Rao Rameshwar R, Vigen Marina L, Peterson Alexis W, Caldwell David J, Putnam Andrew J, Stegemann Jan P
Department of Biomedical Engineering, University of Michigan , Ann Arbor, Michigan.
Tissue Eng Part A. 2015 Feb;21(3-4):530-40. doi: 10.1089/ten.TEA.2013.0740. Epub 2014 Oct 17.
Minimally invasive, injectable bone tissue engineering therapies offer the potential to facilitate orthopedic repair procedures, including in indications where enhanced bone regeneration is needed for complete healing. In this study, we developed a dual-phase tissue construct consisting of osteogenic (Osteo) and vasculogenic (Vasculo) components. A modular tissue engineering approach was used to create collagen/fibrin/hydroxyapatite (COL/FIB/HA) hydrogel microbeads containing embedded human bone marrow-derived mesenchymal stem cells (bmMSC). These microbeads were predifferentiated toward the osteogenic lineage in vitro for 14 days, and they were then embedded within a COL/FIB vasculogenic phase containing a coculture of undifferentiated bmMSC and human umbilical vein endothelial cells (HUVEC). In vitro studies demonstrated homogenous dispersion of microbeads within the outer phase, with endothelial network formation around the microbeads over 14 days in the coculture conditions. Subcutaneous injection into immunodeficient mice was used to investigate the ability of dual-phase (Osteo+Vasculo) and control (Osteo, Vasculo, Blank) constructs to form neovasculature and ectopic bone. Laser Doppler imaging demonstrated blood perfusion through all constructs at 1, 4, and 8 weeks postimplantation. Histological quantification of total vessel density showed no significant differences between the conditions. Microcomputed tomography indicated significantly higher ectopic bone volume (BV) in the Osteo condition at 4 weeks. At 8 weeks both the Osteo and Blank groups exhibited higher BV compared to the Vasculo and dual Osteo+Vasculo groups. These data not only show that osteogenic microbeads can be used to induce ectopic bone formation, but also suggest an inhibitory effect on BV when undifferentiated bmMSC and HUVEC were included in dual-phase constructs. This work may lead to improved methods for engineering vascularized bone tissue, and to injectable therapies for the treatment of orthopedic pathologies in which bone regeneration is delayed or prevented.
微创、可注射的骨组织工程疗法为促进骨科修复手术提供了潜力，包括在需要增强骨再生以实现完全愈合的适应症中。在本研究中，我们开发了一种由成骨（Osteo）和血管生成（Vasculo）成分组成的双相组织构建体。采用模块化组织工程方法创建了含有嵌入的人骨髓来源间充质干细胞（bmMSC）的胶原蛋白/纤维蛋白/羟基磷灰石（COL/FIB/HA）水凝胶微珠。这些微珠在体外向成骨谱系预分化14天，然后嵌入含有未分化bmMSC和人脐静脉内皮细胞（HUVEC）共培养物的COL/FIB血管生成相中。体外研究表明微珠在外相中均匀分散，在共培养条件下14天内在微珠周围形成内皮网络。将构建体皮下注射到免疫缺陷小鼠中，以研究双相（Osteo+Vasculo）和对照（Osteo、Vasculo、空白）构建体形成新血管和异位骨的能力。激光多普勒成像显示植入后1、4和8周时所有构建体均有血液灌注。总血管密度的组织学定量显示各条件之间无显著差异。微型计算机断层扫描表明，在4周时，Osteo条件下的异位骨体积（BV）显著更高。在8周时，与Vasculo组和双Osteo+Vasculo组相比，Osteo组和空白组的BV均更高。这些数据不仅表明成骨微珠可用于诱导异位骨形成，还表明当双相构建体中包含未分化的bmMSC和HUVEC时，对BV有抑制作用。这项工作可能会带来改进的工程化血管化骨组织的方法，以及用于治疗骨再生延迟或受阻的骨科疾病的可注射疗法。
