A comparative study of demineralized freeze-dried bone allograft alone and with 1% metformin in the treatment of intrabony defects in patients with chronic periodontitis: A randomized clinical trial

Abstract

Background:

Periodontal disease is ubiquitous and its treatment requires a detailed treatment plan. Biomaterials are often used along with demineralized freeze-dried bone allograft (DFDBA) for periodontal regeneration. One percent metformin has evolved as a regeneration material. This study was initiated to assess and compare the regenerative potential of DFDBA alone and with 1% metformin in the treatment of intrabony defects in subjects with chronic periodontitis.

Materials and Methods:

20 sites with intrabony defects were diagnosed of which 10 were assigned to Group A (1% Metformin + DFDBA) and 10 to Group B (DFDBA alone). Clinical parameters were measured at baseline, 3, 6, and 9 months postoperatively, whereas radiographic parameters were measured at baseline and 9 months postoperatively, and data were statistically analyzed.

Results:

Both the groups showed a statistically significant improvement in probing pocket depth and relative attachment level at 9 months. Radiographically, a statistically significant reduction in defect depth was seen in both the groups at 9 months. There was no statistically significant difference between the two groups as far as crestal bone loss is concerned. No statistically significant difference was seen for clinical and radiographic parameters between the test and control groups.

Conclusions:

The addition of 1% metformin to DFDBA did not have any additional benefits in the treatment of subjects with defects which are intrabony.

INTRODUCTION

Regeneration entails reconstituting or reproducing a part which may be lost or damaged.[1] Various therapeutic modalities for achieving regeneration have been investigated.[2]

Bone grafts are used as a scaffold, to form new bone and to regenerate periodontium. Routinely, demineralized freeze-dried bone allograft (DFDBA) was either alone or in conjugation with other biomaterials. In controlled human histological studies, DFDBA has been shown to regenerate periodontium.[3]

Metformin HCL (1,1-dimethylbiguanide HCL) (MF) is a biguanide belonging to the second generation which is used to treat diabetes mellitus (Type II).[4] Recently, it has also shown potential to stimulate bone regeneration.[5]

To the best of our knowledge, the combination of 1% metformin + DFDBA has never been compared with DFDBA alone in subjects with intrabony defects. Hence, this study was initiated to evaluate and compare the potential of DFDBA alone and with 1% metformin in intrabony defects in subjects with chronic periodontitis.

MATERIALS AND METHODS

It was a split-mouth, single-blinded, randomized controlled trial study. This clinical trial followed the CONSORT criteria [Figure 1] and the CONSORT checklist attached. Based on the results of the previous study, the sample size was calculated using G*Power 3.0.10. The error was set at 5% (0.05) and the power of the study was set at 80%. Twenty sites were equally divided into two groups. They were assigned randomly to Group A (test group) (DFDBA + 1% metformin) and Group B (control group) (DFDBA) by computer-assisted randomization. This study was done from March 2018 to May 2019, and subjects from the outpatient department of periodontology were chosen. Clearance from the institutional ethical committee was taken, and the trial was registered in the Clinical Trials Registry-India. The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. Written informed consent explaining the nature, duration of the trial, and surgical procedure was obtained from each patient.

Figure 1.

Consort flowchart. n – Sample size

The inclusion criteria included systemically healthy subjects having chronic periodontitis; willing to give informed consent; indicated for flap surgery; and having at least two intrabony defects, one on either side of the same arch with interproximal probing depth of not <5 mm after phase 1 therapy.

The exclusion criteria included (1) female subjects who were pregnant and lactating; (2) subjects using tobacco; (3) subjects in whom periodontal surgery was done in the past 6 months in the same site; (4) subjects with aggressive periodontitis; and (5) any medications are known to affect periodontal therapy outcomes.

The clinical parameters that were recorded presurgically and postsurgically at intervals of 3 months, 6 months, and 9 months were as follows:

1. Plaque index (Silness and Löe)[6]

2. Gingival index (Löe and Silness)[7]

3. Probing pocket depth (PPD)

4. Relative attachment level (RAL) (measured from the lower border of the stent to the base of the pocket).[8]

Radiographic evaluation (i.e., defect depth [DD] and height of alveolar bone) was done using paralleling angle technique at baseline and after 9 months of surgery [Figure 2]. Intraoral periapical (IOPA) radiographs were obtained with a stent in situ by paralleling angle technique using a position-indicating device (XCP Rinn, Dentsply). A radiographic grid of 1 mm² framework was also used on IOPA X-ray film.[9] The depth of the defect was measured from the lower border of the steel ball present interproximally with respect to the concerned tooth to the most apical portion of the defect seen on the radiograph. The distance was measured using a divider and transparent scale. Similarly, the height of the alveolar bone was measured from the lower border of the steel ball present interproximally with respect to the concerned tooth to the crest of the alveolar bone. Although all the radiographs were taken with position indicating device and parallel angle, each radiograph was corrected for enlargement and foreshortening of each stainless steel ball before comparison with the true value. It was done to eliminate magnification defects.

Figure 2.

IOPA-baseline (test) and 9 months postoperative (test). IOPA – Intraoral periapical

The following formula was used.[10]

Fabrication of stent:

Presurgical procedure: In the present study, all subjects underwent a thorough oral prophylaxis (Phase I therapy). Four weeks after the nonsurgical phase, subjects were reevaluated. Subjects maintaining good oral hygiene with intrabony defects on either side of the same arch and with pocket depth ≥5 mm were selected. The subjects were assigned to two groups (computer generated) and were communicated to the surgeon before the surgery by an independent examiner. Group A consisted of 10 sites (test group) where intrabony defects were treated with DFDBA + 1% metformin and Group B consisted of 10 sites (control group) where intrabony defects were treated with DFDBA alone. DFDBA of particle size 500–750 mm was obtained (Tata Memorial Hospital Tissue Bank, Mumbai). Preparation of 1% metformin gel was done as described by Pradeep et al.[11]

Formulation of 1% MF gel: A mixture of distilled water and dry gellan gum powder was mixed for 20 min at a temperature of 95°C with a magnetic stirrer (EMS V-Dent, Shantou, Guangdong, China). Hydrous gellan gum formation was seen. Maintaining the temperature at >80°C, mannitol was added. Following this, the required weighed amount of MF, sucralose, and citric acid was put in. The solution was stirred during the entire procedure. Ten milliliters of distilled water in which sodium citrate was dissolved was then added to this mixture. It was then allowed to cool at room temperature so that it forms into gel.[11] Release kinetics of formulation of 1% MF gel were not studied.

Surgery was carried out by a calibrated periodontist. For all the surgeries, the same periodontal probe (Hu-Friedy PCPUNC 15 mm, Hu-Friedy, Chicago, IL, USA) was used. Preprocedural rinse with 0.12% chlorhexidine was done. Extraoral asepsis was done with Povidone-iodine. After adequate local anesthesia (2% lignocaine with 1:80,000 adrenaline), a crevicular incision using a no. 15 surgical blade was made. A mucoperiosteal elevator was used to raise a full-thickness flap exposing 1–2 mm of healthy bone. The defects were debrided using Gracey curettes (Gracey, Hu-Friedy, Chicago, IL, USA). Scaling and root planing was done using ultrasonic scalers (EMS V-Dent, Shantou, Guangdong, China) and curettes. Presuturing was done for both the groups. DFDBA + 1% metformin [Figure 3] was then placed in the defects in Group A (test group), whereas DFDBA alone was placed in Group B (control group). The flap was sutured with a 4-0 braided black silk suture. Periodontal dressing was applied on the surgical site (Coe-Pak, GC America Inc., Chicago, IL, USA). Sutures and dressing were removed after 7 days, and normal saline was used to irrigate the surgical site. No subjects experienced any allergic reactions.

Figure 3.

One percent metformin + DFDBA. DFDBA – Demineralized freeze-dried bone allograft

Medicines were prescribed that included analgesics and antibiotics for 7 days (500 mg amoxicillin, TDS for 7 days; 400 mg metronidazole, TDS for 7 days; and 800 mg ibuprofen, TDS), and postoperative instructions were given. Subjects were advised to use a 0.2% chlorhexidine digluconate mouthwash for 2 weeks postsurgery.[12,13]

Statistical analysis

All data regarding clinical and radiographic parameters were tabulated and subjected to statistical analysis using the SPSS software (SPSS version 16.0, SPSS, Chicago, IL, USA). Intragroup analysis was done by one-way analysis of variance with post hoc Bonferroni test. Intergroup analysis was done by paired t-test. The level of significance was established at P ≤ 0.05.

RESULTS

Out of 10 subjects (20 sites), no one dropped out [Figure 1]. All subjects showed uneventful healing, and there was no allergic reaction to metformin.

Table 1 shows the mean ± standard deviation values of all clinical and radiological parameters at baseline. Clinical and radiographic parameters of both the groups showed no statistical difference at baseline.

Table 1.

Baseline values

Baseline	Group A	Group B	P
PPD	6.4	6.1	0.737
RAL	10.6	10.3	0.215
DD	7.91	7.84	0.677
Crestal Bone height	6.58	6.58	0.848

P<0.05. PPD - Probing pocket depth (mm); RAL - Relative attachment level (mm); DD - Defect depth; CBH - Crestal bone height; P – P value

The mean plaque and gingival index was 0.689 ± 0.79 and 0.620 ± 0.96, respectively, at baseline which reduced to 0.535 ± 0.99 and 0.531 ± 0.147 at 3 months, 0.533 ± 0.13 and 0.545 ± 0.12 at 6 months, and 0.503 ± 0.16 and 0.523 ± 0.14 at 9 months, respectively. The reduction from baseline to all recall intervals was statistically significant. This shows that good oral hygiene was maintained by all subjects throughout the study [Table 1].

Table 2 shows the probing depth reduction, DD reduction, change in crestal bone height, and RAL gain at all recall intervals.

Table 2.

Reduction and percentage change in probing pocket depth, gain, and percentage change in relative attachment level, mean reduction, and percentage change in defect depth and crestal bone loss at 3, 6, and 9 months

Period	Reduction and percentage change in PPD (mm)			Mean gain and percentage change in RAL (mm)			Mean reduction and percentage change in DD (mm)			Mean reduction and percentage change in CBH (mm)
	Group A (%)	Group B (%)	P	Group A (%)	Group B (%)	P	Group A (%)	Group B (%)	P	Group A (%)	Group B (%)	P
3 months	1.9 (26.9)	1.6 (26.7)	1.066	1.9 (18)	1.6 (15)	0.283	-	-	-	-	-	-
6 months	2.9 (45.1)	2.7 (44.6)	0.673	2.8 (26.41)	2.4 (25.20)	0.591	-	-	-	-	-	-
9 months	3.6 (56.0)	3.2 (52.4)	0.749	3.4 (46.1)	3.0 (42.06)	0.630	1.19 (16.81)	1.05 (16)	0.856	−0.14 (2.12)	−0.35 (5.31)	0.690

P value of <0.05. PPD - Probing pocket depth (mm); RAL - Relative attachment level (mm); DD - Defect depth; CBH - Crestal bone height; P - P value

All clinical and radiological parameters showed improvement at 9 months (P < 0.05) when compared to baseline. The difference was statistically significant. There was a greater reduction in PD in Group A (56%) compared to Group B (52.40%) [Figure 4]. The gain in RAL was more in Group A (46.10%) compared to Group B (42.06%). Furthermore, the reduction in DD was more in Group A (16.81%) compared to Group B (16.00%). Group A also showed less crestal bone resorption compared to Group B [Table 2].

Figure 4.

Percentage change in probing pocket depth, relative attachment level, defect depth, and crestal bone loss at 9 months

However, the difference in improvement of all clinical and radiological parameters between both the groups did not show any significance statistically.

DISCUSSION

Periodontitis results in the destruction of the attachment apparatus of the tooth which then leads to tooth loss.[14]

Heitz-Mayfield and Lang, in 2013, reviewed critical probing depth and they observed that deeper pockets with 5.4 mm depth showed a greater gain of CAL with periodontal surgery. Hence, in our study, teeth with PPD ≥5 mm were selected.[15]

A common approach for the treatment of such defects is periodontal flap approach with bone grafts into the properly debrided bony defects.[16]

Bone replacement grafts help the host’s resident cells to form a scaffold so as to create either osteoconductive or osteoinductive pathways or may have osteogenic factors.[17,18]

DFDBA has gained popularity for regeneration of the PDL tissue when used either alone or in combination with other biomaterials.[19] It has both osteoinductive and osteoconductive activity. It induces the formation of osteoblasts by stimulating undifferentiated mesenchymal cells.[19] It also fills up the space and maintains the volume of the defect.[5,20] According to the consensus report by the World Workshop (1996), DFDBA is considered to fulfill almost all the criteria considered paramount for the promotion of periodontal regeneration.[21] Hence, DFDBA has been used in this trial.

In recent years, new biomaterials along with bone grafts have been researched. One such biomaterial “metformin” has recently shown potential for bone regeneration and hence was used in our study. Metformin HCL (1,1-dimethylbiguanide HCL) (MF) was introduced in 1922 and is routinely used for diabetes mellitus therapy.[8] Bak et al. concluded that metformin helps to form bone in periodontitis induced using ligatures by upregulating osteoblast differentiation.[5] It stimulates osteoprotegerin and reduces receptor activator of nuclear factor-kappa β ligand expression in osteoblasts. It also inhibits osteoclasts and halts the loss of bone in ovariectomized rats.[5]

Our study was a split-mouth randomized controlled trial where 20 intrabony sites in 10 subjects were selected. The split-mouth study design was used because this permits evaluation of the same host response in two different treatment modalities and also it minimizes the bias that would arise due to variation in the healing pattern among individuals. It also eliminates systemic and environmental factor variations.

Subjects with systemic factors such as diabetes mellitus, immunodeficiency virus (HIV), anemia, atherosclerosis, coronary heart disease, smoking tobacco in any form, pregnancy, and lactation were excluded from the study because these factors modify specific modes in the critical pathway of periodontal pathogenesis and healing.[22,23] Subjects with aggressive periodontitis and those who have undergone periodontal surgery in the past 6 months in the same site were also excluded.

In the present study, subjects in both Groups A and B showed improvement in all clinical parameters at all recall intervals when compared to baseline. The difference was statistically significant (P = 0.000, P ≤ 0.005). This was in accordance with the studies performed by Mellonig,[24] Pearson et al.,[25] and Quintero et al.[26] who also concluded that DFDBA + open flap debridement (OFD) resulted in greater clinical improvement than OFD alone.

There was a statistical improvement in PD, RAL, and DD from baseline to all recall intervals even in Group A.

Till date, there is no study which compares 1% metformin + DFDBA and DFDBA alone. However, a similar study by Pradeep et al.[11] compared four groups of subjects who were treated with OFD, OFD with PRF, OFD with 1% metformin, and OFD + PRF + 1% metformin. They found that the mean reduction in PPD and the mean gain in RAL were statistically significant from baseline to 9 months in all the groups.

The above results are in accordance with the results obtained in our study where DFDBA + 1% metformin group showed a statistically significant improvement in PD and RAL at 9 months postoperatively.

In the study by Pradeep et al.,[11] there was a statistically significant reduction in DD in the PRF + 1% metformin group as compared to PRF alone. In our study, although the reduction in DD was more in Group A when compared to Group B, it was not statistically significant.

Research has shown that some amount of alveolar crestal resorption is always seen post-OFD.[27]

According to a study conducted by Mai et al.,[28] metformin inhibits osteoclasts and prevents loss of bone in ovariectomized rats. Hence, in the present study, alveolar bone height was evaluated pre- and postoperatively to determine if metformin has some added benefit in preventing crestal bone resorption.

In our study, the mean crestal bone loss in Group A and B was 0.14 mm and 0.35 mm. Although crestal bone loss was less in Group A compared to Group B, the difference was not statistically significant (P = 0.75). This indicates that the addition of 1% metformin to DFDBA did not have any additional benefit in preventing crestal bone loss. Our study was in accordance with Bak et al. who evaluated the role of metformin in osteoclast differentiation. They concluded that combination treatment of metformin with stimulators such as 1,25(OH) 2D3, LPS, or PGE2 did not influence osteoclast formation. They also concluded that the osteoclast differentiation was not apparent at concentrations of metformin that could exert an osteogenic effect.[5]

According to a study done by Cortizo et al., they investigated the effect of metformin on the growth and differentiation of osteoblasts, two osteoblast-like cell lines (MC3T3E1 and UMR106). They concluded that maximal stimulation was reached at a concentration of 200 μM (115% of basal) for both cell lines. They also examined the effect of metformin on osteoblast differentiation, alkaline phosphatase activity, and Type I collagen production. They show that the metformin significantly stimulated alkaline phosphatase in a dose-dependent manner, with a maximum at 100-μM concentration (145% of basal). In addition, metformin dose dependently stimulated the production of Type I collagenous matrix, and metformin at 500 μM significantly stimulated (385% basal) calcium deposition in mineralized nodules.[29]

Limitations

1. A small sample size could be the limitation of the present study

2. The required concentration and release kinetics of metformin gel used in the study were not carried out. This could be another limitation of the present study

3. More histological and longitudinal studies with a larger sample size need to be conducted to prove the efficacy of metformin in periodontal regeneration.

CONCLUSIONS

To the best of our knowledge, there is no published literature comparing the effect of 1% metformin + DFDBA and DFDBA alone in the treatment of subjects with defects that are infrabony. Although there were statistically significant improvements in all clinical (PD and RAL) and radiological (DD and crestal bone height) parameters at 9 months in both the groups, the addition of 1% metformin to DFDBA showed no additional benefits. However, further research with a greater sample size and histological analysis is needed to confirm our results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.