Platelet Responses to Urethane Dimethacrylate-Based Bone Cements Containing Monocalcium Phosphate/ε-Polylysine: Role of ε-Polylysine in In Vitro Wound Healing Induced by Platelet-Derived Growth Factor-BB

Abstract

Platelets play a pivotal role in initiating bone fracture healing. However, the interaction between platelets and bone cements used for fracture repair remains relatively unexplored. This study investigated the platelet response to recently developed urethane dimethacrylate-based bone cements containing 8% (w/w) monocalcium phosphate monohydrate (MCPM) and/or 5% (w/w) ε-polylysine (PLS). All experimental bone cements achieved final monomer conversions of 75-78%, compared with the 86% conversion of the commercial PMMA bone cement Kyphon. The MCPM and PLS microparticles, varying in size, were dispersed within the glass-filler-incorporated polymer matrix. In contrast to Kyphon, all experimental cements exhibited significantly smoother and more hydrophilic surfaces. Bone cements incorporating PLS, with or without MCPM, effectively activated platelets by inducing cellular adhesion, aggregation, and extracellular-signal-regulated kinase (ERK) activation, comparable to Kyphon. Flow cytometry analysis demonstrated a statistically significant increase in CD62P-positive platelets following exposure to PLS-incorporated bone cements and exogenously administered PLS in a concentration-dependent manner, but not with Kyphon. A wound healing assay revealed a 2-fold enhancement in wound closure within 24 h and exceeding 85% at 48 h by bone cements containing PLS, with or without MCPM, and Kyphon. Notably, platelet-derived growth factor BB (PDGF-BB) secretion was significantly elevated, specifically after platelet exposure to PLS-incorporated bone cements, a phenomenon not observed with Kyphon. Interestingly, PDGF-BB neutralization attenuated wound closure induced by the PLS-incorporated bone cements. In conclusion, the urethane dimethacrylate-based bone cements containing PLS demonstrated a significant enhancement in platelet activation and PDGF-BB secretion, which, at least partly, enhanced in vitro wound closure. The results suggest that PDGF-BB plays a crucial role in the PLS-mediated enhancement of wound healing in these bone cements.

Figure 1

SEM images and corresponding elemental mappings of the cross-sectional surfaces of the 8M5P specimens analyzed by EDX (A) and EPMA (B). Notably, while nitrogen (N) was not detectable by EDX, it was successfully identified by EPMA, with a peak wavelength of 31.3 Å (red arrow).

Figure 2

Surface characterization of the prepared bone cements. (A) SEM micrographs of surface characteristics of bone cements. (B) AFM images of surface characteristics of bone cements and a summary of surface roughness values. (C) Water drop shape images observed on bone cement surfaces and a summary of water contact angles. Data are expressed as mean ± SD (n = 4). In bar graphs, values signed with the same letter indicate that there was no significant difference between them.

Figure 3

Effect of bone cements on the platelet aggregation and the activation of Akt and ERK pathways. Human PRP was cultured on the different bone cements for 30 min, and bone-cement-induced changes of platelets were visualized using SEM (A) and confocal fluorescence microscopy (B). Insets in (A) show a closer view of the platelet and surrounding matrix. In (C), the activation of Akt and ERK signaling pathways in platelets cultured on the bone cements was assessed at 5 min intervals for a period of 20 min following exposure, by quantifying the expression of phosphorylated Akt (p-Akt) and phosphorylated ERK (p-ERK) using Western blot analysis. The expression of β-actin was used as an internal control. The p-Akt/Akt and p-ERK/ERK ratios (vs untreated samples at the same time points) are shown below the Western blot bands. Band intensities of each bone cement sample were normalized to that of the untreated samples at similar time points (set to 1.0 in the untreated group). Ratios in red indicate increases more than 2.0-fold. All results shown are representative from independent experiments using 2 different single-donor PRP samples.

Figure 4

Effect of bone cements on the platelet activation assessed by FCM. Human single-donor PRP (from 4 donors) was cultured on the different bone cements for 30 and 60 min, and the cells were stained for CD42b and CD62P before FCM analysis. The gating strategy to measure the proportion of activated platelets (CD42b⁺CD62P⁺) is shown in (A), and a summary of % CD62P⁺ platelets induced by the different bone cements is shown in (B). Data are expressed as mean ± SD from three different single-donor PRP samples. Plastic surfaces were used for untreated samples. p < 0.05.

Figure 5

Effects of PLS and MCPM on platelet activation assessed by FCM. Human single-donor PRP (from 2 donors) were treated with PLS at 100, 200, and 600 μg/mL (P100, P200, and P600, respectively) and MCPM at 5, 15, and 30 μg/mL (M5, M15, and M30, respectively) for 30 and 60 min, and the cells were stained for CD42b and CD62P before FCM analysis. The gating strategy to measure the proportion of activated platelets (CD42b⁺CD62P⁺) is shown in (A), and a summary of % CD62P⁺ platelets individually induced by PLS and MCPM is shown in (B). Data are expressed as mean ± SD from two different single-donor PRP samples. p < 0.05.

Figure 6

Effect of the bone-cement-exposed conditioned plasma on the in vitro MSC-mediated wound healing. Human PRP samples were incubated with the different bone cements for 1 h, and the cell-free conditioned plasma samples were used in the scratch-based wound healing assay. Representative phase-contrast microscopy images of wound closure after 24–48 h treatment with bone-cement-exposed conditioned plasma samples are depicted in (A), and analysis of the results is summarized in (B). Data are expressed as mean ± SD from three different single-donor PRP samples. p < 0.05.

Figure 7

Effects of bone cements on the cytokine production assessed by ELISA. Human PRP was added to the individual bone cements at 37 °C for 60 min, and the levels of PDGF-BB, TGF-β1, PF-4, and SDF-1α were quantified using ELISA. Data are expressed as mean ± SD from three different single-donor PRP samples. p < 0.05.

Figure 8

Effect of PDGF-BB neutralizing antibody on the bone-cement-exposed conditioned media-induced in vitro MSC-mediated wound healing. Human PRP was incubated with the different bone cements for 1 h, and the cell-free conditioned plasma samples were used in the scratch-based wound healing assay with either isotype control or PDGF-BB neutralizing antibodies for indicated times. Data are expressed as mean ± SD from three different single-donor PRP samples. p < 0.05.