3D-printed microgels supplemented with dentin matrix molecules as a novel biomaterial for direct pulp capping

Abstract

Objectives:

To develop a 3D printed, microparticulate hydrogel supplemented with dentin matrix molecules (DMM) as a novel regenerative strategy for dental pulp capping.

Materials and Methods:

Gelatin methacryloyl microgels (7% w/v) mixed with varying concentrations of DMM were printed using a digital light projection 3D printer and lyophilized for 2 days. The release profile of the DMM-loaded microgels was measured using a bicinchoninic acid assay. Next, dental pulp exposure defects were created in maxillary first molars of Wistar rats. The exposures were randomly capped with (1) inert material - negative control, (2) microgels, (3) microgels + DMM 500 μg/ml, (4) microgels + DMM 1000 μg/ml, (5) microgels + platelet-derived growth factor (PDGF 10 ng/ml) or (6) MTA (n=15/group). After 4 weeks, animals were euthanized, and treated molars were harvested and then processed to evaluate hard tissue deposition, pulp tissue organization, and blood vessel density.

Results:

All the specimens from groups treated with Microgel + 500 μg/ml, Microgel + 1000 μg/ml, Microgel + PDGF, and MTA showed the formation of organized pulp tissue, tertiary dentin, newly formed tubular and atubular dentin, and new blood vessel formation. Dentin bridge formation was greater and pulp necrosis was less in the Microgel + DMM groups as compared to MTA.

Conclusions:

The 3D-printed photocurable microgels doped with DMM exhibited favorable cellular and inflammatory pulp responses, and significantly more tertiary dentin deposition.

Clinical Relevance:

3D Printed microgel with DMM is a promising biomaterial for dentin and dental pulp regeneration in pulp capping procedures.

Introduction

Dental caries is a biofilm-mediated, diet modulated, multifactorial, dynamic disease, which can cause mineral loss of dental hard tissues [1, 2]. Caries affects between 44.1% and 58.9% of children in permanent dentition on continents throughout the world, including 90% of Western adults [3]. Dental caries progression, and/or the occurrence of secondary caries, damages the dentin structure and often leads to pulp tissue exposure, which then requires some form of endodontic treatment [4, 5]. Depending on the extent of the pulp tissue damage, treatment options may include direct pulp capping, partial pulpotomy, full pulpotomy or pulpectomy [4]. Direct pulp capping is a more conservative approach, where treatment includes sealing the exposed dental pulp with a dental cement to induce the formation of reparative dentin and maintain a vital pulp [5]. However, the success of direct pulp capping depends not only on the vitality of the treated dental pulp, but also on patient age, the type and location of the damaged tooth, and the type of restorative material used [6, 7]. If direct pulp capping fails to induce pulp healing, more aggressive approaches will be necessary, including partial or complete extirpation of the dental pulp, which will further compromise tooth response and long-term vitality.

The most widely used material for direct pulp capping is mineral trioxide aggregate (MTA), a calcium silicate cement as recommended by the American Academy of Pediatric Dentistry [8] and the American Association of Endodontists [9]. ProRootMTA was the first commercially available cement and became very popular due to its biocompatibility and ability to set in a moist environment [10]. MTA is placed in the tooth to partially fill the space left by the removal of necrotic dentin and dental pulp. However, despite its capacity to stimulate dentin repair and induce the formation of a mineral barrier above the pulp tissue, MTA fails to promote the regeneration of the native pulp. This shortcoming results from a variety of issues related to silicate cements, such as their compositional and structural dissimilarity to the native dental pulp extracellular matrix, the inability to controllably resorb the material once implanted in the pulp, and the lack of binding sites to allow host cells to adhere to and remodel the material [11]. Moreover, MTA requires long setting times, can present difficulties in handling, and often results in discoloration of the tooth [12].

Dentin is composed of a complex mix of growth factors and cytokines that has been extensively characterized [13]. In addition to hydroxyapatite mineral crystallites and collagen [14], the dentin matrix is comprised of various bioactive proteins and proteoglycans such as transforming growth factor beta (TGF), vascular endothelial growth factor (VEGF), dentin matrix protein-1 (DMP-1), bone sialoprotein, osteocalcin, decorin, and others that are ‘fossilized’ in the mineralized dentin matrix during odontogenesis. [15] As such, these dentin matrix molecules (DMMs) function as bioactive components ready to initiate dental pulp regenerative events after injury of the dentin-pulp complex [14, 15]. DMMs regulate cell growth, migration, differentiation of dental pulp cells and tertiary dentin formation [13, 14]. Thus, there is a stark difference between the functions of truly bioactive molecules and that of MTA, which in and of itself is not regenerative.

To be used for regenerative purposes, DMMs require a vehicle or carrier to provide spatial control of their release in the dental pulp. Toward that end, hydrogels have long been proposed as improved materials to promote the regeneration of soft tissues, due to the fact that they can contain certain properties that mimic the native extracellular matrix (ECM), such as the presence of RGD binding sites, tunable mechanical properties, biological and biocompatibility characteristics, and an ability to function as carriers for growth factors [16, 17]. The use of 3D-printed hydrogels containing DMMs could provide a more regenerative approach to pulp repair/replacement. However, when hydrogel scaffolds are delivered in bulk to a tissue defect site, the diffusion limit of the scaffold’s core and slow pace of remodeling by host cells can pose challenges for regenerating relatively large tissue defects. To overcome the limitations of bulk gels exhibiting low overall surface area, modular strategies have been developed where tissue constructs are built from the bottom-up, as small individual building blocks of high surface area, micro engineered hydrogels, named microgels [18–21]. Microgels are attractive for large tissue regenerative applications due to their advantageous properties of injectability, minimally invasive delivery, well-defined shape and porosity and biocompatibility [22, 23]. Depending on the type of hydrogel and crosslinking method, a variety of fabrication techniques have been used to engineer microgels, including microfluidics [18], batch emulsion [24] and 3D printing [19].

In the present study, we developed a Digital light processing (DLP) 3D printing technology to fabricate photocurable microgels of defined micrometric structure (size and geometry) and that contained embedded DMMs, to provide controlled release of DMMs into the pulp microenvironment, as a new direct pulp capping agent to promote dental pulp regeneration. The use and application of this material in pulp therapy aims to harness the natural ability of dentin to self-repair, and benefits from a self-limiting feature as it is degraded over time, thereby inducing the formation of reparative dentin only at the site of injury. The ability to combine the versatility of 3D-printed microgels with the bioactive properties of DMMs provides a superior product for direct pulp capping and is proposed as a novel strategy for vital pulp therapy. We hypothesized that microparticulate hydrogels supplemented with DMMs would promote increased tertiary dentin formation and dental pulp tissue organization as compared to the current commercially available gold standard, MTA. Toward that end, we have tested this novel material in defects created in maxillary first molars of Wistar rats. Four weeks post treatment, the molars were harvested and evaluated with respect to dental pulp necrosis, pulp tissue organization and tertiary dentin formation.

Materials and Methods

Isolation of dentin matrix molecules (DMM)

As previous studies from our groups showed an equivalence of the proteomic content of human and bovine teeth [13], we used the later as a scalable source of DMM. Thus, bovine incisors were extracted from 30-month-old slaughtered heifers from an USDA-inspected establishment (USDA #M9233/P9223). Harvested teeth were stored in 0.5% chloramine T solution (Sigma-Aldrich, St. Louis, MO, US) at 4°C for no more than one week prior to processing. Next, teeth were cleaned via thorough irrigation with saline, deionized (DI) water and a toothbrush. Soft periodontal and gingival tissue was removed using a scalpel, hard tissue enamel and cementum were removed using a diamond bur under water cooling, and dental pulp tissue was removed using a barbed broach and endodontic file. The remaining dentin was crushed in liquid nitrogen using a 6700 Spex SamplePrep freezer/mill operated via a magnetically driven impactor for 30 min. Pulverized dentin was immersed in an extraction solution of 10% (w/v) ethylenediaminetetracetic acid (EDTA) in Tris buffer, pH 7.2, containing Halt protease inhibitor cocktail (1 μl/mL) (ThermoFisher Scientific, Waltham, MA, US) (Figure 1 A). During the following seven days, the extraction solution was changed daily, and the supernatants were collected and frozen at −80°C. Subsequently, the supernatants were thawed, dialyzed at 4°C for 5 days and lyophilized for 48 hours. The powder containing the DMMs was stored at −80°C until further use.

Fig. 1.

Schematic representation of microgel fabrication.

Microgel printing

GelMA was synthesized using previously published protocols [25]. Briefly, gelatin (10% w/v) was dissolved in 50°C Dulbecco’s phosphate buffered saline (DPBS), and 8% (v/v) methacrylic anhydride was added to the solution in dropwise fashion, allowing the reaction to proceed for 2 hours. The solution was stopped with 5x DPBS and dialyzed against distilled water (DIW) using a 12-14 kDa dialysis tubing at 45±5°C for 5 days. The resulting prepolymer was lyophilized for 5 days and stored at −80^oC until further use. GelMA at a concentration of 7% (w/v) was dissolved in DPBS at 80°C with 0.075% (w/v) lithium phenyl (2,4,6-trimethyl benzoyl) phosphinate (LAP, Tokyo Chemical Industry, L0290) photoinitiator, then vortexed and maintained at 80°C until completely dissolved.

GelMA 3D printing was performed using a Digital Light Processing 3D printer (Ember 3D Printer DLP® by Autodesk, San Rafael, California, USA). Digital light processing (DLP) 3D printing technology employs a conventional light source like an arc lamp with a liquid crystal display pane and a vat with liquid polymer or hydrogel which is exposed to light in safe conditions. The light movement and time are controlled using previously defined CAD files, forming a 3D structure as the build stage is translated vertically, constructing the object layer by layer [26]. Microgels were designed with a 5-pointed flower-like geometry (500 μm width and length) using Fusion 360 (Autodesk). Hydrogel prepolymer was dispensed between two polydimethylsiloxane (Sylgard 184, Dow Corning) sheets, and exposed to a blue light source (405±5 nm) with an irradiance of 20 mW/cm² (Figure 1 B).

In the groups supplemented with DMMs, varying concentrations of the DMM extract (0, 500 or 1000 μg/ml) or PDGF beta (10 ng/ml) were added to the GelMA prepolymer immediately before printing. A design of 5 x 4 clusters of 25 microgels each was used (Figure 1 C). After printing, microgels were washed with 1% penicillin/streptomycin solution (v/v), stored at −80°C overnight, lyophilized for another 24 h, and stored within Eppendorf vials at −20°C until further use.

Release assay

DMMs were added to the GelMA prepolymer and microgels were printed as described above. To measure DMM protein release, 10 mg of lyophilized microgels were placed in 1.5 mL centrifuge tubes, 1 mL of 0.1% (w/v) BSA in PBS was added, and the samples were placed on a horizontal shaker at 4°C overnight. The passive release of proteins into the 0.1% (w/v) BSA in PBS was monitored for seven days at 4°C. On days 1, 2, 3, 5 and 7, the samples were centrifuged at 3000 rpm for 5 min, and the supernatant was collected and replaced with an equivalent volume of fresh 0.1% (w/v) BSA in PBS. The released DMM proteins present in the supernatant were then measured using a Pierce BCA Protein Assay (Thermo Scientific, Waltham, Massachusetts, EUA) following the manufacturer’s protocol.

Study design

All experimental animal procedures were approved by Ethics Committee on Animals Use of the Institution (protocol #6856180618) and performed in accordance with its Animal Care Standards. This study’s methodology was reported in accordance with ARRIVE (Animal research: Reporting in vivo experiments) guidelines for reporting animal research [27].

The study is a comparative animal experiment; rats were divided into four groups according to materials used. A total of 84 maxillary first molars from 84 male Wistar rats were needed for compared (1) clinical gold standard treatment with White MTA (Angelus, Londrina, PR, Brazil) to experimental groups (2) unsupplemented microgels alone (Microgel); (3) microgels supplemented with dentin matrix molecules - 500 μg/ml (Microgel + DMM 500 μg/ml); (4) microgels supplemented with dentin matrix molecules - 1000 μg/ml (Microgel + DMM 1000 μg/ml); and (5) positive control - microgels supplemented with platelet-derived growth factor (PDGF) - 10 ng/ml (Microgel + PDGF 10 ng/ml) and (6) negative control (NC) treated with commercial temporary filling material with inert properties (Zinc Oxide, Zinc Sulphate, Calcium Sulphate, Polyvinyl Acetate, Menthol, Dibutylphthalate – Cotosol, Joinville, SC, Brazil). After 4 weeks the animals were euthanized.

Study setting

This study was conducted at the Central Animal Facility of the Federal University of Ceara. The histological processing of the study was conducted in the Department of Pathology of the Faculty of Dentistry, in the same University.

Sample size

This study was designed to minimize the number of animals required for the experiments. We based our calculation on the study of Tziafa et al., [27] and used G*Power software to calculate sample size and power analyses. Thus, assuming and alpha of 0.05, an effect size of 0.5, a variance of 20% and power of 95% of power, we need at least 12 rats per experimental group. Considering the possibility of sample loss, 10% was added to the sample, totaling 14 rats per study group.

Inclusion and exclusion criteria

Inclusion criteria consisted of healthy, young adult, male Wistar (Rattus norvegicus) rats (n=14/group) weighing 180-220 g and with all maxillary first molars free from caries or fractures. Any rat showing systemic illness, wounds, infections, fractures, carious, or periodontally compromised teeth was excluded. All animals were purchased from the Central Animal Facility of the Federal University of Ceara.

Randomization and allocation concealment

The animals were randomly selected by using the random command in Microsoft Excel (Microsoft Corp., Redmond, WA). Afterward, animals were identified and the position of the cage inside the Animal facility was followed by the previous planning.

Blinding

Only one researcher was aware of the group allocation at the different stages of the experiment. The other researchers were blinded to the animals allocation.

Experimental procedures

A protocol for direct pulp capping previously described by Liu S et al [28] was used in this study (Figure 1, Supplementary Figure 1).

The animal’s teeth were cleaned and disinfected with cotton soaked in 75% ethanol, as well as all instruments and materials were all autoclaved before surgeries[28, 29]. For the anesthesia, injection of a solution of xylazine (100 mg/kg) and ketamine (5 mg/kg) was administered intraperitoneally. The operator used a dental microscope (4.5x magnification) to perform the entire procedure, while an assistant monitored the animal to avoid contamination of the operative field. The defects were prepared on the occlusal surfaces of maxillary first molar teeth using a 0.5 mm diameter round bur (KG Sorensen, Sao Paulo, Brazil) in a high-speed handpiece under continuous irrigation. Dental pulps were exposed using an endodontic file (#15 sterile stainless steel; Dentsply Maillefer, Ballaigues, Switzerland) to push through any remaining thin dentin of each tooth defect [28, 29]. Bleeding was controlled by pressing sterile paper points for a few seconds. MTA was mixed with sterile H2O according to the manufacturer’s instructions and placed using the tip of an explorer into the tooth defect. For microgel containing groups, the material was delivered into the cavity using a pair of tweezers and loosely packed into the dental cavity. Hydration of the lyophilized microgels occurred spontaneously after they were placed within the tooth defect, which also dispersed the material into individual microgels. A thin layer of White MTA was then placed on top of the microgel and other treatments. After the MTA had set, the defects were sealed using self-etching adhesive (Single Bond Universal – 3M ESPE®) and restored with resin composite Z350XT (3M ESPE®) to achieve marginal sealing. The cusp tips of the opposing teeth were cut to minimize occlusal forces on the repaired defect site.

Recovery

After the procedure, rats were returned to their cages to recover from the anesthesia. Post-operative signs of pain were checked by a veterinarian including an inability to sleep normally, rubbing or scratching at an area, a loss of interest in surroundings, and decreased water and food intake [30].

Housing and husbandry

The animals (n = 6/cage) were kept in polypropylene cages (49 x 34 x 16 cm) in a room that was held at a constant ambient temperature (22-24°C) with a 12-h light/dark cycle and easy access to food and filtered water.

Euthanasia

Animals were anesthetized intraperitoneally and sacrificed 4 weeks after (Day 28) surgery, the time point was designed to best observe the response of the dental pulp to treatment. All animals remained in good health from the start of the study through the experiment. Euthanasia was performed with anesthetic overdose.

Histological evaluation

Maxillary first molars were removed with adjacent alveolar bone and fixed with 10% neutral formalin for 24 hours at 4C, then demineralized in 10% EDTA/phosphate-buffered saline solution for one month. After trimming, tooth samples were embedded in paraffin, and serial sectioned in the mesiodistal direction at 4 mm-thickness and stained with hematoxylin & eosin. A trained observer with no previous knowledge of the groups performed a blinded histological evaluation for inflammatory cell response, quality of dentin bridge formation [29], hard tissue formation [31], pulp tissue organization [32] using scoring system shown in Tables 1 and 2. The entire dental pulp was evaluated via serials sections, and the highest inflammation score observed in each tooth was considered. The area of tertiary dentin deposition was quantified using Image J and calculated as a percentage of the dental pulp area under the pulp capping treatment. The results of inflammatory cell infiltration and hard tissue formation were analyzed using the Statistical Package for the Social Sciences (SPSS; version 20.0) and the frequencies of each parameter were compared by Pearson’s chi-square test (p <0.05), Two-way ANOVA with Tukey as post-hoc (α = 0.05) was used for the statistical analysis of the release assay, necrosis, and tertiary dentin formation.

Results

Release assay

The release profile from microgels embedded with DMMs is shown in Figure 2. On day 1, the release rate of the group Microgel + DMMs 500 μg/ml was higher than that of Microgel + DMMs 1000 μg/ml. From day 2 on, Microgel + DMMs 1000 μg/ml increased the release rate over time. Both microgel groups showed the release of the proteins and demonstrated an increasing cumulative release for at least seven days, with the total release from 1000 μg being approximately twice that of the 500μg DMM group

Fig. 2. In vitro release profile of microgel loaded with DMM.

Data is shown as mean ± S.D.

Histological analysis

After 4 weeks of treatment, morphological analysis of the dental pulp showed that most negative control group specimens exhibited dental pulp necrosis and loss of overall dental pulp tissue organization (Figure 3 A, B). Moreover, one third of these samples showed what appeared to be adipocytes close to the injury site (Figure 3 B). The MTA group exhibited severe inflammatory infiltrate below the material (Figure 3 C, D). All microgel samples (unsupplemented and supplemented) showed reestablishment of an organized odontoblast layer adjacent to the microgel, comparably lower levels of inflammation as compared to MTA, and the presence of newly deposited reparative dentin (Figure 3 E–J). Microgel + PDGF 10 ng/ml groups showed large blood vessels filled with erythrocytes in the regenerated pulp (Figure 3 E, F).

Fig.3. Effect of different pulp capping materials on the morphology and organization of the dental pulp.

Red arrowhead depicts the location of the defect in each tooth. (A-L) Micrographs exhibiting a cross-sectional view of representative samples of each group in low and higher magnifications. The presence of newly formed dentin tertiary dentin is visible in all microgel containing groups (G-L) Left side panels: A, B Negative control; C, D MTA; E, F Microgel + PDGF 10ng/ml. Right side panels: G, H Microgel; I, J Microgel + DMM 500 μg/ml; K, L Microgel+ DMM 1000 μg/ml).

The level of dental pulp tissue organization among the groups was quite similar, except for the negative control group which appeared very disorganized (Figure 3). More extensive amounts of tertiary dentin were observed in all microgel treated groups, and in some microgel treated specimens a healthy pulp tissue was observed adjacent to treated defect site (Figure 3).

We used ImageJ to quantify the percent necrosis in dental pulp tissue located under the pulp capping treatment (Figure 4 A) as compared to the total area of the dental pulp. Microgel, Microgel + DMMs 500 μg/ml, Microgel + DMMs 1000 μg/ml groups exhibited the lowest percent necrosis. Conversely, necrosis was increased in samples from the negative control group, and MTA and PDGF. Microgel + DMM 500 μg/ml and Microgel + DMM 1000 μg/ml groups had significantly more newly formed tertiary dentin as compared to MTA after one month (Figure 4 B).

Fig. 4. Percentual of necrosis and tertiary dentin in the pulp chamber.

(A) Quantification of necrosis in the dental pulp tissue under the pulp capping treatment. Data is shown as percentual area of the pulp chamber ± S.D. (B) Quantification of tertiary dentin in the dental pulp tissue under the pulp capping treatment. Data is shown as percentual area of the pulp chamber ± S.D.

Discussion

In this study we demonstrate that novel 3D-printed photocurable microgels of defined micrometric structure (size and geometry), doped with dentin matrix molecules, demonstrate a cumulative release of these molecules to the pulp microenvironment to promote dental pulp tissue regeneration. Our results suggest that the application of this material in the pulp encouraged natural dentin self-repair, and the formation of reparative dentin only at the injury and treatment site. This advanced strategy exploits the versatility of 3D-printed degradable microgels combined with the bioactive properties of DMM as a direct pulp capping agent, capable of inducing both hard tissue dentin deposition and healthy dental pulp tissue formation in direct pulp capping treated teeth.

3D-printed GelMA was chosen as our carrier material for these studies based on its ability to be used to create unique micropatterns, morphologies, and 3D structures controllably, which may be more advantageous for cell migration, attachment, degradation, and remodeling [33]. The ability to create micropatterns is provided by photocrosslinking of associating methacryloyl groups in response to photoinitiator and exposure to light [34]. Our group has been researching the use of gelatin methacryloyl hydrogels photocrossllinked with a dental light for several years, including various applications for regeneration of oral and craniofacial tissues [25, 35–37]. The gelatin composition contains arginine-glycine-aspartic acid (RGD) sequences that promote cell attachment [38], as well as target sequences of matrix metalloproteinases (MMPs) that allow for cell directed remodeling [39]. Furthermore, GelMA is compatible with microfabrication/lithography methods [16]. We optimized the hydrogel composition, identifying that 7% GelMA with 0.075% of photoinitiator (LAP) results in a soft hydrogel (1.5-3.1 kPa) ideal for regeneration of vasculature and soft tissues [25, 36]. We developed a method for fabrication of such hydrogel that could be translatable to the dental clinic, by changing the photoinitiator, so the gels would polymerize using a blue light. We also improved the polymerization of the microgels to occur using a photolithography technique under blue light source for only 20 seconds. Monteiro et al. [25] showed that GelMA hydrogels photopolymerized with blue light created larger and more porous network, higher cross-link density and degradation resistance than GelMA ultra-violet light cured, as well as exhibited the elastic modulus (elasticity) consistence with cell viability. In addition, our group previously demonstrated that five-pointed flower-like GelMA-microgels induced greater cell attachment, invasion, remodeling, and vascularization of engineered constructs, as compared to monolithic gel packing scaffolds [19]. Now, the present study shows that a pool of dentin matrix molecules is a promising source of growth factors to induce rapid tertiary dentin formation in dental pulp capping procedures

The use of DMMs has long been suggested to induce dental pulp regeneration [14, 40–42]. Thus, our hypothesis was that extracting these molecules and loading them into a carrier that can be delivered to dental pulp cells would promote the formation of superior or equivalent new dental pulp tissue, as compared to using growth factors or the gold standard MTA alone. With respect to inflammation, dental pulp tissue organization, and tertiary dentin formation, our findings show that all the microgel containing groups showed lower inflammation and better organized tissue formation as compared to MTA and negative control treated teeth. The DMM-supplemented microgel groups showed extensive hard tissue formation, the presence of tubular or atubular dentin adjacent to the injury site, and organized pulp tissue inside the dental pulp chamber. This capacity of DMMs to harness the regenerative potential of dental pulp cells has been shown in a previous in vitro study, where a direct correlation was found between the concentration of DMMs and the proliferation of undifferentiated mouse dental pulp cells (OD-21), and cells of the rat odontoblast-like cell line (MDPC-23) at 1, 3, and 5 days [40]. Likewise, human dental stem cells from the apical papilla encapsulated in bioprinted hydrogels presented a significantly higher odontogenic differentiation when in contact with bioinks containing at least 100 μg ml⁻¹ of DMMs [43]. A recent clinical trial using a direct capping agent made from an alginate hydrogel scaffold supplemented with small fragments of demineralized dentin showed greater formation of homogenous tubular dentin structures with numerous dentinal tubule lines, as compared to MTA, following pulp injury and treatment for 2-weeks and 2-months [44].

PDGF receptors have been found to be expressed by a variety of cells, including fibroblasts, smooth muscle cells, and dental pulp cells. Dental pulp cells exhibit increased proliferative activity in the presence of PDGF-BB and PDGF appears to play an important role during terminal odontoblast differentiation. Also, it is known to induce angiogenesis by up-regulating VEGF production and modulating the proliferation and recruitment of perivascular cells. This is the reason we chose PDGF as a positive control, however, PDGF alone is not enough to induce a robust formation of tertiary dentin. Conversely, the pool of dentin matrix molecules induces a rapid formation tertiary dentin, suggesting that a biomimetic approach, in which the microgels work as an additional source of growth factors and as a scaffold to provide chemical and physical cues for cells to produce dentin is a more biomimetic and efficient approach than to target only one growth factor.

MTA and other calcium silicate cements are known for promoting the release of DMM such as vascular endothelial growth factor (VEGF), macrophage-colony stimulatory factor (M-CSF), neural growth factor (NGF), insulin-like growth factor (IGF-I), and others [45] into the dental pulp stimulating the migration, proliferation and differentiation of dental pulp cells and dental pulp stem cells into a mineralizing phenotype. However, this effect is not controllable and changes from tooth to tooth as a function of the pulp status. The DMM extract that we use was characterized at the proteomic level, and about 90 proteins were identified in the extract. Those proteins were categorized into 4 groups: 1) phosphorylated proteins, 2) non-phosphorylated proteins, 3) small leucine-rich proteoglycans and 4) superfamilies of growth factors [13]. Those are key contributor to, regeneration, and mineralization. Thus, we expect that using DMM-doped microgels in which the concentration and overall composition of DMM is known, will bring more predictability in the biological responses and, consequently in the clinical results. Also, the combination of MTA (or Biodentine) with such microgels is expected to increase the regenerative stimuli for the pulp.

The use of higher concentrations of DMMs may explain the observed increased mineral deposition found in the dental pulp samples of the Microgel + 1000 μg/ml, since this group released 2x more DMM over the course of one week as compared to the Microgel + 500 μg/ml group. The cocktail of bioactive molecules present in the dentin matrix can promote angiogenesis, odontoblast-like differentiation, and tertiary dentin formation [14, 40, 43]. Consequently, for future regenerative applications, the desired outcomes will guide the choice of DMM concentrations in the hydrogel. It is possible that higher concentrations of DMMs can be used for applications where mineral deposition is needed, while lower concentrations can benefit vasculature and soft tissue formation. Further studies are needed to elucidate additional roles of DMMs in dental and bone regeneration, as well as fine-tuned biofabrication methods and release profiles associated with each method for delivery of these molecules. To distinguish the biological effect of capping materials from the tooth’s capacity to self-repair, Microgel + PDGF 10ng/ml was used as a positive control in this study. PDGF plays an important role not only in promoting DPSC proliferation, viability [46], angiogenesis and dentin matrix protein production, but also in facilitating stem cell homing during repair of the dental pulp [47]. In this study however, samples from the PDGF group showed only moderate tertiary dentin formation, despite the extensive blood vessel formation in the treated pulp (Figure 2).

MTA is currently a gold standard for dental pulp therapy [7, 8, 48]. In our study, we used the white MTA-Angelus, which also exhibited high success rates in direct pulp capping treated teeth after 3-years follow-up [49]. The mineralization process associated with this cement is related to its alkaline nature, and its ability to extract DMMs from adjacent dentin [42, 49, 50]. In the present study, the deposition of tertiary dentin was variable in MTA treated samples, ranging from discrete in some samples to extensive in others, consistent with previously published reports [29, 51]. We also consistently observed inflammation in the pulp tissue beneath MTA treated teeth, which may be caused by multiple factors including high pH, and the presence of heavy metals, which are added to MTA as radiopacifying agents [52, 53]. As such, the regenerative effects of MTA are triggered by a noxious effect on dental pulp that has already been damaged by carious lesions or trauma. In this regard, DMM-loaded microgels offer a much more desirable alternative treatment to regenerate the dental pulp, using protein-based materials that are biocompatible, resorbable and biostimulatory for tertiary dentin and dental pulp formation.

Although the rat animal model used in this study demonstrates in vivo testing required prior to clinical application in humans, we are mindful of the differences in the biological response of rats and humans [54]. The mechanical injury performed on the rat molars induced the stimulation of reparative dentin formation by differentiating odontoblast-like cells [55], similar to what occurs in human molars [56].

The results of this study provide a perspective on how the therapeutic use of DMM-loaded microgels can benefit the dental pulp in cases of trauma without prior exposure to microorganisms. It is established that bacterial products can diffuse through the dentinal tubules, inducing pulpitis even before the pulp exposure, so it was essential to understand the basic mechanisms and biological responses of the dental pulp to this new biomaterial before introducing the complexity of a multispecies infection such as caries. This manuscript represents the first step in the clinical use of DMM-loaded microgels and shows the material development and biological responses in a pre-clinical study, building evidence that this strategy is promising to regenerate the dental pulp. The next step of the study is to evaluate the biological response of these 3D-printed microgels in decayed teeth and understand the effect of DMM-loaded microgels in the context of inflammation and infection. However, while rat dental pulp tissues have the capacity to self-repair with auto-deposition of osteodentin-like matrix, this process is much slower in humans, requiring up to three months to form a dentin barrier [57, 58]. Therefore, additional studies are needed to test this material in clinically relevant larger animals such as pigs, prior to testing in controlled, randomized clinical trials.

Conclusions

According to the experimental conditions and results, this study showed that 3D-printed photocurable microgels with defined micrometric structure (size and geometry), doped with dentin matrix molecules, exhibited favorable cellular and inflammatory pulp responses, and significantly more tertiary dentin deposition as compared to currently used dental pulp capping materials, indicating that this may be a promising new strategy for vital pulp therapy.