Aglycone geniposidic acid, a naturally occurring crosslinking agent, and its application for the fixation of collagenous tissues

A natural compound, aglycone geniposidic acid (aGSA), originated from the fruits of Gardenia jasminoides ELLIS was used for the fixation of collagenous tissues. The presumed crosslinking reaction mechanism of collagenous tissues with aGSA was inferred by reacting aGSA with a bifunctional amine, 1,6-hexanediamine, using a series of (1)H NMR, FT-IR, and UV/Vis spectra analyses. aGSA reacted with 1,6-hexanediamine by a nucleophilic attack on the olefinic carbon atom at C-2 of deoxyloganin aglycone, followed by opening the dihydropyran ring to form heterocyclic amine compounds. It is inferred that aGSA may form intramolecular and intermolecular crosslinks with a heterocyclic structure within collagen fibers in tissues. The degrees of tissue fixation by aGSA at different pH values were investigated by examining the fixation indices and denaturation temperatures of test samples. It was found that the fixation indices and denaturation temperatures of test samples fixed at neutral or basic pH (pH 7.4 or pH 8.5) were significantly greater than at acidic pH (pH 4.0). The results obtained in this study may be used to elucidate the crosslinking mechanism and optimize the fixation process for developing bioprostheses fixed by aGSA.     

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 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.     

Mechanical properties of a porcine aortic valve fixed with a naturally occurring crosslinking agent.

The study investigates the mechanical properties of porcine aortic valve leaflets fixed with a naturally occurring crosslinking agent, genipin, at distinct pressure heads. Fresh and the glutaraldehyde-fixed counterparts were used as controls. Subsequent to fixation, the changes in leaflet collagen crimps and its surface morphology were investigated by light microscopy and scanning electron microscopy (SEM). Also, the crosslinking characteristics of each studied group were determined by measuring its fixation index and denaturation temperature. In the mechanical testing, tissue strips made from each studied group were examined in both the circumferential and radial directions. Histological and SEM comparisons between fresh porcine aortic valve leaflet and those fixed at medium or high pressure revealed that the following changes may occur: elimination of the natural collagen crimping, and extensive loss of the endothelial layer. The denaturation temperatures of the glutaraldehyde-fixed leaflets were significantly greater than the genipin-fixed leaflets; however, their fixation indices were comparable. Generally, fixation pressure did not affect the crosslinking characteristics of the genipin- and glutaraldehyde-fixed leaflets. It was found that fixation of porcine aortic valves in genipin or glutaraldehyde did not alter the mechanical anisotropy observed in fresh valve leaflets. This indicated that the intramolecular and intermolecular crosslinks introduced into the collagen fibrils during fixation is of secondary importance to the presence of structural and mechanical anisotropy in fresh leaflet. Tissue fixation in genipin or glutaraldehyde may produce distinct crosslinking structures. However, the difference in crosslinking structure between the genipin- and glutaraldehyde-fixed leaflets did not seem to cause any significant discrepancies in their mechanical properties when compared at the same fixation pressure. Nevertheless, regardless of the crosslinking agent used, changes in mechanical properties and ruptured patterns were observed when the valve leaflets were fixed at distinct pressures.     

Crosslinking characteristics and mechanical properties of a bovine pericardium fixed with a naturally occurring crosslinking agent.

Currently available crosslinking agents used in fixing bioprostheses are all highly (or relatively highly) cytotoxic, which may induce an adverse inflammatory reaction in vivo. It is therefore desirable to provide a crosslinking agent that is of low cytotoxicty and may form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking agent-genipin-was used by our group to fix biological tissues. Genipin may be obtained from its parent compound, geniposide, which may be isolated from the fruits of Gardenia jasminoides Ellis. In our previous studies, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Also, it was shown that genipin can form stable and biocompatible crosslinked products. The present study further investigates the crosslinking characteristics and mechanical properties of a genipin-fixed bovine pericardium. Fresh and glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was found that the denaturation temperatures of the glutaraldehyde- and genipin-fixed tissues were significantly greater than the epoxy-fixed tissue, although their fixation indices were comparable. The mechanical properties of fresh bovine pericardium are anisotropic. However, fixation tended to eliminate tissue anisotropy. The tendency in the elimination of tissue anisotropy for the genipin-fixed tissue was more remarkable than for the glutaraldehyde- and epoxy-fixed tissues. In addition, the genipin-fixed tissue had the greatest ultimate tensile strength and toughness among all the fixed tissues. Distinct patterns in rupture were observed in the study: The torn collagen fibers of the genipin- and glutaraldehyde-fixed tissues appeared to be bound together, while those of fresh and the epoxy-fixed tissues stayed loose. The results obtained in the study suggests that tissue fixation in glutaraldehyde, epoxy compound, and genipin may produce distinct crosslinking structures. The differences in crosslinking structure may affect the crosslinking characteristics and mechanical properties of the fixed tissues.     

Hemoglobin polymerized with a naturally occurring crosslinking agent as a blood substitute: in vitro and in vivo studies

A naturally occurring crosslinking agent, genipin, extracted from the fruits of Gardenia jasminoides ELLIS was used by our group to chemically modified biomolecules. Genipin and its related iridoid glucosides have been widely used as an antiphlogistic and cholagogue in herbal medicine. Our previous study showed that the cytotoxicity of genipin is significantly lower than glutaraldehyde. The study was to investigate the feasibility of using genipin to polymerize hemoglobin as a blood substitute. The results indicated that the rate of hemoglobin polymerization by glutaraldehyde was significantly faster than that by genipin and it readily produced polymers with molecular masses greater than 500,000 Da. It was found that the maximum degree of hemoglobin polymerization by genipin was approximately 40% if over-polymerization is to be prevented. With increasing the reaction temperature, hemoglobin concentration, and genipin-to-hemoglobin molar ratio, the duration taken to achieve the maximum degree of hemoglobin polymerization by genipin became significantly shorter. The P50 value of the unmodified hemoglobin was 9 mmHg, while that of the genipin-polymerized PLP-hemoglobin increased to 21 mmHg. It was found in a rat model that the genipin-polymerized PLP-hemoglobin resulted in a longer circulation time than the unmodified hemoglobin. In conclusion, the results of the study indicated that the genipin-polymerized hemoglobin solution has a lower oxygen affinity and a longer vascular retention time than the unmodified hemoglobin solution.     

Effects of heparin immobilization on the surface characteristics of a biological tissue fixed with a naturally occurring crosslinking agent (genipin): an in vitro study

Heparinized biomaterials have been used to manufacture blood-contacting prostheses. The present study was intended to characterize the surface properties of a genipin-fixed biological tissue immobilized with heparin using the methods of ionic binding (the /h-i tissue) or covalent binding via multi-point attachment (the /h-m tissue) or end-point attachment (the /h-e tissue). The surface characteristics of test tissues evaluated were water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility. Nonheparinized and the glutaraldehyde-fixed counterparts were used as controls. It was found that immobilization of heparin on the glutaraldehyde- and genipin-fixed tissues increased their hydrophilicity and surface tension and suppressed their mole ratio of adsorbed fibrinogen to adsorbed albumin and the amount of platelets adhered. Among the heparinized tissues, the /h-m tissue was more hydrophobic and had a higher mole ratio of adsorbed fibrinogen to adsorbed albumin and a greater amount of platelets adhered than the /h-i and /h-e tissues. In general, the surface characteristics of the /h-i tissue were comparable to the /h-e tissue. However, it is known that the ionically immobilized heparin may be displaced from the surface by an ion-exchange mechanism when exposed to blood. 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 in comparison with their respective counterparts. However, the cellular compatibility of the genipin-fixed tissues with or without heparinization was significantly superior to its glutaraldehyde-fixed counterparts.     

In vivo evaluation of cellular and acellular bovine pericardia fixed with a naturally occurring crosslinking agent (genipin)

A cell extraction process was employed in the study to remove the cellular components from bovine pericardium, leaving a framework of largely insoluble collagen and elastin. It was hypothesized in the literature that this process may decrease the antigenic load (or increase the biocompatibility) within the material. Additionally, acellular tissues may provide a natural microenvironment for host-cell migration to regenerate the tissue. The study was to evaluate the biocompatibility of cellular and acellular bovine pericardia fixed with a naturally occurring crosslinking agent (genipin) implanted subcutaneously in a growing rat model. Additionally, the tissue regeneration rate in the genipin-fixed acellular tissue was investigated. The glutaraldehyde-fixed counterparts were used as controls. The results indicated that the degrees in inflammatory reaction for the genipin-fixed cellular and acellular tissues were significantly less than their glutaraldehyde-fixed counterparts. Additionally, it was noted that the inflammatory reactions for the glutaraldehyde-fixed cellular and acellular tissues lasted much longer than their genipin-fixed counterparts. The tissue regeneration rate for the genipin-fixed acellular tissue was significantly faster than its glutaraldehyde-fixed counterpart. The calcium content of each studied group, analyzed by atomic absorption. did not change significantly until at the 52nd week, postoperatively. The differences in calcium content between the cellular and acellular tissues were insignificant for both the glutaraldehyde- and genipin-fixed groups throughout the entire course of the study. In summary, the biocompatibility of the genipin-fixed cellular and acellular tissues was superior to their glutaraldehyde-fixed counterparts. The genipin-fixed acellular tissue provided a better microenvironment for tissue regeneration than its glutaraldehyde-fixed counterpart, due to its low cytotoxicity. These results suggested that the genipin-fixed acellular tissue might be used as a tissue-engineering matrix in the clinical applications.     

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.     

Quantification of (+)-calanolide A, a novel and naturally occurring anti-HIV agent, by high-performance liquid chromatography in plasma from rat, dog and human

A HPLC method was validated for quantification of (+)-calanolide A (1), a novel anti-HIV agent, in rat, dog and human plasma. The synthetic intermediate (+/-)-12-oxocalanolide A (2) was found to be a suitable internal standard. Compounds were extracted from plasma using a solid-phase C(18) cartridge and quantified over the assay range of 12.5 to 800 ng/ml. The method was utilized to determine (+)-calanolide A pharmacokinetics in rats, dogs and humans. This is the first report of a validated HPLC assay for determination of (+)-calanolide A concentrations in rat and dog plasma as well as human plasma obtained from clinical trials. There was no evidence of in vivo epimerization of (+)-calanolide A to its inactive epimer (+)-calanolide B (3).     

Use of formaldehyde with pH 8 0 as a fixative for electron and fluorescence microscopy]

For fixation of the material to be investigated at the light and ultrastuctural level the author suggests to use 4% formaldehyde obtained by depolymerization of paraform on 0.1 M phosphate buffer with end pH 8.0 of the solution. This fixative stabilizes well glycogen and tissue structure. The ultrastructural characteristics of cells following continuous (up to 130 days) fixation are comparable with those obtained after fixation with aldehydes by conventional techniques. Results of histological PAS test, Feulgen's reactions and those to acid polycaccharides were identical to the results obtained in fixation with neutralized formalin. The possibility of prolonged storage of tissues makes it possible to perform delayed and repeated ultrastructural investigations of clinical and experimental material.     

Perception of sweetness: the effects of concentration and temperature

The shape of the sweetness function remained unaltered when different concentration ranges of sucrose were evaluated at different temperatures by either one of two psychophysical methods: magnitude estimation or magnitude matching. Results of the sweetness-temperature interaction showed that cool solutions were judged less sweet than warmer solutions. Furthermore the rate of growth of sweetness increased with decreasing temperature, but this effect disappeared when the concentration reached between 0.4 and 0.5 M. That point coincides with the zone in which sweetness functions typically show an inflection point. When Beidler's taste equation was applied to psychophysical data obtained at constant or variable temperature, it was possible to show that sweetness is mediated by an adsorption process rather than an enzymatic reaction.     

Brain temperature and pH measured by 1H chemical shift imaging of a thulium agent

Temperature and pH are two of the most important physiological parameters and are believed to be tightly regulated because they are intricately related to energy metabolism in living organisms. Temperature and/or pH data in mammalian brain are scarce, however, mainly because of lack of precise and non‐invasive methods. At 11.7 T, we demonstrate that a thulium‐based macrocyclic complex infused through the bloodstream can be used to obtain temperature and pH maps of rat brain in vivo by ¹H chemical shift imaging (CSI) of the sensor itself in conjunction with a multi‐parametric model that depends on several proton resonances of the sensor. Accuracies of temperature and pH determination with the thulium sensor – which has a predominantly extracellular presence – depend on stable signals during the course of the CSI experiment as well as redundancy for temperature and pH sensitivities contained within the observed signals. The thulium‐based method compared well with other methods for temperature (¹H MRS of N‐acetylaspartate and water; copper–constantan thermocouple wire) and pH (³¹P MRS of inorganic phosphate and phosphocreatine) assessment, as established by in vitro and in vivo studies. In vitro studies in phantoms with two compartments of different pH value observed under different ambient temperature conditions generated precise temperature and pH distribution maps. In vivo studies in α‐chloralose‐anesthetized and renal‐ligated rats revealed temperature (33–34°C) and pH (7.3–7.4) distributions in the cerebral cortex that are in agreement with observations by other methods. These results show that the thulium sensor can be used to measure temperature and pH distributions in rat brain in vivo simultaneously and accurately with using biosensor imaging of redundant. Copyright © 2008 John Wiley & Sons, Ltd.     

Morphology and structural characteristics of hydroxyapatite whiskers: effect of the initial Ca concentration, Ca/P ratio and pH

Hydrothermal homogeneous precipitation combines the best characteristics of the hydrothermal and homogeneous precipitation methods, and allows long and uniform hydroxyapatite (HA) whiskers, with a high aspect ratio and high crystallinity, to be obtained. Their morphology and structural characteristics depend on the initial Ca/P ratio (iCa/P) and pH (ipH), as well as the initial calcium concentration (i[Ca]). Variation in these values had no effect on constitution, which was crystallographically indistinguishable from HA. Ca/P ratio steadily improved with increases in both ipH and iCa/P, but was independent of i[Ca]. Uniform whiskers were obtained at high iCa/P and low ipH, or at high ipH and low iCa/P. Whiskers with a mean length of 96-140 μm and an aspect ratio of 96-136 were obtained at ipH=2-3 and iCa/P=1.67-2. At a low ipH and low iCa/P, irregular plate-like particles and branch-like whiskers were formed, while a high ipH favoured the formation of lath-like HA at high iCa/P. Preferred growth along the c-axis was more intense at higher iCa/P and ipH as well as at low i[Ca]. However, under these conditions, the crystal growth habit was also changed, showing preferred growth along both the c- and a-axes. The increase in whisker width over the general value obtained was abrupt at low i[Ca] and high iCa/P.     

beta-carotene intestinal absorption: bile, fatty acid, pH, and flow rate effects on transport.

beta carotene absorption in the unanesthetized rat was investigated by recirculating a micellar perfusate that contained beta-carotene through jejunal and ileal intestinal loops. Radioautography revealed extensive distribution of the provitamin throughout the layers of the small bowel. A linear relationship was found between the concentration of beta-carotene in the perfusate and its rate of absorption at perfusate concentrations of 0.5--11 mM. Increases in the perfusate hydrogen ion concentrations, additions of fatty acids of varied chain lengths and degrees of saturation, and an increase in the perfusate flow rate caused higher rates of beta-carotene absorption. Increase in the perfusate sodium taurocholate concentration above 2.5 microM did not change the absorption rate of beta-carotene. These experiments indicate that beta-carotene absorption takes place by passive diffusion. The process of diffusion can be modulated by intraluminal factors that change the physical characteristics of perfusate or stimulate the intracellular cleavage of carotene to retinal.     

Growth of neural transplants in rats: effects of initial volume, growth potential, and fresh vs frozen tissues

Interactions between growth potential (as related to the age of donor embryos and type of tissue), initial volume, and fresh vs frozen conditions of neural transplants were studied in rats. Neural tissues with high growth potential (16-day gestation neocortical tissue) when used fresh yielded the best growth of the transplants, which was positively related to the initial volume of the tissue. At the other extreme, neural tissues with very low growth potential when used following their freezing and thawing yielded the poorest results. Changes in the initial volume of transplants did not seem to improve the final growth. Combination of these variables in between these two extremes yielded transplants of variable sizes.     

Estimation of regional hemoglobin concentration in biological tissues using diffuse reflectance spectroscopy with a novel spectral interpretation algorithm

Both in medical research and clinical settings, regional hemoglobin concentrations ([Hb]) in the microcirculation of biological tissues are highly sought. Diffuse reflectance spectroscopy has been proven to be a favorable method by which to detect regional [Hb]. This paper introduces a new algorithm to retrieve [Hb] information from diffuse reflectance spectra. The proposed algorithm utilizes the natural logarithmic operation and the differential wavelet transform to effectively quench the scattering effects, and then employs the concept of isosbestic wavelength in the transformed spectra to reduce the effects of hemoglobin oxygenation. As a result, the intensity at the defined isosbestic wavelength of the transformed spectra is a good indicator of [Hb] estimation. The algorithm was derived and validated using theoretical spectra produced by Monte Carlo simulation of photon migration. Its accuracy was further evaluated using liquid tissue phantoms, and its clinical utility with an in vivo clinical study of brain tumors. The results demonstrate the applicability of the algorithm for real-time [Hb] estimations from diffuse reflectance spectra, acquired by means of a fiber-optic spectroscopy system.