Download Comparison of Cryopreserved Amniotic Membrane and

Comparison of Cryopreserved Amniotic
Membrane and Umbilical Cord Tissues for
use in Foot and Ankle Reconstructive
Procedures
Howard M. Kimmel1, Ek Kia Tan2, Hua He2, Julie O’Connell3
1Buckeye
Foot Care, Brook Park OH
2TissueTech,
3Amniox
Inc., Miami, FL
Medical, Inc., Atlanta, GA
Disclosure
Kimmel HM – Consultant for Amniox Medical Inc.
Tan EK – Employee of TissueTech Inc.
He H – Employee of TissueTech Inc.
O’Connell J – Employee of Amniox Medical Inc.
Background
• Fetal tissue derived products have demonstrated
success when used clinically as adhesion barriers during
foot and ankle reconstructive procedures
• Many fetal tissue products consist of the amniotic
membrane derived from the amniotic sac of the
placenta proper, with some including the underlying
chorion
• Currently, only one fetal tissue product utilizes amniotic
membrane derived from the umbilical cord
• The purpose of this study was to characterize key
structural, biochemical, and functional differences
between cryopreserved amniotic membrane and
cryopreserved umbilical cord tissues that are
necessary for their therapeutic potential
Materials & Methods
• Materials:
• Cryopreserved amniotic membrane (AM; CLARIXTM 100) and
cryopreserved umbilical cord (UC; CLARIXTM 1K, Amniox
Medical Inc., Atlanta, GA)
• Methods:
• Histology: Samples were stained for hematoxylin and eosin,
Masson’s trichrome, and Safranin-O
• Histochemistry: Tissue matrix hyaluronic acid (HA) content
was measured using HA binding protein (HABP) fluorescent
histochemistry
• Macrophage functional assessment: RAW264.7 macrophage
proliferation and cell death was assessed in cultures on
cryopreserved AM and UC tissues
• Inflammatory cytokine levels: Secreted levels of the proinflammatory cytokine, IL-12, and the anti-inflammatory
cytokine, IL-10, were measured in RAW264.7 cells cultured on
cryopreserved AM and UC tissues
Results
AM
A
UC
AM
B
UC
H&E
HAase (-)
C
D
E
F
MAS
HAase (+)
SafO
• Collagen, non-sulfated glycosaminoglycans, and HA content were similarly distributed
across the AM matrix. However, the additional Wharton’s Jelly matrix considerably
increased the amount of all ECM components
Results
HAase(-)
AM
UC
Total HA/Weight Ratio
HAase(+)
5
AM
HA/Protein Ratio
UC
6
I
HMW HA
II
LMW HA
AM
III
UC
AM
UC
• Cryopreserved umbilical cord
contains approximately 10-fold
higher levels of HA compared to
amniotic membrane
Results
• Cryopreserved AM and UC significantly reduced RAW264.7
macrophage cell proliferation compared to control
• Cryopreserved UC significantly increased macrophage cell death
compared to AM and control
BrdU Proliferation/Protein
AM
UC
Cell Death/Protein
AM
UC
Results
IL-10/IL-12
Ratio
IL-10/Protein
CTL
AM
IL-12/Protein
UC
CTL
AM
UC
• Secreted levels of the pro-inflammatory cytokine IL-12
were significantly reduced in AM and tissues
compared to control. Cryopreserved UC further
decreased IL-12 levels compared to AM.
• Secreted levels of IL-10, an anti-inflammatory cytokine
were increased in UC tissues compared to both control
and AM tissues
CTL
AM
UC
Discussion
• Cryopreserved umbilical cord tissues contain
significantly higher amounts of extracellular matrix
components, including high molecular weight
hyaluronic acid, that are attributed to the antiinflammatory and anti-scarring properties of fetal
tissues that aid in tissue healing
• Cryopreserved umbilical cord outperformed amniotic
membrane in anti-inflammatory functional assays using
RAW264.7 macrophages by reducing macrophage
proliferation and increasing macrophage cell death
compared to amniotic membrane alone
• Levels of pro-inflammatory cytokines are significantly
decreased while levels of anti-inflammatory cytokines
are increased in cryopreserved-UC compared to
cryopreserved-AM
Conclusions
• These results indicate that cryopreserved umbilical
cord may offer physiological advantages within
the wound environment, ultimately translating to
greater clinical efficacy compared to amniotic
membrane alone
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