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Effects of local infiltration of insulin around titanium implants in diabetic rats
Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
Effects of local infiltration of insulin around titanium implants in diabetic rats Baogang Wang a , Yingliang Song a,∗ , Feng Wang a , Dehua Li a , Hepeng Zhang b , Aijie Ma b , Na Huang c a b c
Dept. of Oral Implantology, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, Xi’an, PR China Chemical Department, College of Science, Northwestern Polytechnical University, Xi’an 710072, PR China Dept. of Paediatrics, Xijing Hospital, The Fourth Military Medical University, 169 West Changle Road, Xi’an, PR China
Accepted 10 March 2010 Available online 18 April 2010
Abstract In patients with type 2 diabetes mellitus (DM) there is poorer quality osseointegration than in other patients, and the success of oral implants is less. The aim of the present study was to investigate the influence of local infiltration of insulin at the implant–bone interface after implantation in diabetic rats. We used GK rats (8-week-old Goto-Kakizaki Wistar rats, n = 20) in a newly established model of type 2 DM, and Sprague–Dawley rats were used as controls (n = 10). GK rats were divided into two groups: those with DM alone and those with DM given insulin (INS) (n = 10 in each group). The INS group was given controlled-release insulin at the implant–bone interface. Rats were killed at 2 and 6 weeks after implantation. We evaluated bone–implant contact and bony volume in all rats. Implant–bone contact, osteoid and osteogenic volume, and the amount of newly formed bone in the DM group were significantly less than in the control (p < 0.05) and INS (p < 0.01) groups. Implant–bone contact in the INS group was less than that in the control group, but the amount of newly formed bone was greater. In conclusion, we suggest that although the implant–bone contact in the INS group did not reach the control level, direct infiltration of insulin could improve implant–bone contact. Local infiltration of insulin at the implant–bone interface may have important clinical implications by naturally improving the success of oral implantation in diabetic rats. © 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Titanium implants; Diabetes mellitus; Insulin; Osseointegration; Microspheres; GK rats
Introduction Diabetes mellitus (DM) is characterised by high concentrations of glucose in the blood, and Type 2 DM accounts for about 90–95% of all diagnosed cases of DM. Some studies have shown that DM can affect the bone, which causes osteopenia and impairs the healing of fractures.1,2 Osteopenia is thought to be a contributing factor to the increased risk of fracture in diabetic patients.3–5 Krakauer et al. reported that patients with DM have reduced formation and accumulation of bone during growth, while later in life hyperglycaemia ∗
Corresponding author. Tel.: +86 29 84776451; fax: +86 29 82521217. E-mail address: [email protected] (Y. Song).
leads to increased resorption of bone and osteopenia.6 Some clinical studies have reported that implants in patients with type 2 DM are more likely to fail than those in non-diabetic patients.7–9 Hasegawa et al. reported that bone morphogenesis in diabetic rats was characterised by fragmented bone and extensive soft tissue infiltration, which was not seen in non-diabetic rats.10 Systemic treatment with insulin reversed impaired bony healing in diabetic animals, possibly through improvement in the formation of bone and inhibition of resorption.11 However, that study failed to make clear whether insulin was directly responsible for the improvement in impaired healing in diabetic patients. A previous study12 had shown that insulin is critical to the process of healing of fractures in
0266-4356/$ – see front matter © 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2010.03.006
226
B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
diabetic patients by a direct effect on the callus, and indicated that insulin may have a direct effect on the healing of fractures in healthy animals. There is a continuing search for local insulin delivery systems. Some studies 13,14 have investigated the use of co-polylactic/glycolic acid (PLGA, a biodegradable injectable polymer) microcapsules to make a preparation that releases insulin in a controlled fashion over a long period with no rapid release phases. The biological activity of insulin extracted from the formulation was similar to that of normal insulin.14 In this study, we aimed to deliver insulin from insulincontaining PLGA microcapsules directly to the implant site and find out if it directly improves the implant–bone contact and so increases the success rates of implantation in diabetic rats. To the best of our knowledge this has not been investigated before.
Materials and methods Insulin (INS, 100 U/ml) was obtained from the Chinese branch of Novo Nordisk Pharmaceutical Industries, Inc. PLGA (L:G ratio 50:50), which was synthesised and provided by the Chemical Department, College of Science, Northwestern Polytechnical University, Xi’an, China. Male, 8-week-old GK rats were obtained from Shanghai SLAC Laboratory Animal Co. Ltd., China. Sprague–Dawley rats were bought by Laboratory Animal Resources of The Fourth Military Medical University. Animals We used 20 GK rats and 10 Sprague–Dawley rats. The guidelines of the National Institutes of Health and the Institute of Animal Ethical Committee for the care and use of laboratory animals were respected. The animals were fed with a diet enriched with fat and glucose. Blood samples were drawn from diabetic rats after a week to measure the blood glucose concentration; rats that did not match the required index (fasting blood glucose ≥7.8 mmol/l) were excluded from the study. Sprague–Dawley rats were used as the control group and GK rats were divided into 2 groups (those with DM and given INS, and those with DM alone, n = 10 in each).
for 10 min. The resulting emulsion was stirred at 200 rpm for 8 h with a propeller stirrer to allow evaporation of the solvent and hardening of the microspheres. The microspheres were then isolated by filtration, washed 3 times with deionised water by centrifugation, and freeze-dried. The prepared pulverised microspheres were sterilised and stored at 4 ◦ C in a desiccator. Insulin-containing microspheres were examined under a scanning electron microscope (EM. FEI Quanta 200, Holland). To correspond with the environment of the human blood and body fluids, the insulin-containing microspheres (26 mg) were dispersed in phosphate-buffered saline (PBS, pH = 7.4, 1 ml). The solution was spun continuously (100–120 rpm) at a biochemical incubator at 37 ◦ C (0.5 ◦ C). At preset intervals supernatants were collected after centrifugation, and an equal amount of fresh butter was added and incubated. The release was analysed with an ultraviolet spectrophotometer at 277 nm, and free insulin was used as a standard.16,17 Encapsulation efficiency (%) was calculated as follows (weight of the insulin in microspheres/weight of the feeding insulin) × 100%. The diameter of the particles of microspheres was measured by a fibre-optic particle analyser under scanning EM. Implantation The implants were cleaned in trichloroethylene (99.5%), rinsed in absolute alcohol, and autoclaved at 115 (5)◦ C for 30 min to ensure their sterility. Rats were anaesthetised with an peritoneal injection of 2% sodium pentobarbital (0.3 ml/100 g body weight). A full-thickness incision was made and the implant site prepared by sequential drilling. A considerable amount of sterilised physiological saline solution was used to irrigate the field for cooling and cleaning. We then inserted the implant and confirmed its stability by passive mechanical retention. Simultaneously we mixed the previously prepared pulverised microspheres and blood, and loaded them on the surface of the implant before it was inserted in the rats in the GK group. The microspheres were released immediately directly into the bone around the implants. The wound was closed with conventional sutures. Histological specimens
Insulin-containing microspheres Microspheres were prepared by a modified double emulsion method as reported previously.15 Briefly, polymer (Ingelheim Co., Germany, 0.5 g) and span 80 (sorbitan oleate, Chemical Industry, Beijing) 0.14 g were dissolved in dichloromethane (DCM) 10 ml. In this organic phase, the solution, insulin 0.1 ml, was dissolved in deionised water 0.4 ml, and emulsified using a high-speed homogeniser for 60 s to form the w/o emulsion. This primary emulsion was injected into 100 ml aqueous solution containing 0.2% PVA (polyvinyl alcohol, external phase (Clariant Co., Switzerland) and homogenised
Half of the animals were killed after 2 weeks and the rest after 6 weeks. The tibias were dissected, and blocks containing the experimental specimens were obtained. All specimens were fixed in 10% neutral buffered formalin solution for 4 days, and the specimens were dehydrated using ascending grades of alcohol, infiltrated, and embedded in methylmethacrylate (MMA) without decalcification. The embedded specimens were sawn perpendicular to the exposed implant with a section cutter (LEICA SP1600, Leica Microsystems, Bensheim, Germany). All sections were stained with modified Masson trichrome staining.
B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
227
Histomorphometry of bone For histomorphometric measurements of the bone, images were obtained using an image collection system (Olympus BH2 with S Plan FL2 lens, Tokyo, Japan). The bone–implant contact was measured by using a software program (Leica Imaging System, Cambridge, England). The induced bone density (osteogenic volume, newly formed bone volume) adjacent to the surface of the implant was measured at distances of <1 and 1 mm by assessing the condition of the bone.18,19 Statistical analysis For histomorphometric analyses, data are presented as mean (SD). One-way analysis of variance (ANOVA) was used to assess the significance of differences between the groups with the help of the Statistical Package for the Social Sciences (SPSS version 13.0 for Windows). Probabilities of less than 0.05 were accepted as significant.
Results and discussion Analysis of insulin-containing microspheres The scanning EM image of the insulin-containing microspheres is shown in Fig. 1. The morphology of the surface of the insulin-containing microspheres was uneven, spherules were uniform, and they were scattered about. There was little synechial formation among the microspheres. The size of microspheres measured by a fibre-optic particle analyser under scanning EM was in the range of 1.4–8.9 m. Our microspheres were significantly smaller than those reported in previous studies.14 The encapsulation efficiency of the insulin-containing microspheres was
Fig. 1. Insulin-containing microspheres (scanning electron microscope, original magnification 20 × 1000).
evaluated as 73 (0.5) %. It was anticipated that the insulin was lost during the course of injection and preparation of the microspheres, such as the adherence to the walls of the test tube during rinsing. The figure was a little higher than those given in recent studies.16,20 The reduction in insulin could be avoided by rinsing the syringe over and over again. In previous studies,20,21,22 the in vitro release of microspheres was usually achieved using the centrifuge instead of the dialysis method. The centrifuge method is relatively simple and is able to simulate the pH and temperature of the body, despite the fact that insulin can be released more rapidly than by the dialysis method.20 The insulin-containing microspheres had a well-controlled release effect (Fig. 2). They released almost 30% of the insulin after day 1. However, small amounts of insulin were released during the following days, and there was still an appreciable release of insulin after 10 days. Higher adequate insulin concentrations could be main-
Fig. 2. Release profile of insulin microspheres in vitro. They released 6.0% of insulin after 1 h of incubation; the cumulative insulin release was 14.8% (after 2 h), 20.4% (after 3 h), and 24.7% (after 4 h).
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B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
Fig. 3. Histological appearance of representative titanium implants after 2 weeks in the INS group, the average rate of bone–implant contact was 50.73% (2.80%).
Fig. 5. Histological appearance of representative titanium implants after 2 weeks in the DM group, the average rate of bone–implant contact was 28.82% (5.32%).
tained locally after day 1 because of the controlled release of the microspheres, which had essentially disappeared after 14 days.
(Fig. 5); there was considerable osteoid tissue around the titanium implants in the latter group. Bone–implant contact was noticeable in the implanted area in both the INS and the control group, but not in the DM group. The mean rate of bone–implant contact in the INS group was significantly lower than that in the control group (p < 0.05), but much lower in the DM group (p < 0.01). After 6 weeks, the quantity of newly formed bone was less, and there was a lot of osteogen around the implant in the control and INS groups, but less osteogen and more osteoid tissue in the DM group. The proportion of osteoid in the control group was higher than that in the INS group. The mean (SD) rate of bone–implant contact in the DM group of 51 (3) % was significantly lower than that in the INS group (58 (3) %) (p < 0.01) and in the control group (66 (4) %) (p < 0.01). Bone–implant contact in the INS and control groups also differed significantly (p < 0.01). Insulin can increase the proliferation of osteoblasts as growth factors and in suitable concentrations results in downregulation of P44-42 MAPK signal activity.23 The appropriate concentration of insulin could effectively increase the proliferation of osteoblasts in 1 day, but the effectiveness would lessen during the following days. However, to date we have not had an effective way of maintaining high-enough concentrations of insulin locally over a long period. More time will be required to study the problem. Within 2 weeks, the volume of newly formed bone was stimulated by insulin released from the microspheres in the INS group. However, other rats with DM that were not given insulin had little new bone. In addition, osteogen was detected after 6 weeks at the mineralised new bone adjacent to the surface of the implant. The results showed that the bone–implant contact in the INS group differed from those found in the DM
Histomorphometric analysis After 2 weeks, histological examination showed that new bone had formed beside the titanium implants in the INS group (Fig. 3) and in the control group (Fig. 4). More had formed within the INS group, and less in the DM group
Fig. 4. Histological appearance of representative titanium implants after 2 weeks in the control group, the average rate of bone–implant contact was 56.55% (4.57%).
B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
and control groups after 2 and 6 weeks. Although that in the INS group was not as great as that in the control group, it was significantly different from that in the DM group. Insulin could therefore increase osseointegration after implantation in diabetic rats.
References 1. Jehle PM, Jehle DR, Mohan S, Bohm BO. Serum levels of insulin-like growth factor system components and relationship to bone metabolism in Type 1 and Type 2 diabetes mellitus patients. J Endocrinol 1998;159:297–306. 2. Tuominen JT, Impivaara O, Puukka P, Ronnemaa T. Bonemineral density in patients with type 1 and type 2 diabetes. Diabetes Care 1999;22:1196–200. 3. Ivers RQ, Cumming RG, Mitchell P, Peduto AJ. Diabetes and risk of fracture: The Blue Mountains Eye Study. Diabetes Care 2001;24:1198–203. 4. Nicodemus KK, Folsom AR. Iowa Women’s Health Study. Type 1 and type 2 diabetes and incident hip fractures in postmenopausal women. Diabetes Care 2001;24:1192–7. 5. Vestergaard P, Rejnmark L, Mosekilde L. Relative fracture risk in patients with diabetes mellitus, and the impact of insulin and oral antidiabetic medication on relative fracture risk. Diabetologia 2005;48:1292–9. 6. Krakauer J, McKenna M, Buderer N, Rao D, Whitehouse F, Pafitt A. Bone loss and bone turnover in diabetes. Diabetes 1995;44:775–82. 7. Morris HF, Ochi S, Winkler S. Implant survival in patients with type 2 diabetes: placement to 36 months. Ann Periodontol 2000;5:157–65. 8. Olson JW, Shernoff AF, Tarlow JL, Colwell JA, Scheetz JP, Bingham SF. Dental endosseous implant assessments in type 2 diabetic population: A prospective study. Int J Oral Maxillofac Implants 2000;15:811–8. 9. Moy PK, Medina D, Shetty V, Aghaloo TL. Dental implant failure rates and associated risk factors. Int J Oral Maxillofac Implants 2005;20:569–77. 10. Hasegawa H, Ozawa S, Hashimoto K, Takeichi T, Ogawa T. Type 2 diabetes impairs implant osseointegration capacity in rats. Int J Oral Maxillofac Implants 2008;23:237–46. 11. Beam HA, Parsons JR, Lin SS. The effects of blood glucose control upon fracture healing in the BB Wistar rat with diabetes mellitus. J Orthop Res 2002;20:1210–6.
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12. Gandhi A, Beam HA, O’Connor JP, Parsons JR, Lin SS. The effects of local insulin delivery on diabetic fracture healing. Bone 2005;37:482–90. 13. Takenaga M, Yamaguchi Y, Kitagawa A, Ogawa Y, Mizushima Y, Igarashi R. A novel sustained-release formulation of insulin with dramatic reduction in initial rapid release. J Control Release 2002;79:81–91. 14. Takenaga M, Yamaguchi Y, Kitagawa A, et al. Optimum formulation for sustained-release insulin. Int J Pharm 2004;271:85–94. 15. Frauke Pistel K, Breitenbach A, Zange-Volland R, Kissel T. Brush-like branched biodegradable polyesters, part III. Protein release from microspheres of poly (vinyl alcohol)-graft-poly (d,l-lactic-co-glycolic acid). J Control Release 2002;73:7–20. 16. Balabushevich NG, Larionova NI. Fabrication and characterization of polyelectrolyte microparticles with protein. Biochemistry (Mosc) 2004;69:757–62. 17. Bugamelli F, Raggi MA, Orienti I, Zecchi V. Controlled insulin release from chitosan microparticles. Arch Pharm 1998;331:133–8. 18. Nociti Jr FH, Cesar NJ, Carvalho MD, Sallum EA. Bone density around titanium implants may be influenced by intermittent cigarette smoke inhalation: a histometric study in rats. Int J Oral Maxillofac Implants 2002;17:347–52. 19. Becker J, Kirsch A, Schwarz F, Chatzinikolaidou M, Rothamel D, Lekovic V, et al. Bone apposition to titanium implants biocoated with recombinant human bone morphogenetic protein-2 (rhBMP-2). A pilot study in dogs. Clin Oral Investig 2006;10:217–24 [Erratum: Clin Oral Investig 2006;10:225]. 20. Jian Z, Xiuli Y, Zhifei D, Yang W, Shaoqin L, Xiufeng Y. Novel iron–polysaccharide multilayered microcapsules for controlled insulin release. Acta Biomater 2009;5:1499–507. 21. Fan YF, Wang YN, Fan YG, Ma JB. Preparation of insulin nanoparticles and their encapsulation with biodegradable polyelectrolytes via the layer-by-layer adsorption. Int J Pharm 2006;324:158–67. 22. Dai Z, Heilig A, Zastrow H, Donath E, Mohwald H. Novel formulations of vitamins and insulin by nanoengineering of polyelectrolyte multilayers around microcrystals. Chemistry 2004;10:6369–74. 23. Li-yang L, Min-lian D, Zhe M, Ling-yu H. Effects of insulin on osteoblast proliferation and post-receptor MAP kinase activity. J Sun Yat-Sen Univ 2005;26:268–72.
British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
Effects of local infiltration of insulin around titanium implants in diabetic rats Baogang Wang a , Yingliang Song a,∗ , Feng Wang a , Dehua Li a , Hepeng Zhang b , Aijie Ma b , Na Huang c a b c
Dept. of Oral Implantology, School of Stomatology, The Fourth Military Medical University, 145 West Changle Road, Xi’an, PR China Chemical Department, College of Science, Northwestern Polytechnical University, Xi’an 710072, PR China Dept. of Paediatrics, Xijing Hospital, The Fourth Military Medical University, 169 West Changle Road, Xi’an, PR China
Accepted 10 March 2010 Available online 18 April 2010
Abstract In patients with type 2 diabetes mellitus (DM) there is poorer quality osseointegration than in other patients, and the success of oral implants is less. The aim of the present study was to investigate the influence of local infiltration of insulin at the implant–bone interface after implantation in diabetic rats. We used GK rats (8-week-old Goto-Kakizaki Wistar rats, n = 20) in a newly established model of type 2 DM, and Sprague–Dawley rats were used as controls (n = 10). GK rats were divided into two groups: those with DM alone and those with DM given insulin (INS) (n = 10 in each group). The INS group was given controlled-release insulin at the implant–bone interface. Rats were killed at 2 and 6 weeks after implantation. We evaluated bone–implant contact and bony volume in all rats. Implant–bone contact, osteoid and osteogenic volume, and the amount of newly formed bone in the DM group were significantly less than in the control (p < 0.05) and INS (p < 0.01) groups. Implant–bone contact in the INS group was less than that in the control group, but the amount of newly formed bone was greater. In conclusion, we suggest that although the implant–bone contact in the INS group did not reach the control level, direct infiltration of insulin could improve implant–bone contact. Local infiltration of insulin at the implant–bone interface may have important clinical implications by naturally improving the success of oral implantation in diabetic rats. © 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Keywords: Titanium implants; Diabetes mellitus; Insulin; Osseointegration; Microspheres; GK rats
Introduction Diabetes mellitus (DM) is characterised by high concentrations of glucose in the blood, and Type 2 DM accounts for about 90–95% of all diagnosed cases of DM. Some studies have shown that DM can affect the bone, which causes osteopenia and impairs the healing of fractures.1,2 Osteopenia is thought to be a contributing factor to the increased risk of fracture in diabetic patients.3–5 Krakauer et al. reported that patients with DM have reduced formation and accumulation of bone during growth, while later in life hyperglycaemia ∗
Corresponding author. Tel.: +86 29 84776451; fax: +86 29 82521217. E-mail address: [email protected] (Y. Song).
leads to increased resorption of bone and osteopenia.6 Some clinical studies have reported that implants in patients with type 2 DM are more likely to fail than those in non-diabetic patients.7–9 Hasegawa et al. reported that bone morphogenesis in diabetic rats was characterised by fragmented bone and extensive soft tissue infiltration, which was not seen in non-diabetic rats.10 Systemic treatment with insulin reversed impaired bony healing in diabetic animals, possibly through improvement in the formation of bone and inhibition of resorption.11 However, that study failed to make clear whether insulin was directly responsible for the improvement in impaired healing in diabetic patients. A previous study12 had shown that insulin is critical to the process of healing of fractures in
0266-4356/$ – see front matter © 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bjoms.2010.03.006
226
B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
diabetic patients by a direct effect on the callus, and indicated that insulin may have a direct effect on the healing of fractures in healthy animals. There is a continuing search for local insulin delivery systems. Some studies 13,14 have investigated the use of co-polylactic/glycolic acid (PLGA, a biodegradable injectable polymer) microcapsules to make a preparation that releases insulin in a controlled fashion over a long period with no rapid release phases. The biological activity of insulin extracted from the formulation was similar to that of normal insulin.14 In this study, we aimed to deliver insulin from insulincontaining PLGA microcapsules directly to the implant site and find out if it directly improves the implant–bone contact and so increases the success rates of implantation in diabetic rats. To the best of our knowledge this has not been investigated before.
Materials and methods Insulin (INS, 100 U/ml) was obtained from the Chinese branch of Novo Nordisk Pharmaceutical Industries, Inc. PLGA (L:G ratio 50:50), which was synthesised and provided by the Chemical Department, College of Science, Northwestern Polytechnical University, Xi’an, China. Male, 8-week-old GK rats were obtained from Shanghai SLAC Laboratory Animal Co. Ltd., China. Sprague–Dawley rats were bought by Laboratory Animal Resources of The Fourth Military Medical University. Animals We used 20 GK rats and 10 Sprague–Dawley rats. The guidelines of the National Institutes of Health and the Institute of Animal Ethical Committee for the care and use of laboratory animals were respected. The animals were fed with a diet enriched with fat and glucose. Blood samples were drawn from diabetic rats after a week to measure the blood glucose concentration; rats that did not match the required index (fasting blood glucose ≥7.8 mmol/l) were excluded from the study. Sprague–Dawley rats were used as the control group and GK rats were divided into 2 groups (those with DM and given INS, and those with DM alone, n = 10 in each).
for 10 min. The resulting emulsion was stirred at 200 rpm for 8 h with a propeller stirrer to allow evaporation of the solvent and hardening of the microspheres. The microspheres were then isolated by filtration, washed 3 times with deionised water by centrifugation, and freeze-dried. The prepared pulverised microspheres were sterilised and stored at 4 ◦ C in a desiccator. Insulin-containing microspheres were examined under a scanning electron microscope (EM. FEI Quanta 200, Holland). To correspond with the environment of the human blood and body fluids, the insulin-containing microspheres (26 mg) were dispersed in phosphate-buffered saline (PBS, pH = 7.4, 1 ml). The solution was spun continuously (100–120 rpm) at a biochemical incubator at 37 ◦ C (0.5 ◦ C). At preset intervals supernatants were collected after centrifugation, and an equal amount of fresh butter was added and incubated. The release was analysed with an ultraviolet spectrophotometer at 277 nm, and free insulin was used as a standard.16,17 Encapsulation efficiency (%) was calculated as follows (weight of the insulin in microspheres/weight of the feeding insulin) × 100%. The diameter of the particles of microspheres was measured by a fibre-optic particle analyser under scanning EM. Implantation The implants were cleaned in trichloroethylene (99.5%), rinsed in absolute alcohol, and autoclaved at 115 (5)◦ C for 30 min to ensure their sterility. Rats were anaesthetised with an peritoneal injection of 2% sodium pentobarbital (0.3 ml/100 g body weight). A full-thickness incision was made and the implant site prepared by sequential drilling. A considerable amount of sterilised physiological saline solution was used to irrigate the field for cooling and cleaning. We then inserted the implant and confirmed its stability by passive mechanical retention. Simultaneously we mixed the previously prepared pulverised microspheres and blood, and loaded them on the surface of the implant before it was inserted in the rats in the GK group. The microspheres were released immediately directly into the bone around the implants. The wound was closed with conventional sutures. Histological specimens
Insulin-containing microspheres Microspheres were prepared by a modified double emulsion method as reported previously.15 Briefly, polymer (Ingelheim Co., Germany, 0.5 g) and span 80 (sorbitan oleate, Chemical Industry, Beijing) 0.14 g were dissolved in dichloromethane (DCM) 10 ml. In this organic phase, the solution, insulin 0.1 ml, was dissolved in deionised water 0.4 ml, and emulsified using a high-speed homogeniser for 60 s to form the w/o emulsion. This primary emulsion was injected into 100 ml aqueous solution containing 0.2% PVA (polyvinyl alcohol, external phase (Clariant Co., Switzerland) and homogenised
Half of the animals were killed after 2 weeks and the rest after 6 weeks. The tibias were dissected, and blocks containing the experimental specimens were obtained. All specimens were fixed in 10% neutral buffered formalin solution for 4 days, and the specimens were dehydrated using ascending grades of alcohol, infiltrated, and embedded in methylmethacrylate (MMA) without decalcification. The embedded specimens were sawn perpendicular to the exposed implant with a section cutter (LEICA SP1600, Leica Microsystems, Bensheim, Germany). All sections were stained with modified Masson trichrome staining.
B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
227
Histomorphometry of bone For histomorphometric measurements of the bone, images were obtained using an image collection system (Olympus BH2 with S Plan FL2 lens, Tokyo, Japan). The bone–implant contact was measured by using a software program (Leica Imaging System, Cambridge, England). The induced bone density (osteogenic volume, newly formed bone volume) adjacent to the surface of the implant was measured at distances of <1 and 1 mm by assessing the condition of the bone.18,19 Statistical analysis For histomorphometric analyses, data are presented as mean (SD). One-way analysis of variance (ANOVA) was used to assess the significance of differences between the groups with the help of the Statistical Package for the Social Sciences (SPSS version 13.0 for Windows). Probabilities of less than 0.05 were accepted as significant.
Results and discussion Analysis of insulin-containing microspheres The scanning EM image of the insulin-containing microspheres is shown in Fig. 1. The morphology of the surface of the insulin-containing microspheres was uneven, spherules were uniform, and they were scattered about. There was little synechial formation among the microspheres. The size of microspheres measured by a fibre-optic particle analyser under scanning EM was in the range of 1.4–8.9 m. Our microspheres were significantly smaller than those reported in previous studies.14 The encapsulation efficiency of the insulin-containing microspheres was
Fig. 1. Insulin-containing microspheres (scanning electron microscope, original magnification 20 × 1000).
evaluated as 73 (0.5) %. It was anticipated that the insulin was lost during the course of injection and preparation of the microspheres, such as the adherence to the walls of the test tube during rinsing. The figure was a little higher than those given in recent studies.16,20 The reduction in insulin could be avoided by rinsing the syringe over and over again. In previous studies,20,21,22 the in vitro release of microspheres was usually achieved using the centrifuge instead of the dialysis method. The centrifuge method is relatively simple and is able to simulate the pH and temperature of the body, despite the fact that insulin can be released more rapidly than by the dialysis method.20 The insulin-containing microspheres had a well-controlled release effect (Fig. 2). They released almost 30% of the insulin after day 1. However, small amounts of insulin were released during the following days, and there was still an appreciable release of insulin after 10 days. Higher adequate insulin concentrations could be main-
Fig. 2. Release profile of insulin microspheres in vitro. They released 6.0% of insulin after 1 h of incubation; the cumulative insulin release was 14.8% (after 2 h), 20.4% (after 3 h), and 24.7% (after 4 h).
228
B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
Fig. 3. Histological appearance of representative titanium implants after 2 weeks in the INS group, the average rate of bone–implant contact was 50.73% (2.80%).
Fig. 5. Histological appearance of representative titanium implants after 2 weeks in the DM group, the average rate of bone–implant contact was 28.82% (5.32%).
tained locally after day 1 because of the controlled release of the microspheres, which had essentially disappeared after 14 days.
(Fig. 5); there was considerable osteoid tissue around the titanium implants in the latter group. Bone–implant contact was noticeable in the implanted area in both the INS and the control group, but not in the DM group. The mean rate of bone–implant contact in the INS group was significantly lower than that in the control group (p < 0.05), but much lower in the DM group (p < 0.01). After 6 weeks, the quantity of newly formed bone was less, and there was a lot of osteogen around the implant in the control and INS groups, but less osteogen and more osteoid tissue in the DM group. The proportion of osteoid in the control group was higher than that in the INS group. The mean (SD) rate of bone–implant contact in the DM group of 51 (3) % was significantly lower than that in the INS group (58 (3) %) (p < 0.01) and in the control group (66 (4) %) (p < 0.01). Bone–implant contact in the INS and control groups also differed significantly (p < 0.01). Insulin can increase the proliferation of osteoblasts as growth factors and in suitable concentrations results in downregulation of P44-42 MAPK signal activity.23 The appropriate concentration of insulin could effectively increase the proliferation of osteoblasts in 1 day, but the effectiveness would lessen during the following days. However, to date we have not had an effective way of maintaining high-enough concentrations of insulin locally over a long period. More time will be required to study the problem. Within 2 weeks, the volume of newly formed bone was stimulated by insulin released from the microspheres in the INS group. However, other rats with DM that were not given insulin had little new bone. In addition, osteogen was detected after 6 weeks at the mineralised new bone adjacent to the surface of the implant. The results showed that the bone–implant contact in the INS group differed from those found in the DM
Histomorphometric analysis After 2 weeks, histological examination showed that new bone had formed beside the titanium implants in the INS group (Fig. 3) and in the control group (Fig. 4). More had formed within the INS group, and less in the DM group
Fig. 4. Histological appearance of representative titanium implants after 2 weeks in the control group, the average rate of bone–implant contact was 56.55% (4.57%).
B. Wang et al. / British Journal of Oral and Maxillofacial Surgery 49 (2011) 225–229
and control groups after 2 and 6 weeks. Although that in the INS group was not as great as that in the control group, it was significantly different from that in the DM group. Insulin could therefore increase osseointegration after implantation in diabetic rats.
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