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ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 5  |  Page : 516-520  

Evaluation of inhibitory effect of self-etch adhesives incorporated with proanthocyanidin on cysteine cathepsins present in dentin hybrid layer using gelatin zymography: An In vitro study


1 Department of Conservative Dentistry and Endodontics, Vinayaka Mission's Sankarachariyar Dental College, Vinayaka Mission's Research Foundation (Deemed to be University), Salem, Tamil Nadu, India
2 Private Practitioner, Endodontist and Conservative Dentist, Hosur, Tamil Nadu, India
3 Department of Orthodontics and Dentofacial Orthopedics, Vinayaka Mission's Sankarachariyar Dental College, Vinayaka Mission's Research Foundation (Deemed to be University), Salem, Tamil Nadu, India

Date of Submission19-Sep-2020
Date of Decision17-Oct-2020
Date of Acceptance18-Nov-2020
Date of Web Publication05-Jun-2021

Correspondence Address:
Kaarunya Ravikumar
20/86-1, Logi Street No. 2, Gugai, Salem - 636 006, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.JPBS_535_20

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   Abstract 


Aim: The aim of the study is to evaluate the inhibitory effect of single-bottle and two-bottle self-etch adhesive system after surface pretreatment with 6.5% proanthocyanidin (PA) on cysteine cathepsins (CCs) present in dentin hybrid layer using gelatin zymography. Materials and Methods: Thirty-five samples of dentin measuring 3 mm × 3 mm were obtained from 35 caries-free human third molars which were divided into seven groups. The bonded specimens were cut vertically into 1 mm thick adhesive/dentin interfaces with microtome and were pulverized into powder and subjected to zymography analysis for enzymatic activity. Results: Group VI and VII which were positive control groups had shown thicker clear bands of 50 kDa. Group IV and V which had the administration of chlorhexidine showed thicker clear bands of 50 kDa which indicated slight activity of CCs. Group II in which PA used along with single-bottle system also showed thicker clear band of 59 kDa. CCs could be active due to strong acidic nature of monomer in this group. Group III in which PA used along with two bottle system had no band formation where there is crosslinking of catalytic and allosteric domains of enzymes by PA. Conclusion: Under the limitations of this study, PA proved as a non-specific natural inhibitor of CCs. Two-bottle system showed inhibition of CCs and single bottle system failed to inhibit their activity. Future research have to be done on the bond strength and durability of self-etch adhesives after using PA and its effect on long term survival of the restorations.

Keywords: Dentin bonding agent, gelatin zymography, proanthocyanidin, self-etch adhesives


How to cite this article:
Senthamilselvan A, Ravikumar K, Mallikarjunan D Y, Annadurai T, Samynathan M, Varshini R J. Evaluation of inhibitory effect of self-etch adhesives incorporated with proanthocyanidin on cysteine cathepsins present in dentin hybrid layer using gelatin zymography: An In vitro study. J Pharm Bioall Sci 2021;13, Suppl S1:516-20

How to cite this URL:
Senthamilselvan A, Ravikumar K, Mallikarjunan D Y, Annadurai T, Samynathan M, Varshini R J. Evaluation of inhibitory effect of self-etch adhesives incorporated with proanthocyanidin on cysteine cathepsins present in dentin hybrid layer using gelatin zymography: An In vitro study. J Pharm Bioall Sci [serial online] 2021 [cited 2021 Dec 6];13, Suppl S1:516-20. Available from: https://www.jpbsonline.org/text.asp?2021/13/5/516/317514




   Introduction Top


Hybrid layer is the layer formed during bonding procedure as the result of demineralization of dentinal surface and formation of resin tags into the demineralized dentin collagen and subsequent polymerization.[1] The durability of the hybrid layer is necessary for the success and long-term retention of composite restorations. As the initial bond strength of most bonding agents proved to be more than adequate, literature have shown that it is reduced over a period of time.[2] The expected durability of the esthetic resin restoration is considered to last about only 5.7 years.[3]

The utmost requirement of achieving resin dentin bond is to maintain the hybrid layer with stable collagen fibrils. The durability of resindentin bond is mainly affected by the acidetching procedure that involves uncovering and activation of endogenous dentin proteases in the demineralized dentin matrix which includes gelatinolytic enzymes mainly cysteine cathepsins (CCs) and matrix metalloproteinases.[4],[5],[6]

The mineralized dentin matrix is considered to be found with matrix metalloproteinases mainly MMP 2, 3, 8, 9, 13, and 20. They are released by odontoblasts which induces degradation of extracellular matrix components mainly type I collagen in dentine. CCs are proteolytic enzymes which causes breakdown of collagen in dentine. Cathepsins B and L causes cleavage of collagen in nonhelical telopeptide extensions whereas cathepsin K causes cleavage of triple helical region in collagen.[7],[8],[9] Both MMPs and CCs act in slightly acidic medium and acid activation of cathepsins further activates the matrix-bound MMPs so that both proteases results in breakdown of collagen within the hybrid layer.

The biomechanical properties of dentine can be improved by bio-modifying the demineralized collagen matrices using exogenous cross-linking agents. Bio-modification reduces enzymatic degradation of collagen fibrils which in turn increases the stability of the hybrid layer and the durability of esthetic resin. Cross-linking agents may include glutaraldehyde, genipin, carbodiimide, chlorhexidine (CHX), and proanthocyanidin (PA).[10] A natural collagen cross-linking agent, PA, stimulates intrafibrillar, interfibrillar, and intermicrofibrillar cross-links in the collagen matrix.[10] PA prevents collagen degradation by exogenous collagenase activity and thus improves the mechanical properties of collagen in dentin. An increased bond strength of etch-and-rinse adhesives to both sound as well as caries-affected dentin is facilitated by PA. Literature reveals that there is no studies analyzing the gelotinolytic activity of CCs using zymography in the dentin hybrid layer formed with various self-etch adhesives after surface treatment with PA. The main objective of this study is to evaluate the inhibitory effect of single-bottle and two-bottle self-etch adhesive system after surface pretreatment with 6.5% PA on CCs present in dentin hybrid layer using gelatin zymography.


   Materials and Methods Top


Materials

  1. Self-etch adhesive (Adper Easy One, 3M ESPE)–1


    • pH–0.9×´ strong self-etch adhesives”
    • Components:


      • Acidic component–HEMA phosphates
      • Adhesives-HEMA, bisGMA, modified polyalkenoic acid


  2. Self-etch adhesive (Adhe SE Ivoclar)–2


    • pH – 1.7, intermediary strong self-etchant
    • Components:


      • Primer-phosphoric acid acrylate
      • Bisacrylamide, water,
      • Adhesive-HEMA, dimethacrylate, dispersed silicon dioxide.


  3. 0.2% CHX
  4. PA bottle
  5. Artificial saliva.


Tooth preparation

Thirty-five samples of human third molars which are caries free and extracted within 1 month were used. The teeth were collected after the patient's informed consent and stored in 0.5% chloramine T solution at 48°C. The underlying dentin in the occlusal surfaces of the tooth was exposed by grounding flat, with the help of a slow-speed water-cooled diamond impregnated disk. After removing the enamel rim, 1 mm thick middle/deep coronal dentin measuring 3 mm × 3 mm was obtained with microtome. 600grit silicon carbide paper was used to polish the surface of the obtained dentin.

Sample preparation

Thirty-five samples of dentin were obtained which was divided into seven groups. Each group was assigned with five samples and they were randomly allotted as follows.

  • Group 1 (n = 5): No bonding (Negative control) (NT)
  • Group 2 (n = 5): 6.5% of PA + self-etch adhesive 1 (SB-PA)
  • Group 3 (n = 5): 6.5% of PA + self-etch adhesive 2 (TB-PA)
  • Group 4 (n = 5): CHX 0.2% + self-etch adhesive 1 (SB-CHX)
  • Group 5 (n = 5): CHX 0.2% + self-etch adhesive 2 (TB-CHX)
  • Group 6 (n = 5): self-etch adhesive 1 (positive control) (SBA)
  • Group 7 (n = 5): self-etch adhesive 2 (positive control) (TBA).


The specimens were bonded and the bonded area was sliced vertically into 1 mm thick adhesive/dentin interfaces with microtome. They were pulvurized into powder and are subjected to zymography analysis for enzymatic activity [Figure 1].
Figure 1: Materials of the study. (a) Self-etch adhesive (Adper Easy one, 3M ESPE) - 1. (b) Self-etch adhesive (Adhe SE Ivoclar) - 2. (c) 0.2% chlorhexidine. (d) Proanthocyanidin. (e) Artificial saliva. (f) Sample preparation. (g) Pulverised powder

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Zymographic analysis

Electrophoresis was done with the dentin protein on 7.5% sodium dodecyl sulfatepolyacrylamide gels copolymerized with 2 g/L gelatin from porcine skin (Sigma Chemical) under non-reducing conditions. After electrophoresis, gels were washed in 2.5% Triton X100 with agitation and then incubated for at least 24 h at 37°C in enzyme incubation buffer (50 mM TrisHCl, pH 7.5, containing 5 mM CaCl2, 0.1 mMZnCl2, 0.2% brij nonionic detergent, 100 mM NaCl, 0.01% Triton X100, and 0.002% NaN3). Negative control zymograms were incubated in the presence of 5 mM EDTA and 2 mM 1 and 10phenanthroline for specific inhibition studies. The activation of gelatinase pro forms was achieved with 2 mM paminophenylmercuric acetate at 37°C for 1 h and staining of zymographic gels were done in 0.2% Coomassie Brilliant Blue R250 and destained. The wet gelatin zymograms were scanned at 600 nm with the help of densitometer and image analyzer. This technique identifies the enzymes by the degradation of their preferential substrate and by their molecular weight. The active enzymes degrade the gelatin and the gelatinolytic activity of the enzymes then appear as cleared sharp bands over a dark background.


   Results Top


Zymograms of gelatinolytic activity are shown in the [Figure 2] and [Table 1]. Gelatinase bands were identified by zymography in five samples only. The migration of enzymes which is a characteristic of cathepsinB were observed as Clear bands at molecular mass of 50 kDa as shown in [Figure 2]. After acid etching, zymograms showed clear, thicker bands on 1–7 days' time interval in five groups. Group I which is a negative control group had no formation of band. Group VI and VII which were positive control groups had shown thicker clear bands of 50 kDa. Group IV and V which had administration of CHX showed thicker clear bands of 50 kDa which indicated slight activity of CCs. Group II in which PA used along with single-bottle system also showed thicker clear band of 59 KDa. CCs could be active due to strong acidic nature of monomer in this group. Group III in which PA used along with two-bottle system had no band formation where there is crosslinking of catalytic and allosteric domains of enzymes by PA [Table 1]. This indicated that the CCs were inhibited by PA when used with two bottle system.
Figure 2: Zymography analysis

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Table 1: Zymography analysis

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   Discussion Top


The enzymatic activity of various proteases mainly MMPs and CCs are responsible for degradation and demineralization of dentinal hybrid layer. The host-derived MMPs produces degradation of extracellular matrix components in the hybrid layer and these MMPs are activated by CCs. The activation of both the CCs and MMPs is majorly achieved in mild acidic environment. Degradation of type I collagen in dentine was mainly due to MMPs. During acid-etching of dentine, the inactive matrix-bound MMPs get activated but the acidity of phosphoric acid produces denaturation of it. The inhibitory effect of phosphoric acid on these MMPs is only short term as ensuing application of mildly acidic self-etch[11] and etch-and-rinse adhesives[12] recreates the environment for these MMPs. This results in continuous proteolytic degradation of exposed collagen fibrils within the hybrid layer which may lead to failure of resin dentinal bond over a period of time. Hence, there is a strong and direct association between these host-derived proteases mainly MMPs and CCs and progressive collagen degradation within the hybrid layer.[7],[8],[9] The current research efforts are mainly implicated on the preservation and strengthening of the collagen matrix with the help of MMP inhibitors to achieve resistant and long lasting dentin bonding.

The various cross-linkers in commonly used in dentistry include glutaraldehyde, genipin, carbodiimide, CHX, and PA. PA are condensed tannins which are derived from various plants such as palms, elm tree, and grape fruits. It is also present in minor concentration in edible fruits and vegetables. It behaves as a strong antioxidant and has the ability to form insoluble complexes with carbohydrates and proteins due to its highly hydroxylated structure. It is also an effective cross-linking agent with least cytotoxicity. The interactivity of PA with proline-rich proteins like collagen occurs by covalent bonding, ionic bonding, hydrogen bonding, and hydrophobic bonding interactions.[13],[14],[15] The development of hydrogen bonds between the hydroxyl group of phenols and carbonyl groups of amide proteins helps in stabilizing PA-treated collagen and improving its mechanical properties. In addition, the triple helical structure present in collagen also promotes the availability of the carbonyl oxygen of the peptide which is essential for hydrogen bonding.

Gelatin zymography is mainly used for the detection of gelatinase activities by the degradation of their preferential substrate and by their molecular weight. Although it is one of the most simple and easiest methods, it is an extremely sensitive technique. The innovative approach of this study was to evaluate the inhibitory effect of self-etch adhesives incorporated with PA on CCs present in dentin hybrid layer using gelatin zymography. CHX and PA were used in this study to find out the changes in activity of enzymes at the hybrid layer to prevent the proteolytic degradation of resin-dentin bonds. The dentin proteins of the seven groups were subjected to zymographic analysis and the gelatinolytic activity of zymogens is shown in the [Figure 2] and [Table 1]. Out of seven groups, five groups showed thicker clear bands of gelatinolytic activity. Positive control groups VI and VII showed thicker band of 50 KDa which indicated the activation of Cysteine cathepsins. Due to low acidic nature of monomer, Pro MMPs were produced which further led to the activation of MMPs and CCs.

In this study, CHX was used along with adhesives in group IV and V to evaluate the inhibitory effect of CCs. Previous research studies revealed that CHX when used in the form of aqueous solution produces potent inhibition of MMPs and reduces the solubility of collagen fibrils. When applied over acid-etched dentin, CHX does not have negative impact on the immediate micro-tensile bond strength of adhesive to the substrate. New emerging in vivo and in vitro studies have implicated that the administration of CHX to dentinal surface after acid etching and before the application of adhesive system prevents and reduces the degradation of collagen fibrils exposed at the bottom of the hybrid layer. This helps in development of resistant and durable resin dentin bonding over a period of time.[16],[17],[18],[19],[20] In our study, there is slight activity of CCs due to acidic monomer which is controversy to other studies.

As per many studies, CHX is considered to decrease the degeneration of hybrid layers.[16],[17] However, the newly emerging studies proved that the CHX transiently inhibits the activity of MMPs only for about 9–12 months.[21],[22] It is also proved that it must remain tightly attached to the enzymes at high concentrations for an MMP inhibitor to be potentful against proteolytic enzymes. Since, CHX is water-soluble, it can be easily displaced by competing cations from dentinal fluids and salivary fluids which may hinder the prolonged inhibitory effect of MMPs.[21],[22] Further research is needed in terms of the use of CHX as an MMP inhibitor because of its cytotoxicity to the dentine pulp cells.[23]

Group III in which PA used along with two bottle system had no band formation. Hence, there is no activity of CCs due to crosslinking of catalytic and allosteric domains of enzymes by PA. The activity of collagenase is hindered which lead to impaired production of CCs and MMPs. Hence, the durability of restoration will be improved. When compared with CHX, PA has better advantages as it is non-toxic to pulp, deeper penetration, and detaches collagen bounded MMPs, extended period of action due to hydrogen bonding and inhibits non collagenous proteins such as Feutin-A, DMP-1, and bone sialoprotein.[24]


   Conclusion Top


Under the limitations of this study, PA proved as a non-specific natural inhibitor of CCs. Two-bottle system showed inhibition of CCs and single bottle system failed to inhibit their activity. PA facilitates improved bond strength between etch-and-rinse adhesives and dentin surface. The activity of collagenase is hindered which lead to impaired production of CCs and MMPs. Hence, the durability of restoration will be improved. Future research has to be done on administration of self-etch adhesives along with PA and its effect on biomechanical properties and long-term survival of the restorations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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