Bone affinity fluorescein indirectly marks early angiogenesis during endochondral ossification

doi:10.3969/j.issn.2095-4344.2017.08.008

Abstract

Abstract:

BACKGROUND: There are various methods to observe and detect early angiogenesis in the process of entochondrostosis, but each holds certain deficiencies.
OBJECTIVE: To explore the possibility of tetracycline and alizarin complexone as an indirect marker of angiogenesis in the process of entochondrostosis.
METHODS: New Zealand white rabbit models of bilateral radial bone defects were prepared, followed by β-tricalcium phosphate implantation, and then given the injection of tetracycline and alizarin complexone at 1 and 15 days, respectively. Samples were collected at 28 days, some of which were observed using fluorescence/light microscope after ink perfusion and hard tissue slicing, and the others were decalcified and observed using immunohistochemistry. The uniformity between lumen structures labeled with bone affinity fluorescein and vascular structures marked by immunohistochemistry and ink perfusion was compared.
RESULTS AND CONCLUSION: The lumen structure labeled with bone affinity fluorescein was confirmed to be a CD34 positive vascular structure. Under the fluorescence microscope, the bone affinity fluorescein labeled vascular morphology was consistent with ink perfusion-labeled, and black ink lines could be observed in the lumen structures labeled with bone affinity fluorescein after ink perfusion. In addition, the color of the lumen labeled with fluorescein was more gorgeous, three-dimensional structure more vivid, and the vascular evolution process distinguished more easily by different fluorescein colors, exhibiting unique advantages. Therefore, it is available to detect the early angiogenesis in the process of entochondrostosis

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tetracycline, Fluorescence, Cartilage, Blood Vessels, Tissue Engineering

CLC Number:

R318

Wu Tao, Liu Ying-chao, Guo Zhi-wang, Lv Jun, Xing Jian-zhou, Hou Bing. Bone affinity fluorescein indirectly marks early angiogenesis during endochondral ossification[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(8): 1192-1196.

Figures/Tables 2

2.1 亲骨荧光素标记的管腔结构CD34标记阳性由图1可知，在荧光显微镜下，亲骨荧光素标记的组织呈现出管腔样结构，其中术后第1天四环素标记的组织呈黄色荧光，术后第15天茜素络合酮标记的组织呈现红色荧光，并环绕于黄色荧光周围(图1A，C)。这代表了软骨内成骨期间不同时间段的新骨沉积。免疫组化观察表明，软骨内的管腔结构CD34标记阳性(图1B，D)，二者具有一致性，这说明荧光素标记的钙化组织属于离血管最近的新生骨组织，通过对新骨的示踪可间接标记血管的发生。 2.2 墨汁灌注可在亲骨荧光素标记的管腔结构中显影图2A显示了亲骨荧光素标记的单一血管形态，与墨汁灌注标记的血管形态基本一致(图2B)；经墨汁灌注后的新生血管呈现出网络交织的外观(图2C)，在荧光显微镜下，可见墨汁充盈于黄色荧光标记的管腔结构中央(图2D)，二者具有一致性，这说明在荧光标记的管腔结构中走行的为物理结构的血管组织。图2E是墨汁灌注后软骨内成骨过程中的Masson染色组织切片。图中显示墨汁灌注标记的血管走行于管腔结构的中央，在其周围呈现出环形的红染胶原结构，这代表了最早期的新骨沉积，大量绿色胶原填充于各管腔结构的间隙，代表了后期形成的骨组织及尚未骨化的软骨组织。结合图2A，D，这说明骨组织的沉积是从中央血管周围开始向四周软骨逐渐发生的，标记新生钙化组织能够间接显示软骨内新生的血管结构。 2.3 亲骨荧光素可标记不同阶段的新生血管结构软骨内的血管新生是一个动态的过程，随着时间的推移，血管不断成熟并向深部侵入。伴随着新生血管的侵入，新骨也在其周围沉积。图3A中，术后第15天茜素络合酮标记的红色荧光组织接续术后第1天四环素标记的黄色荧光组织，并形成了新的管腔结构；图3B中，大量黄色荧光沉积于β-磷酸三钙材料外围，这代表了术后第1-14天的血管发生情况。红色荧光环绕在黄色荧光外围，这代表了术后15 d新形成的板层骨，之后红色荧光接续黄色荧光向β-磷酸三钙材料中长入，并单独形成了纤细的管腔样结构，这说明术后第15-28天有新的向材料中侵入的血管生成。这说明在不同时间点采用不同颜色的亲骨荧光素标记新骨可反映不同时间段血管组织的新生。"

References

[1] Wang L, Fan H, Zhang ZY, et al. Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized β-tricalcium phosphate scaffold and mesenchymal stem cells. Biomaterials. 2010;31(36):9452-9461.

[2] 李文革, 郭永智, 王亚军,等. 骨折早期血管生成与骨形成的免疫组化及超微结构研究[J]. 吉林大学学报医学版, 1999, 25(3):251-252.

[3] Khojasteh A, Fahimipour F, Eslaminejad MB, et al. Development of PLGA-coated β-TCP scaffolds containing VEGF for bone tissue engineering. Mater Sci Eng C Mater Biol Appl. 2016;69:780-788.

[4] Bose S, Tarafder S. Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: A review. Acta Biomater. 2012;8(4):1401-1421.

[5] Sukul M, Nguyen TB, Min YK, et al. Effect of Local Sustainable Release of BMP2-VEGF from Nano-Cellulose Loaded in Sponge Biphasic Calcium Phosphate on Bone Regeneration. Tissue Eng Part A. 2015;21(11-12):1822-1836.

[6] Çak?r-Özkan N, E?ri S, Bekar E, et al. The Use of Sequential VEGF- and BMP2-Releasing Biodegradable Scaffolds in Rabbit Mandibular Defects. J Oral Maxillofacial Surg. 2017; 75(1) : 221. e1-221.e14.

[7] 佟玮玮, 王健平, 赵千宁,等. 纳米羟基磷灰石/聚酰胺66盖髓对造牙本质细胞及微血管的影响[J]. 中国组织工程研究, 2016, 20(16):2418-2424.

[8] Ackermann M, Konerding MA. Vascular casting for the study of vascular morphogenesis. Methods Mol Biol. 2015;1214 (1214):49-66.

[9] Wu T, Nan KH, Chen JD, et al. A new bone repair scaffold combined with chitosan/hydroxyapatite and sustained releasing icariin. Chin Sci Bull. 2009;54(17): 2953-2961.

[10] Prisby RD. Bone marrow blood vessel ossification and “microvascular dead space” in rat and human long bone. Bone. 2014;64:195-203.

[11] Fujita Y, Goto S, Ichikawa M, et al. Effect of dietary calcium deficiency and altered diet hardness on the jawbone growth: A micro-CT and bone histomorphometric study in rats. Arch Oral Biol. 2016;72:200.

[12] Wang RK, An L. Doppler optical micro-angiography for volumetric imaging of vascular perfusion in vivo. Opt Express. 2009;17(11):8926-8940.

[13] 简月奎, 吴雪晖, 田晓滨,等. 不同方法显像骨组织微血管的对比研究[J]. 中国矫形外科杂志, 2008, 16(7):534-536.

[14] 王亦璁.骨与关节损伤[M]. 3版.北京:人民卫生出版社,2001: 150.

[15] 胥少汀,葛宝丰,徐印坎.实用骨科学[M]. 3版.北京:人民军医出版社,2005: 330-337.

[16] Berendsen AD, Olsen BR. How Vascular Endothelial Growth Factor-A (VEGF) Regulates Differentiation of Mesenchymal Stem Cells. J Histochem Cytochem. 2014; 62(2):103.

[17] Mark H, Penington A, Nannmark U, et al. Microvascular invasion during endochondral ossification in experimental fractures in rats. Bone. 2004; 35(2):535-542.

[18] Sinder BP, Pettit AR, Mccauley LK. Macrophages: Their Emerging Roles in Bone. J Bone Miner Res. 2015;30(12): 2140-2149.

[19] Raggatt LJ, Wullschleger ME, Alexander KA, et al. Fracture Healing via Periosteal Callus Formation Requires Macrophages for Both Initiation and Progression of Early Endochondral Ossification. Am J Pathol. 2014;184(12): 3192-3204.

[20] 左艳萍, 苑迎娇, 刘昕,等.上颌矢状扩弓的四环素荧光积分光密度分析[J]. 河北医科大学学报, 2014, 35(1):84-86.

[21] 吴拮, 吕荣, 孟国林,等. 钛合金、人工骨材料厚切片再研磨后显示新生骨-荧光双标法[J]. 临床与实验病理学杂志, 2014, 30(10): 1186-1187.

[22] Bensimonbrito A, Cardeira J, Dionísio G, et al. Revisiting in vivo staining with alizarin red S--a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration. BMC Dev Biol. 2016;16(1):1-10.

[23] Wang H, Liu J, Tao S, et al. Tetracycline-grafted PLGA nanoparticles as bone-targeting drug delivery system. Int J Nanomedicine. 2015;10:5671-5685.

[24] Buenzli PR, Pivonka P, Smith DW. Bone refilling in cortical basic multicellular units: insights into tetracycline double labelling from a computational model. Biomech Model Mechanobiol. 2014;13(1):185-203.

[25] Roelofs AJ, Stewart CA, Sun S, et al. Influence of bone affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo. J Bone Miner Res. 2012;27(4):835-847.

[26] 苏畅, 梁林, 李楠,等. 骨靶向化合物研究进展[J]. 武警后勤学院学报医学版, 2007, 16(2):205-208.

[27] 郭建刚，赵然，侯桂英，等.骨组织成分与Masson三色染色反应的关系分析[J].中医正骨,2001,13(11):5-6.

[28] 谭丽思, 林晓萍, 刘尧,等. 组织工程化骨修复犬牙槽骨缺损的组织学观察[J]. 解剖科学进展, 2010, 16(6):492-495.

[29] Jin SK, Lee JH, Hong JH, et al. Enhancement of osseointegration of artificial ligament by nano-hydroxyapatite and bone morphogenic protein-2 into the rabbit femur. Tissue Eng Regen Med. 2016;13(3):284-296.

[30] Sellgren KL, Ma T. Effects of flow configuration on bone tissue engineering using human mesenchymal stem cells in 3D chitosan composite scaffolds. J Biomed Mater Res A. 2014; 103(8):2509-2520.

[31] Hc KD, Jgc W, Phm S, et al. Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits. Biomaterials. 2004;25(11):2123-2132.

[32] Kroese-Deutman HC, Ruhé PQ, Spauwen PH, et al. Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site in rabbits. Biomaterials. 2005;26(10):1131-1138.