Comparison of physical and biological properties of CardioCel® with commonly used bioscaffolds.

OBJECTIVES: Durability of bioscaffolds cross-linked with glutaraldehyde and used in cardiovascular surgery is limited by biomechanical instability, calcification and reduced biocompatibility. This study compares CardioCel®, a bovine pericardial scaffold engineered via the ADAPT® process to ensure optimized biostability and biocompatibility, with the commonly used bioscaffolds. METHODS: Bovine pericardial scaffolds, cross-linked with 0.6% glutaraldehyde (XenoLogiX™, PeriGuard®), dye-mediated photo-oxidized (PhotoFix™) and a non-crosslinked porcine scaffold (CorMatrix®), were compared with CardioCel (decellularized, cross-linked with 0.05% monomeric glutaraldehyde, detoxified) by thermal stability and mechanical tests. Biocompatibility and calcification were assessed in a juvenile subcutaneous rat model at 6 and 12 weeks. RESULTS: CardioCel displayed significantly higher (P < 0.01) cross-link stability (77.99 ± 0.64 °C) than CorMatrix (57.88 ± 0.22 °C) and PhotoFix (53.96 ± 0.41 °C). Tensile strength of CardioCel (8.31 ± 3.36 MPa) was comparable with XenoLogiX (11.00 ± 5.43 MPa, P = 0.734), PeriGuard (16.44 ± 6.69 MPa, P = 0.136), PhotoFix (7.10 ± 6.11, P = 0.399) and CorMatrix (9.75 ± 2.61, P = 0.204). XenoLogiX and PeriGuard recorded the highest Young's modulus (67.01 ± 30.36 vs 95.67 ± 45.91 MPa), while CardioCel (50.21 ± 19.92 MPa) was comparable with CorMatrix (36.78 ± 10.47 MPa, P = 0.204) and PhotoFix (33.50 ± 10.24, P = 0.399). CorMatrix displayed a significantly (P < 0.05) greater stiffness (4.74 ± 0.77 MPa) at 10% strain than PeriGuard (3.73 ± 1.79 MPa), PhotoFix (1.59 ± 0.40 MPa) and CardioCel (3.39 ± 0.83 MPa). Differences in extractable calcium did not reach significance; however, the inorganic phosphorus content of PhotoFix (21.3 ± 9.0 µg/mg) was higher than CardioCel (11.35 ± 0.76 µg/mg, P = 0.004) or PeriGuard (10.7 ± 2.18 µg/mg, P = 0.002) at 12 weeks. CardioCel underwent a typical mild host-graft response with fibroblast infiltration and remodelling. Foreign body reactions were visible in both XenoLogiX and PeriGuard, with isolated fibroblast infiltration. PhotoFix showed severe inflammation and 2 implants were completely degraded at 12 weeks. CONCLUSIONS: CardioCel demonstrated optimized physical properties, minimal mineralization potential and superior biocompatibility. These results may benefit the long-term performance of this bioscaffold for cardiovascular surgery. The favourable characteristics of the comparator products were counterbalanced by less desirable features that may have negative implications on durability and performance when used in cardiovascular procedures.

Evaluation of an established pericardium patch for delivery of mesenchymal stem cells to cardiac tissue.

The present study has evaluated a commercial pericardial material for its capacity to assist as a natural extracellular matrix (ECM) patch for the delivery and retention of mesenchymal stem cells for cardiac repair. The repair of cardiac tissue with cells delivered by an appropriate bioscaffold is expected to offer a superior, long-lasting treatment strategy. The present material, CardioCel®, is based on acellular pericardium that has been stabilized by treatments, including a low concentration of glutaraldehyde, that eliminate calcification after implantation. In the present study, we have assessed this material using human bone marrow mesenchymal stem cells at various cell densities under standard, static cell culture conditions. The initial seeding densities were monitored to evaluate the extent of cell attachment and cell viability, with subsequent cell proliferation assessed up to 4 weeks using an MTS assay. Cell morphology, infiltration, and spreading were tracked using scanning electron microscopy and phalloidin staining. The efficacy of long-term cell survival was further assessed by examining the extent and type of new tissue formation on seeded scaffolds at 70 days; both type I and type III collagens were present in fibrillar structures on these scaffolds indicating that the seeded stem cells had the capacity to differentiate into collagen-producing cells necessary to repair damaged ECM. These data show that the CardioCel® scaffold is an appropriate substrate for the stem cells and has the potential to both retain seeded stem cells and to act as a template for cell propagation and new tissue formation.

New engineering treatment of bovine pericardium confers outstanding resistance to calcification in mitral and pulmonary implantations in a juvenile sheep model.

OBJECTIVE: To conduct a test of noninferiority for CardioCel (Admedus, Brisbane, Australia), a chemically engineered bovine pericardium over autologous pericardium treated intraoperatively with glutaraldehyde in a chronic juvenile sheep model of pulmonary valve (PV) and mitral valve (MV) reconstruction. METHODS: We replaced the posterior leaflet of the MV and of 1 PV cusp with patches in ewes aged 10 months. There were 2 groups: CardioCel (n = 6) and control (n = 4). All valves were competent. Echocardiography was performed before euthanasia. The collected data were function, macroscopy, histology, and calcium contents. The primary end points were thickening and calcium content. RESULTS: All animals survived until sacrifice after 7 months. The valves had normal echo. The macroscopic aspect of the valves was excellent. Examination of the slides for both groups revealed a continuous endothelium on both sides of the patch and a layer of new collagen developed on both sides between patch and endothelium and interstitial cells and smooth muscle cell in these layers. The patch had not thickened but the 2 layers of new collagen for the PV showed a median thickening of 37% in the CardioCel group and 111% in the control group (P = .01), and for the MV a thickening of 108% and 251%, respectively, was seen (P = .01). The median calcium content in the PV was 0.24 µg/mg (range, 0.19-0.30) in the CardioCel group versus 0.34 µg/mg (range, 0.24-0.62) in controls (P = .20). In the MV it was 0.46 µg/mg (range, 0.30-1.0) in the CardioCel group and 0.47 µg/mg (range, 0.29-1.9) in controls (P = 1.0). CONCLUSIONS: In this growing lamb model the CardioCel patch allowed accurate valve repair at both systemic and pulmonary pressure. The mechanical properties of CardioCel after 7 months were preserved with a more controlled healing than the treated autologous pericardium and without calcification.

Evaluation of a tissue-engineered bovine pericardial patch in paediatric patients with congenital cardiac anomalies: initial experience with the ADAPT-treated CardioCel(R) patch.

OBJECTIVES: This study evaluated the safety, efficacy and clinical performance of the tissue-engineered ADAPT® bovine pericardial patch (ABPP) in paediatric patients with a range of congenital cardiac anomalies. METHODS: In this single-centre, prospective, non-randomized clinical study, paediatric patients underwent surgery for insertion of the ABPP. Primary efficacy measures included early (<30 day) morbidity; incidence of device-related complications; haemodynamic performance derived from echocardiography assessment at 6- and 12-month follow-up and magnetic resonance imaging findings in 10 randomly selected patients at 12 months. Secondary measures included device-handling characteristics; shape and sizing characteristics and perioperative implant complications. The Aristotle complexity scoring system was used to score the complexity level of all surgical procedures. Patients completing the 12-month study were eligible to enter a long-term evaluation study. RESULTS: Between April 2008 and September 2009, the ABPP was used in 30 paediatric patients. In the 30-day postoperative period, no graft-related morbidity was observed. In total, there were 5 deaths (2 in the 30-day postoperative period and 3 within the first 6 postoperative months). All deaths were deemed due to comorbid non-graft-related events. Echocardiography assessment at 6 and 12 months revealed intact anatomical and haemodynamically stable repairs without any visible calcification of the patch. Magnetic resonance imaging assessment in 10 patients at 12 months revealed no signs of calcification. Fisher's exact test demonstrated that patients undergoing more complex, higher risk surgical repairs (Aristotle complexity score >8) were significantly more likely to die (P = 0.0055, 58% survival compared with 100% survival for less complex surgical repairs). In 19 patients, echocardiographic data were available at 18-36 months with no evidence of device calcification, infection, thromboembolic events or device failure. CONCLUSIONS: This study demonstrates the safety and efficacy of this engineered bovine pericardial patch as a cardiovascular substitute for surgical repair of both simple and more complex congenital cardiac defects.

The effect of diazepam on myocardial function and coronary vascular tone after endotoxemia in the isolated rat heart model.

OBJECTIVE AND DESIGN: Tumor necrosis factor alpha (TNF-α) has been implicated in the pathogenesis of cardiovascular disease and sepsis-associated cardiac dysfunction. Although initially described solely as a lipopolysaccharide (LPS)-induced macrophage product, evidence exists that cardiac myocytes themselves produce substantial amounts of TNF-α in response to ischemia as well as LPS. The use of phosphodiesterase inhibitors has been shown to decrease LPS-induced TNF-α elaboration. The aim of the present study was to determine the effect of diazepam (Type IV phosphodiesterase inhibitor) on (1) myocardial function and (2) coronary vascular flow after LPS-induced endotoxic shock in an isolated rat heart model. MATERIALS AND METHODS: Endotoxemia was induced by intraperitoneal LPS administration in adult male Wistar rats. Hearts were isolated after 6 h and perfused in a working mode with oxygenated Krebs-Henseleit buffer at 37°C. Diazepam was mixed with Krebs-Henseleit buffer and administered (3.0 µg/ml) for 20 min. RESULTS: LPS-treated hearts showed depressed cardiac function and reduced coronary flow. Myocardial functional parameters (LVDP, +dP/dt, -dP/dt, RPP) and coronary flow (ml/min) were significantly (p < 0.01) improved by diazepam administration. CONCLUSIONS: These findings suggest that diazepam can salvage myocardial function and undo coronary vascular constriction in the endotoxemic rat heart. These findings are clinically relevant to the treatment of cardiovascular depression caused by endotoxic shock.

Mitigation of calcification and cytotoxicity of a glutaraldehyde-preserved bovine pericardial matrix: improved biocompatibility after extended implantation in the subcutaneous rat model.

BACKGROUND AND AIMS OF THE STUDY: Implanted non-crosslinked tissues suffer rapid degeneration, shrinkage and absorption, whereas standard crosslinked tissues cause local cytotoxicity and calcification. Both approaches diminish implant capacity for long-term function. The study aim was to examine the tissue-engineered characteristics (cytotoxicity, calcification potential, biocompatibility) of bovine pericardium, crosslinked with a low concentration of glutaraldehyde (GA) followed by ADAPT anti-mineralization, following prolonged implantation in a subcutaneous rat model. METHODS: Bovine pericardium was decellularized with Triton X-100, deoxycholate, IgePal CA-630, and nucleases. The resulting matrices were allocated to: group I (control, n=10), crosslinked in 0.2% polymeric GA; and group II (treatment, n=10), crosslinked in 0.05% monomeric GA + ADAPT. Cytotoxicity was determined by in vitro cell seeding with human fibroblasts, and quantified using an Alamar Blue assay. The matrices were then implanted in a subcutaneous rat model and retrieved after extended implantation times (26 and 52 weeks). This was followed by further histology, immunohistochemical staining, and measurement of calcium deposition. RESULTS: Complete acellularity and biostability were significantly (p < 0.01) achieved in group II. Inflammatory responses were reduced and beneficial host cell infiltration with neocapillary formation was limited to group II. Fibroblast infiltration was significantly increased from six to 12 months' implantation time. Only group II tested positive for Factor VIII and vimentin. After 52 weeks, extractable calcium levels were significantly (p < 0.001) reduced in group II (2.56 +/- 0.22 microg Ca/mg tissue) compared to group I (136.02 +/- 0.39 microg Ca/mg tissue). CONCLUSION: Acellular bovine pericardium, when crosslinked with a low concentration of GA and treated with ADAPT, retains and improves its integrity with a low immunoreactivity over a prolonged period. Host cell infiltration is also optimized over time. The maintenance of reduced calcification levels in group II suggests that such a biomaterial might have an advanced long-term in vivo potential.

Enhanced biostability and biocompatibility of decellularized bovine pericardium, crosslinked with an ultra-low concentration monomeric aldehyde and treated with ADAPT.

BACKGROUND AND AIM OF THE STUDY: Matrix preparation remains controversial due to incomplete cell removal, inflammatory responses, reabsorption and thrombocyte activation. Previously, crosslinked matrices have been considered unsatisfactory due to cytotoxicity. In the present study, the biostability, biocompatibility and calcification potential of a decellularized matrix crosslinked with a low concentration of monomeric glutaraldehyde (GA) and treated with the ADAPT anti-calcification process were examined. METHODS: Bovine pericardium was decellularized with Triton X-100, deoxycholate, IgePal CA-630 and ribonuclease. The resulting matrices were allocated to either group I (control, n = 5), crosslinked in 0.2% polymeric GA + ADAPT, or to group II (treatment, n = 5), crosslinked in 0.05% monomeric GA + ADAPT. The physical properties, enzymatic degradation, histology and immunohistochemical staining of the tissues were monitored. The matrices were also implanted in the jugular vein of juvenile sheep for 200 days. RESULTS: Complete acellularity was achieved. Biostability was significantly (p <0.01) enhanced in group II, but inflammatory responses were limited in both groups. Host fibroblasts infiltrated the periphery in group I and the entire matrix in group II. The luminal surfaces were free from thrombotic depositions and covered with endothelial cells. Both groups tested positive for Factor VIII, smooth muscle alpha-actin and vimentin. Tissue extractable calcium levels were low (group I = 1.02 +/- 0.39, group II = 0.86 +/- 0.22 microg Ca/mg tissue). CONCLUSION: Low-concentration GA-crosslinked matrices proved to be stable. The immunoreactivity of both groups was low, with host cell infiltration, migration and trans-differentiation being optimized in those grafts crosslinked with an ultra-low monomeric GA concentration. Calcification levels were close to zero in both groups. Enhanced crosslinking and effective anti-calcification produce a biomaterial with advanced in-vivo tissue-engineering properties.

Kangaroo versus freestyle stentless bioprostheses in a juvenile sheep model: hemodynamic performance and calcification behavior.

BACKGROUND: Glutaraldehyde-preserved bioprosthetic heart valve substitutes have limited performance and longevity due to tissue degeneration and calcification. The Freestyle valve (Medtronic Heart Valves, Inc, Minneapolis, MN) combines zero fixation pressure and proportional, variant-amino oleic acid (AOA) as antidegeneration and antimineralization measures. The aim of this study was to compare the calcification behavior of glutaraldehyde-preserved kangaroo aortic valves with Freestyle stentless bioprostheses in a juvenile sheep model. METHODS: Pulmonary artery and valve replacements were performed in juvenile sheep with Freestyle stentless aortic valves (n = 4) or glutaraldehyde-preserved kangaroo stentless aortic valves with no antimineralization measures (n = 6), and explanted at 200 postoperative days. RESULTS: Freestyle stentless valves and stentless kangaroo aortic valves showed normal valve function immediately postoperatively and up to 120 days. Valve leaflets of all valves were macroscopically free of visible calcification with normal histology. Valve leaflet calcification (microg Ca/mg tissue) was less in kangaroo than Freestyle (1.27 +/- 0.43 versus 2.38 +/- 1.37, p = 0.856). Aortic wall tissue calcification was severe in kangaroo and Freestyle (127.93 +/- 12.22 versus 122.19 +/- 11.99, p = 0.596). CONCLUSIONS: We conclude that glutaraldehyde-preserved kangaroo aortic valve leaflets are equal to AOA-treated Freestyle stentless valve leaflets with regard to calcification in juvenile sheep. Both bioprostheses are prone to aortic wall calcification. The low calcification features of the kangaroo aortic valve leaflets without antimineralization treatment may benefit the longevity of the valve.

Evaluation of stentless kangaroo aortic valves in the mitral position of juvenile sheep.

BACKGROUND AND AIM OF THE STUDY: The performance and longevity of bioprosthetic heart valves are limited by tissue calcification and degeneration after implantation. Experimental valve replacement in large animal models forms an important component of the preclinical evaluation of these bioprosthetic heart valves. The study aim was to assess the feasibility of a mitral model for stentless valves and to evaluate the calcification behavior of stentless glutaraldehyde-preserved kangaroo heart valves in the mitral position of a sheep model. METHODS: Medtronic Freestyle (n = 10) and kangaroo (n = 11) stentless aortic valves were implanted in the mitral position of juvenile sheep and retrieved after a maximum of 200 days. Retrieved stentless valves were examined for morphological changes and calcification of the valve tissue, using radiological screening, Von Kossa's staining and atomic absorption spectrophotometry. RESULTS: Four sheep (40.0%) with Medtronic Freestyle and 10 sheep (90.9%) with kangaroo valves could be weaned from bypass and mechanical ventilation. Two animals (20.0%) with Medtronic Freestyle and six animals (54.5%) with kangaroo prostheses survived more than 30 days postoperatively. No significant difference (p >0.05) was seen between the calcification potential of Medtronic Freestyle valve leaflets (3.21 +/- 1.67 microg/mg) after 93 days and kangaroo valve leaflets (2.39 +/- 0.80 microg/mg) after 200 days. CONCLUSION: The present results suggest that implantation of a stentless valve in the mitral position of sheep is possible, but technically difficult. The calcification potential of kangaroo valve tissue is comparable to that of Freestyle valve tissue in the mitral position of sheep.

ADAPT-treated porcine valve tissue (cusp and wall) versus Medtronic Freestyle and Prima Plus: crosslink stability and calcification behavior in the subcutaneous rat model.

BACKGROUND AND AIM OF THE STUDY: The study aim was to compare the crosslink stability and calcification behavior of porcine tissue (cusp and wall), treated with a new antimineralization process (ADAPT) with that of commercially prepared Freestyle and Prima Plus bioprosthetic tissues. METHODS: Porcine aortic roots were divided into two groups: (i) tissue zero pressure-fixed with 0.625% glutaraldehyde (GA) for seven days and stored in 0.25% GA (as control); and (ii) tissue exposed to the ADAPT process for four days and stored in 0.25% buffered GA. These groups were compared with Freestyle and Prima Plus tissues (cusp and wall). Crosslink stability was assessed by shrinkage temperature and resistance to pronase degradation. Calcification behavior was assessed histologically (Von Kossa staining) and by atomic absorption spectrophotometry of explanted tissue after eight weeks. in a subcutaneous rat model. RESULTS: Crosslink stability and calcification potential of ADAPT-treated porcine valve cusps were comparable to those of Freestyle and Prima Plus cusps (p = NS). ADAPT-treated porcine wall tissue showed improved crosslink stability (p <0.05) and significantly (p <0.001) reduced calcification (-95.95%) compared to control (-0.00%), Freestyle (-47.87%) and Prima Plus (-51.95%) tissues. CONCLUSION: The ADAPT process is effective in reducing calcification in both porcine cusp and wall tissues in a subcutaneous rat model, and further suggest that enhanced crosslinking plays an important role in minimizing aortic wall calcification.

Homograft aortic valve replacement--the experience of one unit.

BACKGROUND: Homograft valves offer advantages including avoidance of anticoagulation and less susceptibility to infection especially in the setting of endocarditis. However, there is concern about their durability and possible accelerated degeneration particularly in cases of second time replacement with homografts. AIM: This study aimed to evaluate the pattern of homograft failure and the quality of life in patients after homograft implantation. METHODS: Between 1990 and 1998, 58 patients underwent aortic valve replacement with a homograft (aortic homograft = 47, pulmonary homograft = 11). Evaluation was based on clinical and echocardiographic examination, patient questionnaires and explanted valve pathology. Survival and freedom from cardiac related death were expressed by actuarial methods. RESULTS: Follow up ranged from 1 to 10 years (mean 5.5 years). Analysis of questionnaires revealed 60% of respondents to be in good performance status and 20% in moderate and 20% in poor performance status groups. Eleven patients (18.9%) required subsequent redo valve replacement after initial homograft insertion (pulmonary = 6, aortic = 5) due to either valve dehiscence (n=4) or valve degeneration (n=7). The mean interval of re-replacement was 5.4 years. CONCLUSIONS: Pulmonary homografts have a high failure rate in the aortic position. Overall subjective and clinical improvement after surgery is less than expected for a "physiological" device. In the setting of low availability of homografts the use of off-the-shelf devices such as stentless xenografts may be preferable in most cardiac surgical units in the current era.