Influence of Topological Cues on Fibronectin Adsorption and Contact Guidance of Fibroblasts on Microgrooved Titanium.

The choice of suitable nano- and microstructures of biomaterials is crucial for successful implant integration within the human body. In particular, surface characteristics affect the adsorption of various extra cellular matrix proteins. This work illustrates the interaction of protein adsorption and early cell adhesion on bulk microstructured titanium surfaces with parallel grooves of 27 to 35 µm widths and 15 to 19 µm depths, respectively. In contact with low concentrated fibronectin solutions, distinct adsorption patterns are observed on the edges of the ridges. Moreover, NIH/3T3 fibroblasts cultured in serum-free medium for 1 h, 3 h, and 1 day show enhanced early cell adhesion on fibronectin coated samples compared to uncoated ones. In fact, early adhesion and cell contacts occur mainly on the groove edges where fibronectin adsorption was preferentially detected. Such adsorption patterns support cellular contact guidance on short time scales since the adsorbed fibronectin proteins acted as a chemical boundary superimposing the topographical cues of the grooved microstructure. In fibronectin-free conditions, this chemical boundary is absent after cell seeding and initial cell-surface interaction. Here, cellular fibronectin released by the fibroblasts adsorbs along the grooves after 3 h and contact guidance occurs delayed. After 1 day, cell adhesion and cell morphology on uncoated and fibronectin coated titanium microgrooves were nearly equilibrated. Thus, surface structures can promote directed adsorption of low concentrated fibronectin, which, furthermore, facilitates early cell adhesion. These results give rise to new developments in surface engineering of biomedical implants for improved osseointegration.

Micropatterning of reagent-free, high energy crosslinked gelatin hydrogels for bioapplications.

Hydrogels are crosslinked polymeric gels of great interest in the field of tissue engineering, particularly as biocompatible cell or drug carriers. Reagent-free electron irradiated gelatin is simple to manufacture, inexpensive and biocompatible. Here, the potential to micropattern gelatin hydrogel surfaces during electron irradiation crosslinking was demonstrated as a promising microfabrication technique to produce thermally stable structures on highly relevant length scales for bioapplications. In the present work, grooves of 3.75 to 170 µm width and several hundred nanometers depth were transferred onto gelatin hydrogels during electron irradiation and characterized by 3D confocal microscopy after exposure to ambient and physiological conditions. The survival and influence of these microstructures on cellular growth was further characterized using NIH 3T3 fibroblasts. Topographical modifications produced surface structures on which the cultured fibroblasts attached and responded by adapting their morphologies. This developed technique allows for simple and effective structuring of gelatin and opens up new possibilities for irradiation crosslinked hydrogels in biomedical applications in which cell attachment and contact guidance are favored. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 320-330, 2018.

Cellular Response to Reagent-Free Electron-Irradiated Gelatin Hydrogels.

As a biomaterial, it is well established that gelatin exhibits low cytotoxicity and can promote cellular growth. However, to circumvent the potential toxicity of chemical crosslinkers, reagent-free crosslinking methods such as electron irradiation are highly desirable. While high energy irradiation has been shown to exhibit precise control over the degree of crosslinking, these hydrogels have not been thoroughly investigated for biocompatibility and degradability. Here, NIH 3T3 murine fibroblasts are seeded onto irradiated gelatin hydrogels to examine the hydrogel's influence on cellular viability and morphology. The average projected area of cells seeded onto the hydrogels increases with irradiation dose, which correlates with an increase in the hydrogel's shear modulus up to 10 kPa. Cells on these hydrogels are highly viable and exhibits normal cell cycles, particularly when compared to those grown on glutaraldehyde crosslinked gelatin hydrogels. However, proliferation is reduced on both types of crosslinked samples. To mimic the response of the hydrogels in physiological conditions, degradability is monitored in simulated body fluid to reveal strongly dose-dependent degradation times. Overall, given the low cytotoxicity, influence on cellular morphology and variability in degradation times of the electron irradiated gelatin hydrogels, there is significant potential for application in areas ranging from regenerative medicine to mechanobiology.

Effect of microgrooved surface topography on osteoblast maturation and protein adsorption.

Microgrooved surfaces have been used extensively to influence cell contact guidance. Guiding cell growth, extracellular matrix deposition, and mineralization is important for bone implant longevity. In this study, we investigated the osteoblast response to microgrooved metallic surfaces in serum-supplemented medium. Groove spacing was comparable with the spread osteoblast size. Focal adhesions were observed to confine to the intervening ridge/groove boundaries. Osteoblasts bridged over the grooves and were unable to conform to the concave shape of the underlying grooves. Microgrooved surfaces induced higher osteoblast proliferation and metabolic activity after 14 days in osteogenic medium compared with as-received surfaces, resulting in higher mineralization and alignment of cell-secreted collagen after 28 days. To establish whether preferential cell attachment at the ridge/groove boundaries was influenced by the adhesion proteins contained in the serum-supplemented media, fluorescently labeled fibronectin was adsorbed onto the microgrooved substrates at low concentrations, mimicking the concentrations found in blood serum. Fibronectin was found to selectively adsorb onto the ridge/groove boundaries, the osteoblast focal adhesion sites, suggesting that protein adsorption may have influenced the cell attachment pattern.