Graphical abstract
Abstract
Objective
Dental hypersensitivity has become one of the most common and most costly diseases in the world, even though those maladies are very rarely life threatening. Using amorphous microparticles, fabricated from the natural polymer (polyphosphate), we intend to reseal the dentinal tubules exposed and reduce by that the hypersensitivity.
Methods
Amorphous microparticles (termed aCa-polyP-MP) were prepared from Na-polyphosphate (polyP) and CaCl 2 , then incubated with human teeth. The potential of the microparticles to plug the dentinal tubules was determined by microscopic and spectroscopic techniques.
Results
We demonstrate that, in contrast to polyP, the aCa-polyP-MP efficiently reseal dentinal tubules exposed at the tooth surface. Scanning electron microscopical (SEM) and energy dispersive X-ray spectroscopic (EDX) studies showed that the tooth cement and dentin surfaces, incubated with aCa-polyP-MP, form a nearly homogenous, approximately 50-μm thick solid polyP layer on the tooth cement and dentin surfaces, while no coating on the tooth surface, incubated with Na-polyP [Ca 2+ ], was observed. Determination of the mechanical properties of the polyP coating revealed a Martens hardness of 3.85 ± 0.64 GPa and a reduced elastic modulus of 94.72 ± 8.54 GPa already after a 3 h exposure to the aCa-polyP-MP, which become close to those of the natural enamel (4.33 ± 0.69 GPa and 101.61 ± 8.52 GPa, respectively) after prolonged incubation periods. In addition, aCa-polyP-MP turned out to display morphogenetic activity. Incubation of precursor odontoblasts cultures in the presence of aCa-polyP-MP resulted in a 7-fold increase of the steady-state-expression level of the gene encoding for the alkaline phosphatase (ALP) during a 7 d incubation period.
Significance
Ca-polyP microparticles, consisting of the biocompatible natural polymer polyP, provide a potential sealing material for dentinal tubules on the tooth surface.
1
Introduction
As result of the loss of enamel or cementum dentinal tubules are exposed that cause an increased risk of a series of dental diseases/impairments, like dentin hypersensitivity and pulp inflammation (reviewed in: ). In turn, the patients suffer from sharp and severe pain that can cause anxiety. The causative factor of demineralization of enamel and cementum is biofilm formation by bacterial population; there are especially the acid-producing bacteria, like Streptococcus mutans , that cause demineralization. In addition, the enzyme alkaline phosphatase (ALP) contributes also to the destruction of the hard tissue of teeth. Collagen 1A1 (COL1A1) is a shared marker for tracing of both teeth and bone-forming cell differentiation .
The biominerals of the teeth, of enamel, dentin and cementum, are mainly formed of hydroxyapatite (HA) which is chemically a complex salt of (Ca, Z ) 10 (PO 4 , Y ) 6 (OH, X ) 2 , whereby Z can be Na, Mg, K or Sr and Y is CO 3 or HPO 4 and X is Cl or F. Basically, those mineralic components are build like those found in bone . The formation of those inorganic deposits are based upon a genetic building plan that controls the expression of transcription factors, like the homeobox Msx2 , and structural organic skeletal elements, including amelogenin and ameloblastin, which modulate the developmental fate of the cells or enzymes like ALP and carbonic anhydrase ( ; reviewed in: ). Furthermore, self-assembling peptides are known to form a hierarchal self-assembly into which three-dimensional fibrillar scaffolds are formed that might be involved in the control of the initial mineral deposition ; those layers are impaired during caries formation.
In addition to the genetic component driving hard tissue formation, epigenetic factors control the final chemical and mechanical properties of the HA deposits, especially during maturation of the mineral .
Recently, our group disclosed that during the initial biomineralization Ca-carbonate bioseeds are formed that are the product of an enzymatic catalysis by carbonic anhydrase(s). Importantly, the next step during the maturation of the biomineral the exchange of the anions carbonate by phosphate, is solely mediated by a non-enzymatic process along the thermodynamic gradient .
In the course of investigations with the aim to disclose major sources, besides of bone and teeth, for a physiological supply of ortho-phosphate (P i ) required for hard tissue formation in animals and also in mammals, polyphosphate (polyP) turned out to be a promising candidate (recently reviewed: ). PolyP is found in most human cells and is synthesized and/or stored in larger quantities especially in blood platelets as medium-chain polyP (polyP ≈ 40 P i units; reviewed in: ). After activation of platelets the release of polyP is strongly stimulated, resulting in a polyP blood level of 1–3 μM; even higher are the levels in the surrounding platelet-rich thrombi . Basically not surprising was the finding that polyP acts as a morphogenetically active polymer during bone-formation via osteoblasts (reviewed in: ). Recently, we have been succeeded to fabricate nanoparticles/microparticles from polyP and Ca 2+ whose size could be precisely adjusted by a defined P i : Ca 2+ molar ratio of 1:1 or 1:2 . Importantly, the particles formed retained the amorphous state and hence are prone to enzymatic hydrolysis by ALP. Strong experimental evidence could be presented that after exposure of human cells to polyP their intracellular ATP level increases significantly . Besides of these properties of polyP the polymer has been described to display antibacterial function .
The purpose of this study was to evaluate of the function of Ca-polyP nanoparticles/microparticles, fabricated from highly soluble Na-polyP, as efficient resealing polymer for exposed dentinal tubules. In addition, evidence is given indicating that the strong interaction of the polyP particles with the teeth Ca-phosphate is due to a (partial) interaction of the Ca 2+ ions between the tooth material and the added polyP ( Fig. 1 C ). Moreover, it is shown that the Ca-polyP microparticles, fabricated here, elicit morphogenetic activity in precursor odontoblasts by enhancing the expression of the gene encoding for the ALP enzyme. The existence of this enzyme in the pulp and the dentin layer is well established . As a cell expression model we are using the human multipotent stromal cells/mesenchymal stem cells (hMSC) that might give rise to odontoblast-like cells and predominantly to bone-like cells . In turn, the effects of polyP on hMSC reflect a more general potency of this polymer to stimulate biomineralization.
2
Material and methods
2.1
Materials
Na-polyP with an average chain length of ≈40 phosphate units was obtained from Chemische Fabrik Budenheim (Budenheim, Germany).
2.2
Preparation of amorphous Ca-polyP microparticles
The microparticles, composed of Ca-polyP (termed aCa-polyP-MP), were prepared as described . By application of Fourier transform infrared spectroscopy the polymer characteristics of polyP within the microparticles was verified; X-ray diffraction analysis was used to prove that the material is amorphous . The average size of the particles is 300 nm and they vary within the size range of 100–600 nm ( Fig. 1 A and B).
2.3
In vitro incubation of teeth
In order to determine the efficacy of the aCa-polyP-MP to reseal the dentin layer and to check for the potency of the microparticles to occlude the dentinal tubules human teeth were used. They came from 20- to 50-years-old individuals of the Department of Oral and Maxillofacial Surgery of the University Medical Center of the University Mainz; Germany (gift of Prof. Dr. Bilal Al-Nawas; the permission to collect human teeth was obtained from the Ethical Committee of the “University Medical Center of the Johannes Gutenberg University Mainz” – No. 12-05-2015). Prior to use the teeth were mechanically cleaned from soft tissue, treated for 5 h in 3% Na-hypochlorite to remove tissue remains and then stored at 4 °C in a 100% relative humidity chamber.
Teeth specimens were submersed in saline (0.90% [w/v] NaCl) which contained, as mentioned in the text, either 10 mg/mL of aCa-polyP-MP or Na-polyP, stoichiometrically complexed with Ca 2+ (molar ratio of 2:1/phosphate monomer: Ca 2+ ; ). Incubation was performed at 25 °C. Then the samples were sliced by cutting 1–2 mm thick discs inwards to the pulp as described ; where indicated, the dentin or enamel regions, as well as the cement layer, were included in the measurements.
2.4
Microscopic analyses
Scanning electron microscopy (SEM) was performed with a HITACHI SU 8000 (Hitachi High-Technologies Europe GmbH, Krefeld, Germany), equipped with a low voltage (<1 kV; analysis of near-surface organic surfaces ) detector. Where mentioned the samples have been cut . Digital light microscopy was performed with a VHX-600 Digital Microscope (Keyence, Neu-Isenburg; Germany) equipped with a VH-Z100 zoom lens.
2.5
Energy dispersive X-ray spectroscopy
Energy dispersive X-ray (EDX) spectroscopy was performed with an EDAX Genesis EDX System attached to a scanning electron microscope (Nova 600 Nanolab; FEI, Eindhoven, the Netherlands) operating at 10 kV with a collection time of 30–45 s. Areas of approximately 10 μm 2 were analyzed.
2.6
Mechanical-nanoindentation determinations
The surfaces of either the untreated or the polyP-coated teeth specimens were evaluated at 25 °C by depth-sensing indentation, using a NanoTest Vantage system (Micro Materials Ltd., Wrexham; UK). The methods used have been published previously . In one series of experiments the surfaces of the teeth were brushed using an electric toothbrushes (Braun Oral-B PRO 6000; Procter & Gamble, Cincinnati, OH) at 8000 rpm and 100 g force for 3 min at room temperature, as described .
2.7
Reverse transcription-quantitative real-time PCR analyses
To quantify the expression level of the gene encoding the ALP in the hMSC, the technique of reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) was applied. After incubating the cells for 1, 3 and 7 d in the presence of 30 μg/mL either of Na-polyP [Ca 2+ ] or of aCa-polyP-MP they were harvested, RNA was isolated and qRT-PCR was performed . The primer pairs, matching with the human ALP gene (accession number NM_000478.4 ) Fwd: 5′-TGCAGTACGAGCTGAACAGGAACA-3′ [nt 1141 to nt 1164 ] and Rev: 5′-TCCACCAAATGTGAAGACGTGGGA-3′ [nt 1418 to nt 1395 ; PCR product length 278 bp], as well as the COL1A1 gene (NM_000088.3) Fwd: 5′-GACTGCCAAAGAAGCCTTGCC-3′ [nt 5073 to nt 5093 ] and Rev: 5′-TTCCTGACTCTCCTCCGAACCC-3′ [nt 51196 to nt 5175 ; PCR product length 124 bp] and with the reference gene GAPDH ( glyceraldehyde 3-phosphate dehydrogenase ; NM_002046.3) using the primer pair Fwd: 5′-CCGTCTAGAAAAACCTGCC-3′ [nt 845 to nt 863 ] and Rev: 5′-GCCAAATTCGTTGTCATACC-3′ [nt 1059 to nt 1078 ; 215 bp], were used. The amplification was performed in an iCycler (Bio-Rad, Hercules; CA) with the respective iCycler software.
2.8
Cultivation of the human multipotent stromal cells
The human multipotent stromal cells (hMSC) differentiate into odontoblasts in the presence of conditioned medium from developing tooth germ cells; the conditioned medium was prepared as described . The description of the cultivation procedure for the hMSCs was given recently .
2.9
Statistical analysis
After finding that the values follow a standard normal Gaussian distribution, the results were statistically evaluated using paired Student’s t -test .
2
Material and methods
2.1
Materials
Na-polyP with an average chain length of ≈40 phosphate units was obtained from Chemische Fabrik Budenheim (Budenheim, Germany).
2.2
Preparation of amorphous Ca-polyP microparticles
The microparticles, composed of Ca-polyP (termed aCa-polyP-MP), were prepared as described . By application of Fourier transform infrared spectroscopy the polymer characteristics of polyP within the microparticles was verified; X-ray diffraction analysis was used to prove that the material is amorphous . The average size of the particles is 300 nm and they vary within the size range of 100–600 nm ( Fig. 1 A and B).
2.3
In vitro incubation of teeth
In order to determine the efficacy of the aCa-polyP-MP to reseal the dentin layer and to check for the potency of the microparticles to occlude the dentinal tubules human teeth were used. They came from 20- to 50-years-old individuals of the Department of Oral and Maxillofacial Surgery of the University Medical Center of the University Mainz; Germany (gift of Prof. Dr. Bilal Al-Nawas; the permission to collect human teeth was obtained from the Ethical Committee of the “University Medical Center of the Johannes Gutenberg University Mainz” – No. 12-05-2015). Prior to use the teeth were mechanically cleaned from soft tissue, treated for 5 h in 3% Na-hypochlorite to remove tissue remains and then stored at 4 °C in a 100% relative humidity chamber.
Teeth specimens were submersed in saline (0.90% [w/v] NaCl) which contained, as mentioned in the text, either 10 mg/mL of aCa-polyP-MP or Na-polyP, stoichiometrically complexed with Ca 2+ (molar ratio of 2:1/phosphate monomer: Ca 2+ ; ). Incubation was performed at 25 °C. Then the samples were sliced by cutting 1–2 mm thick discs inwards to the pulp as described ; where indicated, the dentin or enamel regions, as well as the cement layer, were included in the measurements.
2.4
Microscopic analyses
Scanning electron microscopy (SEM) was performed with a HITACHI SU 8000 (Hitachi High-Technologies Europe GmbH, Krefeld, Germany), equipped with a low voltage (<1 kV; analysis of near-surface organic surfaces ) detector. Where mentioned the samples have been cut . Digital light microscopy was performed with a VHX-600 Digital Microscope (Keyence, Neu-Isenburg; Germany) equipped with a VH-Z100 zoom lens.
2.5
Energy dispersive X-ray spectroscopy
Energy dispersive X-ray (EDX) spectroscopy was performed with an EDAX Genesis EDX System attached to a scanning electron microscope (Nova 600 Nanolab; FEI, Eindhoven, the Netherlands) operating at 10 kV with a collection time of 30–45 s. Areas of approximately 10 μm 2 were analyzed.
2.6
Mechanical-nanoindentation determinations
The surfaces of either the untreated or the polyP-coated teeth specimens were evaluated at 25 °C by depth-sensing indentation, using a NanoTest Vantage system (Micro Materials Ltd., Wrexham; UK). The methods used have been published previously . In one series of experiments the surfaces of the teeth were brushed using an electric toothbrushes (Braun Oral-B PRO 6000; Procter & Gamble, Cincinnati, OH) at 8000 rpm and 100 g force for 3 min at room temperature, as described .
2.7
Reverse transcription-quantitative real-time PCR analyses
To quantify the expression level of the gene encoding the ALP in the hMSC, the technique of reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) was applied. After incubating the cells for 1, 3 and 7 d in the presence of 30 μg/mL either of Na-polyP [Ca 2+ ] or of aCa-polyP-MP they were harvested, RNA was isolated and qRT-PCR was performed . The primer pairs, matching with the human ALP gene (accession number NM_000478.4 ) Fwd: 5′-TGCAGTACGAGCTGAACAGGAACA-3′ [nt 1141 to nt 1164 ] and Rev: 5′-TCCACCAAATGTGAAGACGTGGGA-3′ [nt 1418 to nt 1395 ; PCR product length 278 bp], as well as the COL1A1 gene (NM_000088.3) Fwd: 5′-GACTGCCAAAGAAGCCTTGCC-3′ [nt 5073 to nt 5093 ] and Rev: 5′-TTCCTGACTCTCCTCCGAACCC-3′ [nt 51196 to nt 5175 ; PCR product length 124 bp] and with the reference gene GAPDH ( glyceraldehyde 3-phosphate dehydrogenase ; NM_002046.3) using the primer pair Fwd: 5′-CCGTCTAGAAAAACCTGCC-3′ [nt 845 to nt 863 ] and Rev: 5′-GCCAAATTCGTTGTCATACC-3′ [nt 1059 to nt 1078 ; 215 bp], were used. The amplification was performed in an iCycler (Bio-Rad, Hercules; CA) with the respective iCycler software.
2.8
Cultivation of the human multipotent stromal cells
The human multipotent stromal cells (hMSC) differentiate into odontoblasts in the presence of conditioned medium from developing tooth germ cells; the conditioned medium was prepared as described . The description of the cultivation procedure for the hMSCs was given recently .
2.9
Statistical analysis
After finding that the values follow a standard normal Gaussian distribution, the results were statistically evaluated using paired Student’s t -test .
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