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PCL-3D scaffolds (3D Insert PCL, 3D Biotek, New Jersey, USA) were used as substrate. Polycaprolactone pellets with average molecular weight of 80,000 (Sigma-Aldrich CHEMIE GmbH, MO, USA), allylamine (Sigma-Aldrich CHEMIE GmbH, MO, USA), EDC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (Sigma-Aldrich CHEMIE GmbH, MO, USA), NHS (Nhydroxysuccinimide, Sigma-Aldrich CHEMIE GmbH, MO, USA), sodium dihydrogen phosphate and potassium carbonate were purchased from Aldrich. Methyl orange (MO) and hyaluronic acid (HA, TOKYO CHEMICAL INDUSTRY Co., LTD. TOKYO, JAPAN) was purchased from Tokyo Chemical Industry. All other chemicals and bioreagents were used without further purification. The density of amine groups on the PCL films (PCL-F) was quantitatively determined by using MO test. The PCL-F was prepared by dissolving 12.5 g of PCL pellets in 50 mL of chloroform. The polymer solution was then cast onto a glass substrate and the solvent was removed by controlled evaporation at room temperature over a period of 24 h. The obtained pristine PCL-F with a thickness of 40-100 was washed with copious amounts of an alcohol/water (1/1, v/v) mixture. The dried PCL-F was cut into specimen size of 20 mm (diameter) in order to use the samples in this experiment.

The surface modification of the PCL-3D scaffolds was carried out by depositing an ultra-thin polymeric layer containing amine groups through plasma polymerization of AAm using radio frequency (RF) discharge device (model UM1000134-001A mini plasma station, PLASMART, Korea). The system consists of three main parts: (i) a reaction chamber, (ii) a RF generator, and (iii) a vacuum system. The pressure was decreased to 10-3 torr using a vacuum pump and the plasma was generated with a RF power generator. The monomer vapors were supplied to the reactor chamber from liquid allylamine contained in cylindrical flask via a stainless steel line heated at 30°C (20 sccm). Plasma polymerization was carried out at a various discharge power (30, 50, 70, and 100 W) for various deposition times (5, 10, and 15 min). PCL -F and PCL-3D deposited with plasma polymerized AAm thin film are referred to as “PCL-F/NH₂” and “PCL-3D/NH₂”.

A natural polymer HA was immobilized onto the – NH₂ functionalized surface in an EDC-NHS aqueous solution at a concentration of 2.5 mg/mL (EDC/NHS = 4/1 (w/w)) for 2 h with gentle shaking at 0-4 °C (Shi et al., 2007). The samples were then immersed in a 0.3 g/L HA solution for 24 h with mild stirring at 0-4 °C. The primary amine (H₂N-) of the PCL-3D/NH₂ surface was reacted with the COOH group of the HA molecules. The prepared samples were rinsed with distilled water to remove the unbound HA. The PCL-F/NH₂ and PCL-3D/NH₂ immobilized with HA are referred to as “PCL-F/NH₂/HA”, and “PCL-3D/NH₂/HA”.

Methyl orange (MO) is an acid dye which binds with amino group containing substrates, and the dye can be removed again by bases (Gurr, 1971). MO colorimetric measurement can be used to determine the number of amino groups on polymers surfaces. The amino functionalized samples were treated with the acidic methyl orange solution (0.1 mM MO in a 0.1 M solution of sodium dihydrogen phosphate in water, filtered; pH app. 4.7) for 1 h. After rinsing the excess of dye with water the samples were carefully pressed between tissue papers to remove the water. The fixed methyl orange was resolved by 3 mL of a 0.1 M potassium carbonate solution and the concentration determined photometrically at 460 nm (Hartwig and Mulder, 1994). The -NH₂ content was obtained from a calibration plot of the optical density versus dye concentration with a 1:1 ratio assumed between the dye and amino groups (Zuwei et al., 2007).

After plasma treatment, the hydrophilicity of the surface was determined by contact angles using dynamic contact angle measurements (Contact-Angle, GS, Surface Tech. Co. Ltd. Korea). The chemical structure of the coated polymer matrix was analyzed by ATR-FTIR (IFS66V/S & HYPERION 3000, Bruker Optiks, Germany). The surface chemical compositions of the samples were analyzed in X-ray photoelectron spectroscopy (XPS, Multilab 2000 system, SSK, USA). The change of the surface morphology was observed by field emission scanning electron microscopy (FE-SEM, Hitachi, S-4800, Japan).

MC3T3-E1 (ATCC CRL-2953) cell, a clonal preosteoblastic cell line derived from newborn mouse calvarias, were cultured in α-MEM medium and 5% CO₂ at 37 °C with cell seeding density of 1 × 10⁴ cell/mL.

The samples from all groups were prefixed with a mixed solution containing 2.5% glutaraldehyde and 2.5% paraformaldehyde for 3 hours, washed three times for 10 min each in the phosphate buffer, and post-fixed in 1% osmium tetroxide for 30 min. The samples were dehydrated in a graded series of aqueous ethanol solutions (70%, 90%, 95%, and 100%) for 5 min each. The samples were then placed in hexamethyl-di-silazane (Fluka) to remove any alcohol. After 10 min, the samples were removed and allowed to air dry overnight at room temperature. The sample was coated with a thin layer of gold using an automated sputter for 1 min. Representative digital images of each sample were observed using FE-SEM. The proliferation of MC3T3-E1 cells on substrates was examined by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Sigma-Aldrich CHEMIE GmbH, MO, USA) assay after 1, 3, and 5 days of culture. All samples were placed into a 12-well plate and seeded with a density of 2 × 10⁵ cells/mL. MTT assay was described previously (Seo et al., 2010).

The results of the MTT assay were analyzed statistically using a Student’s t test. The statistical significance was considered at p < 0.05.

To investigate the -NH₂ concentration on the plasma treated PCL-F surfaces, MO test was used. By assuming a combination ratio of 1:1 between MO and -NH₂, Hartwig et al. measured the amino group density on ammonium plasma treated polymer surface, which was as low as several nano-mole per square centimeters (Hartwig and Mulder, 1994).

Figure 1 shows the effect of the time and RF input power on the surface density of the amino groups. It was found to increase as a function of the plasma treated time and RF power applied. We determined the optimum condition (100 W, 15 min) of the plasma polymerization process in Figure 1 data and then all samples was treated at optimum condition.

The contact angles of the PCL-F, PCL-F/NH₂, and PCL-F/NH₂/HA surface were 71.6 ± 4.6˚, 45.0 ±7.3˚, and 33.1 ±1.4˚ respectively (n=5). It is clearly shown that the chemical composition of plasma polymers influences the wettability of surfaces. Functional groups like amine- carboxyl-, or hydroxyl groups are known to have a hydrophilic character. Polymers obtained by high-energetic plasma treatment having high amine content were found to be more hydrophilic than others (Hamerli et al., 2002). The PCL-F/NH₂ and PCL-F/HN₂/HA surface shows low contact angle compared with PCL-F surface. Hydrophilic surface shows better cell adhesion on implant materials than hydrophobic surfaces (Foest et al., 2008; Hartwig and Mulder, 1994).

Figure 1 
Surface concentration of amine groups versus RF power (n=3).

Figure 2 shows the ATR-FTIR spectra of the PCL-F, PCL-F/NH₂ and PCL-F/NH₂ /HA surfaces. ATR-FTIR spectrometry is a very powerful technique for detecting the inter-molecular reactions between the two polymers (Kaminśka and Sionkowska, 1996). Several characteristic bands of PCL were observed at 2949 cm^-1 (asymmetric CH₂ stretching), 2865 cm^-1 (symmetric CH₂ stretching), 1727 cm^-1 (carbonyl stretching) (Wan et al., 2009; Ghasemi-Moarakeh et al., 2010). The ATR-FTIR spectrum of PCL-F/NH₂ showed bands at 3380 due to N-H stretching of amide bond, N-H bending at 1543 cm^-1. The broader band between 1550 and 1680 cm^-1 can be assigned to the stretching modes of C=C bonds, imines, amines and amides groups (Lin et al., 2011; Meskinfam et al., 2011). After HA was immobilized onto PCL-F/NH₂ samples, the band near 1500 cm^-1 decreased. The weak peak near 1700 cm^-1 for the PCL-F/NH₂/HA sample was assigned to the carbonyl in amide group derived from the -COOH terminals of HA reacted with the -NH₂ terminals of PCL-F/NH₂ on the surface through covalent bonding. ATR-FTIR confirmed that HA had been immobilized successfully onto the PCL-F/NH₂ surface through crosslinking via EDC.

Figure 2 
ATR-FTIR spectra of (a) PCL-F, (b) PCL-F /NH₂ and (c) PCL-F/NH₂/HA.

Figure 3 and Table 1 show the surface chemical composition of PCL-3D, PCL-3D/NH₂ and PCL-3D/NH₂/ HA surfaces. XPS analysis was used to examine each reaction step because it can provide information on chemical bonds and atomic concentrations. Table 1 lists the change in the chemical components on each surface. The chemical component ratio (O1s/C1s, N1s/C1s) changed and atomic elemental O1s significantly increased after immobilization of the HA. PCL-3D/NH₂/HA surfaces had the higher content of oxygen in its molecules. Thus, the O1s/C1s of PCL-3D/NH₂/HA surface was higher than that of the other. This suggests the formation of the target molecule.

Figure 3 
XPS spectra of (a) PCL-3D, (b) PCL-3D/NH₂ and (c) PCL-3D/NH₂/HA.

The morphology after plasma polymerization and HA immobilization were examined by FE-SEM. It was revealed that the surface morphology of PCL-3D, PCL-3D/NH₂ and PCL-3D/NH₂/HA was not changed significantly with each different treatment, as shown in Figure 4 (a), (b), and (c), respectively.

To observe the initial cell adhesion on the different PCL scaffolds, MC3T3-E1 cells were cultured for one day. Figure 5 shows the morphology of cells grown on the different PCL scaffolds. Cells grown on PCL-3D/NH₂/HA surfaces have a spread out aspect and show multiple microvilli on the surface and long fine cytoplasmic extensions in multiple directions. In addition, these cells demonstrate apparently greater spreading with large lamellipodes indicating an active cell migration. On the HA immobilized samples the cell showed the good cytoplasmic extensions, and the cell almost reached confluence, forming a thick cell layer.

Figure 4 
FE-SEM images of (a) PCL-3D, (b) PCL-3D/NH₂ and (c) PCL-3D/NH₂/HA.

Figure 5 
FE-SEM images of the MC3T3-E1 cells grown on the PCL 3D scaffolds for 1 day of culturing: (a) PCL-3D, (b) PCL-3D/NH₂ and (c) PCL-3D/NH₂/HA.

The samples used were PCL-3D, PCL-3D/NH₂ and PCL-3D/NH₂/HA with the PCL-3D as the control at 1 day and 5 days. The cell viability (proliferation) was assessed using a MTT assay, which is a direct mitochondrial activity assay. Figure 6 shows the viability of MC3T3-E1 seeded on the prepared samples at 1, 3, and 5 days. The cell viability is expressed as a percentage of the control value. The initial adhesion (1 day after cell seeding) of the amino group functionalized sample (PCL-3D/NH₂) and HA immobilized sample (PCL-3D/NH₂/HA) increased to 42% and 48% compared to the PCL-3D. This indicates that –NH₂ groups on the surface can have some positive effects on the implant biocompatibility. In particular, the PCL-3D/NH₂/HA surface showed a significant increase in cell viability after 5 days compared to that of the PCL-3D by approximately 44%, which was attributed to the improved adhesion and spreading ability of HA. At 5 days cell proliferation was significantly higher on the PCL-3D/NH₂/HA than on the PCL-3D. Some studies demonstrated that hyaluronic acid promotes cell adhesion (Arima and Iwata, 2007). The MTT results in the present study indicate that the immobilization of HA can have some positive effects on implant biocompatibility. This confirms that the enhancement of MC3T3-E1 cell proliferation requires the presence of HA. Along with the excellent behavior of the PCL-3D/NH₂/HA, the cell responses showed good cell viability on the PCL-3D/NH₂/HA with a significantly higher level of cell growth than those on the PCL-3D (Figure 5 and 6).

Figure 6 
Cell proliferation of MC3T3-E1 cultured on (a) PCL-3D, (b) PCL-3D/NH₂ and (c) PCL-3D/NH₂ /HA for 1 day, 3 days and 5 days. Data were expressed as the percentage of PCL-3D (n=3). *p<0.05 compared with PCL-3D groups and **p<0.01 compared with PCL-3D groups.