Figure 3
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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Context 1
... 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). ...
Citations
... 75 Although scaffolds for regenerative medicine are not able to successfully support and drive cell behavior to achieve mature and functional tissue, the use of biomaterials in the scaffold composition is able to mimic the physiological electroconductivity of the heart as well as have adequate degradation kinetics for cardiac tissue engineering ( Figure 2). 76,77 These conductive biomaterials include extrinsically conductive materials such as graphene and CNTs, which have chemical and physical properties that can dictate the cytotoxicity and engineered cardiac tissue's outcome in in vitro and in vivo studies. 74 ...
In tissue engineering, the pivotal role of scaffolds is underscored, serving as key elements to emulate the native extracellular matrix. These scaffolds must provide structural integrity and support and supply electrical, mechanical, and chemical cues for cell and tissue growth. Notably, electrical conductivity plays a crucial role when dealing with tissues like bone, spinal, neural, and cardiac tissues. However, the typical materials used as tissue engineering scaffolds are predominantly polymers, which generally characteristically feature poor electrical conductivity. Therefore, it is often necessary to incorporate conductive materials into the polymeric matrix to yield electrically conductive scaffolds and further enable electrical stimulation. Among different conductive materials, carbon nanomaterials have attracted significant attention in developing conductive tissue engineering scaffolds, demonstrating excellent biocompatibility and bioactivity in both in vitro and in vivo settings. This article aims to comprehensively review the current landscape of carbon‐based conductive scaffolds, with a specific focus on their role in advancing tissue engineering for the regeneration and maturation of functional tissues, emphasizing the application of electrical stimulation. This review highlights the versatility of carbon‐based conductive scaffolds and addresses existing challenges and prospects, shedding light on the trajectory of innovative conductive scaffold development in tissue engineering.
... Recent advances in regenerative medicine gave rise to a widespread interest in utilizing decellularized extracellular matrix (ECM) as a biological scaffold to develop novel treatment options [1][2][3][4][5] and suggest the possibility of employing ECM as a scaffold to bioengineer functional organs [4,6,7]. Commonly explored decellularization methods include physical [8,9], biological [10,11] and chemical [12][13][14] techniques. ...
Advancements in regenerative medicine have highlighted the potential of decellularized extracellular matrix (ECM) as a scaffold for organ bioengineering. Although the potential of ECM in major organ systems is well-recognized, studies focusing on the angiogenic effects of pancreatic ECM are limited. This study investigates the capabilities of pancreatic ECM, particularly its role in promoting angiogenesis. Using a Triton-X-100 solution, porcine pancreas was successfully decellularized, resulting in a significant reduction in DNA content (97.1% removal) while preserving key pancreatic ECM components. A three-dimensional ECM hydrogel was then created from this decellularized tissue and used for cell culture. Biocompatibility tests demonstrated enhanced adhesion and proliferation of mouse embryonic stem cell-derived endothelial cells (mES-ECs) and human umbilical vein endothelial cells (HUVECs) in this hydrogel compared to conventional scaffolds. The angiogenic potential was evaluated through tube formation assays, wherein the cells showed superior tube formation capabilities in ECM hydrogel compared to rat tail collagen. The RT-PCR analysis further confirmed the upregulation of pro-angiogenic genes in HUVECs cultured within the ECM hydrogel. Specifically, HUVECs cultured in the ECM hydrogel exhibited a significant upregulation in the expression of MMP2, VEGF and PAR-1, compared to those cultured in collagen hydrogel or in a monolayer condition. The identification of ECM proteins, specifically PRSS2 and Decorin, further supports the efficacy of pancreatic ECM hydrogel as an angiogenic scaffold. These findings highlight the therapeutic promise of pancreatic ECM hydrogel as a candidate for vascularized tissue engineering application.
... Поэтому количество образующихся возможных рецепторов Т-лимфоцитов чрезвычайно велико и способно распознавать огромное количество вероятных антигенов. Среди 56 большого количества Т-лимфоцитов выделяют две основные субпопуляции: CD4-хелперы-индукторы и CD8-цитотоксические лимфоциты (Москалев и др., 2015;Ярилин, 2010;Abbas et al., 2018;Zia et al., 2016). ...
... Чужеродные белки помещаются в сайт молекулы ГКГ I класса, клеточная мембрана инвагинируется, и белок попадает в эндоплазматический ретикулум, а затем транспортируется на поверхность клетки, где находится в сочетании с костимулирующими CD80 и CD86. Этот комплекс распознается CD8-лимфоцитами, имеющими ТКР (корецептор СD3 c гликопротеином CD8), комплементарный белкам ГКГ I класса (Ярилин, 2010;Zia et al., 2016). ...
... Гиперострое отторжение происходит при наличии антител у людей против групповых антигенов полисахаридной природы, так называемого антигена Галили (галак тоза-альфа-1,3-галактоза, Gal-α1-3 Gal-α-Gal эпитоп), который присутствует на эндотелиальных клетках животных, кроме приматов. Могут быть выведены генети-чески модифицированные свиньи, у которых этот эпитоп отсутствует, однако вероятность отторжения трансплантата остается попрежнему высокой Wu et al., 2007;Zia et al., 2016). ...
В монографии приведены основные биолого-физиологические закономерности
развития, функции, механизмы и виды дифференцировки стволовых клеток, их
способность к размножению и генерации потомства на уровне популяции. Дается краткое обоснование двух принципиально разных типов стволовых клеток: плюрипотентных, которые существуют только in vitro, и тканевых, существующих in vivo в послеродовом организме. Показано, что плюрипотентные клетки могут приводить к появлению широкого спектра типов клеток, в отличие от тканевых, которые в нормальных условиях не генерируют клетки, характерные для других типов тканей. Представлены этапы развития плюрипотентных стволовых клеток. Обсуждается роль ключевых маркеров плюрипотентности и факт того, что самым надежным способом идентификации стволовых клеток является определение их фенотипа in vivo. Это свидетельствует о том, что стволовые клетки не несут универсального молекулярного маркера, позволяющего дифференцировать стволовые
клетки от нестволовых. Рассмотрены объекты и современные методы редактирования генома. Охарактеризована иммунная система прокариот и их защитные механизмы, препятствующие целевому редактированию генома в интересах исследователя. Описаны фазы развития эмбриона, начиная с формирования гамет и зародышевых линий, различия в отборе зародышевых и соматических клеток, рассматривается образование истинных зародышевых клеток, их типы, факторы, обеспечивающие их дифференцировку и миграцию. Представлены проблемные и перспективные сведения по использованию стволовых клеток в трансплантологии и другие не менее интересные вопросы, касающиеся стволовых клеток.
Монография предназначена биологам, физиологам, врачам, научным работ-
никам, будет полезна преподавателям, аспирантам и студентам биологических
и медицинских факультетов университетов, академий и институтов, а также
широкому кругу читателей.
... 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. ...
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. ...
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]. ...
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]. ...
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. ...
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]. ...
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]. ...
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.
