clearing alcohols from tissue for implantation

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From:Angel Duty <>
Date:Thu, 20 May 1999 14:56:14 -0400
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Dear Histonetters,

This is not exactly a histology question, but does pertain to the
preservation of tissues...

I am trying to do a bone allograft study in which it is necessary
to store the allografts for several months before reimplantation.
I am concerned only with maintaining the mineral structure.
In fact, I would like to clear the bone of cellular elements
including the marrow.  I think my problem is that I am not
clearing my reagents from the tissue well enough.  I know this
is not a standard question, but perhaps someone has some
suggestions.  If you're intrigued, please read on.....

Here's what I do now:
Basically, I harvest the tissue from one rabbit store in in 70%
EtOH.  Then just prior to reimplantation, I do a defatting
procedure as follows:
1.  3 washes in PBS
2.  3 washes in sterile water
3.  Overnight soak in 1:1 chloroform:methanol
4.  3 washes in methanol
5.  3-5 washes in sterile water
The tissue sets in sterile saline for perhaps two hours before
being reimplanted in a rabbit.

Also, since the graft's orientation (top vs. bottom) must be
maintained, the tissues are inside a foam-lined cassette, so
I'm concerned the rinses way not be clearing everything.
I  implanted these grafts and compared their histology to
that of allografts that had simply been flash frozen and
reimplanted.  The treated grafts had very little bone repair.
In fact, there was very little sign of fibrous tissue or
granulation tissue.  Existing trabeculae had few osteoblasts
lining surfaces and strange areas of ghost cells (for lack of a
better term).  These ghost cells look like fat cells without
nuclei but with fibers throughout.

BTW, the grafts are cylinders of .25" diameter and .25" length.

I appreciate your comments and suggestions.

Thank you!

Angel Duty
Georgia Tech Bioengineering

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