Vascular
Mechanical Evaluation of Decellularized Porcine Thoracic Aorta

https://doi.org/10.1016/j.jss.2011.03.070Get rights and content

Background

Decellularized tissues are expected to have major cellular immunogenic components removed and in the meantime maintain similar mechanical strength and extracellular matrix (ECM) structure. However, the decellularization processes likely cause alterations of the ECM structure and thus influence the mechanical properties. In the present study, the effects of different decellularization protocols on the (passive) mechanical properties of the resulted porcine aortic ECM were evaluated.

Methods

Decellularization methods using anionic detergent (sodium dodecyl sulfate), enzymatic detergent (Trypsin), and non-ionic detergent [tert-octylphenylpolyoxyethylen (Triton X-100)] were adopted to obtain decellularized porcine aortic ECM. Histologic studies and scanning electron microscopy were performed to confirm the removal of cells and to examine the structure of ECM. Biaxial tensile testing was used to characterize both the elastic and viscoelastic mechanical behaviors of decellularized ECM.

Results

All three decellularization protocols remove the cells effectively. The major ECM structure is preserved under sodium dodecyle sulfate (SDS) and Triton X-100 treatments. However, the structure of Trypsin treated ECM is severely disrupted. SDS and Triton X-100 decellularized ECM exhibits similar elastic properties as intact aorta tissues. Decellularized ECM shows less stress relaxation than intact aorta due to the removal of cells. Creep behavior is negligible for both decellularized ECM and intact aortas.

Conclusion

SDS and Triton X-100 decellularized ECM tissue appeared to maintain the critical mechanical and structural properties and might work as a potential material for further vascular tissue engineering.

Introduction

The clinical need for the development of substitute vessels is quite obvious. For example, coronary and peripheral vascular bypass grafting is now performed more than 600,000 times annually in the United States and Europe to treat cardiovascular disease. During a bypass, the vascular surgeon creates a new pathway for blood flow using a natural or artificial substitute vessel. Autologous vessels are preferred as graft material; however, these vessels may not be an option for patients who have undergone previous bypass surgery or who do not have vessels of appropriate quality [1]. Tissue engineered materials have gained great attention in replacement of the malfunctioning or diseased cardiovascular tissues. The challenge of tissue engineering blood vessels lies in the requirement of both mechanical properties of native vessels and also the anti-thrombotics properties [2]. Since decellularization is considered to reduce the immunological response, decellularized tissues have become promising material in the field of tissue engineering for transplant and grafting. They have been successfully used in many preclinical studies 3, 4, 5, 6 and even human clinical applications 7, 8. Decellularization techniques have been applied to many types of tissues/organs, including heart valve 6, 9, bladder [10], ligament [11], tendon [12], vein [13], and artery 14, 15.

Decellularized tissues are expected to have all cellular and nuclear material efficiently removed while maintaining similar composition, biological activity, and mechanical integrity of the extracellular matrix (ECM) [16]. The remaining ECM can be seeded with host’s native cells in vitro before transplantation or in vivo after implantation [17]. Various decellularization protocols have been developed, which may involve a combination of physical, chemical, or enzymatic methods using different detergents or enzymatic agents to remove cells and cellular debris [16]. Among these decellularization protocols, the most commonly used methods include using anionic detergent sodium dodecyle sulfate (SDS) 18, 19, 20, enzymatic agent Trypsin 21, 22, and non-ionic detergent Triton X-100 (tert-octylphenylpolyoxyethylen) 23, 9.

Although decellularization techniques have been broadly applied to native tissues, there is still limited information on the matrix structure and mechanical properties after the decellularization process. It is noted that changes in the ECM structure after decellularization process would affect the mechanical properties of the tissue [16]. Previous studies investigated the matrix structure of decellularized tissue using combined histology and microscopy techniques 24, 25, 26, 27. Several mechanical studies were performed to understand the mechanical integrity of decellularized arteries 4, 14, 15, and decellularized aortic valve leaflet [28]. However, little was found on the time-dependent mechanics of decellularized tissues. Successful decellularization will produce an ECM that possesses anisotropic hyperelasticity, as usually seen in the intact aortas [29]. In a few previous mechanical studies on decellularized arteries 4, 14, 15, loading has been limited to uniaxial stretching, which ignores the multiaxial loading state under physiologic conditions and, thus, the intrinsic anisotropic properties of soft tissue. Planar biaxial tensile test with independent control of load in both perpendicular directions has been used broadly to study the mechanical behavior of various soft biological tissues 29, 30, 31, 32, 33, 34. Although it cannot fully replicate the physiologic loading conditions, biaxial tensile test is sufficient on elucidating the anisotropic mechanical properties of soft tissues with plane stress assumptions. Such capabilities make it a useful tool for in vitro understanding of tissue mechanics.

The objective of the present study is to evaluate three commonly used decellularization protocols with an emphasis on understanding the relationships between structural changes and mechanical alteration. Three decellularization protocols were adopted to obtain the decellularized porcine aortic ECM. SDS was chosen as an anionic detergent, Trypsin as an enzymatic agent, and Triton X-100 as a non-ionic detergent. Scanning electron microscopy (SEM) and histology studies were used to confirm the removal of cells and to investigate the composition and structure of tissue samples. Planar biaxial tensile, biaxial stress relaxation, and creep tests were performed to study the mechanical behavior of decellularized ECM. Results from this study provide insight into the effects of structural changes on the elastic and viscoelastic properties of arteries after decellularization.

Section snippets

Sample Preparation

Fresh porcine descending thoracic aortas were harvested from a local slaughter house and transported on ice to the lab. Immediately after arrival, the aortas were cleaned of adherent tissues and fat, dissected, and rinsed in deionized (DI) water. Square samples of about 2 cm × 2 cm were cut from the middle section of the aortas to minimize the changes of the mechanical properties along the longitudinal position of aorta [33]. The decellularization process of aortic tissue samples was performed

Results

SEM was performed on the cross section along the circumferential direction of the intact and decellularized aortas to examine the morphology and structure. As shown in Figure 1, in the intact artery, a dense structure is present with smooth muscle cells (SMCs) embedded in the cross-linked ECM network of elastin and collagen fibers (Fig. 1A). After the SDS and Triton X-100 decellularization treatment, the ECM network of fibers is better revealed with the removal of SMCs. Images in Figure 1B and

Discussion

The present study examined the efficiency of SDS, Trypsin, and Triton X-100 decellularization protocols on porcine thoracic aortas with an emphasis on understanding the relationships between structural changes and mechanical alteration. The Trypsin decellularization protocol effectively removes all the cells and nuclear components; however, substantial disruption of the ECM is accompanied with this technique. Similar to our findings, a number of previous studies have reported the extensive

Conclusions

In the present study, the elastic and viscoelastic behaviors of decellularized aortic ECM were examined using planar biaxial tensile testing. Three commonly used decellularization protocols were evaluated in porcine thoracic aortas for efficiency of cell removal and ability to maintain mechanical integrity. Changes in mechanical function were related to the structure of the decellularized ECM. SEM and histology studies show that the cells were removed completely in all three decellularization

Acknowledgments

The authors acknowledge financial support from the National Institutes of Health (HL098028) and the CAREER award from the National Science Foundation (CMMI-0954825).

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