In this study, highly tunable synthetic injectable hydrogels were designed with a range of mechanical and biological properties that yielded different cellular responses in vitro. These in vitro data were then used to identify specific formulations that promoted the survival of transplanted cells and/or could provide mechanical stabilization to an injured ventricular wall. In vivo studies confirmed that these fully synthetic systems can be injected directly into the beating ventricle after ischemic injury without causing arrhythmias, and in comparison to non-degradable gels we have previously implanted in the myocardium they remodel with minimal foreign tissue responses and are no longer visible as bulk gel by 6 weeks (unpublished result). These matrices improved the survival of a population of transplanted stem cells and could be useful for providing mechanical stabilization of the ventricle as a stand alone treatment.
While not statistically significant in our study, compared with other published studies using passive materials, we observed similar quantitative increases in ventricle function with the direct injection of the sIPNs. The lack of significance was due to the size and number of comparisons in our study, and the inherent variability in the in vivo
metrics used, as a simple t-test comparing the saline to the sIPN at six weeks was significant on its own (p<0.05). Beneficial effects may initially be mechanical in nature, as even small amounts of added non contractile material in an infarct injured heart may be able to significantly reduce elevated fiber stress according to theoretical models we have developed.22
As high border zone stresses have been implicated in chronic post-MI remodeling,42,43
by reducing this myofiber mechanical dysfunction, the deterioration of the ventricle after a major ischemic event may be minimized or even reversed. The trends we and others16,19,26-28
have observed with injections of the various hydrogels without any stem cells or growth factors provides early support for the published theoretical results, and the hypothesized impact of matrix injection on longer-term LV remodeling. The subgroup analysis of successful border zone injection further strengthens this hypothesis, and is in line with other studies which demonstrate that using biomaterials such as pNIPAAm based polymers, 26,28
in the infarct injured heart improves mechanical behavior and potentially reduces pathological remodeling.
An alternate hypothesis for the noted improvements in ventricle function associated with synthetic materials was originally hypothesized by Fujiwara and co-workers who suggested that postinfarct inhibition of apoptosis might preserve myofibroblasts and endothelial cells in granulation tissue and modulate chronic left ventricular remodeling and heart failure.44,45
Recent experiments using a biodegradable cardiac patch and injectable pNIPAAm hydrogels appear to demonstrate that enhancing the presence of granulation tissue during the inflammatory phase, and temporal extension of this phase, induced by the patch material, leads to improvement in cardiac function.28,46
Furthermore, there may be an angiogenic effect and a tissue generating response from transient inflammation brought about by the host response to an implanted degradable material.28
These effects could help to salvage the organ as any neotissue could provide mechanical support and increased blood perfusion to the at risk area could prevent the continued loss of functional tissue. Although the hypothesis is controversial, its validity cannot be discounted based on experimental evidence, and that it is impossible to avoid a mild inflammatory response to any material implanted in the heart. Further research, including more detailed modeling, in vivo
testing with large animals, and histological tracking of implanted materials is obviously needed to better elucidate the mechanism of improvement.
Although a host of materials have shown promise in stabilization of the ventricle, most materials used have not been not ideal for cardiac tissue engineering applications. Some, such as Matrigel™, are derived biologically, and therefore pose potential problems of disease transmission, purity and reproducibility in large-scale manufacturing. Others have poorly controlled degradation profiles, or, like alginate, require calcium to gel, which may lead to local calcification by themselves or with the use of transplanted cells.47
Most importantly, none of the materials, even the other NIPAAm based formulations,26,28
used in these previous studies lend themselves to systematic optimization both mechanically and biologically, as precise modification of the various substrates is difficult. The material platform used in the present study provides potentially significant advancement in cardiac tissue engineering applications. The wide orthogonal control over both mechanical and biological properties of this injectable material allow for tuning the material for the specific application, presenting bioactive peptides for engagement and angiogenesis to the local tissue, matching the degradation mode of the implant to the specific local MMP production, and choosing the material stiffness to provide an adequate growth structure for transplanted cells.
In contrast to the positive results with the use of a biomaterial alone in our animal model, the transplantation of BMSCs provided a transient improvement at 2 weeks but was associated with a negative trend over the course of the study. Although previous reports of stem cell transplantation using natural matrix products such as Matrigel™ have not described this transient benefit, our data are consistent with early time points from those reports. Kofidis et al
followed hearts for only two weeks after Matrigel™-assisted cell transplantation. We also demonstrated a similar early benefit after cell injection, but found that this benefit was consistently lost at later time points. Furthermore, Kutshka et al.
observed in a working heterotopic heart transplant model that ex vivo
cell transplantation, alone or with either a Matrigel™ or collagen matrix, yielded a trend similar to that observed in this work. In that study, FS was increased at 2 weeks in all four groups undergoing cell transplantation. The average FS in each of these groups, however, decreased by week 4, at a time when the average FS had increased in the groups receiving matrix alone. More recently, Landa et al19
also observed that FS at 8 weeks was not statistically significant from baseline for infarcts treated with neonatal cardiomyocytes, supporting the results in this work.
Although we cannot exclude the possibility that our results are specific to the type of cell used, the transient nature of the functional benefit we observed with cell transplantation is consistent with the published literature, and leads one to question why cell transplantation may not yield a sustained improvement in myocardial performance. One limitation to achieving long-term benefit is the failure to obtain true regeneration of functional tissue through survival, differentiation, and integration of donor cells in the host myocardium. Regression of early benefit may also be related to the rapid loss of the majority of transplanted cells, reported by many groups to occur within days after transplantation. This not only limits the cells available for integration, but also limits their anti-apoptotic effect via paracrine mechanisms.48,49
Death of transplanted cells might even be directly detrimental to the myocardium through exacerbation of inflammatory signals or other biochemical sequelae.
The enhancement of longer-term survival of at least a fraction of cells that we observed with matrix-assisted transplantation provides an intriguing opportunity to overcome these limitations. Although the degree of enhanced survival observed with either natural or first-generation synthetic matrices was adequate to sustain early functional benefit, the tunable, easily engineered nature of synthetic matrices represent a critical advantage over naturally occurring matrices that cannot easily be modified or redesigned. While the complexity of the material presented may make short term clinical translation difficult compared to simpler systems, the orthogonal control of degradation, mechanical properties and biological functionality makes this a material an excellent choice for hypothesis driven testing in cardiac tissue engineering. While clinical delivery may be difficult, requiring cooled catheter based systems due to the stiffening upon warming to body temperature, as a test system, control of a wide latitude of parameters will allow for determining key functionality of cardiac tissue engineering materials, after which the material could be re-engineered into a simpler state containing the key functionality to allow for clinical application both in acute LV treatment and potentially for non-acute injuries such as the globally failing LV.