Comparative study on infection rate of different adeno-associated virus for knee joint cartilage in mice / 中国骨伤

OBJECTIVE@#To explore infection rate of different adeno-associated virus (AAV) on knee joint cartilage in mice and to find a good gene editing tool for mice chondrocytes of knee joint.@*METHODS@#Forty-five 4-week-old SPF C57BL/6 weighed(14.3±0.2) g were selected. According to different injections(6 μl) for right knee joint, mice were divided into 9 different groups, 5 mice in each group. The groups were such as following:control group (normal saline), Vigene 2 group (AAV2 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 5 group (AAV5 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 6 group (AAV6 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 7 group (AAV7 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 8 group (AAV8 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 9 group (AAV9 from vigene biosciences, titer for 1×10¹³ vg/ml), Hanbio DJ group(AAV2-DJ from Hanbio, titer for 1×10¹² vg/ml), Hanbio 5 group (AAV5 from Hanbio, titer for 1×10¹² vg/ml). All AAVs were over-expressed green fluorescent protein(GFP). Knee joint specimens were taken and observed injury of cartilage under stereomicroscope at 30 days after injection, then 10 μm thick frozen sections were prepared. Distribution of green fluorescent protein of meniscus and cartilage of knee joint was observed under fluorescence microscope.@*RESULTS@#Stereomicroscope observation indicated that no obvious lesion was observed in knee joint cartilage of mice after intra-articular injection of AAV. According to frozen sections of knee joints, strong green fluorescence was observed in knee joint cartilage in all AAV experimental groups. Compared with other groups, significantly stronger green fluorescence were observed both in AAV2 and AAV7 groups, whose average fluorescence density was 0.077±0.020 and 0.061±0.022. There were significant differences between two groups and other groups.@*CONCLUSIONS@#AAV could infect chondrocyte of knee joint in vivo by injecting into knee joint cavity. Higher infection efficiency of AAV2 and AAV7 on knee joint cartilage were observed. Local injection of AAV into knee joint cavity could be used as an effective tool for gene editing of knee joint chondrocyte.

Subculture, cryopreservation and recovery of osteoclasts / 中国骨伤

<p><b>OBJECTIVE</b>To explore the feasibility of passage, cryopreservation, and recovery of osteoclasts in order to develop new techniques facilitating osteoclast research.</p><p><b>METHODS</b>Passage of osteoclasts: adult male SD rat(SPF grade, weight of 250 g) was sacrificed and the abdominal aorta was exposed for blood draw. Monocytes isolated from peripheral circulation was treated with RANKL and M-CSF for 2 weeks. After formation of osteoclasts, they were trypsinized with pipetting, centrifuged, re-suspended with α-MEM containing RANKL and M-CSF, and cultured in 6 well-plates and 35 mm culture dishes. Freezing of osteoclasts: trypsinized osteoclasts were centrifuged and resuspended with DMSO, FBS, α-MEM (1:2:7), and were stored in liquid nitrogen(-196 °C). Recovery of osteoclasts: frozen osteoclasts were taken out of liquid nitrogen tank and thawed quickly at 37 °C in water bath. After wash with PBS, the cells were resuspended with α-MEM containing RANKL and M-CSF, and were cultured in 6 well dishes and 35 mm culture dishes. Meanwhile, cells were checked with inverted phase contrast microscope and observed in the live cell station for real time imaging. TRAP staining was performed 3 days after plating.</p><p><b>RESULTS</b>Trypsinization together with pipetting and shaking can detach the adherent osteoclasts, and the resuspended cells can be used for passage and storage in liquid nitrogen. The passaged cells became fully attached to the culture dishes in 2 hours, and the multinucleated feature could be clearly seen. The osteoclasts recovered from liquid nitrogen could completely spread out for 2 to 3 hours so that the multinucleated cells were clearly seen. These cells were still TRAP positive.</p><p><b>CONCLUSIONS</b>Although osteoclasts strongly adhere to the bottom of culture dishes, a large majority of the osteoclasts can be detached after appropriate digestion with trypsin, pipetting and shaking. These cells can be used for passage and cryopreservation. After recovering from liquid nitrogen, these cells still preserve the viability and the feature of osteoclasts. The results provide a new and powerful tool for future study of osteoclast biology.</p>

Clinical experience on postoperative balance of hemostasis and antithrombus for patients with hemophilic arthritis after arthroplasty / 中国骨伤

<p><b>OBJECTIVE</b>To observe the clinical significance of postoperative personalized antithrombotic therapy for patients with hemophilic arthritis (HA) patients after arthroplasty.</p><p><b>METHODS</b>From September 2005 to October 2013, 11 cases of arthroplasty for hemophilic arthritis in hip and knee total operation 14 times,including 1 case of double knees (calculated as one operation), operation in left knees 6 times, operation in right knees 5 times, 2 in hip. All the patients were male and the age ranged from 23 to 57 years old,with an average of (36.1 ± 11.0) years old; the average weight was (64.1 ± 8.9) kg. All the patients were preoperatively diagnosed and classified as hemophilic arthritis with the radiological images and laboratory tests. According to the function of joints, the risk of postoperative venous thromboembolism (VTE), and dynamic observation of Factor VIII:C (FVIII:C) activity, patients were treated with personalized antithrombus by adjusting the dosage of recombinant human coagulation factor VIII (Kogenate FS). All the patients were orderly divided into postoperatively distal joints moving group and none-moving group to observe the coagulation function.</p><p><b>RESULTS</b>The enrolled patients had no postoperative complication of VTE and pulmonary embolism (PE). The APTT and D-2 were different between two groups in the postoperative early stage. Length of hospital day was shorter in the moving group than none-moving group.</p><p><b>CONCLUSION</b>Because of the self-coagulation disorder, patients with HA tended to bleed. However it doesn't mean that there is no risk of postoperative thrombosis. Therefore,it's important to determine how to control the balance between postoperative antithrombus, hemostasis,and coagulation factor replacement therapy after arthroplasty for HA. Postoperative moving has proved helpful for HA, especially in reducing the risk of hemostasis and shortening the time in hospital.</p>

Research advance on intervertebral disc degeneration and cell death / 中国骨伤

Intervertebral disc degeneration is considered as a primary cause of clinical low back pain, however the molecular mechanism is not clear yet. Recently, researches on the molecular basis of intervertebral disc degeneration have become a hotspot. The special structure and biomechanics properties of the disc contribute to its propensity toward degeneration. Intervertebral disc degeneration is associated with the changes of the cytological behavior,including the increase in cell death and the degradation of extracellular matrix. However, the mechanism of cell death including cell apoptosis and autophagy in intervertebral disc degeneration remains unclear. Further study on the molecular mechanism of intervertebral disc degeneration is the foundation of improving and treating the intervertebral disc degeneration in the future. Although some progresses are made in the aspect of biological study, the biological environment of intervertebral disc itself is still a challenge for the development of biological treatment. This article is to review the latest advance on the biological characteristics of normal intervertebral disc and the cell death in the process of the intervertebral disc degeneration.

Different biological characteristics between nucleus pulposus and annulus fibrosus cells in rabbits / 中国骨伤

<p><b>OBJECTIVE</b>To compare biological characteristics between nucleus pulposus and annulus fibrosus cells in vitro model.</p><p><b>METHODS</b>Five New Zealand white rabbits (2 to 3 kg, either gender) were isolated nucleus pulposus and annulus fibrosus under sterilized condition, then cultured in nutrient solution with 15% FBS and DMEM/F12 (1:1) by enzyme digestion combined with tissue block method. When 90% cells fused, subcultring were performed. Cell morphology were observed by inverted phase contrast microscope, cell viability were detected by trypan blue staining, histological were observed by a toluidine blue and HE staining, cell proliferation were tested by MTT method, then the cell morphology, viability, proliferation between nucleus pulposus and annulus fibrosus were compared.</p><p><b>RESULTS</b>There were no obviously differences between nucleus pulposus and annulus fibrosus in original and the first strain. Physalides were appeared in annulus fibrosus on the second generation. The strapping time was later, and activity was lower in nucleus pulposus than annulus fibrosus. The growth of cell proliferation in nucleus pulposus was lower than annulus fibrosus from the ninth day.</p><p><b>CONCLUSION</b>The cell activity in annulus fibrosus is higher than nucleus pulposus. Digenerative disc disease may caused by recession of nucleus pulposus,local biomechnical changes, furether caused structure change and function loss of annulus fibrosus.</p>

Kinetic characteristics of gymnastic vault springboard / 医用生物力学

Objective To analyze the dynamics of vault springboard designated to using in formal competitions by the Federation International de Gymnastique (FIG), and find a new method measuring the reaction force of the springboard, so as to provide scientific supports for diagnosis of its take-off technique. Methods The stiffness values of GYMNOVA soft and hard springboards were derived by method of material mechanics, then the springboards were then tested with static, dynamic experiments and computer simulations. In the static experiment, two video cameras with the sample frequency of 600 Hz were placed at a 90° angle to capture the deformation of the springboards under the loads of 160, 180, 210 and 230 kg, respectively. In the dynamic experiment, a volunteer performed drop jump (DJ) from a 1.25 m high platform onto the hard and soft springboards, respectively, while a camera was employed to capture the deformation at the sample frequency of 300 Hz. All the three cameras were calibrated using a 2-dimensional framework, and the changes of the board height, velocity under different loads and acceleration of center of mass (COM) of the human body in DJ experiment were all obtained by digitizing the videos using SIMI MOTION software. Finally, modeling and computer simulation were performed to simulate the DJ in the dynamic experiment. Results The equation F=kx+c describing force-displacement of both the springboard was obtained. The depressing displacements of the board under different loads in the static experiment were close to those calculated by the equation. The vertical reaction force curve in DJ calculated by the equation was highly correlated to that obtained by acceleration of COM. The coefficient of multiple correlation was all greater than 0.86. Conclusions The study developed a new method for measuring the vertical reaction force of the springboard based on board depressing displacement and velocity with the help of high-speed camera. This method, which can be employed with convenience to rapidly monitor the board reaction force during take-off, provides scientific supports for enhancement of vaulting techniques and plays an active role in prevention of vaulting injuries.

Reseach of oxidative stress induces aging in rabbit intervertebral disc nucleus pulposus cells injured by H2O2 / 中国骨伤

<p><b>OBJECTIVE</b>To study the effect of H2O2 on the morphological pattern,vitality,proliferation,cycle period of rabbit intervertebral disc nucleus pulposus cells.</p><p><b>METHODS</b>Ten New Zealand white rabbits (2 to 3 kg, female) were used for isolating nucleus pulposus cells under sterilized condition. The culture solution with 15% FBS and DMEM/F12 (1:1) was applied for cell cultivation. After 90% cell fusion, the first generation was obtain and stimulated by H2O2 with different concentrations of 0 micromol/L (control group), 130 micromol/L,216 p.mol/L,360 Ipmol/L, 600 micromol/L,and 1000 micromol/L.</p><p><b>RESULTS</b>Compared with the control group, there was little difference of the biological property (P>0.05) in 130 micromol/L and 216 micromol/L H202-treated groups. When the concentration of H2O2 attained 360 micromol/L, 600 micromol/L, and 1 000 micromol/L, the cells suffered aging,with increased cell vacuoles,decreased proliferation,and aging:related increase of 13-galactosidase dyeing. The cell cycle of many nucleus pulposus cells was blocked in G1 stage other than entering S stage. With increasing H2O2 concentrations, the aging degree was increased.</p><p><b>CONCLUSION</b>A certain concentration of H202 could induce early aging of nucleus pulposus cells,resulting in biological abnormalities of these cells.</p>

Imaging observation of live cells originating from osteoclasts of the blood system / 中国骨伤

<p><b>OBJECTIVE</b>To study the application of the live cell imaging method to observe the whole process of osteoclast formation induced by monocyte macrophages in the blood system in order to clarify the origin of osteoclasts and their cytodynamics.</p><p><b>METHODS</b>Blood samples (8 ml) were collected from the abdominal aorta of male SD rats weighing 280 g. Mononuclear cells were obtained by density gradient centrifugation and induced by RANKL and M-CSF. The cells were cultured and divided into four groups: inverted phase contrast microscope (IPCM) group, TRAP group, SEM group and live cell imaging (LCI) group. Images of the IPCM group were captured by a digital microscopic imaging system and recorded daily. The TRAP group was identified by enzyme activity staining after a 21-day cultivation period. The SEM group was SEM-observed after a 21-day cultivation period. The LCI group was consecutively and dynamically observed for 35 days.</p><p><b>RESULTS</b>After 2-week cultivation, IPCM observations showed the formation of numerous apocytes. These cells displayed round, fusiform, fan-shaped, elliptic or irregular gibbous profiles. TRAP staining showed that most apocytes and monocytes had positive(+)reaction. SEM observations showed many bone absorption lacunae, hollows and channels, in which many osteoclasts with absorption activity were observed. Live cell imaging observations found that multinuclear osteoclasts originating from peripheral blood were generated by fusion of monocytes and apocytes and intercross fusion of monocytes and apocytes,which occurred at the adherent stage of the cells. Cytodynamic observations showed that the cell form of osteoclasts was complex and changeable.</p><p><b>CONCLUSION</b>RANKL and M-CSF can induce differentiation and formation from monocytes in rat peripheral blood into multinuclear osteoclasts with bone absorption activity. The osteoclasts were formed by various cell fusion processes at the adherent stage. The adherent property of osteoclasts is important for their survival and function. Osteoclasts have phagocytosis and their morphological structure is dynamically changeable, involving not only apocytes but monocytes. The osteoclast property of multinuclear giant cells formed by cell fusion may be a special biological behavior for their adaptation of functional needs and bone absorption efficiency. This experiment has further evidenced the theory of osteoclast origination in the blood system and provided new experimental clues for clarifying the cytodynamic and cytobiological properties of osteoclasts.</p>

Study on the effect of Youguiyin () on regulating gene expression profile of the rats with femoral head necrosis induced by steroid / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the regulation of Youguiyin (YGY, ) on the gene expression profile of the rat with steroid-induced femoral head necrosis (sFHN), for the sake of investigating its molecular mechanism of sFHN prevention and treatment.</p><p><b>METHODS</b>All the 30 rats were randomly divided into three groups, the normal control group (A), the model control group (B), and the YGY treated group (C), 10 in each group. After rats in Groups B and C were being made into FHN models with steroid injection, they received a daily intragastric administration of saline and YGY respectively in equal volume for a total of 6 weeks, while to the unmodeled normal rats in Group A, saline was administered instead. The rats were sacrificed at the terminal of administration; their mRNA from femoral head tissue was extracted and prepared to cDNA probe through inverse transcription for detecting gene expression profile by microarray, outcomes of which was passing fluorescence quantitative PCR verification, and the differential expressed genes were analyzed adopting gene ontology (GO) method.</p><p><b>RESULTS</b>Compared with Group A, the numbers of differential genes found in Groups B and C were 190 and 92, respectively, but the changing trend in the two groups was opposite, mainly manifested as down-regulating in Group B/Group A (GB/GA) and up-regulating in Group C/Group B (GC/GB). The analysis showed that these differential genes were mainly assigned to cell apoptosis, signal transduction, metabolism, cell proliferation and differentiation, cell cycle, blood coagulation, antioxidant activity, etc.</p><p><b>CONCLUSIONS</b>sFHN was regulated by various genes; the regulation of YGY on expressions of these genes and the intra/extra-cellular signaling processes was possibly the molecular mechanism of YGY for preventing/treating sFHN. This study gave an explanation to the effectiveness of Chinese medicine in preventing/treating FHN from aspects of gene expression and enriched the Chinese medicine theory of "Kidney (Shen) governing bones".</p>

Epimedium-derived flavonoids modulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats via Wnt/β-catenin signal pathway activation / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the function of wnt/β-catenin signal pathway on the process that epimedium-derived flavonoids (EFs) regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, and to provide an experimental evidence for the mechanism of EFs on treating postmenopausal osteoporosis.</p><p><b>METHODS</b>Bone marrow stromal cells from ovariectomized rats were separated and cultivated in the condition of osteoinductive medium or liquid medium for 15 days. Low- (1 μg/mL), medium- (10 μg/mL) and high- (100 μg/mL) dose EFs were administrated correspondingly. Alkaline phosphatase (ALP) staining, ALP activity determination, oil red O staining and realtime polymerese chain reaction (RT-PCR) were used to determine the effect of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats. Moreover, in order to explore the mechanism of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, Dickkopf-related protein 1 (DKK1) was used in the medium group. Enzymelinked immunosorbent assay (ELISA) and RT-PCR were used to determine mRNA levels of β-catenin, low density lipoprotein receptor-related protein 5 (LRP5) and T cell factor (TCF) protein, known as wnt/β-catenin signal pathway related factors.</p><p><b>RESULTS</b>EFs increased mRNA expression levels of ALP and early osteoblast differentiation factors, such as runt-related transcription factor 2 (Runx2), osteocalcin and collagen I, and decreased mRNA expression levels of fat generation factors, such as peroxisome proliferator activated receptor gamma 2 (PPARγ-2) and CCAAT enhancer-binding protein-α (C/EBPα) in a dose-dependent manner. While osteoblast differentiation factors were down-regulated, fat generation factors were up-regulated when DKK1 was applied. Also EFs up-regulated mRNA expression levels of β-catenin, LRP5 and TCF protein which could be blocked by DKK1.</p><p><b>CONCLUSION</b>EFs regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats by activating wnt/β-catenin signal pathway, which may be an important molecular mechanism of EFs on treating postmenopausal osteoporosis.</p>

Genomics study of steroid induced femoral head necrosis of rats / 中华外科杂志

<p><b>OBJECTIVES</b>To identify significantly differentially expression genes of steroid-induced femoral head necrosis (SINFH) of rats by gene chip, and to find out the potential factors and molecular mechanisms that oxidative stress originate or strengthen the SINFH.</p><p><b>METHODS</b>Twenty Wistar rats were divided into experimental group and control group randomly. E. coli endotoxin was given to all rats at a dose of 20 µg/kg body weight by daily i.p. for two times. Then methylprednisolone (40 mg/kg) or saline was daily injected into the left gluteus muscle of the rats in experimental group and control group respectively. Six weeks later, the mRNA was extracted from the femoral head of rats in every group, and the cDNA were obtained by inverse transcript, then carried out microarray detection. The quantitative RT-PCR was used to confirm the result of microarray, and the differentially expressed genes were analyzed for the functional annotation by gene ontology (GO).</p><p><b>RESULTS</b>Compared to the control group, 190 genes in the experimental group were differentially expressed, with 52 up-regulated and 138 down-regulated. Of these genes, 102 are known (have deposited in GeneBank), while 88 of them are unknown. The known genes can be divided into several families according to their biological functions, such as: oxidative stress, apoptosis, signal transduction, angiogenesis, extracellular matrix, lipid metabolism, and gene transcription related genes. The results of quantitative RT-PCR are consistent with gene-chip results.</p><p><b>CONCLUSIONS</b>The occurrence of SINFH is a complicated process affected by multiple factors and signaling pathways. Our findings indicate that many genes which are involved in different signaling pathways were differentially expressed between SINFH rats and normal rats.</p>

The expression and significance of Wnt/beta-catenin signal pathway protein in the effect of bushen huoxue granule containing serum on the osteoblast / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effect of Wnt/beta-catenin signal pathway protein in the effect of Bushen Huoxue Granule (BHG) containing serum on the osteoblast, and to provide necessary experimental reliance for its action of mechanism in treatment of osteoporosis.</p><p><b>METHODS</b>The osteoblast from cranial bones of neonates rat were isolated and cultured in vitro, which was divided into the blank control group and the BHG containing serum group. The alkaline phosphatase (ALP) activities of the osteoblast in each group were quantitatively detected and the ALP staining was performed six days later. The alizarin red staining was performed eighteen days later. At the same time, levels of Wnt/beta-catenin signal pathway protein--beta-catenin, low density lipoprotein correlated protein 5 (LRP 5), and T cell factor (TCF) of osteoblasts in each group were detected by ELISA.</p><p><b>RESULTS</b>BHG containing serum could significantly increase the expression of ALP and promote the formation of mineralizing nodus in the osteoblasts. At the same time it also markedly up-regulated the expressions of p-catenin, LRP 5, and TCF in this process.</p><p><b>CONCLUSION</b>BHG containing serum could markedly increase ossify activities and mineralization of osteoblast. This action was closely correlated with Wnt/p-catenin signal pathway. So it indicated that Wnt/beta-catenin signal pathway played a very important role in the treatment of the osteoporosis by BHG.</p>

Effect of jingui shenqi pill on morphology of injured spinal cell apoptosis in rats caused by brachytherapy / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To study the effect of Jingui Shenqi Pill (JSP) on morphology of spinal cell apoptosis in rats injured by 192Ir irradiation.</p><p><b>METHODS</b>One hundred and twenty rats were randomly divided into four groups: the model group, the JSP group, the prednisone group and the normal group. Corresponding pharmaceutics were given to rats once a day for 14 days respectively. Then except rats in the normal group, the others received 192Ir interstitial irradiation with the dosage of 22 Gy using back-fixing technology. The injured segments of spinal cord were taken out for HE staining, TUNEL examination and observation with electron microscope 8 hrs, 24 hrs and 4 weeks after irradiation.</p><p><b>RESULTS</b>HE staining examination showed no obvious histological change in rats 8 and 24 hrs after irradiation, but pathological changes, as tissue rarefaction and hemorrhage did found in white matter of spinal cord shown by TUNEL 4 weeks later. Electron microscopic examination and TUNEL staining showed that as compared with the model group, the apoptotic index in the JSP and predinisone treated groups was significantly lower (P < 0.01) 8 hrs after radiation, but it showed insignificant difference between groups at the time points of 24 hrs and 4 weeks after radiation (P > 0.05).</p><p><b>CONCLUSION</b>JSP could act against apoptosis of gliocyte in spinal cord of rats in early stage after brachytherapy, indicating that JSP possessing a prednisone-like action.</p>