Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium.

Preparation of cardiac extracellular matrix scaffolds by decellularization of human myocardium

Barbara Oberwallner MSc1, Andreja Brodarac PhD1,3, Yeong-Hoon Choi MD2, Tomo Saric MD PhD2, Petra Anić3, Lars Morawietz MD3, Christof Stamm MD1,3

1

Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.

2

University of Cologne, Cologne, Germany

3

Deutsches Herzzentrum Berlin (DHZB), Berlin, Germany.

Author Disclosure Statement No benefit of any kind will be received either directly or indirectly by the authors.

Short title: Human myocardial ECM Keywords: Heart, decellularization, scaffold, stem cell, regeneration

Correspondence to: Prof. Dr. Christof Stamm Deutsches Herzzentrum Berlin Augustenburger Platz 1 13353 Berlin Germany E-mail: [email protected] Fax: +49 30 4593 2100 Phone: +49 30 4593 2109 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/jbm.a.35000

Journal of Biomedical Materials Research: Part A Oberwallner et al: Human myocardial ECM

Abstract Extracellular 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 defined, 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 X100, 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 2h lysis, 6h SDS, and 3h FBS, but age and pathology of the donor tissue are highly important for producing reproducible, highquality 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. All 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.

2 John Wiley & Sons, Inc.

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1.

Introduction

Extracellular matrix preparations have previously been studied as scaffolds for replacement heart valves1 and for in vitro generation of whole organs.2,3 The need for biomaterials to support cellular regeneration processes in the myocardium has recently been acknowledged, not least because transplantation of exogenous cells alone had very limited success.4 It is assumed that regenerating cells require interaction with extracellular matrix (ECM) components not only for mechanical support but also to acquire and maintain appropriate phenotypic and functional characteristics. Although beneficial effects have been reported when cells for myocardial repair were applied in conjunction with artificial biomaterials,5 currently available polymer preparations are not able to mimic the entire spectrum of required cell-matrix interactions. Biologic ECM produced by decellularization of tissues and organs is supposed to be better suited, and, for instance, myocardial replacement or support patches based on urinary bladder or small intestinal submucosa from animals have been applied in experimental models of heart disease.6-11 Nevertheless, it may be argued that only tissue-specific, i.e. myocardial ECM is ideal for supporting organ repair, and indeed matrix preparations from small and large animal hearts have been developed and benefits regarding their use in myocardial regeneration have been reported.12,13 Whether xenogenic ECM is functionally and immunologically equal to allogenic, i.e. human ECM is currently being investigated,14-17 and such studies as well as possible allogenic therapeutic applications ultimately require the availability of human ECM. Our aim was therefore to generate human cardiac ECM scaffolds for in vitro study of cell-matrix interactions and as a cell delivery platform for cardiac repair. To that aim, we



tested the applicability of several methods that were previously used for decellularization of animal myocardium and developed a novel protocol that provides the best balance between cell removal and ECM integrity. We then assessed the biocompatibility of human myocardial ECM by re-populating it with human mesenchymal stromal cells and murine cardiomyocytes.

2.

Methods

2.1

Decellularization of human myocardial tissue

The study protocol conforms to the ethical principles outlined in the Declaration of Helsinki and was approved by the institutional review board and ethics committee of Charité Medical University. All patients provided written, informed consent for use of heart tissue for experimental studies. Left ventricular (LV) myocardium was obtained from patients undergoing implantation of a mechanical assist device, heart transplantation or resection of a subaortic outflow tract obstruction. Human left ventricular myocardial tissue and, for preliminary experiments, porcine heart tissue was sectioned to 0.3 mm thick sections with a cryostat (CM 3050S, Leica, Wetzlar, Germany). The following decellularization methods were tested in order to develop a protocol suitable for human myocardium: incubation in 0.5% (wt/vol) sodium dodecyl sulphate (SDS) for 9 h, 5% (vol/vol) Triton X-100 for 48 h, 4% (wt/vol) sodium deoxycholate for 40h, all dissolved in PBS, or alternating incubation in 10x and 0.1x PBS (hypo/hypertonic treatment, 2 h each, with 3 changes per day, for 3 days). Samples were incubated in these reagents with orbital shaking at 4°C. Then, three washing cycles in PBS for 10 min each were followed by overnight rinsing in PBS with 4 John Wiley & Sons, Inc.

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penicillin/streptomycin and nystatin added (100U/ml each). For recellularization experiments, a decellularization protocol was chosen based on the results of the ECM analysis, which in the following will be termed the “3-step protocol”. It combines features of two previously published methods,12,13 which we modified for our experimental conditions as follows: 300 µm thick sections were incubated in lysis buffer (10 mM Tris, 0.1% wt/vol EDTA, pH 7.4) for 2 h, followed by solubilization in SDS (0.5% wt/vol in PBS) for 6 h, with orbital shaking at room temperature. After washing with PBS (3 times 10 min plus overnight), residual DNA was removed by incubation in FBS for 3 h at 37°C. Following

a

second

washing

step,

samples

were

stored

in

PBS

plus

penicillin/streptomycin and nystatin at 4°C for up to 2 weeks before recellularization.

2.2

Biochemical analysis of decellularized matrix

DNA in native and decellularized tissue was quantified using the Invisorb Spin Tissue Midi Kit (Invitek, Berlin, Germany). Extracellular matrix components were quantified using a hydroxyproline assay for collagen,

18

the Fastin™ 5,10,15,20-tetraphenyl-

21H,23H-porphine assay for elastin (Biocolor, Carrickfergus, UK), and the Blyscan™ assay for sulfated proteoglycans and glycosaminoglycans (Biocolor, Carrickfergus, UK). Data were expressed as the measured mass of a given component in a sample per weight of the same sample prior to decellularization (i.e. µg collagen/mg myocardium).

2.3

Histology and immunohistochemistry

H&E and Picrosirius Red staining were performed following fixation with 4% (wt/vol) buffered paraformaldehyde, paraffin embedding and microtome sectioning. For all other staining techniques, samples were cryosectioned after fixation and sequential 5 John Wiley & Sons, Inc.

Journal of Biomedical Materials Research: Part A

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Oberwallner et al: Human myocardial ECM

embedding in 15% and 30% (wt/vol) sucrose in PBS, Tissue-Tek O.C.T. compound, and flash-freezing on dry ice/ethanol. Masson’s trichrome staining was carried out using the Accustain

Trichrome

Stain

kit

(Sigma-Aldrich,

Taufkirchen,

Germany).

For

immunohistochemistry, samples were encircled with a delimiting pen (Dako, Hamburg, Germany) and incubated in blocking buffer (PBS with 5% goat serum and 0.1% (vol/vol) Triton X-100 for permeabilization) for 1 h at room temperature. Primary antibody incubation was done for 1 h at room temperature or 4°C overnight, with antibodies against desmin, laminin and fibronectin (Abcam, Cambridge, UK, diluted in blocking buffer) or sarcomeric α-actinin (Sigma-Aldrich). After a washing step with PBS, samples were incubated with biotinylated secondary antibody (Biozol, Eching, Germany, 1:500 in blocking buffer) for 1 h, followed by washing and incubation in horseradish peroxidase streptavidin (Biozol, 1:400 in PBS) for 50 min. Then, samples were washed and incubated for 3 min in diaminobenzidine (1.5 mg in 3 ml PBS with 2 µl 30% H2O2), before they were stained with Mayer’s hemalum (Carl Roth, Karlsruhe, Germany), dehydrated by graded ethanol series, cleared with Roti-Histol (Carl Roth) and sealed with Eukitt mounting medium (Sigma-Aldrich).

2.4

Scanning electron microscopy

Samples were fixed with 2.5% (vol/vol) glutaraldehyde in PBS for 1 h at room temperature. They were washed with PBS, dehydrated by graded acetone series (30%, 50%, 75%, 90%, 100%) and dried by critical point drying with CO2 (CPD 030 Critical Point Dryer, BalTec, Pfäfficon, Switzerland). Critical point drying avoids the phase changes associated with conventional dehydration and is therefore less destructive for


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tissue architecture. Dried samples were fixed on holders, sputter-coated with gold and visualized on a Hitachi S-2700 scanning electron microscope.

2.5

Cell culture

Human umbilical cord blood-derived mesenchymal stem cells (CB-MSC) were isolated, expanded and cryopreserved as previously described.19 After thawing, CB-MSC were cultured in DMEM low glucose (Life Technologies, Darmstadt, Germany) with 10% FBS and 100 U/ml penicillin/streptomycin at 37°C, 5% CO2. Cells were in passage 5 when used for recellularization experiments, and the minimal MSC criteria were confirmed using flow cytometry and differentiation assays for adipogenic, osteogenic and chondrogenic lineages (StemPro kits, Life Technologies). Murine cardiomyocytes were derived from induced pluripotent stem cells expressing puromycin N-acetyltransferase under the control of α-myosin heavy chain promoter as described before, including antibiotic selection to obtain an almost pure cardiomyocyte population.20 Medium consisted of high glucose DMEM (Life Technologies) with 15% FBS, 0.1 mM MEM nonessential amino acids, 50 µM 2-mercaptoethanol and 100 U/ml penicillin/streptomycin. For additional experiments, murine neonatal cardiomyocytes (NMC) were purchased from Provitro and cultured in cardiac myocyte medium (Provitro, Berlin, Germany).

2.6

Recellularization

Decellularized matrix (3-step protocol) was cut with a scalpel to fit into 96 well plates. Cells were counted with a hemocytometer, suspended in medium at the desired cell density and pipetted onto the matrix. Control cells were seeded in empty wells. Samples were incubated at 37°C, 5% CO2, with daily medium change. CB-MSC were seeded on 7 John Wiley & Sons, Inc.


the uncoated, native ECM scaffold, while for repopulation with iPS-CM culture plates and scaffolds were coated with 2.5 µg/ml fibronectin. To assess the myocyte adhesion on ECM with and without additional fibronectin coating, neonatal mouse cardiomyocytes were seeded on both coated and uncoated matrix for comparison.

2.7

Viability assays

Cell viability was analyzed using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Mannheim, Germany). Briefly, cells in 96 well plates with or without matrix were incubated in 100 µl PBS with 5% glucose; 20 µl MTS/PMS solution was added, and the plate was incubated for 4 h at 37°C, 5% CO2, resulting in a color reaction in the presence of viable cells. To measure the viability of cells within the matrix without that of cells attached to the bottom of the well, the matrix was transferred to a new well before incubation. After incubation, 100 µl reaction solution from each well was transferred to a new well to avoid influence of the matrix on spectrophotometry. Relative cell viability was measured as absorbance at 490 nm. Blank values from wells without cells were subtracted from the results.

2.8

Statistical analysis

Values are expressed as means with SEM. Two groups of samples (MTS assays) were compared using Mann-Whitney U test. Three or more groups of samples (DNA extraction, ECM assays) were compared using one-way ANOVA with Games-Howell post-hoc analysis. A p value of ≤ 0.05 was considered statistically significant. Tests were carried out using IBM SPSS Statistics 20 software (IBM, Armonk, NY).


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3.

Results

3.1

Sufficient decellularization of human myocardial tissue with SDS-based methods and serum

Visualization of gross morphology with a stereo microscope showed the typical color change from brown (myoglobin in native tissue) to transparent in the decellularized samples (Figure 1A). Methods involving the detergent SDS in a concentration of 0.5% resulted in removal of nuclei and cellular proteins (Figure 1B & C). By histology, complete decellularization was seen after 9 h shaking at 4°C or, in the 3-step protocol, after 2 h in lysis buffer and 6 h in SDS at room temperature. Treatment with Triton X100, deoxycholic acid, or hypo/hypertonic treatment damaged the tissue architecture but was not sufficient to remove cellular material (Figure 1B & C). When comparing the SDS-based protocols that yielded complete decellularization, more ECM material was preserved in the 3-step protocol than after 9 h SDS at 4°C (Figure 1C, 2B).

To assess the removal of nucleic material quantitatively, we extracted DNA from decellularized matrices and controls and measured DNA concentration by photometry (Figure 2A, Table S1). Native tissue contained 280 ± 28 ng DNA/mg wet weight, and SDS-based decellularization methods reduced DNA levels after shorter incubation periods and to a greater extent than the methods which did not involve SDS. However, without additional treatment the samples were not entirely free of DNA. Therefore, we included a further incubation step with fetal bovine serum (FBS) in our decellularization protocol, a method that has been reported to remove residual DNA from predecellularized samples.13 Indeed, the DNA content after 9 h SDS treatment was reduced from 20 ± 2 ng/mg to 9 ± 1 ng/mg when 3 h incubation in FBS at 37°C was included 9 John Wiley & Sons, Inc.


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(p = 0.04), and the best result was obtained with the 3-step protocol (5 ± 1 ng DNA/mg). Compared to native tissue this represents a 98% reduction in DNA content and is similar to the DNA removal seen after treatment with purified DNAse.12 The complete removal of cellular material by the 3-step protocol was further confirmed by negative immunohistology staining for the muscle cell-specific cytoskeletal protein desmin (Figure 3A). Taken together, we concluded that the most promising decellularization protocols consisted of 9 h SDS plus FBS as well as the 3-step protocol and focused on these during the next steps of our analysis.

3.2

Extracellular matrix preservation in decellularized human myocardium

To study the persistence of ECM components, we quantitatively assessed collagen, elastin and sulphated glycosaminoglycan content before and after decellularization (Figure 2C-E, Table S2-S4). Significantly more ECM components were destroyed by the

9h

SDS/3h

FBS

protocol

than

by

the

3-step

protocol

(p ≤ 0.05

for

glycosaminoglycans, p ≤ 0.01 for collagen and elastin). While the 9 h treatment with SDS showed a pronounced reduction of all three major matrix components (p ≤ 0.01), the collagen content was almost completely preserved in the 3-step protocol compared to that of native tissue. After decellularization with the 3-step protocol, elastin and glycosaminoglycans were reduced but still present (19.2% ± 1.0% sulphated glycosaminoglycans and 27.4% ± 2.4% elastin compared to native tissue). The decellularized

samples

were

also

positive

for

laminin

and

fibronectin

by

immunohistology (Figure 3A), which indicated that all major ECM components were at least partially

preserved. In addition, ECM architecture after decellularization was

studied by electron microscopy. As demonstrated in Figure 3B & C, the orientation of 10 John Wiley & Sons, Inc.

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ECM fibers within a myocardial fiber was largely maintained after cell removal. Taken together, these data led us to concentrate on the 3-step protocol for recellularization experiments, because it provided the best combination of decellularization efficiency and extracellular matrix preservation.

3.3

Differences in the decellularization of normal versus diseased human tissue

We also studied the efficacy of our 3-step decellularization protocol in structurally abnormal human left ventricular myocardium derived from patients who had suffered from myocardial infarction. In the native state before decellularization, the infarcted tissue had a reduced cell content and increased connective tissue deposition compared to normal myocardium. Consequently, the collagen network of the fibrotic ECM was denser than non-infarcted tissue after decellularization, as shown by H&E and Sirius red staining (Figure 4A). For comparison, we included images of native and decellularized porcine myocardium derived from young and completely healthy animals, with no fibrosis whatsoever. Of note, it was impossible to remove the age-related pigment lipofuscin from myocardium of aged human donors (Figures 2B & 4). Figure 4B shows the intense autofluorescence of lipofuscin granules in ECM derived from the heart of a septuagenarian, while all other cellular components have been removed.

3.4

ECM recellularization with MSC

Human CB-MSC displayed the typical immunophenotype and were able to differentiate in adipogenic, chondrogenic and osteogenic lineages (Figure S1, S2). To confirm a linear relationship between cell number and MTS assay data in order to estimate the number of cells present in the matrix at a given time point, we determined cell viability at 11 John Wiley & Sons, Inc.


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different cell doses per well in a separate experiment (Figure S3), and found a nearlinear correlation within a wide range of cell doses. The majority of CB-MSC attached to the matrix and, after initial proliferation on the surface, started to infiltrate the porous scaffold (Figure 5A). When recellularized with 0.5 × 106 CB-MSC/cm2, approximately 80% of the cells attached to the matrix, as estimated by viability measurements after 2 days (Figure 5B). The remaining cells settled on the bottom of the culture plate. We also tested the injection of cells into the matrix with a 20G needle, but cell attachment was not improved (Figure 5B). In other experiments, MTS tests were performed with MSC or iPS-CM seeded in wells with or without matrix and cultivated for 2 days to allow for a direct comparison with the same starting cell number. As seen in Figure 5F, MSC viability/metabolic activity in wells containing matrix was higher than when cells were grown in absence of ECM (p=0.05)

3.5

Recellularization with murine cardiomyocytes

We also recellularized the ECM scaffolds with murine cardiomyocytes derived from induced pluripotent stem cells in vitro. Although these cells attached to a lesser extent than CB-MSC and did not infiltrate the matrix due to their low proliferative and migratory capacity, cells were found to remain viable throughout a 3 week period (Figure 5C). Staining for α-actinin confirmed the cardiomyocyte identity of the iPS-CM on the matrix (Figure 5E). Similar to MSC, IPS-CM in wells with matrix showed a trend toward higher metabolic activity than cells in wells without matrix (p=0.08) (Figure 5F). In these experiments, we coated both matrix and cell culture wells with fibronectin to be able to compare iPS-CM viability of IPS-CM on ECM with that in normal culture plates, where


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fibronectin coating is required for attachment. However, fibronectin coating was not necessary for cardiomyocyte attachment to the matrix, as neonatal mouse cardiomyocytes readily attached to the matrix with or without additional fibronectin (Figure 5D). As expected, the cardiomyocytes contracted synchronously in standard culture, but were also found beating after seeding onto the matrix (Movie S1). The contractions were strong enough to lead to visible movement of the whole matrix sheets.

4.

Discussion

To the best of our knowledge, this study represents the first systematic analysis of decellularization methods for human ventricular myocardium and of its suitability as a scaffold for stem cells and cardiomyocytes. The importance of coordinated cell-matrix interactions for generation of replacement tissues in the cardiovascular system is increasingly being acknowledged.16,21-23 Non-cardiac biologic tissue scaffolds have been used for repair of cardiac defects in animal models,11,24-27 but tissue-specific ECM preparations may provide advantages.2,28-30 Decellularization of myocardial heart tissue from rat or pig has been described,2,13,31-36 and human myocardium has also been used in a recent study.12 The decellularization of ex vivo perfused whole hearts, preserving the architecture of the myocardial vasculature, is possible,2,33,35 and even repopulation of decellularized rat hearts with a cardiac cell mix proved feasible.2 Other than for wholeorgan tissue engineering experiments, tissue-specific ECM may also be useful for cell therapy approaches, providing transplanted cells with a more physiologic environment to facilitate survival, engraftment, and possibly guiding differentiation.37-41



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The quality of tissue decellularization depends on numerous factors such as tissue size and thickness, the incubation protocol (immersion versus perfusion decellularization, temperature) and the species of origin. In contrast to the animal myocardium, the human myocardium was derived from aged donors and contained lipofuscin as well as very mature, cross-linked collagen,42 presumably making it more difficult to decellularize than tissue from young donors. Of the decellularization methods we tested on human myocardium, cellular material was sufficiently removed only when the detergent SDS was used. This finding stands in contrast to two studies in which combined treatment with trypsin and Triton X-100, in one case plus incubation in deoxycholic acid, were used to decellularize porcine myocardium by immersion or perfusion.31,33 Instead, we found that even long-term incubation in high concentrations of Triton X-100 and deoxycholic acid was not sufficient to remove intracellular material in human myocardium. We deliberately avoided the use of trypsin, as this enzyme attacks peptide bonds, including those in ECM proteins such as collagen, and therefore may have detrimental effects on ECM structure and mechanical properties.23 The only other study published to date in which human myocardial tissue was decellularized also included SDS in the decellularization protocol.12 However, fibronectin was lost from the extracellular matrix in that study, whereas we detected preserved fibronectin in the ECM. This discrepancy emphasizes that even small variations in the decellularization protocol can have an important impact on the composition and quality of the resulting ECM product. Initially, we carried out decellularization at 4°C, as we hypothesized that lower temperatures would preserve ECM structure and components better than room temperature. However, complete cell removal at 4°C with SDS took longer (at least


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8-9 h in 0.5% SDS) and eventually destroyed more ECM components than decellularization according to the 3-step protocol at room temperature. The traditional method for depletion of residual DNA in decellularized tissue is DNAse treatment, but commercially available enzyme preparations are costly and it may be difficult to completely remove the enzyme at the end of the protocol so that biocompatibility is not jeopardized. We found that incubation of detergent-decellularized ECM in FBS is an inexpensive, reliable and convenient alternative method to eliminate residual DNA. This is in accordance with the findings of Gui et al.,13 who first suggested the use of FBS in decellularization procedures in various tissues, exploiting the intrinsic DNAse activity of serum.13 Because our human myocardium was derived from aged donors it contained the ageing pigment lipofuscin. None of the decellularization protocols tested completely removed lipofuscin from human myocardium. Lipofuscin is an aggregate of oxidized proteins and lipids formed by oxidative stress in the cytosol of ageing heart cells and other organs and is potentially toxic after intracellular accumulation.43 Primarily intracellular lipofuscin granules are extremely resilient and we were unable to remove them although other cellular components were eliminated. Although cellular re-population of the ECM was successful for at least 3 weeks in vitro, possible long-term toxic or immunogenic effects of lipofuscin cannot be excluded. This emphasizes the need for selection of appropriately healthy and young source tissue for ECM production.44,45 An additional finding that underlines this notion is the result obtained when decellularizing infarcted human myocardium, which maintained its grossly pathologic architecture. As myocardium does not have to be viable for preparation of ECM by immersion



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decellularization, relatively fresh tissue from autopsy specimens without relevant autolysis should, in principle, be sufficient.

In our cellular re-seeding experiments, we could demonstrate that somatic progenitor cells (CB-MSC), neonatal cardiomycytes and mature cardiomyocytes derived from iPS cells are able to repopulate human ECM. In other studies, recellularization of animal myocardial ECM was performed with immature (embryonic or neonatal) cardiomyocytes that still proliferate,2,31,33 or even cardiomyocyte-like cells differentiated from bone marrow MSC.34 The presence of human ECM promoted viability of MSC and showed a similar trend for IPS-CM, but the molecular mechanisms underlying the beneficial ECM effects remain to be determined. Of note, coating of the ECM with an adhesionpromoting substances was not necessary for cell invasion, in contrast to cardiac ECM preparations described by other groups.12

Finally, the question of perfusion vs. immersion decellularization warrants discussion. The group of Taylor et al. pioneered the decellularization of intact rodent hearts,2 and Wainwright et al. demonstrated that this concept can also be applied to human-sized porcine hearts.33 In contrast, Eitan et al. first reported on the preparation of porcine myocardial ECM from tissue slices using detergent immersion techniques followed by lyophilization for long-term storage.31 Both concepts require very different conditions. We chose to develop a detergent immersion process specifically for human myocardium, because the availability of intact human hearts is obviously very limited, and screening tests on ECM composition and behavior require fewer infrastructures and are less costly when done on a small “96-well” scale in vitro. In addition, studies 16 John Wiley & Sons, Inc.

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involving ECM in chronic disease conditions are greatly facilitated when tissue samples from surgical procedures can be processed. Technical reproducibility is expected to be higher with source of a defined thickness and we used 300 µm thick tissue sheets, because oxygen and nutrient supply can still occur by diffusion.46 For therapeutic use, however, thicker ECM scaffolds may be necessary, and Sarig et al. recently described how large parts of the inherent myocardial vasculature were preserved after immersion decellularization and could be repopulated with endothelial cells.36 5.

Conclusion

Decellularized human myocardium with largely preserved ECM composition and complete removal of cellular material can be produced via a 3-step process involving lysis buffer, SDS and serum incubation. Within less than 3 days, ECM scaffolds suitable for studies of cell-matrix interactions or tissue engineering approaches can be prepared. This technology forms the basis for future experiments to compare human with xenogeneic ECM and to test the impact of specific human disease conditions on cellmatrix interactions.

Acknowledgements This study was supported by Charité Universitätsmedizin (FKZ 1315848A) and Helmholtz-Zentrum Geesthacht (FKZ 1315848B). The authors would like to thank Prof. Dr. Rudolph Meyer, Dr. Katharina Wassilew and the pathology department of the DHZB for advice and help with histological stainings. We also thank Dr. Karen Bieback for providing CB-MSC, Dipl. Ing. (FH) Jörg Nissen and Dipl. Ing. Iryna Driehorst for electron microscopy and Anne Gale, ELS, for editorial assistance. 17 John Wiley & Sons, Inc.


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References 1 2 3 4 5 6

7 8 9 10 11 12

13 14 15

16 17

Steinhoff G, Stock U, Karim N, Mertsching H, Timke A, Meliss RR, Pethig K, Haverich A, Bader A. Tissue engineering of pulmonary heart valves on allogenic acellular matrix conduits: in vivo restoration of valve tissue. Circulation 2000;102:III50-III55. Ott HC, Matthiesen TS, Goh S-K, Black LD, Kren SM, Netoff TI, Taylor DA. Perfusiondecellularized matrix: using nature's platform to engineer a bioartificial heart. Nat Med 2008;14:213-221. Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, Ott HC. Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat Med 2013;19:646651. Laflamme MA, Murry CE. Heart regeneration. Nature 2011;473:326-335. Chachques JC, Trainini JC, Lago N, Cortes-Morichetti M, Schussler O, Carpentier A. Myocardial assistance by grafting a new bioartificial upgraded myocardium (MAGNUM trial): clinical feasibility study. Ann Thorac Surg 2008;85:901-908. Turner WS, Wang X, Johnson S, Medberry C, Mendez J, Badylak SF, McCord MG, McCloskey KE. Cardiac tissue development for delivery of embryonic stem cell-derived endothelial and cardiac cells in natural matrices. J Biomed Mater Res B Appl Biomater 100:2060-2072. Wainwright JM, Hashizume R, Fujimoto KL, Remlinger NT, Pesyna C, Wagner WR, Tobita K, Gilbert TW, Badylak SF. Right ventricular outflow tract repair with a cardiac biologic scaffold. Cells Tissues Organs 2012;195:159-170. Ota T, Gilbert TW, Badylak SF, Schwartzman D, Zenati MA. Electromechanical characterization of a tissue-engineered myocardial patch derived from extracellular matrix. J Thorac Cardiovasc Surg 2007;133:979-985. Badylak SF, Kochupura PV, Cohen IS, Doronin SV, Saltman AE, Gilbert TW, Kelly DJ, Ignotz RA, Gaudette GR. The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium. Cell Transplant 2006;15 Suppl 1:S29-S40. Robinson KA, Li J, Mathison M, Redkar A, Cui J, Chronos NA, Matheny RG, Badylak SF. Extracellular matrix scaffold for cardiac repair. Circulation 2005;112:I135-I143. Tudorache I, Kostin S, Meyer T, Teebken O, Bara C, Hilfiker A, Haverich A, Cebotari S. Viable vascularized autologous patch for transmural myocardial reconstruction. Eur J Cardiothorac Surg 2009;36:306-311; discussion 311. Godier-Furnemont AF, Martens TP, Koeckert MS, Wan L, Parks J, Arai K, Zhang G, Hudson B, Homma S, Vunjak-Novakovic G. Composite scaffold provides a cell delivery platform for cardiovascular repair. Proc Natl Acad Sci U S A 2011;108:7974-7979. Gui L, Chan SA, Breuer CK, Niklason LE. Novel utilization of serum in tissue decellularization. Tissue Eng Part C Methods 2010;16:173-184. Bayrak A, Pruger P, Stock UA, Seifert M. Absence of immune responses with xenogeneic collagen and elastin. Tissue Eng Part A Bayrak A, Tyralla M, Ladhoff J, Schleicher M, Stock UA, Volk HD, Seifert M. Human immune responses to porcine xenogeneic matrices and their extracellular matrix constituents in vitro. Biomaterials 31:3793-3803. Badylak SF, Gilbert TW. Immune response to biologic scaffold materials. Semin Immunol 2008;20:109-116. Allman AJ, McPherson TB, Badylak SF, Merrill LC, Kallakury B, Sheehan C, Raeder RH, Metzger DW. Xenogeneic extracellular matrix grafts elicit a TH2-restricted immune response. Transplantation 2001;71:1631-1640. 18 John Wiley & Sons, Inc.

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18 19 20

21 22 23 24

25 26

27 28

29 30 31 32

33

Zeugolis DI, Paul RG, Attenburrow G. Factors influencing the properties of reconstituted collagen fibers prior to self-assembly: animal species and collagen extraction method. J Biomed Mater Res A 2008;86:892-904. Kern S, Eichler H, Stoeve J, Kluter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294-1301. Mauritz C, Schwanke K, Reppel M, Neef S, Katsirntaki K, Maier LS, Nguemo F, Menke S, Haustein M, Hescheler J, Hasenfuss G, Martin U. Generation of functional murine cardiac myocytes from induced pluripotent stem cells. Circulation 2008;118:507-517. Akhyari P, Kamiya H, Haverich A, Karck M, Lichtenberg A. Myocardial tissue engineering: the extracellular matrix. Eur J Cardiothorac Surg 2008;34:229-241. Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials 2007;28:3587-3593. Gilbert T, Sellaro T, Badylak S. Decellularization of tissues and organs. Biomaterials 2006;27:3675-3683. Kelly DJ, Rosen AB, Schuldt AJ, Kochupura PV, Doronin S, Potapova I, Azeloglu EU, Badylak S, Brink PR, Cohen IS, Gaudette GR. Increased myocyte content and mechanical function within a tissue-engineered myocardial patch following implantation. Tissue Eng Part A 2009;15:2189-2201. Kochupura PV, Azeloglu EU, Kelly DJ, Doronin SV, Badylak SF, Krukenkamp IB, Cohen IS, Gaudette GR. Tissue-engineered myocardial patch derived from extracellular matrix provides regional mechanical function. Circulation 2005;112:I144-I149. Potapova IA, Doronin SV, Kelly DJ, Rosen AB, Schuldt AJ, Lu Z, Kochupura PV, Robinson RB, Rosen MR, Brink PR, Gaudette GR, Cohen IS. Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage. Am J Physiol Heart Circ Physiol 2008;295:H2257-H2263. Hata H, Bar A, Dorfman S, Vukadinovic Z, Sawa Y, Haverich A, Hilfiker A. Engineering a novel three-dimensional contractile myocardial patch with cell sheets and decellularised matrix. Eur J Cardiothorac Surg 2010;38:450-455. Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, Dodson A, Martorell J, Bellini S, Parnigotto PP, Dickinson SC, Hollander AP, Mantero S, Conconi MT, Birchall MA. Clinical transplantation of a tissue-engineered airway. Lancet 2008;372:2023-2030. Ozeki M, Narita Y, Kagami H, Ohmiya N, Itoh A, Hirooka Y, Niwa Y, Ueda M, Goto H. Evaluation of decellularized esophagus as a scaffold for cultured esophageal epithelial cells. J Biomed Mater Res A 2006;79:771-778. Sellaro TL, Ravindra AK, Stolz DB, Badylak SF. Maintenance of hepatic sinusoidal endothelial cell phenotype in vitro using organ-specific extracellular matrix scaffolds. Tissue Eng 2007;13:2301-2310. Eitan Y, Sarig U, Dahan N, Machluf M. Acellular cardiac extracellular matrix as a scaffold for tissue engineering: In-vitro cell support, remodeling and biocompatibility. Tissue Eng Part C Methods 2009;16:671-683. Singelyn JM, DeQuach JA, Seif-Naraghi SB, Littlefield RB, Schup-Magoffin PJ, Christman KL. Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering. Biomaterials 2009;30:5409-5416. Wainwright JM, Czajka CA, Patel UB, Freytes DO, Tobita K, Gilbert TW, Badylak SF. Preparation of Cardiac Extracellular Matrix from an Intact Porcine Heart. Tissue Eng Part C Methods 2009;16:525-532.



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34 35 36

37 38 39 40 41 42 43 44 45 46

Wang B, Borazjani A, Tahai M, Curry AL, Simionescu DT, Guan J, To F, Elder SH, Liao J. Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrow mononuclear cells. J Biomed Mater Res A 2010;94:1100-1110. Weymann A, Loganathan S, Takahashi H, Schies C, Claus B, Hirschberg K, Soós P, Korkmaz S, Schmack B, Karck M, Szabó G. Development and Evaluation of a Perfusion Decellularization Porcine Heart Model. Circ J 2011;75:852-860. Sarig U, Au-Yeung GC, Wang Y, Bronshtein T, Dahan N, Boey FY, Venkatraman SS, Machluf M. Thick acellular heart extracellular matrix with inherent vasculature: a potential platform for myocardial tissue regeneration. Tissue Eng Part A 2012;18:2125-2137. Gersh BJ, Simari RD, Behfar A, Terzic CM, Terzic A. Cardiac cell repair therapy: a clinical perspective. Mayo Clin Proc 2009;84:876-892. Hansson EM, Lindsay ME, Chien KR. Regeneration next: toward heart stem cell therapeutics. Cell Stem Cell 2009;5:364-377. Herrmann JL, Abarbanell AM, Weil BR, Wang Y, Wang M, Tan J, Meldrum DR. Cellbased therapy for ischemic heart disease: a clinical update. Ann Thorac Surg 2009;88:1714-1722. Joggerst SJ, Hatzopoulos AK. Stem cell therapy for cardiac repair: benefits and barriers. Expert Rev Mol Med 2009;11:e20. Stamm C, Nasseri B, Choi YH, Hetzer R. Cell therapy for heart disease: great expectations, as yet unmet. Heart Lung Circ 2009;18:245-256. McCormick RJ, Paul Thomas D. Collagen crosslinking in the heart: relationship to development and function. Basic Appl Myol 1998;8:143-150. Jung T, Bader N, Grune T. Lipofuscin: formation, distribution, and metabolic consequences. Ann N Y Acad Sci 2007;1119:97-111. Sicari BM, Johnson SA, Siu BF, Crapo PM, Daly KA, Jiang H, Medberry CJ, Tottey S, Turner NJ, Badylak SF. The effect of source animal age upon the in vivo remodeling characteristics of an extracellular matrix scaffold. Biomaterials 2012;33:5524-5533. Tottey S, Johnson SA, Crapo PM, Reing JE, Zhang L, Jiang H, Medberry CJ, Reines B, Badylak SF. The effect of source animal age upon extracellular matrix scaffold properties. Biomaterials 2011;32:128-136. Laschke MW, Harder Y, Amon M, Martin I, Farhadi J, Ring A, Torio-Padron N, Schramm R, Rücker M, Junker D, Häufel M, Carvalho C, Heberer M, Germann G, Vollmar B, Menger MD. Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes. Tissue Eng 2006;12:2093-2104.


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Figure legends

Figure 1: A-C:

Human

heart

tissue

decellularized

using

different

methods.

Decellularization protocols were hypo/hypertonic treatment for 3 days, 5% Triton X-100 for 48 h, 4% sodium deoxycholate for 40 h, 0.5% SDS for 9 h (without and with ensuing 3 h FBS incubation) and 3-step protocol (lysis buffer, 0.5% SDS for 6 h, FBS). A: Gross morphology. Scale bars = 100 µm. B: H&E staining. Cells (bright red) and nuclei (purple) are only removed in the protocols involving SDS. Scale bars = 50 µm. C: Masson’s trichrome staining. Note that, with the 3-step protocol, the ECM is denser than with the other SDS-based protocols. Scale bars = 50 µm.

Figure 2: A: Results of the DNA extraction. Statistically significant differences are shown for selected protocols (for detailed p-values see Table S1). B: Masson’s trichrome staining of tissue decellularized with 3-step protocol. Arrows point at lipofuscin granules. Scale bar = 20 µm. C-E: Quantification and statistical evaluation of ECM components. Only promising decellularization protocols were analyzed, based on histology and DNA extraction results. For detailed p-values see Table S2-S4. C: Collagen

quantification.

D: Glycosaminoglycan

quantification.

E: Elastin

quantification. The error bars represent SEM.

Figure 3: A: Indirect immunohistochemistry of ECM (decellularized with 3-step protocol) and native tissue. Scale bars = 50 µm. Control: ECM stained without primary antibody. Primary antibodies were directed against desmin (cellular), laminin and fibronectin (ECM proteins). B: Scanning electron microscopy (SEM) of native and decellularized human 21 John Wiley & Sons, Inc.


Page 22 of 53


heart tissue, 1000x and 4000x magnification. Scale bars = 20 µm (1000x), 6 µm (4000x). C: Magnified SEM images of individual myocardial fibers showing that the ECM orientation appears largely preserved after decellularization. Scale bars = 10 µm.

Figure 4: A: Comparison of young porcine (6 months of age), human infarcted and noninfarcted left ventricular heart tissue from an aged human donor before (= native) and after decellularization (= ECM) by H&E and Picrosirius Red staining. Scale bars = 20 µm. Arrows indicate lipofuscin granules. B: Brightfield and fluorescence microscopy of decellularized ECM from aged human donor (stained with H&E), showing bright autofluorescence of lipofuscin granules. Scale bars = 10 µm.

Figure 5: A: Masson’s trichrome staining of matrix recellularized with CB-MSC before (d 0) and 2-10 days after recellularization. Scale bars = 50 µm. B: MTS assay data on day 2 after ECM recellularization with CB-MSC, compared to the same number of cells pipetted into an empty well. As MSC may need more than 24 h to attach, the MTS assays were done on the second day after recellularization. The difference in viability corresponds to the number of cells which settled on the bottom of the plate instead of on the scaffold and was disregarded in the “matrix” samples. C: Masson’s trichrome staining of matrix recellularized with 0.5 M iPS-CM/cm², on day 20 after recellularization. D: Masson’s trichrome staining of murine neonatal cardiomyocytes (NMC) 2 days after seeding on matrix coated with fibronectin (+fib) or without coating (-fib). Scale bars = 20 µm. E: α-actinin immunohistochemistry of iPS-CM on day 20 and controls. Nuclei were counterstained with hematoxylin (purple). Native tissue is positive for α-actinin, but ECM and ECM recellularized with MSC are negative. Scale bar = 20 µm. 22 John Wiley & Sons, Inc.

Page 23 of 53


Arrows point at cells which are enlarged in inserts. F: MTS assay data on day 2 after ECM recellularization with CB-MSC or IPS-CM. Total wells containing matrix were compared to the same number of cells pipetted into an empty well. The p value of a group comparison by Mann-Whitney U test is shown.


Page 49 of 53


Figure_1 63x40mm (300 x 300 DPI)

John Wiley & Sons, Inc.


Figure_2 66x43mm (300 x 300 DPI)


Page 50 of 53

Page 51 of 53


Figure_3 210x435mm (300 x 300 DPI)



Figure_4 128x161mm (300 x 300 DPI)


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Figure_5 159x248mm (300 x 300 DPI)


Supporting information Supplementary tables: Statistical evaluation of cellular and extracellular component quantification compared among different decellularization protocols. The p values were calculated using ANOVA analyses with Games-Howell post-hoc tests. Data are presented for DNA extraction (Table S1), collagen quantification (Table S2), glycosaminoglycan quantification (Table S3) and elastin quantification (Table S4). Table S1: DNA extraction Sample comparison native (N = 9)

3-step (N = 6)

deoxycholate (N = 6)

hypo/hypertonic (N = 6)

p

3-step

.0001

deoxycholate

.0004

hypo/hypertonic

.5248

Triton X

Sample comparison Triton X (N = 6)

p

native

.8900

3-step

.0041

deoxycholate

.0094

.8900

hypo/hypertonic

.9864

SDS 9h+FBS

.0001

SDS 9h+FBS

.0044

SDS8h

.0002

SDS8h

.0060

SDS9h

.0002

SDS9h

.0054

native

.0001

native

.0001

deoxycholate

.0101

3-step

.4595

hypo/hypertonic

.0191

deoxycholate

.0139

Triton X

.0041

hypo/hypertonic

.0205

SDS 9h+FBS

.4595

Triton X

.0044

SDS8h

.0081

SDS8h

.0192

SDS9h

.0128

SDS9h

.0433

native

.0004

native

.0002

3-step

.0101

3-step

.0081

hypo/hypertonic

.0563

deoxycholate

.1055

Triton X

.0094

hypo/hypertonic

.0298

SDS 9h+FBS

.0139

Triton X

.0060

SDS8h

.1055

SDS 9h+FBS

.0192

SDS9h

.0490

SDS9h

.8733

native

.5248

native

.0002

3-step

.0191

3-step

.0128

deoxycholate

.0563

deoxycholate

.0490

Triton X

.9864

hypo/hypertonic

.0265

SDS 9h+FBS

.0205

Triton X

.0054

SDS 9h+FBS (N = 8)

SDS8h (N = 6)

SDS9h (N = 6)

SDS8h

.0298

SDS 9h+FBS

.0433

SDS9h

.0265

SDS8h

.8733

Table S2: Collagen quantification Sample comparison native (N = 7) 3-step (N = 6) SDS 9h+FBS (N = 6)

p

3-step

.9894

SDS 9h+FBS

.0078

native

.9894

SDS 9h+FBS

.0092

native

.0078

3-step

.0092

Table S3: Glycosaminoglycan quantification Sample comparison native (N = 6) 3-step (N = 6) SDS 9h+FBS (N = 6)

p

3-step

.0000005

SDS 9h+FBS

.00000002

native

.0000005

SDS 9h+FBS

.0498

native

.00000002

3-step

.0498

Table S4: Elastin quantification Sample comparison native (N = 6) 3-step (N = 6) SDS 9h+FBS (N = 6)

p

3-step

.00002

SDS 9h+FBS

.00002

native

.00002

SDS 9h+FBS

.0002

native

.00002

3-step

.0002

Supplementary figures:

Figure S1: Images of CB-MSCs in passage 5 after adipogenic, chondrogenic and osteogenic differentiation with StemPro differentiation and control media, 320x magnification. Scale bars = 50 µm. Adipogenic samples were stained with Oil Red O (red lipid droplets), osteogenic samples were stained with silver nitrate and chondrogenic samples were stained with alcian blue. Although only a small proportion of cells underwent adipogenic differentiation, CB-MSCs in passage 5 were

able to differentiate into all three lineages in principle, which is characteristic for MSCs.

Figure S2: Flow cytometry analysis of CB-MSC markers, passage 5. Cells were stained with (anti-) CD 90-APC, CD 73-PE, CD 105-FITC, CD 34-PE, CD 14-FITC, CD 45-FITC and HLA-DR-FITC. The experiment was run on a BD FACSCalibur system and evaluated with FlowJo software. Cells in gate R1 of the FSC/SSC dot blot were analysed, with the exclusion of dead cells positive for propidium iodide (PI, gate R3). When markers appear in several dot blots (e.g. CD 34), the mean % value is presented in the list. The CB-MSCs showed the typical MSC marker expression: They were positive CD 90, CD 73 and CD 105 and negative for the markers CD 34, CD 14, CD 45 and HLA-DR.

Figure S3: These graphs are presented as a validation for the MTS assay in Figure 5C. They depict the relationship between cell number and viability (OD 490 nm) of CB-MSC and IPS-CM, which we observed to see if the assay was in a linear range at the cell number, which we used in our recellularization experiments. The MTS assay was carried out directly after cell seeding and each point shows the mean of 3 wells with SEM. Blank values were not subtracted, but are shown as a cell density of “0”. In a broad range including a cell number of 170 000 cells/well (= 0.5 M/cm²), which was used for recellularization, the relationship between cell number and viability is approximately linear.

Movie S1: Human

cardiac

matrix

recellularized

with

0.5 M

differentiated

iPS-CMs/cm² in a 96 well plate, 5 days after recellularization. IPS-CMs contract spontaneously on the matrix. Magnification: 100x. Format: .avi. Codec: MPEG-4.

Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix.

Crosslinking strategies for preparation of extracellular matrix-derived cardiovascular scaffolds.

Decellularization of fibroblast cell sheets for natural extracellular matrix scaffold preparation.

Influence of pH on extracellular matrix preservation during lung decellularization.

A fast and mild decellularization protocol for obtaining extracellular matrix.

Preparation and characterization of bone marrow mesenchymal stem cell-derived extracellular matrix scaffolds.

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development.

Methods of tissue decellularization used for preparation of biologic scaffolds and in vivo relevance.

Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix liver scaffolds.

Cardiac extracellular matrix-fibrin hybrid scaffolds with tunable properties for cardiovascular tissue engineering.

Decellularization of intact tissue enables MALDI imaging mass spectrometry analysis of the extracellular matrix.

Molecular imaging of the cardiac extracellular matrix.

Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding.

Decellularization of porcine skeletal muscle extracellular matrix for the formulation of a matrix hydrogel: a preliminary study.

Fibroblast extracellular matrix and adhesion on microtextured polydimethylsiloxane scaffolds.

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy.

Mechanisms of extracellular matrix proteoglycan degradation by human neutrophils.

Preparation, Characterization, and Clinical Implications of Human Decellularized Adipose Tissue Extracellular Matrix (hDAM): A Comprehensive Review.

Degradation of extracellular matrix proteoglycans by human neutrophils.

Three-dimensional extracellular matrix scaffolds by microfluidic fabrication for long-term spontaneously contracted cardiomyocyte culture.

Human cardiac fibroblast extracellular matrix remodeling: dual effects of tissue inhibitor of metalloproteinase-2.

The impact of aging on cardiac extracellular matrix.

Extracellular matrix for reconstruction of cardiac structures after tumour resections.

Natural cardiac extracellular matrix hydrogels for cultivation of human stem cell-derived cardiomyocytes.