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Hello All,
I am working with a polymer for tissue engineering applications and was
wondering if any of the list subscribers would have insightful suggestions
on how to breakdown the scaffold for subsequent biochemical analyses.
The polymer I am using is a 5 mm in diameter and 3 mm in height
fiber-reinforced, porous, resorbable scaffolds, composed of a matrix made
from a random copolymer of 75:25 poly(D,L-lactide-co-glycolide) with a glass
transition onset temperature (Tg) of 50 °C, and reinforcing poly(glycolide)
fibers with glass transition onset temperature Tg of 42 °C. The porosity of
the scaffold is between about 65% and 70% by volume.
I am seeding chondrocytes onto these scaffolds and I am interested in
measuring DNA content, Proteoglycan and Collagen (hydroxyproline) content,
as well as isolate RNA for RT-PCR analysis. All of these assays need not be
done on one sample, as I can dedicate certain numbers of scaffolds for each
of the analyses.
To do these analyses, I tried to digest the scaffold (after lyophilizing it)
with 1 milliliter of papain solution (25 mg/mL papain solution in 2mM of
L-cysteine and 2mM of EDTA with Phosphate Buffered Solution as the solvent
base; pH 6.5) at 65°C for 15 to 18 hours overnight. But this treatment
seemed to do nothing on the scaffolds. Of course I could use a harsher
solvent (like Soluene) that would dissolve the polymer but that would also
dissolve the plastic plates I use for the assays I mentioned.
I would really appreciate any input, and will be glad to share the answers I
get with the rest of the group.
Sincerely,
Hani Awad, PhD
Post Doctoral Research Associate
Duke University Medical Center
haniawad@duke.edu
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I am working with a polymer for tissue engineering applications and was
wondering if any of the list subscribers would have insightful suggestions
on how to breakdown the scaffold for subsequent biochemical analyses.
The polymer I am using is a 5 mm in diameter and 3 mm in height
fiber-reinforced, porous, resorbable scaffolds, composed of a matrix made
from a random copolymer of 75:25 poly(D,L-lactide-co-glycolide) with a glass
transition onset temperature (Tg) of 50 °C, and reinforcing poly(glycolide)
fibers with glass transition onset temperature Tg of 42 °C. The porosity of
the scaffold is between about 65% and 70% by volume.
I am seeding chondrocytes onto these scaffolds and I am interested in
measuring DNA content, Proteoglycan and Collagen (hydroxyproline) content,
as well as isolate RNA for RT-PCR analysis. All of these assays need not be
done on one sample, as I can dedicate certain numbers of scaffolds for each
of the analyses.
To do these analyses, I tried to digest the scaffold (after lyophilizing it)
with 1 milliliter of papain solution (25 mg/mL papain solution in 2mM of
L-cysteine and 2mM of EDTA with Phosphate Buffered Solution as the solvent
base; pH 6.5) at 65°C for 15 to 18 hours overnight. But this treatment
seemed to do nothing on the scaffolds. Of course I could use a harsher
solvent (like Soluene) that would dissolve the polymer but that would also
dissolve the plastic plates I use for the assays I mentioned.
I would really appreciate any input, and will be glad to share the answers I
get with the rest of the group.
Sincerely,
Hani Awad, PhD
Post Doctoral Research Associate
Duke University Medical Center
haniawad@duke.edu
---------------------------------------------------------------
To unsubscribe send SIGNOFF BIOMCH-L to LISTSERV@nic.surfnet.nl
For information and archives: http://isb.ri.ccf.org/biomch-l
---------------------------------------------------------------