Sustained Release Of Insulin-like Growth Factor-1 From Poly(lactide-co-glycolide) Microspheres Improves Osseointegration Of Dental Implants In Type 2 Diabetic Rats

Recently, with the rapid development of oral implantology, the use of dental implants as an anchor has become a standard effective treatment modality to restore missing teeth and maxillofacial defects. However, dental implants in medically compromised patients often have a high failure rate, such as diabetes, cardiovascular disease, bone metabolic disorders, bleeding disorders, autoimmune diseases, etc. These compromised conditions exert a great limitation on the application of dental implants. In particularly, diabetes is becoming more and more seriously. Due to a huge transformation of lifestyle and economic postion, the incidence of diabetes in China is increasing year by year. China has become the second largest diabetics'country. Therefore, improvement in the success rate of dental implants in type 2 diabetic patients is a challenge to clinicians. This study will propose a novel way to deal with this tough clinical problem that locally sustained releasing of polypeptide to stimulate new bone formation on a established animal model bears a close resemblance to human type 2 diabetes.Study objectivesFirst, we will establish a animal model similar to human's type 2 diabetes and adapt to implant research. Furthermore, we will evaluate and analyze the effects of type 2 diabetes on the osseointegration of dental implants through this animal model.Second, a novel sustained drug delivery system that PLGA encapsuled rrIGF-1 by a W/O/W double-emulsion solvent evaporation method will be constructed. Then, SEM, CLSM and cumulative-release studies were conducted to analyze the morphology of microspheres and verify the release effect of the microspheres as well as rrIGF-1 bioactivity.Third, established type 2 diabetic rats were divided into two groups equally: a rrIGF-1 MST group and a control group. The rrIGF-1 was encapsulated into PLGA microspheres to produce a sustained-release effect around Ti implants to stimulate new bone formation in the rrIGF-1 MST group. Based on this study, we can perform a next experiment that evaluates sustained release of rhIGF-1 from PLGA microspheres into type 2 diabetic patients'alveolar bone cells in vitro to determine whether this produces a positive effect. This will provide clinicians with a ingenious idea to enhance the success rate of type 2 diabetics. Methods and Results:PartⅠ:Methods: Ten 9-week-old male SPF-level GK rats (type 2 diabetic group) and 10 age-matched normal Wistar rats (control group) were used in this study. They were placed in a same environment. The GK rats were fed forage containing high levels of glucose and grease. Four weeks later, the blood glucose levels of the GK rats were >300 mg/dL (16.7 mmol/L). Thus, we gain ten 13-week-old type 2 diabetic rat models and ten 13-week-old Wistar rats. Then, twenty cylindrical titanium implants (3.3×6mm, surface with a microarc oxidation treating) were placed into the femora of 10 type 2 diabetic and 10 age-matched normal rats. The dissected femur tissues from 5 type 2 diabetic and 5 control rats sacrificed at 4 and 8 weeks were examined histologically by embedding in polyester resin. Each bone sample was mounted on a sawing microtome. Thus, we obtained 30-μm-thick undecalcified sections, which were stained with Ponceau Tri-Chrome stain. Finally, histometric analyses were done between two groups (comprised two variables: BIC and TBV ) .Results:(1) During the whole experiment (at surgery, 4 weeks and 8 weeks after surgery), the blood glucose levels of type 2 diabetic group and control group have a significant difference(P<0.05).(2) At weeks 4 and 8 after operation, the percentage of BIC and TBV between two groups have a significant difference (P<0.05)(with the exception of TBV at 4 weeks after implant placement, P > 0.05).(3) In the control group, an extensive area of bone was in direct contact with the implant, while in the type 2 diabetic rats, the bone was largely intermixed with soft tissue at the implant interface. Meanwhile, the continuity of bone tissue in type 2 diabetic group seems to be more worse than the control group.PartⅡ:Methods: Utilize the rrIGF-1 supplied by Prospec Corp (Israel) as internal aqueous phase (W1), regard PLGA and Span80 as oil phase (O), treat PVA and Tween80 as re-emulsification solution (W2). The microspheres were prepared by a W/O/W double-emulsion solvent evaporation method. SEM and CLSM were conducted to analyze the morphology of microspheres and encapsulation efficiency. The release of rrIGF-1 was determined by the Lowery-Peterson protein determination method. Release profiles were calculated in terms of cumulative release (%) with incubation time. The bioactivity of released rrIGF-1 was confirmed by an MTT bioassay using quiescent MG-63 cells that are known to show a dose-dependent mitogenic response to added rrIGF-1. Finally, we will make a analysis and conclusion based on above data and images.Results:(1) The morphology of the rrIGF-1-loaded PLGA microspheres was shown in Figures in later text. The mean diameter of the rrIGF-1-loaded microspheres, as measured by light scattering, was 1.2525±0.6436μm.(2) A CLSM have demonstrated that PLGA (stained with Nile red in the external oil layer) had successfully encapsulated rrIGF-1 in the internal aqueous solution (W1). The encapsulation efficiency of this PLGA microspheres used in this study was 78.3±2%, which is close to the results of a previous hydrophilic peptide report.(3) The microspheres in this study have a sustained release effect in pharmacodynamics. In vitro release of rrIGF-1 from the PLGA microspheres exhibited an initial burst of approximately 55.3% of the total content within the first 2 days, followed by a continuous release totalling 64.8% after 5 days. More than 85% of the encapsulated rrIGF-1 was released from the PLGA microspheres within 20 days. The remaining 15% was slowly released during days 20–40.PartⅢ:Methods: Twenty type 2 diabetic rats which have established in PartⅠwere divided into two groups: a group receiving recombinant rat insulin-like growth factor 1 microsphere therapy (rrIGF-1 MST) (10 rats) and a control group (10 rats). The implant hole received one of two treatments, randomly distributed among the rats, before implant insertion: (1) placebo PLGA microspheres (control group) in blood clot or (2) 200μg of rrIGF-1 in PLGA microspheres in blood clot (rrIGF-1 MST group). The dissected femur tissues from 5 rrIGF-1 MST group rats and 5 control group rats sacrificed at 4 and 8 weeks were examined histologically by embedding in polyester resin. Each bone sample was mounted on a sawing microtome. Thus, we obtained 30-μm-thick undecalcified sections, which were stained with Ponceau Tri-Chrome stain. Finally, histometric analyses were done between two groups (comprised two variables: BIC and TBV). Meanwhile, compare the interface of bone-implant by a high magnification of SEM between two groups.Results:(1) At weeks 4 and 8 after operation, the percentage of BIC and TBV between the rrIGF-1 MST group and control group have a significant difference (P<0.05)(with the exception of TBV at 4 weeks after implant placement, P > 0.05).(2) In the rrIGF-1 MST group, there was an increase of bone density and new bone formation at the borders of the Ti implants. While in the control group, there was an inconsistent bone reaction around the Ti implants. In some places, bone was loosely organized, and a woven area was seen.(3) Under high magnification of the bone-implant contact area by SEM, there was still a narrow gap between the bone and the Ti implants in the control group. In contrast, several osteoblast-like cells appeared to have integrated with the Ti implants at the implant surface in the rrIGF-1 MST group. Conclusions:(1) Type 2 diabetes can impair and disturb the osseointegration of dental implants in the early post-implantation healing phase obviously. There were many fibrous tissues healing around Ti implants and less continuous bone in type 2 diabetic rats. Therefore, these phenomena lead to a low osseointegration rate in type 2 diabetic rats.(2) The rrIGF-1–loaded PLGA microspheres prepared by a W/O/W double-emulsion solvent evaporation method is a excellent drug sustained delivery system. This novel microspheres have an ideal size, diameter, encapsulation efficiency, sustained delivery ability, peptide bioactivity, which can be used to stimulate osteoblast differentiation and new bone formation.(3) Sustained release of IGF-1 has a positive influence on the osseointegration of dental implants in type 2 diabetes during the early post-implantation healing phase. Furthermore, this study has yielded important evidence on new bone formation around dental implants in type 2 diabetic rats treated with locally sustained release of IGF-1 from PLGA microspheres(4) This study provided us with a basis to perform a next experiment that evaluates sustained release of rhIGF-1 from PLGA microspheres into type 2 diabetic patients'alveolar bone cells in vitro to determine whether this produces a positive effect. All this will make preparations for this novel strategy utilizing in human application in near future.