PGJ₂Nanoparticles In Bone Repair

Part Ⅰ.△12-prostaglandinJ2nanocapsules up-regulates growth factor expression and enhances bone regeneration in ratsObjective:The aim of this study was to explore the effects of local delivery of△12-prostaglandinJ2-loaded poly(lactic-co-glycolic acid)(△12-PGJ2-NC) on growth factors expression and bone formation, which will provide scientific basis for its potential application in the treatment of periodontitis.Methods:△12-PGJ2-NC was prepared by the emulsion solvent diffusion method. The physical and chemical properties of the nanoparticles were evaluated by particle size analysis, transmission electron microscopy, drug-loading ratio and the in vitro release study. Then standardized transcortical defect(5×1.5mm) was conducted in the femur of48male Wistar rats which were randomLy divided into four groups(N=12) and respectively defined as S, K, F, and N.30microliter of saline(S), unloaded nanoparticles(K),△12-PGJ2(F) and△12-PGJ2-NC(N) in a collagen vehicle were delivered inside a titanium chamber fixed over the defect. Then, four subgroups were randomLy divided in each group which were named as D3, D7, D14, and D28(n=3) according to the days3,7,14, and18after the surgery. At days3,7,14, and28, the mRNA expression of the bone morphogenetic proteins-6(BMP-6), platelet-derived growth factor-B (PDGF-B) in defect aera was analyzed by real time quantitive-polymerase chain reaction(qRT-PCR); The protein expression of the BMP-6and EphrinB2was evaluated by Western Blot. HE staining was employed to reveal new bone formation in weeks2and4. Results:△12-PGJ2-NC appeared opalescent white and remained relatively stable, with an average particle size of (135.2±0.85)nm. The images from transmission electron microscopy showed that△12-PGJ2-NC was spherical in shape and homogeneously distributed. The encapsulation efficiency of△12-PGJ2with the PLGA nanocapsules was about92%. The in vitro release of△12-PGJ2-NC at37℃showed a sustained fashion and the average accumulated amount was30%,52%,77%,91%, and98%respectively, at30,60,120,240and360min. Comparing with the animals treated with saline, after dose of100mg/L△12-PGJ2and△12-PGJ2-NC apllication, the mRNA expression level of BMP-6, PDGF-B increased significantly (P<0.05/P<0.01). The protein expression of BMP-6, EphrinB2also up-regulated. Histomorphometry revealed that new bone formation increased at the same dose of100μg/mL. But the unloaded nanoparticles did not behave the same effect(P>0.05).Conclusion:A stable△12-PGJ2loaded nanoparticle has been successfully prepared. When locally administrated, it may upregulate the expression of BMP-6, PDGF-B and EphrinB2, and promote new bone formation in bone defect area. Part Ⅱ.15d-PGJ2nanoparticles in bone repairObjectives:To determine effects of15-deoxy-△12,14-prostaglandinJ2(15d-PGJ2) loaded in poly(D,L-lactide-co-glycolide)(PLGA) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration.Methods:15d-PGJ2-NC were prepared by the emulsion solvent diffusion method. Physical and chemical properties of the nanoparticles were evaluated by particle size analysis, transmission electron microscopy (TEM), and determination of drug-loading ratio and in vitro release. Thirty microliters of saline, unloaded nanoparticles, free15d-PGJ2and15d-PGJ2-NC in a collagen vehicle were delivered inside surgically-created transcortical defects in Wistar rat femurs. Interleukin-6(IL-6), interleukin-lbeta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels in surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time PCR (qRT-PCR) on days1,3,7and14. At the same time, bone morphogenetic protein-6(BMP-6) and platelet-derived growth factor-B (PDGF-B) mRNA in the defect were also examined by qRT-PCR. Masson’s Trichrome staining and immunohistochemistry were employed to observe new bone formation and EphrinB2and osteoprotegerin (OPG) protein expression in the cortical defect area up to28days.Results:15d-PGJ2-NC appeared opalescent white and remained relatively stable on day0,30and60, with average particle sizes of(115.2±0.65),(132.7±0.87) and (134.1±1.61)nm, respectively.15d-PGJ2-NC appeared spherical in shape and homogeneously distributed in TEM images. The encapsulation efficiency of15d-PGJ2in the PLGA nanocapsules was～64%. A sustained release of15d-PGJ2-NC in vitro at37℃was observed with average accumulated amounts of14.7%,21.4%,53.1%,64.8%,86.1%and99.8%at15,30,60,120,240and360min, respectively. Application of15d-PGJ2-NC (100mg/L) in the local bone defect significantly decreased IL-6, IL-1β and TNF-α mRNA and protein, compared with saline-treated controls (P<0.05). Upregulation of BMP-6and PDGF-B mRNA by15d-PGJ2-NC was also demonstrated. New bone formation and increased EphrinB2and OPG protein levels were observed in the cortical defect area in15d-PGJ2-NC-treated animals (100mg/L).Conclusions:Stable15d-PGJ2-NC complexes were successfully prepared and shown to attenuate IL-6, IL-1β and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.