Biomaterial Property Effects on Platelets and Macrophages: An in Vitro Study

Abstract

The purpose of this study was to evaluate the effects of surface properties of bone implants coated with hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) on platelets and macrophages upon implant installation and compare them to grit-blasted Ti and Thermanox used as a control. Surface properties were characterized using scanning electron microscopy, profilometry, crystallography, Fourier transform infrared spectroscopy, and coating stability. For platelets, platelet adherence and morphology were assessed. For macrophages, morphology, proliferation, and polarization were evaluated. Surface characterization showed similar roughness of ∼2.5 μm for grit-blasted Ti discs, both with and without coating. Coating stability assessment showed substantial dissolution of HA and β-TCP coatings. Platelet adherence was significantly higher for grit-blasted Ti, Ti-HA, and Ti-β-TCP coatings compared to that of cell culture control Thermanox. Macrophage cultures revealed a decreased proliferation on both HA and β-TCP coated discs compared to both Thermanox and grit-blasted Ti. In contrast, secretion of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine TGF-β were marginal for grit-blasted Ti and Thermanox, while a coating-dependent increased secretion of pro- and anti-inflammatory cytokines was observed for HA and β-TCP coatings. The results demonstrated a significantly upregulated pro-inflammatory and anti-inflammatory cytokine secretion and marker gene expression of macrophages on HA and β-TCP coatings. Furthermore, HA induced an earlier M1 macrophage polarization but more M2 phenotype potency than β-TCP. In conclusion, our data showed that material surface affects the behaviors of first cell types attached to implants. Due to the demonstrated crucial roles of platelets and macrophages in bone healing and implant integration, this information will greatly aid the design of metallic implants for a higher rate of success in patients.

Figure 1

(A) SEM micrographs of Thermanox, grit-blasted Ti, Ti-HA, and Ti-β-TCP surfaces. (B) X-ray diffraction spectra of grit-blasted Ti and as-deposited and heat treated Ti-HA and Ti-β-TCP coatings. (C) Fourier transform infrared spectra of as-deposited and heat treated Ti-HA and Ti-β-TCP.

Figure 2

(A) Calcium release from as-deposited and heat-treated Ti-HA and Ti-β-TCP coatings over a 28-day incubation period in Milli-Q water (n = 3). * p = 0.036 Ti-β-TCP heat vs Ti-HA heat; ≠ p = 0.037 Ti-β-TCP heat vs Ti-HA heat. (B) Calcium remaining on the coated discs after 28 days in Milli-Q water soaking experiment for as-deposited and heat treated HA and β-TCP coatings (n = 3). Data analysis was performed using Kruskal–Wallis test with a posthoc Dunn test. * p = 0.011.

Figure 3

(A) Scanning electron micrograph of platelets on Thermanox, grit-blasted Ti, Ti-HA, and Ti-β-TCP. Yellow arrows indicate the platelets. (B) LDH activity assay for platelets adhered to Thermanox and grit-blasted Ti, Ti-HA, and Ti-β-TCP (n = 4). Data analysis was performed using Kruskal–Wallis test with a posthoc Dunn test. Results represent mean + SD (n = 4). *Thermanox vs Ti- β-TCP (p = 0.00011); # Thermanox vs Ti- HA (p = 0.0007); θ Thermanox vs grit-blasted Ti (p = 0.0024).

Figure 4

(A) Scanning electron microscopy images of macrophages cultivated on Thermanox and grit-blasted Ti, Ti-HA, and Ti-β-TCP after 1, 4, and 7 days of cultivation. Yellow arrows indicate macrophages (original magnification: 1000×; scale bar: 50 μm).

Figure 5

(A) DNA content (ng) of macrophages cultured on Thermanox and grit-blasted Ti, Ti-HA, and Ti-β-TCP. Results represent mean + SD (n = 4). * p < 0.05; ** p < 0.01; and *** p < 0.001. (B) TNF-α and TGF-β (C) secretion of macrophages after 1, 4, and 7 days of culture on Thermanox and grit-blasted Ti, Ti-HA, and Ti-β-TCP. Results represent mean + SD of 4 experimental groups (n = 4). Data analysis was performed using one-way ANOVA with a posthoc Tukey multiple comparisons test. *p < 0.05; **p < 0.01; and ***p < 0.0001.

Figure 6

Immunostaining for DAPI (nuclei), CCR7 (M1 marker), CD36 (M2 marker), and merged images for macrophages cultured on grit-blasted Ti, Ti-HA, and Ti-β-TCP discs after cultures of 1 (A), 4 (B), and 7 (C) days.

Figure 7

Quantitative immunostaining for CCR7 (M1 marker) and CD36 (M2 marker) for macrophages cultured on Thermanox and grit-blasted Ti, Ti-HA, and Ti-β-TCP discs after cultures of 1 (A), 4 (B), and 7 (C) days (n = 4). Quantitative PCR for INDO and CXCL11 (M1 markers) and MCR-1 and CCL13 (M2 markers) for macrophages cultured on Thermanox and grit-blasted Ti, Ti-HA, and Ti-β-TCP discs after cultures of 1 (D), 4 (E), and 7 (F) days (n = 4). Data analysis was performed using one-way ANOVA with a posthoc Tukey multiple comparisons test. *p < 0.05; **p < 0.01; and ***p < 0.0001.