Preparation of micro/nanopatterned gelatins crosslinked with genipin for biocompatible dental implants

Abstract

Background: Collagen is a basic component of the periodontium and plays an important role in the function of the periodontal unit. Therefore, coating with collagen/gelatin has been applied to enable dental implants to positively interact with peri-implant tissues. Although the micro/nanoscale topography is an important property of the surface of dental implants, smaller collagen/gelatin surface patterns have not been sufficiently developed. Furthermore, only few reports on the behavior of cells on gelatin surfaces with different patterns and sizes exist. In this study, we developed micro/nanometer-scaled gelatin surfaces using genipin crosslinking, with the aim of understanding the use of patterning in surface modification of dental implants. Results: Grooves, holes, and pillars, with widths or diameters of 2 µm, 1 µm, or 500 nm were fabricated using a combination of molding and genipin crosslinking of gelatin. The stability of the different gelatin patterns could be controlled by the degree of genipin crosslinking. The gelatin patterns at 20 mM concentration of genipin and 41% crosslinking maintained a stable, patterned shape for at least 14 days in a cell culture medium. A cell morphology study showed that the cells on groves were aligned along the direction of the grooves. In contrast, the cells on pillars and holes exhibited randomly elongated filopodia. The vinculin spots of the cells were observed on the top of ridges and pillars or the upper surface of holes. The results of a cell attachment assay showed that the number of surface-attached cells increased with increasing patterning of the gelatin surface. Unlike the cell attachment assay, the results of a cell proliferation assay showed that Saos-2 cells prefer grooves with diameters of approximately 2 µm and 1 µm and pillars with diameters of 1 µm and heights of 500 nm. The number of cells on pillars with heights of 2 µm was larger than those of the other gelatin surface patterns tested. Conclusion: These data support that a detailed design of the gelatin surface pattern can control both cell attachment and proliferation of Saos-2 cells. Thus, gelatin surfaces patterned using genipin crosslinking are now an available option for biocompatible material patterning.

Engineering and Functionalization of Gelatin Biomaterials: From Cell Culture to Medical Applications

“…112 Micromolding and nanopatterning are techniques used to modify substrate surfaces to accommodate various geometric patterns and configurations that control the behavior of the cells by imposing spatial restrictions to cell attachment, spreading, etc. [113][114][115] Due to the advent of technology, surface modifications such as micromolding and nanopatterning can be achieved with various techniques such as Owing to its biocompatibility and biodegradability, gelatin has been widely used in both 2D (surface coatings, microsheets) and 3D (hydrogels, microparticles) biomaterial platforms for the enhanced proliferation and assisted differentiation of several types of cells and tissues in vitro.…”

“…Extensive reviews on the different micro and nano surface patterning techniques are available elsewhere. 113,116 Gelatin surface modifications have also been conducted for cell and tissue engineering purposes. 115,117 In one study, mmolded gelatin crosslinked with bacterial glutaminase performed better compared with 2D-coated ECM matrices in maintaining cultures of skeletal myotubes in vitro for 3 weeks.…”

“…85 In another study, nanoembossed gelatin substrates resulted in a significant increase in the proliferation and elongation of NIH3T3 fibroblasts. 118 Other studies include applications of micromolded gelatin in cardiomyocytes, 119 osteosarcoma cells, 113 and other cells. Micro/nanopatterning has immense potential in medicine and tissue regeneration.…”

The Use of Genipin as an Effective, Biocompatible, Anti‐Inflammatory Cross‐Linking Method for Nerve Guidance Conduits

“…[55] Previously, genipin concentrations ranging from 0.02 to 100 mm have been used for cross-linking. [38,[40][41][42][43]45,46,[48][49][50][52][53][54]56,57,[65][66][67][68][69] However, some studies have shown that genipin concentrations of 5 mm and above have cytotoxic effects. [41,51,67] In one study, 1 mm genipin cross-linked samples promoted proliferation and viability of an neural stem cell line (SPC-01) and outgrowth of dissociated adult dorsal root ganglion neurons; these samples also displayed in vivo biocompatibility when injected into focal ischemic lesion in the rat motor cortex.…”

“…Natural cross‐linkers such as genipin have demonstrated equally efficient cross‐linking, while also offering a safer alternative having been FDA approved for use as a food dye (under the name “fruit juice”) 36,37. Genipin has been successfully used for cross‐linking of other collagen‐based materials such as biological tissues,38–44 collagen films,45 gelatin,38,46–50 ECM hydrogels,51 collagen‐chitosan blends,52–55 and other natural polymers;56,57 it also possesses significant anti‐inflammatory properties 58,59. Indeed, a recent study from our group showed that genipin cross‐linking dampens both pro‐ and anti‐inflammatory responses from macrophages,60 one of the first innate immune cells to encounter an implanted biomaterial 61…”