Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrow mononuclear cells

Abstract

Tissue engineered cardiac grafts are a promising therapeutic mode for ventricular wall reconstruction. Recently, it has been found that acellular tissue scaffolds provide natural ultrastructural, mechanical, and compositional cues for recellularization and tissue remodeling. We thus assess the potential of decellularized porcine myocardium as a scaffold for thick cardiac patch tissue engineering. Myocardial sections with 2-mm thickness were decellularized using 0.1% sodium dodecyl sulfate and then reseeded with differentiated bone marrow mononuclear cells. We found that thorough decellularization could be achieved after 2.5 weeks of treatment. Reseeded cells were found to infiltrate and proliferate in the tissue constructs. Immunohistological staining studies showed that the reseeded cells maintained cardiomyocyte-like phenotype and possible endothelialization was found in locations close to vasculature channels, indicating angiogenesis potential. Both biaxial and uniaxial mechanical testing showed a stiffer mechanical response of the acellular myocardial scaffolds; however, tissue extensibility and tensile modulus were found to recover in the constructs along with the culture time, as expected from increased cellular content. The cardiac patch that we envision for clinical application will benefit from the natural architecture of myocardial extracellular matrix, which has the potential to promote stem cell differentiation, cardiac regeneration, and angiogenesis.

Figure 1

Morphology of (a) native myocardium and (b) decellularized myocardial scaffold. Thorough decellularization was achieved after 2.5 weeks treatment.

Figure 2

Mason’s trichrome staining for (a) native myocardium and (b) decellularized myocardial scaffold (blue: collagen, red/pink: cardiomyocytes). (c) H&E staining of the longitudinal and transversal views of the acellular scaffold showed that 3D lacunae housing cardiomyocytes were preserved (red: collagen). (d) Vasculature templates in the decellularized myocardial scaffold (H&E staining). Arrows indicate vasculature channels.

Figure 3

Preservation of vasculature structure was verified by (a) SEM and (b) type IV collagen staining (green color) on the circular structure; (c) transmission light image of the same region shown in (b). Arrows in (a), (b), and (c) indicate the existence of vasculature templates. (d) A combined image of the DAPI staining (blue color) and the transmission light image on the section of the decellularized scaffolds. Lack of DAPI staining verified that no nuclear chromatin fragments remained in the scaffolds. Arrow in (d) showed a minute spot of DAPI stain (blue color).

Figure 4

(a) BMMCs after one week primary culture. (b) Positive staining for stem cell marker CD44 (green color). (c) Cardiomyocyte differentiation after treatment with 5-azacytinine; (d) a higher magnification of the differentiated cells with visible sarcomere alignment. Red staining: sarcomeric α-actinin.

Figure 5

(a) Large pores distributing evenly across the 2 mm thick acellular scaffold (H&E). (b) Edge-to-edge view of thorough recellularization at 2 weeks, in which cells were found to infiltrate and distribute within the myocardial scaffold. (c) Tissue remodeling was observed in the 4-week recellularization tissue construct; even cell distribution was still observed and cell density was found higher than that of the 2-week construct.

Figure 6

(a) Thorough and relatively dense reseeding was observed in tissue construct after 4-week culture (H&E staining). Live/dead cell staining on 4-week tissue construct: (b) surface of tissue construct and (c) side view of cross-sectional cut. Green: living cells; red: dead cells.

Figure 7

Sarcomeric α-actinin staining (red) in the native myocardium (a) and the reseeded patch (b); myosin heavy chain staining (green) in the native myocardium (c) and the reseeded patch (d); cardiac troponin T staining (red) in the native myocardium (e) and the reseeded patch (f). vWF factor (red) was shown in the reseeded patch (g). Cell nucleus: blue.

Figure 8

Biaxial mechanical properties. (a) Native myocardium showed anisotropic nonlinear behavior, in which muscle fiber-preferred direction (PD) is stiffer than cross fiber-preferred direction (XD). (b) PD and XD directions showed stiffer stress-strain curves in decellularized myocardial scaffold (c) 1 week reseeded cardiac patch (d) 2 weeks reseeded cardiac patch. Tissue remodeling caused a recovery of the biaxial mechanical properties after 1-week and 2-week recellularization. The 1-week and 2-week tissue constructs kept nonlinear anisotropic behavior and tissue extensibility more close to native myocardium along both PD and XD directions.

Figure 9

Both (a) uniaxial mechanical properties and (b) tissue failure properties show a recovering tendency after 1-week and 2-week cultures. Note that both loading and unloading stress-strain curves were shown in (a).