The Effect Of Statins On The Process Of Cervical Intervertebral Fusion With Allogenous Cortical Bone Transplantation

OBJECTIVESBone allograft transplantation has been performed in humans for more than 120 years. During the first one hundred years, the major problem in bone allografts transplantation was availability. And during the next twenty years, as allograft began to be used, the major issue was safety. Diseases transmitted during this period include AIDS and hepatitis. Sensitive blood tests and extensive efforts by bone banks to develop ways to clean bone and clear it of infectious agents helped provided safe transplants. With concern of availability and safety receding, the major issue in the future will be the efficacy of the transplant. Future research efforts will be applied to bone allograft transplantation to ensure that bone transplants heal quickly and sufficiently to be able to function as part of the load-bearing skeletal system.Anterior cervical discectomy and fusion has proved to be a reliable surgical procedure for relieving the neural symptoms of cervical spondylosis. When initially described by Robinson and Smith, the procedure used autogeneous bone from iliac crest to obtain fusion. Recently, the use of allograft bone has been increasing. Allogenous cortical bone is attractive principally for its ability to eliminate donor site morbidity, and biomechanical superiority. However, a massive allograft may serve a structural function inthe absence of advanced revascularization and creeping substitution processes.Revascularization and substitution of the original graft bone with new host bone are the aims and key steps in the process of bone grafts healing. Several growth factors are expressed in distinct temporal and spatial patterns during fusion. Of these, BMP-2 and VEGF are of particular interest because of their potential clinical application. In several experimental models, the local application of VEGF or rhBMP-2 coupled with different carriers has demonstrated the ability to promote human bone repair. Understanding and interfering with the process of angiogenesis and osteogenesis are mandatory in promoting allogenous cortical bone incorporation.Statins are HMG-CoA reductase inhibitors that effectively lower serum cholesterol level and are widely prescribed in the treatment of hypercholesterolemia. In 1999, Mundy and colleagues made the seminal observation that statins could increase bone formation in vitro and in rodents. In vitro, statins specifically increase BMP-2 expression and BMP-2 protein in cultured murine and human osteoblasts. In vivo, statins increase osteoblasts proliferation and bone formation when injected subcutaneously over the skull bone of mice. These lines of evidence spurred clinical investigators to reexamined datasets from observational and cohort trials to assess the effect of statin use on fracture risk. Some large population-based case-control clinical studies showed a consistent association between statin use and reduced fracture risk. In addition, it has been proved that statins could promote the proliferation, migration, and survival of endothelial cells and bone marrow-derived endothelial progenitor cells, via which they promote anigogenesis and vasculogenesis.Although the mechanism whereby they promote osteogenesis and angiogenesis is under investigation, statins are showed to be potential anabolic agents in the management of established osteoporosis, pseudarthosis in spinal fusion, as well as delayed union or non-union in bonefracture. This study is based on the clinical data of cervical fusion with allograft bone, and experimental findings in cultured osteoblasts, to investigate the effect of statins on the osteogenesis, angiogenesis and integration of implanted allograft bone. The purpose of this study is to find effective and safe drugs that could promote allograft bone fusion.MATERIALS AND METHODS1. CLINICAL DATAFrom 2001 to 2003, 57 patients of cervical spondylosis underwent graftingby the trephine osteotomy and expansive decompression technique with dual-concave-shaped allogenous cortical bone transplantation, among which 25 cases were supplemented with anterior cervical plate fixation. Clinical follow-up was obtained either by outpatient visit or by questionnaire letter. The average preoperative and postoperative cervical myeolopathy scores and radiographic bony fusion were assessed during hospitalization and again at 3-, 6-, and 12-month intervals. The average duration of follow-up reviewwas 23.1 months (minimum 6 months, maximum 36 months).2. IN VITRO STUDYOsteoblasts, derived from newborn mouse calvaria, were cultured andidentified by alkaline phosphatase staining and mineralization nodules formation induced by ascrobic acid and ï¿¡ -glycerophosphate. Cells were devided into 3 groups according to the different reagents in culture medium: veichle, statins or rhBMP-2. The effect of drugs at different concentration, on primary cultured mouse osteoblasts were investigated with the following methods: MTT assays and BrdU labeling rates were used to detect the cells' proliferation rate; von Kossa staining and image analysis to demonstrate the mineralization ability of clavaria osteoblasts; taken the P -actin as internal control, the relative expression levels of BMP-2, TNSALP and VEGF mRNA were detected through electrophoresis measurement in RT-PCR analysis, and protein levels were detected by cytoplasmic optic density value after immunocytochemicai staining; BMP-2 and BrdU double staining method wasused to show the level of BMP-2 protein in newborn osteoblasts. 3. IN VIVO STUDYThirty male mice were divided into 3 groups randomly: control group,simvastatin group and lovastatin group. Twenty microliters of PBS, simvastatin(20mg ? kg-1 ? d-1) and lovastatin(20mg ? kg-1 ? d-1) were injected subcutaneously over the right calvaria of the three groups for 4 days continuously. One month later, the mice were sacrificed to perform pathological examination. The fine structure of the calvaria in each group was observed and the thickness of the calvaria was measured.Thirty six male Wistar rats, undergone anterior cervical discectomy and fusion with allogenous cortical bone transplantation, were divided into 3 groups randomly: control group, statins group and BMP-2 group. In statins group, rats were fed with simvastatin 20mg per kilogram body weight everyday since the third postoperative day. The implant in the rhBMP-2 group was marinated in the rhBMP-2 solution and treated as control group after operation. The animals were sacrificed to obtain cervical spine specimen at 2-, 4-, 8- and 12-week intervals. The bone mineral density of cervical vertebral body was measured and compared with blank group samples. Bony fusion was assessed by radiographic and pathologic examinations.RESULTS1. CLINICAL RESULTSClinical myeolopathy score was increased by 5.4 points. The cervicallordosis was well preserved. Implant complications occurred in 3 cases within 5 months postoperativeiy, two cases had revisional operations. Revasculation and interface fusion were found at 5 to 6 months postoperativeiy conformed by bone scan and radiography. For all the patients followed up for more thanone year, bony fusion was achieved.2. CELL CULTUREA large amount of osteoblasts, which were positively stained for alkalinephosphatase and be able to form mineralized nodules after 2 weeks' culture,were obtained though enzyme digestion method.MTT assay showed that statins could promote the cell proliferation in a dose dependent manner. Compared with blank group, the increase in the optic density was significant at statins concentration of 2.5 u M , 5 u M and 10 u M. The cell proliferation rates in the statins, rhBMP-2 and control groups are 47.6%, 31.1% and 23.1% respectively. A significant difference among these groups was demonstrated.Simvastatin markedly increase the number of mineralized nodules in culture, which was similar to rhBMP-2. The mineralization area ratios in three groups are (5.44+1.87)% ^ (5.93+1.73)% and (2.13+0.55)% respectively. Ratios in statin group and rhBMP-2 group were statistically higher than that in control group.Taken the P -actin as internal control, semi-quantitive RT-PCR analysis revealed that treatment with simvastatin (10 u M) incuced a significant increase mRNA of BMP-2 (1.18 folds) , TNSALP (7.45 folds) and VEGF(2.4 and 1.09 folds for VEGF 120 and VEGF164 respectively). And the expression level of BMP-2 and VEGF proteins were also augmented in statin group compared with control group. And most importantly, the expression level of BMP-2 protein in newborn osteoblasts (positively stained in BrdU labeling)was significantly improved by simvastatin. 3. ANIMAL EXPERIMENTSThe fine structures of the caivaria were remodeled in both statin groups. Insimvastatin group, marked new vessel formation was observed in the caivaria bone matrix as well as increased new bone formation. While only increased bone formation was observed in the outer lamina of the skull bone in lovastatin group. The mean thickness of the right side of caivaria in lovastain and simvastatin group was increased by 73% and 115% respectively, significantly thicker than that of the control group(P<0.01). Compared with left side, the thickness of the right side caivaria was significantly increased in both statin groups(P<0.01), but no difference was detected between the two sides...