计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合
Summary
自体和异体骨移植物植入构成接受的方法来治疗严重颅面骨量丢失。然而接枝组合物对新血管形成，细胞分化和骨形成之间的相互作用的影响还不清楚。我们提出的目的是阐明血管生成骨形成相互依存的移植接近一个多模式成像协议。
Abstract
一个主要参数确定一个骨移植过程的成功是包围接枝的区域的血管形成。我们假设一个植骨的是植入会诱发更大的骨再生的丰富的血管形成。调查接枝的对新血管形成在缺损部位的影响，我们开发了一种显微计算机断层摄影术（μCT）的方式来表征新形成的血管，其包括具有聚合的造影剂的动物的全身灌流。该方法，能够将其全部器官的血管详细分析。另外，血液灌注是使用血源性荧光剂的荧光成像（FLI）评估。骨的形成是使用羟基磷灰石针对性的探头和μCT分析FLI量化。干细胞募集是由转基因小鼠的生物发光成像（BLI），该表达骨钙蛋白启动子控制下的萤光素酶进行监测。这里，我们描述和展示制备的同种异体移植物的，颅骨缺损手术，对于新血管形成的研究和骨形成分析（包括体内灌注造影剂），并且协议进行数据分析的μCT扫描协议。
脉管系统的三维高分辨率分析表明在动物中植入自体移植显著更大的血管生成，特别是相对于小动脉的形成。因此，血液灌流是由第 7天手术后显著高于自体移植小组。我们在接受自体移植动物中观察到优越的骨矿化和测量更大的骨形成。自体移植诱导驻留干细胞募集到移植物宿主骨缝线，其中所述细胞分化成的第 7和第10日术后天之间骨形成细胞。这一发现意味着，增强骨形成可能是由于扩充血管饲养的特点自体植入。该方法的描述可作为研究骨组织再生紧密界骨骼的形成和新生血管方面的最佳工具。
Introduction
颅面骨量丢失，由于外伤，肿瘤切除，开颅减压术，先天性缺陷很少治愈本身，并提出了明确的未满足的临床需求。自体骨移植物和同种异体骨移植物被广泛地用于治疗这些病症1。
它已被广泛接受的成骨紧密结合与血管生成2,3。因此，提出了治疗骨再生的完整研究应包括血管树整个缺损部位形成的一个全面的调查。有几种可用的方法研究模型来刻画血管。血管树可以通过组织学分析进行调查。自组织学依赖于切片的组织，存在所得到的图像将被扭曲的可能性高。为了解决这个问题，活体显微镜可以执行将图像的完好的血管4;然而，这种方法是限制在一个平面上成像。从灌注造影剂的动物获取的样品μCT扫描允许饲料再生网站5血管网络的3D影像。这种方法允许器官的脉管作为一个整体的一个非常详细的示范，以及血管分布的细致的分析。此外，μCT使多样直径血管，这表征血管的不同亚型之间的分化。
我们假设一个颅骨自体的该植入会诱发比同种异体移植物的植入更大新血管形成，而这增加了新血管形成将导致反过来增强骨formation.To追求这一假设，我们所采用的各种技术。我们通过执行μCT为基础的分析研究了新成立的血管树的模式。我们用血池荧光探针测量血液灌注。接下来，我们的驴SED骨组织矿化羟基磷灰石定向探头和μCT分析FLI。最后，我们监测干细胞招募和分化，在将荧光素酶表达在骨钙素阳性细胞的转基因小鼠进行的BLI。
Protocol
该协议遵循耶路撒冷，以色列（申请号MD-12-13524-4），经批准AAALAC设施和由雪松 – 西奈医学中心的希伯来大学的体制动物护理和使用委员会（IACUC）的指导方针IACUC（申请号3770）。这些动物是在严格遵守美国国立卫生研究院的指导方针进行处理。 1.准备骨移植物安乐死7〜8周龄的Balb / C小鼠，或任何应变来自受体不同，使用的CO 2的吸入或腹腔注射50微升苯巴比妥?…
Representative Results
新血管形成通过μCT体积分析和通过FLI使用荧光血源性剂量化血液灌注进行评估。手术后七天，μCT扫描证实在已经收到自体比在已经收到移植从C57BL / 6（图3A）收获小鼠小鼠显著更高体积的中小直径的血管。有趣的是，移植组中新形成的血管树似乎达到整个缺陷区域，而同种异体移植组中的血管出现穿透从外边缘缺损部位和向内延伸到仅在有限的程度。所述FLI支持了这一观察，示出显?…
Discussion
这里描述的多模成像方法的目的是为了使血管发生成骨轴的颅骨移植的上下文细致的调查。使用μCT协议，这使得血管树的准确高分辨率三维演示喂养整个颅骨缺损新生血管成像。 μCT数据可以使用先进的工具，如IPL的软件很容易分析。例如， 在图3C中示出的厚度的分析显示，自体颅和同种异体移植物之间的主要差别是在容器为0.1至0.08毫米的直径，其表征血液动脉10。这一结果…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge funding from the NIDCR (Grant No. DE019902) and from the Israeli Science Foundation (Grant No. 382/13).
Materials
| C57BL/C Mice | Harlan laboratories | 57 | |
| FVB/n Mice | Harlan laboratories | 862 | |
| Phenobarbital | West waro | NDC 0641-0477-25 | |
| Rodent hair clipper | Wahl animal | 8786-451A | |
| Scalpel 11 | Miltex | 27111504 | |
| Dental micro motor | marathon III | ||
| 5mm trephine | Fine Science tools | 18004-50 | |
| Hair removing cream | Veet | ||
| KetaVed (Ketamine) | Vedco | NDC 50989-996-06 | |
| Domitor | Zoetis | NADA 141-267 | |
| carprofen | Norbrook | 02000/4229 | |
| Eye ointment | Puralube | NDC 17033-211-38 | |
| Operating binocular | Kent scientific | KSCXTS-1121 | |
| Fine scissors | Fine Science tools | 14060-11 | |
| Curve tweezers | Fine Science tools | 11274-20 | |
| Spoon shaped spatula | Fine Science tools | 10090-13 | |
| Tisseel Fibin gel kit | Baxter | 718971 | |
| needle holder | Fine Science tools | 12060-01 | |
| vicryl suture 4-0 | Ethicon | J392H | |
| Antisedan | Zoetis | NADA#141033 | |
| Heparin | Sigma | H3393 | |
| 20ml luerlock | BD | 302830 | |
| 23G scalp vein set (butterfly needle) | BD | 367342 | |
| Hemostat | Fine Science tools | 13008-12 | |
| Syringe pump | Harvard apparatus | PHD 2000 | |
| 3sec gel glue | Scotch | ||
| rodent dissection board | Leica | 38DI02313 | |
| Microfil MV-122 | flow-tech | MV-122 | |
| uCT40 scanner | Scanco | uCT40 | |
| TCA6% | Sigma | T6399 | |
| Osteosense 680 | PerkinElmar | NEV10020EX | |
| Angiosense750 | PerkinElmar | NEV10011 | |
| Oxigen 100% medical grade | |||
| isoflurane (furane) | Baxter | 1001936040 | |
| IVIS kinetics | Xenogen | ||
| Beetle luciferin | Promega | E160A |
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Cite This Article
Cohn Yakubovich, D., Tawackoli, W., Sheyn, D., Kallai, I., Da, X., Pelled, G., Gazit, D., Gazit, Z. Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts. J. Vis. Exp. (106), e53459, doi:10.3791/53459 (2015).