Bioactivity effects of extracellular matrix proteins on apical papilla cells

Abstract Potent signaling agents stimulate and guide pulp tissue regeneration, especially in endodontic treatment of teeth with incomplete root formation. Objective This study evaluated the bioactive properties of low concentrations of extracellular matrix proteins on human apical papilla cells (hAPCs). Methodology Different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96-well plates. Non-treated and pre-treated wells were used as negative (NC) and positive (PC) controls. After seeding the hAPCs (5×103 cells/well) on the different substrates, we assessed the following parameters: adhesion, proliferation, spreading, total collagen/type I collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) (ANOVA/Tukey; α=0.05). Results We observed greater attachment potential for cells on the FN substrate, with the effect depending on concentration. Concentrations of 5 and 10 µg/mL of FN yielded the highest cell proliferation, spreading and collagen synthesis values with 10 µg/mL concentration increasing the ITGA5, ITGAV, and COL1A1 expression compared with PC. LM (5 and 10 µg/mL) showed higher bioactivity values than NC, but those were lower than PC, and COL showed no bioactivity at all. Conclusion We conclude that FN at 10 µg/mL concentration exerted the most intense bioactive effects on hAPCs.


Introduction
The concepts of tissue engineering were introduced to Dentistry in the last decade giving special attention to inducting pulp regeneration. [1][2][3][4] The principles of regenerative endodontics are four: 1. disinfection and detoxification of root canals; 2. use of biomaterials as scaffold for cell adhesion, proliferation, and differentiation; 3. presence of cells with potential to regenerate a new tissue similar to the original; and 4. use of signaling agents to induce cell migration and enhance the bioactive action of the biomaterial. 5,6 Two strategies emerged as the most promising therapies for tissue regeneration: cell approaches and cell-free approaches. 7,8 The first involves implanting pre-cultured cells associated with a biomaterial at the site of injury. The bioactive materials provide a porous three-dimensional structure and act as a temporary extracellular matrix where attached cells can grow to regenerate the tissue. The second, cellfree approaches, involve the use of biomaterials along potent signaling agents. When put into the injury site, these agents stimulate cell migration towards the site of interest, as well as their proliferation and differentiation, favoring tissue healing. 7,8 Cell-free therapies seem to be interesting for teeth with incomplete root formation. A histopathological study of rat teeth with periapical lesion demonstrated a viable apical papilla was maintained even after 90 days of pulp necrosis. 9 The apical papilla is known to have a rich source of stem cells. Therefore, biomaterials associated with potent signaling agents could be used to attract these cells into the root canal. proposed as chemotactic and inducing agents, since scientists have described their proprieties in several biomedical fields. [10][11][12][13][14][15] Previous studies showed FN, either associated with other proteins, or not, can mediate cell adhesion, migration, proliferation, and differentiation; while also helping in tissue formation, remodeling, repair and regeneration. 11,13,15 LM is a complex structure that also regulates cell adhesion, migration and proliferation. 10,12 LM aids in maintaining cellular functions especially in the processes of reeptelialization and angiogenesis. 10 COL is a common protein that surround cells throughout the body, with over 90% of this protein being type I. COL can be used to mimic the natural cell environment, and under specific conditions, is effective in promoting stem cell differentiation and growth. 13,16 One study characterized the dehydrated human umbilical cord (DHUc) in terms of tissue composition and evaluated its in vitro and in vivo effects. 14 Table 1 shows all groups established according to the polystyrene 96-well plates used, as well as the type and concentration of ECMp.
After performing the treatments of wells with ECMp, the hAPCs (5×10 3 cells/well) were seeded in the respective wells and incubated at 37ºC and 5% CO 2 for up to 5 days, with the culture medium being changed every 48 h. Then, the bioactivity parameters were evaluated. All assays were performed twice to ensure the data reproducibility.   After washing the cells with PBS, they were incubated with Hoescht (1:5000; Invitrogen, Carlsbad, CA, USA) for 15 min for nuclear counter-staining. 23 The F-actin assay was then analyzed using a fluorescence microscope (Leica DM 5500B).
Total collagen synthesis: The cells were cultured for 5 days in α-MEM without FBS, and the culture medium was collected and replaced every 48 h. The pool of collected culture medium of each sample was stored at -20ºC until the Sirius Red assay was performed (n=6).
For this purpose, the culture was transferred to 1.5 mL tubes containing Direct Red solution in saturated picric acid (0.1%), and then incubated for 1 hour, under agitation at 400 rpm, in a dry bath at 25°C.
The tubes were centrifuged, the supernatant was discarded, and 0.01 M hydrochloric acid was added.
The tubes were centrifuged again, the supernatant was discarded, followed by the addition of 0.5 M sodium hydroxide to solubilize the precipitated material. 24 The resulting solution from each sample was subjected to absorbance analysis in a spectrophotometer at 555 nm (Synergy H1). 24 The percentage of total collagen synthesis for each sample was calculated based on the mean absorbance values of the negative control group.
Type I collagen synthesis: The medium pool collected in the previous analysis (n=6) was also used to evaluate the type I collagen synthesis (COL-I), which was detected by enzyme-linked immunosorbent The data from cell adhesion, viability, total protein synthesis, type I collagen synthesis, and RT-qPCR assays were evaluated for adherence to the normal curve (Shapiro-Wilk test, p>0.05) and homoscedasticity (Levene test, p>0.05). Then, the data were submitted to the one-way or two-way

Results
Cell adhesion: As demonstrated in Figure 2A and B, the highest attachment potential occurred in Group FN10, followed by Groups FN5, FN1, PC, LM10, and LM5 (p<0.05). No significant difference was observed between Groups LM1, COL1, COL5, and COL10 and Group NC (p>0.05).
Cell proliferation: Figure 3 shows increased cell proliferation occurred over time-points for all groups (p<0.05), except for those treated with type I collagen and Group NC (p>0.05). The best cell viability result was observed in the groups treated with 5 or 10 µg/ mL of FN after intervals of 1, 3 and 5 days (p<0.05).
At the last time-point, this increase was 30x higher when compared with Group NC, 1.7x higher than in Group PC, and 2.7x higher than the group treated with 10 µg/mL LM. The group FN1 also showed higher cell Total collagen synthesis: Figure 5A shows increase in the total collagen synthesis occurred in Groups FN10 and FN5, followed by Groups PC and LM10 in comparison with Group NC (p<0.05).

Gene
Assay ID Encoded protein

Discussion
The application of tissue engineering concepts for the purpose of pulp tissue regeneration has been relevant, especially in endodontic treatment of teeth with incomplete root formation. The thin and fragile walls of the root canal, associated with the short length of the root, make these teeth susceptible to fracturing under the forces to which they are submitted. 7,26 Conservative treatments that aim to stimulate cell migration from apical papilla into the root canal and allow the synthesis of a new pulp-like tissue seem to be an interesting alternative that would allow the return of tooth vitality and continuity of root formation. For this purpose, the use of a potent signaling agent is crucial to stimulate and guide tissue regeneration. 5,6 Studies have shown extracellular matrix proteins in concentrations between 0.5 to 100 µg/mL can act as a bioactive agent on cells from different sources. 27,30 Therefore, in the present study, we assessed the biological activities of low concentrations of ECMp   corroborate with these data previously reported by several researchers. 11,12,13,33,35 This study also showed that the bioactive potential   shown that the association of extracellular matrix proteins with scaffolds approximated the conditions of the in vivo environment. 13,29,33 Thus, they mediated the mechanical-chemical signals in the processes of adhesion, migration, proliferation and differentiation of various cell types, and in the synthesis of a new matrix. 13,29,33 The results obtained in the present study cannot be directly extrapolated to clinical situations.
However, in spite of the limitations of this laboratory investigation, it seems appropriate to conduct further studies to assess techniques using the cell-free approaches proposed by Galler and Widbiller 7 (2020).
In these, the root canals may be filled with biomaterials containing dosages of FN fibronectin capable of stimulating the migration of cells from apical papilla and allowing new pulp-like tissue formation.

Conclusions
According to the methodologies used in the present study, we conclude that 10 µg/mL of fibronectin could act as a potent bioactive agent for human apical papilla cells by inducing cell adhesion, proliferation, spreading and collagen synthesis.