Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.
Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.
The fabrication technique using a biodegradable elastomer described here has several features. (1) We used hyaluronic acid (HA) as a mold release. Since HA is water soluble, scaffold was easily released from the glass mold after soaking it into water. In this report, we used 1.0 wt/vol % of HA solution because low concentration (< 0.5 wt/vol %) of solution is not viscous and flows down so fast when we pouring it on top of glass tubing. To coat HA solution uniformly, we flipped over the glass tubing when the solution flew down the bottom of the tubing and repeated this step. This HA coating is a critical to our fabrication procedure for releasing final scaffolds. (2) We used heat-shrinkable (HS) sleeve for retaining salts in the glass tubing. Since salts were densely packed in the space between the inner wall of glass tubing and the HS sleeve, HS sleeve retained salts after removing mandrel and PTFE ring in the bottom of the tubing. We could remove HS sleeve easily by putting the mold into an oven at 120 °C for 5 min, and then get tubular salt templates. (3) We used the salt fusion method. It is well known that salt fusion method can improve pore interconnectivity and mechanical properties by varying fusion time10. Furthermore, since we used PGS, macro-pores were produced by the salt particles during leaching process, while the micro-pores were likely generated by glycerol vapor formed during PGS curing as we described previously11. Thus, this method has a potential to fabricate porous tubular scaffolds with different macro- and micro-structures by varying salt particles as well as PGS curing condition.
The mechanical conditioning from the bioreactor has provided pulsatile flow perfusion (maximum mean flow = 14 ml/min, maximum shear stress = 15.3 dyne/cm2, frequency = 0.5 – 1.7 Hz) and physiologically relevant pressure with the PGS scaffold, which led to SMC growth and orientation (Fig. 4). These results are consistent with previous studies reporting that cyclic stretch at this frequency and shear stress increases SMC proliferation12, and ECM protein production13,14. In addition to SMC growth and orientation, PGS construct supported ECM protein production, especially circumferentially organized elastic fibers (Fig. 5) within 3-week culture in the bioreactor. Some studies using an elastomeric scaffold as a small-diameter arterial construct have demonstrated mechanical strength and burst pressure comparable to native arteries15, and rapid SMC integration in compliant scaffolds using spinner flask16,17, while no elastic fibers were found in these constructs. Our results suggest that cyclic radial distension from the bioreactor improved mechanical signal transduction more effectively to SMCs in PGS scaffold, which likely contributed to elastin synthesis and organization.
Since vascular SMCs were the only cells that produced ECM proteins in our approach, quiescent endothelium and improve mechanical strength are necessary to develop a clinically successful small-diameter arterial constructs. We have reported that endothelial cells co-cultured with SMCs generated a confluent monolayer and supported phenotype protein expression under our culture conditions and mechanical conditioning9. Therefore, based on our approach described here, modification of co-culture experiment conditions would be a next step to improve the functions of resultant constructs and generate nonthrombogenic, robust, and compliant arterial construct similar to native arteries.
The authors have nothing to disclose.
The author thanks Dr. Jin Gao for PGS synthesis, Dr. Peter Crapo for insightful discussion for bioreactor setup, Drs. Mohamed Ezzelarab and Wei Wu for explanting baboon carotid arteries. This study was supported by a grant from the National Institutes of Health (R01 HL089658).
Name of the reagent | Company | Catalogue number |
---|---|---|
Hyaluronic acid sodium salt | Sigma-Aldrich | H7630 |
Tetrahydrofuran | Sigma-Aldrich | 401757 |
MCDB 131 | Mediatech | 15-100-CV |
Fetal bovine serum | Lonza | BW14-502F |
L-glutamine | Mediatech | 25-005-CV |
Ascorbic acid | Fisher Scientific | A62-500 |
Antibiotic-antimycotic solution | Mediatech | 30-004-CI |
Phosphate Buffered Saline (PBS) | Mediatech | 21-031-CV |
Tissue-Tek optimal cutting temperature compound, 4583 | Sakura Finetek | 25608-930 |