Objective To investigate the feasibil ity of alendronate (ALN) in treating osteoarthritis (OA) by observing the effects of ALN on interleukin 1β (IL-1β) induced chondrocytes of rat in vitro. Methods The chondrocytes of knee articular surface from 15 SD rats (1-month-old, female or male, weighing 100-150 g) were cultured. The chondrocytes were observed by inverted phase contrast microscope and identified by toluidine blue staining and HE staining. The third passage chondrocytes were divided into 3 groups: the chondrocytes were cultured with DMEM for 5 days in group A, with 10 ng/mL IL-1β for 2 days and with DMEM for 3 days in group B, and with 10 ng/mL IL-1β for 2 days and with 1 × 10-6 mol/L ALN for 3 days in group C. Immunocytochemistry and real-time PCR were performed to determine the expression levels of collagen type II (Col II), matrix metalloproteinase 13 (MMP-13), and β-catenin. Results Toluidine blue staining proved that the cultured cells were chondrocytes. The integrated absorbency (IA) value of Col II in group C (10.290 7 ± 0.499 2) was lower than that in group A (15.377 0 ± 0.571 8) and higher than that in group B (5.463 2 ± 0.450 4), showing significant differences (P lt; 0.05). The IA value of MMP-13 in group C (3.068 6 ± 0.205 6) was significantly lower than that in group B (6.998 1 ± 0.329 7, P lt; 0.05), but there was no significant differenc when compared with group A (2.777 5 ± 0.199 6, P gt; 0.05). The IA value of β-catenin in group C (6.611 7 ± 0.381 8) was lower than that in group B (11.799 9 ± 0.348 7) and higher than that in group A (4.390 3 ± 0.551 9), showing significant differences (P lt; 0.05). The mRNA expression of Col II in group C was significantly higher than those in groups A and B (P lt; 0.05), the mRNA expression of MMP-13 in group C was significantly lower than that in group B (P lt; 0.05) but there was no significant difference when compared with group A (P gt; 0.05). The mRNA expression of β-catenin in group C was significantly lower than that in group B (P lt; 0.05) and higher than that in group A (P lt; 0.05). Conclusion ALN can protect rat chondrocyte from OA induced by IL-1β in vitro possibly by upregulating Col II and inhibiting the expression of MMP-13 and β-catenin in the chondrocytes.
Objective To investigate the effects of xianl inggubao (XLGB) on subchondral bone and articular cartilage in the rat osteoarthritis model induced by anterior cruciate l igament transection (ACLT). Methods Twentyfour 3-month-old female SD rats were divided randomly into 3 groups (n=8): Sham group (group A), ACLT group (group B) and XLGB group (group C). The osteoarthritis model was made by ACLT in groups B and C, the joint cave was sutured after exposure of ACL in group A. After 4 days, XLGB was given at 250 mg/(kg·d) in group C and the equivalent amount of sal ine was given in groups A and B. After 12 weeks, the gross appearance of femoral condyles was observed, the degree of cartilagedegeneration was scored by Mankin scoring system. The immunostaining for MMP-13 was performed to investigate the effect of XLGB on prevention of cartilage matrix loss. The bone mineral density (BMD) measurement and bone histomorphometric analysis were done in subchondral bone of right distal femur and proximal tibia after 12 weeks. Results The gross appearance of femoral condyles showed that ulcer in the group C was smaller than that in group B after 12 weeks. The Mankin’s scale and IA value for MMP-13 in group C were markedly lower than those in group B (P lt; 0.05). BMD of the subchondral bone in the group B was significantly lower than those in the groups A and C (P lt; 0.05). The bone mass in group C were significantly higher than that in group B (P lt; 0.05). Conclusion Oral administration of XLGB (250 mg/ kg per day) for 12 weeks could prevent the cartilage degeneration of rats after ACLT, down-regulating MMP-13 and increasing subchondral bone mass might participate in this process.
To evaluate the effects of XiangLingGuBao (XLGB) on femoral fracture heal ing in ovariectomized (OVX) rats. Methods Forty 12-week-old female SD rats weighing (258 ± 14) g were divided randomly into 4 groups (n=10 per group): group A, sham operation by opening the abdominal cavity; group B, bilateral ovariectomy; group C, bilateral ovariectomy, transverse midshaft fracture of the right femur with intramedullary nail fixation, and normal sal ine by gavage; group D, bilateral ovariectomy, transverse midshaft fracture of the right femur with intramedullary nail fixation, and 250 mg/(kg•d) XLGB by gavage. The weight of rabbits in groups A and B was measured 0, 1, 2, 3, 4 and 5 weeks after operation. The right femur of each rat was obtained 5 weeks after operation. Total femur bone mineral density (tBMD), distal femur bone mineral density (dBMD) and middle femur bone mineral density (mBMD) were measured by double energy X-ray absorptiometry CR filming, HE staining and immunohistochemistry staining of groups C and D were performed. Results The weight of rats in group B was obviously higher than that of group A at 3, 4 and 5 weeks after operation (P lt; 0.05), indicating the animal model of postmenopausal osteoporosis was establ ished successfully. CR films showed more callus and obscure fracture l ine in group D, while less callus and distinct fracture l ine in group C. The tBMD and the dBMD of group B were far less than that of group A, the mBMD of group D was significantly higher than that of group C(P lt; 0.05), the tBMD and the dBMD of group D were higher than that of group C, but no significant difference was evidentbetween two groups (P gt; 0.05). Histology observation showed, when compared with group C, most fracture ends in groupD reached bone union, and the introduction of capillaries was evident in the marrow cavity. Immunohistochemistry staining demonstrated that the BMP-2 integrated absorbance (IA) value in groups C and D was 2.236 6 ± 0.181 8 and 3.727 3 ± 0.874 2, respectively, the VEGF IA value in groups C and D was 2.835 5 ± 0.537 0 and 3.839 6 ± 0.223 0, respectively, indicating there were significant differences between two groups (P lt; 0.05). Conclusion XLGB can obviously promote the femoral fracture heal ing in OVX rats, and speed the transformation of woven bone into lamellar bone, which may rely on its role of enhancing expression of BMP-2 and VEGF.
Objective To examine the effects of alendronate (ALN) on IL-1β-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis (OA) induced by anterior cruciate l igament 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: thechondrocytes were cultured in DMEM medium with 10 ng/mL IL-1β for 2 days, subsequently with (ALN group, group A1) orwithout (IL-1β group, group B1) 1 × 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 μg/(kg·d) for 8 weeks. Rabbits of group B2 and C2 received equal normal sal ine 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 appl ied 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 sl ightly 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 lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). Immunocytochemical detection of MMP-13 showed intense staining in group B1, and the intensity of staining was sl ightly 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 lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 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 lowerthan that of group B2 (P lt; 0.05), but significantly higher than that of group C2 (P lt; 0.05). Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2, the intensity of staining was sl ightly 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 gt; 0.05..........