多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复
Summary
在这里, 我们提出了一个协议, 以可视化血管形成在体内和在 real-time 的3D 支架的多显微镜。在小鼠颅骨临界骨缺损模型中研究了基因修饰支架的血管生成。治疗组发现更多的新血管。
Abstract
重度骨缺损的重建仍然是一个严重的临床问题, 因为在修复过程中组织工程支架内血管生成不良, 导致缺乏足够的血液供应, 造成新组织坏死。快速血管化是新的组织生存和与现有宿主组织融合的重要先决条件。在脚手架上的从头到脚生成是使骨骼再生更有效的最重要步骤之一, 允许修复组织成长为支架。为了解决这个问题, 生物材料支架的基因修饰用于加速血管生成和成骨。然而, 在 real-time 和三维 (3D) 支架或新骨组织中, 可视化和追踪体内血管形成仍然是骨组织工程的障碍。多显微镜 (MPM) 是一种新的生物成像模式, 可以获得高分辨率和微创的方式从生物结构的体积数据。本研究的目的是在一个基因修饰的 3 d-PLGA/nHAp 支架中多显微镜下观察血管生成, 用于颅骨临界骨缺损修复. PLGA/nHAp 支架功能化, 以持续交付一个生长因子血小板增殖素 b基因携带慢载体 (LV-pdgfb), 以促进血管生成和促进骨骼再生。在支架植入的颅骨临界骨缺损小鼠模型中, PHp 支架内的血管区 (BVAs) 明显高于 PH 支架。此外, 血小板生长因子-b和血管生成相关基因的表达, vWF和VEGFR2也相应增加。MicroCT 分析表明, 与其他组相比, PHp 组的新骨形成明显改善。据我们所知, 这是第一次用多显微镜进行骨组织工程, 以研究3D 生物降解支架内的血管生成,在体内和 real-time。
Introduction
骨骼是一种高度带血管的组织, 在单个1的生存期内继续进行重塑。由于外伤、骨不连、肿瘤切除或颅面畸形而导致的大骨缺损的快速有效的骨再生是一个复杂的生理过程。传统的治疗方法用于骨缺损修复包括自体移植和同种异体植入, 但其使用涉及几个问题和限制, 如有限的可用性, 严重的供者部位的发病率, 高的感染风险, 和主机免疫排斥2,3。然而, 人工骨移植提供了一种有效的替代方法来缓解这些局限性。它们可以由可生物降解的材料制成, 很容易制造出合适的孔径, 并可进行基因改造4,5。
目前, 在组织工程骨的开发中, 使用了各种组织工程学支架6,7。为了更有效地诱导骨修复和再生, 与生长因子结合的工程生物材料已经出现并取得了良好的效果8,9。不幸的是, 短暂的半衰期, easy-to-lose 活性, 和 supraphysiological 剂量的生长因子的治疗效果限制他们的临床应用10。为了克服这些问题, 将生长因子基因的传递, 而不是生长因子作为一种有效的方法来维持生物活性的治疗骨缺损和疾病11,12。病毒载体是有前途的传递工具, 组织再生由于其高表达效率13。
在生长因子中, 血小板源性生长因子在本研究中被选择, 因为它不仅是间充质和成骨细胞的丝裂原和趋化蛋白, 而且还是一种刺激血管生成的14,15.前期临床研究表明, 血小板生长因子-BB 能安全有效地促进牙周骨缺损的修复,16,17。最近的研究表明, 血小板生长因子-BB 刺激血管内皮细胞迁移和增殖在体内18,19。此外, 血小板生长因子 BB 还能使骨髓间充质干细胞 (mscs) 分化为内皮细胞20, 这进一步凸显了 mscs 在新生血管中的潜在作用。因此, 在骨组织工程支架中, 诱导血管的从头开始形成是修复组织的重要步骤。
骨缺损愈合是一种动态组织形态发生过程, 需要协调成骨和血管生成在修复位置21。血管植入组织工程支架是一个必要的先决条件, 为细胞提供养分和氧气的生长和生存和消除代谢废物。常用的成像方法, 包括 X 射线显微计算机断层扫描 (microCT), 磁共振成像 (MRI), 扫描电子显微镜 (SEM), 光学相干断层扫描 (OCT), 和共焦激光扫描显微镜, 而不是使用组织学检查获取血管生成信息22,23。然而, 这些方法在骨组织工程3D 支架的可视化和测量新生中面临各种障碍。多显微镜 (MPM) 是一种比较新颖的生物成像技术, 具有明显的优势, 同时可视化细胞, 细胞外基质, 和周围的血管网络在体内.它具有一个固有的三维成像能力, 深层组织穿透和导致低光。因此, 在过去的十年中, MPM 在生物医学研究24中得到了很大的关注, 包括神经科学、免疫学和干细胞动力学。然而, 它几乎没有用于骨科研究。
Protocol
Representative Results
Discussion
骨是一种高度带血管的组织, 具有独特的能力, 可以在单个1的整个生命周期中持续地进行修复和重塑。血管化水平对成骨和缺损修复具有重要意义。低血管化限制了组织工程骨的广泛临床应用。根据仿生学的理论构建高度血管化的组织工程骨, 已成为修复大段骨缺损的工具。各种支架已成功地用于修复比较小的骨缺损。然而, 对于严重的骨缺损, 应用支架修复仍然面临的障碍, 主要…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了深圳孔雀计划, 中国 (No. 110811003586331), 深圳基础研究项目 (No。JCYJ20150401150223631, No。JCYJ20150401145529020 和 No。JCYJ20160331190714896), 广东省公共研究和能力建设专项计划 (No. 2015A020212030), 国家自然科学基金 (No. 81501893), 中国国家重点基础研究项目 (2013CB945503), 和先进创新计划为优秀青年研究员 (Y5G010)。
Materials
| Poly(D,L-lactide-co-glycolide) (PLGA) | Sigma | P1941 | L/G ratio 75:25, MW 66000-107000 |
| Hydroxyapatite nanoparticles | Sigma | 702153 | Average diameter < 200nm |
| Chloroquine diphosphate salt | Sigma | C6628 | |
| FITC-conjugated 250-kD dextran | Sigma | FD250S | |
| 1,4-dioxane | lingfeng,Shanghai | 0.45 micron | |
| Stericup filters | Merck Millipore Corporation | SLHV033RB | |
| PDGF-BB Cdna | Sino Biological, Inc | MZ50801-G | |
| Anti-PDGF-BB mouse polyclonal antibody | BioVision, Inc | 5489-30T | |
| PDGF-BB recombinant protein | 4489-50 | ||
| Calcium-phosphate transfection solution | Promega Corporation | E1200 | |
| L-DMEM | Hyclone | SH30021.01 | |
| DPBS | Hyclone | SH30028.01 | |
| Penicillin-Streptomycin, Liquid | Thermo Fisher Scientific | 15140122 | |
| FBS | Thermo Fisher Scientific | 10099-141 | |
| Transwell | Corning | 3422 | |
| Male BALB/c mice | Guangdong Medical Laboratory Animal Center | ||
| sodium pentobarbital | Merck | 1063180500 | |
| multiphoton microscopy | A homemade in Shenzhen Institutes of Advanced Technology to detect two-photon excited fluorescence (TPEF) and second harmonic generation signal (SHG). | ||
| isoflurane | Keyuan, Shandong | 401750169 | |
| TRIzol reagent | Invitrogen | 15596018 | |
| PrimeScript RT Master Mix (Perfect Real Time) | Takara | RR420B | |
| SYBR Premix Ex Taq (Tli RNaseH Plus) | Takara | RR036B | |
| Hematoxylin and eosin | Beyotime | C0105 | |
| Paraffin | Leica | RM2235 | |
| Ultracentrifuge OPtima L-100XP | Beckman Coulter | L-100XP | |
| Low-temperature printer | Tsinghua university | A homemade in Tsinghua university | |
| LightCycler 480 instrument | Roche | 5815916001 | |
| microCT | Bruker | 1176 | |
| commercial software | Bruker |
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