Michael Jan, Schönzart Lena, Israel Ina, Beutner René, Scharnweber Dieter, Worch Hartmut, Hempel Ute, Schwenzer Bernd
Lehrstuhl fur Allgemeine Biochemie, Technische Universitat Dresden, Germany.
Bioconjug Chem. 2009 Apr;20(4):710-8. doi: 10.1021/bc800372e.
A new concept for modular biosurface engineering of titanium implants based on the self-assembly of complementary oligonucleotides was biochemically investigated and optimized. This study describes the synthesis and characterization (RP-HPLC and Sakaguchi assay) of oligodeoxyribonucleotide (ODN) conjugates of the hexapeptide GRGDSP containing the RGD sequence as the recognition motif for cellular adhesion receptors (integrins). The peptide was chosen exemplarily as a model molecule, because it is a simple but potent bioactive molecule and relatively well investigated. The conjugation products must fulfill two main requirements: (I) the ability to hybridize and (II) the preservation of biological activity of the RGD peptide for the enhancement of osteoblast adhesion. In the following text, the term "hybridization" is generally used for Watson-Crick base pairing. The ability of the conjugates to hybridize to surface-immobilized complementary ODN was verified by competitive hybridization with radiolabeled ((32)P) complementary strands and by hybridization experiments using a quartz crystal microbalance (QCM). Surface hybridization was further characterized using different adsorption isotherms (e.g., Freundlich and Frumkin types), since the type of isotherm and the derived thermodynamic parameters may reveal characteristic differences between ODN and conjugates thereof. Biological activity of the conjugates was examined in vitro with osteoblasts. The cells were either cultured directly on the ODN-GRGDSP modified titanium implants or used for competition adhesion studies with dissolved ODN-GRGDSP conjugates. All results support the successful establishment of the new surface modification system. Hybridization of RGD peptide-modified nucleic acids to ODN-modified titanium implant materials is thus a promising method for osteoblast attachment in a modular and self-organizing system on implant surfaces.
基于互补寡核苷酸自组装的钛植入物模块化生物表面工程新概念进行了生物化学研究和优化。本研究描述了含RGD序列的六肽GRGDSP的寡脱氧核糖核苷酸（ODN）缀合物的合成与表征（反相高效液相色谱法和坂口测定法），该序列作为细胞粘附受体（整合素）的识别基序。该肽被示例性地选为模型分子，因为它是一个简单但有效的生物活性分子且研究相对充分。缀合产物必须满足两个主要要求：（I）杂交能力和（II）保留RGD肽的生物活性以增强成骨细胞粘附。在下文，术语“杂交”一般用于沃森-克里克碱基配对。通过与放射性标记的（³²P）互补链进行竞争性杂交以及使用石英晶体微天平（QCM）进行杂交实验，验证了缀合物与表面固定的互补ODN杂交的能力。使用不同的吸附等温线（如弗罗因德利希等温线和弗鲁姆金等温线类型）进一步表征表面杂交，因为等温线类型及其推导的热力学参数可能揭示ODN及其缀合物之间的特征差异。在体外对成骨细胞检测缀合物的生物活性。细胞要么直接培养在ODN-GRGDSP修饰的钛植入物上，要么用于与溶解的ODN-GRGDSP缀合物进行竞争粘附研究。所有结果都支持新表面修饰系统的成功建立。因此，RGD肽修饰的核酸与ODN修饰的钛植入材料的杂交是在植入物表面的模块化和自组织系统中促进成骨细胞附着的一种有前景的方法。
