Yen Chang

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.

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.

Electrical coupling of isolated cardiomyocyte clusters grown on aligned conductive nanofibrous meshes for their synchronized beating

Myocardial infarction is often associated with abnormalities in electrical function due to a massive loss of functioning cardiomyocytes. This work develops a mesh, consisting of aligned composite nanofibers of polyaniline (PANI) and poly(lactic-co-glycolic acid) (PLGA), as an electrically active scaffold for coordinating the beatings of the cultured cardiomyocytes synchronously. Following doping by HCl, the electrospun fibers could be transformed into a conductive form carrying positive charges, which could then attract negatively charged adhesive proteins (i.e. fibronectin and laminin) and enhance cell adhesion. During incubation, the adhered cardiomyocytes became associated with each other and formed isolated cell clusters; the cells within each cluster elongated and aligned their morphology along the major axis of the fibrous mesh. After culture, expression of the gap-junction protein connexin 43 was clearly observed intercellularly in isolated clusters. All of the cardiomyocytes within each cluster beat synchronously, implying that the coupling between the cells was fully developed. Additionally, the beating rates among these isolated cell clusters could be synchronized via an electrical stimulation designed to imitate that generated in a native heart. Importantly, improving the impaired heart function depends on electrical coupling between the engrafted cells and the host myocardium to ensure their synchronized beating.

A genipin-crosslinked gelatin membrane as wound-dressing material: in vitro and in vivo studies

A naturally occurring crosslinking agent (genipin) was used in this study to crosslink gelatin hydrogel to develop a wound-dressing membrane. The study was to investigate the in vitro characteristics of the genipin-crosslinked gelatin membrane. The glutaraldehyde-crosslinked counterpart, at a similar crosslinking degree, was used as control. Additionally, an in vivo experiment was undertaken to study the wound healings covered with the glutaraldehyde- and genipin-crosslinked dressings in a rat model. The in vitro results obtained suggested that crosslinking of gelatin membranes with glutaraldehyde or genipin may produce distinct crosslinking structures. The differences in crosslinking structure can significantly affect the mechanical property, water-vapor-transmission rate, swelling ratio, degradation against enzyme and cellular compatibility of the crosslinked membranes. In the in vivo study, it was found that the degree of inflammatory reaction for the wound treated with the genipin-crosslinked dressing was significantly less severe than that covered with the glutaraldehyde-crosslinked dressing throughout the entire course of the study. Additionally, the healing rate for the wound treated with the genipin-crosslinked dressing was notably faster than its glutaraldehyde-crosslinked counterpart.

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.

Fixation of biological tissues with a naturally occurring crosslinking agent: Fixation rate and effects of pH, temperature, and initial fixative concentration

In an attempt to overcome the cytotoxicity problem of the glutaraldehyde-fixed tissues, a naturally occurring crosslinking agent (genipin) was used by our group to fix biological tissues. The study was intended to investigate the rate of tissue fixation by genipin. Glutaraldehyde was used as a control. In addition, the degrees of tissue fixation by genipin at different pHs (pH 4.0, pH 7. 4, pH 8.5, or pH 10.5), temperatures (4 degrees C, 25 degrees C, 37 degrees C, or 45 degrees C), and initial fixative concentrations (0.250%, 0.625%, or 1.000%) were examined. The results obtained revealed that the rate of tissue fixation by glutaraldehyde was significantly faster than that by genipin. The degree of tissue fixation by genipin may be controlled by adjusting its fixation duration or fixation conditions. The order in degree of tissue fixation by genipin at different pHs, from high to low, was: at nearly neutral pH (pH 7.4 or pH 8.5) > at basic pH (pH 10.5) > at acidic pH (pH 4.0). The degrees of tissue fixation by genipin at different temperatures were about the same, except for that at 4 degrees C. In contrast, the initial fixative concentration did not seem to affect the degree of tissue fixation by genipin, if only the amount of genipin in the fixation solution was sufficient to complete tissue fixation. The concentrations of genipin in the aqueous solutions at different pHs, temperatures, and initial fixative concentrations tended to decrease with time with or without the occurrence of tissue fixation. This indicated that genipin was not stable in the aqueous solution. The instability of aqueous genipin was more remarkable with increasing pH or temperature. The results obtained in this study may be used to optimize the fixation process for developing bioprostheses fixed by genipin.

A Natural Compound (Ginsenoside Re) Isolated from Panax ginseng as a Novel Angiogenic Agent for Tissue Regeneration

No HeadingPurpose.The primary challenge for tissue engineering is to develop a vascular supply that can support the metabolic needs of the engineered tissues in an extracellular matrix. In this study, the feasibility of using a natural compound, ginsenoside Re, isolated from Panax ginseng in stimulating angiogenesis and for tissue regeneration was evaluated.Methods.Effects of ginsenoside Re on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were examined in vitro. Additionally, angiogenesis and tissue regeneration in a genipin-fixed porous acellular bovine pericardium (extracellular matrix; ECM) incorporated with ginsenoside Re implanted subcutaneously in a rat model were investigated. Basic fibroblast growth factor (bFGF) was used as a control.Results.It was found that HUVEC proliferation, migration in a Transwell plate, and tube formation on Matrigel were all significantly enhanced in the presence of bFGF or ginsenoside Re. Additionally, effects of ginsenoside Re on HUVEC proliferation, migration, and tube formation were dose-dependent and reached a maximal level at a concentration of about 30 μg/ml. The in vivo results obtained at 1 week postoperatively showed that the density of neocapillaries and the tissue hemoglobin content in the ECMs were significantly enhanced by bFGF or ginsenoside Re. These results indicated that angiogenesis in the ECMs was significantly enhanced by loading with bFGF or ginsenoside Re. At 1 month postoperatively, vascularzied neo-connective-tissue fibrils were found to fill the pores in the ECMs loaded with bFGF or ginsenoside Re.Conclusions.The aforementioned results indicated that like bFGF, ginsenoside Re-associated induction of angiogenesis enhanced tissue regeneration, supporting the concept of therapeutic angiogenesis in tissue-engineering strategies.

Correctness of multi-detector-row computed tomography for diagnosing mechanical prosthetic heart valve disorders using operative findings as a gold standard

The purpose was to compare the findings of multi-detector computed tomography (MDCT) in prosthetic valve disorders using the operative findings as a gold standard. In a 3-year period, we prospectively enrolled 25 patients with 31 prosthetic heart valves. MDCT and transthoracic echocardiography (TTE) were done to evaluate pannus formation, prosthetic valve dysfunction, suture loosening (paravalvular leak) and pseudoaneurysm formation. Patients indicated for surgery received an operation within 1 week. The MDCT findings were compared with the operative findings. One patient with a Björk-Shiley valve could not be evaluated by MDCT due to a severe beam-hardening artifact; thus, the exclusion rate for MDCT was 3.2% (1/31). Prosthetic valve disorders were suspected in 12 patients by either MDCT or TTE. Six patients received an operation that included three redo aortic valve replacements, two redo mitral replacements and one Amplatzer ductal occluder occlusion of a mitral paravalvular leak. The concordance of MDCT for diagnosing and localizing prosthetic valve disorders and the surgical findings was 100%. Except for images impaired by severe beam-hardening artifacts, MDCT provides excellent delineation of prosthetic valve disorders.

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.

Enhancement of cell retention and functional benefits in myocardial infarction using human amniotic-fluid stem-cell bodies enriched with endogenous ECM

Stem cell transplantation may repair the infarcted heart. Despite the encouraging preliminary results, an optimal cell type used and low retention of the transplanted cells remain to be overcome. In this study, a multiwelled methylcellulose hydrogel system was used to cultivate human amniotic-fluid stem cells (hAFSCs) to form spherically symmetric cell bodies for cellular cardiomyoplasty. The grown hAFSC bodies enriched with extracellular matrices (ECM) were xenogenically transplanted in the peri-infarct area of an immune-suppressed rat, via direct intramyocardial injection. Results of bioluminescence imaging and real-time PCR revealed that hAFSC bodies could considerably enhance cell retention and engraftment in short-term and long-term observations, when compared with dissociated hAFSCs. Echocardiography and magnetic resonance imaging showed that the enhanced cell engraftment in the hAFSC-body group could significantly attenuate the progression of heart failure, improve the global function, and increase the regional wall motion. At the infarct, expressions of HGF, bFGF and VEGF were significantly up-regulated, an indication of the significantly increased vessel densities in the hearts treated with hAFSC bodies. The injected hAFSC bodies could undergo differentiation into angiogenic and cardiomyogenic lineages and contribute to functional benefits by direct regeneration. The aforementioned results demonstrate that hAFSC bodies can attenuate cell loss after intramuscular injection by providing an adequate physical size and offering an enriched ECM environment to retain the transplanted cells in the myocardium, thus improving heart function.