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. 2020 Sep 3;21(17):6406.
doi: 10.3390/ijms21176406.

Phenolic-Enriched Collagen Fibrillar Coatings on Titanium Alloy to Promote Osteogenic Differentiation and Reduce Inflammation

Anna Mieszkowska  1   2 , Harrison Beaumont  3 , Laurine Martocq  3 , Andrey Koptyug  4 , Maria A Surmeneva  5 , Roman A Surmenev  5 , Javad Naderi  6 , Timothy E L Douglas  3   7 , Katarzyna A Gurzawska-Comis  2
Affiliations
Free PMC article

Phenolic-Enriched Collagen Fibrillar Coatings on Titanium Alloy to Promote Osteogenic Differentiation and Reduce Inflammation

Anna Mieszkowska et al. Int J Mol Sci. .
Free PMC article

Abstract

The adsorption of biomolecules on biomaterial surfaces can promote their integration with surrounding tissue without changing their bulk properties. For biomaterials in bone reconstruction, the promotion of osteogenic differentiation and reduction of inflammation are desirable. Fibrillar coatings are interesting because of fibrils' high surface area-volume ratio, aiding adsorption and adhesion. Fibrils also serve as a matrix for the immobilization of biomolecules with biological activity, such as the phenolic compound phloroglucinol (PG), the subunit of marine polyphenols. The aim of this work was to investigate the influence of PG coatings on fibroblast- and osteoblast-like cells to increase the osseointegration of titanium implants. Collagen fibril coatings, containing PG at low and high concentrations, were produced on titanium alloy (Ti6Al4V) scaffolds generated by additive manufacturing (AM). These coatings, especially PG-enriched coatings, reduced hydrophobicity and modulated the behavior of human osteosarcoma SaOS-2 and mouse embryonic fibroblast 3T3 cell lines. Both osteoblastic and fibroblastic cells spread and adhered well on PG-enriched coatings. Coatings significantly reduced the inflammatory response. Moreover, osteogenic differentiation was promoted by collagen coatings with a high PG concentration. Thus, the enrichment of collagen fibril coatings with PG is a promising strategy to improve Ti6Al4V implants for bone contact in orthopedics and dentistry and is worthy of further investigation.

Keywords: Ti6Al4V; coating; collagen fibrils; inflammation; osseointegration; osteoblast differentiation; phenolic.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
FTIR spectra of PG powder and collagen hydrogels containing no PG, 0.333 mg/mL and 1.0 mg/mL of PG solutions (a) in the 4000–500 cm−1, (b) in the 3800–2800 cm−1 and (c) in the 1700–1520 cm−1 wavenumber regions. Col: collagen hydrogel; Col_low: collagen hydrogel with a “low” PG concentration (0.333 mg/mL); Col_high: collagen hydrogel with a “high” PG concentration (1.0 mg/mL).
Figure 2
Figure 2
SEM images of (a) uncoated Ti, and (b) Ti_Col, (c) Ti_Col_low and (d) Ti_Col_high coatings. Ti: titanium alloy Ti6Al4V; Ti_Col: collagen-coated Ti; Ti_Col_low: collagen and low PG concentration-coated Ti; Ti_Col_high: collagen and high PG concentration-coated Ti. Red arrows indicate collagen fibrils.
Figure 3
Figure 3
Atomic composition of uncoated and coated titanium samples. Error bars represent the standard error of the mean. Ti: titanium alloy Ti6Al4V; Ti_Col: collagen-coated Ti; Ti_Col_low: collagen and low PG concentration-coated Ti; Ti_Col_high: collagen and high PG concentration-coated Ti.
Figure 4
Figure 4
Relative expression of (a) COL1A1 and BGLAP genes and (b) RANKL gene in osteoblast-like cells. Ti: titanium alloy Ti6Al4V; Ti_Col: collagen-coated Ti; Ti_Col_low: collagen and low PG concentration-coated Ti; Ti_Col_high: collagen and high PG concentration-coated Ti. Error bars represent the standard error of the mean. One-way ANOVA and Bonferroni corrections for multiple comparisons. A significant level of 0.05 was used throughout the study. * Significant difference between Ti_Col_high and other tested samples/Ti and Tissue Culture Polystyrene (TCPS) controls. ** p < 0.01; *** p < 0.001. # Significant difference between Ti control and tested samples/TCPS control. # p < 0.5, ## p < 0.01.
Figure 5
Figure 5
Relative expression of inflammatory markers in (a) osteoblast-like cells and (b) fibroblast-like cells. Ti: titanium alloy Ti6Al4V; Ti_Col: collagen-coated Ti; Ti_Col_low: collagen and low PG concentration-coated Ti; Ti_Col_high: collagen and high PG concentration-coated Ti. Error bars represent the standard error of the mean. One-way ANOVA and Bonferroni corrections for multiple comparisons. A significant level of 0.05 was used throughout the study. *Significant difference between Ti control and tested samples/TCPS control. ** p < 0.01; *** p < 0.001. #Significant difference between Ti_Col_low and other tested samples/Ti and TCPS controls. ## p < 0.01.
Figure 6
Figure 6
SEM images of (a) osteoblasts on Ti uncoated and on (b) Ti_Col_high coating and (c) fibroblasts on uncoated Ti and on (d) Ti_Col_high coating after 3 days of culture. Circles indicate cells and arrows indicate particles of Ti6Al4V powder (scale bars: 20 μm). Ti: titanium alloy Ti6Al4V; Ti_Col: collagen-coated Ti; Ti_Col_low: collagen and low PG concentration-coated Ti; Ti_Col_high: collagen and high PG concentration-coated Ti.
Figure 7
Figure 7
Diagram of preparation of collagen hydrogels. Collagen hydrogels were prepared by mixing 50 μL of double distilled water or 50 μL PG solution in water with 50 μL 10X MEM Cell Culture Medium and 400 μL of collagen type I solution (3.33 mg/mL). Then, with the neutralization of the solution by adding some drops of 1M NaOH solution, the solution became violet and hydrogels formed.
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