The role of organ level conditioning on the promotion of engineered heart valve tissue development in-vitro using mesenchymal stem cells

Abstract

We have previously shown that combined flexure and flow (CFF) augment engineered heart valve tissue formation using bone marrow-derived mesenchymal stem cells (MSC) seeded on polyglycolic acid (PGA)/poly-L-lactic acid (PLLA) blend nonwoven fibrous scaffolds (Engelmayr, et al., Biomaterials 2006; vol. 27 pp. 6083-95). In the present study, we sought to determine if these phenomena were reproducible at the organ level in a functional tri-leaflet valve. Tissue engineered valve constructs (TEVC) were fabricated using PGA/PLLA nonwoven fibrous scaffolds then seeded with MSCs. Tissue formation rates using both standard and augmented (using basic fibroblast growth factor [bFGF] and ascorbic acid-2-phosphate [AA2P]) media to enhance the overall production of collagen were evaluated, along with their relation to the local fluid flow fields. The resulting TEVCs were statically cultured for 3 weeks, followed by a 3 week dynamic culture period using our organ level bioreactor (Hildebrand et al., ABME, Vol. 32, pp. 1039-49, 2004) under approximated pulmonary artery conditions. Results indicated that supplemented media accelerated collagen formation (approximately 185% increase in collagen mass/MSC compared to standard media), as well as increasing collagen mass production from 3.90 to 4.43 pg/cell/week from 3 to 6 weeks. Using augmented media, dynamic conditioning increased collagen mass production rate from 7.23 to 13.65 pg/cell/week (88.8%) during the dynamic culture period, along with greater preservation of net DNA. Moreover, when compared to our previous CFF study, organ level conditioning increased the collagen production rate from 4.76 to 6.42 pg/cell/week (35%). Newly conducted CFD studies of the CFF specimen flow patterns suggested that oscillatory surface shear stresses were surprisingly similar to a tri-leaflet valve. Overall, we found that the use of simulated pulmonary artery conditions resulted in substantially larger collagen mass production levels and rates found in our earlier CFF study. Moreover, given the fact that the scaffolds underwent modest strains (approximately 7% max) during either CFF or physiological conditioning, the oscillatory surface shear stresses estimated in both studies may play a substantial role in eliciting MSC collagen production in the highly dynamic engineered heart valve fluid mechanical environment.

Figure 1

Details of the TEVC showing (a) leaflet dimensions, (b) final TEVC. Also shown in (c) is the organ-level bioreactor [14] used for dynamic conditioning.

Figure 2

(a) CFF bioreactor geometry taken from [25] showing specimen locations. Note that an entrance length was added to the inlet of the CFD model and 4 specimens were simulated. (b) Representative TEVC leaflet shape under diastolic loading showing the cylindrical shape.

Figure 3

Representative H&E sections of MSC-seeded scaffolds incubated for 3 weeks in: (a) basal culture medium and (b) basal culture medium supplemented with bFGF/AA2P (Scale bar = 500 μm). Representative collagen I immunostained sections of MSC-seeded scaffold incubated for 6 weeks in: (c) basal culture medium and (d) basal culture medium supplemented with AA2P and bFGF (Scale bar = 100 μm). e) Evidence of collagen alignment (by polarized light microscopy) after 6 weeks of static tissue culture.

Figure 4

(a) Effects of bFGF/AA2P supplements to the media on net soluble collagen/DNA (μg collagen/μg DNA) after 3 and 6 weeks of static tissue culture. Also shown are the normalized concentrations of (b) Collagen, (c) GAG and (d) DNA content in TEVC leaflets grown for 3 and 6 weeks in static culture only, along with results for the 3 week static followed by 3 week dynamic tissue culture environment group (dynamic).

Figure 5

A compilation of the temporal evolution of collagen mass production for all groups investigated at the tissue and organ levels. Note that we projected collagen mass formation for the dynamically conditioned (combined modes of cyclic flexure and sub-physiologic shear stress levels (average ~1.1 to 1.8 dynes/cm² [25]) specimens [19] for comparison.

Figure 6

Linearized rates of collagen mass formation in TEVC at physiologically representative pulmonary artery pressure scales and in comparison to specimens conditioned (combined modes of cyclic flexure and sub-physiologic shear stress levels (average ~ 1.1 to 1.8 dynes/cm² [25]) in a CFF bioreactor [19]. Data computed from the information shown in Fig. 5.

Figure 7

CFD analysis performed on CFF bioreactor geometry previously developed by Engelmayr et al, [25] showing the number, location and bent configuration of specimens that were utilized for the CFD model in this study. As illustrated by the friction streamlines on the color maps, flow reversal occurs on bent rectangular specimens. Note the increasing presence of this phenomenon on the inner wall of the CFF specimens as the level of bending in the specimens is increased.

Figure 8

Oscillatory shear index (OSI) plot (as defined by He and Ku [37]) from flow vortex “A” and “B” as shown. Note a large portion of the line connecting the 2 vertices undergoes pure oscillatory flow, i.e., OSI = 0.5.

Figure 9

Wall shear stress magnitudes and friction streamlines previously observed in a native aortic valve [13]. Note the flow vortices on the aortic side of the valve and the ability to reproduce this phenomenon at the CFF bioreactor scale.