Objective To explore the impact of basic fibroblast growth factor (bFGF) and parathyroid hormone-related protein (PTHrP) on early and late chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) induced by transforming growth factor β1 (TGF-β1). Methods BMSCs were isolated from 3 healthy Japanese rabbits (2-month-old, weighing 1.6-2.1 kg, male or female), and were clutured to passage 3. The cells were put into pellet culture system and were divided into 5 groups according to different induce conditions: TGF-β1 group (group A), TGF-β1/bFGF group (group B), TGF-β1/21 days bFGF group (group C), TGF-β1/PTHrP group (group D), and TGF-β1/21 days PTHrP group (group E). At the beginning, TGF-β1 (10 ng/mL) was added to all groups, then bFGF and PTHrP (10 ng/mL) were added to groups B and D respectively; bFGF and PTHrP (10 ng/mL) were added to groups C and E at 21 days respectively. The gene expressions of collagen type I (Col I), Col II, Col X, matrix metalloproteinases (MMP)-13, and alkaline phosphatase (ALP) activity were detected once every week for 6 weeks. The 1, 9-dimethylmethylene blue (DMMB) staining was used to observe the extracellular matrix secretion at 6 weeks. Results The expression of Col I in groups C and E showed a significant downward trend after 3 weeks; the expression in group A was significantly higher than that in groups C and E at 4 and 5 weeks (P lt; 0.05), and than that in groups B and D at 3-6 weeks (P lt; 0.05); and significant differences were found between groups B and C at 3 and 4 weeks, and between groups D and E at 3 weeks (P lt; 0.05). After 3 weeks, the expressions of Col II and Col X in groups C and E gradually decreased, and were significantly lower than those in group A at 4-6 weeks (P lt; 0.05). Groups B and D showed no significant difference in the expressions of Col II and Col X at all time points, but there was significant difference when compared with group A (P lt; 0.05). MMP-13 had no obvious expression at all time points in group A; significant differences were found between group B and groups A, C at 3 weeks (P lt; 0.05); and the expression was significantly higher in group D than in groups A and E (P lt; 0.05). ALP activity gradually increased with time in group A; after 4 weeks, ALP activity in groups C and E obviously decreased, and was significantly lower than that in group A (P lt; 0.05); there were significant differences between groups B and C, and between groups D and E at 2 and 3 weeks (P lt; 0.05). DMMB staining showed more cartilage lacuna in group A than in the other groups at 6 weeks. Conclusion bFGF and PTHrP can inhibit early and late chondrogenic differentiation of BMSCs by changing synthesis and decomposition of the cartilage extracellular matrix. The inhibition is not only by suppressing Col X expression, but also possibly by suppressing other chondrogenic protein.
Objective To investigate and compare the difference between two implants of reconstructing anterior cruciate l igament (ACL) for the early heal ing of implants tunnel interface in terms of biological mechanism. Methods Fiftyfive adult New Zealand rabbits weighing 2.0-2.8 kg were selected. Patellar l igament with tibia-bone block was obtained fromthe left knee joint serving as donor site, right knee joint served as the recipient site of autograft for ACL reconstruction. Thebone block end of implant was bone-bone interface heal ing model, while the l igament end was tendon-bone interface heal ing model. The general condition of rabbits was observed after operation, the gross observation and histology observation were conducted at 2, 4 and 8 weeks after operation (n=5), and biomechanics examination was conducted at 4 and 8 weeks after operation (n=20). Results Rabbits behaved normally after operation. The gross observation indicated that ACL had complete continuity and moderate tension during experiment. Histology observation: most part of bone-bone interface was connected by fibrous tissue, while the tendon-bone interface was mainly filled by granulation tissue 2 weeks after operation; most part of bone-bone interface was bone union, and there were osteogenesis reaction and large quantity of fibroblasts in the tendonbone interface 4 weeks after operation; complete bone union was evident in bone-bone interface, and the appearance of Sharpey fibers and the formation of indirect insertion occured in part of tendon-bone interface 8 weeks after operation. Biomechanics observation: the pull-out rate for tendon-bone interface and bone-bone interface 4 weeks after operation was 85% and 15%, respectively; while it was 95% and 5% 8 weeks after operation, respectively; indicating there was a significant difference between two groups (P lt; 0.001). Conclusion In the early stage after ACL reconstruction, bone-bone interface is better than tendonbone interface in terms of intensity and speed of heal ing.