Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health , AlbaNova University Center, KTH Royal Institute of Technology , SE-106 91 Stockholm , Sweden.
Department of Molecular Biosciences, The Wenner-Gren Institute , Stockholm University , SE-106 91 Stockholm , Sweden.
ACS Appl Mater Interfaces. 2019 Jul 17;11(28):24999-25007. doi: 10.1021/acsami.9b05531. Epub 2019 Jul 5.
Orthopedic and dental implants are associated with a substantial risk of failure due to biomaterial-associated infections and poor osseointegration. To prevent such outcomes, a coating can be applied on the implant to ideally both reduce the risk of bacterial adhesion and support establishment of osteoblasts. We present a strategy to construct dual-functional silk coatings with such properties. Silk coatings were made from a recombinant partial spider silk protein either alone (silk) or fused with a cell-binding motif derived from fibronectin (FN-silk). The biofilm-dispersal enzyme Dispersin B (DspB) and two peptidoglycan degrading endolysins, PlySs2 and SAL-1, were produced recombinantly. A sortase recognition tag (SrtTag) was included to allow site-specific conjugation of each enzyme onto silk and FN-silk coatings using an engineered variant of the transpeptidase Sortase A (SrtA*). To evaluate bacterial adhesion on the samples, was incubated on the coatings and subsequently subjected to live/dead staining. Fluorescence microscopy revealed a reduced number of bacteria on all silk coatings containing enzymes. Moreover, the bacteria were mobile to a higher degree, indicating a negative influence on the bacterial adhesion. The capability to support mammalian cell interactions was assessed by cultivation of the osteosarcoma cell line U-2 OS on dual-functional surfaces, prepared by conjugating the enzymes onto FN-silk coatings. U-2 OS cells could adhere to silk coatings with enzymes and showed high spreading and viability, demonstrating good cell compatibility.
骨科和牙科植入物由于与生物材料相关的感染和骨整合不良而存在很高的失败风险。为了防止这种结果，可以在植入物上施加涂层，理想情况下既可以降低细菌黏附的风险，又可以支持成骨细胞的建立。我们提出了一种构建具有这种特性的双重功能丝质涂层的策略。丝质涂层由重组的部分蜘蛛丝蛋白制成，单独使用（丝质）或与源自纤连蛋白的细胞结合基序融合（FN-丝质）。生物膜分散酶 Dispersin B（DspB）和两种肽聚糖降解内溶素 PlySs2 和 SAL-1 通过重组生产。引入了一种 sortase 识别标签（SrtTag），以允许使用工程变体的转肽酶 Sortase A（SrtA*）将每种酶特异性缀合到丝质和 FN-丝质涂层上。为了评估样品上的细菌黏附，将 孵育在涂层上，然后进行死活染色。荧光显微镜显示，所有含有酶的丝质涂层上的细菌数量都减少了。此外，细菌的迁移程度更高，表明对细菌黏附有负面影响。通过将酶缀合到 FN-丝质涂层上来制备双功能表面，评估了支持哺乳动物细胞相互作用的能力，该表面用于培养骨肉瘤细胞系 U-2 OS。带有酶的丝质涂层可以使 U-2 OS 细胞黏附，并且表现出高的铺展和活力，表明良好的细胞相容性。
