Organ Scaffolds Research Articles

Refunctionalization of Decellularized Organ Scaffold of Pancreas by Recellularization: Whole Organ Regeneration into Functional Pancreas.

Tissue engineering centers on creating a niche similar to the natural one, with a purpose of developing an organ construct. A natural scaffold can replace none while creating a scaffold unique to each tissue in composition, architecture and cues that regulate the character of cells. Whole pancreas from mouse was decellularized using detergent and enzymes, followed by recellularizing with MSC from human placenta. This construct was transplanted in streptozotocin induced diabetic mice. Histopathology of both decellularized and recellularized transplanted pancreas and qPCR analysis were performed to assess its recovery. Decellularization removes the cells leaving behind extracellular matrix rich natural scaffold. After reseeding with mesenchymal stem cells, these cells differentiate into pancreas specific cells. Upon transplantation in streptozotocin induced diabetic mice, this organ was capable of restoring its histomorphology and functioning. Restoration of endocrine (islets), the exocrine region (acinar) and vascular network was seen in transplanted pancreas. The process of functional recovery of endocrine system took about 20days when the mice start showing blood glucose reduction, though none achieved gluconormalization. Natural decellularized scaffolds of soft organs can be refunctionalized using recipient's mesenchymal stem cells to restore structure and function; and counter immune problems arising during transplantation.

Evaluation and preservation of vascular architectures in decellularized whole rat kidneys

Organ transplantation is the gold standard treatment for end-stage organ failure. Due to the severe shortage of transplantable organs, only a tiny fraction of patients may receive timely organ transplantation every year. Decellularization-recellularization technology using allogeneic and xenogeneic organs is currently conceived to be a promising solution to generate functionally transplantable organs in vitro. This approach, however, still faces tremendous technological challenges, one of them being the ability to evaluate and preserve the integrity of vascular architectures upon decellularization and cryostorage of the whole organ matrices so that the off-the-shelf organ grafts are available on demand for clinical applications. In the present study, we report a Micro-CT imaging method for evaluating the integrity of vasculature of the decellularized whole organ scaffolds with/without freezing/thawing. The method uses radiopaque Microfil perfusion and x-ray fluoroscopy to acquire high-resolution angiography of the organ matrix. The whole rat kidney is decellularized using a new multistep perfusion protocol with the combined use of Triton X-100 and DNase. The decellularized kidney matrix is then cryopreserved after the pretreatment with different cryoprotectant solutions. The reconstructed tomographic images from Micro-CT confirm various structural alterations in the vasculature of the whole decellularized kidney matrix with/without frozen storage. The freezing damage to the vascular architectures can be reduced by perfusing cryoprotectant solutions into the whole kidney matrix. Ice-free cryopreservation with the vitrification solution VS83 can successfully preserve the integrity of the whole kidney matrix's vasculature after frozen storage.

Lung Microvascular Niche, Repair, and Engineering

Biomaterials have been used for a long time in the field of medicine. Since the success of “tissue engineering” pioneered by Langer and Vacanti in 1993, tissue engineering studies have advanced from simple tissue generation to whole organ generation with three-dimensional reconstruction. Decellularized scaffolds have been widely used in the field of reconstructive surgery because the tissues used to generate decellularized scaffolds can be easily harvested from animals or humans. When a patient’s own cells can be seeded onto decellularized biomaterials, theoretically this will create immunocompatible organs generated from allo- or xeno-organs. The most important aspect of lung tissue engineering is that the delicate three-dimensional structure of the organ is maintained during the tissue engineering process. Therefore, organ decellularization has special advantages for lung tissue engineering where it is essential to maintain the extremely thin basement membrane in the alveoli. Since 2010, there have been many methodological developments in the decellularization and recellularization of lung scaffolds, which includes improvements in the decellularization protocols and the selection and preparation of seeding cells. However, early transplanted engineered lungs terminated in organ failure in a short period. Immature vasculature reconstruction is considered to be the main cause of engineered organ failure. Immature vasculature causes thrombus formation in the engineered lung. Successful reconstruction of a mature vasculature network would be a major breakthrough in achieving success in lung engineering. In order to regenerate the mature vasculature network, we need to remodel the vascular niche, especially the microvasculature, in the organ scaffold. This review highlights the reconstruction of the vascular niche in a decellularized lung scaffold. Because the vascular niche consists of endothelial cells (ECs), pericytes, extracellular matrix (ECM), and the epithelial–endothelial interface, all of which might affect the vascular tight junction (TJ), we discuss ECM composition and reconstruction, the contribution of ECs and perivascular cells, the air–blood barrier (ABB) function, and the effects of physiological factors during the lung microvasculature repair and engineering process. The goal of the present review is to confirm the possibility of success in lung microvascular engineering in whole organ engineering and explore the future direction of the current methodology.

Development of transplantable porcine liver graft using organ scaffold and regenerative medicine approach

Development of transplantable porcine liver graft using organ scaffold and regenerative medicine approach

Bio-Medical applications of Additive Manufacturing: A Review

Additive Manufacturing (AM) finds multiple applications in a variety of fields such as aviation, defense, manufacturing, automotive sector, industrial, education and research, but most importantly in Bio-Medical, healthcare and pharmaceutical industries because of its ability to fabricate complex customized parts with extensive ease, flexibility and with various biomaterials. This review presents the Bio-medical applications of AM so far. This review concisely highlights the healthcare sectors that are revolutionized by AM, such as bio-printing, Tissue Engineering, Dentistry, printing patient-specific customized implants, Medical devices/Surgical tools, orthopaedics and prosthesis, Drug delivery and also virtual surgical planning. Also, the different bio-inks and Biomaterials utilized for printing cell-seeded tissues, 3DP implants, replacement organ printing and scaffolds, are presented. Thus AM ensures safety, innovation and ease in healthcare sector. Despite these, AM still has tremendous promise and scope for further improvement in pharmaceutical, drug delivery and bio-medicinal sectors.

Decellularized liver scaffolds promote liver regeneration after partial hepatectomy

The resectable liver volume is strictly limited and this reduces the number of patients who may be treated. Recently, “tissue/organ decellularization”, a new approach in bioengineering, has been investigated for its ability to produce a native organ scaffold by removing all the viable cells. Such a scaffold may support the repair of damaged or injured tissue. The purpose of this study was to evaluate the potential contribution of liver scaffolds to hepatic regeneration after hepatectomy. We sutured the partial liver scaffolds onto the surfaces of partially hepatectomized porcine livers and assessed their therapeutic potential by immune histological analysis at various time points. Animals were sacrificed after surgery and the implanted scaffolds were evaluated for the infiltration of various types of cells. Immune histochemical study showed that blood vessel-like structures, covered with CD31 positive endothelial cells and ALB positive cells, were present in all parts of the scaffolds at days 10 and 28. Blood inflow was observed in some of these ductal structures. More interestingly, CK19 and EpCAM positive cells appeared at day 10. These results suggest that the implantation of a decellularized organ scaffold could promote structural reorganization after liver resection.

Utilization of Natural Detergent Potassium Laurate for Decellularization in Lung Bioengineering.

Recent advances in tissue engineering using decellularized organ scaffolds have expanded the possibilities for organ replacement therapy. However, detergent-based decellularization itself damages the extracellular matrix (ECM), which results in failure associated with the transplanted bioengineered organ. This study determined that potassium laurate (PL), a natural detergent, significantly reduces lung ECM damage during the decellularization process compared with protocols using sodium dodecyl sulfate. PL-decellularized lungs showed better microarchitecture preservation and low biological reactions after subcutaneous implantation. PL-decellularized scaffolds supported rat lung endothelial cell attachment/proliferation and the bioengineered lungs significantly reduced lung congestion after transplantation.

Epac agonist improves barrier function in iPSC-derived endothelial colony forming cells for whole organ tissue engineering

Whole organ engineering paradigms typically involve repopulating acellular organ scaffolds with recipient-compatible cells, to generate a neo-organ that may provide key physiological functions. In the case of whole lung engineering, functionally endothelialized pulmonary vasculature is critical for establishing a fluid-tight barrier at the level of the alveolus, so that oxygen and carbon dioxide can be exchanged in the organ. We have previously developed a protocol to efficiently seed endothelial cells into the microvascular channels of decellularized lung scaffolds, but fully functional endothelial coverage, in terms of barrier function and resistance to thrombosis, was not achieved. In this study, we investigated whether various small molecules could favorably impact endothelial functionality after seeding into decellularized lung scaffolds. We demonstrated that the Epac-selective cAMP analog 8CPT-2Me-cAMP improves endothelial barrier function in repopulated lung scaffolds. When treated with the Epac agonist, barrier function of human umbilical vein endothelial cells (HUVECs) improved, and was maintained for at least three days, whereas the effect of other tested molecules lasted for only 5 h. Treatment with the Epac agonist re-organized actin structure, and appeared to increase the continuity of junction proteins such as VE-cadherin and ZO1. Blockade of actin polymerization abolished the effect of the Epac agonist on barrier function and actin reorganization, confirming a strong actin-mediated effect. Similarly, after treatment with Epac agonist, the barrier function in iPSC-derived endothelial colony forming cells (ECFCs) was increased and the enhanced barrier was maintained for at least 60 h. After culture in lung scaffolds for 5 days, iPSC-ECFCs maintained their phenotype by expressing CD31, eNOS, vWF, and VE-Cadherin. Treatment with the Epac agonist significantly improved the barrier function of iPSC-ECFC-repopulated lung for at least 6 h. Taken together, these findings demonstrated that Epac-selective 8CPT-2Me-cAMP activation enhanced vascular barrier in iPSC-ECFC-engineered lungs, and may be useful to improve endothelial functionality for whole organ tissue engineering.

Laminin as a Potent Substrate for Large-Scale Expansion of Human Induced Pluripotent Stem Cells in a Closed Cell Expansion System.

The number of high-quality cells required for engineering an adult human-sized bioartificial organ is greater than one billion. Until the emergence of induced pluripotent stem cells (iPSCs), autologous cell sources of this magnitude and with the required complexity were not available. Growing this number of cells in a traditional 2D cell culture system requires extensive time, resources, and effort and does not always meet clinical requirements. The use of a closed cell culture system is an efficient and clinically applicable method that can be used to expand cells under controlled conditions. We aimed to use the Quantum Cell Expansion System (QES) as an iPSC monolayer-based expansion system. Human iPSCs were expanded (up to 14-fold) using the QES on two different coatings (laminin 521 (LN521) and vitronectin (VN)), and a karyotype analysis was performed. The cells were characterized for spontaneous differentiation and pluripotency by RT-PCR and flow cytometry. Our results demonstrated that the QES provides the necessary environment for exponential iPSC growth, reaching 689.75 × 106 ± 86.88 × 106 in less than 7 days using the LN521 coating with a population doubling level of 3.80 ± 0.19. The same result was not observed when VN was used as a coating. The cells maintained normal karyotype (46-XX), expressed pluripotency markers (OCT4, NANOG, LIN28, SOX2, REX1, DPPA4, NODAL, TDGFb, TERT3, and GDF), and expressed high levels of OCT4, SOX2, NANOG, SSEA4, TRA1-60, and TRA1-81. Spontaneous differentiation into ectoderm (NESTIN, TUBB3, and NEFH), mesoderm (MSX1, BMP4, and T), and endoderm (GATA6, AFP, and SOX17) lineages was detected by RT-PCR with both coating systems. We conclude that the QES maintains the stemness of iPSCs and is a promising platform to provide the number of cells necessary to recellularize small human-sized organ scaffolds for clinical purposes.

Decellularization Concept in Regenerative Medicine.

Decellularized organs and tissues are effectively utilized in a diversity of regenerative medicine purposes, and the decellularization approaches employed differ as broadly as the tissues/organs of concern. Biological scaffold substances formed by extracellular matrix (ECM) are mostly produced with methods that include decellularization of tissues. Conservation of the multifaceted arrangement and three-dimensional (3D) construction of the ECM is very wanted but it is documented that almost every approach of decellularization cause disturbance of the organization and possible forfeiture of surface organization and conformation. The competence of cell elimination from a tissue is reliant on the basis of the tissue and the precise physical, chemical, and enzymatic approaches that are utilized. Here, the most frequently applied and newly developed decellularization techniques are designated, organ engineering with decellularized scaffolds for different organs, recent knowledge in the field are explained.

What will it take before a bioengineered heart will be implanted in patients?

Heart transplantation is the only curative treatment option for end-stage heart failure. However, a shortage of donor organs is a major limitation of this approach. Regenerative medicine targets the goal of increasing the number of available hearts for transplantation. In this review, we highlight the state of the art of building a bioartificial heart. We summarize the components needed, the hurdles, and likely translational steps to make the dream of transplanting a totally functional bioartificial heart a possibility. The therapies being developed in regenerative medicine aim not only to repair, but also to regenerate or replace failing tissues and organs. The engineering of bioartificial hearts utilizing patient-derived cells could theoretically solve the two main complications of heart transplantations: graft rejection and lifelong immunosuppression. Although many hurdles remain, scientists have reached a point in which some of these hurdles have been overcome. Decellularized heart scaffolds have emerged over the past decade as one of the most promising biofabrications. Two possible options for organ scaffolds exist: nontransplantable human hearts and porcine hearts. The use of these scaffolds could lead to the availability of an unlimited number of transplantable organs. The current challenge remains improving processes required for recellularization - including those for cells, bioreactors, and physiologic conditioning. Researchers should focus to solve these hurdles and pave the way toward the dream of in-vivo bioengineered heart maturation. Regenerative medicine has emerged as one of the most promising fields of translational research and has the potential to both minimize the need for donor organs and increase their availability. Meeting the challenge of implanting a totally functional bioengineered heart lies in solving multiple issues simultaneously. Dwarfing the technical hurdles, cost is the largest barrier to success. The scientific hurdles mainly involve scaling up and scaling out of laboratory cell processes, building bioreactors, and delivering cells into every needed region of an organ scaffold. Maintaining sterility and quantifying readiness of the nascent organs are also critical for success.

A Fully Automated High-Throughput Bioreactor System for Lung Regeneration.

This work presents methods for ex vivo lung recellularization and biomimetic culture in a high-throughput and consistent manner. These methods allow for the testing of multiple variables, all of which are simultaneously controlled and monitored on a single fully automated pump system, and subsequent assessment of both epithelial and endothelial repair and tissue regeneration. This system provides a controlled environment for tissue repair, wherein key variables can be modified, monitored, reproduced, and optimized to advance the goal of ex vivo tissue regeneration based on native organ scaffolds.

Molecular dynamics of pancreatic transcription factors in bioengineered humanized insulin producing neoorgan

BackgroundThe present study has been aimed to identify molecular dynamics of pancreatic transcription factors (pTFs) during events of directed trans-differentiation of human hepatic progenitor cells (hHPCs) into insulin producing cells (InPCs) within bioengineered humanized neoorgan. The study demonstrates applicability of acellularized whole splenic scaffold (ASOS) to generate insulin producing humanized transplantable neoorgan through activation of pancreatic transcription factors. MethodsAn efficient acellularization process was developed for xenogeneic rat spleen using change in different gradients of reagents perfusion through splenic artery for varying time points. The acellularized xenogeneic spleen scaffold was characterized thoroughly for preservation of extra-cellular matrix and retention of organ specific vasculature and mechanical properties. Further scaffolds were sterilized and repopulated with hHPCs which were triggered using a stage wise induction with growth factors and hyperglycemic challenge for trans-differentiation into InPCs. Dynamics of pTFs alone or simultaneously during induction process was identified using gene expression analysis and immunological staining. ResultsThe cells within the engineered neoorgan respond to growth factors and extrinsic hyperglycemic challenge and generate large number of InPCs under controlled dynamic regulation of pTFs. Highly controlled regulation of pTFs generates higher percentage of Nkx-6.1+/C-peptide+ cells within the engineered splenic scaffolds. Generation of high percentage of insulin and C-peptide positive cells in three-dimensional organ architecture responded better to hyperglycemic stimuli and produced higher quantity of insulin than 2D-culture system. ConclusionThe present study provides a novel platform for designing effective regenerative strategies using whole organ scaffolds to control hyperglycemia under tight regulation of pTFs using humanized neoorgan system.

The Future of Transplant Biology and Surgery

Although allograft organs remain the gold standard for transplantation, the availability of donor organs has forced us to search for alternatives. Regenerative medicine and tissue engineering offer a solution for this issue. Through the use of synthetic scaffolds and organic substrates, we have been able to generate neoorgans for a variety of different body systems. Although these neoorgans remain largely in the experimental phase, the results are promising, and recent technological developments have made production a realistic endeavor on the large scale. In this review, we highlight recent advances in the field of regenerative medicine and their application to the future of transplant surgery. This review contains 6 figures, 2 tables and 42 references Key Words: bioengineering, biomaterial, bioreactor, decellularized organs, organ scaffolds, regenerative medicine, stem cell, three-dimensional printing

Sodium hydroxide based non-detergent decellularizing solution for rat lung

ABSTRACTLung transplantation is the last option for the treatment of end stage chronic lung disorders. Because the shortage of donor lung organs represents the main hurdle, lung regeneration has been considered to overcome this hurdle. Recellularization of decellularized organ scaffold is a promising option for organ regeneration. Although detergents are ordinarily used for decellularization, other approaches are possible. Here we used high alkaline (pH12) sodium hydroxide (NaOH)-PBS solution without detergents for lung decellularization and compared the efficacy on DNA elimination and ECM preservation with detergent based decellularization solutions CHAPS and SDS. Immunohistochemical image analysis showed that cell components were removed by NaOH solution as well as other detergents. A Collagen and GAG assay showed that the collagen reduction of the NaOH group was comparable to that of the CHAPS and SDS groups. However, DNA reduction was more significant in the NaOH group than in other groups (p < 0.0001). The recellularization of HUVEC revealed cell attachment was not inferior to that of the SDS group. Ex vivo functional analysis showed 100% oxygen ventilation increased oxygen partial pressure as artificial hemoglobin vesicle-PBS solution passed through regenerated lungs in the SDS or NaOH group. It was concluded that the NaOH-PBS based decellularization solution was comparable to ordinal decellularizaton solutions and competitive in cost effectiveness and residues in the decellularized scaffold negligible, thus providing another potential option to detergent for future clinical usage.

Synthesis of Anisotropic Hydrogels and Their Applications.

Owing to their water-rich structures, which are similar to those of biological tissues, hydrogels have long been regarded as promising scaffolds for artificial tissues and organs. However, in terms of the structural anisotropy, most synthetic hydrogels are substantially different from biological systems. Synthetic hydrogels are usually composed of randomly oriented three-dimensional polymer networks whereas biological systems adopt anisotropic structures with hierarchically integrated building units. Such anisotropic structures often play essential roles in biological systems to exhibit particular functions. In this context, anisotropic hydrogels provide an entry point for exploring biomimetic applications of hydrogels. Reflecting these aspects, an increasing number of studies on anisotropic hydrogels have been reported recently. This Minireview highlights the use and perspectives of these anisotropic hydrogels, particularly focusing on their preparation, structures, and applications.

A Comparison of Iron Oxide Particles and Silica Particles for Tracking Organ Recellularization

Reseeding of decellularized organ scaffolds with a patient’s own cells has promise for eliminating graft versus host disease. This study investigated whether ultrasound imaging or magnetic resonance imaging (MRI) can track the reseeding of murine liver scaffolds with silica-labeled or iron-labeled liver hepatocytes. Mesoporous silica particles were created using the Stöber method, loaded with Alexa Flour 647 fluorophore, and conjugated with protamine sulfate, glutamine, and glycine. Fluorescent iron oxide particles were obtained from a commercial source. Liver cells from donor mice were loaded with the silica particles or iron oxide particles. Donor livers were decellularized and reperfused with silica-labeled or iron-labeled cells. The reseeded livers were longitudinally analyzed with ultrasound imaging and MRI. Liver biopsies were imaged with confocal microscopy and scanning electron microscopy. Ultrasound imaging had a detection limit of 0.28 mg/mL, while MRI had a lower detection limit of 0.08 mg/mL based on particle weight. The silica-loaded cells proliferated at a slower rate compared to iron-loaded cells. Ultrasound imaging, MRI, and confocal microscopy underestimated cell numbers relative to scanning electron microscopy. Ultrasound imaging had the greatest underestimation due to coarse resolution compared to the other imaging modalities. Despite this underestimation, both ultrasound imaging and MRI successfully tracked the longitudinal recellularization of liver scaffolds.

Bioengineered Lungs: A Challenge and An Opportunity

Lung biofabrication is a new tissue engineering and regenerative development aimed at providing organs for potential use in transplantation. Lung biofabrication is based on seeding cells into an acellular organ scaffold and on culturing them in an especial purpose bioreactor. The acellular lung scaffold is obtained by decellularizing a non-transplantable donor lung by means of conventional procedures based on application of physical, enzymatic and detergent agents. To avoid immune recipient's rejection of the transplanted bioengineered lung, autologous bone marrow/adipose tissue-derived mesenchymal stem cells, lung progenitor cells or induced pluripotent stem cells are used for biofabricating the bioengineered lung. The bioreactor applies circulatory perfusion and mechanical ventilation with physiological parameters to the lung during biofabrication. These physical stimuli to the organ are translated into the stem cell local microenvironment – e.g. shear stress and cyclic stretch – so that cells sense the physiological conditions in normally functioning mature lungs. After seminal proof of concept in a rodent model was published in 2010, the hypothesis that lungs can be biofabricated is accepted and intense research efforts are being devoted to the topic. The current experimental evidence obtained so far in animal tests and in ex vivo human bioengineered lungs suggests that the date of first clinical tests, although not immediate, is coming. Lung bioengineering is a disrupting concept that poses a challenge for improving our basic science knowledge and is also an opportunity for facilitating lung transplantation in future clinical translation.

Decellularized Liver Matrix-Modified Cryogel Scaffolds as Potential Hepatocyte Carriers in Bioartificial Liver Support Systems and Implantable Liver Constructs.

Recent progress in the use of decellularized organ scaffolds as regenerative matrices for tissue engineering holds great promise in addressing the issue of donor organ shortage. Decellularization preserves the mechanical integrity, composition, and microvasculature critical for zonation of hepatocytes in the liver. Earlier studies have reported the possibility of repopulating decellularized matrices with hepatic cell lines or stem cells to improve liver regeneration. In this work, we study the versatility of the decellularized liver matrix as a substrate coating of three-dimensional cryogel scaffolds. The coated cryogels were analyzed for their ability to maintain hepatic cell growth and functionality in vitro, which was found to be significantly better than the uncoated cryogel scaffolds. The decellularized liver matrix-coated cryogel scaffolds were evaluated for their potential application as a cell-loaded bioreactor for bioartificial liver support and as an implantable liver construct. Extracorporeal connection of the coated cryogel bioreactor to a liver failure model showed improvement in liver function parameters. Additionally, offline clinical evaluation of the bioreactor using patient-derived liver failure plasma showed its efficacy in improving liver failure conditions by approximately 30-60%. Furthermore, implantation of the decellularized matrix-coated cryogel showed complete integration with the native tissue as confirmed by hematoxylin and eosin staining of tissue sections. HepG2 cells and primary human hepatocytes seeded in the coated cryogel scaffolds implanted in the liver failure model maintained functionality in terms of albumin synthesis and cytochrome P450 activity post 2 weeks of implantation. In addition, a 20-60% improvement in liver function parameters was observed post implantation. These results, put together, suggest a possibility of using the decellularized matrix-coated cryogel scaffolds for liver tissue engineering applications.

Recent Advances in 3D Printing of Aliphatic Polyesters.

3D printing represents a valuable alternative to traditional processing methods, clearly demonstrated by the promising results obtained in the manufacture of various products, such as scaffolds for regenerative medicine, artificial tissues and organs, electronics, components for the automotive industry, art objects and so on. This revolutionary technique showed unique capabilities for fabricating complex structures, with precisely controlled physical characteristics, facile tunable mechanical properties, biological functionality and easily customizable architecture. In this paper, we provide an overview of the main 3D-printing technologies currently employed in the case of poly (lactic acid) (PLA) and polyhydroxyalkanoates (PHA), two of the most important classes of thermoplastic aliphatic polyesters. Moreover, a short presentation of the main 3D-printing methods is briefly discussed. Both PLA and PHA, in the form of filaments or powder, proved to be suitable for the fabrication of artificial tissue or scaffolds for bone regeneration. The processability of PLA and PHB blends and composites fabricated through different 3D-printing techniques, their final characteristics and targeted applications in bioengineering are thoroughly reviewed.