Glutaraldehyde-stabilized bovine pericardium is used for clinical application since 1970s because of its desirable features such as less immunogenicity and acceptable durability. However, a propensity for calcification is reported on account of glutaraldehyde treatment. In this study, commercially available glutaraldehyde cross-linked bovine pericardium was evaluated for its in vitro cytotoxic effect, macrophage activation, and in vivo toxic response in comparison to decellularized bovine pericardium. Glutaraldehyde-treated bovine pericardium and its extract were observed to be cytotoxic and it also caused significant inflammatory cytokine release from activated macrophages. Significant antibody response, calcification response, necrotic, and inflammatory response were noticed in glutaraldehyde-treated bovine pericardium in comparison to decellularized bovine pericardium in a rat subcutaneous implantation model. Glutaraldehyde-treated bovine pericardium also failed in acute systemic toxicity testing and intracutaneous irritation testing as per ISO 10993. With respect to healing and implant remodeling, total lack of host tissue incorporation and angiogenesis was noticed in glutaraldehyde-treated bovine pericardium compared to excellent host fibroblast incorporation and angiogenesis within the implant in decellularized bovine pericardium. In conclusion, using in vitro and in vivo techniques, this study has demonstrated that glutaraldehyde-treated bovine pericardium elicits toxic response compared to decellularized bovine pericardium which is not congenial for long-term implant performance.
Calcification; decellularized bovine pericardium; glutaraldehyde-treated bovine pericardium; immunogenicity; toxic response; tissue incorporation
The peritoneum has the same developmental origin as blood vessels, is highly reactive and poorly thrombogenic. We hypothesize that parietal peritoneum can sustain development and regeneration of new vessels.
Methods and Results
The study comprised two experimental approaches. First, to test surgical feasibility and efficacy of the peritoneal vascular autograft, we set up an autologous transplantation procedure in pigs, where a tubularized parietal peritoneal graft was covered with a metal mesh and anastomosed end-to-end in the infrarenal aorta. Second, to dissect the contribution of graft vs host cells to the newly developed vessel wall, we performed human-to-rat peritoneal patch grafting in the abdominal aorta and examined the origin of endothelial and smooth muscle cells. In pig experiments, the graft remodeled to an apparently normal blood vessel, without thrombosis. Histology confirmed arterialization of the graft with complete endothelial coverage and neointimal hyperplasia in the absence of erosion, inflammation or thrombosis. In rats, immunostaining for human mitochondri revealed that endothelial cells and smooth muscle cells rarely were of human origin. Remodeling of the graft was mainly attributable to local cells with no clear evidence of c-kit+ endothelial progenitor cells or c-kit+ resident perivascular progenitor cells.
The parietal peritoneum can be feasibly used as a scaffold to sustain the regeneration of blood vessels, which appears to occur through the contribution of host-derived resident mature cells.
Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, Triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, versus Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-Mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies we have further investigated the effectiveness of MABP in experiments that examined: 1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; 2) Furthermore, recognizing the importance of alkaline phosphatase in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of alkaline phosphatase activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced alkaline phosphatase activity, and in vivo have no significant calcification in long term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.
New glutaraldehyde-treated vascular prosthesis tubes of bovine pericardium were placed in the abdominal aortas of 29 mongrel dogs and observed for 30 to 540 days. Clinical evaluation was performed during follow-up, and histologic and arteriographic studies were done at the end of the study. Patency was shown by arteriography in 27 of 29 tubes (93.1%). Neoendothelialization was found in all grafts, and there was a mixed effect, characterized by both rejection and foreign body reaction in the prostheses from 180 days onward. Both the handling and consistency of the grafts were excellent for implantation. Graft procoagulation and animal anticoagulation were not necessary.
After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron- dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine- receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000- Mr protein binding to the inner face of the subsynaptic membrane.
While a glutaraldehyde crosslinking is most often used to fabricate bioprosthetic heart valves (BHV) using heterograft tissues, it predisposes BHV to calcification and dramatically stiffens the heterograft tissues. Our group previously reported the synthesis and characterization of a novel epoxy-crosslinker, triglycidylamine (TGA). TGA pretreatment of BHV tissues compared to glutaraldehyde results in both calcification resistance in subdermal implants and improved leaflet compliance. In these prior studies, optimal calcification inhibition was noted with the combined use of TGA with mercapto-aminobisphosphonate (MABP). In the present study, we investigated the hypothesis that bovine pericardium cross-linked with TGA-MABP retains these beneficial biomechanical properties in-vivo using a novel mitral valve anterior leaflet (MVAL) ovine valvuloplasty model. Bovine pericardial specimens were crosslinked with either glutaraldehyde or TGA-MABP, from which 1 cm2 sections were implanted in the ovine MVAL after removal of the original tissue of the same size. An array of four sonomicrometry transducers were implanted on the corners and used to compute the complete in-surface strain tensor cardiac cycle over the cardiac cycle at 0 and 4 weeks. Following explant samples were fixed in formalin for histology studies. At 4 weeks both treatment groups experienced no dimensional changes in the unloaded state, indicating no shrinkage. When fully loaded during peak systolic ejection, TGA-MABP valvuloplasty patches were significantly more compliant, which did not change at 4 weeks. In contrast, the glutaraldehyde areal strain increased significantly by 4 weeks. Estimated implant stresses for both treatment groups, based on previously measured biomechanical properties (Biomaterials, vol. 4, 2007), were 40 kPa and 250 kPa in the circumferential and radial directions, respectively, which are comparable to predicted BHV peak stress levels. We conclude that TGA-MABP crosslinked bovine pericardium, when subjected to in-vivo BHV stress levels in a blood contacting environment, maintains stable functionality.
Complete closure of the pericardial sac after open-heart surgery is thought to be a help in avoiding some postoperative complications and in facilitating reoperation.
Since 1977, sixty-six patients who were subjected to open-heart surgery had glutaraldehyde stabilized xenogeneic bovine pericardial patches inserted to achieve closure of their own pericardial sacs. There were no hemodynamic problems related to the implantation of these patches. All 60 surviving patients were clinically well during a follow-up of 145 patient-years.
Reoperation was undertaken in three patients 2½, 3, and 7 years after the initial operation. The presence of the pericardial patch greatly facilitated the reopening of the chest because it prevented the formation of adhesions between the patch and the epicardium. Histologically, the implanted pericardium consisted of laminae of collagen fibrils containing evenly dispersed fibrocyte nuclei and parallel runs of fairly wide, separated elastic fibrils. There was no evidence of immune reaction.
On the basis of our findings that there was no host reaction or complications after its use, glutaraldehyde stabilized xenogeneic bovine pericardium appears to be the material of choice for patch closure of the pericardium when primary closure alone is not feasible.
Bovine pericardium is widely used in surgery and is commonly used for a patch after arteriotomy during cardiovascular surgery. Bovine pericardial patches have several advantages compared to prosthetic patches, including superior biocompatability, easy handling, less suture line bleeding and possibly reduced rates of infection. These advantages of bovine pericardium have led to its common use during carotid endarterectomy. However, long-term clinical results reported after carotid endarterectomy have suggested several issues that may be related to the patch including restenosis, pseudoaneurysm formation, infection, fibrosis, calcification and thrombosis. These complications may diminish the long-term efficacy of carotid endarterectomy and suggest potential areas for improvement of surgical patches. Understanding the mechanisms by which bovine pericardium heals after patch angioplasty may lead to next generation tissue engineered patches.
Glutaraldehyde (GA) is largely used in the cross-linking of collagen matrices to improve their mechanical and biological properties for applications in cardiovascular surgery. However, GA has major drawbacks, including graft degeneration, calcification, and durability. The aim of this study was to test the hypothesis that filling the interstitial space in the bovine pericardium with various space fillers could prevent tissue calcification. GA, genipin, and 1-ethyl-3(-3 dimethyl aminopropyl) carbodiimide hydrochloride fixation with spacefiller treatment have been studied in order to improve the properties of heart valve xenografts. Crosslinking efficiency of GA treated group was better than genipin or 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide/N-hydroxysuccinimide treated group in vitro mechanical, enzymatic degradation resistance tests. Space-filling samples have shown significantly reduced calcification in the rabbit intramuscular implantation model. Regardless of the filling effect, the level of calcification and the cytotoxicity was low in a genipin-treated group compared to levels in the GA-treated group. The results indicated that GA and genipin fixation with space-filler treatment were effective in anticalcification for biological tissue preservation.
Glutaraldehyde (GA) is a widely used cross-linking agent for improving mechanical properties and resistance to enzymatic degradation of collagenous tissue, but it has several drawbacks such as calcification and cytotoxicity. The aim of this study was to find the alternative effective cross-linking methods to GA.
Materials and Methods
Bovine pericardium was processed with GA with ethanol+octanol and glycine detoxification, and polyethylene glycol (PG) space filler, dimethyl 3,3'-dithiobispropionimidate (DTBP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) treatment, and the physical fixation of ultraviolet irradiation were done. The biologic material properties of variously treated pericardial tissues were assessed by biochemical, mechanical and histological tests. Treated pericardial tissues were also implanted subcutaneously or intramuscularly into the rabbit for 10 weeks to assess the xenoreactive antibody response of immunoglobulin G and M, their anti-calcification effect.
The biochemical and mechanical properties of EDC fixed pericardial tissues were comparable to the GA fixed tissue. The cytotoxicity was lowest in space filler treated GA fixed group. In rabbit subcutaneous or intramuscular implantation models, decellularization, space filler, EDC treatment group showed significantly lower calcium content than GA only and DTBP treatment group (p<0.05, analysis of variance). The titer of anti Galα1-3Galβ1-4GlcNAc-R antibodies did not change in the postimplantation serial enzyme-linked immunosorbent assay. Hematoxylin and eosin and von Kossa staining showed that decellularization, space filler, EDC, and ultraviolet treatment had less inflammatory cell infiltration and calcium deposits.
The decellularization process, PG filler, and EDC treatments are good alternative cross-linking methods compared to GA only fixation and primary amine of DTBP treatment for cardiovascular xenograft preservation in terms of the collagen cross-linking stability and in vivo anti-calcification effects.
Pericardium; Bioprosthesis; Polyethylene glycols; Glutaraldehyde; Ultraviolet
A 59-year-old male patient underwent surgery for triple-vessel coronary artery disease and left-ventricular aneurysm in 1994. Four months after coronary artery bypass grafting and classical left-ventricular aneurysmectomy (with Teflon felt strips), a left-ventricular pseudoaneurysm developed due to infection, and this was treated surgically with an autologous glutaraldehyde-treated pericardium patch over which an omental pedicle graft was placed. Two months later, under emergent conditions, re-repair was performed with a diaphragmatic pericardial pedicle graft due to pseudoaneurysm reformation and rupture. A 3rd repair was required in a 3rd episode 8 months later. Sternocostal resection enabled implantation of the left pectoralis major muscle into the ventricular defect. Six months after the last surgical intervention, the patient died of cerebral malignancy. Pseudoaneurysm reformation, however, had not been observed. To our knowledge, our case is the 1st reported in the literature in which there have been 3 or more different operative techniques applied to 3 or more distinct episodes of pseudoaneurysm formation secondary to post-aneurysmectomy infection. We propose that pectoral muscle flaps be strongly considered as a material for re-repair of left-ventricular aneurysms.
Background and Objectives
Glutaraldehyde (GA) has been used as a representative method of tissue preservation in cardiovascular surgery. However, GA has showed limited durability including calcification, mechanical failure and toxicity. To overcome those unsolved problems, we analyzed the crosslinking differences of primary amines, GA and genipin in their mechanical and biochemical properties with a single or double crosslinking agent for clinical application.
Materials and Methods
Samples were divided into 3 groups; control, single crosslinking fixation and double crosslinking fixation after decellurarization using bovine pericardium. For analysis of the biochemical and mechanical properties of each crosslinking method, tensile strength, percentage strain, thermal stability, resistance to pronase, nynhydrin and cytotoxicity test were studied.
Combined hexamethylene diamine and suberic acid in the carbodiimide hydrochloride/N-hydroxysucinimide solution (EDC/NHS) after decellurarization, tensile strength and strain percentage were not statistically significant compared to the single crosslinking treated groups (p>0.05). Tissue crosslinking stability was weak in single treatment of diphenylphosphoryl azide, suberic acid, low concentration of EDC, hexamethylene diamine and procyanidin groups, but thermal stability and resistance to the pronase and ninhydrin were markedly increased in concentrated EDC/NHS or after combined double treatment with low concentration of GA or genipin (p<0.001).
Single or double crosslinking with low concentration of carbodiimide, diphenylphosphonyl azide, procyanidin, suberic acid and hexane diamine were not as effective in mechanical, biochemical, cytotoxic and crosslinking properties compared to GA or genipin fixation, but their mechanical and chemical properties were much improved when combined with low concentrations of GA or genipin in the double crosslinking process.
Calcification is the most frequent cause of clinical failure of bioprosthetic tissues fabricated from GA-fixed porcine valves or bovine pericardium. A multi-factorial approach using different mechanisms was recently developed to reduce the calcification of bioprosthetic tissues. The purpose of the present study was to evaluate the synchronized synergism of using L-arginine and NaBH4, compared with ethanol and L-lysine, in glutaraldehyde treated porcine pericardium from the standpoint of calcification and tissue elasticity.
Materials and Methods
Porcine pericardium was fixed at 0.625% GA (7 days at room temperature after 2 days at 4℃). An interim step of ethanol (80%; 1 day at room temperature) or L-lysine (0.1 M; 2 days at 37℃) or L-arginine (0.1 M; 2 days at 37℃) was followed by completion of the GA fixation. A final step of NaBH4 (0.1 M; 2 days at room temperature) was followed. Their tensile strength, thickness, and thermal stability were measured. Treated pericardia were implanted subcutaneously into three-week-old Sprague-Dawley rats for 8 weeks. Calcium content was assessed by atomic absorption spectroscopy and histology.
L-arginine and NaBH4 pretreatment (1.81±0.39 kgf/5 mm p=0.001, 0.30±0.08 mm p<0.001) significantly increased tensile strength and thickness compared with the control (0.53±0.34 kgf/5 mm, 0.10±0.02 mm). In a thermal stability test, L-arginine and NaBH4 pretreatment (84.25±1.12℃, p=0.023) caused a significant difference from the control (86.25±0.00℃). L-lysine and NaBH4 pretreatment (183.8±42.6 ug/mg, p=0.804), and L-arginine and NaBH4 pretreatment (163.3±27.5 ug/mg, p=0.621) did not significantly inhibit calcification compared to the control (175.5±45.3 ug/mg), but ethanol and NaBH4 pretreatment did (38.5±37.3 ug/mg, p=0.003).
The combined pretreatment using L-arginine and NaBH4 after GA fixation seemed to increase the tensile strength and thickness of porcine pericardium, fixed with GA. Additionally, it seemed to keep thermal stability. However it could not decrease the calcification of porcine pericardium fixed with GA. NaBH4 pretreatment seemed to decrease the calcification of porcine pericardium fixed with GA, but only with ethanol.
Ethanol, L-lysine, NABH4; Bioprosthetic calcification; Porcine pericardium
Eight animals died from 1 to 6 years after the operation, from undetermined diseases, or from pneumonia. Two animals are still alive 7 years after the operation. 1. Condition of the Pleural and Pericardial Cavities.—In the first experiments, extensive pleural and pericardial adhesions were observed 1 year and more after the patching of the pulmonary artery. There was also a great deal of fibrous tissue between the pulmonary artery, the aorta, and the left auricle. In the other experiments, the adhesions of the lungs, pleura, and pericardium were less marked. This was due to some improvements in the technique of handling the viscera. At the time of the operations, it was hoped that no pleural or pericardial adhesions would occur. Great care was taken not to injure the endothelial surfaces by rough handling or by sponging. No blood was allowed to flow into the pleural cavity. The surface of the pericardium was protected by fine silk membranes. The pleural cavity was occluded by thick pads made of cotton and Japanese silk. It seemed that the serous surfaces were almost completely protected against infection and mechanical irritation. The occurrence of primary pleurisy and pericarditis was prevented by this technique. But the development of adhesions in several of the experiments shows that the procedures for the handling of the viscera should be perfected. 2. Condition of the Arterial Wall.—In the experiments in which the orifice was patched, a slight dilatation of the artery was observed. It was not possible to ascertain from the specimen preserved in formaldehyde whether or not there was an insufficiency of the valves. It is probable that there was no leakage, as in none of these cases could any diastolic murmur be heard 6 months after the operation. The only animal which presented clinical evidence of pulmonary insufficiency died during the War. The normal condition of the pulmonary orifice was due to the incision which did not extend far enough on the ventricle, and to the power of redintegration possessed by an organ which is not diseased. The cicatrization of the grafted flap was excellent. Its outline could not be seen on the external side of the wall. Even after opening the artery, the transplant could not be located easily. However, in Experiment 1 the anterior wall of the artery showed a depression about 7 or 8 mm. wide, 18 mm. long, and 2 or 3 mm. deep, behind and above the anterior valve. But the flap was made of human artery, and it is known that a heteroplastic graft always undergoes some dilatation. When transplants of dog tissue were used, no dilatation occurred and the location of the patch could hardly be detected. In Experiment 7, 6 years after the operation, the endothelial surface was smooth, glistening, and no scar could be seen. However, the upper and lower parts of the incision were marked by a slight depression of the wall. The presence of the patch was detected by a distinct thickening of the wall. Although the edges of the incision had not been sutured to the edges of the flap, the endothelial surface was quite smooth. A transverse section of the artery was made through the middle part of the flap in Experiment 4. It showed the width of the arterial opening and the way in which the transplant became adherent to the arterial wall. The examination of these four specimens demonstrated that, in spite of the unfavorable location of the graft, an excellent union had taken place. It showed that homoplastic or heteroplastic tissue can be transplanted onto the pulmonary artery as well as onto the smaller arteries. Where the arterial wall had simply been incised without interposition of a patch, a linear scar was always found. 6 years after the operation, the incision used in the course of an operation for cauterization of the sigmoid and sutured with heavy thread was transformed into a linear scar and the surface of the intima was quite smooth. 3. Condition of the Sigmoid Valves.—In three experiments, the sigmoid valves had been cauterized along their margin and their point of insertion in the artery. One of the animals was still living 7 years after the operation. There was no diastolic murmur. The other animals died 3 and 6 years after the operation. The valves were thin and transparent, and quite normal. However, one of the valves showed two holes, one near the base and the other near the margin. The animal on which the section of the right posterior valve without suture was performed, died 2 or 3 years after the operation. The edges of the incision had not united. They were thickened and the whole valve was rigid. The surface was rough and irregular. No permanent result was obtained by the union of two sigmoid valves by a stitch. There was no stenosis of the orifice, and no union of the valves 4 years after the operation. The stitch had disappeared. There was some scar tissue at the common point of insertion of the posterior valves, which were more rigid and showed thickened edges. 7 years after the section and suture of a sigmoid had been performed, the animal was still living and in good health. No diastolic murmur could be detected.
To determine the expression of vascular endothelial growth factor-A (VEGF-A), macrophage migration inhibition factor (MIF), and matrix metalloproteinase-1 (MMP-1) in the porcine remnant kidney model and quantify renal blood flow and volume using phase contrast magnetic resonance imaging with magnetic resonance angiography (PC MRI/MRA).
Material and methods
In 23 pigs, the left renal artery was completely embolized using polyvinyl acrylide (PVA) particles and the right kidney partially embolized (remnant kidney) while six pigs served as controls. The animals were sacrificed early (day 3, 7, and 14, N=3), day 24 (D24, N=5), day 37 (D37, N=3), day 42 (D42, N=9), and day 84 (D84, N=3). MRI/PC MRA of the kidneys was performed prior to sacrifice. The remnant and control kidneys were harvested for Western blotting of VEGF-A, MMP-1, and MIF. Blood was removed for BUN and creatinine prior to embolization and at time of sacrifice.
The kidney function after the embolization was characterized by chronic renal insufficiency. The renal artery blood flow, volume, and weight of the remnant kidney increased significantly over time when compared to controls. At early time points, there was increased expression of MIF and MMP-1 followed by an increase in the expression of VEGF-A by day 37 (P<0.05 when compared to control). Masson's trichrome staining of the remnant kidney revealed scarring in the tubulointerstitial space.
In this model, renal blood flow and volume increase as the remnant kidney hypertrophies and scars. There is increased expression of MIF, VEGF-A, and MMP-1 in the remnant kidney.
Bovine pericardium is an important biomaterial with current application in glutaraldehyde-fixed bioprosthetic heart valves and possible future application as an unfixed biological scaffold for tissue engineering. The importance of both humoral and cell-mediated rejection responses toward fixed and unfixed xenogeneic tissues has become increasingly apparent. However, the full scope and specific identities of bovine pericardium proteins that can elicit an immune response remain largely unknown. In this study, an immunoproteomic approach was used to survey bovine pericardium proteins for their ability to elicit a humoral immune response in rabbits. A two-stage protein extraction protocol was used to separate bovine pericardium proteins into water- and lipid-soluble fractions. Two-dimensional gel electrophoresis was performed to separate the proteins from each fraction. Western blots were generated from two-dimensional gels of both bovine pericardium protein fractions. These blots were probed with serum from rabbits immunized with bovine pericardium and a secondary antibody was used to assess for IgG positivity. Western blots were compared to duplicate two-dimensional gels and proteins in matched spots were identified by tandem mass spectrometry. Thirty-one putative protein antigens were identified, eight of which are known to be antigenic from previous studies. All of the putative antigens demonstrated progressive staining intensity with increasing days of post-exposure serum. Identified antigenic proteins represented a variety of functional and structural protein types, and included both cellular and matrix proteins. The results of this study have implications for the use of bovine pericardium as a biomaterial in bioprostheses and tissue engineering applications, as well as xenotransplantation in general.
Immunoproteomics; Scaffold; Tissue Engineering; Xenoantigens; Heart Valve
Eighteen Rhesus monkeys underwent lens implantation with Choyce Mark VIII, Binkhorst iridocapsular, and Shearing posterior chamber lenses. They were sacrificed 4 to 28 months following surgery. The eyes were compared clinically and histologically. Controls included unoperated eyes and eyes with lens extraction without implantation. Several histologic findings pertained equally to cataract extraction with or without lens implantation. Late opacification of the posterior capsule was caused by migration and fibrous metaplasia of the lens epithelial cells. These cells appeared to undergo such metaplasia only when exposed directly to aqueous, never when they were in firm apposition to another tissue such as another layer of capsular epithelium, lens cortex, or iris. Also strong fibrous posterior synechiae between the iris and lens remnants occurred only where the anterior lens capsule was missing. All implants were well tolerated clinically. Histologically they showed remarkably little inflammation. The eyes with Binkhorst lenses had a mild tendency to focal loss of iris pigment epithelium and some showed pigmented macrophages in the iris stroma and trabecular mesh. The Choyce lenses frequently displayed marked displacement and thinning of the iris root, and occasionally showed a few chronic inflammatory cells and thin fibrous encapsulation around the implant feet. The Shearing lenses had no effect on the adjacent ciliary body when the loops were well seated in the lens capsule, but when a loop was anterior to the capsule, it eroded into the ciliary body. The loops developed a thin fibrous capsule within the ciliary body with very little chronic inflammatory reaction, but the long term effect of such loops lying in the ciliary body is undetermined. At present it is recommended that, if such a lens is implanted, every effort be made to ensure both loops lie in the capsular envelope. On the basis of this study, it is also recommended that in removing such a lens, one must assume that a loop might lie embedded in the ciliary body and cut the lens free from the loops before removing it.
Biologic valved grafts are important in cardiac surgery, and although several types of graft are currently available, most commercial xenografts tend to cause early disfiguration due to intimal proliferation and calcification. We studied the graft failure patterns on non-fixed and glutaraldehyde-fixed pulmonary xenograft in vivo animal experiment.
Materials and Methods
Pulmonary valved conduits were obtained from the right ventricular outflow tract of eleven miniature pigs. The grafts were subjected to 2 different preservation methods; with or without glutaraldehyde fixation: glutaraldehyde fixation (n=7) and non-glutaraldehyde fixation (n=4). The processed explanted pulmonary valved grafts of miniature pig were then transplanted into eleven goats. Calcium quantization was achieved in all of the explanted xenograft, hemodynamic, histopathologic and radiologic evaluations were performed in the graft which the transplantation period was over 300 days (n=7).
Grafts treated with glutaraldehyde fixation had more calcification and conduit obstruction in mid-term period. Calcium deposition also appeared much higher in the glutaraldehyde treated graft compared to the non-glutaraldehyde treated graft (p<0.05).
The present study suggests that xenografts prepared using glutaraldehyde fixation alone appeared to have severe calcification compared to the findings of non-glutaraldehyde treated xenografts and to be managed with proper anticalcification treatment and novel preservation methods. This experiment gives the useful basic chemical, histologic data of xenograft failure model with calcification for further animal study.
Transplantation, heterologous; Glutaraldehyde
In cardiac surgery, especially in the reconstruction of vascular structures and intracardiac defects, glutaraldehyde has usually been used as the reagent for fixing porcine or bovine pericardial tissues. But the well-known problem of calcification or cytotoxicity of glutaraldehyde motivates the search for a replacement. The aim of this study is to investigate the physical, mechanical, and biochemical characteristics of bovine pericardial tissues fixed with genipin, which is known to be a less toxic and more natural fixing reagent.
Materials and Methods
Bovine pericardial tissues were fixed with different concentrations and conditions of glutaraldehyde and genipin. To determine the physical, mechanical, and biochemical differences among different concentrations and conditions, we divided the tissue into 18 groups by concentration, the addition of organic solvents, and the timing of adding the organic solvents, and compared the characteristics of each group.
Tensile strength, physical activity, and thermal stability tests revealed that the tissues fixed with glutaraldehyde were better with regard to mechanical strength and biochemical durability. However, the difference was not significant statistically.
Genipin can be used as an alternative crosslinking agent for pericardial tissue, considering given its physical, mechanical, biochemical characteristics and low cytotoxicity comparable to glutaraldehyde. However, further studies are needed on the immune reaction and the long term changes in genipin-fixed tissues in the human body.
Xenograft; Glutaraldehyde; Bioprosthesis; Pericardial patch
The permeability properties and the exclusion limits of the crystalline surface layers (S layers) of two selected strains of Bacillus stearothermophilus were investigated. Measurements were performed of passive solute uptake into the intracellular space of native or glutaraldehyde-treated sacculi. Native sacculi were prepared from whole cells by extracting the cytoplasmic membrane with Triton X-100 under conditions which preserved the integrity of the S layer and the peptidoglycan-containing layer. The permeability barrier was found to consist of three adjacent layers, namely, the S layer, the peptidoglycan-containing layer, and an incomplete S layer attached to the inner face of the peptidoglycan-containing layer. In glutaraldehyde-treated sacculi the peptidoglycan was digested after stabilizing the S-layer lattice by chemical cross-linking. The solutes selected for the uptake measurements were mannose, proteins, and dextrans of increasing molecular weights. The S layers of both strains allowed free passage for molecules with a molecular weight of up to 30,000 and showed sharp exclusion limits between molecular weights of 30,000 and 45,000, suggesting a limiting pore diameter of about 4.5 nm.
In this prospective study, the long-term effect of posterior leaflet extension with glutaraldehyde-preserved autologous pericardium and associated mitral valve commissurotomy was investigated in patients with mixed mitral valve disease of rheumatic origin.
Mitral commissurotomy and posterior leaflet extension using a pericardial patch were performed in 25 patients from 1 January 1994 through 31 December 1995 for mixed mitral valve disease. Preoperatively, no patient had chordal rupture or papillary muscle dysfunction. Four patients had left atrial thrombosis. The mean age was 35.7 ± 15.4 years. Associated procedures were tricuspid annuloplasty in 4 patients, aortic annuloplasty in 3, aortic and tricuspid annuloplasty in 1, and aortic homograft replacement in 1.
There were no early deaths. One patient died 2 years after surgery due to noncardiac causes. Mitral valve area increased from 1.53 ± 0.63 cm2 to 2 ± 0.33 cm2 (P = 0.09), and left atrial diameter decreased from 5.8 ± 1 cm to 4.86 ± 1.27 cm (P = 0.07) after 6.1 ± 0.7 years (range, 5.5 to 7.1 years). Mitral insufficiency was reduced significantly, from grade 2.65 ± 0.9 to grade 1.2 ± 0.9 (P = 0.007). Functional capacity improved in all patients (New York Heart Association functional class, 3 ± 0.58 preoperatively vs 1.44 ± 0.82 postoperatively; P = 0.001). Three patients required reoperation and valve replacement.
This type of reconstruction may be a good alternative for patients who are not able to use anticoagulant therapy. Long-term results of this technique are acceptable; however, the risk of reoperation is an important disadvantage in these young patients.
Mitral valve/surgery; mitral valve insufficiency/surgery; pericardium/transplantation; reconstructive surgical procedures; rheumatic heart disease/surgery
Glutaraldehyde-treated bioprosthetic heart valves are commonly used for replacement of diseased heart valves. However, calcification and wear limit their durability, and the development of new and improved bioprosthetic valve designs is needed and must be evaluated in a reliable animal model. We studied glutaraldehyde-treated valves 6 months after implantation to evaluate bioprosthetic valve complications in the mitral position in juvenile pigs.
The study material comprised eight, 5-month old, 60-kg pigs. All pigs received a size 27, glutaraldehyde-treated, stented, Carpentier-Edwards S.A.V. mitral valve prosthesis. After six months, echocardiography was performed, and the valves explanted for gross examination, high resolution X-ray, and histological evaluation.
Five pigs survived the follow-up period. Preexplant echocardiography revealed a median peak and mean velocity of 1.61 m/s (range: 1.17-2.00) and 1.20 (SD = ±0.25), respectively, and a median peak and mean pressure difference of 10.42 mmHg (range: 5.83-16.55) and 6.51 mmHg (SD = ±2.57), respectively. Gross examination showed minor thrombotic depositions at two commissures in two valves and at all three commissures in three valves. High resolution X-ray imaging revealed different degrees of calcification in all explanted valves, primarily in the commissural and belly areas. In all valves, histological evaluation demonstrated various degrees of fibrous sheath formation, limited immunological infiltration, and no overgrowth of host endothelium.
Bioprosthetic glutaraldehyde-treated mitral valves can be implanted into the mitral position in pigs and function after 6 months. Echocardiographic data, calcification, and histological examinations were comparable to results obtained in sheep models and human demonstrating the suitability of the porcine model.
Mitral valve; glutaraldehyde; porcine model; calcification
Most infants with long-gap esophageal atresia receive an esophageal replacement with tissue from stomach or colon, because the native esophagus is too short for true primary repair. Tissue-engineered esophageal conducts could present an attractive alternative. In this paper, circular decellularized porcine esophageal scaffold tissues were implanted subcutaneously into Sprague-Dawley rats. Depending on scaffold cross-linking with genipin, glutaraldehyde, and carbodiimide (untreated scaffolds : positive control; bovine pericardium : gold standard), the number of infiltrating fibroblasts, lymphocytes, macrophages, giant cells, and capillaries was determined to quantify the host response after 1, 9, and 30 days. Decellularized esophagus scaffolds were shown to maintain native matrix morphology and extracellular matrix composition. Typical inflammatory reactions were observed in all implants; however, the cellular infiltration was reduced in the genipin group. We conclude that genipin is the most efficient and best tolerated cross-linking agent to attenuate inflammation and to improve the integration of esophageal scaffolds into its surrounding tissue after implantation.
Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital defect. This anomaly leads to a coronary hypoperfusion phenomenon and to substantial left ventricular dysfunction caused by abnormal perfusion of the left ventricle. The optimal surgical management of such cases is not clearly established.
Here, we report the successful anatomic repair of ALCAPA arising from the non-facing sinus of Valsalva of the pulmonary artery in a 5-kg patient. In order to perform the repair, we created an autologous extrapulmonary tunnel (from a pulmonary artery flap and autologous pericardium), which we implanted into the ascending aorta. Because of post-cardiotomy heart failure, we implanted an extracorporeal membrane oxygenation device during the same procedure. After recovery of the failing heart, the device was easily ex-planted, and the patient was discharged from the hospital on postoperative day 30.
Coronary vessel anomalies/radiography/surgery; infant; extracorporeal membrane oxygenation; heart disease, congenital; heart failure, congestive; pulmonary artery/abnormalities; vascular surgical procedures/methods
The usefulness of endoscope-assisted microsurgical removal of vestibular schwannomas in the internal auditory canal (IAC) was evaluated. Microsurgical removal using the endoscope was done in 28 procedures and microsurgical removal without an endoscope was done in 43 procedures. A retrosigmoid approach was used. The tumor location in the IAC was classified as grade 1 (located up to the mid-portion), 2, 3, or 4 (located up to the fundus with bony destruction) according to the tumor extent, and residual tumor in the IAC was evaluated as grade A (remnant tumor was not observed), B, C, or D (remnant tumor was observed over the mid-portion) according to the extent of remnant tumor. The residual tumor in the IAC was less in the endoscope-assisted group than in the microsurgery group. There was a significant difference only in grade 2, that is, tumor located beyond the mid-portion of the IAC. There was no significant difference in the results of preservation of useful hearing, facial nerve function, and tumor recurrence between the two groups. The benefit of endoscope-assistance microsurgery was shown for those patients whose tumors extended beyond the mid-portion of the IAC but did not reach the fundus.
vestibular schwannoma; endoscopy; retrosigmoid approach; internal auditory canal