Evaluation of Aloe vera as matrix metalloproteinase inhibitor in human dentin with and without dentin-bonding agent: An in vitro study.

Background: Proper hybrid layer formation lays the foundation of resin-dentin bonding. The resin infiltration in demineralized dentin collagen couples with the adhesive/resin composites in the mineralized dentin surface. However, the activation of enzymatic activity in the collagen matrix can degrade the hybrid layer. Over the time, it leads to reduced bond strength. Mainly, the enzymes involved are matrix metalloproteinases (MMPs) which are involved in degrading most of the extracellular matrix components. Aloe vera is an herb with an anti-inflammatory effect, but its role in human dentin as an enzyme inhibitor has not been verified yet. Aims: The purpose of the study was designed for evaluating the inhibitory action of Aloe vera on MMP in human dentin with and without dentin bonding agents. Materials and
Methods: A total of 15 freshly extracted healthy human teeth were collected and stored at 4°C until use. The roots were separated. The enamel and remnant pulp tissue were removed, and collected teeth were pulverized with liquid nitrogen in the minimum volume of 50-mM phosphate buffer to obtain dentin powder extract. The dentin powder extract is the source of MMPs, and therefore, the extract was treated with A. vera solution and incubated to assess the enzyme inhibition by the plate assay method and zymographic analysis.
Results: A. vera treated sample with and without dentin bonding agent showed inhibition of dentin MMP's activity by plate assay method and confirmed by zymogram analysis. Conclusions: A. vera has the potential for inhibiting the MMPs enzyme activity of human dentin collagen with and without dentin bonding agents. Copyright:

INTRODUCTION

The clinical outcomes and viability of composite restoration mainly depend on the resin–dentin bonding. The hybrid layer is a unique combination of demineralized dentin with resin infiltration, which combines with adhesives/resin in the mineralized dentin.[1] The activation of endogenous collagenolytic enzymes present in the collagen matrix degrades the exposed collagen matrix located in the hybrid layer.[23]

The enzymes which are mainly responsible for the degradation are matrix metalloproteinases (MMPs). The identified MMPs in human teeth are MMP-2, 3, 8, 9, and MMP-20. The reduced biodegradation may reinforce the dentin structure and develop a strong and long-lasting bond of dentin–resin interface.[4]

In recent years, more researchers have been taking place in the field of endodontics about the potential for the inhibition of MMP by the substances derived from the natural products. A. barbadensis Miller (Aloe vera) is a short succulent herb resembling a cactus, with green fleshy, spiny, and well-marginated leaves filled with a clear viscous gel. Various studies have shown the potency of A. vera as a strong antibacterial, antifungal, and antiviral properties.[56] It has also been used to relieve thermal burn, sunburn, and promote wound healing[7] and has antimicrobial activity which can help to stimulate the body's immune system.[8] The total leaf extracts contain anthraquinones.[9] Prabhakar et al. conducted a randomized clinical trial in 2015 and advocated the use of A. vera as a cavity disinfectant.[10]

A recent study has revealed A. vera exhibits MMP inhibitory effect against MMP 2 and 9.[11] Proper hybrid layer formation lays the foundation of resin–dentin bonding. The resin infiltration in demineralized dentin collagen couples with the adhesive/resin composites in the mineralized dentin surface. However, the activation of enzymatic activity in the collagen matrix can degrade the hybrid layer.

Over time, it leads to reduced bond strength. Studies have investigated various MMP inhibitors. A. vera is a herb with an anti-inflammatory effect, but its role in human dentin as an enzyme inhibitor still needs to be evaluated due to limited literature. Therefore, the present study was designed for evaluation of the inhibitory effect of Aloe vera on MMP activities with and without bonding agents in human dentin.

MATERIALS AND METHODS

In the present study, 15 freshly extracted healthy impacted third molars were collected from the department of oral and maxillofacial surgery and stored at 4°C until use. The study protocol was approved by the institutional ethical committee (IEC/2019-20/09). In this study, the roots were separated from the specimen tooth using a diamond disk (Markus Ink., Michigan, USA). The enamel from the crown portion and the remnant pulp were removed. The dentin part was pulverized it with liquid nitrogen (BASCO INDIA, Tamil Nadu, India) with the help of a mortar and pestle in an extraction buffer. Centrifugation was carried out to separate the debris done at 10,000 rpm for 20 min at 4°C (Biofuge, Amersham Germany). The supernatant act as enzyme source, and protein concentration was estimated by spectrophotometer (Systronics India) at 660 nm using Lowry's Method.[12]

Preparation of Aloe vera solution

A. barbadenis Miller (A. vera) solution was prepared in 10 mL of distilled water by dissolving 20 mg of A. vera powder (Azyme Biosciences Private Limited, Bangalore, India) with a concentration of 2000 microgram/mL.[13]

Plate assay method

Agar gel 1.5% with the inclusion of gelatin (2%) was prepared in distilled water and sterilized. In a petri plate (10-cm diameter), the hot gel was poured and cooled to solidify for the next 90 min. Two wells of 4-mm size diameter were made on the surface of the gel using a cork borer. The right-hand side well [Figure 1a] was filled with 0.5 ml of gelatinase enzyme source (untreated dentin powder), whereas the left-hand side well [Figure 1b] was filled with enzyme source which was treated with prepared A. vera solution and incubated for the next 24 h. After 24 h, the enzymatic activity of both sides was analyzed by zone of hydrolysis.[14]

Figure 1

Glass plate assay method [a] filled with gelatinase enzyme source (untreated dentin powder), [b] filled with enzyme source which was treated with prepared A. vera solution.

Zymogram analysis

The supernatant solution was collected, precipitated with chilled 40% acetone, and incubation was done for 2 h at -8°C. Then, the precipitate was separated by centrifugation at 6000 rpm for 10 min. The precipitate was resuspended in 50-mM phosphate buffer in minimum soluble volume and used as enzyme source for zymogram analysis. The zymography gel was prepared by with 10% polyacrylamide gel[15] with the inclusion of 0.5% substrate (gelatin), and the native gel electrophoresis (Bio-Rad, Netherlands) was carried out at 80 V for 2 h. The gel was removed and analyzed for hydrolysis of gelatin.

sodium dodecyl–sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-Electrophoresis was carried out at room temperature on 10% polyacrylamide gel (HiMedia, Mumbai, India).[15]

Lane 1-Dye marker

Lane 2-TCA precipitated enzyme

Lane 3-TCA precipitated enzyme.

Native PAGE-Electrophoresis was carried out at 4°C on 10% polyacrylamide gel.

Lane 1-Dye marker

Lane 2-Enzyme source

Lane 3-Enzyme source and A. vera extract solution (2 mg/mL)

Lane 4-Enzyme source, dentin bonding agent (Te-Econom Bond), and A. vera extract

Lane 5-Enzyme source and dentin bonding agent.

The staining of the gel was done with a 0.2% stain Coomassie Brilliant Blue R-250 (Thermo Fischer Scientific, Bangalore, India). The destaining was performed with a prepared destaining buffer of 50% methanol, 10% acetic acid, and 40% water (HiMedia, Mumbai, India).

All the quantitative experiments were carried out in triplicates, and the mean standard deviation was calculated using GraphPad Prism 7 software San Diego, California (USA).

RESULTS

The total enzyme present in the dentin powder extract was estimated by Lowry's method, and protein concentration was found to be 1260 μg/ml. The results were confirmed by triplicates and calculated standard deviation by GraphPad Prism 7 software (USA).

In this method, a zone of hydrolysis was seen around the surrounding area of the right-hand side well-containing enzyme source. On the left-hand side well-containing A. vera treated human dentin powder, no such zone of hydrolysis was appreciated.

Gelatinzymographic analysis

Sodium dodecyl–sulfate-polyacrylamide gel electrophoresis

The SDS PAGE analysis [Figure 2] showed the molecular mass of the enzyme was 80 kDa.

Figure 2

Sodium dodecyl–sulfate-polyacrylamide gel electrophoresis showing zone of hydrolysis of enzyme source

Native polyacrylamide gel electrophoresis

The analysis [Figure 3] showed a zone of hydrolysis in Lane no 2 with only enzyme. In Lane no 3, the specimen with enzyme and A. vera solution showed no zone of hydrolysis. The specimen in Lane no 4 with enzyme, dentin-bonding agent and A. vera solution showed no zone of hydrolysis. Lane no 5 with enzyme and dentin-bonding agent showed the zone of hydrolysis.

Figure 3

Native polyacrylamide gel electrophoresis showing different zones of hydrolysis

DISCUSSION

Mineralized dentin contains MMPs such as MMP-2,-3, 8, and 9. In addition, noncollagen-bound MMPs are also present in saliva, in dentinal tubules, and dentinal fluid.[16] Degradation of resin-sparse collagen fibrils in aged-bonded dentin in vivo has also been linked with the activation of collagen-bound MMPs and/or salivary MMPs by the use of etch-and-rinse adhesives.[17]

Contemporary adhesive dentistry is consistently focusing on the longevity of resin–dentin bond strength which degrades with time. This degradation is mainly caused by enzymatic activity due to the creation of a low pH environment.[2] The enzymes responsible for such degradations are mainly MMPs.[1819] These enzymes cause the breakdown of the hybrid layer and over time lead to decomposed resin–dentin bond.[20] Various studies have shown the inhibition of MMPs can preserve the integrity of the hybrid layer and prevents loss of bond strength.[4]

Chlorhexidine has been found beneficial and has shown improvements in the stability of the hybrid layer. The chlorhexidine was able to preserve the collagen activity for 6 months, but after 1 year, this integrity showed degradation.[21] Chlorhexidine exerts a large molecule and a water-soluble compound, which has chances to leach out of the hybrid layer.[4] Recent studies have demonstrated the toxic effect of chlorhexidine on cell cultures.[22] Therefore, it is relevant to find a substitute for improving bonding efficacy.

In this study, the results defend that the application of A. vera in the specimens inhibited MMPs activity by plate assay method and gelatin zymographic assay with and without dentin bonding agent. In the plate assay method, a zone of hydrolysis was seen around the surrounding area of the right-hand side well-containing enzyme source which indicates the enzymatic activity of the human dentin. On the left-hand side well-containing A. vera treated human dentin powder, no such zone of hydrolysis was appreciated.

In gelatin zymography analysis and in SDS-PAGE were done to analyze the molecular mass of the enzyme. NATIVE PAGE analysis showed a zone of hydrolysis in Lane no 2 with TCA precipitated enzyme demonstrating the enzymatic activity of the human dentin. In Lane no 3, the specimen with TCA precipitating enzyme and A. vera solution showed no zone hydrolysis which indicates the inhibition of enzymatic activity by A. vera. The specimen in Lane no 4 with TCA precipitated enzyme, dentin bonding agent and A. vera also showed no zone of hydrolysis which confirms the dentin gelatinolytic activity was inhibited by A. vera in the presence of dentin bonding agent. Lane no 5 with TCA precipitated enzyme and dentin bonding agent showed the zone of hydrolysis which indicates that enzyme source with dentin bonding agent underwent enzymatic activity.

Gelatinases (MMP-2 and MMP-9) share a unique structural similarity. They have catalytic collagen-binding domains and interact specifically with gelatine substrate and provide positions for cleavage.[4] Gelatin zymography is a quantifiable polyacrylamide gel-based electrophoresis approach to degrade one of their substrates. In this study, under nonreducing conditions, the gelatinases (MMPs) source from human dentin hydrolyzed electrophoretic gel impregnated with gelatin. After the staining and destaining procedure of the obtained gel, the activity of the gelatinases with its substrate was exhibited.

The gelatinolytic activity was exhibited by the formation of a zone of hydrolysis. The gelatinase enzyme source which was treated with A. vera solution showed no activity or clear zone of hydrolysis. For clinical relevance, the activity of the experimental inhibitor with dentin bonding agent was also assayed. The gelatinase enzyme source with A. vera in the presence of dentin bonding agent also successfully inhibited the gelatinolytic activity.

In recent times, the concept of using natural products in dentistry has been promoted. In clinical research, A. vera has gained considerable popularity because of its multiple anti-inflammatories and wound-healing properties. A. vera contains a substance known as aloins. The aloins interact and cause alteration in the levels of calcium ions which are required for the enzymatic activity.[23] The other proposed mechanism is aloins show a noncompetitive inhibition and may attach to sites other than active sites on the enzymes. Aloins also share a structural similarity with tetracyclin which is already known for its MMP inhibitory mechanism.[24] A. vera is an ethanol-based substrate with low-molecular weight when compared with chlorhexidine. Therefore, A. vera may become a better alternative than the presently available products like chlorhexidine in penetration into the dentinal tubules and inhibiting the MMPs activity. Various studies are available with A. vera as a cavity disinfectant; however, this is the first study done wherein A. vera was used with DBA for inhibiting MMPs; further studies are indicated regarding the bond strength and penetration of A. vera in the dentinal tubules.

CONCLUSIONS

The present study suggests the active role of gelatinases (MMP 2, 9) in human dentin. A. vera has the potential to inhibit gelatinases (MMP 2, 9) in dentin collagen. A. vera can be used with a dentin-bonding agent which shows the same inhibitory effects.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.