2018 China guideline for diagnosis and treatment of senile osteoporosis / 中国实用内科杂志

According to the procedures for the development of evidence-based medicine guidelines, a multi-disciplinary guideline development working group was established, after three rounds of discussions by the consensus expert group, a new evidencebased guideline for diagnosis and treatment of senile osteoporosis in China(2018) was developed. The grading of recommendations assessment, development and evaluation(GRADE) system was used to rate the quality of evidence and the strength of recommendations. Recommendations were derived from evidence body, and at the same time considered the balance of benefits and harms as well as values and preferences of Chinese patients. The guideline development working group developed 15 recommendations for the diagnosis and treatment of senile osteoporosis. The guideline covered the screening for senile osteoporosis, risk assessment, diagnosis, basic treatment, multiple anti-osteoporosis drugs, therapeutic effect monitoring and evaluation of senile osteoporosis. This guideline aims to serve as a tool for clinicians and patients for best decisions-making in China.

Effect of erythropoietin on the survival of retinal neurocytes in culture upon serum withdrawal

To clarify whether erythropoietin [EPO] could substitute for the serum component in cultured retinal neurocytes suffering from serum withdrawal. The study was performed in the Shanghai Institute of Traumatology and Orthopedics, Shanghai, China between April 2008 and March 2009. A total of 160 postnatal 2-3 day-old Sprague-Dawley rats were used for this study. After the retinal neurocytes were cultured for 48 hours, the culture media was replaced with serum-free media, and the cells were exposed to 1 U/ml, 3 U/ml, and 6 U/ml EPO for another 24 or 48 hours, the cell body diameter was then assessed using a computerized image-analysis system, and the survival and apoptosis rates of those cells were estimated by method of transcription and translation assay and flow cytometry. Immunocytochemistry was used to detect EPO and erythropoietin receptor [EPOR] expression. The retinal neurocytes had obvious EPO/EPOR expression. The early [p=0.002] and total [p=0.049] apoptosis rates of retinal neurocytes cultured with serum withdrawal were significantly higher than that of neurocytes cultured with serum, and the cell viability of neurocytes cultured with serum withdrawal was significantly lower than that of neurocytes cultured with serum [p=0.047]. The EPO had no effect on the cell body diameter of cultured retinal neurocytes. The cell viability and the apoptosis rates of retinal neurocytes were not significantly different from that of simple serum-withdrawal culture at any EPO concentration. As the addition of EPO immediately after serum withdrawal had no effect in preventing retinal neurocytes apoptosis induced by serum withdrawal, EPO cannot substitute for the serum component

Expression of hypoxia inducible factor-1alpha in long bone development / 中国骨伤

<p><b>OBJECTIVE</b>To observe dynamically the development of fetal long bone and detect the expression and distribution of HIF-1alpha,to investigate the expression pattern and possible effects of hypoxia inducible factor-1alpha (HIF-1alpha) in fetal long bone development of mouse.</p><p><b>METHODS</b>E12.5, E13.5, E14.5, E15.5, E16.5 and E17.5 pregnant C57BL6 mice were sacrificed. After sacrifice, the embryos were delivered by caesarean section. The development of fetal long bone was dynamically observed by stereoscopic microscope, and the distributional expression of HIF-1alpha protein was detected by using method of immunohistochemistry. The expression of HIF-1alpha mRNA and osteoblast marker gene at various stage were also detected by using methods of reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The cartilaginous long bone began to form and joints outline arised at E13.5, then the primary ossification center was observed at E14.5, showing opaque ossification under stereoscopic microscope,and then the osteogenesis expanded and extended to both sides. Immunohistochemistry demonstrated lots of HIF-1alpha protein positive chondrcytes in the center of primary ossification at E14.5, then they decreased dramatically. HIF-1alpha mRNA expressed at high level from E13.5 to E15.5, and then decreased to low level.</p><p><b>CONCLUSION</b>Fetal long bone development pattern appeared to be endochondral osteogenisis process, existing hypoxia microenviroment may increase HIF-1alpha mRNA expression and thus initiate the cascade of endochondral osteogenisis.</p>

Experimental study of core binding factor a1 gene-modified rabbit skin fibroblasts enhance bone defect repair / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts.</p><p><b>METHODS</b>Transfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test.</p><p><b>RESULTS</b>In group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups.</p><p><b>CONCLUSION</b>True bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.</p>

The regulation of hypoxia inducible factor-1alpha on osteoblast function in postmenopausal osteoporosis / 中华外科杂志

<p><b>OBJECTIVE</b>To study the regulation of hypoxia inducible factor-1alpha (HIF-1alpha) on osteoblast function in postmenopausal osteoporosis.</p><p><b>METHODS</b>From October 2004 to May 2006, Cre-Loxp recombinase was used to create mice which the HIF-1alpha gene in osteoblasts was conditional knock-out, 24 female wild-type (WT) mice and 24 female conditional knock-out (CKO) mice of 3 months old were operated on ovariotomy. At 0,4,8 weeks after operation, bone histomorphometry parameters were measured with computer image analysis in HE stain sections and in tetracycline bone double labeling fluorescence sections; Bone density and the trabecular bone architecture parameters were measured by Micro-CT; The mRNA expression of vascular endothelial growth factors (VEGF), RunX2, OC, ALP were detected with quantitative RT-PCR; The protein expression of VEGF and RunX2 were detected with Western-blotting.</p><p><b>RESULTS</b>In CKO mice, the trabecular number, volume, thickness, bone density, mineral apposition rate (MAR), the expression of VEGF, RunX2, OC, ALP on mRNA level and the expression of VEGF, RunX2 on protein level decreased significantly compared with WT mice especially in 8 weeks after operation.</p><p><b>CONCLUSIONS</b>The bone formation ability of osteoblasts in CKO mice was reduced compared with WT mice after ovariotomy. HIF-1alpha can regulate the bone formation ability of osteoblasts in postmenopausal osteoporosis.</p>

Experiment on induction of fibroblasts on 3-D cell-foam structures to express osteoblastic phenotype and its mechanism / 中华外科杂志

<p><b>OBJECTIVE</b>To study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors.</p><p><b>METHODS</b>The fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment.</p><p><b>RESULTS</b>Fibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor.</p><p><b>CONCLUSIONS</b>Fibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.</p>

Expression of Hypoxia Inducible Factor-1? in Vertebra Development of Mouse / 上海第二医科大学学报

Objective To investigate the expression pattern of hypoxia inducible factor-1?(HIF-1?) in fetal vertebra development of the mouse. Methods The development of mouse fetal vertebra was observed dynamically,and the expression of HIF-1? mRNA at various stages was also detected by reverse transcription-polymerase chain reaction. Results The cartilaginous spine column began to form at E13.5.The primary ossification center was observed at E15.5,then the osteogenesis expanded and extended to both sides.HIF-1? mRNA began to express at E13.5,and more significantly at E14.5(P

Expression and regulatory mechanism of hypoxia inducible factor-1? during fracture healing / 上海第二医科大学学报

Objective To explore the expression and regulatory mechanism of hypoxia inducible factor-1?(HIF-1?)during fracture healing. Methods Mouse models of tibia fracture healing were established,and callus samples were collected 1,3,7,14,21 and 28 days after fracture.The development of callus and new bone formation were evaluated with roentgenology,Micro-CT and tetracycline double labeling method,and the expression of HIF-1?,vascular endothelial growth factor(VEGF),Runx2 and ALP in callus were detected with RT-PCR,Western blotting and immunohistochemistry.The relationship between HIF-1? and fracture healing was analysed. Results The expression of HIF-1? was detected in cells in the fracture sites as well as in evolved osteoblasts,chondrocytes and osteocytes in early callus under hypoxia.The highest expression rate of HIF-1? achieved on the 7th day after fracture,lasted for about 7 days,then decreased gradually,and returned to intact level on the 28th day after fracture.The expression tendency of VEGF resembled that of HIF-1?.Bone formation activity was more active in early callus,and the callus volume peaked on the 14th day after fracture and decreased gradually.The mineralization of callus mainly took place in the late healing period(14th to 28th day after fracture).Conclusion Cells involved in fracture healing are hypoxia-responsive cells,which express HIF-1?.HIF-1? can regulate cell state and function,and can promote angiogenesis so as to play a crucial role in fracture healing.

Osteogenesis of rabbit skin fibroblast transfected with core binding factor a1/osteoblast specific transplanting factor-2 gene / 中华外科杂志

<p><b>OBJECTIVE</b>To study osteoblastic phenotype expression of New Zealand rabbit skin fibroblasts transfected with mouse core binding factor a1/osteoblast specific transplanting factor-2 gene (Cbfa1/Osf2).</p><p><b>METHODS</b>Cbfa1/Osf2 gene, engineered into eukaryotic expression vector pSG5, was introduced into New Zealand rabbit skin fibroblasts with catholyte liposomes-Lipofectamine 2000. Meanwhile, those transfected pSG5 and un-transfected were set the control groups. The expression of Cbfa1 gene, osteocalcin (OCN) gene, alkaline phosphatase (ALP) gene and pre-peptide 2 alpha gene of collagen type I were detected by RT-PCR assay. Cbfa1 protein was detected by Western-Blot assay, in-cell ALP activity by p-nitrophenyl phosphate (PNPP) assay and OCN content in the supernatant by radio-immunity method. The ossification nodules was detected by Alizarin-Red staining and scanning electron microscope.</p><p><b>RESULTS</b>Cbfa 1mRNA and Cbfa1 protein were expressed in New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 from the first day to the fifth day, but they were not detected in the control groups. In the pSG5-Cbfa1/Osf2 transfected group, the expression of ALP gene and OCN gene were respectively induced from the third day and the forth day, pre-peptide 2 alpha gene of collagen type I was enhanced from the third day. From the sixth day, ALP activity greatly increased, OCN strongly secreted, and they were maintained at a high level for about 4 weeks, and the difference was significant compared with the control group (P < 0.05). On the forty-second day, ossification nodules were found on the surface of pSG5-Cbfa1/Osf2 gene transfected cells.</p><p><b>CONCLUSIONS</b>New Zealand rabbit skin fibroblasts transfected with pSG5-Cbfa1/Osf2 can express osteogenesis-related genes and proteins, and form ossification nodules on their surface.</p>

Experimental study on the osseointegration of nanophase hydroxyapatite biograde-coated implants / 中华外科杂志

<p><b>OBJECTIVE</b>To study the osseointegration of the nanophase hydroxyapatite biograde-coated implants and host bone.</p><p><b>METHODS</b>Nanophase hydroxyapatite biograde-coated implants, hydroxyapatite biograde-coated implants and noncoated Ti-6Al-4V implants were inserted into both femoral of Beagle canines the tissue response, dynamic osteogensis and SEM were studied at 4, 8 and 12 weeks.</p><p><b>RESULTS</b>The degradation of nanophase hydroxyapatite was slower than hydroxyapatite, dynamic osteogensis and the form of osteoblast were better than the control groups.</p><p><b>CONCLUSION</b>The biological reaction of Nanophase hydroxyapatite biograde-coated implants is better than hydroxyapatite coated implant.</p>

Experiment on fibroblast-PGA complexes cultured in rotary cell culture system / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of RCCS on cell seeding onto 3-D scaffold and cell-scaffold composite culture in vitro.</p><p><b>METHODS</b>Rabbit skin fibroblasts of passage 2 were seeded at 2 x 10(6) cell per cm(3) onto/into polyglycolic acid (PGA) foams by static seeding (dropping a cell suspension onto foams) or dynamic seeding (rotating PGA foams and a cell suspension in RCCS). Attachment of cells in foams was observed by cell-counting after trypsin digestion. The effects of culture condition were next studied by culturing cell-PGA complexes in RCCS versus static culture condition. Distribution and proliferation of cells in foams were investigated with MTT, stereomicroscope and scan electron microscope.</p><p><b>RESULTS</b>Numbers of cells adhering to polymers increased gradually during an initial period of 24 hours. Eight, 12 and 24 hours after seeding, the rates of adhering cells were significantly higher in the dynamic seeding group than in the static seeding group (46.70% + 2.16% vs. 31.50% +/- 3.54%; 56.36% +/- 3.18% vs. 34.28% +/- 3.16%; 66.32% +/- 4.60% vs. 37.38% +/- 4.66%; P < 0.01). The dynamic culture method as compared to the static method resulted in new tissues with a higher cellularity and more uniform cell distribution during a 3 period of weeks.</p><p><b>CONCLUSIONS</b>RCCS has advantages of promoting cell attachment, uniform migration and proliferation in polymer scaffolds and can be used for construction of 3-D cell-polymer tissues in vitro.</p>