Glutaraldehyde-fixed biological tissue calcification: effectiveness of mitigation by dimethylsulphoxide

The conditions defining the extent of dimethylsulphoxide (DMSO) effectiveness in mitigating calcification of glutaraldehyde (GA)-fixed tissue have been evaluated. Exposure of GA-fixed tissue porcine aortic valve cusps to low concentrations of DMSO does not impart calcification inhibitory activity. Mitigation in calcification becomes evident only as the DMSO component nears 100% and is best when neat DMSO is used. In all instances, regardless of the DMSO concentration, exposure to DMSO resulted in an increase in the tissue shrinkage temperature, attributed to further cross-linking in the tissue. Histological examination of samples before implantation indicate some deleterious effects to the tissue, the degree dependent on concentration, time and temperature of DMSO exposure. The results of this study suggest that treatment of GA-fixed tissue with high concentrations of DMSO for a short duration at a lowered temperature could give a bioprosthesis that has good mitigating calcification properties with retention of tissue integrity.     

Evaluation of alternative glutaraldehyde stabilization strategies for collagenous biomaterials

A range of alternative crosslinking conditions based on glutaraldehyde were examined for their effectiveness for stabilizing collagen-based materials using test samples of a collagen-polymer composite tube. Stabilization of collagen was performed with various concentrations of glutaraldehyde at acid pH, in the absence or presence of 0.7 m NaCl to control collagen swelling. For each condition, some samples were further treated at neutral pH. These test samples were compared with samples treated with glutaraldehyde at neutral pH and with samples of Omniflow vascular prosthesis. The effectiveness of the stabilization was examined by amino acid analysis, to assess the extent of modification, isometric tension analysis, to evaluate the extent of crosslinking, compliance and accelerated fatigue testing, to evaluate mechanical properties, and by a rat subcutaneous model to evaluate tissue response and propensity to calcification. The data indicated that effective crosslinking could be achieved at low pH and that this can be increased slightly by the presence of NaCl. At low pH, the extent of calcification was low compared to samples treated at pH 7. Subsequent treatment at pH 7 of samples given an initial low pH glutaraldehyde (GA) treatment generally did not alter shrinkage temperatures although the extent of lysine modification and calcification did increase. In general, a more inflammatory response was observed in samples tanned at low pH, although this was not as severe as responses to untreated tissue implants. The Omniflow vascular prosthesis showed excellent chemical and mechanical properties and did not show any calcification.     

Inhibition of bovine pericardium calcification: A comparative study of Al3+ and lipid removing treatments

The biological heart prostheses present midterm and long term problems owing to the progressive deterioration and calcification of the tissue. In the attempt to study the latter problem, we have compared the effectiveness of anticalcification treatments in calf pericardium samples implanted into female Wistar rats after undergoing the following procedures: Group I, control, treatment with glutaraldehyde; group II, treated with 0.1m Al³⁺ for 24 h; and group III, subjected to lipid removal by chemical treatment with chloroform/methanol for 1 h. Positive results were obtained with both treatments, but the results after 60 days of implantation were more favourable with lipid removal than with Al³⁺ treatment.     

Polyethylene glycol-grafted bovine pericardium: a novel hybrid tissue resistant to calcification

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium (GATBP). An investigation was made of the grafting of different molecular weight polyethylene glycol (PEG 600, 1500, 4000 and 6000) via glutaraldehyde (GA) linkages to bovine pericardium (BP) and of their stability and calcification. The process of the calcification profile was studied by in vitro experiments via incubating pericardial samples in a metastable solution of calcium phosphate. Calcification of bovine pericardium grafted with PEG 6000 was significantly decreased compared to low molecular weight PEG grafts or Sodium dodecyl sulphate- (SDS) and GA-treated tissues. The mechanical properties of these modified tissues after enzyme (Trypsin) digestion and calcification were investigated. The biocompatibility aspects of grafted tissues were also established by monitoring the platelet adhesion, octane contact angle and water of hydration. PEG 6000-grafted tissues retained the maximum strength in trypsin buffer and calcium phosphate solutions. Scanning electron micrographs revealed that the PEG-grafted bovine pericardium had substantially inhibited the platelet–surface attachment and their spreading. It is conceivable that high molecular weight polyethylene glycol-grafted pericardium (a hybrid tissue) may be a suitable calcium-resistant material for developing prosthetic valves due to their stability and biocompatibility. © 1999 Kluwer Academic Publishers     

The influence of chemical treatment and suture on the elastic behavior of calf pericardium utilized in the construction of cardiac bioprostheses

Poor mechanical properties of biological tissue are known to cause wear, leading to the failure of cardiac bioprostheses made of calf pericardium. Different chemical agents such as sodium dodecyl sulfate (SDS) are presently being tested as possible inhibitors of the calcification process. The objective of this report was to determine the mechanical behavior of calf pericardium treated with SDS for 24 h and the influence of the suture on the mechanical properties of the tissue. Forty-eight samples were tested: 24 subjected to a standard treatment with glutaraldehyde (12 sewn with 4/0 silk suture thread) and 24 incubated with SDS for 24 h (12 sewn with the same suture thread). Each sutured and non-sutured sample was cut into two strips to yield paired samples. All were subjected to tensile stress to breaking point. The mean stress at breaking point in the non-sutured series treated with glutaraldehyde alone was 16.42 and 13.85 MPa depending on the region of the pericardium, while in the sutured samples subjected to glutaraldehyde the mean stress was 7.50 and 7.63 MPa, respectively, differences which were statistically significant (p = 0.03 and p = 0.003, respectively) when the means for non-sutured samples from equivalent regions treated with glutaraldehyde were compared. The stress at breaking point was lower in the SDS-treated series, ranging between 2.60 and 3.56 MPa. The mathematical functions that govern the stress/strain or deformation were obtained. In the series of pericardium treated with SDS, deformations of 10% were produced with stresses of under 0.4 MPa, an outcome that is intolerable from the constructive point of view. We established a regression model that enabled us to determine the mechanical behavior of a sutured sample by testing a contiguous piece of tissue, with a high correlation coefficient (r > 0.99). We consider this finding to be of interest in the selection of pericardium for use in the construction of leaflets for cardiac bioprostheses.     

Screening biomaterials with a new in vitro method for potential calcification: Porcine aortic valves and bovine pericardium

Calcification is still a major cause of failure of implantable biomaterials. A fast and reliable in vitro model could contribute to the study of its mechanisms and to testing different anticalcification techniques. In this work, we attempted to investigate the potential calcification of biomaterials using an in vitro model. We purposed to test the ability of this model to screening possible anticalcification efficacy of different biomaterials. Porcine heart valve (PAV) and bovine pericardial (BP) tissues, fixed with glutaraldehyde were immersed into biological mimicking solution, where the pH and the initial concentrations of calcium and phosphoric ions were kept stable by the addition of precipitated ions during calcification. Kinetics of calcification was continuously monitored. The evaluation of biomaterials was carried out by comparing the kinetic rates of formation of calcific deposits. After 24 h, the calcific deposits on PAVs were found to be developed at significant higher rates (ranged from 0.81 x 10(-4)-2.18 x 10(-4)mol/min m2) than on BP (0.19 x 10(-4)-0.52 x 10(-4)mol/min m2) (one-way ANOVA, p < 0.05) depending on the experimental conditions (supersaturation of the solution). Parallel tests for similar biomaterials implanted subcutaneously in animal (rat) model showed after 49 days that significant higher amounts of total minerals deposited on PAV (236.73+/-139.12, 9 animals mg minerals/g dry net tissue) (mean+/-standard deviation) compared with that formed on BP (104.36+/-79.21, #9 mg minerals/g dry net tissue) (ANOVA, p < 0.05). There is evidence that in vitro calcification was correlated well with that of animal model and clinical data.     

The influence of chemical treatment and suture on the elastic behavior of calf pericardium utilized in the construction of cardiac bioprostheses

Poor mechanical properties of biological tissue are known to cause wear, leading to the failure of cardiac bioprostheses made of calf pericardium. Different chemical agents such as sodium dodecyl sulfate (SDS) are presently being tested as possible inhibitors of the calcification process. The objective of this report was to determine the mechanical behavior of calf pericardium treated with SDS for 24 h and the influence of the suture on the mechanical properties of the tissue. Forty-eight samples were tested: 24 subjected to a standard treatment with glutaraldehyde (12 sewn with 4/0 silk suture thread) and 24 incubated with SDS for 24 h (12 sewn with the same suture thread). Each sutured and nonsutured sample was cut into two strips to yield paired samples. All were subjected to tensile stress to breaking point. The mean stress at breaking point in the nonsutured series treated with glutaraldehyde alone was 16.42 and 13.85 MPa, depending on the region of the pericardium, while in the sutured samples subjected to glutaraldehyde the mean stress was 7.50 and 7.63 MPa, respectively, differences which were statistically significant (p=0.03 and p=0.003, respectively) when the means for nonsutured samples from equivalent regions treated with glutaraldehyde were compared. The stress at breaking point was lower in the SDS-treated series, ranging between 2.60 and 3.56 MPa. The mathematical functions that govern the stress/strain or deformation were obtained. In the series of pericardium treated with SDS, deformations of 10% were produced with stresses of under 0.4 MPa, an outcome that is intolerable from the constructive point of view. We established a regression model that enabled us to determine the mechanical behavior of a sutured sample by testing a contiguous piece of tissue, with a high correlation coefficient (r > 0.99). We consider this finding to be of interest in the selection of pericardium for use in the construction of leaflets for cardiac bioprostheses. ©2000 Kluwer Academic Publishers     

HMDC crosslinking of bovine pericardial tissue: a potential role of the solvent environment in the design of bioprosthetic materials

The need for alternative crosslinking techniques in the processing of bioprosthetic materials is widely recognized. While glutaraldehyde remains the most commonly used crosslinking agent in biomaterial applications there is increasing concern as to its biocompatibility-principally due to its association with enhanced calcification, cytotoxicity, and undesirable changes in the mechanical properties of bioprosthetic materials. Hexamethylene diisocyanate (HMDC), like glutaraldehyde, is a bifunctional molecule which covalently bonds with amino groups of lysine residues to form covalent crosslinks. Evidence within the literature indicates HMDC-treated materials are less cytotoxic than glutaraldehyde-treated materials; however, there is limited characterization of the material properties of HMDC-treated tissue. This study uses a multi-disciplined approach to characterize the mechanical, thermal, and biochemical properties of HMDC-treated bovine pericardial tissue. Further, to facilitate stabilization of the HMDC reagent, non-aqueous solvent environments were investigated. HMDC treatment produced changes in mechanical properties, denaturation temperature, and enzymatic resistance consistent with crosslinking similar to that seen in glutaraldehyde treated tissue. The significantly lower extensibility and stiffness observed under low stresses may be attributed to the effect of the 2-propanol solvent environment during crosslinking. While the overall acceptability of HMDC as a crosslinking agent for biomaterial applications remains unclear, it appears to be an interesting alternative to glutaraldehyde with many similar features.     

Thermal and spectrophotometric studies of new crosslinking method for collagen matrix with glutaraldehyde acetals

Despite the many existing crosslinking procedures, glutaraldehyde (GA) is still the method of choice used in the manufacture of bioprosthesis. The major problems with GA are: (a) uncontrolled reactivity due to the chemical complexity or GA solutions; (b) toxicity due to the release of GA from polymeric crosslinks; and (c) tissue impermeabilization due to polymeric and heterogeneous crosslinks formation, partially responsible for the undesirable calcification of the bioprosthesis. A new method of crosslinking glutaraldehyde acetals has been developed with GA in acid ethanolic solution, and after the distribution inside de matrix, GA is released to crosslinking. Concentrations of hydrochloride acid in ethanolic solutions between 0.1 and 0.001 mol/L with GA concentration between 0.1 and 1.0% were measured in an ultraviolet spectrophotometer to verify the presence of free aldehyde groups and polymeric compounds of GA. After these measurements, the solutions were used to crosslink bovine pericardium. The spectrophotometric results showed that GA was better protected in acetal forms for acid ethanolic solution with HCl at 0.003 mol/L and GA 1.0%(v/v). The shrinkage temperature results of bovine pericardium crosslinked with acetal solutions showed values near 85 °C after the exposure to triethylamine vapors.     

Supramolecular structure of human aortic valve and pericardial xenograft material: atomic force microscopy study

Pericardial tissue (bovine or porcine), chemically stabilized with glutaraldehyde (GA), is widely used in cardiovascular surgery in the form of bioprosthetic valves. GA reacts with tissue proteins and creates inter- and intra-molecular cross-links, resulting in improved durability. However, tissue calcification and mechanical damage are still unresolved problems. The purpose of this study was to examine the surface topography of normal human aortic valve and GA-stabilized porcine pericardium tissue in order to gain comparative insight into supramolecular structure of both tissues. The analysis was focused on morphologic evaluation of collagen constituents of the tissues. Atomic force microscopy working in the contact mode in air was employed in the study. Considerable diversity in the spatial orientation of collagen fibrils for the human aortic valve and pericardial tissue were observed. It was found that different forms of collagen fibril packing, i.e. dense and “in phase” or loose, could have an impact on the collagen D-banding pattern. Stabilization with GA introduced significant changes in the surface topography of collagen fibrils and in their spatial organization on the tissue surface. Strong disturbance in the fibril’s D-spacing was observed. It was also suggested, that the observed structural changes at the supramolecular level might make an important contribution to the progressive damage and calcification of the tissue. The presented results demonstrate that the AFM method can be useful for non-destructive structural characterization of heart valves and bioprosthetic heart valve material.     

Bovine pericardium for heart valve bioprostheses:in vitro andin vivo characterization of new chemical treatments

This study is aimed at investigating the bovine pericardium treated with different chemical procedures applied to prevent dystrophic calcification; decellularization of the fresh pericardium (samples B and C); fixation of the pericardium with glutaraldehyde (samples A, B, C, D and E); detoxification with aminoacids (samples A and B) and storage in a solution of benzoic acid esters. Pericardial sacs were harvested and delivered to the laboratories to be submitted to the chemical treatments. The samples E have been treated as the samples D but before the implantation they were exposed to the surgical lamp in order to promote some drying. The samples were tested for their mechanical properties and shrinkage temperature (at 1 week and after 36 months). Thein vivo tests were performed by means of implantation in a paravertebral subcutaneous position in rats. At the explant (2, 4 and 8 weeks) the samples were submitted to histological assay and the calcium content determined by spectroscopic atomic absorption. All the samples showed loss of tensile strength and elongation at 36 months (except for the sample A), as well as a moderate diminution of the shrinkage temperature. The histology showed that the decellularized samples (B, C) were thicker than the others and the collagen fibres were extensively homogenated. The cell colonization was macrophagic for the samples A and D while it was also composed of giant cells in the samples B, C and E at 8 weeks. The von Kossa's staining was positive only for the samples D and E after 4 weeks of implantation. The calcium content of the samples D was 285.3 mg/g at 8 weeks while in E it was 44.4 mg/g dry tissue at 4 weeks; for the remaining samples the calcium content did not increase with the time (2.1–2.3 mg/g at 8 weeks). In conclusion, the pericardium decellularization and detoxification associated with its storage in a glutaraldehyde-free solution is a promising method in order to realize more durable pericardial bioprostheses. The investigated tissue treatments applied to the bovine pericardium permit removal of the calcium nucleation sites, and hence the avoidance of the severe drawback of the aldehyde crosslink, but at the same time maintain the necessary and well known tissue stability.     

The efficacy of a dual anticalcification agent treatment in the prevention of calcification of glutaraldehyde-fixed porcine pericardium

The efficacy of a series of combined anticalcification treatments utilizing a chloroform-methanol mixture or a surfactant, sodium dodecyl sulfate, followed by a trivalent metal ion Fe(III) were evaluated with glutaraldehyde-treated porcine pericardium. The results indicate that the combined treatments are comparable in the reduction of calcification levels to that of the metal ion alone. Furthermore, pretreatment with the organic reagents improves the residence time of Fe(III) in the tissue. The results suggest that the combination approach offers a basis for improved mitigation of calcification in glutaraldehyde-treated tissue over that so far found in single systems.     

Calcification of subcutaneously implanted collagens in relation to cytotoxicity,cellular interactions and crosslinking

In general, calcification of biomaterials occurs through an interaction of host and implanted material factors, but up to now the real origin of pathologic calcification is unknown. In this study we aimed to investigate incidence of calcification of (crosslinked) dermal sheep collagens (DSCs) with respect to their specific properties, during subcutaneous implantation in rats. Three types of DSCs were commercially obtained: non-crosslinked DSC (NDSC), and DSC crosslinked with glutaraldehyde (GDSC) and hexamethylenediisocyanate (HDSC). NDSC, HDSC and GDSC were (enzymatically) tissue culture pretreated to eliminate their cytotoxic products. Beside this, crosslinking methods were modified to optimize mechanical properties and to decrease cytotoxicity, which resulted in HDSC^* and GDSC^*. Furthermore, DSC was crosslinked by activation of the carboxylic groups, i.e. by means of acyl azide and carbodiimide, resulting in AaDSC and CDSC, respectively. After implantation of HDSCs and GDSCs a relation between cytotoxicity and calcification of crosslinked DSC could be made. No relation was found between cellular infiltration of DSCs and calcification. However, from the use of different types and modification of crosslinking methods it might be concluded that calcification is mainly related to stable crosslinks, i.e. to the chemical properties of the obtained material.     

病毒感染对细胞表面超微结构影响的研究

用原子力显微镜（AFM）技术观察汉坦病毒的基本形貌,分别对用戊二醛固定的Vero—E6细胞和用汉坦病毒感染过的Vero—E6细胞进行成像,在原子级或纳米级水平上观测病毒感染后细胞表面超微结构的变化。将病毒直接滴加到云母片上自然风干后进行扫描,可以清晰地观察到病毒的结构大小;用0．5％～2．0％浓度的戊二醛固定细胞,通过成像发现,固定液浓度高时虽然成像质量较好,但对细胞的损伤较大,降低固定液浓度,细胞的形态接近于生理状态,成像质量良好;用不同稀释度的病毒感染细胞后,用合适的戊二醛浓度固定细胞进行观察,发现病毒感染前后细胞的形态结构发生了较大的变化,并且发现其形态的变化与病毒感染的浓度有显著的相关性。     

Calcification and identification of metalloproteinases in bovine pericardium after subcutaneous implantation in rats

The purpose of this study was to evaluate the influence of two anticalcification pre-treatments (chloroform/methanol and ethanol) and serum conditioning of glutaraldehyde-crosslinked bovine pericardium on the calcification degree and the presence of gelatinase activities in a subcutaneous implantation model in rats. Regarding calcification of the implants, glutaraldehyde control treatments showed a significatively higher calcification degree than pericardium treated with anticalcification reagents. Serum conditioning of glutaraldehyde treated tissues did not influence the calcification degree; moreover, no differences were found in these samples with the time of implantation (30 and 90 days). On the other hand, anticalcification treatments resulted in a very significant decrease in the calcium content in the implanted membranes. Gelatinase activities were detected by gelatin zymography in almost all the implanted samples. However, control tissues with and without serum conditioning showed less gelatinase activities than those samples pre-treated with anticalcification treatments. Metalloproteinase (MMP-2) activity was detected in all the samples analyzed but a higher expression of MMP-9 was detected in those implants treated with chloroform/methanol and ethanol. Additional gelatinase activities showing lower molecular weight than MMP-2 were also detected in both anticalcification treated samples. The presence of these gelatinase activities is probably due to host cellular infiltrates and could contribute to the biomaterial degradation.     

Synthesis and characterization of films based on cross linked blends of poly (vinylalcohol) and poly (vinylpyrrolidone) with glutaraldehyde for tissue engineering application

In these recent years, polymer blending is one of the significant interests by tissue engineering experts, since blending could produce biomaterials with enhanced physical and biological properties compare to the parent materials. In this context, present study aims at formation and characterization of polymeric blend of poly (vinylalcohol) and poly (vinylpyrrolidone) produced for tissue engineering application. Both polymers are blended at different concentrations to obtain films and nanofibers using solvent casting and electrospinning method respectively. The successful blending is confirmed by Fourier transform infrared spectroscopy, Field emission scanning electron microscope analysis and X‐ray diffraction. Later glutaraldehyde was added to chemically cross link the polymers and its effect was investigated on swelling and solubility properties of the blend. Experimental results reveal a relevant enhancement in the properties of poly (vinylalcohol) and poly (vinylpyrrolidone) blend when glutaraldehyde was added.     

Fluoropolymer Nanosheet as a Wrapping Mount for High‐Quality Tissue Imaging

In the field of biological microscopy technology, it is still a practical challenge to obtain high‐quality tissue images, due to the tissue desiccation that occurs during observations without an effective sample mounting. Inspired by the use of plastic food wrap, this study proposes the use of polymer thin films (also known as nanosheets) to fix the tissue samples. Water‐repellent nanosheets composed of the amorphous fluoropolymer CYTOP are prepared with adjustable thicknesses and their hydrophobicity, transparency, and adhesion strength are evaluated. They show excellent water‐retention effect and work well for sample fixation. By wrapping cleared mouse brain slices with a 133 nm thick CYTOP nanosheet, this study achieves high spatial resolution neuron images while scanning over a large area for a long period of time. No visible artifacts arising from sample shrinkage can be detected. This study also expects that nanosheet wrapping could be effective over a longer time span by combination with conventional agarose embedding.     

Application of polylactides in spinal cages: Studies in a goat model

Spinal cages are currently made of non-resorbable materials, but they only have a temporary function: after fusion, resorption is desirable both from a biological and mechanical point of view. We studied different polylactides in stand-alone condition in a goat model. Cages were made of 100% poly(L-lactic acid) (PLLA) or 70/30 poly(L/DL-Lactic acid) (PLDLLA); titanium served as control. After six months, all titanium cages showed non-unions comparable to that observed in a clinical retrieval, thus showing validity of the goat model. PLLA cages maintained their mechanical integrity for six months, enough to allow fusion. After that, the material resorbed within 48 months without adverse tissue reactions. Bone formation was faster in PLDLLA cages, but these already failed within three months, thus losing their stabilising function: 50% ended in pseudo-arthrosis. Additional internal fixation provided enough stability for fusion (83%). Biocompatibility of both PLLA and PLDLLA was excellent. The long-term results show that PLLA cages can be used for stand-alone interbody fusion, and that PLLA is an improvement over titanium in terms of fusion rate. PLDLLA showed enhanced bone formation, but also earlier failure of the implant. Chances for spinal fusion were better with additional internal fixation.     

High-frequency ultrasound detection of the temporal evolution of protein cross linking in myocardial tissue

The progressive increase in stiffening of the myocardium associated with the aging process and abetted by comorbid conditions such as diabetes may be linked to an excessive number of collagen cross links within the myocardial extra-cellular matrix. To determine whether ultrasound can delineate changes in the physical properties of heart tissue undergoing cross linking, the authors employed a model in which increased cross linking was induced by treating rat myocardial tissue with specific chemical fixatives. Rat hearts (n=5 each group) were arrested at end-diastole, insonified (30 to 50 MHz) fresh within a few minutes of excision in a phosphate buffered solution, placed in a fixative (10% formalin or 2.5% glutaraldehyde) and insonified at 30-minute intervals thereafter for 24 hours. Ultrasonic attenuation increased in tissues cross linked with formalin (maximal change: 27.2+/-3.4 dB/cm) and glutaraldehyde (maximal change: 40.2+/-5.6 dB/cm) over a 24-hour period. The frequency dependence of the attenuation coefficient increased as a function of the extent of collagen cross links in formalin (maximal change: 0.8+/-0.3 dB/cm-MHz) and glutaraldehyde (maximal change: 0.9+/-0.6 dB/cm-MHz). This study represents the first time that the precise time course of myocardial protein cross linking in situ has been characterized by using real time monitoring, and the physiologic effect has been delineated on microscopic material properties.     

Treatment of a soft tissue calcification in a patient receiving peritoneal dialysis

Chronic Kidney Disease patients suffer from Mineral and Bone Disorder (CKD‐MBD) leading to increased vascular and soft‐tissue calcification. The prevalence of soft tissue calcification in dialysis patients is not well described, and most cases describe such calcifications in hemodialysis patients. We describe a case of a massive soft tissue calcification in the right gluteal region in a peritoneal dialysis patient. The patient had severe pain and were disabled. The treatment was converted to an intensive hemodialysis regimen with a minimal calcium load and high dose of cinacalcet. During the treatment, the calcification diminished rapidly from a diameter of 26.6 to 2.9 cm, and the patient symptoms were relieved, leaving the patient with no pain or restriction in mobilization.