Oxygen tension and formation of cervical-like tissue in two-dimensional and three-dimensional culture

Abstract

Cervical dysfunction contributes to a significant number of preterm births and is a common cause of morbidity and mortality in newborn infants. Cervical dysfunction is related to weakened load bearing properties of the collagen-rich cervical stroma. However, the mechanisms responsible for cervical collagen changes during pregnancy are not well defined. It is known that blood flow and oxygen tension significantly increase in reproductive tissues during pregnancy. To examine the effect of oxygen tension, a key mediator of tissue homeostasis, on the formation of cervical-like tissue in vitro, we grew primary human cervical cells in both two-dimensional (2D) and three-dimensional (3D) culture systems at 5% and 20% oxygen. Immunofluorescence studies revealed a stable fibroblast phenotype across six passages in all subjects studied (n=5). In 2D culture for 2 weeks, 20% oxygen was associated with significantly increased collagen gene expression (p<0.01), increased tissue wet weight (p<0.01), and increased collagen concentration (p=0.046). 3D cultures could be followed for significantly longer time frames than 2D cultures (12 weeks vs. 2 weeks). In contrast to 2D cultures, 20% oxygen in 3D cultures was associated with decreased collagen concentration (p<0.01) and unchanged collagen gene expression, which is similar to cervical collagen changes seen during pregnancy. We infer that 3D culture is more relevant for studying cervical collagen changes in vitro. The data suggest that increased oxygen tension may be related to significant cervical collagen changes seen in pregnancy.

FIG. 1.

Top panel: polytetrafluoroethylene-coated, stainless steel wire was press fit into a polyethylene vial to create the scaffold mold (1). The scaffold was shaped like a cylinder with a central canal (2). The scaffolds were cultured in a spinner flask bioreactor (3). Cervical-like tissue was present on the scaffold exterior and interior after 5 weeks (4). The morphology of cervical-like tissue resembled the stroma of native cervical tissue. Middle panel: Timeline for two-dimensional experiment. At the end of the 2 week period, the cell sheet was scraped from the well and assayed. Bottom panel: Timeline for the three-dimensional experiment. After 9 weeks, cervical-like tissue covered the scaffold and grew into the central canal. 2D, two-dimensional; 3D, three-dimensional. Color images available online at www.liebertonline.com/tea

FIG. 2.

Left panel: cervical cells demonstrated strong immunofluorescence signal for fibroblast markers for all subjects (n=5) and all passages (up to passage 6). Right panel: cervical cells remained viable (green signal) on the silk scaffolds during the 12 week culture period. The cells in the 2D experiment appeared the same as in the 3D experiment (not shown). Color images available online at www.liebertonline.com/tea

FIG. 3.

Two-dimensional culture. Compared with 5% oxygen, 20% oxygen was associated with increased cell proliferation (p=0.01), increased cell metabolism (p=0.007), increased collagen concentration (p=0.046), and increased tissue wet weight (p<0.01). Compared with 5% oxygen in static conditions, 5% oxygen on a lab rotator was associated with increased tissue wet weight (p<0.01) and increased S-GAG (p=0.05). No differences in construct hydration were seen for the three experimental conditions. Data expressed as mean±SD of four replicates. Experimental results are representative of experiments from three subjects. S-GAG; sulfated glycosaminoglycan; SD, standard deviation.

FIG. 4.

Three-dimensional culture. Left panel shows construct histology as a function of time and oxygen tension. Tissue production peaked between the 3 and 6 week time points at both oxygen tensions. At the 6 week time point, histological appearance suggests increased tissue production at 5% oxygen tension. The scale bar is 100 μm. The top, right panel shows gross morphology at the 5 week time point. No gross differences were seen between 5% and 20% oxygen at this time point or any other time point. The bottom right panel shows collagen production as a function of time and oxygen tension. Twenty percent oxygen was associated with nearly 15% decrease in collagen production (p<0.01, two-way analysis of variance). Data expressed as mean±SD of five biological replicates. Color images available online at www.liebertonline.com/tea

FIG. 5.

Real-time quantitative reverse transcription–polymerase chain reaction of matrix associated genes. In the 2D experiment (left panel), COL1A1 and COL3A1 were significantly upregulated in 20% compared with 5% oxygen (p<0.01). There were no differences in collagen gene expression between 5% oxygen in static culture versus 5% oxygen on a lab rotator. In the 3D experiment (right panel). Collagen gene expression was significantly downregulated at all time points compared with the baseline time point. There were no differences in collagen gene expression between 5% and 20% oxygen in 3D culture. No differences were seen in gene expression of Decorin or HAS2. Gene symbols: COL1A1, collagen type I, alpha 1; COL3A1, collagen type III, alpha 1; HAS2, hyaluronan synthase 2; Gene expression was normalized by the geometric mean of two housekeeping genes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta actin. Data are expressed as mean±SEM of four biological replicates. SEM, standard error of the mean.