In Vitro Production of Calcified Bone Matrix onto Wool Keratin Scaffolds via Osteogenic Factors and Electromagnetic Stimulus

Abstract

Pulsed electromagnetic field (PEMF) has drawn attention as a potential tool to improve the ability of bone biomaterials to integrate into the surrounding tissue. We investigated the effects of PEMF (frequency, 75 Hz; magnetic induction amplitude, 2 mT; pulse duration, 1.3 ms) on human osteoblast-like cells (SAOS-2) seeded onto wool keratin scaffolds in terms of proliferation, differentiation, and production of the calcified bone extracellular matrix. The wool keratin scaffold offered a 3D porous architecture for cell guesting and nutrient diffusion, suggesting its possible use as a filler to repair bone defects. Here, the combined approach of applying a daily PEMF exposure with additional osteogenic factors stimulated the cells to increase both the deposition of bone-related proteins and calcified matrix onto the wool keratin scaffolds. Also, the presence of SAOS-2 cells, or PEMF, or osteogenic factors did not influence the compression behavior or the resilience of keratin scaffolds in wet conditions. Besides, ageing tests revealed that wool keratin scaffolds were very stable and showed a lower degradation rate compared to commercial collagen sponges. It is for these reasons that this tissue engineering strategy, which improves the osteointegration properties of the wool keratin scaffold, may have a promising application for long term support of bone formation in vivo.

Keywords: bone tissue engineering; osteogenic factors; pulsed electromagnetic field; wool keratin scaffolds.

Figure 1

(a) Cell growth evaluation has been assessed by means of DNA quantification at day 21 of culture. Bars represent the mean values ± SD (standard deviation) of results (N = 3; n = 3, symbols indicate statistical significance vs. control (*) and vs. PEMF (§)). (b) Cell morphology assessed by SEM on all samples (scale bars = 10 μm; magnification: ctrl = 1020×; PEMF = 1040×; OF = 1050×; PEMF + OF = 1070×). Red stars indicate cell distribution on the wool keratin scaffolds in all conditions. In OF and PEMF + OF the dense layer of extracellular bone matrix (ECM) made difficult to discriminate a cell from another. Representative live cells visualized by fluorescein diacetate (FDA) staining on the scaffold’s surfaces are shown in Figure S3.

Figure 2

Alkaline phosphatase (ALP) activity after 21 days. ALP activity was colorimetrically determined, corrected for the protein content (measured with the BCA Protein Assay kit), and expressed as mM of p-nitrophenol produced per min per μg of protein. Bars express the mean ± SD (N = 3; n = 2, symbols indicate statistical significance vs. control (*), vs. PEMF (§) and vs. OF (°)).

Figure 3

Quantification of inorganic matrix produced for 21 days. (a) Quantification of phosphate content by Phosphate Colorimetric Assay kit. Phosphate is measured in pmol/cell×scaffold; results are presented as mean ± SD (N = 3; n = 2; symbols indicate statistical significance vs. control (*), vs. PEMF (§) and vs. OF (°)). (b) Quantification of calcium content by the Ca²⁺/o-cresolphthalein complexone method. Results are expressed as pg/cell×scaffold and presented as mean ± SD (N = 3; n = 2, symbols indicate statistical significance vs. control (*), vs. PEMF (§) and vs. OF (°)). (c) Scanning Electron Microscopy with Energy Dispersive X-ray Spectroscopy (SEM-EDX) analysis of calcium and phosphorous deposits. Representative SEM pictures show the presence of inorganic deposits (indicated with red dotted circles) in particular in cells cultured in wool keratin scaffolds and treated for 21 days with PEMF + OF. EDX elemental mapping of calcium (green) and phosphorus (red) relative to the SEM pictures (dimension: 64.68 × 43.12 μm). All EDX analyses were conducted with an accelerating voltage of 20 kV and under low vacuum conditions. Wool keratin scaffolds cultured in maintenance medium in absence of cells (w/o cells) and with OF (w/o cells + OF) for 21 days were included as control. In both these conditions, the calcium and phosphorus signals were negligible.

Figure 4

Gene expression of bone-specific markers as determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) at day 7 and 21 of culture. Graphs show the fold change of gene expression relative to the expression in the cells at day 0. Symbols indicate statistical significance vs. day 0 (+), vs. control (*), vs. PEMF (§) and vs. OF (°) as determined by two-way ANOVA (N = 2, n = 3). Abbreviations: OSX, osterix; Runx-2, runt-related transcription factor-2; ALP, alkaline phosphatase; OSC, osteocalcin; DCN, decorin; COL-I, type-I collagen.

Figure 5

Bone osteogenic protein production by cells onto wool fibril sponges in the different conditions after 21 days of culture. (a) Quantification of indicated osteogenic proteins quantified by ELISA assay. Data are expressed as pg/cell×scaffold (N = 3; n = 2, symbols indicate statistical significance vs. control (*), vs. PEMF (§) and vs. OF (°)). (b) Representative orthogonal view of Confocal Laser Scanning Microscope (CLSM) images of bone osteocalcin immunolocalization (green) are shown with xy, yz, and xz planes. Nuclei (blue) were counterstained with Hoechst 33342. White arrows indicate the scaffold areas containing cells. Magnification 20×; the scale bar represents 50 µm. Negative control for non-specific staining of the secondary antibody and Tissue Culture Plates (TCPS) controls are shown in Figure S4.

Figure 6

Compression traces of the wool fibril sponges in the wet state after 21 days of culture in MM with cells and PEMF. Similar behavior was obtained for all other conditions tested.

Figure 7

Degradation rate of the commercial collagen sponge vs. wool fibril sponge.