One of the current challenges in treating age-related macular degeneration (AMD) is the surface modification of the retinal Bruch membrane. In this study, the collagen fibers of the inner collagenous zone of the Bruch membrane were identified as type I and type III. Subsequently, the adsorption of a collagen-binding peptide onto the inner collagenous zone surface was investigated. The collagen-binding peptide was able to bind specifically to the collagen fibers while maintaining the biological activity of the N-terminus biotin tag. These results indicate that the collagen-binding peptide may be used as an anchor to immobilize bioactive molecules on the inner collagenous zone surface of the Bruch membrane.
The small leucine-rich proteoglycans (SLRPs), prevalent in collagenous tissues, regulate collagen fibrillogenesis and provide a host of biochemical cues critical to tissue function and homeostasis. Incorporating SLRPs may enhance tissue engineering designs that mimic the native extracellular matrix, although SLRPs purified from animal sources bear low yields and lack design control. Consequently, we have designed synthetic peptidoglycans, inspired by the native SLRP decorin, that contain a collagen-binding peptide attached to a glycosaminoglycan (GAG) chain. These peptidoglycans modulate collagen fibrillogenesis and decrease fibril diameter in vitro, similarly to decorin, while maintaining the characteristic D-banded fibrils. Application for tissue engineering is demonstrated as these peptidoglycans are incorporated into collagen gels seeded with smooth muscle cells. Gels formed with peptidoglycans and decorin show a faster rate of gel compaction, and one peptidoglycan uniquely increases elastin production. The peptidoglycan design can be tailored with respect to the peptide sequence and GAG identity and is expected to have versatile application in tissue engineering.
Dip‐pen nanolithography is used to directly modify freshly dissected eye tissues with biologically active collagen‐binding peptide molecules. The results address the challenge of surface heterogeneity and utilize dip‐pen nanolithography to control the localization and concentration of molecules on a collagen‐terminated tissue‐derived surface. This method can allow the development of scaffolds for treatment of age‐related macular degeneration.
Collagen is a ubiquitous component of the extracellular matrix environment, and numerous studies have been devoted toward the development of collagen-based tissue scaffolds. These efforts have been primarily focused on synthetic collagenous materials made from purified collagen. In this article, we present a preliminary study toward the development of a technique that can result in a tissue-derived collagen scaffold. The tissue-derived collagenous matrix was isolated from the retinal Bruch's membrane, and dip pen nanolithography was investigated as a mean to modify the collagenous surface. Characterization experiments of the collagenous surface indicate a fairly hydrophobic surface. Minimal swelling (<7%) of the collagen fibers was observed under elevated humidity conditions with negligible rearrangement of the surface. The hydrophobicity and roughness of the surface can pose a barrier for the deposition of molecules via scanning probe lithography. However, deposition of poly(glutamic acid) and polyarginine onto the surface could be achieved under high contact force and elevated relative humidity conditions (above 55%). Under these conditions, the deposition of the polypeptides occurred through molecular deposition with no observable molecular diffusion. On the other hand, the addition of 0.1% (v/v) Tween-20 surfactant to the inking solution facilitated diffusion of polypeptide inks by increasing the wettability of the collagenous surface. As such, deposition of molecule can be observed under lower contact force at ambient relative humidity (37−40%).
Current efforts to reverse loss of visual function due to Age-related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell-adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE-19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells.
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