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British Journal of Oral and Maxillofacial Surgery xxx (2015) xxx–xxx
Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats Zhonghao Liu a,b,1 , Wenjuan Zhou b,c,1 , Stefan Tangl d,e , Shutai Liu b , Xin Xu a,∗ , Xiaohui Rausch-Fan b,c a
Department of Implant Dentistry, School of Stomatology, Shandong University, No.44-1 West culture road, Lixia District, Jinan 250012, P.R. China Department of Dental Implantology, Yantai Stomatological Hospital, No. 142, North Great Str., Zhifu District, Yantai 264008, P.R. China c Orthodontics & Periodontology Research Unit, Bernhard-Gottlieb-University Clinic of Dentistry, Sensengasse 2a-1090 Vienna, Austria d Department of Oral Surgery, Medical University of Vienna, Sensengasse 2a-1090 Vienna, Austria e Austrian Cluster for Tissue Regeneration, Vienna, Austria b
Accepted 28 May 2015
Abstract Our aim was to investigate the impact of diabetes mellitus and different durations of glycaemic control on early osseointegration of dental implants, and to explore possible mechanisms by measuring the expression of integrin ␣51 and fibronectin in bone around the implant. We divided 33 male Zucker diabetic fatty (ZDF) rats aged 3 months into 3 groups. The first group comprised diabetic rats with dental implants (controls); the second group was treated with insulin and implants were placed simultaneously (exenatide alone group); and the third group was treated with insulin until the serum glucose was at a constant concentration (< 16 mmol/L), and implants were then inserted (exenatide+normal glucose group). Rats were killed 7, 14, 30, and 60 days after implants had been inserted. The expression of integrin ␣51 and fibronectin in bone around the implants was detected by immunohistochemical analysis in each group. The expression in the exenatide+normal glucose group was stronger than in the other 2 groups. Fourteen days after implantation, expression of integrin ␣51 in the exenatide alone group was significantly stronger than that in the control group (p=0.027), and 60 days after implantation the expression of fibronectin in the exenatide alone group was also significantly stronger than that among the controls (p=0.001). Both fibronectin and integrin ␣51 participate in the adhesion of osteoblasts and act as signals at the bone/implant interface. Diabetes interferes with the osseointegration of implants by deferring expression of fibronectin and integrin ␣51. © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Dental implant; Osseointegration; Hyperglycemia; Diabetes mellitus; Integrin␣51; Fibronectin; Glycemic Control
Introduction Diabetes mellitus is characterised by hyperglycaemia caused by insufficient action or impaired secretion of insulin. It is well documented as being associated with impaired bony ∗ Corresponding author at: Dean of School of Stomatology, Shandong University, No.44-1 West culture road, Lixia District, Jinan, Shandong, 250012, China. Tel.: +86-535-6215913; fax: +86-535-6228045. E-mail address: [email protected] (X. Xu). 1 as the first author
healing and pathophysiological changes in the skeletal system as a whole. Bony healing plays an important part in implant dentistry, and some authors1,2 have hypothesised that diabetes may have a deleterious effect on the success of dental implants. They have suggested that it could adversely interfere with the process of osseointegration of implants, and even result in loosening of the implant and its ultimate failure as a result of incomplete and delayed bony formation around the implant. Some studies have shown that survival of implants can be improved in diabetic patients when the plasma glucose concentration is brought under control.3,4 If
http://dx.doi.org/10.1016/j.bjoms.2015.05.023 0266-4356/© 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Liu Z, et al. Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.05.023
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induced diabetes affects bone metabolism generally, then specific effects around implanted biomaterials require additional study. One of the key things for initial stability of a dental implant is early attachment of osteoblasts on to the surface of the implant.5 Proteins of the extracellular matrix can stimulate cell adhesion through interactions with a superfamily of cellsurface receptors called integrins such as ␣51 through the integrin-binding arg–gly–asp (RGD) tripeptide motif. These proteins have the potential to bind to the titanium oxide surface when they are present in the surrounding biological milieu. Several recent approaches have emphasised the need to modify the physical and chemical properties on the surface of the implant to increase protein binding, the attraction of appropriate cell types, and integration of the implant.6,7 Integrins are heterodimers of ␣ and  subunits that contain 3 domains: an extracellular one that is responsible for the binding of ligands, and transmembranous and cytoplasmic domains that bind to signalling and structural proteins.8 It has been suggested that integrin could signal through the cell membrane in either direction as the extracellular binding activity of integrins is regulated from the inside of the cell, while binding of the extracellular membrane elicits a cellular response through the activation of cytoplasmic kinases.9 Fibronectin is a large, extracellular, glycoprotein that helps to organise the matrix protein, and its main membrane receptor is an ␣51 integrin subunit in nucleic pulposus cells.10 It acts with RGD to bind integrin receptors, and promote osteoblast binding activity and osteogenesis. In our previous study11 we investigated the effects of exenatide microspheres on the serum bone GLA protein (osteocalcin) concentration and alkaline phosphatase activity in ZDF rats after implantation, and the results indicated that injection of delayed-release microspheres of exenatide on ZDF rats can release exenatide at a steady rate and the blood glucose can be controlled at a constant concentration. Survival of implants could be improved in diabetic subjects when plasma glucose is under control. Following on from that study, we now aimed to investigate the impact of diabetes mellitus and different durations of glycaemic control on early osseointegration of dental implants, and to explore the possible mechanism by expression of integrin ␣51 and fibronectin in peri-implant osseous tissues of ZDF rats.
dental implants (33 rats and 66 implants in total). The first group comprised diabetic rats which were given an injection of saline alone followed by 2 dental implants /rat (control group). In the second group the diabetic rats were given exenatide by subcutaneous injection and 2 implants were placed simultaneously in each rat (exenatide alone group). In the third group the diabetic rats were given 0.74% exenatide 0.1 ml/100 g body weight (Shandong Lvye Pharmaceutical Co., Ltd, Yantai, Shandong, China), until the serum glucose concentration was constant (≤16 mmol/L) and then 2 implants were inserted in each rat (exenatide+normal glucose group).11 Technique of implantation Rats were anaesthetised with an intraperitoneal injection of 4% sodium pentobarbital 0.3 ml/100 g body weight. A fullthickness incision was made on the anteromedial portion of the femur, and the site of the implant was prepared using a drill 2.3 mm in diameter under constant irrigation with sterile physiological saline. We then inserted the implant (SLA coated, screw, 2.5*2 mm, DentiumR , Korea) and confirmed its stability by passive mechanical retention. Postoperatively each rat was given penicillin 0.06 ml/kg intramuscularly daily for 3 days. Preparation of sections Rats were killed at 7 (n=4), 14 (n=6), 30 (n=6), and 60 (n=6) days postoperatively. The femurs were removed, cleaned of soft tissue, and then sectioned into blocks 3 mm mesiodistally from implants by a cutting drill, so that the specimens were formed into rectangle blocks of bone around the implants. After that, the specimens were maintained in a 4% paraformaldehyde solution for 48 hours (4 specimens/group for day 7, and 6 specimens/group for days14, 30, and 60), and were decalcified with 10% ethylene diamine tetra-acetic acid until they resembled soft tissue when probed by a needle. Implants were then removed carefully from the decalcified bone. Specimens were then dehydrated with ethanol solutions in ascending concentrations before being embedded in paraffin. The embedded specimens were sliced transversely and longitudinally into 50-100 m sections around the implants with a section cutter (LEICA RM 2235, Leica Microsystems Ltd. Bensheim, Germany).
Methods Histological examination Animals were kept in a specific pathogen-free facility (Shandong Lvye Pharmaceutical Co., Ltd, Yantai, Shandong, China), and the protocol was approved by the Institutional Animal Use Review Board. We used 33 male ZDF rats 3 months old that weighed 450 g at the beginning of the experiments. Rats were housed with a 12-hour light/dark cycle and allowed a standard pellet diet and free access to tap water throughout the observation period. The animals were divided into 3 groups, each group with 11 rats, and each rat had 2
Haematoxylin and eosin staining was used to identify anatomical details of bone trabeculae, trabecular space, microvessels, fibrous tissue, and marrow. Tartrate-resistant acid phosphatase (TRAP) special staining was used to count osteoclasts. Immunostaining and immunohistochemical analysis of streptavidin-peroxidase12 was used according to the manufacturer’s instructions to detect the expression of integrin ␣51 (Monoclonal anti-integrin ␣51 antibody,
Please cite this article in press as: Liu Z, et al. Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.05.023
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Fig. 1. Histological examination showed an area of activity of osteoclasts (OC) in the exenatide+normal glucose group on day 7 after insertion of the implants. There was abundant infiltration of inflammatory cells surrounding the cells in all 3 groups (haematoxylin and eosin, the original magnification scaler has been given in the pic.).
Sigma-Aldrich) and fibronectin (AntiFN1 polyclonal antibody, Sigma-Aldrich) in peri-implant osseous tissues. Statistical analysis Image-Pro Plus 6.2 software (Media Cybernetics Inc., Bethesda, MD, USA) was used for counting the numbers of osteoclasts that stained for TRAP followed by statistical analysis. The expression of integrin ␣51 and fibronectin in peri-implant osseous tissues was detected by calculating the mean optical density. The significance of differences between groups and within each group were assessed by one-way ANOVA separately and considered significant when the probability was less than 0.05. Levene’s test was used to meet this assumption.
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Fig. 2. Immunolocalisation imaging against fibronectin in the exanatide+normal glucose group on day 30 (immumohistochemical staining, the original magnification scaler has been given in the pic.).
Immunolocalisation imaging against fibronectin and integrin α5β1 Fibronectin and fibronectin receptor were found preferentially in osseous tissue around the surface of implants and osteoclasts, while integrin ␣51 was detected on the surface of osteoblasts. The expression of integrin ␣51 and fibronectin in the exenatide+normal glucose group was stronger than in the other 2 groups (Figs. 2 and 3). Statistical analysis of osteoclasts numbers and mean optical density The number of stained multinuclear cells in the control and exenatide alone groups reached the maximum after 14 days, whereas it was7 days in the exenatide+normal glucose group.
Results Histological examination The histological picture of the bone adjacent to the surface of the implant is shown in Fig. 1. There were more osteoclasts on days 7 and 14 after insertion in all 3 groups. The texture of spongy bone was sparse, and thinner in the control and exenatide alone groups. The peri-implant bone density of these 2 groups was lower, and the bone density of the exenatide+normal glucose group was regular. There was abundant infiltration of inflammatory cells visible in the surrounding cells in all 3 groups, there being most in the control group and least in the exenatide+normal glucose group.
Fig. 3. Immunolocalisation imaging against integrin ␣51 in the exanatide+normal glucose group on day 60 (immumohistochemical staining, the original magnification scaler has been given in the pic.).
Please cite this article in press as: Liu Z, et al. Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.05.023
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Mean opcal density of expression of fibronecn
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Number of osteoclasts
6 5 4 3 2
120 100 80 60 40 20 0
1
7
14
30
60
Days
0
7
14
30
60
Days
Fig. 4. Analysis of numbers of osteoclasts. The numbers of multinuclear cells that stained in the control and exanatide alone groups reached their maximum after 14 days. The number of stained multinuclear cells in the exenatide+normal glucose group reached its maximum on day 7 after implantation. On day 60, the number of stained multinuclear cells in the exenatide alone group seemed to rise, but not significantly so (95% CI). Blue=controls; red=exenatide alone; and green=exenatide+normal glucose.
The mean number of osteoclasts in that group was similar to the numbers in the other 2 groups (Fig. 4). The expression of integrin ␣51 in osseous tissue around the implants in the exenatide+normal glucose group was significantly stronger than in the other 2 groups during the whole study period (p=0.003). Seven days after implantation the expression of integrin ␣51 in the control and exenatide groups was similar, while 14 days after implantation its expression in the exenatide alone group was significantly stronger than that in the control group (p=0.027). After 30 days it seemed to differ between the 2 groups, but not significantly so (Fig. 5). The expression of fibronectin in osseous tissue around the implants in the exenatide+normal glucose group was significantly stronger than that in the other 2 groups during the whole study period (p=0.012). On days 7, 14, and 30 after insertion of the implants, the expression of fibronectin in the exenatide alone group was close to that of the control group, but 60 days later its expression in the exenatide alone Mean opcal density of expression of integrin a5b1
140
160 140 120 100 80 60 40 20 0
7
14
30
60
Days
Fig. 5. Expression of integrin ␣51 in osseous tissue around the implants. The expression of integrin ␣51 in the exenatide+normal glucose group was significantly stronger than that in the control and exenatide alone groups during the whole study period. After 30 days,the control and exenatide alone groups seemed to differ, but not significantly so (95% CI). Blue=controls; red=exenatide alone; and green=exenatide+normal glucose.
Fig. 6. Expression of integrin fibronectin in osseous tissue around the implants. The expression of fibronectin in osseous tissue around the implants in the exenatide+normal glucose group were significantly stronger than in the other 2 groups during the whole study period. On days 7, 14, and 30 after insertion of the implants, the expression of fibronectin in the exenatide alone group was similar to that in the control group, Sixty days later the expression of fibronectin in the exenatide alone group was significantly stronger than that in the control group (95% CI). Blue=controls; red=exenatide alone; and green=exenatide+normal glucose.
group was significantly stronger than that in the control group (p=0.001) (Fig. 6).
Discussion The influence of diabetes mellitus on dental implants has been widely studied in recent years.13–16 Some authors1,2 have stated that it remains a relative contraindication for implant treatment, while others3,4 that survival of implants could be improved if plasma glucose concentrations were brought under control. In our previous study11 our results suggested the latter point of view, and we found that survival of implants could be improved in diabetic subjects when plasma glucose concentrations were controlled. The histological observations in the present study showed that the number of osteoclasts in the exenatide+normal glucose group reached their highest level on day 7 of implantation, while the number of osteoclasts in the exenatide alone group reached their highest level on day 14, which indicated that bony remodelling and regeneration of new bone began earlier when the diabetes was controlled. The histological results also showed that there was less trabecular bone in the control and exenatide alone groups, and the trabecular bone in the exenatide+normal glucose group was arranged in a more regular fashion. The results agreed with those of previous studies, and indicated that implantation with controlled glucose can promote activity of osteoblasts and osteoclasts to a different extent, and can reinforce bony turnover and achieve a high success rate of dental implantation. RGD-integrin is one of the most important ways in which osteoblasts attach themselves.17,18 It has already been known for a number of years that certain peptides such as laminin and fibronectin can increase cellular attachment to synthetic surfaces, and can help to increase the biocompatibility of
Please cite this article in press as: Liu Z, et al. Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.05.023
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these materials. 17,19 The attachment of bone cells to surfaces coated with biomimetic peptides is mediated through different integrin receptors on the cell surface. Integrin ␣51 is one of the most important integrin subunits. 12 It has been reported20 that both periodontal and gingival fibroblasts express ␣51 integrin subunits and adhere to laminin, vitronectin, and bone sialoprotein as part of the cementum. Pegueroles et al., 21 reported that the changes in ␣5 and ␣ v mRNA expression induced by the presence of fibronectin coatings can influence the osteoblasts’ differentiation pathway. Fibronectin is a ubiquitous extracellular matrix protein that is a ligand for several integrins. Cells interact with fibronectin mainly through the ␣51 cell surface integrin, which binds to an RGD sequence. While an RGD has been identified as a key motif, the biological effect of this sequence is only a part of the activity attributed to a larger fragment of fibronectin, within which it is contained.22,23 While fibronectin around dental implants tissues contains a large series of RGD peptide sequences including a receptor-ligand formation established with integrin ␣51, it increases attachment of bone-derived cells of different species as well as increased expression of bone-specific proteins. In the present study immunostaining against fibronectin and integrin ␣51 showed that cells on the titanium surface kept the ability to behave in a bone:cell manner from the beginning of insertion of the implant. Fibronectin with its RGD sequence were found preferentially in osseous tissue around the surface of osteoclasts, from which we deduce that fibronectin blocked the resorption of bone by preventing osteoclasts from adhering to the surface of the bone, so after 7-14 days of implantation when osteoclasts began active, fibronectin was expressed more strongly. At the same time, integrin ␣51 was detected on the surface of osteoblasts, and it can bind to the fibronectin-RGD sequence to mediate the adhesion of the osteoblasts and to promote osseointegration of the implant. Our results have shown that the expression of integrin ␣51 and fibronectin in peri-implant osseous tissues in the exenatide+normal glucose group was significantly stronger than that in the other 2 groups. Fourteen days after implantation expression of integrin ␣51 in the exenatide alonegroup was significantly stronger than that in the control group, which can be explained by the fact that the plasma concentration of glucose was under control, so bony reconstruction could be inproved. All these data confirm our histological observations, which indicated that integrin ␣51 and FN were signals of osseous tissue around the implants. When osteoclasts become activated, the expression of integrin ␣51 and fibronectin in osseous tissue strengthen to prohibit osteoclasts and to promote osseointegration. In conclusion, the present results suggest that both fibronectin and integrin ␣51 participate in the adhesion of osteoblasts and act as a signal at the bone:implant interface. Diabetes interferes with osseointegration to some extent by deferring expression of fibronectin and integrin ␣51. Survival of implants could be improved in patients with
5
diabetes mellitus if the plasma glucose concentration was controlled.
Conflict of Interest We have no conflict of interest.
Ethics statement/confirmation of patients’ permission The care of animals in our studies was in accordance with institutional guidelines, and the protocol was approved by the Institutional Animal Use Review Board.
Acknowledgements The authors thank Professor Wanhui Liu (Chair of Shandong Lvye Pharmaceutical Co., Ltd., Yantai, China) for his general support.
References 1. Marchand F, Raskin A, Dionnes-Hornes A, et al. Dental implants and diabetes: conditions for success. Diabetes Metab 2012;38:14–9. 2. Wang F, Song YL, Li DH, et al. Type 2 diabetes mellitus impairs bone healing of dental implants in GK rats. Diabetes Rese Clin Pract 2010;88:e7–9. 3. Javed F, Romanos GE. Impact of diabetes mellitus and glycemic control on the osseointegration of dental implants: a systematic literature Review. J Periodontol 2009;80:1719–30. 4. Wang B, Song Y, Wang F, et al. Effects of local infiltration of insulin around titanium implants in diabetic rats. Br J Oral Maxillofac Surg 2011;49:225–9. 5. Rinaldi JC, Arana-Chavez VE. Ultrastructure of the interface between periodontal tissues and titanium mini-implants. Angle Orthod 2010;80: 459–65. 6. Rapuano BE, MacDonald DE. Surface oxide net charge of a titanium alloy: modulation of fibronectin-activated attachment and spreading of osteogenic cells. Colloids Surf B Biointerfaces 2011;82:95–103. 7. Martino MM, Tortelli F, Mochizuki M, et al. Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing. Sci Transl Med 2011;3:100ra89. 8. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 1992;69:11–25. 9. Rammelt S, Illert T, Bierbaum S, et al. Coating of titanium implants with collagen, RGD peptide and chondroitin sulfate. Biomaterials 2006;27:5561–71. 10. Gilchrist CL, Chen J, Richardson WJ, et al. Functional integrin subunits regulating cell–matrix interactions in the intervertebral disc. J Orthop Res 2007;25:829–40. 11. Zhou W, Liu Z, Yao J, et al. The effects of exenatide microsphere on serum BGP and ALP levels in ZDF rats after implantation. Clin Implant Dent Relat Res 2013, http://dx.doi.org/10.1111/cid.12184. 12. Liu C, Kaneko S, Soma K. Expression of integrin␣51, focal adhesion kinase and integrin-linked kinase in rat condylar cartilage during mandibular lateral displacement. Arch Oral Biol 2008;53:701–8. 13. Murillo J, Wang Y, Xu X, et al. Advanced glycation of type I collagen and fibronectin modifies periodontal cell behavior. J Periodontol 2008;79:2190–9.
Please cite this article in press as: Liu Z, et al. Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.05.023
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14. Yamamoto T, Ozono K, Miyauchi A, et al. Role of advanced glycation end products in dynamic bone disease in patients with diabetic nephropathy. Am J Kidney Dis 2001;38:S161–4. 15. Santana RB, Xu L, Chase HB, et al. A role for advanced glycation end products in diminished bone healing in type 1 diabetes. Diabetes 2003;52:1502–10. 16. Kim W, Hudson BI, Moser B, et al. Receptor for advanced glycation end products and its ligands: A journey from the complications of diabetes to its pathogenesis. Ann N Y Acad Sci 2005;1043: 553–61. 17. Schliephake H, Scharnweber D, Dard M, et al. Functionalization of dental implant surfaces using adhesion molecules. J Biomed Mater Res B Appl Biomater 2005;73:88–96. 18. Verrier S, Pallu S, Bareille R, et al. Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process. Biomaterials 2002;23:585–96.
19. Bhadriraju K, Hansen LK. Hepatocyte adhesion, growth and differentiated function on RGD-containing proteins. Biomaterials 2000;21: 267–72. 20. Walsh P, Häkkinen L, Pernu H, et al. Expression of fibronectin-binding integrins in gingival epithelium in drug-induced gingival overgrowth. J Periodontal Res 2007;42:144–51. 21. Pegueroles M, Aguirre A, Engel E, et al. Effect of blasting treatment and Fn coating on MG63 adhesion and differentiation on titanium: a gene expression study using real time RT-PCR. J Mater Sci Mater Med 2011;22:617–27. 22. Dufour C, Holy X, Marie PJ. Skeletal unloading induces osteoblast apoptosis and targets ␣51-PI3K-Bcl-2 signaling in rat bone. Exp Cell Res 2007;313:394–403. 23. Xia M, Zhu Y. Fibronectin fragment activation of ERK increasing integrin␣5 and 1 subunit expression to degenerate nucleus pulposus cells. J Orthop Res 2011;29:556–61.
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ARTICLE IN PRESS Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery xxx (2015) xxx–xxx
Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats Zhonghao Liu a,b,1 , Wenjuan Zhou b,c,1 , Stefan Tangl d,e , Shutai Liu b , Xin Xu a,∗ , Xiaohui Rausch-Fan b,c a
Department of Implant Dentistry, School of Stomatology, Shandong University, No.44-1 West culture road, Lixia District, Jinan 250012, P.R. China Department of Dental Implantology, Yantai Stomatological Hospital, No. 142, North Great Str., Zhifu District, Yantai 264008, P.R. China c Orthodontics & Periodontology Research Unit, Bernhard-Gottlieb-University Clinic of Dentistry, Sensengasse 2a-1090 Vienna, Austria d Department of Oral Surgery, Medical University of Vienna, Sensengasse 2a-1090 Vienna, Austria e Austrian Cluster for Tissue Regeneration, Vienna, Austria b
Accepted 28 May 2015
Abstract Our aim was to investigate the impact of diabetes mellitus and different durations of glycaemic control on early osseointegration of dental implants, and to explore possible mechanisms by measuring the expression of integrin ␣51 and fibronectin in bone around the implant. We divided 33 male Zucker diabetic fatty (ZDF) rats aged 3 months into 3 groups. The first group comprised diabetic rats with dental implants (controls); the second group was treated with insulin and implants were placed simultaneously (exenatide alone group); and the third group was treated with insulin until the serum glucose was at a constant concentration (< 16 mmol/L), and implants were then inserted (exenatide+normal glucose group). Rats were killed 7, 14, 30, and 60 days after implants had been inserted. The expression of integrin ␣51 and fibronectin in bone around the implants was detected by immunohistochemical analysis in each group. The expression in the exenatide+normal glucose group was stronger than in the other 2 groups. Fourteen days after implantation, expression of integrin ␣51 in the exenatide alone group was significantly stronger than that in the control group (p=0.027), and 60 days after implantation the expression of fibronectin in the exenatide alone group was also significantly stronger than that among the controls (p=0.001). Both fibronectin and integrin ␣51 participate in the adhesion of osteoblasts and act as signals at the bone/implant interface. Diabetes interferes with the osseointegration of implants by deferring expression of fibronectin and integrin ␣51. © 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Dental implant; Osseointegration; Hyperglycemia; Diabetes mellitus; Integrin␣51; Fibronectin; Glycemic Control
Introduction Diabetes mellitus is characterised by hyperglycaemia caused by insufficient action or impaired secretion of insulin. It is well documented as being associated with impaired bony ∗ Corresponding author at: Dean of School of Stomatology, Shandong University, No.44-1 West culture road, Lixia District, Jinan, Shandong, 250012, China. Tel.: +86-535-6215913; fax: +86-535-6228045. E-mail address: [email protected] (X. Xu). 1 as the first author
healing and pathophysiological changes in the skeletal system as a whole. Bony healing plays an important part in implant dentistry, and some authors1,2 have hypothesised that diabetes may have a deleterious effect on the success of dental implants. They have suggested that it could adversely interfere with the process of osseointegration of implants, and even result in loosening of the implant and its ultimate failure as a result of incomplete and delayed bony formation around the implant. Some studies have shown that survival of implants can be improved in diabetic patients when the plasma glucose concentration is brought under control.3,4 If
http://dx.doi.org/10.1016/j.bjoms.2015.05.023 0266-4356/© 2015 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Liu Z, et al. Potential mechanism for osseointegration of dental implants in Zucker diabetic fatty rats. Br J Oral Maxillofac Surg (2015), http://dx.doi.org/10.1016/j.bjoms.2015.05.023
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induced diabetes affects bone metabolism generally, then specific effects around implanted biomaterials require additional study. One of the key things for initial stability of a dental implant is early attachment of osteoblasts on to the surface of the implant.5 Proteins of the extracellular matrix can stimulate cell adhesion through interactions with a superfamily of cellsurface receptors called integrins such as ␣51 through the integrin-binding arg–gly–asp (RGD) tripeptide motif. These proteins have the potential to bind to the titanium oxide surface when they are present in the surrounding biological milieu. Several recent approaches have emphasised the need to modify the physical and chemical properties on the surface of the implant to increase protein binding, the attraction of appropriate cell types, and integration of the implant.6,7 Integrins are heterodimers of ␣ and  subunits that contain 3 domains: an extracellular one that is responsible for the binding of ligands, and transmembranous and cytoplasmic domains that bind to signalling and structural proteins.8 It has been suggested that integrin could signal through the cell membrane in either direction as the extracellular binding activity of integrins is regulated from the inside of the cell, while binding of the extracellular membrane elicits a cellular response through the activation of cytoplasmic kinases.9 Fibronectin is a large, extracellular, glycoprotein that helps to organise the matrix protein, and its main membrane receptor is an ␣51 integrin subunit in nucleic pulposus cells.10 It acts with RGD to bind integrin receptors, and promote osteoblast binding activity and osteogenesis. In our previous study11 we investigated the effects of exenatide microspheres on the serum bone GLA protein (osteocalcin) concentration and alkaline phosphatase activity in ZDF rats after implantation, and the results indicated that injection of delayed-release microspheres of exenatide on ZDF rats can release exenatide at a steady rate and the blood glucose can be controlled at a constant concentration. Survival of implants could be improved in diabetic subjects when plasma glucose is under control. Following on from that study, we now aimed to investigate the impact of diabetes mellitus and different durations of glycaemic control on early osseointegration of dental implants, and to explore the possible mechanism by expression of integrin ␣51 and fibronectin in peri-implant osseous tissues of ZDF rats.
dental implants (33 rats and 66 implants in total). The first group comprised diabetic rats which were given an injection of saline alone followed by 2 dental implants /rat (control group). In the second group the diabetic rats were given exenatide by subcutaneous injection and 2 implants were placed simultaneously in each rat (exenatide alone group). In the third group the diabetic rats were given 0.74% exenatide 0.1 ml/100 g body weight (Shandong Lvye Pharmaceutical Co., Ltd, Yantai, Shandong, China), until the serum glucose concentration was constant (≤16 mmol/L) and then 2 implants were inserted in each rat (exenatide+normal glucose group).11 Technique of implantation Rats were anaesthetised with an intraperitoneal injection of 4% sodium pentobarbital 0.3 ml/100 g body weight. A fullthickness incision was made on the anteromedial portion of the femur, and the site of the implant was prepared using a drill 2.3 mm in diameter under constant irrigation with sterile physiological saline. We then inserted the implant (SLA coated, screw, 2.5*2 mm, DentiumR , Korea) and confirmed its stability by passive mechanical retention. Postoperatively each rat was given penicillin 0.06 ml/kg intramuscularly daily for 3 days. Preparation of sections Rats were killed at 7 (n=4), 14 (n=6), 30 (n=6), and 60 (n=6) days postoperatively. The femurs were removed, cleaned of soft tissue, and then sectioned into blocks 3 mm mesiodistally from implants by a cutting drill, so that the specimens were formed into rectangle blocks of bone around the implants. After that, the specimens were maintained in a 4% paraformaldehyde solution for 48 hours (4 specimens/group for day 7, and 6 specimens/group for days14, 30, and 60), and were decalcified with 10% ethylene diamine tetra-acetic acid until they resembled soft tissue when probed by a needle. Implants were then removed carefully from the decalcified bone. Specimens were then dehydrated with ethanol solutions in ascending concentrations before being embedded in paraffin. The embedded specimens were sliced transversely and longitudinally into 50-100 m sections around the implants with a section cutter (LEICA RM 2235, Leica Microsystems Ltd. Bensheim, Germany).
Methods Histological examination Animals were kept in a specific pathogen-free facility (Shandong Lvye Pharmaceutical Co., Ltd, Yantai, Shandong, China), and the protocol was approved by the Institutional Animal Use Review Board. We used 33 male ZDF rats 3 months old that weighed 450 g at the beginning of the experiments. Rats were housed with a 12-hour light/dark cycle and allowed a standard pellet diet and free access to tap water throughout the observation period. The animals were divided into 3 groups, each group with 11 rats, and each rat had 2
Haematoxylin and eosin staining was used to identify anatomical details of bone trabeculae, trabecular space, microvessels, fibrous tissue, and marrow. Tartrate-resistant acid phosphatase (TRAP) special staining was used to count osteoclasts. Immunostaining and immunohistochemical analysis of streptavidin-peroxidase12 was used according to the manufacturer’s instructions to detect the expression of integrin ␣51 (Monoclonal anti-integrin ␣51 antibody,
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Fig. 1. Histological examination showed an area of activity of osteoclasts (OC) in the exenatide+normal glucose group on day 7 after insertion of the implants. There was abundant infiltration of inflammatory cells surrounding the cells in all 3 groups (haematoxylin and eosin, the original magnification scaler has been given in the pic.).
Sigma-Aldrich) and fibronectin (AntiFN1 polyclonal antibody, Sigma-Aldrich) in peri-implant osseous tissues. Statistical analysis Image-Pro Plus 6.2 software (Media Cybernetics Inc., Bethesda, MD, USA) was used for counting the numbers of osteoclasts that stained for TRAP followed by statistical analysis. The expression of integrin ␣51 and fibronectin in peri-implant osseous tissues was detected by calculating the mean optical density. The significance of differences between groups and within each group were assessed by one-way ANOVA separately and considered significant when the probability was less than 0.05. Levene’s test was used to meet this assumption.
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Fig. 2. Immunolocalisation imaging against fibronectin in the exanatide+normal glucose group on day 30 (immumohistochemical staining, the original magnification scaler has been given in the pic.).
Immunolocalisation imaging against fibronectin and integrin α5β1 Fibronectin and fibronectin receptor were found preferentially in osseous tissue around the surface of implants and osteoclasts, while integrin ␣51 was detected on the surface of osteoblasts. The expression of integrin ␣51 and fibronectin in the exenatide+normal glucose group was stronger than in the other 2 groups (Figs. 2 and 3). Statistical analysis of osteoclasts numbers and mean optical density The number of stained multinuclear cells in the control and exenatide alone groups reached the maximum after 14 days, whereas it was7 days in the exenatide+normal glucose group.
Results Histological examination The histological picture of the bone adjacent to the surface of the implant is shown in Fig. 1. There were more osteoclasts on days 7 and 14 after insertion in all 3 groups. The texture of spongy bone was sparse, and thinner in the control and exenatide alone groups. The peri-implant bone density of these 2 groups was lower, and the bone density of the exenatide+normal glucose group was regular. There was abundant infiltration of inflammatory cells visible in the surrounding cells in all 3 groups, there being most in the control group and least in the exenatide+normal glucose group.
Fig. 3. Immunolocalisation imaging against integrin ␣51 in the exanatide+normal glucose group on day 60 (immumohistochemical staining, the original magnification scaler has been given in the pic.).
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Mean opcal density of expression of fibronecn
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Number of osteoclasts
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Fig. 4. Analysis of numbers of osteoclasts. The numbers of multinuclear cells that stained in the control and exanatide alone groups reached their maximum after 14 days. The number of stained multinuclear cells in the exenatide+normal glucose group reached its maximum on day 7 after implantation. On day 60, the number of stained multinuclear cells in the exenatide alone group seemed to rise, but not significantly so (95% CI). Blue=controls; red=exenatide alone; and green=exenatide+normal glucose.
The mean number of osteoclasts in that group was similar to the numbers in the other 2 groups (Fig. 4). The expression of integrin ␣51 in osseous tissue around the implants in the exenatide+normal glucose group was significantly stronger than in the other 2 groups during the whole study period (p=0.003). Seven days after implantation the expression of integrin ␣51 in the control and exenatide groups was similar, while 14 days after implantation its expression in the exenatide alone group was significantly stronger than that in the control group (p=0.027). After 30 days it seemed to differ between the 2 groups, but not significantly so (Fig. 5). The expression of fibronectin in osseous tissue around the implants in the exenatide+normal glucose group was significantly stronger than that in the other 2 groups during the whole study period (p=0.012). On days 7, 14, and 30 after insertion of the implants, the expression of fibronectin in the exenatide alone group was close to that of the control group, but 60 days later its expression in the exenatide alone Mean opcal density of expression of integrin a5b1
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Fig. 5. Expression of integrin ␣51 in osseous tissue around the implants. The expression of integrin ␣51 in the exenatide+normal glucose group was significantly stronger than that in the control and exenatide alone groups during the whole study period. After 30 days,the control and exenatide alone groups seemed to differ, but not significantly so (95% CI). Blue=controls; red=exenatide alone; and green=exenatide+normal glucose.
Fig. 6. Expression of integrin fibronectin in osseous tissue around the implants. The expression of fibronectin in osseous tissue around the implants in the exenatide+normal glucose group were significantly stronger than in the other 2 groups during the whole study period. On days 7, 14, and 30 after insertion of the implants, the expression of fibronectin in the exenatide alone group was similar to that in the control group, Sixty days later the expression of fibronectin in the exenatide alone group was significantly stronger than that in the control group (95% CI). Blue=controls; red=exenatide alone; and green=exenatide+normal glucose.
group was significantly stronger than that in the control group (p=0.001) (Fig. 6).
Discussion The influence of diabetes mellitus on dental implants has been widely studied in recent years.13–16 Some authors1,2 have stated that it remains a relative contraindication for implant treatment, while others3,4 that survival of implants could be improved if plasma glucose concentrations were brought under control. In our previous study11 our results suggested the latter point of view, and we found that survival of implants could be improved in diabetic subjects when plasma glucose concentrations were controlled. The histological observations in the present study showed that the number of osteoclasts in the exenatide+normal glucose group reached their highest level on day 7 of implantation, while the number of osteoclasts in the exenatide alone group reached their highest level on day 14, which indicated that bony remodelling and regeneration of new bone began earlier when the diabetes was controlled. The histological results also showed that there was less trabecular bone in the control and exenatide alone groups, and the trabecular bone in the exenatide+normal glucose group was arranged in a more regular fashion. The results agreed with those of previous studies, and indicated that implantation with controlled glucose can promote activity of osteoblasts and osteoclasts to a different extent, and can reinforce bony turnover and achieve a high success rate of dental implantation. RGD-integrin is one of the most important ways in which osteoblasts attach themselves.17,18 It has already been known for a number of years that certain peptides such as laminin and fibronectin can increase cellular attachment to synthetic surfaces, and can help to increase the biocompatibility of
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these materials. 17,19 The attachment of bone cells to surfaces coated with biomimetic peptides is mediated through different integrin receptors on the cell surface. Integrin ␣51 is one of the most important integrin subunits. 12 It has been reported20 that both periodontal and gingival fibroblasts express ␣51 integrin subunits and adhere to laminin, vitronectin, and bone sialoprotein as part of the cementum. Pegueroles et al., 21 reported that the changes in ␣5 and ␣ v mRNA expression induced by the presence of fibronectin coatings can influence the osteoblasts’ differentiation pathway. Fibronectin is a ubiquitous extracellular matrix protein that is a ligand for several integrins. Cells interact with fibronectin mainly through the ␣51 cell surface integrin, which binds to an RGD sequence. While an RGD has been identified as a key motif, the biological effect of this sequence is only a part of the activity attributed to a larger fragment of fibronectin, within which it is contained.22,23 While fibronectin around dental implants tissues contains a large series of RGD peptide sequences including a receptor-ligand formation established with integrin ␣51, it increases attachment of bone-derived cells of different species as well as increased expression of bone-specific proteins. In the present study immunostaining against fibronectin and integrin ␣51 showed that cells on the titanium surface kept the ability to behave in a bone:cell manner from the beginning of insertion of the implant. Fibronectin with its RGD sequence were found preferentially in osseous tissue around the surface of osteoclasts, from which we deduce that fibronectin blocked the resorption of bone by preventing osteoclasts from adhering to the surface of the bone, so after 7-14 days of implantation when osteoclasts began active, fibronectin was expressed more strongly. At the same time, integrin ␣51 was detected on the surface of osteoblasts, and it can bind to the fibronectin-RGD sequence to mediate the adhesion of the osteoblasts and to promote osseointegration of the implant. Our results have shown that the expression of integrin ␣51 and fibronectin in peri-implant osseous tissues in the exenatide+normal glucose group was significantly stronger than that in the other 2 groups. Fourteen days after implantation expression of integrin ␣51 in the exenatide alonegroup was significantly stronger than that in the control group, which can be explained by the fact that the plasma concentration of glucose was under control, so bony reconstruction could be inproved. All these data confirm our histological observations, which indicated that integrin ␣51 and FN were signals of osseous tissue around the implants. When osteoclasts become activated, the expression of integrin ␣51 and fibronectin in osseous tissue strengthen to prohibit osteoclasts and to promote osseointegration. In conclusion, the present results suggest that both fibronectin and integrin ␣51 participate in the adhesion of osteoblasts and act as a signal at the bone:implant interface. Diabetes interferes with osseointegration to some extent by deferring expression of fibronectin and integrin ␣51. Survival of implants could be improved in patients with
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diabetes mellitus if the plasma glucose concentration was controlled.
Conflict of Interest We have no conflict of interest.
Ethics statement/confirmation of patients’ permission The care of animals in our studies was in accordance with institutional guidelines, and the protocol was approved by the Institutional Animal Use Review Board.
Acknowledgements The authors thank Professor Wanhui Liu (Chair of Shandong Lvye Pharmaceutical Co., Ltd., Yantai, China) for his general support.
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