Comparative evaluation of transforming growth factor-beta 1 release from dentin matrix with double antibiotic paste, probiotics, and simvastatin as intracanal medicaments: An in vitro study

Abstract

Aims:

This study evaluates the release of transforming growth factor-beta 1 (TGF-β1) from root canal dentin on the application of double antibiotic paste (DAP), probiotics, and simvastatin as an intracanal medicament (ICM).

Subjects and Methods:

Forty-five extracted premolar teeth (n = 45) were collected and sectioned perpendicular to their long axis to obtain root segments 12 mm long. Access cavity preparation and biomechanical preparation were followed by irrigation with 17% ethylenediaminetetraacetic acid. Teeth were evenly distributed into three groups for different ICMs; Group A: DAP, Group B: Probiotics, Group C: Simvastatin. Samples were maintained at 37°C in 100% relative humidity for 7 days, and then the medicaments were removed with 20 mL of deionized water. TGF-β1 growth factor released was assessed by enzyme-linked immunosorbent assay kit.

Statistical Analysis:

For TGF-β1 release among groups, a one-way analysis of variance was conducted. Tukey’s post hoc analysis was carried out for pairwise comparisons (P < 0.05).

Results:

TGF-β1 release was highest for simvastatin (294.27 pg/ml) followed by probiotics (201 pg/ml) and DAP (112.11 pg/ml).

Conclusions:

A statistically significant difference in TGF-β1 release was observed among the three test groups. Simvastatin and probiotics can effectively be used as an ICM in regenerative endodontics due to their potential to release high amounts of TGF-β1 from the dentin matrix.

INTRODUCTION

Effective regeneration of the pulp-dentin complex requires the seamless integration of all three essential components of the tissue engineering triad: stem cells, growth factors, and scaffolds. The regulation of these stem cells is mediated by growth factors and signaling molecules.[1] Growth factors are bioactive molecules, either proteins or polypeptides, that alter cellular responses through intercellular communication. The dentin matrix incorporates diverse growth-promoting substances, including transforming growth factor-beta (TGF-β), bone morphogenetic proteins (BMP), fibroblast growth factors (FGF), platelet-derived growth factor, and vascular endothelial growth factors (VEGF), which exert powerful biological influences.[2] These factors are primarily produced by odontoblasts during tooth development. Following their production, these growth factors form connections with other components of the extracellular matrix, becoming integrated into the dentin matrix. This integration process is crucial for preserving their biological functionality.[3]

Among these growth factors, TGF-β1 is the most important, as it enhances collagen production by fibroblasts in the dental pulp and is crucial for odontoblast maturation and mineral embedment.[4] During the formation of dentin, inactive TGF β-1 becomes incorporated into the dentin matrix. This latent form can be released when the dentin undergoes demineralization. Various methods can be employed to activate TGF β-1, including exposure to extreme pH levels, heat application, ultrasonic treatment, integrin binding, ionizing radiation, proteolytic enzymes, and the use of chelating agents such as ethylenediaminetetraacetic acid (EDTA).[2]

Diverse intracanal medicaments (ICM) applied between appointments to enhance sterilization of the root canal network may trigger the release of biologically active growth factors from the dentin.[4,5] Root canal disinfection in regenerative endodontic procedures is commonly achieved using medicaments such as calcium hydroxide, triple antibiotic paste (TAP), and double antibiotic paste (DAP). Calcium hydroxide has been commonly used for decades due to its antimicrobial efficacy and capacity to dissolve tissue, but it alters the root canal surface, resulting in decreased fracture resistance and microhardness.[6] TAP is an efficient root canal disinfectant, but it leads to discoloration and cytotoxicity to stem cells at elevated concentrations, reduces growth factor release, and poses challenges in removing from root canals.[4]

DAP, which is composed of metronidazole and ciprofloxacin, causes no discoloration and is believed to have antibacterial properties similar to those of TAP.[7] Previous studies exist documenting TGF β-1 release on the application of TAP, but there is an absence of substantiation regarding the release of growth factors during DAP application.[8] DAP also shows a reduction in root dentin microhardness, prompting research into alternative agents that inhibit bacterial growth while preserving dentin integrity.[9]

Statins have emerged as promising materials for regenerative processes, enhancing the function of odontoblasts, improving dentin formation, and ultimately increasing tooth strength. Statins also exert antimicrobial effects against certain microorganisms, including those responsible for periodontal diseases.[10] Within the statin class, simvastatin has been widely used as an ICM. It is a structural analog of HMG-CoA and a first-line treatment for hyperlipidemia. It has numerous actions, including anti-inflammatory and antibacterial effects, promoting angiogenesis and increasing vascular endothelial cell function. However, literature evidence is inadequate to support TGF-β1 release from the dentin matrix following simvastatin application.[11]

Beneficial microbes known as probiotics confer health advantages to their host organism when ingested in sufficient quantities. They produce hydrogen peroxide, lactic acid, and bacteriocin, which collectively help eliminate harmful bacteria. Research indicates that certain strains of Lactobacillus and Bifidobacterium possess inhibitory properties against Enterococcus faecalis. However, the influence of probiotics on TGF-β1 release from the dentin matrix has not been assessed.[12]

Therefore, the objective of this contemporary research was to compare the release of TGF-β1 from the dentin matrix with the application of DAP, simvastatin, and probiotics as ICM.

SUBJECTS AND METHODS

The research protocol was approved by the Ethical Committee of the institution (GDCH/A1/MDS-Ethics Committee/IEC/45/2024-25)

Teeth selection

Forty-five (n = 45) recently extracted healthy human teeth were collected. Periodontal curettes (GDC Universal) were used to eradicate soft tissue and then these teeth were washed with sterile phosphate-buffered saline. The study only included single-rooted premolars that showed only one root canal on preoperative X-rays and lacked any anatomic abnormalities.

Root canal preparation and application of intracanal medicaments

The teeth were taken and were sectioned perpendicular to their long axis to obtain root segments 12 mm in length. Access opening was done using high-speed endo access (Dentsply Sirona) burs under water cooling. Apical patency of the tooth was checked with No. 10 and No. 15 k-files, and canals were biomechanically prepared to the size of F3 protaper (Dentsply Sirona). Irrigation with sodium hypochlorite solution (5.25%) was carried out between instrumentation. The smear layer was eliminated by applying 17% EDTA (Desmear) for 1 min. To complete the process, 5 ml of saline was utilized for a final rinse.

Samples divided into 3 groups (n = 15) based on ICM applied:

• Group A – DAP

• Group B – Probiotics

• Group C – Simvastatin.

Preparation of double antibiotic paste

Metronidazole (Flagyl 400 mg) and ciprofloxacin (Cipro 500 mg) in a 1:1 ratio were blended with 1 mL sterile water to obtain freshly prepared DAP. It was placed using lentulo spiral fillers (Dentsply Sirona) in the root canal.[13]

Preparation of probiotics

One gram of Bifilac GG mouth melt vanilla granules (Tablets India Ltd.) containing Lactobacillus rhamnosus was quantified on a laboratory scale and added to 10 mL MRS broth. The samples were then incubated at 37°C for 48 h.[14]

Preparation of simvastatin

Simvastatin (Sigma Aldrich 5 mg) powder was added to saline (500 ml) and diluted to a concentration of 1 mg/ml. After it attained a creamy consistency, the paste was placed inside the root canal.[15]

After final irrigation ICMs were placed in the 3 groups, respectively. Two layers of nail polish were applied to the roots. Subsequently, the samples were maintained in an environment with 100% relative humidity at 37°C for 7 days. The medicaments were washed away using 20 mL of deionized water, followed by a 5-min immersion in a fresh ultrasonic bath containing saline solution.

Growth factor quantification

To measure the released growth factor, a TGF-β1 enzyme-linked immunosorbent assay (ELISA) kit (Bioassay Technology Laboratory) was employed [Figure 1]. The test was performed at the Cancer and Stem Cell laboratory, Saveetha Institute of Medical and Technical Sciences. The kit’s provided TGF-β1 was serially diluted to create a standard curve. Following the manufacturer’s protocol, standards and samples were placed in 96-well plates. The optical absorbance at 450 nm was subsequently calculated using a microplate analyzer. Each sample underwent triplicate testing to obtain the final result.

Figure 1.

Transforming growth factor-beta 1 quantification with enzyme-linked immunosorbent assay kit

Statistical analysis

Statistical analysis was conducted using Statistical Package for Social Sciences (SPSS), version 20 developed by IBM in Chicago, Illinois, United States. For TGF-β1 release among groups, a one-way analysis of variance (ANOVA) was conducted. Tukey’s post hoc analysis was carried out for pairwise comparisons (P < 0.05).

RESULTS

The ELISA readings of TGF-β1 release from root canal dentin after using DAP, simvastatin, and probiotics as ICM are outlined in Table 1 and Graph 1. The greatest release of TGF-β1 was noted for simvastatin (294 pg/ml), followed by probiotics (201 pg/ml) and DAP (112 pg/ml). The ANOVA analysis indicated a statistically significant variation among the groups in terms of TGF-β1 release. Post hoc Tukey’s test presented a significant difference between the DAP and probiotics group (P = 0.025), DAP and simvastatin group (P = 0.001), simvastatin and probiotics group (P = 0.020) as depicted in Table 2 and Graph 2.

Table 1.

Comparison of the mean transforming growth factor-beta 1 release among the groups using analysis of variance

Groups	Count	Sum	Average	SD	One-way ANOVA
					F	P
DAP	15	1681.640	112.1093	3.9955	28.060647	0.000901*
Probiotics	15	3015.135	201.0090	11.8837
Simvastatin	15	4414.040	294.2693	50.0399

*Statistically significant. DAP: Double antibiotic paste, SD: Standard deviation, ANOVA: Analysis of variance

Graph 1.

Comparison of the mean transforming growth factor-beta 1 release among the groups using one-way analysis of variance. DAP: Double antibiotic paste

Table 2.

Intergroup comparison using post hoc Tukey’s test

Groups	Count	Sum	Average	SD	Post hoc Tukey’s HSD test
					t	P
DAP	15	1681.640	112.1093	3.9955	−3.655691	0.024755*
Probiotics	15	2496.835	166.4557	48.5990
DAP	15	1681.640	112.1093	3.9955	−7.490699	0.000715*
Simvastatin	15	4414.040	294.2693	50.0399
Probiotics	15	3015.135	201.0090	11.8837	−3.835008	0.02015*
Simvastatin	15	4414.040	294.2693	50.0399

*Statistically significant. DAP: Double antibiotic paste, SD: Standard deviation, HSD: Honestly significant difference

Graph 2.

Intergroup comparison using post hoc Tukey’s test. S: Statistically significant. DAP: Double antibiotic paste

DISCUSSION

The dentin matrix contains various growth-promoting factors, including TGF-β, VEGF, basic fibroblast growth factor (bFGF), and IGF1. These growth factors can aid in recruiting dental stem cells to the injury site, stimulating stem cell differentiation and promoting regeneration.[16] The release of these encased factors from dentin can be initiated during a shift from normal physiological conditions, for instance, carious lesions, or through the conditioning of dentin with different biologically active biochemical agents.[17] Despite ongoing research presenting diverse approaches for growth factor administration mechanisms in dentin-pulp engineering, neither of them has been implemented in clinical practice. Consequently, the dentin matrix, which is rich in growth factors, remains the primary clinically accessible source of essential signaling molecules for regenerative root canal strategies, such as revascularization.

For regeneration procedures, current clinical protocols suggest using a 17% EDTA solution as the final irrigant. However, the use of ICMs in between root canal sessions can influence the release of growth factors. Therefore, the quantity of bio-functional molecules released from the root canal dentin largely depends on the combined influence of irrigating solution and intracanal dressing materials used in regenerative root canal therapy.[4]

There are different methods to check growth factor release ELISA, scanning electron microscopy, immunogold labeling, and Fourier-transformed infrared spectroscopy. Among them, the ELISA test is considered highly sensitive, specific, and more convenient.[18]

TGF-β1 acts as a chemotropic substance, which causes cellular diversification and matrix assembly of odontogenic cells. It causes dental stem cell recruitment, root maturation, and apex closure. Earlier studies have examined growth factor release, but in the current study, emphasis was only on the TGF-β1 release from the root canal space with diverse ICMs.

In the present study, all three ICMs tested caused TGF-β1 release from the dentin matrix, but the highest amount of TGF-β1 released (294 pg/ml) was observed in the simvastatin group. This is because it accelerates the activity of bone and dentin matrix formation markers in human dental pulp stem cells (DPSCs). Markers such as alkaline phosphatase (ALP), OPN, OCN, OSX, Runx-2, DMP-1, and DSP are released by the initiation of the ERK1/2 signaling pathway which in turn increase the release of TGF-β1 from dentin matrix.[19] Moreover, statins have been shown to enhance the expression of bone-forming factors, including VEGF and BMP-2.[11] According to Shroff, et al., simvastatin has shown antimicrobial efficacy comparable to DAP and could be effectively used as an ICM.[15] Alghofaily et al., asserted that simvastatin aids in the healing of preprocedural apical periodontitis. Statins are known to reduce the production of interleukin-6 and interleukin-8, which supports periapical healing.[10,20]

Probiotics are seen as the next key protective mechanism, especially as growing antibiotic resistance threatens the effectiveness of current treatment. By Shaaban et al., the supernatants of probiotics were postulated as a potential alternative to the traditionally employed Ca (OH)2 ICM for combating E. facials.[12] In the current investigation, probiotics released intermediate levels of TGF-β (201 pg/ml). Cell proliferation was enhanced, ALP activity increased, and the expression of BMP-2 and bFGF genes was upregulated as a result of probiotic preconditioning. The literature supports the evidence that bFGF stimulates stem cell proliferation, which initiates the cascade of Phosphoinositide 3-kinase/Protein kinase B (AKT) (PI3K-AKT), Ras/Mitogen-activated protein kinase (RAS-MAPK), Phospholipase C gamma (PLCγ), and Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling pathways. These BMPs are closely linked to TGF-β superfamily.[21] Probiotics, known as lactic acid-producing bacteria, release significant amounts of organic acids into their environment, including lactic acid, butyric acid, acetic acid, and citric acid. The production of these acids facilitates the demineralization of the tooth’s inorganic components, which may promote the release of growth factors.[22]

According to Kim, et al. DAP has demonstrated direct and significant antibacterial effects in immature teeth. Research indicates that these antibacterial properties do not significantly affect the viability, proliferation, or mineralization of DPSCs.[23] Babaahmadi, stated that DAP can release endogenous growth factors from dentin by exposing the dentin organic matrix.[24] However, Gokturk, et al. reported that none of the available irrigation methods can completely eradicate DAP from the root canal.[25] This may block dentinal tubules and prevent growth factors from leaching out. It creates a physical barrier, thereby curtailing further growth factor release. In this study as well, DAP releases the least amount of TGF-β (112 pg/ml). This finding is cohesive with previous well-established literature.

CONCLUSIONS

From the outcomes of this study, it is presumed that simvastatin and probiotics when used as an ICM cause higher concentration of TGF-β1 release from dentin matrix in comparison to DAP. Therefore, they can be an effective alternative in regenerative endodontic procedures due to their good antimicrobial efficacy and potential to release growth factors.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.