EVALUATION OF THE ADHESION AND MOR-PHOLOGY OF STEM CELLS FROM APICAL PA-PILLA IN DIRECT CONTACT WITH MTA AND BIODENTINE

: Purpose: To investigate the use of tricalcium silicate cement Biodentine as an alternative to MTA in regenerative endodontic procedures by assessing the morphology and adhesion of stem cells from apical papilla in direct contact with these materials Materials/Methods: Glass ionomer cement, MTA and Biodentine disks with identical size were created. Stem cells from apical papilla were incubated on these disks, and their size and morphology were assessed with a scanning electron microscope. Results: No adhesion of SCAP was observed when incubating cells with glass ionomer disks, which can be explained by a decrease in pH and a delay in cell proliferation. Cell adhesion was observed in the groups incubated in the presence of MTA and Biodentine discs. Cells adhered to the calcium silicate cements have different morphology and can be round or flat, with several processes. Conclusions: Stem cells from apical papilla adhere to MTA and Biodentinå when in direct contact with discs made of these materials. The cells show different morphology and can be round or flat, with several processes. Calcium silicate cements have biocompatibility and bioactivity and they are suitable materials for the biological treatment of pulp and regenerative endodontics


INTRODUCTION
Cell adhesion plays a crucial role in cellular communication and it is fundamental to tissue development and regeneration. Cell adhesion is the ability of a single cell to attach to another cell or extracellular matrix. Most cells are firmly attached to a substrate in vitro [1]. Cell adhesion is associated with stimulatory signals that regulate cell differentiation, cell cycle, migration and renewal [2]. The affinity of cells to the substrate is extremely important in the creation and implementation of biomaterials. Adhesion is also essential for intercellular signaling and regulation [2].
There are different requirements for cell adhesion depending on the field of application and cell-specific characteristics [3]. Various techniques are used to analyze it in the study of biomaterials, the impact of environmental signals on cell cultures and to determine the adhesion properties of healthy or neoplastic cells [4,5].
Many studies are trying to investigate the complex effects of bioactive materials on living tissues, especially on dental pulp. Calcium hydroxide has been used to treat pulp diseases for decades [6]. Due to its high pH, it causes superficial necrosis and a moderate inflammatory response in the pulp.
The healing process is initiated by resolving the inflammation, stem cell differentiation and formation of tertiary dentin [7]. In recent years, calcium silicate cements (CSCs) have increasingly replaced calcium hydroxide due to fewer adverse effects and a higher clinical success rate [8]. MTA is the gold standard in pulp therapy nowadays. It is also used in the treatment of perforations and for the creation of apical plugs in permanent teeth with incomplete root development [8].
MTA is a calcium silicate cement and it consists mainly of tricalcium silicate, dicalcium silicate and tricalcium aluminate, which form a matrix of calcium silicate hydrate during setting [9]. Its bioactivity is due to apatite precipitates, which are formed upon contact with phosphate-containing liquids and are deposited on the surface of the material [8,9].
MTA is an expensive material with poor handling properties, a long setting time of several hours and a high chance of tooth discoloration, which makes its application challenging despite its proven clinical success [10]. New calcium silicate cements with optimized material properties, such as Biodentine, have been developed, in order to address these disadvantages. In addition to the typical components of tricalcium silicates, it contains fillers of calcium carbonate to improve mechanical stability, as well as zirconium dioxide [11]. The liquid is supplemented with plasticizing polycarboxylate and calcium chloride to improve its handling properties and decrease the setting time [12]. The material is biocompatible and it induces den-https://doi.org/10.5272/jimab.2022284.4655 tin bridge formation with properties comparable to that of MTA [13]. This makes the new CSCs a convenient and affordable alternative to the MTA.
The aim of the present study was to investigate the use of tricalcium silicate cement Biodentine as an alternative to MTA in regenerative endodontic procedures by assessing the morphology and adhesion of stem cells from apical papilla in direct contact with these materials.

MATERIALS AND METHODS
Using the online 3D computer modeling program Tinkercad (Autodesk, San Rafael, California, United States), a plate with 36 holes with a diameter and height of 2 mm was created. The boundaries of the plate were modelled with a height of 4 mm in order to prevent its deformation and bending during the printing process ( Figure 1).
From each group, 20 disks with preserved shape and size were selected, which were sterilized with ultraviolet light in a laminar flow cabin for 30 minutes (Figure 3).  Stem cells from apical papilla (SCAP) were isolated from third molars with incomplete root development, extracted for orthodontic purposes. Informed consent was signed by the patients or their parents. The apical papillae were carefully removed from the root and cut into small pieces with a scalpel blade. Then they were placed in 1ml solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1hat 37 ° C, 5% CO2 and 50% humidity in an incubator. The suspension was centrifuged for 4 min at 3000 rpm and the precipitated cells were resuspended in 1 ml of culture medium. The cells were seeded in 2 cm diameter plastic plates (Greiner Bio-One, Frickenhausen, Germany) with culture medium and 20% fetal bovine serum and cultured at 37 ° C, 5% CO2 and 50% humidity.
The cells from the third passage were incubated on pre-prepared disks of test materials in 96-well plates for 24 hours at 37 ° C, 5% CO2 and 50% humidity. Additionally, several segments of each group were placed in a control medium without cell incubation.
After the incubation period, the samples were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer and observed by scanning electron microscope (SEM). The morphology of the cells and the presence or absence of cell processes were determined. Figure 4 shows representative SEM images of the studied materials without cell incubation.

RESULTS
In each of the plates were prepared 36 disks of the tested materials according to the manufacturers' instructions: • First group -Biodentine (Septodont, Saint-Maurdes-Fossés, France), further condensed with a plugger and ultrasonic tip; • Second group -MTA (MTA Angelus, Angelus, The obtained images show a typical characteristic of cements after immersion in a buffer solution with the presence of irregularly shaped crystals in GIC ( Figure 4A) or fibrillar, rhomboid and hexagonal shaped crystals in MTA and Biodentine ( Figure 4B, C).
No adhesion of SCAP was observed when incubating cells with glass ionomer disks, which can be explained by a decrease in pH and a delay in cell proliferation, as evident by the paler color of the culture medium. Cell adhesion was observed in the groups incubated in the presence of MTA and Biodentine discs (Figure 5, asterisk).
Cells adhered to the CSCs have different morphology and can be round ( Figure 5A) or flat, with several processes ( Figure 5B).

DISCUSSION
The experimental setup in the presented study mimics the clinical protocol in a standard regenerative endodontic procedure, which allowed us to draw our own conclusions. No cell adhesion was observed in the GIC samples, which may be due to impaired SCAP proliferation as a result of acidification of the culture medium or a change in the physicochemical characteristics of the material upon contact with it. Studies have shown that the viscoelastic properties of materials alter the ability of cells to attach, migrate and differentiate significantly [14]. At the same time, some of these materials could stimulate cell adhesion by creating surface roughness. Despite the fact that GIC have pronounced surface roughness, no cell adhesion was observed, which probably confirms the observation that the low pH inhibits cell proliferation.
Ultraviolet light was used in the present study to disinfect the samples, because it does not have a negative effect on the surface properties of dentin and endodontic ma- terials, unlike autoclaving, and this would not interfere with cell proliferation. Dental pulp stem cells proliferate on the surface of MTA, dentin filings and Bio-Oss, with the slowest proliferation on Bio-Oss, showing a much higher ability of MTA and dentin to attract cells. This defines them as good materials for pulp regeneration [15].
The use of new products in the biological treatment of pulp or in regenerative endodontics requires the creation of a barrier for microorganisms and blood clots and necessitates adequate biocompatibility and bioactive properties to induce tissue repair or stimulate cell differentiation and regeneration [16,17]. A high concentration of Biodentine in the culture medium significantly reduces the proliferation of stem cells, which is probably due to the fact that this CSC increases their differentiation [18]. This property may be useful during regenerative endodontic procedures.
Cell adhesion is a good indicator for quality assessment of the used materials, as biocompatible cements main-tain cell adherence [19]. The use of SEM provides valuable information about the behavior of cells in direct contact with biomaterials. Sample preparation involves fixation and dehydration, which may lead to changes in the surface or composition of the materials and related problems for quality and complete observation of the samples [20]. In the present study SCAP adhere to MTA and Biodentine disks, with cells having different morphology. Spindle-shaped or flat cells with processes observed in contact with biomaterials are a good indicator of their low cytotoxicity [19]. Cells of this shape were also observed in this study ( Figure 5).
MTA and Biodentine are known to stimulate the adhesion of dental pulp stem cells and gingival fibroblasts [21]. The materials have very good sealing properties, as Biodentine allows better isolation of the root canal and furcation defects [22]. In some cases, with MTA-based materials, cell adhesion is limited by the presence of several round cells on the surface of the material [17]. However, another study showed that cells in contact with MTA have the same morphology as those that grow only in a culture medium [23]. In contrast, cells in contact with Biodentine are covered with calcium ions and exhibit the morphological characteristics of apoptotic cells. One possible explanation is that the high concentration of calcium ions in this case leads to changes in the cytoskeleton and destruction of microvilli resulting in the onset of a signaling cascade, which can lead to the activation of apoptosis. It is possible that the differences in morphology are due to different types of adhesion that depends on the type of substrate on which the cells grow [23]. The composition of both types of cement should not be neglected. MTA uses bismuth oxide for an opacifier, and Biodentine uses zirconium oxide, which is not toxic to somatic human cells or murine fibroblasts [24].

CONCLUSION
The present study proves that SCAP adhere to MTA and Biodentine when in direct contact with discs made of these materials. The cells show different morphology and can be round or flat, with several processes. The results support the reported in the literature properties of biocompatibility and bioactivity of CSCc and their indisputable indications as suitable materials for biological treatment of pulp and regenerative endodontics.