In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1).
To test the hypothesis that loading conditions can be used to "engineer" ligament autograft behaviors, the effect of cyclic tension on the mRNA levels of matrix molecules and collagenase in in-vivo immobilized and mobilized 6-week rabbit medial collateral ligament (MCL) autografts was examined using an in-vitro system. Femur-[autograft MCL]-tibia complexes were subjected to a tensile stress of 4 MPa at 0.5 Hz for 1 min, followed by 14 min of rest. This 15-min testing cycle was repeated for 4 h. Semi-quantitative reverse transcrip-tase polymerase chain reaction (RT-PCR) was performed on RNA from mechanically treated MCL autografts, using rabbit-specific primer sets for types I and III collagen, biglycan, decorin, fibromodulin, lumican, versican, matrix metalloproteinase-1 (MMP-1, collagenase-1), MMP-13 (collagenase-3), and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, 4 h of culture of normal control MCLs led to increased mRNA levels for MMP-1 (P < 0.05), but there were no significant changes in MMP-13 mRNA levels. Total RNA levels in that normal MCL tissue were, however, decreased after culture (P < 0.05). In-vitro tensile loading of in-vivo mobilized autografts resulted in a significant increase in total RNA (185% of in-vitro non-loaded autografts). On the other hand, in-vitro tensile loading of in-vivo immobilized autografts resulted in no significant changes in total RNA levels compared with levels in non-loaded control grafts. MMP-1 mRNA levels in both the in-vivo mobilized (47% of non-loaded autograft) and in-vivo immobilized (38% of non-loaded autograft) MCL autografts were significantly lower than those in non-loaded control tissue following in-vitro tensile loading, but there were no significant changes in the mRNA levels for the seven other matrix molecules assessed. These results show that it is possible to selectively inhibit MMP-1 mRNA levels in autograft ligaments by supplying mechanical stimuli in vitro. The results also demonstrate that in-vivo immobilization leads to a decrease in the effects of subsequent in-vitro mechanical loading in such autografts with respect to total RNA levels. Collectively, these results demonstrate that both in-vivo and in-vitro loading have implications in the engineering of an ideal ligament graft.
Majima
T
T
McCaig Center for Joint Injury and Arthritis Research, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.
Marchuk
L L
LL
Shrive
N G
NG
Frank
C B
CB
Hart
D A
DA
D000818:Animals; D005260:Female; D007103:Immobilization; D020781:Matrix Metalloproteinase 1 / Q000032:analysis*; D017888:Medial Collateral Ligament, Knee / Q000737:chemistry* / Q000637:transplantation; D009924:Organ Culture Techniques; D012333:RNA, Messenger / Q000032:analysis*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013718:Tensile Strength; D014182:Transplantation, Autologous
D012333:RNA, Messenger; D020781:Matrix Metalloproteinase 1