RAPID 3D CHARACTERIZATION OF CARTILAGE, BONE QUALITY & SCAFFOLD RAPID 3D CHARACTERIZATION OF CARTILAGE, BONE QUALITY & SCAFFOLD

TO SUBMICRON RESOLUTION, WITH A NOVEL MICROCT TO SUBMICRON RESOLUTION, WITH A NOVEL MICROCT TO SUBMICRON RESOLUTION, WITH A NOVEL MICROCT TO SUBMICRON RESOLUTION, WITH A NOVEL MICROCT S H Lau1, M. Chandrasekaran2, V. Fan3 , M. Islam3 , S. Candell1, T. Case1, L. Chitra2, T. Fong1, H. Chang1, W. Broderick1

1Xradia Inc, Concord, CA, USA 2Bio-scaffold International Pte. Ltd, Singapore; 2Bio-scaffold International Pte. Ltd, Singapore;

3 Faculty of Dentistry, National University of SingaporeEmail Contact: shlau@xradia.comEmail Contact: shlau@xradia.com

AbstractAbstract Results & DiscussionResults & DiscussionThe current study is focused on developing a new rapid non invasive

AbstractAbstract Results & DiscussionResults & Discussion

Characterizing Cartilage Thickness Characterizing Cartilage Thickness Human Cancerous Bone Quality EvaluationHuman Cancerous Bone Quality EvaluationThe current study is focused on developing a new rapid non invasive

3D imaging technique for bone quality, bioscaffold, cartilage and its

related drug efficacy evaluation. Bone quality evaluation is critical in

patients suspected with osteoporosis or those treated with synthetic

Characterizing Cartilage Thickness without Contrast Agent in Murine ModelCharacterizing Cartilage Thickness without Contrast Agent in Murine Model

Human Cancerous Bone Quality EvaluationHuman Cancerous Bone Quality Evaluation

Remains of PLGA patients suspected with osteoporosis or those treated with synthetic

scaffolds for restoration of bone. Similarly success of synthetic

scaffolds depends much on the micro architecture of the scaffold.

These evaluation plus those involving cartilage thickness in

Fig 5 compares CT

Remains of PLGA scaffold in trabeculae

These evaluation plus those involving cartilage thickness in

osteoarthritis are mainly carried out with conventional histology, which

requires experienced personnel and time consuming sample

preparation techniques. In addition, histology studies can take up to a

compares CT images of a rat knee joint ( a & c) with the

b5apreparation techniques. In addition, histology studies can take up to a

few weeks, results are often operator dependant, and are only

available as individual 2D slices. While in recent years there are

crack

Fig. 2 MicroCT 3D image of cancerous bone sample extracted for

c) with the corresponding MRI (b )and conventional

several publications on the use of MicroCT for such evaluation, the

biggest deficiency of conventional microCT is the lack of contrast and

resolution to detect fine microstructures on bones and low Z ( low

Fig. 2 MicroCT 3D image of cancerous bone sample extracted for analyzing the quality for implant placement @ 0.7 µµµµm detector resolution

conventional histology slides (d)

dcresolution to detect fine microstructures on bones and low Z ( low

contrast) bioscaffold materials and soft tissue. It is also not possible to

image cartilage without contrast enhancing agents.Fig 6 shows CT

dcPLGA Scaffold remnants

PLGA Scaffold remnants

In the current work we have used a novel microCT system for rapid

virtual histology in 3D for bone quality, cartilage and scaffold

microchannel evaluation to submicron pixel resolution, without contrast

Fig 6 shows CT slices of a rat tibia demonstrating Low Density

bone (gray

Compact bone

microchannel evaluation to submicron pixel resolution, without contrast

agents. Examples using human and murine bones will be illustrated,

including a clinical study involving a human cancerous bone after

bioscaffold implant and its comparison with conventional histology

demonstrating high contrast imaging of cartilage-bone b

bone (gray regions)

Lack of Osteocytes

Compact Bone (bright regions)bioscaffold implant and its comparison with conventional histology

R

C

S

1 µµµµm

cartilage-bone interface (a & b) and the quantitative

Fig 3a microCT slice of bone sample @ 0.7µµµµm 6a

ba b

regions)

MethodsMethodsS

100

quantitative evaluation of cartilage layer thickness

Evaluation of the bone sample shows trabecular bone

structure with a cortical layer similar to Type III bone

Fig 3a microCT slice of bone sample @ 0.7µµµµm resolution, compared with conventional histology (3b)

100 µm

Four different samples are used in this study to test the use of the

novel MicroCT as a possible means for rapid 3D imaging technique for

bone quality and bioscaffold evaluation. These includes:

thickness without the aid of contrast agents (c & d )

structure with a cortical layer similar to Type III bone

structure suited for implant placement.

The Fig 3b shows the connective tissues, the cancerousdc

Evaluation of cartilage degeneration in osteoarthritis

using murine model is possible with a microCT (only with

bone quality and bioscaffold evaluation. These includes:

1.Human Cancerous Bone extracted from a dental alveolar socket after

placement of bioscaffold for 4 months in the socket

agents (c & d )The Fig 3b shows the connective tissues, the cancerous

bone and the cortical layers in the histology which can be

also clearly seen in the CT slice (Fig 3a) where the

cancerous bone is seen as a dull grey region while the

d

contrast agents), with a MRI or through conventional

histology. The novel high contrast CT can image cartilage

structure and its bone interface quickly without contrast

placement of bioscaffold for 4 months in the socket

2.Rat knee joint for cartilage imaging in Osteoarthritis

3.Resorption Pit Assays for Osteoporosis

4.Bioscaffold microchannel and microporosity evaluation after

cancerous bone is seen as a dull grey region while the

cortical boundaries as white regions. The osteoclasts spread

on the cancerous regions (dots seen on the dull grayish

surface) is also seen. The connective tissues are seen as

NOD R

f∆

=

structure and its bone interface quickly without contrast

agent (Fig 5 & 6) [2]. Fig 5 compares the novel CT

imaging at low and high resolution and the equivalent

images from MRI and conventional histology. MRI are

4.Bioscaffold microchannel and microporosity evaluation after

fabrication and post processing.surface) is also seen. The connective tissues are seen as

dark grey regions identical to porosity while scaffold remains

are seen within the connective tissue regions.N

OD Rf

λ

∆=

images from MRI and conventional histology. MRI are

typically very low resolution, while conventional histology

generally takes 2 to 3 weeks to prepare.

.

The patient had a placement of bioscaffold (fig 4) in the socket after

Extraction for about 4 months within which sufficient bone volume was

achieved in the socket. Patient opted for an implant placement and a

are seen within the connective tissue regions.

While the conventional histology required extensive sample

preparation techniques along with staining to identify these

500

.

1 µµµµm

achieved in the socket. Patient opted for an implant placement and a

bone piece was harvested from the proposed implant site to ensure

bone quality and absence of any inflammation. The bone sample was

analyzed both using conventional histology and micro CT which is

Characterizing Bone Resorption pits in Osteoporosis AssaysCharacterizing Bone Resorption pits in Osteoporosis Assays

preparation techniques along with staining to identify these

(up to a few weeks), the microCT was able to provide the

data rapidly (couple of hours) and with very little sample

preparation.500 µm

Characterizing 3D Microchannels in BioscaffoldsCharacterizing 3D Microchannels in Bioscaffolds

analyzed both using conventional histology and micro CT which is

shown in the Figures 2 & 3

Rat knee joints were harvested, fixed in paraffin and imaged at different

preparation.

BioscaffoldsBioscaffoldsRat knee joints were harvested, fixed in paraffin and imaged at different

Resolutions (Fig 5 to 7). Comparison with histology is shown.

In case of bioscaffold, the fabricated samples were analyzed using x-In case of bioscaffold, the fabricated samples were analyzed using x-

ray microCT to characterize the micro porosities and macro channels

present. The slices were taken both in the vertical and horizontal

directions to evaluate the distribution of pores and pore sizes while the7a b

directions to evaluate the distribution of pores and pore sizes while the

3 dimensional images were constructed from the slices to analyze the

macro channels in the scaffold. Conventional imaging technique can

only produce 2 D images which will be giving information on one planeWith detector pixel resolution to < 1 micron range,

microscopic bone resorption pits may be detected in drug

7a b

ConclusionConclusion

only produce 2 D images which will be giving information on one plane

alone (Fig 4).microscopic bone resorption pits may be detected in drug

assays for osteoporosis in murine models ( Fig 7).b

ConclusionConclusionFrom the above figures and discussion, it is evident that

Fig 1: Apparatus: schematic of the

aFrom the above figures and discussion, it is evident that

the novel microCT is an effective tool for characterizing any

solids with very low attenuation factors including human

cancerous bones, murine cartilage and bones and

300 µm

100 µmFig 4 a b & c 3D and 2D CT images of the

Bioscaffold surface and the CT slice image.

schematic of the novel microCT with unique high resolution and

ca

cancerous bones, murine cartilage and bones and

resorbable bio-scaffolds non-invasively. As microCT

technique also requires little or no sample preparation

thereby minimizing the time required while preserving the

Bioscaffold surface and the CT slice image.

It can be clearly seen from the images, that microCT

combines the advantage of conventional optical

microscope, scanning electron microscope and that of a

resolution and high contrast optics

The apparatus used for this study (Fig 1) is based on the Xradia’s

MicroXCT[1], which is capable of submicron detector pixel resolution.

thereby minimizing the time required while preserving the

integrity of sample and information required. It is therefore

envisioned that this technique could supplement

conventional histology for these assays in preclinical and

microscope, scanning electron microscope and that of a

confocal microscope, without the associated sample

preparation requirements. Fig 4a shows the 3 D structure

of the scaffold with macro channel and micro porosity onMicroXCT[1], which is capable of submicron detector pixel resolution.

Unlike conventional MicroCTs which uses point projection technique

where resolution is limited by source spot size and its sample-source

distance- resolution of the novel microCT is not dependant on these

conventional histology for these assays in preclinical and

possibly clinical applications.

References

of the scaffold with macro channel and micro porosity on

the sample surface. The fig 4b clearly shows the macro

channel size and configuration while also reflecting thedistance- resolution of the novel microCT is not dependant on these

parameters. Relatively large biological materials of several mm

diameter may be imaged to 1 micron resolution. With

PhaseEnhancedTM optics, significant increase in contrast is realized,

References[1] S H Lau, et al., : “Virtual Non Invasive 3D Imaging of Biomaterials and Soft Tissue

with a Novel High Contrast CT, with Resolution from mm to sub 30 nm”, Symposiumon Adv Functional Biomaterials, Proceedings of ICMAT, Singapore 2007

channel size and configuration while also reflecting the

microporosity along the sides of the channel. Fig 4c

shows an exploded view of the slice depicting the

microporosity distribution critical for the scaffold. ThePhaseEnhancedTM optics, significant increase in contrast is realized,

making it possible to image inherently low contrast samples such as

cartilage and biomaterials without contrast agents.

on Adv Functional Biomaterials, Proceedings of ICMAT, Singapore 2007[2] S H Lau, et al., : Rapid Virtual Histology in 3D for Cartilage, Soft tissue, Bones, Scaffolds and Cells with a novel micro and nano CT without contrast agents”, Paper # 10, 6TH Combined Meeting, Orthopedic Research Society 2007

microporosity distribution critical for the scaffold. The

ideal architecture desired for a scaffold with about 70-80

% space (pores and channels) and microarchitecture in

terms of size distribution is clearly seen from the CTterms of size distribution is clearly seen from the CT