Douglas A. Kniss

Effects of Filtration Seeding on Cell Density, Spatial Distribution, and Proliferation in Nonwoven Fibrous Matrices

The cell seeding density and spatial distribution in a 3‐D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth‐filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3‐D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast‐like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85−0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth‐filtration seeding method gave a significantly higher initial seeding density (2−4 × 107 vs 4 × 106 cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large‐pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth‐filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth‐filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.

Cyclooxygenases in reproductive medicine and biology.

This review focuses on the role of cyclooxygenases in reproductive medicine and biology. The cyclooxygenase enzymes catalysis the role-limiting reactions for prostaglandins and thromboxame synthesis. Two separate isoforms, COX-1 and COX-2, are present, and differences in their roles are discussed in the context of inflammation and parturition.

Tumor necrosis factor-alpha promotes sustained cyclooxygenase-2 expression: attenuation by dexamethasone and NSAIDs.

Prostaglandin (PG) release is characteristic of most inflammatory diseases. The committed step in the formation of free arachidonic acid into PG products is catalyzed by cyclooxygenase (COX, prostaglandin H2 synthase, PGHS), which exists as two genetically distinct isoforms. COX-1 is constitutively expressed and produces PGs and thromboxane A2 during normal physiologic activities, while COX-2 is an inducible enzyme stimulated by growth factors, lipopolysaccharide, and cytokines during inflammation or cell injury. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) released into the amniotic fluid in the setting of infection have been proposed to signal amnion and decidual cells to produce PGs that may culminate in preterm labor. However, since the molecular control of this phenomenon has not been established, this study used amnion-derived WISH cells to determine if TNF-alpha promoted the formation of PGs through COX-2 activity. Treatment of WISH cells with TNF-alpha (0.1 ng/mL-100 ng/mL) caused a dose-dependent increase in COX-2 expression and the subsequent biosynthesis of PGE2 that persisted for at least 48 hrs. In contrast, COX-1 mRNA and protein levels were unaltered by TNF-alpha treatment as determined by RT-PCR and immunoblot analysis, respectively. TNF-alpha-stimulated COX-2 expression and the subsequent formation of PGE2 were inhibited by dexamethasone (0.1 microM). In addition, indomethacin (1 microM) and the novel COX-2-selective inhibitor, NS-398 (IC50 approximately 1.1 x 10(-9) M), attenuated TNF-alpha-elicited PGE2 production. Results presented here demonstrate that TNF-alpha elicits prolonged and regulatable induction of COX-2 in WISH cells, while COX-1 is constitutively expressed and unchanged in response to TNF-alpha stimulation.

Three-Dimensional Cell-Scaffold Constructs Promote Efficient Gene Transfection: Implications for Cell-Based Gene Therapy

To date, introduction of gene-modified cells in vivo is still a critical limitation for cell-based gene therapy. In this study, based on tissue engineering techniques, we developed a three-dimensional (3-D) transfection system to be cell-based gene delivery vehicle. Human trophoblast-like ED(27) and fibroblastic NIH3T3 cells were used as model cell lines. Cells were seeded onto PET fibrous matrices and plated on polyethylene terephathalate (PET) films as 2-D transfection control. The cell-matrices and cell-films were transfected with pCMV-betagal and pEGFP (green fluorescent protein) reporter gene vectors using LipofectAmine reagent. Gene expression on 3-D versus 2-D growth surface were investigated. The effects of seeding method, seeding density, porosity of the PET matrix, and culturing time of the cell-matrix complex on cDNA transfection and expression in the 3-D cell-matrix complex were also investigated. The beta-gal assay and GFP detection showed that 3-D transfection promoted a higher gene expression level and longer expression time as compared to 2-D transfection. There existed an optimal initial cell seeding density for gene transfection of 3-D cell-matrix complex. Cells seeded on PET matrices with a lower porosity ( approximately 87%) had higher gene expression activities than cells in the matrices with a higher porosity ( approximately 90%). Also, Higher gene expression levels of beta-gal were obtained for the more uniformly seeded matrices that were seeded with a depth-filtration method. The results from this study demonstrate the potential utility of cells seeded onto 3-D fibrous matrices as cell-based gene delivery vehicle for in vitro study of gene expression or in vivo gene therapy.

Human cord cell hematopoiesis in three-dimensional nonwoven fibrous matrices: in vitro simulation of the marrow microenvironment.

Current hematopoietic culture systems mainly utilize two-dimensional devices with limited ability to promote self-renewal of early progenitors. In vivo-like three-dimensional (3-D) culture environments might be conducive to regulating stem cell proliferation and differentiation similar to in vivo hematopoiesis. The few 3-D cultures reported in the literature either produced few progenitors or provided little information about microenvironment. In this study, we constructed a 3-D hematopoietic microenvironment composed of nonwoven matrix and human cord blood (CB) cells to simulate the marrow microenvironment and expand cord progenitors. Nonwoven polyethylene terephthalate (PET) fabric with defined microstructure was used as the 3-D scaffold and the PET surface was modified by hydrolysis to improve cell adhesion. Different cell organizations were formed in a 3-D matrix in a developmental manner, from individual cells and cells bridging between fibers to large cell aggregates. Both stromal and hematopoietic cells were distributed spatially within the scaffold. Compared to two-dimensional (2-D) CD34(+) cell culture, 3-D culture produced 30-100% higher total cells and progenitors without exogenous cytokines. With thrombopoietin and flt-3/flk-2 ligand, it supported two- to three-fold higher total cell number (62.1- vs. 24.6-fold), CD34(+) cell number (6.8- vs. 2.8-fold) and colony-forming unit (CFU) number for 7-9 weeks (n = 6), indicating a hematopoiesis pathway that promoted progenitor production. Culture in 3-D nonwoven matrices enhanced cell-cell and cell-matrix interactions and allowed 3-D distribution of stromal and hematopoietic cells. The formation of cell aggregates and higher progenitor content indicated that the spatial microenvironment in 3-D culture played an important role in promoting hematopoiesis. This 3-D culture system can be used as an in vitro model to study stem cell or progenitor behavior, and to achieve sustained progenitor expansion.

Thermal compression and characterization of three-dimensional nonwoven PET matrices as tissue engineering scaffolds

Nonwoven fibrous matrices have been widely used as scaffolds in tissue engineering, and modification of microstructure of these matrices is needed to organize cells in three-dimensional space with spatially balanced proliferation and differentiation required for functional tissue development. The method of thermal compression of nonwoven polyethylene terephthalate (PET) fabrics was developed and key parameters of temperature, pressure, and compression duration were evaluated in this study. The permanent deformation was obtained at elevated temperature under pressure and the viscoelastic compressional behaviors were observed, characterized by a distinct apparent modulus change in glass transition temperature region. A liquid extrusion method was further employed to analyze both pore size and its distribution for matrices with porosity ranging from 84 to 93%. It is also found that a more uniformly distributed pore size was resulted from thermal compression and the isotropic nature of nonwoven fabrics was preserved because of the proportional reduction of the pore by compression. The thermally compressed fabric matrices with two different pore sizes (15 and 20 microm in pore radius) were used to culture human trophoblast ED27 and NIH 3T3 cells. It was found that cells cultured in the different pore-size PET matrices had different cell spatial organization and proliferation rates. The smaller pores in the matrix allowed cells to spread better and proliferate faster, while cells in the larger pores tended to form large aggregates and had lower proliferation rate. The thermal compression technique also can be applied to other synthetic fibrous matrices including biodegradable polymers used in tissue engineering to modify the microstructure according to their viscoelastic properties.

Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages.

Elevated levels of nitric oxide (NO) produced by expression of inducible nitric oxide synthase (iNOS/NOS type 2) and high levels of prostaglandins (PGs) generated by expression of inducible cyclooxygenase (COX‐2/PGH2 synthase‐2) are important mediators of immune and inflammatory responses. Previous studies have shown that endogenous levels of NO˙ can influence the formation of PGs. We examined the mechanism by which NO˙ regulates PG biosynthesis in macrophages. Treatment of a murine macrophage cell line (ANA‐1) with lipopolysaccharide (LPS, 10 ng/mL) and interferon‐γ (IFN‐γ, 10 U/mL) for 20 h elicited high levels of nitrite (NO2 −) and prostaglandin E2 (PGE2) that were inhibited in a dose‐dependent fashion by the NOS inhibitor, aminoguanidine (AG), with IC50 values of 15.06 and 0.38 μM for NO2 − and PGE2, respectively. Stimulation of cultures with LPS and IFN‐γ for 20 h induced de novo iNOS protein expression that was not altered by the addition of AG (0.1, 10, or 1000 γM). In contrast, treatment of cultures with LPS and IFN‐γ for 20 h promoted COX‐2 mRNA and protein expression that were decreased in a dose‐dependent fashion by AG (P < 0.05 with 10 and 1000 μM). LPS and IFN‐γ‐induced COX‐2 protein expression was not decreased in cultures treated with AG for 2 h, illustrating that AG does not inhibit the formation of COX‐2 protein. Analysis of partially purified enzyme extracts demonstrated that AG did not directly inhibit the enzymatic activity of COX. Additional experiments revealed that NO^ donors (S‐nitroso‐N‐aceytl‐d‐l‐pencillamine, SNAP, at 0.1, 10, and 1000 μM) did not induce de novo COX‐2 protein expression or potentiate COX‐2 expression in cells treated with LPS and/or IFN‐γ. Our results suggest that, while endogenous NO˙ is not required for de novo COX‐2 mRNA and protein expression, NO˙ is necessary for maintaining prolonged COX‐2 gene expression. J. Leukoc. Biol. 65: 792–799; 1999.

Serum and fibroblast growth factor stimulate quiescent astrocytes to re-enter the cell cycle

An in vitro model was used to study the cytokinetics of astroglial cells derived from neonatal rat cerebellum. Confluent monolayers of astrocytes (85% astroglial as assessed by GFAP immunoreactivity) were subcultured at low cell density and after 2-3 days growth were rendered quiescent by shifting them to low serum medium (0.25%) for several days. Cells could be stimulated to re-enter the proliferative compartment by challenging them with high concentrations of fetal bovine serum (5-10% FBS) or fibroblast growth factor (FGF). FGF added alone at a concentration of 25 ng/ml caused quiescent astrocytes to re-enter the cell cycle nearly as effectively as 5-10% serum. Moreover, when FGF (25 ng/ml) was combined with 0.5% serum there was a potentiation of the mitogenic effect seen with FGF alone. This synchronization scheme is an important tool for continuing studies of the growth factor and hormonal requirements for astroglial cell proliferation and differentiation.

Modulation of Cytokine-Induced Cyclooxygenase 2 Expression by PPARG Ligands Through NFκB Signal Disruption in Human WISH and Amnion Cells

Abstract Cyclooxygenase (COX) activity increases in the human amnion in the settings of term and idiopathic preterm labor, contributing to the generation of uterotonic prostaglandins (PGs) known to participate in mammalian parturition. Augmented COX activity is highly correlated with increased COX2 (also known as prostaglandin-endoperoxide synthase 2, PTGS2) gene expression. We and others have demonstrated an essential role for nuclear factor κB (NFκB) in cytokine-driven COX2 expression. Peroxisome proliferator-activated receptor gamma (PPARG), a member of the nuclear hormone receptor superfamily, has been shown to antagonize NFκB activation and inflammatory gene expression, including COX2. We hypothesized that PPARG activation might suppress COX2 expression during pregnancy. Using primary amnion and WISH cells, we evaluated the effects of pharmacological (thiazolidinediones) and putative endogenous (15-deoxy-Δ12,14-prostaglandin J2, 15d-PGJ2) PPARG ligands on cytokine-induced NFκB activation, COX2 expression, and PGE2 production. We observed that COX2 expression and PGE2 production induced by tumor necrosis factor alpha (TNF) were significantly abrogated by 15d-PGJ2. The thiazolidinediones rosiglitazone (ROSI) and troglitazone (TRO) had relatively little effect on cytokine-induced COX2 expression except at high concentrations, at which these agents tended to increase COX2 abundance relative to cells treated with TNF alone. Interestingly, treatment with ROSI, but not TRO, led to augmentation of TNF-stimulated PGE2 production. Mechanistically, we observed that 15d-PGJ2 markedly diminished cytokine-induced activity of the NFκB transcription factor, whereas thiazolidinediones had no discernable effect on this system. Our data suggest that pharmacological and endogenous PPARG ligands use both receptor-dependent and -independent mechanisms to influence COX2 expression.

Effects of pore size in 3‐D fibrous matrix on human trophoblast tissue development

The effects of pore size in a 3-D polyethylene terephthalate (PET) nonwoven fibrous matrix on long-term tissue development of human trophoblast ED27 cells were studied. Thermal compression was used to modify the porosity and pore size of the PET matrix. The pore size distributions in PET matrices were quantified using a liquid extrusion method. Cell metabolic activities, estradiol production, and cell proliferation and differentiation were studied for ED27 cells cultured in the thermally compressed PET matrices with known pore structure characteristics. In general, metabolic activities and proliferation rate were higher initially for cultures grown in the low-porosity (LP) PET matrix (porosity of 0.849, average pore size of 30 microm in diameter) than those in the high-porosity (HP) matrix (porosity of 0.896, average pore size of 39 microm in diameter). However, 17beta-estradiol production and cell differentiation activity in the HP matrix surpassed those in the LP matrix after 12 days. The expression levels of cyclin B1 and p27kip1 in cells revealed progressively decreasing proliferation and increasing differentiation activities for cells grown in PET matrices. Also, difference in pore size controlled the cell spatial organization in the PET matrices and contributed to the tissue development in varying degrees of proliferation and differentiation. It was also found that cells grown on the 2-D surface behaved differently in cell cycle progression and did not show increased differentiation activities after growth had stopped and proliferation activities had lowered to a minimal level. The results from this study suggest that the 3-D cell organization guided by the tissue scaffold is important to tissue formation in vitro.