Experimental Study Of Simvastatin Carried By PLGA/CPC Combined BMSCs On The Effect Of Critical Calvarial Defect

一、BackgroundsBone defects and bone loss caused by trauma, infection, tumor andsurgery affect the living quality of patients. The main treatment of bonedefects were bone grafting, but there were some limitations for differentbone grafting. Autogenous bone grafting for skeletal healing remains oneof the most common surgical techniques in the recent years. However, thepotential donor site morbidity and insufficient donor availabilities limitedits clinical applications, so it was unable to restore the bone defectscompletely in most cases. During the past decades, the development oftissue-engineering technology, has proved to be a promising addition tobone regeneration,has provided a broad way to the restorations of bonedefects. Bone tissue-engineering realized bone restoration byestablishing an artificial biomaterial scaffolds containing appropriate seedcells such as bone marrow stem cells and osteoinductive growth factors,such bone morphogenetic protein (BMP), transforming growth factors(TGF).The early study of our research group has verified1) simvastatinhas promoted the expression of osteoinductive factors, and increased theosteogenic differentiation of BMSCs effectively;2) simvastatin carriedby PLGA/CPC scaffold was an ideal tissue-engineering scaffold;3)0.5mg simvastatin was the best drug concentration to build the tissueengineering bone constructed with simvastatin carried by PLGA/CPC andbone marrow stem cells.In this issue, we try to develop a novel bone substitute, includingthree factors of bone tissue engineering. We will plant BMSCs intosimvastatin/PLGA/CPC scaffolds, this new substitute materials willprovide a new option for a wide range of bone defects. 二、ObjectivesIn order to investigate the possibilities of reconstructingtissue-engineering bone by taking BMSCs as seed cell, simvastatin servedas signal molecule and PLGA/CPC acted as scaffold. Hope to develop anew biological bone substitute.三、Materials and methods1. Cell isolation and cultureWe selected rat bone marrow stem cells as seed cells. Cells were culturedby static adherence method and identify the cells to ensure that they havemultiple differentiation potential.2. Using the solvent casting-the particle leaching technologycombined with phase separation to prepare0.5mg simvastatinPLGA/CPC composite scaffold.3. Seeding the third generation BMSCs stained by dil insynthesized scaffolds, observing the adhesion of cells on the scaffold byscanning electron microscopy and confocal laser scanning microscopy.4. A total of24male rats were divided into three groups atrandom.A midlongitudinal incision was made on the dorsal surface of thecalvarial bone, and care was taken to ensure that the periosteum wascompletely cleared from the surface of the calvarial bone by scraping.Abur was used to create two circular5mm diameter defects in the bothside of rat calvarial bone, the full thickness of the calvarial bone wasremoved. The16calvarial bone defects in each group were thenrandomly divided into four groups that received the following implants:GroupA: simvastatin/PLGA/CPC/BMSCs(n=4), GroupB:simvastatin/PLGA/CPC (n=4), Group C: PLGA/CPC (n=4), Group D:blank control group (n=4). The wound was closed with a continuoussuture. All the rats were sacrificed by an intraperitoneally overdoseinjection of pentobarbital after4,8,12weeks post-operation. Then thecalvarial bone was explanted and fixed in4%phosphate-bufferedformalin solution. The new bone formed in the calvarial bone defectwas assessed using general observation, a Micro-CT, HE andimmunohistochemistry methods. 四、Results1. There was no obvious rejection in the bone defect area after theimplantation of composite scaffolds, that showed a wellhistocompatibility.2. Histological data evidence showed that there were various boneformation in each group except the control group at4,8,13weeks postoperation，the scaffolds showed various degradation, the bone quality andbone formation quantity at the group withsimvastatin/PLGA/CPC/BMSCs were much better than those of the othergroups. At the control group, the bone defect was filled with fibrousconnective tissue.3.The results of immunohistochemistry showed that the positiveexpression of osteocalcin at simvastatin/PLGA/CPC/BMSCs group weremuch higher than that of the other three groups. BMSCs marked withBrdu coming from the allograft showed high rate of survival, andpromoted bone regeneration.五、ConclusionsSimvastatin PLGA/CPC scaffold materials combined BMSCs caninduce new bone formation in critical size calvarial defects，and theosteoblast quality is better than the other three groups, and it also cansignificantly shorten the bone healing time.