Slides for talk at BMES Conference 2011

Novel Biomedical and Biological Applica1ons

using Lab‐based Mul1‐scale CT System

K. Sen Sharma, D. M. Vasilescu, A. S. Kizhakke Puliyakote, E. A. Hoffman, T. Andric, J. W. Freeman, C. Markert,

J. D. SchiMauer, S. Xiao, H. Yu, and G. Wang

Claims

Micro‐ and nano‐CT: 1.  Allow higher resolu1on in tradi1onal biological

applica1ons 2.  Overcome limita1ons of tradi1onal imaging

methods (e.g. SEM, histology) 3.  Allow novel imaging applica1ons not possible

otherwise

Outline

 Sample Applica1ons 1.  Imaging of Vasculature 2.  Tissue Engineering 3.  Imaging of Mouse Lung 4.  Animal Embryo Microfossil

Project objectives: •  To illustrate the advantage of using micro-CT for imaging blood vessel

networks in mouse models Sample: •  Mouse limb, contrast agent: Microfil •  In 10% formalin solution during scan Collaborator: (Wake Forest University) Dr. C. Markert

#1 ‐ Imaging of Vasculature Multiresolution imaging of mouse limb [Duvall et. al. Am J. Heart Circul. Phy. 2004]

Mouse Limb Vasculature

VR of portion of mouse limb – 5µm resolution

0.5mm

VR of entire mouselimb -16µm resolution [Duvall et. al. Am J. Heart Circul. Phy. 2004]

#2 ‐ Tissue Engineering

Scaffolds For bone regeneration

SEM -Only surface imaging -Sectioning required

SOLUTION Micro-CT allows

imaging of internal structure

Bone loss Occurs due to cancer, injury etc.

Project objectives: •  To fabricate fiber scaffolds that

mimic   a single osteon (functional

unit)   cortical bone.

Collaborator: (VT, Rutgers) T. Andric, Dr. J. Freeman

Osteon‐like Scaffolds

Micro-CT for Osteon-Like Scaffolds 1Sen Sharma, K; 1Andric, T; 1Wright, L D; 1Freeman, J W; 1Wyatts, C L; +1Wang, G

+1Virginia Polytechnic Institute and State University

Senior author: [email protected] / [email protected]

INTRODUCTION

Recently, a method has been developed to fabricate fiber scaffolds that

mimic the architectural organization of a single osteon and 3D scaffolds

that mimic the structural organization of the cortical bone. Currently

used assessment techniques like scanning electron microscopy (SEM),

mercury and flow porosimetry etc. are unable to accurately quantify the

internal 3D structure of such scaffolds. Microscale computed

tomography (micro-CT) was used in this study to scan the scaffold at

resolution of 4 !m and acquire 3D images. Micro-CT results obtained

provided information about internal features that could not be obtained

from SEM scans.

METHODS

Osteon-like scaffolds were fabricated using a new electrospinning setup

as described in Andric et al. [1]. Briefly, poly (L-lactide) (PLLA) and

poly (D-lactide) (PDLA) polymer solutions were electrospun onto

rotating poly (glycolide) (PGA) microfibers. The thickness of the

nanofiber layers around a PGA microfiber scaffold was varied by

controlling the volume of the electrospun solution. The osteon-like

sections were then stacked together and wrapped with a sheet of

PLLA/PDLA electrospun mat and placed into a 10 mm cylindrical mold

with 5 mm diameter. These scaffolds were then heat sintered to create

3D scaffolds.

A cross section of the microfiber scaffold was characterized using !"#$

%&'()'$ *++$ !",$emission environmental SEM. Micro-CT scans were

run on Xradia MicroXCT-400. A 3D scaffold was placed in a 0.5 ml

PCR tube secured to a sample holder on the rotary stage of the micro-CT

scanner. X-ray source voltage and current were set at 30 kV and 100 !A

respectively. For each tomogram, 271 projections of 20482 pixels were

acquired between -135º and +135º - a total of 2.3 GB of projection data.

Acquisition time was approximately 5 h/specimen with exposure time of

60 s/projection. Xradia proprietary software (TXM Reconstructor and

TXM 3D Viewer) was used to reconstruct images from the projections

and view the reconstructed results. It took 3 minutes to reconstruct a

10243 image volume.

RESULTS

Figure 1 is an SEM cross-section of a single osteon microfiber with

PGA core and concentric layers of PLLA fibers. It shows extensive

details at the surface but does not provide information about internal 3D

features. Figure 2 is a reconstructed CT slice of a 3D scaffold and shows

the PGA fibers in bright white along with concentric players of PLLA

fibers around each PGA fiber. The voxel size in the tomogram was 2.5

!m. This resolution was sufficient to resolve PGA cores with diameter

95-130 !m. This imaging technique is beneficial to assess the uniformity

of the osteon scaffolds and how well the scaffolds are packed together. It

is seen that the diameter of the osteon scaffolds varies between 190 and

1100 !m. Figure 3 depicts a multi-planar view of the 3D scaffold. The

longitudinal cross-section shows that PGA fibers run along the entire

length of the scaffold. It also shows the layers of PLLA fibers and how

they are packed.

DISCUSSION

Micro-CT data showed that the scaffold engineering procedure was

successful as a proof of concept, but the next step would be to control

the osteon scaffolds better to have a more uniform diameter distribution.

Mineralized scaffolds will also be imaged to view a mineral distribution

throughout the scaffold.

This imaging technique is potentially important to evaluate the

uniformity of the osteon scaffolds and how well the scaffolds are packed

together. It also provides cross sectional images and 3D reconstruction

without fixing or sectioning the scaffold, which is required in classic

methods like SEM. As micro-CT is non-invasive, it might be utilized to

study a single scaffold at different time points of a particular study (e.g.

cell culture, mechanical testing etc). Thus, micro-CT may help build

better scaffolds that have mechanical properties and structures similar to

natural bone in order to promote complete tissue regeneration.

Figure 1. SEM cross section of a microfiber scaffold.

Figure 2. Reconstructed CT slice of the 3D scaffold. Scale width is 2000

!m.

Figure 3. Multiplanar view of the 3D scaffold.

REFERENCES

1. -.$/(01234$5.6.$71289)4$:.$7.$!1;;<'(.$!'=123')2>($>?$

@2(;1'A2B;0$CD);>(EA2F;$G3'??>A0D4$H@"G$/((&'A$@;;)2(8.$

I2))D=&1894$I/4$JG/4$K++L

Multiplanar view of 3d scaffold shows internal structure of individual microfiber scaffolds and

alignment within 3d scaffold.

0.5mm

Osteon‐like Scaffolds

Video showing alignment of microfiber scaffolds within 3d scaffold (Long).

0.5mm 4000 μm

550 μm

Optical microscope (Top) and SEM (Bottom) images of osteon-like scaffold.

[T. Andric et al. Mat. Sci. Eng. C 2011]

Project objectives: •  To develop an imaging protocol that

allows non-destructive multi-resolution imaging.

•  To provide measurements of parenchymal characteristics such as: volume fractions, surface area and alveolar number.

Subject: •  Lungs of mice, fixed in situ by

means of vascular perfusion at 20cmH2O airway pressure.

Collaborators: (University of Iowa) D. M. Vasilescu, A. S. Kizhakke Puliyakote, Dr. E. A. Hoffman

#3 – Imaging of Mouse Lung

Mouse lung imaging and volume calculation [Vasilescu et al. J. Appl. Physiol. 2011]

Acinar Structure of Mouse Lungs

Scanner: Xradia MicroXCT Objective: 0.5x and 10x Voxel size: 13.1µm and 2.0µm

Mouse lung – reconstructed transversal slice (0.5x objective of MicroXCT, 13.1µm voxel); Inset: interior ROI imaged at 10x, 2.04µm voxel.

Imaging series: in vivo (le^, 28µm/voxel), ex‐vivo (middle, 13µm/voxel) and high resolu1on (right, 2µm/voxel) images.

[Vasilescu et al. J. Appl. Physiol. 2011]

Project objectives: To understand the early developmental biology and taphonomy (how fossils are preserved) of the earliest known animals using X-ray CT. Subject: 600 million year old animal embryo fossils with possible nuclei Collaborator: (Virginia Tech) Dr. J. D. Schiffbauer, Dr. S. Xiao

#4 ‐ Animal Embryo Microfossils

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(A) Optical microscope image of microfossil, (E) Microfossil structure, (B-D, F-H) MicroCT images.

[J. W. Hagadorn et al. Science 2006]

Video in which planar sections allow visualization of cell boundaries and nuclues-like structures.

Microfossil in Micro‐CT 0.5mm 250µm

Scanner: Xradia MicroXCT-400 Objective: 20x Voxel size: 0.7µm

Microfossil in Nano‐CT

Scanner: Xradia NanoXCT-100 Objective: NanoXCT LFOV Voxel size: 65nm

10µm

Micro- and nano-CT imaging of internal mold of an early Cambrian shelly fossil

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Conclusion

Higher Resolu1on

Overcome Limita1ons

Novel Applica1ons

Thanks for Your A0en2on from All of Us

(Le^ to Right) Top row: K. Sen Sharma, D. M. Vasilescu, A. S. Kizhakke Puliyakote, E. A. Hoffman, T. Andric Bodom row: J. W. Freeman, C. Markert, J. D. SchiMauer, S. Xiao, H. Yu, G. Wang

Grant Support: NIH SIG grant (RR025667), NSF MRI grant (CMMI0923297), ICTAS-VT and SBES-VT internal funding.

EXTRA SLIDES

Mouse Limb Vasculature

Scanner: Xradia MicroXCT Objective: 4x ROI size: 4.5mm Resolution: 5µm

CT visualization in which both vasculature and bone structures are visible.

0.5mm

Osteon‐like Scaffolds Scanner: Xradia MicroXCT Objective: 4x ROI size: 5mm Resolution: 5µm Subject: •  3D scaffold made of Poly-

L-lactide fibers on polyglycolide fibers.

•  Microfibers spun together by electrospinning; then molded and sintered into the desired shape.

`

SEM image of single microfiber scaffold shows great detail at surface but cannot show

internal structure.

Micro-CT for Osteon-Like Scaffolds 1Sen Sharma, K; 1Andric, T; 1Wright, L D; 1Freeman, J W; 1Wyatts, C L; +1Wang, G

+1Virginia Polytechnic Institute and State University

Senior author: [email protected] / [email protected]

INTRODUCTION

Recently, a method has been developed to fabricate fiber scaffolds that

mimic the architectural organization of a single osteon and 3D scaffolds

that mimic the structural organization of the cortical bone. Currently

used assessment techniques like scanning electron microscopy (SEM),

mercury and flow porosimetry etc. are unable to accurately quantify the

internal 3D structure of such scaffolds. Microscale computed

tomography (micro-CT) was used in this study to scan the scaffold at

resolution of 4 !m and acquire 3D images. Micro-CT results obtained

provided information about internal features that could not be obtained

from SEM scans.

METHODS

Osteon-like scaffolds were fabricated using a new electrospinning setup

as described in Andric et al. [1]. Briefly, poly (L-lactide) (PLLA) and

poly (D-lactide) (PDLA) polymer solutions were electrospun onto

rotating poly (glycolide) (PGA) microfibers. The thickness of the

nanofiber layers around a PGA microfiber scaffold was varied by

controlling the volume of the electrospun solution. The osteon-like

sections were then stacked together and wrapped with a sheet of

PLLA/PDLA electrospun mat and placed into a 10 mm cylindrical mold

with 5 mm diameter. These scaffolds were then heat sintered to create

3D scaffolds.

A cross section of the microfiber scaffold was characterized using !"#$

%&'()'$ *++$ !",$emission environmental SEM. Micro-CT scans were

run on Xradia MicroXCT-400. A 3D scaffold was placed in a 0.5 ml

PCR tube secured to a sample holder on the rotary stage of the micro-CT

scanner. X-ray source voltage and current were set at 30 kV and 100 !A

respectively. For each tomogram, 271 projections of 20482 pixels were

acquired between -135º and +135º - a total of 2.3 GB of projection data.

Acquisition time was approximately 5 h/specimen with exposure time of

60 s/projection. Xradia proprietary software (TXM Reconstructor and

TXM 3D Viewer) was used to reconstruct images from the projections

and view the reconstructed results. It took 3 minutes to reconstruct a

10243 image volume.

RESULTS

Figure 1 is an SEM cross-section of a single osteon microfiber with

PGA core and concentric layers of PLLA fibers. It shows extensive

details at the surface but does not provide information about internal 3D

features. Figure 2 is a reconstructed CT slice of a 3D scaffold and shows

the PGA fibers in bright white along with concentric players of PLLA

fibers around each PGA fiber. The voxel size in the tomogram was 2.5

!m. This resolution was sufficient to resolve PGA cores with diameter

95-130 !m. This imaging technique is beneficial to assess the uniformity

of the osteon scaffolds and how well the scaffolds are packed together. It

is seen that the diameter of the osteon scaffolds varies between 190 and

1100 !m. Figure 3 depicts a multi-planar view of the 3D scaffold. The

longitudinal cross-section shows that PGA fibers run along the entire

length of the scaffold. It also shows the layers of PLLA fibers and how

they are packed.

DISCUSSION

Micro-CT data showed that the scaffold engineering procedure was

successful as a proof of concept, but the next step would be to control

the osteon scaffolds better to have a more uniform diameter distribution.

Mineralized scaffolds will also be imaged to view a mineral distribution

throughout the scaffold.

This imaging technique is potentially important to evaluate the

uniformity of the osteon scaffolds and how well the scaffolds are packed

together. It also provides cross sectional images and 3D reconstruction

without fixing or sectioning the scaffold, which is required in classic

methods like SEM. As micro-CT is non-invasive, it might be utilized to

study a single scaffold at different time points of a particular study (e.g.

cell culture, mechanical testing etc). Thus, micro-CT may help build

better scaffolds that have mechanical properties and structures similar to

natural bone in order to promote complete tissue regeneration.

Figure 1. SEM cross section of a microfiber scaffold.

Figure 2. Reconstructed CT slice of the 3D scaffold. Scale width is 2000

!m.

Figure 3. Multiplanar view of the 3D scaffold.

REFERENCES

1. -.$/(01234$5.6.$71289)4$:.$7.$!1;;<'(.$!'=123')2>($>?$

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I2))D=&1894$I/4$JG/4$K++L

Fixa1on Setup Scheme [Vasilescu et al. J. Appl. Physiol. 2011]

Animal Embryo Microfossils

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MicroCT volume visualization showing nucleus-like structures.

100µm

Zernike phase-contrast mode shows more structural details for this sample.

Microfossils in Nano‐CT 10µm

Slides for talk at BMES Conference 2011

Technology

Transcript of Slides for talk at BMES Conference 2011