The application of biomaterials used in regenerative endodontics should be traceable. In this study, we checked some basic effects of rendering a fibrin hydrogel radiopaque using an iodine-based contrast agent (iodixanol) approved for systemic application. Fibrin hydrogels were prepared from a fibrin sealant (Tisseel) using either an isotonic iodixanol solution (Visipaque 320, test) or Tris buffer (control) as a diluent. Gelation kinetics, radiopacity, and swelling of lyophilized hydrogels were tested using standard methods. Hydrogel structure was evaluated using scanning electron microscopy (SEM). Furthermore, iodixanol release from the test gels was assessed using spectrophotometry, and tissue compatibility was compared between test and control hydrogels using the chick chorioallantoic membrane (CAM) assay. Results were compared using pairwise t-test, p < 0.05. Iodixanol caused a 70-fold delay in gelation to 26 min in the test compared to the control hydrogels (22 ± 1 s). Radiopacity of the test gels was 1.9 ± 0.2 mm Al/mm, compared to zero in the control hydrogels. Lyophilized hydrogel swelling was strongly reduced when iodixanol was added to the hydrogel (p < 0.05). Test hydrogels had an altered SEM appearance compared to controls, and exhibited a reduced porosity. Iodixanol release from the test hydrogels reached 14.5 ± 0.5% after 120 h and then ceased. This release did not have any apparent toxic effect and neither affected the viability, nor the physiology or vascularization of the CAM of fertilized chicken eggs. Iodixanol can render a fibrin hydrogel radiopaque and maintains its tissue compatibility, yet impacts gelation kinetics and hydrogel porosity.

#### Keywords: fibrin gel, regenerative endodontics, iodixanol, contrast agent, tooth

### INTRODUCTION

An exciting new field has emerged in endodontic research over the recent years: Regenerative Endodontics (Hargreaves, 2016). While attempts to attract soft tissues into the necrotic root canal space are not necessarily new (Nygaard Ostby, 1963), the systematic approach in the context of current tissue engineering concepts surely is. Different paths are followed that vary from pure basic science to translational medicine with the development and improvement of clinically applicable protocols. However, the passage to the clinics necessitates specific precautions for tissue engineering concepts and related products (Mao et al., 2012). These include issues such as treatment costs, safety, and also the regulation of medical devices by local administrative bodies.

#### Edited by:

David P. Rice, University of Helsinki, Finland

#### Reviewed by:

Jean-Christophe Farges, Claude Bernard University Lyon 1, France Aránzazu Díaz-Cuenca, Consejo Superior de Investigaciones Científicas (CSIC), Spain

\*Correspondence:

Matthias Zehnder<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology

Received: 11 January 2017 Accepted: 27 February 2017 Published: 15 March 2017

#### Citation:

Hertig G, Zehnder M, Woloszyk A, Mitsiadis TA, Ivica A and Weber FE (2017) Iodixanol as a Contrast Agent in a Fibrin Hydrogel for Endodontic Applications. Front. Physiol. 8:152. doi: 10.3389/fphys.2017.00152

Currently, in the context of pulp tissue engineering, a so-called cell-free or cell-homing approach is seen as favorable (Kim et al., 2010; Lee et al., 2010). This approach involves the conditioning of the root canal wall by EDTA to release molecular cues for the attraction and differentiation of invading pluripotent cells, followed by the application of a scaffold in the pulp space (Galler, 2016). In the current clinical protocol, this scaffold is a blood clot, which is generated by controlled bleeding through the apical foramen (Trope, 2010). Clinical outcomes with this technique vary (Chen et al., 2012), and negative reports have curbed the initial enthusiasm (Nosrat et al., 2012). Discoloration of the crown is a frequent observation, and continued growth of the root is not predictably achieved (Kahler et al., 2014; Simon et al., 2014).

The procedure of "controlled bleeding" per se is problematic from a clinical point of view, mainly for two reasons: Hemorrhage has a negative effect on tooth color (Marin et al., 1997), and the whole procedure and its state-of-the-art execution are hard if not impossible to be monitored. Proper case documentation, however, is the core of good clinical practice. Recent approaches to replace the controlled bleeding step in regenerative endodontics involved the application of a fibrin hydrogel into the empty pulp space in order to create a synthetic, non-staining blood clot (Ruangsawasdi et al., 2014). The fibrin hydrogel could be placed in a controlled manner using a microcannula or a lentulo spiral. However, it would be desirable to obtain a radiopaque hydrogel for better controlling and monitoring of the procedure. In the current study we investigated the effects of a biocompatible iodinated X-ray contrast agent systemically used in angiography on some basic properties of a fibrin hydrogel intended for endodontic applications.

## MATERIALS AND METHODS

### Preparation of Hydrogels

Hydrogels were prepared from a commercially available fibrin sealant (Tisseel, Baxter; Deerfield, IL). The two components of the sealant were diluted to obtain a control fibrin hydrogel as previously described (Ruangsawasdi et al., 2017). To prepare the control hydrogels, the thrombin component and the fibrinogen component were diluted in Tris-buffered saline (TBS) and then mixed as described below. In the test hydrogels, the TBS was replaced by an aqueous isotonic contrast agent (Visipaque 320, GE Healthcare, Cork, Ireland) containing 652 mg of iodixanol per mL of liquid, which equals 320 mg/mL of iodine. The pH of the two solutions was measured using a calibrated electrode (Metrohm, Herisau, Switzerland). The pH of TBS was 7.6, while the pH of Visipaque 320 equaled 7.1.

### Gelation Kinetics

Fibrin hydrogels gelate via fibrin clotting. Clotting time was tested according to the method described by Vermylen et al. (1963). The two hydrogel components were prepared separately in micro-centrifugation tubes in a water bath at 37◦C. For 250 µL of the thrombin solution 2 µL of thrombin were added to 248 µL of TBS or Visipaque; 250 µL of fibrinogen solution were prepared by adding 44 µL of fibrinogen to 206 µL of TBS or Visipaque. Pre-warmed thrombin solution was transferred to the fibrinogen counterpart using this pipette. The agents were mixed using a pipette. The micro-centrifugation tubes were kept in the water bath at 37◦C during the whole experiment. A platinum loop was moved in and out of the clotting mixture until the appearance of the fibrin web, which marked the end point (hydrogelation). The time to reach this point was recorded using a stopwatch. These experiments were performed in triplicates with freshly prepared solutions.

### Assessment of Radiopacity

Test and control hydrogels were filled into polycarbonate molds 10 mm in diameter and 1 mm thick. Three molds were filled per experiment. Slices with a thickness of 1 mm from the crowns of bovine teeth served as control for the clear distinction between material and the surrounding tissue in clinical situations. Specimens were placed on a radiographic sensor (Digora, Soredex, Tuusula, Finland) together with an aluminum step-wedge with a thickness from 0.5 to 6 mm (0.5 and 1 mm increments, **Figures 1a,b**). A Trophy Irix (Trophy, Paris, France) X-ray unit operating at 65 kV, 8 mA, and 0.22 s with a focus-film distance of 25 cm was used. Hydrogels were prepared immediately prior to use. These triplicate measurements were performed three times each with freshly prepared hydrogels. Digital radiographic images were imported with the Digora system, using Digora software version 1.51 for Windows without gray scale correction. Optimas image analysis software (Meyer Instruments Inc., Houston, TX) was used to determine the gray value of the samples and convert these to mm of aluminum equivalent (mm Al). To assess the relative radiopacity of the test fibrin hydrogel in human teeth, the root canals of premolars extracted for orthodontic reasons were prepared using

FIGURE 1 | Digital radiographs of the test (a,c) and the control fibrin hydrogel (b) under investigation. A cross-section of a human incisor crown was used as a reference. A photograph of the set-up is depicted in the insert. The aluminum step-wedge had a thickness of 0.5, 1, 1.5, 2, and then 3 mm Al, followed by 1 mm steps. As can be appreciated from this figure, the test hydrogel (a) had a radiopacity of ∼2 mm Al/mm, whilst the control hydrogel (b) displayed no radiopacity (dotted lines). Freshly mixed test hydrogel containing iodixanol was placed in the root canal system of immature human premolars using a lentulo spiral (c). This test hydrogel apparently filled the whole canal space and could be clearly discerned from root dentin.

the ProTaper system (Sirona Dentsply Endodontics, Ballaigues, Switzerland) and filled with freshly mixed hydrogel using a lentulo spiral. The teeth were radiographed as described above.

### Swelling of Hydrogels

The water-absorbing capacity (swelling) was calculated gravimetrically according to the formula:

Swelling ratio = (ws − wd)/wd

where ws is the weight of the swollen hydrogel and wd is the weight of the dry hydrogel. The hydrogels were prepared with TBS or Visipaque, frozen at −80◦C overnight and freeze-dried under vacuum for 24 h until constant weight in a lyophilization apparatus (Christ, Osterode am Harz, Germany). The dry hydrogels were pre-weighed in a precision balance (PE360; Mettler Toledo, Greifensee, Switzerland) and were immersed in 5 mL of TBS at 37◦C for 5 min, 1, 8, and 24 h. After removing them from the TBS at each time point, the hydrogels were gently wiped with a filter paper and weighted again. Subsequently, the hydrogels were immersed in fresh TBS. The swelling ratio was measured by comparing the weight of hydrogels before and after immersing in TBS according to the equation. These experiments were performed in triplicates.

### Scanning Electron Microscopy

To visualize the microstructure of the test and the control hydrogels under investigation, triplicates of lyophilized specimens were inspected using scanning electron microscopy (SEM). To this end, the freeze-dried hydrogels were attached to sticky carbon pads (Plano, Wetzlar, Germany) on SEM pin stubs. Specimens were sputtered with an 8 nm gold layer (Safematic, Bad Ragaz, Switzerland). Images were obtained at 5 mV acceleration voltage and 12 mm working distance using a secondary electron detector (Zeiss Supra 50VP, Oberkochen, Germany).

### Release of Iodixanol from Test Hydrogels

The UV spectrum of an iodixanol solution in water shows a maximum at 244 nm and a specific absorption coefficient of 320 l g−1 cm−1 at that wavelength (Schroder et al., 1997). To test the release of iodixanol from the test hydrogels, 1 mL of hydrogel was mixed with 49 mL of TBS in 50 mL polypropylene centrifugation tubes (Falcon, Thermo Fisher Scientific, Waltham, MA). These tubes were rotated at room temperature (25◦C) at 6 rpm in an overhead rotator with a radius of 10 cm (Bibby Scientific, Staffordshire, UK). 100 µL of the solution were removed (and not replenished) to be assessed spectrophotometrically for their iodixanol content after 30 min, 1, 3, 8, 24, 48, 72, 120, and finally after 168 h. Experiments were done in triplicates. Iodixanol that washed out from the hydrogel was quantified at 244 nm in a spectrophotometer (Shimadzu, Kyoto, Japan) in quartz cuvettes against a standard curve. Iodixanol wash out is presented as % of total iodixanol originally present in the hydrogel in solution.

### Chick Chorioallantoic Membrane (CAM) Assay

CAM assays were performed to assess the impact of test and control hydrogels on tissue viability and vascularization of fertilized chicken eggs (Beckers et al., 1997). A total of 14 fertilized Lohman white LSL chicken eggs (Animalco AG, Staufen AG, Switzerland) were used for these experiments, seven in the control and seven in the test hydrogel group. According to the local animal care guidelines (Canton of Zurich, Switzerland), no approval was necessary to perform these experiments, as they were executed only until embryonic day (ED) 14. Detailed procedures have been published (Woloszyk et al., 2016). Eggs were pre-incubated for 3 days at 38◦C at a rotational speed of 360◦ /4 h (Bruja 3,000, Brutmaschinen-Janeschitz GmbH, Hammelburg, Germany). On ED 3, the eggs were processed for in ovo cultivation. The eggshell was wiped with 70% ethanol. To lower the developing embryo before opening of the shell using Scotch tape and scissors, 4 mL of albumen were removed through a small hole in the shell. The egg was stabilized in a 60 mm Petri dish base. The opening was covered with a second 60 mm Petri dish base, which was fixed to the bottom base using Scotch tape. Subsequently, the eggs were incubated at 37◦C. On ED 7, hydrogel samples polymerized within a silicone O-ring (from Corning cryogenic vials, Sigma-Aldrich, St. Louis, MO) were gently placed on the CAM (1 per egg), and the eggs were incubated for further 7 days until ED 14. One egg in the test and one in the control group did not survive the 7 days preincubation, leaving 6 viable eggs in the control and the test group, respectively. On ED 14, the hydrogel probes together with the CAM were cut out and after fixing in 4% paraformaldehyde for 60 min at room temperature, the probes were washed and stored in PBS. Pictures were taken using a stereomicroscope (Leica Microsystems AG, Heerbrugg SG, Switzerland). Vascularization within the silicone O-ring was quantified on these images using ImageJ v1.50b.

### Data Presentation

Numerical data is presented as means and standard deviations. Pair-wise comparisons between the test and the control hydrogel were performed using paired t-test. The alpha-type error was set at 5% (p < 0.05).

## RESULTS

The control fibrin hydrogel prepared with TBS clotted quickly. The recorded mean gelation time was 22 ± 1 s. Gelation was strongly delayed by the presence of iodixanol; it occurred after 1,580 ± 75 s (p < 0.05), i.e., 26 min. The iodixanol solution (Visipaque) rendered the test hydrogel radiopaque with a mean radiopacity of 1.9 ± 0.2 mm Al/mm. The test hydrogel could clearly be distinguished from root dentin of human teeth radiographically (**Figures 1a,c**), while the control hydrogel had no detectable radiopacity (**Figure 1b**).

Results showed that swelling was strongly reduced when iodixanol was added to the hydrogel. Values decreased from 203% for control hydrogels prepared with TBS to only

8% for counterparts prepared with Visipaque (time point 5 min, p < 0.05) (**Figure 2a**). Inspection of the lyophilized hydrogels in a scanning electron microscope confirmed that iodixanol influenced their structure. While the control hydrogel showed the typical sponge-like porous structure (**Figure 2b**), the test hydrogel containing iodixanol had a more homogenous appearance with few internal openings (**Figure 2c**).

corresponding image of the test hydrogel containing iodixanol.

Iodixanol release from the hydrogels prepared with Visipaque reached 14.5 ± 0.5% after 120 h and then ceased (**Figure 3**). This release from test hydrogels did not have any apparent effect on the fertilized chicken eggs. All incubated eggs survived the exposure to either the test or the control hydrogel between embryonic day 7 and 14, and vascularization in the chorioallantoic membrane under the hydrogels looked similar (**Figure 4**). Quantification of the vascularization revealed no statistically significant difference between groups (p = 0.34).

### DISCUSSION

This study showed that a radiopaque and tissue-compatible, yet slow setting and less porous fibrin hydrogel is obtained by

diluting a commercially available fibrin sealant with an isotonic contrast agent containing iodixanol rather than TBS. Basic properties of this experimental hydrogel such as hydrogelation

kinetics, radiopacity, swelling, iodixanol release, and overall tissue compatibility were assessed. The current in vitro and in ovo results are preliminary observations and should be seen as such. At this point, it is not possible to draw conclusions regarding the suitability of the test hydrogel for pulp tissue engineering.

The fibrin sealant used in this study (Tisseel), which is designed to achieve hemostasis, to seal or glue tissue, and to support wound healing, has to be diluted in TBS to obtain a fibrin hydrogel that could be invaded by cells during the pulp regeneration process (Ruangsawasdi et al., 2014). This creates an opportunity to replace the TBS used for that purpose with a similar, isotonic product having enhanced contrast properties (Visipaque 320). Opacity of hydrogels by addition of the iodixanol that is contained in Visipaque has recently been tested in chitosan hydrogels for controlled blood vessel embolization (Fatimi et al., 2016). Iodixanol extends blood-clotting time (Bellemain-Appaix et al., 2012), which is concurrent with the results obtained here using a fibrin sealant. It is a non-ionic dimer (Jones and Goodall, 2003). In the current approach, we chose iodixanol because alternative contrast agents such as the nonionic monomer iohexol (Omnipaque, Bayer Pharma AG, Berlin, Germany) and the ionic dimer ioxaglate (Hexabrix, Guerbet, Roissy, France) appear to have an even higher anticoagulant effect than iodixanol (Corot et al., 1996). It has also been shown that the slowing of the hydrogelation process in chitosan hydrogels by iodixanol is dose-dependent (Fatimi et al., 2016). Since teeth are inherently radiopaque structures, we used a high concentration of iodixanol compared to previous experiments on chitosan hydrogels for embolization. However, the radiopacity of permanent sealing materials requested by the ISO norm (2012), which is 3 mm Al/mm, was still not met. Nevertheless, the preliminary filling experiments with human teeth showed that the radiopacity should be sufficient for the intended purpose, i.e., the monitoring of the correct placement of the fibrin hydrogel (**Figure 1c**).

The extended clotting time, which resulted from the presence of iodixanol in the fibrin hydrogel, can be desirable in endodontic applications. The radiopaque fibrin hydrogel under investigation has a working time similar to endodontic sealers (Whitworth, 1999). It can be applied into the root canal system using for example a lentulo spiral. In contrast, the mere fibrin hydrogel diluted with TBS, which is commonly used as a control (Ruangsawasdi et al., 2014, 2017), is not easy to administer into the root canal system because of its immediate gelation.

The swelling of hydrogels is related to the crosslinking ratio in their polymeric network. Highly cross-linked hydrogels tend to swell less than counterparts with a lower crosslinking ratio (Peppas et al., 2000). The effect of iodixanol on swelling of lyophilized hydrogels and on the apparent hydrogel structure in SEM scans strongly suggests that hydrogel porosity is reduced. This is in line with previous studies showing reduction of chitosan hydrogel swelling by increasing their iodixanol concentration (Fatimi et al., 2016). The addition of iodixanol to fibrin-rich clots resulted in shorter, thinner, and more numerous fibrin fibers when compared to counterparts made with ioxaglate or buffer, leading to a much more compact 3D architecture with smaller pores and higher crosslinking of fibrin fibers (Bellemain-Appaix et al., 2012). Furthermore, there was an apparent negative swelling after 5 min with the test hydrogel under the current conditions. This might be indicative of a material degradation process. The iodixanol modified hydrogels hardly took up any TBS fluid, suggesting an important loss of hydrophilicty or material permeability. Both are important characteristics for a biomaterial scaffold in regenerative procedures. Incorporation of iodixanol seems to produce a fiber coalescence process leading to a tensional film-like morphology (**Figure 2**). It thus remains to be investigated how these iodixanol-induced alterations influence the permeability of fibrin hydrogels to migrating cells during regenerative processes.

We used the CAM assay as a measure of tissue compatibility of the new radiopaque hydrogel under investigation. Although this assay is relatively simple, it can be considered suitable for the pre-screening of scaffolds to determine whether they cause any adverse tissue reactions (Baiguera et al., 2012). Our results suggest good tissue compatibility of the test as well as the control hydrogel. However, it needs to be cautioned that the CAM assay is a non-mammalian organism that has not yet developed an immune system. This assay merely determined the effect of the fibrin hydrogels on tissue viability and vascularization. Histological assessment of the hydrogels revealed that no blood vessels grew into the body of the hydrogels (data not shown). This is to be expected, as we did not embed any chemotactic molecules into the hydrogels (Anderson et al., 2011).

In conclusion, the current results revealed the possibility to elaborate radiopaque fibrin hydrogels for potential endodontic applications from materials that are already commercially available. Under the current conditions, tissue compatibility appeared not to be influenced by the addition of iodixanol to the hydrogel. Further in vivo studies should test the effects of different contrast agents on fibrin hydrogel structure combined with cell migration and attraction.

### REFERENCES

### AUTHOR CONTRIBUTIONS

Study design: GH, MZ, TM, and FW. Data collection: GH, AW, and AI. Data analysis: MZ. Drafting manuscript: GH and MZ. Revising manuscript content: GH, MZ, AW, TM, AI, and FW. Approving final version of manuscript: GH, MZ, AW, TM, AI, and FW. MZ takes responsibility for the integrity of the data analysis.

### FUNDING

This research was partly supported by the Swiss National Foundation (SNSF) grant 31003A\_135633 (TM and AW), and a Swiss government scholarship (AI).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Hertig, Zehnder, Woloszyk, Mitsiadis, Ivica and Weber. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Spectroscopic and Mechanical Properties of a New Generation of Bulk Fill Composites

Riccardo Monterubbianesi 1 , Giovanna Orsini 1 \*, Giorgio Tosi 2 , Carla Conti 2 , Vito Librando3 , Maurizio Procaccini 1 and Angelo Putignano1

*1 Department of Clinical Sciences and Stomatology, Polytechnic University of Marche, Ancona, Italy, 2 Department of Materials, Environmental Science and Urban Planning, Polytechnic University of Marche, Ancona, Italy, 3 Department of Chemical Sciences, University of Catania, Catania, Italy*

Objectives: The aims of this study were to *in vitro* evaluate the degree of conversion and the microhardness properties of five bulk fill resin composites; in addition, the performance of two curing lamps, used for composites polymerization, was also analyzed.

#### Edited by:

*Catherine Chaussain, Paris Descartes University, France*

#### Reviewed by:

*Natalina Quarto, University of Naples Federico II, Italy Elisabeth Dursun, Paris Descartes University, France*

\*Correspondence:

*Giovanna Orsini<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology*

Received: *09 October 2016* Accepted: *12 December 2016* Published: *27 December 2016*

#### Citation:

*Monterubbianesi R, Orsini G, Tosi G, Conti C, Librando V, Procaccini M and Putignano A (2016) Spectroscopic and Mechanical Properties of a New Generation of Bulk Fill Composites. Front. Physiol. 7:652. doi: 10.3389/fphys.2016.00652* Materials and Methods: The following five resin-based bulk fill composites were tested: SureFil SDR®, Fill Up!TM , FiltekTM , SonicFillTM , and SonicFill2TM . Samples of 4 mm in thickness were prepared using Teflon molds filled in one increment and light-polymerized using two LED power units. Ten samples for each composite were cured using Elipar S10 and 10 using Demi Ultra. Additional samples of SonicFill2, (3 and 5 mm-thick) were also tested. The degree of conversion (DC) was determined by Raman spectroscopy, while the Vickers microhardness (VMH) was evaluated using a microhardness tester. The experimental evaluation was carried out on top and bottom sides, immediately after curing (t0), and, on bottom, after 24 h (t24). Two-ways analysis of variance was applied to evaluate DC and VMH-values. In all analyses, the level of significance was set at *p* < 0.05.

Results: All bulk fill resin composites recorded satisfactory DCs on top and bottom sides. At t0, the top of SDR and SonicFill2 showed the highest DCs-values (85.56 ± 9.52 and 85.47 ± 1.90, respectively), when cured using Elipar S10; using Demi Ultra, SonicFill2 showed the highest DCs-values (90.53 ± 2.18). At t0, the highest DCs-values of bottom sides were recorded by SDR (84.64 ± 11.68), when cured using Elipar S10, and Filtek (81.52 ± 4.14), using Demi Ultra. On top sides, Demi Ultra lamp showed significant higher DCs compared to the Elipar S10 (*p* < 0.05). SonicFill2 reached suitable DCs also on bottom of 5 mm-thick samples. At t0, VMH-values ranged between 24.4 and 69.18 for Elipar S10, and between 26.5 and 67.3 for Demi Ultra. Using both lamps, the lowest VMH-values were shown by SDR, while the highest values by SonicFill2. At t24, all DC and VMH values significantly increased.

Conclusions: Differences in DC and VMH among materials are suggested to be material and curing lamp dependent. Even at t0, the three high viscosity bulk composites showed higher VMH than the flowable or dual curing composites.

Keywords: bulk fill resin composites, degree of conversion, surface microhardness, curing lamps, spectroscopy

**87**

### INTRODUCTION

In nowadays dentistry, resin-based composites have been and are widely used for dental restorations, even if with some disadvantages: shrinkage, shrinkage stress, micro cracks (in the dental structure or in the resin material), debonding, and secondary caries (Ilie et al., 2007; Ilie and Hickel, 2011; Tantbirojn et al., 2011; El-Saftya et al., 2012; Van Ende et al., 2012; Czasch and Ilie, 2013).

In the time, the multi-layer technique tried to reduce these drawbacks: the fact to place composites in dental cavities, using thin increments of 2 mm or less, allowed a good penetration of the light-curing, thus lowering the shrinkage stress (El-Saftya et al., 2012). Nevertheless, this procedure might afford air bubbles, no linkage between layers, troubles during composites placing, especially in the posterior areas, in which it is sometimes difficult to fill the deep cavities and it is often required a long chair time (Abbas et al., 2003; Sarrett, 2005).

To solve these problems, the manufacturers introduced bulk fill resin composites, consisting in new chemical monomers and fillers with an enhancement of their translucency and, consequently, with the potentiality of obtaining an optimal degree of conversion (DC), even in the bottom of the cavities, where it is more difficult to reach high DC. It has been demonstrated that there are several factors affecting the mechanical properties of a resin composite: chemical composition, amount of emitted radiation, distance from the tip of the light source, and photo-activation mode (Da Silva et al., 2008b). Recent improvements in nanotechnology have led to an optimal filler content of this new generation composites, by the addition of free nanosized spherical particles and clusters, which can act as a single unit, thus significantly improving their mechanical properties, also after finishing and polishing procedures (Beun et al., 2007; Jung et al., 2007; Czasch and Ilie, 2013). Furthermore, the benefit of these materials consists in the fact that they can be cured to a maximal increment thickness of 4–6 mm with a limited shrinkage, due to their high translucency, thus allowing the clinicians to rapidly fill the cavity, shortening the chair time (Van Ende et al., 2012). However, even if the first generation bulk filling materials (introduced by Dentsply with a product called SDR) presented a limited shrinkage stress, they showed unsatisfactory mechanical properties, due to the low percentage of fillers, thus requiring the use of a conventional resin composite, acting as an enamel-top capping layer (Campodonico et al., 2011; Ilie and Hickel, 2011; Van Dijken and Pallesen, 2014).

These limitations were improved with the introduction of high viscosity bulk fill composites, which can fill up the occlusal area in a unique step (as single bulk increment), cured and hence sculpted, without the need of an additional top capping layer. Indeed, innovative composites have been recently available on the market, such as SonicFill, which, for instance, uses the sonic energy to decrease viscosity; once the sonic energy has been removed, the resin composite gradually returns to the starting high viscosity status, assuring good mechanical properties (Ahmad, 2013).

The recent large use of high viscosity bulk fill composites have been due to the following positive factors: simplified procedures, increase of the filler percentage, high depth of cure, acceptable translucency, negligible shrinkage stress after polymerization, and satisfactory cavity adaptation (Ahmad, 2013).

As every modern resin-based system, also bulk fill composites necessitate the light curing process to be polymerized. It is noteworthy that clinicians have the tendency to overestimate the properties of bulk fill resin composites, giving an incorrect evaluation of the volumetric shrinkage, shrinkage stress, and DC. Moreover, other factors such as modulus of elasticity, rate of polymerization, polymerization kinetics, initiator chemistry, gel point, type of filler and monomer, development and intensity of the curing stress have to be taken into account (Ferracane et al., 2014). In fact, even if bulk filling restorations were recommended also in cavities up to 4 mm deep, it turned out that many practitioners are still improperly doubtful on their suitability in the clinical field (Czasch and Ilie, 2013).

The clinical performance of posterior resin-based composite restorations can be strongly affected by various parameters as DC and surface hardness. A high DC can determine good mechanical properties, chemical stability and longevity of the restoration (Da Silva et al., 2008a); it has been measured using different methods, such as Raman, NIR (near-infrared) and MIR (middle-infrared) spectroscopies, in order to evaluate changes of aliphatic/aromatic double bond ratio. A satisfactory polymerization degree is essential for the success of the restoration and can require even more than 24 h (Yoon et al., 2002; Miyazaki et al., 2003; Conti et al., 2005).

The surface hardness (measured as microhardness, by means of Vickers or Knoops tests) is defined as the resistance to permanent indentation or penetration on time, and it has to be evaluated in finishing and polishing phases, or when the resin composites are placed on large areas of masticatory force (Galvao et al., 2013; Tarle et al., 2015).

In order to evaluate the acceptable values of DC and surface microhardness, it is worldwide accepted to refer to the International Standard Test ISO 4049-2009, introduced for dental polymer-based restorative materials. In fact, to consider the suitability of a dental restoration (Flury et al., 2012), it is mandatory to take into account the determination of the depth of cure of a dental composite, referring to both DC and Vickers microhardness (VMH) as fundamental key tests (Flury et al., 2012; Leprince et al., 2012).

The primary aim of this study was to analyse the mechanical and spectroscopic properties of one low viscosity bulk fill composite (SDR), one medium viscosity/dual curing bulk fill composite (Fill Up!), and three high viscosity bulk fill composites (Filtek, SonicFill, SonicFill2), by means of DC and VMH measures. In addition, the potential correlation between DC and VMH was also evaluated.

The secondary aim was to evaluate the performance of two curing lamps in the photo-polymerization process of the different composite samples.

The null hypotheses were: (1) DC values and VMH values do not significantly change within the tested bulk fill composites and there is a correlation between DC and VMH; (2) there are no difference in the performance of the two tested curing lamps.

### MATERIALS AND METHODS

Five commercial (shade 3) bulk fill composites for posterior restorations were tested (**Table 1**):

The two light-curing lamps used for the polymerization of resincomposite samples presented the following technical details. Elipar S10 (3M ESPE) had an energy output 1200 mW/cm2 and spectrum between 430 and 480 nm; on the other hand, Demi Ultra (Kerr Corp.) had variable energy output from 1100 to 1330 mW/cm2 and spectrum between 450 and 470 nm, the energy intensity gradually changing from 1100 until 1330 mW/cm2 .

### Degree of Conversion (DC)

To evaluate DC, homemade Teflon cylinders (of 4 mm in height and 6 mm of internal diameter) were used; for each curing lamp, 10 disk-shaped specimens of each of five resin composites were obtained and photo-polymerized in bulk for 20 s. During the photo-polymerization, to exclude oxygen contamination, each sample was covered with a mylar strip on both surfaces. It has to be noted that the sample mass simulates the amount of resin composite usually used to fill up a dental cavity of recurrent dimensions. All the samples were measured on top side (top) and on bottom side (bottom), immediately once cured (t0), and, only on bottom, after 24 h (t24), too. In addition, for SonicFill2, the DC measurements were performed also in 10 3 mm-thick and 10 5 mm-thick samples, and finally, a last measurement was performed after 240 h (t240) only in SonifFill2 4 mm-thick samples.

A DXR FT Raman spectrometer (Thermo Fischer Scientific, Zug, Switzerland) was used to obtain the Raman spectrum of bulk composites. In Raman determinations, DC was evaluated by comparing the ratio of the alkene carbon-carbon double bond (1638 cm−1 , reaction band B), which was formed during the polymerization, with the one of the aromatic benzene ring (1610 cm−1 ), whose intensity does not change during the polymerization (reference band A). To evaluate the DC, calibration curves were plotted assuming that the ratio A/B on top of the no cured material may represent the 0% of polymerization, while the same ratio on the top at t24, may be taken as 100% of polymerization. For this reason, the top DCvalues will not be described in the results. **Figure 1** shows a typical Raman spectrum with the decrease of olefinic C = C mode at 1635 cm−1 (reaction band) during the curing of both surfaces.

### Vickers Microhardness

Vickers microhardness was determined with Leitz Micro-Hardness (Wetzal GMBH, Wetzlar, Germany) tester on the same samples used for the DC measurements. The method consisted in indenting the sample by a diamond indenter with the form of a right pyramid. In our case, a 50 g load was applied for

*Bis-GMA, bisphenol-Aglycidyldimethacrylate; bis-EMA, ethoxylated bisphenol-A-dimethacrylate; UDMA, urethane dimethacrylate; TMPTMA, trimethylolpropane Trimethacrylate; TEGDMA, triethylene glycol dimethacrylate; TEGDMA, triethylene glycol dimethacrylate; EBADMA, ethoxylated bisphenol-A dimethacrylate; EBPDMA, ethoxylated Bis-GMA; SiO2, silicon dioxide; wt%, weight percentage; vol%, volume percentage.*

15 s. Once the load was removed, it was possible to evaluate (by using a microscope) the corresponding average value of the two indentation diagonals, to get the area of the sloping surface and, hence, to determine the corresponding hardness value.

The measurements of VMH were achieved at t0 and at t24 on the irradiated top surface of the samples. Three measurements were made for each sample: on the middle, on 0.15 mm and on 0.3 mm from the center. Calculations were made by using a computer software (Hardness-Course Vickers/Brinell/Rockwell copyright IBS 2012 version 10.4.4) (Fleming et al., 2008; Roberts et al., 2009; Nagi et al., 2015).

### Statistical Analyses of DC and VMH Data

Statistical analyses were performed by means of R Project for Statistical Computing 3.3.0 (https://www.r-project.org/) and Microsoft Excel 2013. Normality of data distribution and homogeneity of group variances were verified by Kolmogorov-Smirnov test and Levene test, respectively. Differences of DC and VMH-values among groups and at different time intervals were evaluated by two-ways analyses of variance (Two-Ways ANOVA). The Tukey test was applied for post-hoc comparisons. In all analyses, the level of significance was set at p < 0.05.

### RESULTS

Demi Ultra.

Descriptive statistics of DC-values are reported in **Table 2**. Two different curing lamps were used in this study, Elipar S10 and Demi Ultra. At t0, they recorded statistically significant different DC-values between top and bottom sides (p < 0.05).

On top sides, at t0, DC-values of tested composites ranged between 70.40 and 85.56% using Elipar S10. On the other hand, using Demi Ultra lamp, top DC-values at t0 ranged between 82.30 and 90.53%. At t0, top DC-values obtained by Demi Ultra

#### TABLE 2 | Degree of Conversion (DC).

*N, number of samples; DC, Degree of Conversion; SD, Standard Deviation.*

*Mean of DC-values with Standard Deviation at t0, on top and bottom, together with t24 bottom values of samples cured using Elipar S10, and Demi Ultra lamps, respectively.*

were significantly higher than the ones obtained using Elipar S10 (p < 0.05). SDR and SonicFill2 had higher DC-values than the other composites cured using Elipar S10; SonicFill2 recorded the highest DC-values when cured using Demi Ultra.

On bottom side, at t0, DC-values ranged between 65.03 and 84.64%, using Elipar S10. On the other hand, using Demi Ultra, they ranged between 65.45 and 81.52%. At t0, the highest bottom DC-values were recorded by SDR cured by Elipar S10 and Filtek cured by Demi Ultra (p < 0.05). After 24 h, all DC-values significantly increased (p < 0.05). Both at t0 and t24, the values recorded by the two curing lamps were not statistically different (p > 0.05). An additional DC evaluation was performed on SonicFill2 samples of different thickness (3 and 5 mm-thick) at t0, using both lamps. **Figure 2** shows means of DC-values on top side of 3, 4, and 5 mm-thick samples, that were not statistically different (p > 0.05). On the other hand, on bottom side, the mean DC-values in the 3 mm (84.2%) and 5-thick samples (68.7%) were statistically different (p < 0.05). The last evaluation of SoniFill2 samples (4 mm-thick) after 240 h showed no significant difference of DC-values on top between t24 and t240 (p > 0.05).

The VMH-values recorded on the top of the different specimens by using the two curing lamps were statistically different (**Figure 3**). Demi Ultra showed higher VMH-values than Elipar S10 (p < 0.05). The mean of VMH measurements (from t0 to t24) showed the following significant increase (p < 0.05): 51.9 at t0 vs. 61.04 at t24 (using Elipar S10); 53.28 at t0 vs. 61.91 at t24 (using Demi Ultra). SonicFill2 had the highest VMH-values (p < 0.05): being 69.18 ± 3.15 at t0, using Demi Ultra, and 75.2 ± 1.69 at t24; using Elipar S10, 67.3 ± 3.7 at t0 and 71.12 ± 1.52, at t24. On the other hand, SDR showed the lowest VMH-values (p < 0.05): being 28.4 ± 2.34 at t0 and 35.28 ± 1.2 at t24, using Demi Ultra; using Elipar S10, 26.5 ± 3.71 at t0 and 36.73 ± 1.48 at 24.

Finally, Pearson's Test indicated that there is no correlation between DC and VMH, using both curing lamps (0.24 for Demi Ultra, 0.016 for Elipar S10).

### DISCUSSION

Nowadays, it is becoming of growing tendency to use bulk fill resin composite materials because of their simplified procedures for filling in a single increment posterior restorations compared to the multi-increments techniques required by conventional

FIGURE 2 | Degree of Conversion (CD) comparison between top and bottom at different time. Mean of DC top and bottom values at t0 of SonicFill2 in 3, 4, and 5 mm-thick samples, cured using Demi Ultra and Elipar S10 lamps.

resin composites. Indeed, manufacturers and recent scientific reports demonstrate that the main advantages of this restorative procedure consist in an increased depth of cure and a low polymerization shrinkage (Ilie et al., 2013; Leprince et al., 2014). Dental restorative composites polymerize to a certain depth, depending on the light beam penetration of the curing lamp inside the mass (Leloup et al., 2002), and a suitable polymerization of the whole composite mass remains one of the main important factors influencing the clinical success (Czasch and Ilie, 2013). In fact, high DC-values are important to assess optimal physical, mechanical and biological properties of resin composites; on the other hand, when there is not an optimal DC incomplete polymerization (unreacted—dangerous—monomer), marginal microleakage, discoloration, decrease of bonding strength and low mechanical properties can occur (Yap et al., 2004; Kusgoz et al., 2011; Alonso et al., 2013; Galvao et al., 2013). For a clinically acceptable restoration, some authors indicate that the DC-value should reach at least the 55% of it, however, even if resin-based dental materials are properly cured, they generally exhibit a significant amount of unreacted monomers (Soares et al., 2007; Galvao et al., 2013). High values of DC, up to 60%, can be due to improvements in the resin matrix (flexibility and viscosity of the starting monomer), to the composition and size of fillers and to the irradiance intensity (Dickens et al., 2003; Turssi et al., 2005).

In this study, in order to evaluate the DC-values of the tested bulk fill composites, which are based on the use of prepolymerized resins, it was assumed that the ratio 1635/1610 (described above) of the starting material, before the curing, may represent the 0% of polymerization, whereas the ratio of 100% of polymerization is assumed at t24 on the samples top sides (Ferracane, 1995; Blackham et al., 2009). For this reason, at t24, the DC-values on the top were not evaluated. Indeed, due to the overlapping of the reaction bands at 1635 cm−1 at t24 with the one at 240 h (e.g., SonicFill2), it has been hypothesized that after 24 h the polymerization process can be reasonably concluded. Therefore, these two reference points allow us to establish the evolution in time of the polymerization process: it was between t0 and t24, or between 0 and 100%. Raman spectroscopic evaluation shows that different DC-values are recorded between top and bottom at t0 for all composites, using the two curing lamps (p < 0.05). On top and at t0, Demi Ultra seems to cure better than Elipar S10 (p < 0.05), while on bottom and at t0, the two curing lamps are not statistically different to cure SDR and Filtek, which show the highest DC-values using Elipar S10 and Demi Ultra, respectively. After 24 h, bottom DC-values increase using both Elipar S10 and Demi Ultra, with no significant differences between the two curing lamps: once again SDR and Filtek show the best performance. In agreement with literature reports, all 4 mm-thick tested samples show high DC-values, mainly for t0 bottom determinations (Goracci et al., 2014; Leprince et al., 2014; Marovic et al., 2015). In the case of SDR, the high DCvalues can be due to the high fluidity and transparency of this resin, even if, it is noteworthy again to remark, that a flowable resin composite, like SDR, needs an additional hardening top layer. Literature data concerning SonicFill and SDR evidence some discordance and, in general, lower DC-values than our data, mainly for t0 bottom determinations (57.9 and 50.3%, respectively, when using Demi Ultra lamp, with an output of 1100 mW/cm2 ; see Goracci et al., 2014). Indeed some reports show that SDR DC-values on top ranged from the 77% (t0), using a light unit output of 1200 mW/cm2 (Guimaraes et al., 2013), to the 67%, reported by other authors (Van Ende et al., 2013; Marovic et al., 2015). Among the high viscosity samples, SonicFill2 shows the highest DC-value with both curing lamps (at t0, on top), being Demi Ultra the best unit to polymerize it. However, both curing lamps result in a satisfactory performance, being Demi Ultra slightly superior than Elipar S10 in top surfaces curing (p < 0.05).

As mentioned above, DC-values of SonicFill2 have been determined also on 3 and 5 mm-thick samples at t0 (**Figure 2**). The bottom DC mean values of these samples at different thickness are statistically different, thus suggesting that the thickness of samples could condition the DC. However, it is noteworthy to underline that acceptable DC-values are registered even on the bottom side of 5 mm-thick samples.

Finally, the fact that there is no difference in DC SonicFill 2 values between t24 and t240 means that a satisfactory degree of polymerization can be reached after 24 h, in agreement with other literature reports (Ferracane, 1995; Blackham et al., 2009).

Hardness and microhardness measure the resistance to plastic deformation and indicates the resistance to indentation under functional stresses: a high value can be indicative of the ease of finishing and polishing of a restoration (Rahiotis et al., 2004; David et al., 2007). It has already been reported that hardness values may be related to the DC of carbon double bonds of a resin composite; even if with some divergence, it has been shown that a conversion around 90% of the resin may correspond to a bottom/top VMH ratio of 80% (Bouschlicher et al., 2004). Moreover, the microhardness of a resin composite depends also on the thickness: an increase in thickness causes a microhardness lowering.

In recent years, modulator interacting with camphorquinone (as in the case of the dual-curing composite Fill Up!), transparency enhancements and/or technological up grades (as in the case of high viscosity bulk fill composites) have been added to the resin composites, thus resulting in satisfactory microhardness values even in 4 mm-thick samples. Indeed, some authors reported that high viscosity bulk fill composites might exhibit high microhardness values because of the high percentage of fillers content (Barabanti et al., 2015; Nagi et al., 2015). **Figure 3** reports mean and SD of VMH top values at t0 and t24, using the two curing lamps. As expected, after 24 h, all samples show significant VMH increases (p < 0.05). Among the five materials, the lowest VMH mean value is shown by SDR, while a considerable increase is found for the medium viscosity dual curing Fill Up! (57 and 63 vs. 51 VMH, as claimed by the manufacturer Coltene); noteworthy is that at t0, SonicFill2 shows the highest VMH-value, using both curing lamps.

Several studies have tried to find a correlation between the DC and VMH for some authors no correlation may be drawn, while, for others, a negative trend occurs between the two parameters (Ferracane, 1985; De Wald and Ferracane, 1987; Santos et al.,

2007; Da Silva et al., 2008a). In our case, the lack of any correlation is evident (Pearson's correlation coefficient: 0.24 for Demi Ultra, −0.016 for Elipar S10), meaning that, even if high values of two single parameters may point out a satisfactory dental curing, a linear correlation is lacking (**Figure 4**). This fact may be due to the different contents and viscosity of the five composites (**Table 1**).

In the attempt to analyse the behavior of a reasonable range of new bulk fill dental composites, a significant number of samples have been evaluated in the present work, however, other experiments are needed to make definitive remarks and, therefore, future analyses will aim to continue to analyse newly introduced materials. In definitive, differences in DC and VMH-values between our study and previous literature reports can be ascribed to improvements of fillers (composition and particles size distribution, percentage of filler load), of the resin matrix (monomer type and its chemical structure), and also to the enhanced performance of the new curing lamps. Indeed, it is important to underline the great impact that nanotechnology has produced in terms of development and progressing of dental materials science. Therefore, the present study can be considered relevant since there are no previous reports evaluating all the five bulk fill resin composites, using vibrational techniques, such as Raman spectroscopy, and also because this is the first time in which the performance of two new generation curing lamps have been analyzed.

### CONCLUSIONS

In the light of the presented results, the two null hypotheses can be rejected: firstly, because there are differences in DC and VMH within the tested bulk fill composites, and no correlation can be drawn between DC and VMH; secondly, because the behavior of the two curing units is slightly different. Within the limits of the current study, it can be concluded that the five resin composites recorded satisfactory polymerization degree on both top and bottom sides.

To summarize, the flowable SDR shows high DC-values with both lamps, while, among high viscosity samples, SonicFill systems (and especially SonicFill2), combining the advantages of a flowable dental material with a universal resin composite, by using sonic activation, demonstrate excellent DC-values. In particular, SonicFill2 showed the highest DC-value using both curing lamps (at t0, on top), and an acceptable DC, even in case of 5 mm-thick samples.

Significant differences in VMH-values among the five tested materials are found: the lowest value has been evidenced by the flowable SDR, reinforcing the need of a top capping layer, while, in agreement with the literature, an appreciable increase has been found for the dual curing Fill Up!. Among the high viscosity resin composites, a satisfactory performance has been demonstrated by SonicFill and, mainly, by SonicFill2. Both DC and VMH determinations appear clinically significant to make a prevision of the future performances of restorations. The use of medium and, mainly, high viscosity bulk fill materials may also be important to avoid a further capping application. Moreover, this study can be useful to increase the knowledge of clinicians in understanding the curing performance of the tested lamps.

Our upcoming aim will be to increase the number of the tested dental composites, as well as to further study by means of vibrational techniques both dental and composites surfaces after finishing and polishing.

### AUTHOR CONTRIBUTIONS

RM contributed to the research protocol, performed the experiments as part of his Ph.D. project, contributed to the results analyses and writing. GO contributed to the idea, the research protocol, and the writing of the present manuscript. GT contributed to the correct developing of the research idea, the writing, the technical background of the entire manuscript. CC contributed to the technical idea and support all experimental phases. VL contributed to the Raman facilities use and data interpretation, and to the discussion finalization. MP contributed to the idea and the updated results organization of the manuscript. AP contributed to the idea, the findings interpretation, and the editing of the manuscript.

### REFERENCES

### FUNDING

Dental materials were gently donated from the manufacturers.

### ACKNOWLEDGMENTS

This work was supported by institutional funds from the Polytechnic University of Marche (GO, MP and AP). We would like to thank Prof. Luigi Ferrante for statistical advices. The authors are grateful also to Dr. Alberto Catellani and Dr. Gloria Altarocca for the precious experimental support.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer ED and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2016 Monterubbianesi, Orsini, Tosi, Conti, Librando, Procaccini and Putignano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Synchrotron Phase Tomography: An Emerging Imaging Method for Microvessel Detection in Engineered Bone of Craniofacial Districts

Alessandra Giuliani 1 \*, Serena Mazzoni 1 , Luigi Mele2 , Davide Liccardo2 , Giuliana Tromba3 and Max Langer 4

1 Sezione di Biochimica, Biologia e Fisica Applicata, Dipartimento di Scienze Cliniche Specialistiche e Odontostomatologiche, Università Politecnica delle Marche, Ancona, Italy, 2 Sezione di Biotecnologie, Istologia Medica e Biologia Molecolare, Dipartimento di Medicina Sperimentale, Università degli Studi della Campania "L. Vanvitelli", Naples, Italy, 3 Elettra Sincrotrone Trieste S.C.p.A, Trieste, Italy, 4 Centre de Recherche en Acquisition et Traitment d'Images pour la Santé (CREATIS), Centre National de la Recherche Scientifique (CNRS) UMR 5220, Institut national de la santé et de la recherche médicale (Inserm) U1206, Université de Lyon, INSA-Lyon, Villeurbanne, France

Edited by: Thimios Mitsiadis,

University of Zurich, Switzerland

#### Reviewed by:

César Nombela Arrieta, University of Zurich, Switzerland Jean-Christophe Farges, Claude Bernard University Lyon 1, France

> \*Correspondence: Alessandra Giuliani<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology

Received: 27 June 2017 Accepted: 20 September 2017 Published: 29 September 2017

#### Citation:

Giuliani A, Mazzoni S, Mele L, Liccardo D, Tromba G and Langer M (2017) Synchrotron Phase Tomography: An Emerging Imaging Method for Microvessel Detection in Engineered Bone of Craniofacial Districts. Front. Physiol. 8:769. doi: 10.3389/fphys.2017.00769 The engineering of large 3D constructs, such as certain craniofacial bone districts, is nowadays a critical challenge. Indeed, the amount of oxygen needed for cell survival is able to reach a maximum diffusion distance of ∼150–200µm from the original vascularization vector, often hampering the long-term survival of the regenerated tissues. Thus, the rapid growth of new blood vessels, delivering oxygen and nutrients also to the inner cells of the bone grafts, is mandatory for their long-term function in clinical practice. Unfortunately, significant progress in this direction is currently hindered by a lack of methods with which to visualize these processes in 3D and reliably quantify them. In this regard, a challenging method for simultaneous 3D imaging and analysis of microvascularization and bone microstructure has emerged in recent years: it is based on the use of synchrotron phase tomography. This technique is able to simultaneously identify multiple tissue features in a craniofacial bone site (e.g., the microvascular and the calcified tissue structure). Moreover, it overcomes the intrinsic limitations of both histology, achieving only a 2D characterization, and conventional tomographic approaches, poorly resolving the vascularization net in the case of an incomplete filling of the newly formed microvessels by contrast agents. Indeed, phase tomography, being based on phase differences among the scattered X-ray waves, is capable of discriminating tissues with similar absorption coefficients (like vessels and woven bone) in defined experimental conditions. The approach reviewed here is based on the most recent experiences applied to bone regeneration in the craniofacial region.

Keywords: phase tomography, synchrotron radiation, microvessels, craniofacial bone engineering, X-ray phasecontrast imaging

### INTRODUCTION

Nowadays, craniofacial bone defects due to congenital conditions, disease, and injury cause major clinical issues, often solved with tissue replacement by autologous grafting. However, sometimes this procedure is hampered by an extensive donor site morbidity. In these cases, bone engineering protocols could support restoration of the function, or replace damaged or diseased tissues (Alsberg et al., 2001).

Since the long-term function of three-dimensional (3D) bone substitute biomaterials (BSB) is strongly dependent on adequate vascularization after grafting, research in craniofacial bone engineering has recently focused on approaches involving angiogenesis (Auger et al., 2013). These include the delivery of different growth factors to the defect site (Yoo and Kwon, 2013) or the engineering of microvascular networks by using endothelial cells and stem cells (Liu et al., 2013). Nevertheless, current vascularization methods are often not sufficiently rapid for an adequate cellular oxygen supply. Therefore, it is necessary to create microvascular networks within 3D tissue constructs in vitro before grafting (Laschke et al., 2006).

However, full comprehension of the bone vascularization pathways is still hampered by limitations in the use of imaging techniques to monitor these processes in-vivo. Nowadays, new and interesting imaging modes are available. Upputuri et al. (2015) recently classified the vascular imaging approaches into three groups: non-optical techniques (X-ray, magnetic resonance, ultrasound, and positron emission imaging), optical techniques (optical coherence, fluorescence, multiphoton, and laser speckle imaging), and hybrid techniques (photoacoustic imaging). Physical origin of the contrast, levels of resolution achieved, imaging depth and structures resolved (microvessels, bone, etc.) have been described and summarized for each group.

Inside the non-optical group, great interest is focused on Xray imaging methods. They are based on X-ray attenuation of the different tissues and have been successfully used over the past few years to visualize large blood vessels.

In this regard, high resolution tomography (microCT) (Arkudas et al., 2010; Barbetta et al., 2012) is able to provide much higher resolution (∼1µm) imaging, than ultrasound (∼30µm) and MRI (∼100µm), allowing the visualization and quantification of microvasculature. This procedure is normally achieved with the use of contrast agents, which are radio opaque and radio dense fillers. It was recently proved by this approach that the 10µm medium resolution microCT is able to image with success medium and large blood vessels (Nebuloni et al., 2014). Moreover, Langer et al. (Langer et al., 2011) successfully imaged the microvasculature with contrast agent in rat and mouse bone. While they proved that parts of the connectivity were not preserved (due to partial volume effect), a very large part of the contrast agent volume was retrieved.

Also, in recent years, a new method for combined 3D imaging and analysis of microvascularization and bone microstructure has emerged. It is based on the use of synchrotron phase microtomography (PhC-microCT) and it allows, as opposed to conventional 2D techniques like histology, to simultaneously identify in 3D multiple tissue features without using contrast agents. This is due to the increased sensitivity of phase sensitive X-ray imaging techniques. Indeed, due to increased sensitivity, this technique also overcomes the intrinsic limitations of conventional tomographic approaches, often unable to reliably reconstruct the full vascularization network in case of incomplete filling of microvessels by contrast agents (Fei et al., 2010).

In this mini-review, we briefly discuss the very recent advances in synchrotron phase tomography aiming at high-resolution imaging of engineered bone vasculature in craniofacial districts.

### APPROACHING SYNCHROTRON PHASE TOMOGRAPHY METHODS

Differently from the conventional (attenuation-based) microCT in which the contrast is due to attenuation differences within the sample, in PhC-microCT the contrast originates from the phase shift of the X-ray beam passing through the matter (Snigirev et al., 1995; Bravin et al., 2013).

At least three methods have been studied for phase contrast Xray imaging: X-ray grating interferometry (Momose, 1995, 2003; Momose et al., 1996), diffraction enhanced imaging (Davis et al., 1995; Ingal and Beliaevskaya, 1995; Chapman et al., 1997, 1998) and propagation-based imaging (Snigirev et al., 1995; Wilkins et al., 1996; Cloetens et al., 1999).

The first two methods are often confined to synchrotron sources because they require a monochromatic and highly collimated x-ray beam. Indeed, when coherence grating is used at conventional x-ray sources, a detrimental flux reduction is experienced. In turn, this flux reduction would require longer exposure times than at synchrotrons, hampering, as a consequence, the translation of the method to clinical settings (Nesch et al., 2009).

Very recently, grating-based (GB) Talbot interferometry (Weitkamp et al., 2005; Momose et al., 2009) was successfully applied, combined to conventional polychromatic x-ray sources. A third grating (Pfeiffer et al., 2007) was used to study weakly absorbing samples and, using synchrotron facilities, to excellently discriminate soft-tissues in cancerous human liver tissue (Noel et al., 2013) and in atherosclerotic plaques (Hetterich et al., 2013). Other studies performed with polychromatic x-ray sources allowed to achieve an improved imaging of parenchymal lung disease (Yaroshenko et al., 2013), breast lesions (Hauser et al., 2013; Sztrokay et al., 2013), cartilage (Tanaka et al., 2013), atherosclerotic plaques (Saam et al., 2013), and renal ischemia (Velroyen et al., 2014).

Unlike X-ray interferometry, the diffraction enhanced imaging method exploits the benefits of a µrad angular resolution achieved by filtering X-rays deflected by refraction. This is made possible using an analyzer crystal after the sample to select refracted X-rays from the sample.

Instead, the propagation-based method generates contrast by Fresnel or Fraunhofer diffraction: the first is achieved by placing the detector and the sample at a moderate distance.

In practice, at lower resolutions, the X-ray interferometric imaging is thus the preferential approach for revealing soft structures, being able to also sense the shallower phase gradients, compared to the diffraction enhanced and propagationbased imaging methods. This is due to the principles of contrast generation; stronger contrast is generated at structural boundaries where the variations of refractive index are larger due to rapid phase gradients (Momose, 2003).

On the other hand, the propagation-based imaging method is of great advantage in high-resolution imaging because no optical components are needed when a coherent X-ray source is available, like at synchrotron facilities. The chance to get high resolutions has made propagation-based imaging the technique of choice in the study of both human bone ultrastructure and vasculature, with focused pre-clinical and clinical studies on vessels of medium and small thickness.

Concerning the investigation of human bone ultrastructure, X-ray phase nano-tomography, based on the propagation method, was recently shown to provide the appropriate spatial resolution and sensitivity to efficiently visualize and quantify the 3D organization of the lacuno-canalicular pattern (Langer et al., 2012). The study was performed considering several cells in osteonal and interstitial tissue: nanoscale density variations revealed that the cement line separating these tissues is hypermineralized. Moreover, the organization of the collagen fibers was reconstructed in 3D, showing a twisted plywood structure.

Imaging brain microvasculature is crucial for plasticity studies of cerebrovascular diseases. Traditionally, in the brain, absorption-based microCT and microMRI methods are applied, using contrast agents for a better visualization of the vasculature.

Very recently, propagation-based synchrotron PhC-microCT was applied to visualize the whole mouse brain microvasculature. It was carried out at high resolution (∼3.7µm) and without contrast agents (Miao et al., 2016). Microvasculature changes in C57BL/6 mice brain (n = 14) after 14-day reperfusion from transient middle cerebral artery occlusion (tMCAO) were observed. PhC-microCT demonstrated that the branching radius ratio (post- to pre-injury) of small vessels (radius < 7.4µm) in the injury group was significantly smaller than in the sham group. This result revealed active angiogenesis in brain recovery after stroke.

In another recent study, Fratini et al. (2015) showed that PhC-microCT allows the simultaneous visualization of threedimensional micro-vascular network and neuronal systems of exvivo mouse spinal cord. This experiment was carried out at scales spanning from millimeters to hundreds of nanometers, without contrast agents and destructive sample preparation. Images of both the 3D distribution of micro-capillary network and the micrometric nerve fibers, axon-bundles and neuron soma, were obtained, confirming the efficiency of this technique also in pre-clinical studies of neurodegenerative pathologies and spinalcord-injuries.

### ASSESSMENT OF BONE MICROVESSELS DETECTION BY PHASE TOMOGRAPHY

Conventional X-ray microCT is a technique that allows a good visualization of the structure of mineralized bone and biomaterials, but it fails when attempting to discern soft tissues at high resolutions. On the contrary, PhC-microCT, based on propagation-based settings, was demonstrated to present, at high resolution, a better soft tissue contrast than conventional CT, clearly discriminating ligamentous, muscular, neural, and vascular structures (Horng et al., 2014).

These findings led some authors to investigate the 3D vascularization of engineered-bone tissue. Detailed imaging and a quantitative description of the complete vascular network in such constructs is crucial for monitoring the relation between bone formation and vascularization and phase tomography was shown to efficiently discriminate tissues with similar absorption coefficients (Langer et al., 2009).

However, the previous study was still limited by the use of a contrast agent. Indeed, although a new phase-contrast medium—the micro-bubble—was recently successfully applied to angiography applications (Tang et al., 2011), different authors

of the group-D were originally published in Giuliani et al. (2013; http://onlinelibrary.wiley.com/doi/10.5966/sctm.2012-0136/epdf).

TABLE 1 | Summary of high resolution tomography (microCT) imaging modalities for vascular imaging in tissue engineering. Image resolution up to 600 nm (in propagation-based settings).

\* Arkudas et al., 2010; Barbetta et al., 2012.

#Chapman et al., 1998; Connor et al., 2009; Coan et al., 2010.

This fact was confirmed in another recent study (Bukreeva et al., 2015), where synchrotron PhC-microCT was applied to visualize and analyze the 3D micro-vascular networks in boneengineered constructs, made of porous ceramic scaffolds loaded with bone marrow stromal cells (BMSC), in an ectopic bone formation mouse-model. Samples seeded and not seeded with BMSC were compared, with or without the use of contrast agents. The authors achieved the 3D distribution of both vessels and collagen matrix and obtained quantitative information for the different samples, even for those not stained.

### Experience in Craniofacial Districts

Nowadays, autologous bone is still considered the ideal grafting material in the craniofacial district (Yamanichi et al., 2008; Iezzi et al., 2017). Autologous grafts are vascularized and contain viable osteoblasts, organic and inorganic matrices, and growth factors that allow remodeling and structural integration with the host site. However, there are significant limitations associated with the use of autologous grafts, including the availability of donor tissue (since it has to be obtained intraorally), the need of additional surgical procedures and, consequently, increased operating times and costs (Tsigkou et al., 2010; Iezzi et al., 2017). When directly using synthetic and allogeneic grafts, only the periphery of the graft is efficiently vascularized. As a consequence, a central zone of necrosis frequently occurs, resulting in a shell of ossification at the surface, but with low level penetration, due to the limited transport of oxygen and metabolic requirements to the inner cell mass (Tsigkou et al., 2010). Thus, a rapid vascularization of bone grafts is a clinical challenge, based on the fact that the development of a mature and functional vasculature not only depends on migration and proliferation of endothelial cells but also requires cooperation and symbiosis between them and perivascular cells, acting as a potent stabilizer of the engineered blood vessels formed in the porous bone scaffold (Tsigkou et al., 2010).

Therefore a major task is to find advanced imaging techniques that could verify and quantify the mineralization process and neovascularization of grafts, at the early stages of bone formation (Paino et al., 2017). Third-generation synchrotron facilities produce brilliant X-ray photon beams, having high spatial coherence properties. They have been demonstrated to be suitable for several tissue engineering studies, detecting in-vitro the newly formed extracellular matrix (Albertini et al., 2009), the early colonies of endothelial cells (Giuliani et al., 2014), and the early phases of bone mineralization (Manescu et al., 2016; Mazzoni et al., 2017), by introducing X-ray imaging methods based on phase-contrast.

Very recently, some authors (Paino et al., 2017) showed that human dental pulp stem cells (hDPSCs) can lead to a bone tissue ready to be grafted for clinical application. They demonstrated that hDPSCs proliferate in-vitro, differentiate into osteoblasts and express genes associated with angiogenesis factors, such as VEGF and PDGFA. Synchrotron-based X-ray phase-contrast imaging proved that, after 40 days of culture in standard medium, hDPSCs formed woven bone (WB), i.e., fibrous bone with a low level of mineralization. In particular, the PhC-microCT analysis was performed using a polychromatic beam, with a sample-todetector distance of 150 mm, corresponding to a single-distance phase-contrast set-up. The osteogenic potential of WB fabricated after in vitro hDPSC culture was validated by the quantitative data extracted from the 3D PhC-microCT analysis (**Figure 1**). However, it was not possible to detect any vascularization using this technique.

In this regard, the holotomography (HT) technique added fundamental information (Paino et al., 2017). The HT approach differs from the PhC-microCT method based on a single distance in that the acquisition consists of tomographic scans at four different propagation distances, followed by a different reconstruction algorithm (**Figure 2A**). The HT analysis allowed to achieve a 3D reconstruction of the WB (**Figures 2B1,B2**), assessing the presence of new vessels (**Figure 2B3**). Indeed, because of their low attenuation coefficient, these new vessels are transparent in conventional attenuation-based tomographic reconstructions and the PhC-microCT method based on a single distance was still found to be not sufficiently sensitive. Indeed, this HT evidence confirmed deductions derived from the fact that hDPSCs strongly expressed high levels of VEGF and PDGF-A, which explains the vessel formation in the WB. This last finding is of paramount interest for physiology of the bone in craniofacial districts, because it satisfies the need of neoangiogenesis in the engineered site. Thus, these results strongly support the rationale that hDPSCs possess significant differentiation capabilities toward osteoangiogenesis, matching the gold standard for obtaining well-vascularized bone (Paino et al., 2017).

Moreover, some studies (d'Aquino et al., 2009) demonstrated that hDPSCs differentiate into osteoblasts and, when seeded on collagen I scaffolds, efficiently contribute to repair human mandible defects. In this context, another study (Giuliani et al., 2013) showed, by synchrotron HT, the stability and quality of both the regenerated bone and the vessel network 3 years after grafting. It was found that the regenerated tissue in grafted sites was unexpectedly constituted by compact bone, structurally different and denser compared to the healthy native alveolar bone of the same patient. However, in both the human mandible control and the human in vivo SC-treated mandible biopsies analyzed, in agreement with histological analyses, after 3 years the regenerated bone was well structured and vascularized. Two subvolumes of the human in vivo SC-treated mandible are shown in **Figures 2C1,C2**: one, referring to a representative histological slice, was used as a reference (**Figure 2C1**); the other, referring to the 3D reconstruction of the HT scan and the subsequent data analysis (**Figures 2C2,C3**). The unmineralized phases, with the exception of the one representing vessels, were virtually suppressed for a better visualization of bone (gray) and its vascularization (red). As confirmed histologically (**Figure 2C1**) these 3D reconstructions demonstrate that after 3 years bone tissue was well structured and vascularized. In particular, in **Figure 2C3**, only densities compatible with the vessel phase have been visualized, clearly showing a good vascularization. The same information is represented in **Figures 2D1–D3** for human mandible control. It is clearly shown that the bone is a well-structured cancellous structure (**Figures 2D1,D2**), with an homogeneous and fully organized vessel network (**Figure 2D3**): although here the vascularization is more structured than in the human in vivo SC-treated mandible, density signals compatible with neo-vessels were found in several areas of the biopsy retrieved from the treated site, confirming in-vivo that the hDPSCs have significant differentiation capabilities toward osteoangiogenesis (Giuliani et al., 2013).

## CONCLUSIONS

While PhC-microCT was demonstrated to be capable of visualizing the 3D vessel network in in-vitro and ex-vivo conditions and without any sample sectioning and preparation, the use of coherent and highly brilliant Synchrotron X-ray sources was mandatory in order to achieve a higher image quality with sub-micrometer spatial resolution. Indeed, as reported in the literature and summarized in **Table 1**, microvessel detection in engineered bone was carried out mainly in two ways: by attenuation-based microCT, with the use of contrast agents, or by propagation-based PhC-microCT, without any marker. However, the application of the last method has required, up to now, access to synchrotron facilities. This fact constitutes a relevant limitation for a possible future use of phase-contrast imaging in clinical practice, since the radiation dose would be too high. Therefore, we propose this approach as a fundamental tool for angiogenesis studies in preclinical research and bone post-extractive studies in craniofacial districts.

## AUTHOR CONTRIBUTIONS

AG: Concept and design; revision of the whole literature; coordination of the work drafting; final version definition and approval. SM and GT: Design (phase-contrast tomography); data research in literature on PhC-microCT; work drafting; final version approval. LM and DL: Design (physiology concepts); data research in literature on physiological interpretation of data; work drafting; final version approval. ML: Concept and design; data research in literature on PhC-microCT; work drafting; final version definition and approval. All the authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

## REFERENCES

bone: biological and clinical implications. Stem Cells Trans. Med. 2, 316–324. doi: 10.5966/stemcells.2012-0136.

periodontal ligament stem cell xeno-free culture studied by Synchrotron radiation phase-contrast microtomography. Int. J. Mol. Sci. 18:364. doi: 10.3390/ijms18020364

using differential phase-contrast mammography. Invest. Radiol. 46, 801–806. doi: 10.1097/RLI.0b013e31822a585f

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer CA and handling Editor declared their shared affiliation.

Copyright © 2017 Giuliani, Mazzoni, Mele, Liccardo, Tromba and Langer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# An Overview on Current Non-invasive Diagnostic Devices in Oral Oncology

Marco Mascitti1 \*, Giovanna Orsini1 , Vincenzo Tosco1 , Riccardo Monterubbianesi1 , Andrea Balercia2 , Angelo Putignano1 , Maurizio Procaccini1,3 and Andrea Santarelli1,3

1 Department of Clinical Sciences and Stomatology, Marche Polytechnic University, Ancona, Italy, 2 Department of Oral and Head-Neck Surgery, Umberto I General Hospital, Marche Polytechnic University, Ancona, Italy, 3 National Institute of Health and Science of Aging, INRCA, Ancona, Italy

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy, and despite advances in cancer therapies, the overall 5-year survival rate has remained below 50% over the past decades. OSCC is typically preceded by potentially malignant disorders (PMD), but distinguishing high-risk from low-risk PMD is challenging. In the last years, several diagnostic methods as light-based detection systems (LBDS) have been proposed to facilitate the detection of OSCC and PMD. Furthermore, the recent evolution of nanotechnology may provide new opportunities to detect PMD and OSCC at an early stage. Indeed, several preclinical studies showed the potential of nanotechnology to enhance diagnostic accuracy. For these reasons, it is fundamental to conduct studies to evaluate the efficacy of nanotechnology implementation in LBDS. The aim of this article is to review the current literature on LBDS and to provide a summary of the sensitivity and specificity of each technique, and possible future applications of nanotechnologies. The LBDS showed great potential for screening and monitoring oral lesions, but there are several factors that hinder an extensive use of these devices. These devices seem to be useful in assessing lesion margins that must be biopsied. However, to date, conventional oral examination, and tissue biopsy remain the gold standard for OSCC diagnosis. The use of nanotechnologies could be the next step in the evolution of LBDS, thus providing devices that can help clinicians to detect and better monitor oral lesions.

Keywords: light-based detection system, early diagnosis, OSCC, chemiluminescence, autofluorescence, nanotechnology

### INTRODUCTION

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy and the sixth most common tumour worldwide (Warnakulasuriya, 2009). Despite advances in therapies, the overall 5-year survival rate has remained unchanged during the past decades, mainly due to delayed diagnosis (Gomez et al., 2009). OSCC is typically preceded by potentially malignant disorders (PMD), a group of clinically suspicious lesions. Although the majority of PMD do not progress to OSCC, distinguishing high-risk PMD from low-risk PMD is challenging for dental practitioners (Yang et al., 2018). Furthermore, patients treated for OSCC are at risk of developing recurrences and secondary primary tumours, due to field cancerization and/or incomplete surgery (Day and Blot, 1992). Currently, conventional oral examination (COE), consisting in visual and tactile assessment of accessible oral structures, followed by tissue biopsy still constitutes the gold

#### Edited by:

Gianpaolo Papaccio, Università degli Studi della Campania Luigi Vanvitelli, Italy

#### Reviewed by:

Alessandra Pisciotta, Università degli Studi di Modena e Reggio Emilia, Italy Luigi Mele, Università degli Studi di Napoli Federico II, Italy

> \*Correspondence: Marco Mascitti<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology

Received: 30 July 2018 Accepted: 08 October 2018 Published: 25 October 2018

#### Citation:

Mascitti M, Orsini G, Tosco V, Monterubbianesi R, Balercia A, Putignano A, Procaccini M and Santarelli A (2018) An Overview on Current Non-invasive Diagnostic Devices in Oral Oncology. Front. Physiol. 9:1510. doi: 10.3389/fphys.2018.01510

**104**

standard for diagnosis of PMD and OSCC. However, there are some limitations of this procedure, such as sampling bias that can lead to underdiagnosis or misdiagnosis, particularly in multifocal lesions (Yang et al., 2018).

The possibility of making an early diagnosis is crucial for reducing high mortality rate and morbidity of OSCC patients. In the last years, several light-based detection systems (LBDS), based on optical properties of biological tissues, have emerged with claims of enhancing oral mucosal examinations and facilitating the detection of PMD and OSCC.

Furthermore, the recent evolution of nanotechnology may provide new opportunities to detect PMD and OSCC at an early stage (El-Sayed et al., 2005). Several preclinical studies showed the potential to enhance diagnostic accuracy of optical diagnostic technologies (e.g., Raman spectroscopy) or imaging techniques (e.g., Magnetic resonance imaging) (Chen et al., 2018). Among the latter techniques, reflectance confocal microscopy seems to improve the evaluation of oral lesions, by detecting backscattered light from illuminated tissue, producing high resolution tissue map. However, several technological limitations need to be resolved to validate diagnostic accuracy (Lucchese et al., 2016). LBDS showed several advantages compared to the aforementioned approaches, such as low cost and ease of use. For these reasons, it is fundamental to conduct studies to evaluate the efficacy of nanotechnology implementation in LBDS.

The aim of this article is to review the literature on LBDS currently on the market (**Tables 1**, 2), providing clinicians with a better understanding of their advantages and limits, and possible future applications of nanotechnologies.

### ViziLite R

ViziLite R (Zila Pharmaceuticals, Phoenix, AZ, United States) is a chemiluminescence-based detection device designed to facilitate the early identification of PMD and OSCC. In 2002 ViziLite R became the first device approved by FDA for this purpose (Oh and Laskin, 2007).  To activate it, the capsule is bent to break the inner glass vial, triggering the reaction of the chemicals contained in the two compartments. Consequently, a bluish-white light (430–580 nm) is produced,

OL, oral lesions; PMD, potentially malignant disorders; OSCC, oral squamous cell carcinoma; Sens, sensitivity; Spec, specificity; PPV, positive predictive value; NPV, negative predictive value. Where possible, missing data were recalculated.

OL, oral lesions; PMD, potentially malignant disorders; OSCC, oral squamous cell carcinoma; Sens, sensitivity; Spec, specificity; PPV, positive predictive value; NPV, negative predictive value. Where possible, missing data were recalculated.

lasting for 10 min (Liu et al., 2016). A modified version (ViziLite R PLUS) consists of a combination of chemiluminescence and toluidine blue (TB) marking system, an acidophilic dye that selectively stains acidic substances such as DNA. Furthermore, an accessory eyewear has been developed, to allow better isolation of chemiluminescent light (Sambandham et al., 2013). Its clinical use requires a 1-min rinse of 1% acetic acid solution, to desiccate oral tissues, followed by oral examination with 430–580 nm wavelength light. The altered epithelial cells, due to higher nuclear/cytoplasmic ratio, reflect the light and cause the appearance of an "aceto-white" lesion, whereas normal cells appear blue (Nagi et al., 2016).

The first studies regarding ViziLite R , published in 2004–2007, were conducted on subjects with different clinical conditions, ranging from normal mucosa to diagnosed OSCC, with the aim to explore the diagnostic utility of chemiluminescence-based strategies (**Table 2**). In the first reported study, ViziLite R identified a subclinical lesion, suggesting its utility in identifying occult epithelial abnormalities (Huber et al., 2004). In a small cohort of patients with oral lesions, ViziLite R appears to be a better diagnostic tool than TB in detection of OSCC and PMD (Ram and Siar, 2005). Another study highlights the ability of ViziLite R to show brighter and better demarcated lesions than using incandescent light, aiming to enhance the identification of lesions that could be biopsied (Epstein et al., 2006). Unfortunately, these results have not been confirmed, which failed to demonstrate significant improvement in identification and evaluation of oral lesions (Farah and McCullough, 2007; Oh and Laskin, 2007). Interestingly, a cross-sectional study compared ViziLite R and VELscope R to evaluate their clinical utility in diagnosing oral lesions, but the authors failed to demonstrate any superiority to COE (Mehrotra et al., 2010).

For this reason, a new version of this device has been developed (ViziLite R PLUS), aiming to improve the diagnostic power of TB marking system. First results were encouraging, showing that TB reduced the number of false positive cases leaving the false negative rate unchanged (Epstein et al., 2008). On the contrary, ViziLite R PLUS does not seems to be useful to detect malignancies in patients with clearly visible lesions (Mojsa et al., 2012). In fact, some authors described the better diagnostic accuracy of ViziLite R with respect to TB staining alone (Rajmohan et al., 2012; Vashisht et al., 2014), justifying the combined use of these two techniques.

Recently, the results of a clinical study suggested that, although the adjunct of TB to ViziLite R reduced the false positive cases without increasing the number of false negatives, there are little benefits in using this device in general dental practise (Chaudhry et al., 2016).

In conclusion, despite the fact that ViziLite R facilitates the identification of hyperkeratotic areas and may increase the visibility of mucosal lesions, the main limitation is currently the high proportion of false positive and false negative tests, regarding the identification of dysplastic areas rather than hyperkeratosis (Chhabra et al., 2015).

### VELscope R

VELscope R (LED Medical Diagnostics, White Rock, BC, Canada) is a hand-held non-magnifying device for direct visualisation of oral mucosa autofluorescence that became commercially available after FDA approval in 2006 (Ayoub et al., 2015). No need of technical measures, such as the use of dimmed light, pre-rinse or lesion-marking solutions, make VELscope R easy to use. It uses a 120 W arc-lamp and a series of philtres and reflectors optimised for producing 400–460 nm wavelength light. The light emitted reaches oral mucosa and excites endogenous autofluorescence substances, called fluorophores (Yamamoto et al., 2017). Preliminary studies, regarding small groups of patients, gave encouraging results (**Table 1**). In the first reported study, 44 patients with confirmed oral dysplasia or OSCC were evaluated with both COE and VELscope R . The results showed that the device can differentiate PMD and OSCC from normal oral mucosa, with high sensitivity and specificity levels (Lane et al., 2006). These results were confirmed in a small OSCC cohort study, in which the use of autofluorescence-guided examination was able to identify subclinical high-risk fields with cancerous changes (Poh et al., 2006). In a study conducted on 60 patients using a semi-quantitative grading system for autofluorescence, VELscope R demonstrate good sensitivity and a better ability to recognise high-grade lesions than COE (Jayaprakash et al., 2009). Another study evaluated 65 subjects with VELscope R , using a specific algorithm based on the ratio of red-to-green fluorescence. The authors found that 405 nm wavelength light was able to discriminate neoplastic and non-neoplastic tissue with high sensitivity and specificity (Roblyer et al., 2009).

In a cross-sectional study, 175 patients with at least one clinical lesion were evaluated using VELscope R . However, despite the good results, the authors warned that this device could lead to overdiagnosis if used by non-specialists (Paderni et al., 2011). In fact, in the following years several studies on patients with PMD or OSCC reported low specificity values, highlighting this as the primary limitation of VELscope R (Awan et al., 2011a; Koch et al., 2011; Scheer et al., 2011). For these reason, other authors concluded that VELscope R examination alone does not provide significant diagnostic benefit beyond COE in screening for PMD and OSCC, also due to interobserver variability (Farah et al., 2012; McNamara et al., 2012). These results were confirmed by a study on 200 patients, limiting the utility of autofluorescence for OSCC screening (Ganga et al., 2017).

One effort to overcome these shortcomings consists of adding the VELscope R exam to the COE. Indeed, as reported by several authors, the combination of COE and VELscope R examination in patients with oral lesions could provide a significative diagnostic yield (Marzouki et al., 2012; Rana et al., 2012; Hanken et al., 2013). However, these results must be interpreted carefully due to the different inclusion and exclusion criteria used to select the patient cohorts, which can influence both sensitivity and specificity.

Other studies focused on combining VELscope R and other diagnostic tests, aiming to find out better approaches to improve detection of PMD and OSCC. For example, the combination approaches of tissue autofluorescence and salivary protoporphyrin IX levels seems to be effective to distinguish between normal mucosa and high-risk lesions (Kaur and Jacobs, 2015). The use of quantitative analysis of autofluorescence were developed to solve the problem of interobserver variability. Novel methods such as quadratic discriminant analysis or luminance ratio were promising, showing a strong concordance with histopathological diagnosis (Huang et al., 2017; Yamamoto et al., 2017). Recently, a retrospective study based on oral photograph was conducted to find colour distribution patterns related to neoplastic lesions. The fluorescence analysis showed differences in the red-to-green ratios of neoplastic areas, suggesting its clinical utility to detect early OSCC (Burian et al., 2017).

Recently, tissue autofluorescence was used to investigate biological aspects of oral carcinogenesis. In the first in vitro study, VELscope R was used to investigate the autofluorescence in a rat tongue carcinogenesis model. The results showed significant changes in autofluorescence pattern during progression to dysplasia and carcinoma (Ohnishi et al., 2016). In another study, RNA sequencing technique was used to identify molecular differences related to autofluorescence patterns. Results were encouraging, demonstrating that the autofluorescence-based excision was successful in achieving a clear molecular margin when excising PMD (Farah et al., 2018). These results confirmed those previously reported in literature, in which VELscope R demonstrated that the actual sizes of some lesions are significantly larger than they look clinically (Elvers et al., 2015).

In conclusion, several criticisms have been made about VELscope R , mainly focused to the limited capacity to extend the use of this device in general dental practise. Future research directions are aimed at improving the specificity of this device, allowing wider clinical use of VELscope R in routine general practise (Bhatia et al., 2013).

### Identafi R

Identafi R (StarDental - DentalEZ, Lancaster, PA, United States) is a probe-like device designed for multispectral screening of PMD, approved by FDA in 2009 as oral screening device (Vigneswaran et al., 2009). Identafi R has three light sources of different wavelengths: white, violet (405 nm), and green-amber (545 nm) lights, that can be sequentially used in oral examination. While white light provides classical visualisation of oral mucosa, violet light excites endogen fluorophores, enabling the assessment of mucosa autofluorescence, like VELscope R . Green-amber

light, through the reflectance spectroscopy, excites haemoglobin molecules in the blood, with the aim to visualise the vasculature (Messadi et al., 2014). A mirror is attached to the probe to help visualise relatively obscure areas in oral cavity.

The first clinical trial with Identafi R was conducted on 88 patients who were treated previously for OSCC (**Table 2**). Screening results with white, violet, and green lights were compared to each other, showing limited benefits of tissue reflectance and autofluorescence in detecting high-risk lesions (Sweeny et al., 2011). In 2012, was reported a case series of PMD patients with the aim to evaluate the efficacy of Identafi R . Although the results are not clearly described, this device seems to be helpful in identifying characteristics not otherwise visible to the COE (Lane et al., 2012).

In a pilot study, Identafi R was used to evaluate tissue vascularity of PMD and to compare with the histological grading of the lesions using a vascular marker (CD34). The results found a correlation between tissue reflectance and histological assessment of vascular structure, in both OSCC and non-cancerous lesions (Messadi et al., 2014).

Two studies on the effectiveness of Identafi R were conducted on Australian population. In the first one, 342 urban Indigenous community members were screened for oral lesions using reflectance spectroscopy and autofluorescence imaging. Identafi R improved the visibility of oral cavity lesions and was capable to find new lesions not seen during COE, although the prevalence of oral pigmentation in this community could hamper the use of autofluorescence screening systems (Lalla et al., 2015). In the second study, 88 patients were evaluated with Identafi R , showing good specificity, negative predictive value, and accuracy (Lalla et al., 2016).

Taken together, the use of Identafi provide the clinician with more data than COE. Unfortunately, the results interpretation requires high level of experience and clinical training in oral pathology, suggesting that its usage should be limited to reference centres for oral pathology (Lalla et al., 2016).

### OTHER DEVICES

Microlux/DLTM (AdDent Inc., Danbury, CT, United States) is a chemiluminescence-based device which became commercially available after FDA approval in 2005. This device has a diffused blue-white LED light source and a fibre optic light guide (McIntosh et al., 2009). It uses the same principles of ViziLite R : after 1-min rinse with 1% acetic acid, oral examination is performed with 460–555 nm wavelength light. Altered epithelial cells cause the appearance of "aceto-white" lesions, and LED light source makes the lesion more easily recognisable. Furthermore, the use of TB can be used in conjunction with Microlux/DLTM, to enhance the visualisation of dysplastic areas (Ibrahim et al., 2014). In 2009 was conducted a study on 50 patients with oral white lesions to evaluate the efficacy of Microlux/DLTM. The results showed that this device can enhance visualisation of oral mucosa, but no clinical improvement was observed, due to poor ability to distinguish between benign and malignant lesions (McIntosh et al., 2009). Another trial that evaluated the effectiveness of Microlux/DLTM was carried out in 2014. 599 patients were examined with COE and Microlux/DLTM with and without TB, showing high sensitivity but low specificity, indicating that this device is not effective to distinguish between benign and malignant lesions, although seems to be a promising screening test for oral lesions (Ibrahim et al., 2014).

GOCCLES R is a medical device (Pierrel S.p.A, Italy) approved by FDA in 2015. This is a low cost and easy-touse device consisting in a pair of glasses equipped with special optical philtres that allows autofluorescence detection. Indeed, GOCCLES R was created to provide an easy and low cost mean of identification of autofluorescence abnormalities in oral cavity with the use of any dental curing light (Moro et al., 2015). In 2010 was reported the first study on GOCCLES R in a small cohort of selected patients, showing high sensitivity and specificity (Moro et al., 2010). Five years later, a nonrandomised multicentre trial was conducted on patients at risk for OSCC, suggesting the need for further researches to define the diagnostic performance of this device (Moro et al., 2015). Indeed, despite the low cost of GOCCLES R could encourage more careful examinations, its main limitation seems to be the interobserver variability, that could be overcome by proper training.

In recent years, other instruments have been developed and commercialised for facilitate the early identification of oral lesions. Their operating principle is equivalent to the devices described above, using either autofluorescence or chemiluminescence detection. However, their clinical effectiveness is currently hampered by the lack of published studies. For these reasons, they will only be mentioned briefly here. Bio/Screen R (AdDent Inc., Danbury, CT, United States), an instrument with five violet (390–430 nm) high-power LED, designed to enhance the visualisation of mucosal abnormalities through the use of tissue autofluorescence (Kahn et al., 2018).

Orascoptic DKTM system (Orascoptic, Middleton, WI, United States) is another chemiluminescence-based device, designed to improve the visualisation of oral lesions through the use of blue-white LED light and oral rinse of 1% acetic acid solution (Patton et al., 2008).

Sapphire R Plus LD (DenMat Holdings, Lompoc, CA, United States), DentLight DOETM Oral Exam System (DentLight, Richardson, TX, United States), and OralIDTM 2.0 (Forward Science Technologies, Stafford, TX, United States) are other tissue autofluorescence-based devices developed in order to detect oral lesions (Kahn et al., 2018).

### CONCLUSION AND FUTURE PERSPECTIVES

The diagnostic techniques presented here showed great potential for screening and monitoring oral lesions (Liu et al., 2016). Unfortunately, to date several factors hinder an extensive use of these devices: (1) data do not demonstrate clear superiority of these methods compared to COE; (2) there remains the need for well-designed multicentre prospective studies; (3) these devices

exhibit a not-negligible interobserver variability, limiting their use to clinicians with significant experience in oral pathology (Patton et al., 2008; Carreras-Torras and Gay-Escoda, 2015).

However, the current evidence suggests that these devices: (1) seem to be useful in assessing lesion margins that must be biopsied and, therefore, may be useful in surgical management; (2) can be used to investigate biological aspects of oral carcinogenesis, leading to more accurate methods for interpreting data from LBDS; (3) can be enhanced with new approaches used to analyse optical imaging data, with the aim to quantify the results obtained; (4) lowering the costs of these devices could indirectly lead to greater attention for oral lesions among both patients and general dental practitioners, allowing in turn to promote a culture of oral cancer prevention (Carreras-Torras and Gay-Escoda, 2015; Moro et al., 2015); (5) finally, the possibility of implementing LBDS through the use of tissue-marking dyes can in principle allows to develop strategies for the use of nanoparticles. Indeed, nanoparticles can provide molecular targeted imaging, with higher image contrast and resolution. For example, a promising

### REFERENCES

nanotechnology in oral diagnostic research is the quantum dots, consisting in nanometre-sized semiconductor crystals (Walling et al., 2009). The biophysical characteristics of these particles confer several advantages over conventional dyes and fluorescent proteins. The possibility to link the quantum dots to molecules with the ability to target cancer cells make them ideal for diagnostic applications in detecting PMD and OSCC (Chen et al., 2018). Therefore, the use of nanotechnologies could be the next step in the evolution of LBDS, providing devices that can help clinicians to detect and better monitor oral lesions.

### AUTHOR CONTRIBUTIONS

MM, AS, and MP conceived the literature review. MM and VT described the VELscope R system. AS and RM described the ViziLite R system. AB and GO described the Identafi R system. AP, MP, and GO wrote the concluding remarks. All authors discussed and approved the final version of the manuscript.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Mascitti, Orsini, Tosco, Monterubbianesi, Balercia, Putignano, Procaccini and Santarelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Novel Biological and Technological Platforms for Dental Clinical Use

Giovanna Orsini1,2, Pierfrancesco Pagella1 , Angelo Putignano2 and Thimios A. Mitsiadis1 \*

1 Orofacial Development and Regeneration, Institute of Oral Biology, Center of Dental Medicine, Faculty of Medicine, University of Zurich, Zurich, Switzerland, 2 Department of Clinical Sciences and Stomatology, Marche Polytechnic University, Ancona, Italy

Human teeth have a limited capacity to regenerate and thus biological reconstruction of damaged or lost dental tissues remains a significant challange in modern dentistry. Recent efforts focus on alternative therapeutic approaches for partial or whole tooth regeneration that complement traditional dental treatments using sophisticated materials and dental implants. These multidisciplinary approaches are based on the combination of stem cells with advanced tissue engineer products and computing technology, and they hold great promise for future applications in dentistry. The administration to patients of dynamic biological agents composed by stem cells and scaffolds will certainly increase the regenerative capacity of dental pathological tissues. The design of innovative materials for tissue restoration, diagnostics, imaging, and targeted pharmaceutical treatment will significantly improve the quality of dental care and will have a major societal impact. This review depicts the current challenges in dentistry and describes the possibilities for novel and succesful therapeutic applications in the near future.

Edited by:

Catherine Chaussain, Université Paris Descartes, France

#### Reviewed by:

Thomas G. H. Diekwisch, Texas A&M University, United States Ivo Lambrichts, University of Hasselt, Belgium Jean-Christophe Farges, Claude Bernard University Lyon 1, France

\*Correspondence:

Thimios A. Mitsiadis<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Craniofacial Biology and Dental Research, a section of the journal Frontiers in Physiology

Received: 20 June 2018 Accepted: 23 July 2018 Published: 08 August 2018

#### Citation:

Orsini G, Pagella P, Putignano A and Mitsiadis TA (2018) Novel Biological and Technological Platforms for Dental Clinical Use. Front. Physiol. 9:1102. doi: 10.3389/fphys.2018.01102 Keywords: tooth, dental treatment, stem cells, organ-on-chip, organoids, dental implants, dental pulp, periodontium

### INTRODUCTION: THE TOOTH ORGAN

The tooth organ is composed by a unique combination of hard and soft tissues. The outermost layer is constituted by enamel, the most mineralized tissue of the human body, which guarantees protection to the inner elements of the tooth (**Figure 1**). Enamel displays unique physical characteristics, such as complex three-dimensional organization and extremely long hydroxyapatite crystallites, to resist large masticatory forces and continual attacks by acids from food and bacterial sources (Boyde, 1997). Ameloblasts, which are the epithelial cells responsible for enamel formation, and their precursors are lost upon tooth eruption, making human adult teeth inapt of enamel regeneration. The great complexity of enamel, together with the absence of appropriate cells in adult patients, make therapies aiming to enamel regeneration an exciting challenge.

Due to its extremely high mineral content, enamel is very brittle. This property is compensated by dentin, a less mineralized, elastic, avascular tissue (**Figure 1**). Dentin encloses the dental pulp, a soft connective tissue that conveys vascularization and innervation, representing the vital core of the tooth organ (**Figure 1**). The vascular system provides oxygen, nutrients and metabolites, while sensory innervation is fundamental for the perception of pain, heat/cold and mechanosensation that controls biting strength. In the peripheral boundary of the dental pulp

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are situated mesenchymal-derived odontoblasts, which produce and maintain dentin. Dentin is characterized by closely packed tubules traversing its thickness and containing the cytoplasmatic extensions of odontoblasts, as well as sensory nerve terminals, which render dentin highly sensitive to external stimuli. More importantly, dentin can repair itself, due to the activation of the existing odontoblasts or the newly formed odontoblasts derived from pulp stem cells that produce a reactionary mineralized matrix upon injury. However, pulp reaction is not sufficient in case of severe tooth injury and/or extensive infection, and this healing failure often leads to pulp irreversible inflammation followed by necrosis (DeRosa, 2006).

The tooth is anchored to the alveolar bone by the roots, constituted by dentin and cementum. Roots are connected to the alveolar bone by a specialized connective tissue, the periodontal ligament, which ensures tooth stability, provides

within dentin). Abbreviations: d, dentin; e, enamel; p, pulp; td, tertiary dentin.

sensory information and absorbs mechanical stresses during chewing (**Figure 1**). Periodontal disease is the most frequent cause of tooth loss, making periodontal regeneration a pressing need for the dental field (Mitsiadis et al., 2015).

The structural hallmarks of dental hard tissues are strictly dependent on tightly regulated and long developmental processes that cannot be easily reproduced within acceptable therapeutic time frames. Moreover, the oral cavity constitutes a challenging environment for any regenerative approach, as it is constantly exposed to chemical, mechanical and bacterial insults. Despite these difficulties, recent technological advancements are becoming an inherent aspect of dental practice, improving effectiveness of treatments. Similarly, the continuous developments in stem cell research and nanotechnology are paving the way for regenerative approaches in dentistry.

### INNOVATION IN CURRENT DENTAL TREATMENTS: FROM MATERIALS TO TOOTH REGENERATION

The great improvements in computing-related technologies and materials has widened the options to alternative and more precise dental treatments (Beuer et al., 2008; Hancocks, 2017), and helped in establishing more reliable diagnostic tools and therapeutic plans (Levato et al., 2015; Lynch, 2017). Numerous advancements have been made with the advent of novel imaging techniques such as computer-aided design and manufacturing (CAD/CAM) technology, optimized intraoral imaging, digital radiography, and computer aided implant surgery (Hammerle et al., 2009; Levato et al., 2015; Zhou et al., 2018). Apart from its use as a diagnostic tool, imaging contributed to the improvement of the daily dental practice, since treatments benefited from high definition microscopes that permit the detailed visualization of the operative dental field (Del Fabbro et al., 2015).

Material sciences have led the way for the development of therapeutic approaches aiming to substitute damaged or lost dental tissues. Despite limitations in functionality and longevity, biomaterials are still present in dental treatments since nanotechnology has remarkably improved their performance and the clinical outcome of certain procedures. The combination of nanomaterials with advanced technologies has upgraded prosthetic and aesthetic dentistry, which are fields aiming to optimize the functional and aesthetic appearance of dentition. 3D printing systems represent the most innovative nextstep technology, aiming to manufacture customized products based on computer-designed digital tools (Yang et al., 2018). Pain management has also enormously benefited from the advent of these novel technologies (Banerjee et al., 2011). To minimize pain perception, low-level laser therapy and light emitting diode therapy (also known as photobiomodulation) have been used. These processes induce analgesia but also promote tissue healing and reduce inflammation and/or oedema by stimulating cell response (Carroll et al., 2014). Their efficacy has been already demonstrated for the treatment of trigeminal neuralgia, pain management during orthodontic treatment and

following surgeries within the orofacial complex, and dentin hypersensitivity (Kathuria et al., 2015).

However, the most important development of the last decade is the rise of a new dental discipline that is based on the capacity of stem cells to repair or regenerate various impaired tissues. Stem cell-based regenerative dentistry is linked to advanced tissue engineering products and nanotechnology, which have created an important clinical shift toward the functional repair and regeneration of damaged dental tissues.

### Combining Stem Cell Biology and Nanotechnology for Regenerating Dental Tissues

Stem cells are characterized by their potential to self-replicate and their capacity to differentiate into a vast variety of cells populations (Mitsiadis and Graf, 2009). Epithelial and mesenchymal stem cell populations are present in almost all adult human tissues and organs, including teeth. A variety of dental mesenchymal stem cells (DMSCs) populations have been isolated from both deciduous and permanent teeth, characterized, and tested for their potential applications in regenerative dentistry (Gronthos et al., 2000; Gronthos et al., 2002; Miura et al., 2003). Adult DMSCs localized in the dental pulp and periodontal tissue ensure human tooth homeostasis and regeneration (Bluteau et al., 2008), and therefore represent optimal clinical tools for the repair of damaged dental tissues. Actual efforts are oriented toward pulp and periodontal tissue repair, where these tissues can be regenerated by transplantation of stem cells alone or in combination with functionalized scaffolds. More challenging and problematic is, however, the regeneration of tooth enamel using epithelial cells, since neither dental epithelial stem cells (DESCs) nor ameloblasts are present in the crown of adult functional teeth (Mitsiadis et al., 2015; Orsini et al., 2015). More exiting, but greatly perplexing, is the perspective to generate entire brandnew teeth by mixing DESCs and DMSCs. Although very difficult to be realized, several attempts toward this direction have been pursued in animal models (Oshima and Tsuji, 2014).

The success and efficacy of any stem cell-mediated therapy can be evaluated by a set of modern nanotechnology tools, since they allow tracking the migration, fate and regenerative impact of stem cells in vivo. For example, transplanted stem cells can be tracked for long periods with non-invasive imaging techniques using fluorescent dyes (Arbab et al., 2009; Gera et al., 2010), and with magnetic nanoparticles that can be traced by MRI and provide information about their kinetics and fate during dental tissue regeneration (Jimenez-Rojo et al., 2012). This knowledge could be used for designing appropriate scaffolds that will host stem cells before transplantation. Furthermore, it will allow evaluating the therapeutic efficacy of precise dental stem cell populations that have been exposed to specific microenvironments. Indeed, artificial microenvironments, which may direct stem cells toward a precise fate and function, can be achieved through nanotechnology (Bluteau et al., 2008). A big variety of nanoscale biodegradable structures with specific size, surface chemistry and shape can be used for the creation of microenvironments that are adapted for the needs of regenerative dentistry (Mitsiadis et al., 2012). Such biodegradable scaffolds, once transplanted, may act as temporary niches that control stem cell behavior and guide dental tissue repair (Iwatsuki et al., 2006).

It is obvious that the range of dental disciplines that can benefit from the recent advances of stem cell biology, material sciences and nanotechnology is extremely wide. The present mini-review covers current and future therapeutic approaches for managing the (1) damage of the tooth crown, including the harm of enamel and/or dentin-pulp tissues, (2) periodontal insults, and (3) tooth loss.

### TOOTH CROWN DAMAGE

### Current Restorative Treatments

Enamel and dentin of the tooth crown are most often the first tissues to be affected following traumatic injuries or carious lesions (**Figure 1**). Prompt and efficient repair of enamel and/or dentin is fundamental to prevent infection and damage extending toward dental soft tissues (i.e., dental pulp, periodontium) and alveolar bone. The most used approach for treating enamel and dentin harm is the substitution and restoration of the destroyed or lost dental hard tissues by sophisticated composite materials. However, traditional adhesive systems are unstable and fail over time, thus leading to marginal leakage and poor retention of the restoration in the tooth (Breschi et al., 2018). Therefore, a major task of nanotechnology in dentistry is to develop novel durable materials and adhesive systems with improved enamel- and/or dentin-bonding performance in order to increase the longevity of the restorations and prevent repeated treatments. Indeed, the introduction of novel materials such as phosphine oxide initiators and monomethacrylate diluents has led to dental composites with satisfactory and adequate properties (Kilambi et al., 2009). The introduction of nanofillers and nanomaterials led to even more significant advances in terms of optimizing the properties and performance of the composites (Ilie et al., 2013; Goracci et al., 2014; Monterubbianesi et al., 2016). These nanotechnology-based strategies using cross-linking agents and Ca- and P-releasing means, which mimic the process of natural dentin mineralization, have also reduced the degradation of the resin-dentin bonded interface (Mazzoni et al., 2018).

Ceramic-based materials are privileged by dentists for the restoration of damaged tooth crowns, mainly because of their superior aesthetic appearance and biocompatibility (Wittneben et al., 2017; Ozcan and Jonasch, 2018). To overcome the fact that ceramic materials are brittle and prone to cracks propagation, several transformation/toughening mechanisms have been developed, leading to higher aging resistant-ceramics (Zhang et al., 2017) such as zirconia with exceptional toughness and flexural strength (Guazzato et al., 2004).

Nanomodified materials could be also designed for controlling oral microbiota and the formation of dental plaque, and, furthermore, for enhancing the mineralization process in the cases of enamel wear and/or dentin hypersensitivity due to the extensive consumption of acidic drinks (Orsini et al., 2013; Lelli et al., 2014). Indeed, the use of synthetic nanohydroxyapatite particles and other Ca-based nanomodified

materials in dentifrices may offer a protective nanostructured coating on the tooth surface that simultaneously restores the lost minerals from enamel (Orsini et al., 2013; Lelli et al., 2014).

The preservation of the dental pulp, which is a living tissue ensuring tooth physiological function, is of prime importance during the treatment of a damaged tooth crown. In very severe tooth injury, the pulp is also affected and may lose its vitality. Therefore, the endodontic therapy (i.e., pulp tissue removal) is imposed in order to prevent further bacterial progression and damage of the surrounding alveolar bone. This is followed by disinfection of the dental root canals and the replacement of the pulp tissue with inorganic materials. Devitalized teeth are more fragile than normal teeth and consequently are predisposed to postoperative fractures (DeRosa, 2006).

### Challenges in Dentin-Pulp Regeneration

Regenerative endodontics aims at reforming the original pulp tissue morphology and physiology based on tissue engineering principles (Murray and Garcia-Godoy, 2006; Diogenes and Hargreaves, 2017). Nanomaterials can be used either alone or implemented with growth factors and stem cells in order to stimulate and enhance the regenerative capacity of the pulp tissue. Adjustment of biomaterials for dental specific purposes would require adjustments at a nanoscale level, thus allowing multifunctionality within a given small surface, increasing the quality of targeting, and better controlling bioactive molecules delivery (Fioretti et al., 2011; Diogenes and Ruparel, 2017). Nanomaterials developed for endodontic purposes can deliver antibacterial and anti-inflammatory molecules, as well as growth factors that will guide the behavior (e.g., cell migration, proliferation, and differentiation) of the various dental pulp cell populations (e.g., pulp fibroblasts, endothelial cells, neuronal cells, immune cells). Biomimetic scaffolds composed of natural molecules, such as type I collagen, hyaluronic acid and chitosan, combined with nanoassembled materials possessing anti-inflammatory capabilities have been generated to stimulate pulp tissue regeneration and to prevent inflammation (Fioretti et al., 2011). Although such nanofibrous and microporous membranes have provided promising results, significant improvements are still needed to create scaffolds that promote proper pulp regeneration (Yamauchi et al., 2011; Albuquerque et al., 2014).

Numerous attempts using human DMSCs have been made in a variety of animal models in order to achieve complete dental pulp regeneration (**Figure 2A**), a process that also requires neovascularization and re-innervation of this tissue.

Orsini et al. Innovations in Dentistry

Experiments have shown that human DMSCs are capable to differentiate into odontoblasts and to form dentin-like structures when transplanted together with a ceramic powder in immune-compromised mice ex vivo (Gronthos et al., 2000; Gronthos et al., 2002). Similar studies have revealed that human DMSCs seeded on poly-D, L-lactide/glycol scaffolds are able to regenerate vascularized pulp tissue when transplanted into an empty mouse tooth root canal (Volponi et al., 2010; Hayashi et al., 2015). Recently, new experimental strategies have been elaborated, where DMSCs-seeded scaffolds combined with bioactive molecules fulfill the empty pulp chamber immediately after pulp removal (Albuquerque et al., 2014; Piva et al., 2014). Pilot studies in humans have demonstrated the safety and efficacy of DMSCs for complete dental pulp regeneration and new dentin formation (Nakashima and Iohara, 2017; Nakashima et al., 2017). Bone morphogenetic proteins (BMPs) have been commonly used for accelerating and enhancing the production of dentin during dental pulp regeneration (Luiz De Oliveira Da Rosa et al., 2017). While these stem cell-based procedures appear to improve pulp tissue regeneration, their effectiveness for achieving accurate, precise and long-lasting therapies is still unclear. As a matter of fact, most approaches aiming at dental pulp regeneration led to the formation of fibrotic tissues that can undergo degeneration over time or be replaced with bone. The possibility to decellularise healthy human dental pulps (Song et al., 2017) opens new horizons in regenerative dentistry since these decellularised tissues could serve as natural scaffolds for supporting transplanted autologous DMSCs. Decellularised pulps represent ideal biomaterials for hosting stem cells and guiding neovascularization and re-innervation within the regenerating tissues. Moreover, novel 3D printing strategies has been developed to engineer prevascularized pulp-like hydrogel tissue constructs in full-length root canals (Athirasala et al., 2017).

While significant efforts have been produced so far, regenerative procedures have to be further investigated in order to ultimately provide evidence of functional dental pulp regeneration in vivo (**Figure 2A**; Torabinejad and Faras, 2012; Diogenes and Ruparel, 2017).

### Challenges in Enamel Regeneration

De novo formation of enamel in humans is one of the greatest challenges in regenerative dentistry, since amelogenesis is a very complex process and DESCs that could regenerate enamel are very rare in adult human teeth. Very few dental epithelial cells with stem cell properties have been isolated from the periodontal tissue (i.e., epithelial rests of Malassez, ERM). Experiments using porcine ERM have demonstrated that these cells can differentiate into ameloblasts when co-cultured with dental pulp cells in vitro and can form enamel structures after their transplantation in vivo (Shinmura et al., 2008). Although ERM is a potential stem cell source for enamel regeneration, availability of these cells in human teeth is scarce, making thus necessary the identification of other epithelial stem cell populations of non-dental origin that could differentiate into enamel-producing ameloblasts.

Another key issue in generating new enamel is time. The accomplishment of proper enamel formation requires many years, a time frame clearly incompatible with clinical needs. Moreover, mild disturbances during this process could lead to the generation of defective enamel (Cantu et al., 2017). Therefore, any procedure and technique that will be able to considerably accelerate the process of amelogenesis will be of benefit to the patients and dental community.

### PERIODONTAL DISEASES

### Current Periodontal Therapies

Periodontium is a common site of pathologies that severely affect not only the structure of the surrounding tissues (i.e., dental root, alveolar bone) but also tooth functionality. Severe inflammation to the periodontium leads to significant alterations in both the structure and quantity of the alveolar bone, a process that ultimately may cause tooth loss (Lindhe et al., 1983). Contemporary, periodontal therapies include a wide range of surgical procedures along with use of bone grafts as tissue substitutes, barrier membranes for protecting the healing area from undesirable epithelial tissues (Howell et al., 1997; Aghaloo and Moy, 2007), and growth factors for enhancing the healing capacity of the harmed tissues (Lynch et al., 1991b). Bone grafting materials, aiming to stimulate bone augmentation and periodontal regeneration, include intraoral or extraoral autografts, freeze-dried and fresh-frozen bone allografts, animalderived bone deproteinised xenografts, and hydroxyapatite and beta-tricalcium phosphate alloplasts (Pilipchuk et al., 2015; Sheikh et al., 2017). These grafting materials could be used alone or in association with various growth factors. It has been shown that application of these regenerative methods in clinics allowed the formation of novel osseous tissues with similar to the pre-existent native bone characteristics (Scarano et al., 2006; De Angelis et al., 2011; Danesh-Sani et al., 2016; Clark et al., 2018). Even though, these approaches do not always ensure a predictable and desirable outcome of periodontal regeneration and often result in healing with epithelial lining rather than new periodontal tissue formation (Lynch, 1992).

### Challenges in Periodontal Regeneration