Root Canal Obturation by Electrochemical Precipitation of Calcium Phosphates

Featured Use of boron-doped diamond electrodes for root canal obturation. Abstract: Achieving adequate disinfection and preventing reinfection is the major goal in endodontic treatment. Variation in canal morphology and open porosity of dentine prevents achieving complete disinfection. Questionable biocompatibility of materials as well as a lack of sealing ability questions the usefulness of current obturation methods. With a novel disinfection approach based on the use of boron-doped diamond (BDD) electrodes having shown promising results it was the goal of this series of experiments to investigate the possibility of BDD-mediated in situ forming of a biocompatible obturation material. A combination of calcium phosphate and maleic acid was used as precursor solution while Ion Chromatography Mass Spectrometry (IC-MS), Raman spectroscopy (RAMAN), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), dye penetration and micro-computed tomography ( µ CT) were applied for char-acterizing the precipitate. It was possible to achieve a BDD-mediated precipitation of brushite in a clinically applicable timeframe. However, tight sealing of the canal system based on brushite could not be achieved. shown promising results in this study investigating the possibility of BDD-mediated in situ formation of bio-compatible obturation material. A combination of calcium phosphate and maleic acid was used as precursor solution and BDD-mediated precipitation of brushite was observed within a few minutes. The results obtained may provide an excellent basis for the development of a clinically applicable protocol.


Introduction
Current concepts of root canal treatment involve the basic steps of mechanical instrumentation/shaping, cleaning/disinfection and obturation of the canal space. Reinfection of the canal system has to be prevented by sealing the occlusal access. Despite a variety of methods available such as activated rinsing, disinfection of dentin with non-accessible canals and porosities limits the success of endodontic therapy [1][2][3][4][5][6]. Several authors have pointed out that persisting bacteria may regrow and cause periapical inflammation even if proper obturation has been achieved [7][8][9][10]. In this context, the necessity of root canal obturation in general has been questioned [11]. Current experimental approaches are only focusing on the improvement of either disinfection, debridement or obturation [12,13] but do not present a complete treatment solution.

Precipitation Experiments
The precipitation experiments were performed using wound wire BDD electrodes [14,37,38] or glued wire BDD electrodes designed in this study ( Figure 1). The BDD-coated niobium wires (200 µ m diameter, 2.5 cm length) were produced as previously described [14] and glued together using cyanoacrylate glue (Sofortkleber, Bindulin-Werk H.L. Schönleber GmbH, Fürth, Germany) for a narrow cross section of approximately 500 µ m). For precipitation in reaction tubes, electrodes ( Figure 1) were immersed in CaP precursor solution (2 mL). A constant potential was applied to the electrodes (6-16 V, 2 min) using a laboratory power supply (H/H switch power supply PS2002H, Komerci GmbH & Co.KG, Ebern, Germany). Initiation of precipitation was independent of the used electrode type (glued or wound) but strictly dependent on the applied current. After centrifugation (10,000× g, 10 s) the supernatant was removed and stored for IC-MS measurements. The precipitate was washed three times with deionized water, dried under vacuum in a desiccator (65 °C) and stored (RT, desiccator) for further analysis with SEM, EDX, XRD and RAMAN.
To test the applicability of BDD-mediated precipitation in a restricted volume, endodontic training blocks (HyFlex CM Endo Practice Block, Coltene Whaledent, Altstätten, Switzerland) were prepared using standard reciprocating files (Reciproc R50, VDW, Munich, Germany). The apical opening of the simulated root canal was transitionally sealed using commercially available plasticine. CaP precursor solution was added using a syringe and the glued-wire BDD electrode was inserted into the solution. A constant poten- For precipitation in reaction tubes, electrodes ( Figure 1) were immersed in CaP precursor solution (2 mL). A constant potential was applied to the electrodes (6-16 V, 2 min) using a laboratory power supply (H/H switch power supply PS2002H, Komerci GmbH & Co.KG, Ebern, Germany). Initiation of precipitation was independent of the used electrode type (glued or wound) but strictly dependent on the applied current. After centrifugation (10,000× g, 10 s) the supernatant was removed and stored for IC-MS measurements. The precipitate was washed three times with deionized water, dried under vacuum in a desiccator (65 • C) and stored (RT, desiccator) for further analysis with SEM, EDX, XRD and RAMAN.
To test the applicability of BDD-mediated precipitation in a restricted volume, endodontic training blocks (HyFlex CM Endo Practice Block, Coltene Whaledent, Altstätten, Switzerland) were prepared using standard reciprocating files (Reciproc R50, VDW, Munich, Germany). The apical opening of the simulated root canal was transitionally sealed using commercially available plasticine. CaP precursor solution was added using a syringe and the glued-wire BDD electrode was inserted into the solution. A constant potential (9, 11 and 15 V) was applied to the electrode. During precipitation, the precursor solution was constantly added, and the precipitate was condensed using a plugger ( Figure 2). After the apical third was sealed, excess liquid was removed using paper points (Reciproc R 25, VDW GmbH, Munich, Germany). tial (9, 11 and 15 V) was applied to the electrode. During precipitation, the precursor solution was constantly added, and the precipitate was condensed using a plugger ( Figure  2). After the apical third was sealed, excess liquid was removed using paper points (Reciproc R 25, VDW GmbH, Munich, Germany).

Figure 2.
Obturation procedure for the endo practice blocks. The canals were filled with CaP precursor solution and the glued wire BDD prototype was inserted into the canal. The precipitate was dried and condensed before starting the next precipitation cycle until the apical third was filled with precipitate.
As last approach, the roots from three carious-free extracted human teeth were obtained by horizontal cutting at the cementum-enamel junction using a diamond band saw (EXAKT 300, EXAKT Advanced Technologies, Norderstedt, Germany). Root canals were subsequently prepared using reciprocating files (Reciproc R50, VDW, Munich, Germany) and conventional rinsing (sodium hypochlorite, chlorhexidine, ethanol). The prepared teeth were autoclaved (20 min, 121 °C) in distilled water and stored at ambient temperature until use. The roots were then positioned in modeling clay (Figure 3), CaP precursor solution (20 µL) was added using a syringe and the glued-wire BDD electrode was inserted into the canal. A constant potential (12 V) was applied to the electrode. The precursor solution was constantly added (20 µL, multiple times) to the root canal, after drying and plugging the precipitate from the prior precipitation phase until the root canal was filled completely. The filled roots were dried and stored at ambient temperature until µ CT analysis. Obturation procedure for extracted human teeth. The roots were placed in modeling clay, the CaP precursor was added into the canal using a syringe and the BDD-electrode was applied. Scale bar: 1 cm.

Figure 2.
Obturation procedure for the endo practice blocks. The canals were filled with CaP precursor solution and the glued wire BDD prototype was inserted into the canal. The precipitate was dried and condensed before starting the next precipitation cycle until the apical third was filled with precipitate.
As last approach, the roots from three carious-free extracted human teeth were obtained by horizontal cutting at the cementum-enamel junction using a diamond band saw (EXAKT 300, EXAKT Advanced Technologies, Norderstedt, Germany). Root canals were subsequently prepared using reciprocating files (Reciproc R50, VDW, Munich, Germany) and conventional rinsing (sodium hypochlorite, chlorhexidine, ethanol). The prepared teeth were autoclaved (20 min, 121 • C) in distilled water and stored at ambient temperature until use. The roots were then positioned in modeling clay (Figure 3), CaP precursor solution (20 µL) was added using a syringe and the glued-wire BDD electrode was inserted into the canal. A constant potential (12 V) was applied to the electrode. The precursor solution was constantly added (20 µL, multiple times) to the root canal, after drying and plugging the precipitate from the prior precipitation phase until the root canal was filled completely. The filled roots were dried and stored at ambient temperature until µCT analysis.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of 19 tial (9, 11 and 15 V) was applied to the electrode. During precipitation, the precursor solution was constantly added, and the precipitate was condensed using a plugger ( Figure  2). After the apical third was sealed, excess liquid was removed using paper points (Reciproc R 25, VDW GmbH, Munich, Germany).

Figure 2.
Obturation procedure for the endo practice blocks. The canals were filled with CaP precursor solution and the glued wire BDD prototype was inserted into the canal. The precipitate was dried and condensed before starting the next precipitation cycle until the apical third was filled with precipitate.
As last approach, the roots from three carious-free extracted human teeth were obtained by horizontal cutting at the cementum-enamel junction using a diamond band saw (EXAKT 300, EXAKT Advanced Technologies, Norderstedt, Germany). Root canals were subsequently prepared using reciprocating files (Reciproc R50, VDW, Munich, Germany) and conventional rinsing (sodium hypochlorite, chlorhexidine, ethanol). The prepared teeth were autoclaved (20 min, 121 °C) in distilled water and stored at ambient temperature until use. The roots were then positioned in modeling clay (Figure 3), CaP precursor solution (20 µL) was added using a syringe and the glued-wire BDD electrode was inserted into the canal. A constant potential (12 V) was applied to the electrode. The precursor solution was constantly added (20 µL, multiple times) to the root canal, after drying and plugging the precipitate from the prior precipitation phase until the root canal was filled completely. The filled roots were dried and stored at ambient temperature until µ CT analysis. Obturation procedure for extracted human teeth. The roots were placed in modeling clay, the CaP precursor was added into the canal using a syringe and the BDD-electrode was applied. Scale bar: 1 cm. Obturation procedure for extracted human teeth. The roots were placed in modeling clay, the CaP precursor was added into the canal using a syringe and the BDD-electrode was applied. Scale bar: 1 cm.

SEM/EDX Spectroscopy
Dried samples were analyzed by scanning electron microscopy (SEM, FEI Quanta 450, FEI Deutschland GmbH, Frankfurt, Germany). Images were taken by applying the Everhart-Thornley detector (ETD) back-scatter detector (BSE) combination [40]. The precipitate samples and all comparison samples were screened extensively and elemental analysis was performed with energy-dispersive X-ray spectroscopy (EDX) [41].

Analysis of Maleate Degradation
Maleate degradation in supernatants was monitored applying ion chromatography with an ICS 3000 (Dionex, Thermo Fischer Scientific, Wesel, Germany) and ESI/MS/MS detection using a QTrap3200 Triple-Quadrupole mass spectrometer with turbo V ion source (Applied Biosystems, Thermo Fischer Scientific, Wesel, Germany) operated in multiple reaction monitoring mode.

Dye Penetration Test
Dye penetration tests were performed by submerging previously filled endo practice blocks in MIRA-2-TON plaque disclosing solution (Hager & Werken GmbH & Co. KG, Duisburg, Germany) for 24 h. The outcome was assessed via penetration depth measurements and imaged using a stereomicroscope (Nikon SMZ-745T, Nikon Metrology GmbH, Düsseldorf, Germany).

Imaging Analysis of Precipitate Density
SEM imaging was performed as described above. X-ray imaging was performed using a conventional dental X-ray unit (Heliodent Plus, Dentsply Sirona, Bensheim) and intraoral image plates (VistaScan, Dürr Dental, Bietigheim-Bissingen). Micro-CT measurement for sealing quality was performed using a Siemens Inveon PET/SPECT/CT (Siemens Healthcare GmbH, Erlangen, Germany). The X-ray tube voltage and current were set to 80.0 kV and 500 µA, respectively. The resolution was 13.1 µm at a total scan duration of 39.0 min. The Inveon Acquisition Workplace software was used for process controlling and monitoring. The image analysis was performed using the RadiAnt DICOM Viewer (Medixant, Poznan, Poland) to reconstruct 3D data [7,42,43].
For X-ray micro-tomography characterization of material distribution within the root canal, i.e., porosity of filling, µCT scans were performed on a ZEISS Xradia 500 Versa equipped with a 160 kV transmission tube and an indirect detection system made of a scintillator, switchable objective lenses (0.39×, 4×, 20×) and a CCD camera with 2048 × 2048 pixels. Each tooth was attached to the sample holder with the crown down with no specific preparation being needed. The samples were imaged at a source-to-sample distance of 17 mm and a sample-to-detector distance of 12 mm. The 4× objective and a 2-fold detector binning (1024 × 1024) were used for the acquisition, resulting in an effective pixel size of 4 µm. The tomographic scans included 1801 radiographic projection images that were recorded over a sample rotation of 360 • and with an exposure time of 2 s per image. The tube voltage and power were 80 kV and 6 W, respectively. Tomographic reconstruction of the acquired data and further data processing was carried out with the software X-AID (MITOS GmbH, Garching, Germany). The filtered back-projection algorithm for local tomographies was applied in combination with the beam hardening correction for cupping artefacts. The initial datasets were cropped to 850 × 500 × 500 voxels during reconstruction. Thus, the final volumes consist of 850 stacked images of dimensions 2 × 2 mm and cover a total height of 3.4 mm. The gray values of the reconstructed images relate to differences in X-ray attenuation of the respective materials and certain features like the pores within the filling can be segmented based on grayscale thresholds. In the first step, image noise was reduced by using a bilateral post-processing filter to enable better segmentation. Binary volumes masking the root canals were created the following: (i) Segmenting the tooth material by manual thresholding. (ii) Removing segmented structures of similar grey values within the root canals by applying morphological operations like the opening operation. (iii) Inverting the resulting images. (iv) Removing remaining regions outside of the root canals. Binary volumes representing the pores were obtained by Otsu thresholding on the grey value distributions within the root canals. The volume fractions of the pores within the root canals were determined by relating the voxels belonging to the pores in the root canals to the total number of voxels in the root canals. For the analysis, the sample volumes were divided into blocks of 50 images, corresponding to section of 200 µm. Omitting the first and last sections, 15 values were evaluated over a range of 3 mm for each sample.

Maleic Acid as BDD Electrode-Degradable Retardant
Due to its low solubility calcium phosphate tends to precipitate even in low concentrated solution. Stabilization of such solutions can be reached using Ca 2+ chelators. A precursor solution based on maleic acid was stable over a period of one hour, while precipitation from an originally supersaturated solution was achieved by BDD electrode treatment ( Figure 4). acquired data and further data processing was carried out with the software X-AID (MI-TOS GmbH, Garching, Germany). The filtered back-projection algorithm for local tomographies was applied in combination with the beam hardening correction for cupping artefacts. The initial datasets were cropped to 850 × 500 × 500 voxels during reconstruction. Thus, the final volumes consist of 850 stacked images of dimensions 2 × 2 mm and cover a total height of 3.4 mm. The gray values of the reconstructed images relate to differences in X-ray attenuation of the respective materials and certain features like the pores within the filling can be segmented based on grayscale thresholds. In the first step, image noise was reduced by using a bilateral post-processing filter to enable better segmentation. Binary volumes masking the root canals were created the following: (i) Segmenting the tooth material by manual thresholding. (ii) Removing segmented structures of similar grey values within the root canals by applying morphological operations like the opening operation. (iii) Inverting the resulting images. (iv) Removing remaining regions outside of the root canals. Binary volumes representing the pores were obtained by Otsu thresholding on the grey value distributions within the root canals. The volume fractions of the pores within the root canals were determined by relating the voxels belonging to the pores in the root canals to the total number of voxels in the root canals. For the analysis, the sample volumes were divided into blocks of 50 images, corresponding to section of 200 µ m. Omitting the first and last sections, 15 values were evaluated over a range of 3 mm for each sample.

Maleic Acid as BDD Electrode-Degradable Retardant
Due to its low solubility calcium phosphate tends to precipitate even in low concentrated solution. Stabilization of such solutions can be reached using Ca 2+ chelators. A precursor solution based on maleic acid was stable over a period of one hour, while precipitation from an originally supersaturated solution was achieved by BDD electrode treatment ( Figure 4). To validate that degradation of the stabilizer maleic acid is responsible for calcium phosphate precipitation, further experiments were carried out. A quantitative analysis of the maleate concentration in the supernatants after application of different potentials showed a clear BDD electrode-dependent degradation of maleic acid ( Figure 5). As shown To validate that degradation of the stabilizer maleic acid is responsible for calcium phosphate precipitation, further experiments were carried out. A quantitative analysis of the maleate concentration in the supernatants after application of different potentials showed a clear BDD electrode-dependent degradation of maleic acid ( Figure 5). As shown above, calcium phosphates were precipitated with onset of BDD electrode treatment ( Figure 5). As in the case of maleic acid concentration, a direct correlation of BDD electrode treatment and precipitation of calcium phosphates was found ( Figure 5).
above, calcium phosphates were precipitated with onset of BDD electrode treatment (Figure 5). As in the case of maleic acid concentration, a direct correlation of BDD electrode treatment and precipitation of calcium phosphates was found ( Figure 5).

Figure 5.
Relative maleic acid degradation and precipitate mass production in relation to the applied charge quantity. Black data points: Precipitate mass after two minutes of BDD application to two milliliters of the CaP precursor solution. Different potentials (rsp. currents) were applied to the electrode. Interestingly there is a purported decrease of precipitation efficiency at charge quantities higher than 15 As. This may indicate a lower efficiency of the electrode itself due to surface reduction, as the precipitate is blocking cathode space. Another possible explanation could be an increase in oxygen production leading to a lower degradation of maleate via reactive oxygen species. Mean concentration of maleic acid (grey symbols) and CaP mass (black symbols) depending on mean charge quantities applied is shown; the data indicate that the amount of maleate degradation is directly correlated with the precipitate formation of calcium phosphate.

Analysis of Precipitated Calcium Phosphate Salts
Calcium phosphate salts may differ significantly in their specific composition. The precipitates were therefore further analyzed. As a first approach, precipitates were analyzed via SEM and energy dispersive X-ray spectroscopy (EDX) ( Figure 6). Flower-like crystal structures and the observed average Ca/P ratio of 1 may hint to brushite [40,41]. However, statistical analyses (n = 18) of the results obtained revealed a standard deviation of 0.02, leaving room for the presence of other calcium phosphate salts. Figure 5. Relative maleic acid degradation and precipitate mass production in relation to the applied charge quantity. Black data points: Precipitate mass after two minutes of BDD application to two milliliters of the CaP precursor solution. Different potentials (rsp. currents) were applied to the electrode. Interestingly there is a purported decrease of precipitation efficiency at charge quantities higher than 15 As. This may indicate a lower efficiency of the electrode itself due to surface reduction, as the precipitate is blocking cathode space. Another possible explanation could be an increase in oxygen production leading to a lower degradation of maleate via reactive oxygen species. Mean concentration of maleic acid (grey symbols) and CaP mass (black symbols) depending on mean charge quantities applied is shown; the data indicate that the amount of maleate degradation is directly correlated with the precipitate formation of calcium phosphate.

Analysis of Precipitated Calcium Phosphate Salts
Calcium phosphate salts may differ significantly in their specific composition. The precipitates were therefore further analyzed. As a first approach, precipitates were analyzed via SEM and energy dispersive X-ray spectroscopy (EDX) ( Figure 6). Flower-like crystal structures and the observed average Ca/P ratio of 1 may hint to brushite [40,41]. However, statistical analyses (n = 18) of the results obtained revealed a standard deviation of 0.02, leaving room for the presence of other calcium phosphate salts. Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 19 SEM/EDX analyses indicated brushite as precipitated material. To unequivocally identify the precipitated calcium phosphate species, XRD analyses were carried out. Both, SEM/EDX analyses indicated brushite as precipitated material. To unequivocally identify the precipitated calcium phosphate species, XRD analyses were carried out. Both, data base spectra and spectra of commercially available brushite corresponded exactly to the precipitated material (Figure 7). data base spectra and spectra of commercially available brushite corresponded exactly to the precipitated material ( Figure 7). In a last step, RAMAN spectroscopy was applied for a fast and reliable comparison of precipitates obtained after BDD-induced precipitation [45][46][47]. Electrical potentials between 6 and 16 V applied during the precipitation. Again, data base spectra and spectra of commercially available brushite corresponded exactly to the precipitated material independent of the applied charge ( Figure 8). In a last step, RAMAN spectroscopy was applied for a fast and reliable comparison of precipitates obtained after BDD-induced precipitation [44][45][46][47]. Electrical potentials between 6 and 16 V applied during the precipitation. Again, data base spectra and spectra of commercially available brushite corresponded exactly to the precipitated material independent of the applied charge ( Figure 8). Sci. 2021, 11, x FOR PEER REVIEW 10 of 19

Obturation of Root Canals
The volume available in the root canal cannot bear such an amount of precursor solution which is sufficient for root canal obturation. This resulted in the need for repeatedly applying precursor solution following precipitation and removal of fluids. Brushite initially precipitate at the surface of the BDD electrodes ( Figure 9) but continuing treatment leads to deposition of biomaterial in the canal lumen ( Figure 10). Characterization of these brushite depots by SEM revealed a close connection to the wall of the root canal and sealing of dentin tubules (Figure 11).

Obturation of Root Canals
The volume available in the root canal cannot bear such an amount of precursor solution which is sufficient for root canal obturation. This resulted in the need for repeatedly applying precursor solution following precipitation and removal of fluids. Brushite initially precipitate at the surface of the BDD electrodes ( Figure 9) but continuing treatment leads to deposition of biomaterial in the canal lumen ( Figure 10). Characterization of these brushite depots by SEM revealed a close connection to the wall of the root canal and sealing of dentin tubules (Figure 11). i. 2021, 11, x FOR PEER REVIEW 11 of 19      With brushite being an electron-dense material, obturation of root canals can be verified radiographically. When sealing quality was monitored by X-ray and µCT scans, for endodontic training blocks and extracted human teeth, BDD-mediated root canal obturation was visible in conventional with both methods (Figures 12 and 13). In addition, µCT scans revealed maximal distance below 100 µm between precipitate and canal wall ( Figure 12). With brushite being an electron-dense material, obturation of root canals can be verified radiographically. When sealing quality was monitored by X-ray and µCT scans, for endodontic training blocks and extracted human teeth, BDD-mediated root canal obturation was visible in conventional with both methods (Figures 12 and 13). In addition, µCT scans revealed maximal distance below 100 µm between precipitate and canal wall (Figure 12). Appl. Sci. 2021, 11, x FOR PEER REVIEW 13 of 19  For analysis of porosity, tomographic reconstruction of acquired µ CT data and further data processing was carried out with the software X-AID (Figure 14). A total of 15  For analysis of porosity, tomographic reconstruction of acquired µ CT data and further data processing was carried out with the software X-AID ( Figure 14). A total of 15 For analysis of porosity, tomographic reconstruction of acquired µCT data and further data processing was carried out with the software X-AID ( Figure 14). A total of 15 values were evaluated over a range of 3 mm for each sample (n = 3). For the precipitated material, an overall porosity of 11.27% with a standard deviation of ±7.47% was obtained.
. 2021, 11, x FOR PEER REVIEW 14 of 19 values were evaluated over a range of 3 mm for each sample (n = 3). For the precipitated material, an overall porosity of 11.27 % with a standard deviation of ± 7.47 % was obtained. Figure 14. Porosity analysis of BDD-dependent brushite precipitation. Tomographic reconstruction of µ CT data and further data processing was carried out using the X-AID software (MITOS GmbH, Garching, Germany). Pores are indicated in red, solid material in green.

Dye Penetration Test
Dye penetration tests revealed apical leakage of the obturation material as may have been expected due to the hydrophilic nature of Ca-phosphate ( Figure 15) Figure 14. Porosity analysis of BDD-dependent brushite precipitation. Tomographic reconstruction of µCT data and further data processing was carried out using the X-AID software (MITOS GmbH, Garching, Germany). Pores are indicated in red, solid material in green.

Dye Penetration Test
Dye penetration tests revealed apical leakage of the obturation material as may have been expected due to the hydrophilic nature of Ca-phosphate ( Figure 15) Figure 15. Results of dye penetration tests in endo training blocks following BDD-mediated root canal obturation. As expected, complete penetration was visible in all instances.

Discussion
Using maleic acid [36] as chelating agent, it was possible to establish a chemically stable but highly saturated calcium phosphate solution. Application of a BDD electrode led to the oxidation of maleic acid and precipitation of biocompatible brushite on the electrode surface and inside the root canal. A binding to the root canal wall, as seen in Figure  11, may be the effect of maleic acid as self-etching primer [36].
The reaction for the assumed complex formation which is responsible for the retardation effect is shown in Figure 16. The application of a BDD electrode leads to oxidation of the chelating agent and a following complete decomposition to CO2 [31][32][33][34], releasing the calcium ion from the complex. The free calcium ion the forms solid brushite with the hydrogen phosphate anion as soon as the solubility product is reached. An evolution of a significant amount of toxic byproducts is not expected due to a complete degradation of the maleate to CO2 [31,33,34,36].

Discussion
Using maleic acid [36] as chelating agent, it was possible to establish a chemically stable but highly saturated calcium phosphate solution. Application of a BDD electrode led to the oxidation of maleic acid and precipitation of biocompatible brushite on the electrode surface and inside the root canal. A binding to the root canal wall, as seen in Figure 11, may be the effect of maleic acid as self-etching primer [36].
The reaction for the assumed complex formation which is responsible for the retardation effect is shown in Figure 16. The application of a BDD electrode leads to oxidation of the chelating agent and a following complete decomposition to CO 2 [31][32][33][34], releasing the calcium ion from the complex. The free calcium ion the forms solid brushite with the hydrogen phosphate anion as soon as the solubility product is reached. An evolution of a significant amount of toxic byproducts is not expected due to a complete degradation of the maleate to CO 2 [31,33,34,36]. l. Sci. 2021, 11, x FOR PEER REVIEW 16 of 19 Figure 16. Proposed reaction pathway of complex building and BDD mediated decomposition. The formation of a calcium maleate complex prevents the precipitation in the highly supersaturated solution (referred to as brushite and other calcium phosphates).
The amount of formed precipitate can be controlled by the applied current and application time, and the precipitation currently has a purported efficiency maximum at around 30 % ( Figure 5). Also, the applicability of higher potentials and charge quantity is limited due to heat formation during the precipitation process. Therefore, a lower current and longer precipitation period is more useful.
From a handling perspective, the precipitation was achieved within a reasonable timeframe. The root canal obturation required repeated application of precursor solution, BDD application, drying with paper points and plugging.
The obturation material was closely attached to the canal walls and was visible on Xray images. Due to the hydrophilic nature of Ca phosphate, complete penetration of the filling was possible. It may be argued whether this is problematic [11]. Assuming complete disinfection of root canals prior to obturation, bacteria should have also been eliminated from the periapical region. Penetration of body fluids into the obturation material may be not problematic as brushite is biocompatible in contrast to current sealing materials [15,16].

Conclusions
In addition to the previously discussed applications in endodontic and peri-implantitis treatment, BDD electrodes have shown promising results in this study investigating the possibility of BDD-mediated in situ formation of bio-compatible obturation material. A combination of calcium phosphate and maleic acid was used as precursor solution and BDD-mediated precipitation of brushite was observed within a few minutes. The results obtained may provide an excellent basis for the development of a clinically applicable protocol.  The amount of formed precipitate can be controlled by the applied current and application time, and the precipitation currently has a purported efficiency maximum at around 30% ( Figure 5). Also, the applicability of higher potentials and charge quantity is limited due to heat formation during the precipitation process. Therefore, a lower current and longer precipitation period is more useful.
From a handling perspective, the precipitation was achieved within a reasonable timeframe. The root canal obturation required repeated application of precursor solution, BDD application, drying with paper points and plugging.
The obturation material was closely attached to the canal walls and was visible on X-ray images. Due to the hydrophilic nature of Ca phosphate, complete penetration of the filling was possible. It may be argued whether this is problematic [11]. Assuming complete disinfection of root canals prior to obturation, bacteria should have also been eliminated from the periapical region. Penetration of body fluids into the obturation material may be not problematic as brushite is biocompatible in contrast to current sealing materials [15,16].

Conclusions
In addition to the previously discussed applications in endodontic and peri-implantitis treatment, BDD electrodes have shown promising results in this study investigating the possibility of BDD-mediated in situ formation of bio-compatible obturation material. A combination of calcium phosphate and maleic acid was used as precursor solution and BDDmediated precipitation of brushite was observed within a few minutes. The results obtained may provide an excellent basis for the development of a clinically applicable protocol.

Institutional Review Board Statement:
The teeth used in this study were extracted during regular treatments of various patients and stored for research and educational purposes. Ethics committee approval was not required for the study presented.
Informed Consent Statement: Not applicable.