Endoplasmic reticulum stress-induced apoptosis of osteoblasts within the osteolytic craniums

doi:10.3969/j.issn.2095-4344.2014.33.002

Abstract

Abstract:

BACKGROUND: Wear particles-induced osteoblasts apoptosis in vitro has been documented in many studies. However, the apoptosis of osteoblasts in osteolytic bone tissue and the selective mechanisms involved in the pathogenesis of osteolysis have been studied rarely.
OBJECTIVE: To investigate the influence of endoplasmic reticulum (ER) stress on the apoptosis of osteoblasts in osteolytic bone tissue and osteolysis progression.
METHODS: The mouse model of osteolysis was induced with wear particles placed onto the calvaria. The experiment was divided into four groups: blank control group (PBS stimulation); wear particle group (nano-alloy powder suspension stimulation); ER stress positive control group (nano-alloy powder + thapsin stimulation); and ER stress inhibitor group (nano-alloy powder + sodium 4-phenylbutyrate stimulation). The histopathologic change
of osteolysis was assessed by hematoxylin-eosin, toluidine blue and alkaline phosphatase staining. Osteoblast proliferation and differentiation in osteolytic craniums were measured. The expression of ER stress markers in osteolytic craniums was examined by western blot analysis. Osteoblast apoptosis was analyzed by TUNEL staining and immunohistochemistry of Caspase-3 in osteolytic craniums.
RESULTS AND CONCLUSION: Wear particles were capable of inducing osteolysis, aggravating the infiltration of inflammatory cells, and inhibiting the differentiation of osteoblasts in osteolytic craniums. Meanwhile wear particles upregulated the ER stress markers and promote the apoptosis in osteolytic craniums. Blocking ER stress with sodium 4-phenylbutyrate dramatically reduced the severity of osteolysis, significantly reduced bone invasion and inflammatory infiltration, promoted the differentiation of osteoblasts, and dramatically reduced the apoptosis. Along with apoptosis, the expression of ER stress marker was decreased. The present study suggests that the ER stress may be crucial for osteolysis and represent a potential therapeutic target in the prevention and treatment of patients with total joint replacement who are at high risk of early aseptic loosening development.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: osteoblasts, endoplasmic reticulum, Caspases

CLC Number:

R318

Liu Guo-yin, Wang Rui, Dong Lei, Zhang Jun-feng, Zhao Jian-ning. Endoplasmic reticulum stress-induced apoptosis of osteoblasts within the osteolytic craniums[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(33): 5257-5265.

Figures/Tables 3

2.1 实验动物数量分析实验选用小鼠28只，分为4组，实验过程无脱失，全部进入结果分析。 2.2 动物一般情况各组动物造模后送返原笼，麻醉苏醒后常规饲养。各组动物均存活至实验结束，其间动物活动、进食情况基本正常，切口愈合良好，无皮下积液及切口感染等并发症发生。 2.3 内质网应激反应介导的骨溶解结果 2.3.1 小鼠颅骨骨片经甲苯胺蓝染色后置于显微镜下观察见图1A和B。在小鼠颅骨植入磨损微粒纳米合金粉末后，可见大量圆形，椭圆形或梭形的骨吸收陷窝，陷窝深浅不等，着色浓度不一。对骨吸收陷窝面积进行积分显示：相对于空白对照组，纳米合金粉末组骨陷窝吸收率为3.3。还发现，在纳米合金粉末植入的基础上，加入内质网应激的诱导剂毒胡萝卜素后，骨吸收的陷窝最密集，骨溶解症状最严重；经内质网应激的抑制剂(4-苯基丁酸)的治疗后，陷窝的数量和大小均明显减少，骨溶解症状得到明显改善。统计学分析显示，磨损微粒纳米合金粉末组以及纳米合金粉末+毒胡萝卜素组，相对于空白对照组，差异均有显著性意义(P < 0.05)，纳米合金粉末+4-苯基丁酸组相对于纳米合金粉末组差异也有显著性意义(P < 0.05)。结果表明内质网应激参与骨溶解的形成。 2.3.2 小鼠颅骨的苏木精-伊红染色结果根据苏木精-伊红染色结果对颅骨骨溶解和炎症浸润进行评判，见图1C和1D。正常小鼠的颅骨结构清晰，黏膜和骨皮质完整，细胞构成大小、形态相同，排列整齐，极向正常，无炎症细胞浸润；而当小鼠颅骨受到纳米合金粉末的刺激后，表现出严重的浸润破坏，颅骨结构紊乱，黏膜层严重受损，骨皮质受到破坏，细胞构成大小、形态不一，排列多层，极向消失；髓腔大小不一、排列紊乱，炎症细胞浸润至颅深层甚至穿透颅骨，造成髓腔内成骨细胞丢失。经内质网应激的抑制剂(4-苯基丁酸)的治疗后，骨形态完整性得到保持，骨侵蚀和炎症浸润症状明显改善。在纳米合金粉末组中加入内质网应激的诱导剂毒胡萝卜素后，得到相反的结果。毒胡萝卜素加入后，骨形态的完好性受到进一步的破坏，骨侵蚀和炎症浸润程度也急剧加重。结果表明内质网应激在骨溶解的发生发展中起着一定的作用。 2.3.3 小鼠颅骨成骨细胞碱性磷酸酶染色结果见图1E和1F。颅骨中成骨细胞的阳性反应呈现红黑色颗粒或块状沉淀，骨形成隧道呈现深色条带。植入纳米合金粉末后，小鼠颅骨的成骨细胞数量显著减少，骨形成隧道随着磨损微粒纳米合金粉末的加入而逐渐降低。应用4-苯基丁酸后，成骨细胞的数量和骨形成较纳米合金粉末组明显增加；而加入内质网应激的诱导剂毒胡萝卜素后，成骨细胞和骨形成受到进一步损伤。"

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