GTKO rabbit: A novel animal model for preclinical assessment of decellularized xenogeneic grafts via in situ implantation.

Wild type (WT) animals cannot be used to objectively assess the immunogenicity of animal tissue-derived biomaterials when used as recipients due to difference with human in α-Gal expression. The purpose of this study is to compare the differences of immunological responses between the GGTA1 gene-knockout (GTKO) rabbits and WT rabbits after implantation with animal tissue-derived biomaterials. The porcine-derived decellularized bone matrix (natural bone material, NBM) and fresh porcine cancellous bone (PCB) were implanted in GTKO rabbits and WT rabbits, respectively, and sham operation was used as control (Con). At 2- and 6-week post-implantation, the related immunological items including antibody levels, serum-mediated cell lysis, cytokines, lymphocyte subtypes, and histopathological changes were assessed. GTKO rabbits exhibited more sensitive immune responses than WT rabbits after PCB implantation, resulted from a significant increase of antibodies (except total antibodies) and cytokines levels, cell lysis ratios, CD4/CD8 proportions, and inflammatory cells infiltration. Immunological factors and inflammatory cells infiltrate in GTKO rabbits after NBM implantation were significantly lower than those in the PCB group. Among the three groups, the NBM group showed the highest contents of new bone formation elements. In conclusion, the GTKO rabbit is a more sensitive alternative model than WT rabbit for preclinical study of xenografts via in situ implantation. Studies on multiple gene-edited animals are also necessary for more comprehensively evaluating xenoimmunologen risks of animal tissue-derived biomaterials in the future. Additionally, the immunogenicity of NBM was remarkably decreased compared to PCB.

α-Gal antigen-deficient rabbits with GGTA1 gene disruption via CRISPR/Cas9.

BACKGROUND: Previous studies have identified the carbohydrate epitope Galα1-3Galß1-4GlcNAc-R (termed the α-galactosyl epitope), known as the α-Gal antigen as the primary xenoantigen recognized by the human immune system. The α-Gal antigen is regulated by galactosyltransferase (GGTA1), and α-Gal antigen-deficient mice have been widely used in xenoimmunological studies, as well as for the immunogenic risk evaluation of animal-derived medical devices. The objective of this study was to develop α-Gal antigen-deficient rabbits by GGTA1 gene editing with the CRISPR/Cas9 system. RESULTS: The mutation efficiency of GGTA1 gene-editing in rabbits was as high as 92.3% in F0 pups. Phenotype analysis showed that the α-Gal antigen expression in the major organs of F0 rabbits was decreased by more than 99.96% compared with that in wild-type (WT) rabbits, and the specific anti-Gal IgG and IgM antibody levels in F1 rabbits increased with increasing age, peaking at approximately 5 or 6 months. Further study showed that GGTA1 gene expression in F2-edited rabbits was dramatically reduced compared to that in WT rabbits. CONCLUSIONS: α-Gal antigen-deficient rabbits were successfully generated by GGTA1 gene editing via the CRISPR/Cas9 system in this study. The feasibility of using these α-Gal antigen-deficient rabbits for the in situ implantation and residual immunogenic risk evaluation of animal tissue-derived medical devices was also preliminarily confirmed.

Immunological Risk Assessment of Xenogeneic Dural Patch by Comparing with Raw Material via GTKO Mice.

OBJECTIVE: In this study, α-Gal epitope-deficient (GGTA1 knockout (GTKO)) mice were used to assess the immunological risks of xenogeneic dural patch by comparing with raw material. METHODS: The xenogeneic dural patch (T2) was prepared from bovine pericardium (T1, raw material) through decellularization and carboxymethyl chitosan (CMCS) coating. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the collagen fibers and surface microstructural changes in the T1 and T2 samples. The remnant α-Gal epitopes and DNA of implants were detected by standardized method. T1 and T2 were implanted subcutaneously into GTKO mice for 4 and 12 weeks, respectively, and the negative control group (Con) was only performed sham operation. The total serum antibody, anti-Gal antibody, and splenic lymphocyte subtypes were analyzed by ELISA or flow cytometry, and histological analysis of implant-tissue was performed by H&E and Masson stain. RESULTS: TEM and Sirius red staining showed that the collagen fibers in the dural patch were closely arranged, and SEM showed that a loose three-dimensional structure was successfully constructed on the surface of the dural patch after CMCS coating. The remnant DNA in T2 was 24.64 ± 8.73 ng/mg (dry weight), and clearance of α-Gal epitope was up to 99.83% compared to T1. The significant increases in serum total IgM, anti-Gal IgG, and anti-Gal IgM at 4 weeks and the significant changes in anti-Gal IgG and spleen lymphocyte at 12 weeks were observed in the T1 group, but no significant change was observed in the T2 group, compared to the control group. Histological semiquantitative analysis showed severe cell and tissue responses at 4 weeks and a moderate response at 12 weeks in the T1 group, while a moderate response at 4 weeks and a slight response at 12 weeks in the T2 group. CONCLUSIONS: The results demonstrated that the xenogeneic dural patch has a lower and acceptable immunological risk compared to the raw material and control, respectively. On the other hand, it was suggested that GTKO mice are useful experimental model for immunological risk assessment of animal tissue-derived biomaterials.

Immune risk assessment of residual αGal in xenogeneic decellularized cornea using GTKO mice.

The xenogeneic decellularized corneal matrix (DCM) was expected to be used in lamellar keratoplasty in clinic as the substitute of allogeneic cornea. After decellularization treatment, the remaining risk of xenograft rejection needed to be assessed. The galactose-α1,3-galactose, as the most abundant and closely rejection-related xenogeneic antigen, should be one of the important factors concerned in immunological evaluation. In this study, residual αGal in the DCM was first determined by an enzyme-linked immunosorbent assay method with qualified accuracy and specificity. Then the DCM was implanted subcutaneously into the α1,3-galactosyltransferase gene-knockout (GTKO) mice, accompanied by the implantation in the wild-type C57BL/6 mice as a comparison. The total serum antibody levels, anti-Gal antibody levels, inflammatory cytokines and ratios of splenic lymphocyte subtypes were detected and the histopathological analysis of implants were performed to systematically evaluate the immune responses. The experimental result showed the fresh porcine corneal matrix samples had (9.90 ± 1.54) × 1012 αGal epitope per mg while the content of residual αGal in the DCM was (7.90 ± 2.00) × 1012 epitope per mg. The GTKO mice had similar potential of reaction to immune stimulation to that of wild-type C57BL/6 mice. At 4 weeks after implantation of DCM, in WT mice and GTKO mice there were both innate immunity response to the DCM characterized by macrophage infiltration. But the elevations of anti-Gal IgG level and the percentage of splenic natural killer cells were only detected in GTKO mice. These changes were thought to be pertinent to the residual αGal antigen, which could not be detected in WT mice. No further αGal antibody-mediated cellular immunity and significant changes of serum cytokine contents were found in GTKO mice, which perhaps suggested that the immune reactions to the DCM after 4 weeks of implantation were moderate and had minor effect on the survival of the corneal graft.

GGTA1/iGb3S Double Knockout Mice: Immunological Properties and Immunogenicity Response to Xenogeneic Bone Matrix.

BACKGROUND: Animal tissues and tissue-derived biomaterials are widely used in the field of xenotransplantation and regenerative medicine. A potential immunogenic risk that affects the safety and effectiveness of xenografts is the presence of remnant α-Gal antigen (synthesized by GGTA1 or/and iGb3S). GGTA1 knockout mice have been developed as a suitable model for the analysis of anti-Gal antibody-mediated immunogenicity. However, we are yet to establish whether GGTA1/iGb3S double knockout (G/i DKO) mice are sensitive to Gal antigen-positive xenoimplants. METHODS: α-Gal antigen expression in the main organs of G/i DKO mice or bovine bone substitutes was detected via a standardized ELISA inhibition assay. Serum anti-α-Gal antibody titers of G/i DKO mice after immunization with rabbit red blood cells (RRBC) and implantation of raw lyophilized bone substitutes (Gal antigen content was 8.14 ± 3.17 × 1012/mg) or Guanhao Biotech bone substitutes (50% decrease in Gal antigen relative to the raw material) were assessed. The evaluation of total serum antibody, inflammatory cytokine, and splenic lymphocyte subtype populations and the histological analysis of implants and thymus were performed to systematically assess the immune response caused by bovine bone substitutes and bone substitute grafts in G/i DKO mice. RESULTS: α-Gal epitope expression was reduced by 100% in the main organs of G/i DKO mice, compared with their wild-type counterparts. Following immunization with RRBC, serum anti-Gal antibody titers of G/i DKO mice increased from 80- to 180-fold. After subcutaneous implantation of raw lyophilized bone substitutes and Guanhao Biotech bone substitutes into G/i DKO mice, specific anti-α-Gal IgG, anti-α-Gal IgM, and related inflammatory factors (IFN-γ and IL-6) were significantly increased in the raw lyophilized bone substitute group but showed limited changes in the Guanhao Biotech bone substitute group, compared with the control. CONCLUSION: G/i DKO mice are sensitive to Gal antigen-positive xenogeneic grafts and can be effectively utilized for evaluating the α-Gal-mediated immunogenic risk of xenogeneic grafts.

A standardized quantitative method for detecting remnant alpha-Gal antigen in animal tissues or animal tissue-derived biomaterials and its application.

Alpha-Gal (Gal) epitopes present in animal tissues are known to be the key xenoantigens that elicit xenorejection. However, a standardized method to determine Gal epitope in animal tissue-derived biomaterials does not exist. Herein, a standardized method for quantitative detection of Gal antigen was established based on an ELISA inhibition assay with Gal antibody. In this method, the key optimized experimental conditions were: (1) Gal-antigen positive and negative reference materials were developed, and used as positive and negative control in the test system, respectively; (2) A mixture of artificial Gal-BSA antigen plus Gal-negative matrix was used as the calibration standard sample, making it has similar composition with test sample; and (3) The lysis buffer was combined with the homogenate to expose the Gal antigen as much as possible. The results from validation and application experiments showed that the standardized method had good reproducibility (RSD = 12.48%), and the lower detection limit (LDL) is ~7.1 × 1011 Gal epitopes/reaction. This method has been further developed into a detection Kit (Meitan 70101, China), and it has been developed as a standard method for detecting remnant immunogen of animal tissue derived medical devices, and as the industry standard has been released in China. (YY/T 1561-2017).

Gal epitope expression and immunological properties in iGb3S deficient mice.

The Gal antigen is synthesized by glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) or (and) isoglobotrihexosylceramide 3 synthase (iGb3S). However, whether iGb3S deletion changes Gal epitope expression and immunological properties in animals is still not clear. The objective of this study was to develop iGb3S deficient mice, and characterize their Gal epitope expression and Gal epitope-related immunological properties. iGb3S gene knockout mice were generated on the C57BL/6 background using the bacterial artificial chromosome homology region recombination technique. Gal epitope expression in the iGb3S deficient mice was determined by using a monoclonal anti-Gal antibody. Immunological properties were analyzed by enzyme linked immune sorbent assay. It was found that Gal epitope expression was decreased from 5.19% to 21.74% in the main organs of iGb3S deficient mice, compared with that of C57BL/6 wild type mice, suggesting that the iGb3S gene participated to Gal epitope expression. However, iGb3S deletion alone did not cause significant changes in the immunological properties of iGb3S deficient mice with or without exogenous Gal antigen (Rabbit Red Blood Cell) stimulation. The data from this study suggest that the iGb3S gene likely contributes to Gal epitope expression, but may have a very weak effect on immunological properties of the iGb3S deficient mice.

Xenogeneic bone matrix immune risk assessment using GGTA1 knockout mice.

Homeotransplantation of bones for replacement therapy have been demonstrated reliably in clinical data. However, human donor bones applicable for homeotransplantation are in short supply, which facilitates the search for suitable alternatives, such as xenografts grafts. The α-Gal antigen-related immune risk of xenografts directly affects the safety and effectiveness of the biomaterials and limits their applications in the clinic. The immune risk can be prevented by depletion or breaking anti-Gal antibody prior to transplant. Therefore, how to assess the immune risk of the bone substitutes and select the reliable animal research model become extremely important. In this study, we prepared lyophilized bone substitutes (T1) and Guanghao Biotech bone substitutes (T2, animal-derived biomaterials with α-Gal antigen decreased), aimed to assess the immune risk of xenografts bone substitutes on GGTA1 knockout mice. The α-Gal antigen contents of T1 and T2 were firstly detected by ELISA method in vitro. The bone substitutes were then implanted subcutaneously into GGTA1 knockout mice for 2, 4 and 12 weeks, respectively. The total serum antibody levels, anti-α-Gal antibody levels, inflammatory cytokine and splenic lymphocyte surface molecules were detected and histology analysis of skin and thymus were performed to systematically evaluate the immune response caused by the T1 and T2 bone substitutes in mice. In vitro results showed that the amount of α-Gal epitopes in T1 bone substitutes was significantly higher than T2 bone substitutes, and the clearance rate of α-Gal antigen in T2 bone substitutes achieved about 55.6%. Results of antibody level in vivo showed that the T1 bone substitutes group possessed significantly higher total IgG, IgM, IgA and anti-α-Gal IgG levels than T2 and control group, while T2 group showed no significant changes of these indexes compared with control. In terms of inflammatory cytokines, T1 bone substitutes showed evidently higher levels of IL-4, IL-12P70 and IL-10 than T2 and control, while T2 group was comparable to control. No changes in the levels of splenic lymphocyte surface molecules were found in the three groups (T1, T2 and control group) during the experimental periods. The pathological results demonstrated that the inflammatory response in T2 group was lighter than the T1 group, which was in accordance with the inflammatory cytokines levels. The above results indicated that the process of antigen removal effectively reduced the α-Gal antigens content in T2 bone substitutes, which caused little immune response in vivo and could be used as bone healing materials. This study also demonstrated that GGTA1 knockout mice can be used as a routine tool to assess the immune risk of animal-derived biomaterials.

Silver Nanoparticle Exposure Induces Neurotoxicity in the Rat Hippocampus Without Increasing the Blood-Brain Barrier Permeability.

Silver nanoparticles (Ag-NPs) can enter the brain and subsequently induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the hippocampus in vivo are not well understood. To investigate Ag-NP suspension (Ag-NPS)-induced toxicity on the BBB and neurons, Sprague-Dawley rats were randomly divided into 3 groups, and Ag-NPS, Ag-ion, and 5% sucrose solution (vehicle control) were administrated intravenously, respectively. After 24 h exposure to Ag-NPS, the BBB permeability was not significantly changed. However, the Ag concentrations in the brain tissues were only detected in the Ag-NPS group. Gene expression results indicated that the expression of Claudin 4 (tight junction protein) was significantly decreased. Furthermore, astrocyte foot swelling, neuron shrinkage and Ag-NP like particles were observed under transmission electron microscopy. Global gene expression analysis showed that 502 genes were up-regulated and 703 genes were down-regulated in the hippocampi treated with Ag-NPS. In the Ag-NPS-treated group, 78 biological functions were changed based on gene ontology (GO) and 34 signaling pathways were significantly changed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, which were associated with the neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, calcium signaling pathway and MAPK signaling pathway. In comparison, 27 GO and 9 KEGG pathways were changed in the released Ag-ion-treated group. Ag-NPS decreased C1qtnf3 expression and increased Adra1d expression to affect MAPK signaling pathway, which promoted inflammation and apoptosis in the hippocampus. Moreover, Ag-NPS significantly increased Spp1, Cacna1s and Tacr3 mRNAs expression, which may result in intracellular calcium increasing and initiate cell death. Furthermore, Ag-NPS affected calcium signaling pathway and neuroactive ligand-receptor (Grin2a, Drd2, and Adra1d), which are crucial in diverse cellular functions in the brain including cognition and neurodevelopment. These results draw our attention to the importance of Ag-NP-induced toxicity in the rat hippocampus and provide a better understanding of its toxicological mechanisms in vivo.

[Evaluation of Material Permeability of Type I Collagen Hydrogel].

OBJECTIVES: To establish an experimental method for evaluating material permeability of type I collagen hydrogels. METHODS: Using BSA-FITC as an indicator, by combining BSA-FITC with PBS they were used as permeability media, and using transwell load hydrogen sample to detect BSA-FITC transparent rate. RESULTS: In the concentration range of 100 µg·mL-1~0.781 µg·mL-1, the standard curve R2 ≥ 0.99, Lower Limit of Quantity (LLOQ) is 3.125 µg·mL-1, RSD <5%, detection recovery rate is in the range of 80%~120%. CONCLUSIONS: In this study, we established an experimental method for evaluating material permeability of hydrogel. The BSA-FITC transparent rate of type I collagen hydrogel was 100% at 28 h.

A Tiered Experimental Approach for Characterization and Silver Release of Silver-Containing Wound Dressings.

Silver-containing dressings are widely used for wound care owing to their broad-spectrum microbicidal activity. However, the potential adverse effects on human health emerging from exposure to their active ingredients (silver ions or nanoparticles) have resulted in widespread concerns about their use. The release profiles of various chemical forms of silver (Ag) from silver-containing dressing are closely related to their bioavailability and potential adverse effects on the body. In this research, we demonstrated a tiered experimental approach for systematic characterization and assessment of silver-containing wound dressing, which provides information for risk assessments. The combination of scanning electron microscopy, transmission electron microscopy and energy dispersive X-ray, X-ray photoelectron spectroscopy, and X-ray diffraction allowed for systematic characterization of silver-containing dressings including their morphology, size, composition, valence state, and crystal forms, which have a close relationship with their silver release profile, antimicrobial activity, and potential toxicity. We developed an Ag release experiment using a reciprocating holder method and an ultrafiltration membrane column to separate the silver nanoparticles (AgNPs) and silver ions released from the dressing. Furthermore, by adjusting the Ag-ion concentration in the release media (simulated body fluid) we could effectively eliminate the interference from AgCl particles, which have a similar size to the AgNPs. We used our method to analyze the release profiles and the chemical form of the Ag present in three commercial silver-containing dressings. A large variation in the total Ag content, amount of released Ag-ions, and amount of released AgNPs was observed in the three dressings that were tested. The silver release profiles were highly dependent on the crystal structures, surface coating process, and binding modes. The cytotoxicity assays were consistent with the characterization data. This tiered approach provides valuable information of optimized AgNPs usage and proper manufacturing process for further safe applications. This study establishes a systematic characterization methodology for better understanding of risk assessment of nano-embeded consumer products.

Acute toxicity and genotoxicity of silver nanoparticle in rats.

OBJECTIVE: The potential risk of a nanoparticle as a medical application has raised wide concerns, and this study aims to investigate silver nanoparticle (AgNP)-induced acute toxicities, genotoxicities, target organs and the underlying mechanisms. METHODS: Sprague-Dawley rats were randomly divided into 4 groups (n = 4 each group), and AgNP (containing Ag nanoparticles and released Ag+, 5 mg/kg), Ag+ (released from the same dose of AgNP, 0.0003 mg/kg), 5% sucrose solution (vechicle control) and cyclophophamide (positive control, 40 mg/kg) were administrated intravenously for 24 h respectively. Clinical signs and body weight of rats were recorded, and the tissues were subsequently collected for biochemical examination, Ag+ distribution detection, histopathological examination and genotoxicity assays. RESULTS: The rank of Ag detected in organs from highest to lowest is lung>spleen>liver>kidney>thymus>heart. Administration of AgNP induced a marked increase of ALT, BUN, TBil and Cre. Histopathological examination results showed that AgNP induced more extensive organ damages in liver, kidneys, thymus, and spleen. Bone marrow micronucleus assay found no statistical significance among groups (p > 0.05), but the number of aberration cells and multiple aberration cells were predominately increased from rats dosed with Ag+ and AgNP (p < 0.01), and more polyploidy cells were generated in the AgNP group (4.3%) compared with control. CONCLUSION: Our results indicated that the AgNP accumulated in the immune system organs, and mild irritation was observed in the thymus and spleen of animals treated with AgNP, but not with Ag+. The liver and kidneys could be the most affected organs by an acute i.v. dose of AgNP, and significantly increased chromosome breakage and polyploidy cell rates also implied the potential genotoxicity of AgNP. However, particle-specific toxicities and potential carcinogenic effect remain to be further confirmed in a chronic toxicity study.

[Assessment Method of Remnant α-1, 3-galactosyle Epitopes in Animal Tissue-derived Biomaterials].

The aim of this study was to establish an assessment method for determining α-Gal (α-1, 3-galactosyle) epitopes contained in animal tissue or animal tissue-derived biological materials with ELISA inhibition assay. Firstly, a 96 well plate was coated with Gal α-1, 3-Gal/bovine serum albumin (BSA) as a solid phase antigen and meanwhile, the anti-α-Gal M86 was used to react with α-Gal antigens which contained in the test materials. Then, the residual antibodies (M86) in the supernatant of M86-Gal reaction mixture were measured using ELISA inhibition assay by the α-Gal coating plate. The inhibition curve of the ELISA inhibition assay, the R2 = 0.999, was well established. Checking using both α-Gal positive materials (rat liver tissues) and α-Gal negative materials (human placenta tissues) showed a good sensitivity and specificity. Based on the presently established method, the α-Gal expression profile of rat tissues, decellular animal tissue-derived biological materials and porcine dermal before and after decellular treatment were determined. The M86 ELISA inhibition assay method, which can quantitatively determine the α-Gal antigens contained in animal tissues or animal tissue-derived biomaterials, was refined. This M86 specific antibody based-ELISA inhibition assay established in the present study has good sensitivity and specificity, and could be a useful method for determining remnant α-1, 3Gal antigens in animal tissue-derived biomaterials.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

BACKGROUND: Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. METHOD: To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. RESULTS: A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm(2). After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. CONCLUSION: These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

Neurotoxicity of Silver Nanoparticles in Rat Brain After Intragastric Exposure.

It is known that the biological half-life of silver in the central nervous system is longer than in other organs. However, the potential toxicity of silver nanoparticles (NPs) on brain tissue and the underlying mechanism(s) of action are not well understood. In this study, neurotoxicity of silver NPs was examined in rat after intragastric administration. After a two-week exposure to low-dose (1 mg/kg, body weight) or high-dose (10 mg/kg) silver NPs, the pathological and ultrastructural changes in brain tissue were evaluated with H&E staining and transmission electron microscopy. The mRNA expression levels of key tight junction proteins of the blood-brain barrier (BBB) were analyzed by real-time RT-PCR, and several inflammatory factors were assessed in blood using ELISA assay. We observed neuron shrinkage, cytoplasmic or foot swelling of astrocytes, and extra-vascular lymphocytes in silver NP exposure groups. The cadherin 1 (2(-ΔΔCt): 1.45-fold/control) and Claudin-1 (2(-ΔΔCt): 2.77-fold/control) were slightly increase in mRNA expression levels, and IL-4 significantly increased after silver NP exposure. It was suggest that silver NP can induce neuronal degeneration and astrocyte swelling, even with a low-dose (1 mg/kg) oral exposure. One potential mechanism for the effects of silver NPs to the nervous cells is involved in inflammatory effects.

[Overview on the market, supervision and standardization of nanomaterial-contained medical devices].

In this paper, industry development and market tendency, supervision and standardization of nanomaterial-contained medical devices are overviewed comprehensively based on a large number of reference data including national and international information. Furthermore, the consideration about standardization of biological evaluation for nanomaterial-contained medical devices is discussed by combined some works performed in our laboratory.

Toxic responses in rat embryonic cells to silver nanoparticles and released silver ions as analyzed via gene expression profiles and transmission electron microscopy.

After exposing rat embryonic cells to 20 µg/mL of silver nanoparticle (NP) suspension and their released ions for different time periods, silver nanoparticles were found in cellular nuclei, mitochondria, cytoplasm and lysosomes by transmission electron microscopy (TEM). We also observed mitochondrial destruction, distension of endoplasmic reticulum and apoptotic bodies. Global gene expression analysis showed a total of 279 genes that were up-regulated and 389 genes that were down-regulated in the silver-NP suspension exposure group, while 3 genes were up-regulated and 41 genes were down-regulated in the silver ion exposure group. Further, the GO pathway analysis suggested that these differentially expressed genes are involved in several biological processes, such as energy metabolism, oxygen transport, enzyme activities, molecular binding, etc. It is possible that inhibition of oxygen transport is mediated by the significant down-regulation of genes of the globin family, which might play an important role in silver ion-induced toxicity. KEGG pathway analysis showed that there were 23 signal pathways that were affected in the cells after exposure to silver-NP suspension, but not silver ion alone. The most significant change concerned inflammatory signal pathways, which were only found in silver-NP suspension exposed cells, indicating that inflammatory response might play an important role in the mechanism(s) of silver-NP-induced toxicity. The significant up-regulation of matrix metalloproteinases 3 and 9 suggests that silver NPs could induce extracellular matrix degradation via an inflammatory signaling pathway. The significant up-regulation of secretory leukocyte peptidase inhibitor and serine protease inhibitor 2c was considered to be an embryonic cellular defense mechanism in response to silver-NP-induced inflammation.

Cytotoxic and genotoxic effects of silver nanoparticles on primary Syrian hamster embryo (SHE) cells.

Silver-nanoparticles (NPs) have become increasingly common in various applications, raising some safety concerns. In this study, the cytotoxic and genotoxic effects of silver-NPs on primary Syrian hamster embryo (SHE) cells were investigated. Cell viability was assessed using a methyl tetrazolium (MTT) assay, and genotoxic potential was evaluated using a cytokinesis-block micronucleus (CBMN) assay. The results showed that dose-dependent cytotoxicity was induced after 24 h of exposure to silver-NPs. The micronucleation frequency (MNF) also increased significantly in a dose-dependent manner (P < 0.05), suggesting that silver-NPs induce genotoxicity. This is consistent with an increased MNF observed in primary SHE cells. The results of cell cycle analysis indicate that the cell cycles became arrested in the GO/G1 phase and that the S phase shortened after only 8 h of silver-NP exposure, suggesting that DNA replication had been inhibited, which in turn inhibited further cell proliferation. The rate of late-stage apoptosis increased after 12 h of silver-NP exposure, and both early- and late-stage apoptosis were obviously increased after 72 h of exposure than in controls. This study demonstrated that silver-NPs could induce strong cytotoxicity and significant genotoxicity in primary SHE cells and that this is probably due to silver-NP-induced apoptosis and the inhibition of cell proliferation.

[Quantification of residues DNA in animal-derived biological scaffold materials].

Quantification of residual DNA in animal-derived biological scaffold materials is one of technical specifications for evaluating decellularization process and immunotoxicity risk. Up to now, there have been no standard methods available for quantification of residues DNA in animal-derived biological scaffold materials. In this study, a three-step method, including proteinase K digestion, DNA purification and determination of DNA using fluorescence assay, was designed for residual DNA quantification. A parallel recovery experiment of standard DNA using the same protocol to test article determination was used for adjusting final results of residuul DNA amount. DNA purification based on magnetic beads enabled the experiments to get high accuracy and repeatability. The validation experiment showed that the three-step method had high sensitivity up to 6.25ng of DNA per sample with good linearity (recovery curve R2 > 0.99) in the concentration range of 3. 125-100ng, and 25-400ng per sample. This method is useful for determining micro or trace amount DNA remained in the biomaterials.