1

Molecular Interactions between Collagen and Aggrecan from the Cartilage Extracellular Matrix

by

Fredrick P. Rojas

Submitted to the Department of Materials Science and Engineering

in Partial Fulfillment of the Requirements for the Degree of

Bachelor of Science

at the

Massachusetts Institute of Technology

June 2011

© 2011 Fredrick P. Rojas All rights reserved

The author hereby grants to MIT permission to reproduce and to

distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created

Signature of Author ............................................................................................................................ Department of Materials Science and Engineering

May 12, 2011

Certified by ........................................................................................................................................ Christine Ortiz

Professor of Materials Science and Engineering Thesis Supervisor

Certified by ........................................................................................................................................

Alan J. Grodzinsky Professor of Biological, Electrical and Mechanical Engineering

Thesis Supervisor

Accepted by ....................................................................................................................................... Lionel C. Kimerling

Thomas Lord Professor of Materials Science and Engineering Chairman, Undergraduate Thesis Committee

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Molecular Interactions between Collagen and Aggrecan from the Cartilage Extracellular Matrix

by

Fredrick P. Rojas

Submitted to the Department of Materials Science and Engineering on on May 6, 2011 in Partial fulfillment of the

requirements for the Degree of Bachelor of Science in Materials Science and Engineering

Abstract

In this study, colloidal force spectroscopy was utilized to quantify interactions between collagen and aggrecan in physiological and non-physiological aqueous solutions. An aggrecan-functionalized probe tip (R ~ 2.5µm) was indented ~500nm into the surface of a native collagen network, composed of trypsin-treated, proteoglycan-depleted cartilage. The resulting force-indentation curves were used to calculate the adhesion force and adhesion energy of the molecular interaction. Heterogeneous long-range adhesion was observed up to ~ 2.5 µm extension upon retraction after compressing the tip into the sample for a given surface dwell time, t. The adhesion force showed an asymptotic nonlinear increase with t, reaching a maximum value of 3.1 ± 0.2 nN at t = 60 s. Aggrecan-collagen interactions displayed a dependence on ionic strength, with a maximum adhesion force of 4.3 ± 0.3 nN at 1.0 M NaCl (t = 30 s). In addition, aggrecan-collagen interactions showed [Ca2+]-dependence with a maximum adhesion force of 7.4 ± 0.3 nN with a [Ca2+] = 20 mM (t = 30 s, 0.15 M). Molecular interactions between aggrecan and collagen are important in determining structural integrity of the cartilage extracellular matrix and its biological functions, such as energy dissipation, osmotic swelling and hydraulic permeability.

Thesis Supervisor: Christine Ortiz Title: Professor of Materials Science and Engineering Thesis Supervisor: Alan J. Grodzinsky Title: Professor of Biological, Electrical and Mechanical Engineering

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Table of Contents Abstract ...................................................................................................................................... 2 List of Figures and Tables ......................................................................................................... 4 1. Background and Motivation ................................................................................................. 7 1.1 Cartilage Collagen Network .......................................................................................... 7 1.2 Aggrecan .......................................................................................................................... 9 1.3 Reported Collagen-Aggrecan Binding Studies .......................................................... 13 2. Experimental Methods ........................................................................................................ 15 2.1 Sample Preparation ...................................................................................................... 15 2.2 Colloidal Force Spectroscopy ...................................................................................... 16 2.3 Data Processing and Analysis ...................................................................................... 18 3. Results ................................................................................................................................... 20 3.1 Effect of Surface Dwell Time on Aggrecan-Collagen Adhesion ............................... 21 3.2 Effect of Ionic Strength on Aggrecan-Collagen Adhesion ........................................ 24 3.3 Effect of Ca2+ on Aggrecan-Collagen Adhesion ......................................................... 27 4. Discussion ............................................................................................................................. 31 5. Conclusion ............................................................................................................................ 35 6. Acknowledgements .............................................................................................................. 36 7. References ............................................................................................................................. 37

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List of Figures and Tables

Figure 1-1 Schematic representation of the collagen type II, IX, and XI hetero-fibrillar network. Type XI collagen is found in the interior of the type II collagen fibril, while type IX collagen is located throughout the surface of the fibril and crosslinks other type II and IX collagen fibrils (6). ................................................................................................................................. 8  

Table 1-1 Chemical properties and corresponding amino acid compositions of type II collagen from adult porcine articular cartilage at physiological conditions (adapted from (9, 10)). ..... 8  

Figure 1-2 Schematic depicting the varying collagen fiber orientations found in cartilage (11). .. 9  

Figure 1-3 Schematic depicting the regions of aggrecan and the structural subunits of glycosaminoglycan chains (adapted from (15)). .................................................................... 10  

Figure 1-4 (a) Schematic of colloidal force spectroscopy experiment on retraction involving interactions between an aggrecan-functionalized planar substrate and an end-grafted aggrecan spherical probe tip. (b) An example of the resulting data from the experiment shown in (a) plotting the adhesive energy as a function of the time held at maximum compressive load (surface dwell time) and bath ionic strength. These data indicate that aggrecan is capable of undergoing self-adhesion given a sufficient compressive hold time. Statistically significant adhesion values were observed as a function of surface dwell times and ionic strength (4). ............................................................................................................ 12  

Figure 1-5 Schematic showing possible binding mechanisms between neighboring GAG chains while in solution. Dashed arrows represent possible hydrogen bonding, while an example of calcium ion-bridging is shown in the square (adapted from (22)). ........................................ 13  

Figure 1-6 Possible supramoleular assemblies found within the cartilage extracellular matrix. Illustration showing possible pathways of interaction between aggrecan and collagen, along with known dissociation constants (25). ................................................................................ 14  

Figure 2-1 Tapping mode height images of (a) single fetal epiphyseal aggrecan macromolecule on an atomically flat mica surface (adapted from (30)) and (b) air-dried calf femoral condyle knee cartilage surface with aggrecan digested, where the porous structure collapsed while the nanoscale structural features of the collagen fibrils were retained. (c) Schematic of interactions between the collagen network on cartilage surface and the aggrecan end-functionalized spherical probe tip simulating the in vivo situation (3,4,20). ......................... 17  

Figure 2-2 Calibration curves performed on a clean glass slide for an aggrecan functionalized tip in 1 mM and 1 M ionic strength (pH ~5.6). ........................................................................... 18  

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Figure 2-3 Characteristic force-indentation curve of the indentation of a collagen sample with an aggrecan-functionalized probe tip (t =30 s, PBS (IS =0.15 M), pH ~6.4), with appropriately marked variable definitions (35). ........................................................................................... 19  

Figure 3-1. Characteristic force-indentation curves for the indentation (500 nm indentation depth, surface dwell time of t = 30, 0.150 M NaCl, pH ~6.4) of trypsin-treated cartilage samples with colloidal probe tips functionalized with either aggrecan or OH-SAM. ......................... 21  

Figure 3-2. Characteristic force-indentation curves for the indentation of trypsin-treated cartilage samples (500 nm indentation depth, 0.150 M (PBS), [Ca2+]= 0.0 mM, pH ~6.4) with colloidal probe tips functionalized with aggrecan for surface dwell times of 10 and 30 seconds. .................................................................................................................................. 22  

Figure 3-3 The force of adhesion versus surface dwell time for the indentation (500 nm indentation depth, 0.150 M (PBS), [Ca2+]= 0.0 mM, pH ~6.4) of trypsin-treated cartilage samples with colloidal probe tips functionalized with either OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 75). ................................................... 23  

Figure 3-4 The energy of adhesion versus surface dwell time for the indentation (500 nm indentation depth, 0.150 M (PBS), [Ca2+]= 0.0 mM, pH ~6.4) of trypsin-treated cartilage samples with colloidal probe tips functionalized with either OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 75). ................................................... 24  

Figure 3-5 Characteristic force-indentation curves for the indentation (500 nm indentation depth, t = 30 s, ([Ca2+]= 0.0 mM, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with aggrecan in solution ionic strengths of 0.15 M NaCl and 1.0 M NaCl. ...................................................................................................................................... 25  

Figure 3-6 The force of adhesion versus ionic strength for the indentation (500 nm indentation depth, t = 30 s, [Ca2+]= 0.0 mM, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 50). ...................................................................................................... 26  

Figure 3-7 The energy of adhesion versus ionic strength for the indentation (500 nm indentation depth, t = 30 s, [Ca2+]= 0.0 mM, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tip functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 50). ...................................................................................................... 27  

Figure 3-8 Characteristic force-indentation curves for the indentation (500 nm indentation depth, t = 30 s, IS = 0.15 M, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with aggrecan for [Ca2+]= 0.0 mM and 20 mM. ............................................ 28  

Figure 3-9 The force of adhesion versus [Ca2+] for the indentation (500 nm indentation depth, t = 30 s, IS = 0.15 M, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips

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functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 40). ................................................................................................................... 29  

Figure 3-10 The energy of adhesion versus [Ca2+] for the indentation (500 nm indentation depth, t = 30 s, IS = 0.15 M, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 40). ................................................................................................................... 30  

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1. Background and Motivation

Cartilage tissue sustains loads and absorbs shocks between joints during biomechanical

motion. It is a highly hydrated macromolecular fiber-reinforced composite, in which the

extracellular matrix (ECM) is composed of a fibrillar collagen network surrounded by a matrix

of highly negatively charged brush-like aggrecan proteoglycans (1). Currently, relatively little is

known about the molecular details of the interactions between two major cartilage extracellular

matrix (ECM) macromolecular components, the collagen network and the highly negatively

charged aggrecan moiety. This interaction is hypothesized to play a structural and biomechanical

functional role in determining the integrity and tissue function of cartilage (1,2). Toward this end,

in order to elucidate the mechanistic origins of cartilage properties from its molecular

components and structure, we propose to quantify the molecular interactions between aggrecan

and the collagen network under physiologically relevant conditions using atomic force

microscope (AFM)-based colloidal force spectroscopy (3).

1.1 Cartilage Collagen Network

Approximately 60 wt. % of the solid mass found in articular cartilage is composed of

collagen, with type II collagen being the major constituent (4,5). Within a single collagen fibril,

type II collagen is copolymerized in the interior on a template of type XI collagen, while the

fibril surface contains type IX collagen that is capable of cross-linking with type II and IX

collagen neighboring structures (Figure 1-1) (5,6).

8

Figure 1-1 Schematic representation of the collagen type II, IX, and XI hetero-fibrillar network. Type XI collagen is found in the interior of the type II collagen fibril, while type IX collagen is located throughout the surface of the fibril and crosslinks other type II and IX collagen fibrils (6).

The molecular structure of collagen is defined as a triple helix that has the amino acid

sequence of (Gly-X-Y) (7). The amino acid residues at positions X and Y are exposed to the

surrounding solvent and are fully capable of intramolecular and intermolecular interactions (7).

Furthermore, as indicated in Table 1-1, Type II collagen possesses both positively and

negatively charged amino acids. Hence, molecular adhesion with the negatively charged

glycosaminoglycans of aggrecan is a possibility, through van der Waals interactions, hydrogen

bonding, ionic bonding, etc. (8,9).

Chemical Property Amino Acid Composition (Residues/1000 residues)

Positive 92 Negative 138

Polar 46 Nonpolar 542

Hydrophobic 182 Table 1-1 Chemical properties and corresponding amino acid compositions of type II collagen from adult porcine articular cartilage at physiological conditions (adapted from (9, 10)).

In addition, cartilage consists of three distinct regions, each containing a preferred

collagen fibril orientation and morphology (Figure 1-2). The bottom layer consists of a calcified

region and provides the connection from articular cartilage and bone. In the deep zone, the

9

collagen fibers are arranged perpendicular to the subchondral bone. Through the middle zone the

collagen fibers slowly transition to the orientation found in the superficial zone, which is a

layered conformation that is tangential to the articular surface (lamina splendens) (11,12). In this

study, the collagen network was harvested from the top superficial layer of calf knee cartilage,

which remained intact during the microtoming and subsequent chemical treatments to remove

proteoglycans. In the superficial layer, the long fiber axis of collagen fibrils are randomly

oriented in the plane perpendicular to the surface (13).

Figure 1-2 Schematic depicting the varying collagen fiber orientations found in cartilage (11).

1.2 Aggrecan

Aggrecan is the most abundant proteoglycan in cartilage (1) and a major constituent of

the cartilage extracellular matrix, making up approximately 30% of its dry weight (14). As

shown in Figure 1-3, aggrecan consists of a core protein with three globular domains (G1, G2,

and G3) and a “brush-like” region with densely packed chondroitin sulfate glycosaminoglycans

10

CS-GAGs) and keratan sulfate glycosaminoglycans (KS-GAGs) covalently bonded to the core

(1,15). These GAG chains are composed of an alternating sequence of N-acetylgalactosamine

and glucuronic acid, and are negatively charged under physiological conditions. Each aggrecan

molecule is composed of ~100 CS-GAG chains that are densely packed along its core protein,

and are ~40 nm in contour length (16). There are a total of ~104 negative charges per aggrecan

macromolecule [15, 17]. The highly negatively charged aggrecan molecules create a surrounding

of high osmotic pressure that adds to the unique biomechanical properties of cartilage in

compression and shear (1,2).

Figure 1-3 Schematic depicting the regions of aggrecan and the structural subunits of glycosaminoglycan chains (adapted from (15)).

The charged GAG chains also provide a source of electrical double layer repulsive

interactions within and between aggrecan molecules. The implications of the electrical double

layer in an environment with electrolytes are described by the Poisson-Boltzmann equation

∇!Ψ =  !! sinhΨ (1)

Ψ = !  !  !!  !

=   !  !  !!  !

(2)

11

where Ψ is the reduced surface potential, ! is the charge number, ! is Faraday’s constant, ! is

the universal gas constant, ! is the absolute temperature, and ! is the inverse Debye screening

length (3, 18, 19). The Debye Length (!!!) is the distance at which the potential decays to 1 !

of its initial value. For electrostatic interactions, as in the case of the GAG chains in aggrecan,

the full range of effect is approximated as ~5!!!. The Debye-Huckel Approximation provides a

linear approximation for charged surfaces to the Poisson-Boltzmann equation and defines the

Debye length as

!!! =   !  !  !!  !!!!!!

(3)

where !! is the ionic strength of the solution and ! is the permittivity of the solution. The extent

of potential interactions depends only on properties of the bath solution. Of interest is the fact

that increasing the ionic strength decreases the extent of long-range repulsion between two

charged surfaces (3, 18, 19).

In previous nanomechanical studies, we have shown that aggrecan provides compressive

and shear stiffness via electrostatic and steric repulsion between its glycosaminoglycan (GAG)

side chains (3,20,21) and can undergo molecular self-adhesion after compression, which is

hypothesized to be important for the self-assembled architecture and structural integrity of the

cartilage matrix (22). Recently, these nanomechanical force spectroscopy experiments analyzed

interactions between an aggrecan-functionalized planar substrate and an end-grafted aggrecan

spherical probe tip during retraction (Figure 1-4).

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Figure 1-4 (a) Schematic of colloidal force spectroscopy experiment on retraction involving interactions between an aggrecan-functionalized planar substrate and an end-grafted aggrecan spherical probe tip. (b) An example of the resulting data from the experiment shown in (a) plotting the adhesive energy as a function of the time held at maximum compressive load (surface dwell time) and bath ionic strength. These data indicate that aggrecan is capable of undergoing self-adhesion given a sufficient compressive hold time. Statistically significant adhesion values were observed as a function of surface dwell times and ionic strength (4).

In accordance with equation (3), aggrecan self-adhesion was found to have a dependence

on ionic strength of the liquid medium: experiments performed in the presence of calcium ions

measured a nearly four-fold increase in adhesion. These effects were believed to be the result of

ion-bridging of multivalent ions between the monovalent negative charges found in the GAG

chains as depicted in Figure 1-5 (22). As is evident in Figure 1-3 and Figure 1-5, GAG chains

consist of non-polar sugar rings with the following function groups: three hydroxyl (polar), one

sulfate (polar), one carboxyl (polar), and one methyl (nonpolar) (1, 22). This would indicate that

aggrecan is also capable of van der Waals and hydrophobic interactions through its nonpolar

regions and capable of hydrogen bonding through its polar functional groups.

13

Figure 1-5 Schematic showing possible binding mechanisms between neighboring GAG chains while in solution. Dashed arrows represent possible hydrogen bonding, while an example of calcium ion-bridging is shown in the square (adapted from (22)).

1.3 Reported Collagen-Aggrecan Binding Studies

Individual chondroitin sulfate E glycosaminoglycan chains were previously reported to

bind to type II collagen, under physiological pH and varying salt concentrations conditions, with

an equilibrium dissociation constant of 39 nM (23). In addition, isolated segments of keratin

sulfate-rich of aggrecan domains have been found to have a dissociation constant of 1.1 µM with

type I and isolated type II collagen (24), via peptide-collagen interactions.

Recent work also indicates that aggrecan-collagen interactions may be assisted through

two adaptor proteins, matrilin-3 and cartilage oligomeric matrix protein (COMP) (Figure 1-6)

(25). The collagen type II affinity of COMP in the presence of divalent ions was found to have a

dissociation constant of ~10-9 M (26). COMP has also been observed to have a significant

CS-GAG

O

HOOH

OO

O

O

HN

HOO

O

O

SO

O

O

O

HOOH

OO

O

O

HN

HOO

O

O

SO

O

O

O

O

HOOH

OO

O

O

HN

HOO

O

O

SO

O

O

O

HOOH

OO

O

O

HN

HOO

O

O

SO

O

O

O

HOH HOH H

OH HOH

O

OHHO

OO

O OCa2+

14

interaction with matrilin in the form of a dissociation constant on the order of 10-9 M (27).

Furthermore, in addition to interactions with collagen, matrilin is also capable of binding

aggrecan, which may promote the collagen-aggrecan interactions in vivo via collagen-COMP-

matrilin-aggrecan interactions (28, 29). A future endeavor of this project is to determine whether

this adaptor-assisted interaction between aggrecan and collagen is more physically relevant than

the current aggrecan-collagen model.

Figure 1-6 Possible supramoleular assemblies found within the cartilage extracellular matrix. Illustration showing possible pathways of interaction between aggrecan and collagen, along with known dissociation constants (25).

15

2. Experimental Methods

The molecular interactions between aggrecan and the collagen network from the cartilage

ECM were directly measured using colloidal force spectroscopy in aqueous solutions. Aggrecan

was chemically end-grafted to a micrometer-sized spherical probe and the collagen network was

prepared via enzymatic degradation of the superficial region of intact cartilage tissue, using prior

established protocols (3, 22). The functional form of the interaction profile (force versus distance)

on approach and retraction was analyzed and the adhesion force and energy was measured in

physiological and non-physiological aqueous electrolyte solution conditions as a function of

surface dwell time (t), ionic strength (IS), and calcium ion concentration ([Ca2+]). As a means of

comparison, spherical probes that were functionalized with hydroxyl self-assembled monolayers

(OH-SAM) were also tested under the same conditions.

2.1 Sample Preparation

Disks with intact cartilage surface were harvested from the femoropatellar groove of 1 –

2 week old bovine calves by microtoming off the top 1 mm thick layer of 6 mm cartilage plugs,

followed by 0.1 mg/ml trypsin (0.15 M NaCl, 0.05 M sodium phosphate, pH 7.2) digestion for 12

hours at 37°C. The residual plug consisted of an intact collagenous matrix that was devoid of

nearly all non-collagenous components (30), without introducing any significant structural

changes to the fibrillar network either microscopically (31) or macroscopically (32).

Aggrecan was extracted from fetal bovine cartilage that was obtained from the epiphyseal

growth plate region. Aggrecan fractions (A1A1D1D1) were purified through dialysis using 500

volumes of 1M NaCl and deionized water (16). The synthetic cross-linker dithiobis

(sulfosuccinimidyl propionate) (DTSSP) was added to an aggrecan aqueous solution and reacted

16

with aggrecan to form disulfide bonds at the N-terminus end of the aggrecan core proteins.

Dithiothreitol (DTT) was then used to reduce the disulfide bonds to thiol bonds. Thereafter, the

thiol functionalized aggrecan was filtered and diluted to a 1 mg/mL concentration in deionized

water. Gold-coated colloidal spherical probe tips (R = 2.5 µm, nominal spring constant k = 0.5

N/m, Novascan) were end-functionalized with aggrecan by immersion into 100 µL 1 mg/mL

thiol-functionalized aggrecan solution for 48 hours (3). The thiol-gold bonding between the

aggrecan and the colloid resulted in an aggrecan packing density ~50 mg/mL, within the

physiological concentration in the cartilage ECM (3). Control experiments utilized a probe tip

functionalized with a hydroxyl-terminated self-assembled monolayer (OH-SAM, 11-

mercaptoundecanol, HS(CH2)11OH). Identical gold-coated colloidal spherical probe tips were

functionalized by immersion into 5mg/mL ethanol solution of OH-SAM.

2.2 Colloidal Force Spectroscopy

Given that the type II collagen fibrils are randomly aligned in the 2D surface plane within

superficial zone of cartilage (Figure 2-1b) interactions between the aggrecan-depleted cartilage

disk with the intact surface present and the aggrecan functionalized spherical tip can accurately

mimic the interactions between these two components in vivo, without the interference of

artifacts introduced by the disassembled collagen fibrils from the middle-zone cartilage due to

microtoming, (Figure 2-1c) (11). Adhesion between the 2D collagen network and the end-

attached aggrecan layer was measured using the 3D molecular force probe (MFP-3D, Asylum

Research) in electrolyte aqueous solutions at varying ionic strengths (0.01 to 1 M NaCl, with or

without 2 mM [Ca2+]), as seen in Fig. 2-1. Differing the surface holding time conditions from 0

to 60 seconds between the aggrecan tip and collagen substrate at a constant indentation depth

(~500 nm) were also tested via the indenter mode. The cantilever deflection sensitivity (nm/V)

17

was calibrated on a hard mica surface, where the cantilever deflection equals the Z-piezo

displacement in the contact region. The thermal oscillation method was applied to determine the

actual cantilever spring constant (33). The zero contact point was determined via methods

discussed previously (34).

Figure 2-1 Tapping mode height images of (a) single fetal epiphyseal aggrecan macromolecule on an atomically flat mica surface (adapted from (30)) and (b) air-dried calf femoral condyle knee cartilage surface with aggrecan digested, where the porous structure collapsed while the nanoscale structural features of the collagen fibrils were retained. (c) Schematic of interactions between the collagen network on cartilage surface and the aggrecan end-functionalized spherical probe tip simulating the in vivo situation (3,4,20).

As mentioned earlier, ionic strength of the liquid medium has an effect on the long-range

repulsion of aggrecan. During calibrations of aggrecan functionalized cantilever tips, force

spectroscopy was preformed on glass slides, which acted as charged surfaces. Figure 2-2 depicts

two calibration curves performed on a clean glass slide for an aggrecan-functionalized tip in 10.0

mM and 1.0 M NaCl monovalent ion solution. In accordance with Equations (1-3), higher ionic

strength hindered long range repulsion by decreasing the Debye length, allowing the probe tip to

approach closer to the glass slide before significant cantilever bending. Similar calibration curves

were used to ensure that aggrecan was indeed on the surface of the colloidal probe tip before

indentation experiments were performed (18). This test was also performed in between

experimental sets as a measure of sample integrity. Aggrecan functionalized probe tips that lost

the long-range repulsion under low ionic strength conditions were replaced.

100 nm 300 nmNative Collagen Network

(a) (b) aggrecan(c)

18

Figure 2-2 Calibration curves performed on a clean glass slide for an aggrecan functionalized tip in 10 mM and 1 M ionic strength (pH ~5.6).

2.3 Data Processing and Analysis

All data collected from the experiments above were exported to ASCII format and

processed in MatLab (Version 7.1, MathWorks). We quantified the maximum adhesion force,

adhesion distance and adhesion energy from each pair of loading-unloading curves. Relevant

statistical tests (e.g. student’s t-test, analysis of variance, etc.) were performed to examine the

tested effects, including the surface dwell time, ionic strength, and presence of Ca2+. Once the

adhesion between aggrecan and collagen was quantified, molecular interaction theories that

relate the macromolecular components and structures to the interaction magnitudes could be

implemented to explain the observations and elucidate the molecular origins of collagen-

aggrecan interactions.

-­‐1000 -­‐800 -­‐600 -­‐400 -­‐200 0

1

2

3

4

5

6

7

8

9

10

Dis tance  (nm)

Force  (nN)

1.0  M10.0  mM

19

The combination of long range adhesion between aggrecan and collagen during retraction

and the viscoelastic properties of cartilage made it difficult to fit the experimental data collected

for this project to established contact mechanic models to extract adhesion values. For this study,

we utilized the criterion as stated by Lee, which is shown in Figure 2-3 (35). The adhesion

energy was estimated as the area between the loading and unloading curve that corresponds to a

negative force and the force of adhesion was defined as the greatest absolute force during

retraction.

Figure 2-3 Characteristic force-indentation curve of the indentation of a collagen sample with an aggrecan-functionalized probe tip (t =30 s, PBS (IS =0.15 M), pH ~6.4), with appropriately marked variable definitions (35).

-­‐3000 -­‐2500 -­‐2000 -­‐1500 -­‐1000 -­‐500 0 500 1000-­‐10

0

10

20

30

40

Dis tance  (nm)

Force  (nN)

Fad

Ead

20

3. Results

We utilized atomic force microscopy to quantify interactions between collagen and

aggrecan in various aqueous environments. Characteristic force-indentation interaction curves

for the different aqueous solutions are shown below. The adhesion force and energy between

aggrecan and collagen were measured as a function of surface dwell time, ionic strength, and

calcium ion concentration ([Ca2+]). As a means of comparison, we also measured the interactions

between collagen and a hydroxyl terminated self-assembling monolayer (OH-SAM). For all

experimental data sets, each data corresponds to the use of 2-3 bovine joints with a total number

of indentations between 40-75. Two-way analysis of variance (ANOVA) of the data sets showed

no significant differences in adhesion between samples (ANOVA, p > 0.1).

Figure 3-1 depicts characteristic force-indentation curves for the indentation of a

proteoglycan-depleted cartilage sample with both aggrecan functionalized and OH-SAM

functionalized spherical probe tips (500 nm indentation depth, surface dwell time of t = 30, 0.150

M NaCl). The results of these experiments were highly reproducible. The force-indentation

curves displayed some energy hysteresis during retraction due to the viscoelastic properties of

collagen and adhesion (negative forces) caused by the interaction between the native collagen

network and the functionalized spherical probe tips. OH-SAM probe tips often displayed an

overall greater adhesion force and had an adhesive interaction distance of ~1 μm, whereas

aggrecan functionalized probe tips displayed prolonged smaller adhesion forces with adhesive

interaction distances of up to ~2.5 μm.

21

Figure 3-1. Characteristic force-indentation curves for the indentation (500 nm indentation depth, surface dwell time of t = 30, 0.150 M NaCl, pH ~6.4) of trypsin-treated cartilage samples with colloidal probe tips functionalized with either aggrecan or OH-SAM.

3.1 Effect of Surface Dwell Time on Aggrecan-Collagen Adhesion

Characteristic force-indentation curves for aggrecan-collagen interactions (500 nm

indentation depth, 0.150 M (PBS), [Ca2+]= 0.0 mM) under various surface dwell times are shown

in Figure 3-2. Increasing the surface dwell time increased the hysteresis of the reaction curve

and resulted in significantly greater maximum force of adhesion at larger retraction distances.

Though the extent of adhesive interactive distances did not change, the respective adhesion

forces did increase significantly (one-way ANOVA, p < 0.5).

-­‐3000 -­‐2500 -­‐2000 -­‐1500 -­‐1000 -­‐500 0 500 1000-­‐10

0

10

20

30

40

Indentation  Depth  (nm)

Force  (nN)

AggrecanOH-­‐S AM

22

Figure 3-2. Characteristic force-indentation curves for the indentation of trypsin-treated cartilage samples (500 nm indentation depth, 0.150 M (PBS), [Ca2+]= 0.0 mM, pH ~6.4) with colloidal probe tips functionalized with aggrecan for surface dwell times of 10 and 30 seconds.

The overall dwell time dependence of the adhesion force and energy is shown in Figure

3-3 and Figure 3-4. For the control experiment (OH-SAM probe tip), adhesion forces varied

from 1.2 ± 0.1 nN to 5.6 ± 0.3 nN, for t = 0 s and t = 60 s, respectively. The aggrecan probe tip

experienced adhesion forces of 0.8 ± 0.1 nN at t = 0 s and 3.1 ± 0.2 nN at t = 60s s. In both cases,

there was minimal adhesion that trended upward towards an asymptotic horizontal limit with

respect to time. At shorter dwell times, the adhesion force of the two different probe tips were

comparable, but OH-SAM probe tips showed significantly greater adhesion force compared to

the aggrecan tip for t > 0 s (two-way ANOVA, p < 0.5).

-­‐3000 -­‐2500 -­‐2000 -­‐1500 -­‐1000 -­‐500 0 500 1000-­‐10

0

10

20

30

40

Indentation  Depth  (nm)

Force  (nN)

30  sec.10  sec.

23

Figure 3-3 The force of adhesion versus surface dwell time for the indentation (500 nm indentation depth, 0.150 M (PBS), [Ca2+]= 0.0 mM, pH ~6.4) of trypsin-treated cartilage samples with colloidal probe tips functionalized with either OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 75).

Figure 3-4 shows that the OH-SAM probe tips experienced adhesion energy values of 0.6

± 0.1 fJ at t = 0 s to 3.5 ± 0.3 fJ at t = 60 s. The aggrecan-collagen interactions varied from 1.33

± 0.2 fJ for t = 0 s to 3.9 ± 0.3 fJ at t = 60 s (Figure 3-4). In both systems, adhesion energy

increased with surface dwell time in a non-linear fashion. Unlike Figure 3-3, the horizontal

asymptotic limit was less pronounced. The adhesion energy for the aggrecan and OH-SAM

functionalized probe tips were comparable for all dwell times (two-way ANOVA, p > 0.5),

except for the condition of no surface dwell time (t = 0 s).

0 10 20 30 40 50 600

1

2

3

4

5

6

S urfac e  Dwell  T ime  (s )

Adh

esion  Fo

rce  (nN)

AggrecanOH-­‐S AM

24

Figure 3-4 The energy of adhesion versus surface dwell time for the indentation (500 nm indentation depth, 0.150 M (PBS), [Ca2+]= 0.0 mM, pH ~6.4) of trypsin-treated cartilage samples with colloidal probe tips functionalized with either OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 75).

3.2 Effect of Ionic Strength on Aggrecan-Collagen Adhesion

The effect of ionic strength (IS) on the adhesion between aggrecan and collagen at a

constant indentation depth of ~500 nm and a surface dwell time of t = 30 s was investigated in

NaCl aqueous solutions with varying ionic strength (0.01 M, 0.15 M, 1.0 M). The effect of ionic

strength on the aggrecan-collagen interaction is evident in Figure 3-5, which depicts a set of

characteristic force-indentation curves for the aggrecan functionalized tip at a constant surface

dwell time of 30 seconds. Higher ionic strength corresponded to slight increases in adhesion

energy and force, but the adhesive interaction distance was not significantly affected.

0 10 20 30 40 50 600

0.5

1

1.5

2

2.5

3

3.5

4

4.5

S urfac e  Dwell  T ime  (s )

Adh

esion  Ene

rgy  (fJ

)

AggrecanOH-­‐S AM

25

Figure 3-5 Characteristic force-indentation curves for the indentation (500 nm indentation depth, t = 30 s, ([Ca2+]= 0.0 mM, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with aggrecan in solution ionic strengths of 0.15 M NaCl and 1.0 M NaCl.

The indentation of native collagen samples was performed in electrolyte solutions

varying in ionic strength from 0.01 M to 1M NaCl; the overall dependence of adhesion is shown

in Figure 3-6 and Figure 3-7. The adhesion force of the OH-SAM and collagen interactions

varied from 6.4 ± 0.3 nN at 0.01 M NaCl to 5.37 ± 0.3 nN at 1.0 M NaCl. The adhesion force of

the aggrecan-collagen interactions varied from 2.5 ± 0.2 nN at 0.01 M NaCl to 4.3 ± 0.3 nN at

1.0 M NaCl. Interestingly, the difference between 0.01 M and 0.15 M did not lead to a

significant increase in adhesion between aggrecan and collagen. Only at 1M NaCl was there a

significant increase in adhesion (t-test, p < 0.5). In all monovalent conditions, the adhesion force

for the OH-SAM and collagen interactions was significantly greater than the aggrecan-collagen

interactions (one-way ANOVA, p < 0.5).

-­‐3000 -­‐2500 -­‐2000 -­‐1500 -­‐1000 -­‐500 0 500 1000-­‐10

0

10

20

30

40

Indentation  Depth  (nm)

Force  (nN)

0.15  M1.0  M

26

Figure 3-6 The force of adhesion versus ionic strength for the indentation (500 nm indentation depth, t = 30 s, [Ca2+]= 0.0 mM, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 50).

Figure 3-7 shows that the adhesion energy of the OH-SAM and collagen interactions

varied from 6.2 ± 0.4 fJ at 0.01 M NaCl to 6.0 ± 0.6 fJ at 1.0 M NaCl. As for aggrecan-collagen

interactions, the adhesion energy changed from 4.4 ± 0.3 fJ at 0.01 M NaCl to 5.6 ± 0.4 nN at 1.0

M NaCl. The adhesion energy OH-SAM probe tip did not change significantly for the different

ionic strengths (one-way ANOVA, p > 0.05). This may indicate that the slight decrease in

adhesion force of OH-SAM for the greater ionic strength was merely a statistical anomaly.

Similar to the adhesion force, the adhesion energy of the aggrecan-functionalized probe tips only

changed significantly in the 1 M NaCl solution. Adhesion energy values for the aggrecan tip

were still comparable to those experienced by OH-SAM tip for the 0.1 M and 1.0 M NaCl

solutions (t-test, p > 0.05).

0.01 0.15 1.0  0

1

2

3

4

5

6

7

Ionic  S treng th  (M)

Adh

esion  Fo

rce  (nN)

AggrecanOH-­‐S AM

27

Figure 3-7 The energy of adhesion versus ionic strength for the indentation (500 nm indentation depth, t = 30 s, [Ca2+]= 0.0 mM, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tip functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 50).

3.3 Effect of Ca2+ on Aggrecan-Collagen Adhesion

The native collagen networks derived from trypsin treated cartilage samples were also

indented (500 nm indentation depth, surface dwell time of t = 30 s) in aqueous environments

with an overall ionic strength of IS =150 mM, but with varying concentrations of calcium ions in

the form CaCl2 ([Ca2+]= 0.0 mM, 2 mM, 20 mM). Characteristic force-indentation curves for the

aggrecan-functionalized tip under these experimental conditions are shown in Figure 3-8.

Increasing the divalent calcium ion concentration significantly increased the adhesion energy and

adhesion force. The extent of the adhesive interaction distance was not affected by the [Ca2+],

(one-way ANOVA, p > 0.05). Additionally, the effect of [Ca2+] diminished greatly after ~1 µm

retraction from the surface.

0.01 0.15 1.0  0

1

2

3

4

5

6

7

8

Ionic  S treng th  (M)

Adh

esion  Ene

rgy  (fJ

)

AggrecanOH-­‐S AM

28

Figure 3-8 Characteristic force-indentation curves for the indentation (500 nm indentation depth, t = 30 s, IS = 0.15 M, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with aggrecan for [Ca2+]= 0.0 mM and 20 mM.

The overall calcium ion dependence of the adhesion energy and force is shown in Figure

3-9 and Figure 3-10. The adhesion force of the OH-SAM and collagen interactions differed from

4.8 ± 0.4 nN in the absence of Ca2+ to 4.87 ± 0.2 nN with a [Ca2+] = 20 mM. For the OH-SAM

tip, there was no overall dependence of adhesion force on calcium ion concentration (one-way

ANOVA, p > 0.05). The adhesion force of the aggrecan-collagen interactions differed from 2.95

± 0.2 nN in the absence of Ca2+ to 7.4 ± 0.3 nN with a [Ca2+] = 20 mM. The aggrecan-collagen

system experienced drastic changes in adhesion force with respect to [Ca2+] (one-way ANOVA,

p < 0.05). With a [Ca2+] = 20 mM, the aggrecan-collagen interactions were greater than the OH-

SAM and collagen control interactions.

-­‐3000 -­‐2500 -­‐2000 -­‐1500 -­‐1000 -­‐500 0 500 1000-­‐10

0

10

20

30

40

Indentation  Depth  (nm)

Force  (nN)

[C a2+]  =  0.0  mM

[C a2+]  =  20.0  mM

29

Figure 3-9 The force of adhesion versus [Ca2+] for the indentation (500 nm indentation depth, t = 30 s, IS = 0.15 M, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 40).

Figure 3-10 shows that the adhesion energy of the OH-SAM and collagen interactions

varied from 2.5 ± 0.3 fJ in the absence of Ca2+ to 2.9 ± 0.2 fJ with a [Ca2+] = 20 mM. For the

OH-SAM tip, adhesion energy was independent of calcium ion concentration (one-way

ANOVA, p > 0.05). As for aggrecan-collagen interactions, the adhesion energy changed from

3.2 ± 0.7 fJ in the absence of Ca2+ to 4.8 ± 0.4 fJ with a [Ca2+] = 20 mM. There was a significant

increase in adhesion energy for the aggrecan-collagen interactions in aqueous solutions with a

[Ca2+] = 20 mM (t-test, p < 0.05). Furthermore, in contrast to other aqueous conditions, a [Ca2+]

= 20 mM resulted in a greater adhesion energy for the aggrecan tip compared to OH-SAM probe

tip (t-test, p < 0.05).

0.0   2.0   20.00

1

2

3

4

5

6

7

8

C alc ium  Ion  C oncentration  (mM)

Adh

esion  Fo

rce  (nN)

AggrecanOH-­‐S AM

30

Figure 3-10 The energy of adhesion versus [Ca2+] for the indentation (500 nm indentation depth, t = 30 s, IS = 0.15 M, pH ~5.6) of trypsin-treated cartilage samples with colloidal probe tips functionalized with OH-SAM or aggrecan. Error bars correspond to the standard error of measure (n = 40).

0.0   2.0   20.00

1

2

3

4

5

C alc ium  Ion  C oncentration  (mM)

Adh

esion  Ene

rgy  (fJ

)

AggrecanOH-­‐S AM

31

4. Discussion

In this study, aqueous colloidal force spectroscopy was used to quantify interactions

between collagen and aggrecan in aqueous solutions. An aggrecan-functionalized probe tip (R

~2.5μm) was utilized to obtain force-indentation curves on the surface of a native collagen

network, which consisted of trypsin-treated, proteoglycan-depleted cartilage samples (composed

of mostly type II collagen fibrils). The force-indentation curves of the system were analyzed and

used to calculate the adhesion force and energy. Experiments were conducted in physiological

and non-physiological aqueous electrolyte solution conditions, as a function of surface dwell

time, ionic strength, and calcium ion concentration. As a means of comparison, identical tests

were performed with a probe tip functionalized with a hydroxyl self-assembling monolayer.

As previously mentioned, the GAG chains of aggrecan are capable of hydrophobic and

van der Waals interactions by means of their nonpolar regions of methyl functional groups and

sugar rings. Furthermore, the GAG chains are also capable of hydrogen bonding via their

hydroxyl, carboxyl, and sulfate polar functional groups. Similarly, as displayed in Table 1, the

varied amino acid composition of collagen type II enables hydrogen bonding, nonpolar,

hydrophobic, and van der Waals interactions by means of its polar and charged functional groups.

The dependence of surface dwell time for the aggrecan and OH-SAM functionalized

probe tips is evident in Figures 3-3 and 3-4. The adhesion force showed a significant nonlinear

increase with respect to surface dwell time asymptotically approaching a maximum value of 3.1

± 0.2 nN at t = 60 seconds. In addition, there was a nearly four-fold increase in adhesion energy

for the aggrecan-functionalized probe tip for t = 60 s. These results can be attributed to an

increase in molecular interactions: prolonged contact with the collagen network allowed the

32

aggrecan macromolecules to rearrange and spread across the surface of the fibers, effectively

increasing the relative contact area and sites of molecular interactions.

The indentation of the collagen samples with the OH-SAM probe tips resulted in overall

greater adhesion force for all dwell times. This indicates that the interactions between collagen

and individual aggrecan macromolecules were not as strong as those between the hydrophilic

hydroxyl functional groups and collagen. Despite the weaker individual molecular aggrecan-

collagen interactions, the adhesion energy of the two probe tips were statistically similar for t > 0

s. The larger deformability of aggrecan, compared to the OH-SAM (HS(CH2)11OH), enabled

more molecular contacts with the collagen network under the same indentation conditions. This

effect was most evident in the heterogeneous long-range adhesion that was observed at up to

~2.5 μm extension upon retraction for the aggrecan-functionalized probe tips. Given that the

contour length of aggrecan is approximately 400 nm, the extension on the order of microns

suggests that the collagen network was also being pulled during reaction. The different aqueous

conditions did not change the adhesive interaction distance for the aggrecan-functionalized probe

tip.

As seen in Figure 3-5, there was no significant long-range repulsion detectable between

aggrecan and collagen during approach for any ionic strength condition. Given the highly

negative charge of aggrecan, this indicates that the surface of the collagen network did not

display a significant overall charge. However, in Figure 3-6 the adhesion force of the aggrecan

tip nearly doubled between ionic conditions of 10 mM NaCl (2.5 ± 0.2 nN) and 1 M NaCl (4.3 ±

0.3 nN). The increase in adhesion with respect to ionic strength indicates that regions of the

collagen network surface had negatively charged amino acids that were exposed and provided a

source of electrostatic repulsion for individual aggrecan molecules. Given the variation in local

33

charges of the collagen network, shielding of electrostatic repulsion was only experienced at

some molecular interaction sites and thus the adhesion energy only increased ~20% for the 1 M

NaCl solution.

Figure 3-9 and Figure 3-10 show the dependence of adhesion for aggrecan-collagen

interactions on calcium ion concentrations ([Ca2+]). The adhesive interaction distance was

observed to be similar at different [Ca2+], but the adhesion force increased more than two-fold in

magnitude from 0 mM (2.95 ± 0.2 nN) to 20 mM (7.4 ± 0.3 nN). These results are in accordance

with previous experiments that showed the divalent ion-bridging capabilities of aggrecan during

self-adhesion (22,36). Similarly, divalent ion bridging would facilitate an interaction mechanism

between two negative point charges present in aggrecan and the collagen surface. The fact that

the adhesion energy only experienced a 50% increase in the presence of 20mM of CaCl2 denotes

that the divalent ion-bridging was only present in certain (negative) regions of the collagen

network. Moreover, the effect of [Ca2+] on adhesion diminished greatly beyond ~1 µm,

indicating that divalent ion-bridging was present only at shorter lengths. The exact effective

range cannot be estimated, since collagen also contributed to the adhesive interaction distance.

The different ionic conditions provided insight into the collagen-aggrecan interactions

present in vivo. Our study showed there was significant adhesion between aggrecan and the

collagen network at physiological ionic strength of ~150 mM, showing some shielding of

electrostatic repulsion between aggrecan and exposed negatively charged amino acids on the

fibrillar surface of collagen. As would be the case in physiological conditions, a small presence

of calcium ions (~2mM) also increased the adhesion between aggrecan and collagen. The effect

of ionic strength and [Ca2+] on the aggrecan-collagen interaction was similar to that observed in

previous work on aggrecan-aggrecan adhesion. For the physiological condition of 0.15 M ionic

34

strength and [Ca2+] ~2mM, the adhesion of aggrecan-collagen interactions were ~50% greater

than aggrecan-aggrecan interactions.

However, given the increase in adhesion for aggrecan-collagen in 1.0 M ionic strength

solution, there are still some electrostatic repulsion forces present between the two cartilage

extracellular components under physiological ionic concentrations. Therefore, aggrecan is

capable of interacting with collagen in the extracellular matrix but is not immobilized once in

contact with the fibrillar surface of collagen. In turn, this would allow aggrecan to maintain

mobility within the extracellular matrix and continue to provide osmotic pressure and hydraulic

permeability. Furthermore, the adhesion energy between aggrecan and collagen provide another

means of energy dissipation while under loading. As was postulated for the aggrecan-aggrecan

interaction, energy dissipation can help maintain the structural integrity of the cartilage

extracellular matrix (22). Ultimately, we are hopeful that these findings will provide further

insight into the self-assembly of the cartilage extracellular matrix and help develop principles for

tissue regeneration and replacement.

35

5. Conclusion

An aggrecan-functionalized probe tip (R ~2.5µm) was indented ~500nm into the surface

of a native collagen network, composed of trypsin-treated, proteoglycan-depleted cartilage. The

molecular interactions between aggrecan and collagen were quantified in various aqueous

conditions. The aggrecan-collagen adhesion showed a significant ionic strength and [Ca2+]-

dependence. Under the physiological conditions of 0.15 M ionic strength and a [Ca2+] ~2mM,

aggrecan-collagen interactions showed an adhesion force of 5.3 ± 0.9 nN (t = 30 s). Full

electrostatic shielding is evident at 1.0 M ionic strength, indicating there are still some repulsive

electrostatic forces present between aggrecan and collagen at physiological concentrations.

Aggrecan-collagen adhesion was increased through calcium ion-bridging between aggrecan and

negatively charged amino acids exposed at the collagen fibrillar surface. We believe that

aggrecan-collagen adhesion adds to the structural integrity of the cartilage extracellular matrix

and plays a role in the biomechanical properties of cartilage. We are hopeful that these findings

will provide further insight into the self-assembly of the cartilage extracellular matrix and help

develop principles for tissue regeneration and replacement.

The methodologies developed here can be extended to the other underlying layers of

cartilage, where aggrecan content and fibrillar structure vary significantly compared to the

superficial layer. Future work can also incorporate the effects of osteoarthritis, specifically the

degradation of the collagen fibrillar network and its effects on aggrecan-collagen adhesion. In

addition, similar indentation experiments can be performed in the presence of other extracellular

components, such as COMP and matrilin-3, to analyze any possible tertiary effects that are

caused by adaptor mediated interaction mechanisms.

36

6. Acknowledgements

I would like to give a special thanks to Han-Hwa Hung for her assistance with sample

preparation and other laboratory work, Lin Han and Alan Shwartzman for their help with MFP-

3D training and related problems, and Alan Grodzinsky and Christine Ortiz for their helpful

advice and resources.

37

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