半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析
1Skeletal Biotech Laboratory,Hebrew University-Hadassah Faculty of Dental Medicine, 2Department of Surgery,Cedars-Sinai Medical Center, 3Board of Governors Regenerative Medicine Institute,Cedars-Sinai Medical Center, 4Biomedical Imaging Research Institute,Cedars-Sinai Medical Center, 5Department of Orthopedics,Cedars-Sinai Medical Center
Summary
该协议的目的是产生一个裸鼠骨质疏松症相关的椎体压缩骨折模型, 可以纵向评价在体内使用半自动 microcomputed tomography-based 定量结构分析。
Abstract
与骨质疏松相关的椎体压缩骨折 (OVCFs) 是一种常见的和临床未满足的需要随着世界人口年龄的增加而患病率。动物 OVCF 模型对转化组织工程策略的临床前发展至关重要。虽然目前有许多模型存在, 该协议描述了一个优化的方法, 诱导多高度重现性脊柱缺损在一个单一的裸鼠。本文还详细介绍了一种基于纵向半自动 microcomputed 断层扫描 (µCT) 的椎体缺损定量结构分析方法。简单地, 老鼠在术后多时间点被成像。1天扫描被重新定向到标准位置, 定义了标准的兴趣量。随后对每只老鼠的µCT 扫描自动登记到1天扫描, 因此, 同样的兴趣量被分析, 以评估新的骨骼形成。这种通用的方法可以适应各种其他模型, 纵向 imaging-based 分析可以受益于精确的3D 半自动对准。在一起, 本议定书描述了一个容易量化和容易重现的系统骨质疏松和骨骼研究。建议的协议需要4月来诱导裸卵巢大鼠和2.7 和 4 h 之间的骨质疏松症的产生, 图像, 并分析两个椎体缺损, 取决于组织大小和设备。
Introduction
全世界有超过2亿人患有骨质疏松症1。骨密度 (BMD) 和改变的骨微结构的潜在病理降低增加了骨脆性, 因此, 骨折的相对风险为2。骨质疏松症是如此普遍和有害健康, 世界卫生组织已确定它是一个重大的公共卫生问题。此外, 随着世界人口的老龄化, 骨质疏松症有望变得更加普遍。
骨质疏松性椎体压缩骨折是最常见的脆性骨折, 估计在美国每年超过75万。它们与显著的发病率和九倍高的死亡率3有关。在临床试验中, 目前可获得的手术干预, 如椎体成形术和成形, 被发现没有比假治疗更有效4,5, 只留下疼痛管理可供这些患者使用。由于目前的 OVCF 治疗是有限的, 所以必须开发一种能够复制紊乱的动物模型6,7,8。这种动物模型可以促进对目前治疗方法的研究和新疗法的发展, 将转化为临床实践。骨质疏松症已诱导和持续的模型动物通过管理的低饮食 (LCD) 与卵巢切除联合1,9,10,11,12,13,14,15. 为进一步模型骨质疏松与 OVCFs, 椎体骨缺损建立了免疫大鼠的骨骼损伤 16,17,18,19, 20,21,22,23,24。在这项工作中, 一个模型的免疫损伤大鼠骨质疏松症提出。这种新的模型可以用来评估细胞疗法涉及干细胞从各种来源和物种的修复有挑战性的骨折, 如 OVCFs。
骨显像是评价骨折和骨骼疾病的重要组成部分。为了准确评估结构骨的变化和再生策略, 开发了先进的成像方法25。其中, µCT 成像已经成为一种非侵入性的、易用的、廉价的提供高分辨率3D 图像的方法。µCT 成像在评估骨质疏松症患者方面优于其他模式, 因为它提供高分辨率的3D 骨微体系结构26 , 然后可以对其进行定量分析。后者可以用来比较治疗效果的建议治疗。事实上,在体内µCT 成像是一个金标准的脊椎缺损再生监测1,16,27。但是, 很少有出版物28、29、30、31使用了自动注册工具来最小化µCT 的用户依赖性、插值偏差和精度错误imaging-based 分析。最近, 我们是第一个使用注册程序, 以改善骨再生的标准化骨空洞的分析, 如本协议32中所述。
本文所描述的方法可以用来研究新的细胞治疗 OVCFs 的效果, 不受宿主 T 细胞的反应, 可能拒绝异种或同种异体细胞。通过卵巢切除 (卵巢) 和4月的液晶显示器, 诱导幼鼠骨质疏松。年轻的卵巢鼠, 结合 LCD, 我们可以达到一个低峰值骨量, 模仿绝经后骨质疏松, 导致不可逆转的骨质流失。这可以解释部分原因是, 在 LCD 和大约3月的年龄, 大鼠从骨骼模型过渡到重建腰椎椎体的阶段33, 从而增加了维持骨质疏松症的可能性时间.使用幼兽使这种模型更具成本效益, 因为它们成本更低。然而, 它是有限的内在不核算的生物变化的衰老动物。
Protocol
所有动物实验都是根据由雪松-西奈医疗中心的机构动物保育和使用委员会 (IACUC) 批准的协议执行的 (3609 号议定书)。麻醉是为所有的成像和手术程序。所有动物都按照批准的 IACUC 协议安置. 注意: 该协议的实验设计如图 1 所示。购买六周大的老鼠与他们的卵巢手术切除和饲料他们的 LCD 包括0.01% 钙和0.77% 磷酸盐。经过4月的 LCD, 钻 critical-size 脊椎缺损在第四和第五腰椎…
Representative Results
使用这个协议, 你可以图像和量化的再生 n = 8 模型骨质疏松椎体缺损不同时间点。通过注册程序获得的解剖匹配, 可以在所有时间点对同一 VOI 进行分析。这导致了高度准确的纵向3D 计量分析, 即使原始缺陷的边缘不再是可识别的。我们使用五时间点 (天 1, 星期 2, 星期 4, 星期8和星期 12) 作为一个例子为骨骼再生的纵向评估 (图 7)。可通过对2D 剖面和3D …
Discussion
骨质疏松是脊椎压迫性骨折的最常见原因, 脊柱的负荷增加, 导致椎体塌陷。然而, 在真正复制类似椎体塌陷的啮齿动物中, 几乎不可能产生损伤。相反, 研究人员在椎体中心创建一个圆柱形空洞以模拟 OVCFs16,17,18,19,20,21,24<sup…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了加利福尼亚再生医学研究所 (CIRM) (TR2-01780) 的资助。
Materials
| Isoflurane | MWI Animal Health, Pasadena, CA | 501017 | |
| BetadineSolution | MWI Animal Health, Pasadena, CA | 4677 | |
| Chlorhexidine Gluconate2%scrub | MWI Animal Health, Pasadena, CA | 510083 | |
| Isopropyl Alcohol 70%-quart | MWI Animal Health, Pasadena, CA | 501044 | |
| Carprofen | MWI Animal Health, Pasadena, CA | 26357 | |
| Buprenorphine0.3mg/mL | MWI Animal Health, Pasadena, CA | 56163 | |
| Ovariectomized Athymic nude rats | Harlan Laboratories, Indianapolis, IN | Hsd:RH-Foxn1 rnu | |
| Low calcium food | Newco Distributors, Inc., CA | 1814948 (5AV8 AIN-93M w/low calcium) | |
| Phosphate Buffered Saline | Life Technologies Corporation | 14190250 | |
| Dermabond | J AND J ETHICON | DHVM12 | |
| Anesthesia machine | Patterson Scientific | TEC 3EX | |
| Slide Top Induction Chambers | Patterson Scientific | 78917833 | |
| ProStation Heated Workstation | Patterson Scientific | 78914731 | |
| Surgical drape | HALYARD HEALTH INC | 89101 | |
| Magnetic fixator retraction system | Fine Science Tools, Inc., CA | 18200-50 | |
| Dissecting Scissors, 10cm, Curved, SS | World Precision Instruments, FL | 14394 | |
| Iris Scissors, 11.5cm, 45°Angle, Serrated, Sharp/Sharp | World Precision Instruments, FL | 503225 | |
| Forceps, no. 5 | World Precision Instruments, FL | 555048FT | |
| Micro Mosquito Hemostatic Forceps | World Precision Instruments, FL | 503360 | |
| Sterile cotton gauze | Medtronic, MINNEAPOLIS, MN | 9024 | |
| Absorption Spears – Mounted/Sterile | Fine Science Tools, CA | 18105-01 | |
| Syringe, 1 ml | TERUMO TERUMO MED | SS-01T | |
| Needle, 25gauge | BD MED SYS INJECTION SYS | 305127 | |
| Laminar flow hood | Baker | SterilGARD e3-Class II Type A2 Biosafety Cabinet | |
| Thermal Cautery Unit | World Precision Instruments, FL | 501292 | |
| Micro-Drill OmniDrill115/230V | World Precision Instruments, FL | 503598 | |
| Trephines for Micro Drill, 2mm diameter | Fine Science Tools, CA | 18004-20 | |
| 3-0 Vicryl undyed 27” SH taper | J AND J ETHICON | 1663G | |
| 4-0 Ethilon black 18” PC3 conventional cutting | J AND J ETHICON | 1954G | |
| Conebeam in vivo microCT (vivaCT 40) | Scanco Medical | vivaCT 40 | |
| SCANCO Medical microCT systems software suite | Scanco Medical | vivaCT 40 | |
| Analyze software | Biomedical Imaging, Mayo Clinic, Rochester, MN | Analyze 12 | Image analysis software |
| Veterenery eye ointment | |||
References
- Wang, M. L., Massie, J., Perry, A., Garfin, S. R., Kim, C. W. A rat osteoporotic spine model for the evaluation of bioresorbable bone cements. Spine J. 7 (4), 466-474 (2007).
- . Consensus development conference: prophylaxis and treatment of osteoporosis. Am J Med. 90 (1), 107-110 (1991).
- Center, J. R., Nguyen, T. V., Schneider, D., Sambrook, P. N., Eisman, J. A. Mortality after all major types of osteoporotic fracture in men and women: an observational study. Lancet. 353 (9156), 878-882 (1999).
- Buchbinder, R., et al. A randomized trial of vertebroplasty for painful osteoporotic vertebral fractures. N Engl J Med. 361 (6), 557-568 (2009).
- Kallmes, D. F., et al. A randomized trial of vertebroplasty for osteoporotic spinal fractures. N Engl J Med. 361 (6), 569-579 (2009).
- Kado, D. M., et al. Vertebral fractures and mortality in older women: a prospective study. Study of Osteoporotic Fractures Research Group. Arch Intern Med. 159 (11), 1215-1220 (1999).
- Silverman, S. L. The clinical consequences of vertebral compression fracture. Bone. 13, S27-S31 (1992).
- Ross, P. D. Clinical consequences of vertebral fractures. Am J Med. 103 (2A), 30S-43S (1997).
- Saito, T., Kin, Y., Koshino, T. Osteogenic response of hydroxyapatite cement implanted into the femur of rats with experimentally induced osteoporosis. Biomaterials. 23 (13), 2711-2716 (2002).
- Koshihara, M., Masuyama, R., Uehara, M., Suzuki, K. Effect of dietary calcium: Phosphorus ratio on bone mineralization and intestinal calcium absorption in ovariectomized rats. Biofactors. 22 (1-4), 39-42 (2004).
- Martin-Monge, E., et al. Validation of an osteoporotic animal model for dental implant analyses: an in vivo densitometric study in rabbits. Int J Oral Maxillofac Implants. 26 (4), 725-730 (2011).
- Agata, U., et al. The effect of different amounts of calcium intake on bone metabolism and arterial calcification in ovariectomized rats. J Nutr Sci Vitaminol (Tokyo). 59 (1), 29-36 (2013).
- Govindarajan, P., et al. Bone matrix, cellularity, and structural changes in a rat model with high-turnover osteoporosis induced by combined ovariectomy and a multiple-deficient diet. Am J Pathol. 184 (3), 765-777 (2014).
- Govindarajan, P., et al. Implications of combined ovariectomy/multi-deficiency diet on rat bone with age-related variation in bone parameters and bone loss at multiple skeletal sites by DEXA. Med Sci Monit Basic Res. 19, 76-86 (2013).
- Alt, V., et al. A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures. Acta Biomater. 9 (6), 7035-7042 (2013).
- Liang, H., et al. Use of a bioactive scaffold for the repair of bone defects in a novel reproducible vertebral body defect. Bone. 47 (2), 197-204 (2010).
- Liang, H., Li, X., Shimer, A. L., Balian, G., Shen, F. H. A novel strategy of spine defect repair with a degradable bioactive scaffold preloaded with adipose-derived stromal cells. Spine J. 14 (3), 445-454 (2014).
- Fujishiro, T., et al. Histological evaluation of an impacted bone graft substitute composed of a combination of mineralized and demineralized allograft in a sheep vertebral bone defect. J Biomed Mater Res A. 82 (3), 538-544 (2007).
- Sheyn, D., et al. Gene-modified adult stem cells regenerate vertebral bone defect in a rat model. Mol Pharm. 8 (5), 1592-1601 (2011).
- Phillips, F. M., et al. In vivo BMP-7 (OP-1) enhancement of osteoporotic vertebral bodies in an ovine model. Spine J. 6 (5), 500-506 (2006).
- Kobayashi, H., et al. Long-term evaluation of a calcium phosphate bone cement with carboxymethyl cellulose in a vertebral defect model. J Biomed Mater Res A. 88 (4), 880-888 (2009).
- Turner, T. M., et al. Vertebroplasty comparing injectable calcium phosphate cement compared with polymethylmethacrylate in a unique canine vertebral body large defect model. Spine J. 8 (3), 482-487 (2008).
- Zhu, X. S., et al. A novel sheep vertebral bone defect model for injectable bioactive vertebral augmentation materials. J Mater Sci Mater Med. 22 (1), 159-164 (2011).
- Vanecek, V., et al. The combination of mesenchymal stem cells and a bone scaffold in the treatment of vertebral body defects. Eur Spine J. 22 (12), 2777-2786 (2013).
- Geusens, P., et al. High-resolution in vivo imaging of bone and joints: a window to microarchitecture. Nat Rev Rheumatol. 10 (5), 304-313 (2014).
- Genant, H. K., Engelke, K., Prevrhal, S. Advanced CT bone imaging in osteoporosis. Rheumatology (Oxford). 47, 9-16 (2008).
- Kallai, I., et al. Microcomputed tomography-based structural analysis of various bone tissue regeneration models. Nature Protocols. 6 (1), 105-110 (2011).
- Lambers, F. M., Kuhn, G., Schulte, F. A., Koch, K., Muller, R. Longitudinal assessment of in vivo bone dynamics in a mouse tail model of postmenopausal osteoporosis. Calcif Tissue Int. 90 (2), 108-119 (2012).
- de Bakker, C. M., et al. muCT-based, in vivo dynamic bone histomorphometry allows 3D evaluation of the early responses of bone resorption and formation to PTH and alendronate combination therapy. Bone. 73, 198-207 (2015).
- Lan, S. H., et al. 3D image registration is critical to ensure accurate detection of longitudinal changes in trabecular bone density, microstructure, and stiffness measurements in rat tibiae by in vivo microcomputed tomography (μCT). Bone. 56 (1), 83-90 (2013).
- Nishiyama, K. K., Campbell, G. M., Klinck, R. J., Boyd, S. K. Reproducibility of bone micro-architecture measurements in rodents by in vivo micro-computed tomography is maximized with three-dimensional image registration. Bone. 46 (1), 155-161 (2010).
- Sheyn, D., et al. PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries. Mol Ther. 24 (2), 318-330 (2016).
- Lelovas, P. P., Xanthos, T. T., Thoma, S. E., Lyritis, G. P., Dontas, I. A. The laboratory rat as an animal model for osteoporosis research. Comp Med. 58 (5), 424-430 (2008).
- Bouxsein, M. L., et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res. 25 (7), 1468-1486 (2010).
- de Lange, G. L., et al. A histomorphometric and micro-computed tomography study of bone regeneration in the maxillary sinus comparing biphasic calcium phosphate and deproteinized cancellous bovine bone in a human split-mouth model. Oral Surg Oral Med Oral Pathol Oral Radiol. 117 (1), 8-22 (2014).
- Ramalingam, S., et al. Guided bone regeneration in standardized calvarial defects using beta-tricalcium phosphate and collagen membrane: a real-time in vivo micro-computed tomographic experiment in rats. Odontology. 104 (2), 199-210 (2016).
- Leary, S., et al. . AVMA guidelines for the euthanasia of animals: 2013 edition. , (2013).
- Wang, M. L., Massie, J., Allen, R. T., Lee, Y. P., Kim, C. W. Altered bioreactivity and limited osteoconductivity of calcium sulfate-based bone cements in the osteoporotic rat spine. Spine J. 8 (2), 340-350 (2008).
- Liang, H., Li, X., Shimer, A. L., Balian, G., Shen, F. H. A novel strategy of spine defect repair with a degradable bioactive scaffold preloaded with adipose-derived stromal cells. Spine J. 14 (3), 445-454 (2013).
- Sheyn, D., et al. PTH induces systemically administered mesenchymal stem cells to migrate to and regenerate spine injuries. Mol Ther. 24 (2), 318-330 (2015).
- Matthieu, R., et al. A new rat model for translational research in bone regeneration. Tissue Eng Part C Methods. , (2015).
- Turner, A. S. Animal models of osteoporosis–necessity and limitations. Eur Cell Mater. 1, 66-81 (2001).
Citer Cet Article
Shapiro, G., Bez, M., Tawackoli, W., Gazit, Z., Gazit, D., Pelled, G. Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model. J. Vis. Exp. (127), e55928, doi:10.3791/55928 (2017).