含锌煅烧牛松质骨复合骨髓基质细胞构建组织工程骨

目的　探讨含锌煅烧牛松质骨和单纯煅烧牛松质骨复合骨髓基质细胞 (MSCs)构建的组织工程骨在体内的成骨能力。方法诱导培养骨髓基质细胞 ,将其分别接种至含锌煅烧牛松质骨及单纯煅烧牛松质骨 ,植入肌肉内 ,分别于术后 4、8周处死实验动物 5只 ,行组织学观察和新生骨面积测定。结果　术后不同时间组织学观察含锌组新生骨明显多于单纯组 ,新生骨面积测定值含锌组分别为 (17619.14 5± 44 5 9.816)、(2 5 2 3 8.5 3 9± 1995 .164 ) μm2 ,单纯组分别为(80 5 6.5 3 7± 5 67.60 3 )、(10 787.95 7± 92 5 .5 0 9) μm2 。含锌组明显大于单纯组 (P <0 .0 5 )。结论　含锌煅烧骨作为骨组织工程的支架材料优于单纯煅烧骨 ,是一种理想的生物活性支架材料。     

Erythropoietin resistance in the treatment of the anemia of chronic renal failure

Resistance to erythropoietin therapy is a common complication of the modern management of anemia in chronic kidney disease. Iron deficiency, deficiency of other nutrients, toxins, infections, and inadequate dialysis account for the vast majority of episodes of such resistance.     

Cranial repair using BMP-2 gene engineered bone marrow stromal cells

BACKGROUND: Bone grafts, allografts, and biocompatible artificial bone substitutes all have their shortcomings when used for the repair of cranial bone defects. Tissue engineered bone shows promise as an alternative for the repair of these defects. MATERIALS AND METHODS: Rabbit bone marrow mesenchymal stromal cells (MSCs) were separated from iliac crest aspirates and expanded in a monolayer culture 1 month before implantation. These MSCs were then infected with replication-defective adenovirus-human BMP-2 genes 1 week before implantation. Bilateral critical-size cranial defects were created in the animal with removal of osteoinductive periosteum and dura. MSCs were mixed with alginate UP (ultrapure) to form MSC/polymer construct. MSCs used for the control site were infected with adenovirus beta-galactosidase (beta-gal). After 1 week, 6 weeks, and 3 months, five rabbits from each experimental group were sacrificed and the cranial defect site was examined by histology study. RESULTS: Near-complete repair of the large size cranial defects using the tissue engineered MSC/alginate construct was observed. The H&E stain and von Kossa's staining should better regenerate bone at the experiment site. A statistically significant difference in bone formation was noted by 3D CT imaging at 3 months post-BMP-2 treatment of the cranial defects (0.79 +/- 0.06 versus 0.47 +/- 0.05 cm(2), P < 0.001) but not at 6 weeks (0.36 +/- 0.04 versus 0.33 +/- 0.03 cm(2), P = 0.347). CONCLUSIONS: Near-complete repair of large cranial defects can be achieved using tissue engineered bone. The use of newly developed polymers as well as the integration of the stem cell concept with gene medicine is necessary to attain this goal.     

Effect of bone morphogenetic protein-7 transfected bone marrow mesenchymal stem cells on renal injury in rats with chronic renal failure

Objective To observe the bone marrow mesenchymal stem cells (BMSCs) modified by bone morphogenetic protein-7 (BMP-7) gene on the expression of renal BMP-7, transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor (VEGF), further to explore its protective mechanism on renal injury in rats with chronic renal failure (CRF). Methods BMSCs with high expression of BMP-7 gene (BMSCs-BMP-7) and empty vector-BMSCs (BMSCs-EV) were obtained by lentiviral-mediated gene transfection. Thirty male Sprague-Dawley (SD) rats were randomly divided into 5 groups, 6 in each group: normal control (CON) group; PBS intervention (CRF with PBS infusion, CRF+PBS) group; BMSCs intervention (CRF with BMSCs infusion, CRF+BMSCs) group; BMSCs-EV intervention (CRF with BMSCs-empty vector infusion, CRF+BMSCs-EV) group and BMSCs-BMP-7 intervention (CRF with BMSCs-BMP-7 infusion, CRF+BMSCs-BMP-7) group. The CRF model was established by 5/6 nephrectomy. The CON group was a sham operation group. The corresponding 12-weeks interventions of each experimental group were performed after 2 weeks of modeling, the rats in the CON group and the CRF+PBS group were injected with 1 ml of PBS through the tail vein, and the other three groups were injected with 1 ml of the corresponding cell suspension once a week. At the time of sacrifice, blood and renal tissue samples were reserved. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by routine biochemical methods, and the expression of BMP-7, VEGF, TGF-β1 in kidney was assayed by Western blotting. Results At the time of sacrifice, the levels of Scr and BUN in the CRF+PBS group were significantly higher than those in the CON group (all P＜0.01); Compared with the CRF+PBS group, the Scr and BUN of the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were decreased to different extents, the differences were statistically significant (all P＜0.01); the Scr and BUN of the CRF+BMSCs-BMP-7 group were significantly lower than CRF+BMSCs group and CRF+BMSCs-EV group (all P＜0.05). The expression of BMP-7 and VEGF were the lowest in the CRF+PBS group. Compared with the CRF+PBS group, the expression of BMP-7 and VEGF in the CRF+BMSCs group, CRF+BMSCs-EV group and CRF+BMSCs-BMP-7 group were significantly increased respectively (all P＜0.05). The expression of the BMP-7 and VEGF in the CRF+BMSCs-BMP-7 group were higher than those in the CRF+BMSCs group and CRF+BMSCs-EV group (P＜0.01). Compared with the CON group, the expression of TGF-β1 in the CRF+PBS group was significantly increased (P＜0.01); compared with the CRF+PBS group, the expression of TGF-β1 in the CRF+BMSCs group, CRF+BMSCs-EV and CRF+BMSCs-BMP-7 group was significantly decreased (all P＜0.01); the expression of TGF-β1 in the CRF+BMSCs-BMP-7 group was lower than the CRF+BMSCs and CRF+BMSCs-EV group (both P＜0.01). Conclusions BMSCs modified by BMP-7 has a protective effect on CRF rats; its protective mechanism may be related to antagonizing TGF-β1 and up-regulation of renal VEGF expression.     

Considerations for the evaluation of renal function in genetically engineered mice

Transgenic and gene-targeting techniques have opened a new era of physiologic investigation: the field of functional genomics. The nearly exclusive use of the mouse in this discipline has necessitated the development and adaptation of sophisticated techniques for evaluating murine physiology at the cellular, tissue, organ and whole animal levels. Although many of the methodologies for exploring cardiorenal function have been successfully adapted from their use in the rat, there are important limitations and considerations that must be recognized when applying them in the mouse. Investigators have been successful in measuring a wide variety of functional variables at the whole kidney and even single nephron levels. Reviewed here are recent advances in the measurement of blood pressure, renal blood flow, whole kidney electrolyte excretion and clearance rates, single-nephron glomerular filtration rate and transport, and tubuloglomerular feedback.     

Cardiovascular hemodynamic effects of correction of anemia of chronic renal failure with recombinant-human erythropoietin

The results of 8 to 12 weeks of treatment of the anemia of uremia with rHuEPO in patients with chronic renal failure and uremia are: a sustained increased hematocrit; increased RBC mass, and subsequent increased MAP; and increased TPRI. The observed trends of decreased LVEF, and echo Doppler evidence of a trend toward LV systolic and diastolic dysfunction, although not individually statistically significant, represent 3 separate evaluation techniques coupled with hypertension and TPRI increase during administration of rHuEPO to increase the hematocrit and packed red blood cell volume in patients with chronic renal failure and anemia. Increased TPRI and hypertension associated with correction of uremic anemia vasodilation and the increased blood viscosity have been noted in earlier investigations with transfusions. The hypertension and elevated TPRI demonstrated during rHuEPO therapy in patients with progressive chronic renal failure associated with increased hematocrit, and the trends toward systolic and diastolic cardiac dysfunction are noted herein. These changes were associated with the combined increase of packed RBC mass and plasma volume in this study. The natural progressive course of worsening of renal function exhibited by these patients could have limited their ability to regulate plasma volume, making them vulnerable to volume-dependent hypertension and a significant preload adding to potential cardiac dysfunction in addition to the increased TPRI.     

Effect of cimetidine on granulocyte-macrophage colony formation by normal and chronic renal failure bone marrow cells

The effect of cimetidine, an antihistaminic blocker of the H2 receptors, on the development of granulocyte-macrophage colonies (GMC) was examined in vitro on bone marrow cells from healthy individuals and chronic renal failure (CRF) patients. The drug caused a dose-dependent decrease in the number of GMC from bone marrow samples in both healthy subjects and patients, being much more expressed in the latter group. The addition of urea to bone marrow cultures of healthy subjects increased the inhibition of colony formation caused by cimetidine alone. CRF or control serum added to normal bone marrow cells enhanced the colony growth which was significantly more expressed when using a CRF serum sample.     

骨髓基质干细胞在体外向软骨细胞分化

目的观察骨髓基质干细胞(B M SC s)在体外能否分化为软骨细胞。方法利用高密度细胞球培养体系在含转化生长因子-β1(TG F-β1)的培养基中培养B M SC s21d,用免疫组化甲苯胺蓝染色方法分析培养的B M SC s球中蛋白多糖(软骨细胞分泌的主要基质成份)的表达、用免疫组化和R T-P C R方法分析Ⅱ型胶原(软骨细胞特异分泌的主要胶原蛋白)的表达来评估B M SC s是否分化为软骨细胞。结果TG F-β1作用的B M SC s表达了Ⅱ型胶原和蛋白多糖。结论B M SC s在体外特定的培养条件下可分化为软骨细胞,从而可能成为临床上治疗创伤或骨关节炎所致的软骨缺损所需的合适的自体来源的种子细胞。     

血管内皮生长因子基因转染骨髓间充质干细胞对慢性肾衰竭大鼠肾脏的修复作用

目的探讨血管内皮生长因子（vascularendothelialgrowthfactor,VEGF）基因转染骨髓间充质干细胞（mesenchymalstemcelis,MSC）对慢性肾衰竭（chronicrenalfailure,CRF）大鼠肾脏的修复作用。方法体外分离、培养大鼠MSC,Ad—VEGF转染MSC。SD大鼠随机分为假手术组（对照组）、慢性肾衰竭模型组（肾衰组）、慢性肾衰竭大鼠MSC移植组（MsC组）、慢性。肾衰竭竭大鼠AdVEGF注射组（VEGF组）和慢性肾衰竭大鼠Ad—VEGF转染的MSC移植组（转染组）。采用分阶段5／6肾切除术制备大鼠CRF动物模型。对照组与肾衰组于切除右肾之前从该侧肾动脉注射不含血清的DMEM培养液,其他3组分别给予MSC、Ad—VEGF和VEGF基因转染的MSC。8周后检测各组大鼠血肌酐（SCr）、血尿素氮（BUN）和24h尿蛋白,观察各组大鼠残肾组织病理形态,并用免疫印迹和RT—PCR方法检测各组大鼠残肾组织VEGFmRNA和蛋白的表达。结果MSC组和转染组SCr、BUN和尿蛋白均较。肾衰组降低（P〈0．05）;与MSC组比较,转染组SCr、BUN和尿蛋白降低更明显（P〈0．05）。肾衰组残肾组织VEGFmRNA和蛋白的表达较对照组显著减少（P〈0．05）,MSC组和转染组残肾组织VEGFmRNA和蛋白的表达明显增加（与肾衰组比较,均P〈0．05）,转染组残肾组织VEGFmRNA和蛋白的表达增加更明显（与M9C组比较,P〈0．05）。结论VEGF基因转染的MSC移植对CRF大鼠的肾脏有修复作用,这可能与VEGF在肾组织中高表达有关。     

Bone formation following transplantation of genetically modified primary bone marrow stromal cells.

Bone marrow stromal cells contain mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues; in the presence of BMP-2, for example, they differentiate into osteoblasts. We constructed replication-deficient adenoviral vectors encoding human BMP-2 (BMP-2/Ad) or BMP-4 (BMP-4/Ad) and used them to transduce primary bone marrow stromal cells from the femurs of four-week-old female C3H mice, which then expressed and processed functional BMP-2 or BMP-4 protein. Enzyme assays and histochemical staining showed both groups of cells to possess alkaline phosphatase activity, a marker of differentiation into osteoblasts, though the activity was higher in cells transduced with BMP-2/Ad. When BMP-2/Ad-transduced cells were injected into the thigh muscles of immunocompetent C3H mice, ossicle development was detected on radiographs within four weeks after injection. Moreover, histological analysis indicated that newly developed ossicles contain mature osseous components, including cortical bone and bone marrow, within eight weeks. Thus, syngeneic transplantation of genetically modified primary bone marrow stromal cells induced bone formation in immunocompetent mice, perhaps indicating its potential for use in the development of therapeutic protocols aimed at enhancing bone formation.     

Repair of nerve defect with acellular nerve graft supplemented by bone marrow stromal cells in mice

The acellular nerve graft that can provide internal structure and extracellular matrix components of the nerve is an alternative for repair of peripheral nerve defects. However, results of the acellular nerve grafting for nerve repair still remain inconsistent. This study aimed to investigate if supplementing bone marrow mesenchymal stromal cells (MSCs) could improve the results of nerve repair with the acellular nerve graft in a 10-mm sciatic nerve defect model in mice. Eighteen mice were divided into three groups (n = 6 for each group) for nerve repairs with the nerve autograft, the acellular nerve graft, and the acellular nerve graft by supplemented with MSCs (5 × 10(5)) fibrin glue around the graft. The mouse static sciatic index was evaluated by walking-track testing every 2 weeks. The weight preservation of the triceps surae muscles and histomorphometric assessment of triceps surae muscles and repaired nerves were examined at week 8. The results showed that the nerve repair by the nerve autografting obtained the best functional recovery of limb. The nerve repair with the acellular nerve graft supplemented with MSCs achieved better functional recovery and higher axon number than that with the acellular nerve graft alone at week 8 postoperatively. The results indicated that supplementing MSCs might help to improve nerve regeneration and functional recovery in repair of the nerve defect with the acellular nerve graft.     

Erythropoietin and cardiocirculatory condition in aged patients with chronic renal failure

BACKGROUND/AIM: The clearest benefit of recombinant human erythropoietin (rHuEPO) in end-stage renal disease is a substantial reduction in transfusion dependency and an improved quality of life. In this report, we describe the efficacy of weekly subcutaneous administration of rHuEPO in 11 elderly patients with anemia secondary to chronic renal failure. METHODS: The role of rHuEPO therapy in increasing the patient's quality of life and in decreasing the hospitalization rates secondary to cardiac morbidity was verified in 11 elderly patients (age range between 66 and 85 years) with anemia due to chronic renal failure. The mean hemoglobin level at the beginning of the study was 8.2 +/- (SD) 0.7 g/dl, and the serum creatinine concentration was 4.8 +/- 1.36 mg/dl. The patients underwent baseline and annual echocardiography, in addition to an electrocardiogram. RESULTS: Most patients experienced a partial regression of left ventricular hypertrophy, and no congestive heart failure was documented. The mean hemoglobin level during rHuEPO therapy increased to 11.3 +/- 1.2 g/dl, while the mean serum creatinine concentration did not change significantly. CONCLUSIONS: Our results confirm that early anemia correction in aged chronic renal failure patients permits improvement of the quality of life, of exercise performance, and of cognitive functions. Reduced transfusion need and regression of left ventricular hypertrophy favor a minor incidence of cardiac morbidity and contribute to reduce health costs.     

自体骨髓基质干细胞在齿槽裂骨缺损修复中的应用

目的探讨人自体骨髓基质干细胞（human bone marrow stromal cells，hBMSCs）在治疗齿槽裂骨缺损中的可行性。方法2002至2005年，选择齿槽裂骨缺损患者7例（单侧6例，双侧1例），以患者自体骨髓基质干细胞为种子细胞，部分脱钙骨（partly demineralized bone matrix，pDBM）为支架材料构建组织工程骨，治疗齿槽裂骨缺损。从患者髂前上棘穿刺取骨髓，密度梯度离心法分离hBMSCs，经体外成骨诱导和扩增至第3代。将诱导的hBMSCs，复合部分脱钙骨体外培养1周后，手术回植骨缺损区。分别于术后1、3、6、12、24、36个月进行临床外形和三维CT检查随访。结果6例患者头部三维CT检查，结果示术后3个月能形成组织工程化骨，并修复骨组织缺损。术后1～3年的随访表明组织工程骨稳定存在，无明显骨吸收现象，临床治疗效果稳定。1例患者（双侧齿槽裂）植入物外露感染。结论以自体hBMSCs为种子细胞，部分脱钙骨为支架材料，利用组织工程技术可在人体内形成稳定的组织工程化骨组织，并临床修复齿槽裂骨缺损。     

Antileukemic effect of interleukin-7-transduced bone marrow stromal cells in mice following allogeneic T-cell-depleted bone marrow transplantation

Impaired immune reconstitution following allogeneic bone marrow transplantation (BMT) remains a major obstacle to its clinical application. In this study, interleukin (IL)-7-transduced bone marrow stromal cells (MSC-IL7, 1 × 10⁶/mouse) were transfused into lethally irradiated C57BL/6 recipient mice. By day 40 after transplantation, the recipient mice were challenged with the lymphoma cell line EL4. MSC-IL7 cotransplantation protected recipient mice from leukemic mortality (MST >120 days after BMT vs mean survival time (MST) 70 days in the PBS group) It enhance the PFC count and DTH responses of recipients after transplantation. In conclusion, MSC mediated IL-7 gene therapy and may be a more feasible strategy to restore immune function following allo-TCD-BMT.     

Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells

Supraphysiological concentrations of exogenous growth factors are typically required to obtain bone regeneration, and it is unclear why lower levels are not effective. We hypothesized that delivery of bone progenitor cells along with appropriate combinations of growth factors and scaffold characteristics would allow physiological doses of proteins to be used for therapeutic bone regeneration. We tested this hypothesis by measuring bone formation by rat bone marrow stromal cells (BMSCs) transplanted ectopically in SCID mice using alginate hydrogels. The alginate was gamma-irradiated to vary the degradation rate and then covalently modified with RGD-containing peptides to control cell behavior. In the same delivery vehicle, we incorporated bone morphogenetic protein-2 (BMP2) and transforming growth factor-beta3 (TGF-beta3), either individually or in combination. Individual delivery of BMP2 or TGF-beta3 resulted in negligible bone tissue formation up to 22 weeks, regardless of the implant degradation rate. In contrast, when growth factors were delivered together from readily degradable hydrogels, there was significant bone formation by the transplanted BMSCs as early as 6 weeks after implantation. Furthermore, bone formation, which appeared to occur by endochondral ossification, was achieved with the dual growth factor condition at protein concentrations that were more than an order of magnitude less than those reported previously to be necessary for bone formation. These data demonstrate that appropriate combinations of soluble and biomaterial-mediated regulatory signals in cell-based tissue engineering systems can result in both more efficient and more effective tissue regeneration.     

骨形态发生蛋白-2基因转染兔骨髓基质干细胞及复合纳米羟基磷灰石体外构建组织工程骨的研究

目的 探讨腺病毒介导的人骨形态发生蛋白-2表达载体(Ad-BMP-2)转染兔骨髓基质干细胞(rBMSCs)后复合到纳米羟基磷灰石(Nano-HA)体外构建仿生人工骨的可行性. 方法 采用GatewayTM技术构建腺病毒载体,溶胶-絮凝法制备Nano-HA,用Ad-BMP-2转染体外培养的rBMSCs,免疫组化、RT-PCR和蛋白印迹方法检测转染后细胞的BMP-2表达.将转染48 h的细胞均匀接种到Nano-HA支架上,第3、5天取细胞载体复合物扫描电镜观察细胞贴附、生长状况,消化收集贴附支架的细胞,蛋白印迹检测贴附支架细胞BMP-2的表达.结果 转染后BMP-2基金在mRNA水平和蛋白水平均有高表达;扫描电镜见转染细胞在支架材料分布均匀,黏附良好,转染后及复合后Western Blot检测BMP-2表达较高.结论 利用转基因技术,用含目的基因的腺病毒载体转染靶细胞复合到Nano-HA,细胞载体复合物稳定长效地分泌生长因子,体外成功地构建了仿生人工骨.