nanostructure and mechanical property of the …nano res 1 nanostructure and mechanical property of...

Nano Res

1

Nanostructure and mechanical property of the osteocyte

lacunar-canalicular network associated bone matrix

revealed by quantitative nanomechanical mapping

Shuai Zhang1, Fiona Linnea Bach-Gansmo1,2, Dan Xia1, Flemming Besenbacher1, Henrik Birkedal1,2 (), and Mingdong Dong1 ()

Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0825-8

http://www.thenanoresearch.com on June 1, 2015

© Tsinghua University Press 2015

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TABLE OF CONTENTS (TOC)

Nanostructure and

Mechanical Properties of the

Osteocyte

Lacunar-Canalicular

Network Associated Bone

Matrix Revealed by

Quantitative

Nanomechanical Mapping

Shuai Zhang1, Fiona Linnea

Bach-Gansmo1,2, Dan Xia1,

Flemming Besenbacher1,

Henrik Birkedal1,2*, and

Mingdong Dong1,*

1The Interdisciplinary

Nanoscience Center, Aarhus

University 2Department of Chemistry,

Aarhus University

The reduced modulus maps localize and show the heterogeneous nanostructure and

mechanical property of osteocyte lacunar-canalicular network associated bone matrix.

They were captured by nanoindentation and quantitative nanomechnical mapping,

respectively.


Nanostructure and Mechanical Property of the Osteocyte Lacunar-Canalicular Network Associated Bone Matrix Revealed by Quantitative Nanomechanical Mapping

Shuai Zhang1, Fiona Linnea Bach-Gansmo1,2, Dan Xia1, Flemming Besenbacher1, Henrik Birkedal1,2(), and Mingdong Dong1()

1 The Interdisciplinary Nanoscience Center, Aarhus University 2 Department of Chemistry, Aarhus University

Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the publisher)

©The Author(s) 2010. This article is published with open access at Springerlink.com

Received: day month year

Revised: day month year

Accepted: day month year

(automatically inserted by

the publisher)

© Tsinghua University Press

and Springer-Verlag Berlin

Heidelberg 2014

KEYWORDS

Lacunar-canalicular

network, osteocyte,

perilacunar bone matrix,

pericanalicular bone

matrix, reduced modulus,

mechanical properties

ABSTRACT

Osteocytes are the main bone cells embedded in the bone matrix where they

form a large surface-area network called the lacunar-canalicular network (LCN)

by interconnecting their resident spaces, the lacunae, and the canaliculi. More

and more evidences point to osteocytes playing a pivotal role in maintaining

bone quality. On the one hand, osteocytes transmit mechanical strain and

micro-environmental signals through the LCN to regulate the activity of

osteoblasts and osteoclasts, and on the other hand, there are increasing

evidences that the LCN associated bone matrix can be remodeled by osteocytes

in a process called osteocytic osteolysis. However, due to the significant

challenges to assess and characterize the LCN associated bone matrix, little is

known about the structure and corresponding mechanical properties. In this

work, we used quantitative nanomechanical mapping, backscattered electron

imaging and nanoindentation to characterize the LCN associated bone matrix.

The results show that the techniques can be used to probe the LCN associated

bone matrix. Nanoindentation and quantitative mechanical mapping reveal

spatially inhomogeneous mechanical properties of the bone matrix associated

with osteocyte lacunae and canaliculi. The obtained nano-topography and

corresponding nano-mechanical maps reveal altered mechanical properties in

the immediate vicinity of the osteocyte lacunae and canaliculi, which cannot be

explained solely by the topographic change.

Nano Res (automatically inserted by the publisher) DOI (automatically inserted by the publisher)

Review Article/Research Article Research Article

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

3

1. Introduction Osteocytes are the most abundant type of bone cells.

They are embedded within the mineralized bone

matrix. In the bone matrix, the osteocytes reside in

spaces called osteocyte lacunae, with typical lacunar

dimensions ranging between ~3-20 µm. [1-5] The

lacunae are connected by canaliculi of ~200-500 nm

diameter,[6-8] to form a large surface-area

connected network referred to as the

lacunar-canalicular network (LCN). Because the

osteocytes are buried in the mineralized bone matrix,

they are hard to access, and much less is known

about them than about the bone surface associated

osteoblasts and osteoclasts.[9] Recently, osteocytes

have gained massive attentions, as it is becoming

increasingly clear that they play a pivotal role in

maintaining bone quality.[2, 10, 11] It is now

established that osteocytes regulate the bone

remodeling activities of osteoblast and osteoclasts

by sensing mechanical signals and

micro-environmental conditions. [12-16] The

structure of the LCN and the characteristics of the

associated bone matrix have been proposed to

influence the efficiency of mechanosensing, thereby

affecting the bone repair system.[6, 17, 18]

Additionally, osteocytes have been suggested to be

involved in calcium homeostasis in a process called

osteocytic osteolysis, where the osteocytes dissolve

LCN associated bone matrix. [2, 19] While this

capability has been heavily debated in the literatures,

there is now mounting evidence supporting it. [11,

20, 21] Probing the local structure and mechanical

properties of the LCN associated bone matrix

(perilacunar and pericanalicular matrix) is therefore

essential to unravel how osteocytes and their

cellular processes affect the local bone matrix.

The LCN associated matrix and the topography of

osteocyte lacunae have been investigated using

sub-100 nm techniques including ptychography [22],

synchrotron nano-CT, [23] serial focused ion beam

scanning electron microscopy (FIB/SEM) based

backscattered electron imaging (BEI),[8, 24-27] and

serial FIB/SEM tomography on demineralized

samples [8] but detailed information on the

structural and mechanical properties, and the

relation between these are still lacking. Additionally,

high resolution mechanical mapping is crucial for

developing a complete model of the mechanical

properties of bone, covering the relevant length

scales from nano- to millimeter. [28, 29] Hence,

methods sensitive to both structural information

and local mechanical properties of the LCN

associated bone matrix are required.

Atomic force microscopy (AFM) based techniques

allow exploring the relationship between

topography and mechanical properties with

nanoscale resolution. These techniques have already

been used to characterize the structure of biological

materials such as bone.[30-33] The recently

developed AFM-based quantitative nanomechnical

mapping technique allows gathering quantitative

mechanical and morphological information

simultaneously. [34-36] Although it has been widely

applied to soft biomaterials, such as protein/peptide

based ones [37-39], its applicability to hard

biological materials remains less explored. Here we

applied quantitative nanomechanical mapping in

combination with BEI and nanoindentation to

characterize the LCN associated bone matrix.

Quantitative mechanical mapping with nanometer

spatial resolution enables mapping the topography

and mechanical properties of the LCN, and these

results are expected to be essential for

understanding the role of osteocytes and the LCN in

regulating bone quality and calcium homeostasis.

2. Experimental 2.1 Sample preparation

Bone samples from 4 months old female Wistar rats

obtained from a different study[40, 41] have been

measured. Cross sectional samples (400-500 µm

thick) of cortical bone from the mid-femur of rat

hind limbs were sawed (Exakt Apparatebau,

Norderstedt, Germany) and subsequently

embedded in Epofix (Struers, Ballerup, Denmark)

and polished (Knuth Rotor Polishing machine,

Struers, Ballerup, Denmark) using abrasive paper

and diamond grains (3 to 1/4 μm grain size). The

animal experiment was approved by the Danish

Animal Experiments Inspectorate.

2.2 Backscattered Electron Imaging (BEI)

For Backscattered Electron Imaging (BEI)

measurements, the polished cross sections from five

individual animals were rendered electrically

conductive by evaporative carbon coating ((Emitech

K950 evaporator, Ashford, UK, 25 nm thick layer) to

limit charging artefacts. The BEI measurements

were conducted according to the method adapted

by Bach-Gansmo [41] from the work of Roschger et

al.[42]and performed on a Nova NanoSEM600 (FEI


Company, Hillsboro, USA) with a GAD detector

(FEI, Eindhoven, The Netherlands) consisting of two

half circular silicon solid state detectors. The

accelerating voltage of the beam was adjusted to 20

kV, and the working distance was kept at 15 mm.

2.3 Nanoindentation

Indentation measurements were performed using a

Hysitron TriboIndenter (Hysitron, Minneapolis, MN)

with a Berkovitch tip as described by Bach-Gansmo

et al, [41] and the samples from three animals were

tested. The measurements in Fig. 1 consisted of 792

indents placed with 1 µm spacing employing a

maximum load of 500 µN. This combination of

indent spacing and maximum load has previously

been validated in indentation studies of compact

bone.[43] The small indent spacing is necessary to

map changes occurring close to the lacunar surface.

As each indent takes approximately 5 minutes

including 10 s load, 60 s hold time to relax

viscoelastic effects [44] and 10 s to unload and time

for instrument stabilization, the measurement is

laborious and slow. The unloading segment range of

95% to 20% Pmax in the load-displacement diagrams

were used for data fitting, and the reduced elastic

modulus (stiffness, Er) and hardness (H) calculated

using the Oliver-Pharr method.[45] The reduced

elastic modulus is reported due to uncertainties

introduced by estimating Poisson s ratio for

bone.[46] Hence, the reported Er values are relative,

and not absolute values of stiffness for comparison

with table values of Young s modulus. The

measurements were performed in dry conditions.

2.4 Quantitative Nanomechanical mapping

All topography and nanomechnical maps were

captured by MultiMode VIII AFM (Bruker, CA). The

probes used in the experiment is TAP525A (Bruker,

CA) with 8 nm typical tip radius and 200 N/m

typical spring constant. The samples from two

individual animals were tested. The spring constant

of each probe had been further calibrated by the

Thermal Noise method[47]. Offline analysis of

images and force curves were done with Nanoscope

Analysis (Bruker, CA) and Scanning Probe Image

Processor (SPIPTM, Image Metrology ApS, Lyngby,

Denmark).

3. Results and Discussion Osteocyte lacunae and the perilacunar bone matrix

from rat cortical bone were imaged using BEI as

shown in Fig. 1a and 1b. The gray level of the

images captured by BEI effectively maps the local

degree of mineralization, with lighter pixel values

corresponding to a higher degree of mineralization.

[24, 25] Fig. 1a reveals several osteocyte lacunae (L)

and a few blood vessel cavities (V), both having

dark gray levels. The lacunae exhibit a large

variation in size, shape and orientation.

Furthermore, some lacunae have mineral deposits

(labeled as Partly Mineralized Lacunae (PML) or

Mineralized Lacunae (ML) in Fig. 1a), with the

mineral fillings possibly reflecting a near-apoptotic

state of the osteocyte. [48] There is also a

heterogeneity in the local degree of mineralization

of the perilacunar matrix, with some lacunae having

a more highly mineralized perilacunar matrix

(lighter gray level values, one example highlighted

by black dashed circle) than others. By increasing

the imaging magnification, the canalicular (C)

network is partly revealed (Fig. 1b) around a single

lacuna (L). The high degree of porosity in the

perilacunar bone reflects the vast number of

canaliculi connecting the imaged lacuna with its

neighboring lacunae. A few canaliculi can be seen

running more or less parallel to the imaged section,

whereas the numerous circular porosities represent

canaliculi intersecting the image plane. The high

degree of interconnectivity of the LCN is sketched in

a simple manner in Fig. 1c.

BEI provides maps of the local degree of

mineralization at the micro-scale, but does not

provide any information on mechanical properties.

It is well known that the micro- and macro-stiffness

of bone increase with mineral content, but the

mechanical properties are not only sensitive to the

level of mineralization but also to the collagen fibril

orientation. [49, 50] Hence, the mineral content

alone is not a sufficient measure to probe bone

quality. At the nanoscale, interfibrillar

non-collagenous macromolecules are expected to

result in additional mechanical heterogeneity.

Nanoindentation has been widely used in bone

research to map mechanical properties. [51, 52]

Herein, we applied it to map the local mechanical

properties of the bone matrix surrounding osteocyte

lacunae. Fig. 1d shows a representative map of the

reduced modulus (Er) of the bone matrix obtained

with 1 µm indent spacing (which in turn defines the

lateral resolution). The Er is presented by rainbow

color with red and blue representing high and low

reduced moduli, respectively. The map reveals that

the reduced modulus of the perilacunar matrix is


5

smaller than that of the bone matrix further away

from the osteocyte lacuna. The lower panel of Fig.

1d shows the average Er from the area marked by

gray dashed lines in the upper panel of the Fig. 1d.

The average line profile confirms the trend observed

in the upper panel: the bone further away from the

lacuna has the same stiffness on both sides, whereas

the Er of the bone matrix close to the lacuna is more

heterogeneous.

Although nanoindentation allows mapping local

mechanical properties quantitatively, the resolution

of the technique is limited , due to the radius of the

diamond probe and the finite indent depth, which

makes it very challenging to obtain high fidelity

maps at the spatial resolution needed to resolve

local variations in mechanical properties around the

~3-20 µm diameter osteocyte lacunae. The limited

resolution makes it even more difficult to map the

~200-500 nm wide canaliculi and the pericanalicular

matrix, which is an indispensable part of the LCN.

The bone mineral crystals, the collagen fibrils and

non-collagenous proteins are of sub-100 nm size.

These nanoscale structural features determine the

mechanical response of the LCN and the associated

bone matrix. Hence, conventional nanoindentation

averages over several structural features relevant to

the mechanical performance of the LCN associated

bone matrix. To achieve higher resolution structural

and mechanical insights, we applied quantitative

nanomechnical mapping to characterize the

perilacunar and pericanalicular matrices.

A typical AFM topography image displayed in Fig.

2a shows a single osteocyte lacuna and the

associated bone matrix. The osteocytes were

removed during sample preparation; hence the

osteocyte lacuna is shown as a hole in the matrix

(dark contrast). Simultaneously, the in-situ reduced

modulus map has been recorded. It is shown as the

colored skin covering the reconstructed 3D

topography image of Fig. 2a (Fig. 2a’). This

illustrates the correlation between topography and

stiffness of the bone matrix. According to Fig. 2a’,

the perilacunar matrix is softer than the rest of the

matrix (the blue color level indicating lower stiffness,

green color level indicating medium stiffness, red

color level indicating higher stiffness). Additionally,

the perilacunar matrix around the osteocyte is

inhomogeneous in stiffness, with the area to the left

of the osteocyte lacuna being much softer than the

other regions.

Higher resolution maps provide much more detail

on the nature of the perilacunar matrix. Fig. 2b’

shows a higher resolution Er map of the area near

the left bottom corner of the osteocyte lacuna

contour. Blue & green dominate in Fig. 2b with

lower stiffness rather than red & green in Fig. 2c’. By

comparing the mechanical maps with the

topography images in Fig. 2b and 2c, it is obvious

that the variation in Er is not a direct function of the

variation in topography but rather must originate

from variations in matrix nano-structure and

composition.

The resolution of these nanomechnical maps is

remarkably improved compared to nanoindentation

and the standard AFM-based force volume maps.

Fig. 2d shows two typical curves, captured in

mature bone and in the perilacunar matrix,

respectively. The difference is obvious, reflecting a

smaller average stiffness of perilacunar matrix

compared to the bone matrix further away from the

lacuna. The local reduced moduli were determined

using the Derjaguin–Muller–Toporov model [53].

Thereby, the distributions of the logarithm of Er (Fig.

2e) on different areas (Fig. 2b and 2c) could be

constructed. The logarithm of Er is seen to be

normally distributed, reflecting that Er is lognormal

distributed. The peak value of the distribution of the

perilacunar matrix (i.e. data from Fig. 2b), 10.0±2.8

GPa, is much lower than that of the mature bone

matrix, 28.8±10.1 GPa (i.e. data from Fig. 2c) with a

student t-test p value far less than 0.0001. These data

agree well with the nanoindentation results (Fig. 1d),

but with much higher resolution, localization and

structural specificity.

After characterizing the nano-structure and

nano-mechanical properties of the perilacunar

matrix, our attention turned to the pericanalicular

matrix, the other main component of the LCN

associated bone matrix. Fig. 3a shows the

topography of the bone matrix around an osteocyte

lacuna. The contour line is drawn based on the

topography (the black solid line in Fig. 3a)

approximately, and some of the

several-hundred-nanometer wide canaliculi are

clearly identified according to the height (color)

contrast and highlighted by the white arrows

(labeled as canaliculus (C)). Furthermore, one

canaliculus and the surrounding pericanalicular

matrix (rectangular marked area in Fig. 3a) were

imaged at higher resolution (Fig. 3b and 3c). The


canaliculus branching into the pericanalicular bone

matrix is indicated by the blue contour line (Fig. 3b).

Again, the 3D topography map is combined with

the corresponding in-situ stiffness map as skin (Fig.

3c) and the results show that the pericanalicular

matrix is softer than the surrounding bone matrix.

After collecting force-distance curves (Fig. 3d), the Er

of the pericanalicular matrix (20.0±7.5 GPa, Fig. 3e)

was found to have a lower value than the bone

matrix (31.6±5.2 GPa, calculated from Fig. 3e) with

a student t-test p value less than 0.0001, but larger Er

than the perilacunar matrix with a student t-test p

value less than 0.0001. Interestingly, both the

diameter of the canaliculi and the stiffness of the

pericanalicular matrix seemed to vary with distance

from the osteocyte lacuna: the diameter of the

canaliculi decreases while the Er of pericanalicular

matrix increases with increasing distance from the

lacuna.

Not only the Young’s modulus, but also the energy

dissipation maps (Figure S1 and S2 in ES) indicate a

difference between pericanalicular matrix and bone

matrix further away from the canaliculi. The energy

dissipation maps represent the variations of the

energy dissipated during every tapping cycle. It

indicates that bone tissue is able to dissipate

mechanical energy in order to prevent dramatic

damage. Dissipation is influenced by several factors

including capillary forces and viscoelasticity of the

materials.[54] According to Figure S2E, the average

dissipated energy in the pericanalicular matrix is

larger than that of the bone matrix. It proves that the

pericanalicular matrix is different from the bone

matrix.

The present findings of the pericanalicular matrix

being different from the bone matrix further away is

in agreement with the recently published data of

Reznikov et al. who showed that the matrix

structure is remarkably different around canaliculi

both in terms of collagen fibril organization but also

in composition[8]. Herein, we show that this leads to

drastic changes in mechanical properties.

Collagen fiber bundles are the main organic

component in the bone matrix, and in the present

experiment, collagen fibers embedded in the

perilacunar matrix were also captured by the AFM

measurements. Fig. 5a shows bundles of collagen

fibers and individual collagen fibers are

recognizable from the high magnification image (Fig.

5b). The typical d-band periodic structure of type I

collagen fibers is visible with a period of 64.9±3.6 nm.

[41, 55] The correlation between topography and

stiffness maps (Fig. 5d) clearly reveals a much lower

stiffness of the collagen fibers compared to the

mineralized bone. The captured collagen fibers were

found by quantitative analysis (Fig. 5e and 5f) to

have a reduced modulus of 12.6 ±3.5 GPa, which is

in good agreement with previous finding of

mineralized collagen fibers.[56, 57] The mineralized

collagen fibers are proposed to resist LCN

deformation or crack formation/propagation. [32]

Collagen fibers making up bone have sub-micron

diameter and are here clearly localized by

quantitative nanomechnical mapping.

4. Conclusions In this work, the feasibility of applying quantitative

nanomechanical mapping to explore hard

heterogeneous biomaterials with nanoscale

resolution, was demonstrated. The local Young’s

modulus of the bone matrix, which was obtained by

quantitative nanomechnical mapping is of the same

magnitude as that obtained by nanoindentation. But

nanomechanical mapping provides much better

resolution, which allowed unravelling individual

parts of the LCN. Direct measurements of the

mechanical properties of the perilacunar and

pericanalicular bone matrix with nano-resolution

are essential for fully understanding the biological

functions of osteocytes and the LCN in bone. The

mechanical properties in the perilacunar regions

were found to be inhomogeneous and displayed

lower average stiffness than the bone matrix further

away from the osteocyte lacunae. The mapping of

local mechanical properties around and in canaliculi

showed that the local mechanical properties of the

canaliculi may depend on the spatial relation to the

parent osteocyte and/or the canalicular diameter

and in turn to the nanoscale organization and

composition of the extracellular matrix.

The inhomogeneous mechanical properties of the

LCN associate bone matrix will likely impact the

strain sensing capabilities of the LCN. The present

findings are also relevant for modeling work, where

assumptions on homogeneous bone matrix

properties have been employed until now.[6] While

further work is needed to establish the degree to

which these observations influence cell signaling

and whole organ response, such as the mechanical

variations between the bone matrixes from control

and treated animals with induced or inhibited


7

osteocyte osteolysis/perilacunar remodeling, they

clearly demonstrate that bone structure and

mechanics is highly complex and most likely tuned

towards local conditions. The present work

demonstrates that the LCN associated bone matrix is

less stiff than the bone further away from the

osteocyte lacunae. Combining this finding with the

large density of osteocytes in bone and the

mounting evidence that osteocytes are able to

demineralize the perilacunar matrix indicates that

the LCN is likely to play a much more direct role in

bone nano- and micromechanics than hitherto

realized.

Acknowledgements The authors gratefully acknowledge the financial

support from the Interdisciplinary Nanoscience

Center by the Danish National Research Foundation

to the Sino-Danish Center of excellence on “The

Self-assembly and Function of Molecular

Nanostructures on Surfaces” and from the Carlsberg

Foundation. M.D. acknowledges a STENO grant

from the Danish Research Council and the Young

Investigator Program from the Villum Foundation.

H.B. acknowledges the Human Frontiers Science

Program and the Danish Research Councils for

funding.

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Figures

Figure 1 a) BE image of rat cortical bone revealing numerous osteocyte lacunae (L), partially mineralized and mineralized lacunae

(PML & ML), and a few larger blood vessel cavities (V). A variation is seen in degree of mineralization (gray level) of the

perilacunar bone and one example of a more highly mineralized perilacunar matrix is highlighted by a black dashed circle. b) Higher

magnification of BEI reveals the beginning of the canalicular (C) network surrounding a single osteocyte lacuna (L). c) Sketch of

three lacunae (L) and canaliculus (C) to illustrate the LCN, and its high degree of connectivity. d) Reduced modulus map obtained

from nanoindentation measurements of bone matrix around a single lacuna. The osteocyte lacuna is located in the center with void

pixels corresponding to positions where no measurements were made in order to avoid tip crash. The resolution is 1 µm per pixel. A

corresponding line profile is presented in the lower panel, obtained by averaging the values in the area marked by dashed grey lines

in the upper panel.


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Figure 2 Topography images and reduced Young’s modulus maps of the bone matrix around an osteocyte lacuna. a) The overview

topography images. a’) 3D topography reconstruction of a) covered by the corresponding stiffness map. b), b’), c) and c’) Higher

magnification images of the areas indicated by the dashed squares on a) and a’), respectively. d) Two typical force-piezo movement

curves recorded on different areas showing the indent part of the full curves shown in the inset. e) Fitted distributions of the

logarithm of Er of Fig. 2b’ and 2c’.


Figure 3 Topography images and reduced Young’s modulus maps of the bone matrix around lacunae and canaliculi. a) The

topography image of the bone matrix around an osteocyte lacunae; the dark contour line roughly indicates the lacunar surface and the

arrows mark the alignments of example canaliculi. b) Higher magnification image at the position indicated by the dashed square in a)

showing the topography of pericanalicular matrix; the blue contour line highlights the alignment of the canaliculus from the top-left

corner to the bottom-right of the image. c) 3D topography reconstruction of b) covered by the corresponding Er colour map. d) the

indent part of two typical force-distance curves recorded at different positions in c; the inset display the full curves. e) The

distributions of the logarithm of Er with accompanying fits indicated by dashed lines


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Figure 4 Morphology images and Er map of the collagen fibers on the perilacunar matrix, near the osteocyte lacunae in Fig. 2a. a)

The overview morphology image of the collagen fiber-bunch. b) The zoom-in morphology image, showing individual collagen

fibers. c) The line profiles of the corresponding dashed color lines along the fibers in Fig. 4b. d) The 3D morphology reconstruction

of the image zoom-in from Fig. 4a, 4b covered by the corresponding stiffness map. e) The two typical force-piezo movement curves

which were recorded on different areas; zoomed from the inset curves. e) The fitted distributions of the logarithm of Er of selected

collagen fibers.


Electronic Supplementary Material

Nanostructure and Mechanical Property of the Osteocyte Lacunar-Canalicular Network Associated Bone Matrix Revealed by Quantitative Nanomechanical Mapping Shuai Zhang1, Fiona Linnea Bach-Gansmo1,2, Dan Xia1, Flemming Besenbacher1, Henrik Birkedal1,2(), and Mingdong Dong1()

1 The Interdisciplinary Nanoscience Center, Aarhus University 2 Department of Chemistry, Aarhus University

Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)

Figure S1 The topography and corresponding Yong’s modulus maps of LCN. The dashed squares indicate the zoom-in position of Figure S2.


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Figure S2 The zoom-in topography, and corresponding energy dissipation, deformation and Young’s modulus maps of pericanalicular matrix and bone matrix. (E) the distributions of energy dissipation calculated from selected area of Figure S2B with a student t-test p value far less than 0.0001.


Potential cover art, Nanomechnical maps LCN-bone matrix

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