Altered structural and mechanical properties in decellularized rabbit carotid arteries
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.
Section snippets
Tissue harvest
All procedures were performed in accordance with the Institutional Animal Care and Use Committee at Boston University and the NIH Guide for the Care and Use of Laboratory Animals. Healthy male New Zealand white rabbits (2.5–3 kg, Pine Acres Rabbitry, Brattleboro, VT) were euthanized, and carotid arteries were harvested using sterile tools. Vessels were immediately placed in cold Hanks’ Balanced Salt Solution (HBSS: 137 mM NaCl, 5.4 mM KCl, 0.42 mM Na2HPO4, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 10 mM HEPES,
Histology reveals the presence of PG and altered structure of adventitial collagen
Native carotid arteries showed characteristic arterial organization of cells and ECM with distinction between intimal, medial and adventitial layers (Fig. 1A); decellularized arteries also had distinct layers but contained no evidence of cells, and images showed that major ECM components were retained (Fig. 1B). At the magnifications shown, the endothelial cell layer that normally lines the native vessel lumen was not evident, but the internal elastic lamina was clearly observed in both native
Discussion
Decellularized vessels show potential as scaffolds for SDVG in clinical procedures, but often have altered mechanical properties compared with native arteries. Despite the importance of tissue structure to function, few studies have focused on decellularized vessel structure, even though removal of cells is certain to alter the native architecture of the ECM. This study sought to investigate and relate the changes in structural and mechanical properties of decellularized rabbit carotid
Conclusion
It was found that decellularized arteries had a looser, uncrimped collagen network, which could explain increased stiffness and decreased extensibility compared with native arteries. Additionally, increased fiber mobility in decellularized arteries was related to increased stiffness, as fibers could easily rotate toward the direction of strain. High fiber mobility also led to disrupted structural stability due to increased porosity from removal of cells, as well as altered collagen interaction
Acknowledgements
This work was supported by NIH (HL72900) to J.Y.W., AHA pre-doctoral fellowship to C.W., and AHA BGIA (0565346U) to J.L. M.S.S. is an Established Investigator of the AHA. The authors would like to thank Jennifer Debarr, Mark Rubin and Amanda Lawrence for histology, SEM and TEM observations. The authors are also grateful to Erzsebet Bartolak-Suki for insightful discussions and critical review of the manuscript.
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Co-first authors.
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Present address: Department of Chemical and Biochemical Engineering, University of Maryland, Baltimore County, MD 21250, USA.