[In vitro effect of alendronate on chondrocytes and articular cartilage and subchondral bone in rabbit anterior cruciate ligament transection model].
OBJECTIVE
To examine the effects of alendronate (ALN) on IL-1beta-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT).


METHODS
The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups: the chondrocytes were cultured in DMEM medium with 10 ng/mL IL-1beta for 2 days, subsequently with (ALN group, group A1) or without (IL-1beta group, group B1) 1 x 10(-6) mol/L ALN for 3 days; the chondrocytes in vacant group (group C1) were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups (n = 8 per group). The OA model was made by ACLT in ACLT+ALN group (group A2) and ACLT group (group B2); the joint cave was sutured after exposure of ACL in sham group (group C2). After 4 days, the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 microg/(kg x d) for 8 weeks. Rabbits of group B2 and C2 received equal normal saline treatment. After 8 weeks, the rabbits were executed. The macro-pathologic changes of right knee joints were observed, so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was applied to subchondral bone of proximal tibia.


RESULTS
In vitro, the Col II immunocytochemical staining showed intensely positive staining in group C1, and the intensity of staining was slightly decreased in group A1, but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance (IA) value for Col II in group A1 was significantly higher than that of group B1 (P < 0.05), but there was no significant difference between group A1 and group C1 (P > 0.05). Immunocytochemical detection of MMP-13 showed intense staining in group B1, and the intensity of staining was slightly decreased in group A1, but no MMP-13 expression was detected in the group C1. The IA value for MMP-13 in group A1 was significantly lower than that of group B1 (P < 0.05), but significantly higher than that of group C1 (P <0.05). The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 (P < 0.05), but there was no significant difference between group A1 and group C1 (P > 0.05). The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 (P < 0.05), but significantly higher than that of group C1 (P < 0.05). In vivo, the gross appearance of surface of knee joint showed that there was no ulcer in group C2, and there was some ulcers in group A2, but many and all layers ulcers in group B2. Mankin score of group A2 was significantly lower than that of group B2 (P < 0.05), but significantly higher than that of group C2 (P < 0.05). Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2, the intensity of staining was slightly decreased in group A2, and the intensity of Col II immunohistochemical staining was extremely low in group B2, but there was no significant difference between group A2 and group C2 (P > 0.05). The immunohistochemical staining for MMP-13 significantly increased in group B2, the intensity of staining was slightly decreased in group A2, and no MMP-13 expression was detectable in the group C2. The IA value for MMP-13 in group A2 was significantly lower than that of group B2 (P < 0.05), but significant higher than that of group C2 (P < 0.05). Bone histomorphometry showed that in group B2 percent trabecular area and trabecular thickness markedly decreased compared with those in group A2 and group C2 (P < 0.05), but there was no significant difference between group A2 and group C2 (P > 0.05). The trabecular separation, percent labeled perimeter and bone formation rate were significantly elevated in group B2 compared with those in group A1 and group C2 (P < 0.05), but there was no significant difference between group A2 and group C2 (P > 0.05). No significant difference was evident on trabecular number and mineral apposition rate values among groups A2, B2, and C2 (P > 0.05).


CONCLUSIONS
For rabbits OA induced by ACLT, the subcutaneous injections of alendronate can inhibit cartilage degradation, prevent bone loss, and improve microarchitecture of subchondral bone. ALN can partially protect chondrocytes by inhibiting the expression of MMP-13 both in vitro and in vivo.
Hu
Hongyu
H
Department of Orthopaedics, Affiliated Hospital of North China Coal Medical College, Tangshan Hebei, 063000, P.R. China.
Zhang
Liu
L
Li
Bin
B
Zhang
Yinna
Y
Liu
Xiaoning
X
Tian
Faming
F
Cheng
Tan
T
Wang
Zheyan
Z
D019386:Alendronate / Q000494:pharmacology*; D000818:Animals; D016118:Anterior Cruciate Ligament / Q000601:surgery; D002358:Cartilage, Articular / Q000187:drug effects*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects*; D024043:Collagen Type II / Q000378:metabolism; D008297:Male; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D010003:Osteoarthritis / Q000188:drug therapy*; D011817:Rabbits
D024043:Collagen Type II; D053509:Matrix Metalloproteinase 13; D019386:Alendronate