GDF‐5/7 and bFGF activate integrin α2‐mediated cellular migration in rabbit ligament fibroblasts

Abstract

Cellular activities responding to growth factors are important in ligament healing. The anterior cruciate ligament (ACL) has poor healing potential compared to the medial collateral ligament (MCL). To assess the differences, we investigated the proliferation, migration, adhesion, and matrix synthesis responding to growth factors in rabbit ACL and MCL fibroblasts. ACL cell proliferation to basic fibroblast growth factor (bFGF), bone morphogenetic protein-2, growth and differentiation factor (GDF)-5, and GDF-7 treatment was similar to that of MCL cells. GDF-5 enhanced Col1a1 expression in ACL and MCL fibroblasts up to 4.7-and 17-fold levels of control, respectively. MCL fibroblasts showed stronger migration activities in response to bFGF and GDF-5 than ACL cells. GDF-5/7 and bFGF also changed the stress fiber formation and cellular adhesion by modulating the distribution of integrin a2. Functional blocking analyses using anti-integrin a2 antibodies revealed that cellular migration responding to growth factors depended on the integrin a2-mediated adhesion on type I collagen. The expression of integrin a2 was also increased by growth factors in both cells. Our results demonstrate that GDF-5/7 and bFGF stimulate cellular migration by modulating integrin a2 expression and integrin a2-dependent adhesion, especially in MCL fibroblasts. These findings suggest that the different healing potential between ACL and MCL may be caused by different cellular behavior in the integrin a2-mediated cellular migration in response to growth factors. ß

Anterior cruciate ligament-derived cells have high chondrogenic potential

“…The synovial sheath and fat tissue of ligaments were removed. The midsubstance cores of ACL and MCL were digested using collagenase (Sigma) as described [8]. Attached cells (passage 0) were maintained with Dulbecco's modified Eagle's medium (DMEM, Wako) containing fetal bovine serum (HyClone) and penicillin/streptomycin (Sigma) at 37°C in a humidified atmosphere of 5% CO 2 .…”

“…We have previously demonstrated that growth factor-stimulated cellular migration is slower in ACL than in MCL fibroblasts [8]. In addition, we have identified chondroblastic interface cells derived from the ACL-to-bone insertion [9].…”

Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells

“…Inner and outer meniscus cells were prepared, respectively, by collagenase (Sigma, St. Louis, MO) treatment. 30 Attached cells (passage 0) were maintained with Dulbecco's modified Eagle's medium (Wako, Osaka, Japan) containing 10% fetal bovine serum (HyClone, South Logan, UT) and 1% penicillin/ streptomycin (Sigma). Meniscus cells between passage 2 and 4 were used.…”

“…Alexa Fluor 488-conjugated antibody (Invitrogen, Carlsbad, CA) and Alexa Fluor 568 phalloidin (Invitrogen) were used for detection under a fluorescence microscope (1:100 for 20 min). 30 A bovine serum albumin solution without the primary antibody was used as a negative control. Cell extracts were prepared with SDSlysis buffer.…”

Combined use of bFGF and GDF-5 enhances the healing of medial collateral ligament injury

“…PCR amplification was performed in the presence of 10 pmol of each specific primer. The primer sets for rabbit Col1a1, Col3a1, glyceraldehyde-3-phosphate dehydrogenase (G3pdh) were used [14,27]. The cycle number was selected in the linear part of the amplification curve.…”