Download Preparation of ECM Scaffolds

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Preparation of ECM Scaffolds
•  Perfusion decellularization
system was developed.
•  Needle delivery (A) of
decellularization solution into
muscle belly (B)
•  Solution perfused through
muscle and out to waste (C)
•  Flow (ml/hr) controlled to four
units with syringe pump (D)
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Decellularized Skeletal Muscle (DSM)
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Decellularized Skeletal Muscle (DSM)
VML Injury and Repair
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•  Hind limb of mature (3 month)
Fisher 344 rats.
•  Tibialis anterior (TA) VML injury
created using 8mm biopsy punch
(depth = 3mm)
•  Three treatment groups
1.  Unrepaired (VML)
2.  DSM
3.  DSM + minced muscle (MM)
•  Contralateral limb untreated to
serve as a comparative control
(normal)
•  N=8 / treatment group
•  3 month recovery period
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Muscle Electophysiology
•  At 3 months post-op peak TA
contractile force (N) was
measured.
•  Foot was secured to 10N force
force transducer.
•  Peroneal nerve was stimulated
with needle electrodes at
•  5V at 150Hz,
•  Pulse width of 0.1 ms
•  Pulse length 400 ms.
•  Average of 5 contractions, force
normalized to animal weight.
Muscle Electrophysiology
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•  Average contractile force (%
normal)
VML: 62±16%
DSM: 73±13%
DSM+MM: 78±14%
•  Trend: increased contractile
force with repair strategies
•  DSM + MM increased
contractile force by 26%
(compared to unrepaired)
* p<0.05 students t-test
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TA Mass & Gross Anatomy
•  TAs were harvest, weighed,
imaged and then flash frozen
in liquid N2.
•  Treated and control muscles
were all smaller than normal
contralateral muscles.
•  Implant sites were generally
visible.
*
•  Muscle mass (% contralateral)
was modestly increased
following DSM+MM treatment
* p<0.1 students t-test
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Histology
•  Muscle samples cryosectioned (8um).
•  Immunostained for the
presence of collagen type I
(red) and myosin heavy chain
(green).
•  DSM and unrepaired defects
were strongly immunoreactive to collagen type 1,
suggesting connective tissue
healing (scarring).
•  DSM + MM sample defect
sites were less pronounced.