Ibrahim Samir, Joddar Binata, Craps Matthew, Ramamurthi Anand
Department of Bioengineering, Clemson University, 501 Rhodes Research Center, Clemson, SC 29634, USA.
Biomaterials. 2007 Feb;28(5):825-35. doi: 10.1016/j.biomaterials.2006.09.030. Epub 2006 Oct 11.
Crosslinked gels (hylans) containing long-chain (MW>1 x 10(6)Da) hyaluronan (HA), a connective tissue GAG, show exceptional biocompatibility for vascular implantation but poorly interact with vascular endothelial cells (ECs). Previous studies showed in situ fragmentation of HA by UV light to bioactivate hylan gels and elicit enhanced EC responses. Since fragmented HA can be pro-inflammatory, it is important to define an optimal size distribution of HA fragments on the hylan surface that will recruit and support normally functional ECs and limit ulterior responses. Related studies have shown that exogenous models of HA do not necessarily replicate cell responses to HA scaffolds. Since scaffolds cannot be created based on fragmented HA alone, we sought to determine size-specific responses of ECs to HA substrates of defined fragment sizes by creation of HA-tethered culture surfaces. HA (1000, 200, 20 kDa) and an oligomer mixture were tethered onto an aminosilane (APTMS)-treated glass surfaces using a carbodiimide reaction. MALDI-TOF showed the HA digests to contain HA 4-8mers with a 75+/-0.4% w/w of 4mers. Immuno-fluorescence, SEM, AFM and XPS analysis revealed homogeneous amine and HA surfaces. An amine s-SDTB assay and HA fluorophore-assisted carbohydrate electrophoresis (FACE) indicated surface densities of 9+/-3 amine groups/nm(2) and 0.57+/-0.44 microg/cm(2), respectively. HA/HA fragments/oligomers were stable over 21 days of incubation in serum-free culture media. EC proliferation on these surfaces resulted was limited, a possible effect of smooth surface topography, high anionicity, and in case of 4mers, non-interaction with primary HA cell-surface receptors (CD44). This work is significant in that it allows testing of cell responses to substrates composed of single-sized fragments of HA that cannot by themselves be cross-linked into a gel. Future work in our lab will use this model to assess the effects of other HA oligomer sizes on EC behavior.
含有长链（分子量>1×10⁶道尔顿）透明质酸（HA）的交联凝胶（透明质烷），一种结缔组织糖胺聚糖，对血管植入显示出卓越的生物相容性，但与血管内皮细胞（ECs）的相互作用较差。先前的研究表明，通过紫外线对HA进行原位裂解可使透明质烷凝胶生物活化并引发增强的EC反应。由于裂解的HA可能具有促炎作用，因此确定透明质烷表面HA片段的最佳尺寸分布非常重要，该分布将募集并支持正常功能的ECs并限制后续反应。相关研究表明，HA的外源性模型不一定能复制细胞对HA支架的反应。由于不能仅基于裂解的HA来创建支架，我们试图通过创建HA连接的培养表面来确定ECs对具有确定片段大小的HA底物的尺寸特异性反应。使用碳二亚胺反应将HA（1000、200、20 kDa）和低聚物混合物连接到氨基硅烷（APTMS）处理的玻璃表面上。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）显示HA消化产物含有HA 4-8聚体，其中4聚体占75±0.4%（w/w）。免疫荧光、扫描电子显微镜（SEM）、原子力显微镜（AFM）和X射线光电子能谱（XPS）分析显示胺和HA表面均匀。胺s-SDTB测定和HA荧光团辅助碳水化合物电泳（FACE）分别表明表面密度为9±3个胺基团/纳米²和0.57±0.44微克/厘米²。HA/HA片段/低聚物在无血清培养基中孵育21天期间保持稳定。这些表面上的EC增殖受到限制，这可能是光滑表面形貌、高阴离子性的影响，对于4聚体而言，还可能是与初级HA细胞表面受体（CD44）不相互作用的结果。这项工作的意义在于，它允许测试细胞对由不能自身交联成凝胶的单一尺寸HA片段组成的底物的反应。我们实验室未来的工作将使用该模型评估其他HA低聚物尺寸对EC行为的影响。
