Elbjeirami Wafa M, Yonter Edward O, Starcher Barry C, West Jennifer L
Department of Biochemistry & Cell Biology, Rice University, 6100 Main Street, Houston, Texas 77005, USA.
J Biomed Mater Res A. 2003 Sep 1;66(3):513-21. doi: 10.1002/jbm.a.10021.
A number of strategies have been investigated to enhance the mechanical stability of engineered tissues. In this study, we utilized lysyl oxidase (LO) to enzymatically crosslink extracellular matrix (ECM) proteins, particularly collagen and elastin, to enhance the mechanical integrity of the ECM and thereby impart mechanical strength to the engineered tissue. Vascular smooth muscle cells (VSMCs) were liposomally transfected with the LO gene. Both Northern and Western analyses confirmed increased LO expression. Increased LO activity was demonstrated using a fluorescent enzyme substrate assay and by observation of the presence of increased levels of desmosine, a product of LO crosslinking, in the ECM. The mechanical effects of altered crosslink densities within tissue-engineered constructs were demonstrated in a VSMC-populated collagen gel model. When smooth muscle cells transfected with lysyl oxidase were seeded in collagen gels, the tensile strength and elastic modulus in these constructs increased by approximately two-fold compared to constructs seeded with mock-transfected VSMCs. Also, desmosine levels in the LO-populated collagen gels were higher than they were in mock-seeded gels, as demonstrated via immunohistochemical staining. Compositional analysis of the ECM deposited by the transformed cells showed similar collagen and elastin levels, and cell proliferation rates were similar as well, thus attributing increased mechanical properties to ECM crosslinking.
为提高工程组织的机械稳定性，人们已经研究了多种策略。在本研究中，我们利用赖氨酰氧化酶（LO）对细胞外基质（ECM）蛋白，特别是胶原蛋白和弹性蛋白进行酶促交联，以增强ECM的机械完整性，从而赋予工程组织机械强度。用脂质体将LO基因转染到血管平滑肌细胞（VSMC）中。Northern分析和Western分析均证实LO表达增加。使用荧光酶底物测定法并通过观察ECM中LO交联产物异锁链素水平的升高，证明了LO活性的增加。在VSMC填充的胶原凝胶模型中证明了组织工程构建物中交联密度改变的机械效应。当将用赖氨酰氧化酶转染的平滑肌细胞接种到胶原凝胶中时，与接种了mock转染的VSMC的构建物相比，这些构建物的拉伸强度和弹性模量增加了约两倍。此外，通过免疫组织化学染色证明，LO填充的胶原凝胶中的异锁链素水平高于mock接种的凝胶。对转化细胞沉积的ECM进行成分分析，结果显示胶原蛋白和弹性蛋白水平相似，细胞增殖率也相似，因此将机械性能的提高归因于ECM交联。
