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

The peril of a 3-dimensional, robust and sustained myocardial restoration by means of Tissue Engineering is that it still remains a largely experimental approach. Prolific protocols have been developed and tested in small and large animals, but as clinical cardiac surgeons, we have not come to the privilege of utilizing any of them in our clinical practice. The question arises: why? The heart is a unique organ, anatomically and functionally. It is not an easy target to replicate with current techniques, or even to support its viability and function. Currently available therapies fail to reverse the loss of functional cardiac tissue, the fundamental pathology remains unaddressed and a heart transplantation is an ultima ratio treatment option. Owing to equivocal results of cell-based therapies, several strategies have been pursued to overcome 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 the light of the available data, injectable biomaterials suitable for transcatheteter delivery appear to have the highest translational potential,. This article presents a current state-of-the-art in the field of hydrogel-based myocardial restoration therapy.


Introduction
"Stem cells are the future of heart treatment;….and they will always be" Norman Shumway This may constitute a somewhat nihilistic approach, by mouth of an authority in Cardiac Surgery and heart failure treatment, yet holds more or less true to this day, simply taken from the angle of clinical implementation, in form of a comprehensive, recommended, if not guideline supported protocol. Twenty five years into myocardial restoration attempts following myocardial injury, there isn't a single efficient, robust and sustained impact on the injured heart muscle following ischemic insult. Approaches so far have been encompassing 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, but, in brief, they all hold promise and peril.
The unique and complex structure of healthy and injured myocardium Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 April 2021 doi:10.20944/preprints202104.0590.v1 Gerard Buckberg, with his seminal paper "The Helix and the Heart" has awaken many aspired myocardial restorers to the fact that the heart is not a quiescent, homogenously built target, but rather a highly asymmetric, anisotropic and angiotropic organ, featuring an intricate architecture [1]. Not a spot in the heart is built like the other. The heart muscle is not a continuous layer but rather 3 layers superimposed at any given point, which can be folded and unfolded like a ribbon, as demonstrated by Buckberg [1]. This leads to a systematic overlay of 3 layers at any given spot, and the formation of critical intercalations and physical sheer stresses, which are organized in an optimal fashion to A: form an oval shaped vortex, and B: maximize contractile force at the best energy economy. This explains why a systolic diameter increase of only around 8% at the myofiber level translates in a much more disproportionately high fractional shortening, Ejection Fraction and left ventricular wall thickening [2] (Figure 1, B). The arrangement of myofibers, their communications and intercalations, the electrical signal propagation, the fibrous skeleton, the arrangement of supplying arterioles, all render the myocardium a very anisotropic target, where random injection of cells of any kind remains rather unimpressive, in terms of real health gain and symptomatic relief, from a clinician's point of view.

The Vicious Circle of Myocardial Ischemia and the mechanics of Remodelling
Vu et al. had postulated that the acute myocardial injury known as infarct triggers a cascade of events with severe cellular and functional impact [2]. This vicious circle is selfperpetuating, resulting in the so called "non ischemic expansion of the infarct", unrelated and not dependent on further coronary occlusions (Figure 1, A). This is largely due to 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) cause macrophagy and apoptosis [3]. This perpetuates the cell death cycle, stimulation of remodelling mechanisms which result to a 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 remodelling [2], and drop of contractility and Ejection Fraction [1,2] (Figure 1, C), as compared to that in the heart of a healthy individual [6] (Figure 1, B). The outcome is proportional to the extent of tissue loss and dysfunction and may encompass multiple segments of the LV, best to be captured by nuclear scans and MRI.

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 renewal has been recently overthrown by studies demonstrating a low-level proliferation, even in adult hearts [7]. However, the regenerative capacity is minimal and insufficient to overome 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, so far these have not been translated into clinical practice. One 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 the ventricular remodelling. The ensuing hostile and inflammatory environment results in rapid death of injected cells, or lack of integration thereof. It is incomprehensive, and in the vast majority of studies proven that injected cells do not organize in an integrated syncytium, which excites orchestrated contractility. Depending on the type of cells randomly injected, different complications occur [8]. Solid scaffolds, even if adding thickness to the aneurysmatic scar, have not proven themselves as viable solution either, particularly due to the necessity for open heart surgery to implant them ( Figure 2). There is 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 in 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 following key value propositions: -Injectable, hence minimally invasive, administration -Autologous material, not of stem cell nature, to be derived simply, during treatment -Polytherapy approach to address concomitant aspects of the vicious circle of myocardial ischemia (Antioxidants, Purine Metabolism blockers/Anti-inflammatory drugs) -Easy adoption and clinical penetration in the horizon A literature search was performed using the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA) guidelines [9] electronically. We conducted records 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) were used: "Medical Subject Headings" (MeSH) terms were used: 'Hydrogels', 'Extracellular matrix hydrogels', 'Tissue engineering', 'Myocardial infarctions', 'Myocardial infarction therapy', 'Cardiac stem cell therapy', 'Cell-based therapy'. 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.

Cells
The inclusion criteria included any experimental cohort studies in which large animals or patients underwent injectable delivery of hydrogel and/or hydrogel compound analogure for its effect analysis on ischemic heart disease. Furthermore, only studies published after the year 2000 were included to prevent using outdated data. Articles with hydrogel compund 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 and hydrogel characteristics and delivery method, and the outcomes measured. Data extracted with respect to the infarct creation and hydrogel characteristics and delivery method included: method of MI creation, artery involved, cell delivered via hydrogel or its analogues, type of matrix, method of delivery to myocardium. Data extracted with respect to the outcomes measured included: any data related to 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. 61 out of these 70 papers could be retrieved. Following a full-text review of these papers, a final 19 papers were included. 2 papers were added to the final pool via additional sources, leaving a total of 21 papers for inclusion into the present study. The characteristics of the study population are summarised in Table 1. Experimental grouping and aim of the included study of 613 large animals and 15 human subjects has been plotted. The study characteristics did not differ markedly in thier aim but, diversification observes in grouping and use of animal subjects. In studies including Zhou, D et al.
[24] the recipients age and sex were not categorised. Hydrogel characterizaton and its mode of delivery is tabulated in Table 2. Few studies reported the delivery of injectable hydrogel without a celluar componant [11,12,17,21,26,27,30] while the rest chose different composition of cells and 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)  did not measure the direct effect of treatment on LV remodeling. The remaining 16 studies that looked into LV remodeling reported either attenuated or equivocal LV remodeling. This would suggest that injectable hydrogels have the ability to retain the highly complex and intricate architecture of the heart, post-MI, resulting in increased EF [2]. This would be in line with the findings of studies [12,13,16-23, 25,26,27,29,30], which reported both attenuated LV remodeling and an increase in EF.

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 [10,14,15,17,18,20,22-25,29] measured blood vessel density, all of which reported an improved effect, implying that hydrogel treatment can have a positive effect on angiogenesis post-MI. Zhou, D et al [15] 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 myocardium termed "cardiac remodelling" which may result in the development of HF ( Figure 2). The clinical sequelae are encountered in up to three-quarters of patients within 5 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 indicates that approximately half of patients with HF do not survive more than 5 years after the diagnosis [32], meaning that despite advances in cardiac care, survival rates in this patient population are still very poor and 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. One of the most promising therapeutic compounds are hydrogel-based biomaterials that can provide not only a mechanical support for a failing heart, but also can serve as a vehicle for cells, growth factors and drugs. Beacause of their potential for minimally invasive transcatheter delivery, injectable hydrogels appear to be one of the most promising ones in terms of their potential clinical application. Several types of hydrogel- based approaches for cardiac tissue repair have been investigated to date. Each 2 category of hydrogels has its advantages and disadvantages that can influence their 3 potential clinical applicability. There are various types of hydrogels with different 4 properties based on their origin (natural/synthetic), various mechanisms of cross-5 linking, etc. 6 Based on the best evidence, we have observed a diversity of compounds with none 7 of the compositions showing clear superiority (Table 3). There are a number of 8 studies using accelualr hydrogel by changing the matix composition, more focused 9 to investigate whether hydrogel characteristics i.e stiffening enhances therapeutic 10 efficacy to limit LV remodeling and heart failure [26]. Synthetic Hydrogel: Poly 11 (NIPAAm-co-HEMA-co-MAPLA) (Sigma-Aldrich, USA) was used in some studies 12 [12,27], but Hyaluronic acid-based hydrogel was the choice in most cases. Cell Types 13 include skeletal myoblasts (SKMs), CMs, and other progenitor cells capable of 14 differentiation to CMs like embryonic stem cell (ESC), ESC-derived CMs (ESCCMs), 15 and mesenchymal stem cells (MSCs) with limited potentials was investigated. 16 Human umbilical mesenchymal stem cells [hUMSC] are in new focus [13] whereas basic 17 fibroblast growth factor (bFGF); acidic gelatin hydrogel microspheres (AGHM); vascular 18 endothelial growth factor (VEGF) are in use with non-superiority to each other. Vu 27 One of the most important aspects of hydrogel-based myocardial restoration therapy 28 is the mode of delivery. In the context of increasing role of minimally invasive techniques, 29 a particular emphasis has been placed on shifting away from open heart surgery to 30 catheter-based techniques. We doubt that any restoration method involving major 31 surgical trauma can survive as stand-alone treatment, as no patient, cardiologist or 32 surgeon will adopt it. Second, therapy may have to be chronic and repeated, i.e. multiple 33 sessions in the process of time post-MI, as HF chronifies. The patient cannot undergo 34 countless re-dos if the procedure is invasive. This has prompted researchers to develop 35 new devices for pinpoint delivery of therapeutic compounds into a desired area of 36 myocardium. As a result, catheter-based techniques for myocardial restoration therapy 37 have evolved from simple intracoronary injections (which is far from perfect due to rapid 38 wash out of an intravascular compound) to techniques with a more efficient therapeutics' 39 retention. One of the examples is the TransAccess catheter system with a fluoroscopic and 40 intravascular ultrasound guidance which was used for autologous skeletal myoblasts 41 delivery [35]. Currently, the most advanced device for intramyocardial delivery of 42 therapeutic compounds is the NOGA system ( Figure 3). The latter allows to perform 43 three-dimensional electromechanical mapping of LV in order to identify target zones and 44 perform precise transendocardial injections of therapeutics. Available data and the 45 authors' own experience derived from large animal models confirms that the NOGA 46 device is safe and highly effective.

54
Less Invasive procedures, coupled with injectable compounds present a valid 55 platform for a translational restoration protocol, which may be adopted by Interventional 56 Cardiologists and Heart Surgeons. Polytherapeutic adjuvants, such as antioxidants, 57 paracrine-active drugs, antiinflammatory substances, may be added to the protocls, to 58 ensure sustained myocardial restoration effect. 59 As discussed in the present paper, among all biomaterials currently used in cardiac 60 tissue engineering, injectable hydrogels with their potential for minimally invasive 61 delivery, in-vivo breakdown into harmless derivatives, represent the most promising 62 therapeutic option. However, the translational pathway from bench to bedside is 63 challenging and still needs to be explored. It can be anticipated that in the next decades 64 the role of cell-vehicle compounds in the treatment of ischemic HF patients will expand 65 and injectable hydrogels will penetrate into the clinical arena to a higher extent.