在骨缺损的背景下,受损的脉管系统和血液动力学流入的丧失导致血肿形成和低氧张力,其通过HIf-1α途径刺激血管扩张。最重要的是,该途径上调H型血管(CD31hiEmcnhi血管)的发芽。H血管与血管周骨祖细胞(OPCs)直接相互作用,成骨细胞,以及骨形成和重建的破骨细胞。这种血管生成-成骨耦合导致血管和骨组织的同步传播以进行再生愈合。越来越多的文献表明,H血管构成了骨骼固有的成骨愈合能力的很大一部分。我们认为CD31hiEmcnhi血管在牵张成骨(DO)过程中在骨愈合中起作用。DO是利用牵引力促进内源性骨形成的诱导和周围软组织如皮肤的再生的过程,肌肉,肌腱,和神经血管结构。虽然H血管对机械损伤的反应足以促进正常健康组织的愈合,它仍然不足以克服辐射的破坏。我们认为CD31hiEmcnhi血管的破坏在排除DO在辐照骨缺损愈合中的有效性中起作用。我们的目标是,因此,利用H血管的再生能力概括骨愈合的正常途径。我们假设使用去铁胺(DFO)的局部应用将增强H血管介导的对辐射损伤的血管生成反应,并最终在DO期间实现成骨愈合。这一发现可能通过开发转化疗法来利用,有望加速骨形成并缩短DO巩固期。从而潜在地扩大DO在辐照骨愈合中的利用率。Sprague-Dawley大鼠分为三组:DO,使用DO(xDO)的辐射,和辐射与DO和DFO植入(xDODFO)。实验组接受35Gy的辐射。所有组都接受了DO。治疗组在截骨部位注射,每隔一天,从DFO术后第4天(POD)开始。在POD40上处死动物。对于免疫组织化学分析,下颌骨解剖并在4%多聚甲醛中固定48小时,在Cal-ExII中脱钙2天,通过递增浓度的分级乙醇脱水,然后用石蜡包埋.将样品切成7μm厚的纵向定向切片,包括干骨干和骨干。进行CD31和Emcn双免疫荧光染色以评估CD31hiEmcnhi血管形成的程度。然后在4°C下用缀合的抗体过夜染色骨切片。细胞核用Hoechst染色。载玻片也用Osterix和CD31双重染色以研究H血管介导的OPCs募集的量以加速骨愈合。用尼康Ti2宽视场显微镜获取图像,并在NIS-ElementsAdvancedResearch5.41.02软件中进行分析。H型血管的丰度由再生样品内的CD31+Emcn+血管面积的面积分数表示。OPC伴随向牵张间隙的增殖由再生样品内部的Osterix+细胞面积的面积分数表示。DO组的H型血管比xDO组多6倍。与xDO相比,局部DFO使辐照的DO动物的H型血管的丰度显着增加了15倍(p=0.00133531)。此外,H型血管丰度较高的DO和xDODFO组也表现出更好的血管生成和成骨结局.有趣的是,与DO相比,xDODFO组将H血管形成的数量增加了一倍,表明超生理反应(p=0.044655055)。此外,H血管介导的OPCs募集模仿了我们研究组中描述的H血管形成趋势。与DO对照相比,辐照的DO组含有3倍更少的OPCs。对xDO动物的DFO处理通过含有12×Osterix细胞来修复辐照损伤。最后,与DO对照组相比,DFO治疗受辐照动物的骨祖细胞募集到了分散间隙中的四倍。在这项研究中,我们开发了一种新的方法来可视化石蜡切片中的CD31hiEmcnhi以研究DO再生。正常DO表现出H血管形成和相关的血管生成-成骨耦合的显着上调。辐射严重减少了H血管的形成,同时伴随着新骨形成和骨不连的显着减少。DFO管理,然而,导致血管补充和大量CD31hiEmcnhi和OPCs的恢复,概括骨再生和修复的正常过程。DFO治疗修复了与H血管血管生成-成骨耦合增加相关的辐照区域中的新骨形成和骨结合。虽然需要进一步的研究来优化这种方法,这项研究的结果对于期待已久的将局部DFO转化为临床领域非常有希望.
In the setting of bone defects, the injured vasculature and loss of hemodynamic inflow leads to hematoma formation and low oxygen tension which stimulates vascular expansion through the HIf-1α pathway. Most importantly, this pathway upregulates sprouting of type H vessels (CD31hiEmcnhi vessels). H vessels engage in direct interaction with perivascular osteoprogenitor cells (OPCs), osteoblasts, and preosteoclasts of bone formation and remodeling. This angiogenic-osteogenic coupling leads to synchronous propagation of vascular and bony tissue for regenerative healing. A growing body of literature demonstrates that H vessels constitute a large portion of bone\'s innate capacity for osteogenic healing. We believe that CD31hiEmcnhi vessels play a role in bone healing during distraction osteogenesis (DO). DO is a procedure that utilizes traction forces to facilitate induction of endogenous bone formation and regeneration of surrounding soft tissues such as skin, muscle, tendon, and neurovascular structures. While the H vessel response to mechanical injury is adequate to facilitate healing in normal healthy tissue, it remains inadequate to overcome the devastation of radiation. We posit that the destruction of CD31hiEmcnhi vessels plays a role in precluding DO\'s effectiveness in irradiated bone defect healing. We aim, therefore, to recapitulate the normal pathway of bony healing by utilizing the regenerative capacity of H vessels. We hypothesize that using localized application of deferoxamine (DFO) will enhance the H vessel-mediated vasculogenic response to radiation damage and ultimately enable osteogenic healing during DO. This discovery could potentially be exploited by developing translational therapeutics to hopefully accelerate bone formation and shorten the DO consolidation period, thereby potentially expanding DO\'s utilization in irradiated bone healing. Sprague-Dawley rats were divided into three groups: DO, radiation with DO (xDO), and radiation with DO and DFO implantation (xDODFO). Experimental groups received 35 Gy of radiation. All groups underwent DO. The treatment group received injections into the osteotomy site, every other day, beginning on postoperative day (POD) 4 of DFO. Animals were sacrificed on POD 40. For immunohistochemical analysis, mandibles were dissected and fixed in 4% paraformaldehyde for 48 hours, decalcified in Cal-Ex II for 2 days, dehydrated through graded ethanol of increasing concentration, and then embedded in paraffin. Samples were cut into 7-μm thick longitudinally oriented sections including the metaphysis and diaphysis. CD31 and Emcn double immunofluorescent staining were performed to evaluate the extent of CD31hiEmcnhi vessel formation. Bone sections were then stained with conjugated antibodies overnight at 4°C. Nuclei were stained with Hoechst. Slides were also double stained with Osterix and CD31 to study the quantity of H vessel-mediated recruitment of OPCs to accelerate bone healing. Images were acquired with a Nikon Ti2 widefield microscope and analyzed in NIS- Elements Advanced Research 5.41.02 software. The abundance of type H vessels is represented by the area fraction of CD31 + Emcn+ vessel area inside the regenerate sample. OPC concomitant proliferation into the distraction gap is represented by the area fraction of Osterix+ cell area inside of the regenerate sample. There were 6× more type H vessels in DO groups than in xDO groups. Localized DFO significantly increased the abundance of type H vessels of irradiated DO animals compared to xDO by 15× ( p = 0.00133531). Moreover, the DO and xDODFO groups with higher abundance of type H vessels also demonstrated better angiogenesis and osteogenesis outcomes. Interestingly, xDODFO groups doubled the quantity of H vessel formation compared to DO, indicating a supraphysiologic response ( p = 0.044655055). Furthermore, H vessel-mediated recruitment of OPCs mimicked the described H vessel formation trend in our study groups. Irradiated DO groups contained 3× less OPCs compared to DO controls. DFO treatment to xDO animals remediated irradiation damage by containing 12× Osterix+ cells. Finally, DFO treatment of irradiated animals quadrupled osteoprogenitor recruitment into the distraction gap compared to DO controls. In this study, we developed a novel approach to visualize CD31hiEmcnhi in paraffin sections to study DO regeneration. Normal DO demonstrated a significant upregulation of H vessel formation and associated angiogenic-osteogenic coupling. Radiation severely decreased H vessel formation along with an associated significant diminution of new bone formation and nonunion. DFO administration, however, resulted in vascular replenishment and the restoration of high quantities of CD31hiEmcnhi and OPCs, recapitulating the normal process of bony regeneration and repair. DFO treatment remediated new bone formation and bony union in irradiated fields associated with increased H vessel angiogenic-osteogenic coupling. While further studies are required to optimize this approach, the results of this study are incredibly promising for the long-awaited translation of localized DFO into the clinical arena.