INTRODUCTION:

Avulsion is a state in which a tooth has left its socket. Avulsed teeth are an emergency that needs treatment immediately to allow for successful replantation, in which the tooth is returned into the socket^[¹^]. The tooth root is covered by cementum for protection, which is a calcified connective tissue covering the tooth root surface and has a bone-like structure. However, cementum is more resistant to being resorbed than bone.

The function of cementum is to maintain and support the tooth in the alveolar bone by attachment of the periodontal ligament^[²^]. In order to understand the process of cementogenesis, the higher composition of collagen in cementum, as opposed to bone and dentin, is key^[³^].

Many of Indonesia’s natural resources have been used as traditional medicines to deal with a variety of health problems. Aloe vera (L.) Burm.f. is widely available in all areas of Indonesia and has many benefits^[⁴^]. A. vera gel has been used as a traditional medicine in many cultures. Acemannan is a major component of the polysaccharides isolated from A. vera gel, and it can improve various wound-healing mechanism processes such as recruitment of macrophages, collagen synthesis, and wound contraction^[^5,6^]. Another study has also suggested that acemannan stimulates the expression of bone morphogenetic protein-2 (BMP-2) in the periodontal tissue and pulp fibroblasts^[⁷^].

Similarly involved in wound healing, growth factors such as FGF-2 (fibroblast growth factor), TGF-b (transforming growth factor beta), and Smad-3 are biologically active polypeptides which affecting the proliferation, chemotaxis, and differentiation of cells from the epithelium, bone, and connective tissue. They express their action by binding to specific cell-surface receptors present on various target cells including osteoblasts, cementoblasts, and periodontal ligament fibroblasts^[⁸^].

Vera can increase the production of TGF b1 and bFGF, which will stimulate the collagen deposition, fibroblast proliferation, and angiogenesis^[9]. This research was aimed to determine the effect of A. vera gel to the expression of FGF-2, TGF-b, and Smad-3 in the cementum of rat teeth after traumatic a vulsion.

MATERIALS AND METHODS:

Collection of plant material and extraction:

A. vera was collected and extracted in Laboratory of Phytochemical UPT Materia Medica, Batu, Malang. A total of 100mg A. vera powder was put in a 25 ml bottle. The sample was added to 10 ml ethanol and mixed using a macerator for 30 minutes. The mixture was incubated for 4 days and stirred frequently. Then, the solvent was filtered out with filter paper and the residue evaporated by a rotary evaporator at 80°C^[9].

Preparation of the animal models:

This research design used was a post-test randomized controlled group design in a Wistar rats (Rattus norvegicus Berkenhout) model, and was approved by animal ethical clearance by the Ethics Committee of Medical School Brawijaya University (No. 19/EC/KEPK/01/2017). The animals were adapted for a week. For anesthetic prior to the tooth removal operation, ketamine intramuscular 30 mg/kg BW per dose was injected into the right rear foot area.

Treatment:

After the anesthesia, gingiva of the upper jaw was separated, and the right incicivus were luxated with an elevator and extracted with forceps to imitate tooth avulsion. In the non-control group, the rats were administered 2mg/ml A. vera gel to the tooth socket. In both groups, the tooth replanted within 30 minutes by re-insertion into the socket. Sedation was achieved using novalgyn 3mg/kg BW/day for three days. Each group was investigated and sacrificed at day 3, 7 and 14.

Preparation Aloe vera Gel:

A leaf was cut from the plant and immediately washed and sterilized with a solution of calcium hypochlorite, peeled and cut into small pieces and placed in a juicer to obtain the juice from the leaf. Next, to 50ml of A. vera juice was added 200 ml of 96% ethanol.

The mixture of A. vera juice and alcohol was stirred for 10 minutes at 30°C, then sterilized for 10 hours of precipitation at 10°C. The precipitate was separated from the solution by using a filter paper for the precipitate to be deposited onto, under a vacuum dryer at a temperature of 50°C. A total of 0.2 g of methyl-paraben was dissolved into 16.7 g of propylene glycol and then a 3 g carbomer was added to the mixture while stirring rapidly until a gel was formed, then was stored at room temperature for 24 hours before use^[10].

Immunohistochemistry Staining for FGF-2, TGF-b, and Smad3:

The socket was extracted from the sacrificed rats and immediately immersed in formalin buffer before preparing for sectioning and immunostaining. The tissues were decalcified using EDTA 10% for 30 days. Afterwards, tissues were prepared embedded in a paraffin block and cut into 4 µm thick slices, which were placed on a slide. The slides were then assayed for FGF-2, TGF-b, and Smad-3. FGF-2 code sc-79 and TGF-b1 code 3C11 primary antibodies from Santa Cruz Biotechnology, and Smad-3 primary antibodies from DAKO, Carpinteria, USA). The immunohistochemical methods perform were based on the vendor procedure. Each slide was observed under Olympus CX21 microscope (M=400x) for 20 fields of view in different areas and averaged data and each group (three slides)^[^11,12^].

Statistical analysis:

The data were analyzed for normality using the Shapiro-Wilk test. A one-way ANOVA was performed to test for the overall effect of treatment and time for each protein. Comparison of the response variable between groups on days 3, 7 and 14 used the post-hoc Tukey HSD test. The Pearson correlation test was used to analyze the relationship between variables. The statistic were analyzed with SPSS v.22.

RESULTS:

Aloe vera gel induced expression of FGF-2:

The expression of FGF-2 appears on each sample preparation. Based on immunohistochemical staining with specific antibodies (Anti FGF-2 code sc-79) (Figure 1). The number of cementoblast cells expressing FGF-2, statistically increased significantly (p <0.05) in the alovera group (A) day 3 compared to the same day K+ group. Similarly, FGF-2 expression on day 7 and 14 was significantly increased in expression in group A compared with K+ group on the same day. On the other hand, after replantation (K+) on day 3, the expression of FGF-2, decreased significantly compared to the normal group (K-) on the same day. Increasing expression of FGF-2 in the K+ group, when compared with K-, began to appear at day 7, observations and continued to increase by day 14 (Figure 2).

Figure 1. The FGF-2 expression (1000x magnification); A) K- group; B) K+ group; C) Alovera group. The arrow indicates a positive expression (brown: DAB chromogen).

Figure 2. The number of cementoblast cells expressing FGF-2 in K-, K+ and A group.

Aloe vera gel induced expression of TGF-b:

Expression of TGF-β1 appeared on each sample preparation (Figure 3). The number of cementoblast cells expressing TGF-β1, statistically, increased significantly (p < 0.05) in the alovera group (A) day 3 compared to same day + group. Similarly, TGF-β1 expression on days 7 and 14 was significantly increased in expression in group A compared with K + group on the same day. On the other hand, after avulsion (K+) on day 14, TGF-β1 expression increased but was not significant compared to K+ group on day 7. Here there is a tendency that there is a plateau TGF-β1 expression on the avulsion condition on the day 7 and 14 (Figure 4).

Figure 3. The TGF-β1 expression (1000x magnification); A) K- group; B) K+ group; C) Alovera group. The arrow indicates a positive expression (brown: DAB chromogen).

Figure 4. The number of cementoblast cells expressing TGF-β1 in K-, K+ and A group.

Aloe vera gel induced expression of Smad3:

The smad-3 expression was seen in each sample preparation (Figure 5). The number of cementoblast cells expressing smad-3 statistically increased significantly (p < 0.05) In the group alovera (A) day 3 compared with the same day K+ group. Similarly, the smad-3 expression on days 7 and 14 was significantly increased in expression in group A compared with the K + group on the same day. On the other hand after avulsion (K+) on day 3, smad-3 expression decreased significantly compared to normal group (K) on the same day. Increased Smad-3 expression in the K+ group, when compared with K-, began to appear at 7th day observations and continued to increase by day 14 (Figure 6).

Figure 5. The smad-3 expression (1000x magnification); A) K- group; B) K+ group; C) Alovera group. The arrow indicates a positive expression (brown: DAB chromogen).

Figure 6. The number of cementoblast cells expressing Smad-3 in K-, K+ and A group.

DISCUSSION:

Anatomically and physiologically, the periodontium is a very complicated organ containing both soft tissue and hard tissue functioning together to support the teeth in the jaw^[¹³^]. The periodontium refers to the tissues that consists of the gingiva, periodontal ligament, cementum, and alveolar bone. The structure and composition of the periodontium are affected in many acquired and heritable diseases, as well as by traumatic avulsion. The trademarks of the periodontal disorder are the destruction of soft connective tissues, bone loss, and loss of connective tissue attachment to cementum, if left untreated, lead to tooth loss^[¹⁴^].

The successes of periodontal treatment after traumatic injury depends on the periodontal regeneration and new cementum formation. But unfortunately, the proportion of cementum formation is rather slow and unpredictable^[¹²^]. As a previous study has shown, cementoblasts must proliferate to increase cell number, then differentiate, thus secrete growth factors and extracellular matrix and finally induce mineral deposition^[¹³^].

Wound healing involves adhesion, migration, proliferation, and differentiation of several cell types. All these activities are stimulated when polypeptide mediators bind to their cell-surface receptors. Different growth factors regulate different cell functions. For example, in this study, transforming growth factor (TGF-b), and fibroblast growth factors (FGF-2) and their receptors were involved in re-epithelialization^[¹⁵^].

Fibroblast growth factor-2 (FGF-2), a signaling peptide that binds heparin and heparan sulfate, could modulate the expression of the main components of connective tissue including glycosaminoglycans, proteoglycans, collagen, and noncollagenous protein. It has been shown that FGF-2 is involved in the early stage of the wound healing process by the stimulation of migration, growth, and proliferation of the cells derived from pulp and periodontal ligament. Additionally, FGF-2 is reported to be effective in human periodontal regeneration mechanism^[¹^6].

TGF-b1 has been shown to have a role in hard tissue formation, and regulate proliferation and differentiation of osteoblast both in vivo and in vitro^[¹⁷^]. Smad proteins are the central mediators for TGF-β superfamily signaling and are classified into three groups. The first group is the R-Smads, of which Smad1, Smad5, and Smad8 are primarily activated by the BMP-specific type I receptors, whereas Smad3 and Smad2 are activated by the TGF-β–specific type I receptors. The second group is the common mediator Smad (Co-Smad; e.g., Smad4). The third Smad group includes inhibitory Smads (I-Smads; e.g., Smad6 and Smad7)^[18].

Regeneration process requires alveolar bone height restoration to the cementoenamel junction, gingival connective tissue regeneration destroyed by inflammation formation of new acellular extrinsic fiber cementum on previously exposed root surfaces, Sharpey's fibers synthesis, and their insertion into root surfaces, and then re-establishing epithelial seal at the coronal portion^[¹⁹^].

A. vera gel has been suggested to participate in wound healing, correlated to its immune-stimulating effects through the activation of macrophages in the inflammatory phase^[²⁰^]. Acemannan polysaccharide, a major fraction isolated from A. vera gel has been suggested as possibly improving various wound healing process stages, such as the recruitment of macrophages, collagen synthesis, and wound contraction^[¹²^]. This study revealed there were significant differences between the control groups and the treatment groups (p£0.05). Aloe vera gel could promote cementoblasts through the Smad3 signaling as a transduction signaling in the cytoplasm from TGF-b1. TGF-β-induced Smad2/3 and p38 MAPK-dependent signals cooperated to induce cellular events as an expression of tumor suppressor gene Arf in mouse embryo fibroblasts^[16,17]. In previous new study also proved as an in vitro that aloe vera was dose-dependent and time-dependent towards the expression of bFGF and TGFb1 in fibroblast cell. This method could be employed in the prospective treatment of physical lesion^[21].

CONCLUSION:

In this study, A. vera gel can be used as a good model and a new trend in guided tissue regeneration, which is a membrane surface modification mainly by combining adhesion molecules capable of physiologically stimulating cells and tissues. This first step may become used as an application in the specific selection of adhesion molecules for healing and repair of periodontal tissue in general and particularly in dental trauma to teeth.

CONFLICT INTEREST:

The authors declared there is no conflict of interest.

ACKNOWLEDGEMENT:

Authors thank to Head of LPPM of Brawijaya University and the Dean of Medical Science Faculty of Brawijaya University for providing and entrusting the researcher to conduct this research and seminar.

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Received on 02.08.2018 Modified on 27.09.2018

Research J. Pharm. and Tech 2019; 12(9):4405-4409.

DOI: 10.5958/0974-360X.2019.00758.3