Byers Eye Institute at Stanford University School of Medicine , Palo Alto, California 94303, United States.
Department of Materials Science and Engineering, Stanford University , Stanford, California 94305, United States.
Biomacromolecules. 2017 Oct 9;18(10):3185-3196. doi: 10.1021/acs.biomac.7b00838. Epub 2017 Sep 1.
In the treatment of traumatic injuries, burns, and ulcers of the eye, inadequate epithelial tissue healing remains a major challenge. Wound healing is a complex process involving the temporal and spatial interplay between cells and their extracellular milieu. It can be impaired by a variety of causes including infection, poor circulation, loss of critical cells, and/or proteins, and a deficiency in normal neural signaling (e.g., neurotrophic ulcers). Ocular anatomy is particularly vulnerable to lasting morbidity from delayed healing, whether it be scarring or perforation of the cornea, destruction of the conjunctival mucous membrane, or cicatricial changes to the eyelids and surrounding skin. Therefore, there is a major clinical need for new modalities for controlling and accelerating wound healing, particularly in the eye. Collagen matrices have long been explored as scaffolds to support cell growth as both two-dimensional coatings and substrates, as well as three-dimensional matrices. Meanwhile, the immobilization of growth factors to various substrates has also been extensively studied as a way to promote enhanced cellular adhesion and proliferation. Herein we present a new strategy for photochemically immobilizing growth factors to collagen using riboflavin as a photosensitizer and exposure to visible light (∼458 nm). Epidermal growth factor (EGF) was successfully bound to collagen-coated surfaces as well as directly to endogenous collagen from porcine corneas. The initial concentration of riboflavin and EGF as well as the blue light exposure time were keys to the successful binding of growth factors to these surfaces. The photocrosslinking reaction increased EGF residence time on collagen surfaces over 7 days. EGF activity was maintained after the photocrosslinking reaction with a short duration of pulsed blue light exposure. Bound EGF accelerated in vitro corneal epithelial cell proliferation and migration and maintained normal cell phenotype. Additionally, the treated surfaces were cytocompatible, and the photocrosslinking reaction was proven to be safe, preserving nearly 100% cell viability. These results suggest that this general approach is safe and versatile may be used for targeting and immobilizing bioactive factors onto collagen matrices in a variety of applications, including in the presence of live, seeded cells or in vivo onto endogenous extracellular matrix collagen.
在治疗创伤性损伤、烧伤和眼部溃疡时，上皮组织愈合不足仍然是一个主要挑战。伤口愈合是一个复杂的过程，涉及细胞及其细胞外基质之间的时空相互作用。它可能会因多种原因受损，包括感染、血液循环不良、关键细胞和/或蛋白质的丧失以及正常神经信号传递的缺陷（例如神经营养性溃疡）。眼部解剖结构特别容易受到愈合延迟的持久影响，无论是角膜的瘢痕形成或穿孔、结膜黏膜的破坏还是眼睑和周围皮肤的瘢痕形成。因此，临床上迫切需要控制和加速伤口愈合的新方法，尤其是在眼部。胶原蛋白基质长期以来一直被探索作为支持细胞生长的支架，既可以作为二维涂层和基质，也可以作为三维基质。同时，将生长因子固定在各种基质上也被广泛研究作为促进增强细胞黏附和增殖的方法。在此，我们提出了一种新的策略，即使用核黄素作为光增感剂并暴露于可见光（约 458nm），通过光化学将生长因子固定在胶原蛋白上。表皮生长因子（EGF）成功地与胶原蛋白涂层表面结合，也直接与猪角膜中的内源性胶原蛋白结合。核黄素和 EGF 的初始浓度以及蓝光照射时间是成功将生长因子结合到这些表面的关键。光交联反应使 EGF 在胶原蛋白表面的停留时间超过 7 天。在进行短时间的脉冲蓝光照射后，光交联反应仍能保持 EGF 的活性。结合的 EGF 可加速体外角膜上皮细胞的增殖和迁移，并维持正常的细胞表型。此外，处理过的表面具有细胞相容性，光交联反应被证明是安全的，可保持近 100%的细胞活力。这些结果表明，这种通用方法是安全且多功能的，可用于将生物活性因子靶向固定在各种应用中的胶原蛋白基质上，包括在存在活的、接种的细胞或体内内源性细胞外基质胶原蛋白的情况下。
