Comparative Analysis of Biodentine, ProRoot MTA, and Bio-C Repair: Surface Properties and Cytocompatibility with Human Dental Pulp Cells

Abstract

This article provides a comprehensive analysis of the surface morphology, chemical composition, and in vitro cytocompatibility of three prominent calcium silicate-based vital pulp materials: Biodentine, ProRoot MTA, and Bio-C Repair. Tailored for researchers and drug development professionals, the content synthesizes foundational material science, standard methodological approaches for biological evaluation, troubleshooting for material optimization, and a direct comparative validation of biological performance. Evidence from recent studies indicates that while their elemental compositions differ, all three materials demonstrate excellent cytocompatibility, supporting cell attachment and viability of human dental pulp cells (hDPCs), with distinct advantages in specific physicochemical properties.

Decoding the Material Blueprint: Elemental Composition and Surface Characteristics

Core Chemical Constituents Revealed by Energy Dispersive X-ray (EDX) Analysis

Energy Dispersive X-ray (EDX) spectroscopy, also referred to as EDS or EDX, is an indispensable analytical technique for the elemental analysis or chemical characterization of a sample within electron microscopes [1]. In dental materials research, EDX provides critical quantitative data on the core chemical constituents of biomaterials, revealing how their elemental composition influences fundamental properties such as bioactivity and cytocompatibility [2] [3]. This guide objectively compares the chemical compositions of three vital pulp capping materials—Biodentine, ProRoot MTA, and Bio-C Repair—as revealed by EDX analysis, and situates these findings within a broader investigation of their surface cytocompatibility.

Fundamental Principles of EDX Analysis

EDX operates on the principle that each element has a unique atomic structure, leading to a unique set of peaks on its X-ray emission spectrum [1]. The technique is typically integrated with a scanning electron microscope (SEM). When the SEM's focused electron beam strikes the sample, it ejects an electron from an inner shell of a sample atom, creating an electron hole. An electron from a higher-energy, outer shell then fills this vacancy, releasing the energy difference in the form of an X-ray [1] [4]. The energy of this emitted X-ray is characteristic of the element from which it originated. A solid-state silicon drift detector (SDD) collects these X-rays, and software analyzes the signals to identify and quantify all elements present within the sampled volume [1] [4]. This process enables the creation of detailed elemental maps and line scans, providing spatial information about chemical composition [2] [4].

Table 1: Key Components of a Typical SEM-EDX System

Component	Function	Typical Example in Dental Research
Excitation Source	Provides the electron beam that interacts with the sample.	Electron beam in a Scanning Electron Microscope (SEM) [1].
X-ray Detector	Collects and converts X-ray energy into voltage signals.	Silicon Drift Detector (SDD) with Peltier cooling [1].
Pulse Processor	Measures the voltage signals from the detector.	Analyzes signals based on energy and intensity [1].
Analyzer	Displays and interprets the spectral data for the user.	Software for elemental identification and quantification [1] [2].

Comparative EDX Analysis of Pulp Capping Materials

EDX analysis provides direct, quantitative evidence of the differing chemical philosophies behind various bioactive dental materials. The following table summarizes key compositional differences identified via EDX.

Table 2: Core Chemical Constituents of Biodentine, ProRoot MTA, and Bio-C Repair as Revealed by EDX

Material	Major Elements (EDX Weight %)	Key Calcium Silicate Phases	Radiopacifier	Notable Features
Biodentine	Calcium, Carbon, Oxygen [3]	Tricalcium silicate, Dicalcium silicate [5]	Zirconium oxide [5]	High calcium release; forms hydroxyapatite in PBS [5].
ProRoot MTA	Calcium, Carbon, Oxygen [3]	Tricalcium silicate, Dicalcium silicate [5]	Bismuth oxide [5]	Longstanding clinical use; known for bioactivity [5].
Bio-C Repair	Zirconium, Calcium, Oxygen [3]	Calcium silicate-based	Zirconium oxide [3]	Lower concentration of calcium compared to others [3].

The EDX data reveals a fundamental similarity between Biodentine and ProRoot MTA, both being primarily composed of calcium, carbon, and oxygen [3]. This composition is consistent with their classification as calcium silicate cements, where the main components are tricalcium and dicalcium silicate [5]. The setting reaction of these materials involves the formation of calcium hydroxide, which dissociates into calcium and hydroxyl ions, raising the pH and facilitating the release of calcium ions crucial for bioactivity and mineralization [5].

In contrast, Bio-C Repair exhibits a distinctly different elemental profile. While it contains calcium, EDX analysis shows it has a lower concentration of calcium and the highest concentration of zirconium among the three materials [3]. This indicates a significantly different formulation, with a greater proportion of zirconium oxide acting as the radiopacifier compared to the calcium silicate base.

Experimental Protocols for EDX Analysis in Dental Research

To ensure the reliability and comparability of EDX data, researchers adhere to standardized experimental protocols. The following workflow outlines a typical procedure for preparing and analyzing dental materials with SEM-EDX.

Detailed Methodological Steps

The methodology can be broken down into several critical stages, as employed in recent comparative studies:

• Sample Preparation: Specimens of the test materials are typically prepared according to manufacturers' instructions and set under controlled conditions (e.g., 37°C and 95% relative humidity for 24 hours) [5]. For analysis, the set samples are mounted onto aluminum stubs using double-sided adhesive carbon disks or conductive adhesive paste to ensure electrical grounding [2] [6]. The surface is often ground flat and polished in a series of steps using FEPA P1200, P1500, and P4000 grit silicone carbide papers to create a smooth, representative surface for analysis [6]. To prevent charging (a buildup of electrons on non-conductive samples), the specimens are coated with a thin layer of carbon using a sputter coater [2].

• SEM Imaging and EDX Data Acquisition: The prepared samples are placed in the high-vacuum chamber of the SEM. Overview images are first obtained using the secondary electron detector to locate and visualize the areas to be analyzed [6]. For EDX measurement, the electron beam is focused on the region of interest. Studies often use an accelerating voltage of 20 kV and a specific working distance (e.g., 9-16 mm) [2]. Multiple EDX field measurements (e.g., 50 µm x 50 µm areas) and line scans are acquired per sample to ensure data representativeness [6]. The emitted X-rays are collected by a silicon drift detector (SDD), which offers high count rates and better resolution than traditional Si(Li) detectors [1].

• Data Processing and Quantification: The software processes the collected X-ray spectra, identifying elements based on the characteristic energy of their peaks and quantifying them in atomic percent (At%) or weight percent (Wt%) [2] [7] [6]. To ensure accuracy, especially for light elements, modern EDX systems utilize Si₃Nâ‚„ windows which provide higher transmittance for low-energy X-rays [6]. Key ratios, such as the calcium-to-phosphorus (Ca/P) ratio, can be calculated from the results to compare against known stoichiometric values of biological apatite [7] [6].

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for SEM-EDX Experiments

Item	Function/Application	Specific Example from Literature
Scanning Electron Microscope (SEM)	Provides high-resolution imaging and platform for EDX analysis.	FEI Quanta 400 FEG; EVO 18 (Carl Zeiss) [2] [6].
Silicon Drift Detector (SDD)	High-throughput X-ray detector for elemental analysis.	Peltier-cooled SDD [1].
Conductive Mounting Supplies	Ensures electrical conductivity to prevent sample charging.	Aluminum stubs, double-sided carbon tabs, conductive epoxy resin [2] [6].
Polishing Supplies	Creates a flat, artifact-free surface for representative analysis.	Bench grinding machine; FEPA P1200, P1500, P4000 grit papers [6] [5].
Sputter Coater	Applies an ultra-thin conductive layer (carbon) to non-conductive samples.	Carbon coater for dental materials [2].
Storage Solution	Preserves biological specimens (e.g., teeth) before testing.	0.5% chloramine T solution [6].
3-(Trifluoromethyl)quinolin-4-amine	3-(Trifluoromethyl)quinolin-4-amine|Research Grade
6-Methyl-1H-imidazo[1,2-b]pyrazole	6-Methyl-1H-imidazo[1,2-b]pyrazole, CAS:42351-84-8, MF:C6H7N3, MW:121.143	Chemical Reagent

Linking Chemical Composition to Cytocompatibility

The elemental composition revealed by EDX is not merely a fingerprint of the material; it is directly linked to the biological performance observed in cytocompatibility studies. The chemical dynamics initiated by the constituents in Table 2 drive the material's interaction with the biological environment.

• The Role of Calcium Silicate Chemistry: The high calcium content in Biodentine and ProRoot MTA, confirmed by EDX, is fundamental to their bioactivity. When these materials set and come into contact with tissue fluids, the calcium silicates react to form calcium hydroxide [5]. This dissociates into calcium and hydroxyl ions, raising the local pH to approximately 12.5 and creating an environment that fosters odontogenic differentiation [5]. The sustained release of calcium ions is a key signal that promotes the proliferation and mineralization of human dental pulp cells (hDPCs) and human dental pulp stem cells (hDPSCs) [3] [8]. This mechanism underpins the "exceptional level of cytocompatibility" and "advantageous bioactivities" reported for Biodentine [8] and the "excellent cytocompatibility" shared by all three materials [3].

• Hydroxyapatite Precipitation and Sealing Ability: The released calcium ions from Biodentine and ProRoot MTA can further interact with phosphate ions present in physiological fluids (e.g., from PBS buffer or tissue fluid). This interaction leads to the precipitation of hydroxyapatite on the material's surface, which has been observed in solubility tests [5]. This apatite layer is clinically significant as it improves the seal between the material and the tooth structure, reducing microleakage and enhancing long-term success [5]. EDX can be used to monitor the formation of this layer by detecting changes in the surface calcium and phosphorus content over time.

• Impact of Radiopacifiers: While essential for clinical visibility, radiopacifiers like zirconium oxide (in Biodentine and Bio-C Repair) and bismuth oxide (in ProRoot MTA) are largely inert. The high zirconium content detected by EDX in Bio-C Repair suggests a different ratio of radiopacifier to calcium silicate matrix, which could influence the overall rate of calcium ion release and subsequent bioactivity, even if the final cytocompatibility remains excellent [3]. In contrast, bismuth oxide in MTA has been associated with potential tooth discoloration, a drawback not shared by zirconium-based systems [5].

In conclusion, EDX analysis provides an objective, quantitative foundation for understanding the chemical basis of performance in vital pulp therapy materials. The data clearly differentiates the high-calcium silicate chemistry of Biodentine and ProRoot MTA from the zirconium-rich formulation of Bio-C Repair. These compositional differences directly influence their interaction with the biological environment, ultimately supporting their shared, yet mechanistically distinct, pathways to achieving excellent cytocompatibility and clinical efficacy.

Ultrastructural Morphology Visualized via Scanning Electron Microscopy (SEM)

Biocompatibility is an essential property for vital pulp materials that interact with dental pulp tissues, and their surface characteristics play a fundamental role in determining biological responses [9]. The ultrastructural morphology of these materials, when visualized via scanning electron microscopy (SEM), provides critical insights into their surface topography, chemical composition, and ability to support cellular attachment and function—all crucial factors for clinical success in vital pulp therapy [9] [10].

This guide objectively compares the surface properties and cytocompatibility of three calcium silicate-based hydraulic cements—Biodentine, ProRoot MTA, and Bio-C Repair—through the lens of SEM analysis and associated biological assays. The findings presented herein offer researchers and clinicians evidence-based insights for material selection in reparative dentistry.

Material Composition and Surface Characterization

Chemical Composition Analysis

Energy dispersive X-ray analysis (EDX) coupled with SEM reveals distinct elemental profiles for each material, which directly influence their bioactivity and interactions with biological systems.

Table 1: Elemental Composition of Vital Pulp Materials via EDX Analysis

Material	Primary Components	Notable Characteristics	Radiopacifier
ProRoot MTA	Calcium, Carbon, Oxygen (among others) [9]	High calcium ion release [9]	Bismuth oxide [5]
Biodentine	Calcium, Carbon, Oxygen (among others) [9]	Tricalcium silicate base; accelerated cell proliferation [9]	Zirconium dioxide [5]
Bio-C Repair	Low calcium concentration; Highest zirconium concentration [9]	Ready-to-use consistency [9] [10]	Zirconium dioxide [9]

Ultrastructural Morphology

SEM imaging reveals distinct surface characteristics for each material. ProRoot MTA and Biodentine both exhibit crystalline structures with irregular surfaces, which provide favorable sites for cell attachment [9]. Biodentine demonstrates a more homogeneous surface with finer particles, while Bio-C Repair shows significantly smaller particle size, requiring higher magnification (20,000×) for detailed analysis compared to the 2,500× sufficient for the other materials [9].

The surface topography of these materials evolves when exposed to different environments. When stored in saline, both ProRoot MTA and Biodentine display uneven crystalline surfaces with similar hexagonal crystals [11]. However, Biodentine undergoes notable morphological changes in acidic conditions (pH 5.4), transitioning to a relatively smooth surface with more spheroidal crystals [11].

Experimental Protocols for SEM Analysis

Sample Preparation Methodology

Standardized sample preparation is essential for consistent and comparable SEM observations across studies:

• Material Manipulation: Biodentine and ProRoot MTA are mixed according to manufacturer recommendations; Bio-C Repair comes ready-for-use [9] [10].
• Molding: Samples are prepared using silicone molds with 2mm depth and 5mm diameter wells [9].
• Setting Conditions: Samples are allowed to set for 24 hours to 7 days in an incubator at 37°C and 95% humidity to simulate oral cavity conditions [9] [10].
• Cell Seeding for Attachment Studies: Human dental pulp cells (hDPCs) are seeded at a density of 5×10⁴ cells per disk surface and cultured for 72 hours to assess cell-material interaction [9].

SEM Imaging and Analysis Parameters

Consistent imaging parameters ensure reliable comparative analysis:

• Magnification Range: 100× to 50,000×, with Bio-C Repair often requiring higher magnifications (20,000×) due to smaller particle size [9].
• Accelerating Voltage: 5-10 kV using field emission scanning electron microscopes [9].
• Sample Processing: Fixation with glutaraldehyde, dehydration through ethanol series (30%-90% v/v), and sputter coating with gold/palladium before observation [9].

Table 2: Key Experimental Reagents and Their Functions

Research Reagent	Function in Experimental Protocol
Dulbecco's Modified Eagle Medium (DMEM)	Cell culture medium for maintaining hDPCs [9]
Fetal Bovine Serum (FBS)	Serum supplement for cell culture media [9]
Collagenase Type I	Enzyme for isolating hDPCs from dental pulp tissue [9]
MTT Reagent	Tetrazolium salt for assessing cell viability [9]
Glutaraldehyde	Fixative for preserving cell morphology for SEM [9]

Figure 1: Experimental workflow for SEM analysis of dental materials

Cytocompatibility Assessment

Cell Viability and Proliferation

Standardized assays evaluate the biological effects of material eluates on human dental pulp cells:

• MTT Assay: Measures metabolic activity as an indicator of cell viability at 24, 48, and 72-hour intervals [9]
• Flow Cytometry: Quantifies apoptosis and necrosis using Annexin V detection kits [12]
• Immunofluorescence: Assesses actin cytoskeleton organization and cell morphology [9]

Notably, undiluted Biodentine eluates demonstrate significantly higher cell viability than control groups (without eluates) at all time points (p < 0.001) [9]. All three materials show excellent cytocompatibility with no significant cytotoxicity observed [9] [12].

Cell Attachment and Morphology

SEM analysis reveals adequate attachment of hDPCs to all three vital pulp materials without cytoskeletal alterations [9]. Cells appear well-spread with normal morphology when cultured in the presence of material eluates, indicating favorable cell-biomaterial interactions [9].

Similar favorable cell attachment patterns are observed in studies evaluating periodontal ligament fibroblasts on Biodentine and ProRoot MTA, with both materials supporting cell adhesion and spreading [13].

Figure 2: Cell-material interaction mechanism of calcium silicate cements

Comparative Material Properties

Physical and Chemical Properties

Beyond surface morphology, several physicochemical properties influence clinical handling and performance:

• Setting Time: Biodentine sets significantly faster (85.66 ± 6.03 min) compared to ProRoot MTA (228.33 ± 2.88 min) (p < 0.0001) [5]
• Microhardness: Biodentine exhibits significantly higher Vickers microhardness (62.35 ± 11.55 HV) than ProRoot MTA (26.93 ± 4.66 HV) (p < 0.0001), approaching the range of sound dentine (60-90 HV) [5]
• Solubility: Both materials show solubility within ISO 6876:2001 standards (<3%), with ProRoot MTA demonstrating significantly lower solubility than Biodentine (p < 0.0001) [5]
• Radiopacity: ProRoot MTA meets ISO requirements (6.40 ± 0.06 mm Al), while Biodentine falls below the recommended threshold (1.50 ± 0.10 mm Al) (p < 0.0001) [5]

Biological Properties

All three materials demonstrate excellent cytocompatibility with human dental pulp cells, supporting their use in vital pulp therapy [9] [12]. Biodentine shows accelerated dental pulp cell proliferation compared to MTA [9], while Bio-C Repair demonstrates cytocompatibility similar to both established materials despite its different elemental composition [9].

Discussion and Research Implications

The ultrastructural morphology visualized via SEM provides valuable insights into the physical foundations of biological responses to dental biomaterials. The crystalline structures observed in ProRoot MTA and Biodentine, along with their high calcium content, contribute to their bioactivity and ability to promote hydroxyapatite formation [9]. Despite its different elemental composition with lower calcium and higher zirconium concentration, Bio-C Repair demonstrates comparable cytocompatibility, suggesting that multiple factors beyond surface chemistry influence biological responses [9].

For researchers investigating dental biomaterials, these findings highlight several considerations:

• Material surface topography significantly influences cell attachment and spreading
• Calcium ion release appears crucial for bioactivity and stimulation of reparative processes
• Multiple material properties must be balanced for optimal clinical performance

Future research directions should include long-term in vivo studies, investigation of material interactions with inflamed pulp tissues, and development of standardized testing protocols that better simulate clinical conditions.

SEM analysis reveals distinct ultrastructural morphologies and elemental compositions for Biodentine, ProRoot MTA, and Bio-C Repair. Despite these differences, all three materials demonstrate excellent cytocompatibility with human dental pulp cells. Biodentine shows advantages in setting time and cell proliferation, while ProRoot MTA exhibits superior radiopacity and lower solubility. Bio-C Repair, with its ready-to-use format, presents comparable biological performance despite its different composition. These findings provide researchers and clinicians with evidence-based insights for material selection in vital pulp therapy, though clinical decisions should consider the specific requirements of each case alongside these experimental observations.

Within the field of vital pulp therapy and regenerative endodontics, the selection of a bioceramic material is paramount to clinical success. The biological response of dental pulp cells, a core focus of surface cytocompatibility research, is profoundly influenced by the physicochemical characteristics of the material placed in direct contact with the pulp tissue [3]. While final biological outcomes are critical, they are predicated on fundamental physical properties that govern the material's behavior in the clinical environment. This guide provides an objective, data-driven comparison of three prominent calcium silicate-based cements—Biodentine, ProRoot MTA, and Bio-C Repair—focusing on three key physicochemical properties: setting time, porosity, and microhardness. Understanding these properties is essential for researchers and clinicians, as they directly impact a material's sealing ability, durability, and ultimately, its capacity to foster a biocompatible environment conducive to pulp healing and regeneration [14] [15].

Comparative Analysis of Key Properties

The following table summarizes the quantitative experimental data for the key physicochemical properties of Biodentine, ProRoot MTA, and Bio-C Repair, compiled from published research.

Table 1: Comparative Physicochemical Properties of Biodentine, ProRoot MTA, and Bio-C Repair

Property	Biodentine	ProRoot MTA	Bio-C Repair	Significance for Cytocompatibility
Setting Time (Final)	10–12 minutes [14]85.66 ± 6.03 min [5]	165 ± 5 minutes [16]228.33 ± 2.88 min [15]	Similar to MTA [15] (Data specific to Bio-C Repair limited; general class behavior shown)	Faster setting reduces risk of bacterial microleakage and washout, ensuring a stable interface for cell attachment [14].
Porosity (Density)	Lower porosity [14]Density: ~2.260 g/cm³ [14]	Higher porosity [14]Density: ~1.882 g/cm³ [14]	Information not specified in search results	Lower porosity improves mechanical strength and provides a better hermetic seal, preventing ingress of bacteria and toxins [14].
Microhardness (Vickers HV)	62.35 ± 11.55 HV [5]~60 VHN [16]	26.93 ± 4.66 HV [5]	Similar to MTA [15] (Data specific to Bio-C Repair limited; general class behavior shown)	Higher microhardness approximates the properties of natural dentin, providing a stable, fracture-resistant base for restoration and pulp protection [16] [5].
Compressive Strength (24h)	~57 MPa [16]	Significantly less than Biodentine [16]	Information not specified in search results	High early strength allows for earlier restoration of the tooth, reducing treatment time and risk of contamination.
Radiopacity (mm Al)	1.50 ± 0.10 mm Al [5]3.5 mm Al [14] [16]	6.40 ± 0.06 mm Al [5]7.17 mm Al [14]	Information not specified in search results	Adequate radiopacity (≥3 mm Al) is essential for clinical visualization. Biodentine meets the minimum standard, though MTA is more radiopaque [5].

Detailed Experimental Protocols for Property Evaluation

To ensure the reproducibility of data and facilitate future research, this section outlines the standard experimental methodologies used to generate the comparative values for setting time, porosity, and microhardness.

Setting Time Determination

The setting time is typically measured according to international standards such as ANSI/ADA No. 57 or ASTM C266-03 [15].

• Method: Specimens are prepared using ring molds with a set internal diameter (e.g., 10 mm) and height (e.g., 2 mm). The material is mixed according to manufacturer instructions and packed into the mold placed on a glass plate. The assembly is then transferred to an incubator maintained at 37°C and >95% relative humidity.
• Measurement:
  • Initial Set: A Gilmore needle with a specified mass (e.g., 100 g) and tip diameter (e.g., 2.0 mm) is applied vertically to the surface of the specimen at regular intervals. The initial setting time is recorded as the period from the end of mixing until the needle no longer leaves a complete circular impression on the surface.
  • Final Set: The process is repeated with a heavier needle (e.g., 400 g) and a smaller tip diameter (e.g., 1.0 mm). The final setting time is recorded when this needle fails to make a perceptible indentation [15].

Microhardness Testing

Microhardness, a measure of a material's resistance to plastic deformation, is commonly evaluated using the Vickers indentation test.

• Specimen Preparation: Disc-shaped specimens of standardized dimensions are prepared and stored in distilled water at 37°C for predetermined periods (e.g., 1, 7, 28 days) to allow for complete hydration [15].
• Testing Procedure: A diamond indenter in the shape of a right pyramid with a square base is pressed into the polished surface of the set material with a known load (e.g., 100 g) for a specific dwell time (e.g., 10 seconds) [15] [5].
• Calculation: The two diagonals of the resulting indentation are measured under a microscope. The Vickers Hardness Number (VHN or HV) is calculated using the formula: HV = 1.8544 * (Load in kg) / (d² in mm²), where d is the mean length of the diagonals [15].

Porosity and Density Assessment

Porosity, which influences solubility and sealing ability, can be investigated through density measurements or direct porosity tests.

• Density Method: The mass and volume of set specimens are measured to calculate bulk density. Lower density often indicates higher porosity, which can compromise the material's impermeability [14].
• Direct Porosity Method (ASTM C830):
  • The specimen is dried in an oven at 105°C until a constant weight (B) is achieved.
  • The specimen is then immersed in a saturation liquid (e.g., kerosene) to fill the open pores.
  • The saturated weight (C) is recorded, and the porosity percentage is calculated based on the volume of liquid absorbed relative to the total volume of the specimen [15].

The following workflow diagram illustrates the logical relationship between these key properties and their collective impact on the biological response, which is the focus of cytocompatibility research.

The Scientist's Toolkit: Essential Research Reagents and Materials

To conduct standardized and reproducible experiments on bioceramic materials, researchers require a specific set of tools and reagents. The following table details essential items for evaluating physicochemical and biological properties.

Table 2: Key Research Reagent Solutions and Experimental Materials

Item	Function in Research	Application Example
Gilmore Needle Apparatus	To standardly determine the initial and final setting times of hydraulic cements.	Setting time tests per ANSI/ADA Specification No. 57 [15].
Vickers Microhardness Tester	To measure the resistance of the set material to surface deformation using a diamond indenter.	Evaluating dentin-like hardness and mechanical stability over time (e.g., 1-28 days) [15] [5].
Universal Testing Machine	To assess mechanical properties including compressive and push-out bond strength.	Measuring compressive strength at 7 and 28 days of hydration [15].
Scanning Electron Microscope (SEM) with EDX	To analyze ultrastructural surface morphology and perform chemical element characterization.	Studying surface topography and confirming elemental composition (e.g., Ca, Si, Zr) [3].
Induction Medium (DMEM, FBS, Antibiotics)	For the culture and maintenance of human dental pulp cells (hDPCs) in vitro.	Isolating and expanding hDPCs for cytocompatibility assays like MTT and flow cytometry [3] [8].
Phosphate Buffered Saline (PBS)	To provide a phosphate-rich fluid simulating in vivo conditions for solubility and bioactivity studies.	Evaluating hydroxyapatite formation on material surfaces, indicating bioactivity [5].
4-Methyl-5-nitrobenzene-1,2-diamine	4-Methyl-5-nitrobenzene-1,2-diamine, CAS:65958-37-4, MF:C7H9N3O2, MW:167.168	Chemical Reagent
2-(Propan-2-yl)cyclobutan-1-one	2-(Propan-2-yl)cyclobutan-1-one, CAS:27608-63-5, MF:C7H12O, MW:112.172	Chemical Reagent

The experimental data clearly delineate the performance profiles of Biodentine, ProRoot MTA, and Bio-C Repair. Biodentine demonstrates superior handling characteristics with its significantly faster setting time and enhanced mechanical robustness, evidenced by its higher microhardness and lower porosity [14] [5]. These properties are crucial for establishing a immediate and durable seal against bacterial leakage. ProRoot MTA, while a proven material, exhibits a longer setting time and greater porosity, which could influence its initial stability [14] [15]. The search results indicate that Bio-C Repair possesses excellent cytocompatibility similar to its counterparts, though direct comparative data on its physical properties are less extensively documented than for Biodentine [3]. For researchers focusing on surface cytocompatibility, these physicochemical properties are not standalone metrics; they are foundational to creating the stable, sealed, and bioactive environment required for human dental pulp cells to adhere, proliferate, and differentiate, ultimately dictating the success of regenerative therapies [3] [8].

Radiopacifiers are indispensable components in modern dental biomaterials, enabling clinicians to distinguish restorative materials from surrounding tooth structures and bone on radiographs. Within the class of calcium silicate-based cements, including products like Biodentine, ProRoot MTA, and Bio-C Repair, two radiopacifiers dominate contemporary formulations: zirconium oxide (ZrO₂) and bismuth oxide (Bi₂O₃). These compounds serve the critical function of providing sufficient radiographic visibility for clinical assessment while potentially influencing the biological and physicochemical properties of the materials. The selection between zirconium and bismuth-based radiopacifiers represents a significant consideration in material science that extends beyond mere radiopacity, potentially affecting cytocompatibility, discoloration potential, and overall clinical performance. This analysis examines the comparative role of these radiopacifiers within the specific context of emerging research on the surface cytocompatibility of Biodentine, ProRoot MTA, and Bio-C Repair with human dental pulp cells.

Comparative Experimental Data on Radiopacifiers

Radiopacity Performance

Radiopacity, measured in millimeters of aluminum equivalence (mm Al), is a fundamental requirement for dental materials to ensure they are distinguishable from dentin (approximately 1 mm Al) and adjacent structures. The International Organization for Standardization (ISO 6876:2001) stipulates a minimum radiopacity of 3 mm Al for root canal sealing materials.

Table 1: Comparative Radiopacity of Materials with Different Radiopacifiers

Material	Radiopacifier	Radiopacity (mm Al)	ISO 6876:2001 Compliance
ProRoot MTA	Bismuth Oxide	5.72 ± 0.06 [17] [5]	Yes
Portland Cement + Bi₂O₃	Bismuth Oxide	5.88 ± N/A [17]	Yes
Biodentine	Zirconium Oxide	1.50 ± 0.10 [5]	No
Portland Cement + ZrO₂	Zirconium Oxide	3.87 ± N/A [17]	Yes
Bio-C Repair	Zirconium Oxide	Not quantitatively reported [9]	Information missing
Portland Cement + BaSO₄	Barium Sulfate	2.35 ± N/A [17]	No
Portland Cement (pure)	None	1.69 ± N/A [17]	No

Experimental data reveals that bismuth oxide consistently provides superior radiopacity compared to zirconium oxide alternatives. ProRoot MTA, incorporating bismuth oxide, demonstrates significantly higher radiopacity (5.72-6.40 mm Al) compared to Biodentine (1.50 mm Al), which utilizes zirconium oxide [17] [5]. Interestingly, Portland cement combined with zirconium oxide (3.87 mm Al) meets ISO requirements, suggesting that the formulation of Biodentine may utilize a lower concentration of zirconium oxide, resulting in suboptimal radiopacity [17] [5].

Chemical Composition and Surface Characterization

Energy-dispersive X-ray (EDX) analysis provides insights into the elemental composition of these materials, revealing significant differences attributable to their radiopacifiers.

Table 2: Elemental Composition and Biological Properties of Tested Materials

Material	Primary Elements	Radiopacifier	Key Biological Findings
ProRoot MTA	Calcium, Carbon, Oxygen [9]	Bismuth Oxide	Excellent cytocompatibility; promotes cell attachment and viability [9] [3]
Biodentine	Calcium, Carbon, Oxygen [9]	Zirconium Oxide	Higher cell viability than control group; excellent cytocompatibility [9] [3]
Bio-C Repair	Low calcium, highest zirconium concentration [9] [3]	Zirconium Oxide	Excellent cytocompatibility similar to other materials [9] [3]

EDX analysis reveals that ProRoot MTA and Biodentine share similar primary elemental compositions, dominated by calcium, carbon, and oxygen, which are essential for their bioactivity. In contrast, Bio-C Repair exhibits a distinctly different composition with low calcium concentration and the highest relative concentration of zirconium, suggesting a different formulation approach despite utilizing the same radiopacifier type as Biodentine [9] [3].

Experimental Methodologies in Radiopacifier Research

Radiopacity Assessment

The standard methodology for evaluating radiopacity follows ISO 6876:2001 specifications [17] [5]. Researchers typically prepare standardized cement specimens (e.g., 10mm diameter × 1mm thickness or 20mm diameter × 1.6mm height) using stainless steel ring moulds. After setting under controlled conditions (37°C, 95% humidity), specimens are radiographed alongside an aluminum step-wedge with thickness increments from 2-16mm. Digital radiography systems capture images using standardized exposure parameters (e.g., 50 kVp, 10 mA, 33.5cm distance). Software analysis (e.g., Wixwin-2000) compares the radiographic density of materials to the aluminum step-wedge to determine millimeter aluminum equivalence [17]. This standardized approach ensures comparable results across different studies and materials.

Cytocompatibility Testing

Cytocompatibility assessment involves multiple complementary methodologies to evaluate biological responses:

• Cell Viability Assays (MTT/CCK-8): Human dental pulp cells (hDPCs) or apical papilla cells (APCs) are isolated and cultured. Material eluates are prepared according to ISO 10993-5 guidelines by incubating set material specimens in cell culture medium. Cells are exposed to serial dilutions of eluates (1:1, 1:2, 1:4), and viability is quantified using colorimetric assays that measure mitochondrial activity [9] [8] [18].

• Cell Morphology and Attachment: Scanning electron microscopy (SEM) evaluates cell adhesion and morphology on material surfaces. Cells are seeded directly onto material specimens, fixed after incubation, and examined for attachment characteristics, cytoplasmic processes, and filopodia formation [9] [19].

• Apoptosis Assays: Flow cytometry with Annexin V staining quantifies programmed cell death, distinguishing early and late apoptosis and necrosis, providing insight into cytotoxic mechanisms [8] [19].

Material Characterization

• Scanning Electron Microscopy with Energy-Dispersive X-ray Spectroscopy (SEM-EDX): High-resolution imaging reveals surface morphology and microstructure, while EDX provides quantitative elemental composition analysis of material surfaces [9] [18].

• Setting Time and Solubility: Setting time is determined using standardized indenters under controlled conditions. Solubility testing involves measuring mass change after immersion in distilled water or phosphate-buffered saline (PBS) following ISO 6876:2001 specifications [5] [19].

Cytocompatibility in the Context of Biodentine vs ProRoot MTA vs Bio-C Repair

The cytocompatibility of dental materials represents a critical parameter for clinical success, particularly in vital pulp therapies where direct interaction with pulp tissue occurs. Comparative studies examining Biodentine, ProRoot MTA, and Bio-C Repair reveal excellent cytocompatibility profiles across all three materials despite their different radiopacifiers.

Research demonstrates that all three materials support human dental pulp cell attachment, proliferation, and viability without inducing significant cytoskeletal alterations [9] [3]. Interestingly, undiluted Biodentine eluates have been shown to produce higher cell viability than control groups at 24, 48, and 72 hours [9] [3]. Similarly, ProRoot MTA exhibits favorable cytocompatibility, promoting cell attachment and maintaining high viability rates [9] [18].

Bio-C Repair, despite its distinct composition with high zirconium concentration, demonstrates excellent cytocompatibility comparable to both Biodentine and ProRoot MTA [9] [3]. This suggests that while radiopacifiers influence material properties, their specific formulation and integration into the cement matrix may be more critical determinants of biological performance than the radiopacifier type alone.

A notable finding across studies is that zirconium oxide-based materials, particularly Biodentine, occasionally demonstrate enhanced proliferative effects on dental pulp cells compared to controls [9]. This phenomenon may be attributed to the ion release profile and surface characteristics of zirconium-containing materials, though the exact mechanisms require further investigation.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagents and Materials for Radiopacifier Studies

Item	Function	Application Example
Human Dental Pulp Cells (hDPCs)	Primary cell model for cytocompatibility testing	Isolated from third molars; assess biological response to materials [9] [3]
Apical Papilla Cells (APCs)	Stem cell-rich model for regenerative potential	Sourced from teeth with incomplete rhizogenesis; evaluate differentiation capacity [18]
MTT/CCK-8 Assay Kits	Colorimetric measurement of cell viability	Quantify metabolic activity of cells exposed to material eluates [9] [8]
Annexin V Apoptosis Detection Kit	Flow cytometry-based apoptosis assessment	Distinguish viable, early apoptotic, late apoptotic, and necrotic cell populations [8] [19]
Scanning Electron Microscope	High-resolution surface morphology imaging	Visualize cell attachment and material ultrastructure at high magnification [9] [18]
Energy-Dispersive X-ray Spectroscopy (EDX)	Elemental composition analysis	Quantify relative abundance of elements in material composition [9] [18]
Aluminum Step-Wedge	Radiopacity reference standard	Calibrate radiographic density measurements according to ISO 6876 [17] [5]
ISO 6876:2001 Standard	International standard for dental root canal sealing materials	Reference protocol for testing solubility, radiopacity, and setting time [17] [5]
7-Bromo-3,3-difluoroindolin-2-one	7-Bromo-3,3-difluoroindolin-2-one|	7-Bromo-3,3-difluoroindolin-2-one is a versatile fluorinated indolinone building block for antimicrobial and kinase inhibitor research. For Research Use Only. Not for human or veterinary use.
6-bromobenzo[d]isothiazol-3(2H)-one	6-bromobenzo[d]isothiazol-3(2H)-one, CAS:1427079-44-4, MF:C7H4BrNOS, MW:230.08	Chemical Reagent

The comparison between zirconium oxide and bismuth oxide as radiopacifiers in calcium silicate-based dental cements reveals a complex interplay between radiographic performance and biological response. Bismuth oxide demonstrates superior radiopacity, consistently exceeding ISO requirements, while zirconium oxide-based materials may vary in their radiographic performance depending on formulation. Despite these differences in radiopacity, both radiopacifiers can be incorporated into formulations demonstrating excellent cytocompatibility with human dental pulp cells.

The emerging research on Biodentine, ProRoot MTA, and Bio-C Repair indicates that material cytocompatibility depends on multiple factors beyond the radiopacifier alone, including cement chemistry, surface properties, and ion release profiles. While bismuth oxide offers proven radiographic performance, zirconium oxide presents a valuable alternative with potentially favorable biological interactions, particularly in specific formulations like Biodentine that demonstrate enhanced cell viability. These findings underscore the importance of considering both physical properties and biological responses when selecting and developing dental materials with appropriate radiopacifiers for clinical applications.

Bench-to-Bedside Protocols: Standardizing Cytocompatibility Assessment for Pulp Capping Materials

The biological evaluation of medical and dental materials within a risk management process is mandated by the ISO 10993 series of standards [20]. For any device with patient contact, assessing cytocompatibility is a fundamental and required first step [21] [22]. ISO 10993-5, which details test methods for in vitro cytotoxicity, is therefore a cornerstone of the biological safety evaluation [23]. This standard offers a framework to identify potential toxins that can leach from a device and cause adverse effects such as inhibition of cell growth, changes in cell morphology, and cell death [21] [22].

A pivotal component of this assessment is the preparation of material eluates—extracts of the device obtained using a suitable solvent. The elution method is designed to simulate the release of substances from a device under clinical conditions, thereby providing a sensitive means to evaluate the biological response of mammalian cells in vitro [22]. The reliability and relevance of the entire cytotoxicity test are contingent upon the rigor and appropriateness of the eluate preparation protocol. However, the flexibility permitted by ISO 10993-5 in selecting test parameters can lead to significant variability in results between different laboratories, underscoring the need for meticulously detailed and justified methodologies [21].

Framed within a broader investigation comparing the surface cytocompatibility of Biodentine, ProRoot MTA, and Bio-C Repair, this guide details the compliant preparation of material eluates. It provides a standardized protocol to ensure that subsequent biological data on cell viability, morphology, and attachment are generated from a consistent and reproducible foundation, enabling a valid comparative analysis.

Core Principles of ISO 10993-5 and Eluate Preparation

The primary objective of eluate testing is to identify leachable substances from a medical device or material that could cause cytotoxic effects. The basic principle involves exposing the test material to an extraction medium, which is then placed in contact with cultured cells. The biological response of these cells is subsequently evaluated using parameters such as cell viability, proliferation, and morphological changes [22] [23].

ISO 10993-5 intentionally provides a degree of flexibility in test design to accommodate the vast diversity of medical devices and their intended applications. This flexibility, while necessary, introduces critical variables that can profoundly influence the test outcome. A 2023 interlaboratory comparison study highlighted this issue, revealing that when 52 laboratories tested identical materials, only 58% correctly identified the cytotoxic potential of a polyvinyl chloride (PVC) sample. The study concluded that the specifications in ISO 10993-5 are not explicit enough to ensure comparable results across different laboratories for an identical device [21].

This evidence places a strong emphasis on the risk management process mandated by ISO 10993-1, wherein the eluate preparation conditions must be scientifically justified based on the nature and duration of the body contact of the device [20]. The goal is to simulate clinical conditions, sometimes with exaggeration, to ensure a safety margin. Therefore, the parameters chosen for extraction are not arbitrary but must be defensible as part of a comprehensive biological evaluation plan.

Experimental Protocol: Preparation of Material Eluates

The following protocol for preparing eluates from vital pulp materials like Biodentine, ProRoot MTA, and Bio-C Repair is synthesized from established methodologies used in comparative cytocompatibility studies [9] [21]. Adherence to this detailed procedure is essential for generating reliable and comparable data.

Materials and Equipment

Test Materials: Biodentine (Septodont), ProRoot MTA (Dentsply Tulsa Dental Specialties), Bio-C Repair (Angelus). Materials should be mixed according to the manufacturers' instructions under aseptic conditions [9]. Extraction Medium: Dulbecco's Modified Eagle Medium (DMEM) or other suitable cell culture medium, with or without supplementation (e.g., 10% Fetal Bovine Serum - FBS). The choice of serum can significantly impact test sensitivity, as serum proteins can bind leachables and alter the cytotoxic response [21]. Solvent Control: Culture medium alone, processed identically to the test material extracts. Positive Control: A known cytotoxic material, such as a polyvinyl chloride (PVC) tubing with a specific plasticizer, or a solution of phenol, to confirm assay responsiveness [21]. Equipment: Sterile tissue culture plates (e.g., 12-well plates), incubator (37°C, 5% CO₂, 95% humidity), laminar flow hood, sterile forceps and scissors, and a calibrated weighing scale.

Step-by-Step Procedure

• Sample Preparation:

  • Fabricate material samples using sterile silicone molds. A typical sample dimension is 5 mm in diameter and 2 mm in depth, ensuring a standardized surface area [9].
  • Allow the materials to set for 48 hours in an incubator at 37°C and 95% humidity to simulate the oral environment and ensure complete setting prior to extraction [9].

• Aseptic Transfer:

  • Under a laminar flow hood, carefully remove the set material disks from the molds using sterile instruments.
  • Place the samples at the bottom of a sterile 12-well tissue culture plate.

• Defining the Extraction Ratio:

  • Calculate the required volume of extraction medium based on the surface area of the material. The standard ratio recommended by ISO 10993-12 and used in comparative studies is 6 cm²/mL (surface area to volume) [9] [21].
  • For a sample with a total surface area of 50 cm², add 8.3 mL of pre-warmed extraction medium to each well [21].

• Extraction Process:

  • Ensure the material is completely immersed in the medium.
  • Incubate the plates in the dark for 24 hours at 37°C [9] [21]. Agitation is typically not required for this duration.
  • This step simulates the initial leaching of substances from the material into the biological environment.

• Collection and Preparation of Eluates:

  • After the 24-hour incubation, carefully remove the extraction medium (now the "eluate") from each well using a sterile pipette.
  • Filter the eluates through a 0.22 µm sterile filter to remove any particulate matter or microorganisms.
  • These undiluted eluates (considered a 1:1 concentration) can be used directly for cell culture exposure. To assess dose-response, prepare serial dilutions (e.g., 1:2, 1:4) in fresh culture medium [9].
  • Eluates should be used immediately for cytotoxicity assays to maintain the stability of any leached components.

Comparative Experimental Data on Vital Pulp Materials

Applying a standardized eluate preparation protocol is critical for a fair comparison of different materials. The following data, derived from a study that employed the aforementioned methods, highlights the differential biological and chemical profiles of Biodentine, ProRoot MTA, and Bio-C Repair [9].

Table 1: Chemical Composition and Cytocompatibility of Vital Pulp Materials

Material	Key Elements (EDX Analysis)	Cell Viability (MTT Assay)	Cell Morphology & Attachment
Biodentine	Calcium, Carbon, Oxygen [9]	Higher viability than control group with undiluted eluates at 24h, 48h, and 72h (p < 0.001) [9]	Adequate cell attachment; no cytoskeletal alterations observed [9]
ProRoot MTA	Calcium, Carbon, Oxygen (among others) [9]	Excellent cytocompatibility, similar to Biodentine and Bio-C Repair [9]	Adequate cell attachment; no cytoskeletal alterations observed [9]
Bio-C Repair	Low calcium; highest concentration of Zirconium [9]	Excellent cytocompatibility, similar to Biodentine and ProRoot MTA [9]	Adequate cell attachment; no cytoskeletal alterations observed [9]

Table 2: Key Parameter Influence on Eluate Cytotoxicity

Test Parameter	Impact on Results	Recommended Practice for Comparison
Serum Supplementation	10% serum in extraction medium increased test sensitivity for PVC, reducing false negatives [21]	Justify serum content based on the clinical application and the nature of potential leachables.
Extraction Duration	Longer incubation of cells with eluate increased test sensitivity [21]	Align extraction and exposure times with the intended use (e.g., prolonged for permanent implants).
Cell Seeding Density	Affects confluency and exposure to leachables; standardize for assay type (e.g., 5 x 10⁴ cells/sample for SEM) [9]	Optimize and document density to ensure consistent response and reproducible results.

Workflow Visualization

The following diagram illustrates the complete experimental workflow for preparing and testing material eluates, from sample preparation to data analysis.

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for Eluate Studies

Reagent / Material	Function in Experimental Protocol
Dulbecco's Modified Eagle Medium (DMEM)	Serves as the extraction medium and cell culture base, simulating the ionic and nutrient composition of body fluids [9].
Fetal Bovine Serum (FBS)	Common supplement to extraction and culture media; provides proteins, growth factors, and other components that can influence cell health and interact with leachables [21].
Phosphate Buffered Saline (PBS)	A balanced salt solution used for washing cells and samples; can also be used as an alternative extraction solvent [5].
MTT Reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)	A colorimetric assay used to quantify cell viability and metabolic activity. Its reduction to purple formazan crystals by living cells is measured spectrophotometrically [9].
Silicone Molds	Used to fabricate material samples with standardized dimensions (e.g., 5 mm diameter, 2 mm depth), ensuring consistency in surface area for extraction [9].
0.22 µm Sterile Filter	Critical for sterilizing the prepared eluates by removing microbial contamination and particulate matter before application to cell cultures [9].
2-Amino-3,5-dimethyl-4-nitrophenol	2-Amino-3,5-dimethyl-4-nitrophenol|C8H10N2O3
2-[(2-Fluorophenyl)methoxy]pyrazine	2-[(2-Fluorophenyl)methoxy]pyrazine|C11H9FN2O

The preparation of material eluates in compliance with ISO 10993-5 is a foundational step that dictates the quality and reliability of any subsequent in vitro cytocompatibility data. As demonstrated in the comparative analysis of Biodentine, ProRoot MTA, and Bio-C Repair, a standardized and rigorously documented protocol for sample preparation, extraction ratio, medium choice, and incubation conditions is not optional, but essential for generating valid, comparable results. The significant interlaboratory variability reported in the literature serves as a stark warning against ad-hoc methodologies. By adopting a risk-based approach and meticulously controlling the variables outlined in this guide—from the surface area-to-volume ratio to the composition of the extraction medium—researchers can ensure their findings on biological responses are a true reflection of material properties, thereby advancing the development of safer and more effective medical and dental devices.

Human dental pulp stem cells (hDPSCs) have emerged as a critical in vitro model system for evaluating the cytocompatibility of dental biomaterials, particularly those intended for vital pulp therapy [9] [24]. These fibroblast-like, neural crest-derived cells demonstrate multipotent differentiation capacity, including the ability to differentiate into odontoblast-like cells that are essential for reparative dentin formation [25] [24]. Their location at the interface between biomaterials and living tissue makes them ideally suited for assessing cell-biomaterial interactions that determine clinical success [9]. Within the field of regenerative dentistry, hDPSCs represent a biologically relevant testing platform because they are directly involved in the natural healing processes of the dentin-pulp complex [24]. This review utilizes hDPSCs as the primary cellular model to objectively compare the surface cytocompatibility of three calcium silicate-based cements: Biodentine, ProRoot MTA, and Bio-C Repair.

Table 1: Fundamental Characteristics of hDPSCs in Biomaterial Testing

Characteristic	Significance in Cytocompatibility Testing	Reference
Mesenchymal Stem Cell Phenotype	Expresses CD73, CD90, CD105; lacks hematopoietic markers (CD34, CD45) ensuring testing on relevant progenitor cells.	[25] [24]
Odontogenic Differentiation Potential	Capable of differentiating into odontoblast-like cells, directly testing material's ability to promote reparative dentinogenesis.	[25] [24]
High Proliferative Capacity	Enables assessment of material effects on cell expansion and population doubling, key for tissue regeneration.	[25] [24]
Neural Crest Origin	Possess constitutive expression of neural markers (Nestin, GFAP), allowing evaluation of neuro-compatibility.	[25]

Material Composition and Ultrastructural Properties

The chemical composition and surface morphology of calcium silicate cements fundamentally influence their biological interactions with hDPSCs. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis reveal significant compositional differences among the three materials [9].

ProRoot MTA is predominantly composed of calcium, carbon, and oxygen, consistent with its Portland cement-based formulation which includes tricalcium and dicalcium silicate as primary constituents [9] [14]. Biodentine shares a similar chemical profile, being primarily composed of tricalcium silicate (approximately 80.1%) with calcium carbonate as a filler and zirconium oxide as a radiopacifier [9] [14]. In contrast, Bio-C Repair demonstrates a notably different elemental distribution, with a lower concentration of calcium and the highest concentration of zirconium among the three materials according to EDX analysis [9].

At the ultrastructural level, SEM imaging reveals adequate attachment of hDPSCs to all three vital pulp materials, with no observable cytoskeletal alterations when cells are exposed to material eluates [9]. The surface characteristics of these materials create the initial interface for cellular interaction, influencing subsequent cell behaviors including adhesion, spreading, and proliferation.

Table 2: Material Composition and Physical Properties

Parameter	Biodentine	ProRoot MTA	Bio-C Repair
Main Components	Tricalcium silicate (80.1%), Dicalcium silicate, Calcium carbonate, Zirconium oxide [14]	Tricalcium silicate, Dicalcium silicate, Tricalcium aluminate, Bismuth oxide [14]	Calcium silicate compounds, Zirconium (highest concentration) [9]
Principal Elements (EDX)	Calcium, Carbon, Oxygen [9]	Calcium, Carbon, Oxygen [9]	Lower calcium, Highest zirconium [9]
Setting Time (Initial)	6-12 minutes [14] [5]	70 minutes [14]	Information not specified in search results
Microhardness (Vickers)	62.35 ± 11.55 HV [5]	26.93 ± 4.66 HV [5]	Information not specified in search results

Diagram 1: Material composition and cellular response relationships. Biodentine's high tricalcium silicate content correlates with enhanced hDPSC viability, while Bio-C Repair's distinct composition with higher zirconium still supports excellent cytocompatibility.

Experimental Methodologies for hDPSC-Based Cytocompatibility Assessment

Cell Isolation and Culture Protocols

The standardized methodology for hDPSC isolation involves enzymatic digestion of healthy human dental pulp tissues, typically obtained from extracted third molars [9] [25]. The protocol involves immersion in 3 mg/mL collagenase type I solution at 37°C in 5% CO₂ for 90 minutes to obtain single-cell suspensions [9]. Cells are then cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 100 μg/mL penicillin/streptomycin [9]. Researchers typically utilize cells at passages 2-4 for experiments to maintain stem cell properties while achieving sufficient cell numbers [9] [25]. Proper characterization of hDPSCs includes verification of mesenchymal stem cell markers (CD73, CD90, CD105) and absence of hematopoietic markers (CD34, CD45) through flow cytometry [25] [24].

Material Extract Preparation

For cytocompatibility testing, material eluates are prepared according to ISO 10993-5 standards [9]. The materials are mixed under aseptic conditions according to manufacturers' recommendations and allowed to set for 48 hours in an incubator at 37°C and 95% humidity [9]. Subsequently, materials are covered with cell culture medium (DMEM) and incubated in the dark for 24 hours at 37°C [9]. The resulting eluates are typically filtered and used at various dilutions (1:1, 1:2, 1:4) to assess dose-dependent effects on hDPSCs [9].

Cell Viability Assessment (MTT Assay)

The MTT assay measures mitochondrial activity in viable cells through the reduction of yellow tetrazolium salt to purple formazan crystals [9]. In standard protocols, hDPSCs are exposed to material eluates for 24, 48, and 72 hours after seeding [9]. At each interval, 10 μL of MTT reagent (5 mg/mL) is added to 90 μL growth medium per well, followed by incubation at 37°C in 5% CO₂ for 4 hours [9]. The formazan crystals are then dissolved using dimethyl sulfoxide solution, and absorbance is quantified at 570 nm using a multi-well plate reader [9]. This colorimetric measurement directly correlates with the number of viable cells.

Cell Morphology and Attachment Analysis

Scanning electron microscopy (SEM) enables direct visualization of hDPSC attachment to material surfaces [9]. For this analysis, 5 × 10⁴ hDPSCs are seeded directly onto material disks and cultured for 72 hours [9]. Specimens are then fixed with 4% glutaraldehyde in PBS, dehydrated through a graded ethanol series (30%-90% v/v), and coated with sputtered gold/palladium before observation [9]. Cell attachment is typically analyzed at 100×, 300×, and 1500× magnifications [9].

Immunofluorescence assays allow detailed examination of cytoskeletal organization and cell morphology [9]. hDPSCs are seeded at a density of 1.0 × 10⁴ cells/well in medium containing undiluted material extracts, then fixed with 4% paraformaldehyde [9]. Cells are permeabilized and stained for actin filaments and nuclei to visualize overall cell architecture and detect any morphological alterations induced by material components [9].

Diagram 2: Experimental workflow for hDPSC-based cytocompatibility testing. The standardized approach includes cell isolation, material preparation, multiple assessment methodologies, and statistical analysis for comprehensive evaluation.

Comparative Cytocompatibility Performance Data

Quantitative Cell Viability Assessment

The MTT assay provides quantitative data on hDPSC metabolic activity and viability when exposed to eluates from the three materials. Remarkably, the undiluted Biodentine group demonstrated significantly higher viability than the control group (cells without eluates) at 24h, 48h, and 72h time points (p < 0.001) [9]. Both Bio-C Repair and ProRoot MTA also showed excellent cytocompatibility with no significant cytotoxicity observed across various eluate dilutions [9]. The consistency of cell viability across time points indicates stable material composition without leachates that could negatively impact hDPSC metabolism.

Table 3: Comparative Cytocompatibility Assessment on hDPSCs

Test Parameter	Biodentine	ProRoot MTA	Bio-C Repair
Cell Viability (MTT Assay)	Significantly higher than control (p < 0.001) with undiluted eluates [9]	Excellent cytocompatibility, no significant cytotoxicity [9]	Excellent cytocompatibility similar to Biodentine and MTA [9]
Cell Attachment (SEM)	Adequate hDPSC attachment, normal morphology [9]	Adequate hDPSC attachment, normal morphology [9]	Adequate hDPSC attachment, normal morphology [9]
Cytoskeletal Alterations	Not observed in presence of material eluates [9]	Not observed in presence of material eluates [9]	Not observed in presence of material eluates [9]
Inflammatory Response (in vivo)	Information not specified	Information not specified	Mild at 7 days, resolution by 15-30 days [26]

Bioactive Potential and Biomineralization

Beyond basic cytocompatibility, the bioactive potential of these materials influences their ability to stimulate regenerative processes in hDPSCs. Calcium silicate-based materials liberate calcium and hydroxide ions that can form hydroxyapatite on their surface, enabling mineral attachment to dentin [9]. Both Biodentine and ProRoot MTA demonstrate the ability to form apatite-like crystals when immersed in phosphate-containing fluids, which correlates with their bioactivity [5]. In vivo studies with Bio-C Repair have shown formation of von Kossa-positive and birefringent structures, indicating mineralization potential and bioactive properties [26]. These bioactive characteristics are particularly relevant for hDPSC differentiation toward odontoblast-like cells and subsequent reparative dentin formation.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 4: Key Research Reagents for hDPSC-Based Cytocompatibility Testing

Reagent/Material	Function/Application	Specifications/Alternatives
Collagenase Type I	Enzymatic digestion of pulp tissue to isolate hDPSCs	3 mg/mL concentration, 90-minute incubation at 37°C [9]
Dulbecco's Modified Eagle Medium (DMEM)	Base culture medium for hDPSC expansion and maintenance	Supplemented with 10% FBS, 1% L-glutamine, antibiotics [9]
Fetal Bovine Serum (FBS)	Essential growth factors for hDPSC proliferation and viability	Heat-inactivated, typically used at 10% concentration [9]
MTT Reagent	Mitochondrial activity assessment for cell viability	5 mg/mL concentration, 4-hour incubation, measures formazan formation [9]
Dimethyl Sulfoxide (DMSO)	Solvent for dissolving formazan crystals in MTT assay	100 μL/well after medium removal [9]
Glutaraldehyde (4%)	Fixation for SEM sample preparation	In PBS, 4-hour fixation period [9]
Paraformaldehyde (4%)	Fixation for immunofluorescence studies	10-minute fixation, permeabilization required for intracellular staining [9]
1-(Propan-2-yl)piperazine hydrate	1-(Propan-2-yl)piperazine Hydrate|CAS 1221724-78-2
2-Ethyloxetane-2-carboxylic acid	2-Ethyloxetane-2-carboxylic acid, CAS:861534-42-1, MF:C6H10O3, MW:130.143	Chemical Reagent

The comprehensive assessment of Biodentine, ProRoot MTA, and Bio-C Repair using hDPSC culture models demonstrates that all three calcium silicate-based materials exhibit * excellent cytocompatibility* suitable for clinical applications in vital pulp therapy [9]. The significantly enhanced hDPSC viability observed with Biodentine eluates suggests potentially superior biological properties, while Bio-C Repair's performance confirms its viability as a ready-to-use alternative despite its distinct composition with higher zirconium content [9].

These findings underscore the critical importance of standardized hDPSC culture models as predictive tools for evaluating dental biomaterials. The consistent results across multiple assessment methodologies—MTT viability, SEM attachment analysis, and immunofluorescence morphology—provide robust evidence for the safety profiles of these materials. Future research directions should explore long-term hDPSC differentiation capabilities when exposed to these materials and their effects on specific odontogenic marker expression to further elucidate their regenerative potential in clinical applications.

In the rigorous evaluation of vital pulp materials such as Biodentine, ProRoot MTA, and Bio-C Repair, assessing cytocompatibility is a fundamental prerequisite for clinical application. The MTT assay has emerged as a cornerstone colorimetric method for quantifying cell metabolic activity, serving as a reliable indicator of cell viability and proliferation in response to biomaterials [9] [27]. This assay is particularly valuable in dental material research because it measures a crucial functional aspect of cells—their metabolic capacity—which directly reflects the biological safety and potential bioactivity of the tested materials. The protocol is based on the enzymatic reduction of the yellow, water-soluble tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple, water-insoluble formazan crystals by mitochondrial dehydrogenases and other oxidoreductase enzymes in viable cells [28] [27]. The amount of formazan produced is directly proportional to the number of metabolically active cells, providing researchers with quantitative data on material-induced cytotoxicity or proliferation effects [29].

Within the specific context of comparing Biodentine, ProRoot MTA, and Bio-C Repair, the MTT assay provides a critical dataset for understanding how these materials influence the behavior of human dental pulp cells (hDPCs). The assay's utility extends beyond mere toxicity screening; it can reveal proliferative effects, as demonstrated in studies where Biodentine eluates significantly enhanced cell viability compared to control groups [9]. This chapter details the standardized MTT assay protocols, its application in dental material research, and the interpretation of results within a framework of a broader cytocompatibility investigation.

Core MTT Assay Methodology

The execution of a reliable MTT assay requires meticulous attention to protocol. The following section outlines the standardized steps, from reagent preparation to data analysis.

Reagent Preparation

• MTT Stock Solution: MTT powder is dissolved in phosphate-buffered saline (PBS) to a final concentration of 5 mg/mL. The solution is mixed by vortexing or sonication until clear, then filter-sterilized. Aliquots should be stored protected from light at -20°C and are stable for several months. Prolonged storage at 4°C is not recommended [28] [30].
• Solubilization Solution: To dissolve the insoluble formazan crystals, a suitable solvent must be prepared. Common formulations include acidified isopropanol (e.g., 4 mM HCl, 0.1% NP-40 in isopropanol), Dimethyl Sulfoxide (DMSO), or a solution of Sodium Dodecyl Sulfate (SDS) in diluted HCl [28] [30] [27]. The choice of solvent can depend on the cell type and the nature of the experiment.

Step-by-Step Experimental Protocol

The following workflow visualizes the key stages of a standard MTT assay procedure:

Figure 1: Standard workflow for the MTT assay protocol.

• Cell Seeding and Treatment: Cells (e.g., hDPCs) are seeded at an appropriate density (e.g., 10⁴–10⁵ cells/well for a 96-well plate) in culture medium and allowed to adhere. Subsequently, they are exposed to the test conditions. In biomaterial studies, this often involves treating cells with eluates extracted from the materials, prepared according to standards such as ISO 10993-5 [9] [30] [29]. Different dilutions of the eluates (e.g., 1:1, 1:2, 1:4) are typically tested.
• MTT Incubation: After the treatment period, the MTT reagent is added directly to the culture medium. A common approach is to add 10-50 µL of the 5 mg/mL MTT stock solution to each 100 µL of medium present in the well. The plate is then incubated for a defined period, usually 3 to 4 hours, at 37°C in a COâ‚‚ incubator. During this time, metabolically active cells convert MTT to formazan [28] [30].
• Solubilization: After the incubation period, the culture medium containing the MTT reagent is carefully removed without disturbing the formed formazan crystals. For adherent cells, the supernatant is aspirated. Then, a solubilization solution (e.g., DMSO or SDS-HCl) is added to each well to lyse the cells and dissolve the purple formazan crystals. The plate is wrapped in foil and shaken on an orbital shaker for approximately 15 minutes to ensure complete dissolution [28] [29].
• Absorbance Measurement: The absorbance of the solubilized formazan solution is measured using a microplate spectrophotometer. The primary measurement wavelength is 570 nm, with a reference wavelength of 630-690 nm often used to correct for background noise and imperfections in the plate [28] [27]. The reading should be performed within an hour after solubilization to prevent precipitation.

Data Analysis and Interpretation

The raw absorbance data is processed to generate meaningful biological insights:

• Background Subtraction: The average absorbance of background control wells (containing culture medium and MTT reagent but no cells) is subtracted from all sample readings to yield the corrected absorbance [28].
• Viability Calculation: Cell viability is typically expressed as a percentage relative to the untreated control group:

• Proliferation Assessment: To generate a standard curve, a cell titration experiment with a known range of cell numbers can be performed. The amount of formazan, indicated by absorbance, is directly proportional to the number of viable cells, allowing for the assessment of proliferation over time [28] [29].

Application in Dental Material Research: Biodentine, ProRoot MTA, and Bio-C Repair

The MTT assay provides critical quantitative data for direct comparison of novel and established dental materials. The following table summarizes key experimental findings from a study that utilized the MTT assay to compare the cytocompatibility of Biodentine, ProRoot MTA, and Bio-C Repair on human dental pulp cells (hDPCs) over time [9].

Table 1: Comparison of cell viability effects of Biodentine, ProRoot MTA, and Bio-C Repair on hDPCs as measured by MTT assay.

Material Tested	Cell Viability at 24h	Cell Viability at 48h	Cell Viability at 72h	Key Findings
Biodentine	Higher than control [9]	Higher than control [9]	Higher than control [9]	The undiluted eluate group showed significantly higher cell viability than the control group (cells without eluates) at all time points (p < 0.001).
ProRoot MTA	Not significantly different from other materials [9]	Not significantly different from other materials [9]	Not significantly different from other materials [9]	Demonstrated excellent cytocompatibility, with no significant differences observed compared to Bio-C Repair and Biodentine.
Bio-C Repair	Not significantly different from other materials [9]	Not significantly different from other materials [9]	Not significantly different from other materials [9]	Showed excellent cytocompatibility that was similar to Biodentine and ProRoot MTA, despite differences in chemical composition.

Beyond viability, the cited study also integrated other methodologies to provide a comprehensive cytocompatibility profile. Scanning Electron Microscopy (SEM) confirmed that hDPCs attached adequately to all three material surfaces, and immunofluorescence assays showed no cytoskeletal alterations in cells exposed to the material eluates, reinforcing the MTT findings of high biocompatibility [9].

Energy Dispersive X-ray (EDX) analysis revealed differences in the materials' composition, which is crucial for interpreting biological responses. ProRoot MTA and Biodentine were primarily composed of calcium, carbon, and oxygen. In contrast, Bio-C Repair showed a lower concentration of calcium and the highest concentration of zirconium [9]. The fact that Bio-C Repair demonstrated cytocompatibility comparable to the calcium-rich materials, despite its different composition, highlights its potential as a viable alternative.

Essential Research Reagent Solutions

A successful MTT assay relies on a suite of specific reagents and equipment. The following table catalogues the essential components of the "Researcher's Toolkit" for this protocol.

Table 2: Key reagents and equipment required for performing an MTT assay.

Item	Function/Description	Example Protocol Details
MTT Reagent	Yellow tetrazolium salt that is reduced to purple formazan by metabolically active cells.	Prepared as a 5 mg/mL solution in PBS; 10-50 µL added per well [28] [30].
Solubilization Solution	Dissolves the insoluble purple formazan crystals into a colored solution for measurement.	DMSO, acidified isopropanol, or SDS in diluted HCl [28] [27].
Cell Culture Medium	Supports cell health during the assay. Serum-free medium is recommended during MTT incubation.	DMEM, supplemented with FBS, L-glutamine, and penicillin/streptomycin for cell culture [9].
Microplate Reader	Instrument that measures the absorbance of the solubilized formazan to provide quantitative data.	Measures absorbance at 570 nm, often with a reference filter at 630-690 nm [28] [27].
Multi-well Plates	The vessel for culturing cells and performing the assay, compatible with the plate reader.	Typically 96-well plates are used [30] [29].

Critical Considerations and Limitations of the MTT Assay

While the MTT assay is a powerful and widely used tool, researchers must be aware of its limitations and potential confounding factors to avoid misinterpretation of data.

• Metabolic Activity vs. Viability: The MTT assay measures metabolic activity, which is a marker of cell viability but not a direct measure of cell number. Treatments that alter cellular metabolism (e.g., by inducing quiescence or modulating mitochondrial function without causing death) can lead to over- or underestimation of viability [31] [27]. It is therefore recommended to use the MTT assay in conjunction with other methods, such as flow cytometry for apoptosis or direct cell counting, for a more comprehensive assessment [31] [29].
• Interference and Optimization: Several factors can interfere with the assay:
  • Serum and Phenol Red: The presence of serum or phenol red in the culture medium during MTT incubation can generate background absorbance. Using serum-free medium for the incubation step is advised [28].
  • Test Materials: The tested materials themselves can interfere. For instance, colored compounds in material eluates or nanoparticles can absorb light at the measurement wavelength, leading to inaccurate readings [31]. Including appropriate background controls (wells with material eluates and MTT but no cells) is essential to account for this.
  • Formazan Solubilization: Incomplete dissolution of formazan crystals will result in uneven color and inaccurate readings. Ensuring adequate shaking and, if necessary, pipetting the solution is critical [28].
• Assay Conditions: The results are sensitive to cell seeding density, MTT concentration, and incubation time. These parameters must be optimized for each cell type and experimental setup to ensure the assay operates within a linear and dynamic range [31].

The cytocompatibility of vital pulp materials is fundamentally determined by their interaction with dental pulp cells at the cellular level. Cellular morphology and attachment serve as critical indicators of a biomaterial's biological performance, reflecting early cell-material interactions that predate downstream differentiation and mineralization events. Within the field of dental biomaterials research, scanning electron microscopy (SEM) and immunofluorescence techniques have emerged as indispensable tools for qualitatively and quantitatively assessing these interactions.

This guide provides a comparative analysis of cellular responses to three calcium silicate-based materials—Biodentine, ProRoot MTA, and Bio-C Repair—with a specific focus on methodological approaches for evaluating cell morphology and attachment. The experimental data and protocols presented herein are framed within a broader research context investigating the surface properties and cytocompatibility of these commonly used vital pulp materials.

Comparative Analysis of Material Properties and Cellular Responses

Chemical Composition and Surface Characteristics

The surface properties of biomaterials directly influence cell adhesion and spreading. Energy dispersive X-ray (EDX) analysis reveals significant compositional differences among the three materials that likely contribute to their varying surface characteristics and cellular responses.

Table 1: Chemical Composition of Vital Pulp Materials via EDX Analysis [9]

Material	Primary Components	Notable Characteristics	Radiopacifier
ProRoot MTA	Calcium, Carbon, Oxygen (among others)	Traditional calcium silicate composition	Bismuth oxide [5]
Biodentine	Calcium, Carbon, Oxygen (among others)	Tricalcium silicate-based with smaller particle size [32]	Zirconium oxide [5]
Bio-C Repair	Lower calcium concentration, Highest zirconium content	Distinct from traditional calcium silicates	Zirconium oxide [9]

Surface topography analysis indicates that both ProRoot MTA and Biodentine demonstrate time-dependent surface roughness changes in different environmental conditions. In wet conditions, both materials show increased surface roughness after 1 day, which may potentially enhance cell attachment mechanisms. Biodentine specifically exhibits higher surface roughness in blood conditions compared to ProRoot MTA after 28 days [32].

Quantitative Assessment of Cell Viability and Proliferation

Cell viability assays provide crucial quantitative data on material cytocompatibility. The MTT assay, which measures mitochondrial activity in living cells, has been consistently employed to evaluate cellular responses to material eluates.

Table 2: Cell Viability Assessment of Vital Pulp Materials via MTT Assay [9]

Material	24-Hour Viability	48-Hour Viability	72-Hour Viability	Notable Findings
ProRoot MTA	Comparable to control	Comparable to control	Comparable to control	Consistent cytocompatibility profile
Biodentine	Higher than control (p<0.001)	Higher than control (p<0.001)	Higher than control (p<0.001)	Significantly enhanced proliferation
Bio-C Repair	Comparable to control	Comparable to control	Comparable to control	Excellent cytocompatibility similar to other materials

Notably, undiluted Biodentine eluates demonstrated significantly higher cell viability compared to the control group (cells without eluates) at all time points (24h, 48h, and 72h; p<0.001), suggesting potentially stimulatory effects on human dental pulp cells (hDPCs) [9]. Both ProRoot MTA and Bio-C Repair showed excellent cytocompatibility with viability comparable to control groups.

Experimental Methodologies for Morphology and Attachment Assessment

Scanning Electron Microscopy (SEM) for Cell Attachment Analysis

SEM provides high-resolution visualization of cell-material interactions, enabling detailed assessment of cell attachment, spreading, and morphological characteristics on material surfaces.

Protocol: SEM Sample Preparation and Cell Attachment Analysis [9]

• Sample Preparation:

  • Create material discs using silicone molds (2mm depth × 5mm diameter)
  • Allow samples to set for 24-48 hours in incubator (37°C, 95% humidity)
  • Sterilize set samples under ultraviolet radiation for 15 minutes

• Cell Seeding:

  • Seed human dental pulp cells (hDPCs) onto material surfaces at density of 5×10⁴ cells/disc
  • Culture cells for 72 hours in standard culture conditions (37°C, 5% COâ‚‚)

• Fixation and Dehydration:

  • Post-fix samples with 4% glutaraldehyde in PBS for 4 hours
  • Dehydrate through graded ethanol series (30%, 50%, 70%, 90%, 100% v/v)
  • Critical point dry to preserve ultrastructure

• SEM Imaging:

  • Mount samples on stubs and coat with sputtered gold/palladium
  • Image at various magnifications (100×, 300×, 1500×) to assess cell attachment
  • Operate at accelerating voltages of 5-10 kV for optimal resolution

Key Findings: SEM analysis revealed adequate attachment of hDPCs to all three vital pulp materials, with no significant cytoskeletal alterations observed in the presence of material eluates [9]. Cell spreading and morphology appeared normal across all test groups, indicating favorable cell-material interactions.

Figure 1: SEM Sample Preparation Workflow. This diagram illustrates the comprehensive process for preparing and analyzing cell-material interactions using scanning electron microscopy, from initial sample preparation through final imaging and analysis.

Immunofluorescence for Cytoskeletal Organization Analysis

Immunofluorescence techniques allow specific visualization of cytoskeletal components, particularly F-actin filaments, providing insights into cell spreading and morphological characteristics influenced by material surfaces.

Protocol: Immunofluorescence Staining and Cytoskeleton Analysis [9]

• Cell Culture with Material Eluates:

  • Prepare material eluates according to ISO 10993-5 guidelines
  • Seed hDPCs at density of 1.0×10⁴ cells/well on 24-well plates
  • Culture cells in medium containing undiluted material extracts for 24-72 hours

• Cell Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 10 minutes at room temperature
  • Wash twice with phosphate-buffered saline (PBS)
  • Permeabilize cells with 0.1% Triton X-100 in PBS for 5 minutes

• Immunostaining:

  • Incubate with fluorescent phalloidin conjugate (e.g., Alexa Fluor 488-phalloidin) to visualize F-actin
  • Counterstain nuclei with DAPI (4',6-diamidino-2-phenylindole)
  • Include negative controls (no primary antibody) to assess non-specific binding

• Confocal Microscopy and Image Analysis:

  • Image samples using confocal laser scanning microscopy
  • Capture z-stack images for three-dimensional reconstruction
  • Analyze cytoskeletal organization, cell spreading area, and morphological features

Key Findings: Immunofluorescence analysis demonstrated that none of the material eluates induced significant cytoskeletal alterations in hDPCs. Cells maintained normal actin filament organization and cellular morphology, further confirming the cytocompatibility of all three test materials [9].

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Research Reagents for Cell Morphology and Attachment Studies [9]

Category	Specific Reagents/Equipment	Research Function
Cell Culture	Human dental pulp cells (hDPCs), Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Penicillin/Streptomycin	Primary cell culture system for cytocompatibility assessment
Material Preparation	Silicone molds (Elite HD+ Putty), Sterile mixing equipment, Ultraviolet sterilization chamber	Standardized sample fabrication and sterilization
SEM Analysis	Glutaraldehyde, Ethanol series, Critical point dryer, Sputter coater (gold/palladium), Field emission SEM (e.g., Hitachi S-4800)	Sample processing and high-resolution visualization of cell attachment
Immunofluorescence	Paraformaldehyde, Triton X-100, Phalloidin conjugates, DAPI, Confocal microscope	Cytoskeletal staining and morphological analysis
Viability Assays	MTT reagent, Dimethyl sulfoxide (DMSO), Multi-well plate reader	Quantitative assessment of cell metabolic activity
2-Hydroxy-3-methoxypropanenitrile	2-Hydroxy-3-methoxypropanenitrile, CAS:93177-83-4, MF:C4H7NO2, MW:101.105	Chemical Reagent
(2R)-2,6,6-Trimethylheptanoic acid	(2R)-2,6,6-Trimethylheptanoic Acid|High-Purity|RUO

The comprehensive assessment of cellular morphology and attachment through SEM and immunofluorescence techniques provides critical insights into the cytocompatibility of Biodentine, ProRoot MTA, and Bio-C Repair. All three materials demonstrate excellent biocompatibility with human dental pulp cells, supporting their clinical use in vital pulp therapy.

Biodentine shows enhanced cell viability across multiple time points, while Bio-C Repair demonstrates comparable cytocompatibility despite its distinct chemical composition. The experimental methodologies outlined herein provide robust protocols for researchers investigating cell-material interactions in dental biomaterials. These techniques enable precise characterization of cellular responses at the material interface, contributing valuable data for the development and optimization of future bioactive materials for endodontic applications.

Navigating Material Limitations: Solubility, Discoloration, and Clinical Handling

Addressing Long Setting Times and Potential Tooth Discoloration

Hydraulic calcium silicate cements (HCSCs) have transformed vital pulp therapy by enabling genuine bioactivity and pulp tissue regeneration. However, despite their clinical success, first-generation materials like ProRoot MTA present significant challenges including prolonged setting times and potential tooth discoloration, which can compromise treatment outcomes and patient satisfaction. These limitations have driven the development of new formulations like Biodentine and Bio-C Repair, which aim to mitigate these drawbacks while maintaining excellent biological properties.

This comparison guide objectively analyzes the performance of ProRoot MTA, Biodentine, and Bio-C Repair, with particular focus on setting characteristics, discoloration potential, and cytocompatibility. By presenting structured experimental data and detailed methodologies, this resource provides researchers and clinicians with evidence-based insights for material selection in both research and clinical practice, framed within the broader context of surface cytocompatibility research.

Comparative Material Properties Analysis

Table 1: Key Physicochemical Properties of Bio-C Repair, Biodentine, and ProRoot MTA

Property	Bio-C Repair	Biodentine	ProRoot MTA
Primary Composition	Calcium silicates, zirconium oxide [9]	Tricalcium silicate, dicalcium silicate, calcium carbonate, zirconium oxide [14]	Tricalcium silicate, dicalcium silicate, bismuth oxide [14]
Radiopacifier	Zirconium oxide [9]	Zirconium oxide [14]	Bismuth oxide [14]
Initial Setting Time	Data not located in search results	6 minutes [14]	70 minutes [14]
Final Setting Time	Data not located in search results	10.1 minutes [14]	175 minutes [14]
Solubility	Data not located in search results	<3% in 24h (ISO 6876:2001) [5]	<3% in 24h (ISO 6876:2001), significantly lower than Biodentine [5]
Microhardness (Vickers)	Data not located in search results	62.35 ± 11.55 HV [5]	26.93 ± 4.66 HV [5]
Radiopacity (mm Al)	Data not located in search results	1.50 ± 0.10 [5]	6.40 ± 0.06 [5]

Table 2: Biological and Clinical Performance Comparison

Parameter	Bio-C Repair	Biodentine	ProRoot MTA
Cytocompatibility (Cell Viability)	Excellent, similar to Biodentine and ProRoot MTA [9] [3]	Excellent; undiluted eluates showed higher viability than control in hDPCs [9]	Excellent cytocompatibility [9] [3]
Tooth Discoloration Potential	According to manufacturer: does not contribute to discoloration [9]	Significantly lower than MTA [33]	Higher potential for discoloration [33]
Clinical Success Rate (VPT)	Data not located in search results	94.20% at 12 months [33]	Comparable clinical success rates [33]
Key Biological Effects	Promotes DSPP & BSP expression in hDPSCs; enhances mineral deposition [34]	Stimulates dentin bridge formation; high bioactivity [33]	Proven clinical success; induces hard tissue formation [9]

Experimental Evidence and Research Protocols

Cytocompatibility and Cell Response Assessment

Research Objective: To compare the chemical composition, ultrastructural morphology, and biological effects of Biodentine, ProRoot MTA, and Bio-C Repair on human dental pulp cells (hDPCs) [9].

Methodology Overview:

• Material eluate preparation: Materials mixed under aseptic conditions according to manufacturers' recommendations and placed in 12-well tissue culture plates. After setting for 48 hours at 37°C and 95% humidity, materials were covered with Dulbecco's Modified Eagle Medium (DMEM) and incubated for 24 hours in the dark at 37°C. Eluates (1:1) were prepared per ISO 10993-5 recommendations, then filtered and diluted to 1:1, 1:2, and 1:4 concentrations for cell culture experiments [9].
• Cell isolation and culture: hDPCs were isolated from healthy human third molars (n=10) using collagenase type I solution. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine, and penicillin/streptomycin, then incubated at 37°C in 5% COâ‚‚. Cells at passages 2-4 were used for experiments [9].
• Cell viability assay (MTT assay): hDPCs were exposed to material eluates, with viability analyzed at 24, 48, and 72 hours after seeding. MTT reagent was added to culture wells and incubated for 4 hours at 37°C. Formazan crystals were dissolved with dimethyl sulfoxide, and absorbance was quantified at 570nm using a plate reader [9].
• Cell morphology analysis (Immunofluorescence): hDPCs were seeded at 1.0×10⁴ cells/well in culture medium containing undiluted material extracts. Cells were fixed with paraformaldehyde, permeabilized, and stained to visualize cytoskeletal alterations [9].
• SEM and EDX analysis: Material samples were prepared in silicone molds and allowed to set for one week at 37°C and 95% humidity. Samples were visualized under 500× to 50,000× magnifications using field emission scanning electron microscopy. Energy dispersive X-ray analysis was performed at 2500× for elemental characterization [9].

Key Findings:

• EDX analysis revealed ProRoot MTA and Biodentine were primarily composed of calcium, carbon, and oxygen, while Bio-C Repair showed lower calcium concentration and the highest zirconium concentration [9].
• SEM demonstrated adequate attachment of hDPCs to all materials without cytoskeletal alterations in the presence of material eluates [9].
• The undiluted Biodentine group showed higher cell viability than the control group at 24h, 48h, and 72h (p<0.001) [9].
• All three materials demonstrated excellent cytocompatibility with no significant differences in biological response [9] [3].

Odontogenic Differentiation Potential

Research Objective: To compare the ability of Bio-C Repair and Biodentine to promote odontogenic differentiation by evaluating dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) expression in human dental pulp stem cells (hDPSCs) [34].

Methodology Overview:

• Material preparation: Bio-C Repair and Biodentine were pulverized and sterilized according to ISO 10993-5:2009 standards [34].
• Cell culture: hDPSCs from primary cultures (passages 2-3) were characterized for stem cell markers (CD90 98%, CD105 99.7%, CD73 94%, and LinNeg 0.5%) and cultured in osteogenic media with material extracts at concentrations of 1:1, 1:2, or 1:5 [34].
• Odontogenic differentiation assessment: DSPP and BSP expression were analyzed using enzyme-linked immunosorbent assays (ELISA) on days 7 and 14. Mineral deposition was assessed via alizarin red staining on day 21 [34].

Key Findings:

• At 1:1 concentration, significant differences in DSPP and BSP expression were observed between Bio-C Repair and Biodentine [34].
• At concentrations of 1:2 and 1:5, no significant differences in DSPP or BSP expression were observed between materials (p>0.05) [34].
• The highest DSPP and BSP concentrations after 7 and 14 days were observed with both materials at 1:5 concentration (6.6-6.71 and 13.20-13.47 ng/mL) [34].
• Bio-C Repair demonstrated equivalent effectiveness to Biodentine in enhancing DSPP and BSP expression and promoting mineral deposition in hDPSCs [34].

Experimental Pathway for Evaluating Pulp Capping Materials

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for Dental Material Cytocompatibility Studies

Reagent / Material	Function in Research	Experimental Application
Human Dental Pulp Cells (hDPCs)	Primary cell model for biocompatibility testing	Isolated from extracted third molars; used to assess cell viability, attachment, and morphology in response to material eluates [9]
Dulbecco's Modified Eagle Medium (DMEM)	Cell culture medium	Serves as base medium for cell maintenance and as extraction vehicle for material eluates [9]
MTT Reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)	Cell viability assay	Metabolically reduced by viable cells to formazan crystals; quantified spectrophotometrically to determine cell viability [9]
Collagenase Type I	Tissue dissociation enzyme	Digests collagen in pulp tissue to isolate individual cells for primary culture establishment [9]
Alizarin Red Stain	Mineralization detection	Binds to calcium deposits in mineralized nodules; used to assess osteogenic/odontogenic differentiation potential [34]
ELISA Kits for DSPP/BSP	Odontogenic marker quantification	Quantifies expression of dentin sialophosphoprotein and bone sialoprotein; markers of odontogenic differentiation [34]
2-Chloroquinoxaline-6-sulfonamide	2-Chloroquinoxaline-6-sulfonamide, CAS:2091951-23-2, MF:C8H6ClN3O2S, MW:243.67	Chemical Reagent
6-fluoro-2H,3H-furo[2,3-b]pyridine	6-fluoro-2H,3H-furo[2,3-b]pyridine|RUO|Furopyridine

The comparative analysis of ProRoot MTA, Biodentine, and Bio-C Repair reveals a clear trajectory of innovation in hydraulic calcium silicate cement technology. While all three materials demonstrate excellent cytocompatibility, next-generation formulations address two significant limitations of traditional MTA: prolonged setting times and tooth discoloration potential.

Biodentine shows a remarkable reduction in setting time (10.1 minutes final set compared to 175 minutes for ProRoot MTA) while maintaining exceptional bioactivity and potentially reducing discoloration through its zirconium oxide radiopacifier instead of bismuth oxide [5] [14]. Bio-C Repair demonstrates comparable cytocompatibility and odontogenic differentiation potential to both materials, with the practical advantage of a ready-to-use formulation [9] [34].

These advancements represent significant progress in vital pulp therapy materials, offering researchers and clinicians options that balance biological performance with practical clinical handling. The ongoing development of HCSCs continues to focus on optimizing this balance while maintaining the regenerative potential essential for successful long-term outcomes in vital pulp therapy.

Optimizing Solubility and Microleakage for a Perfect Seal

In modern restorative dentistry and endodontics, the long-term success of a procedure hinges on the clinician's ability to achieve a perfect, lasting seal. Solubility and microleakage represent two critical, interconnected properties that directly determine this outcome. Excessive solubility can lead to the gradual degradation of a dental material, while microleakage permits the ingress of bacteria and fluids at the material-tooth interface, both of which are primary causes of secondary caries, postoperative sensitivity, and ultimately, treatment failure. Calcium silicate-based cements have revolutionized dental care by offering bioactive properties that promote healing. Among these, ProRoot MTA has long been the gold standard for various endodontic procedures, while Biodentine was developed as a "dentin substitute" with improved physical characteristics. More recently, Bio-C Repair has entered the market as a ready-to-use bioceramic material. This guide provides a objective, data-driven comparison of these three prominent materials, focusing on the critical parameters of solubility and microleakage to inform material selection and research direction.

Material Properties at a Glance

The fundamental properties of a dental material heavily influence its clinical handling, performance, and ultimate ability to form a seal. The table below summarizes key characteristics of ProRoot MTA, Biodentine, and Bio-C Repair.

Table 1: Comparative Properties of ProRoot MTA, Biodentine, and Bio-C Repair

Property	ProRoot MTA	Biodentine	Bio-C Repair
Primary Component	Tricalcium Silicate [14]	Tricalcium Silicate (80.1%) [14]	Calcium Silicate-based [3]
Radiopacifier	Bismuth Oxide [14] [35]	Zirconium Oxide [14] [36]	Zirconium Oxide [3]
Setting Time (Final)	175-228 minutes [14] [5]	10-45 minutes [14] [16]	Information not specified in search results
Compressive Strength	Lower than Biodentine [16]	~300 MPa after 1 month [14]	Information not specified in search results
Flexural Strength	~14 MPa [14]	~34 MPa [14] [16]	Information not specified in search results
Key Clinical Advantages	Proven bioactivity & biocompatibility [16]	Short setting time, high strength, dentin-like hardness [14] [36]	Ready-to-use convenience [3]

Comparative Analysis of Solubility

Solubility is a double-edged sword for bioactive calcium silicate cements. While excessive solubility compromises the seal, a degree of solubility is necessary for the ion release that drives bioactivity.

Quantitative Solubility Data

Direct comparative studies provide measurable evidence for the dissolution behavior of these materials.

Table 2: Experimentally Measured Solubility of Biodentine and ProRoot MTA

Material	Test Medium	Solubility Result	Significance
Biodentine	Distilled Water (ISO 6876)	< 3% (fulfills ISO standard) [5]	Significantly higher solubility than ProRoot MTA (p < 0.0001) [5]
ProRoot MTA	Distilled Water (ISO 6876)	< 3% (fulfills ISO standard) [5]	Significantly lower solubility than Biodentine (p < 0.0001) [5]
Both Materials	Phosphate Buffered Saline (PBS)	Precipitation of hydroxyapatite observed on material surface [5]	Apatite formation reduces effective solubility and enhances the seal [5]

Experimental Protocols for Solubility Testing

The data in Table 2 were typically generated using a standardized methodology:

• Specimen Preparation: Materials are mixed according to manufacturers' instructions and placed into ring molds (e.g., 20 mm diameter x 1.6 mm height). The specimens are allowed to set in an incubator at 37°C and 95% relative humidity for 24 hours. [5]
• Immersion and Weighing: The initial mass of each set specimen is recorded with a high-precision balance. Specimens are then immersed in a specific volume of test liquid (e.g., 160 mL of distilled water or PBS buffer) at 37°C. The immersion liquid is replaced at set intervals (e.g., weekly) to maintain a consistent driving force for dissolution. [5]
• Solubility Calculation: After the immersion period (e.g., 28 days), specimens are removed, dried, and weighed again. The solubility is calculated as the percentage of mass loss relative to the initial mass. [5]

Comparative Analysis of Microleakage

Microleakage tests evaluate a material's ability to prevent the penetration of bacteria, fluids, and molecules along the interface with the tooth structure, which is a direct measure of its sealing efficacy.

Quantitative Microleakage Data

Various in vitro studies have simulated clinical scenarios to compare the sealing ability of these materials.

Table 3: Experimental Microleakage Comparison Across Different Models

Material	Test Method	Application	Result & Comparison
Biodentine	Dye Extraction (UV Spectrophotometry)	Furcation Perforation Repair	Least dye absorbance, significantly better than RetroMTA; not statistically different from ProRoot MTA [37] [38]
ProRoot MTA	Dye Extraction (UV Spectrophotometry)	Furcation Perforation Repair	Intermediate dye absorbance; no significant difference from Biodentine [37] [38]
Biodentine	Fluid Filtration Technique	Root Canal Obturation	Microleakage: 1.95 ± 1.27 µL/8min; No significant difference from ProRoot MTA (1.83 ± 0.62 µL/8min) [39]
ProRoot MTA	Fluid Filtration Technique	Root Canal Obturation	Microleakage: 1.83 ± 0.62 µL/8min; No significant difference from Biodentine [39]

Experimental Protocols for Microleakage Testing

The referenced microleakage studies often employ the following rigorous approaches:

• Dye Extraction Method:

  • Tooth Preparation: Extracted human teeth are prepared with standardized defects (e.g., furcation perforations). [37] [38]
  • Material Placement: Test materials are used to repair the defects, and the teeth are stored in humid conditions. [37]
  • Dye Challenge: Specimens are immersed in a dye solution (e.g., 2% methylene blue) for a set period (e.g., 48 hours) to allow penetration. [37] [38]
  • Dye Extraction & Quantification: The dye within the specimen is extracted, typically by immersion in nitric acid. The solution is then analyzed using a UV-visible spectrophotometer, which measures the absorbance value, directly quantifying the amount of leaked dye. [37] [38]

• Fluid Filtration Method:

  • Obturation: Root canals of extracted teeth are filled with the test material. [39]
  • System Assembly: The root is connected to a system that applies a constant gas pressure (e.g., 50 kPa) to one end of the canal, forcing liquid through any microscopic gaps. [39]
  • Leakage Measurement: The volume of liquid displaced over a specific time (e.g., 8 minutes) is measured directly using a pipette, providing a quantitative measure of microleakage in microliters. [39]

The following diagram illustrates the logical workflow of the dye extraction method, a key protocol for evaluating microleakage.

Diagram 1: Dye Extraction Microleakage Workflow

The Scientist's Toolkit: Essential Research Reagents

To replicate the experiments cited in this guide or to conduct novel research in this field, the following reagents and materials are essential.

Table 4: Key Reagents for Solubility and Microleakage Research

Reagent / Material	Function in Research	Example Application
Phosphate Buffered Saline (PBS)	Simulates phosphate-containing body fluids; used to study hydroxyapatite precipitation and bioactivity. [5]	Solubility testing in a biologically relevant environment. [5]
Methylene Blue Dye	A tracer molecule used to visually and quantitatively assess the extent of microleakage. [37] [38]	Dye penetration and dye extraction leakage models. [37]
Nitric Acid (HNO₃)	A strong acid used to dissolve tooth structure and extract dye that has penetrated during leakage tests. [37] [38]	Dye extraction method for quantifying total leakage. [37]
UV-Visible Spectrophotometer	Analytical instrument that measures the concentration of a solute in a solution by its light absorption. [37]	Precisely quantifying the amount of dye extracted in leakage studies. [37]

Discussion: Weighing the Evidence for the Perfect Seal

When optimizing for a perfect seal, the data reveal a nuanced picture. ProRoot MTA demonstrates significantly lower solubility in water compared to Biodentine, suggesting a potential advantage for long-term structural stability in environments with constant fluid exposure. [5] However, Biodentine's higher solubility contributes to a greater release of calcium and silicon ions, which promotes the formation of a mineral-rich interfacial layer and hydroxyapatite crystals upon contact with phosphate-rich fluids, thereby enhancing the seal over time. [36] This bioactivity is a shared trait among these calcium silicate cements, but the kinetics may differ.

Regarding the critical parameter of microleakage, multiple studies concur that Biodentine provides a sealing ability equivalent to, and in some models superior to, ProRoot MTA. [37] [39] [38] Biodentine's superior physical properties—such as its lower porosity and higher mechanical strength—likely contribute to this excellent marginal seal. [14] While direct comparative leakage data for Bio-C Repair is limited in the search results, one study noted its excellent cytocompatibility, similar to Biodentine and ProRoot MTA, but with a notably lower concentration of calcium in its composition. [3] This difference in elemental composition could influence its long-term bioactive sealing potential and warrants further direct investigation.

From a clinical perspective, Biodentine's dramatically shorter setting time (10-12 minutes vs. nearly 3 hours for ProRoot MTA) is a significant practical advantage, reducing the risk of material washout or disruption during the critical initial setting phase and contributing to a more reliable immediate seal. [14] [5] Furthermore, Biodentine is formulated without bismuth oxide, a radiopacifier in ProRoot MTA that is associated with tooth discoloration and delayed setting. [14] [36] This makes Biodentine a more aesthetically predictable choice in the anterior region.

In the pursuit of a perfect seal, the choice between ProRoot MTA, Biodentine, and Bio-C Repair involves balancing key material properties. ProRoot MTA remains a proven, highly reliable material with low solubility. However, the body of evidence indicates that Biodentine offers a compelling combination of superior handling characteristics, including a fast setting time and high mechanical strength, coupled with a sealing ability that matches or exceeds that of ProRoot MTA. For researchers and clinicians whose primary focus is on optimizing the immediate and long-term seal to prevent microleakage, Biodentine presents a strong alternative. Bio-C Repair, as a newer material, requires more direct comparative research on its sealing performance. Future studies should focus on long-term in vivo performance and the specific mechanisms of interfacial seal formation for these advanced bioceramic materials.

Balancing Radiopacity Requirements with Biocompatibility

The development of modern bioactive endodontic materials necessitates a delicate balance between essential physical properties and biological performance. Radiopacity is a critical physical property mandated by international standards, enabling clinicians to visualize material placement and assess quality on radiographs. Simultaneously, excellent biocompatibility and the ability to promote cellular responses conducive to healing are fundamental biological requirements for materials interacting with vital pulp tissues. This review objectively compares three calcium silicate-based materials—Biodentine, ProRoot MTA, and Bio-C Repair—focusing on the interplay between their radiopacity, chemical composition, and cytocompatibility with human dental pulp cells (hDPCs). As these materials are increasingly used in vital pulp therapies such as pulp capping, pulpotomy, and perforation repair, understanding how their radiopacifying components influence biological behavior is essential for both clinical practice and future material development.

Material Composition and Radiopacity Profiles

The chemical composition of bioactive endodontic cements directly dictates their physical properties, including radiopacity, and their biological interactions. Radiopacity is conferred by specific radiopacifying agents added to the base material, while biocompatibility is influenced by the entire composition and the ions released during setting and dissolution.

Table 1: Chemical Composition and Radiopacity of Bioactive Endodontic Cements

Material	Primary Components	Radiopacifier	Radiopacity (mm Al)	Meets ISO 6876?
ProRoot MTA	Tricalcium silicate, Dicalcium silicate, Calcium oxide, Silicon oxide [14] [16]	Bismuth Oxide (20%) [14]	6.40 ± 0.06 [5], 7.17 [14]	Yes
Biodentine	Tricalcium silicate (main), Dicalcium silicate, Calcium carbonate [9] [16]	Zirconium Oxide [9] [16]	1.50 ± 0.10 [5], 3.5 [14] [16]	Conflicting Data
Bio-C Repair	Information limited in search results	Zirconium Oxide (High Concentration) [9]	Not fully quantified	Insufficient Data

Energy dispersive X-ray (EDX) analysis provides empirical evidence for these compositions, revealing that ProRoot MTA and Biodentine are predominantly composed of calcium, carbon, and oxygen. In contrast, Bio-C Repair demonstrates a notably low concentration of calcium and the highest concentration of zirconium among the three materials [9]. The radiopacity data reveals a clear distinction. ProRoot MTA's use of bismuth oxide provides radiopacity significantly exceeding the ISO 6876:2001 requirement of 3 mm of aluminum equivalence [5]. Biodentine's radiopacity, dependent on zirconium oxide, is reported with variation across studies. While one study reports a value of 1.5 mm Al, which fails to meet the ISO standard, other reviews cite a value of 3.5 mm Al, which is sufficient [14] [5] [16]. The radiopacity of Bio-C Repair was not quantitatively detailed in the available search results.

Comparative Cytocompatibility and Bioactive Potential

Biocompatibility is an essential property for any material designed to interact with vital pulp tissues. The biological effects of Biodentine, ProRoot MTA, and Bio-C Repair have been extensively evaluated in vitro using human dental pulp cells (hDPCs) and human dental pulp stem cells (hDPSCs).

Cell Viability and Proliferation

Multiple studies confirm that all three materials exhibit * excellent cytocompatibility. In cell viability assays (MTT assay), cells exposed to eluates from these materials showed healthy proliferation rates. Remarkably, the undiluted Biodentine group demonstrated *higher viability than the control group (cells without eluates) at 24, 48, and 72 hours [9]. A separate flow cytometry study using Annexin V staining to detect early apoptosis found that all three materials showed cell viability similar to the control group, with Biodentine exhibiting the highest rates, though not always statistically significant [12]. This suggests that the setting byproducts and ions released from these calcium silicate cements are not cytotoxic and can support cell survival and growth.

Odontogenic Differentiation and Mineralization

Beyond mere survival, the ability of a material to stimulate progenitor cells to differentiate into odontoblast-like cells and form reparative dentin is crucial. Research indicates that both Biodentine and Bio-C Repair effectively promote this odontogenic differentiation. A study evaluating dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) expression found that Bio-C Repair was as effective as Biodentine in enhancing the expression of these key markers in hDPSCs [34]. The highest expression was observed at specific eluate concentrations (1:5), highlighting that material concentration influences cellular response. Furthermore, Alizarin Red staining confirmed that both materials supported significant mineral deposition, a functional indicator of differentiated cell activity [34]. Another study on Bio MTA Plus, a material with a similar classification, also showed high expression of odontogenic markers (RUNX2, DMP1, DSSP), reinforcing the bioactive potential of this newer generation of calcium silicate cements [40].

Table 2: Summary of Key Biological Properties from In Vitro Studies

Material	Cell Viability / Cytocompatibility	Effect on Odontogenic Markers	Mineral Deposition
ProRoot MTA	Excellent, non-cytotoxic [12]	Supports differentiation [40]	Promotes biomineralization [41]
Biodentine	Excellent, often superior to control [9] [12]	Induces DSPP, BSP, DMP1 [34] [40]	Stimulates mineralized tissue formation [34]
Bio-C Repair	Excellent, similar to Biodentine & MTA [9]	Enhances DSPP & BSP expression [34]	Promotes significant mineral deposition [34]

Experimental Methodologies for Key assays

To ensure reproducibility and critical evaluation of the data, understanding the core experimental protocols used in these studies is vital.

Material Eluate Preparation (ISO 10993-5)

The preparation of material eluates for cell culture follows standardized biocompatibility testing. Typically, materials are mixed according to manufacturers' instructions and allowed to set for 24-48 hours in an incubator at 37°C and 95% humidity to simulate oral conditions. The set materials are then immersed in a cell culture medium, such as Dulbecco's Modified Eagle Medium (DMEM), for 24 hours to create a concentrated extract (often considered a 1:1 eluate). This eluate is subsequently filtered to remove particulates and may be diluted (e.g., 1:2, 1:4) with fresh medium to create a concentration gradient for testing [9] [34]. This method assesses the effects of soluble components released by the materials.

Cell Viability and Cytotoxicity Assays

• MTT Assay: This colorimetric assay measures cellular metabolic activity as an indicator of cell viability. Cells are seeded in plates and exposed to material eluates. After incubation, the MTT reagent is added, which is reduced by living cells to purple formazan crystals. These crystals are dissolved, and the solution's absorbance is measured spectrophotometrically. Higher absorbance correlates with greater cell viability [9].
• Flow Cytometry with Annexin V: This assay quantitatively distinguishes viable, apoptotic, and necrotic cells. Cells treated with material eluates are stained with Annexin V (which binds to phosphatidylserine exposed on the surface of apoptotic cells) and a viability dye like Propidium Iodide (PI, which enters necrotic cells). The cell population is then analyzed using a flow cytometer to determine the percentages of cells in each stage of cell death [12].

Assessment of Odontogenic/Bioactive Potential

• Gene Expression Analysis (RT-PCR/qPCR): This technique measures the mRNA levels of specific odontogenic genes (e.g., DSPP, DMP1, BSP, RUNX2). Cells are cultured under odontogenic conditions with material eluates. RNA is extracted, reverse-transcribed to cDNA, and then amplified using gene-specific primers. The amount of PCR product corresponds to the initial level of the target gene, indicating the degree of differentiation [40].
• Alizarin Red Staining: This assay visualizes and quantifies calcium-rich deposits or nodules formed by differentiated cells. After a culture period (e.g., 21 days), cells are fixed and stained with Alizarin Red S, which binds to calcium salts. The stained nodules can be observed microscopically, and the dye can be extracted and quantified to provide a measure of mineralization [34].
• Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray (EDX): SEM provides high-resolution images of surface morphology, including cell attachment and the formation of interfacial layers or apatite. When coupled with EDX, the technique allows for elemental analysis of the surface, such as determining the calcium-to-phosphorus (Ca/P) ratio in precipitates, which can indicate the formation of bioactive hydroxyapatite [9] [41].

Diagram 1: Experimental workflow for evaluating cytocompatibility and bioactivity of dental materials, showing the path from sample preparation through specific assays to data analysis.

Signaling Pathways in Odontogenic Differentiation

The bioactive properties of calcium silicate-based materials are largely mediated through the release of ions, particularly calcium (Ca²⁺) and silicate (SiO₄⁴⁻), which initiate a cascade of cellular events leading to tissue regeneration. The following diagram summarizes the key signaling pathways involved in this process.

Diagram 2: Signaling pathways in odontogenic differentiation induced by bioactive materials, showing key ions, cellular responses, and genetic markers.

The Scientist's Toolkit: Key Research Reagents

To conduct rigorous research in this field, specific reagents and tools are essential for evaluating both the physical and biological properties of these materials. The following table details some of the key solutions and their functions.

Table 3: Essential Research Reagents for Material Cytocompatibility Testing

Reagent / Solution	Primary Function in Research	Exemplary Use Case
Dulbecco's Modified Eagle Medium (DMEM)	Cell culture medium for growing hDPSCs/hDPCs and preparing material eluates.	Serves as the base for creating the liquid extract of set materials to test soluble component effects [9].
MTT Reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)	Colorimetric assay to quantify cell viability and metabolic activity.	Added to cells after exposure to material eluates; measured absorbance indicates number of viable cells [9].
Annexin V Detection Kit	Flow cytometry-based detection of apoptosis (programmed cell death).	Used with Propidium Iodide to distinguish viable, early apoptotic, and necrotic cell populations after material treatment [12].
Osteogenic/Odontogenic Induction Medium	Culture medium supplemented with factors (e.g., ascorbic acid, β-glycerophosphate) to promote differentiation.	Creates an environment that pushes hDPSCs toward an odontoblast-like lineage for differentiation studies [34] [40].
Alizarin Red S	Histochemical dye that binds to calcium salts, staining mineralized nodules.	Used to quantify and visualize in vitro mineralization, a key endpoint of successful odontogenic differentiation [34].
Phosphate Buffered Saline (PBS) / Simulated Body Fluid (SBF)	Ionic solutions mimicking physiological conditions for bioactivity tests.	Used in solubility studies and to observe the formation of hydroxyapatite on material surfaces, indicating bioactivity [5] [41].

The comparative analysis of ProRoot MTA, Biodentine, and Bio-C Repair reveals a complex landscape where radiopacity and biocompatibility are intrinsically linked to material composition. ProRoot MTA, with its bismuth oxide radiopacifier, consistently provides high radiopacity, a reliable choice when visualization is paramount. Biodentine demonstrates exceptional cytocompatibility, often enhancing cell viability and promoting strong odontogenic differentiation. Bio-C Repair, while potentially differing in its elemental profile with a high zirconium content, establishes itself as a cytocompatible material with bioactive potential comparable to its predecessors. The choice between these materials in clinical practice and research should therefore be guided by a balanced consideration of the specific physical property requirements and the desired biological outcome, acknowledging that the radiopacifier type and concentration are non-negligible factors in the overall bioactivity profile. Future research should continue to elucidate the long-term biological effects of these radiopacifying agents and strive to develop new formulations that optimize both clinical practicality and regenerative potential.

Strategies for Improving Handling Properties and Clinical Application

The evolution of calcium silicate-based cements has revolutionized vital pulp therapy and endodontic repair. Among these, ProRoot MTA, Biodentine, and Bio-C Repair are prominent, yet each presents distinct handling characteristics and clinical performance. ProRoot MTA, a long-standing gold standard, faces challenges like difficult manipulation and prolonged setting time. Newer materials like Biodentine and the ready-to-use Bio-C Repair have been developed to mitigate these issues while maintaining excellent biocompatibility and bioactivity [14] [42]. This guide objectively compares these materials based on physicochemical properties and biological performance data, providing a scientific resource for researchers and clinicians in material selection and application strategy development.

Comparative Analysis of Key Properties

The clinical application of a bioceramic material is directly influenced by its fundamental physicochemical properties. The table below summarizes key characteristics for ProRoot MTA, Biodentine, and Bio-C Repair.

Table 1: Comparative Physicochemical and Handling Properties of Vital Pulp Materials

Property	ProRoot MTA	Biodentine	Bio-C Repair
Main Composition	Tricalcium silicate, Dicalcium silicate, Bismuth oxide [14]	Tricalcium silicate (80.1%), Calcium carbonate, Zirconium oxide [9] [14]	Calcium silicate, Zirconium oxide [9]
Setting Time (Final)	165 ± 5 minutes [42] to 228 ± 3 minutes [5]	10-12 minutes [42] to 45 minutes [42]	Information not explicitly stated in search results
Compressive Strength	Significantly lower than amalgam and IRM at 24 hours [42]	~72 MPa (24 hours); increases to ~300 MPa after one month [14]	Information not explicitly stated in search results
Microhardness (Vickers)	26.93 ± 4.66 HV [5]	62.35 ± 11.55 HV [5]	Information not explicitly stated in search results
Radiopacity (mm Al)	6.40 ± 0.06 [5]	1.50 ± 0.10 [5] to 3.5 [42]	Information not explicitly stated in search results
Solubility	<3% (in accordance with ISO 6876) [5]	<3% but significantly higher than ProRoot MTA [5]	Information not explicitly stated in search results
Key Handling Feature	Difficult manipulation, long setting time [14]	Easy mixing (trituration), fast setting [36]	Ready-to-use format [9]

Experimental Data on Biological Properties

Biocompatibility and the ability to promote tissue repair are paramount. The following table compares the biological effects of these materials based on in vitro studies.

Table 2: Comparative Biological Properties and Cytocompatibility

Biological Property	ProRoot MTA	Biodentine	Bio-C Repair
Cytocompatibility (Cell Viability)	Excellent cytocompatibility, similar to Biodentine and Bio-C Repair [9]	Higher cell viability than control in undiluted eluates at 24h, 48h, 72h [9]	Excellent cytocompatibility, similar to Biodentine and ProRoot MTA [9]
Cell Attachment	Adequate attachment of human dental pulp cells observed via SEM [9]	Adequate attachment of human dental pulp cells observed via SEM [9]	Adequate attachment of human dental pulp cells observed via SEM [9]
Odontogenic Differentiation (DSPP/BSP Expression)	Used as a positive control in studies, promotes differentiation [40]	High expression of DSPP and BSP, especially at 1:5 concentration [43]	Similar DSPP/BSP expression to Biodentine at 1:2 & 1:5 concentrations; effective at promoting differentiation [43]
Bioactivity (Apatite Formation)	Forms hydroxyapatite upon contact with phosphate-containing fluids [5]	Forms hydroxyapatite crystals; releases high levels of Ca2+ and Si4+ ions [36]	Information not explicitly stated in search results

Detailed Experimental Protocols for Assessment

Sample Preparation and Eluate Extraction

This protocol is critical for standardizing in vitro biological testing and is adapted from methodologies used to evaluate all three materials [9].

• Material Placement: Under aseptic conditions, place the mixed material at the bottom of a tissue culture plate. For pre-set materials like Bio-C Repair, use a silicone mold to create samples (e.g., 2mm depth x 5mm diameter).
• Setting Phase: Allow materials to set for 48 hours in an incubator at 37°C and 95% humidity to simulate oral conditions.
• Eluate Preparation: Cover set materials with a culture medium, such as Dulbecco's Modified Eagle Medium (DMEM). Incubate in the dark for 24 hours at 37°C to create a "stock" eluate.
• Dilution Series: Filter the stock eluate through a 0.22 µm sterile filter. Prepare serial dilutions (e.g., 1:1, 1:2, 1:4) using fresh culture medium for subsequent cell culture experiments [9] [43].

Cell Viability and Cytocompatibility Assay (MTT Assay)

The MTT assay is a standard colorimetric method for assessing cell metabolic activity, a marker of cell viability and proliferation [9].

• Cell Seeding: Seed isolated human dental pulp cells (hDPCs) into a multi-well plate at a standardized density and allow them to attach.
• Application of Eluates: After 24 hours, replace the standard culture medium with the prepared material eluates (including various dilutions). Cells cultured in standard medium serve as the negative control.
• Incubation and MTT Addition: Incubate cells for predetermined periods (e.g., 24, 48, 72 hours). At each time point, add MTT reagent to each well and incubate for 4 hours. Living cells reduce the yellow MTT to purple formazan crystals.
• Quantification: Dissolve the formazan crystals in a solvent like dimethyl sulfoxide (DMSO). Measure the absorbance of the solution at 570 nm using a multi-well plate reader. Higher absorbance correlates with greater cell viability and metabolic activity [9].

Analysis of Odontogenic Differentiation

This protocol evaluates the material's ability to induce stem cells to differentiate into odontoblast-like cells, which is crucial for dentin repair [43].

• Cell Culture with Eluates: Culture human dental pulp stem cells (hDPSCs) in an osteogenic/odontogenic medium supplemented with material eluates at different concentrations.
• Gene/Protein Expression Analysis:
  • ELISA: On days 7 and 14, assess the expression of key odontogenic proteins like Dentin Sialophosphoprotein (DSPP) and Bone Sialoprotein (BSP) using Enzyme-Linked Immunosorbent Assays (ELISA).
  • RT-PCR: Analyze the expression of odontogenic genes (e.g., RUNX2, DMP1, DSPP) via Real-Time Quantitative Polymerase Chain Reaction (RT-PCR) to understand early differentiation events.
• Mineralization Assessment (Alizarin Red Staining): On day 21, fix the cells and stain with Alizarin Red S, which binds to calcium deposits. Quantify the stained mineralized nodules by eluting the dye and measuring absorbance or by analyzing images with software like ImageJ [43] [40].

Diagram 1: Experimental workflow for material comparison.

The Scientist's Toolkit: Key Research Reagents

Table 3: Essential Reagents for Vital Pulp Material Research

Reagent/Material	Function in Research	Example Application
Human Dental Pulp Stem Cells (hDPSCs)	Primary cell model for assessing cytocompatibility, proliferation, and odontogenic differentiation potential [43] [40]	Isolated from extracted third molars; used in viability, gene expression, and mineralization studies.
Dulbecco's Modified Eagle Medium (DMEM)	Base culture medium for cell maintenance and as a solvent for preparing material eluates [9] [43]	Used to create extracts of set materials for cell treatment.
MTT Reagent (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)	Colorimetric indicator of cell metabolic activity and viability [9]	Added to cell cultures to quantify the number of viable cells after exposure to material eluates.
Osteogenic/Odontogenic Supplements	Induces and supports stem cell differentiation towards a mineralizing lineage.	Typically includes ascorbic acid, beta-glycerophosphate, and dexamethasone [43].
ELISA Kits (e.g., for DSPP, BSP)	Quantify the expression levels of specific non-collagenous proteins critical to dentin formation [43]	Used to measure odontogenic differentiation in cell cultures after 7 and 14 days.
Alizarin Red S	Histochemical dye that binds to calcium salts, identifying and quantifying mineralized nodule formation in vitro [43]	Used to stain cell cultures after ~21 days to confirm mineralization potential.

The choice between ProRoot MTA, Biodentine, and Bio-C Repair involves a careful consideration of their respective handling properties and biological performance. ProRoot MTA remains a biologically excellent material but is hampered by its difficult handling. Biodentine offers a significant improvement in handling and setting time while maintaining high compressive strength and the ability to stimulate odontogenic differentiation. The newer, ready-to-use Bio-C Repair presents a compelling option with its simplified application and demonstrated cytocompatibility and bioactivity comparable to its predecessors. Future research should focus on long-term clinical studies and further exploration of the signaling pathways activated by these materials to fully elucidate their mechanisms of action in pulp repair and regeneration.

Diagram 2: Signaling and tissue regeneration pathways.

Head-to-Head Biological Performance: A Direct Comparison of Cytocompatibility and Bioactivity

Within the field of vital pulp therapy, the biological response of dental pulp cells to restorative biomaterials is a critical determinant of clinical success. The cytocompatibility of these materials, defined by their ability to support cell viability, proliferation, and function without inducing adverse effects, is paramount for promoting pulp tissue healing and reparative dentin formation. This guide provides a systematic comparison of the cell viability profiles of three calcium silicate-based hydraulic cements—Biodentine, ProRoot MTA, and Bio-C Repair—as assessed by the MTT assay at 24, 48, and 72-hour intervals. The MTT assay, a colorimetric method for measuring the activity of mitochondrial enzymes in living cells, serves as a fundamental in vitro tool for preliminary biocompatibility screening, reflecting cellular metabolic activity and overall health [3] [10]. Framed within broader research on the surface cytocompatibility of these materials, this analysis aims to furnish researchers and dental material scientists with objective, data-driven insights to inform material selection and future investigative work.

Experimental Protocols and Methodologies

The comparative data presented herein are primarily derived from a standardized in vitro study model that isolated and cultured human dental pulp cells (hDPCs) from healthy third molars [3] [10]. The key methodological steps are summarized below.

Material Preparation and Eluate Collection

• Material Mixing: Biodentine (Septodont), ProRoot MTA (Dentsply Tulsa Dental Specialties), and Bio-C Repair (Angelus) were prepared under aseptic conditions according to the manufacturers' instructions [10].
• Eluate Preparation: The set materials were immersed in Dulbecco's Modified Eagle Medium (DMEM) and incubated for 24 hours at 37°C to obtain undiluted (1:1) extracts, following the principles of ISO 10993-5 for biological evaluation of medical devices [10] [44]. Subsequent dilutions (e.g., 1:2, 1:4) were prepared from this stock eluate using fresh culture medium.

Cell Culture and Treatment

• Cell Source: hDPCs were isolated via enzymatic digestion using 3 mg/mL collagenase type I [10].
• Culture Conditions: Cells between passages 2-4 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin in a humidified incubator at 37°C with 5% COâ‚‚ [10].
• Viability Assay: The MTT assay was performed by seeding cells in 96-well plates and exposing them to material eluates. At each time point (24, 48, 72 h), the MTT reagent was added to the wells. Metabolically active cells reduce the yellow tetrazolium salt MTT to purple formazan crystals. The crystals are then solubilized, and the absorbance of the resulting solution is measured at a specific wavelength (typically 570 nm), which is directly proportional to the number of viable cells [10] [44].

The following diagram illustrates this experimental workflow.

Comparative Cell Viability Data

The cell viability profiles, expressed as a percentage relative to the control group (cells cultured without material eluates), are summarized in the table below. Data reflects findings from the undiluted (1:1) eluates, which represent the most concentrated bioactive challenge to the cells.

Table 1: Comparative Cell Viability (%) of hDPCs Exposed to Undiluted Material Eluates

Material	24 Hours	48 Hours	72 Hours	Key Observations
Biodentine	>100% [3] [10]	>100% [3] [10]	>100% [3] [10]	Consistently promoted cell viability exceeding the control, indicating a proliferative effect.
ProRoot MTA	High cytocompatibility [3] [10]	High cytocompatibility [3] [10]	High cytocompatibility [3] [10]	Supported excellent cell viability, performing at a level similar to the control.
Bio-C Repair	High cytocompatibility [3] [10]	High cytocompatibility [3] [10]	High cytocompatibility [3] [10]	Demonstrated excellent cytocompatibility that was statistically similar to Biodentine and ProRoot MTA.

A separate study investigating the cytotoxicity of these materials on stem cells from human exfoliated deciduous teeth (SHED) highlighted that the effect can be concentration-dependent. For instance, while MTA Repair HP showed high viability even at a 1:1 dilution, Bio-C Repair and Biodentine exhibited higher cytotoxicity at 1:1 dilution, which significantly decreased at a 1:2 dilution, resulting in high cell viability [44].

The Scientist's Toolkit: Essential Research Reagents

Successful replication of these cytocompatibility studies requires the use of specific, high-quality reagents and materials. The following table details key components used in the referenced protocols.

Table 2: Essential Research Reagents for Dental Material Cytocompatibility Assays

Reagent / Material	Function in the Experiment	Specification / Note
Human Dental Pulp Cells (hDPCs)	Primary cell model for assessing biocompatibility.	Isolated from healthy third molars via enzymatic digestion [10].
Dulbecco's Modified Eagle Medium (DMEM)	Base culture medium providing essential nutrients for cell growth.	Supplemented with 10% FBS, antibiotics, and L-glutamine [10].
Collagenase Type I	Enzyme for digesting pulp tissue to isolate individual hDPCs.	Typically used at 3 mg/mL concentration [10].
MTT Reagent	Colorimetric indicator of cell viability and metabolic activity.	(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) [10] [44].
Fetal Bovine Serum (FBS)	Provides growth factors, hormones, and other vital components for cell proliferation.	Standardly used at 10% concentration in culture medium [10].
Test Materials	Subjects of the biocompatibility investigation.	Biodentine, ProRoot MTA, and Bio-C Repair must be mixed aseptically per manufacturer guidelines [10].

Interpretation of Findings and Underlying Mechanisms

The quantitative data from MTT assays reveal distinct biological profiles for each material. Biodentine's performance, characterized by viability rates exceeding 100% of the control, suggests it does not merely preserve cell health but actively stimulates cellular proliferation [3] [10]. This is likely driven by its significant release of calcium ions, which are known to play a crucial role in cell signaling and metabolism [45].

Both ProRoot MTA and Bio-C Repair demonstrate excellent cytocompatibility, supporting cell viability at levels comparable to the negative control group throughout the 72-hour period [3] [46] [10]. This indicates a high degree of biocompatibility without significant cytotoxic effects. Energy dispersive X-ray (EDX) analysis provides a clue to the difference in their bioactive potential; ProRoot MTA and Biodentine are rich in calcium, whereas Bio-C Repair has a lower calcium content and incorporates zirconium as a radiopacifier [3] [10]. The released ions from the materials influence the microenvironment, including pH modulation, which is a critical factor affecting cell behavior and viability [45].

The relationship between material composition, ion release, and cellular response is a key signaling pathway in cytocompatibility, which can be visualized as follows.

This comparative analysis elucidates that while Biodentine, ProRoot MTA, and Bio-C Repair all exhibit a high level of cytocompatibility suitable for clinical applications in vital pulp therapy, their cellular interactions are distinct. Biodentine demonstrates a proactive bioactivity, enhancing metabolic activity and promoting the proliferation of human dental pulp cells. ProRoot MTA serves as a reliable benchmark, consistently supporting cell viability. Bio-C Repair, as a newer ready-to-use cement, shows excellent and comparable cytocompatibility despite its differing chemical composition. These findings, derived from standardized MTT assays, provide a robust foundation for researchers to further investigate long-term bioactivity, mineralization potential, and the specific molecular mechanisms underpinning these observed cell viability profiles.

ASSESSMENT OF CELL ATTACHMENT AND CYTOSKELETAL ORGANIZATION ON MATERIAL SURFACES

Assessment of Cell Attachment and Cytoskeletal Organization on Material Surfaces

A Comparative Guide to Biodentine, ProRoot MTA, and Bio-C Repair

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The success of vital pulp therapy (VPT) is fundamentally dependent on the response of dental pulp cells to the bioactive material placed in direct contact with the underlying tissue. A critical aspect of this response is the initial cell attachment and the subsequent organization of the cytoskeleton, which are primary indicators of a material's cytocompatibility. These early cellular events dictate downstream processes, including proliferation, differentiation, and the formation of reparative dentin. Consequently, the surface properties of hydraulic calcium silicate cements—such as their chemical composition, morphology, and ion release profile—are pivotal in determining their clinical performance [9] [10].

This guide provides an objective, data-driven comparison of three prominent VPT materials: Biodentine (Septodont), ProRoot MTA (Dentsply), and Bio-C Repair (Angelus). The focus is squarely on their performance in in vitro models assessing cell attachment and cytoskeletal organization of human dental pulp cells (hDPCs), providing researchers with a clear analysis of the available experimental evidence.

Material Properties and Biological Context

The biological performance of a material is intrinsically linked to its physicochemical characteristics. The table below summarizes the key properties of the three materials, which provide context for their biological interactions.

Table 1: Comparative Physicochemical Properties of Biodentine, ProRoot MTA, and Bio-C Repair

Property	Biodentine	ProRoot MTA	Bio-C Repair
Primary Composition	Tricalcium silicate, Calcium carbonate, Zirconium oxide [14] [36]	Tricalcium silicate, Dicalcium silicate, Bismuth oxide [14]	Calcium silicate-based; lower calcium, highest zirconium [9]
Setting Time (Initial)	9-12 minutes [36]	~70 minutes [14]	Information not specified in search results
Main Radiopacifier	Zirconium oxide [36]	Bismuth oxide [14]	Zirconium oxide [9]
Key Biological Ions Released	High Ca²⁺ and Si⁴⁺ ions [36]	Ca²⁺ and OH⁻ ions [5]	Information not specified in search results

These material properties create the biological microenvironment that cells encounter. The release of calcium ions (Ca²⁺) is particularly crucial, as it is known to stimulate dental pulp cell proliferation and differentiation, promoting dentin bridge formation [36]. Furthermore, the surface topography observed via Scanning Electron Microscopy (SEM) provides the physical scaffold for cell adhesion.

Experimental Data on Cytocompatibility

Direct experimental evidence is essential for comparing the cytocompatibility of these materials. The following table summarizes quantitative and qualitative findings from a key study that evaluated the effects of these materials on human dental pulp cells (hDPCs) [9] [10].

Table 2: Experimental Cytocompatibility Data on hDPCs

Assessment Method	Biodentine	ProRoot MTA	Bio-C Repair
Cell Viability (MTT Assay)	Higher viability than control with undiluted eluates at 24h, 48h, and 72h (p < 0.001) [9]	Favorable cell viability, similar to control levels [9]	Excellent cell viability, comparable to Biodentine and ProRoot MTA [9]
Cell Attachment (SEM Analysis)	Adequate cell attachment with well-spread morphology observed [9]	Adequate cell attachment with well-spread morphology observed [9]	Adequate cell attachment with well-spread morphology observed [9]
Cytoskeleton Organization (Immunofluorescence)	No cytoskeletal alterations were observed in the presence of material eluates [9]	No cytoskeletal alterations were observed in the presence of material eluates [9]	No cytoskeletal alterations were observed in the presence of material eluates [9]
Chemical Composition (EDX)	High concentrations of Calcium (Ca), Carbon (C), and Oxygen (O) [9]	High concentrations of Calcium (Ca), Carbon (C), and Oxygen (O) [9]	Lower concentration of Calcium (Ca); highest concentration of Zirconium (Zr) [9]

The data demonstrates that all three materials support cell health, with Biodentine showing a stimulatory effect on viability. Critically, none of the materials induced adverse changes to the cytoskeleton, a key indicator of healthy cell attachment.

Detailed Experimental Protocols

To facilitate the replication and critical evaluation of the data presented, this section outlines the core methodologies used in the cited studies.

Cell Culture and Material Eluate Preparation

• Cell Source: Human dental pulp cells (hDPCs) were isolated from healthy third molars extracted for surgical reasons. The pulp tissue was digested in a solution of 3 mg/mL collagenase type I for 90 minutes to obtain single-cell suspensions [9] [10].
• Culture Conditions: Isolated hDPCs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and penicillin/streptomycin. Cells were maintained at 37°C in a 5% COâ‚‚ atmosphere, and passages 2-4 were used for experiments to ensure consistency [9] [10].
• Eluate Preparation: Biodentine, ProRoot MTA, and Bio-C Repair were mixed according to manufacturers' instructions and allowed to set for 48 hours under sterile conditions at 37°C and 95% humidity. The set materials were then covered with cell culture medium (DMEM) and incubated for 24 hours to create "undiluted" extracts. These eluates were subsequently filtered and diluted (e.g., 1:1, 1:2, 1:4) for treatment [9].

Assessing Cell Viability (MTT Assay)

The MTT assay measures cellular metabolic activity as a marker of cell viability and proliferation [9].

• Cell Seeding: hDPCs are seeded into well plates.
• Treatment: Cells are exposed to the eluates from the test materials for specified periods (e.g., 24, 48, 72 hours).
• MTT Incubation: MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is added to each well and incubated for 4 hours. Viable cells with active metabolism reduce the yellow MTT to purple formazan crystals.
• Solubilization & Quantification: The formazan crystals are dissolved using a solvent like dimethyl sulfoxide (DMSO).
• Absorbance Measurement: The absorbance of the solution is quantified at a wavelength of 570 nm using a multi-well plate reader. Higher absorbance correlates directly with a greater number of viable cells [9].

Analyzing Cell Attachment and Morphology (SEM)

Scanning Electron Microscopy provides high-resolution images to visualize cell attachment and morphology on material surfaces [9].

• Cell Seeding: hDPCs are directly seeded onto the surface of pre-set material disks and cultured for 72 hours.
• Fixation: Specimens are fixed with 4% glutaraldehyde to preserve cell structure.
• Dehydration: Samples are dehydrated through a graded series of ethanol solutions (e.g., 30% to 90% v/v) to remove all water.
• Coating: The dehydrated samples are mounted and coated with a thin layer of a conductive material, such as gold/palladium.
• Imaging: Coated samples are observed under an SEM at various magnifications (e.g., 100×, 300×, 1500×) to assess cell attachment, spreading, and interaction with the material surface [9].

Visualizing Cytoskeletal Organization (Immunofluorescence)

Immunofluorescence allows for the specific staining and visualization of cytoskeletal components, such as actin filaments.

• Cell Treatment & Fixation: hDPCs grown on coverslips and treated with material eluates are fixed with 4% paraformaldehyde.
• Permeabilization: Cells are permeabilized with a detergent (e.g., 0.1% Triton X-100) to allow antibodies to enter.
• Staining: Samples are incubated with a fluorescently-labeled phalloidin probe, which selectively binds to filamentous actin (F-actin).
• Mounting & Imaging: Stained samples are mounted on slides and visualized using a confocal laser scanning microscope. The resulting images reveal the organization and integrity of the actin cytoskeleton, where a well-organized network indicates healthy, adherent cells [9].

The workflow below summarizes the key experimental steps for assessing cell attachment and cytoskeletal organization.

Experimental Workflow for Cytocompatibility Assessment

The Scientist's Toolkit: Essential Research Reagents

The following table lists key reagents and materials required to conduct the experiments described in this guide.

Table 3: Essential Research Reagents and Materials

Reagent / Material	Function / Application	Example / Note
Human Dental Pulp Cells (hDPCs)	Primary cell model for in vitro cytocompatibility testing. Isolated from healthy third molars [9].	Cells at passages 2-4 are recommended for experiments [9].
Dulbecco's Modified Eagle Medium (DMEM)	Standard culture medium for maintaining hDPCs in vitro [9].	Supplemented with 10% FBS and antibiotics [9].
Collagenase Type I	Enzyme for digesting pulp tissue to isolate individual hDPCs [9].	Used at 3 mg/mL concentration for isolation [9].
MTT Reagent	A colorimetric assay for quantifying cell metabolic activity and viability [9].	Yellow MTT is reduced to purple formazan by viable cells [9].
Phalloidin Probe (Fluorophore-conjugated)	High-affinity stain for visualizing filamentous actin (F-actin) in the cytoskeleton [9].	Used in immunofluorescence assays.
Glutaraldehyde	A cross-linking fixative agent used for SEM sample preparation to preserve cell ultrastructure [9].	Typically used at 4% concentration [9].
Scanning Electron Microscope (SEM)	High-resolution imaging system for visualizing surface morphology of materials and attached cells [9].	Often coupled with EDX for elemental analysis [9].

Based on the current body of in vitro evidence, Biodentine, ProRoot MTA, and Bio-C Repair all demonstrate excellent cytocompatibility, supporting adequate cell attachment and healthy cytoskeletal organization in human dental pulp cells without inducing adverse morphological changes [9] [10].

The primary distinction lies in their material composition and its functional impact. Biodentine is characterized by a high calcium ion release, which correlates with its observed stimulatory effect on cell viability [9] [36]. ProRoot MTA, the historical gold standard, provides reliable biocompatibility but with a notably longer setting time [14]. Bio-C Repair presents a distinct elemental profile with lower calcium and higher zirconium content, yet this does not appear to compromise its cytocompatibility, which is similar to the other materials [9].

For researchers, the choice of material for experimental or clinical use may therefore depend on the specific property of interest: Biodentine for enhanced bioactivity and faster setting, ProRoot MTA for its well-documented clinical history, or Bio-C Repair as a ready-to-use alternative with comparable biocompatibility. This analysis underscores that all three materials are viable options for applications requiring superior surface cytocompatibility.

Within the field of vital pulp therapy (VPT), the biological response of dental pulp cells to a biomaterial is paramount for successful treatment outcomes. The ideal material should not only be cytocompatible but also actively promote the natural healing processes of the pulp, specifically the differentiation of resident stem cells into odontoblast-like cells and the subsequent formation of reparative dentin. This process, known as odontogenic differentiation and mineralization, is the cornerstone of pulp regeneration. Calcium silicate-based cements have become the gold standard for these procedures due to their well-documented bioactivity. Among them, Biodentine, ProRoot MTA, and the newer Bio-C Repair are widely used, yet a detailed comparison of their capacity to induce odontogenic events is crucial for clinical and research decision-making. Framed within broader research on their surface cytocompatibility, this guide objectively compares the bioactive potential of these three materials, supported by experimental data on cell viability, differentiation markers, and mineralization outcomes.

Comparative Bioactive Properties of Pulp Capping Materials

The following tables summarize key experimental findings from in vitro studies, providing a direct comparison of the biological effects of Biodentine, ProRoot MTA, and Bio-C Repair on human dental pulp cells.

Table 1: Cytocompatibility and Cell Viability Profile

Material	Test Method	Cell Type	Key Findings on Viability/Attachment	Citation
Biodentine	MTT Assay	hDPCs	Significantly higher cell viability than the control group at 24h, 48h, and 72h.	[9] [3]
ProRoot MTA	MTT Assay	hDPCs	Showed excellent cytocompatibility, with no significant cytotoxicity observed.	[9] [47] [3]
Bio-C Repair	MTT Assay, Immunofluorescence	hDPCs	Exhibited excellent cytocompatibility, similar to Biodentine and ProRoot MTA; no cytoskeletal alterations.	[9] [3]
All Three Materials	SEM for Cell Attachment	hDPCs	All materials demonstrated adequate attachment of human dental pulp cells.	[9] [10]

Table 2: Odontogenic Differentiation and Mineralization Capacity

Material	Assay Type	Key Findings on Differentiation & Mineralization	Citation
Biodentine	Alizarin Red Staining, ALP Activity, Gene Expression	Releases high levels of Ca²⁺ and Si⁴⁺, stimulating dental pulp cell differentiation and dentin bridge formation; induces mineralization.	[48] [36]
ProRoot MTA	Alizarin Red Staining, RT-PCR (RUNX2, DMP1, DSPP)	Demonstrates significant osteoinductivity and stimulates hard tissue formation; promotes odontogenic marker expression.	[47] [8]
Bio-C Repair	Alizarin Red Staining, Gene Expression	Shows excellent cytocompatibility and promotes mineralization, though with a lower calcium content than MTA and Biodentine.	[9] [3]
Bio MTA+ (Reference)	CCK-8, Annexin V, RT-PCR, Alizarin Red	In a 2025 study, demonstrated superior proliferation, less apoptosis, and higher expression of RUNX2, DMP1, and DSPP vs. ProRoot MTA.	[8]

Table 3: Physicochemical Properties Relevant to Bioactivity

Property	Biodentine	ProRoot MTA	Bio-C Repair	Clinical Significance
Primary Composition	Tricalcium silicate [36]	Calcium Silicate [5]	Calcium Silicate (lower Ca, high Zr) [9]	Basis for ion release and bioactivity.
Main Radiopacifier	Zirconium oxide [36]	Bismuth oxide [5]	Zirconium oxide [9]	Biodentine's radiopacity may be insufficient per ISO [5].
Setting Time	~12 minutes [36]	~228 minutes [5]	Information Not Specified	Shorter setting minimizes contamination risk.
Calcium Ion Release	High [36]	High [5]	Lower [9]	High Ca²⁺ release is crucial for bioactivity and apatite formation.

Detailed Experimental Protocols for Key Assays

To ensure the reproducibility of the comparative data, this section outlines the standard experimental methodologies employed in the cited studies.

Material Eluate Preparation

This protocol is used to create a liquid extract of the test materials for cell culture experiments, assessing the effects of leachable components [9] [10].

• Preparation: Materials (Biodentine, ProRoot MTA, Bio-C Repair) are mixed according to manufacturers' instructions under aseptic conditions.
• Setting: The mixed materials are plated and allowed to set for 48 hours in an incubator at 37°C and 95% humidity.
• Extraction: After setting, materials are covered with a cell culture medium, such as Dulbecco's Modified Eagle Medium (DMEM).
• .Incubation: The setup is incubated in the dark for 24 hours at 37°C.
• .Filtration: The resulting liquid (eluate) is filtered to remove any particulate matter. Serial dilutions (e.g., 1:1, 1:2, 1:4) are typically prepared for dose-response assays.

Cell Viability and Cytocompatibility Assay (MTT)

The MTT assay measures cellular metabolic activity as an indicator of cell viability and proliferation [9] [10].

• Cell Seeding: Human Dental Pulp Cells (hDPCs) are seeded onto sterile tissue culture plates and incubated until a monolayer forms.
• Treatment: The culture medium is replaced with the material eluates at various concentrations. Cells in a standard culture medium serve as the negative control.
• MTT Incubation: After the treatment period (e.g., 24, 48, 72 hours), MTT reagent is added to each well and incubated for several hours. Living cells reduce the yellow MTT to purple formazan crystals.
• Solubilization: The medium is removed, and a solvent like Dimethyl Sulfoxide (DMSO) is added to dissolve the formazan crystals.
• Quantification: The absorbance of the solution is measured at a specific wavelength (typically 570 nm) using a multi-well plate reader. Higher absorbance correlates with greater cell viability.

Odontogenic Differentiation and Mineralization

These assays evaluate the material's potential to induce stem cell differentiation into odontoblast-like cells and the subsequent formation of a mineralized matrix.

• Alkaline Phosphatase (ALP) Activity: ALP is an early marker of odontogenic/osteogenic differentiation. Cells are cultured in an odontogenic induction medium with material eluates. ALP activity is measured using a biochemical assay that detects the conversion of a substrate into a colored product, which is quantified spectrophotometrically [49].
• Alizarin Red Staining (ARS): This assay detects late-stage mineralization by staining calcium deposits. After a culture period of 14-21 days in induction medium, cells are fixed and stained with Alizarin Red S solution. The stained nodules can be visualized under a microscope. For quantification, the bound dye is solubilized with a solution like cetylpyridinium chloride, and the absorbance is measured [47] [49] [8].
• Gene Expression Analysis (qRT-PCR): This technique quantifies the expression levels of genes specific to odontogenic differentiation. Key markers include:
  • DSPP (Dentin Sialophosphoprotein): A definitive marker for odontoblast phenotype.
  • DMP-1 (Dentin Matrix Acidic Phosphoprotein 1): Essential for dentin mineralization.
  • Runx-2 (Runt-related transcription factor 2): A key transcription factor in osteo/odontogenic differentiation. RNA is extracted from treated cells, converted to cDNA, and amplified using gene-specific primers in a quantitative PCR machine. The results indicate the fold-change in gene expression compared to control groups [49] [8].

Signaling Pathways in Odontogenic Differentiation

The bioactivity of calcium silicate materials is intrinsically linked to their ability to release ions, such as calcium (Ca²⁺), which influence key cellular signaling pathways that drive odontogenic differentiation. The Wnt/β-catenin pathway is a principal regulator of this process. The following diagram illustrates the core mechanism of how these materials and novel bioactive compounds stimulate dentin regeneration.

Figure 1: Mechanism of Odontogenic Differentiation Induction. This diagram illustrates the core signaling pathway through which calcium silicate-based materials (like Biodentine and ProRoot MTA) and novel small molecule or peptide inhibitors (like GSK-3βi) promote dentin regeneration. The key step is the activation of the Wnt/β-catenin pathway, leading to the expression of genes responsible for odontogenic differentiation and mineralization [48] [36] [49].

The Scientist's Toolkit: Essential Research Reagents

The following table catalogues key reagents and materials essential for conducting in vitro research on the odontogenic potential of dental biomaterials.

Table 4: Key Reagents for Odontogenic Differentiation Research

Reagent/Material	Function in Research	Example Application
Human Dental Pulp Stem Cells (hDPSCs)	Primary cell model for evaluating biomaterial-cell interactions; possess multi-lineage differentiation potential.	Isolation from extracted third molars for use in viability, differentiation, and mineralization assays. [9] [49] [8]
Odontogenic/Osteogenic Induction Medium	Culture medium supplemented with agents to induce differentiation (e.g., ascorbic acid, β-glycerophosphate, dexamethasone).	Used as a positive control or base medium to test the material's ability to enhance differentiation. [47] [49]
MTT Reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)	Tetrazolium salt reduced by metabolically active cells to a purple formazan product; measures cell viability/proliferation.	Cytocompatibility assessment of material eluates over time (24, 48, 72 hours). [9] [10]
Alizarin Red S	A dye that binds to calcium salts, staining calcium deposits red; used to detect and quantify in vitro mineralization.	Quantification of calcium nodule formation after 2-3 weeks of culture in induction medium. [47] [49] [8]
qRT-PCR Reagents & Primers	Tools for quantitative real-time polymerase chain reaction; used to measure mRNA expression levels of target genes.	Analysis of odontogenic gene expression markers (e.g., DSPP, DMP-1, Runx-2). [49] [8]
GSK-3β Inhibitors (e.g., L803-mts, Phenamil)	Small molecules or peptides that inhibit GSK-3β, leading to β-catenin stabilization and activation of Wnt signaling.	Used as experimental bioactive compounds to directly stimulate odontogenic differentiation and compare with material-induced effects. [48] [49]

Vital pulp therapy (VPT) aims to preserve and regenerate the dentin-pulp complex following injury, a process highly dependent on the bioactivity of the pulp capping material used [9] [10]. A key mechanism of this regeneration is the odontogenic differentiation of human dental pulp stem cells (hDPSCs) into odontoblast-like cells, which are responsible for secreting reparative dentin [50]. This differentiation is characterized by the expression of specific non-collagenous proteins, primarily dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) [50]. DSPP is a critical regulator of dentin formation, while BSP plays a significant role in the mineralization of hard tissues like dentin and bone [50]. Consequently, the analysis of DSPP and BSP expression in hDPSCs serves as a fundamental in vitro method for evaluating and comparing the bioactivity and regenerative potential of hydraulic calcium silicate-based cements. This guide provides a structured comparison of three such materials—Biodentine, ProRoot MTA, and Bio-C Repair—focusing on their effects on dentin marker expression and cytocompatibility.

Quantitative Comparison of Dentin Marker Expression & Mineralization

The odontogenic potential of pulp capping materials can be quantitatively assessed by measuring protein expression and subsequent mineral deposition. The following tables consolidate key experimental findings from comparative studies.

Table 1: DSPP and BSP Expression in hDPSCs Treated with Material Extracts

Material	Concentration	DSPP Expression (ng/mL)	BSP Expression (ng/mL)	Observation Period
Biodentine (BD)	1:5	6.71	13.47	14 days [50]
Bio-C Repair (BC)	1:5	6.60	13.20	14 days [50]
Biodentine (BD)	1:1	Not Highest	Not Highest	7 & 14 days [50]
Bio-C Repair (BC)	1:1	Not Highest	Not Highest	7 & 14 days [50]

Table 2: Summary of Cytocompatibility and Mineralization Findings

Material	Cell Viability & Proliferation	Cell Attachment	Mineral Deposition (Alizarin Red)
Biodentine	High viability; superior to control in undiluted form [9] [3]	Adequate cell attachment observed [9] [10]	Significant mineral deposition, highest at 1:5 concentration [50]
ProRoot MTA	Excellent cytocompatibility [9] [3]	Adequate cell attachment observed [9]	Not specifically reported in search results
Bio-C Repair	Excellent cytocompatibility, similar to Biodentine and ProRoot MTA [9] [3]	Adequate cell attachment observed [9] [10]	Significant mineral deposition, highest at 1:5 concentration [50]

Experimental Protocols for Odontogenic Differentiation Analysis

To ensure reproducible and standardized research on hDPSC differentiation, the following core experimental protocols are detailed.

hDPSC Culture and Characterization

• Cell Source: hDPSCs are typically isolated from healthy human third molars extracted for orthodontic or surgical reasons [9] [51]. The pulp tissue is digested using collagenase type I (e.g., 3 mg/mL) to obtain a single-cell suspension [9] [51].
• Culture Conditions: Cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) or α-MEM, supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin [50] [52]. Incubation is performed at 37°C in a humidified atmosphere of 5% COâ‚‚ [52].
• Characterization: Prior to experiments, the mesenchymal stem cell identity of hDPSCs is confirmed via flow cytometry. Cells must show high positive expression for markers like CD90 (98%), CD105 (99.7%), and CD73 (94%), and negative expression for hematopoietic markers such as CD34 and CD45 [50] [51] [53]. Cells from passages 2-4 are recommended for experimental use [50] [52].

Material Extract Preparation

• Sample Fabrication: Materials are mixed according to manufacturers' instructions and placed in molds (e.g., 5 mm diameter, 2 mm depth) [50] [9]. Samples are allowed to set for 48 hours at 37°C and 95% humidity to simulate oral conditions [9].
• Eluate Generation: Set material disks are sterilized, crushed, and pulverized. A 1:1 concentration extract is prepared by immersing 3.5 mg of material in 3.5 mL of DMEM for 24 hours at 37°C [50]. The solution is then filtered through a 0.22 µm sterile filter.
• Dilution Series: Further dilutions (e.g., 1:2 and 1:5) are prepared from the 1:1 extract using DMEM and the standard dilution formula M₁V₁ = Mâ‚‚Vâ‚‚ [50].

Analysis of Dentin Marker Expression and Mineralization

• Enzyme-Linked Immunosorbent Assay (ELISA):
  • Procedure: hDPSCs are seeded in 96-well plates and cultured with material extracts. On days 7 and 14, the culture supernatant is collected, and DSPP and BSP expression levels are quantified using a microplate reader at 405 nm, following the manufacturer's protocol for specific ELISA kits [50].
• Alizarin Red Staining (ARS):
  • Procedure: After 21 days of culture in osteogenic/odontogenic media, cells are fixed and stained with Alizarin Red S to detect calcium-rich mineralized nodules [50]. The stained nodules are visualized under a microscope, and the red areas are quantified using image analysis software like ImageJ to determine the percentage of mineral deposition [50].

Signaling Pathways in Odontogenic Differentiation

The differentiation of hDPSCs into odontoblasts is a tightly regulated process. Understanding the involved signaling pathways is crucial for interpreting experimental data. The Rho/ROCK pathway, for instance, has been identified as a key regulator.

The Scientist's Toolkit: Essential Research Reagents

The following table outlines key reagents and their critical functions in conducting hDPSC differentiation studies.

Table 3: Essential Research Reagents for hDPSC Differentiation Studies

Reagent / Material	Function in Experimental Protocol
Collagenase Type I	Enzymatic digestion of pulp tissue to isolate hDPSCs [9] [51].
CD90, CD105, CD73 Antibodies	Positive identification of mesenchymal stem cell markers via flow cytometry [50] [51].
CD34, CD45 Antibodies	Negative identification to rule out hematopoietic stem cell origin [51] [53].
Dulbecco's Modified Eagle Medium (DMEM)	Base culture medium for maintaining hDPSCs [50] [9].
Fetal Bovine Serum (FBS)	Essential supplement for cell culture media, providing growth factors and nutrients [50] [52].
Osteogenic Supplements (β-glycerophosphate, Ascorbic Acid, Dexamethasone)	Key components added to base media to induce odontogenic/osteogenic differentiation [50].
DSPP & BSP ELISA Kits	Quantitative measurement of dentin marker expression levels in cell culture supernatants [50].
Alizarin Red S	Histochemical staining for detecting and quantifying calcium-rich mineralized nodules formed by differentiated cells [50].
0.22 µm Sterile Filter	Sterilization of material eluates before application to cell cultures [50] [9].

Based on the synthesized experimental data, Biodentine and Bio-C Repair demonstrate comparable and significant efficacy in promoting the expression of key dentin markers DSPP and BSP, as well as facilitating mineral deposition in hDPSCs [50]. The 1:5 concentration of material extracts appears to be particularly effective for maximizing this odontogenic response [50]. Furthermore, all three materials—Biodentine, ProRoot MTA, and Bio-C Repair—show excellent cytocompatibility, supporting cell attachment and viability [9] [3]. These in vitro findings provide a robust scientific foundation for their use in vital pulp therapy, with Biodentine and Bio-C Repair showing specific promise in driving the regenerative processes critical for dentin-pulp complex repair.

Conclusion

The collective evidence affirms that Biodentine, ProRoot MTA, and Bio-C Repair are highly cytocompatible materials suitable for vital pulp therapy, effectively supporting the attachment, proliferation, and viability of human dental pulp cells. Despite variations in their chemical composition—notably the lower calcium and higher zirconium content in Bio-C Repair—all three materials perform favorably in biological assays. Key differentiators lie in their physicochemical properties; Biodentine offers a significantly faster setting time and higher initial microhardness, while ProRoot MTA demonstrates lower solubility. Future research should focus on long-term in vivo studies, the development of standardized 3D culture models that better mimic the pulp microenvironment, and the engineering of next-generation materials that amalgamate the optimal handling, mechanical, and bioactive properties of these benchmarks to further advance regenerative endodontics.

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