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: Bone morphogenetic protein 2 and transforming growth factor β are important factors in bone regeneration, increasing the expressions of bone morphogenetic protein 2 and transforming growth factor β can promote the osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To construct the lentivirus vector carrying bone morphogenetic protein 2 and transforming growth factor β3, and to observe the expression of lentivirus vector in bone marrow mesenchymal stem cells. METHODS: The recombinant lentiviral vectors carrying transforming growth factor β3, bone morphogenetic protein 2 and green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect the passage 3 rabbit bone marrow mesenchymal stem cells in vitro cultured (transfection group). The bone marrow mesenchymal stem cells transfected with single gene lentivirals (single gene transfection group) carrying transforming growth factor β3 and bone morphogenetic protein 2 or single lentivirals were as control (control group). At 1 week after trasfection, the total RNA and protein were extracted from each group for detection. RESULTS AND CONCLUSION: The green fluorescence bone marrow mesenchymal stem cells transfected with transforming growth factor β3 and bone morphogenetic protein 2 gene for 3 days could be observed under fluorescence microscope, and the transfection efficiency was over 90%. Reverse transcription-PCR and Western blot results showed the mRNA and protein expressions of transforming growth factor β3 and bone morphogenetic protein 2 in the transfection group were higher than those in the single gene transfection group and the control group. The results indicate that lentivirus can successful y transfect transforming growth factor β3 and bone morphogenetic protein 2 into the bone marrow msenchymal stem cells and achieve its high expression, and these two genes have the synergistic effect of promoting expression.

BACKGROUND:Mesenchymal stem cel s are the seed cel s for tendon tissue engineering which can be obtained in large quantities, but how to induce in vitro is a key technology.
OBJECTIVE:To explore the effect of platelet-rich plasma on the proliferation and col agen production of in vitro cultured mesenchymal stem cel s.
METHODS:The rabbit mesenchymal stem cel s were separated ad cultured. The high-dose platelet-rich plasma group, middle-dose platelet-rich plasma group and low-dose platelet-rich plasma group were set to induce the mesenchymal stem cel s, and the blank control group was set as control.
RESULTS AND CONCLUSION:The proliferation of mesenchymal stem cel s in the high-dose platelet-rich plasma group, middle-dose platelet-rich plasma group and low-dose platelet-rich plasma group was high, and in rapid growth with big increase amplitude, and there was no significant difference in the proliferation when compared with the blank control group (P<0.05). The effect was positively correlated with the culture time, and after cultured for a certain time, the effect was in dose-dependent manner, as higher dose platelet-rich plasma had more significant effect on the proliferation of the cel s. The results indicate that platelet-rich plasma can significantly promote the synthesis of col agen type Ⅰ and Ⅲ of mesenchymal stem cel s, the higher the dose, the more significant the effect on the col agen.

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.

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: Due to lack of the distribution of vessels and nerve, self-repairing capability of articular cartilage tissue is poor after inflammatory erosion.OBJECTIVE: To evaluate the effects of melatonin combined with compound betamethasone on the articular cartilage of osteoarthritis (OA) in rats.METHODS: Thirty Sprague-Dawley rats received intra-articular injection of papain solution for establishing knee OA models.Meanwhile, 20 of them underwent constant intensive light condition for establishing pinealectomy models. Ten rats that under pinealectomy were administered melatonin combined with compound betamethasone. Another 10 normal control rats receiving no treatment served as controls. After 4 weeks of treatment, serum melatonin concentrations at 2 a.m. (highest melatonin concentration within circadian rhythms) and 2 p.m. (lowest melatonin concentration within circadian rhythms) were detected by ELISA. At the same time, all rats were sacrificed to collect femoral condyle cartilage for gross observation.After decalcification and toluidine blue staining, articular cartilage lesions were evaluated based on Mankin scores.RESULTS AND CONCLUSION: After OA model was created, cartilage surface was uneven, lost their luster, the chondrocytes were poorly arranged, severe loss of staining was observed, serum level of melatonin was decreased, and circadian change was unobvious. Constant intensive light condition further aggravated cartilage damage. After treatment by melatonin combined with compound betamethasone, softened cartilage disappeared, there were more regular chondrocytes arrangement, and dispersed chondrocytes and loss of staining were gradually decreased. In addition, there was significant difference in Mankin scores of toludine blue staining among groups (P ＜ 0.05). These findings indicate that melatonin combined with compound betamethasone can restrain the progression of cartilage damage.

BACKGROUND: Transforming growth factor beta (TGF-β) has an important role in tendon healing and adhesion formation.Inhibiting TGF-β and its receptor expression may prevent adhesions after tendon open.OBJECTIVE: To study the effects of mannose-6-phosphate, a natural inhibitor of TGF-β, on TGF-β and its receptor production in tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes of rabbit flexor toes.METHODS: Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendon and cultured separately. All these cells were divided into 2 groups at random, experiment group supplemented with mannose-6-phosphate and control group without mannose-6-phosphate. The expression of TGF-β and TGF-β receptor was quantified with enzyme-linked immunosorbent assay. The expression of TGF-β1 was also assessed with in situ hybridization and immunohistochemistry.RESULTS AND CONCLUSION: The expression of TGF-β and TGF-β receptor in experiment group was significantly lower than that in control group (P ＜ 0.05). In experimental group, the positive expression of TGF-β1 mRNA and the expression level of intracellular TGF-β1 mRNA in all tendon cells demonstrated significantly lower than those in the control group (P ＜ 0.05).Immunohistochemical staining showed expression of TGF-β1 were significantly lower in all three types of tendon cell cultured with mannose-6-phosphate.

BACKGROUND: Self-repairing capability of articular cartilage tissue is poor, due to lack of the distribution of vessels and lymph.OBJECTIVE: To concisely describe the research progress of autologous chondrocyte implantation (ACI), including matrix-induced autologous chondrocyte implantation (MACI), in vivo scaffolds, and related tissue engineering technologies, and to prospect the future developments.METHODS: A search across the databases of ISI Web of Knowledge and PubMed (1979 to February 2009) was performed, with key words of "articular cartilage, transplantation, stern cells, tissue engineering". As well, a search in the database of CNKI (1979 to Febraruy 2009) was performed with the key words of "articular cartilage, repair, tissue engineering". Contents referring to ACI,MACI, in vivo scaffolds and related tissue engineering technologies were included, while contents regarding to the clinical imaging of articular cartilage defects, intracellular signaling pathways in chondrocytes, or gene therapy for articular cartilage defects were excluded.RESULTS AND CONCLUSION: 824 articles were obtained from the preliminary search across the databases. Based on the nominated evaluation criterions to the outcome, analysis focusing on ACI, MACI, in vivo scaffolds and related tissue engineering technologies was performed. As the most successful treatment for articular cartilage defects in the past decade, ACI has undergone a significant development. Recent improvements of ACI include MACI, in vivo scaffolds and related tissue engineering technologies, which exhibit relatively more success in engineering and clinical practice. Nonetheless, limitations still exist and therefore, further researches are required. As a promising alternative of ACI, MACI is more and more widely used in clinical practice for treating articular cartilage defects these years. The long-term curative effect of MACI, however, requires further clinical data to confirm. In addition, other improvements of ACI, in terms of material science, cytology and molecular biology, have been also provided by the developments of in vivo scaffolds and related tissue engineering technologies.

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: Studies have showed that transforming growth factor-β1 (TGF-β1) could yield to the collagen synthesis and adhesion formation of tendon cells at the process of healing. OBJECTIVE: To investigate the preventive effect of TGF-β1 neutralizing antibody on the collagen production and adhesion formation of flexor tendon. DESIGN, TIME AND SETTING: Randomized grouping observational experiments were performed in the Experimental Animal Center of Tongji Medical College between September 2005 and June 2006. MATERIALS: New Zealand white rabbits aged 2-5 months, weighing 3.5-4.5 kg. TGF was offered by Santa Cruz Biotechnology, USA. METHODS: Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Cells were divided into two groups at random. In the experiment group, each cell culture was supplemented with 1 μg/L of TGF-β at increasing dose (0.1, 0.5, 1.0 mg/L) of TGF-β1 neutralizing antibody. No reagents were given in the control group. Collagen Ⅰ production was measured by enzyme-linked immunoabsorbent assay. Eighty-four adult New Zealand white rabbit forepaws underwent sharp transection of middle toe flexor digitorum profundus, followed by immediate repair. Thirty-six adult New Zealand white rabbit were divided into three groups randomly (n=12), injecting with the saline, 1.0 mg/L TGF-β1 neutralizing antibody and 2.0 mg/L TGF-β1 neutralizing antibody into tendon sheath respectively. Tendons were harvested at 4 and 8 weeks to conduct adhesion detection, biomechanical testing, histological evaluation and scanning electron microscopy observation. The remaining 48 New Zealand white rabbits were divided into two groups randomly (n=24), undergoing the saline and 1,0 mg/L TGF-β1 neutralizing antibody injection in tendon sheath respectively. Tendons were harvested at an increasing time interval (1, 2, 4, 8 weeks) and analyzed by in situ hybridization to determine the mRNA expression of TGF-β1 and collagen Ⅰ. MAIN OUTCOME MEASURES: Collagen production and adhesion of rabbit tendon cells. RESULTS: ELISA exhibited that TGF-β1 increased collagen Ⅰ production and the addition of neutralizing antibody significantly reduced TGF-β-induced collagen Ⅰ production in all cell cultures. The effect between antibody and collagen Ⅰ was dose dependent. At 4 and 8 weeks after operation, the gliding excursion ratio of the tendon was shortened and the simulated active flexion ratio were less in saline group compared with 1.0 and 2.0 mg/L TGF-β1 groups (P < 0.05). The tendon anastomosis breaking strength was shown no significant differences among 3 groups (P > 0.05). Scanning electron microscopy and histological observation showed that collagen fibers arranged irregularly in saline group, but arranged regularly in 1.0 and 2.0 mg/L TGF-β1 groups at 4 and 8 weeks after operation. The in situ hybridization examination revealed that TGF-β1 and collagen Ⅰ mRNA expression in 1.0 mg/L TGF-β1 group was lower than that in saline group at each time (P < 0.05). CONCLUSION: TGF-β1 neutralizing antibody can inhibit the function of the TGF-β1 effectively following the flexor tendon injury and repair, and can prevent adhesion formation.