Early responses of blood platelets and immunoinflammatory cells (macrophages) to titanium (Ti) bone implants affect the subsequent biological healing of implants by modulating early tissue healing-microenvironments via the formation of temporary fibrin matrix scaffolds for stem cell migration and production of growth factors and cytokines. This study investigated the effects of nanoscale surface topography and calcium ion (Ca2+) modification of Ti surfaces on biocompatibility regulated by blood platelets and macrophages, for the future surface design of Ti bone implants with enhanced early osteogenic capacity. A nanostructured Ti surface with or without Ca2+ enrichment was prepared using the hydrothermal treatment. Immediate and early functions of platelets and macrophages modulated by modified Ti surfaces were investigated by morphological observation of platelet spreading and fibrin matrix formation, platelet growth factor release, immunostaining of macrophage phenotypes, and macrophage inflammatory cytokine production. The results showed that surface nanoscale topographical modification of Ti promotes blood platelet activation and suppresses the inflammatory response of macrophages. In addition, surface chemistry modifications with Ca2+ enhanced the platelet response-modulating function of the nanostructured Ti surface, which accelerated immediate fibrin matrix formation and platelet-derived growth factor-AB release. Thus, nanotopographical and Ca2+ modifications of implant surfaces are expected to be effective approaches that favor the initial phase of wound healing around the Ti bone implants through positive modulation of immediate blood platelet function and early macrophage immunoinflammatory response.
Blood-implant contact is the first biological events that occurs in the wound healing process after the implantation of biomaterials. The immediate early response of platelets to implanted materials affects the biological healing of titanium (Ti) bone implants by causing the release of bioactive molecules and fibrin matrix formation [
Recently, nanotopographical and bioactive chemistry have garnered much attention for the surface modification of Ti bone implants [
In this study, the effects of nanostructured and bioactive surface modifications (in this study, Ca2+) on the immediate and early responses of platelets and macrophages were investigated, and it was determined if Ca2+ modifications of implant surfaces clearly enhance these early biological events at the nanostructured Ti implant surface. To this end, Ti samples with surface nanotopography and Ca2+ chemistry were prepared by wet chemical treatment. Then the
Disk-shaped Ti samples 15 mm in diameter and 2 mm thick were made from commercially pure Ti rods (ASTM grade 4). Ti samples were wet-abraded to #1200 grit silicon carbide paper and ultrasonically cleaned in acetone, alcohol, and double-distilled water (control Ti surface). To prepare nanostructured Ti samples with surface Ca2+ modification (Nano/Ca surface) and without Ca2+ modification (Nanosurface), wet-abraded Ti disks were hydrothermally treated according to a previously described method [
The surface nanotopography of the investigated samples was evaluated by atomic force microscopy ([AFM] XE-100; Park Systems, Suwon, Korea) using a 10 nm AFM tip in the noncontact mode (scan rate of 0.5 Hz). The surface roughness values of the samples were measured over a 2.5
Whole blood drawn from a healthy volunteer was used to prepare platelet-rich plasma (PRP) by a double centrifugation technique according to the method described elsewhere [
Field emission-scanning electron microscopy (FE-SEM, S-4800; Hitachi, Tokyo, Japan) was used for the morphological evaluation of immediate platelet adhesion and fibrin matrix formation on the samples after 5 and 20 min of culture. At the indicated incubation time points, adherent platelets on Ti disks were sequentially fixed in 2% glutaraldehyde and 1% osmium tetroxide. After dehydration using an ascending series of alcohols and critical point drying and gold-palladium coating, fibrin matrix formation and adherent platelet morphology on the surfaces were observed.
Platelet growth factor release was measured using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit (R&D systems, Minneapolis, MN, USA). Quantification of transforming growth factor-
Mouse macrophage J774.A1 cells (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 U/mL Fungizone under 100% humidity and 5% CO2, at 37°C.
Immunocytochemistry was used to investigate early local expression levels of proinflammatory M1 marker (CCR7) and the proregenerative M2 marker (MR; CD206) in macrophage cells grown on the investigated surfaces. J774.A1 cells were seeded on Ti disk samples at an initial seeding density of 1 × 104 cells/Ti disk. At 24 h of culture, cells adhered to Ti samples were fixed and permeabilized with 0.05% Triton X-100 (Thermo Fisher Scientific, Waltham, MA, USA) according to a previously described method [
The concentrations of inflammation-related cytokines (tumor necrosis factor
Three independent cell culture experiments were performed. Statistical analysis was performed using one-way analysis of variance with Tukey’s multiple comparison tests.
Figure
Representative 3D and 2D AFM images of the unmodified wet-abraded Ti, hydrothermally obtained pure nanostructured Nano and Ca2+-containing nanostructured Nano/Ca surfaces.
Thin-film X-ray diffraction patterns of the Nano and Nano/Ca samples.
The average surface roughness (
Surface characteristics of the investigated surfaces. (a)
The Ti sample with a surface anatase TiO2 structure and nanoscale topography (Nanosurface) exhibited a more hydrophilic surface property than the unmodified Ti and Ca2+-incorporated nanostructured (Nano/Ca) samples (Figure
The surface chemical compositions of the investigated samples, as determined by XPS analysis, are shown in Table
Chemical composition of investigated surfaces by X-ray photoelectron spectroscopy (atomic %).
Group | Ti2p | O1s | C1s | Ca2p | Na1s | N1s |
---|---|---|---|---|---|---|
Ti | 18.4 | 53.7 | 25.1 | 1.7 | 1.1 | |
Nano | 22.2 | 55.4 | 21.7 | < 0.1 | 0.7 | |
Nano/Ca | 12.2 | 51.3 | 22.1 | 12.8 | 0.8 | 0.8 |
The morphologies of adherent blood platelets on the investigated surfaces, as evaluated by FE-SEM, are shown in Figures
FE-SEM images showing the morphology of adherent blood platelets in the investigated samples after 5 min of incubation. Platelets in the Ti samples with surface nanostructure (Nano and Nano/Ca samples) and Ca2+ chemistry (Nano/Ca sample) had more dendritic spread than the unmodified Ti sample.
FE-SEM images showing the morphology of adherent platelets in the investigated samples after 20 min of incubation. Platelets at the Nano and Nano/Ca surfaces had denser fibrin matrix formation than the unmodified wet-abraded Ti sample.
At 20 min, FE-SEM observation showed more evident clot formation patterns on all of the investigated surfaces than at 5 min of incubation (Figure
The dense fibrin network formed on implant surfaces serves as a temporary scaffold to promote homing of osteogenic MSCs to the implant surface [
After investigating platelet activation of the investigated samples by morphological evaluation using SEM observation, the concentrations of growth factors (TFG-
ELISA results for detection of TGF-
TGF-
Our results showed that surface chemistry modification with Ca2+ promoted platelet activation at the nanostructured Ti surface, with an effect that was superior to that of the pure anatase TiO2 structure with better surface hydrophilicity. These findings are somewhat in agreement with the results of other studies reporting that surface hydrophilicity, nanoscale topography, and crystalline TiO2 structure affect platelet activation on the Ti surface [
CLSM images of macrophage phenotype expression in J774.A1 cells induced by surface modification modalities are shown in Figure
(upper panel) CLSM images showing expression of the proinflammatory M1 phenotype (CCR7, in green) in macrophages in the investigated samples (lower panel). Merged immunofluorescent images of macrophages expressing proinflammatory M1 and proregenerative M2 phenotypes (CD206, in red) in the investigated samples after 24 h incubation.
Early protein production levels of proinflammatory cytokines (TNF
ELISA results of TNF
These findings indicate that both the surface nanotopographical and Ca2+ modifications suppress inflammatory M1 macrophage phenotype development at the Ti implant surface in the early healing stage. Positive modulation of macrophage phenotype expression by surface nanostructure and Ca2+ modification at this stage may induce subsequently favorable osteogenesis outcome regulated by osteogenic MSCs at the implant surface [
In this study, we investigated the effects of nanoscale surface topographical and Ca2+ modifications on the immediate and early responses of platelets and macrophages at the Ti implant surface. Understanding the biological mechanism underlying the bone healing capacity of modified Ti implants in the initial phase of healing (i.e., proceeding the osteogenic cell-governed healing phase) is essential for the future surface design of Ti bone implants with enhanced osteogenic capacity. Our results demonstrate that nanotopographical surface modification of Ti accelerates platelet activation, which promotes fibrin matrix formation and PDGF-AB release. Surface Ca2+ modification enhanced immediate platelet activation at the nanostructured Ti surface. Surface nanotopographical and Ca2+ modifications strongly suppressed inflammation-related macrophage phenotype at an early incubation time, which in turn significantly decreased production of the inflammatory cytokines, TNF
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Korea government [NRF-2017R1A2A2A05001442].