Yan jiu jing da ji liang shuang lin suan yan yu chu li hou de da shu gu she yu he mo shi bing wei fei dian xing xing gu gu gu she jian li xin xing zhi liao ce lue

Ph.D.Bisphosphonates (BPs) are potent bone resorption inhibitors widely prescribed for preventing osteoporosis and subsequent osteoporotic (OP) fractures. However, long-term BPs treatment is associated with a high risk of developing a new kind of fracture at non-trabecular dominant regions of femora, named atypical femoral fracture (AFF). Despite AFF patients commonly received surgical fixation with intramedullary nail (IMN) as recommended by the ASBMR (American Society for Bone and Mineral Research), the non-union rate of AFF is as high as 30%, suggesting that pre-fracture BPs treatment may impair fracture healing, and novel devices are urgently needed to help escape from this dilemma.To date, the mechanism by which long-term BPs treatment induces delayed fracture healing of AFF is not fully understood. Interestingly, some recent evidence suggests that long-term BPs exposure decreases the production of calcitonin gene-related peptide (CGRP) in sensory nerve endings of bone tissues. On the other hand, CGRP as a neuropeptide has proven indispensable for normal fracture healing process; depletion of CGRP results in reduced bone formation as well as impaired fracture repair. Therefore, we speculated that long-term BPs usage delayed fracture healing of AFF by suppressing CGRP production. In addition, we also proposed that approaches that elevate CGRP could improve AFF healing.Magnesium (Mg)-based orthopedic implant may well fulfil this requirement to enhance AFF healing. We have previously developed an innovative Mg-ions-releasing IMN that could enhance femoral fracture repair in ovariectomized (OVX) rats. More importantly, Mg ions released from the implant have been proved to boost endogenous CGRP releases from the sensory nerve endings to periosteal region at the fracture gap, resulting in an accelerated fracture healing. Based on these advantages, it is desirable to investigate the potential benefits of our innovative Mg-IMN in treating clinical AFF.In this study, we postulated that 1) long-term pretreatment of BPs would lead to impaired fracture healing in a rat when fixed by stainless-steel IMN without Mg; 2) Reduction in CGRP may be the key factor limiting healing; 3) Mg ions released from Mg-IMN fixation hybrid nail system would facilitate AFF healing via upregulating neuropeptide CGRP at fracture sites.In the first part, we investigated if BPs pre-treatment could induce delayed fracture healing in rats, with similar pathological features as observed in clinical AFF specimens. We first administrated zoledronate (ZOL) at a high (0.3 mg/kg/week) or low dose (0.1 mg/kg/3 months) to OVX rats prior to fractures. Zoledronate was chosen because it was one of the most potent and commonly prescribed BPs clinically. After 3 or 6 months of treatment, unilateral femoral closed fracture was performed and fixed with stainless-steel IMN (SS-IMN). As a result, approximately 40% (low dose) and 60% (high dose) of ZOL-treated rats showed non-bridged callus on X-rays at week 12 post-fracture. We next treated 6-month-old non-OVX rats with a high dose ZOL (0.3 mg/kg/week) for 3 months before fractures. This regimen produced a more consistent phenotype of delayed healing compared to OVX rats: the callus non-union rate was up to 90% at week 12 post-fracture. Such ZOL-induced fracture nonunion was demonstrated to be characterized by abundant fibrous tissues in the fracture gap, phenocopying clinical specimens of AFFs. Moreover, it is proved that these fibrous tissues hindering callus bridging was attributed to ZOL-induced pathologic differentiation of BMSCs into fibroblasts.CGRP was suppressed in the delayed healing model created in BPs-pretreated rats. Compared to control, ZOL-treated rats displayed remarkably suppressed expressions of CGRP in both the fracture callus and dorsal root ganglion (DRG). Moreover, flow cytometry confirmed the proportion of CGRP-positive stem cells in the fracture callus of ZOL group was also lower than that in control. Finally, in vitro treatment of DRG with ZOL showed that ZOL dose-dependently inhibited CGRP gene expression as indicated by the reverse transcription polymerase chain reaction (RT-PCR). These data suggest an inhibitory effect of BPs on CGRP production during fracture healing.Local delivery of CGRP facilitated AFF healing via suppressing fibrous tissues formation. We next injected CGRP to the fracture gap of stainless steel IMN-fixed and BPs-pretreated rats for 14 days in order to test if replenishing CGRP was beneficial to AFF repair. X-ray results showed CGRP treatment improved the callus bridging rate from 10% to 50% at week 12. This improvement was accompanied by an increased fraction of CGRP-positive stem cells, while a decreased fraction of fibroblasts in the fracture calluses as assessed by flow cytometry. Notably, BMSCs isolated from CGRP-treated rats presented with an attenuated fibrogenic potential as compared to those from ZOL group. This evidence together with others collectively suggests that delivery of CGRP may enhance AFF healing via suppressing fibrogenesis of BMSCs.Mg-IMN attenuated ZOL-impaired fracture healing of AFF via upregulating CGRP expression. We have developed a hybrid magnesium fixation system (Mg-IMN) to enhance fracture healing via boosting endogenous CGRP expression. Accordingly, we investigated the efficacy of this novel device in treating AFF in our animal model. Encouragingly, Mg-IMN improved the callus bridging rate to an extant as local CGRP injection did: 47% of Mg-IMN treated rats displayed united calluses at week 12 post-fracture, in comparison with 5.88% of those fixed by SS-IMN while with neither CGRP nor Mg treatment. We next tested CGRP expression in Mg-treated rats. As expected, Mg-IMN reversed the BPs-suppressed CGRP expressions in both the callus and DRG as assessed by immunostaining and/or flow cytometry. Notably, this upregulated CGRP expression was accompanied by less fibrosis tissues and fibroblasts in the fracture callus as assessed by histological examination and flow cytometry, respectively. In addition, BMSCs from Mg-treated rats showed downregulated fibrogenic potential compared to those from ZOL group. These data collectively suggest that Mg-IMN may upregulate CGRP expression to attenuate the increased fibrogenic potential of BMSCs secondary to BPs treatment. Finally, we performed in vivo knockdown of CGRP receptor by injecting adenovirus-carried Ramp1 shRNA into the Mg-IMN-fixed femora. As a result, the optimized healing by Mg-IMN was abrogated, indicating that CGRP is required for the beneficial effects Mg-IMN on healing AFF.Clinical impact of research outcomes: The present study has elaborated pathologic features of delayed healing of AFF in detail, demonstrating that fibrosis is the key detrimental factor. This sheds light on finding potential therapeutic target not only to treat AFF, but also accelerate regular fracture repair. This study would provide valuable information on potential translation of Mg-IMN to bedside, with the hope of providing an effective approach to accelerate AFF healing. This will to some extent relief individual’s suffering from potential delayed fracture healing or even fracture non-union, which in turn will reduce our healthcare and socioeconomic burdens.雙磷酸鹽類藥物因為具有較強的抗骨吸收能力，較為廣泛的被應用於預防骨質疏鬆性骨折。然而，長期使用雙磷酸鹽類藥物會增加一種名為“非典型性股骨骨折”的骨折風險。美國骨礦鹽研究協會指南建議，大部分患該類型骨折的病人應接受髓內針內固定手術治療。然而，大約有百分之三十的手術治療的非典型性股骨骨折患者出現了骨折不愈合。這說明，長期使用雙磷酸鹽類藥物對骨折愈合有不利作用。研發更有效的骨內固定物用以治療該類型骨折的需求迫切。目前，雙磷酸鹽導致骨折愈合延遲的機制未明。近期的一些研究表明，雙磷酸長期使用後，分佈于骨組織中感覺神經里的降鈣素基因相關肽(CGRP)產生減少。另外一方面來說，CGRP是骨生成和骨折愈合過程中不可或缺的物質；缺少該物質可以引起骨生成減少，骨折愈合受損。因此，我們推測由雙磷酸鹽使用而導致的CGRP減少，是引起臨床非典型性股骨骨折愈合障礙的重要原因。我們還推測，補充缺失的CGRP，可以促進非典型股骨骨折愈合。基於鎂金屬的骨內固定物可以很好的滿足以上需求。我們在以往的研究中，發明了一種創新性鎂離子緩釋內固定髓內針(Mg-IMN)用來促進骨質疏鬆性大鼠骨折愈合。關鍵點在于，該內固定釋放的鎂離子能夠促進機體的內源性CGRP由感覺神經末梢向骨組織釋放，進而優化骨折愈合。因此，我們相信，研究該鎂金屬固定物對於非典型性股骨骨折的治療作用，有重大意義。本研究中，我們有以下幾方面假設：1）骨折前長期使用雙磷酸鹽，可以引起大鼠股骨骨折愈合障礙；2）CGRP的產生不足，是引起該骨折不愈合的主要因素；3）通過Mg-IMN釋放的鎂離子來增加內源性CGRP的釋放，可以促進非典型性股骨骨折愈合。在本研究的第一部分，我們研究了骨折前長期使用雙磷酸鹽是否可以導致大鼠的骨折延遲愈合，并將其與非典型性股骨骨折的臨床病理標本進行對比。我們首先用高(0.3 mg/kg/week)或者低(0.1 mg/kg/3 months)劑量的zoledronate （ZOL）對切除卵巢的大鼠進行皮下注射,治療時間為3或6個月。該藥物(ZOL)是臨床上最常用的雙磷酸鹽類藥物之一。隨後，我們建立了大鼠單側股骨閉合性骨折，并用不鏽鋼髓內針固定（SS-IMN）骨折端。結果顯示，大約有40%的低劑量，和60%的高劑量ZOL注射的大鼠在術後第12週出現了骨折骨痂不連接。這之後，我們使用高劑量ZOL對6月齡的健康大鼠進行3個月的給藥，然後建立單側股骨閉合性骨折，并用不鏽鋼髓內針固定。結果顯示，高達90%的接受該治療方式的大鼠，在術後12週骨痂仍然不愈合。病理切片提示，不愈合的大鼠的骨痂中含有大量纖維組織。我們進一步證明，雙磷酸鹽的長期使用會促進骨髓間充值幹細胞分化為纖維細胞，進而形成這些影響骨痂連接的纖維組織。雙磷酸鹽使用后的大鼠，CGRP表達量下降。與對照組相比，ZOL治療過的大鼠，無論是骨痂，或是脊髓背根神經節，CGRP的表達均下降。流式細胞檢測提示，ZOL組大鼠骨痂中，CGRP陽性表達的幹細胞數目也減少。隨後，我們把新鮮脊髓背根神經節組織與ZOL進行體外共培養。RT-PCR提示，ZOL劑量依賴性的抑制CGRP的基因表達。以上結果提示，雙磷酸鹽對CGRP的表達具有抑制作用。局部注射降鈣素基因相關肽減少纖維組織生成，從而促進非典型性骨折大鼠模型愈合。我們接下來把CGRP在骨折后14天連續局部注射至骨折端，以觀察其對改善雙磷酸影響骨折愈合的副作用的能力。通過術後X射線觀察，CGRP治療可以把骨折後第12週的骨折愈合率由10%提升至50%。通過流式細胞技術，我們發現接受CGRP治療的大鼠骨痂中，CGRP表達陽性的幹細胞的比例增加，同時纖維細胞比例下降。我們把骨髓間充質幹細胞從接受過CGRP注射的大鼠股骨中提取出來，進行體外培養。我們發現其成纖維分化能力相對於非治療組明顯下降。以上證據表明，局部補充CGRP通過抑制骨髓幹細胞的成纖維分化，促進非典型性股骨骨折的愈合。Mg-IMN通過提高CGRP而改善ZOL導致的骨折不愈合。我們在之前的研究中已經發明了一種含鎂金屬髓內針(Mg-IMN)，其通過增加CGRP的釋放而促進骨折愈合。因此，我們把該內固定物應用於我們的建立的ZOL導致的骨折不愈合的模型上，以測試其對非典型性股骨骨折的治療功效。令人鼓舞的是，該含鎂髓內針對於促進骨折愈合，可以達到與局部直接注射CGRP同等的療效。47%的Mg-IMN治療過的大鼠，骨痂在術後第12週達到連接。而對於長期使用過ZOL但未接受過CGRP或Mg-IMN治療的大鼠，僅有5.88%達到了骨痂連接。接下來，我們測量了Mg-IMN治療過的大鼠組織中CGRP的含量。通過免疫組化和流式細胞技術，我們發現Mg-IMN可以逆轉ZOL對於骨組織和脊髓背根神經節中CGRP的抑製作用。同時，ZOL導致的骨折端過度纖維化在Mg-IMN治療后也得到了緩解。另外，從Mg-IMN治療過的大鼠股骨所提取的骨髓間充質幹細胞，其成纖維分化能力也較ZOL組降低。以上證據表明我們設計的含鎂髓內針可以通過增加CGRP釋放，減少纖維化，從而改善雙磷酸鹽導致的骨折延遲愈合。另外，我們把腺病毒包裹的shRNA注射至Mg-IMN固定的大鼠股骨內，以在骨折愈合過程中下調CGRP受體Ramp1。結果顯示，鎂金屬的療效也隨之下調，這說明，含鎂髓內針改善雙磷酸鹽所導致的骨折延遲愈合副作用的功效依賴於CGRP。本研究的臨床重要性：目前的研究詳細闡述了雙磷酸鹽長期使用導致的非典型性股骨骨折愈合的病理特征，發現了骨折端過度纖維化可能是造成其延遲愈合的病理機制。此發現不僅有利於針對非典型性骨折開發新的治療方案，而且還可以用以促進常規性的骨折愈合。我們的研究還為含鎂髓內針的臨床轉化提供了有價值的基礎醫學證據。我們期望該研究最終可用於臨床治療非典型性骨折，使患者受益，減少社會經濟負擔。Zheng, Nianye."December 2019."Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 228-262).Abstracts also in Chinese.Title from PDF title page (viewed on 20 May, 2021).Zheng, Nianye