Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds

doi:10.1016/j.biomaterials.2004.04.036

Biomaterials

Volume 26, Issue 12, April 2005, Pages 1405-1411

https://doi.org/10.1016/j.biomaterials.2004.04.036 Get rights and content

Abstract

Blood vessels are subjected in vivo to mechanical forces in a form of radial distention, encompassing cyclic mechanical strain due to the pulsatile nature of blood flow. Vascular smooth muscle (VSM) tissues engineered in vitro with a conventional tissue engineering technique may not be functional, because vascular smooth muscle cells (VSMCs) cultured in vitro typically revert from a contractile phenotype to a synthetic phenotype. In this study, we hypothesized that pulsatile strain and shear stress stimulate VSM tissue development and induce VSMCs to retain the differentiated phenotype in VSM engineering in vitro. To test the hypothesis, rabbit aortic smooth muscle cells (SMCs) were seeded onto rubber-like elastic, three-dimensional PLCL [poly(lactide-co-caprolactone), 50:50] scaffolds and subjected to pulsatile strain and shear stress by culturing them in pulsatile perfusion bioreactors for up to 8 weeks. As control experiments, VSMCs were cultured on PLCL scaffolds statically. The pulsatile strain and shear stress enhanced the VSMCs proliferation and collagen production. In addition, a significant cell alignment in a direction radial to the distending direction was observed in VSM tissues exposed to radial distention, which is similar to that of native VSM tissues in vivo, whereas VSMs in VSM tissues engineered in the static condition randomly aligned. Importantly, the expression of SM α-actin, a differentiated phenotype of SMCs, was upregulated by 2.5-fold in VSM tissues engineered under the mechano-active condition, compared to VSM tissues engineered in the static condition. This study demonstrates that tissue engineering of VSM tissues in vitro by using pulsatile perfusion bioreactors and elastic PLCL scaffolds leads to the enhancement of tissue development and the retention of differentiated cell phenotype.

Introduction

A number of studies have shown that mechanical environments regulate the characteristics of tissue-engineered vascular smooth muscle (VSM). Vascular smooth muscle cells (VSMCs) in vivo typically reside in mechanically dynamic environments, align in a specific direction, and exist in a contractile, differentiated phenotype [1], which is critical for contractile functions of VSM [2]. However, VSM tissues engineered in vitro using conventional tissue engineering techniques may not be functional, because VSMCs cultured in vitro usually do not align [3] and revert from a contractile, differentiated phenotype to a synthetic, nondifferentiated phenotype [4], [5]. Several studies have shown that mechanical signals significantly regulate the phenotype of VSMCs in two-dimensional [6], [7], [8], [9], [10] or three-dimensional culture systems [2], [11], [12], [13], [14], [15]. Engineering VSM or blood vessels under mechanical active conditions resulted in enhanced mechanical strength, collagen production, or blood vessel patency [11], [16].

The scaffolds for tissue engineering should have an interconnected porous structure so that the cells seeded can migrate into the scaffolds. In addition, it would be necessary to develop highly elastic scaffolds if one wishes to engineer VSM tissues or blood vessels under mechanically active conditions. For these applications, the scaffolds must be rubber-like elastic and able to deliver mechanical signals to VSMCs adherent onto the scaffolds. The scaffolds should also degrade properly and be replaced by newly forming tissues. In previous reports, we developed highly elastic, biodegradable poly-(glycolide-co-caprolactone) (PGCL) [17] and poly-(lactide-co-caprolactone) (PLCL) scaffolds [18]. These scaffolds were flexible and rubber-like elastic to show a complete recovery under cyclic stretching in culture media for up to 2 weeks. Furthermore, PLCL scaffolds exhibited good biocompatibility for VSMCs and proper degradation in vivo [18], [19].

The aim of this study was to investigate the effect of a mechanical stimulation on the proliferation and phenotype of VSMCs. We hypothesized that a radial distention induces the phenotype of VSMCs in in vitro engineered tissues to be similar to that of SMCs in native tissues in vivo. To test the hypothesis, aortic SMCs were seeded onto elastic three-dimensional scaffolds and subjected to a pulsatile perfusion system (bioreactor). Very elastic PLCL scaffold were applied to take the advantage of rubber-like elasticity capable to deliver the mechanical signal during the cell culture. For the comparison, VSMCs on scaffolds were cultured statically too. The proliferation and cellular alignment of VSMCs, collagen synthesis, and the expression of SM α-actin and SM myosin heavy chain, phenotypic markers of VSMCs, were examined in the engineered VSM tissues.

Section snippets

Preparation of PLCL scaffolds

PLCL was synthesized and tubular scaffolds were fabricated from the PLCL using methods described previously [18], [19]. Briefly, l-lactide (100 mmol) and ε-caprolactone (100 mmol) were polymerized at 150°C for 24 h in the presence of 1,6-hexanediol (0.5 mmol) and stannous octoate (1 mmol) as catalysts. PLCL tubular scaffolds were fabricated by an extrusion-particulate leaching technique. PLCL was dissolved in chloroform (1% w/v), mixed with by NaCl particles (100–200 μm), and extruded into tubes (in 4

Results

Tubular PLCL scaffolds fabricated by an extrusion-particulate leaching technique showed an interconnected porous structure (Fig. 2). The average pore size and porosity of scaffolds were about 150±50 μm and 90%, respectively, as determined with SEM and mercury porosimetry. In dry states, the scaffold exhibited the tensile strength and the elongation at break 0.81 MPa and 210%, respectively. The porous scaffolds showed a recovery over 97% after elongation up to 130%. Mechanical properties and

Discussion

Engineered tissues must be functional to be utilized clinically, and mechanical stimulation may be critical for tissue engineering of functional SM. An important function of the SM element in many tissues (e.g., blood vessels and intestines) is contracting to regulate blood pressures or transfer fluids. SMCs in vivo normally align in a specific direction and exist in a contractile or differentiated state, which is critical for contractile functions of SM [1], [2]. However, SMCs cultured in

Acknowledgements

This work was supported by Korean Ministry of Science & Technology, National Research Labs Program (Grants No. 2N23480, 2N24620 and 2N26670).

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Mechano-active tissue engineering of vascular smooth muscle using pulsatile perfusion bioreactors and elastic PLCL scaffolds

Abstract

Introduction

Section snippets

Preparation of PLCL scaffolds

Results

Discussion

Acknowledgements

Int Rev Cytol

J Urol

J Vasc Surg

Biomaterials

Anal Biochem

Euro J Vasc Endovasc Surg

The mechanical environment of the artery

Tissue engineering of smooth muscle under a mechanically dynamic condition

J Microbiol Biotechnol

Orientation response of arterial smooth muscle cells to mechanical stimulation

Eur J Cell Biol

Mechanotransduction by vascular smooth muscle

J Vasc Res

Mechanical strain induces growth of vascular smooth muscle cells via autocrine action PDGF

J Cell Biol