重组人骨保护素对聚乙烯颗粒刺激破骨细胞功能的影响

目的通过观察重组人骨保护素（recombinant humanoste oprotegerin，rhOPG）对聚乙烯颗粒刺激破骨细胞功能的影响，探讨rhOPG防治人工关节无菌性松动的可行性。方法取出生24h内新西兰白兔5只，体重80～100g，雌雄不限。分离培养四肢长骨破骨细胞，以1×10^5／mL密度均匀接种于大小为10mm×10mm玻片和8mm×8mm×50μm骨片的24孔板内。根据加入rhOPG及聚乙烯颗粒浓度和密度的不同，玻片培养板和骨片培养板再分别分为1×10^9／mL聚乙烯颗粒培养组（聚乙烯组）、1×10^9／mL聚乙烯颗粒和100ng／mL rhOPG共培养组（聚乙烯／rhOPG组）及空白对照组。于培养1、3、5、7d取细胞玻片行HE、甲苯胺蓝染色，抗酒石酸酸性磷酸酶（trafrate resistant acid phosphatase，TRAP）染色计数阳性细胞数：皮质骨片行甲苯胺蓝染色并用扫描电镜观察骨片吸收陷窝形态。结果TRAP染色后破骨细胞呈玫瑰红色：培养后5、7d，聚乙烯组阳性染色的破骨细胞与对照组、聚乙烯／rhOPG组比较明显增多，差异均有统计学意义（P〈0．05）；对照组与聚乙烯／rhOPG组比较，差异无统计学意义（P〉0．05）。骨片吸收陷窝甲苯胺蓝染色呈蓝紫色：培养5、7d后，聚乙烯组骨片上的吸收陷窝计数与对照组比较明显增多（P〈0．05）；培养后1、3、5、7d，聚乙烯／rhOPG组骨片上的吸收陷窝计数与聚乙烯组比较，差异均有统计学意义（P〈0．05）。结论rhOPG可抑制聚乙烯颗粒对破骨细胞的刺激作用，可用于防治人工关节置换术后的无菌性关节松动。

EFFECT OF RECOMBINANT HUMAN OSTEOPROTEGERIN ON OSTEOCLASTS STIMULATED BY POLYETHYLENE PARTICLES

OBJECTIVE: To observe the effect of recombinant human osteoprotegerin (rhOPG) on osteoclasts which were stimulated by polyethylene particles and to investigate the feasibility of applying rhOPG for the prosthetic aseptic looseness. METHODS: The osteoclasts were isolated from the long bones of 5 New Zealand rabbits born within 24 hours, weighing 80-100 g, male or female, and were plated into the 24-well coverslips (10 mm x 10 mm) and bone slices (8 mm x 8 mm x 50 microm) at the density of 1 x 10(5)/mL. Based on the different concentration and density of rhOPG and polyethylene particles, the plates of culture were divided into 3 groups: the group with polyethylene particles of 1 x 109/mL (polyethylene group), the group co-cultured with polyethylene particles of 1 x 10(9)/mL and rhOPG of 100 ng/mL (polyethylene/rhOPG group) and the control group. The glass cover slips and bone slices were exposed to HE, toluidine blue and tartrate-resistant acid phosphatase (TRAP) staining at 1, 3, 5 and 7 days, and TRAP positive multinucleated cells and bone resorption tips were counted. Scanning electron microscope was used to observe the pits of bone resorption. RESULTS: The osteoclast was rose-red when exposed to TRAP staining. For the number of the TRAP-positive osteoclasts, the polyethylene group witnessed an obviouse increase compared with the control group and the polyethylene/rhOPG group after 5 and 7 days of culture (P < 0.05). And no significant difference between the control group and the polyethylene/rhOPG group was evident (P > 0.05). The pits of bone resorption was blue-purple when exposed to toluidine blue staining. For the number of bone resorption pits in the bone slice, significant difference was evident between the polyethylene group and the control group after 5 and 7 days of culture (P < 0.05), and there was significant different between the polyethylene/rhOPG group and the polyethylene group 1, 3, 5 and 7 days after culture (P < 0.05). CONCLUSION: rhOPG could inhibit the stimulated effect of polyethylene particles on osteoclasts, and might be used to prevent the prosthetic aseptic looseness after artificial joint substitution.