In sonoporation, the cell membrane is broken-up temporarily by ultrasound mediated microbubbles, which is promoting drug or gene into the cell. In current literatures, there are numerous studies of single microbubble dynamics in sonoporation. However till now, little studies have been focused on the sonoporation incidence caused by more than one microbubble. In this article, the dynamic model of two adjacent microbubbles in stable cavitation has been introduced. By the model, the forces including secondary Bjerknes force on cell membrane given by microbubbles and their effects on sonoporation have been numerically studied. According to the experimental parameters, we numerically studied (1) effects of the ultrasound and microbubble parameters on the secondary Bjerknes forces; (2) the forces exerted on cell membrane by microbubble, including the secondary Bjerknes force; (3) the sonoporation possibility caused by those forces produced by microbubble. In this article, the ultrasound and microbubbles' parameters range were found to produce sonoporation by two adjacent microbubbles. Furthermore, it is the first time to found that the microbubbles' parameters are more important than ultrasound parameters on sonoporation.

Objective To investigate the correlation between disease severity and pleural effu sion in patients with acute pancreatitis(AP).Methods A retrospective analysis was conducted on a prospectively collected database.The demographic,clinical,and laboratory data of 246 consecutive cases of AP in patients admitted to the Affiliated Union Hospital of Fujian Medical University between January 2008 to December 2012 were reviewed.Acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score and computed tomography severity index (CTSI) were used to evaluate the disease severity of AP.The relationship between the severity and pleural effusion was analyzed.Receiver operator characteristic (ROC) curve was used to compare the values of APACHE Ⅱ score and CTSI in predicting the prognosis of patients with pleural effusion.Results Among the 246 patients,there were 184 patients with pleural effusion and 62 patients without pleural effusion.The incidence of pleu ral effusion in AP was 74.8％.Further study showed that the difference in the incidences of pleural effusion between the severe group and the mild group was significant (P＜0.01).There was a trend that the more serious the patient's condition,the more the pleural effusion.Moreover,the levels of pleural effusion were significantly and positively correlated with the APACHE Ⅱ score (r=0.775,P＜0.01) and CTSI (r=0.525,P＜0.05).Logistic regression analysis showed that the factors significantly associated with pleural effusion formation were a high APACHE Ⅱ score and a high CTSI.Areas under the ROC curve of the APACHE Ⅱ score,CTSI and combined assessment were 0.798,0.687 and 0.812 for predicting mortality of the patients with pleural effusion.Through comparison of the areas under the ROC curve,there was a significant difference between the APACHE Ⅱ score and CTSI as well as combined assessment and CTSI (P＜0.05).Conclusions The disease severity was closely related to pleural effusion in patients with AP.Combining the two scoring systems to evaluate the disease severity and providing active treatment were important to improve the prognosis of patients with pleural effusion.

Objective To explore labeling efficiency and appropriate conditions of Superpara magnetic iron exide nanopaticles (SPIO) nanoparticles for Bone marrow stromal cells(BMSCs). Methods BMSCs were aquired from skeletally mature dogs via iliac crest aspiration and separated by adherent cell cytopheresis.BMSCs were cultured and incubated with SPIO at different concentrations in vitro. The labeling efficiency of BMSCs with different labeling concentrations SPIO nanoparticles as well as detection of characteristics and signal attenuation rules were evaluated by MRI at 1.5T in vitro. Results BMSCs were efficiently labeled by SPIOin vitro and has no alterations to viability and proliferation profiles at this labeling concentration. BMSCs loaded with SPIO can be detected by MRI at certainly cell quantity in vitro(5 × 104). The quantity of SPIO in cells gradually reduced as cell culture time prolonged, with no statistically significant changes in cell death(P＞ 0.05). Conclusion The results demonstrated the potential application of SPIO as a wonderful cell tracer in vitro.

BACKGROUND: The study of isolation, purification, culture, cell labeling, inducing factors, effects of gene transfection on cytobiology, cell carrier construction, and time window for back transplantation of cell compound pertaining to marrow stromal cells (MSCs) is still in its infancy. OBJECTIVE: To search for an in vitro culture method that can be simply and effectively obtained. DESIGN, TIME AND SETTING: The present cytological in vitro experiment was performed at the Beijing Institute of Genome, Chinese Academy of Sciences between June 2006 and July 2007. MATERIALS: Eight specific pathogen-free New Zealand rabbits, aged 6 weeks, were provided by the Laboratory Animal Center, Institute of Genetics and Development, Chinese Academy of Sciences. METHODS: Under sterile condition, 1 mL rabbit bone marrow was taken and diluted with D-Hanks solution. Following centrifugation and subsequent supernatant removal, bone marrow was re-suspended using dulbecco's modified eagle's medium (DMEM) for single cell suspension. Next, single cell suspension was dropped onto the liquid surface of equal-volume lymphocyte separation medium (density: 1.077). Subsequent to centrifugation, cloudlike mononuclear cell layer was collected and re-suspended with DMEM containing 20% fetal bovine serum. The cells were inoculated at lxl0/cm2 and purified by adherent method. When 70%-80% of flask bottom was covered, cell digestion and passage was performed.MAIN OUTCOME MEASURES: Cell growth was observed with an operating microscope. Surviving cells were counted by Trypan blue viability test. Cell identification was performed by hematoxylin-eosin staining. Through the use of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, cell viability was detected to observe the cellular resuscitation of the cultured cells following cryopreservation. RESULTS: Twenty-four hours after inoculation, cells began to adhere to the wall, exhibiting short shuttle- or triangle-shaped appearance with different sizes of cellular processes. Three days later, adherent cells began to divide, and cell clusters could be found in some areas; One week later, most of cells exhibited scattered fibroblast-like growth; After passage, cells were evenly distributed with long shuttle-shaped appearance and arranged orderly. Following 3 passages, there wound be 5×106-1×107 adherent cells in 1 mL MSC suspension. Approximately 90% of MSCs survived and identified as mononuclear cells. Cells vigorously grew at days 1-6 after inoculation and reached a peak level at day 8, followed by a viability decline. After 56 days of resuscitation, frozen cells displayed a good and rapid growth. CONCLUSION: Highly purified MSCs can be acquired by gradient centrifugation of lymphocyte separation medium. Enough seeded cells can be obtained by in vitro culture and the cellular viability does not change after frozen preservation and resuscitation.

BACKGROUND: SOX-9 plays an important role in occurrence and development of cartilaginous tissues,enhances agglutination of mesenchymal cells,has structural domains of transcriptional activation,and can directly regulate the transcription,OBJECTIVE: To construct pDC316-SOX-9 plasmid for transfection of rabbit bone marrow stromal cells (BMSCs) using SOX-9 gene,and to investigate the effects of SOX-9 gene on growth characteristics of BMSCs and product expression. DESIGN,TIME AND SETTING: The cell gene engineering in vitro experiment was performe,d at the Beijing Institute of Genome, Chinese Academy of Scienees from June 2006 to January 2007.MATERIALS: Eight SPF New Zealand rabbits aged 6 weeks were used for culture of BMSCs.METHODS: pDC316-SOX-9 plasmid was used for transfection of rabbit BMSCs by liposome mediated method,Cell transfection included a SOX-9 plasmid transfection group,a blank plasmid group and a blank control group (only treatment of liposome). MAIN OUTCOME MEASURES: Cell morphology; growth activity; The SOX-9 protein expression in rabbit BMSCs were detected by immunohistochemistry,hematoxylin-eosin staining,reserve transcriptase-polymcrase chain reaction (RT-PCR) and enzyme-labeled immunosorbent assay (ELISA). RESULTS: Some cell colonies were detected at 4 days after pDC316-SOX-9 plasmid transfection.Spindle-shaped cells were collected after clone amplification.Cells in the blank control group gradually died over time.Cell activities in the SOX-9 plasmid transfeetion group and the blank plasmid group significantly prolonged,reached a peak at 2 weeks of transfection,and then gradually decreased.At 6 days,BMSCs were yellow in the SOX-9 plasmid transfection group following immunohistochemistry,expressing SOX-9 protein.Hematoxyliu-eosin staining showed many dikaryocytes,rich cell proliferation,similar to chondroblasts.No SOX-9 protein expression and unproductive cell proliferation in the blank plasmid group.SOX-9 mRNA was detected in the SOX-9 plasmid transfection group,but SOX-9 mRNA was not detected in the blank plasmid group and blank control group.SOX-9 levels were significantly higher in the SOX-9 plasmid transfection group than in the blank plasmid group and blank control group at 24,48 and 72 hours,1,2 weeks (P＜ 0.01).CONCLUSION: Rabbit BMSCs were successfully transfected with pDC316-SOX-9 plasmid to enhance cell growth activity and to persistently stably secrete SOX-9 protein,resulting in the differentiation of BMSCs into cartilages.

BACKGROUND: The bone marrow stromal cells in red bone marrow (RBM) are the non-specific bone growth factors,and the target cells of bone morphogenetic protein (BMP), and they have the activities of osteoinduction and osteogenesis.Injectable calcium alginate gel (CaAG) is a degradable biomaterial with good histocompatibility,it provides scaffold for the proliferation and adhesion of osteoblasts and chondroblasts,and it is good for the proliferation of new capillaries.OBJECTIVE:To prepare CaAG-BMP-RBM gel compound,and observe its osteoinductivity.DESIGN:A randomized controlled animal trial.SETTINGS:Department of Orthopaedics,Shenzhen People's Hospital;Department of Orthopaedics,Union Hospital affiliated to Tongji Medical College,Huazhong University of Science and Technology.MATERIALS:The experiments were carried out in Shenzhen People's Hospital from February 2002 to February 2003.Twenty-seven pure SD rats, either male or female,weighing (200±20) g,were purchased from the Animal Experimental Center of Southern Medical University (No.SCXK2002-99).BMP was bought from Xijing Hospital affiliated to the Fourth Military Medical University of Chinese PLA.METHODS:The rats were randomly divided into three groups:CaAG-BMP-RBM group(n=9),CaAG.BMP group (n=9) and CaAG group (n=9).In the CaAG-BMP-RBM group,the rats were anesthetized,then 0.1 mL RBM collected from trochanteric section of fetour was added into the prepared CaAG-BMP compound (0.4 mL),sufficiently mixed,and then injected into posterior femoral muscle.The rats in the CaAG-BMP group and CaAG group were implanted with CaAG-BMP and CaAG into bilateraI posterior femoral muscles respectively.①The conditions of becoming conscious after anesthesia, incision healing, diet and activities were observed, the size and consistency of the implants and distribution of vessels were also observed.②The indexes were detected at 1,2 and 4 weeks after model establishment respectively,and 3 rats were used for each time point.The sections were observed under light microscope.and the activity of alkaline phosphatase (ALP) was determined.MAIN OUTCOME MEASURES: ①General conditions of animals and gross observation of the implants;②Histopathological observation;③ALP activities.RESULTS: All the 27 SD rats were involved in the analvsis of results.The rats became conscious at 4.6 hours after anesthesia,and they could eat normally;Hematom at incision disappeared.and the rats could move normally at 24 hours;No sign of infection at incision was observed at 72 hours;The incisions healed completely and suture shed after 2 weeks.The incisions were stage Ⅰ healing in all the 27 rats.①Results of gross observation of the implants:At 1 week after implantation.the size of implant did not decrease and vessels in-grew in both the CaAG-BMP-RBM group and CaAG-BMP group;At 2 weeks,lhe quality was hard, and the section appeared as gray and white with deposition of bone-like tissues;At 4 weeks,a great amount of vessels in-grew,and there were many depositions of hard bone-like tissues.②Results of the histopathological observation under light microscope:In the CaAG-BMP.RBM group.many mesenchymal cells aggregated, osteoblasts and chondroblasts proliferated actively al 1 week;The chondrocytes tended to mature with cartilage-like and bone-like matrixes at 2 weeks; Many osteocytes were observed and fibrous bone formed at 4 weeks.which were more then those in the other two groups.③Results of ALP activity:At 1.2 and 4 weeks after implantation.the ALP activities in the CaAG group [(0.179±0.018),(0.058±0.017).(0.027±0.018)IU/g] were lower than those in the CaAG-BMP-RBM group[(0.922±0.226),(1.169±0.249),(0.431±0.081)IU/g,P＜0.01);At 1 and 4 weeks,the ALP activities in the CaAG-BMP group[(0.447±0.015),(0.276±0.081)IU/g]were lower than those in the CaAG-BMP-RBM group (P＜0.05).CONCLUSION:The compound of CaAG-BMP-RBM possesses stable and lasting osleoinductivity.

BACKGROUND: During recent years, mononucleotide polymorphism of some genes is possibly related to affectability of osteoarthritis (OA). However, previous researches mainly compare the gene expression of synoviocytes between OA and rheumatoid arthritis (RhA); therefore, the correlation of gene expression between synovioblast and fibroblast in other tissues should be further studied as compared with OA.OBJ ECTIVE: To observe the differences of gene expression between OA synovioblast and skin fibroblast.DESIGN: Observational contrast analysis.SETTING: People's Hospital of Shenzhen City.PARTICIPANTS: Synovium tissue was derived from OA patients who received replacement of knee joint in the Department of Orthopaedics, People's Hospital of Shenzhen City. All OA patients met the diagnostic criteria of osteoarthritis established by American College of Rheumatology in 1995. Three patients including 1 male and 2 females aged more than 65 years old and they did not have cardiac and pulmonary disease and diabetes mellitus. Three male normal volunteers who aged 25 to 35 years did not have rheumatic disease, osteoarthritis and dermatosis. All subjects provided a confirmed consent. The main reagents were detailed as follows: RPMI1640 culture medium, fetal bovine serum and TRIZOL agent (Invitrogen Life Technologies Company, USA); pGEM-T pUC (Progema Company, USA);Display PROFILE-BASIC and Display PROFILE Probe kits (Qbiogen Company, USA).METHODS: The experiment was carried out in People's Hospital of Shenzhen City from January to June 2005. Synovium of OA patients were treated with primary culture to obtain synovioblast; meanwhile, skin fibroblast treated with primary culture from normal subjects was regarded as the control group. Restricted enzyme section differential display was used to separate the different-expressed genes of synovioblast and skin fibroblast in OA patients. In addition, blast technique was used to compare the resulted ranks with Genbank ranks.MAIN OUTCOME MEASURES: Differences of gene expression between synovioblast and skin fibroblast in OA patients.RESULTS: Gene expressions of superoxide dismutase (SOD), TFPI2, CXCL2, CXCL6 and transforming growth factor (TGF) were high in synovioblast of OA patients as compared with those in skin fibroblast of normal subjects.CONCLUSION: Gene expressions of SOD, TFPI2, CXCL2, CXCL6 and TGF are high in synovioblast of OA patients as compared with those in skin fibroblast of normal subjects. This suggests that gene may play a certain role in onset of OA.

BACKGROUND:There is lack of an objective and standardized animal model for assessing the therapeutic effect of physical and medication treatment on bone defoct, the effectiveness of operation, as well as the role of bone substitute in the repairing of bone defects.DESIGN:Verified study on the experimental model of bone nonunion in rabbitsSETTING: Department of Orthopaedics in Shenzhen people' s Hospital MATERIALS:Twenty common grade pure New Zealand rabbits of either gender were selected with body mass of (2.5±0.5)kg,aged 6 to 8 months.METHODS :This experiment was carried out at the experimental animal center of Shenzhen people's Hospital between May and August 1999. 1.5cm bone segment (including periosteum)was cut off in the middle of forearm radius in 20 common grade pure NewZealand rabbits,the broken ends were covered with bone wax, 10 weeks later, the bone nonunion status was assessed by macropathological observation, pathohistological and X-ray examination.MAIN OUTCOME MEASURES:Observations on rabbit forearm radius defects by macropathological observation, pathohistological and X-ray examination.RESULTS :Twenty rabbits(40 side radius)were enrolled in this study and weeks later, bone defect region was found filled with fibrous cicatricial tissue without osseous connection ,bone wax was not absorbed, capitulum was ossified with medullary cavity blocked,a small amount of callus formed at both broken ends of fractural bone ,length of bone defect ranged from 0.8 to blocked under optical microscope,chondrocyte and osteocyte could be observed arranging disorderly and covered with fibrous membrane,defect reosseous connection could be detected at defect region at week 10,broken end was ossified and medullary cavity was blocked ,there was small amount of callus appeared at both broken ends displaying irregular shape.CONCLUSION:Bone nonunion experimental animal was successfully established on rabbits in this study, with pathological changes meeting the need of bone nonunion and displaying typical properties,which can be used as reliable and feasible experimental animal model.

Objective To discuss the results of treatment of proximal humeral fractures in elderly patients with a locking proximal humerus plate. Methods 35 aged patients with proximal humeral fracture were treated with a locking proximal humerus plate from January 2001 to January 2004. Early rehabilitation after operation was performed. Results 35 cases were followed up for 13.2 months on the average. The mean time for bony union was 8.3 (7 to 12) weeks. 1 patient developed an avascular necrosis of the humeral head. According to the Constant Score, the average for all fractures was 81.4 (39 to 95). The excellent and good rate was 85.7%. Conclusion The locking proximal humeral plate proves to be safe and can be recommended for the treatment of proximal humeral fracture in elderly osteoporotic patients.

Objective To discuss clinical results of treatment of trimalleolar fractures by minimally invasive surgical osteosynthesis. Methods From January 2002 to Doctober 2005, twenty-eight cases (mean age: 38.7 years) of trimalleolar fracture were treated by minimally invasive surgical osteosynthesis. Their lateral and posterior ankle joints were exposed through the Gatellier-Chastang incision. The sequence of reduction and fixation of ankle fracture was firstly the posterior ankle, then the medial and lateral malleolus, and distal tibiofibular syndesmosis lastly. Postoperatively, all the patients were fixed externally from 3 to 4 weeks with plaster splint. Results Follow-ups of 18 months on average revealed that all the cases healed. The healing time ranging from 2.8 to 4.5 months averaged 3.2 months. According to the Baird-Jackson scoring system, the results were rated as excellent in 16 cases, good in eight cases, moderate in three cases, and poor in one case, with the good-excellent rate being 85.7% . Conclusions The anatomical reduction and firm internal fixation are key factors in treatment of trimalleolar fractures. The minimally invasive surgical osteosynthesis is a good method due to the minimal invasion, a high rate of union, and fewer complications it results in.