OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.
Objective To study the effect of autogenous bone marrow on guided bone regeneration (GBR),and evaluate the repairing ability of GBR in bone defect with autogenous bone marrow. Methods Ten mm segmental defects were produced in both radii of 18 rabbits. The defect was bridged with a silicon tube. Autogenous bone marrow was injected into the tube on the experimental group at 0, 2,4 weeks after operation, and peripheralblood into the control group at thesame time. The X-ray, gross, histological and biochemical examinations were observed invarious times. Results The new bone formation of experimental group was prior to that of control group; calcium and alkaline phosphatase of experimental groupwere higher than those of control group. The experimental group had all been healed at the tenth week, but no one healed in control group. Conclusion It can be conclude that autogenous bone marrow can stimulate bone formation and facilitate GBR in bone defect.
【摘要】 目的 探討LIM礦化蛋白（LIM mineralization protein，LMP）-1和LMP-3雙基因共轉染骨髓間充質干細胞（bone mesenchymal stem cells，BMSC）的表達情況。 方法 采用人工設計合成人LMP-1和LMP-3基因片段，分別與質粒pEGFP-N2連接，經酶切、測序鑒定后。分離培養新西蘭兔BMSC，用脂質體包裹轉染BMSC，按轉染情況分為5組:未轉染組（A組）、轉染空載體組（B組）、轉染LMP-1基因組（C組）、轉染LMP-3基因組（D組）、LMP-1與LMP-3雙基因共轉染組（E組）。采用實時聚合酶鏈反應（real-time polymerase chain reaction，RT-PCR）和蛋白質印跡法檢測LMP-1和LMP-3的表達。 結果 酶切及測序表明真核表達質粒pEGFP-N2-LMP-1和pEGFP-N2-LMP-3構建成功。E組可同時較高水平表達LMP-1和LMP-3分子。對RT-PCR及蛋白質印跡法檢測結果行灰度值測量并行統計學分析顯示:LMP-1 mRNA及蛋白水平的表達，5組間差異有統計學意義（Plt;0.05），但E組與C組的差異無統計學意義（Pgt;0.05）；LMP-3 mRNA及蛋白水平的表達，5組間差異有統計學意義（Plt;0.05），且E組與D組差異也有統計學意義（Plt;0.05）。 結論 雙基因共轉染的BMSC能在體外同時表達LMP-1與LMP-3，為基因修復骨缺損帶來新思路。【Abstract】 Objective To study the expression of LIM mineralization protein (LMP)-1 and LMP-3 genes after cotransfecting them into bone mesenchymal stem cells (BMSC) of rabbit in vitro. Methods Fragments of LMP-1 gene and LMP-3 gene were gained through artificial synthesis, and were constructed respectively into the plasmid vector pEGFP-N2. The inserted target genes in plasmid were verified by nucleotide sequencing and enzymes. The plasmids carrying LMP-1 and LMP-3 genes were cotransfected into chondrocytes by liposome method. According to the transfected situation, the BMSC were divided into 5 groups: the non-transfected group (Group A), the group transfected by empty vector (Group B), the group transfected by LMP-1 (Group C), the group transfected by LMP-3 (Group D) and the group transfected by both LMP-1 and LMP-3 (Group E). The expressions of LMP-1 and LMP-3 were detected by RT-PCR and western bloting technique. Results The plasmid pEGFP-N2-LMP-1 and pEGFP-N2-LMP-1 were obtained successfully by cloning technique and verified by nucleotide sequencing and enzymes. The LMP-1 and LMP-3 molecules were both expressed at a high level in Group E. The results of RT-PCR and western bloting were measured with the grey value. For the expression of LMP-1 mRNA and protein of LMP-1, the differences between groups A, B and groups C, D, E were significant (P＜0.05), while the difference between groups C and E was not significant (P＞0.05); For the expression of LMP-3 mRNA and protein of LMP-3, the differences between groups A, B and groups C, D, E were significant (P＜0.05), and the difference between groups D and E was also significant（P＜0.05). Conclusion LMP-1 and LMP-3 genes can be expressed effectively after being cotransfected into BMSC, which provides a basis for gene therapy for treating bone defects.
Objective To monitor the stem cell migration into the bone defect following an injection of the labeled mesenchymal stem cells (MSCs) by the enha nced green fluorescent protein (EGFP)technology and to provide insights into an application of MSCs for the fracture healing. Methods Isolated MSCs from the rabbit femur marrow were culture-expanded and were labeled by the transfection with the recombinant retrovirus containing the EGFP gene. Then, some labeled MSCs were cultured under the osteogenic differentiation condition and the phenotype was examined. After the fracture of their bilateral ulna, 18 rabbits were divide d into two groups. The labeled MSCs were injected into the aural vein at 1×107 cells／kg in the experimental group and the unmarked MSCs were injected in the control group 24 hours before surgery, and 1 and 24 hours after surgery, res pectively. Necropsies were performed 2 days after surgery in the two groups. The sections from the left defects were observed under the fluorescence microscope and the others were analyzed by the bright-field microscopy after the HE staining. Results The EGFP did not affect the MSCs viability. After the labeled cells were incubated in the osteogenic medium alkaline phosphatase, the calcium nodule s were observed. All the rabbits survived. The tissue of haematoma was observed in the bone defects and the fluorescent cells were found in the experimental gr oup, but no fluorescent cells existed in the control group. Conclusion The EG FP labeled MSCs can undergo osteogenic differentiation in vitro and can mig rate into bone defects after their being injected into the peripheral vein.
Objective To investigate the effect of the synthetic bone morphogenetic protein 2 (BMP-2)derived peptide on the osteogenic induction in the marrow mesenchymal stem cells (MSCs）and to evaluate the osteoinductivity and dosedependence of the BMP-2 derived peptide in vitro. Methods MSCs of 4-week old Wistar rats were separated and cultured. In the 3rd passage, the conditional culture medium was changed, in which the BMP-2-derived peptide in the following doses was added: 300,200, 100, 50, and 0 μg/ml, respectively (Groups A-E). The activity of alkaline phosphatase (ALP）and the amount of calciumdeposition were meassured at 5，10，15 and 20 days during the culture with the conditional culture medium. The real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) was performed to measure the mRNA expressions of collagen type Ⅰ, osteopontin (OPN), and osteocalcin（OCN）and to measure the osteoinductivity of the BMP-2-derived peptide in the different concentrations.Results Under the inverted phase contrast microscope, MSCs cultured in the conditional culture medium for 3-4 days were changed in shape, from long fusiform to short fusiform or polygon. As the concentration of the BMP-2-derived peptide increased, the time for MSCs to change into the osteoblasts decreased. There was a significantly greater level of the ALP activity and amount of the calcium deposition in Groups A and B than in the other groups（Plt;0.05）. However,there was no significant difference between Group A and Group B （Pgt;0.05）. Theresult of FQPCR showed that after MSCs were cultured in the different doses of theconditional culture medium for 14 days, the mRNA expressions of collagen type Ⅰ, OPN andOCN were at higher levels. An increasing order in the level of the cycle threshold (Ct) was found in the following groups: Agt;Bgt;Cgt;D. Almost no expression was found in Group E. The Ct levels were significantly greater in Groups A and B thanin Groups C and D（Plt;0.05）. However, there was no significant difference between Group A and Group B （Pgt;0.05）.ConclusionThe BMP-2-derived peptide can greatly promote differentiation of MSCs into the osteoblasts, the promotion of osteogenesis has a dosedependent pattern, and the best inducing dosage is 200 μg/ml.
Objective To investigate the method and clinical effect of free iliac flap grafting in repairing the tibia traumatic osteomyelitis complicated withboneskin defect. Methods From June 2001 to February 2006，28 patients with tibia traumatic osteomyelitis complicated with boneskin defect were treated with free iliac flap grafting at stageⅠ. There were 18 males and 10 females, with an average of 32.5 years(1868 years). There were traffic injury in 11 cases, bruise in 6 cases, explosive injury in 5 cases, machinery injury in 4 cases, and falling injury in 2 cases. The disease courses of patients were 1-6 months. All patients had been treated by 26 operations. The wounds located at the mid and upper tibia in 13 cases, and the inferior tibia in 15 cases. The length of free iliac was0.5-6.0 cm and the size of the flap ranged from 4.5 cm×3.5 cm to 28.0 cm×16.0 cm.The external fixation were applied in 18 cases, and steel plate were applied in 10 cases. The donor sites were sutured directly. Results All of the flaps survived completely. The wounds healed by first intention in 26 cases and by second intention in 2 cases. The donorsites healed by first intention. Twentyeight patients were followed up for 6 to 56 months(mean, 30 months).The appearances of the flaps were satisfactory and the colour was similar to recipient site. All grafted bone united 2-14 months (mean，4.6 months) after operation according to X-ray examination. In 20 patients who did not achieved union before operation, fracture healed 2 to 6 months after operation(mean, 3.2 months). Osteomyelitis recurred 12 months after operation in 2 cases and healed by nidus clearing. Conclusion Free iliac flap which used to repair tibia traumatic osteomyelitis complicated with boneskin defect, can repair the defect at stageⅠand enhance the antiinfectious ability. It isone of appropriate and effective clinical methods.
目的 觀察藏醫滋補方“巴桑母酥油丸”對放射線-化學復合損傷小鼠骨髓不同細胞群增殖能力的影響，探討其促進放射線-化學復合損傷機體外周血象恢復的機制。 方法 采用造血祖細胞集落分析方法、流式細胞術，檢測灌胃巴桑母酥油丸后放射線-化學復合損傷小鼠骨髓細胞中早期紅系祖細胞（CFU-E）、晚期造血祖細胞（BFU-E）、粒-巨噬系祖細胞（CFU-GM）、巨核系祖細胞（CFU-Meg）集落產率、骨髓細胞增殖周期各時相細胞比例、造血干細胞抗原-1（Sca-1）免疫表型陽性細胞數變化情況。 結果 巴桑母酥油丸組骨髓細胞S+G2/M期細胞比例高于生理鹽水組（P＜0.05）和自然恢復的空白組（P＜0.01）；CFU-E、BFU-E、CFU-GM集落產率高于生理鹽水組和空白組（P＜0.05）；CFU-Meg集落產率、Sca-1+細胞數在各組間差異無統計學意義（P＞0.05）。 結論 巴桑母酥油丸對放射線-化學復合損傷小鼠骨髓細胞具有促進增殖的作用，這可能是其促進放射線-化學復合損傷機體外周血象恢復的途徑，但對不同階段、不同系別的造血細胞其促進作用不同。
Objective To observe the effect of cationic liposomal ceftazidime (CLC) combined with nano-hydroxyapatite/β-tricalcium phosphate (n-HA/β-TCP) in the treatment of chronic osteomyelitis of rabbits. Methods Thirty healthy New Zealand white rabbits (4-6 months old; weighing, 2-3 kg) were selected to prepare the chronic osteomyelitis models. After 4 weeks, the gross observation, X-ray examination, and bacteriological and histopathological examinations were done; the models were made successfully in 27 rabbits. Of 27 rabbits, 24 were randomly divided into 4 groups (n=6): only debridement was performed in group A; ceftazidime was given (90 mg/kg), twice a day for 8 weeks after debridement in group B; ceftazidime and n-HA/β-TC were implanted after debridement in group C; and CLC and n-HA/β-TCP were implanted after debridement in group D. Before and after treatments, X-ray examination was done, and Norden score was recorded. At 8 weeks after treatment, the specimens were harvested for gross observation and for gross bone pathological score (GBPS) using Rissing standard; half of the specimens was used for histological observation and Smeltzer scoring, the other half for bacteriological examination and calculation of the positive rate of bacteria culture. Results At 8 weeks after treatment, Norden score of group D was significantly lower than that of groups A, B, and C (P lt; 0.05), but no significant difference was found among groups A, B, and C (P gt; 0.05). At 8 weeks after treatment, sinus healed in groups C and D, but sinus was observed in groups A and B; the GBPS scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The Smeltzer scores of groups C and D were significantly lower than those of groups A and B (P lt; 0.05). The positive rates of bacteria culture of groups C (0) and D (0) were significantly lower than those of group A (25.0%) and group B (16.7%) (P lt; 0.05). Conclusion CLC combined with n-HA/β-TCP has good effect in treating chronic osteomyelitis of rabbits, and it has better effect in treating chronic osteomyelitis of rabbits than ceftazidime with n-HA/β-TCP.
Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.