Growth differentiation factor-15 as predictor of periodontal treatment effects in patients with periodontitis: a randomized-controlled clinical trial

doi:10.21203/rs.3.rs-2581177/v1

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Growth differentiation factor-15 as predictor of periodontal treatment effects in patients with periodontitis: a randomized-controlled clinical trial

https://doi.org/10.21203/rs.3.rs-2581177/v1

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published 21 Aug, 2023

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Background: It has been shown that periodontal treatment could potentially decrease the risk of developing cardiovascular disease (CVD) and that growth differentiation factor 15 (GDF-15) is a valuable biomarker of early CVD risk. This study evaluated the efficacy of periodontal treatment on growth differentiation factor 15 (GDF-15) and related circulating systemic risk biomarkers such as glutathione peroxidase 1 (GPx-1), c-reactive protein (hs-CRP), and surfactant protein D (SP-D) and explored whether patients with high GDF-15 at baseline showed increased clinical benefits with two non-surgical treatments protocols at 6-months follow-up.

Methods: Patients with periodontitis were randomized to receive quadrant (Q-SRP, n=23) or full-mouth disinfection (FMD, n=23) protocols. Clinical and periodontal parameters were recorded in all enrolled patients. Serum concentrations of GDF-15, GPx-1, hs-CRP, and SP-D concentrations were assessed at baseline and 30, 90, and 180-days follow-up through enzyme-linked immunosorbent assay (ELISA) and nephelometric assay techniques.

Results: Q-SRP was more effective than FMD in lowering periodontal parameters, and the mean proportions of GDF-15 (p=0.005), GPx-1 (p=0.025), hs-CRP (p<0.001), and SP-D (p=0.042) after 6 months of treatment. Periodontal parameters were significantly associated with concentrations of GDF-15, GPx-1, hs-CRP, and SP-D at 6-months follow-up. Furthermore, the ANOVA analysis revealed that Q-SRP significantly influenced the upregulation of GPx-1 and on downregulation of GDF-15, and SP-D and substantially impacted the effectiveness of periodontal therapy at 6 months follow-up.

Conclusion: The results evidenced that, in comparison with FMD, Q-SRP was more effective at reducing periodontal variables and positively impacting GDF-15, GPx-1, hs-CRP, and SP-D concentrations, although patients who exhibited higher GDF-15 and SP-D and lowered GPx-1 at baseline, more benefited the effects of periodontal treatment at 6-month follow-up.

Periodontitis

treatment outcome

growth differentiation factor 15

related circulating systemic risk

glutathione peroxidase 1

c-reactive protein

and surfactant protein D

serum

periodontal debridement

Full mouth disinfection

clinical trial

Periodontitis is a chronic, multifactorial inflammatory illness produced by infectious biofilm that might lead to the deterioration of tooth-supporting tissues and tooth loss if not prevented and treated appropriately [1]. Several large-cohort studies have acknowledged in recent decades that periodontitis may afflict more than half of the world's population and negatively impact a number of systemic disorders, including cardiovascular disease (CVD) [2], diabetes [3], metabolic syndrome [4] that impact the lower overall low quality of life [5]. Some evidence suggests that the development and progression of periodontitis is connected with the dysregulation of certain pro-inflammatory mediators, including as interleukins (ILs), prostaglandins, and c-reactive proteins (hs-CRP) and other inflammatory mediators that are released into the bloodstream [6]. This might indicate an elevated risk of chronic systemic inflammation, endothelial dysfunction, and cardiovascular disease via a particular oxidative stress route [7, 8]. In this regard, a significant association between periodontitis, heart failure and CVD has been assessed in several large cohort studies in which periodontitis patients were more commonly related with early myocardial infarction risk [9, 10].

Recently some cohort clinical trials have shown that the growth differentiation factor 15 (GDF-15), a biomarker released in cardiac and blood wall tissues in response to heart volume expansion or abnormal pressure load, is associated with early diagnosis and bad outcomes in patients with CVD and heart failure [11]. Evidence suggests that elevated serum GDF-15, together with 1-antitrypsin and glutathione peroxidase 1 (GPx-1) levels, are associated with a high risk of the acute coronary syndrome, a phenomenon that occurs in early heart failure in asymptomatic patients and for the risk stratification of patients with acute coronary syndrome, endothelial dysfunction and CVD [12, 13]. Furthermore, GDF-15 and GPx-1 have been utilized as inclusion criteria in a number of large-population studies to investigate the association between CVD therapy impact and various heart failure overall outcomes [14, 15].

Previous reports have shown that during periodontitis, the upregulated GDF-15 levels support the inflammatory response of periodontal ligament fibroblasts stimulated by P. gingivalis [16], and was also that upregulated GDF-15 concentrations were correlated with the degree of periodontal tissue loss [17]. In addition, throughout the past several decades, few exploratory investigations have highlighted the favorable effects of periodontal therapy on systemic inflammatory indicators. This research intended to demonstrate that periodontal therapy might result in considerable reductions of certain CVD biomarkers as hs-CRP [18] and surfactant protein D (SP-D) [19], notwithstanding variation in reported results among trials. In patients with periodontitis, full-mouth disinfection (FMD) protocol, conducted with the adjuvant use of chlorhexidine, was more successful than traditional quadrant scaling and root planing (Q-SRP) in decreasing various inflammatory biomarkers, particularly in the short-term run [20–22].

Finding mediators that might improve the predictive accuracy of CVD and endothelial risk, as well as assessing the impact of periodontal therapy on early indicators of CVD and heart failure, are of increasing interest. The purpose of the present study was to evaluate the effect of periodontal therapy performed with Q-SRP or FMD protocols on GDF-15, GPx-1, hs-CRP, and SP-D in patients with periodontitis during a 6-month follow-up period. In addition, it was analyzed the impact of periodontal therapy on serum GDF-15, GPx-1, hs-CRP, and SP-D, and if patients who harboured high baseline GDF-15 and related biomarkers benefited the efficacy of periodontal treatment. The null hypothesis to be rejected was that at 6-months follow-up, there were no significant changes in serum GDF-15, GPx-1, hs-CRP, and SP-D among enrolled patients treated with different periodontal treatment protocols.

2.1 Study design and sample

The present study was designed as a randomized clinical trial. The study was done according to CONSORT criteria and the 2016 Helsinki Declaration on medical research. Before the study, all participants completed a written permission form outlining the risks and features of the research. Prior to patient enrolment, the local International Review Board of the University of Catania in Italy granted ethical approval (22–149 PO). The study was retrospectively registered on clinicaltrials.gov (NCT05720481–09/02/23).

For this study, consecutive patients with a diagnosis of periodontitis [23] were recruited at the Unit of Periodontology of the Dental School of the University of Catania, Italy. The inclusion criteria were (1) good general health, (2) a minimum of six teeth per quadrant, (3) at least two teeth in each quadrant with a probing depth (PD) 5 mm and a clinical attachment level (CAL) 4 mm, (4) at least 40% of periodontal sites with bleeding on probing (BOP), (5) no involvement of teeth furcations, and (6) at least 2 sites with radiographically verifiable alveolar bone loss (ABL) [24]. The exclusion criteria were as follows: (1) periodontal therapy in the previous 12 months prior to the study; (2) use of antibiotics in the previous 6 months prior to the study; (3) pregnancy or lactation; (4) presence of systemic condition that could affect the study results; (5) use of mouthwash containing antimicrobials in the previous 3 months prior to the study; (6) use of anti-inflammatory, immunosuppressive, or contraceptive drugs; (7) alcohol consumption.

At baseline, in order to obtain an equal gender and age proportion, male and female patients aged between 30 and 65 years were enrolled, ensuring that at least 50% of them were female. For the present study, 208 patients with periodontitis were first screened. Following the first patient selection, a total of 162 patients were finally excluded because they did not fully meet the study criteria (n = 97), refused to participate in the study (n = 36) or were absent at the periodontal examination at baseline (n = 27). The present study enrolled 48 patients and categorized them as stage III periodontitis. However, 2 patients in each group were lost during the follow-up sessions, and a total number of 46 patients were finally analyzed (Fig. 1).

After identification, all eligible individuals receive a medical history and demographic factors such as age, gender, body mass index (BMI), comorbidities (if present), medicines, and levels of education are recorded. By dividing the patient's weight by the cube of their height, the BMI (kg/m²) was computed. In addition, the patient's socioeconomic status (SES) was noted based on prior employment and economic and social situations. Following the interview, each patient was categorized as having a high, moderate, or low SES [25]. On the basis of patient’s smoking history, each participant was categorized as current smokers, ex-smokers (patients who stopped smoking ≥ 5 years), and non-smokers.

Each recruited patient had a comprehensive dental and periodontal examination by a masked examiner. Using a conventional periodontal probe, six locations per tooth were examined for periodontal health (UNC-15, Hu-Friedy, Italy), recording PD, BOP, ABL, and plaque index score (PI) and setting CAL as a primary variable of periodontitis extent severity.

2.2 Sample size and reliability analysis

The power sample analysis was calculated using statistical software (G* POWER, Universität Düsseldorf, Germany). The sample size was obtained by setting serum GDF-15 as a primary outcome variable [11] and considering two groups of patients, an effect size of 0.30, a 2-sided level of 0.05, a standard deviation of 1.5, and a power level of 80%. Therefore, it was fixed a priori that at least 22 patients per group were needed. However, to avoid potential dropouts during the 6-months follow-up, 23 patients were enrolled, so the primary variable (CAL) achieved a power value of 0.80.

The inter- and intra-examiner reliability analysis was achieved using CAL as reference values for the reliability analysis and using the intraclass correlation coefficient (ICC). The ICC analysis showed good agreement among examiners for CAL (ICC = 0.817). More specifically, the first examiner had good reliability for CAL (ICC = 0.823) and the second examiner (ICC = 0.825).

2.3 Randomization

Through a permuted block design, the randomization was performed by a single clinician, not involved in the subsequent trial stages, which generated a random assignment of a treatment using a sequence 1:1 ratio by a computer random-number generator.

Each patient was allocated to receive Q-SRP or FMD. The allocation was concealed to the doctor who administered the therapy using serially numbered, sealed envelopes, and the sequence details were concealed from all other clinicians. Before each treatment, a clinician who was not engaged in data processing assigned the patient's treatment to a sealed envelope bearing the treatment's name and initials.

Just prior to each treatment session, another clinician opened an envelope containing the allocated therapy and the operator who would administer it. To eliminate bias in the experimental data evaluation, the same clinician with ten years of expertise in periodontics performed all the procedures while blinded to previously recorded data.

2.4 Treatment

The primary outcome was the analysis of serum GDF-15 expression changes after 6-months after treatment between the two groups. Furthermore, GPx-1, hs-CRP, and SP-D concentration changes were analyzed before and after 6 months of periodontal treatment. The secondary objective was to examine the influence and interaction of the periodontal treatment protocol (Q-SRP) and treatment timing on GDF-15, GPx-1, hs-CRP, and SP-D changes, as well as whether high baseline levels of GDF-15, GPx-1, hs-CRP, and SP-D influenced the efficacy of periodontal treatment after 6 months of follow-up.

Shortly after the baseline assessments, each enrolled patient received oral hygiene instructions.

Patients allocated to the FMD group received a full mouth SRP in one side of the mouth for each session, within 24h in two separate sessions, on two consecutive days with the adjunctive use of local antiseptic in accordance with Quirynen et al. [26]. SRP was performed by using both hand and ultrasonic instrumentation by tips No. 5/6/7 (Satelec Ultrasonics, Acteon, VA, Italy), which was used with constant water irrigation with a 20.000 Hz of frequency. Two right quadrants were instrumented in the morning, while the other two were instrumented in the afternoon, along with brushing the dorsum of the tongue with 1% chlorhexidine gel for 1 minute, rinsing twice with 0.2% chlorhexidine solution for 1 minute, and applying 0.2% chlorhexidine spray twice per day (GlaxoSmithKline, Milan, Italy) to the tonsils, subgingival irrigation of all pockets with 1% chlorhexidine gel (three times within 5–10 minutes), which was repeated one week later. Patients were instructed to rinse twice a day for 1 min with 0.2% chlorhexidine solution and to spray the tonsils twice a day with 0.2% chlorhexidine spray over a period of 2 months. Treatments were recorded in minutes and performed under local anaesthesia only if necessary.

Patients allocated to the Q-SRP group received quadrant scaling in four different sessions with an interval of 1 week. In each patient, the first session started in the upper right maxillary quadrant. For the control group, the same periodontal treatment performed in the test group was provided upon completion of the study. At the conclusion of each type of treatment, every patient was informed and encouraged to practice oral hygiene.

2.5 Sampling

The same examiner took baseline, 3- and 6-month blood samples from all patients between 8:00 and 10:00 a.m., prior to any periodontal examination. Urgently following collection, serum samples were centrifuged at 4°C (1000x g for 2 min). The serum GDF-15, GPx-1, and SP-D levels were obtained using a specific kit according to the manufacturer's instructions and evaluated using human-specific enzyme-linked immunosorbent assay (ELISA) kits. The hs-CRP levels were obtained by a nephelometric assay kit.

2.6 Statistical Analysis

Numerical data are expressed by mean ± standard deviation (SD), while categorical variables were reported as numbers and percentages. Because most of the analyzed variables were not normally distributed, as verified by the Kolmogorov-Smirnov test, a non-parametric approach was applied. The Mann-Whitney test was used for the numerical data comparison between groups, while the Chi-Square test was applied for the comparisons between categorical variables. The single patient was set as a test unit. For intragroup comparisons, the Friedman test was used to compare numerical variables over four-time intervals (baseline, 30, 90, and 180 days), whereas the Wilcoxon test was used for two-by-two comparisons across dependent groups. For multiple comparisons, Bonferroni's correction was applied, and the alpha level of 0.050 was divided by the number of potential comparisons (baseline, 3-months, 6-months) to get an adjusted significance level of 0.017 (0.050/3). The Spearman's correlation test was used to examine a potential substantial dependency between GDF-15, GPx-1, hs-CRP, and SP-D and all studied variables at the 6-month of therapy.

To analyze the effect of the treatment protocol on GDF-15, GPx-1, hs-CRP, and SP-D (as continuous variables), after checking the conditions for its applicability, a two-way ANOVA was used to estimate whether the mean of the quantitative variable (GDF-15, GPx-1, hs-CRP, and SP-D) changes based on the levels of two categorical variables, treatment and timing of treatment. Specifically, it was evaluated how the two independent variables (treatment protocols and timing), alone and in combination, influenced serum GDF-15 concentration changes. The same models were applied for the secondary outcomes GPx-1, hs-CRP, and SP-D changes. Statistical analyses were performed using IBM SPSS version 22 Statistical software for Windows (Armonk, NY, IBM corp). A significant P-value was set as < 0.05.

3.1 Patient characteristics

In the analyzes sample, gender (p = 0.331), age (p = 0.596), HbA1c (p = 0.541), lipids and the number of smokers (p = 0.417) were well matched (Table 1). There were no changes in the periodontal treatment times between the Q-SRP (41.3 ± 5.7 min) and FMD (39.8 ± 4.6 min) groups (p = 0.247).

Table 1

Characteristics of the study sample at baseline. Values are reported as frequency, median and IQR, and interquartile range (1st ;3rd ). IQR, Interquartile Range; BMI, Body Mass Index. FMD, Full-mouth disinfection; Q-SRP, Quadrant scaling and root planing.
Characteristics	FMD (n = 23)	Q-SRP (n = 23)	P-value
Male/female, n°	10/13	11/12	0.331
Age, median (IQR)	50 (48.6–52.4)	51 (47.6–54.9)	0.596
Race Caucasians, n. (%) Black, n. (%)	21 (91.3) 2 (8.7)	21 (91.3) 2 (8.7)	0.912
Education level			0.326
Primary School, n. (%)	11 (47.8)	12 (52.2)
High School, n. (%)	7 (30.4)	7 (30.4)
University, n. (%)	5 (21.8)	4 (17.4)
HbA1c %, median, (IQR)	5.1 (4.8–5.2)	5 (4.7–5.2)	0.541
Total cholesterol (mg/dl)	175 (122; 189)	172 (142–191)	0.332
HDL cholesterol (mg/dl)	42 (35–49)	43 (32–51)	0.285
LDL cholesterol (mg/dl)	78 (71–86)	79 (68–93)	0.105
BMI (kg/m²), median, (IQR)	20.7 (18.2–22.1)	20.2 (17.7–23.1)	0.425
Smoking			0.417
Current smokers, n. (%)	1 (4.4)	1 (4.4)
Former smokers, n. (%)	-	1 (4.4)
Non-smokers, n. (%)	22 (95.6)	21 (91.2)
Teeth at baseline median, (IQR)	21 (17.8–24.4)	22 (18.7–23.9)	0.254

Table 2 shows the periodontal features of the study sample. Compared with FMD, treatment with Q-SRP resulted in a significant decrease in mean CAL at 3- (Q-SRP, 3.29 ± 0.2 mm; FMD, 4.04 ± 0.2 mm, p = 0.049) and at 6-months after therapy (Q-SRP, 2.68 ± 0.5 mm; FMD, 3.66 ± 0.4 mm, p = 0.015), in mean PD at 3- (Q-SRP, 3.15 ± 0.5 mm; FMD, 3.68 ± 0.2 mm, p = 0.025) and at 6-months (Q-SRP, 2.82 ± 0.3 mm; FMD, 3.47 ± 0.6 mm, p = 0.013), as well as in the % of PD ≥ 4 mm at 6-months after therapy (Q-SRP, 18.5 ± 10.5%; FMD, 25.3 ± 12.1%, p < 0.001). Moreover, in comparison with FMD, treatment performed with Q-SRP also significantly decreased at 6-months after treatment, BOP (p = 0.016), and PI (p = 0.026).

Table 2

Periodontal characteristics of the sample at baseline and at each follow-up session. Values are reported as mean ± standard deviation (SD). PD, Probing Depth; CAL, Clinical Attachment Loss; BOP, Bleeding on Probing; PI, Plaque Index. FMD, Full-mouth disinfection; Q-SRP, Quadrant scaling and root planing.
Variable	FMD (n = 23)	Q-SRP (n = 23)	P-value
PD, mm
Baseline	4.87 ± 0.2	4.79 ± 0.5	0.524
30 days	4.08 ± 0.3 ^a	3.58 ± 0.6 ^a	0.044
90 days	3.68 ± 0.2 ^b	3.15 ± 0.5 ^b	0.025
180 days	3.47 ± 0.6 ^c	2.82 ± 0.3 ^{c, e}	0.013
CAL, mm
Baseline	5.04 ± 0.2	5.22 ± 0.5	0.331
30 days	4.37 ± 0.2 ^a	3.95 ± 0.5 ^a	0.058
90 days	4.04 ± 0.2 ^{b, d}	3.29 ± 0.2 ^b	0.049
180 days	3.66 ± 0.4 ^{c, f}	2.68 ± 0.5 ^c	0.015
% sites with PD ≥ 4 mm
Baseline	37.8 ± 11.4	36.1 ± 12.6	0.278
30 days	29.5 ± 12.3 ^a	27.9 ± 12.2	0.017
90 days	26.5 ± 12.2 ^b	21.9 ± 12.6 ^b,d	0.066
180 days	25.3 ± 12.1 ^c	18.5 ± 10.5 ^c	< 0.001
BOP, %
Baseline	49.5 ± 16.2	48.5 ± 15.2	0.315
30 days	36.7 ± 14.3 ^a	30.5 ± 14.5 ^a	0.028
90 days	28.4 ± 15.8 ^{b, d}	21.7 ± 12.5 ^{b, d}	0.025
180 days	25.6 ± 16.7 ^c	18.5 ± 11.5 ^{c, e}	0.016
PI, %
Baseline	37.9 ± 11.6	38.2 ± 12.5	0.331
30 days	33.4 ± 12.5 ^a	24.5 ± 12.4 ^a	0.027
90 days	25.8 ± 15.3 ^{b, d}	22.3 ± 11.2 ^{b, d}	0.105
180 days	23.1 ± 14.7 ^{c, f}	18.7 ± 12.3 ^{c, f}	0.036
^a significance between baseline and 30 days, ^b significance between baseline and 90 days; ^c significance between baseline and 180 days; ^d significance between 30 and 90 days; ^e significance between 30 and 180 days; ^f significance between 90 and 180 days. P-value significant < 0.008 (Bonferroni’s correction).

3.2 Primary outcome

In comparison with the FMD, Q-SRP produced, at 6-months after therapy, a significant reduction of the GDF-15 levels (p = 0.012) (Table 2). Furthermore, in comparison with the FMD, the Q-SRP treatment determined a significant reduction, at 6-months of therapy, in GPx-1 (p = 0.011), hs-CRP (p = 0.019), and in SP-D (p = 0.015) concentrations (Fig. 2).

The correlation analysis evidenced that, at 6-months of therapy, there was a correlation between serum GDF-15 and smoking (negative, p = 0.046), pockets with PD ≥ 4 mm (p = 0.022), number of teeth (p = 0.031) and BOP (p = 0.039); between serum GPx-1 and smoking (negative, p = 0.044), HbA1c (p = 0.045), LDL-cholesterol (p = 0.044), number of teeth (p = 0.041), CAL (p = 0.025) and BOP (p = 0.027); between hs-CRP and HbA1c (p = 0.048) no. of pockets with PD ≥ 4mm (p = 0.032), BOP (p = 0.045), PI (p = 0.039), and LDL cholesterol (negative, p = 0.019); between SP-D and smoking (p = 0.025), no. of teeth (p = 0.035); CAL (p < 0.001), BOP (p = 0.016), and PI (p = 0.011) (Table 3).

Table 3

Correlation analysis among GDF-15, GPx-1 and the analyzed variables at 6-months of treatment. For sex, males served as a reference. GDF-15, growth differentiation factor 15; GPx-1, glutathione peroxidase 1; hs-CRP, c-reactive proteins; SP-D, surfactant protein D. HbA1c, Glycated hemoglobin; BMI, Body Mass Index, PD, Probing Depth; CAL, Clinical Attachment Loss; BOP, Bleeding on Probing; PI, Plaque Index.
Variable	GDF-15		GPx-1
Variable	Rs coeff.	p-value	Rs coeff.	p-value
Age	0.362	0.104	0.256	0.548
Sex	-0.155	0.335	0.331	0.317
Smoking	-0.108	0.045	-0.331	0.044
Education	-0.058	0.274	0.258	0.095
HbA1c	0.105	0.081	-0.258	0.045
BMI	0.205	0.663	0.309	0.524
Total cholesterol	0.105	0.048	0.201	0.332
HDL cholesterol	0.184	0.205	0.147	0.201
LDL cholesterol	-0.332	0.336	0.196	0.044
Pocket PD ≥ 4mm	0.305	0.035	-0.109	0.085
No. of teeth	0.157	0.028	-0.215	0.041
CAL	0.274	0.065	-0.297	0.025
BOP	0.356	0.044	-0.365	0.027
PI	0.269	0.045	-0.357	0.548
	hs-CRP		SP-D
	Rs coeff.	p-value	Rs coeff.	p-value
Age	0.302	0.085	0.205	0.325
Sex	-0.128	0.105	-0.241	0.066
Smoking	-0.204	0.256	0.336	0.025
Education	-0.394	0.104	0.185	0.108
HbA1c	0.258	0.048	0.332	0.278
BMI	0.325	0.587	0.245	0.358
Total cholesterol	-0.258	0.056	-0.658	0.221
HDL cholesterol	0.365	0.065	0.365	0.365
LDL cholesterol	-0.274	0.019	0.478	0.258
Pocket PD ≥ 4mm	0.331	0.032	-0.108	0.069
No. of teeth	0.247	0.066	0.335	0.035
CAL	0.285	0.068	0.297	< 0.001
BOP	0.369	0.045	-0.244	0.016
PI	-0.105	0.039	0.428	0.011

3.3 Secondary outcome

The estimation of models aimed to determine the effect of Q-SRP and treatment timing on GDF-15, GPx-1, hs-CRP, and SP-D concentration changes after 6 months of treatment using a two-way ANOVA test revealed that Q-SRP had a significant effect on the reduction of serum GDF-15 (p = 0.015), GPx-1 (p = 0.045), and SP-D (p = 0.045) together with the timing of treatment for GDF-15 (p = 0.028) and GPx-1 (p < 0.001) concentrations. People who harboured high concentrations of GDF-15, GPx-1, and SP-D gained more benefits from Q-SRP treatment at 6-month follow-up (Table 4).

Table 4

Results of two-way ANOVA for the dependent variable GDF-15, GPx-1, hs-CRP, and SP-D. Q-SRP as a reference for group. GDF-15, growth differentiation factor 15; GPx-1, glutathione peroxidase 1; hs-CRP, c-reactive proteins; SP-D, surfactant protein D.
	GDF-15
Source of variation	MS	F	p-value
Group	885.66	205.12	0.015
Timing	698.36	356.21	0.028
*GroupTiming**	21.24	3.35	0.002
Within	4.33
	GPx-1
Source of variation	MS	F	p-value
Group	265.55	136.12	0.045
Timing	247.36	105.41	< 0.001
*GroupTiming**	4.26	4.33	0.026
Within	3.28
	hs-CRP
Source of variation	MS	F	p-value
Group	389.36	196.54	0.105
Timing	205.56	158.45	0.331
*GroupTiming**	6.65	5.66	0.206
Within	2.41
	SP-D
Source of variation	MS	F	p-value
Group	415.41	205.44	0.026
Timing	248.66	306.56	0.226
*GroupTiming**	3.98	4.87	0.045
Within	2.66
MS: Mean of Square. F: Fisher test; Group*Timing: Interaction term

In the past few decades, more evidence of an inflammatory component in the etiology of atherosclerosis and endothelial dysfunction has provided support for the hypothesis that systemic inflammation is the link between periodontitis and CVD. Due to these factors, new non-surgical instrumentation techniques have been created. Periodontal therapy regimens that use separate periodontal treatment procedures to avoid reinfection of treated sites from untreated pockets and other intraoral habitats are increasing [22]. In this regard, the purpose of the present study was to examine the effect of periodontal therapy on GDF-15 with GPx-1, hs-CRP and SP-D and determine the impact of Q-SRP and timing of periodontal treatment on concentration changes GDF-15, GPx-1, hs-CRP and SP-D six months following periodontal treatment.

Both groups of patients demonstrated a significant decrease of GDF-15 hs-CRP and SP-D levels and an increase of GPx-1 compared to baseline. Compared to the FMD group, Q-SRP produced a greater decrease in serum GDF-15, as measured by periodontal parameters reduction together with GDF-15 reduction. Several studies suggest that imbalanced serum GDF-15 levels may change the innate host defences against periodontal pathogens during periodontitis [17]. During the active phases of periodontitis, bacterial LPS and related periodontal fibroblast has been discovered to produce an increase in serum GDF-15 levels via a process involving the synthesis of proteolysis products [16]. A further investigation revealed that fibroblasts from persons with periodontal disease had higher levels of serum GDF-15, whose expression was tightly correlated with the periodontal inflamed surface area even in the earliest stages in a rat model of periodontal disease [27].

The therapeutic significance of the statistical difference between treatments of about 1 mm mean of PD or 10 percent greater PD closed pockets is not well understood regarding periodontal therapy. In this context, a recent meta-analysis comparing the efficacy of adjuvant systemic antimicrobials to subgingival scaling in randomized controlled trials demonstrated an additional 0.48 mm decrease in PD at six-month follow-up following periodontal treatment [28]. Consideration of pocket closure as the most relevant periodontal variable leaves the question of clinical significance unresolved. A recent workshop deemed the pocket closure to be a more significant clinical finding than mean probing depth, as shallow sites (4 mm) without > 30% bleeding are one of the strongest indicators of periodontal stability [29]. In particular, the improvement in PD pocket closure does not be approached with higher antibiotic consumption [28]. In this regard, some recent studies and meta-analyses [30, 21] confirmed the previous findings [31] that periodontal therapy influences clinical parameters and that there is no clinical variation across SRP methods. In this regard, the present study may have achieved statistically significant results that were more favorable results due to the differing number of patients included in the present study compared to other similar ones [21]. Suppose a baseline reveals a negligible baseline PD; in this instance, the difference between baseline and post-treatment PD is often negligible, even though the probing error may be independent of the magnitude of the PD [32]. Furthermore, this study evidenced that there was a positive correlation between serum GDF-15, GPx-1, hs-CRP and SP-D with the extent of periodontitis. Moreover, serum GDF-15 and GPx-1 changes have been related to the activation of the several inflammatory pathways and have also been linked to the extent of PD patients compared with healthy subjects [33]. Therefore, the reduction in serum GDF-15, hs-CRP and SP-D levels 6 months after periodontal therapy with Q-SRP implies that this therapy determines a more effective breakdown of the bacterial biofilm and a superior host response compared to baseline and to periodontal treatment performed with FMD. In this regard, both GDF-15 [34, 16], GPx-1 [35], hs-CRP [6, 36, 37] and SP-D [19] have not only levels been implicated in regulating the inflammatory process, but also alveolar bone homeostasis and resorption related to the extent of periodontal disease. More specifically, the expression of GPx-1 and surfactant proteins have been associated to alterations in bone homeostasis and osteoclast differentiation [38, 39]. Furthermore, during CVD, the overexpression of serum GDF-15, hs-CRP levels have been connected with the decrease of endothelial dysfunction and high risk of CVD, validating the current study's findings [34, 15].

In accordance, the two-way ANOVA analysis of the present investigation demonstrated that Q-SRP significantly improved and impacted serum GDF-15, GPx-1 and SP-D after six months of therapy. In addition, the ANOVA analysis demonstrated that, at six months follow-up, patients who harboured high levels of GDF-15, and SP-D and low levels of GPx-1 before periodontal treatment positively benefited the periodontal treatment efficacy at 180 days follow-up. In this regard, it has been demonstrated, in accordance with the current literature, that periodontal treatment can induce a short-term inflammatory response, resulting in a progressive and consistent reduction in systemic inflammation and an improvement in endothelial functions in patients with periodontitis [40]. Research that included120 patients with severe periodontitis, Furthermore, a graded and independent association between tooth loss and GDF-15, suggested that tooth loss and its underlying mechanisms may be involved in multiple pathophysiological pathways also implicated in the development and prognosis of CVD [11].

The present study had some limitations that need to be addressed as the number of patients involved. For this investigation, a larger sample size would have improved the ability to identify clinical and serum variations in the analyzed biomarkers across treatment groups. This larger database might have also allowed for more distinct outcomes. In addition, the monocentric design and ratings for plaque, CAL and bleeding scores were not fully accomplished. A longer monitoring period would be beneficial to assess if the differential clinical results have a positive long-term impact on stable periodontal outcomes.

In conclusion, the findings of the present randomized controlled study demonstrated that both Q-SRP and FMD were advantageous for all patients who were treated. However, periodontal therapy with Q-SRP decreased clinical periodontal parameters and improved GDF-15, GPx-1, hs-CRP and SP-D in individuals with periodontitis. In addition, patients with high GDF-15 and SP-D and lower GPx-1 concentrations at baseline had larger clinical improvements from periodontal therapy during the 6-month follow-up. The results of this study are still early, and further research is required to better comprehend the benefits of non-surgical periodontitis treatments on blood GDF-15 in patients with periodontitis.

Ethics approval and consent to participate: International Review Board of the University of Catania approved the study protocol (22-149 PO). All patients signed a an appropriate written informed consent for the study participation.

Consent for publication: Not Applicable.

Availability of data and materials: Data are available from corresponding authors upon reasonable request.

Competing interests: The authors declare that they have no conflict or competing interests in the present study.

Funding: The present research was funded by Departmental funding only.

Authors' contributions: G.I. conceived the research, planned and performed the experimental procedures and wrote the manuscript. G.M.T., A.L.G. and S.S. performed the procedures. A.C. and A.M. validated the experimental results and revised the manuscript.

Acknowledgements: The authors acknowledge the staff of the Unit of Periodontology of the University of Catania for their support.

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No competing interests reported.

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Growth differentiation factor-15 as predictor of periodontal treatment effects in patients with periodontitis: a randomized-controlled clinical trial

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Abstract

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1. Introduction

2. Materials And Methods

2.1 Study design and sample

2.2 Sample size and reliability analysis

2.3 Randomization

2.4 Treatment

2.5 Sampling

2.6 Statistical Analysis

3. Results

3.1 Patient characteristics

3.2 Primary outcome

3.3 Secondary outcome

4. Discussion

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