Effect of cell seeding density on the mechanical and structural maturation of collagen gel-based tubular scaffolds for vascular tissue engineering

Camasao, Dimitria B (LBB/CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)
Pezzoli, Daniele (LBB/CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)
Loy, Caroline (LBB/CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)
Levesque, Lucie (LBB/CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)
Mantovani, Diego (LBB/CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)

Introduction

Vascular tissue engineering usually relies on the cellularization of scaffold materials with vascular cells with the ultimate aim to produce artificial living artery equivalents overcoming the drawbacks of current vascular grafts, especially for small diameter vessels. Type I collagen, main component of native vessel extracellular matrix (ECM), is a promising scaffold material owing to its favorable biological properties and to the possibility of adding cells during gel preparation. Conversely, cellularized collagen gels lack of mechanical properties, which are achieved in native vessels due to other ECM components (e.g. elastin) and to their proper structural organization. Smooth muscle cells (SMCs) populate the media layer of vascular wall and are responsible of ECM deposition and remodeling. These cells can adopt both contractile and synthetic phenotype in response to biochemical, mechanical and physical factors. When cultured in 3D systems, especially in collagen gels, SMCs are reported to shift to a more synthetic phenotype and the presence of fibroblasts also promotes a similar behavior1. In addition, SMCs compact and remodel the collagenous matrix thus increasing collagen density and construct mechanical properties. Generally, low cell seeding densities are reported in literature in collagen-based systems and the role of cell density on construct maturation has never been evaluated. In this light, herein, the influence of SMC density in cellularized tubular collagen gels on compaction, mechanical properties and gene expression was investigated.

Materials and Methods

Human umbilical SMCs (HUASMCs) were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of FBS, 1% of PenStrep, 0.05% insulin, 0.1% EGF and 0.1% FGF. Collagen type I was extracted from rat tail tendon, solubilized in acetic acid (0.02N) at 4mg/mL according to a protocol previously reported1. Cell-seeded collagen gels were prepared by mixing the collagen solution with a basic stock solution (60nM NaOH, 3.4X DMEM and 80mM Hepes) and cell suspensions in complete medium (DMEM supplemented with 10% of FBS and 1% of PenStrep) to final cell densities of 0.5, 1.5 and 4.5×106 cells/mL. The final solution was poured in tubular molds and left 1 hour at r.t. to allow gelification before addition of complete medium and incubation at 37oC. The mechanical properties were evaluated through circumferential stress-relaxation tests (5 progressive stress-relaxation cycles of 10% strain) and the equilibrium elastic modulus EEq was calculated. For IHC, samples were fixed in 3.7% formaldehyde, embedded in paraffin and cut in sections of 10 μm. Deparaffinised sections were stained by a Masson's trichrome procedure. Weigert's iron hematoxylin, acid fuchsin with xylidine ponceau and light green SF yellowfish were used to stain nuclei (dark brown), cytoplasm (red) and collagen (green), respectively. For real time PCR, RNA was isolated from constructs using TRIzol® reagent and retrotranscribed to cDNA (QuantiTect® RT kit). Real-time PCR was performed using the TaqMan® Gene Expression assay for GAPDH as an endogenous control, collagen, elastin and calponin. All the analyses were performed after 1, 3 and 7 days of static maturation. Comparisons among groups were performed by one-way ANOVA with post-hoc Tukey test. Significance was retained when 0.05>p.

Results

As expected, the SMC-mediated compaction of collagen gels (Figure 1) increased with time and cell density, but no difference could be observed between 1.5 and 4.5×106 cells/mL after 7 days of maturation. In parallel, also mechanical properties (Figure 1), evaluated in terms of EEq in stress-relaxation tests, increased with cell density but the ratio between EEq values at different cell densities did not decrease with time, demonstrating that mechanical reinforcement is not solely due to collagen compaction. Histological staining (Figure 2) confirmed the differences in cell density and compaction of the structure. Finally, real time PCR analyses showed an upregulation of collagen and elastin expression and a slightly downregulation of calponin for higher cell densities already after 1 day of maturation.

Discussion and Conclusion

The time course of compaction of collagen gels cellularized with SMCs showed a similar behavior as reported for fibroblasts, with a rapid initial contraction phase followed by a slow one2. Sample volume tended to achieve a plateau and, the higher the cell density, the faster the plateau was reached. Histological stainings at day 7 confirmed the denser structure of the highest density construct. In this experimental condition, not only water was expelled faster but also cell-mediated ECM remodeling led to superior mechanical properties, with an EEq of ca. 0.065 MPa, two- and more than three-fold higher than for 1.5 and 0.5 ×106 cells/mL and approaching values reported in literature for coronary arteries3. These results may suggest that high SMC densities can stimulate ECM remodelling and deposition by promoting a shift towards a more synthetic phenotype. The PCR results supported this hypothesis as the expression by SMCs of important components of the ECM such as elastin and collagen was upregulated by cell density. Altogether, this study demonstrates that, by profoundly influencing gel compaction, phenotype, ECM remodeling and deposition, cell density plays a key role in the process of maturation of SMC-cellularized collagen gels and that high densities should be preferred in vascular tissue engineering approaches.


Figure 1: Volumetric compaction (%) and equilibrium elastic modulus (MPa) for each cell seeding density condition of tubular collagen gels after 1, 3 and 7 of static maturation. *0.05>p, 4.5 millions cells/mL vs. 1.5 millions cells/mL and 0.5 million cells/mL.

Figure 2: Histological staining by Masson Trichrome after 7 days of static maturation. Nuclei are stained in dark brown, cytoplasm in red and collagen in green. Scale bar 200µm.

Acknowledgements

DBC and DP were awarded a MSc and a Postdoctoral Scholarship, respectively, from NSERC CREATE Program in Regenerative Medicine (www.ncprm.ulaval.ca). This work was partially supported by NCPRM, NSERC, CFI, FQRS, and FQRNT.

References

1. Loy, C.; Meghezi, S.; Lévesque, L.; Pezzoli, D.; Kumra, H.; Reinhardt, D.; Kizhakkedathu, J. N.; Mantovani, D. A planar model of the vessel wall from cellularized-collagen scaffolds: focus on cell-matrix interactions in mono-, bi- and tri-culture models, Biomater Sci. 2016, 5, 153–162.

2. Nishiyama, T.; Tominaga, N.; Nakajima, K.; Hayashi, T. Quantitative Evaluation of the Factors Affecting the Process of Fibroblast-Mediated Collagen Gel Contraction by Separating the Process into Three Phases, Coll Relat Res. 1988, 8(3), 259-273.

3. Garcia, M.; Kassab, G. S. Right coronary artery becomes stiffer with increase in elastin and collagen in right ventricular hypertrophy, J Appl Physiol. 2009, 106(4), 1338-1346.

Copyright ©1990 - 2018
Web Development by CrookedBush.com Inc.

Close Drag