Human ECM Research Articles

SAT-397-HIC-5 and GARP expression is upregulated by hydrogen peroxide and TGF beta in primary human hepatic stellate cells cultured on decellularized human liver 3D ECM scaffolds

SAT-397-HIC-5 and GARP expression is upregulated by hydrogen peroxide and TGF beta in primary human hepatic stellate cells cultured on decellularized human liver 3D ECM scaffolds

Tissue Specific Extracellular Matrix Extract Promotes Salivary Gland Organoid Formation with Human Salivary Progenitors

Hypofunction of the salivary glands (SGs) has several detrimental effects. Several approaches can be used to restore the function of damaged SGs, such as tissue engineering based on stem cells and biomaterials. Among these, three dimensional (3D) culture without biomaterials (termed organoids) can recapitulate the characteristics of the native organ in vivo. However, despite the substantial progress made, most organoids which are formed from stem cells or cell aggregates still lack a specific tissue niche. We hypothesized that tissue specific ECM proteins are required for formation and maintenance of functional organoids. Thus, we evaluate the effect of human tissue specific ECM extracts on the formation of functional SG organoids containing human SG epithelial and mesenchymal cells. We prepared human salivary gland ECM (hSG-ECM) with a growth factor-enriched supplement and the prepared hSG-ECM was introdeced in a uniform high-throughput 3D platform. First, we determined the optimum culture conditions; organoids comprising 5,000 cells at a mesenchymal: epithelial cell ratio of 1:9 exhibited a low frequency of apoptosis, high proliferation capacity, and a structure resembling that of SGs. Introduction of hSG-ECM extract with proper concentration resulted in maintenance of the stem cell niche and promotion of stem cell differentiation. Moreover, treatment of the organoids with decellularized supplements increased their sensitivity to neurotransmitters. In conclusion, we developed functional human SG organoids using salivary progenitors and ECM extract. The SG organoids have potential for SG tissue repair, drug development, and disease modeling.

FRI-107 - Primary human HSC cell phenotype is differently regulated by pro-fibrogenic and pro-inflammatory stimuli in cirrhotic and healthy human liver 3D ECM scaffolds

FRI-107 - Primary human HSC cell phenotype is differently regulated by pro-fibrogenic and pro-inflammatory stimuli in cirrhotic and healthy human liver 3D ECM scaffolds

Porous Scaffolds Derived from Devitalized Tissue Engineered Cartilaginous Matrix Support Chondrogenesis of Adult Stem Cells.

ECM-derived scaffolds have previously been developed from devitalized native cartilage and successfully used in tissue engineering. Such ECM-based biomaterials are commonly derived from animal tissue, which may not represent the ideal source for applications in human. Native human ECM can be used as an alternative to xenogeneic tissue; however, its supply may be limited, leading to the need for a more readily available source of such biomaterials. The objective of this study was to compare devitalized native and tissue engineered cartilaginous ECM as chondro-permissive scaffolds for tissue engineering. To this end, porous scaffolds were produced using ECM derived from porcine articular cartilage and cartilaginous sheets engineered using human bone marrow stem cells. An identical process was used to produce scaffolds from three different types of devitalized ECMs, namely that derived from porcine cartilage (Native), human engineered cartilaginous sheets (Eng), and human engineered cartilaginous sheets generated in the presence of growth factor releasing microspheres (Eng-MS). Scaffolds produced using both devitalized engineered and native ECM possessed similar mechanical properties, pore size and GAG content, although were compositionally distinct. After being seeded with human infrapatellar fat pad stem cells, the engineered ECM-derived scaffolds (no Microspheres) supported less robust cartilage matrix deposition than native ECM scaffolds. However, more chondro-permissive scaffolds could be generated using cartilaginous ECM engineered in the presence of TGF-β1 releasing microspheres. Eng-MS scaffolds supported comparable levels of GAG synthesis to native ECM scaffolds. These results demonstrate that engineered ECM can be used to produce scaffolds for cartilage tissue engineering, overcoming stock limitations and other barriers associated with native autogeneic, allogeneic, and xenogeneic tissues. Such engineered ECM holds significant promise as an off-the-shelf chondro-permissive scaffold for articular cartilage repair.

Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution.

Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.

Nitinol-Based Nanotubular Arrays with Controlled Diameters Upregulate Human Vascular Cell ECM Production.

Current approaches to reducing restenosis do not balance the reduction of vascular smooth muscle cell proliferation with the increase in the healing of the endothelium. Building on our previous work, we present our study on the effects of Nitinol-based nanotubular coatings with different nanotube diameters on the reduction of restenosis. Here, we demonstrate that the nanotubular coatings reduced primary human aortic smooth muscle cell (HASMC) proliferation and increased the migration (by more than 4 times), collagen (by 2-3 times per cell) and elastin (by 5-8 times per cell) production of primary human aortic endothelial cells (HAEC). Furthermore, a significant increase in elastin and soluble collagen production of HAEC was observed with an increase in nanotube diameter. Our findings suggest that nanotubes-coated Nitinol may provide a surface conducive for HAEC reendothelialization while reducing the proliferation of HASMC.

Hydrogel Derived from Human Cardiac ECM Microparticles Exerts Superior Cytoprotective Effects

Hydrogel Derived from Human Cardiac ECM Microparticles Exerts Superior Cytoprotective Effects

Human ECM particles produced in vitro are injectable and support angiogenesis and adipogenesis in vivo

Event Abstract Back to Event Human ECM particles produced in vitro are injectable and support angiogenesis and adipogenesis in vivo Nicolas L'Heureux1, 2*, Adam D. Young3*, Nathalie Dusserre1*, Sonya B. Sonnenberg3*, Todd D. Johnson3*, Ricardo L. Rodriguez4*, Todd N. Mcallister1* and Karen L. Christman3* 1 Cytograft Tissue Engineering, United States 2 Université de Bordeaux, U1026 BioTis, France 3 University of California, San Diego, Department of Bioengineering, Sanford Consortium for Regenerative Medicine, United States 4 CosmeticSurg, United States Cell-synthesized ECM assembled by cultured cells has emerged as a promising biological scaffold for tissue engineering[1]. Tissue-engineered blood vessels built using rolled sheets of cell-assembled matrix (CAM) have shown unprecedented long-term patency in humans[2]. In this study, the CAM was mechanically processed into particles to produce an injectable form of the material suitable for many applications including aesthetic treatments. The goal of this study was to assess 1) the composition, 2) the injectability, 3) the stability, and 4) the host’s response to the CAM. Adult human dermal fibroblasts were cultured to promote ECM production and assembly in sheets for 8 weeks as previously described[3]. Dried sheets were milled into particles that passed through a #60 sieve (250µm mesh) and sterilized by gamma irradiation. Particles composition was analyzed using mass spectroscopy, immunolabeling and biochemical testing. Particle suspensions in saline were injected subcutaneously in nude mice along with two commercially available dermal fillers: Juvéderm Ultra XC® (cross-linked hyaluronic acid-based product) and Radiesse® (hydroxyapatite-based product). Each of 12 animals received four injections (upper shoulders and thighs) and was sacrificed after 2, 4, 8 and 12 weeks (4 biopsies per material per time point). Analysis of the ECM revealed that the particles contained numerous components including fibrillar and other collagens, elastic fiber components, fibrogenesis-associated and signaling components as well as glycosaminoglycans. The particle at 175 mg/ml produced a thick solution that could be injected through a 25 Ga needle. Subcutaneous injections were easily performed and, after diffusion of the excess injected fluids (≈24hrs), a palpable and localized bolus was observed. Histologically, the collagen rich particles were easily recognizable and generated a very mild inflammatory reaction with a generally thin fibrous layer around the injection site and only sparse immune cells. Rapid fibroblastic cell migration (2 weeks) was observed between the particles followed by progressive (2-12 week) perfusion by small blood vessels (also between particles). Adipocytes were also observed between particles within the injection bolus as early as 2 weeks but were more abundant as time went by. Cells did not appear to penetrate the particles and no signs of active degradation were observed. On the other hand, Juvéderm® did not form a well-delineated injection bolus and did not elicit an active remodeling process. Radiesse generated a very significant inflammatory response throughout the study period with abundant immune cell migration, giant cell formation, thicker fibrous capsule formation and extensive angiogenesis. These results clearly demonstrate the feasibility of injecting human CAM particles, at a clinically relevant concentration, using a clinically relevant system. Further more, these results support the idea that CAM is not targeted by the non-specific immune system. Finally, CAM particles supported both angiogenesis and adipogenesis. As a whole, these results illustrate the potential of this injectable material as a new tool for long-term tissue reconstruction and rejuvenation. While previous studies have shown great clinical successes using CAM-based tissue engineering[2], this is the first study to provide safety and remodeling data in a controlled setting. Marissa Peck, David Gebhart, Sunny Virk, Casey Mount, and Heinz Scheuenstuhl; Sophia Suarez; Rebecca Braden; Jessica Ungerleider; Dr. Kent Osborn; ASEE/NSF Engineering Innovation Fellows Program; National Science Foundation Graduate Research Fellowship Program

The type II histidine triad protein HtpsC is a novel adhesion with the involvement of Streptococcus suis virulence

Streptococcal histidine triad proteins HTPs are widely distributed within the Streptococcus genus. Based on the phylogenetic relationship and domain composition, HTPs are classified into type I and type II subfamilies. Previous studies revealed that several pathogenic streptococci contain more than one htp gene. We found that the highly virulent strain of Streptococcus suis 2 (S. suis 2), 05ZYH33 encodes 3 HTPs, designated HtpsA (previously described as HtpS), HtpsB, and HtpsC. Among them, HtpsC is the only member that contains leucine-rich repeat (LRR) domains at the C-terminal. In this study, we demonstrated that the recombinant HtpsC could bind to 2 different components of human ECM complex laminin and fibronectin in vitro, suggesting that it is a novel adhesin of S. suis 2. Having constructed an htpsC mutant, we evaluated its role in the pathogenesis of the highly virulent S. suis 2 strain 05ZYH33. Our data showed that inactivation of htpsC significantly affected adherence of S. suis 2 to Hep-2 cells and shortened the survival of the bacteria in whole blood. Furthermore, deletion of htpsC significantly attenuated the virulence of S. suis 2 in mice. These results demonstrated that htpsC was involved in the pathogenesis of the highly virulent S. suis 2 strain 05ZYH33. In line with the observation, immunization with HtpsC significantly prolonged mice's survival after S. suis 05ZYH33 challenge, indicating its potential use in the vaccine development against S. suis.

Retention of the stemness of mouse adipose-derived stem cells by their expansion on human bone marrow stromal cell-derived extracellular matrix.

Mesenchymal stem cells (MSCs) usually lose their stemness during in vitro expansion as they are deprived of their niche environment. Cell-extracellular matrix (ECM) interaction is known to play important roles in preserving the stemness of the cells in their stem cell niche environment. Previously, coating with bone marrow MSC (BMSC)-derived ECM was found able to maintain the differentiation potential of in vitro cultured MSCs. This study aimed to determine if this ECM coating could also maintain the stemness of cultured murine adipose-derived stem cells (ASCs) using a regular culture flask as a control. Cells were expanded in ECM-coated and ECM-noncoated flasks for two and four passages and then harvested for various analyses. The results showed that ASCs exhibited fibroblast-like spindle morphology in ECM-coated flasks, whereas ASCs gradually spread and enlarged in the ECM-noncoated flasks. After three and five passages, both groups of cells exhibited similar cytokinetics in the MSC culture medium (MesenPRO RS™ Medium). However, when cultured in Dulbecco's modified Eagles medium (DMEM) plus 10% fetal bovine serum, coating group cells exhibited more potent proliferation than control group cells with a significant difference in both passages 3 and 5 (p<0.01). When seeded at low density (500 cells/10-cm dish), coating group cells formed significantly more and larger sized cell colonies than control group cells with significant difference in cell colony numbers between two groups (p<0.05). In addition, coated colony cells were much smaller and more compactly arranged compared to control colony cells. Furthermore, ASCs expanded in coated flasks exhibited greater potentials for adipogenic, osteogenic, and chondrogenic differentiations than the cells expanded in regular flasks. Quantitatively, the Oil Red O staining area, Alizarin staining area, and Toluidine Blue staining area were all significantly larger than the respective staining areas of control cells (p<0.05). Real-time polymerase chain reaction also revealed significantly higher gene expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein (C/EBP), Runx2, osteocalcin, Sox9, collagen II, and aggrecan in ECM-coated group cells than in control group cells (p<0.05). Collectively, these results suggest that human BMSC decellular ECM coating helps to preserve the stemness of cultured murine ASCs.

Human microvascular pericyte basement membrane remodeling regulates neutrophil recruitment.

Neutrophil extravasation at post-capillary venules, consisting of EC, PC, and the shared ECM, increases following fibrotic remodeling in the lung, liver, and skin. The role of fibrotic pericyte-derived ECM in regulating EC activation and neutrophil recruitment remains unexplored. To elucidate the role of human pericyte-derived ECM in EC activation, we characterized PC-derived ECM following transforming growth factor-β1, IL-1β, CCL2, or bleomycin activation, and examined surface adhesion molecule expression and neutrophil recruitment by EC cultured on PC-ECM. Pro-inflammatory activation of PC-induced deposition of compositionally distinct ECM compared with non-activated control. Bleomycin activation induced fibronectin-rich and collagen-poor ECM remodeling by PC, facilitating increased neutrophil transendothelial migration when compared with non-activated pericyte ECM (49.9±3.4% versus 29.7±1.4%). Increases in fibronectin compared to collagen I, are largely responsible for ECM-regulated neutrophil recruitment, as EC cultured on fibronectin supported increased neutrophil transmigration compared to collagen I (51.6±6.2% versus 28.0±4.8%). We attribute this difference to increased expression of ICAM-1 and its redistribution to EC borders. This is the first demonstration of human pericyte sensitivity to inflammatory stimuli, inducing fibrotic matrix deposition that regulates EC adhesion molecule expression and neutrophil recruitment.

Abstract 4855: Proteomic characterization of the extracellular matrix in multiple myeloma

Abstract Background: The extracellular matrix is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM) ECM components, such as integrins, fibronectin and collagens, have been shown are critical to the pathogenesis of MM and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Recent advances in proteomics have led to the characterization of the ECM and ECM-related proteins (“matrisome”) in normal human tissues and tumors in a systematic and comprehensive approach. Methods: Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x106) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after two weeks in order to mimic early tumor development (luminescence= 1x105 p/sec/cm2/sr). and compared to mice demonstrating high tumor burden (1x108 p/sec/cm2/sr) 5 weeks post injection. Human bone marrow aspirates; Whole bone marrow was obtained from MM patients (newly diagnosed n=9, relapsed n=9) and healthy human donors (n=9) following written informed consent. Sequential extractions of whole pooled bone marrow from mice and individual bone marrow humans was performed using the CNMCS(Cytosol/Nucleus/Membrane/Cytoskeleton) Compartmental Protein Extraction Kit (Cytomol, CA). Following this, proteins underwent reversed-phase high performance liquid chromatography followed by tandem mass spectrometry (MS). Identified peptide spectra were counted as a semi quantitative measure of abundance. Results: We detected a total of 1202, 982 and 329 unique proteins from enriched whole bone marrow samples from relapsed patients, newly diagnosed patients and mice, respectively. Of these, critical ECM components such as laminins, matrix metalloproteinases and collagens were found to be enriched in human MM ECM in comparison to healthy donors with increased abundance apparent with disease progression in mice. Specifically Bone Marrow Proteoglycan and Proteoglycan 3 were amongst the ECM proteins significantly enriched in the ECM of newly diagnosed patients. PRG3 is a p53 responsive gene that has been demonstrated to be upregulated in apoptotic cells in several cancers, including CLL. The ECM protein Elastin, the expression of which is closely associated with the invasive/metastatic potential of various cancer types, was also upregulated in the MM ECM where it may play an important role in the tumor-ECM interaction. Conclusions: We have profiled the ECM in MM using mass spectrometry with a view to determining the specific components that may be important in MM disease biology. Through this approach molecular mechanisms that influence MM development and progression can be uncovered and potential targets for therapy identified. Citation Format: Siobhan V. Glavey, Alexandra Naba, Manuela Gambella, Alberto Rocci, Antonio Sacco, John Asara, Antonio Palumbo, Richard O. Hynes, Aldo M. Roccaro, Irene M. Ghobrial. Proteomic characterization of the extracellular matrix in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4855. doi:10.1158/1538-7445.AM2014-4855

ECM Scaffolds From Discarded Human Kidneys Retain Growth Factors Along With Intact Microstructural Morphologies of the Innate Vasculature.

Introduction and purpose: Renal transplantation is the only curative treatment for end stage renal disease patients but it's limited by the shortage of transplantable organs. Regenerative medicine has the potential to initiate a shift in transplantation with its aim to fabricate a functional bioengineered organ. Acellular human renal ECM scaffolds (hrECMs) can be generated from discarded human kidneys by decellularization. Our aim was to assess biological and anatomical status of the hrECMs evaluating that major growth factors and 3D microstructural properties were preserved. Methods: A cytokine antibody array was performed to analyze relevant growth factors (GFs) retain in hrECMs compared to control human kidneys (5 vs. 5). Microvasculature status was assessed performing 16 renal endocast of discarded human kidneys and acellular human renal scaffolds (8 vs. 8).Figure: No Caption available.Morphometric measurements of the glomerulus were taken from SEM cast images and underwent statistical studies. Results: A significant level of GFs remains post-decellularization. Compared to controls, levels of TGF-α, FGF-6 and IGFBP-3 in the scaffolds were 23.54%, 20.23%, and 19.71% respectively. HB-EGF, IGFBP-6, NT-3, P1GF, TGF-β, VEGF and VEGF-D were moderately (10-13%) presentFigure: No Caption available.Microvascular analysis of the casts showed that glomerular shape was near-perfectly preserved but its volume was reduced by 46.92% of the initial volume (control). Conclusion: hrECMs maintain growth factor levels along with intact microstructural morphologies after cells removal. These findings indicate that their potential use as a platform for cellular seeding in the context of organ engineering merits further consideration.

ECM Scaffolds From Discarded Human Kidneys Perform as Bio-System When Seeded With Multipotent Progenitor Cells.

Background and purpose: Regenerative medicine has the potential to meet the need for a potentially inexhaustible source of organs and for immunosuppression-free transplantation. In the attempt to bioengineer kidney organoids for transplantation, acellular ECM scaffolds are being generated from porcine and discarded human kidneys. Such scaffolds constitute platforms to be reseeded with autologous cells to facilitate regeneration and recapitulation of native biological and morphological properties. Given the lack of an in-depth understanding of the interactions between ECM and cells, our study aims to understand the mechanisms of interaction between multipotent progenitor cells and the ECM scaffold obtained from discarded kidneys. Methods: 300 μm-thick, 1cm2 sections of human renal ECM scaffolds (hrECMs) were seeded with human amniotic fluid stem cells (hAFSCs) and cultured. Sections were collected for histology at days 7, 14, 28 and 35. Samples of the culture media were collected at the same time points to assess secretion of molecules involved in matrix remodeling, inflammation and angiogenesis. Study endpoints were hAFSCs attachment, proliferation, and migration; cell turnover, studied by immunohistochemical analysis for Bcl-associated X protein and PCNA; expression of genes of early kidney development; secretion of MMPs, TIMPs, cytokines and interleukins, which was determined in hAFSC prior to and throughout culture on hrECMs. Results: The various staining showed that hAFSCs attach, proliferate and migrate throughout the 3D framework of hrECMs. The cells were viable and metabolically functional, as demonstrated by their ability to synthesize molecules involved in the remodeling of the matrix, pro-inflammatory signals, angiogenic factors, integrins, and collagen 4alpha. Significant proliferation and reduced apoptosis of hAFSCs were observed within the first 14 days; this trend reversed at the end of the study period (5 weeks). Notably, hrECMs were able to induce the expression of genes associated with early kidney development Conclusion: When seeded with progenitor cells, hrECMs perform as a “bio-system” in that a dynamic interplay of proliferative signaling and cellular migration with virtually no exogenous induction is observable.

Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium

AbstractExtracellular matrix (ECM) derived by tissue decellularization has applications as a tissue engineering scaffold and for support of cellular regeneration. Myocardial ECM from animals has been produced by whole‐organ perfusion or immersion processes, but methods for preparation of human myocardial ECM for therapy and research have not been compared in detail, yet. We analyzed the impact of decellularization processes on human myocardial ECM, and tested its ability to serve as a scaffold for cell seeding. Sodium dodecyl sulfate (SDS)‐based decellularization, but not treatments based on Triton X‐100, deoxycholate or hypo/hypertonic incubations, removed cells satisfactorily, and incubation with fetal bovine serum (FBS) eliminated residual DNA. ECM architecture was best preserved by a protocol consisting of 2 h lysis, 6 h SDS, and 3 h FBS, but age and pathology of the donor tissue are highly important for producing reproducible, high‐quality scaffolds. We also studied ECM repopulation with mesenchymal stem cells (CB‐MSC), cardiomyocytes derived from induced pluripotent stem cells (iPS‐CM), and naïve neonatal mouse cardiomyocytes. Cells attached to the matrix and proliferated and displayed higher viability than in standard culture. We conclude that human cardiac ECM sheets may be suitable scaffold for cell‐matrix interaction studies and as a biomaterial for tissue regeneration and engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3263–3272, 2014

Decellularizing Human Hearts: Characterizing Native Cardiac Matrix for Clinical Translation

Purpose Nearly 22 million people suffer from heart failure (HF) worldwide. Heart transplantation is the only viable treatment for HF, but donor organ shortage fails to meet clinical demand. A bioartificial heart could overcome this limitation. The first step towards development is the generation of a perfusable cardiac matrix. Herein, we introduce a standard protocol for decellularizing human hearts and characterize the resulting scaffolds. Methods and Materials Ten human hearts unsuitable for transplantation were retrieved for research, 4 of which were donation after cardiac death (DCD) donors. Applying antegrade coronary perfusion, hearts were decellularized using sodium dodecyl sulfate and subsequent washes. Valve competency and coronary angiographies were performed with a pressure-controlled pump in a fluoroscopy suite. ECM characterization was done by immunohistochemistry (IHC) for matrix proteins and cardiac cell markers, scanning electron microscopy (SEM), and equibiaxial mechanical testing. Results Human hearts can be decellularized using antegrade coronary perfusion. IHC and SEM analysis revealed a lack of cells, negative staining for cardiac markers, and intact basement membranes. Valve competency showed all four heart valves exhibited higher regurgitant flow during physiological pressures, though decelled mitral valves showed statistically higher flow. Coronary angiography confirmed preservation of patent coronaries. IHC analysis showed decelled human hearts maintain ECM protein content and structure. Equibiaxial testing of decelled myocardium exhibited non-linear behavior and similar elastic modulus along both axes. Hearts from DCD donors were successfully decellularized, with similar outcomes to non-DCD hearts. Conclusions Decellularization of human hearts renders a native cardiac matrix with ECM-protein retention and perfusable coronary conduits. Successful decellularization of DCD hearts may provide a significant source of new donor grafts, from organs previously considered unsuitable for transplant.

Thick Acellular Heart Extracellular Matrix with Inherent Vasculature: A Potential Platform for Myocardial Tissue Regeneration

The decellularization of porcine heart tissue offers many opportunities for the production of physiologically relevant myocardial mimetic scaffolds. Earlier, we reported the successful isolation of a thin porcine cardiac extracellular matrix (pcECM) exhibiting relevant bio-mechanical properties for myocardial tissue engineering. Nevertheless, since native cardiac tissue is much thicker, such thin scaffolds may offer limited regeneration capacity. However, generation of thicker myocardial mimetic tissue constructs is hindered by diffusion limitations (~100 μm), and the lack of a proper vascular-like network within these constructs. In our present work, we focused on optimizing the decellularization procedure for thicker tissue slabs (10-15 mm), while retaining their inherent vasculature, and on characterizing the resulting pcECM. The trypsin/Triton-based perfusion procedure that resulted in a nonimmunogenic and cell-supportive pcECM was found to be more effective in cell removal and in the preservation of fiber morphology and structural characteristics than stirring, sonication, or sodium dodecyl sulfate/Triton-based procedures. Mass spectroscopy revealed that the pcECM is mainly composed of ECM proteins with no apparent cellular protein remains. Mechanical testing indicated that the obtained pcECM is viscoelastic in nature and possesses the typical stress-strain profile of biological materials. It is stiffer than native tissue yet exhibits matched mechanical properties in terms of energy dissipation, toughness, and ultimate stress behavior. Vascular network functionality was maintained to the first three-four branches from the main coronary vessels. Taken together, these results reaffirm the efficiency of the decellularization procedure reported herein for yielding thick nonimmunogenic cell-supportive pcECM scaffolds, preserving both native tissue ultra-structural properties and an inherent vascular network. When reseeded with the appropriate progenitor cells, these scaffolds can potentially serve as ex vivo screening platforms for new therapeutics, as models for human cardiac ECM, or as biomedical constructs for patch or transmural transplantation strategies.

Construction of scaffold with human extracellular matrix from adipose tissue

To investigate the feasibility of constructing scaffold for tissue engineering with human extracellular matrix from adipose tissue. Fresh human adipose tissue was obtained by liposuction in 7 women who undergone liposuction. One part of the fat was used to isolate the adipose-derived stem cells (ADSCs), the other part was used to extract human extracellular matrix powder. After removing blood and oil components, the tissue was homogenized, centrifuged, freeze-dried, and crushed to powder by instrument. The structure of human ECM powder was observed with electron microscopy. The ADSCs were seeded and attached to the human extracellular matrix powder before and after labeled with fluorescent DiI, respectively. The adhesion rate was detected. The adhesion and growth of ADSCs were observed with Fluorescence microscope. The adhesion rate before and after DiI labeling was analyzed statistically with two-sample test of SPSS 13.0. The ADSCs and human extracellular matrix powder were obtained successfully from adipose tissue. The ADSCs could be differentiated into adipose cells, bone cells and chondrocytes. SEM images showed that the power had both rugged and smooth surface with a porous structure characteristics. ADSCs could adhere to the scaffold easily, and the adhesion rate was (88.81 +/- 4.81)% and (86.48 +/- 4.58)% before and after DiI labeling. There was no difference between two groups. DiI labeled ADSCs were adhered to extracellular matrix scaffold and could grow in good condition. Human adipose tissue extracellular matrix powder was easy to obtain, with diversity in size and shape which provided excellent substrates for cell adhesion and growth. It could be an ideal adipose tissue engineering scaffold.

Development of a Human Extracellular Matrix for Applications Related with Stem Cells and Tissue Engineering

The recent progress in stem cell biology has created new approaches for their study as well as their application to the treatment of human diseases [1–3]. The success of stem-cell based technologies in the clinical setting [4, 5] has emphasized the need to improve the standards of quality for all phases of cell therapy, particularly the development of culture methods that circumvent products of animal origin since these might provoke infections or immune rejection following transplantation in patients. Indeed, since Martin et al. (2005) [6] demonstrated that hESC cultured with animal or serum products retained non-human sialic acid which was immunogenic when transplanted in humans, the establishment of animal-free conditions to support the maintenance and differentiation of human stem cells has been a major goal of the field of regenerative medicine [7]. Thus, chemically-defined culture systems that are devoid of non-human substances will greatly facilitate the use of stem cells in regenerative strategies. The concept of a niche is crucial for the organization of stem cells. A niche is consider as a subset of tissue cells and extracellular substrates (matrix and soluble factors) that can support stem cells and control their self-renewal in vivo [8]. Extracellular matrices help to structure niches spatially and modulate the concentration of adhesive and signalling molecules locally. The ECM is a molecular complex that contains collagens and other glycoproteins, hyaluronic acid, proteoglycans, glycosaminoglycans (GAGs), and elastins. Additionally, the ECM harbours growth factors or cytokines to protect against degradation [9]. ECM components are responsible for adhesion during the majority of cell interactions and are implicated in the maintenance of embryonic induction during development as well as stem cell differentiation in vitro [10]. Thus, local changes in ECM can dramatically modulate the proliferation and migration of stem cells and may participate in the specification of lineages. hESC have provided invaluable tools for gaining insight into the developmental origins of human tissues. However, to realize the full biological and clinical potential of hESC, certain problems related with the routine culture of these cells must be solved. Mouse embryonic fibroblasts (MEFs) and murine derivatives such as Matrigel are widely used in the maintenance and differentiation of hESC. Recently, considerable effort has been dedicated to the elimination of animal-derived reagents from the culture of hESC and in parallel, to the control of cell growth parameters by avoiding human feeder cells. For example, in 2006 Ludwig et al. reported the use of conditioned media and high doses of FGF to maintain the undifferentiated state in hESC cultured on plastic, but some abnormalities were detected at passage 20 under these conditions [11, 12]. The use of human feeders complicates the growth and molecular analysis of both pluripotency and differentiation since experimental data may reflect the combined effects of hESC and feeder cells in the culture. Given these considerations, the use of an ECM of human or synthetic origin would provide many advantages. Indeed, there have been attempts [13, 14] to produce such a tool but the results have not been satisfactory because the products were unable to maintain hESC in the undifferentiated state over time. Matrix proteins have been used as coating for in vitro cultures of human stem cells but they have usually been applied as undefined protein mixtures [11] of animal origin [15] with undefined media [13], serum [16] or a synthetic mixture [17, 18]. However, most of these human biological reagents are expensive to manufacture and thus, are cost-prohibitive for many laboratories. Decellularization procedures have been used traditionally to isolate ECM from cells in culture, tissues or organs [19]. The goal of decellularization protocols is to efficiently remove cellular and nuclear material while minimizing any adverse effect on the composition, biological activity and mechanical and structural integrity of the remaining ECM [20, 21]. Decellularized human scaffolds have facilitated the remodelling of various tissues in both animal models and humans [4, 22]. However, any biochemical procedure employed to remove cells may also alter the native three-dimensional architecture of the ECM and thus, a balance must be achieved between chemical and physical treatments during the decellularization process [4, 5, 23]. Given the potential importance of hESC in translational research and regenerative medicine, the aim of the present study was to develop a simple, efficient protocol for the production of a human ECM that is both safe and economical. Here we report that hypotonic lysis of human foreskin fibroblasts (HFF) generates a human ECM that retains protein components which are essential for attachment and cell-cell interaction. This hffECM was capable of maintaining the pluripotency of hESC and supporting their differentiation when used with the appropriate medium. Therefore, our results reveal hffECM as a novel tool which may facilitate the clinical application of hESC-based technologies.

Response of human corneal fibroblasts on silk film surface patterns.

Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37-342 nm depths and 445-3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions.