Micro-CT based analysis of Subchondral Vessel Density in a Model by housework


									Micro-CT based analysis of
Subchondral Vessel Density in a
Model of Osteoarthritis


             Kasun Laksandu Attanapola


               Dr. David Holdsworth


                   April 13th, 2009

I would like to thank the following people for their support and guidance, given during the time
spent on this research.

   •   My supervisor, Dr. David Holdsworth for his positive influence and help in making this
       research experience beneficial and educational
   •   Dr. Ian MacDonald for his thorough explanations of the project outlines and
       stereological methods.
   •   PhD Student David McErlain for his invaluable technical support and time in making this
       project a success.



Microfil is a silicone-rubber based compound used as a computed tomography (CT)- contrast

agent. Microfil, fills and opacifies microvasculature, and other spaces of non-surviving animals.

In this experiment mice were first injected with fluoroescein isothiocyanate Dextran (FITC-


which is a fluorescent dye added to the plasma to visualize the vascular space. Then saline

was injected to flush all the blood out. After that, Microfil was perfused to cast vessels of

subchondral bone. Images were recorded after Microfil polymerization, by using exvivo micro-

CT imaging, and then analyzed using point counting stereological methods. Ex-vivo micro-CT

imaging was used due to its very high resolution that cannot be matched by any other imaging

modality. For imaging subchondral bone a voxel size of 0.028mm was chosen.


Osteoarthritis is a degenerative disease that causes erosion in articular cartilage and

subchondral bone. Clinical symptoms include joint pain, tenderness, stiffness, inflammation

and creaking and locking of joints. It’s the most common form of arthritis and it’s usually

associated with old age. 4 million Canadians between ages 16-64 are affected by this disease,

and currently there is no known cause or early cure. The only form of therapy at the moment is

surgical intervention once disease has reached its final stage. The purpose of this study is to

investigate the affects of osteoarthritis on bone vasculature, and to find a cause for this

common disease.


In this experiment effects of osteoarthritis were studied on a rat model, since rats have similar

physiological features to humans. Rats were grouped into batches, and all but the control group

were induced with osteoarthritis. Osteoarthritis was induced by severing the anterior cruciate

ligament (ACL) in the knee joint to increase shear stress within the joint. Rats were then left for

set amounts of time (2 weeks, 4 weeks, 8 weeks and 20 weeks) before their joints were imaged.

Prior to imaging rats were injected with a contrast agent Microfil (MV 122) to add contrast to

the bone vasculature.

After images were acquired, they were opened in MicroView (General Electric Health Care,

London, Canada) and VFFview for analysis. Vessel volume fraction analysis was performed at

the Medial Joint compartment since this was the joint compartment that was operated on. At

each joint point counts were performed at 3 different slices(center, anterior and posterior) to

increase accuracy.

Choosing the central plane-

After an image was loaded on MicroView the center of the joint was located on the Sagittal

plane. From there, the center of the Medial joint compartment was located using the Coronal

plane, and finally the minimum joint space from the Sagittal plane was located. This location

was chosen as the central slice. Approximately 1mm to the anterior and posterior from the

central slice lies the anterior and posterior slices, in which the vessel volume fraction was


Measuring Vessel Volume Fraction-

To do a point count, a grid of 5 x 10 was laid on the Coronal plane at the three slices mentioned

above. Bone and Vessels on Micro-CT images have the same contrast and appear white while

tissue appears black. And so distinguishing between bone and vessels is a tedious task. For this

we opened the same image on Vffview to pan in and out of the coronal plane. Since vessels are

continuous throughout the volume and bone end abruptly, any high contrast continuous

structure was counted as a vessel, or if otherwise bone. 3 point counts were done at each slice.

                                                             The total fractions for the joint at a
                                                             certain time point were then
                                                             averaged from the 9 counts.

                                                                 Axial plane

                                                                 Sagittal plane


                                                                 Coronal Plane

Posterior slice selected before a point count.

                                                                        Medial joint compartment

Figure 2

Image showing a 5 X 10mm grid with 1mm line spacing which is laid on the medial joint

compartment before a point count. Grid was chosen to cover the medial joint compartment

because this area was where the surgery was performed.


                                                                                  Figure 3

Graph shows the vessel volume fractions for healthy control rats and rats with Osteoarthritis

(OA). Dots and squares represent the individual vessel volume fractions (VVF) obtained for each

joint while the straight line represent the mean vessel volume fraction for the rat knee joint at 2

time points. This graph represents a drop in vessel volume fraction for rats infected with OA.


  •   Table Analyzed Unpaired, two-tailed t-test

  •   Column A      Control

  •   vs            vs

  •   Column B      OA

  •   Unpaired t test

  •    P value =0.0008                     Significance level 0.05

  •    P value summary ***

  •   Are means significantly. different? - (P < 0.05) Yes

  •    One- or two-tailed P value? - Two-tailed

      T test comparing the two averages gives an extremely low P value of 0.008.

Figure 4                                           Figure 5
               20 weeks Control                                    20 weeks OA

Figure 4 shows that OA significantly degrades bone. Image taken after 20 weeks of

OA (figure 5) shows a lot more, low contrast (black) areas compared to the control image. Black

low contrast areas represent tissue in the knee joint.


From the results obtained, it’s clear that there is a significant difference in the intra-osseus

vessel volume fraction between healthy joints and joints infected with osteoarthritis. The 0.008

P value confirms this statistically. Causes for Osteoarthritis and decrease in vessel perfusion can

be due to reasons illustrated in the figure below.

Findlay DM. “Vascular pathology and osteoarthritis”, Rheumatology. 46(12):1763-8, 2007
The major causes are summarized as follows:


Embolism occurs when an object migrates from one part of the body and causes a blockage

in another part of the body. In this case it is most likely that a blood clot is blocking vessels in

the subchondral bone. When vessels get blocked there will be no perfusion through them, and

this will block contrast agents flowing into these blocked areas. Blocked vessels will limit

diffusion of oxygen into the subchondral bone stunting re-growth of cells and degrading bone

causing ischemia, which in turn may explain the degradation of bone associated with

osteoarthritis. This will also result in the decrease of vessel volume fraction.


This is a syndrome that affects arteries where the accumulation of white blood cells and low

density lipo -proteins will form a plaque on the artery walls hardening the vessel. This plaque

breaks off easily and can result in blood clots. Atherosclerosis occurring in the subchondral

bone can deprive the bone of oxygen and nutrients resulting in ischemia and osteoarthritis. As a

result of the vessels being blocked they will not be imaged on Micro CT images reducing the

vessel volume fraction.

Venous Stasis

This is a condition that arises due to low blood flow in the veins. When the blood flow of veins

is slow or stopped blood clots can form and block perfusion. If venous stasis occurs in the

vessels within the subchondral bone there is a risk of blood clots blocking off perfusion away

from the bone and resulting in blood pooling. This will again result in ischemia and induce

osteoarthritis. Blocked perfusion will reduce vessel volume fraction due to reasons mentioned


Reperfusion Injury

This refers to damage caused to tissue when blood supply returns to the tissue after a long

period of ischemia. The lack of Oxygen and nutrients creates a condition in which the

restoration of perfusion will cause inflammation and oxidative damage. If there is an ischemic

condition due to reasons mentioned above there is a chance that reperfusion injury can

degrade the bone and cause osteoarthritis. Reperfusion injury will however, not have an impact

on the vessel volume fraction.

Osteoarthritis can occur due to one or all the reasons mentioned above depending on the

health of an individual. From our results and the figure above we see a clear link between

osteoarthritis and bone vessel volume fractions. From this we can say that vessel volume

fractions and risk of osteoarthritis appear inversely proportional to each other.

                                         FUTURE WORK

From the 2 time points we have obtained, it’s clear that OA has an impact on vessel volume

fraction within bone. The low P value calculated confirms this statistically. As future work we

should first test how vessel volume fractions change with time. Vessel volume fractions taken at

2 points will not be enough to validate a point. Therefore it is best to take volume fractions at

intermediate time points as well, ex- 1 week, 2 weeks, 4 weeks etc.

If practical, testing the effects of OA on another mammal can be also tested to see if this is a

common phenomenon.

Most importantly the effects of increasing perfusion in subchondral bone should be studied.

Perfusion is mostly reduced by vessel blockages due to reasons mentioned above. By injecting a

drug that reduces blood viscosity, non functional vessels could be re-perfused and the supply of

nutrients and Oxygen to the bone can be restored. After treating diseased rats with the drug,

vessel volume fractions should be taken at set time points using exact methods listed above. If

there is a change, and the effects of OA are reversed, a cure for OA can be developed.

                                         ERROR ANALYSIS

Since bone and vessel have the same contrast it’s difficult to differentiate between the two

while point counting. Even after panning between views in the coronal plane on VFFview there

is a chance of missing some vessels, oriented transverse to the coronal plane. Very small vessels

are also easy to miss while point counting. Accuracy however, can be increased by taking an

average of a large number of readings.


Since an average of 9 point counts were taken for one knee joint and an average of 45 point

counts were taken at given time points it’s safe to assume that the error mentioned above is

minimal. Therefore in conclusion, it is clear that Osteoarthritis reduces vessel volume fractions

in subchondral bone.


•                                                                       Flow Tech Inc.
    1999. 13 Apr. 2009 <http://flowtech-inc.com/microfil.htm>.

•                                                                      "Definition of
    Emboli." MedicineNet.com. 29 Dec. 2000. MedicineNet, Inc. 13 Apr. 2009

•                                                                         " What Is
    Atherosclerosis?" National Heart, Lung and Blood Institute: Diseases and
    Conditions Index. Nov. 2007. U.S Department of Health and Human Services. 13 Apr.
    2009 <http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis

•                                                                         "Venous Stasis
    Disease: Pain Associated with Pooling of Blood, Venous Insufficiency."
     Associatedcontent.com. 21 Dec. 2007. 13 Apr. 2009 <http://www.associatedcontent
    .com/ article/484425/venous_stasis_disease_pain_associated.html?cat=70>.

•                                                                       "Reperfusion
    Injury in Stroke." emedicine.medscape.com. 17 Jan. 2007. 13 Apr. 2009


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