Cardiovascular diseases (CVDs) are disorders of the heart and the blood vessels which are the leading cause of morbidity and mortality worldwide and related with 31% global deaths every year.[64] Although pharmacological and interventional treatments are well-established to relief the symptoms and complications (e.g. arrhythmia) to prevent sudden cardiac death, these strategies mainly delay the death with a poor long-term prognosis rather than provide functional recovery of cardiovascular system.[1b, 65] Due to the limited regenerative capacity of mature caridomyocytes (CMs), stem cell based therapies are considered as one of the most promising treatments to restore the compromised function and regenerate lost tissue for cardiovascular system. Although clinical trials using various types of stem cells such as MSCs,[66] hematopoietic stem cells (HSCs),[67] and bone marrow-derived mononuclear cells (BMMNCs)[68] showed modest improvement in functional recovery of myocardium and blood vessels, the effect of cell treatment is still limited due to the low survival and retention of the transplanted cells. Therefore, development of more efficient delivery strategies for applications of stem cell-based therapies in CVDs treatment is urgently needed.
The heart pumps blood with rhythmic contractions to support circulatory system which is crucial for vertebrate life.[69] Most cardiac diseases, such as myocarial infarction (MI), are caused by insufficient supply of oxygen and nutrients occurs during ischemic conditions resulted from blockage of coronary artery. These are fatal and will result in irreversible damage of CMs. Stem cell-based therapies hold great promise of effective treatment to recover cardiac function and attenuate adverse ventricular remodeling. However, there are still many obstacles to regenerate cardiac tissue via stem cell-based therapies including cell survival and maturity, anisotropic structure and alignment of CMs, electromechanical integration of transplanted cells with the native myocardium, and angiogenesis of neonatal cardiac tissue.
One of the major hurdles associated with current clinical application of stem cell therapy for cardiac regeneration is the low survival and retention during and after transplantation. To overcome the issues, diverse biomaterials have been developed and utilized in preclinical trails especially for MI treatment. The initial concern was providing mechanical protection to prevent cell necrosis caused by shear and extensional forces during injection process. Polymer- and protein-based shear-thinning hydrogels such as hyaluronic acid shear-thinning hydrogel was shown to be an effective delivery biomaterial to salvage cell viability, thus improve myocardium remodeling and functional recovery.
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Stimuli-sensitive polymers which are viscoelastic liquid during injection but can be rapid polymerized under physiological conditions also act as an excellent candidate for stem cell delivery in cardiac regeneration. Combination of shear-thing and stimuli-responsive properties can increase the mechanical strength and providing controllable degradation which is critical for cell integration.[47b] Single cell encapsulation represents another major technique for cell protection with advantages of higher encapsulation efficiency and permeability of nutrients compared to conventional hydrogel delivery.[53, 72] A recent research reported by Davis group encapsulated single explant-derived cardiac stem cells (EDCs) in nanoporous gel (NPG) cocoons consist of ECM and low melt agarose with an average diameter around 60 μm (Figure 2).
The ECM component provided the adhesive site which protected cells through integrin-dependent pro-survival signaling. Meanwhile, incorporation of ECM component promoted cell migration potential which would benefit integration of the delivered cells to the host myocardium. Increasing agarose concentration from 2% to 3.5% in NPG promoted cell survival in short time by reducing cell apoptosis and enhance cell proliferation, which attributed to the increased stiffness of the matrix. Although the increased stiffness did not further improve long-term cell retention, it enhanced the exosome production and secretion of cytokines including interleukin-6 (IL-6) and beta fibroblast growth factor (bFGF) from EDCs which play critical roles in post infarct healing.[74] Intramyocardial injection of NPG capsule compositions indicated the importance of matrix stiffness in the cardiac regeneration as the 3.5% NPG-EDCs showed significant decrease in infarct size, and increase in ejection fraction and peri-infarct vessels formation compared to suspended EDCs and 2% NPG-EDCs.
Besides the mechanical stress during delivery, the specific local microenvironments of MI region also induce diverse stresses which limited the survival and retention of transplanted cells. The oxidative stress occurred post MI, especially after reperfusion, induces damage of cell membrane and causes cell death. Therefore, biomaterials with antioxidant properties which could reduce reactive oxygen species (ROS) can also improve stem cell survival and cardiac regeneration after delivery. For instance, a recent study from Wang group delivered the brown adipose-derived stem cells (BADSCs) using a fullerenol/ alginate hydrogel for MI treatment.
The water soluble fullerenol nanoparticles had great antioxidant property due to the abundance of electron-deficient position which is capable to absorb reactive oxygen species (ROS) and quench the ROS via electron transfer.[76] This antioxidant property promoted cell survival under ROS rich environment. The fullerenol nanoparticles increased expression level of phosphorylated ERK and p38 while inhibited expression of phosphor-JNK, which reduced the apoptosis of BADSCs induced by H2O2. Meanwhile, activation of p38-MAPK pathway by fullerenol nanoparticles facilitated the cardiomyogenic differentiation of BADSCs with higher expression of cTnT, α-actinin, and Cx-43 which are cariomyocyte-specific markers. In vivo experiments further proved that the fullerenol/ alginate hydrogel improved survival and retention of BADSCs at the injection site, which contributed to the remodeling of myocardium by reducing infarct size, enhancing ventricular wall thickness of MI zone, and promoting angiogenesis leading to a functional recovery of the MI hearts.
Immune responses from host tissue is also a critical point to affect the efficacy of delivered stem cells. Infiltration of immune cells such as macrophages, neutrophils and T-cells will attack transplanted stem cells leading to cell death and granuloma formation.[77] A recent delivery of stem cells using a thermo-responsive nanogel with coploymer of poly(N-isopropylacrylamine-co-acrylic acid) (P(NIPAM-AA)) into immunocompetent mice avoided the elicit of immunoresponse, as the circulating level of pro-inflammatory factors and presence of immune cells including T-cells and macrophages were significantly reduced compared to hCSCs injected with PBS.[78] Besides synthetic polymer, nature polymer were also observed with anti-inflammatory property to salvage transplanted cells. An alginate-chitosan micromatrix (ACM) which could degrade within 3 d after transplantation were used to encapsulate ESC aggregates.[29] The ACM layer minimized the host immune response to the implanted cell aggregates within short time which allowed the cells to adapt to the MI microenvironment, and avoided the teratoma formation. This strategy enhanced the cell survival and retention after in vivo delivery compare to saline treated, single cells treated, bare cell aggregates treated and ACM treated groups.
Another important factor for functional recovery of myocardium hinges on the structure of regenerated cardiac tissue. Native CMs have a cylindrical shape with length approximately 100 μm, diameter of 10-25 μm, and assemble anisotropically.[79] The key features of native extracellular matrix (ECM) structure need to be taken into account when regenerate cardiac tissue to acquire native tissue equivalent functions. Linear and honeycomb-like geometries have been widely used to mimic the native ECM structures to guide anisotropic alignment of CMs.
More advanced strategies allow fabrication of subcellular-structures mimicking native microenvironment in cardiac tissue. For instance, a recent study fabricated a substrate with multiscale topography that replicated from primary human mature CMs to construct the submicrometer structure of myofibrils in CMs (Figure 3).[81] A micropatterned PDMS substrate with a dimension similar to mature human CMs were firstly prepared and seeded with CMs. When majority of CMs expressed mature cardiomyocyte markers, cells were fixed and further used as template for a second PDMS substrate fabrication. The fully micropatterned surfaces inherited the submicrometer topography features of primary CMs and improved the differentiation and maturation of iPSC-CMs.
According to another report from Zhang group, translation of cardiac patches with a native-like cardiac ECM architecture further proved the influences of structure on cardiac regeneration in vivo.[82] The multiphoton-excited (MPE) 3D printing were used to fabricate the gelatin scaffolds replicating the size and distribution of ECM (mainly fibronectin) features that mapped from native myocardial tissue of mouse with resolutions of less than 1 μm. Human iPSC-derived cardiac linage cells cultured on the 3D printed scaffold showed similar morphology and alignment as the primary CMs, which leading to a stable and robust contraction, calcium transients, and electrical signal propagation among the engineered human-induced pluripotent stem cell-derived cardiac muscle patch (hCMP). Transplantation of hCMP improved the remodeling and functional recovery of infarcted myocardium, indicating regulation of engineered cardiac tissue morphogenesis would significantly improve the efficacy of stem cell treatment for ischemic myocardial injury.
Electromechanical integration of delivered cells to host tissue is another issue that hindered the application of stem cells therapies in cardiac regeneration. Mismatch of electrical and mechanical support will cause complications including heart failure, diastolic dysfunction and arrhythmias.[83] Association of conductive nanomaterials[84] and conjugation of conductive polymers[85] have been widely used to improve conductivity of biomaterials. Enhanced bioelectrical signal propagation contributes to the differentiation and maturation of stem cell-derived cardiomyocytes, and improves infarcted myocardium recovery. Meanwhile, more and more researches have focused on the influence of the mechanical properties on cell functions. A consensus has emerged that the mechanical properties of the biomaterials are critical to tissue regeneration especially for ischemic heart treatment.[80c, 83a, 86] The native cardiac tissue has anisotropic mechanical properties and dynamic Young’s modulus in systole or diastole. The unique mechanical properties can be achieved by designing of microstructured cardiac patch. For instance, a recent research from Stevens group designed a bow-tie shaped cardiac patch based on chitosan-polyaniline complex.[85c] The conductive polyaniline was deposited on a chitosan film which provided mechanical support. The two positive charged component were ionically crosslinked by phytic acid which also acted as dopant.[85b] By designing a bow-tie shape, the auxetic patch showed native tissue comparabled mechanical properties. And in vivo data showed that the cardiac patches attenuated the hypertrophy of the MI hearts.
Although the electromechanical properties can be easily achieved, the thoracic surgery for cell delivery to myocardium is considered more invasive which results in additional stresses. However, a recent report from Radisic group invented a flexible shape-memory film which gave a hint on development of novel injectable cardiac patches.[61a] The patch was fabricated using poly(octamethylene maleate (anhydride) citrate) (POMAC) elastomer (Figure 4). The citrate-based biopolymers have excellent anti-oxidant properties[87] and the abundant carboxyl groups can be easily functionalized with conductive properties[85d], which makes it one of the suitable biomaterials for cardiac regeneration. Micropatterning of the POMAC film with a diamond-like design enabled returning of its original shape after folding. This shape-memory property allowed injection of the cardiac patches into heart tissue without any compromise in cell viability. Meanwhile, the anisotropic structure of the patches leaded to different elasticity in longitudinal and transversal directions which mimic the anisotropic elasticity of native heart tissue. Delivery of the ESC-CMs seeded cardiac patch into rat and porcine infarcted cardiac tissue significantly improved the recovery of a functional myocardium.
