Kamal Hany Hussein

Biocompatibility evaluation of tissue-engineered decellularized scaffolds for biomedical application.

Biomaterials based on seeding of cells on decellularized scaffolds have gained increasing interest in the last few years and suggested to serve as an alternative approach to bioengineer artificial organs and tissues for transplantation. The reaction of the host toward the decellularized scaffold and transplanted cells depends on the biocompatibility of the construct. Before proceeding to the clinical application step of decellularized scaffolds, it is greatly important to apply a number of biocompatibility tests in vitro and in vivo. This review describes the different methodology involved in cytotoxicity, pathogenicity, immunogenicity and biodegradability testing for evaluating the biocompatibility of various decellularized matrices obtained from human or animals.

Fabrication of novel high performance ductile poly(lactic acid) nanofiber scaffold coated with poly(vinyl alcohol) for tissue engineering applications

Poly(lactic acid) (PLA) nanofiber scaffold has received increasing interest as a promising material for potential application in the field of regenerative medicine. However, the low hydrophilicity and poor ductility restrict its practical application. Integration of hydrophilic elastic polymer onto the surface of the nanofiber scaffold may help to overcome the drawbacks of PLA material. Herein, we successfully optimized the parameters for in situ deposition of poly(vinyl alcohol), (PVA) onto post-electrospun PLA nanofibers using a simple hydrothermal approach. Our results showed that the average fiber diameter of coated nanofiber mat is about 1265±222 nm, which is remarkably higher than its pristine counterpart (650±180 nm). The hydrophilicity of PLA nanofiber scaffold coated with a PVA thin layer improved dramatically (36.11±1.5°) compared to that of pristine PLA (119.7±1.5°) scaffold. The mechanical testing showed that the PLA nanofiber scaffold could be converted from rigid to ductile with enhanced tensile strength, due to maximizing the hydrogen bond interaction during the heat treatment and in the presence of PVA. Cytocompatibility performance of the pristine and coated PLA fibers with PVA was observed through an in vitro experiment based on cell attachment and the MTT assay by EA.hy926 human endothelial cells. The cytocompatibility results showed that human cells induced more favorable attachment and proliferation behavior on hydrophilic PLA composite scaffold than that of pristine PLA. Hence, PVA coating resulted in an increase in initial human cell attachment and proliferation. We believe that the novel PVA-coated PLA nanofiber scaffold developed in this study, could be a promising high performance biomaterial in regeneration medicine.

Sterilization using electrolyzed water highly retains the biological properties in tissue-engineered porcine liver scaffold

Purpose The aims of this study were to investigate the effects of sterilization with peracetic acid (PAA) and ethanol on the biological activity of porcine liver scaffolds and to develop a new technique for sterilization using slightly acidic electrolyzed water (SAEW). Methods Decellularization of liver slices was performed using 0.1% sodium-dodecyl-sulfate, then evaluated by histological and polymerase chain reaction analyses. Decellularized slices were treated with either PAA or ethanol or SAEW, and then DNA content was quantified. We determined sterilization efficiency by culturing scaffolds in culture medium and on blood agar. We next analyzed the glycosaminoglycan and collagen contents of the scaffolds. Finally, we tested the cytotoxicity of the scaffolds as well as the effects of sterilization on host cell attachment and proliferation. Results Complete cell and antigenic epitopes removal emphasized the decellularization efficiency. PAA and SAEW treatments achieved the highest efficiency of sterilization compared to that of the ethanol treated scaffolds, and were able to remove a considerable fraction of DNA from decellular-ized livers. The retained glycosaminoglycan content decreased in all treatments in the following order: SAEW, ethanol, and PAA. Ethanol caused a significant loss in collagen content compared to the other groups. A cytotoxicity evaluation revealed that all scaffolds were nontoxic. SAEW-treated scaffolds supported cell attachment and proliferation at a significantly higher rate than other groups. Conclusions These data suggest that SAEW is highly efficient for sterilizing scaffolds and allowed the scaffolds to retain their bioactivity in addition to its high efficiency for cell remnant removal.

Three dimensional culture of HepG2 liver cells on a rat decellularized liver matrix for pharmacological studies.

Three-dimensional in vitro tumor models are needed to obtain more information about drug behavior in tumors. The aim of this study is to establish a new model for hepatocellular carcinoma (HCC) using decellularized rat livers. After generating the rat liver scaffolds, HepG2 liver cancer cells were perfused via the portal vein and placed in a bioreactor for 10 days. Histology was performed to analyze cell distribution within the scaffolds. Function and tumor-related gene expression were examined by polymerase chain reaction (PCR). We evaluated the function of HepG2 cells grown on scaffolds in the presence of a well-known anti-cancer drug to investigate the potential application of our system for drug screening. The scaffolds were devoid of cellular materials and preserved extracellular matrix components. HepG2 cells grew well on the scaffolds. The PCR results showed that the cells maintained function and invasion ability at significantly higher levels than cells grown on two-dimensional (2-D) dishes or spheroids on Matrigel. Unlike the 2-D cultures, albumin secretion and alpha-fetoprotein expression in three-dimensional cultures were less susceptible to lower concentrations of the drug. Cells grown in scaffolds seemed to respond to the drug in an analogous manner to its known activity in vivo. These findings strengthen the potential use of rat liver scaffolds for screening HCC drugs.

Construction of a biocompatible decellularized porcine hepatic lobe for liver bioengineering.

Objective One of the major obstacles in applying decellularized organs for clinical use is the recellularization step, during which huge numbers of cells are required to develop whole livers. We established a simple protocol for constructing a bioartificial hepatic lobe and investigated its biocompatibility. Methods The right lateral lobe of porcine liver was decellularized using 0.1% sodium dodecyl sulfate through the right branch of the portal vein. Decellularized lobes were evaluated by histological and biochemical analyses. DNA content was quantified to validate the decellularization protocol. The presence of immunogenic and pathogenic antigens was checked to exclude potential rejection and thrombosis after xenotransplantation. Xeno-reactivity of decellularized tissue against human peripheral blood mononuclear cells was examined. Cytotoxicity was evaluated against hepatocarcinoma cells. Finally, scaffolds were incubated in collagenase for biodegradation testing. Results The decellularized lobe preserved the three-dimensional architecture, ultrastructure, extracellular matrix components, and vasculature. Scaffolds were almost depleted of DNA in addition to antigenic and pathogenic antigens, which are considered barriers to xenotransplantation. The human immune response against scaffolds was considered non-significant. Our matrices were biocompatible and biodegradable. Conclusions We successfully developed a non-cytotoxic, non-immunogenic, and biodegradable porcine hepatic lobe for future liver regeneration and bioengineering.

Amorphous apatite thin film formation on a biodegradable Mg alloy for bone regeneration: strategy, characterization, biodegradation, and in vitro cell study

Bioactive films with a nanoplate structure were prepared on the surface of a biodegradable AZ31B magnesium (Mg) alloy via anodization in simulated body fluid (SBF) as an electrolyte to control Mg biodegradability and improve surface bioactivity. The effect of the electrolyte temperature and pH values on the formation of the biomimetic film were studied. The electrolyte was set at three different temperatures of 37, 50, and 80 °C, with pH values ranging from 7.4 to 8 for the lower electrolyte temperature and 11.5–12 for the two higher levels of temperature. The apatite films on the different samples were characterized using X-ray diffraction spectroscopy (XRD), field emission scanning electron microscopy (FE-SEM), EDS element mapping, X-ray photon spectroscopy (XPS), and FTIR spectroscopy. The water contact angles of the different surfaces were evaluated, moreover, the corrosion behaviors of the different samples were studied using electrochemical potentiodynamic DC, electrochemical impedance spectroscopy (EIS), and immersion tests. The human fetal-osteoblast cell line hFOB 1.19 was used in a cell culture test, and the biological response and cell function were evaluated in vitro using DNA and PCR. The decomposition of the apatite film was affected by the anodization electrolyte temperature, resulting in an amorphous structure. It is observed that the apatite structure has nanoplates at electrolyte temperature (37 to 50) °C and these have a tendency to disappear at 80 °C.

Hepatic Differentiation of Porcine Embryonic Stem Cells for Translational Research of Hepatocyte Transplantation

Porcine embryonic stem cells (ES) are considered attractive preclinical research tools for human liver diseases. Although several studies previously reported generation of porcine ES, none of these studies has described hepatic differentiation from porcine ES. The aim of this study was to generate hepatocytes from porcine ES and analyze their characteristics. We optimized conditions for definitive endoderm induction and developed a 4-step hepatic differentiation protocol. A brief serum-free condition with activin A efficiently induced definitive endoderm differentiation from porcine ES. The porcine ES-derived hepatocyte-like cells highly expressed hepatic markers including albumin and α-fetoprotein, and displayed liver characteristics such as glycogen storage, lipid production, and low-density lipoprotein uptake. For the first time, we describe a highly efficient protocol for hepatic differentiation from porcine ES. Our findings provide valuable information for translational liver research using porcine models, including hepatic regeneration and transplant studies, drug screening, and toxicology.

Kidney injury molecule-1 is involved in the chemotactic migration of mesenchymal stem cells

A better understanding of the organ specific factors that regulate the migration of mesenchymal stem cells (MSCs) into the target organ is essential for optimization of strategies to improve the repair after injury. In the present study, we showed that the kidney injury molecule-1 (KIM-1), a well-known kidney-specific biomarker, enhanced the in vitro migration capacity of MSCs as a potent kidney-specific chemo-attractant or an inducer. The in vitro roles were verified by migration assay using KIM1-PK1 cell lines, the mouse proximal tubular epithelial cells (mPTEs) and recombinant human KIM-1 proteins (rhKIM-1). Immunofluorescence staining displayed specific ectodomain binding of KIM-1 on the surface of MSCs. Upregulation of chemokine receptor type 4 (CXCR4) protein when treated with tumor necrosis factor alpha (TNF-α) was shown. The effect of KIM-1 on migration of MSCs was augmented by TNF-α pretreatment in a dose-dependent manner, and reduced by AMD3100, an antagonist of CXCR4. These results suggest that KIM-1 is a potential chemo-ligand of CXCR4 and may play an important role in kidney-specific migration of MSCs via interaction between KIM-1 and CXCR4.

Ultrasonicated graphene oxide enhances bone and skin wound regeneration

In the present study, we investigated the applications of ultrasonicated graphene oxide (UGO) for bone regeneration and skin wound healing. Ultrasonication of a GO suspension increased the dispersion and stability (by increasing the zeta potential) of the GO suspension. UGO has fewer oxygen-containing groups but still displays excellent water dispersion. The UGO supension showed high biocompatibility for human fetal osteoblast (hFOB cells), human endothelial cells (EA.hy 926 cells), and mouse embryonic fibroblasts. Importantly, UGO could support cell attachment and proliferation, in addition to promoting the osteogenesis of seeded cells and the promotion of new bone formation. In addition, a 1% UGO supension enhanced cell migration in an in vitro skin scratch assay and promoted wound closure in an in vivo rat excisional skin defect model. These results showed that UGO offers a good environment for cells involved in bone and skin healing, suggesting its potential application in tissue regeneration.

A novel mouse model of diabetes mellitus using unilateral nephrectomy.

Diabetes mellitus (DM) is a major cause of morbidity and mortality worldwide, and its complications are prominent public health issues. Many experimental models of streptozotocin (STZ)-induced and high-fat diet (HF)-induced DM have been used to study this disease. Studies have indicated that unilateral nephrectomy (UN) accelerates the development of diabetic nephropathy. We hypothesized that UN stimulates HF and STZ combination-induced DM in mice. Seventy-two female C57BL/6J mice were divided into four treatment groups: HF; HF + STZ120 (HF and STZ, 120 mg/kg); UN + HF + STZ120 (UN, HF and STZ, 120 mg/kg); and HF + STZ200 (HF and STZ, 200 mg/kg). Onset of DM, survival rate, blood pressure, urine glucose level, and pancreatic histology were investigated. Additionally, renal function was evaluated in the UN + HF + STZ120 group after STZ injection. DM was induced in the UN + HF + STZ120 and HF + STZ200 groups within one week. The UN + HF + STZ120 group had lower mortality than the HF + STZ200 group and greater pancreatic destruction than the HF and HF + STZ120 groups. Two weeks after STZ injection, blood pressure was not significantly different among the groups. Nephrotoxicity associated with the combination of UN and STZ was not observed. In conclusion, the combination of these three techniques – UN, HF and STZ induced DM rapidly and effectively.