OBJECTIVE: To investigate the effect of stretch on long non-coding RNA taurine upregulated gene 1 (TUG1)-mediated miR-545-3p/cannbinoida receptor 2 (CNR2) pathway regulating bone regeneration in the distraction area of rats during distraction osteogenesis. METHODS: Thirty-six 10-week-old male Sprague Dawley rats were randomly divided into 3 groups ( n=12 in each group): group A (femoral fracture+injection of interfering RNA), group B (distraction osteogenesis+injection of interfering RNA), and group C (distraction osteogenesis+injection of TUG1). Groups A and B were injected with 60 μg of interfering RNA at the beginning of incubation period (immediate after operation), the beginning of distraction phase (7 days after operation), and the end of distraction phase (21 days after operation), and group C was injected with 60 μg of synthetic TUG1 in vivo interfering sequence at the same time. The general situation of rats in each group was observed during the experiment. The mineralization of fracture space or distraction area was observed by X-ray films at 21, 35, and 49 days after operation. At 49 days after operation, the samples of the distraction area were taken for HE staining to observe the mineralization, and real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expressions of osteoblast-related genes such as TUG1, miR-545-3p, CNR2, alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). Blood samples were collected from the abdominal aorta of the rats, and the expressions of ALP and C terminal telopeptide of type Ⅰ (CTX-Ⅰ) protein were detected by ELISA assay. RESULTS: The results of X-ray film and HE staining observations showed that osteogenesis in group C was superior to groups A and B at the same time point. The results of qRT-PCR showed that the relative mRNA expressions of TUG1, CNR2, ALP, OCN, and OPN in group C were significantly higher than those in group A and group B, and the relative mRNA expression of miR-545-3p in group C was significantly lower than that in group A and group B ( P<0.05). The relative mRNA expressions of TUG1 and ALP in group B were significantly higher than those in group A, and the relative mRNA expression of miR-545-3p in group B was significantly lower than that in group A ( P<0.05). There was no significant difference in the relative mRNA expressions of CNR2, OCN, and OPN between group A and group B ( P>0.05). The results of ELISA showed that the expressions of ALP and CTX-Ⅰ protein were significantly higher in group C than in group A and group B, and in group B than in group A ( P<0.05). CONCLUSION Under the action of stretch, the expression of TUG1 in the femoral distraction area of rats increases, which promotes the expression of CNR2 by inhibiting the expression of miR-545-3P, which is helpful to the mineralization of the extension area and osteogenesis.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Male ; Osteogenesis, Distraction/methods* ; Rats ; RNA, Long Noncoding/metabolism* ; Osteopontin/genetics* ; Osteogenesis ; Bone Regeneration ; RNA, Small Interfering/genetics* ; Osteocalcin/genetics* ; Alkaline Phosphatase/metabolism* ; Osteoblasts/cytology* ; Signal Transduction ; Femoral Fractures/surgery*

Reports describing significant health risks due to inadequate vitamin D status continue to generate considerable interest amongst the medical and lay communities alike. Recent research on the various molecular activities of the vitamin D system, including the nuclear vitamin D receptor and other receptors for 1,25-dihydroxyvitamin D and vitamin D metabolism, provides evidence that the vitamin D system carries out biological activities across a wide range of tissues similar to other nuclear receptor hormones. This knowledge provides physiological plausibility of the various health benefits claimed to be provided by vitamin D and supports the proposals for conducting clinical trials. The vitamin D system plays critical roles in the maintenance of plasma calcium and phosphate and bone mineral homeostasis. Recent evidence confirms that plasma calcium homeostasis is the critical factor modulating vitamin D activity. Vitamin D activities in the skeleton include stimulation or inhibition of bone resorption and inhibition or stimulation of bone formation. The three major bone cell types, which are osteoblasts, osteocytes and osteoclasts, can all respond to vitamin D via the classical nuclear vitamin D receptor and metabolize 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D to activate the vitamin D receptor and modulate gene expression. Dietary calcium intake interacts with vitamin D metabolism at both the renal and bone tissue levels to direct either a catabolic action on the bone through the endocrine system when calcium intake is inadequate or an anabolic action through a bone autocrine or paracrine system when calcium intake is sufficient.
Calcium/metabolism ; Fractures, Bone/metabolism/pathology ; Humans ; Osteoporosis/metabolism/pathology ; Protein Binding ; Receptors, Calcitriol/genetics/metabolism ; Vitamin D/analogs & derivatives/*metabolism

Osteoporotic fracture is the most destructive ending of osteoporosis, many interacting risk factors play a role in its occurrence. With age, the bodies function decline gradually. Symptoms like soreness of the waist, back pain, cramp of the lower limb and weakness appears. These are related to TCM syndrome factors like kidney deficiency, liver deficiency, spleen deficiency and blood stagnation. Symptoms can act as a warning of the occurrence of osteoporotic fracture in the early stages. The risk assessment tool of osteoporotic fracture is mostly developed on the basis of modern risk factors and thus lacks syndrome differentiation, that may limit its utilization. If the relevant content of syndrome differentiation is integrated into the forecasting tool and a risk assessment tool of osteoporotic fracture which complies with the demographics of China is built up, population risk assessment for high of risk could be established and scientific evidence for the adoption of effective interventions could be applied.
Bone Density ; China ; epidemiology ; Humans ; Osteoporotic Fractures ; epidemiology ; genetics ; metabolism ; physiopathology ; Risk Assessment ; Risk Factors

OBJECTIVE: To explore the difference of bone formation potential between hypertrophic nonunion tissue and atrophic nonunion tissue, which may be beneficial to nonunion therapy. METHODS: From October 2010 to March 2014, 40 nonunion tissue samples were collected in Department of Orthopedics, Second Xiangya Hospital. The samples were divided into a hypertrophic nonunion group (n=20) and an atrophic nonunion group (n=20) according to nonunion character; or a 20 to 35 years old group (n=18), a 36 to 50 years old group (n=18), a more than 50 years old group (n=4) according to different ages; or a 9-12 months group (n=21), 13-24 months group (n=14) and a more than 24 months group (n=5) according to different nonunion time. Semi-quantification was performed by SP immunohistochemical method and IPP6.0 was used to analyze the expression of bone morphogenetic protein-2 (BMP-2) through measuring the mean optical density. RESULTS: The mean optical density of BMP-2 was 0.1540±0.0408 in hypertrophic nonunion tissue, 0.1372±0.0372 in atrophic nonunion tissue, there was no significant difference between the 2 groups (P>0.05). The mean optical density of BMP-2 was 0.1477±0.0379 in the 20 to 35 years old group, 0.1419±0.0399 in the 35 to 50 years old group, 0.1456±0.0595 in the more than 50 years old group, there was no significant difference among the three groups (P>0.05). The mean optical density of BMP-2 was 0.1449±0.0366 in the 9-12 months group, 0.1472±0.0400 in the 13-24 months group, 0.1445±0.0541 in the more than 24 months group, there was no significant difference among the 3 groups (P>0.05). CONCLUSION The present results suggest that the hypertrophic nonunion tissue share similar osteogenic potential with the atrophic nonunion tissue.
Adult ; Bone Morphogenetic Protein 2 ; genetics ; metabolism ; Fracture Healing ; Fractures, Ununited ; genetics ; metabolism ; Humans ; Middle Aged ; Osteogenesis ; Young Adult

Osteogenesis imperfecta (OI) comprises a heterogeneous group of disorders characterized by bone fragility, frequent fractures, and low bone mass. Dominantly inherited COL1A1 or COL1A2 mutations appear to be causative in the majority of OI types, but rare recessively inherited genes have also been reported. Recently, SERPINF1 has been reported as another causative gene in OI type VI. To date, only eight SERPINF1 mutations have been reported and all are homozygous. Our patient showed no abnormalities at birth, frequent fractures, osteopenia, and poor response on pamidronate therapy. At the time of her most recent evaluation, she was 8 yr old, and could not walk independently due to frequent lower-extremity fractures, resulting in severe deformity. No clinical signs were seen of hearing impairment, blue sclera, or dentinogenesis imperfecta. In this study, we describe the clinical and radiological findings of one Korean patient with novel compound heterozygous mutations (c.77dupC and c.421dupC) of SERPINF1.
Bone Density/genetics ; Child ; Collagen Type I/genetics ; Eye Proteins/*genetics ; Female ; Fractures, Bone/genetics ; Humans ; Nerve Growth Factors/*genetics ; Osteogenesis Imperfecta/diagnosis/*genetics ; Serpins/*genetics

There is increasing evidence of a biochemical link between lipid oxidation and bone metabolism. Paraoxonase 1 (PON1) prevents the oxidation of low-density lipoprotein (LDL) and metabolizes biologically active phospholipids in oxidized LDLs. Here, we performed association analyses of genetic variation in PON1 to ascertain its contribution to osteoporotic fractures (OFs) and bone mineral density (BMD). We directly sequenced the PON1 gene in 24 Korean individuals and identified 26 sequence variants. A large population of Korean postmenopausal women (n = 1,329) was then genotyped for eight selected PON1 polymorphisms. BMD at the lumbar spine and femoral neck was measured using dual-energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained for vertebral fracture assessment, and the occurrence of non-vertebral fractures (i.e., wrist, hip, forearm, humerus, rib, and pelvis) was examined using self-reported data. Multivariate analyses showed that none of the polymorphisms was associated with BMD at either site. However, +5989A>G and +26080T>C polymorphisms were significantly associated with non-vertebral and vertebral fractures, respectively, after adjustment for covariates. Specifically, the minor allele of +5989A>G exerted a highly protective effect against non-vertebral fractures (OR = 0.59, P = 0.036), whereas the minor allele of +26080T>C was associated with increased susceptibility to vertebral fractures (OR = 1.73, P = 0.020). When the risk for any OFs (i.e., vertebral or non-vertebral) was considered, the statistical significance of both polymorphisms persisted (P = 0.002-0.010). These results suggest that PON1 polymorphisms could be one of useful genetic markers for OF risk in postmenopausal women.
Aged ; Alleles ; Aryldialkylphosphatase/*genetics ; Bone Density ; Female ; Gene Frequency ; Gene Order ; Genetic Markers ; Genetic Predisposition to Disease ; Haplotypes ; Humans ; Korea/epidemiology ; Linkage Disequilibrium ; Male ; Middle Aged ; Molecular Typing ; Osteoporotic Fractures/epidemiology/*genetics ; *Polymorphism, Genetic ; *Postmenopause ; Risk Factors

OBJECTIVETo observe the effects of the three-period treatment theory of bone fracture in TCM (Traditional Chinese Medicine) on VEGF and VEGF mRNA expression in the issues of outer periosteum, endosteum and bone marrow of rabbits, and to explore the rationality of phasing method in TCM in treating fracture.

METHODS3 mm bone defection were made at lower one third part of both radius in 140 male healthy rabbits. The rabbits were randomly divided into four groups, including three-period treatment group (TTG), one-period treatment group(OTG), positive medicine treatment group(PTG) and model control group (MCG). The rabbits in TG were treated with three-period treatment, rabbits in OTG were treated with one-period treatment, rabbits in PTG were fed by Guzhe-Cuoshangsan (a Chinese patent medicine which was used to treat bone fracture), rabbits in model control group were given no prescription or drug but distilled water as same dose as that of other groups. At day 3, 6, 9, 14, 28, 42 and 56, five rabbits from every group were randomly selected and were killed by aeroembolism. The left radiuses were taken out as the research object. Immunohistochemistry stain and in situ hybridization stain were performed to examinate the VEGF and VEGF mRNA expression in the outer periosteum, endosteum and bone marrow.

RESULTSThe VEGF and VEGF mRNA expression of all TCM treatment groups were enhanced in the outer periosteum, endosteum and bone marrow at different time points in fracture healing. The VEGF and VEGF mRNA expression in the three tissues of TTG had the tendency of higher than that of the other groups at the most time points after operation.

CONCLUSIONTreating fracture in stages has more predominant effect on the expression of VEGF and VEGF mRNA in the outer periosteum, endosteum and bone marrow than that of treating fracture with single prescription or drug.

Animals ; Bone Marrow ; metabolism ; Fractures, Bone ; metabolism ; therapy ; Male ; Medicine, Chinese Traditional ; Periosteum ; metabolism ; RNA, Messenger ; analysis ; Rabbits ; Vascular Endothelial Growth Factors ; analysis ; genetics

To compare the effects between gene therapy and gradual release carrier for bone morphogenetic protein-2 (BMP-2) in repairing bone defects, bone defects for 15 mm were created.on the bilateral radius in rabbits and treated with four kinds of implantations, ie, composite of transgeneic MSCs and PLA/PCL (Group A), composite of MSCs and gradual release carrier for BMP-2 (Group B), composite of MSCs and PLA/PCL (Group C), and PLA/PCL alone (Group D). After 4, 8, and 12 weeks of the operations, X-ray, histological examination, biomechanics analysis, and bone density measurement were conducted. Results showed that both osteoblasts and mesenchymal cells displayed strongly positive expression of BMP-2 in Group A after 4 weeks of the operation, the speed and quality of bone formation in Group A were much better than those in Group B. After 12 weeks of the operations, bone defects were completely repaired in Group A. BMP-2 gene therapy is really a good method to repair segmental bone defects.
Animals ; Bone Morphogenetic Protein 2 ; genetics ; therapeutic use ; Caprolactam ; analogs & derivatives ; chemistry ; Fracture Healing ; Genetic Therapy ; Implants, Experimental ; Lactic Acid ; chemistry ; Mesenchymal Stem Cell Transplantation ; methods ; Osteogenesis ; Polyesters ; Polymers ; chemistry ; Rabbits ; Radius Fractures ; therapy ; Tissue Scaffolds

Adenovirus carrying BMP-2 gene, after being mixed with fibrinous gel, was siphoned off on biodegradable scaffolds (PLA/PCL). The composite was used to repair 1.5 cm long radius defect in rabbits. Four methods were in use in the experiments: Ad-BMP-2 plus fibrinous gel and PLA/PCL (Group A), reconstructed hBMP-2 plus fibrinous gel and PLA/PCL (Group B), Ad-Lacz plus fibrinous gel and PLA/PCL (Group C), and fibrinous gel and PLA/PCL (Group D). Results showed that the defects treated in Group A were repaired with much more new bone regenerated, bridged earlier and stronger than those in Group B 12 weeks after operation. The defects treated in the other two groups could not attain osseous tissue healing. BMP-2 gene carried by biodegradable scaffold and fibrinous gel is easy to conduct and has very strong osteoinduction ability. It is really a good method to repair segmental bone defects.
Absorbable Implants ; Adenoviridae ; genetics ; metabolism ; Animals ; Biocompatible Materials ; Bone Morphogenetic Protein 2 ; biosynthesis ; genetics ; Bone Substitutes ; Caprolactam ; analogs & derivatives ; chemistry ; Fracture Healing ; Gels ; Genetic Therapy ; Humans ; Implants, Experimental ; Lactic Acid ; chemistry ; Polyesters ; Polymers ; chemistry ; Rabbits ; Radius Fractures ; therapy ; Tissue Engineering ; Tissue Scaffolds

OBJECTIVETo observe the effects of the three-period treatment theory of bone fracture in traditional Chinese medicine (TCM) on VEGF and VEGF mRNA expression in the issues of callus of rabbits. And to explore the rationality of phasing method in TCM in treating fracture.

METHODone hundred and forty male and healthy rabbits were made 3 mm wide bone defection at lower one third part of both radius as fracture healing model. Then those rabbits were divided into four groups randomly, which are three-period treatment group (TTG), one-period treatment group (OTG), positive medicine treatment group (PTG) and model control group (MCG). Those rabbits in TTG were treated with three-period treatment. Those in OTG were treated with one-period treatment. Those in PTG were feed by guzhecuoshangsan, a Chinese patent medicine which is used to treat bone fracture. Those in model control group were given no prescription or drug but distilled water as same dose as that of other groups. At day 3, 6, 9, 14, 28, 42 and 56, five rabbits were selected from every group randomly and were killed by aeroembolism respectively. Their radius were taken out and the left one was taken as the research object. Immunohistochemistry stain and in situ hybridization stain were performed to examinate the VEGF and VEGF mRNA expression in the haematoma, fibrous callus and soft callus.

RESULTAll TCM treatment groups can enhance the VEGF and VEGF mRNA expression in the haematoma, fibrous callus and soft callus at different time points in fracture healing. The VEGF and VEGF mRNA expression in the three issues of TTG had the tendency of higher than that of the other groups at the most time points after operation.

CONCLUSIONTCM can promote the VEGF and VEGF mRNA expression in the haematoma, fibrous callus and soft callus. Different Chinese medicines play various roles on VEGF and VEGF mRNA expression at different stage of fracture healing. Treating fracture in three-period treatment has more predominant effect on the expression of VEGF and VEGF mRNA in the haematoma, fibrous callus and soft callus than that of treating fracture with single prescription or drug. It is necessary to treat fracture in stages.

Animals ; Bony Callus ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Fractures, Bone ; drug therapy ; metabolism ; Male ; Medicine, Chinese Traditional ; Phytotherapy ; methods ; RNA, Messenger ; metabolism ; Rabbits ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics