Novel porous aortic elastin and collagen scaffolds for tissue engineering

Abstract

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress–strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 μm into elastin scaffolds and about 40 μm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.

Introduction

Tissue engineering offers the potential to create functional and viable tissue constructs for patients requiring organ replacement. One potential approach for creating tissue constructs is to isolate cells from the patient, expand the cell population, culture cells in vitro on a scaffold, and then implant the resulted tissue engineered construct back into the patient. In this approach, a three-dimensional (3-D) scaffold is necessary to serve as a template to guide cell growth and tissue development. Formation of the new tissue is greatly influenced by the chemical composition, porosity and 3-D structure of the scaffold [1]. Several requirements have been identified to be crucial to tissue engineered scaffolds: (1) biocompatibility, to prevent unwanted host tissue responses to the implant; (2) appropriate surface chemistry to promote cell attachment and proliferation; (3) interconnected pores with proper size to favor cell infiltration and vascularization; (4) controlled biodegradability to facilitate the formation of new tissue; (5) adequate mechanical properties to maintain the structure and function immediately after implantation and during remodeling of the implants [2]. Currently, biodegradable synthetic polymers such as polyglycolic acid and polylactic acid have been used as scaffold materials in tissue engineering. However, polymeric scaffolds may not interact with cells in a desired manner since their surface chemistry does not promote adequate cell adhesion [3] and may induce toxic and inflammatory reactions [4]. In addition, it is difficult to construct a 3-D synthetic polymer scaffold that resembles the structure of natural tissues.

An alternative to synthetic polymers is the use of natural materials. Scaffolds composed of purified extracellular matrix (ECM) proteins have been used extensively in tissue engineering. The majority of these studies have focused on the fabrication of collagen scaffolds, combined with various procedures for the formation of pores followed by seeding with cells [5], [6], [7]. Even though the biochemical composition of scaffolds resembles those of natural tissues, they display very poor mechanical properties [6], [8], [9]. This may be due to the fact that reconstituted ECM molecules are not sufficiently structured and lack proper orientation. To exploit the 3-D ECM networks of natural tissues, processed vascular tissues have been employed for tissue engineering. This involved removal of cells followed by moderately successful attempts to repopulate the decellularized tissues with cells in vitro [10]. Sheep studies also showed that decellularized porcine aorta exhibited a low potential for repopulation in vivo [11]. These results may be due to the dense structure of the aorta and the lack of sufficient porosity. The aorta is made of concentric layers of elastin sheets interspersed with a collagen fiber network and populated by smooth muscle cells [12]. It is apparent that the aorta, even after decellularization, lacks the required porosity necessary for cell infiltration.

In the current study we selectively removed one of the two major structural components (elastin or collagen) from decellularized aorta for the purpose of creating interconnected pores with adequate sizes for cell infiltration and repopulation. Two types of scaffolds derived from porcine aorta, namely aortic elastin scaffolds and aortic collagen scaffolds were prepared and characterized. Their usefulness for the development of cell-populated constructs was investigated in vitro by culturing fibroblasts on the two scaffolds.