Cardiac Extracellular Matrix Scaffold Generated Using Sarcomeric Disassembly and Antigen Removal

Abstract

Xenogeneic cardiac extracellular matrix (cECM) scaffolds for reconstructive cardiac surgery applications have potential to overcome the limitations of current clinically utilized patch materials. A potentially ideal cECM scaffold would be immunologically acceptable while preserving the native cECM niche. Production of such a scaffold necessitates removal of cellular and antigenic components from cardiac tissue while preserving cECM structure/function properties. Existing decellularization methodologies predominantly utilize denaturing detergents which might irreversibly alter cECM material properties. To overcome potential deficiencies of current approaches, the effect of sarcomere relaxation and disassembly on resultant cECM scaffold cellularity was investigated. Additionally, the ability of sequential differential protein solubilization (antigen removal-AR) to reduce cECM scaffold antigenicity was examined. Sarcomeric relaxation and disassembly were necessary to achieve scaffold acellularity. All groups in which AR was employed displayed statistically significant decreases in residual antigenicity regardless of their degree of acellularity. AR combined with sarcomeric disassembly preserved structural, biochemical, mechanical and recellularization properties of the cECM scaffold. However, sodium dodecyl sulfate significantly altered cECM properties. This study demonstrates the importance of solubilizing cellular elements and antigenic components in a stepwise manner for production of a potentially ideal cECM scaffold and may have implications for future tissue engineering and regenerative medicine applications.

Skeletal Extracellular Matrix Supports Cardiac Differentiation of Embryonic Stem Cells: a Potential Scaffold for Engineered Cardiac Tissue

“…Based on published literatures, we employed pretreatment/SDS/TritonX-100 three-step-method with shorter SDS treatment to obtain sECM. The whole decellularization procedure was much shorter than other reported procedure of sECM [16] and cECM [7]. Our results showed that the residual DNA contents of sECM in PreST group were less than 50 ng/mg, which is similar to the criteria proposed by Crapo and colleagues [30].…”

“…Meanwhile, the collagen fibers of sECM presented typical transverse periodic striations [31] with an average diameter far less than 0.1 µm, which is similar to the diameter of cECM [4]. SECM in the present study showed a honeycomblike endomysium structure and perimysium fibers, which is similar to the microstructure of cECM [7]. …”

“…They provide tissue-specific cues to support cell attachment, proliferation and differentiation [5, 6]. Generally, cECMs acquired by decellularization procedure are constituted of numerous of coiled and aligned fibril bundles and exhibit complex meshwork of pores [7]. Accumulated data have demonstrated that cECMs enhance the survival, proliferation, migration and cardiac differentiation of Sca-1 + [4] or c-kit + [8] cardiac progenitor cells.…”

Small Diameter Xenogeneic Extracellular Matrix Scaffolds for Vascular Applications

“…AR has been proposed as a alternative tissue processing method, employing protein chemistry principles of differential solubilization to achieve stepwise removal of antigens with shared physiochemical properties (i.e., hydrophile solubilization through application of reducing conditions and salting in, followed by lipophile solubilization through zwitterionic detergent use and nuclear antigen solubilization through nucleic acid digestion), showing promise in dramatically reducing resultant scaffold antigen content while maintaining native ECM structure-function properties. 116,194 Bovine percardium (BP) treated with AR approach has yielded scaffolds with native ECM protein macromolecular structure. Encouragingly, AR methods significantly reduced a-gal, MHC I, and total minor histocompatibility antigens in BP, resulting in local graft-specific adaptive immune response, which was significantly less than that toward GTAfixed BP.…”

“…Encouragingly, AR methods significantly reduced a-gal, MHC I, and total minor histocompatibility antigens in BP, resulting in local graft-specific adaptive immune response, which was significantly less than that toward GTAfixed BP. 116,194 Furthermore, BP-AR scaffolds avoided proinflammatory innate immune response on in vivo implantation, ameliorating fibrosis and calcification associated with traditional decellularized BP. 116,194 Whether the result obtained for AR applied to BP would translate into small diameter vessel ECM scaffolds remains to be determined.…”