Residual Sodium Dodecyl Sulfate Research Articles

Reconstruction of the human nipple-areolar complex: a tissue engineering approach.

Introduction: Nipple-areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization-recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast. Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7days, while human keratinocytes were seeded on the scaffold epidermal side for 10days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis. Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10days of culture. Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

Analysis of the Effects of Surfactants on Extracellular Polymeric Substances

Reservoirs after chemical flooding usually have residual chemicals, which can affect the driving effect of subsequent microbial drives. Among them, the effect of surfactants on the metabolites of oil-recovering bacteria is the most obvious. Therefore, this paper investigates the influence mechanism of sodium dodecyl sulfate (SDS) on the nature and structure of Extracellular Polymeric Substances (EPS) produced by metabolism of Enterobacter cloacae, through a variety of characterization to analysis the components and structure of EPS under SDS stress. The results showed that Enterobacter cloacae was identified as a glycolipid-producing strain, the main components of EPS were polysaccharides and proteins. The polysaccharide composition (%: w/w) was glucosamine, 37.2; glucose, 31.5; rhamnose, 26.3; xylose, 1.7; and unidentified sugar, 3.3; and the main component of proteins was polyglutamic acid. EPS under the stress of SDS showed an increase in the content of functional groups such as -C=O and -COOH and an increase in the cellular particle size, and production of EPS increased by 10.69 × 103 mg/L when the SDS concentration was 2.5 × 102 mg/L; 3D-EEM results showed that the components of all three types of EPS The 3D-EEM results showed that all three types of EPS fractions contained tryptophan and protein-like substances, humic acid-like substances were only distributed in the solubilized extracellular polymers (SL-EPS), and aromatic proteins were only present in the loosely bound type (LB-EPS) and tightly bound type (TB-EPS). In addition, the peaks representing humic-like substances showed a blue shift, indicating that SDS had the greatest effect on SL-EPS. This study provides a guidance for refining the mechanism of strain EPS response to reservoir residual surfactant SDS, and provides a more comprehensive and in-depth understanding of surfactant-protein interactions.

Preparation of extracellular matrix of fish swim bladders by decellularization with supercritical carbon dioxide

Fish swim bladders used to be considered as byproducts or waste in fishery; however, they are potential materials for biological medicine with abundant collagen. In this work, an efficient noncytotoxic decellularization process using sodium dodecyl sulfate (SDS) ternary system assisted with supercritical carbon dioxide (scCO2) as the green extraction fluid and ethanol (ET) as the cosolvent has been developed to harvest acellular fish swim bladders (AFSBs). The experimental results show that the tissue treated by SDS assisted with scCO2 and ethanol at 37 °C and 25 MPa can be decellularized thoroughly and maintains intact fibers and uniform pore distribution, which resulting in a tensile strength of 5.61 MPa and satisfactory biocompatibility. Meanwhile, the residual SDS content in scCO2/SDS/ET ternary system is 0.0122% which is significantly lower than it in scCO2/SDS system due to the enhanced mass transfer rate of SDS in tissues by scCO2 with ethanol. The synergy between SDS and ethanol can enhance the diffusion coefficient and the solubility of SDS in scCO2, which reduced the contact time between SDS and tissues. Meaningfully, the results obtained in this work can not only provide a novel strategy to produce acellular matrix with superior properties, but also offer a further understanding of the decellularization through scCO2 extraction processing with the synergy of suitable detergent/cosolvent.Graphical

Decellularized vascularized bone grafts: A preliminary in vitro porcine model for bioengineered transplantable bone shafts.

Introduction: Durable reconstruction of critical size bone defects is still a surgical challenge despite the availability of numerous autologous and substitute bone options. In this paper, we have investigated the possibility of creating a living bone allograft, using the perfusion/decellularization/recellularization (PDR) technique, which was applied to an original model of vascularized porcine bone graft. Materials and Methods: 11 porcine bone forelimbs, including radius and ulna, were harvested along with their vasculature including the interosseous artery and then decellularized using a sequential detergent perfusion protocol. Cellular clearance, vasculature, extracellular matrix (ECM), and preservation of biomechanical properties were evaluated. The cytocompatibility and in vitro osteoinductive potential of acellular extracellular matrix were studied by static seeding of NIH-3T3 cells and porcine adipose mesenchymal stem cells (pAMSC), respectively. Results: The vascularized bone grafts were successfully decellularized, with an excellent preservation of the 3D morphology and ECM microarchitecture. Measurements of DNA and ECM components revealed complete cellular clearance and preservation of ECM's major proteins. Bone mineral density (BMD) acquisitions revealed a slight, yet non-significant, decrease after decellularization, while biomechanical testing was unmodified. Cone beam computed tomography (CBCT) acquisitions after vascular injection of barium sulphate confirmed the preservation of the vascular network throughout the whole graft. The non-toxicity of the scaffold was proven by the very low amount of residual sodium dodecyl sulfate (SDS) in the ECM and confirmed by the high live/dead ratio of fibroblasts seeded on periosteum and bone ECM-grafts after 3, 7, and 16days of culture. Moreover, cell proliferation tests showed a significant multiplication of seeded cell populations at the same endpoints. Lastly, the differentiation study using pAMSC confirmed the ECM graft's potential to promote osteogenic differentiation. An osteoid-like deposition occurred when pAMSC were cultured on bone ECM in both proliferative and osteogenic differentiation media. Conclusion: Fully decellularized bone grafts can be obtained by perfusion decellularization, thereby preserving ECM architecture and their vascular network, while promoting cell growth and differentiation. These vascularized decellularized bone shaft allografts thus present a true potential for future in vivo reimplantation. Therefore, they may offer new perspectives for repairing large bone defects and for bone tissue engineering.

Effect of ethanol washing on porcine pulmonary artery wall decellularization using sodium dodecyl sulfate.

To determine the effectiveness of ethanol (EtOH) washing on porcine pulmonary artery (PA) wall decellularization using sodium dodecyl sulfate (SDS), we compared three different washing methods (phosphate-buffered saline [PBS], pH 9 alkali, and EtOH washing). Fresh porcine PA walls were decellularized using 0.5% SDS and 0.5% sodium deoxycholate (SDC). The decellularized tissues were rinsed using three different washing techniques. Histological, biochemical, and mechanical analyses were conducted. Implantation into the subcutaneous tissue of rats and patch implantation into the carotid artery of dogs were performed as preliminary in vivo studies. The decellularization protocol based on SDS and SDC effectively removed the cells. The major extracellular matrix (ECM) structures (collagen, elastic fiber, and glycosaminoglycan) were properly preserved with the 75% EtOH-washing method. Significantly reduced residual SDS content was identified in EtOH-washed tissues compared to that in the other methods. No significant difference in the mechanical strength test was observed between the washing methods, and the EtOH-washing method showed better results in the metabolic activity test compared to the PBS-washing method. In the rat study model, no acute rejection or massive calcification was observed. The in vivo preliminary canine study showed better cell repopulation in the EtOH-washed group. EtOH washing of SDS-based decellularized porcine PA wall can reduce the residual SDS content and preserve ECM structures, especially the elastin content, and could also enhance cell repopulation after re-implantation.

Automated Electrokinetic Platform for High-Throughput Sodium Dodecyl Sulfate Depletion Ahead of Proteome Analysis by Mass Spectrometry.

Sodium dodecyl sulfate (SDS) provides numerous benefits for proteome sample preparation. However, the surfactant can be detrimental to downstream mass spectrometry analysis. Although strategies are available to deplete SDS from proteins, each is plagued by unique deficiencies that challenge their utility for high-throughput proteomics. An optimal approach would rapidly and reproducibly achieve less than 10 ppm residual SDS while simultaneously maximizing analyte recovery. Here, we describe improvements to a simple electrokinetic device termed transmembrane electrophoresis, which we previously reported for automated, rapid SDS depletion of proteome samples. Voltage-driven transport of SDS across a molecular weight cutoff membrane is enhanced at higher electric fields, which is herein achieved by integrating an active cooling mechanism to mitigate the impacts of Joule heating. We report 99.9% reduction of SDS (final concentration < 5 ppm) in 5 min. The device is employed in a detergent-based proteomic workflow for analysis of an enriched yeast membrane proteome extract, demonstrating quantitative protein recovery (>98%) and increasing the number of identifications by liquid chromatography-tandem mass spectrometry.

Detection of the residual concentration of sodium dodecyl sulfate in the decellularized whole rabbit kidney extracellular matrix.

To optimize rabbit kidney decellularization protocol, using sodium dodecyl sulfate (SDS) as a commonly used detergent, a methylene blue based assay was employed for detecting the minimum nontoxic SDS level for future cell seeding. The rabbit kidney tissues were decellularized with the perfusion-based method and underwent several investigations to determine the efficacy of decellularization in preserving the extracellular matrix (ECM) and cell removal. SDS detection was performed by incubating with methylene blue and subsequent extraction with chloroform. MTT (3-(4, 5-dimethylthiazol-2-yr)-2,5-diphenyltetrazolium bromide) assay and SDS release were also evaluated during the entire process. After the first washing cycle, SDS concentration was 0.036, in 500 mL of the washing liquid, which slowly decreased and reached to 0.009 % after at the end of seventh washing cycle. In the 9th cycle, SDS was gradually decreased and reached to 0.003 %. SDS was significantly released after one week of incubation which ceased after ten washing cycles. The results of MTT assay demonstrated that different cells exhibited various sensitivity levels when exposed to serial concentrations of SDS. Human embryonic kidney cells (HEK293) with 0.003 % threshold for cellular toxicity and 87.4 % cell viability were more resistant compared with mesenchymal stem cells with 0.001 % threshold and 85.4 % cell viability. Colorimetric assay with methylene blue is a straightforward and non-invasive method to detect residual SDS present in tissue and can also prevent ECM destruction after several washings for detergent removal from decellularized tissues.

An innovative method to obtain porous porcine aorta scaffolds for tissue engineering.

Decellularized porcine aorta (PA) is a promising biomaterial for vascular substitutes. However, decellularized PAs suffer from mechanical weakness and have less pores, which limit cellular ingrowth into the grafts and hinder the remodeling. In this study, PAs were decellularized by vacuum-freeze-thawing cycles and 0.3% of sodium dodecyl sulfate (SDS) buffer (VLS). Results showed that the application of vacuum-freeze-thawing significantly improved the decellularizationefficiency of SDS while effectively preserved the mechanical function of PA tissues, decreased residual SDS, and minimized cytotoxicity. Furthermore, scanning electron microscopy (SEM) examination demonstrated that VLS generated interconnected pores with uniform distribution. In vivo subcutaneous implantation assay further demonstrated that VLS implants had less calcification and adverse inflammatory response. Moreover, VLS treatment markedly enhanced ingrowth of myofibroblasts and endothelial cells, and thereby promoted synthesis of extracellular matrix and vascularization. These results suggest that the application of vacuum-freeze-thawing into thedecellularization process may produce a promising vascular graft candidate for tissue engineering application.

Evaluation of vacuum washing in the removal of SDS from decellularized bovine pericardium: method and device description

AimsThe aim of this study was to present a new method for removing Sodium dodecyl sulfate (SDS) detergent from decellularized bovine pericardium using vacuum. Materials and MethodsThe cows’ pericardia were collected and decellularized. The samples were incubated with SDS1% for 48 h at 40 °C. To perform vacuum washing (VW: negative pressure was used to wash and remove detergents), every decellularized tissue was cut in 75mm diameter and fixed via a stainless-steel ring with 60mm diameter in the center of filtration Buchner Funnel which was connected to glass filtration flask The system was connected to a vacuum pump by a hose, and a negative pressure of -100 mmHg was applied for 15 min. Then, the samples were shaken and washed at 40-rpm in 100 ml of distilled water for 45 min. This process was repeated for samples of each group (6 times for sample VW6h, 12 times for sample VW12h, and 24 times for sample VW24h). At the end of every cycle, the effluent was collected to take a sample for SDS measurement. The normal washing (NW) group containing distilled water (NWd) and PBS (Phosphate buffered saline) (NWp) were used to wash and remove detergents. SDS measurements, MTT Assay, histological and tensile test, to compare two methods were used. ResultsThe highest SDS in the effluent was in groups VW12h and VW24h (P ≤ 0.001) and the lowest residual SDS in scaffold was in two groups of VW12h and VW24h (P ≤ 0.001). MTT assay showed that cell survival in the VW12h and VW24h groups was higher than other groups and there' was no significant difference between cell survival in the VW12h and VW24h groups. Histological study showed destruction of tissue in the VW24h group. The results of the tensile test were shown that the native group had the highest module and the lowest amount was the VW24h sample which was reported with P ≤ 0.001 significance for all groups. ConclusionVW12h can be used as an effective method for SDS removal from decellularized pericardium which morphologically demonstrated a good structure in ECM.

Quantitation of residual sodium dodecyl sulfate in meningococcal polysaccharide by gas chromatography-mass spectrometry

Sodium dodecyl sulfate (SDS) is a commonly used surfactant in protein solubilization and also during the polysaccharide purification. A GC-MS method has been developed to quantitate residual SDS in meningococcal polysaccharide serogroups A,C,W,Y and X circumventing the need of spectroscopic assays and HPLC based methods which are either unstable or requires the confirmation by MS. The developed method is based on quantitative conversion of SDS to 1-dodecanol at elevated temperature. Meningococcal polysaccharides and SDS standards were treated with methanolic-HCl and extracted in n-Hexane. The conversion of SDS to 1-dodecanol was confirmed by mass spectra and separation was achieved using a DB-5ms column. The mass spectral analysis of 1-dodecanol showed characteristic ions at m/z 168, 140 and 125. The GC-MS method validation performed on intermediate and purified meningococcal polysaccharides showed linearity with r2 > 0.99 over the concentration range of 2.5–200 μg/ml with LOD and LOQ of 1.27 and 3.85 respectively. The method was found to be precise, robust and accurate with spike recovery ranging 83–117%. The GC-MS method can be used in the quantitation of residual SDS during polysaccharide purification and provides valuable information about consistency of polysaccharide manufacturing process for development of pentavalent meningococcal conjugate vaccine.

Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo

Detergents such as sodium dodecyl sulfate (SDS) are commonly used to extract cells from tissues in a process called "decellularization". Residual SDS is difficult to completely remove and may lead to an undesirable host response towards an implanted biomaterial. In this study, we developed a modification for SDS cell extraction from muscle equally efficient to previous methods but leading to significantly less residual SDS remnants in the matrices. Muscle-derived matrices were prepared via 2 SDS-based decellularization methods, which led to removal of either 81.4% or 98.4% of the SDS. In vitro, matrices were seeded with thp1 macrophages and primary human foreskin fibroblasts. By Day 2, both matrices demonstrated similar macrophage polarization; however, fibroblasts cultured on matrices with greater residual SDS expressed higher levels of mRNA associated with fibroblast activation: α-smooth muscle actin and connective tissue growth factor. In vivo, Collagen I gels spiked with increasing concentrations of SDS displayed a corresponding decrease in cell infiltration when implanted subcutaneously in rats after 4days. Finally, as a model for muscle regeneration, matrices produced by each method were implanted in rat latissimus dorsi defects. At POD 30 greater levels of IL-1β mRNA were present in defects treated with matrices containing higher levels of SDS, indicating a more severe inflammatory response. Although matrices containing higher levels of residual SDS became encapsulated by POD 30 and showed evidence of a foreign body response, matrices with the lower levels of SDS integrated into the defect area with lower levels of inflammatory and fibrosis-related gene expression.

Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves.

Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis.

A novel approach for methylene blue removal by calcium dodecyl sulfate enhanced precipitation and microbial flocculant GA1 flocculation

A novel approach was proposed for the removal of methylene blue (MB), a soluble cationic dye, from aqueous solution by calcium dodecyl sulfate (Ca(DS)2) enhanced precipitation which was based on the solubilization of sodium dodecyl sulfate (SDS) on MB and Ca2+ effect on SDS micelles, and microbial flocculant GA1 (MBFGA1) flocculation. The independent and interactive effects of factors, such as SDS, Ca2+ and MBFGA1 dosages, on the MB removal and interaction between SDS and Ca2+ were investigated. The response surface methodology (RSM), environmental scanning electron microscope (ESEM) and energy dispersive spectrometer (EDS) analysis were employed to discuss the interaction mechanism between SDS and Ca2+, and MB removal mechanism. The results showed that MB and SDS removal efficiency could reach 98.63% and 88.97%, respectively, with pH (10), MB (50mg/L), SDS (8mM), Ca2+ (5mM) and MBFGA1 (4mL/L). Under the optimal conditions, residual SDS and Ca2+ concentrations in the upper phase were 0.88mM and 1.27mM, respectively, which reached the Ksp of Ca(DS)2. The concentration consumption ratio between SDS and Ca2+ was 2.0. The interaction between SDS and Ca2+ was depended on the SDS-Ca2+ concentration ratio in aqueous solution rather than the CMC of SDS. When Ca2+ concentration was relatively sufficient, SDS micelles containing the solubilized MB (SDS-MB micelles) would disassemble to generate MB loaded Ca(DS)2 particles (CDS-MB particles) which would be flocculated by MBFGA1. Whereas, when SDS concentration was superfluous relatively, SDS micelles formed in the upper phase would redissolve the CDS-MB particles in flocs.

Using Hemolysis as a Novel Method for Assessment of Cytotoxicity and Blood Compatibility of Decellularized Heart Tissues.

Developing patient-specific transplantable organs is a promising response to the increasing need of more effective therapies for patients with organ failure. Advances in tissue engineering strategies have demonstrated favorable results, including the use of decellularized hearts as scaffolds for cardiac engineering; however, there is a need to establish methods to characterize the cytotoxicity and blood compatibility of cardiac extracellular matrix (cECM) scaffolds created by decellularization. In this study, porcine hearts were decellularized in an automated perfusion apparatus utilizing sodium dodecyl sulfate (SDS) detergent. Residual SDS was measured by a colorimetric assay. Phosphate-buffered saline, distilled water (DW), and Triton X-100 washes were used to remove SDS. The efficiency of detergent removal was measured as a function of time. It was observed that using Triton-X 100 can nearly double the rate of SDS removal. An assay based on human blood hemolysis was developed to measure the remaining cytotoxicity of the cECM. The results from the hemolysis cytotoxicity assay were consistent with a standard live/dead assay using MS1 endothelial cells incubated with the cECM. This study demonstrated an effective, reliable, and relatively inexpensive method for determining the cytotoxicity and blood compatibility of decellularized cECM scaffolds.

Detergent decellularization methods affect the surface molecular functionality of biologic scaffolds

Detergent decellularization methods affect the surface molecular functionality of biologic scaffolds

Surface characterization and protein interaction of a series of model poly[acrylonitrile-co-(N-vinyl pyrrolidone)] nanocarriers for drug targeting

The surface properties of intravenously injected nanoparticles determine the acquired blood protein adsorption pattern and subsequently the organ distribution and cellular recognition. A series of poly[acrylonitrile-co-(N-vinyl pyrrolidone)] (PANcoNVP) model nanoparticles (133–181nm) was synthesized, in which the surface properties were altered by changing the molar content of NVP (0–33.8mol%) as the more hydrophilic repeating unit. The extent of achieved surface property variation was comprehensively characterized. The residual sodium dodecyl sulfate (SDS) content from the synthesis was in the range 0.3–1.6μgml−1, potentially contributing to the surface properties. Surface hydrophobicity was determined by Rose Bengal dye adsorption, hydrophobic interaction chromatography (HIC) and aqueous two-phase partitioning (TPP). Particle charge was quantified by zeta potential (ZP) measurements including ZP–pH profiles. The interaction with proteins was analyzed by ZP measurements in serum and by adsorption studies with single proteins. Compared to hydrophobic polystyrene model nanoparticles, all PANcoNVP particles were very hydrophilic. Differences in surface hydrophobicity could be detected, which did not linearly correlate with the systematically altered bulk composition of the PANcoNVP nanoparticles. This proves the high importance of a thorough surface characterization applying a full spectrum of methods, complementing predictions solely based on bulk polymer composition.

Adsorption and recognition of protein molecular imprinted calcium alginate/polyacrylamide hydrogel film with good regeneration performance and high toughness

Protein imprinted calcium alginate/polyacrylamide hydrogel film (CA/PAM MIP) with high toughness was prepared using bovine serum albumin (BSA) as template molecule, sodium alginate and acrylamide as functional monomers, N,N′-methylenebisacrylamide (MBAA) as the covalent cross-linker and CaCl2 as the ionic cross-linker via UV radiation-reduced polymerization. Factors affecting the adsorption capacity and imprinting efficiency of the BSA-imprinted CA/PAM hydrogel films were investigated, such as ratio of polyacrylamide/sodium alginate, film thickness, MBAA concentration and CaCl2 concentration. Results showed that the CA/PAM MIP exhibited an obvious improvement in terms of adsorption capacity for BSA compared with non-imprinted polymer (NIP). The adsorption capacity of MIP for BSA reached 22.49mg/g, which was 2.7times higher than NIP. The regeneration property of the BSA-imprinted CA/PAM hydrogel was distinctly improved and the imprinting efficiency of CA/PAM MIP maintained 77.95% of the initial value after five repetitions. Single and binary proteins rebinding indicated that the CA/PAM MIP exhibited good recognition performance. Cell culture experiments showed CA/PAM MIP was more suitable for cell culture than CA/PAM NIP. The residual sodium dodecyl sulfate (SDS) in the elution process leaded to the death of mouse fibroblast cells (L929) after 3days. A moderate elution solution without residue eluent should be used to prepare MIP for cell culture.

Successful re-endothelialization of a perfusable biological vascularized matrix (BioVaM) for the generation of 3D artificial cardiac tissue.

Generating cellularized 3D constructs with clinical relevant dimensions is challenged by nutrition supply. This is of utmost importance for cardiac tissue engineering, since cardiomyocytes are extremely sensitive to malnutrition and hypoxia in vitro and after implantation. To develop a perfusable myocardial patch, we have focused on seeding a decellularized biological vascularized matrix (BioVaM) with endothelial cells. BioVaM is produced by decellularization of porcine small intestinal segments with preserved arterial and venous pedicles, which can be connected to a perfusion system in vitro or the host vasculature in vivo. The BioVaM vessel bed was re-seeded with porcine primary endothelial cells (pCEC). Seeding efficiency was influenced by detergent composition used for decellularization (sodium dodecyl sulfate (SDS) and/or Triton X-100) and the medium composition used for re-seeding. After decellularization, residual SDS was detected in the matrix affecting the survival of pCEC which showed a low tolerance to SDS and Triton X-100. Sensitivity to detergents was attenuated by supplementation of the medium with bovine serum albumin (BSA) or fetal calf serum (FCS). Pre-conditioning of the BioVaM with 20% FCS was not sufficient to attain pCEC survival in the vascular bed. However, re-cellularization was achieved by prolonged FCS supplementation during cultivation, resulting in a perfusable, re-endothelialized matrix of 11 cm2 in size. This achievement represents a promising step towards engineering of perfusable, 3D cardiac constructs with clinically relevant dimensions.

Up-Scaling Decellularization and Whole Organ Culture for Human Lung Regeneration

Purpose Organ engineering is a theoretical therapy for organ failure. Whole organ matrices can be created by detergent perfusion, generating acellular scaffolds with the capacity for recellularization. This study aimed to up-scale this technology to clinically relevant porcine and human lungs, and to test their recellularization potential in biomimetic culture. Methods and Materials Rat, pig, and human lungs were perfused with detergent via the pulmonary artery at constant pressures, followed by washing. DNA was quantified by PicoGreen. Residual SDS was quantified by Stains-All assay. Soluble collagen, elastin, and glycosaminoglycans were quantified by colourimetric assays. Proteomic analysis by mass spectrometry (LC-MS/MS) generated spectral counts as a measure of abundance. Perfusion recellularization experiments of human lungs used standard organ culture techniques derived from rodent protocols. Results Rat lungs decellularized using (1) Sodium Dodecyl Sulfate (SDS), (2) Sodium deoxycholate, or (3) CHAPS showed loss of DNA but greatest preservation of ECM in SDS decellularized lungs. Histology and proteomics also confirmed greater matrix preservation in SDS lungs. The SDS-based decellularization protocol was up-scaled to porcine (n=7) and human (n=3) lungs with greater SDS concentration and perfusion pressures, and extended decellularization time. Removal of residual DNA and SDS was confirmed. Analysis of 10 areas from each decellularized lung showed intact ECM and lung architecture. Proteomics of human decellularized lungs further demonstrated matrix preservation. Recellularization of isolated lobar scaffolds used endothelial (HUVEC) and lung epithelial (SAEC) lines delivered to the appropriate organ compartment. Throughout biomimetic organ culture matrix integrity was maintained, while cell retention was observed by H&E and DAPI. Conclusions SDS decellularization can generate organ scaffolds with preserved ECM and architecture in whole porcine and human lungs. The results confirm clinical scale lung scaffolds can support recellularization for organ regeneration.

Biocompatibility and Potential of Acellular Human Amniotic Membrane to Support the Attachment and Proliferation of Allogeneic Cells

The aim of this study was to determine the biocompatibility of an acellular human amniotic membrane biomaterial, which may have clinical utility for cell delivery. Human amniotic membrane was decellularized using 0.03% (w/v) sodium dodecyl sulfate (SDS), with hypotonic tris buffer and protease inhibitors and nuclease treatment. The membrane was terminally sterilized using an optimal concentration of peracetic acid. Residual SDS present within the acellular membrane was quantified using radio-labeled C14 SDS. In vivo biocompatibility was assessed by implantation of acellular human amniotic membrane subcutaneously into mice for 3 months and comparison with fresh and glutaraldehyde-fixed tissue. Cellular infiltrate into the explanted tissues was characterized using monoclonal antibodies against the following cell surface markers: CD3, CD4, CD34, and F4/80. Calcification was determined using the Von Kossa's stain. The potential of acellular human amniotic membrane to support the attachment and proliferation, and maintain viability of primary human dermal fibroblasts and primary human dermal keratinocytes was assessed in vitro, using a static culture system. Peracetic acid at a concentration of 0.1% (v/v) was sufficient for the sterilization of acellular amniotic membrane. Levels of SDS present within the acellular tissue were 0.62 +/- 0.13 microg/mg. Analysis of explanted samples from the mice indicated that acellular amniotic membrane contained low numbers of T-cells and high numbers of fibroblastic cells, macrophages, and endothelial cells, indicative of a wound-healing response. There was no evidence of calcification present within explanted acellular amniotic membrane compared to explanted glutaraldehyde-fixed amniotic membrane. Acellular amniotic membrane was shown to be capable of supporting the attachment and proliferation of primary human fibroblasts and keratinocytes. The viability of the cells was maintained for up to 4 weeks. Cell-seeded acellular amniotic membrane has the potential for delivering autologous or allogeneic cells to treat a variety of conditions, including diabetic foot ulcers, corneal defects, and severe skin burns.