Biomaterials and Stem Cell Therapies for Injuries Associated to Skeletal Muscular Tissues - Advances in Biomaterials Science and Biomedical Applications - page 354

the plasma and sarcolemmal membrane; the mitochondria (size, density and shape); T-tu‐
bule; amount of lipid; nucleus; phagocytic granules and amount of glycogen. EM is extreme‐
ly useful in some cases: to identify inclusions primarily found by light microscopy; to help
in the characterization of stored material found on light microscopy and define its intracel‐
lular localization; to analyze structural abnormalities found by light microscopy; can assist
in the diagnosis of mitochondrial myopathy or seeking evidence to support a diagnosis of
dermatomyositis (EM can be used to look for tubuloreticular inclusion in endothelial cells
when light microscopic fails to reveal it).
5.6. Contraction force measurement
To obtain an estimate of total TA muscle strength reduced by the injury and possibly recov‐
ered by the cell/vehicle implants, contractile force due to electrical stimulation can be meas‐
ured before injury, after injury and at the time of sacrifice (at different time points) for non-
implanted and implanted animals. This can be accomplished with the animals under
general anesthesia and by anchoring the knee joint using a custom clamping system anch‐
ored to the floor of the surgical stereomicroscope stand and attaching a silk ligature to the
cleft between digits 1 and 2 that must be anchored to a transducer at the other end. This can
also be executed by cutting the TA tendon just before the insertion at the ankle and tying it
with a 4.0 nylon suture attached to the isometric transducer. The exposed muscle is stimulat‐
ed using 2 custom needle electrodes placed at the proximal muscle surface. Electrical stimu‐
lation of the TA muscle is applied at 5 volts, 4 ms pulse duration, at 500 ms intervals and the
resultant tetanic force recorded (200 points(s) using a BioPac MP-100 (Harvard Apparatus)
and accompanying software (Acknowledge TM ). The muscle must be kept hydrated during
the procedure using sterile saline. Maximum tetanic force is measured by reducing the stim‐
ulation interval to 20 ms, generating continuous stimulation simulating tetanus condition.
Another method of applying the electrical stimulation can be obtained by exposing the sciat‐
ic nerve with an incision in the hamstring region The tibial nerve is cut just after the sciatic
nerve splits into the tibial and peroneal nerves to eliminate any contraction from the gastro‐
cnemius muscle causing background in the force data. The exposed sciatic nerve is then laid
over two electrodes with a small piece of parafilm and should also be kept moist with peri‐
odic treatment of mineral oil. Stimulation is made using a supra-maximal square-wave
pulse of 0.1 ms duration. Measurements are performed at the length at which maximal ex‐
tension is obtained during the twitch and the data should be recorded for sub-maximal and
maximal isometric force. Specific maximal force should be quantified by correcting for mus‐
cle mass [40, 98].
Acknowledgements
The authors would like to thank the support by Dr. José Manuel Correia Costa, from INSRJ,
Porto, Portugal; and Biosckin, Molecular and Cell Therapies SA for the umbilical cord units
supply and access to the GMP cell culture room (Scientific Protocol between Porto Universi‐
ty and Biosckin, Molecular and Cell Therapies SA). This work was supported by Fundação