Calcification of tissue heart valve substitutes: progress toward understanding and prevention

Calcification plays a major role in the failure of bioprosthetic and other tissue heart valve substitutes. Tissue valve calcification is initiated primarily within residual cells that have been devitalized, usually by glutaraldehyde pretreatment. The mechanism involves reaction of calcium-containing extracellular fluid with membrane-associated phosphorus to yield calcium phosphate mineral deposits. Calcification is accelerated by young recipient age, valve factors such as glutaraldehyde fixation, and increased mechanical stress. Recent studies have suggested that pathologic calcification is regulated by inductive and inhibitory factors, similar to the physiologic mineralization of bone. The most promising preventive strategies have included binding of calcification inhibitors to glutaraldehyde fixed tissue, removal or modification of calcifiable components, modification of glutaraldehyde fixation, and use of tissue cross linking agents other than glutaraldehyde. This review summarizes current concepts in the pathophysiology of tissue valve calcification, including emerging concepts of endogenous regulation, progress toward prevention of calcification, and issues related to calcification of the aortic wall of stentless bioprosthetic valves.     

Evaluation of a new disinfectant for dialyzer reuse

Glutaraldehyde has been proposed to be as effective as formaldehyde as a disinfectant for reprocessing capillary hemodialyzers. Formaldehyde has become the standard to which all disinfectants are compared. The two products are compared for microbiological efficacy, reuse, membrane integrity, biocompatibility, performance, residual binding and ease of removal, environmental hazards, and immunogenicity. Glutaraldehyde (0.8%) is as effective as 4% formaldehyde in its microbiocidal effect. The disinfectants are comparable except in the following areas: the use of glutaraldehyde leads to lower reuse rates than formaldehyde, significantly less glutaraldehyde than formaldehyde remains in the dialyzer following standard predialysis rinse procedures, and less glutaraldehyde than formaldehyde is found in environmental air.     

Nerve and conduction tissue injury caused by contact with BioGlue

BACKGROUND: BioGlue-a surgical adhesive composed of bovine albumin and glutaraldehyde-is commonly used in cardiovascular operations. The objectives of this study were to determine whether BioGlue injures nerves and cardiac conduction tissues, and whether a water-soluble gel barrier protects against such injury. MATERIALS AND METHODS: In 18 pigs, diaphragmatic excursion during direct phrenic nerve stimulation was measured at baseline and at 3 and 30 min after nerve exposure to albumin (n = 3), glutaraldehyde (n = 3), BioGlue (n = 6), or water-soluble gel followed by BioGlue (n = 6). Additionally, BioGlue was applied to the cavoatrial junction overlying the sinoatrial node (SAN), either alone (n = 12) or after application of gel (n = 6). RESULTS: Mean diaphragmatic excursions in the BioGlue and glutaraldehyde groups were lower at 3 min and 30 min than in the albumin group (P < 0.05). Mean excursions in the gel group were similar to those of the albumin group (P = 0.9). Five BioGlue pigs (83%) and one gel pig (17%) had diaphragmatic paralysis by 30 min (P < 0.05 and P = 0.3 versus albumin, respectively). Coagulation necrosis extended into the myocardium at the cavoatrial junction in all 12 BioGlue pigs but only two gel pigs (33%, P < 0.01). Two BioGlue pigs (17%), but no gel pigs, had focal SAN degeneration and persistent bradycardia (P < 0.01). CONCLUSIONS: BioGlue causes acute nerve injury and myocardial necrosis that can lead to SAN damage. A water-soluble gel barrier is protective.     

In vivo evaluation of polyester arterial grafts coated with albumin: the role and importance of cross-linking agents

Previous in vitro studies have predicted that the type of chemical used to cross-link albumin-coated polyester arterial prostheses may influence the rate of bioerosion of the albumin layer in vivo. This study has confirmed that the healing process of this type of compound prosthesis does indeed depend on the nature and concentration of the cross-linking agent used. Four series of implantations in the thoracic aorta of dogs for scheduled periods for 4 h up to 6 months were conducted using 1.6% glutaraldehyde, 2.5% glutaraldehyde and 0.2 M carbodiimide as the alternative cross-linking agents plus a nonalbuminated preclotted polyester prosthesis which served as the control. The pathology of the explanted grafts revealed that in the short and medium term the rate of healing and the extent of tissue ingrowth was dependent initially on the presence of and later on the rate of bioerosion of the albumin layer. After 3 months in situ, the prostheses coated with albumin cross-linked with 1.6% glutaraldehyde and carbodiimide had healed more rapidly and were invaded by more extensive tissue ingrowth than the one cross-linked with 2.5% glutaraldehyde or the preclotted control. Moreover, the migration of cells over the carbodiimide-treated surface was the most fully developed and most regularly organized of all four series. Immunostaining revealed that the presence of glutaraldehyde induced an inflammatory response which failed to support the growth of normal luminal cells with the endothelial phenotype.     

Pathological Effects of Processed Bovine Pericardial Scaffolds—A Comparative In Vivo Evaluation

The objective of the present study was to assess the biocompatibility and regenerative potential of decellularized bovine pericardial scaffold in comparison with glutaraldehyde‐treated and fresh bovine pericardial implants using short‐term intramuscular implantation testing in a rat model. The inflammatory and immune responses were assessed using histopathological examination, special stains for connective tissue, histomorphometric evaluation, and immunohistochemistry. The decellularized pericardium showed an active tissue remodeling response with complete cellular invasion, minimum connective tissue encapsulation, extensive fibrovascular tissue formation, and collagen deposition. On the contrary, the glutaraldehyde‐treated pericardial implants showed incomplete degradation and cellular invasion, while the fresh pericardial implants elicited a severe foreign body reaction. The results of immunohistochemical staining revealed a minimum T helper (CD4+) lymphocyte response in decellularized pericardial implants compared with its glutaraldehyde‐treated and fresh counterparts. The decellularized bovine pericardium was better accepted as a prosthetic scaffold, which permitted maximum collagen deposition and active tissue remodeling by invading host cells and showed good tissue integration in vivo compared with glutaraldehyde‐treated and fresh/untreated pericardium.     

Calcification of glutaraldehyde cross-linked collagen in bladder neck injections in children with incontinence: a long-term complication

PURPOSE: We report the incidence of calcifications developing at the bladder neck/urethra in pediatric patients treated with glutaraldehyde cross-linked collagen for urinary incontinence. MATERIALS AND METHODS: We reviewed charts of patients treated with glutaraldehyde cross-linked collagen injections for urinary incontinence between 1994 and 1999. Etiology of incontinence, pertinent medical history, operative details and postoperative imaging were examined. RESULTS: Of 31 patients 4 (13%) had development of submucosal calcifications in the bladder neck/urethra. All 4 patients had received multiple injections of glutaraldehyde cross-linked collagen for incontinence secondary to neurogenic bladder. The calcifications were confirmed surgically. Pathology reports available for 2 of 4 patients showed chronic inflammation without dysplasia or malignant changes. Mean followup was significantly different between calcified and noncalcified cases (10.3 vs 7.2 years, p = 0.009), as was total volume of collagen injected (21 vs 12 cc, p = 0.012). Mean time to diagnosis of calcifications was 8.8 years (range 7 to 11) after first injection. A total of 24 patients without calcification underwent bladder imaging at a mean of 6.8 years (+/-2.2) after glutaraldehyde cross-linked collagen injection, which was not significantly different than the time to diagnosis (p = 0.089). The number of injections was not significantly different between the 2 groups (p = 0.426). CONCLUSIONS: Of our patients 13% had development of calcifications at the site of prior glutaraldehyde cross-linked collagen injections for incontinence. These calcifications were surrounded by chronic inflammation. Patients who have undergone glutaraldehyde cross-linked collagen injections may benefit from long-term followup with bladder imaging to detect and follow calcifications at prior injection sites.     

Glutaraldehyde affects biocompatibility of bioprosthetic heart valves.

A marked release of glutaraldehyde from commercially available pericardial bioprosthetic heart valve (BHV) material in washing solutions was found by high performance liquid chromatography (up to 1.8 ppm of glutaraldehyde per gram of dry tissue). In vitro endothelial cell proliferation rate was impaired dose-dependently in the presence of increasing glutaraldehyde concentrations of the cultivation medium (r = 0.9; p less than 0.05). Cultivation of endothelial cells was impossible on the surface of commercially available BHV material, but successful and uninhibited when toxic glutaraldehyde ligands of the BHV material were antagonized by treatment with L-glutamic acid.     

Kangaroo vs. porcine aortic valves: calcification potential after glutaraldehyde fixation

The aim of this study was to evaluate and compare the calcification potential of kangaroo and porcine aortic valves after glutaraldehyde fixation at both low (0.6%) and high (2.0%) concentrations of glutaraldehyde in the rat subcutaneous model. To our knowledge this is the first report comparing the time-related, progressive calcification of these two species in the rat subcutaneous model. Twenty-two Sprague-Dawley rats were each implanted with two aortic valve leaflets (porcine and kangaroo) after fixation in 0.6% glutaraldehyde and two aortic valve leaflets (porcine and kangaroo) after fixation in 2% glutaraldehyde respectively. Animals were sacrificed after 24 h and thereafter weekly for up to 10 weeks after implantation. Calcium content was determined using inductively coupled plasma-mass spectrometry and confirmed histologically. Mean calcium content per milligram of tissue (dry weight) treated with 0.6 and 2% glutaraldehyde was 116.2 and 110.4 microg/mg tissue for kangaroo and 95.0 and 106.8 microg/mg tissue for porcine valves. Calcium content increased significantly over time (8.8 microg/mg tissue per week) and was not significantly different between groups. Regression analysis of calcification over time showed no significant difference in calcification of valves treated with 0.6 or 2% glutaraldehyde within and between the two species. Using the subcutaneous model, we did not detect a difference in calcification potential between kangaroo and porcine aortic valves treated with either high or low concentrations of glutaraldehyde.     

Compliance changes in glutaraldehyde-treated arteries

Glutaraldehyde tanning of carotid arteries was used to develop a model for studying the effects of compliance on arterial graft performance, independent of other graft parameters. Canine carotid segments were filled with dilute phosphate-buffered glutaraldehyde (0-0.5%, pH 7.4), maintained at physiological pressure, and then immersed in either saline or 10.0% glutaraldehyde for up to 1 hr. After rinsing with saline, compliance was measured in vitro. All vessels which were immersed in 10% glutaraldehyde exhibited a significant reduction in compliance compared to native artery control [C = 11.8 +/- 1.3 (mean +/- SEM), % radial change/mm Hg X 10(-2), measured at 100 mm Hg], but maximum stiffness (C = 1.1 +/- 0.3) required that the lumen be specifically exposed to at least 0.025% glutaraldehyde in addition to simple immersion of the vessel segment in 10% fixative. Exposing the artery to 0.5% glutaraldehyde internally, without immersion of the entire structure, caused a decrease in compliance similar to that obtained after immersion in 10% glutaraldehyde, with only saline present in the lumen. Matched pairs of stiff and compliant grafts were generated by exposing the lumen to 0.025% glutaraldehyde and immersing the vessels in 10% fixative or saline, respectively. Light and scanning electron microscopy, internal reflection spectroscopy, and measurements of critical surface tension revealed nearly identical wall morphology and lumenal surface chemistry for these matched pairs. Differential tanning of the internal and external surfaces of carotid arteries thus provides a good model of arterial prostheses, wherein a substantial compliance mismatch can be studied without the complicating influences of varying diameter or differing flow surface properties.     

The Cross-Linking and Structure Modification of the Collagen Matrix in the Design of Cardiovascular Prosthesis

Glutaraldehyde cross-linking 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 cross-links. Implants of collagenous materials cross-linked with glutaraldehyde are subject long-term to calcification, biodegradation, and low-grade immune reactions. We have attempted to overcome these problems by enhancing cross-linking through (a) bridging of activated carboxyl groups with diamines and (b) using glutaraldehyde to cross-link the epsilon-NH2 groups in collagen and the unreacted amines introduced by aliphatic dismines. This cross-linking reduces tissue degradation and nearly eliminates humoral antibody induction. Covalent binding of diphosphonates, specifically 3-amino-1-hydroxypopane-1, 1-diphosphonic acid (3-APD), and to a lesser extent chondroitin sulfate to collagen or to the cross-ling-enhanced collagen network reduces its potential for calcification. Platelet aggregation also is reduced by glutaraldehyde cross-linking and nearly eliminated by the covalent binding of chondroitin sulfate to collagen. The cytotoxicity of residual glutaraldehyde can be minimized by chemical neutralization and thorough rough rinsing.     

Glutaraldehyde-preserved venous valve transplantation in the dog

Transplantation of femoral vein grafts was performed on 33 mongrel dogs to assess graft patency and valvular function after storage in glutaraldehyde. The grafts were removed from the donor, flushed with room temperature heparinized lactated Ringer's solution, and then stored in a 0.2% glutaraldehyde solution for 16 hr. At the time of grafting, the veins were again flushed with lactated Ringer's and anastomosed orthotopically to the recipient. An arteriovenous fistula was also created. Postoperatively the animals received daily doses of aspirin (2 mg/kg) and dipyridamol (50 mg). The following groups were studied: Group I (n = 10) served as controls and received fresh autografts. Group II (n = 13) received autografts stored for 16 hr in 0.2% glutaraldehyde. Group III (n = 10) received allografts stored similarly in glutaraldehyde for 16 hr. The grafts were monitored for evidence of patency. All grafts were removed for histological evaluation when patency was no longer detected or at the end of 7 weeks. Of the fresh and glutaraldehyde-preserved autografts (Group 1), 100% were patent at 7 weeks, and generally retained valve function. Patency of allografts was only slightly inferior but valve function was disappointingly poor at 7 weeks.     

Complete atrioventricular block due to a hydatid cyst located in the interventricular septum: a case report

We report a case of complete atrioventricular block and tricuspid valve insufficiency due to a cardiac hydatid cyst located in the interventricular septum that occurred in a 19-year-old male patient. Surgery was carried out with the help of cardiopulmonary bypass. The cyst was excised, and the interventricular septum was closed with an autologous pericardial patch treated with glutaraldehyde. The tricuspid valve insufficiency was repaired with annuloplasty. A dual-chamber epicardial pacemaker was implanted. The postoperative course was uneventful, and the patient was doing well 6 months after discharge. This is the first report of a cardiac hydatidosis case for which the repair of the interventicular septum, tricuspid annulopasty, and permanent pacemaker implantation were performed during the same operation.     

紫外线与戊二醛双重交联法构建真皮支架的研究

目的 比较不同预冻温度及交联方法对胶原膜内部超微结构及成纤维细胞增殖的影响. 方法 将牛Ⅰ型胶原溶液(10 g/L)分别在-20℃和-80℃预冻12 h后,放入-70℃冻干机内冷冻干燥48 h.利用扫描电镜测量2种预冻温度下胶原膜的内部孔径,比较戊二醛交联法及紫外线+戊二醛双重交联法对胶原孔径的影响,通过噻唑蓝(MTT)法检测人成纤维细胞在不同交联法制备的胶原膜中的增殖情况. 结果 -20℃预冻胶原膜的孔径为(172±37)μm,-80℃预冻胶原膜孔径为(99±24)μm,选择后者进行后续实验.经戊二醛交联后胶原膜孔径缩小,种植后第8天,成纤维细胞的吸光度值为1.534±0.013;紫外线联合戊二醛双重交联后胶原膜原有孔径不变,种植后第8天成纤维细胞的吸光度值为3.778±0.010,与前者比较,差异有统计学意义(P<0.05). 结论 -80℃预冻后经紫外线+戊二醛双重交联法构建的胶原膜,可作为体外真皮支架替代物.     

Brain injury following neuroleptic malignant syndrome: case report and review of the literature

Objective: To report a case of brain injury following neuroleptic malignant syndrome (NMS) and review the literature for similar documented cases.

Case report: A 30-year old woman presented to the ER with psychotic features and was treated with several anti-psychotics. Subsequently, she developed neurological symptoms and was diagnosed with neuroleptic malignant syndrome. Following a prolonged course in an acute care facility, she was admitted to a rehabilitation ward, where cognitive and physical examinations revealed significant findings. These included marked dysarthria, difficulties comprehending commands, attention problems, as well as abnormalities in her muscle tone, power, reflexes, gait, co-ordination and sensory function.

Conclusion: Literature reviews reveal few documented cases of brain injury following neuroleptic malignant syndrome. A further exploration of the effects of NMS on the brain is warranted to elicit whether cerebellar damage is indeed common following neuroleptic malignant syndrome. Such research could eventually lead to therapeutic interventions aimed at preventing permanent brain injury in persons with NMS.     

The Chemical Protecting Group Concept Applied in Crosslinking of Natural Tissues with Glutaraldehyde Acetals

This work describes the results of the controlled crosslinking of collagen matrices by glutaraldehyde based on a double protection strategy, glutaraldehyde acetals and lysine protonation due to the acidic conditions of acetal formation. Materials crosslinked by this approach were characterized by thermal stability comparable to those obtained by conventional procedures with mechanical properties expected for bioprosthesis manufacture and with a higher stability toward collagenase hydrolysis. The integrity of the microfibrillar structure was confirmed by optical and scanning electronic microscopy. The results indicate that the glutaraldehyde acetals procedure may be of potential use for the crosslinking of bovine pericardium used in the manufacture of bioprosthetic devices. Advantages may be related to the production of materials with homogeneous crosslinking distributions, associated with better definition in the nature of the chemical link that they introduce, due to a better distribution of glutaraldehyde within the tissue matrix before the crosslinking reaction is allowed to occur. As a result, materials with improved biological and mechanical properties are expected.     

Meniscal material properties are minimally affected by matrix stabilization using glutaraldehyde and glycation with ribose.

Knee meniscus replacement holds promise, but current allografts are susceptible to biodegradation. Matrix stabilization with glutaraldehyde, a crosslinking agent used clinically to fabricate cardiovascular bioprostheses, or with glycation, a process of crosslinking collagen with sugars such as ribose, is a potential means of rendering tissue resistant to such degradation. However, stabilization should not significantly alter meniscal material properties, which could disturb normal function in the knee. Our objective was to evaluate the effects of glutaraldehyde- and glycation-induced matrix stabilization on the material properties of porcine meniscus. Normal untreated meniscus specimens were tested in confined compression at one of three applied stresses (0.069, 0.208, 0.347 MPa), subjected to either a glutaraldehyde or glycation stabilization treatment, and then re-tested to measure changes in tissue aggregate modulus, permeability, and compressive strain at equilibrium. Changes in these properties significantly increased with glutaraldehyde concentration and exposure time to ribose. One glutaraldehyde and three glycation treatments did not alter aggregate modulus or compressive strain at equilibrium compared to controls (p > 0.10). However, all treatments increased permeability by at least 108% compared to controls (p < 0.001). This study reveals a dose-dependent relationship between meniscal material properties and certain stabilization conditions and identifies treatments that minimally affect these properties. Further research is necessary to determine whether these treatments prevent enzymatic degradation before and after surgical implantation in the knee.     

Acellular Endocardium as a Novel Biomaterial for the Intima of Tissue‐Engineered Small‐Caliber Vascular Grafts

We aimed to investigate whether acellular endocardium can be used as a useful biomaterial for the intima of engineered small‐caliber vascular grafts. Fresh endocardium was harvested from the swine left atrium and was decellularized by digestion with the decellularization solution of Triton X‐100 and SDS containing DNase I and RNase A. Surface morphological characteristics and Young's modulus were evaluated. To analyze the effect of mechanical characteristics on cell adhesion, the decellularized endocardium was stiffened with 2.5% glutaraldehyde. Small‐caliber vascular grafts were constructed using decellularized endocardium treated with or without glutaraldehyde as the intima. CD34+ cells were seeded onto the luminal surface of the vascular grafts and linked to bioreactors that simulate a pulsatile blood stream. Acellular endocardium had distinct surface morphological characteristics, which were quite different from those of other materials. The compliance of acellular endocardium was higher than that of other materials tested by Young's modulus. CD34+ cells formed a monolayer structure and adhered to the inner face of the acellular endocardium. The glutaraldehyde treatment stiffened the acellular endocardium but had little impact on the surface morphological characteristics or static adhesiveness of the cells. Data from the bioreactor study showed that the detachment of the cells from the surface of glutaraldehyde‐treated acellular endocardium increased dramatically when the pressure was equal or higher than 40 mm Hg, while the cells on the untreated acellular endocardium remained well and formed confluent monolayers and tight junctions under the same pressure. Acellular endocardium has distinct structures and mechanical characteristics that are beneficial for CD34+ cell adhesion and retention under dynamic fluid perfusion. Thus, it can be used as a useful biomaterial for the construction of the intima of engineered small‐caliber vascular grafts.     

Port-site tuberculosis after laparoscopy

In light of the explosive increase in laparoscopic surgery, there is concern about the effectiveness of sterilizing reusable laparoscopic instruments by immersion in 2% glutaraldehyde. This article describes the clinical features of eight patients who presented with biopsy-proven tuberculosis at the port-site unassociated with other clinical features of tuberculosis. Three of the eight patients had positive cultures for Mycobacterium tuberculosis. The port-site sinuses healed with antituberculous chemotherapy. There is conflicting information in the literature regarding the effectiveness of a 20-min instrument soak in 2% glutaraldehyde to clear M. tuberculosis. In light of the preceding information, the current practice of glutaraldehyde disinfection for reusable laparoscopes needs to be reexamined.     

Dexamethasone in severe head injuries

Summary The results of a double-blind-study on the effects of placebo, a low dose and a high dose of dexamethasone on severe closed head injury are presented. 95 patients were selected and carefully analyzed according to mortality, neurological course and symptoms, midbrain lesions and final outcome. The results demonstrate that dexamethasone, particularly given in high doses, reduces mortality and improves the neurological course. The steroid treatment seems to improve chances as well as quality of survival. Apart from the dose, timing of administration is of great importance.

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Zusammenfassung Es werden die Ergebnisse einer Doppel-Blind-Studie über die Auswirkungen von Placebo, niedriger und hoher Dosierung von Dexamethason bei schweren gedeckten Schädel-Hirn-Verletzungen mitgeteilt. Die Daten von 95 Patienten wurden sorgfältig analysiert in bezug auf Mortalität, neurologischen Verlauf und neurologische Symptome, Mittelhirn-Läsionen und Endzustand. Die Ergebnisse zeigen, daß Dexamethason, besonders in hoher Dosis, die Mortalität senkt und den neurologischen Verlauf bessert. Die Steroid-Behandlung scheint sowohl die Überlebens-Chance wie auch -Qualität zu verbessern. Neben der Dosierung kommt dem Zeitpunkt der Verabreichung große Bedeutung zu.

     

Alternative fixation of bioprostheses

The glutaraldehyde fixation technique for bioprosthetic tissue valves has been used for about 30 years. It is more or less generally recognized, however, that a solution to the ongoing problems of calcification and immunomodulated inflammation can only be achieved with the development of alternative fixation techniques that differ from fixation with glutaraldehyde. This review reports on the chemistry behind newly developed tissue-fixation processes and the preliminary results achieved with these.