Basic responses of mesenchymal stem cells exposed to bovine biomaterial and platelet rich fibrin

The scaffolds and their interaction with mesenchymal stem cells are objects of study in bioengineering and tissue repair. Mechanisms such as surface adhesion, proliferation, viability, and cytotoxicity are essential for the development of therapies. The present study analyzed the influence of platelet-rich fibrin (PRF) in viability, cytotoxicity, and proliferation of stem cells from human exfoliated deciduous teeth (SHED) exposed to bovine biomaterial surfaces. The studied groups were divided and analyzed as follows: (S) only SHED as control Group; (SB) SHED + biomaterial; (SBP) SHED + biomaterial + PRF. Analyses of cells seeded in 24-well plates were performed after 24, 48 and 72 hours. Individual groups were subjected to viability, cytotoxicity and cell proliferation tests using neutral red, MTT and crystal violet, respectively; and in the 72-hour group, scanning electron microscopy (SEM) was performed to record cell ultramorphology. Data were submitted to statistical analysis by two-factor ANOVA with a significance level of 5%. The results demonstrated a better performance in the viability/cytotoxicity and proliferation of stem cells for the group (SBP) in comparison to the group (SB) and the group (S). The applied statistical tests showed that the biomaterial factor, time, and interaction between them gave rise to results with statistical significance. SHED submitted to bovine biomaterial were more viable, proliferative and with lower toxicity when associated with PRF. PRF seemed to activate the metabolism of stem cells in culture, indicating that such an association can bring an effective benefit in clinical outcome.


Introduction
Tissue engineering is the use of a combination of multidisciplinary approaches with the aim to improve or replace biological tissues (Huawei et al. 2019) and it is highly applied in reconstructive surgery procedures such as bone augmentation.
The essential elements needed are osteoconductive scaffolds, osseoinductive growth factors and progenitor cells.
Autologous mesenchymal stem cells from the adjacent tissues to the bone, such as periosteum and bone marrow, represent the first source of undifferentiated cells for bone reconstruction. However, mesenchymal stem cells can be obtained from other sources, such as adipose, stromal fraction of bone, dental pulp tissues (Strauer et al., 2008) and stem cells from human exfoliated deciduous teeth (SHED) (Miura et al., 2003), for use in regenerative medicine for bone tissue repair. Nevertheless, SHED represent a viable source for mesenchymal stem cells obtained, once the young human being has twenty opportunities for collection, because of the deciduous teeth natural process of exfoliation. Another important element to be considered is cell interaction with osseocondutor mineralized biomaterials when acting the basic components of scaffolds and play a fundamental role in bone tissue engineering (Huawei et al., 2019). Xenogenic sources, such as bovine, are extensively (Precheur, 2007) due to their ultrastructural characteristics, which can drive the development of new bone tissue. In addition to that, osteoconductive materials are biocompatible of a bone neoformed structure (Moraschini et al., 2015).
Finally, the mesenchymal stem cells need signalizing specific growth factors and cytokines signaling to promote transduction, gene transcription and protein synthesis, modulating chemotaxis, migration, proliferation, and cell differentiation.
The autologous platelet-rich fibrin (PRF) is an additional scaffold and can act as a continuum medium between cells and the biomaterial surface. The PRF possesses supraphysiologicalgrowth factors concentrations, glycoproteins with adhesives properties, and contains live and functional platelets and leukocytes and a variety of cell compounds like immunoglobulins and ligand molecules (Miron et al., 2017;Oliveira et al., 2020). Based on these properties, the PRF can promote induction proliferation and differentiation of osteoprogenitor cells and improve bone regeneration in reconstruction surgeries (He et al., 2009;Dohan et al., 2009).
Thus, the present study aims to qualitatively evaluate the influence of PRF in the levels of cytotoxicity, viability, and proliferation status of mesenchymal stem cells associated with a bovine bone grafting biomateria l.

Individuals and ethical aspects
The present study observed all ethical standards for scientific research with humans in conformity with the Declaration of Helsinki (World Medical Association Recommendation 2013). The mesenchymal stem cells were obtained from a twelveyear-old donor after the signed an informed consent form. A healthy tooth, without caries, with complete rhizolysis, was used at the time of natural tooth exfoliation. The blood donation (single donor, thirty-year-old) for PRF preparation followed the same ethical steps. The Research Ethics Committee of Universidade Positivo approved the study under number 3.6665.172/2019. The methodology herein presented followed de Oliveira et al. (2020), Lisboa et al. (2021) and Stroparo et al. (2021). Research, Society andDevelopment, v. 10, n. 11, e46101119134, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19134 4

Stem Cell Culture
After oral prophylaxis, the exfoliated tooth was inserted in a container with sterile medium (DMEM [Dulbecco's Modified Eagle's Medium] + 10% Fetal Bovine Serum + 1% streptomycin/penicillin + 1% amphotericin). Explant cells process and primary culture was obtained.
All cells were kept in a CO2 incubator at 37ºC with the same medium. The cells were cultivated until they reached 80% confluence at the 4 th passage, observed in the inverted microscope. Trypsin was used and 4 × 10 4 cells were seeded in three wells (24-well plate) for each group, at 24, 48 and 72 hours. Mesenchymal stem cells were characterized by Dominici protocols (2006). This methodology was describeb by Lisboa et al. (2021) and Stroparo et al. (2021).

Obtaining the PRF matrices by centrifugation
A healthy and non-smoker adult voluntarily donated a total of 80 ml of blood, collected in 9,0 ml sterile polystyrene vacuum tubes (Greiner Bio-One, Brazil) for PRF production. Blood samples immediately transferred to fixed-angle centrifuge FibrinFuge25 (Montserrat, Brazil), centrifuged with 200 × g for 10 minutes, in conformity with de Oliveira et al. (2020). The methodology can be seen in Figure 1.

Experimental groups
Cells from culture flasks were washed with PBS at 37ºC, treated with trypsin and resuspended with 1 ml of culture medium; a small aliquot was counted in Neubauer chamber and 4 × 10 4 cells that were plated in the experiment wells. In vitro tests were performed on 24-well plates (Greiner, Germany), in doble triplicates. The groups were divides as follows.

1) Group C (Control, SHED only):
in the wells of the control group, the SHED was seeded and maintained only in supplemented DMEM.

Cell Viability -Neutral Red
The Neutral Red method was performed according to the methodologies described by Borenfreund and Puerner (1985), Chaim et al. (2006) and Fotakis and Timbrell (2006). At 24, 48 and 72 hours of culture, the cells were incubated with 37.5 µl of a neutral red stock solution (1100 µg / ml, Sigma-Aldrich / Merck, Darmstadt, Germany) diluted in PBS, and then diluted in 337.5 µl of the culture medium, thus obtaining a final concentration of 100 µg / ml of neutral red. The absorbance was read in a microplate reader (540 nm, Biotek, Winooski, USA).
The Formazan crystals were dissolved in 200 µl of DMSO, then the contents of the wells were removed, and the absorbance was read in a microplate reader (BIOTEK, Winooski, USA) at a wavelength of 570 nm.

Scanning Electron Microscopy Analysis
The PRF clot fragments obtained by 200 × g / 10 minutes were separated and conditioned in plastic tubes that were free of additives (Greiner Bio-One, Brazil). Immediately after PRF production and after 24, 48 and 72 h at 37°C, the specimens were sectioned (body, buffy coat 3 of 12 proximal sediment) and fixed in 2% glutaraldehyde solution and 2% paraformaldehyde in 0.1M sodium cacodylate buffer, pH 7.2. After being fixed, they were washed in 0.1 M sodium cacodylate buffer, post-fixed in osmium tetroxide (1%) in sodium cacodylate buffer, then the specimens were dehydrated for 15 minutes in solutions with increasing concentrations of acetone (30%, 50%, 70%, 90% and 3x 100%). Afterwards, the specimens were dried to the critical point with CO2 and metalized with gold. For analyzes by scanning electron microscopy (JEOL 7001F, Tokyo, Japan), the images (20 to 20000 × of magnification) were analyzed by using GIMP 2.10 software (Cockroach Labs, USA). For the morphometric study the diameter of twenty fibers was evaluated using the digital planimeter of Autocad software (Autodesk, USA).

Statistical analysis
The data used for the different tests of cell viability, cytotoxicity and cell proliferation were analyzed statistically by two-way ANOVA and Tukey's test with a significance level of 5%.

Cell Viability -Neutral Red
Despite being non-toxic, in 24 h the Group B samples showed a lower viability than the control, with results between 38 to 58%; while in the Group BP the viability was close to 60 %. In 48 h, the experimental groups showed an improvement in viability in relation to the 24 h test, with viability between 40 to 70% in Group B, and viability greater than 60% in Group BP.
The results within 72 h showed even more improvement in comparison to 24 and 48 h, while Group (B) showed viability greater than 50%, the Group BP presented viability greater than 70%, with results surprisingly higher than the control. The results are shown in Figure 2 (1A, 1B, 1C).
The means and standard deviations for the groups that were evaluated indicated statistically significant differences for the biomaterial factor (p = 0.001) and time (p <0.001). The double interaction was statistically significant (p = 0.037), as shown in Table 1.

Cell Cytotoxicity -MTT
The MTT results for 24 h indicate no toxicity, in which the Group B had 100% viable cells; however, the Group BP showed a small decrease at this MTT assay. In 48 hours, none of the groups presented toxicity, with 100% viable cells; a similar result was observed in the 72-hour period. The results are shown in Figure 2 (2A, 2B, 2C).
Therefore, the results indicated statistically significant differences for the biomaterial factor (p = 0.032) and time (p <0.001). The double interaction was not statistically significant (p = 0.480), as shown in Table 2.

Cell proliferation -Crystal Violet Assay
It can be seen in 24 h that Group B has an anti-proliferative effect with a decrease in cell density. However, a slight stimulus was observed in the Group BP. In 48h, Group B had a more pronounced anti-proliferative effect in comparison to the same group in 24h. Meanwhile, the Group BP showed a greater stimulus in proliferation, like that of the control. Finally, at 72 h, the Group B had a greater anti-proliferative effect in comparison to the 24 and 48 h. Notably, the Group BP had a pronounced proliferation stimulating effect, with results greater than the control. The results are shown in Figure 2 (3A, 3B, 3C).
The results indicated statistically significant differences for the biomaterial factor (p = 0.001) and time (p <0.001).
Double interaction was not statistically significant (p = 0.001), as shown in Table 3.

Scanning Electron Microscopy -SEM
By SEM, it was possible to verify the adhesion to the surface at the Group BP after 72 h of treatment, which presents fusiform morphology (Figures 3 and 4). The SEM refers to sparse cells still with a fibroblastic characteristic after 72 hours of culture with stabilization of cell morphology and increased proliferation.

Discussion
Isolated from sources such as those of primary tooth pulp, human MSC appears as a model closer to the reality found in the clinic for testing new techniques and products, prior to animal tests or clinical trials. As well as stem cells, additional elements have shown valuable properties for application in the clinical and surgical context, such as PRF, which has great biological potential in wound healing (Massuda et al., 2020). Considering these facts, the objective of this study was the analysis In the evaluation of proliferation by means of the crystal violet assay, it was observed that after 72h that Group B had an anti-proliferative effect; in an opposite way, a stimulating effect of proliferation was found in Group BP, suggesting that the association of SHED + biomaterial + PRF influences SHED to multiply with greater intensity. Studies point to such an association as a strong adjunct in the treatment of various diseases (Orlic et al., 2002;Oliveira et al., 2017).
The SEM images of the SHED were obtained from samples of Group BP, at 72 hours, because in this group and at this time they were more proliferative. Sparse cells, with fusiform aspect and characteristics of stabilization of cell morphology and increased proliferation were observed. These results are in line with different studies, such as those described by Yamada and Watari (2003), Lendeckel et al. (2004), Ratnayake and Currie (2017), as well as in the study proposed by Amaral (2006), who investigated the morphological characteristics of mesenchymal stem cells and their differentiation capacity. Naz et al. (2019) and Nakajima et al. (2019) emphasize the need for using standardized and adequate protocols for this type of assessments to comply with the necessary requirements for the biocompatibility process (Sanada et al., 2003;Rosa et al., 2000). In this study, the followed methodology has proven to be viable and reproducible for future research, suggesting future studies with longer periods and immune characterization, which can further disclose more cell behavior towards biomaterial.

Conclusion
Although the results from viability, cytotoxicity, and proliferation among all groups seemed to converge, while exposed to the bovine biomaterial used in this study, SHED decreased their performance. However, when the biomaterial was associated with PRF, a proliferative and stimulatory effect was observed on SHED, indicating that such association can bring an effective benefit in clinical outcomes .