Altered structural and mechanical properties in decellularized rabbit carotid arteries

Abstract

Recently, major achievements in creating decellularized whole tissue scaffolds have drawn considerable attention to decellularization as a promising approach for tissue engineering. Decellularized tissues are expected to have mechanical strength and structure similar to the native tissues from which they are derived. However, numerous studies have shown that mechanical properties change after decellularization. Often, tissue structure is observed by histology and electron microscopy, but the structural alterations that may have occurred are not always evident. Here, a variety of techniques were used to investigate changes in tissue structure and relate them to altered mechanical behavior in decellularized rabbit carotid arteries. Histology and scanning electron microscopy revealed that major extracellular matrix components were preserved and fibers appeared intact, although collagen appeared looser and less crimped after decellularization. Transmission electron microscopy confirmed the presence of proteoglycans (PG), but there was decreased PG density and increased spacing between collagen fibrils. Mechanical testing and opening angle measurements showed that decellularized arteries had significantly increased stiffness, decreased extensibility and decreased residual stress compared with native arteries. Small-angle light scattering revealed that fibers had increased mobility and that structural integrity was compromised in decellularized arteries. Taken together, these studies revealed structural alterations that could be related to changes in mechanical properties. Further studies are warranted to determine the specific effects of different decellularization methods on the structure and performance of decellularized arteries used as vascular grafts.

Introduction

Decellularized tissues have gained significant attention in the field of tissue engineering, especially for their promise in whole organ transplant and grafting [1], [2]. Although most applications are still far from clinical use, numerous tissue types have been successfully decellularized, including heart [3], heart valve [4], [5], [6], [7], bladder [1], [8], blood vessel [9], [10], [11], skeletal muscle [12], [13], tendon [14] and ligament [15], [16]. A major motivation for using decellularized tissues is that they are expected to mimic closely the complex 3D structure and mechanical properties of the native tissues from which they are derived [17], [18]. It is well established that the mechanical properties of a tissue are intimately linked to its structure [19], and this relationship is especially important for load-bearing tissues such as the artery [20].

Decellularized blood vessels have been studied extensively [9], [10], [11], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], mostly for their potential as scaffolds for small diameter vascular grafts (SDVG). There is a great need for tissue-engineered SDVG, as many patients do not have autologous vessels available and synthetic grafts are prone to failure in small diameter applications [35]; in many cases, this has been linked to inappropriate structure and/or mechanical performance [36], [37]. If decellularized vessels do indeed maintain native tissue architecture and mechanical properties, these challenges could be overcome.

A majority of the literature regarding decellularized vessels tends to focus on cell seeding and implantation; while such studies are important and encouraging, there is still a lack of fundamental study of decellularized vessel structure–function relationships. Interestingly, many studies have reported that decellularized vessels have significantly altered mechanical characteristics compared with native vessels [10], [11], [21], [22], [23], [29]. Histology and/or electron microscopy images are often included, but changes (or similarities) in extracellular matrix (ECM) structure are not necessarily evident. While these imaging techniques are commonly used and do provide some useful information on structure, they have limitations: specifically, they do not reveal whether fiber–fiber interactions, fiber orientation or fiber mobility changes as a result of decellularization, which are important to the structural integrity of tissue.

Preservation of ECM does not necessarily correspond to preservation of tissue architecture. Although the intention of most decellularization procedures is to minimize disruption to the ECM, the removal of cells inevitably results in changes to native ECM structure [17]. Therefore, the goal of the present study was to use a variety of characterization techniques to investigate tissue structure in decellularized rabbit carotid arteries and to relate structural changes to altered mechanical properties. Histology, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to confirm removal of cells and to assess qualitatively ECM composition and ultrastructure. Mechanical properties were determined using stress–strain analysis and stress relaxation tests; additionally, opening angle studies were used as a measure of residual stress in the vessel wall. Finally, small-angle light scattering (SALS), a quantitative technique that measures the average local fiber orientation throughout the tissue thickness [38], was used to determine gross fiber architecture and changes in ECM fiber kinematics (e.g., fiber mobility and organization). SALS was included to elucidate changes in structural integrity that would not be revealed from histology and EM alone. Together, these data provided insight into how altered structural properties could be related to changes in mechanical properties as a result of decellularization.