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Published in final edited form as: J Oral Maxillofac Surg. 2018 Mar 1;76(8):1677–1687. doi: 10.1016/j.joms.2018.02.021

Effect of obesity/metabolic syndrome and diabetes on osseointegration of dental implants in a miniature swine model. A Pilot Study

Paulo G Coelho 1,*, Benjamin Pippenger 2, Nick Tovar 3, Sietse-Jan Koopmans 4, Natalie Plana 5, Dana T Graves 6, Steve Engebretson 7, Heleen MM van Beusekom 8, Paula GFP Oliveira 9, Michel Dard 10
PMCID: PMC6064394  NIHMSID: NIHMS947603  PMID: 29572133

Abstract

Purpose

The increasing prevalence of obesity/metabolic syndrome (O/MS) and diabetes mellitus (DM) remains a global health concern. Clinically relevant and practical translational models mimicking human characteristics of these conditions are lacking. This study aimed to demonstrate proof of concept of the induction of stable obesity/metabolic syndrome (O/MS) and type-2 diabetes mellitus (DM) in a Göttingen minipig model and validate both of these disease-adjusted Göttingen mini-pig models as impaired healing models for the testing of dental implants.

Materials and Methods

9 minipigs were split into 3 groups: control (normal diet), obese (cafeteria diet) and diabetic (cafeteria diet + Streptozotocin), and followed by placement of dental implants. Inflammatory markers including TNF-α, C-reactive protein, and cortisol were recorded for each study group. Removal torque and histomorphometric analysis (bone to implant contact (BIC) and bone area fraction occupancy (BAFO)) were performed.

Results

O/MS pigs showed, on average, a 2-fold increase in plasma C-reactive protein (p<0.05) and cortisol (p<0.09) concentrations compared to controls; DM pigs showed, on average, a 40-fold increase in plasma TNF-α (p<0.05) and a 2-fold increase in cortisol (p<0.05) concentrations compared to controls. The impact of O/MS and DM on implants was determined. Torque to interface failure was highest in control (200 Ncm), and significantly lower in O/MS (90 Ncm) and DM (60 Ncm) groups (p<0.01). Bone formation around implants was significantly greater in control than O/MS and DM (p<0.02).

Conclusions

Both O/MS and DM minipigs express human-like disease phenotype and both presented bone healing impairment around dental implants. No significant difference between type-2 diabetes and obesity/metabolic syndrome on bone formation around implants provides evidence that further investigation of the impact of obesity/metabolic syndrome is warranted.

1. Introduction

Nearly 40 and 10% of the global population suffers from obesity and diabetes, respectively, with a projected 20–30% increase in diabetes prevalence by 2050 1–3. Among the many consequences of these diseases, poor wound healing remains a primary concern to oral health practitioners. These patients carry an increased risk of alveolar bone loss 4 and periodontal diseases 5. While it is recognized that diabetes negatively impacts implant treatment, less is known about the impact of obesity and metabolic syndrome 6. Current understanding of bone pathology in obesity/metabolic syndrome (O/MS) and its progression to type 2 diabetes mellitus (DM) is limited to studies suggesting that adipose-derived pro-inflammatory cytokines may be responsible for the degeneration of oral health in these populations 7. Thus, studies investigating the metabolic effects on dentition are warranted, given the paucity of clinically relevant and translational O/MS and DM animal models for studying disease effect on dental implants.

Swine are increasingly the preferred alternative to dogs or non-human primates for nonrodent biomedical and food research 8, 9. Similarities to humans permit a close replication of complex pathophysiology, with recent developments in obese and diabetic swine models demonstrating similar complications to their human counterparts, including hyperglycemia, hyperlipidemia, hypertension, insulin resistance, a pro-inflammatory state 10–12, delayed wound healing 13, and reduced bone mineralization 14. The reproducibility of these complications, which are known to challenge the long-term success of dental implants in humans, makes the obese/diabetic pig particularly useful for preclinical studies on dental surgery and periodontitis 15. Swine models also allow for the assessment of implants used in humans, and thereby yield relevant translational data. However, adult obese swine are difficult to handle due to their size (reaching 300 – 400 kg body weight) and do not express an extreme obese phenotype, thereby citing a need for a more manageable model.

Göttingen minipig models have been successfully used in oral surgery translational research. Metabolic syndrome Göttingen minipig models have already been developed through a high energy feeding diet for short periods of up to 3 months 16, 17. Minipigs can achieve body mass index levels characteristic of O/MS while maintaining a far more manageable adult body weight that rarely exceeds 80–90 kg. A type 2 diabetes-like state has also been induced in obese Göttingen minipigs by low dosage administration of streptozotocin 17, 18, demonstrating the potential to study the progression of obesity to diabetes, closely resembling human metabolic compromise. To date, studies have not reported stable pathological changes to the Göttingen minipig in long-term models 16–18. In this pilot study we aimed to: 1) demonstrate proof of concept of the induction of stable, obesity/metabolic syndrome (O/MS) and type-2 diabetes mellitus (DM) in a Göttingen minipig model and 2) validate both of these disease-adjusted Göttingen mini-pig models as impaired healing models for the testing of dental implant osseointegration.

2. Materials and Methods

2.1 Establishment/Maintenance of obese/metabolic syndrome and diabetic minipig models

Animal selection, surgery protocol and study management were approved by the Animal Care and Use Committee and followed ARRIVE guidelines 19. A total of 9 female Göttingen minipigs (Ellegaard, Dalmose, Denmark) 18 months of age were used for this study. Minipigs were split into 3 groups: (1) control (normal diet) (n=3), (2) obese (cafeteria diet) (n=3) and (3) diabetic (cafeteria diet + Streptozotocin) (n=3). Normal diet was characterized as being low in fat Standard Diet (SDS Standard Diet Service, UK# 801586), while a diet high in saturated and hydrogenated fats/cholesterol/sugar was defined as “cafeteria” diet by RDS Cafeteria Diet (Research Diet Services NL) (obese/metabolic syndrome and diabetic groups). The total weight of raw material between the two diets were equivalent20 (Table 1). Animals were fed twice a day. Diet progression was split into 3 phases: conversion, growth, and maintenance phases. To induce O/MS, minipigs (n = 6; O/MS and DM groups) were gradually introduced to the cafeteria diet over a period of 4 weeks (conversion phase, 25% decrease in normal diet on a weekly basis and restricted feeding to two 500g meals per day), after which time they remained at 100% cafeteria diet for 8 months and were allowed to feed ad libitum (growth phase). Once O/MS and DM animals approximately doubled their original weight, the cafeteria diet was then halved and combined with control diet (maintenance phase). Control animals were fed the control diet. Minipigs assigned to the DM group (n = 3) were induced by slow injection of filter-sterilized streptozotocin solution (STZ, Enzo Life Sciences, Raamsdonksveer, the Netherlands) (20 mg/kg in 0.1 mol/L Na-citrate, pH 4.5) on two consecutive days after overnight fasting18. STZ-injected swine were given 25g glucose to offset insulin release from dying β-cells, thereby preventing hypoglycemia.

Table 1.

Basic formulation of the control and cafeteria diets

Raw Material Control diet Cafeteria diet
Soya beans, extracted (cf<50g/kg) 164.5
Potato protein (ash<10g/kg) 50 50
Wheat gluten meal 8.7 106
Barley 396.2
Wheat 500
Porcine fat (lard) 100
Hydrogenated soya bean oil 100
Hydrogenated coconut oil 50
Soy bean oil 17.3
Fructose 200
Sucrose 200
Limestone 13 9.6
Mono calcium phosphate 6.9 10.8
NaCl 4 4.7
Mineral/vitamin.Premix 2 2
L-lysine HCl 1.9 2.4
Cholesterol (extra) 10
Total (g/kg) 1000 1010
Gross energy (GE; MJ/kg) 17.3 23.5
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The following criteria were used to characterize and validate the induction of O/MS and DM in this study: animal weight, blood analysis including glucose, ketones, tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), plasma cortisol, plasma insulin levels, and pancreatic histology. Animal weight was measured at animal reception, 8 weeks, 14 weeks and subsequently every 2 weeks for the remainder of the study. Additionally, at time of euthanasia, internal organs were independently weighed (wet weight) including the heart (right and left ventricles independently), liver, spleen, and kidneys. The pathophysiology associated with metabolic syndrome in humans and swine models are presented in Table 2. 21–36

Table 2.

Pathophysiology related to Metabolic Syndrome.

Weight/
Abdominal
circunference		 Glucose Ketones TNF-
α		 CRP Plasma
cortisol		 Plasma
insulin
levels		 Pancreatic
histology		 Wet
weight
organs
Göttingen Minipig Current study + + + + + + + + +
Humans Mantizoros, 200621 + + N/A + + N/A + N/A N/A
Ervin, 2009 22 + + N/A N/A N/A N/A N/A N/A N/A
Yang et al., 2014 23 + + N/A N/A N/A N/A N/A N/A N/A
Aguilar et al., 2015 24 + + N/A N/A N/A N/A N/A N/A N/A
Aronson et al., 2005 25 + + N/A N/A + N/A N/A N/A N/A
Raffaitin et al., 2009 26 + + N/A N/A N/A N/A N/A N/A N/A
Birdsill et al., 2013 27 + + N/A N/A N/A N/A N/A N/A N/A
Hanson et al., 2002 28 + + N/A N/A N/A N/A + N/A N/A
Lind, 2008 29 + + N/A N/A N/A N/A N/A N/A N/A
Göttingen Minipig Pedersen et al., 2013 30 + + N/A N/A N/A N/A + N/A N/A
Johansen et al., 2001 16 + + N/A N/A N/A N/A + N/A +
Christoffersen et al., 2007 31 + + N/A N/A N/A N/A + N/A N/A
Christoffersen et al., 2013 32 + + N/A N/A N/A N/A N/A N/A N/A
Swines Huang et al., 2013 33 + + N/A N/A N/A N/A + N/A +
Phillips-Eakley et al., 2015 34 + + N/A N/A N/A N/A N/A N/A N/A
Westover et al., 2016 35 + + N/A N/A N/A N/A N/A N/A N/A
Bratz et al., 2008 36 + + N/A N/A N/A N/A + N/A +
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+ - Available

N/A – Not available

Fasting plasma glucose and ketone levels were monitored weekly using Glucomen LX (A. Menarini Diagnostics, Germany). Plasma TNF-α, c-reactive protein (CRP), insulin and cortisol levels were tested at the time of implantation and termination (late maintenance phase weeks 43 and 47, respectively) through commercially available kits: TNF-α (Invitrogen Elisa Kit for Swine TNF-α Assay, Invitrogen Corporation, USA), CRP (Porcine C-reactive Protein Assay, Tridelta Development Ltd, Ireland), cortisol (Radioimmunoassay Coat-a-count cortisol, Siemens Healthcare Diagnostics Inc., CA, USA), and insulin (Porcine Insulin ELISA kit, Mercodia AB, Uppsala, Sweden).

To determine the amount of insulin-producing cells, tissues were immersion-fixed in 4% buffered formaldehyde processed for paraffin embedding. Immunohistochemistry was performed using a polyclonal guinea-pig anti-swine insulin antibody (DAKO, Glostrup, Denmark), with diaminobenzidine/H2O2 as a chromogen to visualize the horseradish peroxidase-labeled secondary (rabbit anti-guinea-pig) antibody. The insulin stain was quantified by determining the total surface of insulin-producing cells as a percentage of the total area in that section.

2.2 Dental Implant Surgical Placement and Implant Analyses

Surgeries were performed following previously described methodology37. At the initiation of the study, and prior to O/MS and DM induction, mandibular premolars and first molars (P1, P2, P3 and M1) were extracted. Healing was allowed for 3 months prior to metabolic disease induction. After induction and stabilization of O/MS and DM conditions, custom designed 4.2 × 6 mm implants were placed bilaterally (2 per side) and allowed a 4-week implant healing period prior to euthanasia.

After euthanasia, the right mandibular implants were subjected to removal torque in counterclockwise rotation to the implant axis at a rate of 0.1 degree/second and maximum torque was recorded as previously described 38. The left mandibular implants underwent non-decalcified histologic processing as previously described 39. Photographs were taken from all samples at 200X. Each histologic section was assessed for bone-to-implant contact (BIC) and bone area fraction occupancy (BAFO) within the implant healing chambers as previously described 40.

2.3 Statistical Analysis

Results are expressed as means ± SD and the criterion of statistical significance was set at p < 0.05. All of the data (except biomechanical and histomorphometric) were subjected to the analysis of variance procedure (ANOVA) followed by the student’s t-test of Genstat 5 (Payne RW, Lane PW, Ainsley AE: Genstat 5 Reference Manual Oxford, UK: Oxford University Press; 1987) for determination of differences between the three pig groups. A linear mixed model was utilized to evaluate biomechanical and histomorphometric data.

3. Results

Animal and Organ Weights

At initiation of the study, Göttingen minipigs presented with an average weight of 28kg. Upon initiation of respective diets the control animals continued to grow for about 14 weeks when weight plateaued at ~45 kg. Weights of both groups of pigs receiving the cafeteria diet (DM and O/MS) increased over time to ~73 and ~83kg at termination, respectively (Figure 1A). Macroscopically, necropsy revealed excessive fatty deposits surrounding the internal organs, with particularly thick deposits around the heart and subcutaneous tissue. Organ wet weights showed that O/MS group with heavier left ventricle and right ventricle relative to control and DM groups (Figure 1B). The DM group presented higher lung, liver, and kidney weights compared to O/MS and control groups (Figures 1B and 1C). Spleen weights decreased from control to O/MS to DM (Figure 1C).

Figure 1. Progressive weight gain in Göttingen minipigs and critical organ wet weights.

Figure 1

Figure 1

Figure 1

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A) Gross weight of control, O/MS, and DM pigs throughout the experiment demonstrating rapid weight gain for animals receiving the cafeteria diet (O/MS and DM) in the first phase, followed by weight stabilization in DM group and slower increase in O/MS group. STZ indicates the time of Streptozotocin administration. Time represents weeks after dental extraction. B) Average Heart (left and right ventricles weighed separately) and lung weights at 47 weeks (sacrifice) demonstrating higher average weights for critically affected organs in O/MS and DM animals. C) Average weights of liver, spleen, and left and right kidneys of each group at time of sacrifice.

Blood Assays

Following STZ administration, the DM group presented average blood glucose levels 5-times greater (~12 mmol/L) than the O/MS and control groups (average of 3 mmol/L). These values remained relatively constant throughout the experiment (Figure 2A). Ketone levels similarly demonstrated an increase in the DM group after STZ administration at an average of 0.5 mmol/L, with fluctuation, while the control and O/MS groups presented close to undetectable levels (Figure 2B). TNF-α plasma levels were significantly higher in the DM group (p<0.05) compared to both control and O/MS groups (Figure 2C). Plasma CRP levels were greatest in the O/MS group, compared to controls, values that were significant (p<0.05) at the time of animal sacrifice (O/MS: 3.1±0.6 mg/L, control: 1.0±0.2 mg/L) (Figure 2D). The average plasma cortisol levels at implantation were not statistically different between groups. However the levels were significantly (p<0.05) increased for the DM group compared to the control group at euthanasia. The O/MS group showed a tendency (p<0.09) towards increased cortisol levels compared to controls (Figure 2E). Plasma insulin levels were slightly greater in the O/MS group, but were not significantly different among groups at implantation or termination (Figure 2F).

Figure 2. Blood marker profiles demonstrate effective induction of metabolic syndrome and a DM phenotype.

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

Figure 2

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A) Blood glucose levels following STZ administration. Glucose levels are particularly elevated in DM animals while O/MS animal levels are comparable to control. B) Blood ketone levels are elevated in the DM group. Obese and control groups remain at basal levels. C–F) Average plasma levels of Tumor necrosis factor-α (TNF-α), Creactive protein (CRP), Cortisol and Insulin taken at the time of implantation and termination. Overall pro-inflammatory status is evident in the O/MS and DM groups. G–J) Immunohistochemical staining and quantification of insulin on pancreatic tissue sections demonstrate significantly increased staining in (H) O/MS pigs compared to (G) controls and (I) DM, however no (J) statistical differences was detected between controls and DM. The number of asterisks denotes statistically homogeneous groups.

Pancreatic Insulin Stain

Quantitative immunohistochemical staining demonstrated that O/MS pigs had a significantly higher levels of insulin staining compared to control and DM animals (p<0.02). Insulin staining was not significantly different between the DM and control groups (p>0.15) (Figures 2G–J).

Biomechanical Testing and Histomorphometric Analysis

The torque to interfacial failure was significantly (p<0.001) decreased in the O/MS group and the DM group compared to controls (Figure 3A). Qualitative histologic examination demonstrated increased amounts of bone growth around implants placed in control group animals compared to metabolically compromised groups (Figures 4A–C). No bone morphologic difference between groups was evident. Histomorphometric analysis showed that the control group exhibited significantly higher values of BIC (bone-to-implant contact) compared to O/MS (p<0.01) and DM (P<0.001) (Figure 3B). Similarly, the control group exhibited significantly higher values (p<0.02) of BAFO (bone area fraction occupancy) measurements compared to metabolic disease groups (Figure 3C). There was no significant difference between the O/MS and DM groups in both BIC and BAFO measurements.

Figure 3. Biomechanical and histomorphometric measurements.

Figure 3

Figure 3

Figure 3

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A) Maximum torque-out values for implants at time of sacrifice. Lower torque-out vales demonstrate that the osseointegration of implants in O/MS and DM animals is significantly less than in the control group. There are no significant differences between O/MS and DM groups. B) Histomorphometric analysis of tissue/implant sections. BAFO corresponds to the new bone area per total area within a defined region of interest (ROI), here defined as the total area from the defect border to the implant surface. BIC corresponds to the total bone to implant contact and is expressed as a percentage of the bone physically attached to the implant surface as compared to the total implant surface. Histological evidence supports the biomechanical measurements and demonstrates the significantly less new bone formation around dental implants is O/MS and DM groups as compared to the control group. The number of asterisks denotes statistically homogeneous groups.

Figure 4. Histological sections demonstrate bone formation surrounding implants.

Figure 4

Figure 4

Figure 4

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Hematoxylin and eosin stained sections of mandibular implant sites of A) Control, B) O/MS and C) DM pig groups demonstrating considerable amounts of newly formed bone encompassing dental implants seen as a darker pink compared to existing bone seen in lighter pink.

4. Discussion

Compromised healing around endosteal implants due to metabolic conditions such as O/MS and DM are prominent health concerns that remain unaddressed 41. The present investigation aimed to demonstrate proof of concept of the induction of stable, obesity/metabolic syndrome (O/MS) and type-2 diabetes mellitus (DM) in a Göttingen minipig model and determine whether obesity induced metabolic syndrome had the same impact on the initial stability of dental implants as type-2 diabetes and to determine whether mechanistically it was caused by reduced bone formation around the implant.

While bone healing impairment around implants was observed upon induction of a stable diabetic state, we also observed that in the obese state impaired bone healing reached comparable impairment levels to those found in uncontrolled diabetic animals. The O/MS minipigs had high weight gain, high levels of systemic inflammation as evidenced by elevated CRP, and high levels of insulin production in the pancreas. Weight gain through a diet high in saturated and hydrogenated fats and sugars resulted in disease induction phenotypically similar to human in these animals. Animal weight increase and stabilization without reversing the disease process strongly suggests that Göttingen minipigs manifest a similar disease process to humans 42, 43. The DM group followed the natural progression of obesity with a mild state of diabetes (detectable glucose metabolism deficiency) successfully achieved with a mild STZ regimen that has been reported to have no metabolic effect on Göttingen minipigs. Yet, when following diet induced O/MS, such STZ regimen effectively induced a disease phenotype similar to type-2 diabetes mellitus, 17, 44 indicating that O/MS Göttingen minipigs are predisposed to type-2 diabetes and the modest additional damage to beta-cells by low dose STZ is sufficient to induce a type 2 DM phenotype. Importantly, following induction of DM, animals reached diagnostic levels of hyperglycemia.

Previous studies 41, 45 have used swine as a systemically compromised and clinically relevant model for dental implant testing. However, these differed from the current investigation as they utilized the substantially larger domestic pig model using STZ to induce a diabetic state without prior O/MS induction resulting in a Type I diabetes phenotype (no obesity induction was performed prior STZ diabetes induction).

In the context of insulin-resistance in both O/MS and DM 46, elevated cortisol levels are expected given the known insulin antagonistic effects of cortisol 47. Conversely, and in agreement with previous reports 48, TNF-α levels were not significantly elevated in the obese phenotype but were elevated in the DM group, suggesting that further inflammatory state evolution is induced by the diabetic state, contributing to clinical sequelae prevalent in this metabolic disease. A previous study has shown that inflammatory factors are not as affected in obese mice relative to the values observed in the present investigation.49

For both metabolic conditions, initial bone formation around implants was significantly less pronounced compared to controls and affected the biomechanical stability of implants. Such histomorphometric and biomechanical results in diabetic animals have been explained by previous investigations and include reduced osteoblast expression, reduced osteoid production, impaired bone apposition to implants,5, 50–52 and decreased expression of bone matrix proteins 53, 54. While no reports are currently available regarding impaired osseointegration of implants in O/MS subjects, recent work in humans has demonstrated reduced bone mineral density in adolescents.

5. Conclusion

Obesity/metabolic syndrome and diabetes have known associations with higher dental implant faiure and are regarded as significant risk-factors for implant therapy 55. In this study, we present a highly translational large animal model of O/MS where a pro-inflammatory state is established in the early phases of obesity and persists upon induction of diabetes. Moreover, we showed the equally compromised bone healing around dental implants in O/MS and uncontrolled DM pigs compared to controls. Given the increasing prevalence of these progressive metabolic conditions, the animal models described may be a useful in future translational development of preventive/therapeutic approaches that minimize oral rehabilitation morbidity related to O/MS and DM.

Acknowledgments

The authors acknowledge and valuable help and input from Marcel Obrecht. The implants utilized in the study were kindly donated by Straumann AB, Switzerland. Funds for the current project originated from a grant (926/2013) from the ITI Foundation (Switzerland), NIH/NIDCR DE023649-01A1, and DE023649-02S1.

Footnotes

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Conflict of interest

The authors declare no conflict of interest.

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