A Comparative Evaluation of Efficacy of Root Surface Biomodification using MTAD, MTAD+I-PRF on Adhesion of Fibrin Clot to Dentin Sem Study

ABSTRACT

Root biomodifiers help in removing the smear layer following mechanical debridement. In this context, we evaluated and compared the in vitro efficacy of MTAD, MTAD+I-PRF, and phosphate-buffered saline-conditioned dentin surfaces by examining the distribution of the fibrin network using scanning electron microscopy. It was concluded that MTAD can serve as a potentially useful root conditioner/biomodifier. Further, the adjunct of MTAD+I-P resulted in more fibrin network linkage on the dentinal surface when compared to MTAD alone which can be of great utility in Advanced Regenerative Therapy.

INTRODUCTION

The primary goal of periodontal therapy is to eliminate dental calculus and bacterial deposits. However, this leads to the creation of smear film resulting in impaired periodontal healing. Many studies have examined various chemical conditioning techniques since the possibility of periodontal regeneration could rise with the successful removal of the smear layer.[1] In practice, such smear layers over the root can be dissolved by subjecting the root to chemical treatment using agents such as citric-acid-4.25%, Ethylenediaminetetraacetic (EDTA)-17%, Tetracycline hydrochloride acid-10% and phosphoric acid-37%. After dissolving the smear layer, the collagen is exposed on the root surface, thereby facilitating the attachment of fibroblasts to the root.[2] Torabinejad et al. in 2003 introduced Biopure MTAD (Dentsply Tulsa Dental, USA). The composition of MTAD is 4.25% citric acid, 3% doxyclinehyclate (an isomer of tetracycline), and 0.5% polysorbate 80 (a foaming agent). This MTAD is bactericidal to Enterococcus faecalis and effective in the removal of the intracanal smear layer and thereby helpful for use in periodontal conditioning therapy.[3] Choukron et al., in 2001, first described the utility of Platelet-Rich Fibrin (PRF) using patients’ own plasma that can facilitate tissue regeneration through its effect on vascularization.[4] A liquid form of PRF is the Injectable PRF called I-PRF which contains fibronectin. The application of fibronectin on root surfaces enhances cellular proliferation from the periodontal ligament to the supracrestal parts.[5] The use of I-PRF results in increased expression of various growth factors like PDGF, TGF-β, and collagen which induces higher fibroblast migration.[6] Now the question arises is that if MTAD significantly removes the smear layer from the root surface,[7] then how effective it is on fibrin clot adhesion? In this context, by using a scanning electron microscope, we evaluated and compared the in vitro efficacy of MTAD, MTAD+I-PRF, and phosphate buffered saline [PBS]-conditioned dentin surfaces by examining the dentin surface for the distribution of the fibrin network.

MATERIALS AND METHODS

In the present in-vitro study, ten chronic periodontitis patients with no systemic disease requiring extraction of teeth having hopeless prognoses were identified. The samples were taken as per the guidelines in the article published by Fabio Renato.[8] The data set was analyzed by following a non-parametric test, according to CRIS guidelines.[9] Inclusion Criteria for tooth extraction: 1. Single rooted teeth with advanced periodontal disease 2. Teeth with attachment loss of more than 5 mm or 50% bone loss 3. Teeth with Grade- 3 mobility 4. Teeth with hopeless prognosis. Exclusion Criteria for tooth extraction: 1. Fractured teeth 2. Teeth having root resorption 3. Teeth with root caries 4. Teeth having undergone endodontic treatment or prosthodontic restoration 5. Teeth having anomalies of root (e.g. Tori, Hypercementosis, Enamel pearl) 6. Those who received antibiotics in 6 months/or NSAIDs in the last 3 months.

Sample Preparation – The extracted teeth were washed with 0.9% saline and debris was removed with curettes. Using the diamond disc at the cementoenamel junction, the crown portion of the tooth and the apical one-third of each root were separated. The residual midfoot was sectioned through the root canal in a longitudinal plane followed by flattening of the root surface using high-speed fissure burr. From each root, three dentin blocks with approximate size of 4 mm × 3 mm × 1 mm were prepared. So essentially, a sum of 30 specimens of dentin blocks were created from 10 extracted teeth.

Preparation of I-PRF and Specimen – Peripheral venous blood of 10 ml was collected in two sterile vacutainer plastic tubes of 10 ml with no anticoagulant and centrifuged immediately (Remi R-8C, 16 × 15 ml Laboratory Centrifuge, India) at standard room temperature and relatively low centrifuge speed of 700 rpm for 3 mins. At the end of the spin, the supernatant is a liquid (i-prf) and stays liquid for at least around 10–12 mins. The upper 1 ml was collected as i-prf using a 21 G needle mounted on a syringe of the preparation layer was removed using 1 ml syringe. The block specimens were then treated with clinical agents like PBS, MTAD, and MTAD + I-PRF using a cotton pellet. After rinsing the specimen for 3 minutes, the block specimens were left for air drying for 20 minutes followed by fixation at ambient temperature using 2.5% glutaraldehyde (pH 7.2) for a duration of 30 minutes. After fixation, the saline wash was done thrice for five minutes each. The specimens were then subjected to desiccation by placing them for five minutes in a varying concentration of aqueous ethanol (25%, 50%, 70%, 90%, twice in 95%) in each concentration. Later, further desiccation was done twice for 3 minutes each using hexamethyl disilazine. Using a sputter coater, a tiny layer of the conductive substance, gold palladium, was then coated onto the specimens, which had been mounted in SEM stubs.

Scanning Electron Microscope Analysis – The specimens were examined using SEM (Voltage of 15-20 KV) under 5000X magnification with no tilt angles. To get an overall sense of the surface topography, every dentin block was carefully scanned and examined for the placement of the fibrin strands and the adsorption and adherence of the blood component. The scoring of the degree of fibrin network linkage to the conditioned root surface was done as follows:

Score 0: Absence of fibrin network linkage to dentin surface

Score 1: Scarce fibrin network linkage to dentin surface

Score 2: Moderate fibrin network linkage to dentin surface

Score 3: Dense fibrin network linkage to dentin surface

Statistical analysis

The master chart study data in Microsoft Excel was uploaded into the IBM SPSS 20.0 version software for statistical analysis. The mean and standard deviation were calculated for the descriptive statistical analysis whereas the Mann–Whitney U test and Kruskal–Wallis test ranks were used for qualitative data analysis. To determine statistical significance, a P value of less than 0.05 was taken as significant.

RESULTS

We found that the fibrin network formed on the dentin surfaces within each treatment group had consistent characteristics with little intragroup variations as shown in Tables 1–3.

Table 1.

Scores-wise distribution of dentin blocks (specimens)

Scores	Group-A		Group-B		Group-C
	Number	%	Number	%	Number	%
0	8	80.0	1	10.0	0	0.0
1	2	20.0	7	70.0	0	0.0
2	0	0.0	2	20.0	2	20.0
3	0	0.0	0	0.0	8	80.0
Total	10	100.0	10	100.0	10	100.0

Table 3.

Pairwise comparison between the study groups using Mann–Whitney U test with Bonferroni correction

Assessment observations	Descriptive statistics			Mann–Whitney U test ranks			Test statistics
	Groups	Mean±SD	n	Mean rank	Sum of ranks	U	Z	P	Significance
Comparison Group A v/s B	Group-A	0.20±0.40	10	6.80	68.50	13.00	3.091	P=0.004	HS
	Group-B	1.10±0.53	10	14.20	142.0
Comparison Group A v/s C	Group-A	0.20±0.40	10	5.50	55.0	0.000	4.047	P=0.000	VHS
	Group-C	2.80±0.40	10	15.50	155.0
Comparison Group B v/s C	Group B	1.10±0.53	10	5.70	57.0	2.000	3.852	P=0.000	VHS
	Group C	2.80±0.40	10	15.30	153.0

NS=Not significant, S=Significant, HS=Highly significant, VHS=Very highly significant

Table 2.

Comparison of scores of fibrin network between the groups

Groups	Treatment	Descriptive statistics		Kruskal–Wallis test ranks Mean rank	Test statistics
		Mean±SD	n		Test value	P	Significance
Group-A	PBS (pH 7.4)	0.20±0.40	10	6.80	24.106	P=0.000	VHS
Group-B	MTAD	1.10±0.53	10	14.40
Group-C	MTAD + I-PRF	2.80±0.40	10	25.30

NS=Not significant, S=Significant, HS=Highly significant, VHS=Very highly significant

DISCUSSION

The main intent of any periodontal therapy is to achieve adequate regeneration of the periodontal attachment which is damaged due to the periodontal disease. We know that the mechanical debridement done as part of periodontal therapy leaves a smear layer covering the debrided root surface.[1,10] Various chemicals like citric acid, EDTA, and tetracycline plus techniques like laser have shown successful eradication of the smear layer.[11,12] As a final endodontic irrigant to clean the canal and eliminate the smear layer, Torabinejad et al. created BIOPURETM MTAD (a combination of tetracycline, acid, and detergent).[3]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.