Detergent-based Protocol Research Articles

Decellularized tissue exhibits large differences of extracellular matrix properties dependent on decellularization method: novel insights from a standardized characterization on skeletal muscle

Decellularized matrices are an attractive choice of scaffold in regenerative medicine as they can provide the necessary extracellular matrix (ECM) components, signals and mechanical properties. Various detergent-based protocols have already been proposed for decellularization of skeletal muscle tissue. However, a proper comparison is difficult due to differences in species, muscle origin and sample sizes. Moreover, a thorough evaluation of the remaining acellular matrix is often lacking. We compared an in-house developed decellularization protocol to four previously published methods in a standardized manner. Porcine skeletal muscle samples with uniform thickness were subjected to in-depth histological, ultrastructural, biochemical and biomechanical analysis. In addition, 2D and three-dimensional cytocompatibility experiments were performed. We found that the decellularization methods had a differential effect on the properties of the resulting acellular matrices. Sodium deoxycholate combined with deoxyribonuclease I was not an effective method for decellularizing thick skeletal muscle tissue. Triton X-100 in combination with trypsin, on the other hand, removed nuclear material but not cytoplasmic proteins at low concentrations. Moreover, it led to significant alterations in the biomechanical properties. Finally, sodium dodecyl sulphate (SDS) seemed most promising, resulting in a drastic decrease in DNA content without major effects on the ECM composition and biomechanical properties. Moreover, cell attachment and metabolic activity were also found to be the highest on samples decellularized with SDS. Through a newly proposed standardized analysis, we provide a comprehensive understanding of the impact of different decellularizing agents on the structure and composition of skeletal muscle. Evaluation of nuclear content as well as ECM composition, biomechanical properties and cell growth are important parameters to assess. SDS comes forward as a detergent with the best balance between all measured parameters and holds the most promise for decellularization of skeletal muscle tissue.

Identification of transient receptor potential melastatin 3 proteotypic peptides employing an efficient membrane protein extraction method for natural killer cells.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer’s recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150–200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.

Smooth muscle matrix bioink promotes myogenic differentiation of encapsulated adipose-derived stem cells.

Hydrogels derived from decellularized extracellular matrices (dECM) can mimic the biochemical composition of the native tissue. They can also act as a template to culture reseeded cells in vitro. However, detergent-based decellularization methods are known to alter the biochemical compositions, thereby compromising the bioactive potential of dECM. This study proposes a facile detergent-free method to achieve dECM from smooth muscle tissue. We have used the muscle layer of caprine esophageal tissue and decellularized using hypo and hyper-molar sodium chloride solutions alternatingly. Then, a hydrogel was prepared from this decellularized smooth muscle matrix (dSMM) and characterized thoroughly. A comparative analysis of the dSMM prepared with our protocol with the existing detergent-based protocol suggests successful and comparable decellularization with minimal residual DNA content. Interestingly, an 8.78-fold increase in sulfated glycosaminoglycans content and 1.62-fold increased collagen content indicated higher retention of ECM constituents with NaCl-based decellularization strategy. Moreover, the dSMM gel induces differentiation of the encapsulated adipose-derived mesenchymal stem cells toward smooth muscle cells (SMCs) as observed by their expression of alpha-smooth muscle actin and smooth muscle myosin heavy chain, the hallmarks of SMCs. Finally, we optimized the process parameter for productive bioprinting with this dSMM bioink and fabricated 3D muscle constructs. Our results suggest that dSMM has the potential to be used as a bioink to engineer personalized esophageal tissues.

Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells

Membrane proteins constitute around 20–30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of “omics” techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers.

Decellularization and Recellularization Methods for Avian Lungs: An Alternative Approach for Use in Pulmonary Therapeutics.

The shortage of compatible allogeneic organs and an increase in the number of patients requiring long-term lung assist devices while waiting for lung transplantation have motivated scientists to explore alternatives to bioengineer new lungs, including through decellularization and recellularization processes. A novel approach for bioengineering an extracorporeal membrane oxygenator is based on the parenchymal structure of avian lungs which utilizes a cross-current unidirectional flow of air and blood rather than bidirectional airflow, and thus eliminates dead-space ventilation. This provides more efficient gas exchange than mammalian lungs. The novel approach utilized is to decellularize avian lungs and then to recellularize with patient-derived human lung epithelial and vascular endothelial cells with the goal of creating a fully functional structure that can be used as a gas-exchange device. Here, we present avian lung decellularization and recellularization methods for chicken and emu lungs, in order to study both small- and large-scale avian lung models. For decellularization, a detergent-based protocol is utilized, and different techniques are used to validate the de- and recellularization of those lungs, including microscopy, mass spectrometry, and immunohistochemical analyses. For recellularization, techniques for seeding different human lung cell types into the decellularized scaffolds are presented.

Comparative study of decellularization techniques to obtain natural extracellular matrix scaffolds of human peripheral-nerve allografts.

We hypothesize that adding sonication cycles to the process of decellularization of cadaveric human peripheral nerves will increase the removal of cell debris and myelin sheath, increasing their utility as allografts. Our aim of this study was to develop a decellularization protocol that allows the removal of cells and myelin sheath without detrimental effects on nerve architecture. Segments of ulnar and median nerves from human donors, isolated both before and after cardiac arrest, were subjected to two methods of decellularization: two-detergent-based (M1) and the same method with sonication added (M2). We evaluated the histology of unprocessed and decellularized nerves (n = 24 per group) for general morphology, presence of cell nuclei, nuclear remnants, collagen fibers, and myelin. We performed immunohistochemistry to verify the removal of Schwann cells associated with histomorphometry. We used scanning electron microscopy (EM) to evaluate the ultrastructure of both native and decellularized nerves. The efficacy of decellularization was assessed by analysis of genomic DNA. Histology confirmed that both decellularization protocols were adequate and maintained natural nerve architecture. Scanning EM showed that 3D ultrastructural architecture also was maintained. Histomorphometric parameters showed a more complete removal of the myelin with the M2 protocol than with M1 (p = 0.009). Fiber diameter and density were not modified by decellularization methods. Sonication can be a complementary method to decellularization of peripheral nerve allografts with sonication increasing the effectiveness of detergent-based protocols for the removal of unwanted cellular components from peripheral nerve allografts.

Photocrosslinkable liver extracellular matrix hydrogels for the generation of 3D liver microenvironment models

Liver extracellular matrix (ECM)-based hydrogels have gained considerable interest as biomimetic 3D cell culture environments to investigate the mechanisms of liver pathology, metabolism, and toxicity. The preparation of current liver ECM hydrogels, however, is based on time-consuming thermal gelation and limits the control of mechanical properties. In this study, we used detergent-based protocols to produce decellularized porcine liver ECM, which in turn were solubilized and functionalized with methacrylic anhydride to generate photocrosslinkable methacrylated liver ECM (LivMA) hydrogels. Firstly, we explored the efficacy of two protocols to decellularize porcine liver tissue using varying combinations of commonly used chemical agents such as Triton X-100, Sodium Dodecyl Sulphate (SDS) and Ammonium hydroxide. Then, we demonstrated successful formation of stable, reproducible LivMA hydrogels from both the protocols by photocrosslinking. The LivMA hydrogels obtained from the two decellularization protocols showed distinct mechanical properties. The compressive modulus of the hydrogels was directly dependent on the hydrogel concentration, thereby demonstrating the tuneability of mechanical properties of these hydrogels. Immortalized Human Hepatocytes cells were encapsulated in the LivMA hydrogels and cytocompatibility of the hydrogels was demonstrated after one week of culture. In summary, the LivMA hydrogel system provides a simple, photocrosslinkable platform, which can potentially be used to simulate healthy versus damaged liver for liver disease research, drug studies and cancer metastasis modelling.

Preliminary assessment of an injectable extracellular matrix from decellularized bovine myocardial tissue.

The goal of this study was to develop an injectable form of decellularized bovine myocardial tissue matrix which could retain high levels of functional ECM molecules, and could gel at physiological temperature. Dissected ventricular tissue was processed by a detergent-based protocol, lyophilized, enzymatically-digested, and neutralized to form the injectable myocardial matrix (IMM). Histochemical analysis, DNA quantification, and agarose gel electrophoresis demonstrated the efficiency of the applied protocol. Chemical, thermal, morphological, and rheological characterization; protein and sulfated glycosaminoglycan (sGAG) content analysis were performed, in vitro biological properties were evaluated. An in vivo histocompatibility and biodegradability study was performed. Histochemistry revealed complete removal of myocardial cells. DNA content analysis revealed a significant decrease (87%) in the nuclear material, while protein and sGAG contents were highly preserved following decellularization. Soluble IMM was capable of turning into gel form at ∼37°C, indicating selfassembling property. In vitro findings showed the biomaterial was noncytotoxic, nonhemolytic, and supported the attachment and proliferation of mesenchymal stem cells. In vivo study demonstrated IMM was well-tolerated by rats receiving subcutaneous injection. This work demonstrates that the IMM from decellularized bovine myocardial tissue has the potential for use as a feasible regenerative biomaterial in prospective tissue engineering and regenerative medicine studies.

Decellularization of porcine carotid arteries using low-concentration sodium dodecyl sulfate.

The decellularized scaffold is a promising material for producing tissue-engineered vascular grafts (TEVGs) because of its complex, native-like three-dimensional structure and mechanical properties. Sodium dodecyl sulfate (SDS), one of the most commonly used decellularization reagents, appears to be more effective than other detergents for removing cells from dense tissues. The concentrations of SDS used in previous studies and their effects on decellularization are not consistent. In this study, porcine carotid arteries were decellularized using detergent-based protocols using Triton X-100 followed by SDS at different concentrations and exposing time. Cell removal efficiency and composition were evaluated by histological analysis, and DNA and collagen quantification. Ultrastructure, mechanical properties, pore size distribution, and in vivo biocompatibility of decellularized arteries were also evaluated. The DNA content of decellularized scaffolds treated with 0.3% SDS for 72 h or 0.5% SDS for 48 h was significantly less than that treated with 1% SDS for 30 h. There was a significant loss of soluble collagen after treatment with 1% SDS relative to native arteries. The extensive loss of elastin and glycosaminoglycans was observed in decellularized arteries treated with 0.5% SDS or 1% SDS. The basement membrane and biomechanics were also damaged by these two protocols. Moreover, decellularized scaffolds became more porous with many large pores after treatment with 0.3% SDS. Low-concentration SDS could be a suitable choice for artery decellularization. Decellularized porcine carotid arteries, prepared using Triton X-100 followed by 0.3% SDS, may be a promising biological scaffold for TEVGs.

Lipid Scrambling Induced by Membrane-Active Substances

The functional roles of the lipid asymmetry of biomembranes are attracting increasing attention. This study characterizes the activity of surfactants to induce transmembrane flip-flop of lipids and thus “scramble” this asymmetry. Detergent-induced lipid scrambling of liposomes mimicking the charge asymmetry of bacterial membranes with 20 mol % of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol in the outer leaflet only was quantified by ζ-potential measurements for octaethylene glycol dodecyl ether (C12EO8), octyl glucoside (OG), and dodecyl maltoside. Membrane leakage was separately measured by the fluorescence lifetime-based calcein leakage assay and the onset of the membrane-to-micelle transition by isothermal titration calorimetry. Partition coefficients and partial molar areas were obtained as well. For the quickly membrane-permeant C12EO8 and OG, leakage proceeds at a rather sharp threshold content in the membrane, which is well below the onset of solubilization and little dependent on incubation time; it is accompanied by fast lipid scrambling. However, unlike leakage, flip-flop is a relaxation process that speeds up gradually from taking weeks in the detergent-free membrane to minutes or less in the leaking membrane. Hence, after 24 h of incubation, 10 mol % of C12EO8 or 50 mol % of OG in the membrane suffice for virtually complete lipid scrambling, whereas leakage remains below 10% for up to 14 mol % of C12EO8 and 88 mol % of OG. There is thus a concentration window in which lipid scrambling proceeds without leakage. This implies that lipid scrambling must be considered a possible mode of action of antimicrobial peptides and other membrane-active drugs or biomolecules. A related, detergent-based protocol for scrambling the lipid asymmetry of liposomes and maybe cells without compromising their overall integrity would be a very valuable tool to study functions of lipid asymmetry.

Avian lungs: A novel scaffold for lung bioengineering.

Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics. To initially assess this in both small and large avian lung models, chicken (Gallus gallus domesticus) and emu (Dromaius novaehollandiae) lungs were decellularized using modifications of a detergent-based protocol, previously utilized with mammalian lungs. Light and electron microscopy, vascular and airway resistance, quantitation and gel analyses of residual DNA, and immunohistochemical and mass spectrometric analyses of remaining extracellular matrix (ECM) proteins demonstrated maintenance of lung structure, minimal residual DNA, and retention of major ECM proteins in the decellularized scaffolds. Seeding with human bronchial epithelial cells, human pulmonary vascular endothelial cells, human mesenchymal stromal cells, and human lung fibroblasts demonstrated initial cell attachment on decellularized avian lungs and growth over a 7-day period. These initial studies demonstrate that decellularized avian lungs may be a feasible approach for generating functional lung tissue for clinical therapeutics.

Decellularized amnion scaffold with activated PRP: a new paradigm dressing material for burn wound healing.

Direct application of amnion has greater risk of immunological rejection and infection. Decellularization is an effective method to lower the risk of immune complications and infections. The bioreactor assembly with multiple cassettes was designed for decellurization of multiple amnions with different cell types simultaneously in single run. A detergent-based protocol was modified to remove all cellular components from amnion and diminish the DNA content to render it non-immunogenic. Amnion (n=10) were treated with 2% sodium dodecyl sulphate (SDS), 5% dimethyl sulfoxide (DMSO) and 2% sodium deoxycholeate (SD). Decellularized amnion samples were analyzed by haematoxylin-eosin staining (HE), Alcian blue pH 1 (AB-pH-1), 4,6-diamnionidino-2-phenylindol (DAPI), Massion's trichrome stain, DNA quantification, mechanical testing and scanning electron microscopy (SEM). Histological analysis showed complete removal of cellular components and the histoarchitecture of scaffold remained intact. Amnion scaffold activated with platelet rich plasma (PRP) and calcium chloride composition supported better adherence to the wound than amnion alone. Only single application showed good healing. In vivo assessment of activated amnion revealed stable dressing. It has good promising outcome. At day 7, histologically the wounds treated with activated amnion were almost closed without scarring and showed well differentiated epidermis, proliferation of keratinocytes, hair follicles and basement membrane as compared to controls and silver nitrate gel dressings in a mouse (Mus musculus). Cryopreservation had no adverse effect on the mechanical properties of the amnion scaffold. Cryopreservation of decellularized amnion by Dulbecco's modified eagle medium (DMEM) was expected to prepare off-the-shelf skin substitutes and preserve them to be immediately available upon request of patients' needs.

Detergent-Based Decellularization of Bovine Carotid Arteries for Vascular Tissue Engineering

Vascular diseases are an increasing health issue, and common alloplastic, allogenic or autologous vascular grafts show frequent complications. The aim of this study is to develop an acellular, xenogenic bypass-graft from a bovine carotid artery (BAC) using detergent-based protocols. We compared decellularization with sodium desoxycholate (DOA), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), sodium dodecyl sulfate (SDS), and Triton X100 and improved suitable methods by variation of concentration, buffer system, incubation time, temperature, rinsing, and flow rate. All processes were evaluated systematically based on cellular residues, biocompatibility, structural and mechanical integrity. Decellularization with SDS and Triton X100 was not sufficient for the removal of cellular components. We optimized protocols using 1% DOA and Chaps by a buffered system at 37°C with extended decellularization and rinsing. Decellularization with DOA depleted DNA to 0.5±0.1% and soluble proteins to 0.6±0.2%. Using Chaps, DNA was reduced to 0.2±0.2% and proteins to 0.6±0.3%. The improved protocols eliminated RNA completely from the matrix, and no cytotoxic effects were detected. Mechanical and structural integrity of decellularized tissues was comparable to non-decellularized controls. Our method effectively removed cellular components from the extracellular matrix while preserving the structural and mechanical integrity of the tissue. Decellularized BACs could be a promising alternative for vascular replacement therapy.

Application of a novel bioreactor for in vivo engineering of pancreas tissue.

Type 1 diabetes is characterized by autoimmune destruction of pancreatic cells. Organ transplantation is an acceptable treatment for native organ failure. However, it is associated with several problems due to a number of reasons, such as the lack of appropriate donors and immunosuppression. In our present study, a novel model is presented for in vivo recellularization of acellular pancreas by implanting between the host pancreas and the adjacent omental flap. In this study, the pancreases were harvested and cannulated via the common bile duct and then, the scaffolds were acellularized by a detergent-based protocol. After that, the abdomens of 35 rats were opened and the spleen was extracted with the adjacent omentum, and placed outside the abdomen. The acellularized scaffold was stretched over the host pancreas and the omentum was wrapped around it to make a sandwich-like structure, which was then fixed with Chromic Sutures 6-0 and marked with Prolene 4-0 on four sides. All samples were biopsied at 14, 30, 60, 90, and 120 days post-transplantation. The result showed marked recellularization of acellularized pancreas with visible neovascularization and neoβ-cells with minimal inflammatory response. This study provides a new approach to produces a normal-like pancreas by allograft transplantation for pancreas tissue engineering. We observed that in vivo transplantation of acellularized pancreas can promote recellularization, proliferation, and differentiation by blood circulation. These findings support that in vivo studies can contribute to finding faster solutions for the treatment of diabetes.

Effects of Detergent-Based Protocols on Decellularization of Corneas With Sclerocorneal Limbus. Evaluation of Regional Differences.

In this work, we decellularized whole porcine corneas including the sclerocorneal limbus (SCL) and we evaluated regional differences in order to identify an efficient method to decellularize whole corneas for future clinical use. We analyzed the efficiency of four decellularization protocols based on benzalkonium chloride (BAK), Igepal, sodium dodecyl sulfate (SDS), and Triton X-100 detergents on whole porcine corneas. Results showed that the decellularization efficiency of most protocols was low, with the specific protocol resulting in more efficient levels of decellularization being 0.1% SDS for 48 hours, especially in the medium and posterior cornea regions. A significant correlation was found between the decellularization efficiency and the concentration of agent used (P = 0.0174; r = 0.1540), but not for the incubation time (P > 0.05). The analysis of cornea components preservation demonstrated that all protocols were able to preserve the integrity of the Bowman's layer and Descemet's membrane. Although the collagen structure was partially altered, the global decellularization groups showing highest preservation of the ECM collagen contents and orientation were Igepal and SDS, with differences among the three regions of the cornea. All global groups showed high levels of proteoglycan and glycoprotein preservation after decellularization, with the best results were found in the SDS group followed by the Igepal group. These results suggest that very powerful protocols are necessary for whole-cornea decellularization. For the generation of lamelar corneas for clinical use, decellularization regional differences should be taken into account. Decellularized whole corneas may be potential therapeutic agents for lamelar keratoplasty.

TISSUE ENGINEERING OF THE ATRIOVENTRICULAR VALVE: DECELLULARIZED MATRIX IN OVINE MITRAL ALLOGRAFT MODEL

Aim. Development of the tissue engineered matrix for the atrioventricular valve graft in a large animal model. Materials and мethods. The study was performed on ovine mitral valves (n = 100). The decellularization of the whole mitral valve grafts was performed using 21 different detergent-based protocols. The effectiveness of the decellularization was investigated by immunofluorescent staining for DNA (as a marker of intracellular antigens) and α-GAL (as a marker of membrane-associated antigens), as well as DNA quantification. The mechanical properties and the repopulation potential were analyzed. The structure and repopulation rate analysis included hematoxylin-eosin, Movat’s pentachrome and Van Gieson histological staining, and immunofluorescent staining for collagen I and IV, as well as endothelial cell markers. Results. The protocols that included the gradual rise of the detergent solution were shown to be the most effective for the decellularization of the whole mitral valve graft. At the same time, the detergents alone failed to remove α-GAL. The reducing agent enhanced the decellularization, which resulted in complete removal of DNA and α-GAL, according to immunofluorescent staining. DNA quantification showed a 96.4% reduction in DNA content. The mechanical properties were shown to be comparable to the native tissue. The reseeding of the decellularized tissue showed a potential for the repopulation. Conclusions. A new matrix for the atrioventricular valve substitute could be developed using the decellularization as the initial step of the valve tissue engineering. The detergent combined with a reducing agent showed significantly better results compared to the detergents alone for the mitral valve decellulrization. The matrix demonstrated mechanical properties, comparable with the native one, and the potential for the repopulation.

Evaluation of decellularization protocols for production of tubular small intestine submucosa scaffolds for use in oesophageal tissue engineering

Small intestine submucosa (SIS) has emerged as one of a number of naturally derived extracellular matrix (ECM) biomaterials currently in clinical use. In addition to clinical applications, ECM materials form the basis for a variety of approaches within tissue engineering research. In our preliminary work it was found that SIS can be consistently and reliably made into tubular scaffolds which confer certain potential advantages. Given that decellularization protocols for SIS are applied to sheet-form SIS, it was hypothesized that a tubular-form SIS would behave differently to pre-existing protocols. In this work, tubular SIS was produced and decellularized by the conventional peracetic acid–agitation method, peracetic acid under perfusion along with two commonly used detergent–perfusion protocols. The aim of this was to produce a tubular SIS that was both adequately decellularized and possessing the mechanical properties which would make it a suitable scaffold for oesophageal tissue engineering, which was one of the goals of this work. Analysis was carried out via mechanical tensile testing, DNA quantification, scanning electron and light microscopy, and a metabolic assay, which was used to give an indication of the biocompatibility of each decellularization method. Both peracetic acid protocols were shown to be unsuitable methods with the agitation-protocol-produced SIS, which was poorly decellularized, and the perfusion protocol resulted in poor mechanical properties. Both detergent-based protocols produced well-decellularized SIS, with no adverse mechanical effects; however, one protocol emerged, SDS/Triton X-100, which proved superior in both respects. However, this SIS showed reduced metabolic activity, and this cytotoxic effect was attributed to residual reagents. Consequently, the use of SIS produced using the detergent SD as the decellularization agent was deemed to be the most suitable, although the elimination of the DNase enzyme would give further improvement.

In vitro generation of atrioventricular heart valve neoscaffolds.

Tissue engineering of cardiovascular structures represents a novel approach to improve clinical strategies in heart valve disease treatment. The aim of this study was to engineer decellularized atrioventricular heart valve neoscaffolds with an intact ultrastructure and to reseed them with umbilical cord-derived endothelial cells under physiological conditions in a bioreactor environment. Mitral (n=38) and tricuspid (n=36) valves were harvested from 40 hearts of German Landrace swine from a selected abattoir. Decellularization of atrioventricular heart valves was achieved by a detergent-based cell extraction protocol. Evaluation of the decellularization method was conducted with light microscopy and quantitative analysis of collagen and elastin content. The presence of residual DNA within the decellularized atrioventricular heart valves was determined with spectrophotometric quantification. The described decellularization regime produced full removal of native cells while maintaining the mechanical stability and the quantitative composition of the atrioventricular heart valve neoscaffolds. The surface of the xenogeneic matrix could be successfully reseeded with in vitro-expanded human umbilical cord-derived endothelial cells under physiological flow conditions. After complete decellularization with the detergent-based protocol described here, physiological reseeding of the xenogeneic neoscaffolds resulted in the formation of a confluent layer of human umbilical cord-derived endothelial cells. These results warrant further research toward the generation of atrioventricular heart valve neoscaffolds on the basis of decellularized xenogeneic tissue.

Transcatheter treatment of tricuspid regurgitation by caval valve implantation—experimental evaluation of decellularized tissue valves in central venous position

Caval valve implantation has been suggested for transcatheter treatment of severe tricuspid regurgitation (TR). Combining the interventional technique with the promising surgical experience with decellularized valves, we sought to evaluate the functional and structural outcome of decellularized pericardial tissue valves (dTVs) in the low-pressure venous circulation in a chronic model of TR. Sixteen pericardial tissue valves were heterotopically implanted in the inferior and superior vena cava in a sheep model (54-98 kg; median 74.5 kg, n = 8) of severe TR. The devices were assembled using self-expanding nitinol stents and bovine pericardia decellularized by a detergent-based protocol (group dTV; n = 8). Glutaraldehyde-fixed pericardial tissue valves served as control (GaTV, n = 8). After 6 months, device function and structural maturation were analyzed using echocardiographic, histologic, immunohistologic, and electron microscopic approaches. After implantation, cardiac output increased significantly from 3.7 ± 1.1 l/min to 4.8 ± 1.1 l/min (P < 0.05) and competent valve function was verified by angiography. At 6 months, angiographic and echocardiographic evaluation revealed moderate to severe regurgitation in all GaTV. In contrast, five of the eight dTVs functioned well with only minor regurgitation. In these animals, autopsy revealed preserved valve structure with tender leaflets without signs of thrombosis or calcification. Conversely, GaTV showed severe degeneration with large calcification areas. Microscopic and histologic analysis confirmed endothelial repopulation in both valve types. However, additional interstitial reseeding was observed in decellularized valves. In the venous circulation in severe TR, decellularized valves show superior functional performance compared to Ga-fixed tissue valves. Macroscopic and microscopic analyses suggest preserved structural integrity and advanced endothelial and interstitial repopulation with evidence of less degradation in dTV. © 2014 Wiley Periodicals, Inc.

Development and Evaluation of a Novel Combined Perfusion Decellularization Heart-Lung Model

Purpose The benefit of decellularized native tissues for study of regeneration has been widely demonstrated. The aim of this study was to evaluate the potential of a novel perfusion decellularization model for combined heart-lung tissue engineering. Methods and Materials Heart and Lung from 3-month-old male Sprague-Dawley rats were excised en bloc with trachea. After preparation, native heart-lung scaffolds were mounted and perfused with an ionic detergent-based perfusion protocol. Analysis of decellularization success was performed with histology and quantitative analysis of collagen and elastin content. Data regarding the presence of residual DNA within the decellularized heart-lung scaffolds was measured with spectrophotometric quantification and compared to controls. Results The decellularization method resulted in full removal of native cells while the mechanical stability and the original composition with micro- and macroarchitecture of the heart-lung neoscaffolds was successfully maintained. As a first step, the resulting neoscaffolds could successfully be reseeded with neonatal rat cardiomyocytes, assessing their biocompatibility for three-dimensional in vitro studies. Conclusions Combined heart-lung decellularization can be achieved using the novel method described in this investigation. This work demonstrates the feasibility of producing a decellularized heart-lung scaffold that can be used to study matrix biology and mechanics, independent of the effects of cellular components.