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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
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
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Animal Embryo Microfossils
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Zernike phase-contrast mode shows more structural details for this sample.
Microfossils in Nano‐CT 10µm