硫酸乙醯肝素酶對小鼠骨癒合之影響

Abstract

骨折不癒合或是延遲癒合一直是醫學所面對的難題，雖然近年來有許多新的促進骨癒合方式被發表，如幹細胞或生長因子等，但都尚未成為標準的治療方式。 硫酸乙醯肝素蛋白聚醣 (heparan sulphate proteoglycan, HSPG) 是廣泛存在於生物體內的物質，除了分布於細胞表面，更是組成細胞外基質 (extracellular matrix, ECM) 最主要的物質之一。HSPG藉由其上的硫酸乙醯肝素 (heparan sulfate, HS) 支鏈，與體內各種不同的生長因子、激素或蛋白質接合，除了作為這些因子的儲存庫外，更具有調節這些因子的能力。硫酸乙醯肝素酶 (heparanase) 是哺乳動物體內切割HSPG最主要的酵素，可以在HS上特定的位置切割，釋放HS片段及其所接合的生長因子，除了發現具有促進血管新生和皮膚傷口癒合的效果外，近年也發現有調節造骨細胞分化的效果。本實驗使用小鼠骨缺損模式，以評估外源性heparanase在骨癒合上的效果。 本實驗使用90隻C57BL/6小鼠，分別以手術製造股骨遠端外側幹骺端的骨缺損後，依分組以植入式連續給藥膠囊局部連續給予高劑量heparanase、低劑量heparanase、高劑量heparanase inhibitor、低劑量heparanase inhibitor或溶劑 (控制組) ，並在骨缺損後第7、14及21天犧牲，並以微電腦斷層及組織學切片評估癒合程度。 不論是微電腦斷層對於骨小樑的生成、型態上的變化或是組織學切片下皮質骨的生成變化，heparanase都表現出顯著具有促進骨癒合的效果。有趣的是，本研究結果發現，雖不如heparanase如此顯著，但高劑量的heparanase inhibitor似乎也具有促進骨癒合的效果。這個結果可能暗示著在造骨 (造軟骨) 前驅細胞分化時，不論heparanase存在或是受到大量抑制時，都會使平衡偏向骨質新生的方向。

Treatments for nonunion or delay union following bone fracture are important issues in clinical medicine. Many researches focused on promoting the bone regeneration, including stem cells and growth factors therapy. However, with various reasons, these methods have not been routinely applied as standard treatments in clinical cases. Heparan sulphate proteoglycans (HSPG) are abundant components of extracellular matrix and cell surface. Heparan sulphate (HS) chains in HSPG bind a variety of growth factors, chemokines and proteins, and can not only act as a depository of growth factors but also regulate their bioactivities. Heparanase is the sole mammalian endoglycosidase that cleaves heparan sulfate. Heparanase has been proved to promote angiogenesis and skin wound healing by releasing the HS fragments and growth factors. Recently, its effect on osteogenesis regulation was also reported. The aim of this study is to investigate the effect of exogenous heparanase in bone healing. A total of 90 C57BL/6 mice were randomly assigned into 5 groups. After surgically created a bone defect at the distal femoral metaphysis, high-dose heparanase, low-dose heparanase, high-dose heparanase inhibitor, low-dose heparanase inhibitor or 1% DMSO as vehicle control was delivered via an osmotic pump to provide the continuous topical infusion of the treatment. The bone healing process was evaluated by micro computed tomography and histological examination on 7, 14 and 21 days after the bone defect was created. The heparanase showed significant effect on promoting bone healing based on the trabecular formation, remodeling and cortical bone regeneration. Interestingly, in the current study, the high dose of heparanase inhibitor also showed has positive effect on bone healing. These results suggested that under the condition of either externally supplying heparanase or suppressing it with high dose of inhibitor, the differentiation of osteochondroblastogensis precursor cells will shift toward to the osteogenesis.