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Calcification of Leaflets from Porcine Aortic Valves Crosslinked by Ultraviolet Irradiation
Abstract
Glutaraldehyde (GA)-pretreated porcine aortic valves are generally used as a bioprosthetic valve, but gradual calcification of the leaflets often occurs. It has been hypothesized that the crosslinking agent, GA, stabilizes and perhaps modifies phosphorus-rich calcifiable structures in the bioprosthetic tissue. This is supported by our findings that calcium deposition is induced rapidly in GA-pretreated leaflets in comparison with ultraviolet (UV) irradiated leaflets. After 3 days of in vitro calcification test, calcium levels were 257.6 ± 23.5 μg/cm3 in GA-pretreated, 57.7 ± 10.2 μg/cm3 in the control, and 108.6 ± 7.6 μg/cm3 in 24 h UV irradiated leaflets. The calcium level in all test groups increased as time proceeds and the calcium level of GA-pretreated leaflets was significantly higher than the control and UV irradiated leaflets during test. This rapid calcium deposition on the GA-pretreated leaflets may be due to residual aldehyde groups after pretreatment. The exposure time of UV irradiation was not significantly correlated with the extent of calcification. After 14 days of the test, calcium levels in leaflets UV irradiated for 1, 2, 4, and 24 h were 502.6 ± 12.3 μg/cm3, 547.5 ± 34.1 μg/cm3, 564.3 ± 26.1 μg/cm3, and 543.0 ± 55.5 μg/cm3. In all test groups, [Ca]/[P] molar ratio decreased toward that of hydroxyapatite as the predominant mineral phase as time proceeds. This study suggests that UV irradiation can be considerable as an efficient crosslinking method to surmount the side effects induced by GA-pretreatment and may endow tissue with mechanical property.
... Collagen has been modified chemically to improve its biological and physical properties [39]. For example, collagen-based biomaterials are usually stabilized either by physical or chemical crosslinking to control the rate of biodegradation of the material [40,41], and succinylated collagen has been applied in cardiovascular implant coating in order to let the implants resistant for thrombogenesis [42]. However, the effect of these chemical treatments on molecular structural characteristics of collagen related to induce cell function has not been well investigated. ...
Collagen is regarded as one of the most useful biomaterials. The excellent biocompatibility and safety due to its biological characteristics, such as biodegradability and weak antigenecity, made collagen the primary source in biomedical application. Collagen has been widely used in the crosslinked form to extend the durability of collagen. The chemical treatment influences the structural integrity of collagen molecule resulting in the loss of triple helical characteristic. The structural characteristic of collagen is importantly related to its biological function for the interaction with cell. In this study, structural stability of collagen was enhanced thought EGCG treatment, resulting in high resistance against degradation by bacterial collagenase and MMP-1, which is confirmed by collagen zymography. The triple helical structure of EGCG-treated collagen could be maintained at 37 degrees C in comparison with collagen, which confirmed by CD spectra analysis, and EGCG-treated collagen showed high free-radical scavenging activity. Also, with fibroblasts culture on EGCG-treated collagen, the structural stability of EGCG-treated collagen provided a favorable support for cell function in cell adhesion and actin filament expression. These observations underscore the need for native, triple helical collagen conformation as a prerequisite for integrin-mediated cell adhesion and functions. According to this experiment, EGCG-treated collagen assumes to provide a practical benefit to resist the degradation by collagenase retaining its structural characteristic, and can be a suitable biomaterial for biomedical application.
... As a result GAGs are unremittingly lost from BHVs during in vitro fatigue experiments, storage, as well as when implanted in vivo345. Alternative fixation techniques using carbodiimides [1], epoxides [6], acyl azides [7], dye-mediated fixation [8], ultraviolet irradiation [9] and sodium periodate [10] been used to over come the inadequacies associated with GLUT. Only periodate crosslinking has been investigated specifically for GAG-targeted fixation. ...
Numerous crosslinking chemistries and methodologies have been investigated as alternative fixatives to glutaraldehyde (GLUT) for the stabilization of bioprosthetic heart valves (BHVs). Particular attention has been paid to valve leaflet collagen and elastin stability following fixation. However, the stability of glycosaminoglycans (GAGs), the primary component of the spongiosa layer of the BHV, has been largely overlooked despite recent evidence provided by our group illustrating their structural and functional importance. In the present study we investigate the ability of two different crosslinking chemistries: sodium metaperiodate (NaIO(4)) followed by GLUT (PG) and 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) followed by GLUT (ENG) to stabilize GAGs within BHV leaflets and compare resulting leaflet characteristics with that of GLUT-treated tissue. Incubation of fixed leaflets in GAG-degrading enzymes illustrated in vitro resistance of GAGs towards degradation in PG and ENG treated tissue while GLUT fixation alone was not effective in preventing GAG loss from BHV leaflets. Following subdermal implantation, significant amounts of GAGs were retained in leaflets in the ENG group in comparison to GLUT-treated tissue, although GAG loss was evident in all groups. Utilizing GAG-targeted fixation did not alter calcification potential of the leaflets while collagen stability was maintained at levels similar to that observed in conventional GLUT-treated tissue.
High resolution solid state NMR spectroscopy is a powerful tool for understanding bone structures, bone substitutes and implants. In particular it can be used to estimate osteoformation via bioceramic bone colonisation. Pathological calcification occurring in bioprosthetic heart valves and breast prostheses can be characterised.
Pathologic calcification of implants is a detrimental condition that can severely impact device performance and ultimately lead to implant failure. Although calcification affects a wide variety of medical implants, both synthetic and biologically derived, the pathogenesis of the disease is not well understood. In biologically derived implants, such as bioprosthetic heart valves, the major mechanisms of and factors contributing to calcification are chemical crosslinking, cellular damage, extracellular matrix composition, patient factors, and mechanical stress on the device. However, in synthetic-material implants, for example, intraocular lenses, pacemakers, and vascular replacements, calcification is largely due to surface conditions, such as material porosity, surface defects, and protein adsorption. This chapter investigates the mechanisms of implant calcification and discusses anti-calcification strategies, using specific medical devices as examples.
Bioprosthetic heart valves (BPHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, the majority of bioprostheses fail clinically because of calcification and degeneration. We have recently shown that glycosaminoglycan (GAG) loss may be in part responsible for degeneration of glutaraldehyde-crosslinked bioprostheses. In the present studies, we used a mild reaction of periodate-mediated crosslinking to stabilize glycosaminoglycans in the bioprosthetic tissue. We demonstrate the feasibility of periodate reaction by crosslinking major components of extracellular matrix of bioprosthetic heart valve tissue, namely type I collagen and hyaluronic acid (HA). Uronic acid assay of periodate-fixed HA-collagen matrices showed 48% of HA disaccharides were bound to collagen. Furthermore, we show that such reactions are also feasible to fix glycosaminoglycans present in the middle spongiosa layer of bioprosthetic heart valves. The periodate reactions were compatible with conventional glutaraldehyde crosslinking and showed adequate stabilization of extracellular matrix as demonstrated by thermal denaturation temperature and collagenase assays. Moreover, uronic acid assays of periodate-fixed BPHV cusps showed 36% reduction in the amount of unbound GAG disaccharides as compared with glutaraldehyde-crosslinked cusps. We also demonstrate that calcification of BPHV cusps was significantly reduced in the periodate-fixed group as compared with the glutaraldehyde-fixed group in 21-day rat subdermal calcification studies (periodate-fixed tissue Ca 72.01 ± 5.97 μg/mg, glutaraldehyde-fixed tissue Ca 107.25 ± 6.56 μg/mg). We conclude that periodate-mediated GAG fixation could reduce structural degeneration of BPHVs and may therefore increase the useful lifetime of these devices. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 478–486, 2001
Bioprosthetic heart valves (BPHVs) derived from glutaraldehyde-crosslinked porcine aortic valves are frequently used in heart valve replacement surgeries. However, the majority of bioprostheses fail clinically because of calcification and degeneration. We have recently shown that glycosaminoglycan (GAG) loss may be in part responsible for degeneration of glutaraldehyde-crosslinked bioprostheses. In the present studies, we used a mild reaction of periodate-mediated crosslinking to stabilize glycosaminoglycans in the bioprosthetic tissue. We demonstrate the feasibility of periodate reaction by crosslinking major components of extracellular matrix of bioprosthetic heart valve tissue, namely type I collagen and hyaluronic acid (HA). Uronic acid assay of periodate-fixed HA-collagen matrices showed 48% of HA disaccharides were bound to collagen. Furthermore, we show that such reactions are also feasible to fix glycosaminoglycans present in the middle spongiosa layer of bioprosthetic heart valves. The periodate reactions were compatible with conventional glutaraldehyde crosslinking and showed adequate stabilization of extracellular matrix as demonstrated by thermal denaturation temperature and collagenase assays. Moreover, uronic acid assays of periodate-fixed BPHV cusps showed 36% reduction in the amount of unbound GAG disaccharides as compared with glutaraldehyde-crosslinked cusps. We also demonstrate that calcification of BPHV cusps was significantly reduced in the periodate-fixed group as compared with the glutaraldehyde-fixed group in 21-day rat subdermal calcification studies (periodate-fixed tissue Ca 72.01 +/- 5.97 microg/mg, glutaraldehyde-fixed tissue Ca 107.25 +/- 6.56 microg/mg). We conclude that periodate-mediated GAG fixation could reduce structural degeneration of BPHVs and may therefore increase the useful lifetime of these devices.
Polytetrafluoroethylene (PTFE), polyurethane (PU) and silicone are widely known biocompatible polymers which are commonly used for vascular grafts. However, in vitro and in vivo calcifications of these polymers have been found to seriously compromise their quality as biomaterials. In consideration of this problem, the present study compared the calcification rate and extent of PTFE, PU and silicone. Using the in vitro flow-type method, PTFE, PU and silicone films were tested for 1, 4, 7, 10, 14 and 21 days. After 21 days of in vitro calcification test, the calcium levels on PTFE, PU and silicone were 35.89 +/- 5.01 microg/cm2, 23.73 +/- 0.68 microg/cm2 and 19.86 +/- 5.28 microg/cm2, respectively. The higher observed calcium level for PTFE may be due to the effect of the rough surface of PTFE in accumulating calcium ions on the polymer surface. From the 7th day of test, the [Ca]/[P] molar ratio started to decrease over time, and PTFE showed a faster calcification process. This decreasing [Ca]/[P] molar ratio demonstrated the typical calcification mechanism consisting of phosphorus ion accumulation following calcium ion accumulation. This study concluded that PU and silicone are less calcified than PTFE film, a finding in good agreement with previously published studies.
High-resolution solid state NMR spectroscopy appears to be a powerful method for a better understanding of bone structures and bone substitutes and implants. It is particularly efficient to estimate osteoformation via bioceramic bone colonization. Pathological calcification occurring in bioprosthetic heart valves and breast prostheses can be characterized as a complement to X-ray spectrometry.
Surgical replacement with artificial devices has revolutionised the care of patients with severe valvular diseases. Mechanical valves are very durable, but require long-term anticoagulation. Bioprosthetic heart valves (BHVs), devices manufactured from glutaraldehyde-fixed animal tissues, do not need long-term anticoagulation, but their long-term durability is limited to 15 - 20 years, mainly because of mechanical failure and tissue calcification. Although mechanisms of BHV calcification are not fully understood, major determinants are glutaraldehyde fixation, presence of devitalised cells and alteration of specific extracellular matrix components. Treatments targeted at the prevention of calcification include those that target neutralisation of the effects of glutaraldehyde, removal of cells, and modifications of matrix components. Several existing calcification-prevention treatments are in clinical use at present, and there are excellent mid-term clinical follow-up reports available. The purpose of this review is to appraise basic knowledge acquired in the field of prevention of BHV calcification, and to provide directions for future research and development.
Bioprosthetic heart valve tissue and associated calcification were studied in their natural state, using environmental scanning electron microscopy (ESEM). Energy dispersive X-ray micro-analysis, X-ray diffraction, Fourier-transform infrared and Raman spectroscopy were used to characterize the various calcific deposits observed with ESEM. The major elements present in calcified valves were also analyzed by inductively coupled plasma-optical emission spectroscopy. To better understand the precursor formation of the calcific deposits, results from the elemental analyses were statistically correlated. ESEM revealed the presence of four broad types of calcium phosphate crystal morphology. In addition, two main patterns of organization of calcific deposits were observed associated with the collagen fibres. Energy dispersive X-ray micro-analysis identified the crystals observed by ESEM as salts containing mainly calcium and phosphate with ratios from 1.340 (possibly octacalcium phosphate, which has a Ca/P ratio of 1.336) to 2.045 (possibly hydroxyapatite with incorporation of carbonate and metal ion contaminants, such as silicon and magnesium, in the crystal lattice). Raman and fourier-transform infrared spectroscopy also identified the presence of carbonate and the analyses showed spectral features very similar to a crystalline hydroxyapatite spectrum, also refuting the presence of precursor phases such as beta-tricalcium phosphate, octacalcium phosphate and dicalcium phosphate dihydrate. The results of this study raised the possibility of the presence of precursor phases associated with the early stages of calcification.
Tensile testing of tissue strips has been used to examine the effect of simple fixation in glutaraldehyde and formaldehyde on the viscoelastic properties of bovine pericardium. To assess tissue anisotropy, tissue strips were cut at 0 degree, 30 degrees, 60 degrees, and 90 degrees relative to the base-to-apex direction. Fresh anterior pericardium was modestly anisotropic, being least extensible in the base-to-apex direction; however, fixation removed this anisotropy. Fixation also produced a marked change in the response of the material to initial cyclic loading during preconditioning. Overall extensibility of the fixed material was significantly greater than that for the fresh tissue, consistent with a 10.7% shrinkage in aldehydes calculated from strain at fracture data. Reductions in stress relaxation and creep after fixation were noted as well, consistent with intrafibrillar crosslinking. Cyclic hysteresis and ultimate tensile strength were unaffected. Since the observed changes in the stress-strain response were largely attributable to shrinkage, control of shrinkage by physical means would allow for engineering modification of bovine pericardial mechanics for controlled anisotropy.
Although collagen-containing implants are widely used in various surgical applications, there has been relatively little attention paid to the possibility that this type of biomaterial may undergo pathologic calcification which could compromise its function. The present study reports for the first time the calcification of a series of implants of purified collagen sponges prepared with graded degrees of aldehyde-induced cross-linkages (assessed by shrinkage-temperature, wetting time, and collagenase digestibility). Type I collagen sponges were pretreated with either glutaraldehyde (0.1% to 2.0% aqueous solution, for 5-180 minutes) or formaldehyde (as vapors for 15 minutes to 15 hours), and implanted subcutaneously for 21 days in weanling rats. Although specimens not pretreated with either aldehyde reagent and the formaldehyde sponges pretreated for 15 minutes were resorbed without evidence of calcification, all other aldehyde-pretreated implants mineralized. The degree of calcification did not correlate with extent of cross-linking. Formaldehyde-pretreated implants calcified more extensively (Ca2+ = 87.8 +/- 2.8 micrograms/mg, mean +/- standard error of the mean; n = 58) than did glutaraldehyde-pretreated implants (Ca2+ = 40.9 +/- 1.4 micrograms/mg; n = 52). It is concluded that both glutaraldehyde- and formaldehyde-pretreated Type I collagen sponges calcify after subdermal implantation in young rats. Although aldehyde pretreatment of Type I collagen sponge implants is a prerequisite for their eventual mineralization, the threshold level of aldehyde-induced cross-linking required to potentiate their maximal pathologic calcification is low.
The porcine bioprosthetic heart valve has been commercially available since 1970 and has been the prosthetic heart valve of choice in our institution since 1971. Since that time 817 patients with 951 porcine valves have been discharged from the hospital and were available for long-term follow-up. Patient survival rates, with operative mortality excluded, were 80% ± 1.7% (standard error) at 5 years and 68% ± 2.7% at 10 years. Survival rates for patients with aortic valve prostheses were 78% ± 2.8% at 5 years and 57% ± 5.4% at 10 years; for patients with mitral valve prostheses, survival rates were 80% ± 2.2% at 5 years and 69% ± 3.2% at 10 years. Freedom from thromboembolism for aortic valves was 93% ± 1.4% at 5 years and 88% ± 2.6% at 10 years; for mitral valves the freedom from thromboembolism was 89% ± 1.5% at 5 years and 84% ± 2.2% at 10 years. Freedom from degeneration or primary tissue failure for aortic valves was 97% ± 1.3% at 5 years and 71% ± 7.6% at 10 years; for mitral valves these figures were 96% ± 1.2% at 5 years and 71% ± 4.1% at 10 years. Valves in patients 35 years of age and below had a significantly greater rate of degeneration (p < 0.001). After 12 years’ experience the porcine bioprosthetic valve has performed well with regard to patient survival and low rate of thromboembolism. For patients older than 35 years the freedom from primary tissue failure is 80% at 10 years.
Physical chemical observations have been made on the α-chymotrypsin-glutaraldehyde system during formation of an insoluble, enzymically active product. Lightscattering measurements indicate a two-step reaction, the second step being a linear condensation polymerization reaction. The effects of pH and ionic strength on the rate of the second-step reaction are best explained in terms of an acid shift in the pKa of the ϵ-amino groups of glutaraldehyde-modified lysine residues and a decrease in attractive forces between enzyme particles with increasing ionic strength, respectively. The large cross-linked particles formed appear to be branched flexible coils. Time-dependent ultraviolet spectra demonstrate an apparent hyperchromism due to increased scattering with no new absorption bands. Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements show no gross unfolding of the α-chymotrypsin molecule upon cross-linking, although small local changes in conformation could be indicated by changes in the CD spectrum at 255 and 229 nm. Measurements on small soluble polymers show that there is a large loss in activity (60%–70%) during formation of these relatively small derivatives, suggesting that reaction of glutaraldehyde with primary amino groups and not intermolecular cross-link formation could be the main reason for the loss in activity.
We previously demonstrated that ultraviolet (UV) or dehydrothermal (DHT) crosslinking partially denatured fibers extruded from an insoluble type I collagen dispersion. In this study denaturation effects were evaluated by measuring collagen-fiber sensitivity to trypsin. Shrinkage-temperature measurements and sensitivity to collagenase served as indices of crosslinking. UV or DHT crosslinking increased the collagen-fiber shrinkage temperature, resistance to degradation in collagenase, and durability under load in collagenase. However, in trypsin solutions, solubility was significantly increased for UV (≈11%) or DHT (≈15%) crosslinked fibers compared with uncrosslinked fibers (≈4%). Size-exclusion chromatography indicated that no intact collagen α-chains were present in the soluble fraction of fibers exposed to trypsin (MW <1 kD). Interestingly, UV-crosslinked collagen fibers remained intact an order of magnitude longer (4840 ± 739 min) than DHT-crosslinked (473 ± 39 min) or uncrosslinked (108 ± 53 min) fibers when placed under load in trypsin solutions. These data indicate that mechanical loading during incubation in a trypsin solution measures denaturation effects not detected by the trypsin-solubility assay. Our results suggest that DHT-crosslinked collagen fibers should not be used as load-bearing implants. UV-crosslinked fibers may retain more native structure and should exhibit greater resistance to nonspecific proteases in vivo. © 1996 John Wiley & Sons, Inc.
A dynamic flow type testing to study calcification was self-designed to investigate calcification in bioprosthetic heart valves. The apparatus consists of a container into which leaflets from a porcine aortic valve are placed, a chamber that contains calcium solution, and a peristaltic pump that provides a continuous supply of the solution toward the container. Efficacy of the apparatus was compared with the conventional batch type calcification testing at 37°C through measuring the amount of calcium and phosphate deposited by inductively coupled plasma (ICP) and scanning electron microscope (SEM). After 14 days, calcium levels detected from the calcified deposit on leaflets were 470.4 ± 37.0 μg/cm3 in the flow type testing whereas in the batch type testing levels were 81.0 ± 6.7 μg/cm3. Though the calcium level on the leaflet increased as the exposure time to calcium solution increased in both testings, the rate and the tendency of calcification could be assessed very rapidly by flow type testing in comparison with batch type testing. [Ca]/[P] molar ratio decreased over time, and after 14 days, the ratio was close to 1.83 ± 0.18 in the flow type testing. The ratio could not be determined in the batch type testing because the deposit was too small to assess. The descending rate of [Ca]/[P] molar ratio demonstrates that deposited calcium-complex at the earliest stage may interact with inorganic phosphate ions to create a calcified deposit mineral precursor. This in vitro dynamic flow type calcification testing was a favorable tool for rapid investigation of calcification.
This review presents various considerations on the construction of a synthetic burn dressing, based mainly on collagen protein. Membranous wound covers are compared with sponge-felt types, monocomponental with composite. The importance of collagen crosslinking agent and the nonextractibility of any component from the dressing material are discussed. According to the type of the burn the dressing should be used dry or wet, plain or medicated, and changed often to reduce substantially the presence of necrotic tissue, inflammatory cells of the granulation tissue, and bacterial contamination.
Calcification of cardiovascular prosthetic implants is a common and important problem. This review provides an update based upon the Conference on Cardiovascular Implant Calcification held as part of the 13th World Congress of the International Society for Heart Research, 1989. A variety of cardiovascular prostheses are affected clinically by calcification, including bioprosthetic heart valves, aortic homografts and trileaflet polymeric valve prostheses. In addition, experimental studies have demonstrated calcification of artificial heart devices in ventricular assist systems in long-term calf studies. The pathophysiology of this disease process is incompletely understood. A common element between the various types of cardiovascular implant calcification is the localization of calcific deposits to devitalized cells and membranous debris. Prevention of cardiovascular implant calcification by either biomaterial modifications or regional drug therapy (controlled release) is being investigated.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Degenerative alterations of two different glutaraldehyde (GA)-fixed bioprosthetic heart valve materials were investigated in subcutaneous rat implants: Bovine pericardium, prepared according to clinically used bioprosthetic heart valve material (BHV) was compared to alternatively preserved pericardium (APHV), which was fixed in GA and treated with L-glutamic acid. Following 63 days of subcutaneous implantation, calcification of APHV implants was significantly lower as compared to BHV implants (13 +/- 6 versus 158 +/- 18 micrograms Ca/mg dry weight tissue; p less than 0.05). In BHV implants ultrastructural investigations showed nucleation of plate-shaped hydroxyapatite crystals at the surface of collagen fibrils and in remnants of connective tissue cells; no signs of calcification could be detected in APHV implants. The time-course of the inflammatory reaction was determined by quantification of immunohistochemical stained mononuclear host-cells invading the implants. In both preparation groups inflammatory reaction reached maximum 42 days after implantation. However, infiltration rate of inflammatory cells was markedly decreased in APHVs as compared to BHVs (p less than 0.05).
Crosslinking of collagenous biomaterials currently employs the use of glutaraldehyde. The putative enhancement of glutaraldehyde crosslinking by lysine was investigated in three model systems: bovine pericardium, collagen membranes, and bovine serum albumin. Repetitive sequential treatment of bovine pericardium with glutaraldehyde and lysine and finally with formaldehyde produced a matrix which, by the two criteria used (shrinkage temperature and urea/SDS soluble collagen), was shown to be more highly crosslinked than pericardium fixed in glutaraldehyde alone. Essentially the same results were obtained when membranes prepared from pepsin-soluble pericardial collagen were subjected to sequential glutaraldehyde and lysine treatments, reaching shrinkage temperatures of more than 90 degrees C. Heart valves prepared from lysine-enhanced glutaraldehyde crosslinked bovine pericardium were tested in vitro in an accelerated fatigue tester and have been shown to behave satisfactorily after 300 million cycles. These additional crosslinks proved to be stable in saline at 37 degrees C. Studies on bovine serum albumin attempted to get an insight into the mechanisms of lysine enhancement of glutaraldehyde crosslinking by treating sequentially albumin with glutaraldehyde and lysine and analysis of the products by gel filtration and SDS-PAGE. These studies suggest that free amino groups exposed by proteins are initially reacted with glutaraldehyde and then bridged by the diamino compound (lysine) producing more extensive intermolecular crosslinking than glutaraldehyde alone.
The reversibility of glutaraldehyde crosslinks has been suggested as a reason for failure of long-term bioprosthetic implants. The stability of such crosslinks was investigated in tendons and model compounds. Small but cytotoxic levels of glutaraldehyde were still released from crosslinked tendons even after these tendons were extensively rinsed for up to 6 months. The toxic effect was evidenced by the death of fibroblasts surrounding a midsection piece of rinsed crosslinked tendon, while the end section pieces did not show toxic effects. The formation and stability of glutaraldehyde modified [14C]-L-lysine derivatives were investigated. The polymerization of glutaraldehyde with amino compounds was initially fast but continued to proceed slowly for months. Degradation of high-molecular-weight soluble polymers was detected by gel filtration chromatography. Low-molecular-weight soluble materials were also released from insoluble products which were formed when high concentrations of glutaraldehyde and radioactive lysine were reacted. These chemical and biological studies suggest that local cytotoxicity of glutaraldehyde crosslinked bioprostheses may be due to unstable glutaraldehyde polymers that persist in the interstices of crosslinked tissues.
High-strength bioactive glass-ceramic A-W was soaked in various acellular aqueous solutions different in ion concentrations and pH. After soaking for 7 and 30 days, surface structural changes of the glass-ceramic were investigated by means of Fourier transform infrared reflection spectroscopy, thin-film x-ray diffraction, and scanning electronmicroscopic observations, in comparison with in vivo surface structural changes. So-called Tris buffer solution, pure water buffered with trishydroxymethyl-aminomethane, which had been used by various workers as a "simulated body fluid," did not reproduce the in vivo surface structural changes, i.e., apatite formation on the surface. A solution, ion concentrations and pH of which are almost equal to those of the human blood plasma--i.e., Na+ 142.0, K+ 5.0, Mg2+ 1.5, Ca2+ 2.5, Cl- 148.8, HCO3- 4.2 and PO4(2-) 1.0 mM and buffered at pH 7.25 with the trishydroxymethyl-aminomethane--most precisely reproduced in vivo surface structure change. This shows that careful selection of simulated body fluid is required for in vitro experiments. The results also support the concept that the apatite phase on the surface of glass-ceramic A-W is formed by a chemical reaction of the glass-ceramic with the Ca2+, HPO4(2-), and OH- ions in the body fluid.
This study involves comparison of the mechanical properties of reconstituted collagen fibres with those of collagen fibres obtained from rat tail tendons. Reconstituted collagen fibres were cross-linked in the presence of glutaraldehyde vapour for 2 and 4 d or using a combination of severe dehydration and carbodiimide treatment. Ultimate tensile strengths for reconstituted fibres cross-linked with glutaraldehyde ranged from 50 to 66 MPa while those cross-linked by severe dehydration and carbodiimide treatment had ultimate tensile strengths between 24 and 31 MPa. Rat tail tendon fibres had tensile strengths that ranged from 33 to 39 MPa. These results indicate that high-strength collagen fibres can be reconstituted in vitro and that these fibres may be useful in repair of dermal, dental, cardiovascular and orthopaedic defects.
Glutaraldehyde crosslinking of native or reconstituted collagen fibrils and tissues rich in collagen significantly reduces biodegradation. Other aldehydes are less efficient than glutaraldehyde in generating chemically, biologically, and thermally stable crosslinks. Tissues crosslinked with glutaraldehyde retain many of the viscoelastic properties of the native collagen fibrillar network which render them suitable for bioprostheses. Implants of collagenous materials crosslinked with glutaraldehyde are subject long-term to calcification, biodegradation, and low-grade immune reactions. We have attempted to overcome these problems by enhancing crosslinking through bridging of activated carboxyl groups with diamines and using glutaraldehyde to crosslink the epsilon-NH2 groups in collagen and the unreacted amines introduced by aliphatic diamines. This crosslinking reduces tissue degradation and nearly eliminates humoral antibody induction. Covalent binding of diphosphonates, specifically 3-amino-1-hydroxypropane-1, 1-diphosphonic acid (3-APD), and chondroitin sulfate to collagen or to the crosslink-enhanced collagen network reduces its potential for calcification. Platelet aggregation is also reduced by glutaraldehyde crosslinking and nearly eliminated by the covalent binding of chondroitin sulfate to collagen. The cytotoxicity of residual glutaraldehyde--leaching through the interstices of the collagen fibrils or the tissue matrix--and of reactive aldehydes associated with the bound polymeric glutaraldehyde can be minimized by neutralization and thorough rinsing after crosslinking and storage in a nontoxic bacteriostatic solution.
Calcification is the principal mode of failure of bioprosthetic heart valves (BPHV) fabricated from glutaraldehyde-pretreated porcine aortic valves or bovine pericardium. Covalent binding of aminopropanehydroxy-diphosphonate (APDP) to residual glutaraldehyde in pericardial BPHV tissue was studied as an approach for the inhibition of calcification. BPHV tissue was preincubated in 0.14 M APDP at pH 7.4, 9.0, and 11.0 for various durations (1 hour to 8 days). The need for NaBH4 stabilization of the tissue-bound APDP was also examined in vitro. The bound APDP was determined using 14C-labeled APDP. APDP uptake was dependent on incubation duration and pH. Calcification of APDP-pretreated BPHV was studied using 21-day rat subdermal implants. Calcification inhibition was directly related to the amount of tissue APDP incorporation. Inhibition of calcification to less than 15% of control was achieved with a concentration of bound APDP of greater than or equal to 30 nM/mg dry tissue with more than 1 hour of incubation at pH 11.0 (bound APDP, 33.55 nM/mg; BPHV calcium content = 3.1 +/- 0.9 micrograms/mg). No adverse effects such as rat growth inhibition or disruption of bone architecture were observed after any treatment. Additionally, in vitro, NaBH4 stabilized tissue-bound APDP. In conclusion, APDP covalently bound to residual aldehyde functions markedly inhibited calcification of BPHV tissue. This inhibition was dependent on the amount of APDP incorporated. NaBH4 stabilized APDP-glutaraldehyde covalent bonds.
Bovine pericardium, a dense collagenous connective tissue, was crosslinked with glutaraldehyde using different modalities of fixation. The degree of crosslinking was evaluated as a function of the ability of CNBr and pronase to solubilize collagen. Our results suggest that glutaraldehyde fixes primarily the surface of the fibers and creates a polymeric network which hinders the further crosslinking of the interstitium of the fiber. When a low concentration of glutaraldehyde was used, a slow time-dependent crosslinking process was observed. This slow process is maintained over a long period of time, greatly beyond that required for the actual penetration of glutaraldehyde to occur.
Deposition of calcium-containing apatite mineral occurs widely in association with cardiovascular and noncardiovascular medical devices and biomaterials, is the leading cause of failure of contemporary bioprosthetic heart valves, and limits the functional lifetime of experimental (and potentially clinical) mechanical blood pumps and polymeric heart valves. Calcification of bioprosthetic tissue is primarily intrinsic, related to cuspal connective tissue cells and fragments, and collagen. In contrast, the predominant site of calcific crystals on flexing polymeric surfaces in blood pumps or valve prostheses is extrinsic, associated with adherent cells, thrombus, or pseudointima. Pathologic calcification shares key features with physiologic skeletal mineralization, including crystal initiation through the mediation of cell membranes, usually in the form of extracellular vesicles. This suggests a unified hypothesis for normal and abnormal mineralization. Several approaches are being studied experimentally for the inhibition of bioprosthetic heart valve calcification. Controlled-release diphosphonate therapy, perhaps in conjunction with an anticalcification cuspal pretreatment, appears most effective. Research objectives in biomaterial-associated calcification include (1) development of animal models, (2) determination of initial crystal nucleation events and sites, (3) elucidation of the relative roles of host, implant, and mechanical determinants, and (4) development of approaches for the inhibition of mineralization.
The porcine bioprosthetic heart valve has been commercially available since 1970 and has been the prosthetic heart valve of choice in our institution since 1971. Since that time 817 patients with 951 porcine valves have been discharged from the hospital and were available for long-term follow-up. Patient survival rates, with operative mortality excluded, were 80% +/- 1.7% (standard error) at 5 years and 68% +/- 2.7% at 10 years. Survival rates for patients with aortic valve prostheses were 78% +/- 2.8% at 5 years and 57% +/- 5.4% at 10 years; for patients with mitral valve prostheses, survival rates were 80% +/- 2.2% at 5 years and 69% +/- 3.2% at 10 years. Freedom from thromboembolism for aortic valves was 93% +/- 1.4% at 5 years and 88% +/- 2.6% at 10 years; for mitral valves the freedom from degeneration or primary tissue failure for aortic valves was 97% +/- 1.3% at 5 years and 71% +/- 7.6% at 10 years; for mitral valves these figures were 96% +/- 1.2% at 5 years and 71% +/- 4.1% at 10 years. Valves in patients 35 years of age and below had a significantly greater rate of degeneration (p less than 0.001). After 12 years' experience the porcine bioprosthetic valve has performed well with regard to patient survival and low rate of thromboembolism. For patients older than 35 years the freedom from primary tissue failure is 80% at 10 years.
A consecutive series of 706 mitral valve replacements was performed from January, 1972, to January, 1984. The follow-up ranged from 6 to 150 months with a mean of 50 and a median of 43 months. Seven percent (50) of the patient were lost to follow-up. There were 243 men and 463 women, whose ages ranged from 17 to 86 years (mean 58). A porcine bioprosthetic valve was implanted in 528 patients (514 Hancock and 14 Carpentier-Edwards valves) and a prosthetic disc valve in 178 patients (102 standard disc Björk-Shiley, 34 Beall, and 42 Harken disc valves). Seven patients were in Functional Class II, 325 in Class III, and 374 in Class IV. A concomitant operative procedure was performed in 253 of the 706 patients (36%). Mitral regurgitation was the primary hemodynamic lesion in 363 and mitral stenosis in 343. Operative mortality figures were as follows: 77 of 706 (11%) for the overall group, 34 of 453 (7.5%) for isolated mitral valve replacement, 30 of 169 (17.5%, p = 0.001) for mitral replacement plus coronary bypass, 49 of 528 (9%) for the bioprosthetic valve group, and 28 of 178 (16%) for the prosthetic disc valve group (p = 0.01). After the operation, 262 patients were in Functional Class I, 99 in Class II, and 18 in Class III. The long-term survival rate was significantly lower in patients who had an associated procedure (45% +/- 6%), who had mitral regurgitation rather than mitral stenosis (53% +/- 5% versus 67% +/- 4%) (p = 0.002), who were in Functional Class IV rather than Classes I to III (51% +/- 4% versus 70% +/- 4%) (p = 0.001), and who received a prosthetic disc valve rather than a bioprosthesis (40% +/- 6% versus 67% +/- 4%) (p = 0.001). Thromboembolic rates were significantly higher with prosthetic valves than with bioprosthetic valves (4.6% +/- 0.22% versus 2.4% +/- 0.5% per patient-year of follow-up), and the incidence of anticoagulant-related hemorrhage was significantly higher in the prosthetic valve group (1.65% versus 0.43% per patient-year). Primary valve dysfunction was significantly more common in the bioprostheses (1.23% versus 0.40% per patient-year).(ABSTRACT TRUNCATED AT 400 WORDS)
THE chief product of calcification in bone is usually considered to be a variant of hydroxyapatite, Ca10 (PO4)6 (OH)2 (ref. 1), the unit cell of the crystal lattice containing all eighteen ions of the formula. A tacit assumption is made that the two OH- ions come from the water in the calcifying solution. Because the equilibrium constant for water (Kw) is only 10-14 (mole/l.) (ref. 2) at 25° C, however, the dissociation of water would require more energy to increase the OH- concentration than is readily available in the surrounding mineral matter2.
1. A study has been made of the effect of ultraviolet irradiation, in the presence of air and nitrogen, on the conformational changes taking place in cooled solutions of gelatin prepared from thermally denatured neutral-salt-soluble collagen. 2. The increase in negative rotation and viscosity at 15 degrees for irradiated and thermally denatured solutions of collagen becomes less as the irradiation time is increased. 3. The principal effect of ultraviolet irradiation is the fission of the primary collagen peptide chains, eventually yielding chain lengths incapable of stabilizing a helical structure. 4. Irradiation in both air and nitrogen results in a loss of tyrosine, histidine and phenylalanine, with more denaturation occurring in the presence of nitrogen.
Aortic valve replacement was performed in 912 consecutive patients from January, 1972, to January, 1983. The 616 male and 296 female patients, whose ages ranged from 16 to 95 years (mean 60.6 years and median 63 years), received 663 bioprosthetic valves and 249 tilting disc valves. A higher incidence of Functional Class IV heart disease and ascending aortic aneurysms was noted in the group receiving the tilting disc valve. Six hundred fifty-seven patients had primarily aortic stenosis and 255 had primarily aortic regurgitation. Associated procedures were done in 308 patients (33%): 233 had coronary bypass grafting, 46 had replacement of ascending aortic aneurysms, and 29 had miscellaneous procedures. The overall operative mortality was 6.4% (59/912). The operative mortality was 4.5% (29/640) for isolated aortic valve replacement, 4.2% (21/233) for valve replacement plus coronary bypass, and 17% (8/46) for valve replacement plus replacement of an ascending aortic aneurysm. The mortality was 4.2% (20/663) for the group receiving bioprostheses and 12.4% (31/249) for those receiving tilting disc valves. The operative mortality for 1983 for all aortic valve replacement procedures was 2.1%; for isolated valve replacement, 1%; for valve replacement plus coronary bypass, 4.4%; and for valve replacement plus aortic aneurysm replacement, 0%. The long-term follow-up was analyzed as of Jan. 1, 1984, so that there was a minimum follow-up of 12 months (mean 55 months and median 51 months). The actuarial survival rate at 108 months for all patients was 67% +/- 2%; for valve replacement alone, 71% +/- 3%; for valve replacement plus coronary bypass, 58% +/- 7%; for valve replacement plus ascending aortic aneurysm replacement, 45% +/- 10%; for aortic stenosis, 70% +/- 3%; for aortic regurgitation, 61% +/- 4%; for Functional Classes I to III, 77% +/- 3%; for Class IV, 53% +/- 4%; for age less than 63 years, 75% +/- 3%; and for age greater than 63 years, 57% +/- 4%. At 108 months, the probability of freedom from thromboembolism was 85% +/- 3% after bioprosthetic valve replacement and 83% +/- 3% after replacement with a tilting disc valve (p = NS). The probability of freedom from hemorrhage at 108 months was 98.6% +/- 7% for the bioprosthetic valve group and 89% +/- 2% for the tilting disc valve group (p less than 0.001). The valve thrombosis rate was 0.34% per patient-year for the tilting disc valves and 0.07% per patient-year for the bioprostheses.(ABSTRACT TRUNCATED AT 400 WORDS)
Bioprosthetic cardiac valve calcification is a frequent complication after long-term valve replacement. In this study the authors sought to examine the biologic determinants of this type of dystrophic calcification using subcutaneous implants of glutaraldehyde-preserved porcine aortic valve leaflets (GPVs) in rats. GPVs and clinical valvular bioprostheses were prepared identically. Retrieved implants were examined for calcification and the deposition of osteocalcin (OC), a vitamin K-dependent, bone-derived protein, that is found in other dystrophic and ectopic calcifications. GPVs implanted in 3-week-old rats calcified progressively (GPV Ca2+, 122.9 +/- 6.0 micrograms/mg) after 21 days, with mineral deposition occurring in a morphologic pattern comparable to that noted in clinical retrievals. Calcified GPVs accumulated osteocalcin (OC, 183.4 +/- 19.4 ng/mg); Nonpreserved porcine aortic leaflet implants did not calcify (Ca2+ + 5.6 +/- 1.0 micrograms/mg). Millipore diffusion chamber (0.45-mu pore size enclosed GPV implants accumulated calcium and adsorbed osteocalcin despite the absence of attached host cells. GPVs implanted for 21 days in 8-month-old rats calcified less (GPV Ca2+, 22.4 +/- 5.0 micrograms/mg) than did GPVs implanted in 3-week-old rats (see above). High-dose warfarin therapy (80 mg/kg) did not alter GPV calcification (GPV Ca2+, 39.6 +/- 2.9 micrograms/mg) in 72-hour subcutaneous implants in 3-week-old male rats, compared with control rats (GPV Ca2+, 40.8 +/- 4.8 micrograms/mg).
Data from 366 patients with mitral valve replacement (250 single and 116 multiple) who received pericardial xenografts between 1971 and 1981 were analyzed. Cumulative duration of follow-up was 1,151 patient-years, with a maximum duration of 10.7 years. Actuarial survival at 11 years is 71.6 +/- 14.2%. Pericardial valve failure occurred in 7 patients (0.6 episodes per 100 patient-years). Actuarial freedom from valve failure at 11 years is 90.4 +/- 9.1% for the entire series. Although 275 (75.1%) patients were in chronic atrial fibrillation, anticoagulants were not used in any patient beyond the first 6 postoperative weeks. The incidence of emboli was 0.6% per year. Six episodes occurred following single mitral valve replacement and 1 after multiple valve replacement (5 early and 2 late). The actuarial freedom from embolism in 96.4 +/- 1.5% at 6 and 11 years postoperatively. Valve thrombosis has not been encountered. This analysis has shown a low incidence of valve dysfunction and a very low risk of embolic complications without long-term anticoagulation. The pericardial xenograft is a safe substitute for the mitral valve, with predictable behavior during the first decade of follow-up.
Collagen fibers used in a scaffolding device for ligament reconstruction must be thin, strong, and degradable. The purpose of this study was to determine the effects of fiber diameter (20, 50, or 90 microns), crosslinking agent (uncrosslinked, dehydrothermal-cyanamide, or glutaraldehyde), and hydration on the initial mechanical properties, biocompatibility, and subcutaneous degradation rates of fibers extruded from an acidic dispersion of insoluble type I collagen. The wet tensile strength of extruded collagen fibers was significantly improved by decreasing the fiber diameter. Low-diameter, crosslinked fibers had wet tensile strengths ranging from 75-110 MPa. In contrast, high diameter fibers had wet strength values of about 30 MPa. The degradation rate of the implanted fibers, in contrast, was not significantly prolonged by changing the initial fiber diameter. This result is important because prolonged degradation of the fibers can lead to implant encapsulation instead of neoligament formation. By minimizing the diameter, fiber strength can be increased without prolonging the fiber degradation rate. Low-diameter, dehydrothermal-cyanamide crosslinked fibers have greater tensile strength and a more rapid degradation rate than medium-diameter, glutaraldehyde crosslinked fibers, and are therefore more suitable for use in a degradable ligament reconstruction device.
The purpose of this study was to characterize the physicochemical properties of calcific deposits that cause the failure of tissue-derived heart valve bioprostheses. This was done in an effort to understand the mechanism of pathologic biomineralization in the cardiovascular system and potentially prevent deterioration of bioprostheses. Calcific deposits taken from 10 failed bioprosthetic valves that had been implanted in patients for 2-13 years were characterized by chemical analysis, x-ray diffraction, FTIR spectroscopy, scanning electron microscopy, polarized light microscopy, and solubility measurements. The combined results identified the biomineral as an apatitic calcium phosphate salt with substantial incorporation of sodium, magnesium and carbonate. The average Ca/PO4 ratio for this "young" pathologic biomineral was approximately 1.3, considerably lower than approximately 1.7 found in mature atherosclerotic plaque biomineral and mature skeletal biomineral, both of which approximate hydroxyapatite in composition. Deproteinated calcific deposits from bioprostheses had thermodynamic solubilities comparable to those of both atherosclerotic plaque, typical pathologic biomineral and hydrolyzed octacalcium phosphate (OCP, Ca4H(PO4)3 x 2.5 H2O), a proposed precursor phase to biomineral apatite. This later finding, together with chemical composition and structural details of the bioprosthetic deposits themselves, supports a mechanism of cardiovascular calcification in which OCP plays a crucial role in the formation of the final apatitic phase. This suggests an approach toward prevention of bioprosthetic tissue calcification through control of the formation of the kinetically favored OCP precursor and/or its transformation into bioapatite.
To develop an artificial bone substitute that would be gradually degraded and replaced by the regenerating natural bone, a carbonate apatite type I atelocollagen (82/12 in v/v%) composite was designed. The carbonate apatite synthesized at 58 degrees C demonstrated a crystallinity similar to that of natural bone. Type I atelocollagen was purified from human umbilical cords. Carbonate apatite-atelocollagen composite rods (diameter 6 mm x height 10 mm) were irradiated by ultraviolet ray (wave length 254 nm) for 4 hr to increase the collagen fibrillar cross links. Rabbit tibiae were dissected to prepare an artificial total bone defect (length 10 mm). The composites and porous hydroxyapatite rods, sintered at 1200 degrees C, were implanted into the defects and the tibiae were fixed by external osseofixators. The implanted composites were gradually degraded in the lesions, and the regenerated bone totally replaced the defects within 6 weeks, while the hydroxyapatite rod implanted lesions were not replaced by bone. No specific histologic abnormalities appeared in either the hydroxyapatite or composite rod implanted lesions.
The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. We compared the effects of two physical crosslinking methods, ultraviolet irradiation (UV) (254 nm) and dehydrothermal (DHT) treatment, on the mechanical properties and molecular integrity of collagen fibers extruded from an acidic dispersion of type I bovine dermal collagen. Collagen fibers exposed to UV irradiation for 15 min had ultimate tensile strength (54 MPa) and modulus (184 MPa) values greater than or equivalent to values for fibers crosslinked with DHT treatment for 3 or 5 days. UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen fibers. Characterization of collagen extracted from the crosslinked samples by dilute acetic acid and limited pepsin digestion indicate that both UV and DHT treatments cause fragmentation of at least a portion of the collagen molecules. Partial loss of the native collagen structure may influence attachment migration, and proliferation of cells on collagen fiberbased ligament analogs. These issues are currently being addressed in our laboratory.
Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction.
Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87 +/- 0.29 micrograms/mg tissue compared with control calcium level, 236.00 +/- 6.10 micrograms/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22 +/- 2.94 micrograms/mg tissue; control calcium level, 32.50 +/- 11.50 micrograms/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid.
Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.
The effectiveness of ethanol pretreatment on preventing calcification of glutaraldehyde-fixed porcine aortic bioprosthetic heart valve (BPHV) cusps was previously demonstrated, and the mechanism of action of ethanol was attributed in part to both lipid removal and a specific collagen conformational change. In the present work, the effect of ethanol pretreatment on BPHV aortic wall calcification was investigated using both rat subdermal and sheep circulatory implants. Ethanol pretreatment significantly inhibited calcification of BPHV aortic wall, but with less than complete inhibition. The maximum inhibition of calcification of BPHV aortic wall was achieved using an 80% ethanol pretreatment; calcium levels were 71.80+/-8.45 microg/mg with 80% ethanol pretreatment compared to the control calcium level of 129.90+/-7.24 microg/mg (p = 0.001). Increasing the duration of ethanol exposure did not significantly improve the inhibitory effect of ethanol on aortic wall calcification. In the sheep circulatory implants, ethanol pretreatment partly prevented BPHV aortic wall calcification with a calcium level of 28.02+/-4.42 microg/mg compared to the control calcium level of 56.35+/-6.14 microg/mg (p = 0.004). Infrared spectroscopy (ATR-FTIR) studies of ethanol-pretreated BPHV aortic wall (vs. control) demonstrated a significant change in protein structure due to ethanol pretreatment. The water content of the aortic wall tissue and the spin-lattice relaxation times (T1) as assessed by proton nuclear magnetic resonance spectroscopy did not change significantly owing to ethanol pretreatment. The optimum condition of 80% ethanol pretreatment almost completely extracted both phospholipids and cholesterol from the aortic wall; despite this, significant calcification occurred. In conclusion, these results clearly demonstrate that ethanol pretreatment is significantly but only partially effective for inhibition of calcification of BPHV aortic wall and this effect may be due in part to lipid extraction and protein structure changes caused by ethanol. It is hypothesized that ethanol pretreatment may be of benefit for preventing bioprosthetic aortic wall calcification only in synergistic combination with another agent.
1. The effect of ultraviolet irradiation on acid-soluble and neutral-salt-soluble calf-skin collagen was studied by chromatography, gel filtration, amino acid analysis and sedimentation of the sub-units, and the reaction kinetics of degradation were obtained from viscosity and optical rotation measurements. 2. It was demonstrated that, whereas the structure of neutral-salt-soluble calf-skin collagen may be represented by the formula (alpha(1))(2)alpha(2), the acid-soluble extract has the formula alpha(1).(alpha(2))(2). The acid-soluble collagen is also unusual in containing a large amount of a component that could be beta(22). 3. Ultraviolet irradiation causes the progressive degradation of the collagen molecule into smaller molecular fragments that subsequently lose their helical nature. The rate constants show that the denaturation of soluble collagens by ultraviolet irradiation is much slower, under the conditions used, than denaturation by heat or enzymes.
Considerations on manufacturing principles of synthetic burn dressing: A review
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