Biocompatibility assessment of polytetrafluoroethylene/wollastonite composites using endothelial cells and macrophages

The aim of the study was to prepare a composite of polytetrafluoroethylene/wollastonite (PTFE/W) and evaluate its biocompatibility with endothelial cells. A composite of PTFE with wollastonite in the proportion 90/10 w/w was prepared. The dynamic storage modulus of composite is found to increase from 260 to about 453 MPa at room temperature while a marginal increase is observed in the compressive modulus. Higher values of storage modulus of PTFE/W relative to pristine PTFE over a range of temperature indicated the contribution of wollastonite in improving the rigidity of PTFE. Electron microscopic visualization of composite surface indicates suitable morphology for cell growth with the cross-section showing no evidence of bonding between PTFE and wollastonite. The water contact angle of the composite indicates increased hydrophilicity over native PTFE due to the presence of wollastonite. A direct-contact test did not show any deleterious effects on endothelial cell morphology and viability, indicating its compatibility. Leached-out products (LOP) from the composite were determined to be non-toxic as tested by tetrazolium (MTT) and Neutral red uptake (NRU) assays. Mouse peritoneal macrophages cultured in the presence of the composites did not show upregulation of activation markers such as CD11b/CD 18 (Mac-1), CD45, CD 14, and CD86 (B7.2) in comparison to macrophages cultured in contact with PTFE alone, indicating its non-activating nature. LOP did not induce proliferation of mouse splenic lymphocytes suggesting its immuno-tolerance. In static incubation assay contact with composite did not lead to hemolysis thus exhibiting preliminary hemocompatibility of the material. Suitable physico-chemical properties and well tolerance by endothelial cells and macrophages make this composite a prospective biomaterial. One could foresee the applications of this composite in areas where materials need to possess high rigidity and are subject to elevated temperatures.     

Preparation, characterization and in vitro biocompatibility evaluation of poly(butylene terephthalate)/wollastonite composites

The aim of the study was to prepare composites of poly(butylene terepthalate)/wollastonite (PBT/W), evaluate their properties and in vitro biocompatibility. Composites of PBT with wollastonite in two different proportions, viz. 70/30 (PW-30), 50/50 (PW-50) were prepared. The DSC studies indicate marginal changes in the melting behavior and enhanced crystallization in PBT/W composites. The mechanical properties of the composites such as tensile modulus shows remarkable improvement as a result of incorporation of wollastonite. SEM studies of fractured surfaces of impact samples showed no evidence of bonding between PBT and wollastonite. Water contact angle of PW30 and PW50 was 73.7 and 78.7, respectively. In vitro biocompatibility of PW-30 was evaluated as a representative composite. Direct cell contact test did not show deleterious effects on NIH3T3 fibroblast morphology and DNA integrity indicating its compatibility. Leach out products (LOP) of PW-30 were evaluated non-toxic as tested by MTT assay. Mouse peritoneal macrophages in contact with PW-30 showed comparable expression of CD 11b/18, CD45, CD14 and B7.2 to macrophages in contact with PTFE control indicating its non-activating nature. LOP did not induce proliferation of mouse splenic lymphocytes suggesting its immuno-tolerance. PW-30 also exhibited preliminary blood compatibility. These physical properties and biocompatibility of PBT/W composites show their suitability as potential biomaterials.     

Polyamide 6 composite membranes: properties and in vitro biocompatibility evaluation

The aim of the present study was to develop polyamide 6 membrane blended with gelatin and chondroitin sulfate using the phase precipitation method and evaluate its in vitro biocompatibility. Morphology of membranes was studied by laser scanning confocal microscopy which allowed the nondestructive visualization of internal bulk morphology of membranes. Membranes exhibited porous morphology with pores spanning across the membrane width with interconnections at various depths. Membranes showed adequate mechanical properties with tensile strengths of 20.10 +/- 0.64 MPa, % strain of 3.01+/-0.07, and modulus of 1082.50+/-23.50 MPa. In vitro biocompatibility of membranes by direct contact test did not show degenerative effects on NIH3T3 cells and also its leach-out products (LOP), as determined by tetrazolium (MTT) and neutral red uptake (NRU) assay. Mouse peritoneal macrophage cultured in contact with membranes and PTFE control showed comparable expression of activation markers such as CD11b/CD18, CD45, CD14, and CD86 suggesting the membranes' non-activating nature. Membrane LOP did not induce excessive proliferation of mouse splenocytes suggesting its non-antigenic nature. Preliminary blood compatibility of membranes was observed with no detectable hemolysis in static incubation assay. Taken collectively, the present data demonstrate that polyamide 6 composite membranes are biocompatible and prospective candidates for tissue engineering applications.     

Polyamide 6 composite membranes: Properties and in vitro biocompatibility evaluation

The aim of the present study was to develop polyamide 6 membrane blended with gelatin and chondroitin sulfate using the phase precipitation method and evaluate its in vitro biocompatibility. Morphology of membranes was studied by laser scanning confocal microscopy which allowed the nondestructive visualization of internal bulk morphology of membranes. Membranes exhibited porous morphology with pores spanning across the membrane width with interconnections at various depths. Membranes showed adequate mechanical properties with tensile strengths of 20.10 ± 0.64 MPa, % strain of 3.01±0.07, and modulus of 1082.50±23.50 MPa. In vitro biocompatibility of membranes by direct contact test did not show degenerative effects on NIH3T3 cells and also its leach-out products (LOP), as determined by tetrazolium (MTT) and neutral red uptake (NRU) assay. Mouse peritoneal macrophage cultured in contact with membranes and PTFE control showed comparable expression of activation markers such as CD11b/CD18, CD45, CD14, and CD86 suggesting the membranes' non-activating nature. Membrane LOP did not induce excessive proliferation of mouse splenocytes suggesting its non-antigenic nature. Preliminary blood compatibility of membranes was observed with no detectable hemolysis in static incubation assay. Taken collectively, the present data demonstrate that polyamide 6 composite membranes are biocompatible and prospective candidates for tissue engineering applications.     

Modulation of angiogenic functions in human macrophages by biomaterials

We examined the ability of polyvinylchloride (PVC), polytetrafluorethylene (PTFE) and tissue culture polystyrene (TCPS) to affect angiogenic functions in human monocyte-derived macrophages by measuring the mRNA expression of genes encoding angiogenic and anti-angiogenic molecules including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and thrombospondin-1 (Tsp-1). The angiogenic activity of the corresponding macrophage conditioned media (CM) was measured by the proliferation of endothelial cells and the sprouting of new capillaries from fragments of human placental blood vessels. We determined that bFGF was not expressed in macrophages while VEGF and Tsp-1 mRNAs were expressed constitutively. Ang-1 was expressed in macrophages cultured up to 7 days on PTFE and TCPS independent of the culture stage. In contrast, macrophages cultured on PVC did not produce detectable amounts of Ang-1 mRNA after 7 days. CM from macrophages cultured either on PTFE or TCPS stimulated angiogenesis whereas CM from macrophages cultured on PVC inhibited it. The results demonstrate that polymers can cause differential expression of the angiogenic molecule Ang-1 in macrophages. They also induce different phenotypes of macrophages, which can either stimulate or inhibit angiogenesis suggesting a material-dependent influence on neovascularization.     

Effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells in PHBV/wollastonite composite scaffolds

In this study, the effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells (hBMSCs) have been investigated based on a polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/ wollastonite (W) composite scaffolds system. Cell morphology, proliferation, and differentiation were measured. The results showed that the incorporation of wollastonite benefited hBMSCs adhesion, proliferation, and differentiation rate. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composite scaffolds increased from 10 to 20 wt%. Based on our previous studies on PHBV/W composite discs, the differentiation measurements in this paper further proved that the wollastonite itself can stimulate the hBMSCs to differentiate toward osteoblasts without any osteogenic medium, and the ionic products (Ca and Si) released from wollastonite might contribute to this advantage. It is also suggested that the osteogenic differentiation of the hBMSCs can be affected by adjusting the wollastonite content in the composite scaffolds.     

Plasma sprayed wollastonite/TiO2 composite coatings on titanium alloys

Wollastonite/TiO2 composite coatings were prepared using plasma spraying technology onto Ti-6Al-4V substrate. The composite coatings exhibit obvious lamellar structure with alternating wollastonite coating and TiO2 coating. No obvious cracks exist on the interface between coatings and substrate. In the case of composite coatings, the primarily crystalline phases of the coatings are wollastonite and rutile, indicating wollastonite and TiO2 did not react during plasma spraying process. Some of rutile in the powders transforms into anatase due to plasma spraying. The mean bond strength of the composite coatings is higher than 30 MPa. The Vickers microhardness of coatings increase with the increase in the content of TiO2. Wollastonite/TiO2 composite coatings were soaked in simulated body fluid to examine their bioactivity. Carbonate-containing hydroxyapatite (CHA) layer was formed on the surface of the wollastonite and W7T3 coatings soaked in simulated body fluid, while was not formed on the surface of the TiO2 and W3T7 coatings after immersion. In addition, a rich-silica layer appeared at the interface of CHA and wollastonite and W7T3 coatings. In order to investigate the cytocompatibility of the coatings, osteoblast was seeded onto the surface of the coatings. The scanning electron microscopy observation showed that the addition of wollastonite promote the proliferation of osteoblast. It is enough to prove that the wollastonite and wollastonite/TiO2 composite coatings possess excellent cytocompatibility.     

Is it possible to obtain "true endothelial progenitor cells" by in vitro culture of bone marrow mononuclear cells?

In vitro-cultured bone marrow cells have been shown to contain some low-density lipoprotein (LDL) uptake-positive cells. Although a small portion of LDL uptake-positive cells had expression for endothelial markers, all of them demonstrated a phagocytosis function similar to monocyte/macrophages and expression of the panleukocyte surface marker CD45 and monocyte marker CD14. These LDL uptake-positive cells did not show significant proliferative capacity and died out gradually in long-term culture. In contrast, the bone marrow-derived LDL uptake-negative cells showed strong proliferation and expression of typical mesenchymal surface markers CD29 and CD44. Although cultured under endothelial promoting conditions, these mesenchymal stem cells (MSCs) did not show any sign of differentiation toward endothelial cells. In conclusion, adult bone marrow-derived LDL uptake-positive cells that have been reported so far actually are monocytes/macrophages that can express some endothelial markers but are not "true endothelial progenitor cells" (EPCs). MSCs, which are the only cell type that shows strong proliferation during long-term adherent culture for bone marrow cells, do not differentiate toward the endothelial lineage when grown under endothelial promoting conditions.     

Suitability of cellulose molecular dialysis membrane for bioartificial pancreas: in vitro biocompatibility studies

The success of immunoisolation devices for islet transplantation depends on the properties and biocompatibility of semipermeable immunobarrier membranes. In the present study, we have evaluated the in vitro biocompatibility of the cellulose membrane Spectra/Por 2 (MW no larger than 12- 14,000) for its possible application in islet immunoisolation. The membrane was found to be hydrophilic (octane contact angle: 153.2+/-0.66 degrees) and exhibited decreased protein adsorption. It showed mechanical stability after 1 month of storage in PBS (pH 7.4) with tensile strength, percent elongation, and Young's modulus of 88.88 MPa, 36.22, and 291.8 MPa, respectively. It allowed regulated transport of glucose and insulin in an in vitro diffusion assay. The high viability of NIH3T3 fibroblasts and the inability of lymphocytes to proliferate in vitro on exposure to the membrane leach-out products suggested its noncytotoxic and nonimmunogenic nature. Macrophages, when cultured on membranes, did not show increased expression of inflammatory surface marker such as CD11b/CD18, CD45, CD14, and B 7.2. Image analysis studies showed integrity and intact morphology of mouse islets cultured on and inside the membranes with high viability (91%, 89.7%). These islets also retained their functionality, as judged by insulin secretion. The present study provides sufficient documentation to consider cellulose molecular dialysis membrane Spectra/Por 2 (MW no larger than 12-14,000) as a potential candidate for immunoisolation of islets.     

IL-10 prevents the differentiation of monocytes to dendritic cells but promotes their maturation to macrophages

Human monocytes cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-13 for 7 days differentiate into cells with the morphology and function of dendritic cells (DC). We have investigated the effect of IL-10 on this differentiation pathway. In the presence of IL-10 cells did not develop DC morphology, did not express CD1a and had lower levels of MHC class II. IL-10 promoted the differentiation of large cells with the morphology, cytochemistry and membrane phenotype of macrophages, including staining for nonspecific esterase and high levels of CD14, CD16 and CD68. The effect of IL-10 was dose dependent and was best appreciated when the cytokine was added at the initiation of the culture, as addition on day 3 was less inhibitory. When added to already differentiated DC on day 6, IL-10 caused only a modest reduction of MHC class II and CD1a expression, and no acquisition of the macrophage markers CD14, CD16 and CD68. Prolonged incubation up to 5 days with IL-10 did not induce a shift of differentiated DC to macrophages. On the other hand, the macrophages obtained by culturing for 7 days with GM-CSF+IL-13+IL-10 did not shift to DC upon removal of IL-10 for up to 3 days. Thus, the effect of IL-10 on monocyte differentiation, occurs only at the precursor level and confers an irreversible phenotype. From a functional point of view, cells cultured in the presence of IL-10 were poor stimulators of allogeneic cord blood T cells in mixed lymphocyte reaction (MLR) and presented tetanus toxin (TT) to specific T cell lines with much less efficiency than control DC. In contrast, IL-10-cultured DC showed 7 times greater endocytosis of FITC-dextran. This increased endocytosis was mostly mediated via the mannose receptor, as demonstrated by blocking with unlabeled mannose. In conclusion, IL-10 inhibits DC differentiation from monocytes and, in a substantial proportion of the cells, promotes the differentiation to mature macrophages. Intriguingly, IL-10 inhibits antigen presentation while it stimulates endocytic activity.     

Early infiltration of CD8+ macrophages/microglia to lesions of rat traumatic brain injury

Local inflammatory responses play an important role in mediating secondary tissue damage in traumatic brain injury. Characterization of leukocytic subpopulations contributing to the early infiltration of the damaged tissue might aid in further understanding of lesion development and contribute to definition of cellular targets for selective immunotherapy. In a rat traumatic brain injury model, significant CD8+ cell accumulation was observed 3 days post-injury. The CD8+ cells were strictly distributed to the pannecrotic areas and around the pannecrotic perimeter. The morphology, time course of accumulation and distribution of CD8+ cells were similar to that of reactive ED1+ and endothelial monocyte-activating polypeptide II+ microglia/macrophages, but different from W3/13+ T cells. Further double-labeling experiments confirmed that the major cellular sources of CD8 were reactive macrophages/microglia. Both the location of these CD8+ macrophages/microglia to the border of the pannecrosis and their co-expression of endothelial monocyte-activating polypeptide II and P2X4 receptor suggest they might have a central role in lesion development and might thus be candidates for development of immunotherapeutic, anti-inflammatory strategies.     

Atheromatous plaque macrophages produce plasminogen activator inhibitor type-1 and stimulate its production by endothelial cells and vascular smooth muscle cells.

The capacity of macrophages to influence directly and indirectly fibrinolytic processes in atherosclerosis was studied using macrophages isolated from atherosclerotic plaques of patients undergoing surgical repair of distal aortic and femoral arteries. These cells were characterized by their morphology, adherence, esterase positivity, and expression of CD14 antigen. Production of plasminogen activator inhibitor type-1 (PAI-1) by plaque macrophages (6.7 +/- 2.7 ng/10(5) cells/24 hours [mean +/- SEM]) was significantly greater than PAI-1 production by blood monocytes isolated simultaneously from the same patients (1.8 +/- 1.5 ng/10(5) cells/24 hours). Production of tissue type plasminogen activator and urokinase type was not augmented compared to blood monocytes. Conditioned medium from cultured plaque macrophages significantly increased production of PAI-1 by endothelial cells (85 +/- 11% above basal) and vascular smooth muscle cells (25 +/- 10%) in vitro. This response was significantly greater than the response to monocyte-conditioned medium (endothelial cells 38 +/- 11%, vascular smooth muscle cells 2.5 +/- 2.0%). Stimulation of endothelial cell PAI-1 production by macrophage-conditioned medium was partially inhibitable by a monoclonal antibody to transforming growth factor-beta. Tissue type plasminogen activator production by endothelial cells and vascular smooth muscle cells was not affected by plaque macrophage- or monocyte-conditioned medium. Urokinase type plasminogen activator production by endothelial cells and vascular smooth muscle cells was undetectable in control medium and was augmented to similar levels in response to plaque macrophage- and monocyte-conditioned media. These results demonstrate upregulation of PAI-1 production by macrophages in atheromatous plaques and the capacity of soluble products from plaque macrophages to upregulate PAI-1 production by endothelial cells and vascular smooth muscle cells in vitro. These data suggest that macrophages in atherosclerotic plaques may inhibit thrombolysis both directly and indirectly by effects of their soluble products on endothelial cells and vascular smooth muscle cells.     

大鼠肺微血管内皮细胞培养及其粘弹性研究

为了建立肺微血管内皮细胞培养方法 ,研究肺微血管内皮细胞粘弹性。我们取大鼠肺周边组织 (宽度不应大于 1.5 mm) ,将组织剪成 1.5 mm× 1mm× 1mm的组织块 ,贴入无菌的 2 5 cm3培养瓶 ,每瓶 10～ 15块 ,同时加入含 2 0胎牛血清、肝素 90 U/ml、L-谷氨酰胺 4mmol、青霉素 10 0 U/ml和链霉素 10 0 μg/ml的 DMEM培养基 3ml,放入 37℃二氧化碳培养箱中静置培养 ;8h后翻转培养瓶 ,6 0 h后取出肺组织块 ,接着继续培养 2～ 4d后进行传代。最后消化分离肺微血管内皮细胞 ,用微管吸吮系统研究肺微血管内皮细胞粘弹性。结果显示 :肺微血管内皮细胞通过倒置相差显微镜观察 ,细胞呈鹅卵石镶嵌状排列 ,状如梭形或多角形 ,大小均匀 ,胞核清晰 ,呈卵圆形 ,胞浆丰富 ; 因子相关抗原免疫荧光染色呈阳性 ;肺微血管内皮细胞弹性模量 K1 =49.3± 9.2 Pa、K2 =73.2±2 4.8Pa、粘性系数 μ=19.2± 7.2 Pa.s。这些结果表明用组织块法培养肺微血管内皮细胞是可行的 ,肺微血管内皮细胞表现出较大的刚性     

CD40-CD154 interactions between macrophages and natural killer cells during sepsis are critical for macrophage activation and are not interferon gamma dependent

Natural killer (NK) cell interactions with macrophages have been shown to be important during bacterial sepsis in activating macrophages to improve bacterial clearance. The mechanism for this increased activation, however, is unclear. This study determines the relative roles of interferon (IFN)-gamma and CD40/CD154 direct cell interactions on macrophage and NK cell activation in an experimental model of sepsis. Splenic NK cells and peritoneal macrophages were isolated and cultured alone or in coculture, with and without LPS. CD69 expression on NK cells, phagocytosis ability of macrophages, and cell cytokine production was assessed at 24 and 48 h. Coculture of NK cells and macrophages significantly increased activation levels of both cell types, and through experiments culturing NK cells with supernatants from stimulated macrophages and macrophages with supernatants from stimulated NK cells, this activation was determined to be cell-contact-dependent. Similar experiments were conducted using NK cells from IFN-gamma deficient (-/-) mice, as well as anti-IFN-gamma neutralizing antibody. These experiments determined that IFN-gamma is not required for NK or macrophage activation, although it did augment activation levels. Experiments were again repeated using peritoneal macrophages from CD40-/- mice or splenic NK cells from CD154-/- mice. CD40/CD154 interactions were important in the ingestion of bacteria by macrophages, but did not affect NK cell activation at 24 h. There was, however, a protective effect of CD40/CD154 interactions on NK cell activation-induced cell death that occurred at 48 h. CD40/CD154 interactions between macrophages and NK cells are therefore important in macrophage phagocytosis, and are not dependent on IFN-gamma.     

Biocompatible hydrogel supports the growth of respiratory epithelial cells: possibilities in tracheal tissue engineering

Extensive tracheal defect reconstruction is a major challenge in plastic and reconstructive surgery. The lack of an epithelial lining on the luminal surfaces of tracheal prostheses is among the major causes of their failure. Chitosan-gelatin hydrogels were synthesized for the development of biocompatible, growth-supportive substrata for respiratory epithelial cells. We employed J774 macrophages to test the immunocompatibility of this gel. The hydrogel did not exert a cytotoxic effect on macrophages, as confirmed by tetrazolium reduction and neutral red uptake assay. Flow cytometric analysis of macrophages cultured on the hydrogel showed a comparable expression of activation markers CD11b/CD18, CD45, and CD14 to the control. Semiquantitative RT-PCR results showed an absence of upregulation of interleukin-6 (IL-6) and TNF-alpha in these macrophages with respect to the controls. Primary human respiratory epithelial cells cultured on the hydrogel showed proper attachment, normal morphology, and growth. A small proportion of cells on the hydrogel showed synchronously beating cilia. RT-PCR analysis showed that cells on the hydrogel expressed mucins 2 and 5 and cytokeratin 13, which are markers for secretory goblet and squamous cells, respectively. All these results demonstrate that the hydrogel supports the growth of a mixed population of differentiated epithelial cells. This hydrogel is suitable as a culture substratum for respiratory epithelial cells and could be used as a potential candidate for coating tracheal prostheses.     

LNFPIII/LeX-stimulated macrophages activate natural killer cells via CD40-CD40L interaction

Lacto-N-fucopentaose III (LNFPIII) is a human milk sugar containing the biologically active Lewis X (LeX) trisaccharide. LNFPIII/LeX is also expressed by immunosuppressive helminth parasites, by bacteria, and on a number of tumor/cancer cells. In this report, we first demonstrate that LNFPIII activates macrophages in vitro as indicated by upregulation of Gr-1 expression on F4/80(+) cells. Further, we investigated the effect of LNFPIII-activated macrophages on NK cell activity. We found that LNFPIII-stimulated F4/80(+) cells were able to activate NK cells, inducing upregulation of CD69 expression and gamma interferon (IFN-gamma) production. The experiments show that NK cell activation is macrophage dependent, since NK cells alone did not secrete IFN-gamma in response to LNFPIII. Furthermore, we found that activation of NK cells by glycan-stimulated macrophages required cell-cell contact. As part of the cell-cell contact mechanism, we determined that CD40-CD40L interaction was critical for IFN-gamma secretion by NK cells, as the addition of anti-CD40L antibodies to the coculture blocked IFN-gamma production. We also demonstrated that LNFPIII-stimulated macrophages secrete prostaglandin E(2), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha) but a very low level of IL-12. Interestingly, addition of anti-TNF-alpha, anti-IL-10, or anti-IL-12 monoclonal antibodies did not significantly alter NK cell activity. Our data show that these soluble mediators are not critical for LNFPIII-stimulated macrophage activation of NK cells and provide further evidence for the importance of cell-cell contact and CD40-CD40L interactions between macrophages and NK cells.     

Flow and High Affinity Binding Affect the Elastic Modulus of the Nucleus,Cell Body and the Stress Fibers of Endothelial Cells

Cell mechanical properties are important in the adhesion of endothelial cells to synthetic vascular grafts exposed to shear flow. We hypothesized that the local apparent elastic modulus of the nucleus and the cell body would increase to a greater extent for cells adherent via the dual ligand (integrin-fibronectin/avidin-biotin) and exposed to flow, than for cells treated with either ligand alone. High affinity avidin-biotin bonds and in vitro flow exposure were used to improve adhesion to grafts thereby altering the mechanical properties of endothelial cells. Introduction of the dual ligand chemistry at the cell-substrate interface increased the apparent elastic modulus of the cells as compared to cells adherent with the fibronectin-integrin bonds only. Cells cultured on the dual ligand surface exhibited higher elastic moduli of the nucleus and cell body relative to cells cultured on fibronectin alone. Exposure of cells to flow increased the apparent elastic modulus of the cell body, nucleus, and stress fibers of cells adherent to the fibronectin surface. A similar effect was seen for cells adherent to the dual ligand surface, although there was little effect on the elastic modulus of the nucleus. While the dual ligand surface produces an increase in adhesion strength, focal contact area and elastic modulus, the change in elastic modulus after exposure to flow is due only to an increase in stress fibers and not an increase in contact area.     

Fabrication and characterization of bioactive wollastonite/PHBV composite scaffolds

Composite scaffolds of polyhydroxybutyrate-polyhydroxyvalerate (PHBV) with bioactive wollastonite were fabricated by a compression moulding, thermal processing, and salt particulate leaching method. Structure and mechanical properties of the scaffolds were determined. The bioactivity of the composites was evaluated by soaking in a simulated body fluid (SBF), and the formation of the hydroxyapatite (HAp) layer was determined by Scanning Electron Microscope (SEM) and Energy-Dispersive Spectrometer (EDS). The results showed that the wollastonite/PHBV composites were bioactive as it induced the formation of HAp on the surface of the composite scaffolds after soaking in SBF for 14 days. In addition, the measurements of the water contact angles suggested that incorporation of wollastonite into PHBV could improve the hydrophilicity of the composites and the enhancement was dependent on the wollastonite content. Furthermore, the pH and ion concentration changes of SBF solutions with composite scaffolds showed that the composites released Ca and Si ions, which could neutralize the acidic by-products of the PHBV and stabilize the pH of the SBF solutions between 7.2 and 7.8 within a 3-week soaking period. All of these results suggest that the incorporation of wollastonite was a useful approach to obtain composite scaffolds with improved properties.     

Human monocytes differentiate into macrophages under the influence of human KPB-M15 conditioned medium

Culture medium conditioned by the L929 murine fibroblast cell line contains macrophage colony-stimulating factor (M-CSF), providing an alternative to recombinant M-CSF for in vitro generation of murine macrophages. No such alternative has been described for in vitro studies requiring human macrophages. We tested the differentiation of human blood monocytes into mature macrophages by culturing in media conditioned by the human KPB-M15 cell line, which produces M-CSF and interleukin 6 (IL-6). The phenotypes of macrophages cultured in KPB-M15 conditioned media and recombinant M-CSF were compared by examining viability, expression of cell surface markers, phagocytic/pinocytic activity, and cytokine/chemokine secretion in response to bacterial lipopolysaccharide (LPS). In conditioned medium, monocytes differentiated into a homogeneous population of large cells that exhibited higher expression of CD14 and the macrophage mannose receptor (CD206) than did M-CSF-cultured cells. Cells matured in KPB-M15 conditioned medium exhibited macrophage morphology, were phagocytic, and were activated in response to LPS. These data demonstrate that KPB-M15 conditioned medium can be used to differentiate human blood monocytes into macrophages in vitro.     

Ultrastructure of IL2-stimulated Tumor-infiltrating Lymphocytes Showing Cytolytic Activity Against Tumor Cells

Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD87CD57- and CD8⁺/CD57⁺, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing. Acta Pathol Jpn 41: 94-105, 1991.