Recellularization Of Scaffolds Research Articles

An update on a technical method of cartilage decellularization: (a physical-based protocol)

Introduction: Despite advances in orthopedic surgery, the lack of effective conventional treatment for cartilage defects has led to research in cartilage tissue engineering. One of the interesting topics is the use of decellularized extracellular matrix (ECM) as a suitable natural scaffold that supports the growth and function of cells cultured in it. A concern with decellularization protocols, especially those with high detergent concentrations, is the disruption of native ECM, which has deleterious effects on subsequent scaffold recellularization. Therefore, this study focused on optimizing cartilage decellularization by physical methods without the use of ionic detergents. Methods: The bovine tracheal cartilage fragments were decellularized by a combination of 8 cycles of freeze-thaw and ultrasound techniques. Then, the tissues were immersed and shaken in 0.25% trypsin for 24 hours. Efficient cell removal and preservation of ECM were confirmed by histological and cytocompatibility assessments. The in-vivo studies were performed to evaluate the biocompatibility and bioactivity of the scaffold. Results: The histological assessments indicated the appropriate cytocompatibility and the fibroblast cell culture study demonstrated that cells were able to proliferate and migrate on the decellularized cartilage. In-vivo evaluation also showed a reduced adverse immune response, including leukocyte infiltration into the ECM. Conclusions: These results suggest that a cartilage scaffold created using a physical decellularization protocol that efficiently removes cells while preserving the native ECM can be a suitable scaffold for cartilage reconstruction. The main advantage of this protocol is the absence of potentially toxic chemicals in the tissues.

Regional and disease-specific glycosaminoglycan composition and function in decellularized human lung extracellular matrix.

Decellularized lung scaffolds and hydrogels are increasingly being utilized in ex vivo lung bioengineering. However, the lung is a regionally heterogenous organ with proximal and distal airway and vascular compartments of different structures and functions that may be altered as part of disease pathogenesis. We previously described decellularized normal whole human lung extracellular matrix (ECM) glycosaminoglycan (GAG) composition and functional ability to bind matrix-associated growth factors. We now determine differential GAG composition and function in airway, vascular, and alveolar-enriched regions of decellularized lungs obtained from normal, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) patients. Significant differences were observed in heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) content and CS/HS compositions between both different lung regions and between normal and diseased lungs. Surface plasmon resonance demonstrated that HS and CS from decellularized normal and COPD lungs similarly bound fibroblast growth factor 2, but that binding was decreased in decellularized IPF lungs. Binding of transforming growth factor β to CS was similar in all three groups but binding to HS was decreased in IPF compared to normal and COPD lungs. In addition, cytokines dissociate faster from the IPF GAGs than their counterparts. The differences in cytokine binding features of IPF GAGs may result from different disaccharide compositions. The purified HS from IPF lung is less sulfated than that from other lungs, and the CS from IPF contains more 6-O-sulfated disaccharide. These observations provide further information for understanding functional roles of ECM GAGs in lung function and disease. STATEMENT OF SIGNIFICANCE: Lung transplantation remains limited due to donor organ availability and need for life-long immunosuppressive medication. One solution, the ex vivo bioengineering of lungs via de- and recellularization has not yet led to a fully functional organ. Notably, the role of glycosaminoglycans (GAGs) remaining in decellularized lung scaffolds is poorly understood despite their important effects on cell behaviors. We have previously investigated residual GAG content of native and decellularized lungs and their respective functionality, and role during scaffold recellularization. We now present a detailed characterization of GAG and GAG chain content and function in different anatomical regions of normal diseased human lungs. These are novel and important observations that further expand knowledge about functional GAG roles in lung biology and disease.

Kidney Bioengineering for Transplantation.

The kidney is an important organ for maintenance of homeostasis in the human body. As renal failure progresses, renal replacement therapy becomes necessary. However, there is a chronic shortage of kidney donors, creating a major problem for transplantation. To solve this problem, many strategies for the generation of transplantable kidneys are under investigation. Since the first reports describing that nephron progenitors could be induced from human induced pluripotent stem cells, kidney organoids have been attracting attention as tools for studying human kidney development and diseases. Because the kidney is formed through the interactions of multiple renal progenitors, current studies are investigating ways to combine these progenitors derived from human induced pluripotent stem cells for the generation of transplantable kidney organoids. Other bioengineering strategies, such as decellularization and recellularization of scaffolds, 3-dimensional bioprinting, interspecies blastocyst complementation and progenitor replacement, and xenotransplantation, also have the potential to generate whole kidneys, although each of these strategies has its own challenges. Combinations of these approaches will lead to the generation of bioengineered kidneys that are transplantable into humans.

Decellularized Vascular Scaffolds Derived from Bovine Placenta Blood Vessels.

Diseases associated with the circulatory system are the main causes of worldwide morbidity and mortality, implying the need for vascular implants. Thus, the production of vascular biomaterials has proven to be a promising alternative to therapies used in studies and research related to vascular physiology. The present project aims to achieve the artificial development of blood vessels through the recellularization of vascular scaffolds derived from bovine placental vessels. The chorioallantoic surface of the bovine placenta was used to produce decellularized biomaterials. For recellularization, 2.5 x 104 endothelial cells were seeded above each decellularized vessel fragment during three or seven days, when culture were interrupted, and the fragments were fixed for cell attachment analysis. Decellularized and recellularized biomaterials were evaluated by basic histology, scanning electron microscopy, and immunohistochemistry. The decellularization process produced vessels that maintained natural structure and elastin content, and no cells or gDNA remains were observed. Endothelial precursor cells were also attached to lumen and external surface of the decellularized vessel.Conclusion: Our results show a possibility of future uses of this biomaterial in cardiovascular medicine, as in the development of engineered vessels.

Evaluation of decellularization process for developing osteogenic bovine cancellous bone scaffolds in-vitro.

Current immunological issues in bone grafting regarding the transfer of xenogeneic donor bone cells into the recipient are challenging the industry to produce safer acellular natural matrices for bone regeneration. The aim of this study was to investigate the efficacy of a novel decellularization technique for producing bovine cancellous bone scaffold and compare its physicochemical, mechanical, and biological characteristics with demineralized cancellous bone scaffold in an in-vitro study. Cancellous bone blocks were harvested from a bovine femoral head (18-24 months old) subjected to physical cleansing and chemical defatting, and further processed in two ways. Group I was subjected to demineralization, while Group II underwent decellularization through physical, chemical, and enzymatic treatments. Both were then freeze-dried, and gamma radiated, finally producing a demineralized bovine cancellous bone (DMB) scaffold and decellularized bovine cancellous bone (DCC) scaffold. Both DMB and DCC scaffolds were subjected to histological evaluation, scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS), fourier-transform infrared spectroscopy (FTIR), quantification of lipid, collagen, and residual nucleic acid content, and mechanical testing. The osteogenic potential was investigated through the recellularization of scaffolds with human osteoblast cell seeding and examined for cell attachment, proliferation, and mineralization by Alizarin staining and gene expression. DCC produced a complete acellular extracellular matrix (ECM) with the absence of nucleic acid content, wider pores with extensive interconnectivity and partially retaining collagen fibrils. DCC demonstrated a higher cell proliferation rate, upregulation of osteogenic differentiation markers, and substantial mineralized nodules production. Our findings suggest that the decellularization technique produced an acellular DCC scaffold with minimal damage to ECM and possesses osteogenic potential through the mechanisms of osteoconduction, osteoinduction, and osteogenesis in-vitro.

Advances in Recellularization of Decellularized Liver Grafts with Different Liver (Stem) Cells: Towards Clinical Applications.

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.

Recellularization of Acellular Xeno Kidney Scaffold: An In Vivo Method to Generate Bioartificial Kidney.

A significant hurdle for kidney tissue engineering is reproducing the complex three-dimensional structure of the kidney. In our study, a stepwise approach of generating a reproducible Xeno kidney scaffold from a goat kidney is described, which can be implanted and recellularized by host cells. We have proposed a combination of sodium dodecyl sulfate and Triton-X-100-based protocol to generate a reproducible Xeno kidney scaffold, which was then analyzed by histology, DNA quantification, SEM, and renal angiography. Further, a small portion from the cortico-medullar region of the acellular scaffold was implanted in the rat's kidney subcapsular pocket for a period of 1 month, to check the recruitment of host cells into the scaffold. Post implantation, the extracellular matrix of the scaffold was well preserved and it did not induce any damage or inflammation in the native kidney. Implantation of the Xeno scaffold resulted in apparent early vascularization which helped in the recruitment of the host cells, which was characterized by histology, immunohistochemistry, and scanning electron microscopy. Implanted Xeno scaffold showed AQP-1, Nephrin, α-SMA, and VEGF expression in proximal tubules and renal glomerulus. Importantly, Ki-67 and WTAP-expressing cells were also observed near proximal tubules suggesting a high level of proliferation in the scaffold. Thus, showing the potential of Xeno kidney development that can be recellularized by the host cell to engineer into a functional kidney.

Establishment of a human 3D pancreatic adenocarcinoma model based on a patient-derived extracellular matrix scaffold

Pancreatic cancer is likely to become one of the leading causes of cancer-related death in many countries within the next decade. Surgery is the potentially curative treatment for pancreatic ductal adenocarcinoma (PDAC), although only 10%–20% of patients have a resectable disease after diagnosis. Despite recent advances in curative surgery the current prognosis ranges from 6% to 10% globally. One of the main issues at the pre-clinical level is the lacking of model which simultaneously reflects the tumour microenvironment (TME) at both structural and cellular levels. Here we describe an innovative tissue engineering approach applied to PDAC starting from decellularized human biopsies in order to generate an organotypic 3D in vitro model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence, mechanical analysis, and scanning electron microscopy. Mass spectrometry confirmed a different extracellular matrix (ECM) composition between decellularized healthy pancreas and PDAC by identifying a total of 110 non-redundant differently expressed proteins. Immunofluorescence analyses after 7 days of scaffold recellularization with PANC-1 and AsPC-1 pancreatic cell lines, were performed to assess the biocompatibility of 3D matrices to sustain engraftment, localization and infiltration. Finally, both PANC-1 and AsPC-1 cells cultured in 3D matrices showed a reduced response to treatment with FOLFIRINOX if compared to conventional bi-dimensional culture. Our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the pancreatic cancer microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of pancreatic cancer response to chemotherapy agents.

An Effective Method for Decellularization of Human Foreskin: Implications for Skin Regeneration in Small Wounds.

Objective Acellular matrices of different allogeneic or xenogeneic origins are widely used as structural scaffolds in regenerative medicine. The main goal of this research was to optimize a method for decellularization of foreskin for skin regeneration in small wounds. Materials and Methods In this experimental study, the dermal layers of foreskin were divided into two sections and subjected to two different decellularization methods: the sodium dodecyl sulfate method (SDS-M), and our optimized foreskin decellularization method (OFD-M). A combination of non-ionic detergents and SDS were used to decellularize the foreskin in OFD-M. The histological, morphological, and biomechanical properties of both methods were compared. In addition, human umbilical cord mesenchymal stem cells (hucMSCs) were isolated, and the biocompatibility and recellularization of both scaffolds by hucMSC were subsequently determined. Results We observed that OFD-M is an appropriate approach for successful removal of cellular components from the foreskin tissue, without physical disturbance to the acellular matrix. In comparison to SDS-M, this new bioscaffold possesses a fine network containing a high amount of collagen fibers and glycosaminoglycans (GAG) (P≤0.03), is biocompatible and harmless for hucMSC (viability 91.7%), and exhibits a relatively high tensile strength. ConclusionWe found that the extracellular matrix (ECM) structural integrity, the main ECM components, and the mechanical properties of the foreskin are well maintained after applying the OFD-M decellularization technique, indicating that the resulting scaffold would be a suitable platform for culturing MSC for skin grafting in small wounds.

MP22-13 OFF THE SHELF ACELLULAR FETAL SKIN SCAFFOLD AS A NOVEL ALTERNATIVE TO BUCCAL MUCOSA GRAFT: THE DEVELOPMENT AND CHARACTERIZATION OF HUMAN TISSUE ENGINEERED FETAL DERMAL MATRIX IN RABBIT MODEL OF HYPOSPADIASIS

You have accessJournal of UrologyCME1 May 2022MP22-13 OFF THE SHELF ACELLULAR FETAL SKIN SCAFFOLD AS A NOVEL ALTERNATIVE TO BUCCAL MUCOSA GRAFT: THE DEVELOPMENT AND CHARACTERIZATION OF HUMAN TISSUE ENGINEERED FETAL DERMAL MATRIX IN RABBIT MODEL OF HYPOSPADIASIS Abdol-Mohammad Kajbafzadeh, Soheila Sobhani, and Fahimeh Jafarnezhad-Ansariha Abdol-Mohammad KajbafzadehAbdol-Mohammad Kajbafzadeh More articles by this author , Soheila SobhaniSoheila Sobhani More articles by this author , and Fahimeh Jafarnezhad-AnsarihaFahimeh Jafarnezhad-Ansariha More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002561.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To introduce a novel alternative to buccal mucosal graft from the acellular human fetal skin for management of hypospadias in rabbit model. To optimize the acellularization protocol for development and characterization of human tissue engineered fetal dermal matrix as an “off the shelf” natural biomaterial. METHODS: Human fetal skin were obtained (from the therapeutic abortion fetuses) with gestational age of 16-19 weeks with respect to a singed informed consent from parents following receiving the university ethical committee permission. The dissected full thickness fetal skin tissues were placed into SDS and Triton X-100 in different dosages to achieve the optimum decellularization protocol. Histopathology of the fetal acellular matrix were assess by hematoxcillin & eosin, DAPI staining for confirmation of removal of all cell materials, trichrome staining for collagen evaluation DNA quantification for confirmation of DNA content, scanning electron microscopy for evaluation of scaffold microstructure. Specific dermal markers (Vimentin, type I collagen, CK14) were depict by IHC. The prepared dermal scaffolds were then grafted into the induced model of hypospadias in 8 male rabbits. The rabbits were followed up for 1, 2, 3 and 6 months post operatively. RESULTS: In order to show the success of decellularization process, we characterized it with H&E, trichrome-Masson staining, DAPI and SEM which confirmed the significant removal of cells; meanwhile the ECM was completely preserved. At the time of biopsy after 2, 4 and 6 moths, no evidence of inflammation, fibrosis, necrosis or rejection was observed and the grafted dermal scaffolds appeared histologically and anatomically normal histologic appearance by recellularization of scaffold by circulating CD 34 + bone marrow stem cells “represent the body as a natural bioreactor”. CONCLUSIONS: The use of this novel “off the shelf” natural fetal skin scaffold without cell seeding is an excellent alterative to buccal mucosal graft and pave the road for decreasing surgical time (at least one hour) in complex hypospadias reconstruction. This method is safe, feasible with excellent angioneogenesis and cell seeding in due course without cell culture and cell seeding or shrinkage of the graft, no creation of oral mucosal wound and in addition, there will be no limitations of graft size for staged hypospadias cripple surgery. Source of Funding: There was no source of funding for this study © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e376 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Abdol-Mohammad Kajbafzadeh More articles by this author Soheila Sobhani More articles by this author Fahimeh Jafarnezhad-Ansariha More articles by this author Expand All Advertisement PDF DownloadLoading ...

Functional Recellularization of Acellular Rat Liver Scaffold by Induced Pluripotent Stem Cells: Molecular Evidence for Wnt/B-Catenin Upregulation.

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.

QS4: Optimizing The Decellularization Of The Rodent Epigastric Free Flap: A Comparison Of Automated SDS-based Protocols

Purpose:Raising flaps to cover complex wounds with exposed critical structures are lengthy operations that result in donor site morbidity. Tissue engineering research is developing with great promise to build replacement tissues without morbidity. Decellularization removes whole cells and cell debris, and is the initial step to create a scaffold with an intact vascular network. Benchmark measurement of the overall cellular level is quantification of the DNA content, where 50 ng/mg is classically considered as a threshold. Although perfusion decellularization and recellularization approaches have shown exceptional promise in whole organ engineering, there is minimal crossover into the microsurgical field. Sodium dodecyl sulfate (SDS)-based protocols are known to have deleterious effects on the ultrastructure and capillary network of the scaffolds, but remain the predominant choice for decellularization protocols. This study aims to optimize the SDS exposure protocol for automated decellularization by comparing different SDS perfusion times to gain better understanding of the balance between decellularization and scaffold preservation.Methods:A 3D-printed closed-system bioreactor capable of continuously perfusing fluid throughout the vasculature was used for decellularization of free flaps. 2x2 cm fasciocutaneous free flaps from the epigastric region of the rat were harvested, and the vascular pedicle was isolated as a single artery and vein. Three flaps were evaluated in each group. The artery (inflow) and vein (outflow) were cannulated to monitor preservation of the vasculature. 1% SDS solution was perfused in different durations (3, 5 and 10 days) based on several protocols found in the literature. Automated SDS perfusion was followed by 1 day of 1% Triton X-100 and 1 day of 1xPhosphate-buffered saline (PBS), all of which were at the perfusion pressure of 120 mmHg.Results:For vasculature analysis, continual perfusion into the artery and out of the vein within the bioreactor was assessed throughout the decellularization process. H&E, Masson’s trichrome, and Verhoeff-Van Gieson staining were performed to assess architecture and locale of residual nuclei. Residual DNA was quantified by the fluorescent marker PicoGreen. 5 days of 1% SDS solution had the least residual DNA content (1.309±0.807 ng/mg) followed by 10 days (12.684±14.184 ng/mg) and 3 days (82.387±71.595 ng/mg), p<0.001. The DNA content ratio of skin over subcutaneous tissues was consistent across all protocols with skin having twice as much residual DNA after each protocol. The vascular network was visualized for qualitative assessment with the perfusion of hardening cast to create contrast against soft tissue to visualize with microCT imaging.Conclusion:A decellularization protocol of 5 days of 1% SDS solution followed by 1 day of 1% Triton X-100 and 1 day of 1xPBS was the most successful to keep the residual DNA content at a minimum, while preserving the structural integrity of the tissues. The bioreactor is capable of running automated and repeatable protocols using several solutions and continuously perfusing fluid throughout the vasculature of a free flap. This compact and integrated system can decrease hands-on time and be used in the future for further recellularization of scaffolds to bioengineer soft tissue replacement without donor site morbidity.

TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds

The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.

A decellularized human corneal scaffold for anterior corneal surface reconstruction

Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

Pre-coating decellularized liver with HepG2-conditioned medium improves hepatic recellularization

Liver transplantation from compatible donors has been the main therapy available for patients with irreversible hepatic injuries. Due to the increasing shortage of organs suitable for transplantation, tissue engineering technologies are important alternatives or surrogate approaches for the future of human organ transplantations. New bioengineering tools have been designed to produce decellularized organs (i.e. scaffolds) which could be recellularized with human cells. Specifically, there is an unmet need for developing reproducible protocols for inducing better cellular spreading in decellularized liver scaffolds. The aim of the present work was to investigate the possibility to improve liver scaffold recellularization by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium (CM). Furthermore, we evaluated the capability of commercial human liver cells (HepG2) to adhere to several types of extracellular matrices (ECM) as well as CM components. Wistar rat livers were decellularized and analyzed by histology, scanning electron microscopy (SEM), immunohistochemistry and residual DNA-content analysis. Human induced pluripotent stem cells (hiPSCs)-derived mesenchymal cells (hiMSCs), and human commercial hepatic (HepG2) and endothelial (HAEC) cells were used for liver scaffold recellularization with or without CM pre-coating. Recellularization occurred for up to 5weeks. Hepatic tissues and CM were analyzed by proteomic assays. We show that integrity and anatomical organization of the hepatic ECM were maintained after decellularization, and proteomic analysis suggested that pre-coating with CM enriched the decellularized liver ECM. Pre-coating with HepG2-CM highly improved liver recellularization and revealed the positive effects of liver ECM and CM components association.

Preseeding of Mesenchymal Stem Cells Increases Integration of an iPSC-Derived CM Sheet into a Cardiac Matrix.

Cell sheet technology has demonstrated great promise in delivering a large amount of therapeutic cells for tissue repair, including in the myocardium. However, the lack of host integration remains one of the key challenges in using cell sheets for cardiac repair. Paracrine factors secreted by mesenchymal stem cells (MSCs) have been reported to facilitate tissue repair and regeneration in a variety of ways. It has been demonstrated that paracrine factors from MSCs could enhance scaffold recellularization and vascularization. In this study, we used an in vitro cardiac matrix mimic platform to examine the effects of hMSCs preseeding on the interactions between cell sheets and cardiac matrix. The fabricated human induced pluripotent stem cells-derived cardiomyocyte sheets were attached to a decellularized porcine myocardium slice with or without preseeding of hMSCs. The hMSCs preseeding significantly enhanced the interactions between cardiomyocyte sheets and cardiac matrix in terms of cell migration distance, cell distribution, and mature vascular and cardiomyocyte marker expressions in the matrix. Growth factor and matrix metalloproteinases array analysis suggested that hMSCs- induced vascularization and MMPs regulation are the two possible mechanisms that lead to the improved CMs and cardiac matrix interactions. Further examination of these two mechanisms will enable the development of new approaches to facilitate transplanted cells for tissue repair.

The Role of Autologous Mesenchymal Stem Cell Recellularization in Rescuing the Xenoreactive Immune Response

Emerging evidence suggests that current xenograft valve replacements provoke an intense cell-mediated and humoral immune response that is a major regulator of time-dependent structural valve deterioration (SVD). Autologous recellularization may improve valvular durability by masking the xenograft scaffold from immune recognition. The purpose of this study was to directly address the role of autologous recellularization of xenograft scaffolds, in the regulation of the xenoreactive immune response in an in-vitro human model. To study the human xenoreactive immune response, we collected bone marrow, human pericardium and isolated peripheral blood mononuclear cells (PBMCs) from adult patients undergoing elective cardiac surgery. Decellularized bovine pericardium underwent recellularization with human mesenchymal stem cells (MSCs). Grafts were tested as either non-decellularized (fresh) bovine pericardium, decellularized bovine pericardium and recellularized bovine pericardium. We show that when decellularized bovine pericardium was recellularized with human MSCs and exposed to autologous human PBMCs in-vitro, there was a significant reduction in TNF-α and IFN-γ expression, along with a significant decrease in T-cell proliferation when compared to both wild-type and decellularized bovine pericardium (p<0.01). Importantly, recellularized bovine pericardium had an equivalent expression of TNF-α, IFN-γ and T-cell proliferation when compared to human pericardium exposed to autologous human PBMCs. Our data suggest that autologous MSC recellularization of xenogenic tissue abrogates the xenoreactive immune response with subsequent reduction in pro-inflammatory cytokine production and T-cell proliferation in an in-vitro human model. As such, autologous MSC recellularization of an acellular xenogenic scaffolds may be an effective approach to decrease the progression of bioprosthetic SVD.

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.

A Hepatic Scaffold from Decellularized Liver Tissue: Food for Thought.

Allogeneic liver transplantation is still deemed the gold standard solution for end-stage organ failure; however, donor organ shortages have led to extended waiting lists for organ transplants. In order to overcome the lack of donors, the development of new therapeutic options is mandatory. In the last several years, organ bioengineering has been extensively explored to provide transplantable tissues or whole organs with the final goal of creating a three-dimensional growth microenvironment mimicking the native structure. It has been frequently reported that an extracellular matrix-based scaffold offers a structural support and important biological molecules that could help cellular proliferation during the recellularization process. The aim of the present review is to underline the recent developments in cell-on-scaffold technology for liver bioengineering, taking into account: (1) biological and synthetic scaffolds; (2) animal and human tissue decellularization; (3) scaffold recellularization; (4) 3D bioprinting; and (5) organoid technology. Future possible clinical applications in regenerative medicine for liver tissue engineering and for drug testing were underlined and dissected.

Amphiregulin modulates murine lung recovery and fibroblast function following exposure to agriculture organic dust.

Exposure to agricultural bioaerosols can lead to chronic inflammatory lung diseases. Amphiregulin (AREG) can promote the lung repair process but can also lead to fibrotic remodeling. The objective of this study was to determine the role of AREG in altering recovery from environmental dust exposure in a murine in vivo model and in vitro using cultured human and murine lung fibroblasts. C57BL/6 mice were intranasally exposed to swine confinement facility dust extract (DE) or saline daily for 1 wk or allowed to recover for 3-7 days while being treated with an AREG-neutralizing antibody or recombinant AREG. Treatment with the anti-AREG antibody prevented resolution of DE exposure-induced airway influx of total cells, neutrophils, and macrophages and increased levels of TNF-α, IL-6, and CXCL1. Neutrophils and activated macrophages (CD11c+CD11bhi) persisted after recovery in lung tissues of anti-AREG-treated mice. In murine and human lung fibroblasts, DE induced the release of AREG and inflammatory cytokines. Fibroblast recellularization of primary human lung mesenchymal matrix scaffolds and wound closure was inhibited by DE and enhanced with recombinant AREG alone. AREG treatment rescued the DE-induced inhibitory fibroblast effects. AREG intranasal treatment for 3 days during recovery phase reduced repetitive DE-induced airway inflammatory cell influx and cytokine release. Collectively, these studies demonstrate that inhibition of AREG reduced, whereas AREG supplementation promoted, the airway inflammatory recovery response following environmental bioaerosol exposure, and AREG enhanced fibroblast function, suggesting that AREG could be targeted in agricultural workers repetitively exposed to organic dust environments to potentially prevent and/or reduce disease.