Perfusion decellularization of whole rat hearts. (A)–(C): Photographs of cadaveric rat hearts mounted on a Langendorff apparatus. Retrograde perfusion of cadaveric rat heart over 12 h using, (A) PEG, (B) Triton-X-100 and (C) SDS. The heart becomes more translucent as cellular material is washed out from the right ventricle, then the atria and finally the left ventricle. (D)–(F) Corresponding H&E staining of thin sections from LV, showing complete decellularization in (F) and incomplete decellularization in (D) and (E). (D) and (E) Hearts treated with PEG or Triton-X-100 retained nuclei and myofibers. Scale bars, 200 mm. (F) H&E staining of SDS-treated heart showing no intact cells or nuclei. Scale bar, 200 mm. All three protocols maintain large vasculature conduits (black asterisks). Note: Ao, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Source: Adapted with permission from Ott et al. (2008). 

Perfusion decellularization of whole rat hearts. (A)–(C): Photographs of cadaveric rat hearts mounted on a Langendorff apparatus. Retrograde perfusion of cadaveric rat heart over 12 h using, (A) PEG, (B) Triton-X-100 and (C) SDS. The heart becomes more translucent as cellular material is washed out from the right ventricle, then the atria and finally the left ventricle. (D)–(F) Corresponding H&E staining of thin sections from LV, showing complete decellularization in (F) and incomplete decellularization in (D) and (E). (D) and (E) Hearts treated with PEG or Triton-X-100 retained nuclei and myofibers. Scale bars, 200 mm. (F) H&E staining of SDS-treated heart showing no intact cells or nuclei. Scale bar, 200 mm. All three protocols maintain large vasculature conduits (black asterisks). Note: Ao, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Source: Adapted with permission from Ott et al. (2008). 

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Abstract Whole-organ decellularization and tissue engineering approaches have made significant inroads during recent years. If proven to be successful and clinically viable, it is highly likely that this field would be poised to revolutionize organ transplantation surgery. In particular, whole-heart decellularization has captured the attention and...

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... Ott et al. (2008) carried out antegrade coronary perfusion of cadaveric rat hearts on a modified Langendorff apparatus and subsequently used histological evaluation of nuclei or contractile elements to compare the decellularization efficacy amongst three chemicals were present. Histological stains clearly revealed that the use of SDS yielded a fully decellularized construct and gave better results than polyethylene glycol (PEG), Triton- X100 or enzyme-based protocols (Ott et al., 2008) ( Figure 3). ...

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... Four decellularization methodswhich used either Trypsin or NH 4 OH as the main lysing agent-were employed to void BTM of cells (Figure 1). Trypsin is known to remove cells from the tissue by cleavage of the cell cytoskeleton from the ECM through hydrolysis of peptide bonds [47], and as an alkaline agent, NH 4 OH causes disruption to the cell membrane leading to osmotic swelling and cell lysis [48]. The main difference among the four methods trialed for decellularization of BTM involved adjusting the time associated with the lysing agent (24 h or 2 h) to assess the ability to remove cells whilst limiting damage to the tissue's structure. ...
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Glaucoma is linked to raised intraocular pressure (IOP). The trabecular meshwork (TM) plays a major role in regulating IOP by enabling outflow of aqueous humor from the eye through its complex 3D structure. A lack of therapies targeting the dysfunctional TM highlights the need to develop biomimetic scaffolds that provide 3D in vitro models for glaucoma research or as implantable devices to regenerate TM tissue. To artificially mimic the TM’s structure, we assessed methods for its decellularization and outline an optimized protocol for cell removal and structural retention. Using bovine TM, we trialed 2 lysing agents—Trypsin (0.05% v/v) and Ammonium Hydroxide (NH4OH; 2% v/v). Twenty-four hours in Trypsin caused significant structural changes. Shorter exposure (2 h) reduced this disruption whilst decellularizing the tissue (dsDNA 26 ± 14 ng/mL (control 1970 ± 146 ng/mL)). In contrast, NH4OH lysed all cells (dsDNA 25 ± 21 ng/mL), and the TM structure remained intact. For human TM, 2% v/v NH4OH similarly removed cells (dsDNA 52 ± 4 ng/mL (control 1965 ± 233 ng/mL)), and light microscopy and SEM presented no structural damage. X-ray computed tomography enabled a novel 3D reconstruction of decellularized human TM and observation of the tissue’s intricate architecture. This study provides a new, validated method using NH4OH to decellularize delicate human TM without compromising tissue structure.
... There are two classifications of grafts used by cardiac tissue engineers: allogeneic and xenogeneic (Zia et al., 2016). Allografts are samples derived from human tissue, meaning, ECM composition and specialization are ideal for therapeutic use. ...
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3D cardiac engineered constructs have yielded not only the next generation of cardiac regenerative medicine but also have allowed for more accurate modeling of both healthy and diseased cardiac tissues. This is critical as current cardiac treatments are rudimentary and often default to eventual heart transplants. This review serves to highlight the various cell types found in cardiac tissues and how they correspond with current advanced fabrication methods for creating cardiac engineered constructs capable of shedding light on various pathologies and providing the therapeutic potential for damaged myocardium. In addition, insight is given towards the future direction of the field with an emphasis on the creation of specialized and personalized constructs that model the region-specific microtopography and function of native cardiac tissues.
... The quality control of an acellular scaffold has the full attention of researchers due to the lack of standard methods for nondestructive characterization. Histology and scanning electron microscopy (SEM) provide excellent feedback regarding the decellularized scaffold quality, but they require destructive protocols for the samples [27]. ...
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Whole organ decellularization techniques have facilitated the fabrication of extracellular matrices (ECMs) for engineering new organs. Unfortunately, there is no objective gold standard evaluation of the scaffold without applying a destructive method such as histological analysis or DNA removal quantification of the dry tissue. Our proposal is a software application using deep convolutional neural networks (DCNN) to distinguish between different stages of decellularization, determining the exact moment of completion. Hearts from male Sprague Dawley rats (n = 10) were decellularized using 1% sodium dodecyl sulfate (SDS) in a modified Langendorff device in the presence of an alternating rectangular electric field. Spectrophotometric measurements of deoxyribonucleic acid (DNA) and total proteins concentration from the decellularization solution were taken every 30 min. A monitoring system supervised the sessions, collecting a large number of photos saved in corresponding folders. This system aimed to prove a strong correlation between the data gathered by spectrophotometry and the state of the heart that could be visualized with an OpenCV-based spectrometer. A decellularization completion metric was built using a DCNN based classifier model trained using an image set comprising thousands of photos. Optimizing the decellularization process using a machine learning approach launches exponential progress in tissue bioengineering research.
... Although the engineered heart could generate macroscale contractions under perfusion culture and electrical stimulation, its pump function was reported to be only about 2% of adult heart. This is partly associated with the challenge to introduce cells back into the decellularized scaffold at physiological density [36]. To leverage the excellent biological properties of dECM, an increasingly popular strategy is using dECM as bioinks for 3D printing cardiac tissues, providing excellent microenvironment for cell growth and tissue function [37]. ...
Article
The field of cardiac tissue engineering has advanced over the past decades; however, most research progress has been limited to engineered cardiac tissues (ECTs) at the microscale with minimal geometrical complexities such as 3D strips and patches. Although microscale ECTs are advantageous for drug screening applications because of their high-throughput and standardization characteristics, they have limited translational applications in heart repair and the in vitro modeling of cardiac function and diseases. Recently, researchers have made various attempts to construct engineered cardiac pumps (ECPs) such as chambered ventricles, recapitulating the geometrical complexity of the native heart. The transition from microscale ECTs to ECPs at a translatable scale would greatly accelerate their translational applications; however, researchers are confronted with several major hurdles, including geometrical reconstruction, vascularization, and functional maturation. Therefore, the objective of this paper is to review the recent advances on bioengineering approaches for fabrication of functional engineered cardiac pumps. We first review the bioengineering approaches to fabricate ECPs, and then emphasize the unmatched potential of 3D bioprinting techniques. We highlight key advances in bioprinting strategies with high cell density as researchers have begun to realize the critical role that the cell density of non-proliferative cardiomyocytes plays in the cell–cell interaction and functional contracting performance. We summarize the current approaches to engineering vasculatures both at micro- and meso-scales, crucial for the survival of thick cardiac tissues and ECPs. We showcase a variety of strategies developed to enable the functional maturation of cardiac tissues, mimicking the in vivo environment during cardiac development. By highlighting state-of-the-art research, this review offers personal perspectives on future opportunities and trends that may bring us closer to the promise of functional ECPs.
... Several aspects, such as cell density, matrix density, thickness and tissue morphology affect the decellularization process and consequently the physicochemical and mechanical properties of the obtained dECM. In fact, the decellularized structure is intended to preserve its 3D geometry and leave the whole organ intact: thereby, tissue-engineered heart [70], lungs [71], urethra [72] and bladder [73] were developed in recent years. ...
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The history of biomaterials dates back to the mists of time: human beings had always used exogenous materials to facilitate wound healing and try to restore damaged tissues and organs. Nowadays, a wide variety of materials are commercially available and many others are under investigation to both maintain and restore bodily functions. Emerging clinical needs forced the development of new biomaterials, and lately discovered biomaterials allowed for the performing of new clinical applications. The definition of biomaterials as materials specifically conceived for biomedical uses was raised when it was acknowledged that they have to possess a fundamental feature: biocompatibility. At first, biocompatibility was mainly associated with biologically inert substances; around the 1970s, bioactivity was first discovered and the definition of biomaterials was consequently extended. At present, it also includes biologically derived materials and biological tissues. The present work aims at walking across the history of biomaterials, looking towards the scientific literature published on this matter. Finally, some current applications of biomaterials are briefly depicted and their future exploitation is hypothesized.
... The decellularization process involves washing out all the cells and cellular remnants such as deoxyribonucleic acid (DNA), membranes and cytosolic materials [11,12]. Different decellularization methods have been used over time, including physical, chemical and enzymatic methods and combinations of them [13,14]. ...
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Heart transplantation remains the only curative treatment for end-stage heart failure. This life-saving option continues to be limited by the low number of organ donors, graft rejection and adverse effects of immunosuppressants. Engineering bioartificial hearts from acellular native-derived scaffolds and stem cells has gained attention because of its potential to overcome these limitations. In this study, rat hearts (n = 20) were decellularized by means of coronary perfusion with 1% sodium dodecyl sulfate (SDS) in a modified Langendorff device. The electrical field behavior of the SDS molecule was studied and it was assumed that when applying an alternating current, the exposure time of the tissue to the detergent might decrease. To repopulate the decellularized extracellular matrix (ECM), human mesenchymal stem cells (hMSCs) were used, induced to differentiate into cardiomyocytes (CMs) with 5-azacytidine (5-aza). The results showed no cellular debris and an intact ECM following decellularization. Decellularization in the presence of an electric field proved to be faster, decreasing the potential risk of ECM damage due to the detergent. After cell seeding and culturing of eight scaffolds with hMSCs, the recellularization process was analyzed using optic microscopy (OM), which showed cells suggestive for CMs. This study presents a novel and efficient decellularization protocol using an electric field and suggests that hMSCs can be useful in the generation of a bioartificial heart.
... Initially, concepts were developed, based on synthetic filamentous matrices fixed with glass fibre [31]. Furthermore, re-population of extracellular matrix derived from decellularization of murine hearts has been shown to be effective in generating 3D structures (reviewed in Zia et al., 2016) [32]. To date, 3D cultures like engineered heart muscle (EHM) or engineered heart tissue (EHT) are mostly generated by solidification of defined cell/matrix solutions (e.g. on a fibrinogen or collagen basis) in moulds around posts of defined stiffnesses ( Fig. 1), also holding the potential of force measurement deducted from post bending [11,33]. ...
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Purpose of Review Heart failure is among the most prevalent disease complexes overall and is associated with high morbidity and mortality. The underlying aetiology is manifold including coronary artery disease, genetic alterations and mutations, viral infections, adverse immune responses, and cardiac toxicity. To date, no specific therapies have been developed despite notable efforts. This can especially be attributed to hurdles in translational research, mainly due to the lack of proficient models of heart failure limited translation of therapeutic approaches from bench to bedside. Recent Findings Human induced pluripotent stem cells (hiPSCs) are rising in popularity, granting the ability to divide infinitely, to hold human, patient-specific genome, and to differentiate into any human cell, including cardiomyocytes (hiPSC-CMs). This brings magnificent promise to cardiological research, providing the possibility to recapitulate cardiac diseases in a dish. Advances in yield, maturity, and in vivo resemblance due to straightforward, low-cost protocols, high-throughput approaches, and complex 3D cultures have made this tool widely applicable. In recent years, hiPSC-CMs have been used to model a wide variety of cardiac diseases, bringing along the possibility to not only elucidate molecular mechanisms but also to test novel therapeutic approaches in the dish. Summary Within the last decade, hiPSC-CMs have been exponentially employed to model heart failure. Constant advancements are aiming at improvements of differentiation protocols, hiPSC-CM maturity, and assays to elucidate molecular mechanisms and cellular functions. However, hiPSC-CMs are remaining relatively immature, and in vitro models can only partially recapitulate the complex interactions in vivo. Nevertheless, hiPSC-CMs have evolved as an essential model system in cardiovascular research.
... Hydrogels composition is chosen by researcher and depends on the purpose of the investigation: they are prepared with hydrophilic polymers, natural or synthetic, can absorb a great amount of fluids and generate a cross-linked matrix that can mimic the native ECM [105]. Besides artificial matrices, it is also possible to obtain a completely natural scaffold from decellularized tissue deriving from animal hearts or from plants, subsequently repopulated with CMs and ECs [106,107]. This approach can be adapted emulsifying the remaining natural ECM and reconstituting it as decellularized myocardial matrix hydrogel [108]. ...
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The availability of appropriate and reliable in vitro cell models recapitulating human cardiovascular diseases has been the aim of numerous researchers, in order to retrace pathologic phenotypes, elucidate molecular mechanisms, and discover therapies using simple and reproducible techniques. In the past years, several human cell types have been utilized for these goals, including heterologous systems, cardiovascular and non-cardiovascular primary cells, and embryonic stem cells. The introduction of induced pluripotent stem cells and their differentiation potential brought new prospects for large-scale cardiovascular experiments, bypassing ethical concerns of embryonic stem cells and providing an advanced tool for disease modeling, diagnosis, and therapy. Each model has its advantages and disadvantages in terms of accessibility, maintenance, throughput, physiological relevance, recapitulation of the disease. A higher level of complexity in diseases modeling has been achieved with multicellular co-cultures. Furthermore, the important progresses reached by bioengineering during the last years, together with the opportunities given by pluripotent stem cells, have allowed the generation of increasingly advanced in vitro three-dimensional tissue-like constructs mimicking in vivo physiology. This review provides an overview of the main cell models used in cardiovascular research, highlighting the pros and cons of each, and describing examples of practical applications in disease modeling.
... The scaffolds utilized in tissue engineering must be biocompatible with negligible immune reactions and inflammatory responses [128,129]. They also should exhibit controllable biodegradability and proper mechanical and architectural characteristics which provide an appropriate microenvironment with interconnected pores for growth, proliferation, and differentiation of the cells [130][131][132]. ...
Article
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... The decellularized material could keep the whole organ intact or could be further processed by cutting or digesting into the liquid to form a coat or ECM-containing hydrogel. By using detergents or mechanical manipulations, natural 3D scaffolds such as heart [58], lungs [59], urethra [60] and bladders [61] are decellularized and functionalized by re-implanting host-specific stem cells (re-cellularized) together with additional growth factors [62]. The sources for creating ECM scaffolds include native devitalized and decellularized human or animal (porcine, bovine) organs and tissues or de novo synthesized ECM from autologous, allogenic or xenogenic cells. ...
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Considering the advantages and disadvantages of biomaterials used for the production of 3D scaffolds for tissue engineering, new strategies for designing advanced functional biomimetic structures have been reviewed. We offer a comprehensive summary of recent trends in development of single- (metal, ceramics and polymers), composite-type and cell-laden scaffolds that in addition to mechanical support, promote simultaneous tissue growth, and deliver different molecules (growth factors, cytokines, bioactive ions, genes, drugs, antibiotics, etc.) or cells with therapeutic or facilitating regeneration effect. The paper briefly focuses on divers 3D bioprinting constructs and the challenges they face. Based on their application in hard and soft tissue engineering, in vitro and in vivo effects triggered by the structural and biological functionalized biomaterials are underlined. The authors discuss the future outlook for the development of bioactive scaffolds that could pave the way for their successful imposing in clinical therapy.