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。     

Loading-induced changes in synovial fluid affect cartilage metabolism

The purpose of this study was to determine whether changes in the synovial fluid (SF) induced by in vivo loading can induce an alteration in the metabolic activity of chondrocytes in vitro. Therefore, SF was collected from ponies after a period of box rest and after they had exercise for a week. Normal, unloaded articular cartilage explants were cultured in 20% solutions of these SFs for 4 days and chondrocyte activity was determined by glycosaminoglycan (GAG) turnover. In explants cultured in post-exercise SF, GAG synthesis was enhanced and GAG release was diminished when compared to cultures in pre-exercise SF. SF analysis showed that levels of insulin-like growth factors (IGF- I and IGF-II) tended to be higher in post-exercise SF, while no differences were found in metalloproteinase activity, hyaluronic acid and protein concentrations. This study showed that anabolic effects of joint loading on cartilage are, at least partially, mediated by alterations in the SF.      

Detection of residual lymphoma cells by polymerase chain reaction in peripheral blood is significantly less predictive for relapse than detection in bone marrow

Polymerase chain reaction (PCR) amplification of the t(14;18) has been shown to be a highly sensitive method to detect minimal residual disease in patients with non-Hodgkin's lymphoma (NHL) whose tumors bear this translocation. The ideal tissue source to detect residual lymphoma would be from a previously involved lymph node. However, lymphoid tissue is rarely available once patients achieve complete remission. Although PCR amplification has been used to detect residual lymphoma cells in both bone marrow (BM) and peripheral blood (PB) of patients in complete remission, it is presently unknown whether BM and PB are equivalent tissue sources to detect residual disease. In the present study, we compared the clinical utility of the detection of residual lymphoma in both the BM and the PB of patients with advanced-stage non- Hodgkin's lymphoma before, at the time of, and after high-dose therapy and autologous BM transplantation (ABMT). The detection of residual lymphoma in either the BM or PB was associated with decreased disease- free survival. However, in the present study, 44% of patients who relapsed had no evidence of circulating lymphoma cells in their PB. At the time of BM harvest, PCR-detectable residual lymphoma cells were detected in 211 of 212 patients; although, in a subset of these patients analyzed, lymphoma cells were detected in the peripheral blood of only 49% of patients. When residual lymphoma cells within the autologous BM are infused into the patient these cells are rapidly detectable circulating in the PB in the patient. These cells continue to circulate during the immediate posttransplant period and be detectable in the PB in the majority of patients who are infused with marrow containing residual lymphoma. We conclude that BM is a more informative tissue source than PB in detecting minimal residual disease at the time of and after ABMT, and that contamination of PB early after ABMT appears to be the consequence of reinfusion of lymphoma cells within autologous marrow.     

Human immunodeficiency virus infection in patients with leukemia

Eighteen human immunodeficiency virus (HIV)-seropositive patients were found among 211 previously treated adult patients with a variety of leukemias who had been multiply transfused before April 1985. Patients known to be homosexual or intravenous drug users were excluded from this study. The spouse of one HIV-seropositive patient became HIV infected and subsequently developed the acquired immune deficiency syndrome. Patients with leukemia who were multiply transfused before the availability of screening of blood products for HIV antibody should be counseled regarding their individual risks of HIV infection and the risk to sexual contacts.     

Bedside filtration of blood products in the prevention of HLA alloimmunization--a prospective randomized study. Alloimmunisation Study Group

To test the efficacy of poststorage bedside leucodepletion of blood products in the prevention of primary HLA alloimmunization and its clinical sequelae, 172 patients with hematologic malignancy requiring intensive red blood cell and platelet support were randomized to receive either standard or filtered red blood cells and platelets. Quality control of bedside filtration was explored by sequential sampling downstream of the filter, but this did not predict the total number of leucocytes transfused. After exclusions, 123 evaluable patients were assessed every two weeks until the end of therapy. HLA antibodies developed in 21 of 56 (37.5%) nonfilter (NF) and 15 of 67 (22%) filter (F) patients (risk ratio estimate, 0.60 [95% confidence interval, 0.34 to 1.05]; P = .07). Patients with acute myeloid leukemia (AML; n = 53) had higher alloimmunization rates in both arms of the study, with a greater effect of filtration (62.5% NF and 31.0% F; P = .025). Bedside filtration did not affect the overall incidence of febrile transfusion reactions (FTRs; 37% NF and 34% F; P = .71) or of platelet refractoriness assessed in 50 patients (30% NF and 26% F), despite an association between broad HLA reactivity and both FTRs and refractoriness. However, FTRs were also seen in 28 patients without HLA antibodies. Five alloimmunized refractory patients (2 F and 3 NF) required HLA-selected platelets. This report, the first prospective study of bedside filtration, has failed to show clear clinical benefit. Methodological limitations may account in part for this failure, notably the difficulties in accurately assessing the number of leucocytes transfused.     

Purification of common acute lymphoblastic leukemia antigen positive cells from normal human bone marrow

Mononuclear cells expressing the common acute lymphoblastic leukemia antigen (CALLA) were purified from normal adult human bone marrow, where they constitute a small fraction of the total population. This was accomplished by a two-step purification from Ficoll-Hypaque- isolated mononuclear cells. Isolated mononuclear cells were first labeled with a mixture of monoclonal antibodies (MoAb) specific for myeloid and erythroid precursor cells, and immune rosettes were then formed with sheep erythrocytes coated with rabbit anti-mouse antibodies (R/M-SRBC). Sedimentation through Ficoll-Hypaque then eliminated the majority of mature myeloid cells. The second step consisted of labeling the remaining rosette-negative cells with CALLA-specific MoAb and purifying CALLA+ cells by fluorescence activated cell sorting. Alternatively, CALLA+ cells were purified in a second R/M-SRBC rosette sedimentation step. The purified CALLA+ cells, which morphologically were medium to large lymphoid cells, were subsequently studied using dual fluorescence techniques to identify surface markers as well as intracytoplasmic staining to detect terminal deoxynucleotidyl transferase enzyme (TdT) and intracytoplasmic mu. While the CALLA+ cell suspensions contained very few mature myeloid cells or T lymphocytes, the finding that 5% to 11% of them were cyto-mu+ and 13% to 22% expressed the B1 differentiation antigen clearly indicated that at least some of these cells were B cell precursors. Because 48% to 63% of the cells were TdT+ and practically all of them expressed Ia antigen, it appears that these cells are a mixture of very early lymphoid precursor cells as well as more differentiated pre-B cells. The phenotype of these normal cells is very similar to that of common ALL cells. Differences in the surface marker phenotypes between adult and fetal CALLA+ cells that have previously been purified were also identified.     

Hemoglobin-spectrin complexes: interference with spectrin tetramer assembly as a mechanism for compartmentalization of band 1 and band 2 complexes

The irreducible complexation of hemoglobin with spectrin is a natural phenomenon of red blood cell aging, positively correlating with increasing cell density and decreasing cell deformability. The current study begins to address the role of these complexes in the disruption of membrane skeletal physiology and structure. The effect of bound hemoglobin on spectrin dimer self-association was investigated in vitro. The extent of conversion of isolated spectrin dimers to tetramers was evaluated as a function of peroxide-induced globin complexation before the conversion incubations. The incremental accumulation of tetramer was observed to decrease with increasing peroxide concentration used in the globin complexation step. The role of oxidized heme in this process was made apparent by the inability of carboxyhemoglobin to inhibit tetramer accumulation. A Western blot analysis of naturally formed globin-spectrin conjugates demonstrated irreducible complexes of globin with both bands 1 and 2. The complexes are tentatively designated "h1" and "h2". This analysis also demonstrated that h1 is completely extractable from cell ghosts, whereas h2 is only 50% extractable. These findings are incorporated into a hypothesis linking globin-spectrin complexation and the consequent inhibition of spectrin dimer self-association to the clustered band 3 senescence antigen (Low et al, Science 227:531, 1985).     

Heterogeneity of hepatitis C virus genotypes in hemophilia: relationship with chronic liver disease

In this study we have determined the hepatitis C virus (HCV) serotype and genotype in a cohort of 96 HCV-infected hemophiliacs and have examined the relationship between HCV genotype and severity of chronic liver disease as determined by liver biopsy. HCV serotype was determined by specific enzyme-linked immunosorbent assays (ELISAs) and genotype by restriction fragment length polymorphism (RFLP) and HCV viral sequencing. The pattern of genotype distribution was quite unlike that of HCV-infected United Kingdom (UK) blood donors in that five of the six known HCV genotypes were represented, 50% were type 1, 13% type 2, and 18% type 3. An unexpected observation was the presence of HCV genotype 4 in four patients and type 5 in two patients. An additional feature was the presence of mixed infection, detected in 14% and 7% by serotype and genotype analysis, respectively. Liver biopsies were available from 51 patients. Cirrhosis was present in five of 27 (19%) of individuals with type 1, in 2 of 9 (22%) with type 2, and 5 of 8 (63%) of those with type 3. The heterogeneous pattern of HCV genotype distribution in this cohort of patients and the observed relationship between the severity of the related liver disease and specific HCV genotype may have important implications with respect to the natural history and treatment of HCV-related chronic liver disease in infected hemophiliacs worldwide.     

Quercetin inhibits the growth of leukemic progenitors and induces the expression of transforming growth factor-beta 1 in these cells

We previously showed that quercetin (3,3',4',5,7 pentahydroxyflavone) inhibits in a dose-dependent manner the growth of acute leukemias and is able to enhance the antiproliferative activity of cytosine arabinoside. We show here that quercetin inhibits the clonogenic activity of 20 of 22 acute leukemias (AL; 4 M1-AML, 3 M2-AML, 2 M3-AML, 3 M4-AML, 3 M5-AML, and 7 ALL). In the present report, we show that the induction of transforming growth factor-beta 1 (TGF-beta 1) in leukemic blasts is one of the growth-inhibitory mechanisms of quercetin in these cells. This observation was supported by the following data. (1) Quercetin-sensitive leukemic blasts, when treated with quercetin, secrete large amounts of TGF-beta 1 in the medium and show positivity for TGF-beta 1-immunoreactive material in the cytoplasm. (2) At a concentration of 8 mumol/L, antisense TGF-beta 1 oligonucleotides prevent the growth-inhibitory action of quercetin. (3) Anti-TGF-beta 1 neutralizing monoclonal antibodies can prevent almost completely the growth-inhibitory activity of quercetin. The analysis of quercetin- resistant cases confirmed as well the central role of TGF-beta 1 in the growth-inhibitory activity of quercetin. In conclusion, quercetin can act as a cytostatic agent for leukemic cells by modulating the production of TGF-beta 1.