Effects of Nano-Hydroxyapatite/Polyetheretherketone-Coated, Sandblasted, Large-Grit, and Acid-Etched Implants on Inflammatory Cytokines and Osseointegration in a Peri-Implantitis Model in Beagle Dogs.

BACKGROUND This study explored the effects of nano-hydroxyapatite/polyetheretherketone (n-HA/PEEK)- coated sandblasted, large-grit, and acid-etched (SLA) implants on inflammatory cytokines and osseointegration in peri-implantitis model beagle dogs. MATERIAL AND METHODS Peri-implantitis models were established. Eight beagle dogs were randomly and evenly assigned into SLA tied, SLA + n-HA/PEEK tied, SLA untied, or SLA + n-HA/PEEK untied groups. A special periodontal probe was used to detect the plaque index (PLI), probing depth (PD), and modified Sulcus Bleeding Index (mSBI). Gingival crevicular fluid was collected and an ELISA kit was utilized to detect IL-1, IL-6, and IL-17 levels. The colony-forming units were counted and the maximum shear strength of implants was tested using the axial pullout test. HE staining was used to detect the inflammation of peri-implant bone tissues. Osseointegration was observed through toluidine blue staining. Bone-to-implant contact (BIC) was obtained through histological observation and the mineral apposition rate (MAR) was calculated after immune fluorescent double staining. RESULTS The SLA tied group demonstrated higher levels of PLI, PD, mSBI, IL-1, IL-6, and IL-17 and a higher degree of inflammation than the SLA + n-HA/PEEK tied group. The tied groups also displayed similar results over the untied groups at the same time point. The maximum shear strength, BIC, and MAR in the SLA tied group were significantly lower than in the SLA + n-HA/PEEK tied group. CONCLUSIONS Our findings demonstrate that SLA + n-HA/PEEK implants can promote osseointegration and relieve the inflammation response of peri-implantitis in beagle dogs.

Background

Peri-implantitis is an inflammatory process which affects the functioning of soft and hard supporting tissues around an implant through the loss of supporting bone. Peri-implantitis is a common biological implantation complication and is a main cause of implant failure [1]. Peri-implant mucositis is defined as reversible inflammatory changes of peri-implant soft tissues with no bone loss [2]. The prevalence rates of peri-implantitis and peri-implant mucositis range from 1~47% and 19~65%, respectively [2]. With the exception of higher long-term implant survival, maintaining functionally loaded implants with adequate health and aesthetics has become a prerequisite for implant restoration success [3]. As the number of patients who have implant-supported dentures increases, dentists are increasingly dealing with peri-implant soft and hard tissues diseases [4]. As there are large differences between dental implants and natural teeth, their maintenance is critical for the success and longevity of osseointegrated implants [3].

Evidence shows that soft tissue sealing on the implant surface has a significant role in preventing peri-implantitis; however, unanimity is an unavoidable problem [5]. Transmucosal elements are suggested to accompany polished surfaces to prevent biofilm adhesion [6,7]. A coating is therefore suggested to be applied in order to reduce bacterial activity in the tissues surrounding a peri-implant [5]. It is reported that hydroxyapatite (HA)-coated femoral stems can provide durable fixation and good clinical outcomes in patients with osteoporotic fractures [8]. Polyetheretherketone (PEEK) is recommended in dental and orthopedic applications because it has similar mechanical properties to natural bone. However, its bioinertness and inferior osteoconduction have been reported to hamper various clinical applications [9]. Osseointegration is of vital importance when trying to achieve maximal stability for spinal fusion implants. However, common PEEK implants appear to be limited in osseointegration potential because of the fibrous soft tissue forming along the implant-bone interface [10]. Nano-hydroxyapatite (nHA) combined with polyetheretherketone (PEEK) has been the target of much research in recent years as a coating material [11,12]. One of the most important current problems relating to dental implant therapy is how to preserve the osseointegration and reduce inflammatory cytokines. Sandblasted, large-grit, and acid-etched (SLA) implants can provide predictable long-term results in the treatment of partially or fully edentulous patients [13]. Therefore, our study explores the effect of n-HA/PEEK-coated SLA implants on osseointegration and the inflammation response following peri-implantitis in beagle dogs.

Material and Methods

Experimental animals and grouping

The experiment started in May 2015 and ended in June 2016. Eight beagle dogs (age 16~18 months; mean weight 15.0±0.18 kg) were obtained from the Laboratory Animal Center of Zhongshan University (Certification No. 0027932). The inclusion criteria were: 1. male beagle dog (their mandibles are superior to females’ in both height and width); 2. moderate-figured dog with gentle temperament and stable genetic character; 3. no genetic nerve system disease. We collected 36 SLA standard implants (4.1×10 mm; Straumann, Basel, Switzerland) for this study. We randomly selected 24 SLA implants and their surfaces were coated with the n-HA/PEEK composite (thickness within 50 μm). The n-HA/PEEK composite was provided by the Wuhan Institute of Technology (Wuhan, China). This experiment was conducted in line with the pain trial on conscious animal guidelines proposed by the International Association for the Study of Pain (IASP) and was approved by the Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai Key Laboratory of Stomatology.

Dental implant and model establishment

Dogs were injected with 3% pentothal sodium at a dose of 25 mg/kg (CQ6475000, Shanghai First Traditional Pharmaceutical Factory, Shanghai, China) in a hindlimb vein. After general anesthesia, 8 beagle dogs were randomly divided into the SLA group (n=4) or SLA + n-HA/PEEK group (n=4). The second, third, and fourth premolars in the bilateral mandible (6 teeth in total) were removed using a minimally invasive method. During the 8th week of wound healing, the mucoperiosteal flap was exposed to implant the SLA or SLA + n-HA/PEEK implants using the South Korea Dentium or iCTmotor implant system. At an interval of 1.5 cm, the implant neck tip and alveolar bone tip were kept at the same level. After surgery, dogs were intravenously administered with antibiotics (penicillin, 8×105 IU/day for 3 days, Shanghai Xianfeng Pharmaceutical Co., Ltd, Shanghai, China) and fed a soft diet for 10 days. On the 10th day, sutures were removed and the dogs were fed normally, followed by oral hygiene procedures. After 10 weeks, the second surgery was conducted to connect the pile, and rehabilitation started 2 weeks later. In the second, third, and fourth premolar teeth on the left side, a 4-0 thread (Shanghai Pudong Jinhuan medical supplies Co., Ltd., Shanghai, China) was twined around the neck of the implant abutment 3 times and then inserted into the caries trench along the direction of the root tip. Dogs were fed a soft diet to develop dental plaque and inflammation. The second, third, and fourth premolar implants on the right side were used as controls. According to different treatments, the dogs were divided into 4 subgroups: SLA tied group, SLA + n-HA/PEEK tied group, SLA untied group, and SLA + n-/HA/PEEK untied group. The dental plaque was controlled on the right side of the implants and the thread around the neck of the implant abutment was checked on the left side of implants. Dogs were sacrificed through air embolism in the 9th week. Dogs were injected with tetracycline hydrochloride (25 mg/kg, for 3 days, Sigma-Aldrich Chemical Company, USA) 10 days and 3 days before death.

Measurement of clinical index

In the 2nd, 4th, 6th, and 8th week after rehabilitation, the plaque index (PLI), probing depth (PD), and modified Sulcus Bleeding Index (mSBI) of soft tissue around the implants were tested 3 times according to the Silness and Loe standards [14] using a periodontal-specific probe (Shanghai Cambridge Dental Medical Instrument Factory, Shanghai, China) to obtain the mean value. The condition of soft tissue around the implant was observed and the implants were gently hit with a small hammer to observe the implant mobility.

Enzyme-linked immunosorbent assay (ELISA)

All implants were moisture protected. A Whatman 3# filter (2×20 nm; Whatman, Co., UK) was inserted in the mesial, distal, buccal, and lingual side of implants to avoid blood and saliva contamination. If any resistance occurred, the operation was stopped immediately, and 30 s later the dampened area was measured using a Vernier caliper and recorded. Subsequently, the drying filter paper was removed and placed into a 1.5-mL Eppendorf tube (containing 200 μ1 0.1% BSA-PBS, pH=7.4, Costar Co., USA) and immediately stored in a −70°C freezer for later detection. The dog serum containing constituents similar to gingival crevicular fluid (GCF) was added in drops over filter paper and the Vernier caliper (METTLER TOLEDO, USA) was used to measure the dampened area. The linear correlation and t test were applied for statistical analysis. The results indicated that there was a significant correlation between serum amount and dampened area, and the standard curve was obtained from this. The GCF was then collected using the same type of filter paper. The GCF content was calculated based on the dampened area. IL-1, IL-6, and IL-17 protein expression was detected using an ELISA kit (product specification 96T/48T, Shanghai Bang Yi Biotechnology Co., Ltd., Shanghai, China). In strict accordance with the kit manual, the reagent was maintained at room temperature for 30 min and the standard was diluted for later experiments. The GCF was defrosted at normal temperature for 30 min, followed by sample uploading, liquid preparation, washing, color development, and termination. The optical density (OD) value was finally obtained using a microplate reader (model SAF-680T, Shanghai Bajiu Co., Ltd., Shanghai, China) at a wavelength of 405 nm. The standard curve was drawn to calculate the protein concentration of sample IL-1, IL-6, and IL-17 [15].

Colony-forming units

At the end of the 8th week, the dental plaque around the implant was collected, smeared, and anaerobically incubated for 72 h. Subsequently, the colony-forming units in each implant were counted.

Axial pullout test

The bone mass with implants was embedded using a piece of plastic package. Each surface of the package was perpendicular to or parallel with the long axis of the implant. A bone saw was utilized to take out bones with implants and to remove extra soft tissue until the bone nut was exposed. Finally, the bone nut was ligatured using a thread to penetrate the nut hole. The axial pullout test was conducted at a loading speed of 5 mm/min using the Instron model 1122 universal mechanical testing instrument. The force of implant-bone interface F (N) and the area of the implant-bone cortex were recorded using the formula: shear strength (Pa)=shear force/area [16].

Decalcified tissue specimen

Bilateral mandibular bones were collected and those bone masses with a single implant were sliced using a low-speed slicing machine. The soft tissue around the implant was removed and the peri-implant bone tissues were trimmed to be bone masses with implants (1×1×1.5 cm). Six implant-bone masses were randomly chosen from each group and fixed in 4% polyformaldehyde (Takara Co., Japan). One week later, the decalcified tissue sections and undecalcified bone slices had been created. Residual implant-bone masses of each group were stored in normal saline for further axial pullout testing. Implant-bone tissues were decalcified in 15% ethylene diamine tetraacetic acid (EDTA) for 1 month and then peri-implant bone tissues were trimmed to be bone masses about 1.0 cm3 in size. Subsequently, they were decalcified for another 2 months, had masses embedded, and were sliced into sections with a thickness of 7 mm using a slicing machine (EXAKT400, Germany). Undecalcified bone slices were prepared after the implant-bone masses were dehydrated using gradient ethanol (Disinfectant Factory of Qiqihar) and embedded with methyl methacrylate (guarantee reagent, Beijing Chemical Reagent Co., Ltd., Beijing, China). Sections were sliced longitudinally in a buccolingual direction (parallel to the longitudinal axis of the implants) with a saw microtome (Leica SP 1600, Germany). Three slices with a thickness of approximately 200 mm were randomly selected from each mass and polished to be 30 mm thick using a grinding machine (EXAKT300 CP, Germany) with 800 and 1200 mesh imported sandpaper.

Hematoxylin-eosin (HE) staining

Decalcified tissue sections were stained using HE. These sections were then observed under an optical microscope (OLYMPUS BX43, Olympus Optical Co., Ltd., Tokyo, Japan), and 10 high-power fields (×400) of each slice were selected and photographed using a digital camera (COOLPIX 5000, Nikon) and image analysis was conducted.

Toluidine blue staining

After being polished and heavily load for 12 h, the tissue sections were stained with 2% toluidine blue dye ethanol (Amresco, Co., USA) for 5 min. Sections were then differentiated with 95% alcohol and washed. This was followed by gradient ethanol dehydration (70%, 80%, 90%, and 100%), xylene transparency, and gum sealing. The implant-bone interface osseointegration condition was observed under an optical microscope.

Histological measurement of bone-to-implant contact (BIC)

The undecalcified bone specimens were observed and photographed under an optical microscope. The images were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Silver Spring, MD) to measure the bone-implant interface bonding length and total interface length. The ratio of bonding length to total length was expressed as BIC.

Immunofluorescent double staining

Five fields from each specimen were selected under a fluorescence microscope (360~400 nm exciting light; Olympus-BX51; Olympus Optical Co., Ltd., Tokyo, Japan). The vertical distance between the tetracycline and double-labeling thread was recorded, and the ratio of the mean vertical distance and the number of interval days was identified as the mineral apposition rate (MAR).

Statistical analysis

Data analysis was carried out using SPSS v. 21.0 statistical software (SPSS Inc., Chicago, IL, USA). Measurement data are presented as mean ± standard deviation (SD). The normal distribution was tested using the Shapiro-Wilk normality test and Levine’s test was used to analyze the homogeneity of variance. One-way analysis of variance (ANOVA) was performed to see if the variance was equal; otherwise, the Welch algorithm was used for adjustment. Independent samples t tests were used for comparison of data between 2 groups. P<0.05 indicates statistical significance.

Results

Baseline characteristics of beagle dogs among the 3 groups

All 8 beagle dogs survived the experiment. The beagle dogs had good appetite and spirit after the operation and no implant incisions were infected or ulcerated, without abnormal secretion. Eight weeks later, the tied group (the SLA tied group and the SLA + n-HA/PEEK tied group) plaque calculus was attached to the implant base, the peri-implant gingivae swelled, ulcerations had overflow pus, but no implant loosened or shed. The implant base of dogs in the untied group (the SLA untied group and the SLA + n-HA/PEEK untied group) was clean without loosening or shedding implants and the color and texture of the peri-implant gingivae was normal.

The PLI, PD, and mSBI of beagle dogs among the 3 groups

As shown in Table 1, there was no obvious difference between the 2 untied groups. Compared with the untied groups, the PD and mSBI gradually increased in the corresponding tied groups from the 2nd, 4th, 6th, and 8th week after model establishment. The PLI also gradually elevated from the 4th, 6th, and 8th week onwards. From the 2nd week on, the PLI, PD, and mSBI of the SLA untied group were significantly higher than those in the SLA + n-HA/PEEK tied group (all P<0.05). From the 6th week on, the PD value of the SLA tied group was greater than 4 mm (a clinical index of peri-implantitis). This result was 2 weeks earlier than in the SLA + n-HA/PEEK tied group, which occurred in the 8th week. These results indicate that the reaction time of inflammation in dogs with SLA implants was shorter than that of dogs with SLA + n-HA/PEEK implants.

Table 1

Comparison of PLI, PD and mSBI of beagle dogs among the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups.

	SLA tied	SLA + n-HA/PEEK tied	SLA untied	SLA + n-HA/PEEK untied
PLI
2nd week	0.92±0.10	0.88±0.06*	0.59±0.06*#	0.56±0.05*#
4th week	1.79±0.18	1.62±0.06*	0.40±0.03*#	0.38±0.03*#
6th week	2.39±0.20	2.19±0.10*	0.40±0.04*#	0.38±0.04*#
8th week	2.84±0.38	2.34±0.09*	0.42±0.03*#	0.36±0.05*#
PD
2nd week	2.23±0.07	1.79±0.05*	1.69±0.02*#	1.63±0.06*#
4th week	3.19±0.02	2.64±0.04*	1.72±0.04*#	1.70±0.09*#
6th week	4.33±0.05	3.47±0.12*	1.70±0.04*#	1.75±0.05*#
8th week	4.33±0.43	4.15±0.24*	1.71±0.03*#	1.67±0.03*#
mSBI
2nd week	1.85±0.34	0.87±0.09*	0.17±0.04*#	0.20±0.05*#
4th week	3.19±0.19	2.28±0.07*	0.10±0.05*#	0.15±0.08*#
6th week	3.97±0.18	2.74±0.12*	0.16±0.04*#	0.14±0.03*#
8th week	3.97±0.18	2.74±0.12*	0.16±0.04*#	0.14±0.03*#

P<0.05, compared with the SLA tied group;

P<0.05 compared with the SLA + n-HA/PEEK tied group;

PLI – plague index; PD – probing depth; mSBI – modified sulcus bleeding index; SLA – Sandblasted, large-grit and acid-etched; Implants; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone.

The IL-1, IL-6, and IL-17 levels of beagle dogs among the 3 groups

As shown in Table 2, there was no significant difference between the 2 untied groups. From the 2nd week on, the levels of inflammatory cytokines (IL-1, IL-6, and IL-17) in the tied group were higher than those in the untied group. Furthermore, they were higher in the SLA tied group than in the SLA + n-HA/PEEK tied group. The IL-1, IL-6, and IL-17 levels in the SLA tied group were also significantly different from those in the SLA + n-HA/PEEK tied group at different time points (all P<0.05).

Table 2

Comparison of PLI, PD and mSBI of beagle dogs among the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups.

	SLA tied	SLA + n-HA/PEEK tied	SLA untied	SLA + n-HA/PEEK untied
IL-1 (ng/ml)
2nd week	24.52±1.27	18.62±1.60*	14.83±0.92*#	14.38±0.15*#
4th week	26.91±1.75	20.44±1.71*	16.62±1.87*#	15.95±1.03*#
6th week	30.33±3.13	20.94±1.43*	18.41±0.87*#	16.45±1.54*#
8th week	36.43±6.66	25.26±2.72*	18.38±1.80*#	16.78±0.49*#
IL-6 (ng/ml)
2nd week	24.49±1.89	19.32±2.95*	14.87±3.46*#	15.10±1.56*#
4th week	27.78±1.46	20.54±2.68*	16.02±0.58*#	16.36±0.29*#
6th week	32.46±2.80	23.15±2.58*	16.66±1.45*#	15.61±0.46*#
8th week	37.83±1.63	26.73±2.00*	18.74±3.15*#	15.60±1.85*#
IL-17 (ng/ml)
2nd week	54.19±1.84	43.04±4.43*	31.04±1.38*#	31.26±1.42*#
4th week	55.62±4.28	48.07±2.73*	36.29±3.45*#	34.29±3.86*#
6th week	61.07±3.77	55.19±0.83*	33.45±5.88*#	33.41±5.17*#
8th week	68.16±1.58	54.70±2.54*	34.84±3.12*#	32.10±1.14*#

P<0.05 compared with the SLA tied group;

P<0.05 compared with the SLA + n-HA/PEEK tied group;

SLA – Sandblasted, large-grit and acid-etched; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone; IL – interleukin.

Colony-forming units of beagle dogs among the 3 groups

The number of colony-forming units in the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups were (14.89±0.83)×104, (14.82±0.99)×104, (7.99±0.80)×104, and (4.61±0.49)×104, respectively, in the 8th week. No evident difference was observed between 2 tied groups. However, the numbers of colony-forming units in the untied groups were significantly lower than in the tied groups. The number of colony-forming units in the SLA untied group was also obviously higher than that of the SLA + n-HA/PEEK untied group (all P<0.05) (Figure 1).

Figure 1

Comparison of number of colony-forming units in beagle dogs among the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups in the 8th week. * P<0.05 compared with the SLA tied group; # P<0.05 compared with the SLA untied group; SLA – Sandblasted, large-grit, and acid-etched; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone.

The maximum shear strength of beagle dogs among the 3 groups

The maximum shear strength in the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups were 1.75±0.10 MPa, 2.19±0.2 MPa, 3.45±0.33 MPa and 3.62±0.12 MPa, respectively, in the 8th week. No notable difference was found between 2 untied groups. However, the maximum shear strength of the untied groups was significantly higher than in the tied groups. The SLA tied group also showed a lower maximum shear strength than in the SLA + n-HA/PEEK tied group (all P<0.05) (Figure 2).

Figure 2

Comparison of maximum shear strength of implants in the beagle dogs among the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups in the 8th week. * P<0.05 compared with the SLA tied group; # P<0.05 compared with the SLA untied group; SLA – Sandblasted, large-grit, and acid-etched; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone.

Inflammation degree of beagle dogs among the 3 groups

Eight weeks after the bone tissue of 8 beagles was stained with HE, the 2 tied groups showed a large number of inflammatory cells. They also showed neutrophils, T lymphocytes, and plasma cell infiltration in the junctional epithelium and subepithelial connective tissues of the peri-implantitis site. Inflammation extended along the epithelium towards the lateral and root side. Furthermore, the subepithelial connective tissue degeneration was accompanied by a loss of collagen structure. In addition, inflammation in the SLA tied group was much higher than in the SLA + n-HA/PEEK tied group. Fewer inflammatory cells were observed in the untied group (Figure 3).

Figure 3

Inflammation response in bone tissues of beagle dogs after HE staining (× 400) in the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups in the 8th week. (A) The SLA tied group; (B) the SLA + n-HA/PEEK tied group; (C) the SLA untied group; (D) the SLA + n-HA/PEEK untied group; SLA – Sandblasted, large-grit, and acid-etched; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone; HE – hematoxylin-eosin; NC – new cementum; NP – new connective tissue; NB – new alveolar bone.

Inflammatory cell infiltration and bone reabsorption in the beagle dog implants among the 3 groups

As shown in Figure 4, the newly-formed trabecular bone on the surface of implants was stained using toluidine blue. Under the optical microscope, the newly-formed trabecular bone was seen as dark blue in color, the original bone was pale purple, and the bone calcification front was a light blue with purple granules. Under the optical microscope, the SLA+ n-HA/PEEK + tied group exhibited a peri-implant alveolar bone reabsorption with more micro-gaps and a disorganized and sparse trabecular bone. Inflammatory cell infiltration and bone reabsorption in the SLA+ n-HA/PEEK tied group, however, was less severe than in the SLA tied group. In the SLA tied group, peri-implant alveolar bone reabsorption was clearly visible and the peri-implant trabecular bone was disordered and sparse. It also showed osteoclast hyperplasia, a decrease in bone density, and inflammatory cells infiltration, indicating an inflammatory reaction. In the SLA untied and SLA + n-HA/PEEK untied groups, no obvious bone reabsorption was observed in the peri-implant neck. The peri-implant trabecular bone was also closely and neatly arranged, and a small amount of trabecular bone was visible and was arranged disorderly on the implant-bone interface. The medullary cavity also showed an irregular size and capillaries were observable. In the SLA + n-HA/PEEK untied group, a few bone tissues on the implant-bone interface directly and continuously touched the implant surface. This mainly included the osteoid or woven bone and osteoblasts with some lacuna.

Figure 4

Histological observation in implant-bone interface of beagle dogs in the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups in the 8th week. (A) The SLA tied group; (B) The SLA + n-HA/PEEK tied group; (C) The SLA untied group; (D) The SLA + n-HA/PEEK untied group; SLA – Sandblasted, large-grit, and acid-etched; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone.

Comparison of BIC of beagle dogs among the 3 groups

The BIC of the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups were 58.53%, 67.98%, 76.98%, and 78.82%, respectively. No significant difference was observed between the 2 untied groups. The BIC of the tied groups were significantly lower than those of the untied groups. The SLA tied group also showed a lower BIC than the SLA + n-HA/PEEK tied group (all P<0.05) (Figure 5).

Figure 5

Comparison of BIC of implants in beagle dogs among the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups. * P<0.05 compared with the SLA tied group; # P<0.05 compared with the SLA + n-HA/PEEK tied group; SLA – Sandblasted, large-grit, and acid-etched; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone; BIC – bone-to-implant contact.

The MAR of beagle dogs among the 3 groups

As shown in Figure 6, the results of fluorescence staining revealed that tetracycline labeling (repeated twice) under the fluorescence microscope displays continuous yellowish-green fluorescence bands. This represented newly-formed bone tissue during the 7 days. Around the implant, a bright yellow fluorescent band indicated that bone mineral deposition was active. The MAR of the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups were 1.24 ± 0.21 μm/day, 1.61 ± 0.12 μm/day, 2.52±0.13 μm/day, and 2.71±0.16 μm/day, respectively. The MAR of the SLA tied group was evidently lower than that of the SLA + n-HA/PEEK tied group (P<0.05).

Figure 6

Comparison of MAR of peri-implant bone tissues in beagle dogs among the SLA tied, SLA + n-HA/PEEK tied, SLA untied, and SLA + n-HA/PEEK untied groups. The SLA tied group; the SLA + n-HA/PEEK tied group; the SLA untied group; the SLA + n-HA/PEEK untied group; * P<0.05 compared with the SLA tied group; # P<0.05 compared with the SLA + n-HA/PEEK tied group; SLA – Sandblasted, large-grit, and acid-etched; n-HA/PEEK – nano-hydroxyapatite/polyetheretherketone; MAR – mineral apposition rate; white arrow – implant.

Discussion

In recent years, some peri-implantitis strategies such as preventing bone loss around implants, new implant designs to reduce bone remodeling following osseointegration, and modern implant abutment connection to minimize bacterial filtration have been developed. However, as bacterial contamination is impossible to completely eliminate, how the formation of subgingival plaque leads to peri-implantitis still remains unknown. Most treatment still reverts to mechanical debriding, antibiotic treatment, and osseous regeneration [1]. The present study explored the effects of n-HA/PEEK-coated SLA implants on inflammatory cytokines and osseointegration in peri-implantitis models. Inflammatory cytokines that occur at implants subjected to experimental peri-implantitis were also analyzed. Our findings demonstrate that the SLA + n-HA/PEEK implants can relieve the inflammation response following peri-implantitis in beagle dogs and promote osseointegration.

First, we found that PLI, PD, and mSBI in the SLA tied group were higher than in the SLA + n-HA/PEEK tied group at the same point in time. They were also significantly higher in the tied groups than in the untied groups. This indicates that the severity of peri-implantitis in patients with SLA implants is greater than those with SLA + n-HA/PEEK implants. PLI, PD, and mSBI are the most popularly used periodontal parameters to assess periodontal condition and monitor soft tissue status after dental implants [17,18]. The measurement of PD at implant and teeth provides different information, and minor alterations in PD at implants can reflect changes in soft tissue inflammation. More specifically, an increasing PD may be an indicator of peri-implant diseases [3]. It has been reported that PLI is significantly associated with different subgingival plaque bacterial morphotypes, and subjects with chronic periodontitis have ≥4 mm PD and/or attachment loss [19]. Second, our findings also indicated that the expression of IL-1, IL-17, and IL-6 in the tied implants groups were significantly lower than in the SLA untied implants groups. We also discovered that these expressions were significantly higher in the SLA tied groups than in the SLA + n-HA/PEEK tied group. This indicates that the SLA + n-HA/PEEK implants can relieve the inflammation response following peri-implantitis in beagle dogs. IL-1 is a proinflammatory cytokine produced by CD8+ T lymphocytes and can lead to the activity of local osteoclasts and macrophages, resulting in local destruction. IL-1 is reported to be a prognostic marker of implant failure [20]. IL-17 plays a critical role in stress granulopoiesis during microbial invasion [21] and is closely related to peri-implantitis [22]. Furthermore, cytokine IL-6 has proinflammatory functions and can induce bone reabsorption [23]. An increased IL-6 expression is correlated with peri-implantitis [15]. Severino et al. reported that the expression of IL-17 is remarkably higher in patients with peri-implantitis than in patients without peri-implantitis [24]. Yaghobee et al. demonstrated that implants with peri-implantitis show higher levels of proinflammatory cytokines such as IL-β and IL-6 in the peri-implant crevicular fluid than in healthy implants [25]. Third, this study showed that the SLA + n-HA/PEEK tied group exhibited a decrease in colony-forming units compared to the SLA tied group. Consistent with our results, Tamura et al. found that peri-implantitis sites have approximately a 10-fold higher mean number of colony-forming units (per milliliter) than healthy implant sites [26]. Collectively, these results show that SLA + n-HA/PEEK implants play an inhibitory role in the development of peri-implantitis inflammation response.

Another main finding of the present study was that the SLA tied group displayed less osseointegration and MAR than in the SLA + n-HA/PEEK tied group. Although dental implants have been clinically used, peri-implantitis is a high-incidence dental implant complication that greatly limits the longevity of the dental implant [27]. Current treatments for peri-implantitis are limited and it is hard to obtain a useful degree of re-osseointegration. Evidence from animal studies reported by Miyata et al. [28] and Hoshaw [29] and clinical studies conducted by Sanz et al. [30] and Quirynen et al. [31] indicate that occlusal load may lead to a loss of marginal peri-implant bone. SLA surface is recognized to be easily contaminated during storage, and the chemical state of SLA surfaces is often changed by different sterilization methods. This causes an increase in surface hydrophobicity and results in osseointegration degradation [32]. PEEK is a kind of bioinert material and its implantation in the human body can lead to the encapsulation of fibrous tissues, which can isolate the implant from the surrounding bone tissues [33,34]. A study on intervertebral fusion by Toth et al. reported that PEEK-Cage showed lower osseointegration efficiency than Ti-Cage [35]. Webster et al. also demonstrated that the bone contact ratio of implanted machined PEEK of a skull defect is only approximately 8% [36]. Two strategies currently available to improve PEEK bioactivity are surface treatment/coating with physical/chemical methods and composite preparation (mixing bioactive materials into PEEK) [37]. HA material is bioactive and biocompatible; therefore, it is clinically used as a critical bone substitute [38]. Consistent with our results, Abu et al. evaluated the biological responses and tissue ingrowth of HA/PEEK with the histological results in the presence of fibrovascular tissue and mature bone formation, reporting that the HA/PEEK composite showed favorable osseointegration [39]. Additionally, Ma et al. implanted a HA/PEEK composite in rat femurs and found that the new bone tissues grew faster around the composite implant and had a higher HA content. They also reported that the interface between the HA/PEEK composite and new bones were seamless 3 months after implantation, indicating a reliable biological fixation [40]. Lower osseointegration in the tied groups may be related with a lower degree of sterilization.

Conclusion

In conclusion, our study provides evidence that SLA implants coated with n-HA/PEEK can promote osseointegration and relieve the inflammation response following peri-implantitis in beagle dogs. Our study is a systematic investigation on the effects of the SLA + n-HA/PEEK implants, and further studies will be conducted in the future to verify the findings of the present study.