ABSTRACT
Transplantation of Pulsatile Myocardial Tissue by Using Scaffolds and Cardiac Cells The trials of pulsatile 3D myocardial tissue reconstruction are discussed. Three-dimensional myocardial tissue can be also reconstructed by using biodegradable 3D scaffolds, hydrogels, or decellularized native tissues and beating cardiac cells. Li et al. have reconstructed 3D myocardial tissue by seeding rat cardiac cells into a biodegradable gelatin mesh [18]. Engineered myocardial tissue has contracted spontaneously and constantly as a real heart tissue for a long time (at least 2 months) in vitro cultivation. A myocardial tissue graft contracts spontaneously and regularly even after transplantation into a subcutaneous rat tissue, and the
pulsatile tissue can be clearly identified at the site of implantation by echocardiography. The transplanted myocardial tissue shows a native myocardium-like histology, and multiple blood vessels are also observed within the tissue. Leor et al. have used rat cardiac cells and 3D alginate porous scaffolds [19]. Cardiac cells are located within the scaffold pores, and most of cells are arranged in small, viable 3D aggregates. Some of the myocardial aggregations pulsate spontaneously and constantly,
and cardiac cells isolated from the scaffolds are also contracting after reseeding onto tissue culture dishes. Reconstructed myocardial tissue has also been transplanted into rat myocardial scars. The histology of transplanted tissue and the host heart at nine weeks after transplantation shows (1) intensive neovascularization from coronary networks; (2) well-formed myofibers with typical striation between collagen bundles, gap junctions (GJs), and multiple blood vessels within the tissue graft; (3) good integration between transplanted cells and host cells; and (4) disappearance of the scaffold. While sham control rats develop a significant left ventricular (LV) dilatation accompanied by the progressive deterioration of LV contractility (fractional shortening [FS] from 47 ± 2% at the baseline to 33 ± 4%), myocardial graft-treated rats show an attenuation of LV dilatation and the preservation of LV contractility (FS from 53 ± 4% to 47 ± 5%). Zimmermann et al. have reconstructed ring-shaped 3D myocardial tissue by mixing neonatal rat cardiac cells with liquid collagen type I, Matrigel, and a serum-containing culture medium [20, 21]. A fabricated myocardial tissue is cultured with a chronic mechanical load using a static device, and after six to seven days’ cultivation, a vigorous spontaneous and macroscopic beating is observed in the myocardial tissue like real heart tissue. Engineered myocardial tissue is found to have characteristically histological features that are more similar to the native adult myocardium than immature heart tissue. Cardiac cells within engineered tissue show highly organized sarcomeres along the longitudinal cell axis and have specialized cell-cell junctions, including adherens junctions, GJs, and desmosomes; a well-developed T-tubular system and dyad formation with the sarcoplasmic reticulum; and a basement membrane surrounding cardiac myocytes. Engineered myocardial tissue shows the contractile and electrophysiological characters of a working myocardium. Zimmermann et al. have also succeeded in the fabrication of more powerful and large-size myocardial tissue (thickness: 1-4 mm; diameter: 15 mm), which is a multiloop type and is fabricated by staking loop-type myocardial tissues [22]. The force of engineered myocardial tissue is enhanced by cultivation (1) using an elevated ambient O2, (2) using an auxotonic load, and (3) using a culture medium containing insulin. The enhancement of the tissue force is paralleled by the increase of calsequestrin protein. The reconstructed powerful myocardial tissue has been transplanted
into rat infarction models. At four weeks after transplantation, the engineered myocardial tissue shows compact and well-differentiated tissue-derived cardiac muscle, whose thickness is 443 ± 32 µm, covering the infarcted myocardium. Well-organized sarcomeres and blood vessels, including endothelial cells (ECs) and smooth muscle cells, which must be of donor origin, are observed in the implanted grafts, and erythrocytes are also detected in the vessels, showing that the vessels are connected to the host vasculature and the connections must contribute to the long-term survival of implanted tissue grafts. A point stimulation for transplanted myocardial tissues, expect noncontractile myocardial tissue, which is observed in their formaldehyde-fixed myocardial tissue or noncardiomyocyte grafts, propagates as an electrical response to the remote myocardium, showing the establishment of electrical coupling between the host myocardium and the implanted graft. Because implanted grafts can be uncoupled after acidification (pH 6.7) of the heart, the electrical coupling between the implanted tissue graft and the host myocardium is direct and normal but not in subnormal graft-host coupling. In addition, telemetric electrocardiographic recordings show that the transplantation of a myocardial tissue graft hardly increases arrhythmias, and the circadian rhythms of recipient rats are restored after transplantation. After infarction treatment, while in the sham-operation control group, the increase of (1) LV end-diastolic dimension (LVEDD), (2) LV end-diastolic pressure (LVEDP), and (3) relaxation (tau) and (4) the decrease of fractional area shortening (FAS) are observed, the engineered-tissue-grafted group shows (1) maintenance of low-level values of LVEDD, LVEDP, and tau and (2) improvement of FAS. In addition, the therapeutic effects of engineered-myocardial-tissue transplantation are higher than those of noncontractile-graft transplantation. These results show that large-size pulsatile myocardial tissue shows electrical coupling with the host myocardium without arrhythmia and survives for a long time via functional anastomosis with the host vessels after transplantation and contributes to myocardial regeneration and cardiac function improvements, which is higher than expected efficiency from paracrine effects. Ott et al. have reconstructed 3D myocardial tissue by using neonatal rat cardiac cells and a rat whole myocardium, which are decellularized by coronary perfusion using a modified Langendorff apparatus with detergents [23]. Sodium dodecyl sulfate (SDS)
treatment gives better results than those by polyethylene glycol, Triton-X 100, or enzyme-based treatment for decellularizing, and a fully decellularized whole myocardial construct is obtained by antegrade coronary SDS perfusion over 12 hours. Treated myocardial tissue shows histologically no nuclear or contractile element, and the measured amount of deoxyribonucleic acid (DNA) decreases less than 4% of that in cadaveric myocardium, while the amount of glycosaminoglycan is unchanged. In addition, there remain collagen types I and III, laminin, and fibronectin within the decellularized tissue, and the fiber composition and orientation of myocardial ECM are conserved. After the reseeding of cardiac cells, the recellularized myocardial tissue is cultivated by coronary perfusion in a bioreactor that simulates and provides a myocardial physiological condition. At day 4, the synchronous and macroscopic beatings of the tissue are observed, and at day 8, with a physiological load and electrical stimulation, the tissue can generate its pumping function, which is comparable to approximately 2% of adult rat heart function and 25% of 16-week fetal rat heart function, and the myocardial tissue survives in in vitro cultivation up to 28 days. Immature cross-striated contractile fibers begin to organize, and the many expressions of GJ-related protein, connexin-43, within recellularized myocardial tissue are histologically detected at 8-10 days. The synchronous beating of the tissue at four days shows that expressed connexin-43 is functional. In addition, re-endothelialization tissue, which forms single-EC layers in both larger and smaller coronary vessels, is also fabricated by reseeding ECs into a decellularized construct. Zhao et al. have fabricated 3D myocardial tissue with characteristic features similar to the native myocardium by examining (1) the optimal cell densities of cardiac cells and (2) the concentration of hydrogel [24]. Engelmayr et al. have prepared a material having an accordion-like honeycomb microstructure from poly(glycerol sebacate) (PGS), fabricated a porous elastomeric 3D scaffold with controllable stiffness and anisotropy from the material, and showed the feasibility of scaffolds in the reconstruction of myocardial tissue [25]. Reconstructed myocardial tissue using scaffolds and neonatal rat cardiac cells shows (1) closely matched mechanical properties compared to the native myocardium with adequate stiffness controlled by PGS curing time, (2) cardiac cell contractility being inducible by an electric field stimulation with directionally dependent electrical excitation thresholds, and (3) greater cardiac cell alignment than the control isotropic scaffolds.
