Bozzini Sabrina, Giuliano Liliana, Altomare Lina, Petrini Paola, Bandiera Antonella, Conconi Maria Teresa, Farè Silvia, Tanzi Maria Cristina
Bioengineering Department, Biomaterials Laboratory, Politecnico di Milano, Milan, Italy.
J Mater Sci Mater Med. 2011 Dec;22(12):2641-50. doi: 10.1007/s10856-011-4451-z. Epub 2011 Oct 13.
The use of polymers naturally occurring in the extracellular matrix (ECM) is a promising strategy in regenerative medicine. If compared to natural ECM proteins, proteins obtained by recombinant DNA technology have intrinsic advantages including reproducible macromolecular composition, sequence and molecular mass, and overcoming the potential pathogens transmission related to polymers of animal origin. Among ECM-mimicking materials, the family of recombinant elastin-like polymers is proposed for drug delivery applications and for the repair of damaged elastic tissues. This work aims to evaluate the potentiality of a recombinant human elastin-like polypeptide (HELP) as a base material of cross-linked matrices for regenerative medicine. The cross-linking of HELP was accomplished by the insertion of cross-linking sites, glutamine and lysine, in the recombinant polymer and generating ε-(γ-glutamyl) lysine links through the enzyme transglutaminase. The cross-linking efficacy was estimated by infrared spectroscopy. Freeze-dried cross-linked matrices showed swelling ratios in deionized water (≈2500%) with good structural stability up to 24 h. Mechanical compression tests, performed at 37°C in wet conditions, in a frequency sweep mode, indicated a storage modulus of 2/3 kPa, with no significant changes when increasing number of cycles or frequency. These results demonstrate the possibility to obtain mechanically resistant hydrogels via enzymatic crosslinking of HELP. Cytotoxicity tests of cross-linked HELP were performed with human umbilical vein endothelial cells, by use of transwell filter chambers for 1-7 days, or with its extracts in the opportune culture medium for 24 h. In both cases no cytotoxic effects were observed in comparison with the control cultures. On the whole, the results suggest the potentiality of this genetically engineered HELP for regenerative medicine applications, particularly for vascular tissue regeneration.
使用细胞外基质（ECM）中天然存在的聚合物是再生医学中一种很有前景的策略。与天然ECM蛋白相比，通过重组DNA技术获得的蛋白具有内在优势，包括可重现的大分子组成、序列和分子量，以及克服与动物源性聚合物相关的潜在病原体传播问题。在模拟ECM的材料中，重组弹性蛋白样聚合物家族被提议用于药物递送应用和受损弹性组织的修复。这项工作旨在评估重组人弹性蛋白样多肽（HELP）作为再生医学交联基质基础材料的潜力。通过在重组聚合物中插入交联位点谷氨酰胺和赖氨酸，并通过转谷氨酰胺酶生成ε-（γ-谷氨酰基）赖氨酸连接来实现HELP的交联。通过红外光谱估计交联效率。冻干的交联基质在去离子水中显示出溶胀率（约2500%），在长达24小时内具有良好的结构稳定性。在37°C潮湿条件下以频率扫描模式进行的机械压缩试验表明，储能模量为2/3 kPa，当循环次数或频率增加时无显著变化。这些结果证明了通过HELP的酶促交联获得机械抗性水凝胶的可能性。使用跨孔滤室对人脐静脉内皮细胞进行1 - 7天的交联HELP细胞毒性试验，或在合适的培养基中用其提取物进行24小时的细胞毒性试验。在这两种情况下，与对照培养物相比均未观察到细胞毒性作用。总体而言，结果表明这种基因工程化的HELP在再生医学应用中具有潜力，特别是对于血管组织再生。
