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.     

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 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.     

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。     

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.     

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.     

Adsorption and reactivity of human fibrinogen and immunoglobulin G on two types of hemodialysis membranes

Phase contrast microscopic examination of Cuprophane and polyacrylonitrile. After clinical ex vivo use by uremic patients revealed extensive cellular deposition of erythrocytes, platelets, and leukocytes on Cuprophane but not on polyacrylonitrile. In vitro studies with 125I-labeled human fibrinogen or immunoglobulin G (IgG) showed that the adsorption of fibrinogen or IgG was greater on polyacrylonitrile than on Cuprophane. Further studies on the reactivity of fibrinogen adsorbed on polyacrylonitrile surface indicated that the absorbed fibrinogen: (a) was not desorbed readily from the surface, (b) was not appreciably displaced by other plasma proteins such as albumin, IgG, and fibrinogen, (c) was not readily accessible for reaction with 125I-antifibrinogen-IgG, and (d) did not promote the adhesion of 51Cr-labeled platelets. IgG adsorbed on the surface produced essentially no effect on platelet adhesion to polyacrylonitrile and promoted only slightly on the adhesion of granulocytes to the same material. On the other hand, fibrinogen and IgG augmented greatly the platelet adhesion to Cuprophane and IgG enhanced the granulocyte adhesion moderately. These data indicate that fibrinogen and IgG, though at high concentration on polyacrylonitrile, may adsorb in a biologically inactive form. Our observations suggest that the thrombogenicity of an artificial surface may not be assessed entirely by the types and amount of the various protein species adsorbed but is likely determined by the reactivity of the specific protein species adsorbed on the material.     

Crosslinking of Bovine Pericardia Using Procyanidins

The aim of this study was to evaluate the crosslinking effect of a naturally crosslinking reagent-procyanidins (PA)-on the materials of bioprosthetic heart valves. After fixing bovine pericardial tissues by procyanidins, crosslikng characteristics, mechanical properties, in vitro enzymatic degradation resistance, the hydrophilicity and hemolysis tests were examined. The results showed that the fixation of biological tissue with glutaraldehyde (GA) or procyanidins increased its denaturation temperature, the surface hydrophilicity and mechanical properties as well as in vitro enzymatic degradation resistance. There were no significant differences in denaturation temperature, mechanical properties, the hydrophilicity and the in vitro enzymatic degradation between the glutaraldehyde and procyanidins fixed tissues. However, the ultimate tensile strength of the procyanidins fixed tissues was significantly superior to the glutaraldehyde fixed tissues. The hemolysis tests showed that hemolysis rate of the procyanidins fixed tissues was lower than that of the glutaraldehyde fixed tissues. This study shows that procyanidins can crosslink which bovine pericardiaa effectively without toxicity. Our results suggest that this method might be a useful approach for the preparation of bioprosthetic heart valve.     

Postadsorptive transitions in fibrinogen adsorbed to biomer: changes in baboon platelet adhesion, antibody binding, and sodium dodecyl sulfate elutability

Residence-time-dependent changes in fibrinogen after its adsorption to Biomer were examined by measuring platelet adhesion and antibody binding to the adsorbed protein, and the amount of adsorbed fibrinogen which could be eluted by sodium dodecyl sulfate (SDS). Baboon fibrinogen was first adsorbed (from either pure solution or dilute plasma) to Biomer, which was then stored in either buffer or buffered albumin solution prior to testing. Subsequently, the adherent protein layer was either probed for fibrinogen capable of mediating platelet adhesion using 111In radiolabeled, washed platelet suspensions under both static and shearing conditions, or for fibrinogen capable of binding antibody using a direct enzyme linked immunosorbent assay (ELISA). Alternatively, the surface with the adsorbed protein layer was soaked in a 3% SDS solution, and the amount of 125I radiolabeled fibrinogen retained was measured. Decreases in platelet and antibody binding, and in the SDS elutability of the adsorbed fibrinogen after it was stored in buffer were detected, although different rates of decrease were observed for each method. When the protein-coated surfaces were stored in buffered albumin solution rather than buffer, the decrease in the reactivity of fibrinogen was prevented. While each of the three assays measures a different property of adsorbed fibrinogen, this study suggests that the adherent protein undergoes time dependent conformational changes which render it less reactive toward platelets and antibodies, and more resistant to elution by SDS.     

Study on the prevention of surface-induced platelet activation by albumin coating

To understand how albumin on the surface inhibits surface-induced platelet activation, we adsorbed albumin on dimethyldichlorosilane-coated glass (DDS-glass) and modified the adsorbed albumin by three different methods. The adsorbed albumin was crosslinked with glutaraldehyde, dried and rehydrated, or digested with trypsin. Surface albumin concentration did not change by crosslinking; however, it decreased by about 15% by a simple dry-and-rehydration process. Trypsin digestion reduced the surface albumin concentration by 50%. Platelets were found to adhere and activate on albumin coated DDS-glass, if the adsorbed albumin was modified. The extent of platelet activation was quantified with two numeric parameters, the spread area and circularity. Fibrinogen adsorption to the dried or digested albumin layer resulted in enhancement of platelet activation, while adsorption of more albumin inhibited platelet activation. The results suggest that albumin can inhibit platelet activation as long as it covers the surface completely and remains flexible on the surface. This study indicates that steric repulsion is one of the mechanisms of surface passivation by albumin.     

Study on the prevention of surface-induced platelet activation by albumin coating.

To understand how albumin on the surface inhibits surface-induced platelet activation, we adsorbed albumin on dimethyldichlorosilane-coated glass (DDS-glass) and modified the adsorbed albumin by three different methods. The adsorbed albumin was crosslinked with glutaraldehyde, dried and rehydrated, or digested with trypsin. Surface albumin concentration did not change by crosslinking; however, it decreased by about 15% by a simple dry-and-rehydration process. Trypsin digestion reduced the surface albumin concentration by 50%. Platelets were found to adhere and activate on albumin coated DDS-glass, if the adsorbed albumin was modified. The extent of platelet activation was quantified with two numeric parameters, the spread area and circularity. Fibrinogen adsorption to the dried or digested albumin layer resulted in enhancement of platelet activation, while adsorption of more albumin inhibited platelet activation. The results suggest that albumin can inhibit platelet activation as long as it covers the surface completely and remains flexible on the surface. This study indicates that steric repulsion is one of the mechanisms of surface passivation by albumin.     

Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers

The effect of surface wettability on fibrinogen adsorption, platelet adhesion and platelet activation was investigated using self-assembled monolayers (SAMs) containing different ratios of longer chain methyl- and shorter chain hydroxyl-terminated alkanethiols (C15CH3 vs. C11OH) on gold. Protein adsorption studies were performed using radiolabeled human fibrinogen (HFG). Platelet adhesion and activation studies with and without pre-adsorbed fibrinogen, albumin and plasma were assessed using scanning electron microscopy (SEM) and a glutaraldehyde-induced fluorescence technique (GIFT). Results demonstrated a linear decrease of HFG adsorption with the increase of OH groups on the monolayer (increase of the hydrophilicity). Platelet adhesion and activation also decrease with increase of hydrophilicity of surface. Concerning SAMs pre-immersed in proteins, fibrinogen adsorption was related with high platelet adhesion and activation. The passivant effect of albumin on platelet adhesion and activation was only demonstrated on SAMs contained C11OH. When all the blood proteins are present (plasma) platelet adhesion was almost absent on SAMs with 65% and 100% C11OH. This could be explained by the higher albumin affinity of the SAMs with 65% C11OH and the lower total protein adsorption associated with SAMs with 100% C11OH.     

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.     

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.     

Glow discharge plasma deposition of tetraethylene glycol dimethyl ether for fouling-resistant biomaterial surfaces.

The glow discharge plasma deposition (GDPD) of tetraethylene glycol dimethyl ether is introduced as a novel method for obtaining surfaces that are resistant to protein adsorption and cellular attachment. Analysis of films by x-ray photoelectron spectroscopy and several biological assays indicate the formation of a fouling-resistant, PEO-like surface on several substrata (e.g., glass, polytetrafluoroethylene, polyethylene). Adsorption of 125I-radiolabelled proteins (fibrinogen, albumin and IgG) from buffer and plasma was very low (typically less than 20 ng/cm2) when compared to the untreated substrata, which exhibited much higher levels of protein adsorption. Not all coated substrata adsorbed equal amounts of protein (e.g., coated glass samples typically adsorbed more protein than coated polyethylene or coated polytetrafluoroethylene samples), suggesting that the substratum used may affect the amount of protein adsorbed. Measurement of dynamic platelet adhesion, using epifluorescent video microscopy, and endothelial cell attachment further demonstrates the short-term nonadhesiveness of these surfaces.     

Adsorption of baboon fibrinogen and the adhesion of platelets to a thin film polymer deposited by radio-frequency glow discharge of allylamine.

Platelet adhesion under static and flow conditions from a washed platelet suspension containing albumin to a polymer deposited by radio-frequency glow discharge of allylamine vapour on a poly(ethylene terephthalate) substrate was measured. Electron spectroscopy for chemical analysis was used to characterize the surface. Fibrinogen adsorption from a series of dilute plasma solutions to radio-frequency glow discharge/allylamine, measured using 125I radiolabelled baboon fibrinogen, increased with decreasing plasma dilution to a level much higher than that previously observed on polyurethanes. Elutability by sodium dodecyl sulphate of fibrinogen adsorbed from dilute plasma also increased with increasing plasma concentration, but fibrinogen preadsorbed from plasma became non-elutable when surfaces were stored in buffer for 5 d before contact with sodium dodecyl sulphate. Platelet adhesion to substrates which had been pre-adsorbed with dilute plasma was measured using baboon platelets radiolabelled with 111In. Adhesion greatly decreased as the plasma concentration used for preadsorption increased, suggesting that non-specific platelet binding to the bare surface occurs when protein coverage is incomplete. Non-specific platelet binding was inhibited to varying degrees by preadsorption of different proteins to the surface. Platelet adhesion to surfaces preadsorbed with dilute (1.0%) baboon and human plasmas lacking fibrinogen (i.e. serum, heat-defibrinogenated plasma and congenitally afibrinogenemic plasma) was diminished compared with normal plasma. Addition of exogenous fibrinogen to the deficient plasma partially restored platelet adhesion to normal levels. Adhesion to surfaces preadsorbed with human plasma deficient in von Willebrand factor was comparable to that observed with normal plasma. The plasma preadsorption studies with fibrinogen deficient media suggested that adsorbed fibrinogen is necessary for platelet adhesion to the radio-frequency glow discharge/allylamine substrate at high protein coverage. However, since adhesion was greatly reduced when the plasma preadsorbed substrate was stored in buffer before platelet contact, the conformation of adsorbed fibrinogen is also important in mediating platelet adhesion to radio-frequency glow discharge.     

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.     

Small diameter blood vessel prostheses from blends of polyethylene oxide and polypropylene oxide

Studies on the relationship between blood platelet adhesion and type and amount of polyether segments in copolyetherurethanes report a reduced platelet adhesion with increasing polyether content. We therefore assumed that combinations of polyethylene oxide (PEO) and polypropylene oxide (PPOX) might give materials with a good blood compatibility. Water-soluble PEO was attached to PPOX by u.v.-initiated crosslinking. Films were tested for hydrophilicity, mechanical properties, protein adsorption and blood compatibility. The hydrophilicity was determined by swelling experiments. A compromise between hydrophilicity (PEO) and mechanical strength (PPOX) was met at a swelling of 0.5 (PPOX/PEO ratio: 90/10). In protein adsorption studies only small amounts of adsorbed proteins were found. Three blood material interaction in vitro tests gave good results: a low platelet adhesion and kallikrein generation and a high APTT value. Porous tubings (inner diameter 1.3 mm) were fabricated, by spinning from solution, for implantation in the abdominal aorta of rats. Stress-strain diagrams were comparable to those reported for natural blood vessels.     

Variations in the ability of adsorbed fibrinogen to mediate platelet adhesion to polystyrene-based materials: a multivariate statistical analysis of antibody binding to the platelet binding sites of fibrinogen

Platelet adhesion to the surfaces of biomaterials preadsorbed with plasma previously has been shown to be mediated exclusively by surface-bound fibrinogen and does not seem to involve the other adhesion proteins in plasma (Tsai et al., J Biomed Mater Res 2002;60:348-359). In this study, the influence of surface-bound fibrinogen on platelet adhesion to five different types of polystyrene-based microtiter plates preadsorbed with plasma was analyzed relative to the amount of adsorbed fibrinogen and monoclonal antibody binding to the adsorbed fibrinogen. There was no significant correlation between platelet adhesion and the absolute amount of adsorbed fibrinogen. However, platelet adhesion was positively correlated to the ability of the adsorbed fibrinogen to bind three types of monoclonal antibodies. The antibodies used bound to the sites on fibrinogen thought to be involved in platelet binding (the two gamma chain C-terminal dodecapeptides and the RGDF and RGDS sequences in each of the Aalpha chains). A partial least-squares calibration model was used to analyze the relative importance of these binding sites in fibrinogen to platelet adhesion. The gamma chain C-terminal dodecapeptide was shown to be the most important site in adsorbed fibrinogen in mediating platelet adhesion.