Underlying this work is the hypothesis that, first, it is possible to fabricate a pulmonary valve and artery in vitro by delivering autologous cells onto a temporary scaffold, and, second, that after implantation into an animal model, this structure will assume the tissue organization characteristic of a normal semilunar valve and artery by processes of remodeling and repair. A crucial early step in this process is the delivery of cells onto the naked scaffold. This article explores ways of achieving these ends.
Previously, our approach had been to make valves on an individual basis by delivering cells in a concentrated paste onto scaffolds held in an open dish. These constructs were left for an arbitrary period of time for cell attachment before being immersed in the cell culture medium. 1 Two theoretical problems are intrinsic to this approach: 1) obtaining an even distribution of cells is difficult to achieve by this method, and 2) there is the potential for cell death through the depletion of metabolites and the accumulation of toxic waste products in this extremely concentrated paste during the time allowed for cell attachment.
An alternative technique is to deliver cells by immersing the entire scaffold in a cell suspension. However, if the suspension were to remain static, then cells that fail to encounter the scaffold would deposit onto the base of the container and be effectively lost. Therefore, a means for maintaining these cells in suspension is required. Shaker and spinner flasks have been used for this purpose 2; however, the physical forces created by such violent movements have the disadvantage of tending to disrupt nascent cell-polymer interactions.
We wanted to investigate slow rotation as a means of keeping unattached cells in suspension while minimizing shear stress on cell-polymer interactions. Our group had previously demonstrated that the slow rotation of heart valve scaffolds, which were immersed in a cell suspension and contained in closed tubes, is superior to previous methods, in relation to distribution of cells over the construct (unpublished data). However, the absence of gas exchange in this system necessitates a daily change of medium and the resuspension of cells. This exposes the constructs to fluctuating pH and other tissue culture conditions. We, therefore, sought an alternative system that retained the characteristics of slow rotation but incorporated a means for gas exchange. Such a system is the rotating wall vessel (RWV), developed by National Aeronautics and Space Administration (NASA) scientists for the terrestrial study of the effects of diminished gravitational forces on cells in culture. In this system, scaffolds may be placed in the annular space between the cylindrical outer wall and the central core membrane (Figure 1). In terms of tissue engineering applications, the RWV has been used successfully to mature small pieces of polyglycolic acid (PGA) mesh that had been seeded with chondrocytes. 3 These constructs exhibited superior tissue formation to similar constructs that had been grown in either static or mixer flasks. 4 This paper describes an empiric comparison between rotating individual sealed tubes (RISTs) and the RWV as means for delivering cells to scaffolds for tissue engineering (Figure 1).
Scaffold Design and Construction
Polymer scaffolds were constructed using a nonwoven PGA mesh at 65 mg/ml density (Albany International Research, Mansfield, MA), dip-coated in a 1% solution of poly-4-hydroxybutyrate (Typha Inc., Cambridge, MA), as was previously described by our group. 1 A 40 mm length of mesh was wrapped over a 10 mm mandrel, and the overlap was joined by a heat application welding technique. A polystyrene collar (Kontes Glass Company, Vineland, NJ) was sutured to each end, with 5-0 PDS (Ethicon, Somerville, NJ), to add buoyancy and to maintain the structural form of the conduits. Constructs were sterilized with exposure to a cold cycle of ethylene oxide.
Establishing Primary Cell Cultures
A 1 cm segment of carotid artery was removed from an adult Dover sheep at the time of interposition grafting. The vessel lumen was flushed with phosphate buffered saline (PBS) to remove all traces of blood, and it was transferred to the laboratory in PBS. The segment of artery was minced with scissors into small pieces (<1 mm) that were placed on polystyrene tissue culture dishes (Becton Dickinson Labware, Franklin Lakes, NJ) under sterile coverslips. The primary cultures were grown in Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY), which was supplemented with 10% fetal bovine serum (Life Technologies), 1% antibiotic-antimycotic solution (Life Technologies), and 2 ng/ml human fibroblast growth factor B (Sigma-Aldrich, St. Louis, MO). The cultures were incubated in a humidified atmosphere that had been enriched with 5% CO2 at 37°C. Cells expanded in a 1:4 ratio into successively larger plates.
Characterization of Smooth Muscle Cells
Smooth muscle cells were characterized by the presence of α-smooth muscle actin. Contaminating endothelial cells were detected by the surface expression of CD31. Ovine endothelial cells, derived from the jugular vein, served as a positive control for the CD31 marker. Cells were passaged and plated onto 12 mm coverslips (Fisher Scientific, Pittsburgh, PA) and fixed with methanol. Mouse anti-smooth muscle actin immunoglobulin G (IgG) (Sigma) and goat anti-CD31 IgG (Santa Cruz Biotechnology, CA) were used as primary antibodies. Fluorescein conjugated anti-mouse IgG and Texas red conjugated anti-goat IgG (Vector Laboratories, Burlinghame, CA) were used as secondary antibodies. A mixture of purified mouse IgG and purified goat IgG served as a negative control. Coverslips were exposed to the primary antibody solution for 45 minutes, were washed, and were then exposed to the secondary antibody solution for a further 45 minutes. The coverslips were then washed, mounted onto slides, and viewed under an inverted phase contrast microscope (Carl Zeiss Inc., Thornwood, NY).
Delivery of Cells to Naked Scaffolds
Cells from passage 7 were trypsinized and resuspended in a fresh medium. The common suspension was then divided proportionally between the RWV (5 ml) and the RISTs (1 ml to each of five tubes). The same absolute number of smooth muscle cells was, therefore, available to each scaffold (770,000 cells/cm2). The RWV was filled to its 500 ml capacity with fresh medium and each RIST was filled to its 15 ml capacity. The RWV and the RISTs were attached to their respective rotating systems, and were placed in an incubator enriched with 5% CO2 at 37°C. At that point, rotation commenced. Medium in the RISTs was changed daily, and unattached cells were resuspended in the fresh medium.
Measurement of Physiologic Parameters and DNA Assay
Daily estimations of basic physiologic parameters pH, pO2, pCO2, and lactate concentration were performed on samples of medium from the RWV and discarded medium from the RISTs using a blood gas analyzer (Nova Biomedical, Waltham, MA).
After 6 days, conduits were removed from their respective systems. Each sample was divided into three equal segments, and the segments were assayed for DNA content using the phenol extraction technique. Results were expressed either as the weight of DNA per mg, the dry weight of the sample (μg/mg), or as the total DNA per conduit (μg).
Physiologic data and comparisons of total DNA per conduit were compared using a two-tailed Student’s t-test with equal variance assumption. The regional distribution of DNA between the center and each end was compared using a paired, two-tailed Student’s t-test with equal variance assumption.
All cells that were used to seed the constructs expressed α-smooth muscle actin and none of the cells stained positive for the endothelial cell surface marker CD31.
Total DNA per conduit was 226 ± 7 μg for the rotating individual sealed tubes and 396 ± 18 μg for the RWV (Figure 2). Overall, 75% more DNA was detected on the constructs that had been seeded within the RWV than on those that had been seeded in the RISTs (p < 0.001). The mean difference in the DNA content among samples taken from the central portion and samples taken from each end (Figure 2) was 0.6 ± 0.3 μg/mg dry weight for the RWV (p = 0.09) and 0.15 ± 0.11 μg/mg dry weight for the RISTs (p = 0.28).
Results for pH, pCO2, pO2, and lactate concentrations are shown in Figure 3. The pH, pCO2, and pO2 remained stable in the RWV at each interval sampled throughout the 6 day period, while lactate gradually increased. Except on the first day, the pO2 measurement was not significantly different between the RIST and RWV (p = 0.001, 0.381, 0.546, 0.483, 0.259). However, the pH measurement was consistently lower, and both the pCO2 and the lactate concentration measurements were consistently higher in the RIST system than in the RWV at each of the time points measured (p < 0.001).
This study was an empirical comparison between two cell delivery systems. The chosen end-point was their ability to deliver cells onto biodegradable scaffolds. The total amount of DNA per conduit was used as a biochemical surrogate for the total number of cells that attached to each sample conduit. On conduits that had been seeded in the RWV, 75% more DNA existed than on those conduits that had been seeded in the RISTs (p < 0.001). This suggests that the RWV is superior to the RISTs for delivering cells to cardiovascular scaffolds under these conditions. It is possible that some of the changes observed may be attributable to cell proliferation over the 6 day time frame that we allowed for cell delivery. Unfortunately, because a finite time is required for cells to attach to polymer scaffolds, it was not possible to separate out the two processes of cell delivery and cell proliferation in the study design. The results also suggest that it is possible to distribute cells evenly along the length of tubular constructs using both systems. This latter result is an important prerequisite for even tissue formation over the final construct.
Differences in the physiologic parameters that were demonstrated between the two systems were largely intuitive and follow from the reduced volume and available buffering capacity in the RISTs, as well as from the absence of a mechanism for gas exchange in the RISTs. They were measured in an attempt to explain expected differences in total DNA content demonstrated between the systems.
The difference in the total DNA content between conduits seeded within the two systems is a consequence of at least three variables: 1) differences in the total volume of distribution between the two systems and, therefore, in the available buffering capacity and supply of nutrients, 2) the presence of a mechanism for gas exchange in one system, and 3) differences in the physical forces experienced by cells and scaffolds within each system. It is not possible to distinguish which of these factors is the most important, but the data suggest that a mechanism for gas exchange is more important than merely concentrating cells around the scaffold.
Clearly, it will be important to look next at cell distribution over more complex geometric forms, such as that of a valved conduit scaffold, and to substantiate these findings with data of a more histologic type. Work is currently underway to devise a system that combines the benefits of gas exchange afforded by the RWV with a scaffold-enclosing membrane that restricts the movement of cells to the vicinity of the scaffold, yet allows the free movement of media, nutrients, and dissolved gases with the much larger volume of the RWV.
In the longer term, the ability to generate a group of at least two scaffolds with a cell density that falls within the relatively small sample variation demonstrated by this study will be a prerequisite to the design of experiments that integrate later steps in the tissue engineering process.
This work was supported by the Pappas Foundation and a grant (5RO1 H1 6,043-03) from the National Institutes of Health. Dr. Sutherland is supported by a Fellowship from the British Cardiac Society/Merck, Sharpe, and Dohme and by the Pathological Society of Great Britain and Ireland.
1. Hoerstrup SP, Sodian R, Daebritz S, et al: Functional living trileaflet heart valves grown in vitro
. Circulation 102 (Suppl 3): III44–III49, 2000.
2. Kim BS, Putnam AJ, Kulik TJ, Mooney DJ: Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices. Biotechnol Bioeng 57: 46–54, 1998.
3. Freed LE, Vunjak-Novakovic G, Langer R: Cultivation of cell-polymer cartilage implants in bioreactors. J Cell Biochem 51: 257–264, 1993.
4. Vunjak-Novakovic G, Martin I, Obradovic B, et al: Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res 17: 130–138, 1999.