| OBJECTIVETo investigate the release profile in vitro and the biocompatibility in vivo of calcium phosphate cement/poly(lactic-co-glycolic acid)microsphere composite incorporated RANKL.To observe the effect of RANKL release on the material and the vicinity of the material,to put forward new ideas on promoting the degradation of calcium phosphate material,to provide scientific basis for the development of new strategies of this kind of biomaterials.METHODSThe experiment consists of two parts: in vitro and in vivo.1.RANKL release from CPC / PLGA microsphere composite and RANKL activity in vitroPLGA microspheres were prepared by W / O / W double-emulsion method,scanning electron microscopy was used to observe and take microphotos of PLGA microspheres,Image-Pro Plus software was used to calculate the particle size of the microspheres.Respectively,5 ml group A)0.5 ug / ml,group B)1.0ug / ml,group C)1.5 ug / ml RANKL solution were mixed with 0.2 g PLGAmicrospheres and freeze-dried.0.8g CPC and 0.2 g PLGA microspheres of group A,group B,and group C were mixed with 0.35 ml setting liquid,moulded in the cylinder with a diameter of 6 mm and a height of 2 mm.Samples of group A,B,and C which were pre-set 24 h were soaked in 1.5 ml PBS solution and incubated at 37?.Samples were taken by collecting all of PBS after 1 hour and7,14,21,28,35,42 days,and the quantitive of RANKL was detected by ELISA.The remaining samples were concentrated by a centrifugal ultra-filtration unit and were reconstituted in 50 ml with 10% fetal bovine serum + DMEM culture medium with 100ng/ml RANKL according to the result of the release.RAW264.7 was cultured with the above culture medium.Tartrate acid phosphatase staining was performed after 8 days.2.Subcutaneous implantation of CPC / PLGA microsphere composite incorporated RANKLThe groups were divided into group A(CPC/PLGA incorporated 0.5ug/ml RANKL),group B(CPC/PLGA incorporated 1.0ug/ml RANKL),group C(CPC/PLGA incorporated 1.5 ug/ml RANKL),and group D(CPC/PLGA without RANKL).The SD subcutaneous implantation model was established with four samples were implanted subcutaneously on the back of each rat.Rats were sacrificed after 4,8 and 12 weeks of implantation.the specimens were harvested and decalcification.HE stain stain was performed to observe the histologic changes in the surrounding tissue of the material.Using the immunohistochemistry method to detect the expression of RANKL in the surrounding tissue of the material.RESULTS1.The PLGA microspheres prepared by W/O/W double emulsion method were spherical or ellipsoidal,the surface of which was rounded,and the average size was 19 ± 8 um.2.Result of RANKL release in vitro showed that: the release of group A,B,C was fast within the first 7 days,then it entered the slow release stage.RANKL was detected within 1 h,and a release peak was detected within the first 7days,but RANKL was not detected after 35 days in each group.The release percentage to the total release amount of RANKL was 62.7% of group A,62.5%of group B and 52.4% of group C after 7 days.There was no statistical difference among these groups(P> 0.05).And it was 98.1% of group A,97.2%of group B and 96.1% of group C,and there was no statistical difference among these groups(P> 0.05).The total release of RANKL to the input in group A,B and C was 31.0%,17.6% and 14.7% respectively.There was significant difference between group A and group B,as well as group A and group C(P<0.05).3.Result of Tartrate resistant acid phosphatase(TRAP)staining showed that: on the 8th day of the cultivation of RAW 264.7 cells,osteoclast-like cells that stain positive for TRAP could be observed in group CPC/PLGA/RANKL.These osteoclast-like cells are large and irregular,with red or deep red stained particles in the cytoplasm,and the density is relatively sparse.There was no TRAP(+)OLC(Osteoclast-like cell,OLC)observed in the control group.4.Result of the HE staining showed that: at 4th week,a few scattered lymphocytes and plasmacytes were observed in the surrounding soft tissue of the material,with capillary and fibroblast proliferation which formed fibrous capsule around the material.OLCs could be observed between the material and the capsule.There was no obvious difference among group A,B,C,D in the number of OLCs,the nucleus number of OLC,inflammatory cell response and capsule sac grade.At 8th week,the number of OLC and the number of OLC nucleus of all groups were higher than those at 4th week(P <0.05),and there was no significant difference among all groups in the number of OLCs.The nucleus number of OLCs between group A and group D was statistically different at 8th week.There was no significant difference among all groups in the inflammatorycell response and the capsule sac grade.There was no significant difference among all groups in the number of OLCs at 12 th week.There was no significant difference between the 12 th week and the 8th week.For the nucleus number of OLCs,there was significant difference between group A and group D as well as group B and group D.The nucleus number at 12 th week of group A is higher than the 4th and 8th week.There was no significant difference among all groups in the inflammatory cell responses and the capsule sac grade at 12 th week.5.Result of immunohistochemistry staining showed that: at 4th week,the positive expression of RANKL in group A,B and C was stronger than that in control group,there was no significant difference between group A and group B,C,D(P>0.05),there was statistical difference between group B and D as well as group C and D(P<0.05).At 8th week,the positive expression of RANKL in group A,B and C were stronger than that in control group,and were stronger than week 4's,there was no significant difference between group A and group B,C,D,there was statistical difference between group B and D as well as group C and D.At 12 th week,the positive expression of RANKL in group A,B and C was weaker than that at 8th week,there was no statistical difference in the differences among all groups.CONCLUSIONThe RANKL –loaded CPC/PLGA microspere composite could induce the formation of osteoclast-like cells and enhance the activity of them,and it could also increase the expression of RANKL of the surrounding tissues. |
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