Search Results (21)

The field of reconstructive surgery faces significant challenges in addressing limb loss and disfigurement, with current organ preservation methods limited by short storage times. Decellularization offers a promising solution for generating engineered alternatives for reconstructive surgery by removing cellular material while preserving the extracellular matrix (ECM) and providing scaffolds for tissue regeneration. In this study, we developed a robust protocol for decellularizing whole digits from long-term freezer storage, achieving the successful removal of cellular material with intact ECM. Digit angiography confirmed the preservation of vascular integrity, facilitating future perfusion for recellularization. Quantitative analysis revealed significantly lower DNA content in decellularized tissues, indicating effective decellularization. Furthermore, extracellular matrix analysis showed the preservation of collagen, elastin, and glycosaminoglycans (GAGs) contents. Histological examination confirmed the reduction in cellularity and maintenance of tissue architecture in decellularized digits. Mechanical strength testing of decellularized digit tendons proved consistent with that of native digits. Our findings highlight the potential of decellularized digits as versatile platforms for tissue engineering and regenerative medicine. Moving forward, further optimization of protocols and collaborative efforts are essential for translating these findings into clinical practice, offering innovative solutions for reconstructive surgery and limb transplantation. Full article

Liver transplantation is the only curative option for end-stage liver disease and is necessary for an increasing number of patients with advanced primary or secondary liver cancer. Many patient groups can benefit from this treatment, however the shortage of liver grafts remains an unsolved problem. Liver bioengineering offers a promising method for expanding the donor pool through the production of acellular scaffolds that can be seeded with recipient cells. Decellularization protocols involve the removal of cells using various chemical, physical, and enzymatic steps to create a collagenous network that provides support for introduced cells and future vascular and biliary beds. However, the removal of the cells causes varying degrees of matrix damage, that can affect cell seeding and future organ performance. The main objective of this review is to present the existing techniques of producing decellularized livers, with an emphasis on the assessment and definition of acellularity. Decellularization agents are discussed, and the standard process of acellular matrix production is evaluated. We also introduce the concept of the stepwise assessment of the matrix during decellularization through decellularization cycles. This method may lead to shorter detergent exposure times and less scaffold damage. The introduction of apoptosis induction in the field of organ engineering may provide a valuable alternative to existing long perfusion protocols, which lead to significant matrix damage. A thorough understanding of the decellularization process and the action of the various factors influencing the final composition of the scaffold is essential to produce a biocompatible matrix, which can be the basis for further studies regarding recellularization and retransplantation. Full article

The shortage of tissues and damaged organs led to the development of tissue engineering. Biological scaffolds, created from the extracellular matrix (ECM) of organs and tissues, have emerged as a promising solution for transplants. The ECM of decellularized auricular cartilage is a potential tool for producing ideal scaffolds for the recellularization and implantation of new tissue in damaged areas. In order to be classified as an ideal scaffold, it must be acellular, preserving its proteins and physical characteristics necessary for cell adhesion. This study aimed to develop a decellularization protocol for pig ear cartilage and evaluate the integrity of the ECM. Four tests were performed using different methods and protocols, with four pig ears from which the skin and subcutaneous tissue were removed, leaving only the cartilage. The most efficient protocol was the combination of trypsin with a sodium hydroxide solution (0.2 N) and SDS (1%) without altering the ECM conformation or the collagen architecture. In conclusion, it was observed that auricular cartilage is difficult to decellularize, influenced by material size, exposure time, and the composition of the solution. Freezing and thawing did not affect the procedure. The sample thickness significantly impacted the decellularization time. Full article

Transplantation of decellularized uterus tissue showed promise in supporting regeneration following uterine injury in animal models, suggesting an alternative to complete uterus transplantation for uterine factor infertility treatment. However, most animal studies utilized small grafts, limiting their clinical relevance. Hence, we used larger grafts (20 × 10 mm), equivalent to nearly one uterine horn in rats, to better evaluate the bioengineering challenges associated with structural support, revascularization, and tissue regeneration. We analyzed histopathology, employed immunohistochemistry, and investigated gene expression discrepancies in growth-related proteins over four months post-transplantation in acellular grafts and those recellularized (RC) with bone marrow-derived mesenchymal stem cells (bmMSCs). RC grafts exhibited less inflammation and faster epithelialization and migration of endogenous cells into the graft compared with acellular grafts. Despite the lack of a significant difference in the density of CD31 positive blood vessels between groups, the RC group demonstrated a better organized myometrial layer and an overall faster regenerative progress. Elevated gene expression for Vegf, Cd44, and Itgb1 correlated with the enhanced tissue regeneration in this group. Elevated Tgfb expression was noted in both groups, potentially contributing to the rapid revascularization. Our findings suggest that large uterine injuries can be regenerated using decellularized tissue, with bmMSCs enhancing the endogenous repair mechanisms. Full article

Type 1 diabetes mellitus (T1DM) is a chronic condition primarily managed with insulin replacement, leading to significant treatment costs. Complications include vasculopathy, cardiovascular diseases, nephropathy, neuropathy, and reticulopathy. Pancreatic islet transplantation is an option but its success does not depend solely on adequate vascularization. The main limitations to clinical islet transplantation are the scarcity of human pancreas, the need for immunosuppression, and the inadequacy of the islet isolation process. Despite extensive research, T1DM remains a major global health issue. In 2015, diabetes affected approximately 415 million people, with projected expenditures of USD 1.7 trillion by 2030. Pancreas transplantation faces challenges due to limited organ availability and complex vascularization. T1DM is caused by the autoimmune destruction of insulin-producing pancreatic cells. Advances in biomaterials, particularly the extracellular matrix (ECM), show promise in tissue reconstruction and transplantation, offering structural and regulatory functions critical for cell migration, differentiation, and adhesion. Tissue engineering aims to create bioartificial pancreases integrating insulin-producing cells and suitable frameworks. This involves decellularization and recellularization techniques to develop biological scaffolds. The challenges include replicating the pancreas’s intricate architecture and maintaining cell viability and functionality. Emerging technologies, such as 3D printing and advanced biomaterials, have shown potential in constructing bioartificial organs. ECM components, including collagens and glycoproteins, play essential roles in cell adhesion, migration, and differentiation. Clinical applications focus on developing functional scaffolds for transplantation, with ongoing research addressing immunological responses and long-term efficacy. Pancreatic bioengineering represents a promising avenue for T1DM treatment, requiring further research to ensure successful implementation. Full article

Several lung diseases can cause structural damage, making lung transplantation the only therapeutic option for advanced disease stages. However, the transplantation success rate remains limited. Lung bioengineering using the natural extracellular matrix (ECM) of decellularized lungs is a potential alternative. The use of undifferentiated cells to seed the ECM is practical; however, sterilizing the organ for recellularization is challenging. Photobiomodulation therapy (PBMT) may offer a solution, in which the wavelength is crucial for tissue penetration. This study aimed to explore the potential of optimizing lung recellularization with mesenchymal stem cells using PBMT (660 nm) after sterilization with PBMT (880 nm). The lungs from C57BL/6 mice were decellularized using 1% SDS and sterilized using PBMT (880 nm, 100 mW, 30 s). Recellularization was performed in two groups: (1) recellularized lung and (2) recellularized lung + 660 nm PBMT (660 nm, 100 mW, 30 s). Both were seeded with mesenchymal stem cells from human tooth pulp (DPSc) and incubated for 24 h at 37 °C and 5% CO₂ in bioreactor-like conditions with continuous positive airway pressure (CPAP) at 20 cmH₂O and 90% O₂. The culture medium was analyzed after 24 h. H&E, immunostaining, SEM, and ELISA assays were performed. Viable biological scaffolds were produced, which were free of cell DNA and preserved the glycosaminoglycans; collagens I, III, and IV; fibronectin; laminin; elastin; and the lung structure (SEM). The IL-6 and IL-8 levels were stable during the 24 h culture, but the IFN-γ levels showed significant differences in the recellularized lung and recellularized lung + 660 nm PBMT groups. Greater immunological modulation was observed in the recellularized groups regarding pro-inflammatory cytokines (IL-6, IFN-γ, and IL-8). These findings suggest that PBMT plays a role in cytokine regulation and antimicrobial activity, thus offering promise for enhanced therapeutic strategies in lung bioengineering. Full article

The recellularization of tissues after decellularization is a relatively new technology in the field of tissue engineering (TE). Decellularization involves removing cells from a tissue or organ, leaving only the extracellular matrix (ECM). This can then be recellularized with new cells to create functional tissues or organs. The first significant mention of recellularization in decellularized tissues can be traced to research conducted in the early 2000s. One of the landmark studies in this field was published in 2008 by Ott, where researchers demonstrated the recellularization of a decellularized rat heart with cardiac cells, resulting in a functional organ capable of contraction. Since then, other important studies have been published. These studies paved the way for the widespread application of recellularization in TE, demonstrating the potential of decellularized ECM to serve as a scaffold for regenerating functional tissues. Thus, although the concept of recellularization was initially explored in previous decades, these studies from the 2000s marked a major turning point in the development and practical application of the technology for the recellularization of decellularized tissues. The article reviews the historical advances and limitations in organ recellularization in TE over the last two decades. Full article

Biomaterial templates play a critical role in establishing and bioinstructing three-dimensional cellular growth, proliferation and spatial morphogenetic processes that culminate in the development of physiologically relevant in vitro liver models. Various natural and synthetic polymeric biomaterials are currently available to construct biomimetic cell culture environments to investigate hepatic cell–matrix interactions, drug response assessment, toxicity, and disease mechanisms. One specific class of natural biomaterials consists of the decellularized liver extracellular matrix (dECM) derived from xenogeneic or allogeneic sources, which is rich in bioconstituents essential for the ultrastructural stability, function, repair, and regeneration of tissues/organs. Considering the significance of the key design blueprints of organ-specific acellular substrates for physiologically active graft reconstruction, herein we showcased the latest updates in the field of liver decellularization–recellularization technologies. Overall, this review highlights the potential of acellular matrix as a promising biomaterial in light of recent advances in the preparation of liver-specific whole organ scaffolds. The review concludes with a discussion of the challenges and future prospects of liver-specific decellularized materials in the direction of translational research. Full article

Tissue engineering has emerged as an indispensable tool for the reconstruction of organ-specific environments. Organ-derived extracellular matrices (ECM) and, especially, decellularized tissues (DCL) are recognized as the most successful biomaterials in regenerative medicine, as DCL preserves the most essential organ-specific ECM properties such as composition alongside biomechanics characterized by stiffness and porosity. Expansion of the DCL technology to cancer biology research, drug development, and nanomedicine is pending refinement of the existing DCL protocols whose reproducibility remains sub-optimal varying from organ to organ. We introduce a facile decellularization protocol universally applicable to murine organs, including liver, lungs, spleen, kidneys, and ovaries, with demonstrated robustness, reproducibility, high purification from cell debris, and architecture preservation, as confirmed by the histological and SEM analysis. The biomechanical properties of as-produced DCL organs expressed in terms of the local and total stiffness were measured using our facile methodology and were found well preserved in comparison with the intact organs. To demonstrate the utility of the developed DCL model to cancer research, we engineered three-dimensional tissue constructs by recellularization representative decellularized organs and collagenous hydrogel with human breast cancer cells of pronounced mesenchymal (MDA-MB-231) or epithelial (SKBR-3) phenotypes. The biomechanical properties of the DCL organs were found pivotal to determining the cancer cell fate and progression. Our histological and scanning electron microscopy (SEM) study revealed that the larger the ECM mean pore size and the smaller the total stiffness (as in lung and ovary), the more proliferative and invasive the mesenchymal cells became. At the same time, the low local stiffness ECMs (ranged 2.8–3.6 kPa) did support the epithelial-like SKBR-3 cells’ viability (as in lung and spleen), while stiff ECMs did not. The total and local stiffness of the collagenous hydrogel was measured too low to sustain the proliferative potential of both cell lines. The observed cell proliferation patterns were easily interpretable in terms of the ECM biomechanical properties, such as binding sites, embedment facilities, and migration space. As such, our three-dimensional tissue engineering model is scalable and adaptable for pharmacological testing and cancer biology research of metastatic and primary tumors, including early metastatic colonization in native organ-specific ECM. Full article

The pancreas is a complex organ consisting of differentiated cells and extracellular matrix (ECM) organized adequately to enable its endocrine and exocrine functions. Although much is known about the intrinsic factors that control pancreas development, very few studies have focused on the microenvironment surrounding pancreatic cells. This environment is composed of various cells and ECM components, which play a critical role in maintaining tissue organization and homeostasis. In this study, we applied mass spectrometry to identify and quantify the ECM composition of the developing pancreas at the embryonic (E) day 14.5 and postnatal (P) day 1 stages. Our proteomic analysis identified 160 ECM proteins that displayed a dynamic expression profile with a shift in collagens and proteoglycans. Furthermore, we used atomic force microscopy to measure the biomechanical properties and found that the pancreatic ECM was soft (≤400 Pa) with no significant change during pancreas maturation. Lastly, we optimized a decellularization protocol for P1 pancreatic tissues, incorporating a preliminary crosslinking step, which effectively preserved the 3D organization of the ECM. The resulting ECM scaffold proved suitable for recellularization studies. Our findings provide insights into the composition and biomechanics of the pancreatic embryonic and perinatal ECM, offering a foundation for future studies investigating the dynamic interactions between the ECM and pancreatic cells. Full article

Diabetes mellitus and pancreatitis are common pancreatic diseases in dogs, affecting the endocrine and exocrine portions of the organ. Dogs have a significant role in the history of research related to genetic diseases, being considered potential models for the study of human diseases. This review discusses the importance of using the extracellular matrix of the canine pancreas as a model for the study of diabetes mellitus and pancreatitis, in addition to focusing on the importance of using extracellular matrix in new regenerative techniques, such as decellularization and recellularization. Unlike humans, rabbits, mice, and pigs, there are no reports in the literature characterizing the healthy pancreatic extracellular matrix in dogs, in addition to the absence of studies related to matrix components that are involved in triggering diabetes melittus and pancreatitis. The extracellular matrix plays the role of physical support for the cells and allows the regulation of various cellular processes. In this context, it has already been demonstrated that physiologic and pathologic pancreatic changes lead to ECM remodeling, highlighting the importance of an in-depth study of the changes associated with pancreatic diseases. Full article

Decellularization of organs creates an acellular scaffold, ideal for being repopulated by cells. In this work, a low-cost perfusion system was created to be used in the process of liver decellularization and as a bioreactor after recellularization. It consists of a glass chamber to house the organ coupled to a peristaltic pump to promote liquid flow through the organ vascular tree. The rats’ liver decellularization was made with a solution of sodium dodecyl sulfate. The recellularization was made with 10⁸ mesenchymal stromal/stem cells and cultivated for seven days. The decellularized matrices showed an absence of DNA while preserving the collagen and glycosaminoglycans quantities, confirming the efficiency of the process. The functional analyses showed a rise in lactate dehydrogenase levels occurring in the first days of the cultivation, suggesting that there is cell death in this period, which stabilized on the seventh day. Histological analysis showed conservation of the collagen web and some groups of cells next to the vessels. It was possible to establish a system for decellularization and a bioreactor to use for the recellularization method. It is easy to assemble, can be ready to use in little time and be easily sterilized. Full article

Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized. Full article

Cardiovascular diseases are the leading cause of global mortality. Over the past two decades, researchers have tried to provide novel solutions for end-stage heart failure to address cardiac transplantation hurdles such as donor organ shortage, chronic rejection, and life-long immunosuppression. Cardiac decellularized extracellular matrix (dECM) has been widely explored as a promising approach in tissue-regenerative medicine because of its remarkable similarity to the original tissue. Optimized decellularization protocols combining physical, chemical, and enzymatic agents have been developed to obtain the perfect balance between cell removal, ECM composition, and function maintenance. However, proper assessment of decellularized tissue composition is still needed before clinical translation. Recellularizing the acellular scaffold with organ-specific cells and evaluating the extent of cardiomyocyte repopulation is also challenging. This review aims to discuss the existing literature on decellularized cardiac scaffolds, especially on the advantages and methods of preparation, pointing out areas for improvement. Finally, an overview of the state of research regarding the application of cardiac dECM and future challenges in bioengineering a human heart suitable for transplantation is provided. Full article

Background. Liver transplantation remains the only viable therapy for liver failure but has a severely restricted utility. Here, we aimed to decellularize rat livers to form acellular 3D bio-scaffolds suitable for seeding with induced pluripotent cells (iPSCs) as a tool to investigate the role of Wnt/β-catenin signaling in liver development and generation. Methods. Dissected rat livers were randomly divided into three groups: I (control); II (decellularized scaffolds) and III (recellularized scaffolds). Liver decellularization was established via an adapted perfusion procedure and assessed through the measurement of extracellular matrix (ECM) proteins and DNA content. Liver recellularization was assessed through histological examination and measurement of transcript levels of Wnt/β-catenin pathway, hepatogenesis, liver-specific microRNAs and growth factors essential for liver development. Adult rat liver decellularization was confirmed by the maintenance of ECM proteins and persistence of growth factors essential for liver regeneration. Results. iPSCs seeded rat decellularized livers displayed upregulated transcript expression of Wnt/β-catenin pathway-related, growth factors, and liver specification genes. Further, recellularized livers displayed restored liver-specific functions including albumin secretion and urea synthesis. Conclusion. This establishes proof-of-principle for the generation of three-dimensional liver organ scaffolds as grafts and functional re-establishment. Full article