alter the culture environment tissues were then cultured in dulbecco 39 s modified eagle 39 s medium dmem supplemented with 10 fetal bovine serum nonessential amino acids 0 1 mm l gutamine 4 mm penicillin 100 u ml streptomycin 100 u03bcg ml and fungizone 0 25 u03bcg ml guanethidine or sterile pbs was added to media for the treated and control tissues respectively the concentration of guanethidine in media was selected to be the same as the concentration of guanethidine present in the blood during treatment using normative rat data for blood volume 5 ml blood 100 g body weight guanethidine was prepared and dissolved in sterile pbs and added to the media controls received equal volumes of sterile pbs the cultures were maintained in an incubator at 37 u00b0 c and 5 co 2 media were changed every 48 hours at which time tissues were rinsed with sterile pbs to reduce drug carryover into the fresh media containing the treatment drug or equivalent volume of carrier organ culture was maintained for 10 days after which time the mcls were tested mechanically in the same manner as the in vivo treatment group described below mechanical testing immediately after death animal hindlimbs for biomechanical testing n 8 control n 8 treated mcls were disarticulated at the hip joint and stored at u2212 80 u00b0 c until testing storage by freezing does not significantly affect the biomechanical properties of ligament on the day of testing hindlimbs were thawed at room temperature and tissue harvest and testing were performed using methods similar to those previously.