Preparation of a pure autologous biodegradable fibrin matrix for tissue engineering

Parallel to the growing role of tissue engineering, the need for cell embedding materials, which allow cells to stabilise in a three-dimensional distribution, has increased. Although several substances have been tested, fibrin is thus far the only one that permits the clinical application of cultured tissue. To date, can cause severe immunological side effects. The objective of this study was to explore the practicability of obtaining autologous thrombin from a single patient in an adequate concentration and amount. Fibrinogen was cryoprecipitated from 200 ml of freshly-frozen plasma. Thrombin was isolated from the supernatant through ionexchange chromatography. The thrombin was first bound to Sephadex A-50 and then eluated using 2ml of a salt buffer (2.0M NaCl in 0.015M trisodiumcitrate, pH 7.0). The activity of the thrombin (51 NIH ml⁻¹ to 414 NIH ml⁻¹) reached levels comparable to those in commercially available fibrin glues (4–500 NIH ml⁻¹). The study has shown that it is possible to obtain a sufficient amount of autologous thrombin from a single donor to create a fibrin matrix of high efficiency without the risk of immunological and infectious side effects.     

Stromal reaction in cancer tissue: Pathophysiologic significance of the expression of matrix-degrading enzymes in relation to matrix turnover and immune/inflammatory reactions

Cancers are characterized by invasive growth and distant metastasis. Cancer cells not only destroy the pre-existing extracellular matrix, but cancer invasion per se usually induces new matrix formation by activation of stromal cells; that is, desmoplastic reaction. This process includes both matrix production and degradation; that Is, the remodeling process. The similarity between desmoplastic reactions in cancer stroma and the wound healing process has already been pointed out, and it has been well documented that matrix-degrading processes are actively involved In the wound healing process. A recent study revealed that most matrix-degrading enzymes, generally considered to be one of the main mechanisms of cancer invasion and metastasis, are originated from stromal cells. Based on these preconditions, the present review postulates that the abundant expression of matrix-degrading enzymes by fibroblasts, coupled with the abundant expression of type I procollagen, is involved in the matrix remodeling processes occurring in cancer stroma; that is, the mechanism similar to the wound healing process. Next, macrophages distributed along the invasive margin are known to express matrix-degrading enzymes/factors. Data from past studies of colon carcinoma indicate that the tissue expression of matrix metalloproteinase-9 and urokinase-type plas-mlnogen activator receptor Is inversely associated with simultaneous liver metastasis and infiltrating growth pattern. Previous clinicopathologic data have indicated that immune/Inflammatory cells are one of the factors for a favorable prognosis. This suggests that the expression of matrix-degrading enzymes/factors by these host cells may be involved in host immune/inflammatory reactions, and that the net function of these cells can be defensive towards the host. Data from past studies of colon carcinoma on the expression of the intercellular adhesion molecule-1 suggest that the interaction between macrophages, lymphocytes, and the phenotypes of venules distributed along the Invasive margin, further support the pro-inflammatory milieu there. Therefore, the matrix degradation process in cancer tissue is multifunctional: besides the Involvement in cancer invasion and metastasis, the matrix degradation process is also involved in the tissue remodeling process and in the immune/inflammatory reaction occurring in the stroma.     

Evaluation of EDTA and fish skin extract in primary culture of fish liver cells

The isolation, primary culture and attachment of liver cells to the substratum from a tropical catfish (Heteropneustes fossilis) using ethylenediaminetetraacetic acid (EDTA) as isolating agent of liver cells and skin extract (SE) from fish as attachment substrate for the primary culture of liver cells has been standardised. A suitable temperature for such cultures has also been determined. Attachment efficiency of the liver cells in culture and their intracellular lactate dehydrogenase (LDH) activity have been taken as parameters for characterization of the primary culture. Disaggregation of liver cells with EDTA is very potent to isolate substantial rumber of cells from the liver of H. fossilis. An ideal concentration of EDTA for liver cell isolation has been standardized. Matrix prepared from carp and catfish skin at different pH (2.0, 4.0, 6.0 and 8.0) were also evaluated for liver cell culture by considering the attachment efficiency of the cells over the substratum as well as retention of intracellular LDH enzyme after 48 hours of seeding. Matrix of carp skin was compared with that of catfish as suitable substrate for primary culture of fish liver cells. It has been found that the SE prepared at pH 4.0, both from carp and catfish skin, performed better than those at other pHs. At the same time, the matrix of carp skin was found to be better than that of catfish skin. Cultures were incubated separately at 17 and 23 °C in air atmosphere. Incubation temperature at 23 °C was found to be more suitable than that at 17 °C. The percent of detached/unattached cells showed only marginal variation between two temperatures but LDH-activity recorded drastic reduction (between 50 to 75%) depending upon the pH of the matrix during preparation. Our finding establishes despensibility of enzyme (collagenase/trypsin) for cell isolation in catfish. Our studies also exhibit that carp skin extract performs better than catfish skin extract in terms of attachment efficiency as well as intracellular LDH activity. This study indicates that no species/generic barrier exists in matrix between catfish and carp.     

The hepatic extracellular matrix

The unique nature of the hepatic extracellular matrix (ECM) is predicted by the special configuration of the space of Disse. Whereas other epithelial organs have two basement membranes (BM) and a substantial ECM interposed between endothelial and epithelial cells, the liver lobule has no BM and only an attenuated ECM, consisting mostly of fibronectin (FN), some collagen type I, and minor quantities of types III, IV, V, and VI. This configuration, together with the abundant fenestrations and gaps of the sinusoidal endothelial cells, seems ideally suited to facilitate the rapid bidirectional exchange of macromolecules normally taking place between plasma and hepatocytes. During organogenesis, the liver anlage is vascularized by continuous capillaries with BM, but by day 13.5 of development (in the rat) the vessels in the immediate proximity of hepatocytes become fenestrated, lacking specialized junctions and BM, suggesting that the hepatocytes produce signals capable of modulating the endothelial phenotype. In regeneration, hepatocyte proliferation precedes vascular proliferation resulting in the formation of hepatocyte clusters that, temporarily, lack sinusoids. Eventually, vascular proliferation follows and the normal hepatocyte-vascular relationships are restored. During this period laminin synthesis by Ito cells is prominent. As soon as hepatocytes become stable, secretion of the sinusoid phenotype-maintaining factors resumes and laminin synthesis and secretion terminates. The interplay between extracellular matrix and liver cells is essential for normal homeostasis and its modification results in derranged hepatic function.Parts of this editorial have been adapted from: A. Martinez-Hernandez, P.S. Amenta. Morphology, localization and origin of the hepatic extracellular matrix. In: Zern MA, Reid L (eds) Extracellular matrix: chemistry, biology, and pathology with emphasis on the liver. Marcel Dekker, New York, (in press)     

HBV基因组转染对小鼠NIH3T3细胞内基质金属蛋白酶组织抑制因子的转录调控作用

ObjectiveTo understand the mechanism of liver cirrhosis after the infection of hepatitis B virus.MethodsMouse fibroblast NIH3T3 cells were transfected with 3.2 kb HBV DND by exposure of the cells to calcium phosphate.The change of the levels of mRNA for tissue inhibitor of metalloproteinase 1and 2(TIMP1,2) was detected in mouse fibroblast NIH3T3 cells and the cells of transfection with HBV Genome by in situ hybridization.ResultsThe levels of mRNA for TIMP1 and TIMP2 were increased significantly.ConclusionHBV infection can induce the expression of the mRNA for TIMP1 and TIMP2.     

Importance of Metalloproteinases and Macrophages in Viper Snake Envenomation–Induced Local Inflammation

The inflammatory action of jararhagin, a hemorrhagic metalloproteinase from Bothrops jararaca venom, was studied in mice using dorsal air pouches. The injection of the toxin in 6-day-old air pouches resulted in a leukocyte accumulation comparable to that induced by LPS and whole venom. Polymorphonuclear and mononuclear cells were present in this infiltrate, with a predominance of neutrophils. Treatment of jararhagin with 1,10-phenantroline abolished its proteolytic activity and reduced the pro-inflammatory effect in approximately 50%. Cell influx was not observed when jararhagin was injected into 1-hr air pouches devoid of macrophages, except when it was injected together with 10⁶ syngeneic peritoneal macrophages. Supernatants of macrophages stimulated in vitro with jararhagin did not induce leukocyte influx in 1-hr air pouches; the influx occurred after injection of the pellets of stimulated cultures. In summary, jararhagin is an important pro-inflammatory component of B. jararaca venom, and its activity is dependent upon the proteolytic activity and the presence of macrophages.     

Endocrinology and paracrinology: Roles of growth factors during peri-implantation development

Several growth factor ligand and receptor gene products havebeen shown to play roles during preimplantation mammalian development.Genes for insulin-like growth factors (IGFs), transforming growthfactors (TGFs), fibroblast growth factor (FGF), platelet-derivedgrowth factor (PDGF) and receptors for insulin, IGF, PDGF, TGFand epidermal growth factor (EGF) are expressed by early embryosof several species including mouse, rat, cow and sheep. Rolesof growth factors during early development have been demonstratedby addition of purified growth factors to culture medium orby molecular genetic techniques that interfere with gene expression.In this way, it has been shown that successful development ofthe blastocyst is dependent on the action of epidermal growthfactor (EGF) and leukaemia inhibitory factor (LIF). Recent experimentsshow that both LIF and EGF stimulate secretion of urokinase-typeplasminogen activator (uPA) and gelatinase B/ matrix metalloproteinase-9(MMP-9) in day 7 mouse blastocyst outgrowths. At the same time,tissue inhibitors of MMPs (TIMPs) are also expressed by embryonic,decidual and uterine tissues during the implantation process.It appears that LIF may act directly or indirectly, by inducingthe expression of other cytokines, to regulate the temporaland spatial production and activity of proteases and proteaseinhibitors to create a favourable environment for implantation.     

凝血酶对大鼠脑微血管内皮细胞的影响

目的 :探讨凝血酶 (Thrombin ,TM )对脑微血管内皮的影响。方法 :将大鼠脑微血管内皮细胞进行培养 ,培养液中加入 10U的TM或10U的TM + 0 .4mU的组织蛋白酶G(CaspethsinG ,CATG ) ,相差显微镜动态观察内皮细胞形态的变化 ,免疫组织化学技术检测基质金属蛋白酶 2 (MatrixMetalloproteinase 2 ,MMP 2 )表达的改变。 结果 :TM使内皮细胞发生收缩 ,细胞收缩程度具有时间依赖性 ,使内皮细胞MMP 2表达水平明显增加。TM +CATG加入培养液后 ,细胞形态、MMP 2表达与对照组比较均无明显统计学差异 (P >0 .0 5 )。结论 :TM通过激活蛋白酶激活受体 1(proteaseactivatedreceptor 1,PAR 1) ,使内皮细胞发生收缩 ,促进MMP 2表达 ,是TM增加血脑屏障 (BloodBrainBarrier,BBB)通透性的可能机制。     

Sensitivity of prostate tumors to wild type and M protein mutant vesicular stomatitis viruses

Because of its potent ability to induce apoptosis, vesicular stomatitis virus (VSV) is an attractive candidate as an oncolytic virus for tumor therapy. Previous studies have suggested that VSV selectively infects tumor cells due to defects in their antiviral responses making them more susceptible to VSV infection than normal cells. We tested this hypothesis in the prostate tumor system by comparing LNCaP and PC-3 prostate tumor cells to benign human prostatic epithelial cells from patient prostatectomy specimens. We compared the cell killing ability of a recombinant virus containing a wild-type (wt) M protein (rwt) and an isogenic M protein mutant virus (rM51R-M) that induces interferon (IFN) in infected cells and should display a greater selectivity for tumor cells. Our results showed that in single-cycle infection experiments, LNCaP cells were sensitive to killing by both wt and mutant viruses, while PC-3 cells were highly resistant to VSV-induced cell killing. LNCaP and benign prostate cells were similarly susceptible to both viruses, indicating that normal prostate cells are not inherently resistant to killing by VSV. In each of the cell lines, the rM51R-M virus induced similar levels of apoptosis to rwt virus, showing that the M protein does not play a significant role in apoptosis induction by VSV in these cells. In multiple-cycle infection experiments, LNCaP cells were more sensitive than benign prostatic epithelial cells to virus-induced cell killing by rM51R-M virus, but not rwt virus. Both viruses were equally effective at reducing LNCaP tumor volume in vivo following intratumoral and intravenous inoculation in nude mice, while PC-3 tumors were resistant to VSV treatment. None of the mice treated with rM51R-M virus died as a result of virus infection, while 50-71% of mice treated with rwt virus succumbed to virus infection. Similarly, when inoculated by the more sensitive intranasal route, the rM51R-M virus was less pathogenic than the rwt virus from which it was derived. These results indicate that M protein mutant viruses are superior candidates as oncolytic viruses for therapies of prostate tumors, but future strategies for use of VSV will require testing individual tumors for their susceptibility to virus infection.     

Plasminogen modulation of IL-1-stimulated degradation in bovine and human articular cartilage explants. The role of the endogenous inhibitors: PAI-1,α 2-antiplasmin,α 1-PI,α 2-macroglobulin and TIMP

The studies described here examine the involvement of the fibrinolytic cascade and its endogenous inhibitors in the regulation of activity of matrix metalloproteinases and cartilage degradation related to non-inflammatory joint disease, like osteoarthritis. An interleukin-1-induced model of degradation using [³⁵S]-labeled bovine and human articular cartilage explants was utilized. One goal of these studies was to compare the responses of bovine and human articular cartilage. Degradation was not inhibited by ₁-PI, PAI-1, ₂-macroglobulin, ₂-antiplasmin or TIMP-2, when IL-1 alone was added. Addition of human plasminogen to bovine explants, at concentrations found in human synovial fluid, increased degradation by three to fourfold. Under these conditions, the degradation was inhibited effectively by all of the endogenous inhibitors tested, indicating the presence of a cascade where activated chondrocytes are a source of u-PA. Plasminogen activated by u-PA gives plasmin, which is known to further activate pro-stromelysin. Stromelysin is essential for activation of collegenase. Not only TIMP, but also inhibitors at earlier steps of activation like PAI-1, ₂-antiplasmin, ₁-PI and ₂-macroglobulin inhibited degradation, and could provide cartilage protection in vivo. An experiment with human articular cartilage explants showed that very little or no degradation occurred when human articular cartilage explants were stimulated with interleukin-1 alone. Addition of human plasminogen (at physiologically relevant concentrations) resulted in significant degradation, which was inhibited in the same manner as in bovine explants, by inhibitors of the fibrinolytic cascade and TIMP. TIMP is much more efficient in human explants, indicating the limited participation of human plasmin in the degradation of human cartilage. Although speculative, it is possible that in vivo, cartilage degradation could be promoted not only by TIMP/MMP imbalance, but also accelerated by decreased levels of certain serpins in synovial fluid (e.g. PAIs, ₂-antiplasmin and ₁-PI).accepted by W. B. van den BergWork supported by OsteoArthritis Science Inc., One Kendall Square, Bldg. 200, Cambridge, MA 01139, USA.