The influence of Aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats

Abstract Objective This study aimed to evaluate the inflammatory effect and bone formation in sterile surgical failures after implantation of a collagen sponge with mesenchymal stem cells from human dental pulp (hDPSCs) and Aloe vera. Material and Methods Rattus norvegicus (n=75) were divided into five experimental groups according to treatment: G1) control (blood clot); G2) Hemospon®; G3) Hemospon® in a culture medium enriched with 8% Aloe vera; G4) Hemospon® in a culture medium containing hDPSCs and G5) Hemospon® in a culture medium enriched with 8% Aloe vera and hDPSCs. On days 7, 15 and 30, the animals were euthanized, and the tibia was dissected for histological, immunohistochemistry and immunofluorescence analyses. The results were analyzed using nonparametric Kruskal-Wallis test and Dunn’s post-test. Results On days 7 and 15, the groups with Aloe vera had less average acute inflammatory infiltrate compared to the control group and the group with Hemospon® (p<0.05). No statistically significant difference was found between the groups regarding bone formation at the three experimental points in time. Osteopontin expression corroborated the intensity of bone formation. Fluorescence microscopy revealed positive labeling with Q-Tracker® in hDPSCs before transplantation and tissue repair. Conclusion The results suggest that the combination of Hemospon®, Aloe vera and hDPSCs is a form of clinical treatment for the repair of non-critical bone defects that reduces the inflammatory cascade’s effects.


Introduction
Bone defects are one of the most dangerous problems found in patients seeking oral rehabilitation.
These defects cause difficulty for treating and restoring the patient's smile and functionality, especially if the defect is critical. Many studies involving bone repair were undertaken to gather new knowledge, techniques and materials to solve the problem. [1][2][3] Among these, stem cell research has grown exponentially in recent years due to the recognition that this therapy has great potential in regenerative medicine. 4 The repair of bone defects is characterized by three overlapping phases: the formation of blood clots, bone formation, and bone remodeling. During the second phase, osteoprogenitor cells migrate to the wound site, proliferate and differentiate into osteoblasts that segregate local growth factors, the extracellular matrix and induce mineralization. Therefore, the tissue's regeneration depends on the availability of these precursor cells and on the presence of stimuli required to recruit and stimulate these cells. 1,3 Thus, the tissue's regeneration can be improved if the material has osteoinductive characteristics. With this purpose, studies involving stem cells have grown exponentially due to the recognition that this therapy has great potential in regenerative medicine. 4 Dental pulp stem cells (DPSCs) are a vital source of osteoprogenitor cells that have a fundamental role in the mechanisms related to the pulp tissue's plasticity.
They are also involved in inducing tissue regeneration based on their differentiation ability in culture and expression of mesenchymal stem cell (MSC) markers. [5][6][7] These characteristics were observed for the dental pulp of permanent teeth (hDPSCs) 5,6 and exfoliated deciduous teeth (SHED) 7

in humans.
The pursuit of care and improvements in the tissue repair process and in the pharmacodynamic anti-inflammatory and immunoregulatory properties of natural products have drawn attention, and many of them were tested on tissues. The plant species of the Aloe (Liliaceae) genus were extensively studied due to their medicinal properties. Among them is Aloe vera, a species that contains many biologically active substances 8 that are able to provide significant anti-inflammatory, antibacterial, hypoglycemic, immunomodulating, wound healing and regenerative properties. 9 Additionally, acemannan, a polysaccharide extracted from Aloe vera, is a biomolecule with potential for tissue regeneration, playing a significant role in cell proliferation, extracellular matrix synthesis and mineralization. 1,[10][11][12] Therefore, an important question became the focus of this work: could Aloe vera enhance and accelerate bone repair? This study aimed to evaluate bone repair in non-critical defects of rat tibias after implantation of a collagen sponge (Hemospon ® ) colonized with mesenchymal stem cells from human dental pulp (hDPSCs) and Aloe vera. When they reached 80% confluence, the cultured cells were subcultured, avoiding the induction of cell differentiation by contact. The culture was subjected to trypsinization with 1 mL of trypsin-EDTA and incubated for 5 minutes. The addition of 2 ml of the culture medium deactivated trypsin. The mixture was added to a 15-ml conical tube and centrifuged at 20°C at 1500 rpm for 10 minutes. The supernatant was discarded.

Material and methods
The pellet obtained was re-suspended in 3 ml of culture medium and transferred to a 25-cm² culture flask.
The cultures were expanded in 25 cm 2 bottles until the fourth passage, replicated with twice the original area to reach 80% confluence, and cryopreserved.
Each culture was washed with PBS, subjected to an enzymatic dissociation process using trypsin-EDTA digestion, and re-suspended in a freezing medium (40% FBS, 50% DMEM low glucose and 10% dimethylsulfoxide, Sigma-Aldrich, St. Louis, Missouri, USA). After this process, the cells were transferred to freezing tubes (cryo-tubes, TPP) in 1.0x10 6 cells/ml and kept at -196°C in liquid nitrogen.
As the specific isolation of stem cells was not performed, the cell culture used in the present study represents a mixed culture of pulp cells with the presence of a subpopulation of stem cells.  The four antibodies were each placed in a tube (CD14 -10 µL, CD45 -10 µL, CD105 -5 µL). One tube was used as the control. The tubes were left in an oven at 37°C with 5% CO 2 and 95% humidity for 30 minutes without light. After this period, two centrifugations at 462 g were performed for 5 minutes. 400 µL of PBS was added to the resulting pellet. After this, they were transferred to a cytometer tube, where data acquisition was conducted followed by analysis in a flow cytometer.

Kinetics and cell viability
The results were plotted in a histogram for evaluation.

Kinetics and cell viability in the presence of Aloe vera
To evaluate the kinetics and cellular viability in a   The hDPSCs demonstrated the potential of multilineage differentiation after being induced in a specific medium in vitro. Figure 4A shows the adipogenic, osteogenic and chondrogenic differentiation from left to right. Regarding the phenotypic profile of the hDPSCs using flow cytometry, the results showed that approximately 60% of the cells expressed CD105, a mesenchymal cell indicator. In relation to CD14 and CD45, no marking was found as expected, excluding the possibility that these are hematopoietic cells ( Figure 4B).

Histological and immunohistochemical analysis
The acute inflammatory infiltrates decreased as time passed for the Hemospon ® group and the Hemospon ® and hDPSCs groups. After 7 and 15 days, the Aloe vera groups had a lower average with statistically significant difference (p<0.05) between the control and Hemospon ® groups. After 30 days, lower averages were found for the groups that had Hemospon ® and Aloe vera, Hemospon ® and hDPSCs, and Hemospon ® , hDPSCs, and Aloe vera, with statistically significant differences between the first two compared to the control group (p<0.05) ( Figure 5).
During the seven-day period, the group with Hemospon ® , hDPSCs, and Aloe vera featured repair, based on the presence of primary bone tissue. The bone    In the presence of a medium enriched with 8% Aloe vera, the hDPSCs continued to experience exponential growth during ten days of cultivation, like the control group. In both groups, the formation of colonies and of a fibroblast morphology that is characteristic of MSCs was observed. 5,7 Furthermore, the results demonstrated that the component was not toxic to the cell culture since there was no greater number of dead cells observed during the viability analysis.
SCs in bone reconstitute the presence of a biocompatible, resorbable, radiolucent, and porous support material with osteoconductive capability that is necessary. 19 Collagen sponges are currently used in various therapeutic practices due to their high availability, secure purification, biocompatibility and non-toxicity. 20 Additionally, their use as a "scaffold" in bone defects has shown positive results whether they are colonized with SCs or not. 21 In the study, we opted for a Hemospon ® collagen sponge (Technew, Rio de Janeiro, RJ, Brazil), a dental material for clinical use, which is easy to access and has a web of collagen fibers that allows the adhesion of cells. It is also capable of being reabsorbed within four weeks. 22 This short period could be a disadvantage for the treatment of critical bone defects, but it would be a good alternative to accelerate the repair process in non-critical defects.  The results obtained in this study allow us to suggest that Hemospon ® can be used as a "scaffold" for SCs in tissue repair.
The Aloe vera was used in the study as an antiinflammatory factor to aid in bone repair. 11 where OPN expression was present after seven days, indicating that these groups already had proliferation and osteoblastic activity.
For a complete evaluation of bone defect repair mediated using SCs, it is necessary to make sure that the transplanted cells remained in the defected area and contributed to bone healing. 29 The culture of cells with fluorescent markers is an easy method that is commonly used to track cells in vivo and for monitoring them. 30 Once inside the cells, Qdots ® provide a stable and robust fluorescence that can be traced back for several generations while not being transferred to adjacent cells in a population. In our study, the labeling of cells before transplantation with Qdots ® and in regenerated tissue in the groups treated with hDPSCs in the presence or absence of Aloe vera was confirmed, indicating that bone regeneration was guided by the hDPSCs (Figure 8).

Conclusions
These results suggest that Hemospon ® , Aloe vera, and hDPSCs are a promising clinical procedure to repair non-critical defects and speed up the repair process, reducing the inflammatory cascade's effects.

Conflict of interest
None declared.

Statement
We declare that all those involved in the work participated in the development of the project.
The influence of Aloe vera with mesenchymal stem cells from dental pulp on bone regeneration: characterization and treatment of non-critical defects of the tibia in rats