Structural and dynamical studies of acid-mediated conversion in amorphous-calcium-phosphate based dental composites

Abstract

Objective

To investigate the complex structural and dynamical conversion process of the amorphous-calcium-phosphate (ACP)-to-apatite transition in ACP based dental composite materials.

Methods

Composite disks were prepared using zirconia hybridized ACP fillers (0.4 mass fraction) and photo-activated Bis-GMA/TEGDMA resin (0.6 mass fraction). We performed an investigation of the solution-mediated ACP-to-apatite conversion mechanism in controlled acidic aqueous environment with in situ ultra-small angle X-ray scattering based coherent X-ray photon correlation spectroscopy and ex situ X-ray diffraction, as well as other complementary techniques.

Results

We established that the ACP-to-apatite conversion in ACP composites is a two-step process, owing to the sensitivity to local structural changes provided by coherent X-rays. Initially, ACP undergoes a local microstructural rearrangement without losing its amorphous character. We established the catalytic role of the acid and found the time scale of this rearrangement strongly depends on the pH of the solution, which agrees with previous findings about ACP without the polymer matrix being present. In the second step, ACP is converted to an apatitic form with the crystallinity of the formed crystallites being poor. Separately, we also confirmed that in the regular Zr-modified ACP the rate of ACP conversion to hydroxyapatite is slowed significantly compared to unmodified ACP, which is beneficial for targeted slow release of functional calcium and phosphate ions from dental composite materials.

Significance

For the first time, we were able to follow the complete solution-mediated transition process from ACP to apatite in this class of dental composites in a controlled aqueous environment. A two-step process, suggested previously, was conclusively identified.

Introduction

Amorphous calcium phosphate (ACP) is a unique form of calcium phosphate minerals in organisms . As suggested by its name, the atomic structure of ACP lacks the long-range periodic order of crystalline calcium phosphates. ACP, a metastable phase, is formed as the initial solid phase that precipitates from a highly supersaturated calcium phosphate solution , and is known to be capable of converting to more stable crystalline hydroxyapatite (HAP) phases through a few different transition pathways .

ACP has drawn much attention since its discovery due to its importance in biomineralization research. For example, ACP has been identified as a component of bone along with crystalline apatites . The content of ACP in bone is found to correlate with the age of bone . More recently, several groups of authors have employed Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray absorption near-edge structure micro-spectroscopy, and scanning and transmission electron microscopy to present evidence for ACP being a transient precursor phase to crystalline biominerals in a wide variety of animal systems, including larval and adult echinoderm skeletons , radular teeth of chitons , larval mollusk shells , crustacean cuticles , and the fin bones of (vertebrate) zebrafish . Moreover, studies of nucleation of apatite crystals in vitro suggest that transient ACP is a required intermediate step for the formation of HAP nanocrystal . Despite all this progress, we note that the role of ACP as a precursor phase in biomineralization remains inconclusive due to lack of unquestionable proof. Regardless of this ambiguity, ACP is currently among the most widely studied and used biomineralization agents.

It has long been recognized that HAP is the primary inorganic component of mineralized tooth tissues . Due to the strong connection between ACP and HAP, ACP compounds have been explored as restorative and adhesive dental materials designed to promote remineralization of mineral deficient teeth . For dental applications, ACP has been shown to possess benefits such as better in vivo osteoconductivity and biodegradability than tricalcium phosphate and HAP, good bioactivity, and no cytotoxicity . ACP has also been shown to increase alkaline phosphatase activities of mesoblasts, enhance cell proliferation and promote cell adhesion . The unique role of ACP during the formation of mineralized tooth tissue makes it a promising candidate for dental materials.

To assess these benefits of ACP and make it more relevant to general dentistry, ACP has been incorporated as a filler phase in bioactive polymer composites . In these preventive or restorative dental materials, ACP is encapsulated in a polymer binder, and is capable of slowly releasing in aqueous environments substantial amounts of calcium and phosphate ions in a sustained manner through the transition from ACP to apatitic phases , where the polymer resin serves to slow down the transition, as well as providing the mechanical integrity of the composite material. These composites have been shown to promote the recovery of mineral deficient tooth structure in in vitro situations such as remineralization of artificially produced, caries-like lesions in bovine enamel . The bioactivity of these materials originates from the propensity of ACP, once exposed to oral fluids with fluctuating pH, which include those with acidic low pH, to release calcium and phosphate ions in a sustained manner while spontaneously converting to thermodynamically stable apatitic structures such as HAP . It has also been demonstrated that the released calcium and phosphate ions in saliva milieus create local calcium- and phosphate-enriched super-saturation conditions favorable for the regeneration of tooth mineral lost to decay or wear because these ions can be deposited into tooth structures as apatitic mineral, which is similar to the HAP found naturally in tooth and bone . These features of the ACP-based composite make them very attractive bioactive dental restoration materials, but the detailed structural aspects of this kinetic transformation have yet to be elucidated.

Our previous studies of ACP-based dental materials have been primarily focused on the design of bioactive, non-degradable, biocompatible polymeric composites derived from dental acrylic resins and ACP fillers rendered by photochemical or chemically activated polymerization . While the unambiguous potential of this class of composite materials has clearly been demonstrated through our efforts and the efforts of others, unlike the transformation from ACP to HAP where there is a reasonable understanding of the conversion process, it still remains unclear how polymer encapsulated ACP converts to crystalline calcium phosphate phases in these composite materials. The objective of this work is to improve the understanding of the structural evolution and the fundamental process that governs ACP stability. In particular, we focus our study on the transition of ACP in acidic environments where it is speculated that acidic oral fluids with fluctuating pH values act to assist the conversion of ACP in these composites materials. In practical terms, this is also important because acidic oral challenges (lactic acid from bacteria generated biofilms, citric acid from foods, etc. ) are generally accepted as the primary cause of tooth mineral loss in humans .

To achieve this set of goals, we primarily employ X-ray techniques including X-ray photon correlation spectroscopy (XPCS), ultra-small angle X-ray scattering (USAXS), and X-ray diffraction (XRD) to evaluate the effect of acidic challenges on structural changes that occur in ACP in both composites and as a filler phase without the polymer matrix. These X-ray results were complemented by in situ Fourier transform infrared spectroscopy (FTIR) studies, where special attention was given to a region between 630 cm −1 and 500 cm −1 , where a peak associated with phosphate bending can be found. We focused on this region because it is most sensitive to the conversion. It is our hope that such “in-depth” structural studies performed on the ACP polymer composite, ACP filler alone, and composite thin films under identical experimental regimens will provide better understanding of the underlying mechanisms that govern demineralization/remineralization phenomena associated with dental hard tissue structural decay and its healing.

In the next section, we will briefly introduce the details of the material synthesis and the characterization techniques. We will then discuss the data reduction and analysis procedure, followed by a presentation of our detailed experimental results and finally provide some concluding remarks.

Materials and methods

Materials

Zirconia modified ACP fillers were synthesized following the protocol of Eanes . Pyrophosphate-stabilized ACP was precipitated at room temperature during rapid mixing of equal volumes of an 800 mmol/L Ca(NO 3 ) 2 solution and a solution of 536 mmol/L Na 2 HPO 4 incorporating 1.37 mmol/L fraction Na 4 P 2 O 7 and an appropriate volume of a 250 mmol/L ZrOCl 2 solution (0.1 mole fraction of ZrOCl 2 based on the Ca reactant). Zr was found to effectively hinder the transformation of ACP to HAP . For the purpose of comparison, pure ACP powders were also synthesized following the same protocol without the addition of ZrOCl 2 . We identify these unmodified ACP powders as pure ACP.

The polymer matrix was formulated from commercially available dental monomers and photo-initiators used for visible light polymerization. The monomers 2,2-bis[(p-2′-hydroxy-3′-methacryloxypropoxy)phenyl]-propane (Bis-GMA) and triethyleneglycol dimethacrylate (TEGDMA) were used in a 50:50 mass ratio as base and diluent monomer, respectively. In this formulation Bis-GMA acts to reduce photopolymerization-induced volumetric shrinkage and enhance resin reactivity, while TEGDMA enables improved vinyl double bond conversion . Bis-GMA/TEGDMA resin was photo-activated by the inclusion of camphorquinone (mass fraction 0.002) and ethyl-4,4-N,N-dimethylaminobenzoate (mass fraction 0.008). The names, acronyms, sources of the monomers, components of the photo-initiator system and fillers are listed in Table 1 . 1

1 Certain commercial materials and equipment are identified in this paper only to specify adequately the experimental procedure. In no case does such identification imply recommendation by NIST nor does it imply that the material or equipment identified is necessarily the best available for this purpose.

Table 1
Chemical names, acronyms, and sources of the composite components.
Acronym Name Source
Bis-GMA 2,2-bis[(p-2′-hydroxy-3′-methacryloxypropoxy)phenyl]-propane Esstech, Essington, PA, USA
TEGDMA Triethyleneglycol dimethacrylate Esstech, Essington, PA, USA
CQ Camphorquinone Sigma–Aldrich, St. Louis, MO, USA
4EDMAB Ethyl-4,4-N,N-dimethylaminobenzoate Sigma–Aldrich, St. Louis, MO, USA
ACP Amorphous calcium phosphate Volpe Research Center
HAP Hydroxyapatite Volpe Research Center

Composite pastes were formulated by hand-mixing photo-activated Bis-GMA/TEGDMA resin (0.6 mass fraction) and filler (0.4 mass fraction). The pastes were mixed until a uniform consistency was achieved (with no remaining large aggregates of the filler particles visible) and then kept under moderate vacuum (2.7 kPa) overnight to remove air trapped inside the pastes during mixing. The pastes were molded to form disks (≈ 10 mm diameter, ≈ 1 mm in thickness) by filling the circular openings of flat Teflon molds. The filled molds were covered with Mylar™ films and glass slides, and then clamped tightly with spring clips. The composite disks were polymerized by means of a 120 s photo-polymerization procedure, using a commercial visible light source (Triad 2000, Dentsply International, York, PA, USA). A minimum of ten disks were made for each type of specimen so that measurements performed on samples from the same batch were directly comparable. Pure Bis-GMA/TEGDMA resin disks (without fillers) were also prepared following the same specimen preparation procedure. The established synthesis protocol was described in detail elsewhere.

Characterization Methods

Ultra-small angle X-ray scattering measurements

Ultra-small angle X-ray scattering studies were conducted using the USAXS instrument at sector 15-ID at the Advanced Photon Source (APS), Argonne National Laboratory, IL . The schematic of the USAXS instrument is shown in Fig. 1 . This instrument employs Bonse–Hart-type double-crystal optics to extend the scattering vector q range of small-angle X-ray scattering (SAXS) down to 1 × 10 −4 −1 (where q = (4 π / λ )sin( θ )), where λ is the X-ray wavelength, and 2 θ is the scattering angle. We used collimated monochromatic X-rays in transmission geometry to measure the scattering intensity as a function of q . The X-ray energy was 10.5 keV, corresponding to an X-ray wavelength of 1.18 Å. The instrument was operated in 2D collimated mode with beam-defining slits set at 0.5 mm × 0.5 mm . USAXS measurements were performed in the q range from 10 −4 −1 to 10 −1 −1 . The q resolution was approximately 1.5 × 10 −4 −1 and the incident photon flux on the sample was on the order of 1 × 10 12 photons s −1 . Data were collected at 150 points. Data points were logarithmically distributed through the q range, and data collection time for each data point was 1 s.

Fig. 1
Schematic of the USAXS and USAXS-XPCS configurations at the APS USAXS instrument. Both configurations make use of the same set of Si (2 2 0) optics consisting of both vertical and horizontal collimating and analyzing crystal pairs. We used four reflections off horizontal crystal pairs and two reflections off vertical crystal pairs. The main difference resides in the pre-optics slits. In the case of USAXS, the slits define the beam while for USAXS-XPCS, the slits serve as a secondary coherent source which provides the partially coherent illumination as received by the sample.

For USAXS and subsequent ultra-small angle X-ray scattering – X-ray photon correlation spectroscopy (USAXS-XPCS) measurements, we cut a piece of sample from the sample disk. The sample dimension was approximately 1 mm × 1 mm × 10 mm. We placed this cut sample inside a glass capillary with an outer diameter of 1.5 mm. We also filled the capillary with HCl + H 2 O solution of chosen strength while establishing that the sample was fully submerged inside the acid. We sealed the filled capillary with wax and placed it vertically in the beam. We further aligned the X-ray beam at the center of the sample for our scattering measurements.

USAXS-XPCS measurements

We conducted the USAXS-XPCS measurements with the USAXS instrument at the APS. The instrumental configuration for this type of coherent scattering experiment was described previously . Most notably, as illustrated in Fig. 1 , we placed a pair of 15 μm × 15 μm coherence-defining slits in front of the collimating crystals as a secondary coherent source. Samples were loaded into the capillaries as described above. We followed an established procedure to monitor the nonequilibrium dynamics of samples using a scan mode of USAXS-XPCS, which is detailed in Supplementary Materials.

X-ray diffraction

The X-ray diffraction (XRD) measurements of the composites were made using an X-ray diffractometer (D8, Bruker, Woodlands, TX, USA) utilizing Cu K α radiation (X-ray energy = 8.047 keV, wavelength = 1.541 Å) and incorporating a 2D position-sensitive multiwire detector (Hi-Star, Bruker, Woodlands, TX, USA). Zr-modified ACP/polymer resin composite samples, in the form of thin disks, approximately 1 mm thick and 10 mm in diameter, were soaked for a specified time in a selected HCl acid molar concentration, rinsed with water and dried, mounted and held on a glass slide by vacuum on the D8 vertical sample stage. Measurements were made in a reflection geometry optimized for a mean position sensitive detector (PSD) scattering angle, 2 θ = 42.5°, and with the normal to the sample plane bisecting this angle. The PSD collected data over the scattering angle range: 25° < 2 θ < 60° ( q = 3.45–7.06 Å −1 ) over a 1 h count time for each sample run. The incident slit collimation was a 1 mm pinhole, and each sample was oscillated by ±2 mm within its own plane (perpendicular to q ) in both the horizontal and vertical directions to ensure a statistically representative volume was sampled during each XRD measurement.

The XRD measurements of powder ACP samples were made using a Siemens D500 X-ray diffractometer equipped with a Johansson monochromator that eliminates the K α 2 component of the Cu K α radiation. We set the incident slit size at 0.68° and the receiving slit size at 0.15°. For both the pure ACP and Zr-modified ACP powders, 1 mL of 0.10 M HCl was added to (50 ± 1) mg of powder and the mixture was left overnight to react. We chose the duration of this reaction to be similar to the time span of the XPCS measurements. The acid was syphoned off, and the residue was dried at 100 °C overnight. The fractional mass loss caused by this processing was similar (0.28 for pure ACP and 0.25 for Zr-ACP). The resulting powder was compacted into disk-shaped sample cells. XRD measurements were conducted over the scattering angle range of 20° < 2 θ < 70° ( q = 1.42–4.68 Å −1 ) with a fixed step size of 0.025°, over a 1 h count time for each sample run. In addition, semi-quantitative energy dispersive spectroscopy (EDS) was carried out on the treated powders.

Materials and methods

Materials

Zirconia modified ACP fillers were synthesized following the protocol of Eanes . Pyrophosphate-stabilized ACP was precipitated at room temperature during rapid mixing of equal volumes of an 800 mmol/L Ca(NO 3 ) 2 solution and a solution of 536 mmol/L Na 2 HPO 4 incorporating 1.37 mmol/L fraction Na 4 P 2 O 7 and an appropriate volume of a 250 mmol/L ZrOCl 2 solution (0.1 mole fraction of ZrOCl 2 based on the Ca reactant). Zr was found to effectively hinder the transformation of ACP to HAP . For the purpose of comparison, pure ACP powders were also synthesized following the same protocol without the addition of ZrOCl 2 . We identify these unmodified ACP powders as pure ACP.

The polymer matrix was formulated from commercially available dental monomers and photo-initiators used for visible light polymerization. The monomers 2,2-bis[(p-2′-hydroxy-3′-methacryloxypropoxy)phenyl]-propane (Bis-GMA) and triethyleneglycol dimethacrylate (TEGDMA) were used in a 50:50 mass ratio as base and diluent monomer, respectively. In this formulation Bis-GMA acts to reduce photopolymerization-induced volumetric shrinkage and enhance resin reactivity, while TEGDMA enables improved vinyl double bond conversion . Bis-GMA/TEGDMA resin was photo-activated by the inclusion of camphorquinone (mass fraction 0.002) and ethyl-4,4-N,N-dimethylaminobenzoate (mass fraction 0.008). The names, acronyms, sources of the monomers, components of the photo-initiator system and fillers are listed in Table 1 . 1

1 Certain commercial materials and equipment are identified in this paper only to specify adequately the experimental procedure. In no case does such identification imply recommendation by NIST nor does it imply that the material or equipment identified is necessarily the best available for this purpose.

Table 1
Chemical names, acronyms, and sources of the composite components.
Acronym Name Source
Bis-GMA 2,2-bis[(p-2′-hydroxy-3′-methacryloxypropoxy)phenyl]-propane Esstech, Essington, PA, USA
TEGDMA Triethyleneglycol dimethacrylate Esstech, Essington, PA, USA
CQ Camphorquinone Sigma–Aldrich, St. Louis, MO, USA
4EDMAB Ethyl-4,4-N,N-dimethylaminobenzoate Sigma–Aldrich, St. Louis, MO, USA
ACP Amorphous calcium phosphate Volpe Research Center
HAP Hydroxyapatite Volpe Research Center

Composite pastes were formulated by hand-mixing photo-activated Bis-GMA/TEGDMA resin (0.6 mass fraction) and filler (0.4 mass fraction). The pastes were mixed until a uniform consistency was achieved (with no remaining large aggregates of the filler particles visible) and then kept under moderate vacuum (2.7 kPa) overnight to remove air trapped inside the pastes during mixing. The pastes were molded to form disks (≈ 10 mm diameter, ≈ 1 mm in thickness) by filling the circular openings of flat Teflon molds. The filled molds were covered with Mylar™ films and glass slides, and then clamped tightly with spring clips. The composite disks were polymerized by means of a 120 s photo-polymerization procedure, using a commercial visible light source (Triad 2000, Dentsply International, York, PA, USA). A minimum of ten disks were made for each type of specimen so that measurements performed on samples from the same batch were directly comparable. Pure Bis-GMA/TEGDMA resin disks (without fillers) were also prepared following the same specimen preparation procedure. The established synthesis protocol was described in detail elsewhere.

Characterization Methods

Ultra-small angle X-ray scattering measurements

Ultra-small angle X-ray scattering studies were conducted using the USAXS instrument at sector 15-ID at the Advanced Photon Source (APS), Argonne National Laboratory, IL . The schematic of the USAXS instrument is shown in Fig. 1 . This instrument employs Bonse–Hart-type double-crystal optics to extend the scattering vector q range of small-angle X-ray scattering (SAXS) down to 1 × 10 −4 −1 (where q = (4 π / λ )sin( θ )), where λ is the X-ray wavelength, and 2 θ is the scattering angle. We used collimated monochromatic X-rays in transmission geometry to measure the scattering intensity as a function of q . The X-ray energy was 10.5 keV, corresponding to an X-ray wavelength of 1.18 Å. The instrument was operated in 2D collimated mode with beam-defining slits set at 0.5 mm × 0.5 mm . USAXS measurements were performed in the q range from 10 −4 −1 to 10 −1 −1 . The q resolution was approximately 1.5 × 10 −4 −1 and the incident photon flux on the sample was on the order of 1 × 10 12 photons s −1 . Data were collected at 150 points. Data points were logarithmically distributed through the q range, and data collection time for each data point was 1 s.

Fig. 1
Schematic of the USAXS and USAXS-XPCS configurations at the APS USAXS instrument. Both configurations make use of the same set of Si (2 2 0) optics consisting of both vertical and horizontal collimating and analyzing crystal pairs. We used four reflections off horizontal crystal pairs and two reflections off vertical crystal pairs. The main difference resides in the pre-optics slits. In the case of USAXS, the slits define the beam while for USAXS-XPCS, the slits serve as a secondary coherent source which provides the partially coherent illumination as received by the sample.

For USAXS and subsequent ultra-small angle X-ray scattering – X-ray photon correlation spectroscopy (USAXS-XPCS) measurements, we cut a piece of sample from the sample disk. The sample dimension was approximately 1 mm × 1 mm × 10 mm. We placed this cut sample inside a glass capillary with an outer diameter of 1.5 mm. We also filled the capillary with HCl + H 2 O solution of chosen strength while establishing that the sample was fully submerged inside the acid. We sealed the filled capillary with wax and placed it vertically in the beam. We further aligned the X-ray beam at the center of the sample for our scattering measurements.

USAXS-XPCS measurements

We conducted the USAXS-XPCS measurements with the USAXS instrument at the APS. The instrumental configuration for this type of coherent scattering experiment was described previously . Most notably, as illustrated in Fig. 1 , we placed a pair of 15 μm × 15 μm coherence-defining slits in front of the collimating crystals as a secondary coherent source. Samples were loaded into the capillaries as described above. We followed an established procedure to monitor the nonequilibrium dynamics of samples using a scan mode of USAXS-XPCS, which is detailed in Supplementary Materials.

X-ray diffraction

The X-ray diffraction (XRD) measurements of the composites were made using an X-ray diffractometer (D8, Bruker, Woodlands, TX, USA) utilizing Cu K α radiation (X-ray energy = 8.047 keV, wavelength = 1.541 Å) and incorporating a 2D position-sensitive multiwire detector (Hi-Star, Bruker, Woodlands, TX, USA). Zr-modified ACP/polymer resin composite samples, in the form of thin disks, approximately 1 mm thick and 10 mm in diameter, were soaked for a specified time in a selected HCl acid molar concentration, rinsed with water and dried, mounted and held on a glass slide by vacuum on the D8 vertical sample stage. Measurements were made in a reflection geometry optimized for a mean position sensitive detector (PSD) scattering angle, 2 θ = 42.5°, and with the normal to the sample plane bisecting this angle. The PSD collected data over the scattering angle range: 25° < 2 θ < 60° ( q = 3.45–7.06 Å −1 ) over a 1 h count time for each sample run. The incident slit collimation was a 1 mm pinhole, and each sample was oscillated by ±2 mm within its own plane (perpendicular to q ) in both the horizontal and vertical directions to ensure a statistically representative volume was sampled during each XRD measurement.

The XRD measurements of powder ACP samples were made using a Siemens D500 X-ray diffractometer equipped with a Johansson monochromator that eliminates the K α 2 component of the Cu K α radiation. We set the incident slit size at 0.68° and the receiving slit size at 0.15°. For both the pure ACP and Zr-modified ACP powders, 1 mL of 0.10 M HCl was added to (50 ± 1) mg of powder and the mixture was left overnight to react. We chose the duration of this reaction to be similar to the time span of the XPCS measurements. The acid was syphoned off, and the residue was dried at 100 °C overnight. The fractional mass loss caused by this processing was similar (0.28 for pure ACP and 0.25 for Zr-ACP). The resulting powder was compacted into disk-shaped sample cells. XRD measurements were conducted over the scattering angle range of 20° < 2 θ < 70° ( q = 1.42–4.68 Å −1 ) with a fixed step size of 0.025°, over a 1 h count time for each sample run. In addition, semi-quantitative energy dispersive spectroscopy (EDS) was carried out on the treated powders.

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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Structural and dynamical studies of acid-mediated conversion in amorphous-calcium-phosphate based dental composites
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