Elsevier

Biomaterials

Volume 28, Issue 17, June 2007, Pages 2738-2746
Biomaterials

Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization

https://doi.org/10.1016/j.biomaterials.2007.02.012Get rights and content

Abstract

Biodegradable synthetic matrices that resemble the size scale, architecture and mechanical properties of the native extracellular matrix (ECM) can be fabricated through electrospinning. Tubular conduits may also be fabricated with properties appropriate for vascular tissue engineering. Achieving substantial cellular infiltration within the electrospun matrix in vitro remains time consuming and challenging. This difficulty was overcome by electrospraying smooth muscle cells (SMCs) concurrently with electrospinning of a biodegradable, elastomeric poly(ester urethane) urea (PEUU) small-diameter conduit. Constructs were cultured statically or in spinner flasks. Hematoxylin and eosin (H&E) staining demonstrated qualitatively uniform SMCs integration radially and circumferentially within the conduit after initial static culture. In comparison with static culture, samples cultured in spinner flasks indicated 2.4 times more viable cells present from MTT and significantly larger numbers of SMCs spread within the electrospun fiber networks by H&E image analysis. Conduits were strong and flexible with mechanical behaviors that mimicked those of native arteries, including static compliance of 1.6±0.5×10−3 mmHg−1, dynamic compliance of 8.7±1.8×10−4 mmHg−1, burst strengths of 1750±220 mmHg, and suture retention. This method to rapidly and efficiently integrate cells into a strong, compliant biodegradable tubular matrix represents a significant achievement as a tissue engineering approach for blood vessel replacement.

Introduction

Given the incidence of coronary and peripheral arterial disease—and the complications associated with treatment—the need for engineered tissue replacements for small-diameter blood vessels is clear. Research has been pursued by a wide variety of groups aimed at creating a blood vessel tissue equivalent based upon cells and extracellular matrix (ECM) alone (cultured into sheets and rolled) [1], [2], cells combined with natural materials (e.g. fibrin and collagen) [3], [4], [5], [6], and cells combined with synthetic matrices (e.g. hydrolytically labile polyesters) [7], [8], [9], [10]. Common to many of these approaches, particularly those that possess higher cell densities are inadequate vessel mechanical properties. Although a synthetic or processed natural matrix can provide mechanical support, this usually comes at the expense of long culture times, which can be on the order of months [1], [2], [9].

A critical requirement of blood vessel replacements is accurate replication of the original vessel compliance. Compliance mismatch is a complex phenomenon involving the mechanical response of blood vessel walls to pulse waves. At the interfaces between native arteries and graft (anastomoses) [11] the flow can be perturbed causing altered-shear stress areas, which favor platelet deposition and thrombosis. These complications can further lead to myointimal hyperplasia and consequent graft failure. Therefore, in developing an ideal blood vessel replacement, it is necessary to not only create a non-thrombogenic luminal surface but to also closely replicate the elastic properties of the vessel wall.

Biodegradable elastomers represent ideal scaffolding materials to utilize for replication of native vessel compliance behaviors. In particular, thermoplastic biodegradable polyurethanes offer a high degree of tunability achievable by appropriate selection of hard and soft segments. An elastomeric biodegradable poly(ester urethane) urea (PEUU) developed in our laboratory based on a poly(ε-caprolactone) diol soft segment and a hard segment of 1,4-diisocyanatobutane and putrescine has been shown to be cytocompatible, produce non-toxic degradation products, and be processable into scaffolding by an electrospinning technique [12], [13].

Electrospun PEUU nanofibers are attractive in that they possess ECM of similar architecture appropriate to serve as cell scaffolding [13]. Moreover, the electrospun PEUU matrix can be produced with the appropriate tissue mimicking mechanical anisotropy appropriate for tissue scaffolding [14]. To produce a highly cellularized construct, but to also provide substantial elastomeric mechanical support, we were the first to develop an approach wherein a meshwork of submicron elastomeric fibers was built during cellular placement [15]. This process, termed “microintegration,” utilized electrohydrodynamic atomization to simultaneously electrospray cells while electrospinning PEUU nanofibers. This process overcame inherent challenges in obtaining cell infiltration into the small pore sizes of an electrospun scaffold in vitro by literally surrounding cells with the fiber matrix as it was constructed. The resulting scaffold sheets were strong and flexible with large numbers of spread cells infiltrated throughout their bulk after only a few days of perfusion culture [15].

In this work, the microintegration process has been extended to fabrication of small-diameter tubular conduits appropriate for blood vessel tissue engineering. Vascular SMCs were electrosprayed during electrospinning of a small-diameter biodegradable and elastomeric PEUU tubular conduit. The objective was to utilize this combined electrohydrodynamic atomization method to produce highly cellularized small-diameter conduits after only a few days in culture that possessed mechanical properties similar to native vessels. These constructs were cultured under appropriate static or spinner flask conditions and subsequently characterized for their cellularity and mechanical properties. Viable cell numbers were measured at each timepoint and cell morphologies within construct cross-sections were examined. Mechanical properties of the constructs such as static and dynamic compliance, stiffness index, burst pressure, ultimate tensile stress, stretch to failure, modulus, and suture retention were calculated as appropriate for vascular tissue engineering.

Section snippets

Polymer synthesis

Solvents dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF) were dried over 4-A molecular sieves. Polycaprolactone diol (PCL, Aldrich, MW=2000) was dried under vacuum for 48 h at 50 °C. Putrescine (Sigma) and 1,4-diisocyanatobutane (Fluka) were distilled under vacuum. Stannous octoate (Sigma) catalyst and hexafluoroisopropanol (HFIP, Oakwood) solvent were used as received. Biodegradable and biocompatible PEUU was synthesized from BDI and PCL with putrescine chain extension as described

Conduit microintegration

In order to extend the technology of cellular microintegration to small-diameter tubes, a 4.7 mm diameter stainless-steel mandrel was used in the place of the previously employed 19 mm diameter mandrel for sheet microintegration [15]. To manufacture reproducible highly cellular and defect free small-diameter tubular constructs, it was necessary to slightly decrease electrospraying distance from 5.0 to 4.5 cm and lower the mandrel negative charge from −10 to −3 kV from previous methods. During

Discussion

Efforts have been extensive in the tissue engineering community to develop a highly cellular and functional blood vessel replacement [1], [2], [5], [21], [22], [23]. Central to this theme of functionality was development of grafts that have adequate burst strength and compliance to function in an in vivo environment. Electrospinning technology, in particular, has been studied for its ability to fabricate small-diameter tubular constructs with fiber sizes and architectures that mimic the ECM [24]

Conclusions

Cellular microintegration offers a means to simultaneously seed cells and fabricate nanofibrous tubular scaffolding. More specifically, SMCs were electrosprayed concurrent with PEUU electrospinning to produce a hybrid tissue engineered blood vessel construct that was highly cellular and reinforced with an elastomeric fiber matrix. These conduits were demonstrated to be cytocompatible, strong, and possess compliance values similar to native vessels. These methods represent a significant advance

Acknowledgements

This work was supported by the Commonwealth of Pennsylvania and the National Institutes of Health, (#HL069368). We sincerely acknowledge Drs. Jianjun Guan, Alejandro Nieponice, and Michael Sacks for their support and guidance on this project. In addition, we thank Dan McKeel for his assistance in refining the rotating target used during fabrication.

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    These authors contributed equally to this manuscript.

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