Rong-Nan Huang

In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation

A recognized drawback of the currently available chemical cross-linking reagents used to fix bioprostheses is the potential toxic effects a recipient may be exposed to from the fixed tissues and/or the residues. It is, therefore, desirable to provide a cross-linking reagent which is of low cytotoxicity and may form stable and biocompatible cross-linked products. To achieve this goal, a naturally occurring cross-linking reagent -- genipin -- which has been used in herbal medicine and in the fabrication of food dyes, was used by our group to fix biological tissues. The study was to assess the cytotoxicity of genipin in vitro using 3T3 fibroblasts (BALB/3T3 C1A31-1-1). Glutaraldehyde, the most commonly used cross-linking reagent for tissue fixation, was used as a control. The cytotoxicity of the glutaraldehyde- and genipin-fixed tissues and their residues was also evaluated and compared. The observation in the light microscopic examination revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde. Additionally, the results obtained in the MTT assay implied that genipin was about 10000 times less cytotoxic than glutaraldehyde. Moreover, the colony forming assay suggested that the proliferative capacity of cells after exposure to genipin was approximately 5000 times greater than that after exposure to glutaraldehyde. It was noted that the cells seeded on the surface of the glutaraldehyde-fixed tissue were not able to survive. In contrast, the surface of the genipin-fixed tissue was found to be filled with 3T3 fibroblasts. Additionally, neocollagen fibrils made by these fibroblasts were observed on the genipin-fixed tissue. This fact suggested that the cellular compatibility of the genipin-fixed tissue was superior to its glutaraldehyde-fixed counterpart. Also, the residues from the glutaraldehyde-fixed tissue markedly reduced the population of the cultured cells, while those released from the genipin-fixed tissue had no toxic effect on the seeded cells. In conclusion, as far as cytotoxicity is concerned, genipin is a promising cross-linking reagent for biological tissue fixation.

Evaluation of gelatin hydrogel crosslinked with various crosslinking agents as bioadhesives: in vitro study.

Bioadhesives are used for tissue adhesion and hemostasis in surgery. A gelatin-resorcinol mixture crosslinked with formaldehyde (GRF glue) and/or glutaraldehyde (GRG) is used for this purpose. Although the bonding strength of the GRF glue to tissue is satisfactory, concerns about the cytotoxicity of formaldehyde are reported in the literature. It was suggested that the cytotoxicity problem of the GRF glue may be overcome by changing its crosslinking method. The study was therefore undertaken to assess the feasibility of using an epoxy compound (GRE glue), a water-soluble carbodiimide (GAC glue), or genipin (GG glue) to crosslink with a gelatin hydrogel as new bioadhesives. GRF glue and GRG glue were used as controls. The results of our cytotoxicity study suggested that the cellular compatibility of the GAC and GG glues was superior to the GRF, GRG, and GRE glues. The gelation time for the GG glue was relatively longer than the GRF and GRG glues, while no gelation time could be determined for the GAC glue. Additionally, it took approximately 17 h for the GRE glue to become adhesive. The GRF and GRG glues had the greatest bonding strengths to tissue among all test adhesives, while the bonding strengths of the GAC and GG glues were comparable. In contrast, there was almost no bonding strength to tissue for the GRE glue. However, the GRF and GRG glues were less flexible than the GAC and GG glues. Subsequent to the bonding strength measurement, each test adhesive was found to adhere firmly to the tissue surface and underwent cohesive failure during the bond breaking. In conclusion, the GRF and GRG glues may be used as tissue adhesives when their ability to bind tissue rapidly and tightly is required; the GAC and GG glues are preferable when the adhesive action must be accompanied with minimal cytotoxicity and stiffness; and the GRE glue is not suitable for bioadhesion in clinical applications.

Feasibility study of a natural crosslinking reagent for biological tissue fixation

Bioprostheses derived from biological tissues must be chemically modified and subsequently sterilized before they can be implanted in humans. Various crosslinking reagents, including formaldehyde, glutaraldehyde, dialdehyde starch, and epoxy compound, have been used to chemically modify biological tissues. However, these synthetic crosslinking reagents are all highly (or relatively highly) cytotoxic. It is therefore desirable to provide a crosslinking reagent suitable for use in biomedical applications that is of low cytotoxicity and that forms stable and biocompatible crosslinked products. This study evaluates the feasibility of using a naturally occurring crosslinking reagent--genipin--to chemically modify biological tissues. Genipin and its related iridoid compounds, extracted from gardenia fruits, have been used in traditional Chinese medicine for the treatments of jaundice and various inflammatory and hepatic diseases. In this feasibility study, the cytotoxicity of genipin and the crosslinking characteristics of genipin-fixed biological tissues were investigated. Fresh porcine pericardia procured from a slaughterhouse were used as raw materials. Glutaraldehyde and an epoxy compound (ethylene glycol diglycidyl ether), which has been used extensively in developing bioprostheses, were used as controls. It was found that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde and the epoxy compound. The amino acid residues in the porcine pericardium that may react with genipin were lysine, hydroxylysine, and arginine. Additionally, the genipin-fixed tissue had a mechanical strength and resistance against enzymatic degradation comparable to the glutaraldehyde-fixed tissue. This suggests that genipin can form stable crosslinked products. The results of this in vitro study demonstrate that genipin is an effective crosslinking reagent for biological tissue fixation.

Stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin)

The study was undertaken to investigate the stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin) at distinct elapsed storage durations. The glutaraldehyde-fixed counterpart was used as a control. Porcine pericardia procured from a slaughterhouse were used as raw materials. After fixation, the fixed tissues were sterilized in a graded series of ethanol solutions and thoroughly rinsed in phosphate buffered saline for 1 day, and then stored in a jar containing sterilized water. The samples were taken out and tested for their stability during the durations of 1day through 6 months after storage. The stability of each study group was tested by measuring its tensile strength, free-amino-group content, and denaturation temperature. Additionally, the cytotoxicity of each test sample and its corresponding storage solution were investigated in vitro using 3T3 fibroblasts. The results were examined using a microscope and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the stability of the genipin-fixed tissue during storage was superior to its glutaraldehyde-fixed counterpart. The differences in stability between the genipin- and glutaraldehyde-fixed tissues during storage may be caused by their differences in crosslinking structure. There was no apparent cytotoxicity for both the genipin-fixed tissue and its corresponding storage solution throughout the entire course of the study, whereas significant cytotoxicity was observed for both the glutaraldehyde-fixed tissue and its storage solution. However, the cytotoxicity of the glutaraldehyde-fixed tissue decreased with increasing elapsed storage duration, whereas that of its corresponding storage solution increased. This suggested that the toxic residues remaining in the glutaraldehyde-fixed tissue leached out slowly into its corresponding storage solution during the course of storage.

In vitro evaluation of a chitosan membrane cross-linked with genipin

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties, antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.

In vitro evaluation of the genotoxicity of a naturally occurring crosslinking agent (genipin) for biologic tissue fixation.

The objective of the present study was to evaluate in vitro, using Chinese hamster ovary (CHO-K1) cells, the genotoxicity of genipin, a naturally occurring crosslinking agent. Glutaraldehyde, the most commonly used crosslinking agent for biologic tissue fixation, was employed as a reference chemical. The selected procedures for this evaluation were the micronucleus (MN) and sister chromatid exchange (SCE) assays with or without the addition of a metabolic activation system (S9 mix). Before starting the genotoxicity assays, the maximum noncytotoxic amounts of glutaraldehyde and genipin were determined using the MTT assay. The results obtained in the MTT assay revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde with or without S9 mix. The frequencies of MN observed in the cases drugged with varying concentrations of glutaraldehyde or genipin were not statistically different from those seen in the negative controls (blank) in the presence or absence of S9 mix. However, it was noted that glutaraldehyde significantly inhibited the cell-cycle progression while the cells drugged with genipin did not result in cell-cycle delay. In the SCE assay, the numbers of SCE per cell observed in the cases drugged with varying concentrations of glutaraldehyde were significantly greater than those found in the negative controls with or without S9 mix. Nevertheless, these numbers were still low compared to the numbers of SCE induced by the strong mutagens used as our positive control substances. This suggests that glutaraldehyde may produce a weakly clastogenic response in CHO-K1 cells. In contrast, the numbers of SCE per cell obtained in the cases drugged with genipin were comparable to those observed in the negative controls in those that were except drugged with the highest dose (50 ppm). This suggests that genipin does not cause clastogenic response in CHO-K1 cells provided its concentration is lower than 50 ppm. In conclusion, as far as cytotoxicity and genotoxicity are concerned, genipin is a promising crosslinking agent for biologic tissue fixation.

In vitro surface characterization of a biological patch fixed with a naturally occurring crosslinking agent

The study was designed to characterize the surface properties (including water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility) of a biological patch fixed with genipin, a naturally occurring crosslinking agent. Fresh and glutaraldehyde-fixed counterparts were used as controls. It was found that both glutaraldehyde and genipin are effective crosslinking agents for biological tissue fixation. Fixation of biological tissue with glutaraldehyde or genipin significantly increased its hydrophilicity and surface tension and reduced its mol ratio of adsorbed fibrinogen to adsorbed albumin as well as the amount of adhered platelet. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues. However, the cellular compatibilities of fresh and the genipin-fixed tissues were significantly superior to the glutaraldehyde-fixed tissue.

PPARgamma rescue of the mitochondrial dysfunction in Huntington's disease.

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The resultant mutant Htt protein (mHtt) forms aggregates in the brain (e.g., cortex and striatum), and causes devastating neuronal degeneration. Transcriptional dysfunction caused by mHtt is critical for HD. We recently demonstrated that a crucial transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) played a major function in the energy homeostasis observed in HD and that PPARγ is a potentially neuroprotective target for this disease. We report here that the transcript level of PPARγ was markedly downregulated in the cortex of a transgenic mouse model of HD (R6/2). Treatment of R6/2 mice with an agonist of PPARγ (thiazolidinedione, TZD) resulted in a beneficial effect on PPARγ. By reducing Htt aggregates and thereby ameliorating the recruitment of PPARγ into Htt aggregates, TZD treatment also elevated the availability of PPARγ level and subsequently normalized the expression of downstream genes (including PGC-1α and several mitochondrial genes) in the cortex. The above protective effects appeared to be exerted by a direct activation of the PPARγ agonist (rosiglitazone) because rosiglitazone protected a neuroblastoma cell line (N2A) from mHtt-evoked mitochondrial deficiency. Our results reveal that TZD and rosiglitazone may play a protective role in HD, and support the view that PPARγ is a potential therapeutic target in HD.

Identification of galectin I and thioredoxin peroxidase II as two arsenic-binding proteins in Chinese hamster ovary cells.

In this study, we report the identification of two arsenic-binding proteins from Chinese hamster ovary (CHO) cells. The crude extract derived from CHO and SA7 (arsenic-resistant CHO cells) was applied to a phenylarsine oxide-agarose affinity column, and after extensive washing, the absorbed proteins were eluted with buffers containing 20 mM 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). Three differentially expressed proteins, galectin 1 (Gal-1; in the 2-ME-eluted fraction from CHO cells), glutathione S-transferase P-form (GST-P) and thioredoxin peroxidase II (TPX-II), respectively in the 2-ME- and DTT-eluted fractions from SA7 cells, were identified by partial amino acid sequence analysis after separation by SDS/PAGE. The GST-P protein has been previously shown to facilitate the excretion of sodium arsenite [As(III)] from SA7 cells. TPX II was detected predominately in SA7 cells [routinely cultured in As(III)-containing medium], but not in CHO or SA7N (a revertant of SA7 cells cultured in regular medium) cells. In contrast, Gal-1 was specifically identified in CHO and SA7N cells, but not in SA7 cells. The preferential expression of Gal-1 in CHO cells and TPX-II in SA7 cells was further illustrated by quantitative PCR analysis. The binding of Gal-1 and TPX-II with As(III) was further verified by both co-immunoprecipitation and co-elution of Gal-1 and TPX-II with As(III). It is suggested that Gal-1 and TPX-II are two proteins that serve as high-affinity binding sites for As(III) and thus both may be involved in the biological action of As(III).

Quantitative Peptidomics Study Reveals That a Wound-Induced Peptide from PR-1 Regulates Immune Signaling in Tomato

CAPE1, a conserved peptide elicitor derived from tomato PR-1, was induced by wounding and found to regulate immune responses against biological threats. As PR-1 is highly conserved across many organisms and the putative peptide from AtPR1 was also found to be bioactive in Arabidopsis, the results suggest that this peptide may be useful for enhancing resistance to stress in other plant species. Many important cell-to-cell communication events in multicellular organisms are mediated by peptides, but only a few peptides have been identified in plants. In an attempt to address the difficulties in identifying plant signaling peptides, we developed a novel peptidomics approach and used this approach to discover defense signaling peptides in plants. In addition to the canonical peptide systemin, several novel peptides were confidently identified in tomato (Solanum lycopersicum) and quantified to be induced by both wounding and methyl jasmonate (MeJA). A wounding or wounding plus MeJA-induced peptide derived from the pathogenesis-related protein 1 (PR-1) family was found to induce significant antipathogen and minor antiherbivore responses in tomato. This study highlights a role for PR-1 in immune signaling and suggests the potential application of plant endogenous peptides in efforts to defeat biological threats in crop production. As PR-1 is highly conserved across many organisms and the putative peptide from At-PR1 was also found to be bioactive in Arabidopsis thaliana, our results suggest that this peptide may be useful for enhancing resistance to stress in other plant species.