7 6 7 8 0 4 9 6 0 1 0 1

2 A re a (µ m )

Figure IV-6. Maximum height determined (Sz) for area in the range 0.01 to 10000 µm2. Incremental Sz values are found at higher areas of observation.

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The detailed Anova-test results are shown in Appendix 1. The analysis revealed a significant difference between groups independently of the roughness parameter studied.

IV.3.1 Comparison of Roughness Average (Sa)

Multiple comparison analysis showed no significant differences between the polished specimens at all scales of measurement analysed (Table IV-2). The effect of brushing produced variable rougher surfaces compared to the polished materials, except for

Ceram X duo brushed, which was significantly rougher than its polished control.

Among the brushed samples only at 25 µm2 CeramX had a significant difference in roughness to Tetric EvoCeram (Figure IV-4). Interestingly, Tetric EvoCeram was not affected by brushing at any scale to produce significant differences in Sa respect to the polished samples (Table IV-2 and Figure IV-4); and Venus Diamond brushed was rougher than polished samples only at 10,000 µm2 measurement (Table IV-2).

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Table IV-2. Multiple comparison analysis (Tukey test) of mean Sa results of resin- composites at three different dimensions. 5x5 µm 20x20 µm 100x100 µm Subset for alpha = Subset for alpha = Subset for alpha = Material N 0.05 0.05 0.05 1 2 1 2 1 2

VDiamond 3 10.754 18.9547 31.32 polished Tetric 3 15.743 20.028 39.5187 polished CeramX 3 35.4203 35.773 35.773 137.0627 polished Tetric 3 52.4913 126.803 126.803 215.1597 215.1597 brushed VDiamond 3 90.2913 90.2913 253.0713 253.0713 357.7927 brushed CeramX 3 170.1357 262.134 366.2473 brushed Sig. 0.054 0.053 0.058 0.069 0.078 0.186

Means for groups in homogeneous subsets are displayed (nm)

In general, at higher scale of observation higher values of Sa were obtained. A more pronounce effect of this relationship was observed in rougher samples (Figure IV-4).

Interestingly, the Sa ratio brushed:polished per sample was variable where higher variation for the three materials was found at 400 µm2 (Table IV-3).

Table IV-3. Roughness Average Ratio (Brushed:Polished) at different scales of measurement Ceram X duo Vdiamond Tetric EvoCeram Scale (µm) 5x5 20x20 100x100 5x5 20x20 100x100 5x5 20x20 100x100 Brushed 170.14 262.13 366.25 90.29 253.07 357.79 52.49 126.80 215.16 Polished 35.42 35.77 137.06 10.75 18.95 31.32 15.74 20.03 39.52 Ratio 4.80 7.33 2.67 8.40 13.35 11.42 3.33 6.33 5.44 Values of Sa expressed in nm.

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IV.3.2 Comparison of Root Mean Square Roughness (Sq)

Similarly to Sa, Tukey test analysis show no significant differences among the polished specimens. Despite the size analysed and brushing, differences were only observed between Ceram X brushed at 5x5 µm and 100x100 µm, and Venus Diamond brushed at 100x100 µm with the polished materials (Table IV-4). Regarding the brushed specimens, only the measurement at 25 µm2 proved to produce a significant difference between CeramX duo and Tetric EvoCeram.

Table IV-4. Multiple comparison analysis (Tukey test) of mean of Sq results of resin- composites at three different dimensions

5x5 µm 20x20 µm 100x 100 µm Subset for Subset for alpha = Subset for alpha = Material N alpha = 0.05 0.05 0.05 1 2 1 2 3 1 2 3

VDiamond 3 13.6 25.1 39.5 polished Tetric 3 20.9 25.6 25.6 49.5 49.5 polished CeramX 3 47.4 46.7 46.7 46.7 174.9 174.9 polished Tetric 3 66.9 160.9 160.9 160.9 267.1 267.1 brushed VDiamond 3 114.6 114.6 322.8 322.8 470.8 brushed CeramX 3 218.1 329.9 458.6 brushed Sig. 0.068 0.060 0.652 0.050 0.065 0.388 0.061 0.086

Means for groups in homogeneous subsets are displayed (nm)

The ratio variation between brushed and polished Sq values per sample was not maintained through different scales of measurement and the biggest difference was observed at 400 µm2 (Table IV-5).

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Table IV-5. RMS roughness Ratio (brushed:polished) at different scales of measurement Ceram X Vdiamond Tetric

Scale (µm) 5x5 20x20 100x100 5x5 20x20 100x100 5x5 20x20 100x100

Brushed 218.08 329.87 458.55 114.60 322.83 470.83 66.86 160.90 267.06

Polished 47.40 46.66 174.88 13.56 25.05 39.52 20.89 25.62 49.51

Ratio 4.60 7.07 2.62 8.45 12.89 11.92 3.20 6.28 5.39

IV.3.3 Comparison of the Maximum height (Sz)

This height parameter analysed with Tukey’s test showed no significant differences among polished materials at any observation scale. The highest values consistently with significant differences were found between the CeramX and Venus Diamond brushed specimens and the polished materials (Table IV-6). For Tetric EvoCeram brushed, the values were grouped with the polished specimens at 25 and 400 µm2 observations (Table IV-6). In addition, the values for Tetric EvoCeram brushed were only significantly smaller than Ceram X brushed at 25 or 10000 µm2.

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Table IV-6. Multiple comparison analysis (Tukey test) of mean Sz results of resin- composites at three different dimensions 5x5 20x20 100x100 Subset for alpha Subset for alpha = 0.05 Subset for alpha = 0.05 Material N = 0.05 1 2 3 1 2 1 2 3 4

VDiamond 3 116.6 349.8 486.7 polished Tetric 3 161.8 231.4 492.5 polished CeramX 3 433.7 433.7 462.2 1437.4 1437.4 polished Tetric 3 487.1 487.1 1340.1 1340.1 2160.3 2160.3 brushed VDiamond 3 758.7 758.7 1963.0 2848.4 2848.4 brushed CeramX 3 1275.1 2144.4 3532.7 brushed Sig. 0.33 0.45 0.09 0.113 0.359 0.114 0.316 0.363 0.368

Means for groups in homogeneous subsets are displayed (nm)

Ratio (brushed:polished) for Sz parameter was found to be dependent of the scale of measurement. But similarly to Sa and Sz, the biggest difference between polished and brushed sample was found at 20x20 µm (Table IV-7).

Table IV-7. Maximum height ratios (brushed:polished) at different scales of measurement CeramX Vdiamond Tetric 100x 100x 100x Scale (µm) 5x5 20x20 100 5x5 20x20 100 5x5 20x20 100 Brushed 1275.1 2144.4 3532.7 758.9 1963.0 2848.4 487.1 1340.1 2160.3 Polished 433.66 462.21 1437.44 116.58 349.77 486.67 161.78 231.42 492.53 Ratio 2.94 4.64 2.46 6.51 5.61 5.85 3.01 5.79 4.39 Sz values expressed in nm.

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IV.4 Discussion

The surface texture of resin-composites is an important factor for the selection of this materials (283). Surfaces presenting higher roughness increase the risk of periodontal tissue inflammation, secondary caries and wear of the opossing teeth (240-243). The surface roughness can be measured using several devices (237). However, AFM has been mentioned as more accurate and having superior texture definition (244).

Similarly, there are several methods to measure roughness. From these, the amplitude parameters are commonly used (284). In dentistry, roughness average (Ra) is the most frequently used indicator of surface texture (285, 286). However, this 2D parameter may not represent correctly the surface topography, which is in nature tri- dimensional (284). In contrast, areal parameters such as those used in this study, provide a more accurate representation of the surface topography (287), allowing more stable results (284). The surface roughness has been commonly used to compare the effect of different mechanical treatments on the dental composites materials (238, 241, 242, 245-248). This study identified the consequences of the surface area on the surface texture of three different commercial resin-composites by the analysis of 3 commonly used parameters, i.e. Roughness average (Sa), root mean square height (Sq) and maximum height (Sz) according to ISO 25178-2:2012 (282). As expected the surfaces submitted to brushing presented a higher roughness compared to polished surfaces. In general, the roughness measurement at larger areas increased.

The difference was critical especially for the brushed specimens. From these results, the paramount importance of stating clearly the method and the dimension used to obtain the results for the comparison of different studies (237, 241) is reaffirmed. 121

According to Rashid (288), different methods for roughness evaluation can produce different results because these systems have different resolutions. But even using the same system dissimilar results can be obtained. Here, it is presented clear evidence of the roughness variation as a consequence of the area analysed, irrespective of the experimental parameter. Interestingly, the variation observed did not followed the same ratio and rougher surfaces were more influenced by the area of observation.

Frequently in dentistry, studies analysing roughness do not consider different scales of measurement for the determination of roughness. However, in order to characterise accurately surfaces, based on these results, instead of recommending an optimal area for roughness measurement, different areas should be considered.

The optimisation of dental procedures in order to improve perfomance is widely recommended (289). Therefore in this case, reaching an agreement of the area(s) in which surface roughness should be measured and used universally otherwise, suitable comparisons across the literaure are difficult. Until agreement is not reached, a clear description of the procedure including area must be clearly stated.

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IV.5 Conclusion

This study confirms that the choice of measurement area is a critical factor in characterising the topography of dental material surfaces. However, it is not possible to determine an optimal area for analysis of roughness. Thus, it is reafirmed that stating clearly the area and method used to measure roughness within publishing literature and ultimately an universal agreement for this is essential. In addition, the

AFM is a reliable instrument to distinguish the topographical consequences of brushing of the resin-composite surfaces.

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Chapter V. Discussion

The evolution of the restorative procedures in dentistry has been possible due to the

increasing understanding of the dental tissues and how they interact with the

materials (251). However, dentine continues to be a challenging tissue (290). The

hybrid layer is a critical part of dentine bonding and is considered to be paramount to

the clinical longevity of a resin-composite restoration. A significant amount of

research, over the last 15 years, has been devoted to Matrix Metalloproteinase

enzymes (MMPs) that are part of dentine and can degrade the hybrid layer by

attacking collagen (17, 18). Thus, would undermine the restoration and contribute

towards a clinical failure. Most evidence is indirect via bond strength studies. There is

little evidence on how the collagen is modified through all these changes. This study

investigated how type I collagen is affected by dental bonding procedures. Therefore,

to establish a collagen model was the first step. Dentine cannot be used as a starting

point for the evaluation of such changes, as the collagen extraction from this tissue

involves necessarily a chemical intervention (179). Thus, this study used collagen

from rat tail as a good alternative for initial evaluations. This source of collagen, in

contrast to reconstituted collagen possesses cross-links able to provide stronger

proteolytic resistance and mechanical strength (291). Apart from setting a collagen

model, the novel element of this study was to employ advance imaging techniques

that offer nanometre scale resolution. By retaining the classic characteristic of fibrils

rather than ribbons, it allowed re-evaluation of the same area by atomic force

microscopy after several procedures such as acid etching, applying chlorhexidine and

primer, used as part of a dentine bonding agent. Although this study demonstrated, 124

for the first time, structural changes especially as a consequence of acid treatment, these results are yet to be confirmed to occur in dentine. According to Venturoni et al.

(276), the presence of calcium or sodium ions could prevent the dissolution of fibrils immersed in acid solutions. However, only the D-banding and the height difference gap-overlap were considered as a parameter to establish their structural intactness

(276). Although the presence of these ions may avoid major structural changes, even small alterations could have a great effect in the distribution of chemical charges on the collagen surface. Another novel element was the use of Kelvin Probe Force

Microscopy to monitor electrostatic changes after different treatments on collagen.

As demonstrated here, diluted solutions in brief contact to native collagen resulted in an increase value of surface potential. Thus, within the limits of this exploratory study, the first hypothesis “Bonding procedures induce structural and surface potential changes on collagen type I” was confirmed. Certainly, more research is required to fully understand these changes before learning how to overcome or use these modifications in favour of achieving more stable bonds between dentine and resin-composite materials.

Finally, moving to the macro scale, the same imaging techniques were utilised to provide accurate surface measurement. The surface of the material is also important to achieve long-lasting resin-composite restorations. Resin-composite is primarily an aesthetic direct restorative material and rougher topographies will affect their aesthetic appearance. They will also contribute towards further clinical problems such as inflammation of the periodontal tissues, secondary caries, and wear of opposing teeth (233, 240-243). Thus, roughness measurements are used for the

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comparison of different materials. Although the 2-D parameter roughness average

(Ra) is most commonly used to describe roughness (285, 286), area roughness parameter offer a more accurate representation of the surface (284, 287). Regardless of the parameter, the device used and the measurement area are fundamental since they determine the relative roughness measured (237, 241). Surface roughness of resin-composites has been extensively studied and there are numerous articles that refer to this property. However, the lack of a standard procedure limits comparison across the scientific literature (289). It can also lead to misleading results when the same material is compared under two different conditions. This study was unsuccessful to determine an optimal area for the measurement of roughness using

AFM. On the contrary, the evaluation on dissimilar scales showed that the difference found at a certain scale was not found necessarily at others. As a result, the significant finding was that for some areas roughness is dependent on size area and it should be clearly described in all roughness measurements. Therefore, it seems worthy using different dimensions for the accurate description of a surface, at least until no agreement is achieved. This suggestion is especially important on rougher surfaces, as these presented greater variations at different scales. In consequence, the second hypothesis “The surface roughness of resin-composites are different depending on the observation area” was also accepted.

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Future work

This thesis investigated two aspects related to the failure of resin-composites restoration, i.e. effects of bonding agents on collagen and dependence of the roughness surface of resin-composites on the area. Therefore, some recommendations for further research are proposed:

o Assessment of the effects of bonding agents on more complex 3-D model using

AFM and KPFM.

o Combine data with FTIR spectroscopy mapping (Nano-IR) from same zones to

allow characterisation of chemical changes in collagen and their interaction

with monomers.

o Increase of sample size to evaluate if the differences found are significant is

essential.

o Explore the use of quantitative AC–KPFM instead of classic KPFM, which could

offer higher control sensitivity compared to classic KPFM by avoiding the use

of a disturbing DC-bias.

o Assessment of the effects of bonding agents on dentine samples using same

techniques mentioned before, i.e. AFM, quantitative AC-KPFM, Nano-IR.

o Explore different strategies to achieve better interaction between monomers

and collagen using the knowledge acquired.

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Appendix 1

156

Descriptives for Roughness Average (Sa)

N Mean Std. Std. 95% Confidence Interval Minim Maxim Deviation Error for Mean um um

Lower Bound Upper Bound

CeramX 3 170.14 66.96 38.66 3.79 336.48 112.35 243.52 brushed CeramX 3 35.42 13.98 8.07 .68 70.16 21.12 49.07 polished Tetric brushed 3 52.49 16.08 9.28 12.56 92.43 36.04 68.17 25 µm2 Tetric polished 3 15.74 2.36 1.36 9.88 21.61 14.22 18.46

VDiamond 3 90.29 15.97 9.22 50.61 129.97 71.86 99.99 brushed VDiamond 3 10.75 1.57 .91 6.85 14.66 8.94 11.68 polished Total 18 62.47 61.63 14.53 31.83 93.12 8.94 243.52 CeramX 3 262.13 100.44 57.99 12.62 511.65 160.08 360.88 brushed CeramX 3 35.77 13.93 8.04 1.17 70.38 20.52 47.82 polished Tetric brushed 3 126.80 27.31 15.77 58.96 194.65 108.23 158.16 400 Tetric polished 3 20.03 3.74 2.16 10.73 29.32 16.71 24.08 µm2 VDiamond 108.0 3 253.07 187.13 -211.78 717.92 85.98 455.26 brushed 4 VDiamond 3 18.95 2.48 1.43 12.80 25.11 16.10 20.40 polished Total 18 119.46 130.13 30.67 54.75 184.17 16.10 455.26 CeramX 3 366.25 120.46 69.55 67.00 665.49 260.22 497.24 brushed CeramX 3 137.06 102.18 58.99 -116.76 390.89 29.51 232.84 polished

Tetric brushed 3 215.16 64.99 37.52 53.72 376.60 148.33 278.14 10000 Tetric polished 3 39.52 13.67 7.89 5.55 73.49 30.37 55.24 µm2 VDiamond 3 357.79 48.90 28.23 236.31 479.27 319.06 412.74 brushed VDiamond 3 31.32 2.62 1.51 24.81 37.83 29.43 34.31 polished Total 18 191.18 152.42 35.93 115.39 266.98 29.43 497.24

157

ANOVA Sa

Sum of Squares df Mean Square F Sig. Between Groups 54165.413 5 10833.083 12.497 .000 25 µm2 Within Groups 10402.097 12 866.841 Total 64567.510 17 Between Groups 195760.011 5 39152.002 5.100 .010 400 µm2 Within Groups 92129.922 12 7677.494 Total 287889.933 17 Between Groups 331405.325 5 66281.065 12.521 .000 10000 µm2 Within Groups 63521.076 12 5293.423 Total 394926.401 17

158

Descriptives for Root mean square roughness (Sq)

N Mean Std. Std. 95% Confidence Interval Minimu Maximu Deviation Error for Mean m m Lower Upper Bound Bound

CeramX 3 218.08 89.60 51.73 -4.51 440.66 136.99 314.28 brushed CeramX 3 47.40 19.27 11.12 -.47 95.26 27.07 65.40 polished Tetric brushed 3 66.86 19.50 11.26 18.42 115.29 46.18 84.92

25 µm2 Tetric polished 3 20.89 3.55 2.05 12.08 29.69 18.26 24.92 VDiamond 3 114.60 17.62 10.17 70.83 158.38 94.92 128.92 brushed VDiamond 3 13.56 2.04 1.18 8.48 18.63 11.21 14.97 polished Total 18 80.23 79.13 18.65 40.88 119.58 11.21 314.28 CeramX 3 329.87 125.60 72.51 17.86 641.87 205.96 457.09 brushed CeramX 3 46.67 18.89 10.91 -.26 93.59 26.19 63.41 polished Tetric brushed 3 160.90 38.18 22.04 66.05 255.75 135.55 204.82 400 Tetric polished 3 25.62 4.54 2.62 14.34 36.90 21.40 30.42 µm2 VDiamond 3 322.84 230.21 132.91 -249.05 894.72 110.93 567.79 brushed VDiamond 3 25.05 3.42 1.97 16.57 33.54 21.35 28.08 polished Total 18 151.82 163.33 38.50 70.60 233.04 21.35 567.79 CeramX 3 458.55 128.65 74.28 138.97 778.14 343.46 597.44 brushed CeramX 3 174.88 128.38 74.12 -144.04 493.81 37.64 292.04 polished Tetric brushed 3 267.06 72.24 41.71 87.62 446.51 189.71 332.77 10000 Tetric polished 3 49.51 16.85 9.73 7.66 91.36 38.32 68.89 µm2 VDiamond 3 470.83 45.67 26.37 357.37 584.28 434.69 522.16 brushed

VDiamond 3 39.52 3.05 1.76 31.94 47.09 36.99 42.91 polished Total 18 243.39 192.29 45.32 147.77 339.01 36.99 597.44 159

ANOVA (Sq)

Sum of Squares df Mean Square F Sig. Between Groups 88220.528 5 17644.106 11.624 .000 25 µm2 Within Groups 18215.293 12 1517.941 Total 106435.822 17 Between Groups 312247.990 5 62449.598 5.306 .008 400 µm2 Within Groups 141240.917 12 11770.076 Total 453488.907 17 Between Groups 547295.177 5 109459.035 16.164 .000 10000 µm2 Within Groups 81261.011 12 6771.751 Total 628556.188 17

160

Descriptives for Maximum height (Sz)

N Mean Std. Std. 95% Confidence Minimum Maximum Deviation Error Interval for Mean Lower Upper Bound Bound

CeramX 3 1275.12 440.41 254.27 181.08 2369.17 823.16 1703.00 brushed CeramX 3 433.66 195.35 112.78 -51.61 918.93 209.54 567.84 polished Tetric brushed 3 487.11 168.71 97.40 68.02 906.20 302.98 634.25

25 µm2 Tetric polished 3 161.78 22.08 12.75 106.93 216.62 136.30 175.30 VDiamond 3 758.68 68.81 39.73 587.75 929.62 692.54 829.89 brushed VDiamond 3 116.58 25.38 14.65 53.53 179.63 91.32 142.08 polished Total 18 538.82 441.05 103.96 319.49 758.15 91.32 1703.00 CeramX 3 2144.37 524.83 303.01 840.60 3448.13 1565.30 2588.70 brushed CeramX 3 462.21 204.06 117.82 -44.71 969.13 273.75 678.93 polished Tetric brushed 3 1340.07 265.81 153.46 679.76 2000.37 1033.40 1504.40 400 Tetric polished 3 231.42 19.70 11.38 182.47 280.36 211.00 250.32 µm2 VDiamond 3 1963.03 971.22 560.73 -449.60 4375.67 1069.80 2996.90 brushed VDiamond 3 349.77 135.09 78.00 14.18 685.35 267.67 505.68 polished Total 18 1081.81 892.62 210.39 637.92 1525.70 211.00 2996.90 CeramX 3 3532.67 309.96 178.96 2762.68 4302.65 3189.50 3792.30 brushed CeramX 3 1437.44 723.05 417.45 -358.72 3233.59 638.61 2047.10 polished Tetric brushed 3 2160.30 209.14 120.75 1640.76 2679.84 2022.00 2400.90 10000 Tetric polished 3 492.53 121.77 70.30 190.03 795.02 364.30 606.61 µm2 VDiamond 3 2848.43 526.77 304.13 1539.86 4157.01 2285.10 3328.80 brushed

VDiamond 3 486.67 204.08 117.82 -20.29 993.62 331.79 717.91 polished Total 18 1826.34 1221.66 287.95 1218.82 2433.85 331.79 3792.30 161

ANOVA (Sz)

Sum of Squares df Mean Square F Sig. Between Groups 2773985.669 5 554797.134 12.493 .000 25 µm2 Within Groups 532905.484 12 44408.790 Total 3306891.153 17 Between Groups 10845703.942 5 2169140.788 9.643 .001 400 µm2 Within Groups 2699295.759 12 224941.313 Total 13544999.701 17 Between Groups 23378345.821 5 4675669.164 28.150 .000 10000 µm2 Within Groups 1993160.509 12 166096.709 Total 25371506.329 17

162