Amine modification of calcium phosphate by low-pressure plasma for bone regeneration

Materials and reagents

Dense and porous disks of β-TCP [Ca₃(PO₄)₂] were provided by Coors Tek KK (Tokyo, Japan). All the dense and porous disks used in this study had the same dimensions of 5 mm in diameter and 2 mm in height (φ 5 mm × h 2 mm). The porous disks had well-organized interconnected structures with a porosity of 72–78%, an average pore diameter of 150 μm, and an average diameter of interconnected passages of 40 μm¹⁰.

The following culture media were used for in vitro experiments: (1) growth medium (GM) comprising α Eagle’s minimal essential medium (α-MEM, Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic–antimycotic solution (Sigma-Aldrich); (2) osteogenic differentiation medium (ODM) consisting of GM supplemented with 50 μg/ml l-ascorbic acid 2-phosphate (Sigma-Aldrich), 10 mM β-glycerol phosphate (Merck KGaA, Frankfurt, Germany), and 10 nM dexamethasone (Sigma-Aldrich).

Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Kumamoto, Japan) was used for the cell proliferation assay. LabAssay ALP (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) was used for the evaluation of alkaline phosphatase (ALP) activity and BCIP/NBT Color Development Substrate (Promega Corp., Madison, WI, USA) was used for ALP staining. M-PER and Pierce Rapid Gold BCA Protein Assay Kits (Thermo Fisher Scientific) were used for total protein extraction and quantification, respectively. K-CX AT solution (Falma Co., Tokyo, Japan) was used for decalcification of in vivo specimens.

Plasma polymerization of β-TCP disks

Plasma polymerization was performed with a bipolar pulsed-plasma deposition system, as shown in Fig. 1A. The details of the system are described elsewhere^11,12,13,14. The β-TCP disks were placed on the bottom metal (molybdenum) electrode, 190 mm in diameter, connected to the bipolar high-voltage power supply. The applied bipolar pulse voltages were 1.1 and − 1.1 kV (peak-to-peak voltage of 2.2 kV) and the pulse duration was 1 μs for each positive or negative pulse. The power, repetition frequency, and duty cycle were 15 W, 5 kHz, and 1%, respectively. The time lapse between a positive pulse and the subsequent negative pulse was 100 μs. The upper metal (aluminum) electrode, 80 mm in diameter, was grounded and the distance between the two electrodes was 38 mm. For plasma polymer deposition, the discharge was generated in a CH₄/N₂/He gas mixture with flow rates of 10, 20, and 10 sccm, respectively, and a gas pressure of 70 Pa. For biological (i.e., in vitro and in vivo) experiments, dense disks were treated only on one side for 30 min whereas porous disks were treated on both sides for 60 min (i.e., 30 min each). This is because dense disks were used only for in vitro experiments in this study, where cells were places only on one side of the disk, whereas porous disks were used only for in vivo experiments in this study, where all sides of the disk were exposed to the animal tissues. In either one-side or both-side plasma treatment, the sidewall of a disk was exposed to the plasma and therefore plasma treated. For non-biological experiments (e.g., physical or chemical characterization of plasma-polymerized films), the disk was plasma treated only on one side for 30 min and the film deposited on the plasma-facing surface or the inner pore surfaces was examined.

Plasma polymer film characterization

The chemical compositions of untreated or plasma-treated β-TCP surfaces were analyzed by X-ray photoelectron spectroscopy (XPS) using ESCA-850 (Shimadzu Co., Kyoto, Japan) with a non-monochromatized Mg-K_α (1253.6 eV) X-ray source at Osaka University, for which the pass energy was 75 eV, the photoelectron take-off angle was 90°, and the spot diameter was 8 mm (90% uniformity), or by high-resolution XPS using PHI Quantera II (ULVAC-PHI, Inc., Chigasaki, Japan) with a monochromatic Al-Kα (1486.6 eV) X-ray source at the Foundation for Promotion of Material Science and Technology of Japan (MST), for which the pass energy was 112 eV, the photoelectron take-off angle was 45°, and the spot diameter was 100 μm.

The thickness of a deposited polymer film was evaluated by the standard ellipsometry^15,16, using data acquired with the V-VASE Ellipsometer (J.A. Woolam, Lincoln, NE, USA) at Central European Institute of Technology (CEITEC), Brno University of Technology, in the spectral range from 0.75 to 6.5 eV at four angles of incidence 60°, 65°, 70° and 75°.

The detection of primary amine groups (–NH₂) on plasma-treated disk surfaces was performed using the standard derivatization with 4-trifluoromethyl-benzaldehyde (TFBA), according to the published method^17,18. The derivatization reactions of TFBA vapors with plasma-treated disk surfaces were allowed to occur in an Ar atmosphere (Ar flow rate of 60 sccm at atmospheric pressure) at room temperature (approximately 25 °C) for 4 h inside the glove box. The relative concentrations of primary amines on the sample surfaces were determined by the detection of fluorine (F) atoms of TFBA with ESCA-850 XPS analysis after the derivatization reactions.

The surface morphologies of untreated and plasma-treated porous β-TCP disks were observed with a scanning electron microscope (SEM) (S-4800, Hitachi, Ltd., Tokyo, Japan) at Osaka University.

Cells

Rat BMSCs were obtained from the bone shafts of the femora of four 3-week-old green fluorescent protein (GFP)-transgenic male Sprague–Dawley rats (SD-Tg (CAG-EGFP) rats, Japan SLC, Hamamatsu, Japan). Following the sacrifice of the rats using CO₂ inhalation, both ends of the femur were removed from the epiphysis; the marrow was flushed out using 10 ml of GM expelled from a syringe through a 21-gauge needle according to the previously described method¹⁹. The released cells were collected in two 100 mm culture dishes containing 15 ml of GM. The medium was changed after 24 h to remove hematopoietic cells and renewed twice weekly. Cultures were maintained in a humidified atmosphere of 95% air with 5% CO₂ at 37 °C. When the cells reached 80–90% confluency they were washed with phosphate-buffered saline (PBS) and trypsinized with 1% trypsin-ethylenediaminetetraacetic acid (EDTA). Following centrifugation for 5 min at 400g, the cells were resuspended and plated at a density of 3.6 × 10⁴/cm². After again reaching confluency, cells were collected and stored at − 80 °C (Passage 1). Prior to in vitro experiments, stocked cells were thawed and resuspended in 15 ml of GM, then plated in a 100 mm dish and cultured for three days to reach 80–90% confluency (Passage 2).

Cell adhesion assay

A centrifugation cell adhesion assay was performed according to a published method²⁰. Cell suspension (5 × 10³ cells/35 μl GM) was gently dropped on each dense β-TCP disk surface to form a centroclinal water drop and incubated for 30 min in a 24 well culture plate to initiate adhesion. Then, 1 ml of PBS was gently added to each well containing a cell-adhered disk and macro fluorescence photos were taken with the Leica AF6000 Fluorescence Imaging System (Leica Microsystems, Wetzlar, Germany. Exposure: 1000 ms; Gain: 2.7; Binning 2 × 2; Magnification: 12.6×) to quantify the initial adherent cells. After this, the cell adhered disks were embedded into a 48-well culture plate containing 100 μl of Vaseline (KENEI Pharmaceutical Co., Ltd., Osaka, Japan) in each well. After filling each well with PBS, the plate was sealed with a sealing tape and set upside-down on a centrifuge (PlateSpinII, Kubota Corp., Tokyo, Japan) and centrifuged at 10g for 5 min to detach weakly adherent cells. After the centrifugation, the detached cells were slowly aspirated and each well was carefully filled with 200 μl of PBS. Macro fluorescence photos of the plate were taken again under the same conditions as those prior to the centrifugation. Automatic cell counting was performed with the macro fluorescence photos and ImageJ software²¹. The adhesion rate is defined as the ratio of the number of cells attached to the surface after centrifugation to that before centrifugation.

Morphology analysis of adhered cells

Cell suspension (5 × 10³ cells/35 μl GM) was gently dropped on dense β-TCP disks and incubated for 3 h in a 24-well culture plate to initiate adhesion. Then, the wells were slowly filled with 1 ml of GM, and the incubation was continued for another 24 h. Macro fluorescence photos of random areas of the culture wells containing incubated cells were then obtained. The open-source software CellProfiler (www.cellprofiler.org)²² was used for sorting cells and cell morphology analysis. In particular, the cell area was measured to quantify the spreading of attached BMSCs. Two cell-shape descriptors (circularity and solidity) were investigated; the circularity indicates the closeness of the cell shape to a perfect circle, and the solidity is an index to quantify the amount and size of concavities of the cell²³:

where A is the cell area and P is the perimeter.

where ConvexA is the area of the smallest convex hull that contains the cell.

Cell proliferation assay

To balance the initial cell count of adherent cells on untreated and plasma-treated β-TCP disks, two different concentrations (5 × 10³ cells/35 μl GM for the untreated group and 3 × 10³ cells/35 μl GM for the plasma-treated group) of cell suspension were dropped on dense untreated and plasma-treated β-TCP disks in a 48-well plate. After incubating for 30 min, 500 μl of GM was slowly added to each well. At day 1, 3, 5, 7, 11, and 14, 50 μl of CCK-8 solution was added to each well and the plate was incubated for 2 h, then 100 μl of GM from each well was transferred into a 96-well plate. The optical density at 450 nm was measured using a spectrophotometer (Multiskan GO, Thermo Fisher Scientific).

Osteogenic differentiation assay

Cell suspension (2 × 10⁴ cells/35 μl GM ) was dropped on dense β-TCP disks and incubated for 30 min in a 48-well plate to initiate adhesion. Then, 500 μl of GM was slowly added into each well, incubated for 24 h, and the culture medium was replaced with ODM for osteogenic differentiation. The subculture was maintained for another four days. After washing the disks twice with PBS, the attached cells were fixed using 500 μl of 4% paraformaldehyde (PFA) for ALP staining, according to the manufacturer’s instructions (Promega Corp.). For ALP activity, 60 μl of M-PER was added to each well and the cells were detached from the disks using a mini scraper. After lysing the cells for 5 min, the supernatant was collected for ALP and total protein assays. An ALP activity unit was defined as the release of 1 nmol p-nitrophenol per min of incubation at 37 °C for each β-TCP disk. The total protein content of each sample was measured to standardize the ALP activity values.

Rat calvarial defect model

A total of 20, 8-week-old male Sprague–Dawley rats (Charles River Laboratories Japan, Yokohama, Japan) were used to generate the calvarial defect model. Anesthesia was maintained by intraperitoneal injection of a mixture of 0.15 mg/kg medetomidine, 2.0 mg/kg midazolam, and 2.5 mg/kg butorphanol after introducing anesthesia by inhalation of 5% isoflurane. A 1.5 cm longitudinal incision was made at the center of the vertex and two full-thickness bone defects with a diameter of 5 mm were then carefully created using a high-speed trephine burr under constant irrigation with saline to avoid heat injury of the surrounding tissue. The plasma-treated and untreated β-TCP disks were implanted in the right and left defects, respectively. The rats were given free access to water and food after the surgery. The rats were sacrificed at postoperative 3 (n = 5) and 6 (n = 15) weeks by CO₂ inhalation. Microfocus computed tomography (micro-CT) and histological analyses were performed to evaluate new bone formation in the inner pores of porous β-TCP disks at 3 and 6 weeks postoperatively.

Microfocus computed tomography (micro-CT)

Harvested specimens were fixed in 10% buffered formalin, dehydrated and degreased using a graded ethanol series, and stored in 70% ethanol at 4 °C for micro-CT scanning (Skyscan 1272 micro-CT, Bruker, Kontich, Belgium), which was performed with the following parameters: camera binning = 2 × 2, source voltage = 80 kV, source current = 125 μA, image pixel size = 4 μm, rotation step = 0.6°, and filter = Al 1 mm. Image analysis was performed using CTAN software (Version 1.18.8.0+, Brucker). Micro-CT images of the specimens were compared with the results of histological evaluation of the specimens, discussed below, and the difference in image intensities between the newly formed bone and residual β-TCP was identified. Using this information, three-dimensional (3D) images of newly formed bones inside the porous β-TCP disks were reconstructed from the Micro-CT images of the specimens.

Histological evaluation

After micro-CT scanning, the specimens were demineralized using K-CX AT solution at 4 °C, cleared in xylene, and embedded in paraffin. Then, several sections of 4 μm thickness were cut off from the center of each specimen and stained with hematoxylin and eosin (H&E).

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 7.04 for Windows (GraphPad Software, San Diego, CA, USA) applying the Mann–Whitney U test for non-parametric data, Student’s or Welch’s t-test for parametric data, and Wilcoxon matched-pairs signed-rank test for in vivo results. The values are presented as mean ± standard deviation (SD). The differences were considered statistically significant for p value < 0.05.

Ethic declarations

All animal work was approved by The Animal Experimental Committee of Osaka University Graduate School of Medicine (01-070-000) and restrictedly followed ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals²⁴.