Biomimetic Coating of Hydroxyapatite on Glycerol Phosphate-Conjugated Polyurethane via Mineralization.

In this study, glycerol phosphate was introduced into polyurethane (PU) to promote the coating stability of hydroxyapatite (HA) during its mineralization on the PU surface. Glycerol phosphate was successfully conjugated with the PU chain during polymerization. Phosphate groups in glycerol phosphate accelerated the nucleation of HA under calcium phosphate ion-rich conditions (concentrated simulated body fluid), resulting in the enhancement of structural stability. The robust interface between HA and PU also improved mechanical properties. Hydrophilic phosphate groups and bioactive HA improved in vitro cellular responses in terms of the attachment and proliferation of L929 fibroblasts and MC3T3-E1 preosteoblasts. Thus, the highly elastic and bioactive PU-gp-HA could be a promising candidate for tissue engineering applications that experience frequent deformation, including diverse cartilage replacements.

Introduction

Elastomers have been widely adopted in long-term tissue engineering fields, such as breast implants, tissue expanders, and cartilage replacements, because most of these materials resist degradation in body fluids, maintaining their inherent viscoelastic mechanical properties.[1−3] One of the representative elastomers for biomedical applications is polyurethane (PU), which exhibits rubber-like elasticity and has an inherent porous structure.[4,5] In addition, the molecular stability of PU is essential to permanent implants for nonregenerative tissues, including breast prosthesis surrounded by soft tissues, and the cartilage or bone of elderly individuals.[6−8] However, the only limitation of PU for use in permanent implants is its low biocompatibility, reflecting its hydrophobic polymer chain.[9] To enhance the biological property of PU, several bioactive coatings were deposited using hydrogels or bioceramics.[10,11]

HA is one of the most qualified biocompatible calcium phosphate minerals, and it has a high affinity to biopolymers and thermodynamic stability in body fluids.[12,13] Thus, there have been numerous efforts to improve the biological property of polymers using various HA-incorporating methods, including the solvent-compression method, simple powder mixing, and mineralization.[14−16] Among these techniques, mineralization is favorable to improve the coating stability and biological properties because HA nucleation is induced at specific functional groups (hydroxyl, carboxyl, and phosphate groups) with strong binding forces.[17,18] However, as PU has no specific sites for interactions with hydrophilic coating layers, coating stability had not been achieved without any further processes. Thus, Yuk et al. attempted to generate a robust interface between elastomers and hydrogels through the application of chemical crosslinking processes.[19] Kim et al. provided nucleation sites on substrates for accelerating apatite growth.[20] Chemically bonded amorphous calcium phosphates grew into hydroxyapatite (HA), forming a stable and homogeneous coating layer.

In this study, glycerol phosphate was introduced as a new conjugate to improve HA coating stability on PU to provide nucleation sites for calcium phosphate ions and accelerate HA mineralization. Glycerol phosphate is a derivative of glycerol, which is a chain extender of PU.[21] Thus, glycerol phosphate, which has a structure similar to that of glycerol, can be easily conjugated with PU through addition without any complex conditions. In addition, the exposed phosphate groups on the polymer chain act as a link between mineralized HA and PU, inducing stable coating systems.

This study addresses an efficient method to fabricate a structurally stable and biologically improved PU-HA composite incorporating glycerol phosphate. The surface morphology, chemical bonding, and crystalline phase were analyzed, and the mechanical and biological properties were also investigated. Moreover, its potential uses, including cartilage or bone replacements, were introduced.

Results and Discussion

Fabrication of PU-gp-HA

PU-gp-HA was synthesized through two steps, PU modification and HA mineralization, as schematically represented (Figure a). First, glycerol phosphate was conjugated with the PU chain during polymerization. Isocyanates in PU prepolymer reacted with hydroxyl groups of water (H—OH), forming a urea linkage, which is the main bonding of the polymer chain. Phosphate functional groups were also exposed on the surface of the polymer chains as a result of another reaction between the isocyanates and hydroxyl groups (R—OH) of glycerol phosphate. Through the competitive reaction between two hydroxyl groups, PU-gp was successfully fabricated and characterized using 31P NMR (Figure b). The peak was shifted from the zero point of standard phosphoric acid to 6.68 ppm, indicating the presence of the phosphate monoester (R-O-PO(OH)2) of glycerol phosphates, whereas no peaks were detected in pure PU, as this compound has no P source or linkage.[22]

Figure 1

(a) Schemes of (1) PU-gp and (2) PU-gp-HA formation; (b) solid-state 31P NMR results according to the existence of glycerol phosphate with chemical structure of designed PU-gp-HA and (c) scanning electron microscope (SEM) images of surfaces of PU-gp-HA at different precipitation times (0, 4, and 12 h) (scale bar = 10 μm).

Second, PU-gp-HA was fabricated through treatment in concentrated simulated body fluid (SBF) solutions for 4 and 12 h. During precipitation, amorphous CaP nanoparticles reversibly formed in the solution. The phosphate groups in PU-gp provided nucleation sites for reversibly existing CaP nanoparticles and calcium and phosphorus ions. The nanoparticles were anchored onto nucleation sites and grown into microspheres with a diameter of 1 μm, which were homogeneously distributed during the 4 h treatment. After 12 h, spherical CaP particles were fully deposited on the substrate, forming flat HA crystal films (Figure c).

X-ray diffraction (XRD) analysis was performed according to the precipitation time (4 and 12 h) and the amounts of glycerol phosphate (0, 2.5, and 10 wt % to polymer) (Figure ). As pure PU has no nucleation sites that gathered CaP nanoparticles, no specific peaks were detected after the 4 h precipitation (see Supporting Information). After 12 h, HA peaks were observed on pure PU, resulting from physically stacked HA sediments. However, as the amounts of glycerol phosphate increased, PU-gp accelerated CaP nucleation and HA crystal growth, which is thermodynamically more stable at a given pH and ion concentration than CaP.[23] Broad HA peaks were observed only for the 4 h treated PU-gp, reflecting the fast nucleation and crystallization process. After 12 h, highly crystalline HA was clearly obtained, showing a high and narrow peak.

Figure 2

XRD peaks of PU-gp-HA (4 and 12 h treated specimens). The asterisk indicates the characteristic peaks of HA. Additionally, the surface morphology change depending on the precipitation time and the amounts of glycerol phosphate was briefly depicted in the Supporting Information.

Mechanically Reinforcing Effects of HA

The mechanical properties of PU, PU-gp, and PU-gp-HA were investigated to understand the effect of conjugated glycerol phosphates and HA on the mechanical properties of PU. A representative stress–strain curve was obtained from the tensile test (Figure ). Failure occurred in the elastic deformation region for all groups. The ultimate tensile strength (UTS), elongation at break, and stiffness are summarized in Table to determine the glycerol phosphate effects on the mechanical properties of PU. Pure PU showed a highly elastic performance, deforming 100.82 ± 8.98%. However, as the concentration of glycerol phosphate increased, the overall mechanical properties decreased. A 2.5 wt % of glycerol phosphate did not deteriorate the innate property of pure PU, whereas a 10 wt % of glycerol phosphate negatively influenced the mechanical properties of PU. UTS decreased to 0.41 ± 0.04 MPa, and the elongation at break simultaneously decreased to 68.62 ± 1.66% in PU-10 wt % gp. The competitive reaction of water and glycerol phosphates primarily contributed to the attenuated mechanical properties. A very large amount of glycerol phosphate hindered chain propagation compared with pure PU, leading to a short polymer chain, and the final product lost its elastic property similar to PU-10 wt % gp, although 2.5 wt % conjugation did not unfavorably affect PU. However, incorporating the HA layer enhanced the strength of PU-gp. The stiffness of PU-2.5 wt % gp significantly increased from 0.69 ± 0.04 MPa (PU-2.5 wt % gp) to 0.80 ± 0.02 MPa (PU-2.5 wt % gp-HA), an even higher value than that of pure PU (0.69 ± 0.05 MPa), demonstrating that the sturdy HA layer partially dissipates the deformation energy. It has been suggested that the robust interface between elastomers and bioceramics induced higher stiffness and strength. Thus, these materials were not just physically stacked, but they electrically interacted with the phosphate groups of PU-gp. In addition, even in PU-10 wt % gp, the HA layer compensated for the weakened strength and stiffness leading to values comparable to those of pure PU. In summary, 2.5 wt % of glycerol phosphate was optimum for improving mechanical properties without any undesirable effects.

Figure 3

Stress–strain curve of PU, PU-x wt % gp, and PU-x wt % gp-HA (x = 2.5, 10) (n = 3).

Table 1

Mechanical Properties (Strength, Strain at Failure, and Stiffness) of PU, PU-gp, and PU-gp-HA

	PU	PU-2.5 wt % gp	PU-2.5 wt % gp-HA	PU-10 wt % gp	PU-10 wt % gp-HA
strength (MPa)	0.69 ± 0.03	0.66 ± 0.12	0.72 ± 0.05	0.41 ± 0.04	0.52 ± 0.06
strain at failure (%)	101 ± 8.98	105 ± 16.4	88.1 ± 10.0	68.6 ± 1.66	64.8 ± 5.08
stiffness (MPa)	0.69 ± 0.05	0.69 ± 0.04	0.80 ± 0.02	0.57 ± 0.04	0.65 ± 0.08

HA Coating Stability

The coating technique is one of the most widely used methods to integrate two or more compounds for improvement of the biological properties of substrates.[24] However, fabricating a durable and long-lasting coating layer is essential and also difficult, particularly in biomedical applications, reflecting frequent deformation under physiological conditions.[25] Thus, additional treatments, such as conjugating another biocompatible agent with the original polymer framework, have been adopted for high affinity with other biolayers.[26] In effect, glycerol phosphate successfully enhanced the coating stability, even in harsh environments. To demonstrate the coating stability, the durability was assessed when exterior forces were applied to PU-gp-HA and PU-HA. The initial amounts of HA were significantly different according to the existence of glycerol phosphate (8.22 ± 2.28% of HA in PU-HA and 14.73 ± 1.94% of HA in PU-gp-HA) (Figure a). After the sonication test, little HA debris was detected on the PU-HA surface, where 80% of HA was detached because the turbulence of sonication was sufficient to delaminate physically deposited HA from PU (Figure b). However, most HA (12.13 ± 0.02% of HA) remained in PU-gp-HA, as the sonication energy was insufficient to disrupt the robust interface between PU-gp and HA. Thus, the coating stability and durability of this compound are advantageous for bioimplants, which should bear frequent movements or impacts, such as meniscal replacements.[27]

Figure 4

(a) Remaining HA amount of PU-HA and PU-gp-HA before and after sonication for 30 min, calculated based on TGA results. Surface morphology of (b) PU-HA and PU-gp-HA after sonication. The asterisk indicates statistically significant p-values of **p < 0.01 with n = 3 (scale bar = 10 μm).

In Vitro Biological Properties of PU, PU-gp, and PU-gp-HA

To demonstrate the potential uses in tissue engineering applications, the in vitro biological properties of PU, PU-gp, and PU-gp-HA were evaluated using L929 fibroblasts (Figure a–c) and MC3T3-E1 preosteoblasts (Figure d–f). On pure PU, the cells were not spread and showed a spherical shape, reflecting the bioinert nature of PU (Figure a,d). However, the cells stretched their filopodia, forming focal adhesions with the phosphate groups in PU-gp (Figure b,e) and bioactive HA of PU-gp-HA (Figure c,f). The degree of proliferation, obtained using an MTS assay, is also indicated in Figure g,h. On day 3, cell viability was not significantly different in all samples because the stage of proliferation was too early to show a clear difference in the number of viable cells. The cell proliferation levels on Pu-gp at day 5 showed relatively low effectiveness of PU-gp in promoting cell proliferation. However, the cells on PU-gp-HA were significantly higher than those on PU-gp at day 5, suggesting that glycerol phosphate provided a hydrophilic and biocompatible surface for cells to attach with elongated or stellate shapes. HA not only provided suitable surfaces for cell adhesion but also enhanced cell viability remarkably. Thus, PU-gp-HA is promising for use in diverse tissue engineering applications, including both soft tissue and hard tissue from in vitro cell tests.

Figure 5

In vitro fibroblast (a–c) and osteoblast (d–f) attachments cultured for 1 day (scale bar = 10 μm) and MTS analysis for 3 and 5 days (g and h). The asterisk indicates statistically significant p-values of *p < 0.05 and **p < 0.01 with n = 3.

Conclusions

The results of this study demonstrated that elastomer-modifying techniques using glycerol phosphate can induce homogeneous HA precipitation, enhance adhesion stability between PU and HA, and improve the inherent biological properties of PU. During precipitation, CaP nanoparticles spontaneously gathered to form phosphate functional groups, acting as nucleation sites for HA. Rapid HA formation and the robust interface reinforced the mechanical properties of PU. The coating stability was also improved through interactions between covalently bonded functional groups and HA. Furthermore, outstanding in vitro biocompatibility indicated a great potential for use in tissue engineering applications, including auricular cartilage replacement and bone regeneration scaffolds.

Materials and Methods

Preparation of PU-gp

Glycerol phosphate solutions were prepared after dissolving glycerol phosphate disodium salt hydrate (C3H7Na2O6P·xH2O, Sigma Aldrich) in distilled water with a weight ratio of 1:80 and 1:20, respectively. The prepolymer of PU (HYPOL 2002; Dow Chemical Co., Ltd., U.K.) and glycerol phosphate solutions were mixed at a weight ratio of 1:2 at room temperature to adjust final compositions (weight ratio of PU/gp = 1:0.025 and 1:0.1). In addition, pure PU was also fabricated as a comparison group through a reaction between distilled water and the prepolymer without glycerol phosphate.

HA Mineralization on PU-gp

For accelerating HA mineralization, highly concentrated ionic solutions were prepared after sequentially dissolving sodium chloride (NaCl, Sigma Aldrich), potassium chloride (KCl, Sigma Aldrich), calcium chloride (CaCl2, Sigma Aldrich), magnesium chloride (MgCl2, Sigma Aldrich), and sodium phosphate monobasic (NaH2PO4, Sigma Aldrich) in water to reach the concentration of 10-fold SBF,[23] PU-gp was immersed in solution for 4 and 12 h after adding sodium bicarbonate (NaHCO3, Sigma Aldrich) to increase the pH to 6.5 at 37 °C.

Characterization of PU, PU-gp, and PU-gp-HA

The chemical structure of PU-gp was investigated using a 500 MHz solid NMR spectrometer (Bruker Advance II) equipped with 4 mm MAS BB-1H. The specimens were prepared as fine particles with dimensions of 60–70 mesh and dried in a vacuum chamber. 31P nuclear detecting channel was acquired at 202.46 MHz using exponential multiplication (EM) window functions. After confirmation of PU-gp synthesis, to verify the effects of glycerol phosphate on HA nucleation, the surface morphology and crystalline structure of PU-gp-HA with different amounts of glycerol phosphate were detected using a field-emission SEM (SUPRA 55 VP; Carl Zeiss, Germany) and XRD with Cu sources (XRD, D8-advance; Bruker Co., Germany) at a scanning rate of 2°/min from 20 to 60°, respectively.

Mechanical Properties of PU, PU-gp, and PU-gp-HA

The mechanical properties of PU, PU-gp (weight ratio of PU/gp was 2.5 and 10 wt % each), and PU-gp-HA (precipitation time was 12 h) were measured using Instron in the tensile mode (Model 5565; Instron Corp., Danvers, MA). The specimens were prepared in rectangular shape with a dimension of 4 mm × 5 mm × 50 mm. The experiments were performed with a constant strain rate of 1 mm/min. The UTS, elongation at break, and stiffness of each group were determined from the stress–strain curves.

Stability Test of PU-gp-HA

To demonstrate the effect of glycerol phosphate on the stability of HA, PU-HA (control) and PU-gp-HA were immersed in water and sonication was performed for 30 min. Specimens with and without sonication were combusted under N2 conditions up to 600 °C with a heating rate of 5 °C/min. The ratio of the remaining HA in PU-HA and PU-gp-HA was calculated using the following equationwhere WHA,r is the weight ratio of HA in PU-HA and PU-gp-HA, WHA is the weight of HA after combustion, Wcomp is the initial weight of PU-gp and PU-gp-HA. Moreover, the surface morphology of PU-HA and PU-gp-HA after sonication was observed using SEM to determine the degree of HA detachments.

Biological Properties of PU, PU-gp, and PU-gp-HA

The biological properties of PU, PU-gp, and PU-gp-HA were analyzed using a preosteoblast cell line (MC3T3-E1, ATCC, CRL-2593) and a fibroblast cell line (L929, derivative of strain L, Mus musculus, mouse). Before the cell tests, the specimens were washed with sterilized distilled water for 3 days in a clean bench. Preosteoblasts and fibroblasts were seeded onto each sample at densities of 2 × 104 and 5 × 104 cells/mL, respectively, for the attachment test. The cells were cultured in an α-minimum essential medium (Welgene, Korea) containing fetal bovine serum (5% for preosteoblasts and 10% for fibroblasts) and 1% penicillin–streptomycin and incubated for 1 day in air containing 5% CO2 at 37 °C. After a day of culturing, the cells were fixed using 2.5% glutaraldehyde after rinsing with phosphate-buffered saline (PBS). Subsequently, the cells were sequentially dehydrated using 75, 95, and 100% ethanol. Finally, the cells were treated with 1,1,1,3,3,3-hexamethyldisilazane for 10 min and dried in a fume hood. The post-treated specimens were observed using SEM. Cell viability was also verified by MTS assay (CellTiter 96 Aqueous One Solution; Promega) using preosteoblasts and fibroblasts. After 3 and 5 days of culturing, post-treatment was conducted after the following steps. The specimens were washed with PBS, transferred to new 24-well tissue culture plates, immersed in α-minimum essential medium with tetrazolium compound dye and incubated at 37 °C for 2 h. The quantity of formazan product as a result of mitochondrial activity in live cells was measured as the absorbance at 492 nm using a microreader (Model 550; Bio-Rad).

Statistical Analysis

All experimental data were expressed as mean ± standard deviation (SD), using three specimens for each group (n = 3). The statistical analysis was performed using one-way analysis of variance (ANOVA). A p-value of <0.05 was considered statistically significant (*p < 0.05, **p < 0.01).