Decellularization Strategies for Regenerating Cardiac and Skeletal Muscle Tissues.

Cardiovascular disease is the leading cause of death worldwide and is associated with approximately 17.9 million deaths each year. Musculoskeletal conditions affect more than 1.71 billion people globally and are the leading cause of disability. These two areas represent a massive global health burden that is perpetuated by a lack of functionally restorative treatment options. The fields of regenerative medicine and tissue engineering offer great promise for the development of therapies to repair damaged or diseased tissues. Decellularized tissues and extracellular matrices are cornerstones of regenerative biomaterials and have been used clinically for decades and many have received FDA approval. In this review, we first discuss and compare methods used to produce decellularized tissues and ECMs from cardiac and skeletal muscle. We take a focused look at how different biophysical properties such as spatial topography, extracellular matrix composition, and mechanical characteristics influence cell behavior and function in the context of regenerative medicine. Lastly, we describe emerging research and forecast the future high impact applications of decellularized cardiac and skeletal muscle that will drive novel and effective regenerative therapies.

Transplantation of insulin-like growth factor-1 laden scaffolds combined with exercise promotes neuroregeneration and angiogenesis in a preclinical muscle injury model.

The regeneration of skeletal muscle can be permanently impaired by traumatic injuries, despite the high regenerative capacity of native muscle. An attractive therapeutic approach for treating severe muscle inuries is the implantation of off-the-shelf engineered biomimetic scaffolds into the site of tissue damage to enhance muscle regeneration. Anisotropic nanofibrillar scaffolds provide spatial patterning cues to create organized myofibers, and growth factors such as insulin-like growth factor-1 (IGF-1) are potent inducers of both muscle regeneration as well as angiogenesis. The aim of this study was to test the therapeutic efficacy of anisotropic IGF-1-releasing collagen scaffolds combined with voluntary exercise for the treatment of acute volumetric muscle loss, with a focus on histomorphological effects. To enhance the angiogenic and regenerative potential of injured murine skeletal muscle, IGF-1-laden nanofibrillar scaffolds with aligned topography were fabricated using a shear-mediated extrusion approach, followed by growth factor adsorption. Individual scaffolds released a cumulative total of 1244 ng ± 153 ng of IGF-1 over the course of 21 days in vitro. To test the bioactivity of IGF-1-releasing scaffolds, the myotube formation capacity of murine myoblasts was quantified. On IGF-1-releasing scaffolds seeded with myoblasts, the resulting myotubes formed were 1.5-fold longer in length and contained 2-fold greater nuclei per myotube, when compared to scaffolds without IGF-1. When implanted into the ablated murine tibialis anterior muscle, the IGF-1-laden scaffolds, in conjunction with voluntary wheel running, significantly increased the density of perfused microvessels by greater than 3-fold, in comparison to treatment with scaffolds without IGF-1. Enhanced myogenesis was also observed in animals treated with the IGF-1-laden scaffolds combined with exercise, compared to control scaffolds transplanted into mice that did not receive exercise. Furthermore, the abundance of mature neuromuscular junctions was greater by approximately 2-fold in muscles treated with IGF-1-laden scaffolds, when paired with exercise, in comparison to the same treatment without exercise. These findings demonstrate that voluntary exercise improves the regenerative effect of growth factor-laden scaffolds by augmenting neurovascular regeneration, and have important translational implications in the design of off-the-shelf therapeutics for the treatment of traumatic muscle injury.

Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration.

Traumatic skeletal muscle injuries cause irreversible tissue damage and impaired revascularization. Engineered muscle is promising for enhancing tissue revascularization and regeneration in injured muscle. Here we fabricated engineered skeletal muscle composed of myotubes interspersed with vascular endothelial cells using spatially patterned scaffolds that induce aligned cellular organization, and then assessed their therapeutic benefit for treatment of murine volumetric muscle loss. Murine skeletal myoblasts co-cultured with endothelial cells in aligned nanofibrillar scaffolds form endothelialized and aligned muscle with longer myotubes, more synchronized contractility, and more abundant secretion of angiogenic cytokines, compared to endothelialized engineered muscle formed from randomly-oriented scaffolds. Treatment of traumatically injured muscle with endothelialized and aligned skeletal muscle promotes the formation of highly organized myofibers and microvasculature, along with greater vascular perfusion, compared to treatment of muscle derived from randomly-oriented scaffolds. This work demonstrates the potential of endothelialized and aligned engineered skeletal muscle to promote vascular regeneration following transplantation.

Engineering Biomimetic Materials for Skeletal Muscle Repair and Regeneration.

Although skeletal muscle is highly regenerative following injury or disease, endogenous self-regeneration is severely impaired in conditions of volume traumatic muscle loss. Consequently, tissue engineering approaches are a promising means to regenerate skeletal muscle. Biological scaffolds serve as not only structural support for the promotion of cellular ingrowth but also impart potent modulatory signaling cues that may be beneficial for tissue regeneration. In this work, the progress of tissue engineering approaches for skeletal muscle engineering and regeneration is overviewed, with a focus on the techniques to create biomimetic engineered tissue using extracellular cues. These factors include mechanical and electrical stimulation, geometric patterning, and delivery of growth factors or other bioactive molecules. The progress of evaluating the therapeutic efficacy of these approaches in preclinical models of muscle injury is further discussed.

Big bottlenecks in cardiovascular tissue engineering.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges.

Rehabilitative exercise and spatially patterned nanofibrillar scaffolds enhance vascularization and innervation following volumetric muscle loss.

Muscle regeneration can be permanently impaired by traumatic injuries, despite the high regenerative capacity of skeletal muscle. Implantation of engineered biomimetic scaffolds to the site of muscle ablation may serve as an attractive off-the-shelf therapeutic approach. The objective of the study was to histologically assess the therapeutic benefit of a three-dimensional spatially patterned collagen scaffold, in conjunction with rehabilitative exercise, for treatment of volumetric muscle loss. To mimic the physiologic organization of skeletal muscle, which is generally composed of myofibers aligned in parallel, three-dimensional parallel-aligned nanofibrillar collagen scaffolds were fabricated. When implanted into the ablated murine tibialis anterior muscle, the aligned nanofibrillar scaffolds, in conjunction with voluntary caged wheel exercise, significantly improved the density of perfused microvessels, in comparison to treatments of the randomly oriented nanofibrillar scaffold, decellularized scaffold, or in the untreated control group. The abundance of neuromuscular junctions was 19-fold higher when treated with aligned nanofibrillar scaffolds in conjunction with exercise, in comparison to treatment of aligned scaffold without exercise. Although, the density of de novo myofibers was not significantly improved by aligned scaffolds, regardless of exercise activity, the cross-sectional area of regenerating myofibers was increased by > 60% when treated with either aligned and randomly oriented scaffolds, in comparison to treatment of decellularized scaffold or untreated controls. These findings demonstrate that voluntary exercise improved the regenerative effect of aligned scaffolds by augmenting neurovascularization, and have important implications in the design of engineered biomimetic scaffolds for treatment of traumatic muscle injury.

Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells.

Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun microfibrous polycaprolactone scaffolds with either randomly oriented (14 µm fiber diameter) or parallel-aligned (7 µm fiber diameter) microfiber arrangement and co-seeded the scaffolds with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. Here we demonstrated that aligned microfibrous scaffolds induced iCM alignment along the direction of the aligned microfibers after 2 days of iCM seeding, as well as promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Furthermore, the benefit of scaffold anisotropy was evident in the significantly higher maximum contraction velocity of iCMs on the aligned scaffolds, compared to randomly oriented scaffolds, at 12 days of culture. Co-seeding of iCMs with iECs led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that maintain iCM organization and contractile function.

Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat.

Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.

Nanoscale Patterning of Extracellular Matrix Alters Endothelial Function under Shear Stress.

The role of nanotopographical extracellular matrix (ECM) cues in vascular endothelial cell (EC) organization and function is not well-understood, despite the composition of nano- to microscale fibrillar ECMs within blood vessels. Instead, the predominant modulator of EC organization and function is traditionally thought to be hemodynamic shear stress, in which uniform shear stress induces parallel-alignment of ECs with anti-inflammatory function, whereas disturbed flow induces a disorganized configuration with pro-inflammatory function. Since shear stress acts on ECs by applying a mechanical force concomitant with inducing spatial patterning of the cells, we sought to decouple the effects of shear stress using parallel-aligned nanofibrillar collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging, we tracked the alignment, migration trajectories, proliferation, and anti-inflammatory behavior of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly, ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils, despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore, in stark contrast to ECs cultured on randomly oriented films, ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation, while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly, it provides new insight into how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues.

Bilayered vascular graft derived from human induced pluripotent stem cells with biomimetic structure and function.

BACKGROUND: We developed an aligned bi-layered vascular graft derived from human induced pluripotent stem cells (iPSCs) that recapitulates the cellular composition, orientation, and anti-inflammatory function of blood vessels. MATERIALS & METHODS: The luminal layer consisted of longitudinal-aligned nanofibrillar collagen containing primary endothelial cells (ECs) or iPSC-derived ECs (iPSC-ECs). The outer layer contained circumferentially oriented nanofibrillar collagen with primary smooth muscle cells (SMCs) or iPSC-derived SMCs(iPSC-SMCs). RESULTS: On the aligned scaffolds, cells organized F-actin assembly within 8º from the direction of nanofibrils. When compared to randomly-oriented scaffolds, EC-seeded aligned scaffolds had significant reduced inflammatory response, based on adhesivity to monocytes. CONCLUSION: This study highlights the importance of anisotropic scaffolds in directing cell form and function, and has therapeutic significance as physiologically relevant blood vessels.

Aligned-Braided Nanofibrillar Scaffold with Endothelial Cells Enhances Arteriogenesis.

The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000-1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies.

Near-infrared II fluorescence for imaging hindlimb vessel regeneration with dynamic tissue perfusion measurement.

BACKGROUND: Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths. METHODS AND RESULTS: Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy. CONCLUSIONS: The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease.

Role of extracellular matrix signaling cues in modulating cell fate commitment for cardiovascular tissue engineering.

It is generally agreed that engineered cardiovascular tissues require cellular interactions with the local milieu. Within the microenvironment, the extracellular matrix (ECM) is an important support structure that provides dynamic signaling cues in part through its chemical, physical, and mechanical properties. In response to ECM factors, cells activate biochemical and mechanotransduction pathways that modulate their survival, growth, migration, differentiation, and function. This Review describes the role of ECM chemical composition, spatial patterning, and mechanical stimulation in the specification of cardiovascular lineages, with a focus on stem cell differentiation, direct transdifferentiation, and endothelial-to-mesenchymal transition. The translational application of ECMs is discussed in the context of cardiovascular tissue engineering and regenerative medicine.

Multi-cellular interactions sustain long-term contractility of human pluripotent stem cell-derived cardiomyocytes.

Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.

Tissue specificity of decellularized rhesus monkey kidney and lung scaffolds.

Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (1) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human embryonic stem cells (hESCs) for recellularization in order to explore cellular differentiation in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of growth factors and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC differentiation by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix.

Renal tissue engineering with decellularized rhesus monkey kidneys: age-related differences.

New therapies for severely damaged kidneys are needed due to limited regenerative capacity and organ donor shortages. The goal of this study was to repopulate decellularized kidney sections in vitro and to determine the impact of donor age on recellularization. This was addressed by generating decellularized kidney scaffolds from fetal, juvenile, and adult rhesus monkey kidney sections using a procedure that removes cellular components while preserving the structural and functional properties of the native extracellular matrix (ECM). Kidney scaffolds were recellularized using explants from different age groups (fetal, juvenile, adult) and fetal renal cell fractions. Results showed vimentin+ cytokeratin+ calbindin+ cell infiltration and organization around the scaffold ECM. The extent of cellular repopulation was greatest with scaffolds from the youngest donors, and with seeding of mixed fetal renal aggregates that formed tubular structures within the kidney scaffolds. These findings suggest that decellularized kidney sections from different age groups can be effectively repopulated with donor cells and the age of the donor is a critical factor in repopulation efficiency.

Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering.

The goal of this study was the production of a decellularized kidney scaffold with structural, mechanical, and physiological properties necessary for engineering basic renal structures in vitro. Fetal, infant, juvenile, and adult rhesus monkey kidney sections were treated with either 1% (v/v) sodium dodecyl sulfate or Triton X-100 followed by quantitative and qualitative analysis. Comparison of decellularization agents and incubation temperatures demonstrated sodium dodecyl sulfate at 4 degrees C to be most effective in preserving the native architecture. Hematoxylin and eosin staining confirmed the removal of cellular material, and immunohistochemistry demonstrated preservation of native expression patterns of extracellular matrix proteins, including heparan sulfate proteoglycan, fibronectin, collagen types I and IV, and laminin. Biomechanical testing revealed a decrease in the compressive modulus of decellularized compared to fresh kidneys. Layering of fetal kidney explants on age-matched decellularized kidney scaffolds demonstrated the capacity of the scaffold to support Pax2+/vimentin+ cell attachment and migration to recellularize the scaffold. These findings demonstrate that decellularized kidney sections retain critical structural and functional properties necessary for use as a three-dimensional scaffold and promote cellular repopulation. Further, this study provides the initial steps in developing new regenerative medicine strategies for renal tissue engineering and repair.