Feasibility study using a natural compound (reuterin) produced by Lactobacillus reuteri in sterilizing and crosslinking biological tissues

Bioprostheses derived from biological tissues have to be fixed and subsequently sterilized before they can be implanted in humans. Currently available crosslinking agents and sterilants used in the fixation or sterilization of biological tissues such as glutaraldehyde and formaldehyde are all highly cytotoxic, which may impair the biocompatibility of bioprostheses. Therefore, it is desirable to provide an agent suitable for use in biomedical applications that is of low cytotoxicity and may form sterile and biocompatible crosslinked products. To achieve this goal, a natural compound (reuterin), produced by Lactobacillus reuteri in the presence of glycerol, was used by our group. It is known that reuterin has antibacterial, antimycotic, and antiprotozoal activities. Additionally, as in the case with formaldehyde, reuterin may react with the free amino groups in biological tissues by using its aldehyde functional group. Therefore, it was speculated that reuterin could be used as a crosslinking agent and a sterilant for biological tissues in the same way as glutaraldehyde and formaldehyde. In the study, the production of reuterin, produced by Lactobacillus reuteri under control conditions, was reported. Preparative chromatography was used to purify reuterin. Also, the minimal inhibitory concentration and minimal bactericidal concentration of reuterin and its antimicrobial activity on a contaminated tissue were investigated. In addition, the cytotoxicity of reuterin was evaluated. Glutaraldehyde, the most commonly used sterilant in the sterilization of biological tissues, was employed as a control. Furthermore, the feasibility of using reuterin as a crosslinking agent in fixing biological tissues was studied. Fresh and the glutaraldehyde-fixed tissues were used as controls. The results obtained in the minimal inhibitory concentration and minimal bactericidal concentration studies and in the sterilization study of a contaminated tissue indicated that the antimicrobial activity of reuterin is significantly superior to its glutaraldehyde counterpart. In addition, the results obtained in the 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide assay showed that reuterin is significantly less cytotoxic than glutaraldehyde. Additionally, it was found that reuterin is an effective crosslinking agent for biological tissue fixation. The reuterin-fixed tissue had comparable free amino group content, denaturation temperature, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. In conclusion, the results obtained in this study indicate that reuterin is an effective agent in the sterilization and fixation of biological tissues.     

A natural compound (reuterin) produced by Lactobacillus reuteri for biological-tissue fixation

The study was undertaken to examine the degree of tissue fixation by reuterin, a natural compound produced by Lactobacillus reuteri, at distinct fixation conditions (pH, temperature, and fixative concentration). Additionally, the rate of tissue fixation by reuterin was investigated using glutaraldehyde as a control. It was found by the Fourier transformed infrared spectroscopy and nuclear magnetic resonance spectroscopy that both mono- and di-aldehyde reuterin oligomers may be present in the acidic and basic aqueous reuterin solutions. Therefore, reuterin may crosslink biological tissues as glutaraldehyde (a di-aldehyde agent). The degree of tissue fixation by reuterin is significantly affected by its fixation conditions. Generally, with increasing the pH, temperature, or fixative concentration, the reduction in free-amino-group content, denaturation temperature, tensile strength, and resistance against enzymatic degradation of the reuterin-fixed tissue increased significantly. Also, the rate of tissue fixation by reuterin is significantly slower than that by glutaraldehyde. However, after fixation, it was noted that the reuterin-fixed tissue has comparable free-amino-group content, denaturation temperature, tensile strength, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue.     

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.     

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 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.     

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.     

氧化海藻酸钠交联改性脱细胞基质材料及其细胞相容性研究

运用高碘酸钠氧化法制备新型的生物交联剂—氧化海藻酸钠(ADA)并将之用于交联改性脱细胞基质材料,通过检测一些交联指标及交联后材料的性能特征来研究ADA交联改性脱细胞基质材料的特点。实验中采用了目前典型的两类交联剂(戊二醛(GA)和京尼平(GP))作为实验对照组。用三种交联剂处理血管组织15min～72h,测定交联过程中的交联指数,并对彻底交联后材料的力学性能以及细胞相容性进行研究。结果表明,ADA交联脱细胞基质材料的交联速率、交联程度不亚于GA,明显优于GP;交联后的材料也具有更适宜的力学性能;在细胞相容性方面,ADA交联的材料也具有明显优于GA、与GP相当的这样非常理想的效果。综上所述,ADA是一种具有很大发展前景的生物组织交联剂。     

Reduced calcification of bioprostheses, cross-linked via an improved carbodiimide based method

Glutaraldehyde fixation of bioprosthetic tissue has been used successfully for almost 40 years. However, it is generally recognized that glutaraldehyde fixation of bioprostheses is associated with the occurrence of calcification. Accordingly, many efforts have been undertaken to develop techniques for the fixation of bioprostheses, which will not lead to calcification. Here we describe a new improved carbodiimide based cross-linking method. Rather than cross-linking the tissue through its free primary amine groups, these groups were first blocked with butanal and the tissue was then cross-linked by means of carbodiimide activation of tissue carboxylic acid groups followed by a reaction with a poly(propylene glycol)bis 2-(aminopropyl) ether, (Jeffamine trade mark ). It was demonstrated that cross-linked porcine leaflets had a calcification of less than 1mg/g tissue after 8 weeks sub-dermal implantion in rats. Furthermore, aortic wall calcification was reduced to 50mg/g, compared to standard glutaraldehyde fixed tissue, which showed 120mg/g tissue calcification in the 8 weeks calcification model used.     

Periodate-mediated glycosaminoglycan stabilization in bioprosthetic heart valves

Bioprosthetic heart valves (BPHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, the majority of bioprostheses fail clinically because of calcification and degeneration. We have recently shown that glycosaminoglycan (GAG) loss may be in part responsible for degeneration of glutaraldehyde-crosslinked bioprostheses. In the present studies, we used a mild reaction of periodate-mediated crosslinking to stabilize glycosaminoglycans in the bioprosthetic tissue. We demonstrate the feasibility of periodate reaction by crosslinking major components of extracellular matrix of bioprosthetic heart valve tissue, namely type I collagen and hyaluronic acid (HA). Uronic acid assay of periodate-fixed HA-collagen matrices showed 48% of HA disaccharides were bound to collagen. Furthermore, we show that such reactions are also feasible to fix glycosaminoglycans present in the middle spongiosa layer of bioprosthetic heart valves. The periodate reactions were compatible with conventional glutaraldehyde crosslinking and showed adequate stabilization of extracellular matrix as demonstrated by thermal denaturation temperature and collagenase assays. Moreover, uronic acid assays of periodate-fixed BPHV cusps showed 36% reduction in the amount of unbound GAG disaccharides as compared with glutaraldehyde-crosslinked cusps. We also demonstrate that calcification of BPHV cusps was significantly reduced in the periodate-fixed group as compared with the glutaraldehyde-fixed group in 21-day rat subdermal calcification studies (periodate-fixed tissue Ca 72.01 +/- 5.97 microg/mg, glutaraldehyde-fixed tissue Ca 107.25 +/- 6.56 microg/mg). We conclude that periodate-mediated GAG fixation could reduce structural degeneration of BPHVs and may therefore increase the useful lifetime of these devices.     

Glutaraldehyde-fixed kangaroo aortic wall tissue: histology,crosslink stability and calcification potential

Stentless aortic heart valve substitutes, manufactured from biological tissues, are fixed with glutaraldehyde to cross-link collagen, reduce antigenicity, and sterilize the tissue. Despite improved cross linking, reduced antigenicity, and various anticalcification measures, the aortic wall tissue present in these prostheses tends to calcify. The aim of this study was to assess the morphology, collagen cross-link stability, and calcification potential of glutaraldehyde-preserved kangaroo aortic wall tissue as opposed to porcine aortic wall tissue. Porcine and kangaroo aortic wall tissues were fixed in 0.625% buffered glutaraldehyde. Histology and cross-link stability were examined. Calcification potential was determined in the subcutaneous rat model. Kangaroo aortic wall tissue was significantly (p < 0.01) less calcified than porcine aortic wall tissue (26.67 +/- 6.53 versus 41.959 +/- 2.75 microg/mg tissue) at 8 weeks. In conclusion, the histological differences between kangaroo and porcine aortic wall tissue correlate well with the reduced calcification potential of kangaroo aortic wall tissue. The reduced calcification potential could result in improved long-term durability of stentless kangaroo heart valves as bioprostheses.     

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.     

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.     

Calcification of chemically treated bovine pericardium.

One of the most important problems arising in cardiac bioprostheses made with bovine pericardium and, more generally, with biologically-derived tissues is tissue calcification. The present study assessed four chemical treatments on patches of bovine pericardium, intended to avoid or minimize calcification. Pericardium specimens were treated with: A) 0.5% glutaraldehyde; B) 0.5% glutaraldehyde + 4% formaldehyde; C) same as A, but with a further neutralization treatment; D) acylation of fresh bovine pericardium. Circular samples of 1 cm diameter were subcutaneously implanted in the abdominal region of three groups of six rats. The explants were retrieved after 2, 4 and 8 weeks. The calcium content and the histological results showed better behaviour for C and D samples than with the commonly used fixation methods (A and B). The lowest calcification was observed with treatment D, even though its morphological structures were somewhat modified with homogenation of collagen bundles. Among the glutaraldehyde-based treatments, treatment C appears to be the most promising because the pericardium shows slower calcium accumulation with a diffusive pattern.     

Extracellular matrix degrading enzymes are active in porcine stentless aortic bioprosthetic heart valves

Glutaraldehyde-fixed porcine aortic valve tissues are widely used for heart valve replacement surgery in the form of bioprosthetic heart valves (BHVs). The durability of BHVs in the clinical setting is limited by tissue degeneration, mechanical failure, and calcification. BHVs rely on the putative ability of glutaraldehyde to render biologic tissues metabolically inert and fully resistant to enzymatic attack. In the present study, we detected and partially characterized the activity of collagen and elastin-degrading enzymes in unimplanted, glutaraldehyde-fixed porcine aortic cusp and wall tissues and compared enzyme activities with those extracted from fresh tissues. Active enzymes capable of degrading extracellular matrix were found to be present in soluble form as well as immobilized on glutaraldehyde-crosslinked tissue matrix. Total levels of collagenolytic activities were evaluated to approximately 0.25 microg of degraded collagen/mg of dry tissue/24 h for both glutaraldehyde-fixed wall and cusp tissues. A major finding of this study was the ability of soluble tissue enzymes to partially degrade glutaraldehyde-fixed collagen and particularly large amounts of glutaraldehyde-fixed elastin. These calcium-dependent gelatinases share many biochemical similarities with matrix metalloproteinases. These data strongly indicate that glutaraldehyde-fixed porcine valvular tissues are not metabolically inert and are not entirely resistant to enzymatic attack, thereby rendering BHVs vulnerable to biologic degeneration and subsequent chronic failure.     

Effects of treatment protocols and subcutaneous implantation on bovine pericardium: a Raman spectroscopy study

Using Raman microspectroscopy, we have studied mineral deposition on bovine pericardia, fixed according to three different protocols and either implanted subcutaneously or not implanted (controls). A lightly carbonated apatitic phosphate mineral, similar to that found in bone tissue, was deposited on the surface of a glutaraldehyde-fixed, implanted pericardium. Implanted pericardia fixed in glutaraldehyde followed by treatment in either an 80% ethanol or a 5% octanol/40% ethanol solution did not mineralize on implantation. Collagen secondary structure changes were observed on glutaraldehyde fixation by monitoring the center of gravity of the amide I envelope. It is proposed that the decrease in the amide I center of gravity frequency for the glutaraldehyde-fixed tissue compared to the nonfixed tissue is due to an increase in nonreducible collagen cross-links (1660 cm(-1)) and a decrease in reducible cross-links (1690 cm(-1)). The amide I center of gravity in the glutaraldehyde/ethanol-fixed pericardium was higher than the glutaraldehyde-fixed tissue center of gravity. This increase in center of gravity could possibly be due to a decrease in hydrogen bonding within the collagen fibrils following the ethanol pretreatment. In addition, we found a secondary structure change to the pericardial collagen after implantation: an increase in the frequency of the center of gravity of amide I is indicative of an increase in cross-links.     

A novel chemical modification of bioprosthetic tissues using L-arginine

A novel chemical modification of biological tissues was developed by the direct coupling of bioactive molecule, L-arginine to bovine pericardium (BP). The modification involves pretreatment of BP using GA and followed by grafting arginine to BP by the reaction of residual aldehyde and amine group of L-arginine. BP was modified by direct coupling of bioactive molecules and the effect of L-arginine coupling on calcification and biocompatibility was evaluated in vitro and in vivo.Modified BPs were characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro plasma protein adsorption and platelet adhesion, and in vivo calcification. Thermal and mechanical properties showed that the durability of arginine treated tissue increased as compared with fresh tissue and GA treated tissue. Resistance to collagenase digestion revealed that modified tissues have greater resistance to enzyme digestion than did fresh tissue and GA treated tissue. Lower protein adsorption and platelet adhesion were observed on modified tissue than non-modified tissue. In vivo calcification study demonstrated much less calcium deposition on arginine treated BP than GA treated one. Obtained results attest to the usefulness of L-arginine treated BP for cardiovascular bioprostheses.     

Physical properties of a porcine internal thoracic artery fixed with an epoxy compound

This study aimed to investigate the physical properties of a porcine internal thoracic artery fixed with an epoxy compound. Fresh and glutaraldehyde-fixed porcine arteries were used as controls. Both the epoxy-fixed and glutaraldehyde-fixed porcine arteries showed significant increases in fixation index and denaturation temperature as compared to the fresh one. Additionally, the resistance of porcine arteries against bacterial collagenase In vitro degradation increased significantly after cross-linking with the epoxy compound or glutaraldehyde. The reduction of the tensile strength of the epoxy-fixed artery after bacterial collagenase degradation was more severe than in its glutaraldehyde-fixed counterpart. Generally, with increasing pH, temperature or fixative concentration, the fixation indices and denaturation temperatures of the epoxy-fixed porcine arteries increased. The results obtained in this study may be used to optimize the fixation process for developing a small-diameter biological vascular graft fixed by an epoxy compound.     

Improved calcification resistance and biocompatibility of tissue patch grafted with sulfonated PEO or heparin after glutaraldehyde fixation

A novel chemical modification of biological tissues was developed aimed at improving biocompatibility and calcification resistance. This method involved the additional grafting of sulfonated PEO (PEO-SO(3)) or heparin after conventional glutaraldehyde (GA) fixation of bovine pericardium (BP). The amino groups of PEO-SO(3) or heparin were utilized to react to the GA residues to block them. The PEO-SO(3) or heparin grafted tissues demonstrated a slightly higher shrinkage temperature and tensile strength, but greater resistance to collagenase digestion, than GA treated ones. These results suggest that modified tissues have improved durability due to the grafting and filling effect of PEO-SO(3) or heparin in addition to the GA cross-linking. At the direct contact cytotoxicity test in vitro, PEO-SO(3) or heparin grafted tissue was shown to be nontoxic, while relatively significant cytotoxicity was observed for the GA treated tissues, possibly due to the release of GA. From the in vivo calcification study, calcium contents deposited on the modified tissues were much less than those on GA treated tissues. Such a decreased calcification might be explained by the decrease of residual GA groups during the additional treatment, and the space-filling effect and the nonadhesive property and/or the blood compatibility of PEO-SO(3) or heparin grafted covalently. The newly modified tissue patch was observed to show improved pathological assessibility including less inflammation and tissue reactions. This simple modification method may be useful for calcification-resistant and blood-compatible tissue patches for cardiovascular implants.     

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.     

Tissue regeneration patterns in acellular bovine pericardia implanted in a canine model as a vascular patch

It was noted in our previous study that acellular tissues can provide a natural microenvironment for host cell migration and proliferation to accelerate tissue regeneration. The purpose of this study was to further investigate the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined. It was found that the degree of inflammatory reaction observed for the genipin-fixed acellular patch was significantly less than its glutaraldehyde-fixed counterpart. At 1 month postoperatively, intimal thickening was found on the inner surfaces of both studied groups. The intimal thickening observed on the glutaraldehyde-fixed acellular patch was significantly thicker than its genipin-fixed counterpart. An intact layer of endothelial cells was found on the intimal thickening of the genipin-fixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaraldehyde-fixed acellular patch. Additionally, fibroblasts with neocollagen fibrils and myofibroblasts were observed in the acellular patches for both studied groups, an indication of tissue regeneration. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart. At 6 months postoperatively, foci of chondroid and/or bony metaplasia were found in each retrieved sample for both studied groups. The observed adverse response of chondroid metaplasia may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk after implantation or a lack of angiogenesis in the regenerated tissue observed at 1 month postoperatively. Bony metaplasia may then develop as in other chondroid tissues. It was reported that ischemia is a usual cause of metaplasia.