Comparison of Osteoimmunological and Microbiological Parameters of Extra Short and Longer Implants Loaded in the Posterior Mandible: A Split Mouth Randomized Clinical Study.

OBJECTIVES: This study aimed to evaluate the levels of TNF-α, PGE2, RANKL, RANK, OPG, the markers of periimplant bone loss in peri-implant crevicular fluid obtained around standard and extra short implants. Moreover, the levels of putative oral pathogens were investigated in the submucosal biofilm samples.
MATERIAL AND METHODS: The implants were divided into two groups according to their lengths: standard (≥8 mm) and extra short (4 mm). A total of 60 implants were researched in 30 patients. The probing depth (PD), clinical attachment level (CAL), presence of bleeding on probing (BOP), 3-year survival rate (CSR), and bone loss (BL) were measured.
RESULTS: No statistically significant difference was found in the values of PD, CAL, BOP, CSR, and BL between the groups (P> 0.05). Total amounts of PGE2, TNF-α, RANKL, RANK, OPG, and RANKL/OPG were not statistically significantly different between the groups (P> 0.05). The abundance of F. nucleatum, T. forsythia, P. intermedia, P. gingivalis, S. oralis and T. denticola was compared between the groups and the results were not statistically significant (P> 0.05).
CONCLUSION: The results of this study suggested that PGE2, TNF-α, RANKL, RANK, OPG, and RANKL/OPG in PICF, as well as microbiological parameters in submucosal biofilms, were similar between standard (≥8 mm) and extra short (4 mm) implants. Therefore, the implant length does not seem to influence the bone loss, levels of osteoimmunological and microbiological markers in the peri-implant tissues and survival rates.

Introduction

Dental implants are usually considered an alternative treatment option in edentulous patients. Following tooth extraction, the vertical and horizontal areas in the alveolar socket are significantly reduced (). Clinicians have to consider more complex and time-consuming techniques for reconstruction of the maxilla and mandible, such as a sinus lift or vertical guided bone regeneration procedures (, ); however, these procedures must be precise and are often accompanied by high costs, high morbidity, and intra- and postsurgical complications. Short dental implants have been suggested as a less invasive, cheaper, and faster alternative to prevent the disadvantages of surgical techniques and for the rehabilitation of toothless areas (-). A large number of randomized controlled clinical trials demonstrated that the long-term success and survival rates of short implants were similar to those of standard long implants (-).

Accumulation of microbial dental plaque around the implant is the most important cause of implant loss (). If the microbial attachment is not removed, diseases such as peri-implant mucositis and peri-implantitis may occur and result in implant loss in the long term (). Peri-implant mucositis is an inflammatory disease that affects soft tissues surrounding the implant; it is reversible with proper treatment. Peri-implantitis is a microbial inflammatory disease characterized by the resorption of the supportive bone surrounding the implant in function (). Gram-negative anaerobic bacteria predominate around the implant sites affected by the disease. While they resemble chronic periodontal infections, they have a more complex microbiological character (). Predominant species around a peri-implantitis implant are red complex (T. denticola, T. forsythia and P. gingivalis) and orange complex bacteria (P. intermedia and F. nucleatum) described by Socransky ().

Apse et al. () have identified what they termed “peri-implant crevicular fluid” (PICF), which surrounds the peri-implant sulcus and has properties similar to those of the gingival crevicular fluid. PICF contains several inflammatory mediators, the levels which provide information on the inflammatory state of the tissue, including the activation of mechanisms of bone destruction (). Prostaglandins, especially prostaglandin E2 (PGE2), are considered as a potent mediator of alveolar bone destruction in periodontitis. A large number of studies reported an increase in PGE2 levels from healthy state to periodontitis (). Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine regulating the Gram-negative bacterial response. The TNF-α concentration is an indicator of bacterial load and degree of inflammation (). In areas where peri-implantitis is active, the presence and activity of osteoclasts are necessary for bone destruction to occur. Osteoclast formation and functions are regulated when the following three TNFs are activated: osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (RANK) and receptor activator of nuclear factor kappa B ligand (RANKL). Soluble receptor activator of nuclear factor- кB ligand (sRANKL) and OPG have been suggested as molecular determinants of bone resorption. RANKL is a ligand required for osteoclast generation, RANK is the receptor for RANKL, and osteoprotegerin (OPG) is a decoy receptor for RANKL () Osteoclast differentiation and activation occur with the binding of RANKL to RANK over the surface of osteoclasts and precursors. OPG, which is a soluble protein of TNF receptors, antagonizes RANK–RANKL interaction and increases bone formation by inhibiting osteoclastogenesis (). The levels of proinflammatory cytokines, such as PGE2, TNF-α, IL-1, IL-6 and RANKL/OPG rates, which allow the determination of osteoclastic activity, change in the case of peri-implantitis ().

The aim of this study was to evaluate the levels of PGE2, TNF-α, RANK, RANKL and OPG in extra short and standard dental implants after a 36- month monitoring period. An additional aim was to to investigate the levels of putative oral pathogens P. intermedia, P. gingivalis, F. nucleatum, S. oralis, T. denticola and T. forsythia in submucosal biofilm samples.

Material and methods

This study involved a prospective, randomized, and split-mouth design clinical trial. A total of 30 extra-short implants (intrabony length = 4 mm) and 30 standard implants (intrabony length ≥ 8 mm) in 30 periodontally healthy subjects were randomly placed according to the design to receive both implant systems in posterior mandibular edentulous sites.

Patient selection and study design

This study was carried out by recalling individuals whose bilateral partial tooth losses were treated with implant-supported fixed restorations and whose implants had been functioning for at least 3 years after prosthetic rehabilitation.

The study was conducted according to the principles of the Declaration of Helsinki (Clinical Researches Ethical Board with the 28. 09. 2016 and 2016/009 decision numbered approval). This study is in compliance with the CONSORT Statement. The study protocol was registered with clinicaltrials.gov (registration number NCT04475406) prior to its commencement. Similar methodology and design have been used in the study of other authors ().

Sufficient bone height for a 4-mm implant and sufficient bone width for at least a 5.5-mm implant with no augmentations and no history of periodontitis.

Implants placed bilaterally by the same periodontologist (E.Ö.) functioning for at least 3 years

Placed implants having the same brand (Straumann SLA Active; Institute Straumann AG, Basel, Switzerland). Patients with cemented implant prosthesis in which standard abutment was used in the mandibular posterior region ().

The inclusion criteria for the study were as follows:

Patients with any systemic diseases and smokers with poor oral hygiene (plaque score >20%) and having a parafunctional habit were excluded.

A total of 31 patients met the abovementioned inclusion criteria. One patient did not continue the study. 60 implants were researched in 30 patients (16 female and 14 male). The bilateral regions of patients with a standard implant and an extra short implant were grouped into two ().

Control group: Standard implant, intra-bone length ≥8 mm (30 implants)

Test group: Extra Short implant, intra-bone length ≤4 mm (30 implants)

Collection of clinical and radiological data

A single examiner performed all clinical measurements (B.K.), including the presence of bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL), 3-year survival rate (CSR), and bone loss (BL).

The values of PD and BOP were measured from four sites of each implant (mesial, distal, buccal, and lingual) with a Williams type (Hue Friedy, Switzerland) plastic periodontal probe. The PD was recorded as the distance from the base of the peri-implant to the side of the gum in millimeters. BOP was evaluated according to the presence (+) or absence (–) of bleeding within the first 30 s following the measurement of PD ().

Digital periapical radiographs were taken using the paralleling technique. Similarly, in line with previous studies, site-specific bone loss around the implants was measured at mesial aspects (). Therefore at baseline and 36 months after prosthetic loading, the distance between the implant shoulder and first bone contact point was measured.

Collection of PICF and subgingival plaque samples

Cotton rolls were used to isolate the implants, and they were dried with an air spray. Then, the plaque and soft attachments around the implants were removed. The PICF were obtained from the mesio-buccal aspect of the implants by the paper strips (Oraflow Inc, NY, USA). Paper strips were placed 1–2 mm in the peri-implant sulcus and left in place for 30 s. Strips contaminated with saliva or blood were discarded.

After collecting the PICF, the supragingival plaque was removed by a sterile scaler and subgingival plaque samples were collected from the mesio-buccal aspect of the implants by a sterile plastic Gracey curette (Hu-Friedy, Switzerland) for 30 s. The samples collected were transferred to sterile Eppendorf tubes containing 200 µL of PBS. The tubes were stored at –80°C until the laboratory analyses.

PICF analyses

PICFs were eluted from the strips by placing them in 200 µL PBS (pH 7.2) containing an EDTA-free protease inhibitor (Roche Applied Science, Basel, Switzerland). The total protein content of PICF was quantified using a Qubit Protein Assay kit (Elabscience Biotechnology Co., Ltd, Wuhan, China). according to the manufacturer’s instructions.

Commercial enzyme-linked immunosorbent assay kits were used for measuring the levels of TNF-α, PGE2, RANKL, RANK, and OPG according to the manufacturer’s recommendations (Elabscience Biotechnology Co., Ltd, Wuhan, China). The measuring ranges were as follows: TNF-α, 7.81–500 pg/mL; PGE2, 31.25–2000 pg/mL; RANKL, 0.16–10 pg/mL; RANK, 0.16–10 pg/mL; and OPG, 0.16–10 pg/mL. Optical density was measured at 450 nm, and the samples were compared with standards. Biochemical data were measured as the total amount (pg/30 s).

Genomic DNA preparation

An extraction kit was used according to the manufacturer’s instructions to purify the DNA in the collected plaque samples (GF-1 bacterial DNA extraction kit, Vivantis, Malaysia). Standards were used for total DNA in the target bacteria. Genomic DNA was obtained and stored at 4°C.

Real-time polymerase chain reaction

Primary probes were determined to define each bacterium and observe the proliferation curves using real-time polymerase chain reaction (PCR) (Table 1). A real-time PCR system (Roche Light Cycler 480 Instrument II, Switzerland) using a master mix (SYBR Green Master Mix; Life Technologies, CA, USA) was used to perform the procedures. PCR cycles were as follows: 10 min at 95°C, 40 cycles at 95°C for 30 s and 2 min at 60°C. DNA contents were calculated using standard curves.

Table 1

Primers/probes and DNA sequences of bacterial species

1 Total bacteria
Forward: 5′-CGCTAGTAATCGTGGATCAGAATG-3′
Reverse: 5′-TGTGACGGGCGGTGTGTA-3′
Probe: 5′-FAM-CACGGTGAATACGTTCCCGGGC-TAMRA-3′
2. P. intermedia
Forward: 5′- CGG TCT GTT AAG CGT GTT GTG-3′
Reverse: 5′- CAC CAT GAA TTC CGC ATA CG-3′
Probe: 5′-FAM-TGG CGG ACT TGA GTG CAC GC-TAMRA-3′
3. T. forsythia
Forward: 5′-GGG TGA GTA ACG CGT ATG TAA CCT-3′
Reverse: 5′-ACC CAT CCG CAA CCA ATA AA-3′
Probe: 5′-FAM-CCC GCA ACA GAG GGA TAA CCC GG-TAMRA-3′
4. T. denticola
Forward: 5′-GTTGTTCGGAATTATTGG-3′
Reverse: 5′- GATTCAAGTCAAGCAGTA-3′
Probe: 5′-Cy5.5-TCACACCAGGCTTACC-3′-BHQ 2
5. F. nucleatum
Forward: 5′-GGCTTCCCCATCGGCATTCC-3′
Reverse: 5′-AATGCAGGGCTCAACTCTGT-3′
Probe: 5′-Cy5-TCCGCTTACCTCTCCAG -3′- BHQ 2
6. P. gingivalis
Forward: 5′-CTGCGTATCCGACATATC-3′
Reverse: 5′-GGTACTGGTTCACTATCG-3′
Probe: 5′-Texas Red ACCATAGACGACGGAGCACC-3′-BHQ 2
7. Streptococcus oralis glucosyltransferase (gtfR) gene
Forward: 5′-GCGTAAGGCAGACAAGAAGTA--3
Reverse: 5′-CCATAGTAGACCCGAGTGATAGA -3′
Probe: 5′ FAM-ATCCCAACTGCTCATGCCCTCAT -3′ -TAMRA

Statistical analysis

The SPSS 19.0 (IBM Inc., IL, USA) was used for the statistical analyses. To determine normally distribution, Kolmogorov–Smirnov and Shapiro–Wilk tests were used. The level of significance was used as 0.05 while commenting on the results.

The independent-samples t test was used for normally distributed variables, while the nonparametric Mann–Whitney U test was used for the variables which were not normally distributed. The chi-square analysis was used while examining the relationships between the groups of nominal variables. The survival rate (CSR) was calculated according to the number of short and standard implants placed.

Results

Sixty implants were randomly placed into 30 periodontally healthy subjects (16 women and 14 men) with a mean age of 35–66 years and bilaterally posterior mandibular edentulous sites using the split-mouth design (Table 2). The mean ± SD bone resorption based on the radiographs was 0.00 ± 0.50 in the extra-short implant group and 0.33 ± 0.60 in the standard-length implant group with no significant difference between the groups (P > 0.05) (Table 3). A 3-year implant survival rate was 100% in both implants. PD values (mean SD) of both groups were within healthy limits (PD: 1.99 ± 0.14 vs. 3.03 ± 0.20 at baseline and 2.20 ± 0.21 vs. 3.32 ± 0.30 at 36 months in Test group and Control group, respectively). BOP scores are presented in Table 4. BOP values (full mouth) of both groups were low with no significant difference (3% vs. 4% at baseline and 2%vs 2% at 36 months in Test group and Control group, respectively). The BOP means ± SDs for both groups were below 10% with no significant difference between the groups (Table 4). PI scores of both groups were measured below 2 both at baseline and at 36 months. CAL values of both groups were within healthy limits also, and there was no significant change in CAL level at 36 months (CAL: 2.10 vs. 3.89 at baseline and 2.30 vs. 3.50 at 36 months in Test group and Control group, respectively).

Table 2

Comparison of probing depth and clinical attachment level according to implant groups

		Group	n	Mean	SD	P
Probing depth (mm)	Baseline	Test	30	1.99	0.14	0,12
		Control	30	3.03	0.20
	3-year	Test	30	2.20	0.21	0,24
		Control	30	3.32	0.30
Clinical attachment level (mm)	Baseline	Test	30	2.10	0.05	0,22
		Control	30	3.89	0.32
	3-year	Test	30	2.30	0.15	0,43
		Control	30	3.50	0.50

Table 3

Mean radiographic Marginal Bone Loss ± SD (mm) at 3-year examination

MBL	Test	Control	p
Mesial	0.11 ± 0.2	0.25 ± 0.5	0,25
Distal	0.14 ± 0.7	0.43 ± 0.7	0,32

Table 4

Comparison of bleeding on probing according to implant groups (%)

	Baseline	3-year
Test	3%	4%
Control	2%	2%
Significance	0,08	0,15

Immunological results

When the samples taken from extra short and long implants functioning for 3 years were evaluated, no statistically significant difference was found between the groups in terms of total amounts of PGE2, TNF-α (P > 0.05) (Table 3). The total amounts of RANKL, RANK and OPG and the RANKL/OPG ratio in PISF samples in each group are presented in Table 3. No statistically significant difference was found between the groups (Table 5).

Table 5

Comparison of immunological results according to implant groups after 3 years

	Group	n	Mean ± SD	P
PGE2 (ng/30 s)	Test	30	26.25 ± 4.94	0.90
	Control	30	25.94 ± 6.17
TNF ALFA (ng/30 s)	Test	30	33.96 ± 2.5	0.92
	Control	30	34.61 ± 1.85
OPG (ng/30 s)	Test	30	1.60 ± 0.02	0.91
	Control	30	1.60 ± 0.02
RANKL (ng/30 s)	Test	30	0.80 ± 0.02	0.72
	Control	30	0.81 ± 0.03
RANKL/OPG	Test	30	0.50 ± 0.01	0.479
	Control	30	0.51 ± 0.01
RANK (ng/30 s)	Test	30	0.59 ± 0.17	0.536
	Control	30	0.63 ± 0.09

Microbiological results

Submucosal biofilm samples were assessed using qPCR for six individual bacterial species and for total bacterial counts (Table 4). When microbial plaque samples taken from functioning implants were evaluated, the amounts of P. intermedia, F. nucleatum, T. denticola, T. forsythia, P. gingivalis and S. oralis did not reveal statistically significant differences between the groups (P > 0.05) (Table 6).

Table 6

Comparison of microbiological results according to implant groups after 3 years

	Group	n	Mean	SD	P
F. nucleatum	Test	30	2.7 × 104	2.4 × 104	0.094
	Control	30	2.6 × 104	2.6 × 104
T. forsytia	Test	30	5.3 × 103	3.1 × 103	0.06
	Control	30	6.7 ×103	4.2 × 103
P. intermedia	Test	30	0.9 × 103	1.3 × 102	0.19
	Control	30	1.1 × 103	1.8 × 102
P. gingivalis	Test	30	2.4 × 104	1.1 × 104	0.73
	Control	30	3.9 × 104	2.7 × 104
S. oralis	Test	30	1.4 × 102	2.7 × 103	0.161
	Control	30	4.2 × 102	1.3 × 103
T. denticola	Test	30	5.4 × 104	3.1 × 104	0.094
	Control	30	4.4 × 104	5.3 × 104

Discussion

The present randomized, clinical trial showed that 36 months after the prosthesis loading, peri-implant bone loss surrounding short- and standard-length implants was comparable. Clinical peri-implant parameters (PD, CAL, BOP, and CSR), in terms of the levels of total PGE2, RANK, RANKL, OPG and TNF-α, RANKL/OPG ratio, and microbiological findings both types of implants were similar and showed similar favorable results.

The RANK–RANKL–OPG system is vital in the bone remodeling mechanism in the bone and implant interface. RANK/RANKL/OPG interaction, TNF-α, and PGE2 are components of a complex process, and systemic health, hormonal and metabolic states. (). Previous studies showed no differences between standard and short implants with a rough surface in terms of BL. The present study also demonstrated no significant difference in total amounts of RANKL, RANK, OPG, TNF-α, and PGE2 between the groups ().

Lamster et al. () stated that the total amount of gingival crevicular fluid was a better indicator compared with concentration. The concentration is directly affected by sample volume, and the total amount provides more objective results. In this study, the total amount of PICF was evaluated and found to be similar between the groups.

Bacterial colonization surrounding implants was observed sometime after mouth penetration. The authors of previous studies have noticed that during implantation, the microflora in the oral cavity was also affected. In addition, periodontal pathogens were often present in the implants of patients who had a history of periodontal disease (). The present study suggested that a low abundance of periodontal pathogens might be related to the absence of periodontitis history in patients. In addition, a decrease in the presence of bacteria was related to the position of the implant in the bone (). In the present study, the bacteria load was relatively low in all implants placed in the bone.

The results of recent studies indicated that the survival rates of the 6-mm short, micro-rough implants were similar to those of standard-length implants (, ). The CSR% range for 6-mm short implants (93.7%–97.6%) was also found to be compatible with long-term survival rates of standard-length implants published in previous studies (, ). In our study, neither group experienced enhanced bone resorption or pathological destruction.

No consensus has been reached on the performance of short implants compared with standard implants (). Short implants are associated with higher failure rates than standard-length implants because of their reduced contact with bone and high primary stability causing decreased osseointegration. In addition, the high crown-to-root ratio may cause increased occlusal stresses on the periimplant bone. (, ). However, a large number of studies showed similar success rates with standard implants (). In the study of Guarni et al. short and standard implants had similar survival rates, MBL, and peri-implant soft tissue conditions over the observation period of 3 years (). Studies conducted in recent years have emphasized the advantages of short implants (, , , ).

One of the limitations of this study was a small sample size. This might be the reason why the difference between the total amounts of RANKL, RANK, OPG, TNF-α, and PGE2 and the abundance of F. nucleatum, P. gingivalis, P. intermedia, S. oralis, T. forsythia and T. denticola between the groups was not significant. Another limitation was that it was not possible to distinguish between the numbers of living or nonliving bacteria because the bacterial study was conducted with PCR.

When bilaterally placed extra short and standard implants were compared, similar clinical, immunological, and microbiological results with standard implants were obtained in short implant sites after a 3-year functioning period.

Conclusions

In conclusion, the present study showed that the level of PGE2, TNF-α, RANKL, RANK, OPG, and RANKL/OPG ratio in PICF was similar between standard (≥8 mm) and extra short (4 mm) implants after a 36-month monitoring period. Both implant types had favorable clinical results with similar osteoimmunological and microbiological responses of the peri-implant tissues. Within the limitations, we can say that placement of extra-short implants (4 mm) is an option to standard-length implants in treating patients with an atrophic posterior mandibular arch as observed during a 3-year follow-up examination.

In patients that require bone augmentation, extra-short implants can be an alternative to standard-length implants. It was observed that with 4-mm implants, the rehabilitation of posterior atrophic mandible was faster and cheaper.

The present results should be verified by additional studies that include longer follow-up periods. Additionally, more samples are required to validate the findings of the present study.