原花青素交联牛心包材料的研究

本研究针对天然交联剂原花青素处理牛心包材料的性能进行研究.采用原花青素交联牛心包组织,制备人工生物心脏瓣膜材料,并对其交联特性、力学特性、抗酶降解性能、亲疏水性能、细胞毒性试验以及溶血试验等进行分析.结果显示:原花青素或戊二醛处理的牛心包组织变性温度、力学特性、表面亲水性能和抗酶降解能力都明显提高;与传统交联剂戊二醛相比组织稳定性、亲水性能和抗酶降解能力未见显著性差异,但是最大断裂强度显著提高(原花青素交联组织最大断裂强度(13.863 0 MPa)明显高于戊二醛交联组织(10.784 2 MPa);溶血试验结果表明原花青素处理固定组织的溶血率(2.61%)远低于戊二醛处理的组织(12.54%);细胞毒性试验结果表明原花青素交联组织在1、3、5 d的细胞增殖率分别为76.19%、88.96%、100.12%,而戊二醛在相同的时间点的细胞增殖率分别为63.15%、57.28%、48.74%.原花青素交联的瓣膜材料组织结构稳定、亲水性好、毒性小且能维持较好的力学性能,作为人工生物瓣膜材料具有很好的研究前景.     

Study on Characteristics of Acellular Bovine Pericardium Crosslinked with Genipin

The study used a naturally occurring crosslinking reagent-genipin to chemically modify acellular bovine pericardium, prepare cardiac valve tissue engineering scaffold material,and evaluated genipin crosslinked acellular matrix of bovine pericardium by investigating the physical and chemical properties of the tissues, such as the surface properties, crosslinking characteristics, mechanical properties, resistance to enzymatic capacity in vitro, and hemolysis tests. The results showed that acellular bovine pericardium matrix crosslinked with genipin was strong hydrophilicity, high crosslinking index, and stable structure, which can maintain good mechanical properties. As a kind of scaffold material for valve tissue engineering, it has wide application prospect.     

Crosslinking of decellularized porcine heart valve matrix by procyanidins

Heart valve diseases have a significant high mortality, and the valve replacement using glutaraldehyde crosslinked porcine heart valves is one of the main curing techniques. But its application is limited due to poor durability, calcification of the valves and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of procyanidins on porcine heart valve matrix. After crosslinking of the decellularized porcine aortic heart valves by procyanidins, the tensile strength, the in vitro enzymatic degradation resistance, procyanidins release from the crosslinked materials and the cytotoxicity of procyanidins to heart valvular interstitial cells were examined. The results showed that the tensile strength of procyanidins crosslinked valve matrix was higher than that of glutaraldehyde crosslinked valve matrix. Valve matrix crosslinked by 10 mg/ml procyanidins could be stored in D-Hanks solution for at least 45 days without any decline in ultimate tensile strength and maintained the elasticity as the fresh valves. Furthermore, procyanidins was found to release when the crosslinked tissue stored in D-Hanks solution. The release rate was high during the first 4 days and then dramatically decreased thereafter. During releasing phase, the concentration of procyanidins was no toxicity to heart valve interstitial cells. In vitro enzymatic degradation revealed that crosslinked matrix could resist the enzymatic hydrolysis, and the resistant capacity was approximately the same as glutaraldehyde crosslinked valve matrix. This study shows that procyanidins can crosslink porcine heart valves effectively without toxicity. Our results suggested that this method might be a useful approach for preparation of bioprosthetic heart valve.     

Neomycin prevents enzyme-mediated glycosaminoglycan degradation in bioprosthetic heart valves

Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, BHVs have limited durability and fail either due to degeneration or calcification. Glycosaminoglycans (GAGs), one of the integral components of heart valve cuspal tissue, are not stabilized by conventional glutaraldehyde crosslinking. Previously we have shown that valvular GAGs could be chemically fixed with GAG-targeted chemistry. However, chemically stabilized GAGs were only partially stable to enzymatic degradation. In the present study an enzyme inhibitor was incorporated in the cusps to effectively prevent enzymatic degradation. Thus, neomycin trisulfate, a known hyaluronidase inhibitor, was incorporated in cusps via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemistry followed by glutaraldehyde crosslinking (NEG). Controls included cusps crosslinked with either EDC/NHS followed by glutaraldehyde (ENG) or only with glutaraldehyde (GLUT). NEG group showed improved resistance to in vitro enzymatic degradation as compared to GLUT and ENG groups. All groups showed similar collagen stability, measured as a thermal denaturation temperature by differential scanning calorimetry (DSC). The cusps were implanted subdermally in rats to study in vivo degradation of GAGs. NEG group preserved significantly more GAGs than ENG and GLUT. NEG and ENG groups showed reduced calcification than GLUT.     

Crosslinking of biological tissues using genipin and/or carbodiimide

The study was to investigate the crosslinking characteristics, mechanical properties, and resistance against enzymatic degradation of biological tissues after fixation with genipin (a naturally occurring crosslinking agent) and/or carbodiimide. Fresh tissue was used as a control. It was found that both genipin and carbodiimide are effective crosslinking agents for tissue fixation and genipin crosslinking is comparatively slower than carbodiimide crosslinking. Additionally, tissue fixation in genipin and/or carbodiimide may produce distinct crosslinking structures. Carbodiimide may form intrahelical and interhelical crosslinks within or between tropocollagen molecules, whereas genipin may further introduce intermicrofibrillar crosslinks between adjacent collagen microfibrils. The stability (denaturation temperature and resistance against enzymatic degradation) of the fixed tissue is mainly determined by its intrahelical and interhelical crosslinks. In contrast, intermicrofibrillar crosslinks significantly affect the mechanical properties (tissue shrinkage during fixation, tensile strength, strain at break, and ruptured pattern) of the fixed tissue. Moreover, the degree of enzymatic degradation of the fixed tissue may be influenced by three factors: the availability, to the enzyme, of recognizable cleavage sites, the degree of crosslinking, and the extent of helical integrity of tropocollagen molecules in 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.     

Quercetin-crosslinked porcine heart valve matrix: Mechanical properties,stability, anticalcification and cytocompatibility

Bioprosthetic heart valves, prepared by glutaraldehyde (GA) crosslinking, have some limitations due to poor durability, calcification and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of a natural product, quercetin, on decellularized porcine heart valve extracellular matrix (ECM). After crosslinking, the mechanical properties, stability, anticalcification and cytocompatibility were examined. The results showed that the tensile strength of quercetin-crosslinked ECM was higher than that of GA-crosslinked ECM. After crosslinking with quercetin, the thermal denaturation temperature of ECM was clearly increased. Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any loss of ultimate tensile strength and elasticity. After soaking in D-Hanks solution for 36 days, there was only 11.55% non-crosslinked excess quercetin released and no further release thereafter. Cell culture study shows that no inhibition on proliferation of vascular endothelial cells occurred when the quercetin concentration was lower than 1 μg ml^?1. This non-cytotoxic concentration was 100 times higher than that of GA. The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of GA-crosslinked ECM. An in vitro anticalcification experiment showed that quercetin crosslinking could protect ECM from deposition of minerals in simulated body fluid. The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively, which suggests that quercetin might be a new crosslinking reagent for the preparation of bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.     

Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern

It was reported that acellular biological tissues can provide a natural microenvironment for host cell migration and may be used as a scaffold for tissue regeneration. To reduce antigenicity, biological tissues have to be fixed with a crosslinking agent before implantation. As a tissue-engineering scaffold, it is speculated that the crosslinking degree of an acellular tissue may affect its tissue regeneration pattern. In the study, a cell extraction process was employed to remove the cellular components from bovine pericardia. The acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completely degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.     

Effect of alternative crosslinking methods on the low strain rate viscoelastic properties of bovine pericardial bioprosthetic material

Early failures of bovine pericardial heart valves have been due to leaflet perforation/tearing and calcification. Since glutaraldehyde fixation has been shown to produce marked changes in leaflet mechanics and has been linked to the development of calcification, alternative crosslinking techniques have been suggested as means to overcome these obstacles. We have examined the low strain rate viscoelastic behavior of bovine pericardium: (1) fresh; (2) chemically treated with glutaraldehyde, cyanimide, or polyglycidyl ether; or (3) physically treated by freeze-drying or heat-drying. Shrinkage temperature tests were conducted to assess intrahelical crosslinking. Polyglycidyl ether and glutaraldehyde both produced substantial crosslinking, with the shrinkage temperature rising above 80 degrees C. Mechanical changes were nearly equivalent, both showing decreased stress relaxation and increased extensibility consistent with intrahelical crosslinking and shrinkage during fixation. Cyanimide, known to crosslink pure collagen materials, showed no evidence of crosslinking intact tissue. Heat-drying, also effective in pure collagen preparations, produced an increase in UTS and tissue modulus, but otherwise left the tissue unchanged. Freeze-drying had no mechanical effect, and therefore provides an attractive means for the storage of connective tissues for later mechanical testing.     

In vivo biomechanical assessment of triglycidylamine crosslinked pericardium

While glutaraldehyde crosslinking is most often used to fabricate bioprosthetic heart valves (BHV) using heterograft tissues, it predisposes BHV to calcification and dramatically stiffens the heterograft tissues. Our group previously reported the synthesis and characterization of a novel epoxy-crosslinker, triglycidylamine (TGA). TGA pretreatment of BHV tissues compared to glutaraldehyde results in both calcification resistance in subdermal implants and improved leaflet compliance. In these prior studies, optimal calcification inhibition was noted with the combined use of TGA with mercapto-aminobisphosphonate (MABP). In the present study, we investigated the hypothesis that bovine pericardium cross-linked with TGA-MABP retains these beneficial biomechanical properties in vivo using a novel mitral valve anterior leaflet (MVAL) ovine valvuloplasty model. Bovine pericardial specimens were crosslinked with either glutaraldehyde or TGA-MABP, from which 1cm2 sections were implanted in the ovine MVAL after removal of the original tissue of the same size. An array of four sonomicrometry transducers were implanted on the corners and used to compute the complete in-surface strain tensor cardiac cycle over the cardiac cycle at 0 and 4 weeks. Following explant samples were fixed in formalin for histology studies. At 4 weeks both treatment groups experienced no dimensional changes in the unloaded state, indicating no shrinkage. When fully loaded during peak systolic ejection, TGA-MABP valvuloplasty patches were significantly more compliant, which did not change at 4 weeks. In contrast, the glutaraldehyde areal strain increased significantly by 4 weeks. Estimated implant stresses for both treatment groups, based on previously measured biomechanical properties [Connolly JM, Alferiev I, Clark-Gruel JN, Eidelman N, Sacks M, Palmatory E, et al. Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms. Am J Pathol 2005;166(1):1-13], were 40 and 250 kPa in the circumferential and radial directions, respectively, which are comparable to predicted BHV peak stress levels. We conclude that TGA-MABP crosslinked bovine pericardium, when subjected to in vivo BHV stress levels in a blood-contacting environment, maintains stable functionality.     

Glucose stabilizes collagen sterilized with gamma irradiation

Gamma irradiation sterilization (gamma-irradiation) fragments and denatures collagen, drastically decreasing critical physical properties. Our goal was to maintain strength and stability of gamma-irradiated collagen by adding glucose, which in theory can initiate crosslink formation in collagen during exposure to gamma-irradiation. Collagen films prepared with and without glucose were gamma-irradiated with a standard dose of 2.5 Mrad. Relative amounts of crosslinking and denaturation were approximated based on solubility and the mechanical properties of the films after hydration, heat denaturation, or incubation in enzymes (collagenase and trypsin). After exposure to gamma-irradiation, collagen films containing glucose had significantly higher mechanical properties, greater resistance to enzymatic degradation, and decreased solubility compared with control films. The entire experiment was repeated with a second set of films that were exposed first to ultraviolet irradiation (254 nm) to provide higher initial strength and then gamma-irradiated. Again, films containing glucose had significantly greater mechanical properties and resistance to enzymatic degradation compared with controls. Gel electrophoresis showed that glucose did not prevent peptide fragmentation; therefore, the higher strength and stability in glucose-incorporated films may be due to glucose-derived crosslinks. The results of this study suggest that glucose may be a useful additive to stabilize collagenous materials or tissues sterilized by gamma-irradiation.     

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.     

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.     

Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.     

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。     

Tannic acid treatment enhances biostability and reduces calcification of glutaraldehyde fixed aortic wall

Progressive degeneration and calcification of glutaraldehyde (Glut) fixed tissues used in cardiovascular surgery restrict their long-term clinical performance. This limited biological stability may be attributable to the inability of Glut to adequately protect certain tissue components such as elastin from enzymatic attack. The aim of our studies was to develop novel tissue-processing techniques targeted specifically at elastin stabilization by using tannic acid (TA), a plant polyphenol capable of protecting elastin from digestion by specific enzymes. In present studies we demonstrated that Glut does not adequately protect porcine aorta from elastase-mediated degradation in vitro. The addition of TA to the Glut fixation process increased the stability of Glut-fixed aorta to elastase digestion by 15-fold and also decreased calcification in the rat subdermal model by 66%. TA was found to be chemically compatible with Glut fixation and did not hinder collagen crosslinking as shown by minor changes in thermal denaturation temperatures, resistance to collagenase and mechanical properties. In vitro and in vivo studies also revealed that TA binding to aortic wall was stable over an extended period of time. TA-mediated elastin stabilization in Glut-fixed cardiovascular implants may significantly extend the clinical durability of these tissue replacements.     

Mechanisms of the in vivo inhibition of calcification of bioprosthetic porcine aortic valve cusps and aortic wall with triglycidylamine/mercapto bisphosphonate

Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, vs. Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies, we have further investigated the effectiveness of MABP in experiments that examined: (1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; (2) Furthermore, recognizing the importance of alkaline phosphatase (ALP) in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of ALP activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced ALP activity, and in vivo have no significant calcification in long-term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.     

The effect of temperature and glutaraldehyde fixation on the mechanical properties of bovine pericardial tissues

A comparison is made of the elastic response of fresh pericardial tissues at 25, 37, and 42 degrees C, and also of fresh and glutaraldehyde-fixed tissues. Strips of bovine pericardial tissues cut perpendicular to the base-apex axis of the heart were used. An Instron machine was used for uniaxial tensile tests, and the strain-rate used was 666.7% X min-1. No significant differences in tissue mechanical properties were observed for temperature values of 25, 37, and 42 degrees C. However tissues fixed in a glutaraldehyde solution were more extensible than fresh tissues. The elastic responses of tissue preserved for 1 day in glutaraldehyde are not very different from those preserved for up to 10 days.     

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.     

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.