The DOPA-based Conducting Cryogel For Regenerative Cardiac Tissue Patch In A Rat Infarct Model

BackgroundHigh morbidity, mortality and high recurrence, myocardial infarction (MI) is still a challenge due to the poor regeneration ability of the adult cardiomyocytes. And worse, MI drives progressively the injured cardiac tissue to lose cardiomyocytes and to form the fibrous scar tissue. In order to address these concerns, the repair or replacement of the injured myocardium is suggested. The proposed strategy is to inject different types of cells with differential capability, such as neonatal cardiomyocytes and cardiac progenitor stem cells, directly into damaged cardiac tissue. However, these cells can’t anchor in-situ to the injured tissue and quickly leak into the ventricular cavity or vasculature, and MI caused ischemia aggravates survival rates of cells. This compromises the therapeutic function of cell itself in MI.The engineered cardiac patch (ECP) is a promising strategy to promote the efficiency of the cell therapy by combining cells into scaffolds. The scaffolds should be able to replicate the structure and function of natural myocardium, which could induce the cardiomyocytes attachment and growth and facilitate the cardiomyocytes functionalization in vitro, and could repair the infarcted myocardium leading to the elevation of the cardiac function in vivo. An ideal cardiac patch should have these properties as follows:1) resilience in mechanics which could better mimic the dynamic contractive properties of the nature myocardium facilitating the cardiomyocytes alignment and elongation, and resist the damage from the beating heart than the stiff scaffolds; 2) great cardiac cells’ affinity and retention ability to meet the long time repair process for infracted myocardium in the preclinical experiments in the animal models and avoid the cell detachment from the scaffold because of the high-speed and high-strength beating on the surface of the infracted heart in vivo; and 3) the synchronous contraction ability to well conduct the bioelectric signals and avoid the severe arrhythmia in vivo. The current majority of the cardiac scaffolds addressed one or two properties listed above, but not all. For instance, the usage of natural polymers (gelatin, alginate, collagen type Ⅰ and fibrin) to develop the cardiac patches have the well biocompatibility while having the weak and slowly resilience. A macroporous chitosan-gelatin cryogels possessed an elastically property but have a weak conductive property. Further, conducting polymers were incorporated into scaffolds to promote the contraction ability but ignoring the compliance after they were implanted in vivo.Herein, we report a mussel-inspired conductive cryogel, which could simultaneously address the aforementioned three properties of the ideal cardiac scaffold. Cryogels, which was synthesized by the novel cryogelation technology at sub-zero temperature, show the interconnected macroporous networks and the excellent compressed property. The well compliance of the cryogel mainly results from the interreaction of the methacryoyl groups during cryogelation. Recently reported stiffness of methacrylated-alginate or methacrylated-gelatin (MA-G) cryogels is much lower than that of the nature cardiac tissue. Therefore, we introduced poly (ethylene glycol) diacrylate (PEGDA) which often used to improve the mechanical properties of the substrate into the methacrylated-gelatin for cryogels. As to synchronous contraction in ECP, the native heart induces the synchronous contraction of the cardiac cells via conducting the electrical signals from the sinoatrial node throughout the whole myocardium. The electrical signals are an important stimulation to advance the contraction behavior and the expression of the cardiac-specific proteins. The conductive scaffolds hence can greatly improve the performance of the cardiac patch by strengthening the cell-cell of myocardium communication and the electric contraction coupling. Especially after it is implanted in vivo, the conductive scaffolds permit the host heart and the cardiac patch as a unit through the electrical pulse conduction and thus support the reestablishment of the synchronous contraction in the damaged heart. Among the conducting polymers, the polypyrrole (Ppy) was often used in ECP and was proven to induce the isolated cardiomyocytes by stimulating the expression of the gap junction protein connexin 43 (CX-43) and enhancing the Ca2+signal conduction.In order to improve the integrity of Ppy nanoparticles and cryogel, we introduced mussel-inspired dopamine as a crosslinker for MA-G/PEGDA cryogel. Polydopamine, derived from the mussel foot protein, exhibits the strong and robust adhesion to almost all types of substrates. The catechol group plays a dominant role in the mussel-mimicking properties. Polydopmamine interacts the substrates which contain amine and/or thiol groups via Michael addition, while it plays its effective adhesion via the noncovalent binding (such as chelating, hydrogen bonding, π-π stacking and quinhydrone charge-transfer complexes) under the ambient condition. Recently, we successfully fabricated the dopamine-MA-G/PCL nanofibers using the dopamine-N’N’-methylene-bis-acrylamide (MBA) crosslinker while maintaining the strong adhesion of dopamine. Further, the dopamine-derived materials are proved safe and compatible for cellular adherence, spread and proliferation.Objective1. designed an engineered cardiac patch and in vitro characterization and biological properties.2. Cardiomyocytes-loaded ECP was implanted onto the infarct myocardium in the myocardial infarction rat models and assess the function of myocardial infarction.Methods1. Synthesis and Characterization of cardiac patch tissue engineering scaffold.Preparation of dopamine cross-linker(DOPA-MBA),gelatin with double bond (MA-G) and MA-G-Ppy. The prepolymer was synthesized from various combinations of DOPA-based MA-G/PEGDA/Ppy cryogel, MA-G/PEGDA/Ppy cryogel, DOPA-based MA-G/PEGDA cryogel, MA-G/PEGDA cryogel.1H NMR was to calculate the percentage of double bond congregated to gelatin., all the electrical conductivity tests of samples are measured by Multifunction Digital Four-probe Tester, The mechanical properties of cryogels were tested by Instron 5965.2. Cardiac patch constructed in vitro characterization and biological describe the morphology of detection.1-3 day-old SD rats, collect sufficient cardiomyocytes and cardiac fibroblasts which were seeded on different cryogels. we use live/dead cell staining and CCK-8 to detected the cell viability which cultured on different cryogels. The ultra-microstructural observation by Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM). In vitro immunofluorescence and Western blot detection of myocardial cell connexin 43 (CX-43) and a-actinin in DOPA-based MA-G/PEGDA/Ppy cryogel and DOPA-based MA-G/PEGDA cryogel.3. Tissue engineering cardiac patch vitro were verified myocardial infarction in SD rats.Male Sprague-Dawley (SD) rats (250±20 g) were anesthetized with isoflurane and were performed by permanent LAD ligation according to the procedure described previously.14 days after formation of MI, the survived rats underwent an echocardiographic examination. The rats with FS<30% were selected and were randomly divided into MI group and MI+patch group. The left ventricular function of animals from all groups was assessed using an IE33 echocardiographic system (Philips Medical Systems, Nederland B.V) equipped with a 15-MHz (s12) transducer. 4 weeks after cardiac patch transplantation, The LV internal dimensions at both diastole and systole (LVIDd and LVIDs, respectively), fractional shortening (FS) and EF (ejection fraction) were measured. At 4 weeks after the patch transplantation, the animals were anesthetized, sacrificed, and the hearts were harvested. The hearts were cut into 2-4 mm slices The infarct size were calculated by Masson trichrome measure as the percentage of scar tissue occupied in the LV free-wall surface area by measure the inner perimeter. Immunofluorescence was detection a-actinin protein and tracking CM-DiI stained cardiomyocytes.4. Statistical analysis.Data are presented as means±standard deviations. All results were compared using SPSS 13.0 software. One-way analysis of variance (ANOVA) in conjunction with post hoc comparison of more than 2 means by the Bonferroni method. Values of P less than 0.05 were considered significant.ResultsScaffold Characterization. The DOPA-based MA-G/PEGDA/Ppy cryogel was like that of the nature myocardium with 200-500 kPa at the end of diastole. Further, it could be compressed freely and recovered to its original shape immediately. Our results showed the conducting polymer, Ppy, could improve the mechanical strength of the biomaterials. Further, our results also proved that the dopamine crosslinker group participated in modulating the stiffness of the cryogel. The cryogelation technology, Ppy and the dopamine group had the synergetic effect on the gel resulting in the well resilience and the biomimetic Young’s modulus.As to the ultrastructure of the different cryogels, the SEM results revealed that all the MA-G/PEGDA cryogel, DOPA-based MA-G/PEGDA cryogel, MA-G/PEGDA/Ppy cryogel, and the DOPA-based MA-G/PEGDA/Ppy cryogel had the uniform porous structure. The pore sizes in all of the groups were uniform with well-distributed. Further, we found that the pore sizes of the cryogels were consistent with their stiffness. The smallest pore size (25.6±3.78μm) was presented in the MA-G/PEGDA/Ppy cryogel with the highest Young’s modulus. The mean pore sizes of the basic MA-G/PEGDA cryogel was 37.5±5.02μm. Similar to the trend of the Young’s modulus among the different cryogels, the DOPA crosslinker group increased the pore sizes, that is, the mean pore sizes of the DOPA-based MA-G/PEGDA cryogel (45.6±6.03μm) were more bigger than the basic MA-G/PEGDA cryogel, and the pore sizes in the MA-G/PEGDA/Ppy cryogel were significantly increased to 72.3±8.06μm by the DOPA crosslinker.The polypyrrole homogeneously distributed on the surface of the DOPA-based MA-G/PEGDA/Ppy cryogel, while the polypyrrole agminated and scattered on the surface of the MA-G/PEGDA/Ppy cryogel. The results suggest that the DOPA-MBA as a crooslinker play an efficiently effect on the dispersion for the MA-G-Ppy in the cryogel. The similar finding was presented that dopamine provided the effective immobilization of some biomoleculers and even magnifies their functions in some dopamine-coated biomaterials.The DOPA-Based Cryogel Are Benign for The Cardiac Cells Attachment, Spreading and Cells Viability. The cells attachment and spreading in the scaffolds were observed through live/dead detection after 6 hrs and 24 hrs post-seeding respectively. The viability of the cardiomyocytes in the gels was detected in 3 days,7 days,14 days and 21 days post-seeding respectively using CCK-8 method. From live/dead cell assay, most cardiomyocytes presented a more spreading status on the DOPA-based cryogels for 6 hrs, whereas cardiomyocytes on the DOPA-absent cryogels displayed a round or narrow shape, which was the typical non-spreading cell morphology. Obviously, the excellent cytocompatibility of the mussel-inspired cryogels is related to the appearance of mussel-crosslinker. It might be due to the following two evidences:1) The mussel-crosslinker coordinated for obtaining the appropriate modulus and well compressed properties of the cryogels for ECP, which provide the excellent flexibility for promoting the cardiomyocytes attatchment, growth, alignment and elongation. Annabi et al developed a highly elastic substrate, the methacrylated tropoelastin, as the scaffold of the ECP and found the highly elastic substrates possessed stronger ability for the cell attatchment, spreading and function of the cardiomyocytes than that of MA-G hydrogel. In this study, we developed a flexible and simple way by adding the DOPA crosslinker into the basic cryogel to obtain the optimized elastic property for ECP, accompanying with the better cardiac cells’ growth status.2) The mussel-crosslinker its own strong affinity for cells attachment, spread and proliferation, which is resulting from DOPA’s adsorption of the serum proteins providing the sites for cell-adhesion, through the stable and tightly covalent bonding.The Incorporated Ppy in the Mussel-Inspired Cryogel Enhance the Cardiomyocytes Functionalization in vitro. The cardiac-specific proteins expression in the cardiomyocytes in DOPA-based MA-G/PEGDA cryogel, DOPA-based MA-G/PEGDA/Ppy cryogel and the tissue culture plates (TCPs). Compared with the cardiomyocytes in DOPA-based MA-G/PEGDA cryogel, more a-actinin-positive cardiomyocytes were observed and high level of CX-43 proteins appeared along the cell plasmalemma and the adjacent site between cells in the DOPA-based MA-G/PEGDA/Ppy cryogel on day 3. While less expression of the a-actinin proteins and CX-43 proteins were observed in the DOPA-based MA-G/PEGDA cryogel. Such phenomenon was not found in the TCPs groups. On day 8, the stronger expression of the a-actinin protein as well as a larger number of sarcomeres existed in cardiomyocytes in the DOPA-based MA-G/PEGDA/Ppy cryogel than that in the DOPA-based MA-G/PEGDA cryogel, while there seemed no obviously difference in CX-43 expression between the two samples. As the control group, the expression of the cardiac-specific proteins were just scattered on the TCPs. Furthermore, western blotting analysis revealed that inclusion of Ppy in DOPA-based MA-G/PEGDA cryogel significantly increased the expression of the a-actinin proteins and the CX-43 proteins in the cardiomyocytes on day 3 and day 8. As for the cardiomyocytes in the DOPA-based MA-G/PEGDA cryogel, the higher expression of the cardiac-specific proteins was detected till at day 8 compared to that in the TCPs. We compared their ultra-microstructure using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) on day 8. Interestingly, the Ppy nanoparticles were spontaneously gathered and adhered to the cytomembrane surface of the cardiomyocytes from the surface of the DOPA-based MA-G/PEGDA/Ppy cryogel while the cytomembrane surface of the cardiomyocytes in the DOPA-based MA-G/PEGDA cryogel were smooth. It was noticeable that, the well-aligned sarcomeres composed of the Z bands were obviously observed in most of the cardiomyocytes in the DOPA-based MA-G/PEGDA/Ppy cryogel under TEM field. Meanwhile, the Ppy directly contacted and fused with the membrane of the cardiomyocytes was also observed in the DOPA-based MA-G/PEGDA/Ppy cryogel under TEM field. On the contrary, the above ultrastructures were not observed and few predominant orientated myofibrils appeared in the cardiomyocytes in the DOPA-based MA-G/PEGDA cryogel.Functional Improvement and Tissue Repair in infracted Left Ventricle after ECPs Transplantation in vivo. A series of echocardiographic detection were performed to assess the cardiac function among the sham group, MI group and MI+ECP group. The fractional shortening (FS) and ejection fraction (EF) significantly decreased and the left ventricle internal dimensions at systole (LVIDs) significantly increased in MI rats after the left anterior descending (LAD) occlusion when the cardiac function was quantified by echocardiography, indicating the progressive deterioration of cardiac function and left ventricle enlargement. The cardiomyocytes were co-cultured with DOPA-based MA-G/PEGDA/Ppy cryogel for 8 days to form cells-scaffold ECPs. Subsequently, the cardiac function of the left ventricle significantly improved in 4 weeks after the ECPs were implanted on the epicardium of the infarcted hearts. The images of echocardiography showed that the ECPs-implanted groups exhibited the enhanced contractile activity of the free wall in left ventricle. Simultaneously, the ECPs-implanted group elicited the great reversal changes of the cardiac function compared to that of the MI group, although the function of the left ventricle did not yet recover to the normal level. All of the results suggested that the ECPs implantation could efficiently prevent the left ventricle enlargement and elevate the cardiac function. The cardiac morphology was investigated among the sham group, MI group and MI+ECP group using Masson’s Trichrome staining method. Notably, the myocardium in ventricle wall in MI rats was almost substituted by the fibrous tissue, while much more myocardium was found in the ECP-implanted heart. The neomyocardium in left ventricle in the ECP group was gradually spreading into the infarct area from the normal myocardium area. Additionally, scar size was quantified according to the percentage of the fibrous area by the Masson’s detection. The scar size of infarct area in ECP group was much smaller than that in MI rats. The cardiomyogenesis in the infarct area in 4 weeks after the patch implantation was assessed by a-actinin expression. The CM-Dil stain was used to track the donor cells. As shown in Fig. a large amount of Dil+ cells was found in the patch, and the superimposed views of the Dil positive red fluorescence and the a-actinin positive green fluorescence in the donor cells indicated that the well retention capability of the patch to the cardiomyocytes. On the other hand, some of the DiI+ cells were found to intersperse in the host myocardium, indicating the migration of the donor cardiomyocytes from the patch to the host tissue. Furthermore, a small a-actinin positive myocardium there existed beneath the endocardium in the MI heart, while much more a-actinin positive myocardium were found in the infarct area in the patch-implanted heart. Therefore, all of these results suggested that the patch composed of the mussel-inspired conductive cryogel and the cardiomyocytes was beneficial for the donor cells retention and migration, as well as stimulating the infarct tissue to generate the neomyocardium.ConclusionDopamine-MBA was taken as the crosslinker to fabricate the DOPA-based conductive MA-G/PEGDA cryogel successfully. This kind of the conductive biomaterials exhibits super-elasticity, similar Young’s modulus with the nature myocardium and well conductivity. Furthermore, this conductive cryogel had the strong ability for cardiac cell affinity and the enhancement of the cardiomyocytes functionalization. Consequently, after the engineered cardiac patch with the mussel-inspired conductive cryogel loading the cardiomyocytes was implanted onto the rat infarct myocardium, it displayed the excellent performance for the donor cells retention and migration, the neomyocardium formation in the infarct area and the improvement of the cardiac function. Accordingly, the DOPA-based MA-G/PEGDA/Ppy cryogel can be taken as a promising cardiac scaffold for the infarct tissue repair. In addition, the results in this study also confirmed that the dopamine took the vital effect on the cell affinity and the Ppy nanoparticles dispersion. The migration and fusion of the conductive Ppy nanoparticles into the cardiomyocytes from the scaffold was benefit to the improvement of the infarct cardiac function.