Expression Of Soluble Receptor Activator Of Nuclear Factor-κb Ligand And Osteoprotegerin In Adolescent Idiopathic Scoliosis Patients At The Osteoblast Level

Expression of soluble receptor activator of nuclear factor-κB ligand and osteoprotegerin in adolescent idiopathic scoliosis patients at the osteoblast level1. Isolation , cultivation , amplification and phenotype identification of osteoblasts from adolescent subjects in vitroObjective To establish the methods of the isolation,cultivation and amplification of osteoblast (OB) from adolescent subjects in vitro.Methods This study covered 28 subjects,including patients with AIS and normal age-matched non-scoliosis adolescents who underwent spinal surgery. AIS group was 20 patients(15 females and 5 males,mean age of 14.75±2.7 years),Normal group was 8 cases(2 females and 6 males,mean age of 15.25±3.2 years). The average Cobb angle of AIS group was 59.65°(ranged from 40°to 96°). The cancellous bone was harvested from the posterior superior iliac spine of the subjects. The chipped explants were cultured to obtain the osteoblasts. The incremet time of each generation were recorded. The second generation (P2) cells were scraped to count.Formation of calcium nodules,staining of alkaline phosphatase and osteocalcin mRNA expression detected by RT-PCR were used to confirm the osteoblast phenotype.Results Cell outgrowths appeared after 5-6 days,and then turned to be fusiform in shape and confluent cultures after 4 weeks. 0.5×107 cells could be obtained in each P2 generation culture flask. The positive results of staining of alkaline phosphatase, formation of calcium nodules and expression of osteocalcin confirmed that the P2 cells were typical osteoblasts. There was no statistical difference between AIS and normal group about increment time of osteoblasts. Conclusion Osteoblast from adolescent idiopathic scoliosis have the same capacity of growth and proliferation in vitro as that of the normal group. Key words Adolescent;Idiopathic scoliosis;2. The Relationship between RANKL/OPG and the Decreased Bone Mass in Adolescent Idiopathic Scoliosis PatientsObjective To investigate the relationship between RANKL/OPG and the decreased bone mass of adolescent idiopathic scoliosis patients at the osteoblast level.Methods This study covered 28 subjects (aged from 11 to 19),including patients with AIS and normal age-matched non-scoliosis adolescents who underwent spinal surgery. AIS group was 20 patients (15 females and 5 males,mean age of 14.75±2.7 years), Normal group was 8 cases (2 females and 6 males,mean age of 15.25±3.2 years). The average Cobb angle of AIS group was 55.7°(ranged from 40°to 95°). BMD of the lumbar spine and proximal femur was measured using dual energy X-ray absorptiometry in both groups. The cancellous bone was harvested from the posterior superior iliac spine of the subjects. The chipped explants were cultured to obtain the osteoblasts. Expressive intensity of Receptor activator of NF-κB ligand (RANKL) and Osteoprotegerin (OPG) of P2 generation osteoblasts from two groups were detected by using RT-PCR and Western blotting.Results BMD of the lumbar spine (L2~L4) and proximal femur was obviously lower in AIS group than that of the control group. RT-PCR and Western blotting results showed that:1) Expression of RANKL of osteoblasts had increased obviously in AIS group as compared with the control group (P<0.01). 2) Expression of OPG did not have significant difference. 3) RANKL to OPG ratio in patients with AIS were increased compared with that in control subjects (P<0.05). Conclusion These findings mean that the imbalance and the disturbed interaction of RANKL and OPG may be an important cause and pathogenesis in reduced BMD in AIS.