Quantitative characterization of mineralized silk film remodeling during long-term osteoblast-osteoclast co-culture

Abstract

The goal of this study was to explore quantitative assessments of mineralized silk protein biomaterial films by co-cultures of human mesenchymal stem cell-derived osteoblasts and human acute monocytic leukemia cell line-derived osteoclasts during long-term culture (8-32 weeks). The remodeled films were quantitatively assessed using three different techniques during this extended cultivation to provide more comprehensive insight into the impact of co-cultures on surface remodeling. Scanning electron microscopy (SEM) with three dimensional surface reconstructions was used to quantitatively determine various surface morphological features and measures of roughness indicative of remodeling by the cells. Additionally, reconstructed surfaces were converted to depth images for Fourier analysis to quantify the potential fractal organization of biomineralization. The long-term remodeled films were also imaged using confocal reflectance microscopy and micro-computed tomography (micro-CT) to further quantify morphological changes. Films remodeled in co-culture demonstrated increased roughness parameters, fractal organization, and volume compared to films remodeled by osteoblasts alone. The combination of these techniques to quantify remodeling of mineralized protein films shows promise for quantifying processes related to mineralized surfaces.

Keywords: Bone remodeling; Bone tissue engineering; Confocal microscopy; Scanning electron microscopy; Silk; Surface topography.

Figure 1

Experimental schematic. Silk films were seeded with osteoblasts, osteoclasts, or a co-culture of the two cell types. Film remodeling was assessed by SEM-based surface metrology, confocal reflectance, and micro-CT after 8, 16, 24, and 32 weeks of culture.

Figure 2

SEM, micro-CT, and confocal reflectance images of films at time zero, prior to cell seeding. Films appear microscopically flat by SEM with a scan resolution of 0.15 μm per pixel. Micro-CT reveals smooth films with some macroscopic curvature at a resolution of approximately 10 μm, and confocal reflectance microscopy reveals smooth films with some artifacts from casting and drying with a scan resolution of 0.38 μm per pixel at 1.5 μm increments along the Z-axis.

Figure 3

SEM images of remodeled film surfaces at 8, 16, 24, and 32 weeks. ECM deposition is particularly pronounced on co-culture films.

Figure 4

Average height, ten point height, and developed interfacial ratios of silk films remodeled by osteoblasts, osteoclasts, or co-cultures at 8, 16, 24, and 32 weeks. Average height is relative to the reference plain, ten point height is a robust measure of peak height, and developed interfacial ratio is the increase in surface area as a result of roughness. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Power law exponent and goodness of fit of SEM-based reconstructions of films remodeled by osteoblasts, osteoclasts, or co-cultures at 8, 16, 24, and 32 weeks. The power law exponent quantifies the level of clustering of mineralization, while the goodness of fit quantifies the degree to which the surface exhibits fractal properties. ** p < 0.01, *** p < 0.001.

Figure 6

Average projection of confocal reflectance from remodeled films at 8, 16, 24, and 32 weeks. Osteoclast films are visually distinct at early time points, but film appearance across groups is more similar at later time points.

Figure 7

Power law exponent and goodness of fit of confocal reflectance microscopy-based reconstructions of films remodeled by osteoblasts, osteoclasts, or co-cultures at 8, 16, 24, and 32 weeks. The power law exponent quantifies the level of clustering of mineralization, while the goodness of fit quantifies the degree to which the surface exhibits fractal properties. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Micro-CT of remodeled films at 8, 16, 24, and 32 weeks. Co-culture films at 24 and 32 weeks exhibit macroscopically visible ECM deposition.

Figure 9

Volume of reconstructed silk films determined by micro-CT with a threshold to exclude surrounding material. * p < 0.05, *** p < 0.001.