Cyclic Mechanical Stimulation of SMCs: A Comparative Study between 2D and 3D Models

Bono, Nina (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)
Levesque, Lucie (BB, CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)
Loy, Caroline (BB, CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)
Candiani, Gabriele (Dept. of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Milan, Italy)
Fiore, Gianfranco B (µBS Lab, Dept. Electronics, Information and Bioengineering, Politecnico di Milano, Milan, Italy)
Mantovani, Diego (LBB, CRC-I, Dept. Min-Met-Materials Eng and CHU de Québec, Laval University, Quebec City, Canada)


With the ageing of the population worldwide and the increasing of the life expectancy, the demand for new reliable models to study physiological and pathological processes occurring in vascular tissues is growing. Vascular smooth muscle cells (VSMCs) populate the tunica media of blood vessels, where they are arranged concentrically inside the extracellular matrix (ECM). In physiologic conditions, VSMCs are in contractile phenotype and contribute to the maintenance of vascular tone by regulating vasoconstriction and have also a key role in the remodeling of the vessel wall. However, as response to vascular injuries or altered mechanical stimuli in vivo they can dedifferentiate into a synthetic phenotype. This plasticity is fundamental for vascular repair but it can also contribute to the development of vascular pathologies. It is well known that mechanical cues play pivotal roles in the regulation of SMCs phenotype and behaviors, and understanding the mechanisms underlying these processes would be essential for a better understanding of the etiology of vascular diseases. To now, both 2D and 3D in vitro models have been used to study the effects of mechanical solicitation (especially cyclic strain) on SMCs behavior but a direct comparison between the two culture conditions has never been reported in literature. This direct comparison is the purpose of this work.

Materials and Methods

Type I collagen was extracted from rat-tail tendon as previously described [1]. Primary Human Umbelical Artery SMCs (HUASMCs) were cultured in complete DMEM containing 10% FBS, 2 ng/ml FGF and 0.5 ng/ml EGF. After trypsinization, cells were resuspended in complete DMEM without growth factors and, for 2D cultures, seeded at a density of 3x10^4 cells/cm^2 in flexible silicone elastomer-bottomed 6-well culture plates (UniFlex®, Flexcell®) coated with collagen I. For 3D cultures, collagen gels were prepared by mixing 2 volumes of 4 mg/ml collagen I solution in 20 mM acetic acid, 1 volume of 60 mM NaOH in DMEM 3.5X and 1 volume of cell suspension, to a final cell density of 5x10^5 cells/ml. The cell-collagen mixture (3 ml) was cast in the central region experiencing uniaxial strain of UniFlex® plates using ad hoc made molds and let jellify for 1 hour at 37°C. Then complete DMEM without growth factors was added. Specifically designed anchoring systems were used to secure the gel to the elastic membrane. The experimental set up is shown in Fig. 1. After 24 hours, a computer-controlled vacuum-based system [2] was used to apply uniaxial 7% cyclic strain at 1 Hz for 2 and 5 days. Static controls were grown in the same conditions without any stretch. For histology, 3D constructs were fixed in formaldehyde, paraffin-embedded and cut into 10-μm thick sections and stained with Masson’s trichrome. For immunofluorescence of both monolayers and histology sections, cells were permealized with saponine, blocked with BSA and stained with phalloidin-rhodamine and DAPI. For Western blot analysis, both monolayers and gels were lysed in RIPA buffer, proteins quantified by BCA assay, separated in 4-15% gradient agarose gels by SDS-PAGE and then transferred to nitrocellulose membranes that were then stained against β-actin, α-actin and calponin.


The collagen matrix in 3D cell cultures was significantly remodeled all along the duration of the experiment, leading to construct compaction both in static and dynamic conditions. After 2 and 5 days of cyclic mechanical stimulation, HUASMCs cultured in monolayer aligned in the direction perpendicular to the strain, as shown in Fig. 2B, where the cytoskeleton of SMCs is stained. Oppositely, HUASMCs cultured inside collagen gel demonstrated alignment in the direction of the strain (Fig. 2D and 3B). The evaluation of the expression of α-actin and calponin is ongoing, but preliminary results suggest that dynamic stimulation in 2D cultures had minimal influence on the synthesis of contractile phenotype markers while a significant effect could be observed for 3D models.

Discussion and Conclusion

The behavior of HUASMCs in 2D and 3D models of cyclic uniaxial strain were compared. Collagen gels were selected as scaffolds for 3D cultures since they can directly and uniformly be seeded with cells during the fabrication process and, accordingly, collagen I coated supports were used for monolayer cultures. A completely opposite response to cyclic strain by HUASMCs was observed: cells aligned perpendicular to the strain direction in 2D cultures, parallel in 3D ones. The first behavior is quite peculiar, since it is completely opposite to what occurs in arteries in vivo, where SMCs are aligned circumferentially, thus orientated parallel to the strain while it is similar to what happens during intima formation [3]. Therefore, our results confirm the influence of mechanical stimuli on SMCs behaviors, but also highlight that the dimensionality of the culture support can strongly affect their final response. This might be attributed to the different surrounding environment and to the different mechanisms of transmission of the mechanical cues to the cells. Concluding, the comparative approach investigated herein, together with further investigation of VSMCs mechanobiology, will allow to identify the mechanisms involved in the response of VSMCs to dynamic stimulation in both 2D and 3D models. This is potentially susceptible to contribute to the design of in vitro models conceived ad hoc for the study of specific physiological and pathological conditions.

Fig. 1. Schematic of representation of 3D cell-gels (left) and cell monolayers (right) on UniFlex® flexible plates under stretch conditions.

Fig. 2. Immunofluorescence images of HUASMCs cultured in monolayers (2D; A, B) and inside collagen-gels (3D; C, D) for 5 days in static (A, C) or cyclic dynamic (B, D) conditions. Cytoskeleton was stained with phalloidin-rhodamine (red), nuclei with DAPI (blue). Arrows indicate the strain direction.

Fig. 3. Histology images of HUASMCs culture models. HUASMCs were cultured inside collagen-gels for 5 days in static (A) or cyclic dynamic (B) conditions. Histological sections were stained with Masson’s trichrome. Arrows indicate the strain direction.


Nina Bono was awarded of a PhD Scholarship from the Italian Ministry of Education and completed with a mobility scholarship from Italian InterPolitecnica School. Daniele Pezzoli was awarded a postdoctoral scholarship from NSERC CREATE program for regenerative medicine (NCPRM). This work was partially supported by NCPRM, NSERC, CFI, FQRS, and FQRNT.


1. Rajan N., Habermehl J., Cote M.-F., Doillon C. J., Mantovani D. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications, Nat Protoc. 2007, 1(6), 2753-8. 2. Levesque L., Mantovani D. The Effect of Dynamical Strain on the Maturation of Collagen-Based Cell-Containing Scaffolds for Vascular Tissue Engineering, Advanced Materials research. 2012, 409, 152-7. 3. Kockx M. M., Wuyts F. L., Buyssens N., Vandenbossche R. M., Demeyer G. R., Bult H., et al. Longitudinally orientated smooth-muscle cells in rabbit arteries, Virchows Archiv a-Pathological Anatomy and Histopathology.

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