快速成型骨组织工程支架的结构及力学仿生性能特征

目的:利用显微CT的成像技术及材料力学测试,观察多孔双相磷酸钙陶瓷支架的三维结构和力学仿生性能特征。方法:实验于2004-06/2005-12在清华大学新型陶瓷与精细工艺国家重点实验室、香港中文大学威尔士亲王医院骨与关节肌肉研究室和解放军总医院骨科研究所完成。利用三维凝胶叠层成型技术和发泡法复合的方法,制备仿骨多孔双相磷酸钙陶瓷支架,经显微CT扫描得到分辨率为20μm的断层图像,并按骨形态计量方法计算三维计量参数,并与急性脑死亡年轻人股骨头(由解放军总医院骨组织库提供,已签署捐献同意书)标本负重区松质骨样本的三维参数进行统计比较。最后对双相磷酸钙陶瓷支架两个互相垂直方向和松质骨样本的长轴方向进行压缩强度试验,计算压缩强度和弹性模量,并进行统计分析。结果:①双相磷酸钙陶瓷支架的小梁平均宽度和间距低于松质骨小梁(P<0.05);支架的小梁数目和各项异性程度高于松质骨样本(P<0.05);说明支架小梁在排列上呈现更为明显的各向异性特征。②双相磷酸钙陶瓷支架的体积分数、表面积体积比及结构模型指数与松质骨样本相比差异无显著性(P>0.05),两组样本均呈明显的板层结构。③力学测试结果显示双相磷酸钙陶瓷支架材料长轴方向的强度和弹性模量高于垂直方向(P<0.05),说明支架材料具有明显的方向性。长轴方向的强度低于正常松质骨样本,但弹性模量仅比松质骨样本弹性模量高20%(P<0.05)。结论:支架材料在空隙率和表面积方面具有很好的仿生性,利于细胞的黏附和长入。同时具有明显的方向性,提高了其在排列方向上的强度。弹性模量接近股骨头松质骨,具有较好的应力顺应性。     

大鼠骨髓间充质干细胞体外分离、纯化与培养适宜条件的筛选

目的:为获得大量、高纯度的骨髓间充质干细胞,筛选体外分离、纯化和培养的适宜条件。方法:实验于2006-08/2007-07在解放军兰州军区兰州总医院进行。①实验材料:2月龄SD大鼠,雄性,体质量(100±20)g,由解放军兰州军区兰州总医院实验动物科提供。②实验方法:分别采用全骨髓法(贴壁筛选法)和密度梯度离心法分离和纯化大鼠骨髓间充质干细胞。将采用密度梯度离心法获得的单个核细胞按1×107,1×108,1.5×108,2×108,1×109,2×109L-1的接种密度分别分成1～6组,接种在24孔板内,每组接种6孔。比较细胞出现伸展时间、原代培养时间。于细胞接种后第1,2,3,4,5,6天进行首次换液。将换下的部分细胞置于新的培养皿中进行培养,至少培养3d,观察是否还有新的贴壁细胞出现,以确定首次换液时间。观察体积分数为0.10,0.12,0.15,0.18,0.20血清对原代及传代后细胞生长的影响,比较不同浓度血清中细胞数量、集落形成率。集落形成率=集落数目/接种细胞数目×100%。采用流式细胞仪检测骨髓间充质干细胞细胞周期。选取第5代扩增的骨髓间充质干细胞,接近完全融合后进行成骨和成脂肪诱导培养。诱导剂分别为:10nmol/L地塞米松、0.05nmol/L抗坏血酸及10nmol/Lβ-甘油磷酸钠;1μmol/L地塞米松、0.5mmol/LIBMX、0.01mmol/L人胰岛素、0.01mmol/L吲哚美辛。并设未加诱导剂培养液培养的骨髓间充质干细胞对照。③实验评估:采用碱粒酶测定成骨诱导后细胞,采用油红O染色鉴定成脂肪诱导后细胞。结果:①细胞出现伸展时间、原代培养时间:密度梯度离心法培养细胞出现伸展时间与全骨髓法相比,差异无显著性意义。密度梯度离心法原代培养时间较全骨髓法长[(9.41±1.11),(14.73±2.86)d,P<0.05]。②不同接种密度对密度梯度离心法所获细胞生长影响:1×109L-1组细胞出现伸展时间及原代培养细胞融合时间较早。1×107L-1组培养20d内不能传代。③首次换液时间对细胞生长的影响:第5天开始在换下的液体中,很少有再贴壁的细胞出现。因此首次换液时间应为第5天。④不同体积分数血清对细胞生长的影响:在其他条件相同的前提下,骨髓间充质干细胞在含体积分数为0.12胎牛血清中细胞集落形成率最高。⑤成骨及成脂诱导:成骨诱导的骨髓间充质干细胞经碱粒酶测定,结果钙化结节呈蓝色。成脂诱导的骨髓间充质干细胞经油红O染色,苏木精-伊红复染后胞内脂滴呈桔红色,核呈蓝色。结论:采用密度梯度离心法分离纯化的骨髓间充质干细胞以1×109L-1密度接种,在含体积分数为0.12胎牛血清的MEM培养基中培养,生长状况良好,增殖速度快。     

Hypermethylation of the 5' region of the calcitonin gene is a property of human lymphoid and acute myeloid malignancies

An abnormal increase in numbers of CCGG sites methylated in the 5' region of the human calcitonin (CT) gene occurred in tumor cell DNA samples from 90% (17 of 19) of patients with non-Hodgkin's T and B cell lymphoid neoplasms and in 95% (21 of 22) of tumor cell DNA samples from patients with acute nonlymphocytic leukemia (ANLL). The changes were not seen in patients with chronic myelogenous leukemia (0 of 9). The abnormal methylation patterns appear to be a property only of transformed or malignant cells since they were not found in DNA from nonneoplastic adult tissues including sperm, early myeloid progenitor cells, benign lymphoid hyperplasia, peripheral lymphocytes stimulated to divide, or early myeloid progenitor cells (obtained by immunoaffinity using anti-My-10 antibody), but they did appear after Epstein-Barr virus transformation of lymphocytes. Moreover, during the course of therapy in patients with ANLL, the hypermethylation pattern reflects the presence of the leukemic clone even in normal-appearing granulocytes derived from this clone. The increased methylation of the CT gene may then provide an important molecular marker for biologic events in human cell transformation or tumor progression and may prove clinically useful in monitoring patients with lymphoid and acute myelogenous neoplasms.     

Thrombocytopenia associated with pregnancy in a patient with type IIB von Willebrand's disease

Thrombocytopenia may accompany variant (type IIB) von Willebrand's disease (vWD) and is thought to result from binding of the abnormal von Willebrand factor (vWF) to the patient's platelets with subsequent platelet aggregate formation and clearance. We have studied a patient with type IIB vWD who became thrombocytopenic during two pregnancies. During the third trimester of pregnancy, her platelet counts dropped to 20,000 to 30,000/microL, and an increase in the intermediate-sized vWF multimers was seen on agarose gel electrophoresis. During this time her platelet-rich plasma showed spontaneous platelet aggregation, and her plasma caused spontaneous aggregation of normal washed platelets. Antibody to platelet glycoprotein Ib completely blocked the spontaneous platelet aggregation, while antibody to platelet glycoprotein IIb/IIIa did not block the response at the concentrations used. Inhibitors of platelet function that elevate platelet cyclic AMP also blocked the response, but aspirin had no effect on the spontaneous platelet aggregation. The patient illustrates that the platelet counts in one individual can vary greatly in type IIB vWD and that the thrombocytopenia that occurs can appear under physiologic conditions that stimulate the endogenous production of the patient's abnormal vWF. The mechanisms leading to spontaneous platelet aggregation and thrombocytopenia appear to be similar to those described for other patients with type IIB vWD.     

Epileptic spasms without hypsarrhythmia in infancy and childhood: tonic spasms as a seizure type

Epileptic spasms were defined by the International League Against Epilepsy Task Force on Classification and Terminology in 2001 as a specific seizure type. Epileptic spasms without hypsarrhythmia have been described in some series of patients, occurring either in infancy or childhood. More prolonged epileptic spasms without hypsarrhythmia were previously defined as a different seizure type, and referred to as “tonic spasm seizures”. Here, we present a 5‐year‐old boy who started having epileptic spasms without hypsarrhythmia at 8 months of age, effectively treated with oxcarbazepine. With the withdrawal of medication, epileptic spasms returned. Video‐EEG monitoring revealed high‐voltage slow waves superimposed by low‐voltage fast activity, followed by an electrodecremental phase and a burst of asymmetric fast activity, time‐locked to clinical tonic spasm seizures. Brain MRI showed left temporal atrophy with temporal pole grey/white matter junction blurring and ictal PET‐CT showed left basal frontal hypermetabolism. Seizures were refractory to several AEDs and vigabatrin was introduced with seizure cessation. Despite efforts to classify epileptic spasms, these are still considered as part of the group of unknown seizure types. In some cases, a focal origin has been suggested, leading to the term “periodic spasms” and “focal spasms”. In this case, epileptic spasms without hypsarrhythmia, associated with tonic spasms, may be a variant of focal spasms and might be considered as an epileptic syndrome. [Published with video sequence]     

Biologic and prognostic significance of the presence of more than two mu heavy-chain genes in childhood acute lymphoblastic leukemia of B precursor cell origin

We examined the arrangement of the mu heavy-chain immunoglobulin (Ig) genes in the leukemic blast cell DNA of 93 children with acute lymphoblastic leukemia (ALL). All cases met morphologic and cytochemical criteria for ALL, lacked detectable T cell surface antigens, and expressed HLA-DR (Ia) antigens. Eighty-three of the 93 patients (89%) were positive for the common acute lymphoblastic leukemia antigen (CALLA), and 20 of 91 (22%) tested had detectable cytoplasmic immunoglobulin. As expected, the heavy-chain lg gene was rearranged in all cases, and the pattern of rearrangements was variable; 23 had one allele rearranged and one in the germ line configuration; 15 had one rearranged and one deleted; and 37 had two rearranged. Unexpectedly, in 18 patients the presence of more than two mu gene-hybridizing bands was detected. Combinations of enzymes and heavy-chain gene probes were used to confirm that the extra bands were not the result of underdigestion of the DNA or DNA restriction site polymorphism. In eight of the 18 patients, we identified an extra chromosome 14 as a possible cause of the extra bands' hybridizing to the mu heavy-chain constant-region probe. In the remaining ten patients, the presence of three or four bands hybridizing with the mu probe suggests the presence of two populations of leukemic cells that may have arisen either by separate leukemic transformation events or by clonal evolution of one clone into two related lines. Although preliminary (2-year follow-up), our data suggest that childhood ALL of B lineage with more than two mu heavy-chain genes, but without extra copies of chromosome 14, may be more resistant to therapy.     

Erythropoietic stress, macrocytosis, and hemoglobin switching in HbAA sheep

Hemoglobin switching and macrocytosis were studied in homozygous hemoglobinAA sheep. An abrupt initiation of erythropoietic stress, accompanied by a pulsed elevation of circulating erythropoietin levels, was induced by phlebotomy. Sequential blood samples were separated according to density on Stractan gradients to isolate cells newly entering the circulation from the marrow each day. Analysis of hemoglobin phenotype and cell volume distribution in these young reticulocytes revealed a distinct temporal separation in the appearance of hemoglobin C and increased cell volume. The appearance of macrocytes within 24 hr of erythropoietin elevation suggests that macrocytosis could be the result of the action of erythropoietin during the late stages of erythroid maturation. The 72-hr delay in the appearance of hemoglobin C indicates that commitment to a particular hemoglobin phenotype occurs at an early stage of differentiation and involves immature erythroid stem cells. The results of this study show that these consequences of erythropoietic stress are initiated at two different developmental stages, resulting in the production of macrocytosis and hemoglobin switching.