BACKGROUND: Under special conditions, bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteoblasts and chondroblasts. However, MSCs are few in bone marrow. How to harvest, purity and rapidly proliferate in vitro is a foundation of application in tissue engineering technique. OBJECTIVE: To optimize, collect, purity, assess rabbit BMSCs and to observe the biological character of BMSCs. DESIGN, TIME AND SETTING: The observational study was performed at the Animal Experimental Center of Tongji Medical College from September 2005 to July 2006. MATERIALS: One female New Zealand rabbits aged 2 months were used for MSC collection and primary culture. METHODS: Bone marrow solution was purified by density gradient centrifugation and adherence screening method. Culture solution was obtained. BMSCs were incubated in phosphate buffered solution (PBS), supplemented with 2.5 g/L trypsin (3.0 mL), and placed in an incubator at 37 ℃ for two or three minutes. Cell morphology was observed using an inverted microscope. The digestion was stopped when cytoplasm recovery, long and thin cells with large intercellular space, and few round cells appeared. Subsequently, BMSCs were incubated in serum-free L-DMEM, and placed in a plastic culture flask at 1.0×108/L. MAIN OUTCOME MEASURES: MSC morphology, ultrastructura and surface marker; Proliferation of the first, third, fifth, eighth and tenth passages of BMSCs; Cell growth curve was drawn. RESULTS: BMSCs was pure following density gradient centrifugation and adherence screening method. The third and fifth passage of cells had typical whirlpool-shape. Transmission electron microscope demonstrated that round or oval MSCs possessed large nuclei, big nucleus proportion, a few cellular organ. These were low-differentiated cells. Growth curve of cultured MSCs was "S" shape. The first, third and fifth passage cells had strong reproductive capability. The eighth and tenth passage of cells had significantly reduced proliferation. Cells isolated were positive for CD44 and CD90, but negative for CD34. These were low-differentiated cells under the electron microscope. CONCLUSION: Isolated cells are MSCs, with the property of stem cells. The third and fifth passage cells are pure, with strong reproductive capability.

BACKGROUND:The immunological rejection between host and graft is the leading cause of organ
transplantation failure. The traditional immunosuppressive agents have been unable to meet the needs of clinical treatment. Antibody-drug conjugate, as a type of new drugs, may be hope for the treatment of immune rejection. OBJECTIVE:To comprehensively analyze the composition of antibody-drug conjugates, mechanism of action, clinical research progress as wel as the development trend.
METHODS:A computer-based online retrieval was performed to search papers in CNKI and PubMed database using the key words of ADCs, immunosuppressive agents, immunotoxins, organ transplantation, graft rejection in Chinese and English. Recently published or published in the prestigious journals were selected in the same field. After excluding objective-independent papers and repeated studies, 42 papers were included for further analysis. RESULTS AND CONCLUSION:Antibody-drug conjugates, as highly effective and lowly toxic immunosuppressant, have achieved a breakthrough in treatment of targeting tumor, while the role of it in anti-immune rejection is stil at the exploratory stage. For islet transplantation, novel antibody-drug conjugates are required to block CD8+T effector by CD103/E-Cadherin pathway, and wil probably serve as a potential drug intervention for al ograft rejection.

[Objective]To compare the early clinical outcome of ACL reconstruction using the ligament advanced reinforcement system and the ?-ray irradiation allograft.[Method]From January 2006 to January 2008,55 cases of ACL reconstruction were studied.According to the indication of LARS ligament and allograft,suggestion from surgeon and patient's aspiration,one of them were chosen as grafts for ACL reconstruction,including 25 LARS ligaments with titanium screw fixation and 19 allografts with absorptive screw fixation.After reconstruction,both groups of patients carried on functional exercise.Each patient was evaluated with Lysholm-Tegner score and KT-2000 measurements preoperatively and postoperatively at 3 months,6 months,9 months,12 months,15 months and 18 months.[Result]Fifty-two cases were followed up,3 cases were dismissed.The Lysholm -Tegner score in cases using the LARS ligament were higher than that in cases using the allograft 3 months post-operation,6 months post-operation,9 months post-operation.Contemporary comparison of the score gap between the LARS ligament group and the allograft group showed a trend of decrease.During the follow up,the allograft group displayed significantly more laxity in KT-1000 measurements at all time points than the LARS ligament group.And the measurements gap between the two groups showed a trend of increase.[Conclusion]The knee anterior-posterior stability of the patients using the LARS ligament were better than that of patients using the allograft.The knee function of the patients using the LARS ligament recovered earlier than that of patients using allograft at nonage,but long-term effect is almost the same.

BACKGROUND: Bone defect is commonly treated in clinic with auto graft, allograft and artificial bone graft, but with the disadvantages to various extents. Therefore, how to better repair bone tissue in bone defect becomes the hot spot in surgical field.OBJECTIVE: The complex of marrow stromal stem cell (MSC) and calcium-phosphates artificial bone (CPAB) was prepared and the repair of bone defect was observed in animal experiment.DESIGN: Randomized controlled experiment was designed.SETTING: Department of Traumatic Surgery, Affiliated Hospital of Qingdao University Medical College.MATERIALS: The experiment was performed in Animal Experimental Center of Affiliated Hospital of Qingdao University Medical College from May 2002 to September 2003, in which, 24 New Zealand health normal big white adult rabbits were employed, aged varied from 6 to 14 months, mass weighted from 9 to 3.5 kg, of either sex.METHODS: (① Rabbit MSCs were differentiated and proliferated and CPAB was taken as carrier to prepare the complex of CPAB and MSCs. ②24 rabbits (48 laterals) were prepared as model of bone defect, divided into 3 groups, named complex group (24 laterals), in which, unilateral bone defect of 24 rabbits were implanted with cell + carrier complex; The rtificial bone group (12 laterals), in which, CPAB was implanted contralaterally;and blank control (12 laterals), in which, no any management was done contralaterally in bone defect of 12 rabbits. In 8, 16 and 24 weeks after surgery, 4 animals were collected at each time spot in each group for radionuclide monitoring; and the animals were sacrificed under anesthesia for X-ray picturing and histological staining analysis.MAIN OUTCOME MEASURES: ① Gross observation of bone defect in each group. ② Results of X-ray examination. ③ Results of histomorphologic observation. ④ Results of radionuclide monitoring.RESULTS: 24 rabbits all entered result analysis. ① Gross observation of bone defect in each group and results of X-ray examination: In complex group of 24 hours, the fractutred ends were connected partially, the bounder was not clear between the regenerated bone and material and reopened medullary cavity was not visible. In simple artificial bone group, the implanted material was integrated with the cross end and material volume did not change obviously compared with the initial implanting stage. In bland control, ununion fracture presented and medullary cavity was closed. ②Results of histomorphological observation: In 24 hours, the bone defect was smaller in size and the union happened on the defect area in complex group. In artificial bone group, the regenerated bone was integrated tightly with the material and grew inside partially. In blank control group,medullary cavity was closed and bones were not connected. ③ Results of radionuclide monitoring: It was remarkably distinguished with naked eye that radioactive concentration was in tendency of obvious increasing in both complex group and artificial bone group in 8-24 weeks.CONCLUSION: The complex of CPAB and MSCs regenerates bone tissue and repair radial defect in whole. In addition, the repair ability is superior remarkably to simple CPAB.

BACKGROUND: Hormone use has become the primary cause of steroid-induced avascular necrosis of femoral head (SANFH). OBJECTIVE: This study used a combination of injection of horse serum and a large dose of corticosteroid to develop a hormone-induced rabbit model of early avascular necrosis of femoral head (ANFH), and preliminary discussed the pathogenesis of ANFH. METHODS: New Zealand rabbits were randomly divided into three groups. Methylprednisolone with horse serum group: horse serum (10 mL/kg) was injected. Three weeks later, 6 mL/kg of horse serum was injected. Two weeks later, 45 mg/kg of methylprednisolone was daily injected for 5 consecutive days. Methylprednisolone group: 45 mg/kg of methylprednisolone was daily injected for 5 consecutive days. Control group: no treatment was given. Serum cholesterol and triacylglycerol levels were detected at 1, 3, 7 and 14 days before and after hormone injection. MRI and histopathological detection was done in femoral head at 2, 4 and 8 weeks after hormone injection. RESULTS AND CONCLUSION: The serum triglyceride and total cholesterol in methylprednisolone with horse serum group and methylprednisolone group were higher than control group at 1 and 3 days after hormone injection (P < 0.01). MRI results displayed abnormal signal in femoral head at 4 weeks in methylprednisolone with horse serum group, but in the methylprednisolone group at 8 weeks. Histological detection results exhibited that at 4 weeks, some trabeculae were broken into fragments, and the empty bone lacunae increased. At 8 weeks, the trabeculae showed thinning and broken. There were large amount of empty bone lacunae with bone cell atrophy and larger fat cells which were fused into bubbles. In methylprednisolone group, the level of necrosis was lighter than methylprednisolone with horse serum group during each period. Results suggest that hormone combined with horse serum can successfully prepare early-stage hormone-induced ANFH.

BACKGROUND:Artificial knee joint replacement in older patients often combines with basic diseases, such as hypertension and diabetes. Perioperative blood loss is an important factor affecting the safety of replacement. OBJECTIVE: To explore the effect of the early closure of drainage tube on blood loss after primary total knee arthroplasty. METHODS: We randomly selected 90 patients with osteoarthritis of the knee who underwent primary total knee arthroplasty in the Affiliated Hospital of Qingdao University from January 2014 to July 2015. The patients were randomly divided into three groups (n=30). In the 4-hour occlusion group, the drainage tube was closed for 4 hours in early stage of replacement. In the 2-hour occlusion group, the drainage tube was closed for 2 hours in early stage of replacement. In the control group, the drainage tube was not closed. Because of the use of tourniquet during surgery, the amount of intraoperative blood loss was considered as 0 mL. Drainage blood loss after surgery was recorded. Total blood loss was calculated according to Gross formula through patient height, weight and preoperative and postoperative hematocrit. Hidden blood loss was gotten by subtracting the visible blood loss from total loss. Under the observation of postoperative joint sweling and subcutaneous ecchymosis, knee Hospital for Special Surgery score was recorded at 6 weeks after replacement, and compared among groups. RESULTS AND CONCLUSION:Statistical analysis indicated that significant differences in total blood loss and dominant blood loss were detected among the three groups (P < 0.05), indicating that both occlusion for 2 hours and 4 hours could reduce total blood loss and dominant blood loss, but the range of reduction was greater in occlusion for 4 hours. At 6 months after replacement, no significant difference in knee Hospital for Special Surgery score and hidden blood loss was detectable among three groups (P > 0.05). The incidence of joint sweling and subcutaneous ecchymosis was increased in the 4-hour occlusion group (P < 0.05). Above results confirmed that drainage tube occlusion can decrease total blood loss and dominant blood loss after total knee arthroplasty, but cannot reduce hidden blood loss. 2-hour occlusion after total knee arthroplasty is an ideal choice, but the amount of hidden blood loss should be carefuly considered.

BACKGROUND: Mesenchymal stem cells (MSCs) exist in umbilical cord blood (UCB), currently, there is not a method to in vitro separate, culture and amplificate human UCB-MSCs effectively. OBJECTIVE: To explore factors that influence yields of UCB-MSCs. METHODS: The relationship between the success rate of yielding UCB-MSCs and several factors, such as gestational ages (≥40 weeks, 37 weeks and ≤32 weeks), the number of mononuclear cells (MNCs) in UCB (≥2.5×109/L, <2.5×109/L), the inoculum density of MNCs (1×107, 1×109, 1×1011/L), the concentration of fetal bovine serum (FBS, 5%, 10%, 15%, 20%) in culture medium, and whether the culture flask being coated with FBS or not beforehand, as well as relationships among these factors were investigated. RESULTS AND CONCLUSION: The success rate of yielding UCB-MSCs was up to 58.3%. The success rate decreased as the gestational ages increasing (P < 0.01). The success rate could be enhanced to 76.9% when the MNCs count was more than 2.5×109/L, and there was significant difference when comparing to that of the group (36.4%) with MNCs count less than 2.5×109/L (x2=8.07, P=0.005). There was a negative correlation between the MNCs count and the gestational ages in the specimens with the same volume of UCB (r=-0.95, P < 0.01). In the group with the cell inoculum density of 1×1011/L, the growth and proliferation of primary and subculturing MSCs were better than that of the groups with the cell inoculum density lower than 1×1011/L. The adherence of MSCs in the group with the culture medium containing 5% FBS happened much later than other 3 groups, while the purity of MSCs in this group was much higher. When comparing the passage rate, there was no significant difference among the 4 groups with different concentration of FBS. In the group of culture flask being coated with FBS beforehand, the purity and proliferation ability of MSCs was higher than that in the groups with culture flask not being coated. It is suggested that culture of UCB-MSCs was influenced by several factors. The success rate could be increased by choosing the fetus with relative lower gestational ages, collecting enough volume of UCB, inoculating cells with a higher density, choosing the medium with lower concentration of FBS, and coating the culture flask with FBS beforehand.

BACKGROUND: Up to now, there are few reports addressing the biological properties and differentiation potential of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs). OBJECTIVE: To observe the biological characteristics, osteogenic and adipogenic differentiation potential of UCB-MSCs. METHODS: MSCs were harvested and cultured from UCB at various gestational ages (GA). Harvested UCB-MSCs were cultured primarily and subcultured, and then induced to differentiate into osteoblasts and adipocytes. RESULTS AND CONCLUSION: Under the inverted phase contrast microscope, UCB-MSCs adhered to the wall, showing fibroblast like morphology and whirlpool like growth alignment. Observation of the ultramicrostructure under transmission electron microscope showed that UCB-MSCs had a big cellnucleus, fewer cellular organelles and big karyoplasmic ratio.Allofthe growth curves of primary and passaged UCB-MSCs presented S-shaped. The 3rd and 5th passages of MSCs showed the strongest proliferation activity. The count of colony forming unit fibroblasts varied with GA, significant difference was found among the three GA groups (P < 0.05), and the lower GA group had a higher count of colony forming unit fibroblasts than that in the older GA group. Flow cytometry showed that these cells expressed CD29, CD44 and CD90 positively, but they failed to express hematopoiesis related molecules such as CD34 and CD45. When the MSCs were induced to osteogenic and adipogenic differentiation for 3 weeks, strong expression of alkaline phosphatase was found and the formation of a mineral extracellular matrix was detected by alizarin red staining were detected; and neutral lipid vacuoles were detected by oil red O staining. UCB-MSCs have similarmorphologicaland biological characteristics and cell surface molecule markers with MSCs derived from bone marrow, both of which have great capability of proliferation and regeneration. UCB-MSCs can be induced to osteoblasts and adipocytes in a suitable condition in vitro.

Objective To compare the clinical outcomes of irradiated versus non-irradiated deepfrozen bone-patellar tendon-bone (B-PF-B) ullograft in anterior cruciate ligament (ACL) reconstruction. Methods A total of 66 patients undergoing arthroecopic ACL reconstruction were prospectively random-ized consecutively into two groups, ie, Group A ( irradiated deep-frozen allograft, n = 34) and Group B ( non-irradiated deep-frozen allograft, n = 32). All ACL reconstructions were done by the same senior surgeon with the same arthroscopic technique. Before and after surgery, the clinical results were compared in aspects of general conditions, range of motion ( ROM), Pivot shift test, Lachman and Anterior Drawer Test (ADT), Daniel's one-leg hop and Harner's vertical jump tests, overall international knee docu-mentation committee (IKDC) rating and KT-2000 arthrometer testing. Results Of all, 63 patients in-eluding 32 patients in Group A and 31 in Group B were available for a follow-up of average 38 months ( mean 38.3 months in Group A and mean 37.7 months in Group B) and three lost follow-up. There was one patient with late septic infection. While there was no statistical difference in aspects of general condi-tions including hospital stay, duration of fever and complications ( P > 0. 05 ), but there was a trend that the patients in Group A had a longer postoperative duration of fever ( mean 8.9 days ) than Group B (mean 7.8 days). Physical examinations showed no statistical difference upon ROM in both groups ( P > 0. 05), while there was statistical difference between Lachman test and ADT ( P < 0.05 ). The positive Pivot shift test was found in 12 patients in Group A and 3 in Group B, with statistical difference ( P < 0. 05 ). KT-2000 testing showed a side-to-side difference of less than 3 mm in 26 patients in Group B and only 8 in Group A, and a side-to-side difference of more than 5 mm in two patients in Group B and 12 in Group A, with statistical difference (P < 0.05 ). The anterior and rotational stability was decreased sig-nificantly in Group A. No statistical difference was found between two groups in overall IKDC rating, Daniel' s one-leg hop and Harner' s vertical jump tests ( P > 0.05 ). However, the function and IKDC score tended to decrease in Group A. Conclusion The short term clinical outcomes of the ACL reconstruction with irradiated B-PT-B allograft are adversely affected and unsatisfactory, indicating a cautious use.

Objective To evaluate the efficacy of sequential psychotherapy combined with selective serotonin reuptake inhibitors(SSRI) s on refractory obsessive-compulsive disorder(OCD). Methods 50 patients with refractory OCD were given to sequential psycho therapy in the original basis of the SSRIs drugs treatment. The patients were given psychoanalysis,cognitive behavioral therapy and reality therapy for 8 weeks at the end of in the first stage, second stage and the third stage. The efficacy was assessed by the Yale-Brown Obsessive Compulsive Scale(Y-BOCS) before treatment,8 weeks, 16 weeks, and 24 weeks after treatment, respectively. Results The total score of the YBOCS before the treatment and at the 24th week after treatment was (21.10 ±4.69) and ( 10.18±6. 14). According to the score-reducing rate of the YBOCS , recovery rate was 34.0%, effective rate was 42.0%. Conclusion Sequential psychotherapy combined with SSRIs is an effective treatment for refractory OCD.