Decellularized Human Sclera; an Optimized Biomaterial for Scleral Reconstruction.

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.

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.

Various detergent-based protocols are used to remove cells and cellular debris in porcine aortic valve (PAV). However, the removal of antigenic cellular components has not been thoroughly elucidated to date. In this study, we used 4 detergent-based protocols to decellularize PAVs and aimed to evaluate their effects on removing antigenic cellular components. Porcine aortic valves were decellularized using sodium dodecyl sulfate (SDS), SDS in combination with sodium deoxycholate (SDS/SD), Triton X-100, and Triton X-100 in combination with SD (Triton X-100/SD), respectively. Untreated PAVs were used as controls. Immunohistochemical and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed to determine the removal of antigenic protein components. Histological, biochemical, and biomechanical analyses were performed to determine the preservation and mechanical properties of the extracellular matrix. PAV tissues were implanted subcutaneously in Sprague Dawley rats to evaluate the host immune response. Implanted PAVs were taken out at the indicated time points for histological and immunohistochemical examinations. All 4 protocols effectively removed the membrane antigenic proteins major histocompatibility complex I molecule and galactose-α-1,3 galactose. The SDS/SD protocol was the most effective method to remove the cytoplasmic cytoskeletal proteins, vimentin and α-SMA, and SDS alone partly removed vimentin protein. The SDS/SD protocol was the most effective method to remove nuclear DNA with residual DNA below 50ng/mg, followed by the SDS protocol with residual DNA of 74.9ng/mg. The SDS protocol was the most effective method to remove proteins ranged from 10 to 55kDa, followed by the SDS/SD protocol. SDS and SDS/SD PAV implants attracted fewer neutrophils in vivo on postoperative day 3. The infiltration of macrophages and T lymphocytes was significantly lower in SDS and SDS/SD implants on days 14 and day 28. All of the decellularized protocols that were examined greatly reduced the contents of collagen, elastin, and glycosaminoglycan compared to controls. Biomechanical analysis revealed significant differences in ultimate tensile strength and Young's modulus between control PAVs and decellularized PAVs generated using SDS, SDS/SD, Triton X-100, or Triton X-100/SD. These results indicated that SDS-based protocols more effectively removed antigenic cellular components compared to Triton X-100-based protocols. These results are clinically significant because complete removal of antigenic determinants is critical to decrease adverse immune-mediated and inflammatory responses to a PAV when used in xenogeneic application.

Porcine kidneys as a source of ECM scaffold for kidney regeneration.

Atherosclerosis and its complications still represent the leading cause of death in the developed countries. While autologous blood vessels may be regarded as the best solution for peripheral and coronary bypass, they are unavailable in most patients. Even though tissue engineering techniques are often applied to the development of small-diameter vascular grafts, limiting factors of this approach are represented by the lack of essential extracellular matrix proteins and/or poor biomechanical properties of the scaffolds used. Along these lines, the aim of this study was to develop a decellularization protocol for ovine carotids to be used as suitable small-diameter vascular grafts. Samples were treated either with sodium dodecyl sulphate (SDS) or with Trypsin and Triton X-100; a final nuclease digestion was performed for both protocols. Morphological analyses demonstrate complete removal of nuclei and cellular components in treated vessels, also confirmed by significant reduction in wall thickness and DNA content. Essential extracellular matrix proteins such as collagen, elastin, and fibronectin are well preserved after decellularization. From a mechanical point of view, Trypsin and Triton X-100 treated arteries show elastic modules and compliance comparable to native carotids, whereas the use of SDS makes samples stiffer, with a significant decrease in the compliance mean value and an increase in longitudinal and circumferential Young’s modules. It is demonstrated that the treatment where Trypsin and Triton X-100 are combined guarantees complete decellularization of carotids, with no significant alteration of biomechanical and structural properties, thus preserving a suitable environment for adhesion, proliferation, and migration of cells.

Amyloid fibrillation by hen egg white lysozyme (HEWL) under the influences of two common surfactants, sodium dodecyl sulfate (SDS) and Triton X-100 (TX-100), was investigated with atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. Detailed AFM investigations indicated that both SDS and TX-100 were able to induce some significant morphological changes of HEWL amyloid fibrils. Both SDS and TX-100 could induce a morphological change which was characterized with alternating fibril regions with increased thicknesses along the fibril axis. In addition, TX-100 was also able to induce a morphological change which was characterized with fibril branching. In contrast, the positively charged cetyltrimethylammonium bromide displayed no such effect as compared with the negatively charged SDS and the nonionic TX-100. These intriguing modulation effects of SDS and TX-100 on amyloid fibrillation were hypothesized to be due to surfactant-induced surface-catalyzed secondary nucleation owing to the noncovalent interaction between the surfactants and HEWL. The proposed hypothesis was further supported by FTIR spectroscopic investigation, which demonstrated the formation of a SDS-amyloid fibril complex and TX-100-amyloid fibril complex. Detailed FTIR analysis suggested that the tail group of SDS associated with amyloid fibrils is more disordered similar to that of SDS in aqueous solution, while the headgroup of SDS appears to interact with amyloid fibrils strongly similar to that of SDS in the solid state, and the ether groups of TX-100 associated with amyloid fibrils experienced a different microenvironment as compared with that of neat TX-100. Additional control experiments with FTIR spectroscopy revealed that the interactions of SDS/TX-100 with HEWL amyloid fibrils were different from those of SDS/TX-100 with HEWL amorphous aggregates. In addition, the β-sheet structures of the HEWL amyloid fibrils were found to be increased due to the influences of SDS and TX-100. Our work provides new insight into the modulation effects of surfactants on amyloid fibrillation.

Objectives: The purpose of this study is to evaluate the optimum conditions for oral mucosal irritation testing using the buccal pouch of hamsters. Methods: Test materials were applied to the buccal pouch of seven-week old male Syrian hamsters (SLC, Japan) four times at one-hour intervals and macroscopic changes were examined at 24 hours after final treatment. After sacrifice, the buccal pouches were removed and prepared for histopathological evaluation. In order to set the exposure time, we performed exposure tests of 5, 12, 18 and 23 minutes using sodium lauryl sulfate (SLS) 1% and set the treatment volume from the test results at 2, 3, or 4 ml treatment using SLS 1%, Triton X-100 1% and ethanol. After setting the experimental conditions, seven groups of materials [sodium lauryl sulfate (SLS) (1%), Triton X-100 (1%), hydrogen peroxide (3%), ethanol (100%), chlorhexidine (0.2%, 2%), phosphate buffer saline (PBS)] were assessed. Results: Experimental conditions of material exposure time were fixed as 18 minutes from the exposure tests of 5, 12, 18 or 23 min using sodium lauryl sulfate (SLS) 1%. Treated volume was set as 4 ml per each pouch from the test results of 2, 3, or 4 ml treatments using SLS 1%, Triton X-100 1% and ethanol. The results in terms of irritation degree were in the order of sodium lauryl sulfate (SLS) (1%) > Triton X-100 (1%) <TEX>${\fallingdotseq}$</TEX> hydrogen peroxide (3%) > ethanol (100%) <TEX>${\fallingdotseq}$</TEX> chlorhexidine (0.2%, 2%) > phosphate buffer saline (PBS). Conclusion: From this study, suitable conditions for hamster mucosal irritation testing were suggested and this method was verified through materials commonly used on oral mucosal membranes.

Nano- and micro-sized vesicular and colloidal structures mediate cell-cell communication. They are important players in the physiology of plants, animals, and humans, and are a subject of increasing interest. We investigated the effect of three surfactants, N-cetylpyridinium chloride (CPC), sodium dodecyl sulfate (SDS), and Triton X-100 (TX100), and two anionic polyelectrolytes, sodium polystyrene sulfonate (NaPSS) and sodium polymethacrylate (NaPMA), on nanoliposomes. In addition, the effect of SDS and TX100 on selected biological membranes (erythrocytes and microalgae) was investigated. The liposomes were produced by extrusion and evaluated by microcalorimetry and light scattering, based on the total intensity of the scattered light (Itot), hydrodynamic radius (Rh), radius of gyration (Rg), shape parameter p (=Rh/Rg,0), and polydispersity index. The EPs shed from erythrocytes and microalgae Dunaliella tertiolecta and Phaeodactylum tricornutum were visualized by scanning electron microscopy (SEM) and analyzed by flow cytometry (FCM). The Rh and Itot values in POPC liposome suspensions with added CPC, SDS, and TX100 were roughly constant up to the respective critical micelle concentrations (CMCs) of the surfactants. At higher compound concentrations, Itot dropped towards zero, whereas Rh increased to values higher than in pure POPC suspensions (Rh ≈ 60-70 nm), indicating the disintegration of liposomes and formation of larger particles, i.e., various POPC-S aggregates. Nanoliposomes were stable upon the addition of NaPSS and NaPMA, as indicated by the constant Rh and Itot values. The interaction of CPC, SDS, or TX100 with liposomes was exothermic, while there were no measurable heat effects with NaPSS or NaPMA. The SDS and TX100 increased the number density of EPs several-fold in erythrocyte suspensions and up to 30-fold in the conditioned media of Dunaliella tertiolecta at the expense of the number density of cells, which decreased to less than 5% in erythrocytes and several-fold in Dunaliella tertiolecta. The SDS and TX100 did not affect the number density of the microalgae Phaeodactylum tricornutum, while the number density of EPs was lower in the conditioned media than in the control, but increased several-fold in a concentration-dependent manner. Our results indicate that amphiphilic molecules need to be organized in nanosized particles to match the local curvature of the membrane for facilitated uptake. To pursue this hypothesis, other surfactants and biological membranes should be studied in the future for more general conclusions.

Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system

Tumor suppressor p53 is known to activate certain sets of genes while suppressing others. However, whether p53 can both activate and suppress the same gene is unclear. To address this question, concentration-dependent p53 effect on the manganese superoxide dismutase (MnSOD) gene was investigated. By transfecting p53 in PC-3 cells, we demonstrate that low concentrations of p53 increase while high concentrations suppress MnSOD expression. The physiological relevance of this effect was determined in vitro and in vivo using combined UVB-mediated activation and small interference RNA-mediated suppression of p53. Results were consistent with the bi-directional effect of p53 on MnSOD expression. MnSOD-promoter/enhancer analysis demonstrates that p53 is suppressive to the promoter activity regardless of the presence or absence of putative p53 binding sites. However, a low level of p53 increases MnSOD gene transcription in the presence of the intronic-enhancer element, and this effect is dependent on nuclear-factor kappaB (NF-kappaB) binding sites. Expression of p53 enhances nuclear levels of p65 with corresponding increase in the DNA-binding activity of NF-kappaB as detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. Transfection of p65 small interference RNA reduces the positive effect of p53 on MnSOD gene transcription. These data suggest that p65 can overcome the negative effect of p53 on MnSOD expression. However, when the level of p53 was further increased, the suppressive effect of p53 outweighed the positive effect of p65 and led to the suppression of MnSOD gene transcription. These results demonstrated that p53 can both suppress and induce MnSOD expression depending on the balance of promoter and enhancer binding transcription factors.

The innate extracellular matrix (ECM) scaffolds can be a promising scaffold for regeneration of complex organ such as heart, liver and kidney. They possess intact 3-dimentional architecture and biochemical components that allow to access to the organ’s capillary network. In this study we have developed a porcine renal ECM scaffold and analyzed its physical and biochemical characteristics including biocompatibility for human kidney regeneration. A segmented porcine kidney cortex was obtained and treated with 1% (v/v) Triton X-100 (Triton) or sodium dodecyl sulfate (SDS) in a shaking chamber, and rinsed with distilled water. After confirmation of decellularization with H&E stain, the matrix was lyophilized and sterilized. Scanning Electron Microscope (SEM) analysis showed that both scaffolds were preserved with proper architecture including porosity for cell adhesion and composition of the renal ECM. The water uptake ability of the Triton treated scaffold was higher than that of SDS treated one. The maximum compressive strength of Triton was lower than SDS treated scaffolds and that correlates with the results of porosity and water uptake analysis. In ATR-IR analysis, both scaffolds showed a peak at 3445–3446 cm−1 and that indicates the presence of amide II (-NH). Triton treated scaffold demonstrated that there are richer contents of ECM proteins and growth factors compared to SDS treated one. When scaffolds were seeded with primary human kidney cells, Triton treated scaffold showed 2.66 times higher number of adherent cells than SDS treated one at 24 hrs post-seeding. On a CCK-8 analysis, the Triton treated scaffold showed significantly higher cell viability and proliferation rate than that of SDS treated one. Both scaffolds had no tumorigenecity for 8 weeks in vivo analysis. In conclusion, we successfully developed porcine renal ECM scaffold and confirmed that there is a great potential of porcine renal ECM scaffold to be used as human kidney regeneration. We also verified that 1% Triton X-100 is more suitable decellularizing agent than SDS regarding structural, biochemical integrity and biocompatibitilty of the scaffold. To support our findings and human application of practical regeneration, we are planning to perform in vivo experiment for kidney regeneration near future.

Terpolymers composed of acrylic acid, methacrylamide, and DiC6, DiC8, or DiC10 twin-tailed hydrophobic monomers have been synthesized using micellar polymerization methods, and the interactions of these polymers with surfactants were investigated. These surfactants include sodium dodecyl sulfate (SDS), cetyl trimethylammonium bromide (CTAB), and Triton X-100. Viscosity measurements of DiC6AM and DiC8AM mixtures indicate little interaction with SDS, gelation with CTAB, and hemimicelle formation followed by polymer hydrophobe solubilization with Triton X-100. The DiC10AM terpolymer shows similar interaction behavior with CTAB and Triton X-100. However, the enhanced hydrophobic nature of the DiC10 polymer allows complex formation with SDS as confirmed by surface tensiometry. Fluorescence measurements performed on a dansyl-labeled DiC10AM terpolymer in the presence of increasing amounts of each of the surfactants indicate relative interaction strengths to be CTAB > Triton X-100 > SDS.

Mesenchymal stem cells (MSCs) are essential in regenerative medicine. However, conventional expansion and harvesting methods often fail to maintain the essential extracellular matrix (ECM) components, which are crucial for their functionality and efficacy in therapeutic applications. Here, we introduce a bone marrow-inspired macroporous hydrogel designed for the large-scale production of MSC-ECM spheroids. Through a soft-templating approach leveraging liquid-liquid phase separation, we engineer macroporous hydrogels with customizable features, including pore size, stiffness, bioactive ligand distribution, and enzyme-responsive degradability. These tailored environments are conducive to optimal MSC proliferation and ease of harvesting. We find that soft hydrogels enhance mechanotransduction in MSCs, establishing a standard for hydrogel-based 3D cell culture. Within these hydrogels, MSCs exist as both cohesive spheroids, preserving their innate vitality, and as migrating entities that actively secrete functional ECM proteins. Additionally, we also introduce a gentle, enzymatic harvesting method that breaks down the hydrogels, allowing MSCs and secreted ECM to naturally form MSC-ECM spheroids. These spheroids display heightened stemness and differentiation capacity, mirroring the benefits of a native ECM milieu. Our research underscores the significance of sophisticated materials design in nurturing distinct MSC subpopulations, facilitating the generation of MSC-ECM spheroids with enhanced therapeutic potential.

Tissue engineering is a promising approach for the production of small-diameter vascular grafts; however, there are limited data directly comparing the suitability of applicable cell types for vessel biofabrication. Here, we investigated the potential of adult smooth muscle cells (SMCs), placental mesenchymal stem cells (MSCs), placental endothelial colony-forming cells (ECFCs), and a combination of MSCs and ECFCs on highly porous biocompatible poly(ɛ-caprolactone) (PCL) scaffolds produced via melt electrowriting (MEW) for the biofabrication of tissue-engineered vascular grafts (TEVGs). Cellular attachment, proliferation, and deposition of essential extracellular matrix (ECM) components were analysed in vitro over four weeks. TEVGs cultured with MSCs accumulated the highest levels of collagenous components within a dense ECM, while SMCs and the coculture were more sparsely populated, ascertained via histological and immunofluorescence imaging, and biochemical assessment. Scanning electron microscopy (SEM) enabled visualisation of morphological differences in cell attachment and growth, with MSCs and SMCs infiltrating and covering scaffolds completely within the 28-day culture period. Coverage and matrix deposition by ECFCs was limited. However, ECFCs lined the ECM formed by MSCs in coculture, visualised via immunostaining. Thus, of cells investigated, placental MSCs were identified as the preferred cell source for the fabrication of tissue-engineered constructs, exhibiting extensive population of porous polymer scaffolds and production of ECM components; with the inclusion of ECFCs for luminal endothelialisation, an encouraging outcome warranting further consideration in future studies. In combination, these findings represent a substantial step toward the development of the next generation of small-diameter vascular grafts in the management of cardiovascular disease.

In this study, we sought to explore an efficient decellularization protocol for bovine pericardia with better extracellular matrix preservation and good biocompatibility. Bovine pericardia were decellularized by sodium dodecyl sulphate (SDS), SDS + sodium deoxycholate (SD), Triton X-100 (TX), TX + SD (TS), freeze-thaw cycles + SDS + SD (FSS) and freeze-thaw cycles + TX + SD (FTS), respectively. Untreated pericardia were used as native control. Histological examination, residual cellular content analysis, biochemical and biomechanical evaluations and cytotoxicity assay were performed to investigate decellularization efficiency, xenoantigens removal, extracellular matrix preservation and biocompatibility. In vivo biocompatibility was evaluated using a subcutaneous implantation method in rats. Among these protocols, FSS and FTS protocols were the most effective methods to remove both the DNA material and the galactose-α-1,3-galactose antigen. TX, TS and FTS bovine pericardia maintained the collagen content and had no cytotoxicity to human umbilical vein endothelial cells. The contents of elastin and glycosaminoglycan were lost to different degrees after decellularization, with the highest content of preservation with TX, followed by TS and FTS. Consistently, no significant difference was found between native bovine pericardia and TX, TS or FTS bovine pericardia. In vivo, FTS implants had minimal infiltration of macrophages and T-lymphocytes, with no histological evidence of peri-implant necrosis and calcification. These results suggested that the FTS protocol showed optimal decellularization results with better extracellular matrix preservation and good biocompatibility. It may be a suitable protocol for producing a suitable scaffold for heart tissue engineering.