Engineering small-caliber arterial models in a biomechanical in vitro platform

Bono, Nina (LBB, Laval University, Quebec City, Canada and μBS Lab, Politecnico di Milano, Milan, Italy)
Meghezi, Sebastien (LBB, Laval University, Quebec City, Canada)
Soncini, Monica (μBS Lab, Politecnico di Milano, Milan, Italy)
Fiore, Gianfranco Beniamino (μBS Lab, Politecnico di Milano, Milan, Italy)
Mantovani, Diego (LBB, Laval University, Quebec City, Canada)

Introduction

Vascular tissue engineering has made significant advancement over the past decades toward the definition of optimal scaffolds, cell sources, and biomechanical culture parameters for obtaining engineered tissue (ET) products that resemble native tissues. Although a great effort has been made for the generation of functional ETs, there are still numerous challenges and hurdles before widespread clinical use is achieved. In the meantime, and together with the goal of generating grafts for patient implantation, vascular ETs could provide reliable models, allowing to investigate physiological and pathological cellular mechanisms and structure–function relationships in a controlled environment [1]. Accordingly, this work represents the first step aimed at engineering small-caliber arterial models which will be obtained by: i) fabricating ETs by direct mixing of collagen gels with cells in a one-step process; ii) maturation of the constructs by means of a previously developed bioreactor [2] able to stimulate cell growth, and the regeneration of ETs.

Materials and Methods

Architecture of the bioreactor. The bioreactor (Fig. 1, B) consists of: i) a culture chamber; ii) hydraulic circuit and actuators (pump and solenoid pinch-valve); iii) a monitoring and control system (M/C). Arterial model fabrication and culture. Small-caliber arterial models were fabricated by the direct assembling of rat-tail collagen and porcine smooth muscle cells (pSMCs) in a tubular shape with the use of mandrel-molding technique as reported in Fig. 2. The mold was prepared by inserting a 3-ml syringe over a silicone sleeve (2.8mm ID x 3.2mm OD x 4mm long), mounted within the bioreactor, that served as a mandrel for the constructs. Type I collagen was extracted from rat-tail tendon [3], solubilized in acetic acid solution (0.02 N), sterilized, and mixed with 25% (v/v) of buffer solution (15 mM NaOH, and 20 mM Hepes in deionized water) and 25% (v/v) of the cell suspension (final concentration 1x10^6 cells/ml) in DMEM supplemented with 10% FBS, 10% PS, 1% pen-strep. Afterwards, the cells-collagen mixture was poured into the mold-equipped bioreactor, and let gel for 1h at room temperature. After the gelation occurred, the molds were removed,and the constructs were then transferred into the reservoir of the bioreactor filled with 40 mL of culture medium and incubated (T = 37 °C, 5% CO2, 100% humidity) for different time steps (1, 3, 7 days). Histological analyses. Immediately after gelation and 1, 3, 7 days of static culture, samples were harvested, were fixed with 3.7% formaldehyde, paraffin-embedded and cut into 10-μm thick sections. Sections were stained with Masson's trichrome (nuclei stain dark brown, muscle stain red, collagen stains green). Thickness measurements were performed on stained sections whose digital images were acquired using a light microscope (magnification 20x).

Results

Biological tests were carried out in order to evaluate tissue viability and integrity of the ETs. Histological analysis showed that the pSMCs were distributed homogeneously throughout the thickness of the constructs (Fig. 3). Moreover, the constructs exhibited high circumferential compaction around 72% of their molded dimensions at day 1, and the constructs contract to 77% at day 7, due to the cell-driven early remodeling of collagen gel matrix.

Discussion and Conclusion

In this study we present a strategy for the in vitro fabrication of vascular ETs in a one-step process by mixing collagen gels with cells. Herein, the feasibility of using the bioreactor in combination with cellularized collagen-based ETs has been demonstrated. In fact, the device was used since the early fabrication step supplying a monitored and controlled sterile environment where constructs can grow and regenerate. The device, conceived as a laboratory-oriented tool for stimulating the regeneration of vascular ETs, is also able to replicate in vitro the physiological hemodynamic forces, while integrating a mechanical testing operating mode for the evaluation biomechanical status of hosted samples. Further experiments have been conceived and planned with the aim i) to investigate how different biomechanical forces impact the architecture of the ETs; ii) to provide insights into the interplay between cells and ECM during growth and remodeling in response to mechanical stimuli.


Fig. 1. A) 3D CAD model of the culture chamber. The chamber includes a commercial reservoir (50-ml falcon tubing) and a housing, which is integrated with the reservoir cap. B) Layout of the bioractor: blue lines, hydraulic circuit; gray line, monitoring and control (M/C) signal.


Fig. 2 Fabrication of the constructs in sterile conditions. The cells and collagen mixture was poured into the mold-equipped bioreactor (A), and let gel for 1 h at room temperature (B). Afterwards, the mold was removed (C), the bioreactor was assembled (D) and incubated for 1, 3, or 7 days.


Fig. 3 Masson’s trichrome-stained sections of the harvested constructs at day 1, 3, 7 of culture (left panel: low power of magnification at 5x; right panel: high power of magnification at 20x).

Acknowledgements

Nina Bono was awarded of a PhD Scholarship from the Italian Ministry of Education and completed with a mobility scholarship from Scuola Interpolitecnica di Dottorato, Italy. This work was partially supported by NSERC, CFI, FQRS, and FQRNT.

References

1. Gibbons, M. C., Foley, M. A. & Cardinal, K. O. Thinking Inside the Box: Keeping Tissue-Engineered Constructs In Vitro for Use as Preclinical Models. Tissue Eng Part B: Reviews 19 (1), 14-30, doi:10.1089/ten.teb.2012.0305 (2013). 2. Piola, M., Prandi, F., et al. A compact and automated ex vivo vessel culture system for the pulsatile pressure conditioning of human saphenous veins. J-TERM, doi:10.1002/term.1798 (2013). 3. Rajan, N., et al. Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nature Protocols 1 (6), 2753-2758, doi:10.1038/nprot.2006.430 (2007).

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