Multiple growth factors acted synergistically in biphasic compound.
Controlled release of multiple growth factors from nCS/CS/HAp cement.
Mineralization analysis of nCS/CS/HAp/TGF-β1/VEGF in vivo and in vitro .
Vital pulp therapy aims to treat reversible pulpal injuries via protective dentinogenesis and to preserve more tooth structure. Mineral trioxide aggregate (MTA)-based capping materials demonstrate prolonged setting time increases the risk of pulpal infection during multi-visit treatment. Their non-degradable property occupies pulp space and limits dentin-pulp regeneration. This study reports an inorganic degradable biomaterial that presents a short initial setting time and acts as a growth factor reservoir to promote reparative dentinogenesis.
We synthesize nanocrystalline calcium sulfate hemihydrate (nCS), hydroxyapatite (HAp) and calcium sulfate hemihydrate (CS) as a reservoir to which transforming growth factor-beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF) are added (denoted as nCS/HAp/CS/TGF-β1/VEGF). In vitro biocompatibility and mineralization (the activity and expression of alkaline phosphatase, ALP) were evaluated. Rat animal model was created to test in vivo efficacy.
Cultured human dental pulp cells (HDPCs) showed that nCS/HAp/CS/TGF-β1/VEGF cement has excellent biocompatibility and the potential to elevate the activity and expression of ALP. The in vivo efficacy (rat animal model) indicates protective dentin by micro-computed tomography (μ-CT) measurements and histological analyses. The 3D μ-CT non-destructive analysis also determines volume changes during pulpotomy, suggesting that the degraded space of the nCS/HAp/CS/TGF-β1/VEGF cement is repaired by the formation of dentin-pulp tissue.
These findings demonstrate that nCS/HAp/CS cement acts as a potent reservoir for the sustained release of growth factors, and that nCS/HAp/CS/TGF-β1/VEGF cement has a high potential to form the reparative dentinogenesis in vivo .
Teeth with deep caries, traumatic injuries or other damage may lead to dental pulp inflammation or exposure. The traditional treatment for tooth pulp injury is root canal therapy. Although the success rate of root canal therapy is nearly 90% in a 2- to 4-year follow-up study , the access opening and shaping of root canal will lose certain amount of hard tissue structure of tooth during the treatment or post space fabrication which may increase the risk of tooth fracture . Thus, preservation of remaining pulp vitality via direct/indirect/pulpotomy vital pulp therapy can be regarded as minimally invasive dentistry . Direct pulp capping therapy is an operation on the floor of cavities after the removal of deep carious lesions or after traumatic exposure, whereas pulpotomy therapy operates on the radicular pulp after removal of the coronal tissue. Three factors are crucial for successful vital pulp therapy: (1) remove harmful challenges to control the infection; (2) adopt a capping biomaterial to protect and stimulate the pulp dentinogenic response; and (3) provide a good seal to prevent bacterial microleakage . Direct vital pulp therapy uses dressing or capping biomaterials to form a protective layer over the exposed vital pulp tissue in pulp capping procedures or pulpotomies. These protective biomaterials should possess specific biological ability and biocompatibility, to promote dental pulp cell activity and form the reparative dentin .
The dental pulp contains proliferative stem/progenitor cells, which possess self-renewal and differentiation abilities. These cells may differentiate into odontoblast-like cells and secrete dentin matrix when dental pulp cells are affected by extracellular matrix molecules or external stimulation . It is well recognized that throughout the process, growth factors in the extracellular matrix are critical for inducing odontoblast differentiation in dentinogenesis . Therefore, transforming growth factor-beta 1 (TGF-β1) and angiogenic growth factors, such as vascular endothelial growth factor (VEGF), have been used as potential inducing factors for dentin-pulp complex engineering . The major effect of TGF-β1 on odontoblasts was to trigger differentiation and improve dentin or dentin-like hard tissue generation during the healing process. The key role of VEGF was to induce human dental pulp cells to develop into endothelial cells and undergo osteogenetic differentiation . However, the short half-life of growth factors prevents the expected biological effect from being realized, as they can easily be degraded by enzymes . Thus, research aimed at prolonging the half-life of growth factor is being widely pursued . Studies have proven that biodegradable polymers acting as carriers can effectively control the release of growth factors . Yuan et al. found that biocompatibility, toxicity, and degradation are crucial considerations in choosing polymers for the capping material . Approaches to vital pulp therapy using organic ( e.g. , silk fibroin, dentin matrix) or inorganic cements as pulp capping agents have been applied for different purposes . Organic materials were less irritating and exhibited good biocompatibility, but most of them were provided as scaffolds with stem cells added to vitalize pulp cells. Inorganic materials may either provide strong mechanical properties and good bacterial sealing or cause pulp inflammation and reduce dentin-pulp complex formation. For example, conventional calcium hydroxide-based materials have been clinically used as pulp capping materials for reparative dentinogenesis for approximately 50 years. However, high solubility and fast dissolution are typical drawbacks that make them clinically inadequate for covering bleeding points and hinder their long-term viability . Mineral trioxide aggregate (MTA) cements, a well-known type of calcium silicate-based cements in dentistry, are hydraulic self-setting materials mainly composed of dicalcium and tricalcium silicates. Their peculiar intrinsic properties, forming calcium hydroxide during their hydration process and setting in the presence of blood and other biological fluids, ensuring a good seal, make them suitable for clinical use . However, their unfavorable healing properties, prolonged setting time, and insufficient ability to promote dentin-pulp regeneration limit their application .
In this study, we synthesized an inorganic biomaterial containing nanocrystalline calcium sulfate (nCS), 20–50 μm particles of hydroxyapatite (HAp) and calcium sulfate (CS) – denoted as nCS/HAp/CS – to carry growth factors (TGF-β1 and VEGF) and to provide a substantial release reservoir. HAp, the major inorganic component of enamel and dentin, is well known for its high biocompatibility and improves the material’s mechanical strength . CS is a highly biocompatible material and is used as a drug release material for antibiotics, growth factors and pharmacological agents . In addition, to increase the growth factor adsorption, we nanosized calcium sulfate to form nanoparticles with diameters of 80–100 nm. The purpose of our study is to take advantage of each material to maintain pulp vitality, seal the infected channel to avoid irritation from microorganisms and regenerate the dentin-pulp complex. We investigated the efficacy of this synthesized biomaterial by examining its biocompatibility and bioactivity in vitro as well as its new reparative dentinogenesis in vivo .
Materials and methods
Preparation of nCS/HAp/CS/TGF-β1/VEGF biomaterials
The synthesis of nanocrystalline calcium sulfate (nCS) was based on the cryo-vacuum method. A dilute solution of calcium sulfate (CS, CaSO 4 ·2H 2 O) was prepared using 1 g of 99.9% pure dihydrate CS (J.T. Baker, 1452-01) per 500 ml of distilled deionized water, well distributed by stirring for at least 24 h at room temperature. After 24 h of stirring, the CS solution was placed in centrifuge tubes and quick-frozen by full immersion in liquid nitrogen (−196 °C) for 2 min. For freeze-drying, frozen CS was lyophilized by a freeze dryer (FDU-1200, EYELA, Tokyo, Japan) at a temperature below −55 °C and a pressure of 10 Pa for at least 72 h. After 72 h, the nanocrystalline dihydrate CS was dehydrated in a high-temperature furnace at 140 °C for 30 min, yielding β-hemihydrate nCS. Hydroxyapatite (HAp) powder was prepared via the chemical precipitation technique. All reactions were performed at 85 °C. First, 0.5 M Ca(OH) 2 was vigorously stirred and heated. Then, 0.3 M H 3 PO 4 was dropped into the Ca(OH) 2 solution and the pH was adjusted to 8.5 by NH 4 OH for 2 h. After standing for 20 h, the reaction mixture was centrifuged at 6000 rpm and 25 °C. The precipitate was then washed with distilled-deionized water. After three repetitions of the centrifugation and wash procedure, the precipitate was lyophilized by a freeze dryer at a temperature below −55 °C and a pressure of 10 Pa for 72 h. The raw HAp was ground into fine particles by a 25–53 μm sieve. The nCS/HAp/CS cement was produced using synthesized nCS and HAp through the addition of gypsum, CS (CaSO 4 ·1/2H 2 O) at a 1:1:1 ratio by weight with distilled-deionized water containing 6 ng/ml of recombinant human TGF-β1 (R&D System, 240-B) and 0.5 ng/ml of recombinant human VEGF (R&D System, 293-VE).
Microstructure examination, physical and chemical property analyses
Field emission scanning electron microscopy (FESEM) and energy dispersive X-ray spectrometer (EDS)
The dehydrated nCS, HAp powders and nCS/HAp/CS (CaSO 4 ·1/2H 2 O) cements were coated with gold using a coating machine (Q150R Rotary-Pumped Sputter Coater, Quorum Technologies) at a setting of 25 mA/90 s. The surface and fracture sections were examined using a FESEM with EDS (Nova NanoSEM™ 230, FEI Company, Eindhoven, Netherlands).
The ASTM C266-99 Standard Test Method was used to measure the setting time of each cement. The experimental cements were placed in molds with a diameter of 10 mm and a height of 1 mm: nCS/HAp/CS powder at ratios of 1:1:1 and 1:1:0.5, HAp/nCS powder at a ratio of 1:1, HAp/CS powder at a ratio of 1:1 by weight percentage (wt%), and ProRoot MTA (DENTSPLY, Tulsa, OK, USA). The powders and distilled-deionized water were mixed at a powder/liquid ratio of 1.0 g/ml except for MTA, which was mixed at a powder/liquid ratio of 3.0 g/ml. The initial setting times of the specimens were tested at 5–10 min time intervals after setting at 37 °C and 100% relative humidity using a mass of 113.4 ± 0.5 g by a Gillmore needle. The final setting times were obtained using a mass of 453.6 ± 0.5 g by a Gillmore needle until no appreciable indentation was produced on the material surface.
The nCS/HAp/CS/TGF-β1/VEGF cement was placed in stainless steel molds with a diameter of 6 mm and height of 2 mm. The specimens were removed from the molds after setting at 37 °C and 100% relative humidity for 1 day. The cements were weighed accurately to determine W o (initial weight) and then incubated in 50 ml of PBS (pH 7.4) at 37 °C with 100% relative humidity for 1, 7, 14 and 28 days with the PBS changed at intervals of 2 days. W r (final weight) was measured at a given time after the cement was dehydrated. The weight loss was expressed as a percentage and calculated using the following equation:
Calcium release and pH value measurement
The nCS/HAp/CS cement was placed in stainless steel molds with a diameter of 6 mm and a height of 2 mm. The specimens were removed from the mold and immersed in PBS (pH 7.4) at 0.1 g/ml for 0.5, 1, 2, 4, 12, and 24 h and 2, 3, 7, and 14 days at 37 °C with 100% relative humidity. The solutions were used for calcium ion release and pH measurements with a multi-function water quality meter (MM-60R, TOA-DKK Co., Japan).
TGF-β1 and VEGF release
To determine the release of TGF-β1 and VEGF from the experimental cements, 0.1 g of each experimental cement was placed in a centrifuge tube containing 1 ml of PBS (pH 7.4). All samples were maintained at 37 °C until each time point (0.5, 1, 2, 3, 4, 12, and 24 h and 2, 3, 7, 14, 21, and 28 days). The tubes were centrifuged (12,000 rpm, 5 min), and the supernatant was collected and frozen at −20 °C for analysis. The samples were refreshed with new PBS and vortexed for 5 s. The TGF-β1 and VEGF released from the developed biomaterial compound at each time point were measured in triplicate using a commercially available sandwich-assay enzyme-linked immunosorbent assay (ELISA) kit (Quantikine Kit, R&D Systems, USA) according to the manufacturer’s instructions. The TGF-β1 and VEGF results were expressed as percentages of the initial concentration.
Extract liquid preparation
The extract liquids of the experimental cements were used for the biocompatibility and bioactivity tests in this study in accordance with ISO10993. All of the materials were pre-sterilized using ethylene oxide gas. Experimental cement pastes were extracted with serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) at 4 °C for 24 h and 4, 7, 11, 14, and 17 days at a ratio of 0.1 g/ml, except for MTA, which was extracted at a ratio of 0.033 g/ml. The extracts were centrifuged at 14,000 rpm for 5 min, and the supernatant was filtrated through syringe filters with a pore diameter of 0.22 μm. Then, 4% FBS was added to each extract liquid of the experimental cements.
Primary culture of human dental pulp cells (HDPCs)
HDPCs were selected from adults (20–50 years old) with the approval of the Ethics Committee of National Taiwan University Hospital. Healthy premolars and third molars were extracted from patients undergoing orthodontic treatment and immediately stored in DMEM at 4 °C. The teeth were cleaned with 70% ethanol and then split with a hammer to obtain pulp tissues. The pulp tissues were minced (into approximately 1 mm 3 pieces) using ophthalmic scissors and digested with 3 mg/ml collagenase type I (Sigma, C0130) and 4 mg/ml dispase II (Sigma, D4693) at 37 °C for 1 h. Cell suspensions were cultured in DMEM (Gibco, 11965-084) containing 10% FBS (Gibco, 10437-028), 1× Antibiotic-Antimycotic (Gibco, 15240-062) and 2 mM l -glutamine (Gibco, 25030-081) and plated onto 6-cm plates at 37 °C in 5% CO 2 . After the pulp cells migrated to reach 80% confluence, they were subcultured at a ratio of 1:3. Cultured cells between the 3rd and 8th passages were used for in vitro experiments.
HDPCs (5 × 10 3 cells/well) were seeded on disinfected nCS/HAp/CS cements in the presence or absence of TGF-β1/VEGF in 48-well plates at 37 °C in 5% CO 2 . On the following day, the cements were washed three times with PBS and dehydrated in a graded series of ethanol. The preparations were then further dehydrated using the K850 critical point dryer (Quorum Technologies). After gold coating, the cements were examined by FESEM.
Cell viability and cytotoxicity
The viability of HDPCs was measured by 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) reagent (Roche, 1 644 807) in accordance with the manufacturer’s protocol. HDPCs (5 × 10 3 cells/well) were seeded onto 96-well plates. On the following day, the medium was replaced with the extract liquids of the experimental cements at time intervals of 1 and 3 days. The absorbance value of each well was measured using a microplate reader (Thermo Multiskan Spectrum, USA) at 490 nm. The cytotoxicity of HDPCs was determined using a CytoTox 96 ® Lactate dehydrogenase (LDH) Kit (Promega Corp., G1780) in accordance with the manufacturer’s protocol. HDPCs (5 × 10 3 cells/well) were seeded onto 96-well plates the day prior to treatment with the extract liquids at time intervals of 1 and 3 days. After treatment, cell medium supernatants were collected for LDH activity analysis, and the absorbance values were measured using a microplate reader at 490 nm.
Analysis of mineralization ability
ALP activity assay
After 5 and 10 days of incubation with the extract liquids in 24-well plates, HDPCs were lysed with 200 μl of extraction buffer (0.1% Triton X-100 and 2 mM MgCl 2 in distilled-deionized H 2 O) for 15 min at 37 °C. ALPase activity in the lysates was measured in alkaline buffer solution (Sigma, A9226) with alkaline phosphate yellow liquid substrate (Sigma, P7998) for 30 min at 37 °C. Reactions were halted by the addition of 3 N NaOH solution, and the absorbance of the reactions was read at 405 nm. The total protein quantitation was assayed using a bicinchoninic acid (BCA) protein assay kit (Santa Cruz Biotechnologies, sc-202389), and ALPase activity was normalized to the protein concentration (nmol/μg of protein/30 min).
Alkaline phosphatase (ALP) staining
HDPCs (1 × 10 4 cells/well) were seeded onto 24-well plates and cultured in DMEM containing 10% FBS until they reached 80% confluence. On the following day, the HDPCs were treated with the extract liquids. HDPCs were collected after 10 days, washed in PBS, and then fixed with 4% paraformaldehyde at room temperature for 30 min and stained with substrate solution (3 mg of Naphthol AS phosphate (Sigma), 10 mg of Fast Blue BB salt (Sigma), and 0.05 ml of N,N-dimethylformamide (Sigma) in 10 ml of 0.1 M Tris buffer [pH 9.1]) for 30 min in the dark. The morphologies and ALP staining of the HDPCs were examined using a light microscope (Olympus Co., Tokyo, Japan).
Male rats (Wistar, 8 weeks old, body weight of 250–300 g, BioLASCO Taiwan Co., Ltd) were used as rodent models for vital pulp therapy. All experimental protocols were approved by the guidelines of the Institutional Animal Care and Usage Committees at the College of Medicine, National Taiwan University. A total of 36 maxillary right and left first molars from 18 rats were used. Prior to pulpotomy, male rats were anesthetized with Zoletil (20–40 mg/kg) and Rompun (10–20 mg/kg), injected intraperitoneally. The oral cavity was made on the occlusal aspect of two maxillary first molar teeth after cleaning and disinfection with beta-iodine. Coronal pulp tissues were removed using a #1/2 round bur (diameter of 0.8 mm) and an ultrasonic device, with sterile saline irrigation to prevent damage from heat. The amputated pulp surface was rinsed with normal saline. Hemorrhage was controlled with sterile paper points and cotton pellets. The exposed pulp was dressed with one of two test materials: nCS/HAp/CS/TGF-β1/VEGF or nCS/HAp/CS. After the dressing materials were placed and compacted, the access cavities were restored using self-etch bonding agent (Adper™ Easy One, 3M ESPE, St Paul, Minnesota) and light-cured flowable composite resin (PermaFlo ® Purple, Ultradent, South Jordan, Utah, USA). Experimental animals were sacrificed 1, 2 and 4 weeks ( n = 3) after pulpotomy by 20 min of 100% CO 2 inhalation.
The tooth samples were fixed with 10% formalin at room temperature and then placed in 14% ethylenediaminetetraacetic acid (EDTA) decalcified solution. After decalcification (approximately 45 days), the specimens were embedded in paraffin and sliced to a thickness of 4 μm. The sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Zeiss Axiovert 200M).
Micro-computed tomography (μ-CT) image analysis
Non-destructive measurement timeframes were 0, 1, 2, and 4 weeks after the pulpotomy procedures. After anesthesia by isoflurane, the rat was set on the object stage of the μ-CT system (SkyScan 1176 in vivo μ-CT, Kontich, Belgium). The head of the rat was scanned at a resolution of 18 μm and scan settings of 70 kV and 278 μA, ensuring that all molar teeth and the surrounding alveolar bone of the posterior maxilla were encompassed. The μ-CT data were visualized and reconstructed by DataViewer ® , and the 3D reconstructed images were analyzed by CTAn ® . The calcium standard samples (0.25 g/cm 3 and 0.75 g/cm 3 ) were provided by Bruker Skyscan using radiodensity calibration (Hounsfield Unit) to calculate the HU values from each image. The color 3D reconstruction of the μ-CT images was performed by CTVox ® .
The differences between the control and experimental groups were analyzed by one-way or two-way analysis of variance (ANOVA) and the post hoc Scheffe’s method or Tukey’s test using the IBM SPSS 20.0 software for Windows (SPSS Inc., Chicago, IL). p < 0.05 was considered to indicate a statistically significant difference between groups.