Targeted Myocardial Restoration with Injectable Hydrogels—In Search of The Holy Grail in Regenerating Damaged Heart Tissue

A 3-dimensional, robust, and sustained myocardial restoration by means of tissue engineering remains an experimental approach. Prolific protocols have been developed and tested in small and large animals, but, as clinical cardiac surgeons, we have not arrived at the privilege of utilizing any of them in our clinical practice. The question arises as to why this is. The heart is a unique organ, anatomically and functionally. It is not an easy target to replicate with current techniques, or even to support in its viability and function. Currently, available therapies fail to reverse the loss of functional cardiac tissue, the fundamental pathology remains unaddressed, and heart transplantation is an ultima ratio treatment option. Owing to the equivocal results of cell-based therapies, several strategies have been pursued to overcome the limitations of the current treatment options. Preclinical data, as well as first-in-human studies, conducted to-date have provided important insights into the understanding of injection-based approaches for myocardial restoration. In light of the available data, injectable biomaterials suitable for transcatheter delivery appear to have the highest translational potential. This article presents a current state-of-the-literature review in the field of hydrogel-based myocardial restoration therapy.


"Stem Cells Are the Future of Heart Treatment, and They Will Always Be" Norman Shumway
This may constitute a somewhat nihilistic approach, from the mouth of an authority in cardiac surgery and heart failure treatment, yet holds more or less true to this day-simply taken from the perspective of clinical implementation-in the form of a comprehensive, recommended, if not guideline-supported, protocol: after 25 years into myocardial restoration attempts following myocardial injury, there has not been a single efficient, robust, and sustained impact on the injured heart muscle following ischemic insult. Approaches so far have encompassed various types of cells, cell products or derivatives, scaffolds of various physical conditions, as well as multiple administration routes. It would be beyond the scope of the present paper to revisit them all; however, in brief, they all hold promise and peril.

Cell-Based Therapy: Unfulfilled Hopes or Misguided Expectations? Why Not Only Cells?
The prevailing dogma suggesting that adult mammalian cardiomyocytes are postmitotic cells with no ability to renew has been recently overthrown by studies demonstrating a low level of proliferation, even in adult hearts [7]. However, the regenerative capacity is minimal and insufficient to overcome the loss of cardiac cells following MI. The inability of the adult heart to regenerate has yielded several preclinical and clinical studies focused on different cell-based therapies. Despite very promising preclinical results, these results have so far not been translated into clinical practice. Some of the major challenges limiting their clinical application are low retention and survival rates; very limited trans-differentiation into cardiomyocytes; safety; and, in some cases, ethical concerns.
Over the last few decades, cell therapy has been applied in clinical myocardial restoration. Though the result is non-conclusive, some studies have shown the attenuation of ventricular remodeling. The ensuing hostile and inflammatory environment results in the rapid death of injected cells, or lack of integration thereof. It is incomprehensive, and the vast majority of studies have proven that injected cells do not organize in an integrated syncytium, which excites orchestrated contractility. Depending on the type of cells that are randomly injected, different complications occur [8]. Solid scaffolds-even when adding thickness to the aneurysmatic scar-have not proven themselves as a viable solution either, particularly due to the necessity of open heart surgery to implant them ( Figure 2

The Vicious Circle of Myocardial Ischemia and the Mechanics of Remodeling
Vu et al. had postulated that the acute myocardial injury, known as infarct, triggers a cascade of events with severe cellular and functional impacts [2]. This vicious circle is self-perpetuating, resulting in the so-called "non-ischemic expansion of the infarct", unrelated to and not dependent on further coronary occlusions ( Figure 1A). This is largely due to a mechanical shift of the myocardial plates and a series of biological phenomena with architectural sequelae. When acute myocardial ischemia and injury manifest, cell death ensues. Enzymatic damage to the tissue is next, with the release of so-called "danger signals" (derivatives of purine metabolism, free radicals, etc.), causing macrophagy and apoptosis [3]. This perpetuates the cell death cycle, stimulating remodeling mechanisms that result in scar formation. As a result, the affected myocardium thins out, while the surrounding myocardium may become temporarily dysfunctional as well. When the LV wall thins out, the modified Laplace law [4] of the oval of the heart takes effect, thus leading to extreme circumferential wall stress, more cell death [5] and architectural remodeling [2], and a drop of contractility and ejection fraction [1,2] (Figure 1C), as compared to that in the heart of a healthy individual [6] ( Figure 1B). The outcome is proportional to the extent of tissue loss and dysfunction and may encompass multiple segments of the LV; this is best captured by nuclear scans and MRIs.

Cell-Based Therapy: Unfulfilled Hopes or Misguided Expectations? Why Not Only Cells?
The prevailing dogma suggesting that adult mammalian cardiomyocytes are postmitotic cells with no ability to renew has been recently overthrown by studies demonstrating a low level of proliferation, even in adult hearts [7]. However, the regenerative capacity is minimal and insufficient to overcome the loss of cardiac cells following MI. The inability of the adult heart to regenerate has yielded several preclinical and clinical studies focused on different cell-based therapies. Despite very promising preclinical results, these results have so far not been translated into clinical practice. Some of the major challenges limiting their clinical application are low retention and survival rates; very limited trans-differentiation into cardiomyocytes; safety; and, in some cases, ethical concerns.
Over the last few decades, cell therapy has been applied in clinical myocardial restoration. Though the result is non-conclusive, some studies have shown the attenuation of ventricular remodeling. The ensuing hostile and inflammatory environment results in the rapid death of injected cells, or lack of integration thereof. It is incomprehensive, and the vast majority of studies have proven that injected cells do not organize in an integrated syncytium, which excites orchestrated contractility. Depending on the type of cells that are randomly injected, different complications occur [8]. Solid scaffolds-even when adding thickness to the aneurysmatic scar-have not proven themselves as a viable solution either, particularly due to the necessity of open heart surgery to implant them ( Figure 2).  There is an obvious need for a targeted, less invasive myocardial restoration trea ment, which does not add too much stand-alone trauma to the patient and can be int grated into a viable clinical protocol, to be adopted by cardiologists as well. Arising fro the above pain-points, we have long shifted our focus from stem cells to liquid com pounds, with the following key value propositions: 1. Injectable, hence minimally invasive, administration 2. Autologous material, not of stem cell nature, to be derived simply during treatmen 3. A polytherapy approach to address concomitant aspects of the vicious circle of my cardial ischemia (antioxidants, purine metabolism blockers/anti-inflammatory drug 4. Easy adoption and clinical penetration in the horizon

Materials and Methods
A literature search was performed electronically using the Preferred Reporting Item for Systematic Reviews and MetaAnalyses (PRISMA) guidelines [9]. We conducted reco scrutiny on Medline (via PubMed), Embase, and Web of Science from inception to 31 There is an obvious need for a targeted, less invasive myocardial restoration treatment, which does not add too much stand-alone trauma to the patient and can be integrated into a viable clinical protocol, to be adopted by cardiologists as well. Arising from the above pain-points, we have long shifted our focus from stem cells to liquid compounds, with the following key value propositions:
Autologous material, not of stem cell nature, to be derived simply during treatment 3.
A polytherapy approach to address concomitant aspects of the vicious circle of myocardial ischemia (antioxidants, purine metabolism blockers/anti-inflammatory drugs) 4.
Easy adoption and clinical penetration in the horizon.

Materials and Methods
A literature search was performed electronically using the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) guidelines [9]. We conducted record scrutiny on Medline (via PubMed), Embase, and Web of Science from inception to 31st March 2021. A repetitive and exhaustive combination of the following 'medical subject headings' (MeSH) terms were used: "hydrogels", "extracellular matrix hydrogels", "tissue engineering", "myocardial infarctions", "myocardial infarction therapy", "cardiac stem cell therapy", and "f'cell-based therapy". This study protocol was registered with PROSPERO #CRD42021250140. The full search strategy can be found in the supplementary materials (Supplementary Figure S1). Relevant articles were screened and systematically assessed with inclusion and exclusion criteria applied.
The inclusion criteria included any experimental cohort studies in which large animals or patients underwent an injectable delivery of hydrogel and/or hydrogel compound analogure for an effect analysis on ischemic heart disease. Furthermore, only studies published after the year 2000 were included to prevent using outdated data. Articles with hydrogel compound processing (lab experiment) and in-vitro experiments, small animal studies, and case reports were excluded. Additionally, any studies that were not written in the English language were excluded. Three authors (E.L., W.W., and F.S.) independently abstracted details of the study characteristics, the myocardial infarct (MI) creation, the hydrogel characteristics, the delivery method, and the outcomes measured. Data extracted, with respect to the infarct creation, hydrogel characteristics, and the delivery method, included: method of MI creation, the artery involved, the cell delivered via hydrogel or its analogues, the type of matrix, and the method of delivery to the myocardium. Data extracted, with respect to the outcomes measured, included: any data related to the functional and morphological outcomes of the heart. Outcomes were then grouped according to the modality they were measured with. All outcomes are expressed as the treatment group outcome when compared to the control group.

Findings
The systematic search revealed a total of 28,704 papers. After 13,775 duplicates were excluded, 14,929 papers remained for screening. Based on the title and abstract, irrelevant articles were excluded, leaving 70 papers for full-text review. Out of these 70 papers, 69 could be retrieved. Following a full-text review of these papers, 19 papers remained for inclusion. Additional sources provided 2 papers that were added to the final pool, resulting in a total of 21 papers for inclusion into the present study (Supplementary Figure S2). The characteristics of the study population are summarised in Table 1. The experimental groupings and aims of the included studies, including 613 large animals and 15 human subjects, have been plotted. The study characteristics did not differ markedly in their aim, but there was diversity observed in the groupings used and in the use of animal subjects. In some studies, including Zhou et al. [10] and Liu et al. [11], the recipients' age and sex were not categorized. The hydrogel characterization and its mode of delivery are tabulated in Table 2. Few studies reported the delivery of injectable hydrogel without a cellular component [12][13][14][15][16][17][18], while the rest chose a different composition of cells and type of matrix.

Treatment with Hydrogel Improves Systolic and Diastolic Cardiac Function
Out of the 21 studies, 17 measured systolic function via LVEF and 4 via SV (Table 2). Yamamoto et al., 2001 [21], Chang et al. [25,28], and Li et al. [27] also measured diastolic function via LV EDP. All of the studies that measured LVEF reported an increase, with the exception of the Yamamoto et al. [21] study, which reported an equivocal outcome. These parameters were frequently measured via three main modalities: echocardiography, magnetic resonance imaging (MRI), or ventricular catheterization. Out of the 21 studies, only Leor et al. [12] did not measure functional outcomes of the treatment group.

Treatment with Hydrogel Reduces Cardiac Fibrosis
Cardiac fibrosis was frequently quantified via scar size and the extent of fibrosis. These parameters were measured via immunohistochemistry, Masson's trichrome stain, MRI, and computed tomography (CT). Out of the 21 studies, only 5 studies [10,13,14,23,28] measured the effect of treatment on fibrosis, with Zhou et al. [10] reporting equivocal scar size and the rest reporting reduced fibrosis with treatment.

Treatment with Hydrogel Supports Angiogenesis Post-Infarction
The degree of angiogenesis was mainly quantified using blood vessel density via immunohistochemistry staining. Out of the 21 studies, only 11 [10,11,14,19,21,23,[25][26][27][28]30] measured blood vessel density; each of the 11 studies reported an improved effect, implying that hydrogel treatment can have a positive effect on angiogenesis post-MI. Zhou et al. [10] particularly focuses on the density of specific blood vessels-namely arterioles, small vessels, and larger arterioles-all of which show an increase in density.

Post-MI Survivability
In the vast majority of cases, MI is a consequence of a vulnerable plaque rupture and a subsequent intracoronary thrombosis. The process initiates maladaptive changes in the myocardium, termed "cardiac remodeling", which may result in the development of HF (Figure 2). The clinical sequelae are encountered in up to three-quarters of patients within five years after their first coronary event [31]. Importantly, HF has not only a significant impact on patients' functional capacity and quality of life, but the disease also significantly affects their life expectancy. Available data indicate that approximately half of the patients with HF do not survive for more than five years after the diagnosis [32], meaning that despite the advances in cardiac care, survival rates in this patient population are still very poor and are comparable to those observed in many types of cancer [33,34]. Given the above, more still needs to be done to tackle the burden of the disease more efficiently, thus triggering alternative mono-or poly-therapeutic treatments using viable matter and scaffolds.

Injectable Hydrogel-Based Approach for Cardiac Tissue Engineering
Owing to the intricate myocardial architecture and function, we believe that the triple approach (i.e., enhancing viability, counteracting inflammation, and stabilizing the diminishing architectural integrity of the left ventricle) yields the best restorative effect. Some of the most promising therapeutic compounds are hydrogel-based biomaterials that can not only provide mechanical support for a failing heart, but can also serve as a vehicle for cells, growth factors, and drugs. Because of their potential for minimally invasive transcatheter delivery, injectable hydrogels appear to be one of the most promising types of compounds in terms of their potential clinical application. Several types of hydrogel-based approaches for cardiac tissue repair have been investigated to date. Each category of hydrogels has its advantages and disadvantages that can influence their potential clinical applicability. There are various types of hydrogels with different properties based on their origin (natural/synthetic), various mechanisms of cross-linking, etc.
Based on the best evidence, we have observed a diverse range of compounds with none of the compositions showing clear superiority (Table 3). There are a number of studies, using acellular hydrogel by changing the matrix composition, more focused to investigate whether hydrogel characteristics (i.e, stiffening) enhances therapeutic efficacy to limit LV remodeling and heart failure [16]. Synthetic hydrogel: poly (NIPAAm-co-HEMAco-MAPLA) (Sigma-Aldrich, St. Louis, MO, USA) was used in some studies [13,17], but hyaluronic acid-based hydrogel was used in most cases. Cell types, including skeletal myoblasts (SKMs), CMs, and other progenitor cells capable of differentiation to CMs, like embryonic stem cells (ESCs), ESC-derived CMs (ESCCMs), and mesenchymal stem cells (MSCs) with limited potentials, were investigated. Human umbilical mesenchymal stem cells [hUMSC] are a new focus [20], whereas basic fibroblast growth factor (bFGF), acidic gelatin hydrogel microspheres (AGHM), and vascular endothelial growth factor (VEGF) are in use with non-superiority to each other. Vu et al. used hyaluronic acid-based hydrogel coupled with PRP and showed improved host-cell viability [30]; while Traverse et al. [18] reported the first-in-human study with VentriGel TM (ECM from the decellularized porcine myocardium) in patients with 1st STEMI treated by PCI within a period of post-intervention between 60 days and 3 years, and found MRI evidence of LV remodeling and a clinical improvement in the study subgroup.

Less Invasive Administration Modes
One of the most important aspects of hydrogel-based myocardial restoration therapy is the mode of delivery. In the context of the increasing role of minimally invasive techniques, a particular emphasis has been placed on shifting away from open heart surgery to catheterbased techniques. We doubt that any restoration method involving major surgical trauma can survive as a stand-alone treatment, as no patient, cardiologist, or surgeon will adopt it. Second, therapy may have to be chronic and repeated (i.e., multiple sessions during the process of time post-MI as HF chronifies). The patient cannot undergo countless re-dos if the procedure is invasive. This has prompted researchers to develop new devices for pinpointing the delivery of therapeutic compounds into a desired area of the myocardium. As a result, catheter-based techniques for myocardial restoration therapy have evolved from simple intracoronary injections (which are far from perfect, due to the rapid washout of an intravascular compound) to techniques with more efficient therapeutic retention. One of the examples is the TransAccess catheter system with fluoroscopic and intravascular ultrasound guidance, which was used for autologous skeletal myoblast delivery [35]. Currently, the most advanced device for intramyocardial delivery of therapeutic compounds is the NOGA system ( Figure 3). The latter allows for the performance of 3D electromechanical mapping of the LV in order to identify target zones and perform precise transendocardial injections of therapeutics. Available data and the authors' own experience, derived from large animal models, confirm that the NOGA device is safe and highly effective. can help the electromechanical assessment of the myocardium. NOGA shows viability on the left column; dense scarring is visible at the apex and the antero-septal wall (red); scar area (<0.5 mV) = RED; viable tissue (>1.5 mV) = PURPLE. Comparing the bipolar and unipolar maps, NOGA is able to define border zone areas better.

Conclusions
Less Invasive procedures, coupled with injectable compounds, present a valid platform for a translational restoration protocol, which may be adopted by interventional cardiologists and heart surgeons. Polytherapeutic adjuvants, such as antioxidants, paracrine-active drugs, and anti-inflammatory substances, may be added to the protocols to ensure a sustained myocardial restoration effect.
As discussed in the present paper, among all biomaterials currently used in cardiac tissue engineering, injectable hydrogels, with their potential for minimally invasive delivery and in-vivo breakdown into harmless derivatives, represent the most promising therapeutic option. However, the translational pathway from bench to bedside is challenging and still needs to be explored. It can be anticipated that in the next few decades the role of cell-vehicle compounds in the treatment of ischemic HF patients will expand, and injectable hydrogels will penetrate into the clinical arena to a higher extent.  can help the electromechanical assessment of the myocardium. NOGA shows viability on the left column; dense scarring is visible at the apex and the antero-septal wall (red); scar area (<0.5 mV) = RED; viable tissue (>1.5 mV) = PURPLE. Comparing the bipolar and unipolar maps, NOGA is able to define border zone areas better.

Conclusions
Less Invasive procedures, coupled with injectable compounds, present a valid platform for a translational restoration protocol, which may be adopted by interventional cardiologists and heart surgeons. Polytherapeutic adjuvants, such as antioxidants, paracrineactive drugs, and anti-inflammatory substances, may be added to the protocols to ensure a sustained myocardial restoration effect.
As discussed in the present paper, among all biomaterials currently used in cardiac tissue engineering, injectable hydrogels, with their potential for minimally invasive delivery and in-vivo breakdown into harmless derivatives, represent the most promising therapeutic option. However, the translational pathway from bench to bedside is challenging and still needs to be explored. It can be anticipated that in the next few decades the role of cell-vehicle compounds in the treatment of ischemic HF patients will expand, and injectable hydrogels will penetrate into the clinical arena to a higher extent.