Perioperative care in infective endocarditis.

Patients undergoing surgery for acute infective endocarditis are among those with the highest risk. Their preoperative condition has significant impact on outcomes. There are specific issues related with the preoperative situation, intraoperative findings, and postoperative management. In this narrative review, focus is placed on the most critical aspects in the perioperative period including the management and weaning from mechanical ventilation, the management of vasoplegia, the management of the chest open, antithrombotic therapy, transfusion, coagulopathy, management of atrial fibrillation, the duration of antibiotic therapy, and pacemaker implantation.

Significance and current approaches to vascular graft infection.

Vascular graft/endograft infection (VGEI) is a constant in cardiovascular surgery with published rates between 1 and 5%. Every graft type and anatomical location is a potential target for infectious complications. These patients are sick patients with high frailty burden. Management of VGEI entails a multidisciplinary and multimodality approach. Here we review some aspects of the problem of VGEI including prevention, diagnosis, and surgical therapy with focus on recent developments in the field.

Development and testing of a transcatheter heart valve with reduced calcification potential.

Introduction: Patients from developing countries who require heart valve surgery are younger and have less access to open heart surgery than those from developed countries. Transcatheter heart valves (THVs) may be an alternative but are currently unsuitable for young patients because of their inadequate durability. We developed and tested a THV utilizing two new types of decellularized bovine pericardial leaflets in an ovine model. Methods: The two decellularized tissues [one with a very low dose (0.05%) of monomeric glutaraldehyde (GA) fixation and detoxification (DF) and the other without glutaraldehyde (DE)] were compared to an industry standard [Glycar-fixed with the standard dose (0.625%) of glutaraldehyde]. THVs were manufactured with the three tissue types and implanted in the pulmonary position of nine juvenile sheep for 180 days. Baseline and post-explantation evaluations were performed to determine the hemodynamic performance of the valves and their dynamic strength, structure, biological interaction, and calcification. Results: Heart failure occurred in one animal due to incompetence of its Glycar valve, and the animal was euthanized at 158 days. The gradients over the Glycar valves were higher at the explant than at the implant, but the DE and DF valves maintained normal hemodynamic performance throughout the study. The DF and DE tissues performed well during the mechanical testing of explanted leaflets. Glycar tissue developed thick pannus and calcification. Compared to Glycar, the DF tissue exhibited reduced pannus overgrowth and calcification and the DE tissue exhibited no pannus formation and calcification. All tissues were endothelialized adequately. There was a striking absence of host ingrowth in the DE tissue leaflets, yet these leaflets maintained integrity and mechanical function. Conclusion: In the juvenile sheep THV model, Glycar tissue developed significant pannus, calcification, and hemodynamic deterioration. Using a very low dose of monomeric GA to fix the decellularized bovine pericardium yielded less pannus formation, less calcification, and better hemodynamic function. We postulate that the limited pannus formation in the DF group results from GA. Bovine pericardium decellularized with our proprietary method resulted in inert tissue, which is a unique finding. These results justify further development and evaluation of the two decellularized tissue types in THVs for use in younger patients.

Contemporary risk factors associated with ischaemic heart disease in central South Africa: a single-centre study.

BACKGROUND: Deaths from non-communicable diseases have increased in sub-Saharan Africa over the years, with limited data on coronary artery disease (CAD) and the risk factors thereof. The objective of this study was to investigate modifiable and non-modifiable risk factors in central South Africa in patients with CAD. METHODS: Patients with angiographically confirmed CAD who were evaluated in the catheterisation laboratory for the first time over a two-year period (2016 - 2017) were included. Data were extracted from the patients' medical records. RESULTS: Four hundred and eighty-two patients met the inclusion criteria, presenting at a mean age of 58.4 ± 10.8 years, and were predominantly male (66%). Females were significantly older than the males (60.3 ± 9.6 vs 57.4 ± 11.1 years; p < 0.05). The mean age at presentation was comparable between ethnic groups, except Asian patients who presented at a significantly younger age compared to Caucasians (49.8 ± 10.5 vs 59.1 ± 10.8 years; p < 0.05). Hypertension (91%) was the most common risk factor, followed by smoking (67%) and obesity (41%). Black Africans demonstrated a higher incidence of hypertension when compared to Caucasians (96 vs 87%; p < 0.05). Smoking was more prevalent in Caucasians than black Africans (68 vs 55%; p < 0.05) and occurred more commonly in males than females (73 vs 55%; p < 0.05). Most patients presented with acute coronary syndrome (ACS) (72%), mainly with ST-elevation myocardial infarction (STEMI) (36%). The majority of patients presenting with ACS were in the age group 51 - 60 years. The ACS risk-factor profile was similar to that of the total study group. CONCLUSION: CAD was present in all ethnic groups, and modifiable and non-modifiable risk factors were similar to the classical risk factors described worldwide. Minor interracial differences were observed and hypertension was the most prevalent risk factor recorded in central South Africa. Most patients with CAD presented with ACS, particularly STEMI. Recognition of the risk factors associated with CAD would contribute to improved planning of healthcare systems and increased awareness of CAD.

Monomeric glutaraldehyde fixation and amino acid detoxification of decellularized bovine pericardium for production of biocompatible tissue with tissue-guided regenerative potential.

The effect of monomeric glutaraldehyde fixation and amino acid detoxification on biocompatibility and tissue-guided regenerative potential of decellularized bovine pericardium was evaluated. The degree of cross-linking, porosity, enzymatic degradation, alpha-galactosyl content, the efficacy of detoxification, and cytotoxicity towards human epithelial cells were assessed. Tissue was subcutaneously implanted for eight weeks in male juvenile Sprague-Dawley rats, and mechanical properties, host cell infiltration, and calcification were evaluated. Three groups were compared i) decellularized tissue, ii) decellularized, monomeric glutaraldehyde fixed and amino acid detoxified tissue, and iii) commercial glutaraldehyde fixed non-decellularized tissue (Glycar®) (n = 6 rats per group). The fixation process gave a high degree of cross-linking (>85%), and was resistant to enzymatic degradation, with no significant effect on porosity. The detoxification process was effective, and the tissue was not toxic to mammalian cells in vitro. Tissue from both decellularized groups had significantly higher (p < 0.05) porosity and host cell infiltration in vivo. The process mitigated calcification. A non-significant decrease in the alpha-galactosyl content was observed, which increased when including the alpha-galactosidase enzyme. Mechanical properties were maintained. The fixation and detoxification process adequately removes free aldehyde groups and reduces toxicity, preventing enzymatic degradation and allowing for host cell infiltration while mitigating calcification and retaining the mechanical properties of the tissue. This process can be considered for processing decellularized bovine pericardium with tissue-guided regeneration potential for use in cardiovascular bioprostheses; however, methods of further reducing antigenicity, such as the use of enzymes, should be investigated.

Transcatheter heart valve interventions for patients with rheumatic heart disease.

Rheumatic heart disease [RHD] is the most prevalent cause of valvular heart disease in the world, outstripping degenerative aortic stenosis numbers fourfold. Despite this, global resources are firmly aimed at improving the management of degenerative disease. Reasons remain complex and include lack of resources, expertise, and overall access to valve interventions in developing nations, where RHD is most prevalent. Is it time to consider less invasive alternatives to conventional valve surgery? Several anatomical and pathological differences exist between degenerative and rheumatic valves, including percutaneous valve landing zones. These are poorly documented and may require dedicated solutions when considering percutaneous intervention. Percutaneous balloon mitral valvuloplasty (PBMV) is the treatment of choice for severe mitral stenosis (MS) but is reserved for patients with suitable valve anatomy without significant mitral regurgitation (MR), the commonest lesion in RHD. Valvuloplasty also rarely offers a durable solution for patients with rheumatic aortic stenosis (AS) or aortic regurgitation (AR). MR and AR pose unique challenges to successful transcatheter valve implantation as landing zone calcification, so central in docking transcatheter aortic valves in degenerative AS, is often lacking. Surgery in young RHD patients requires mechanical prostheses for durability but morbidity and mortality from both thrombotic complications and bleeding on Warfarin remains excessively high. Also, redo surgery rates are high for progression of aortic valve disease in patients with prior mitral valve replacement (MVR). Transcatheter treatments may offer a solution to anticoagulation problems and address reoperation in patients with prior MVR or failing ventricles, but would have to be tailored to the rheumatic environment. The high prevalence of MR and AR, lack of calcification and other unique anatomical challenges remain. Improvements in tissue durability, the development of novel synthetic valve leaflet materials, dedicated delivery systems and docking stations or anchoring systems to securely land the transcatheter devices, would all require attention. We review the epidemiology of RHD and discuss anatomical differences between rheumatic valves and other pathologies with a view to transcatheter solutions. The shortcomings of current RHD management, including current transcatheter treatments, will be discussed and finally we look at future developments in the field.

Anastomotic Urethroplasty with Double Layer Continuous Running Suture Re-Anastomosis Versus Interrupted Suture Re-Anastomosis for Infective Bulbar Urethral Strictures: A Prospective Randomised Trial.

INTRODUCTION: The objective of this study was to compare a double-layer running suture re-anastomosis urethral stricture repair with early catheter removal to the conventional interrupted suture re-anastomosis after excision of a bulbar urethral stricture. METHODS: A consecutive series of patients with bulbar urethral stricture were enrolled in the study. The patients were randomized into two groups according to an odd/even serial number distribution. Patients' medical records were analyzed for demographics, stricture characteristics, and lower urinary tract obstructive symptoms. The outcomes were based on the presence/absence of obstructive voiding symptoms, and retrograde urethrography (RGU) performed on the first post-operative day in Group 1 and in both groups (Groups 1 and 2) at six weeks after surgery. Flexible urethroscopy was only performed on specific cases where RGU was unclear both pre- and post-operatively or when clinical recurrence was suspected. The minimum follow-up (FU) was 18 months. Success was defined as no need for subsequent dilatation, direct vision internal urethrotomy (DVIU), or urethroplasty. RESULTS: A total of thirty-six patients with a mean age of 45 years (range 20 to 69 years) with bulbar urethral stricture were included in this study. Group 1 and Group 2 included 19 and 17 patients, respectively. Two patients were lost during randomization and subsequently to FU. The average stricture lengths were comparable between the two groups according to the retrograde urethrogram: 1.20 cm (range 0.6 to 2) in Group 1 and 1.27 cm (range 0.5 to 2.4) in Group 2, respectively (p = 0.631). The success rate for Group 1 was 90% after a mean follow-up of thirty-six months (range 20 to 40), which was clinically significant compared to the 71% in Group 2 after a mean FU of thirty-three months (range 19 to 40; p = 0.0218; 95% CI: 0.462-41.5766). CONCLUSIONS: Anastomotic urethroplasty (AR) performed with a double layer re-anastomosis had a cure rate comparable to the conventional anastomosis with interrupted sutures after a follow-up of eighteen months and longer. The urethral catheter can be safely removed within twenty-four hours after the excision of stricture and double-layer re-anastomosis.

Impact of Three Different Processing Techniques on the Strength and Structure of Juvenile Ovine Pulmonary Homografts.

Homografts are routinely stored by cryopreservation; however, donor cells and remnants contribute to immunogenicity. Although decellularization strategies can address immunogenicity, additional fixation might be required to maintain strength. This study investigated the effect of cryopreservation, decellularization, and decellularization with additional glutaraldhyde fixation on the strength and structure of ovine pulmonary homografts harvested 48 h post-mortem. Cells and cellular remnants were present for the cryopreserved group, while the decellularized groups were acellular. The decellularized group had large interfibrillar spaces in the extracellular matrix with uniform collagen distribution, while the additional fixation led to the collagen network becoming dense and compacted. The collagen of the cryopreserved group was collapsed and appeared disrupted and fractured. There were no significant differences in strength and elasticity between the groups. Compared to cryopreservation, decellularization without fixation can be considered an alternative processing technique to maintain a well-organized collagen matrix and tissue strength of homografts.

Comparison of the function and structural integrity of cryopreserved pulmonary homografts versus decellularized pulmonary homografts after 180 days implantation in the juvenile ovine model.

Homograft availability and durability remain big challenges. Increasing the post-mortem ischaemic harvesting time beyond 24 h increases the potential donor pool. Cryopreservation, routinely used to preserve homografts, damages the extracellular matrix (ECM), contributing to valve degeneration. Decellularization might preserve the ECM, promoting host-cell infiltration and contributing towards better clinical outcomes. This study compared the performance of cryopreserved versus decellularized pulmonary homografts in the right ventricle outflow tract (RVOT) of a juvenile ovine model. Homografts (n = 10) were harvested from juvenile sheep, subjected to 48 h post-mortem cold ischaemia, cryopreserved or decellularized and implanted in the RVOT of juvenile sheep for 180 days. Valve performance was monitored echocardiographically. Explanted leaflet and wall tissue evaluated histologically, on electron microscopical appearance, mechanical properties and calcium content. In both groups the annulus diameter increased. Cryopreserved homografts developed significant (¾) pulmonary regurgitation, with trivial regurgitation (») in the decellularized group. Macroscopically, explanted cryopreserved valve leaflets retracted and thickened while decellularized leaflets remained thin and pliable with good coaptation. Cryopreserved leaflets and walls demonstrated loss of interstitial cells with collapsed collagen, and decellularized scaffolds extensive, uniform ingrowth of host-cells with an intact collagen network. Calcific deposits were shown only in leaflets and walls of cryopreserved explants. Young fibroblasts, with vacuoles and rough endoplasmic reticulum in the cytoplasm, repopulated the leaflets and walls of decellularized scaffolds. Young's modulus of wall tissue in both groups increased significantly. Cryopreserved valves deteriorate over time due to loss of cellularity and calcification, while decellularized scaffolds demonstrated host-cell repopulation, structural maintenance, tissue remodelling and growth potential.

Synergy in a detergent combination results in superior decellularized bovine pericardial extracellular matrix scaffolds.

Decellularization involves removal of cellular material from tissue which results in a scaffold material consisting of only the extra cellular matrix (ECM). The effect of each individual decellularizing detergent on the final ECM scaffold and how that may differ from the combined use of these detergents is currently a gap in decellularization methodologies. This study evaluates the hypothesis that a synergistic effect exists when commonly used decellularization detergents are combined. This was evaluated with regard to decellularization efficiency, tissue strength, and collagen structure. Bovine pericardium was decellularized using a combination of 0.5% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate (SDC) and 1% TritonX-100, and compared to the use of each detergent individually. The combined detergent decellularization protocol showed effective decellularization (p = .004), with minimal effects on tissue strength (p = .21) and structure (p = .21). Use of detergents individually, resulted in detrimental effects on tissue structure and integrity or ineffective decellularization. This study shows a synergistic relationship between SDS, SDC and TritonX-100 when combined at specific concentrations. The use of detergents in combination instead of individually appears to be superior, as it results in less ECM damage and improved decellularization effectivity.

Alternative mechanical heart valves for the developing world.

Due to the prevalence of rheumatic heart disease in the developing world, mechanical heart valves in the younger patient population remain the prostheses of choice if repair is not feasible. Despite their durability, mechanical valves are burdened by coagulation and thromboembolism. Modern design tools can be utilized during the design process of mechanical valves, which allow a more systematic design approach and more detailed analysis of the blood flow through and around valves. These tools include computer-aided design, manufacturing, and engineering, such as computational fluid dynamics and finite element analysis, modern manufacturing techniques such as additive manufacturing, and sophisticated in-vitro and in-vivo tests. Following this systematic approach, a poppet valve was redesigned and the results demonstrate the benefits of the method. More organized flow patterns and fewer complex fluid structures were observed. The alternative trileaflet valve design has also been identified as a potential solution and, if a similar design approach is adopted, it could lead to the development of an improved mechanical heart valve in the future. It is imperative that researchers in developing countries continue their search for a mechanical heart valve with a reduced thromboembolic risk, requiring less or no anticoagulation.

Cadaver donation: structural integrity of pulmonary homografts harvested 48 h post mortem in the juvenile ovine model.

Cryopreserved pulmonary homograft (CPH) implantation remains the gold standard for reconstruction of the right ventricular outflow tract (RVOT). Harvesting homografts < 24-h post mortem is the international norm, thereby largely excluding cadaveric donors. This study examines the structural integrity and stability of ovine pulmonary homografts harvested after a 48-h post mortem period, cryopreserved and then implanted for up to 180 days. Fifteen ovine pulmonary homografts were harvested 48-h post mortem and cryopreserved. Five CPH served as a control group (group 1; n = 5). CPH were implanted in the RVOT of juvenile sheep and explanted after 14 days (group 2; n = 5) and 180 days (group 3; n = 5). Leaflet integrity was evaluated by strength analysis, using tensile strength (TS), Young's modulus (YM) and thermal denaturation temperature (Td), and morphology, including haematoxylin and eosin (H&E), Picrosirius red staining, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and von Kossa stains. Echocardiography confirmed normal function in all implants. In explants, no reduction in TS, YM or Td could be demonstrated and H&E showed mostly acellular leaflet tissue with no difference on Picrosirius red. TEM demonstrated consistent collagen disruption after cryopreservation in all three groups, with no morphological deterioration during the study period. von Kossa stains showed mild calcification in group 3. No deterioration of structural integrity could be demonstrated using strength or morphological evaluations between the controls and implant groups over the study period. Extending the post mortem harvesting time of homografts beyond 24 h did not appear to negatively affect the long-term performance of such transplanted valves in this study.

Tissue Engineered Small Vessel Conduits - The Anti-Thrombotic Effect of Re-Endothelialization of Decellularized Baboon Arteries: A Preliminary Experimental Study.

BACKGROUND The use of decellularized biological scaffolds for the reconstruction of small-diameter vascular grafts remains a challenge in tissue engineering. Thrombogenicity is an important cause of obstruction in these vessels due to decellularization. Seeding of the decellularized vascular constructs with endothelial cells is therefore a prerequisite for the prevention of thrombosis. The aim of this study was to seed decellularized baboon arteries with endothelial cells and to compare the thrombogenicity to that of decellularized arteries after circulation of blood. MATERIAL AND METHODS Carotid, radial, and femoral arteries (12 arteries in total) were harvested from 2 Papio ursinus baboons. Ten arteries were decellularized. Normal morphology was confirmed in the control vessels. The effect of re-endothelialization was studied in the vessel scaffolds using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Decellularization resulted in vessel scaffolds with well-preserved extracellular matrix and intact basal membranes. Six of the decellularized vessel scaffolds were seeded with viable human umbilical vein endothelial cells (HUVEC). Luminal endothelialization was established after 7 days in a bioreactor and SEM confirmed confluency. Two control, 4 decellularized, and 6 decellularized re-endothelialized vessel scaffolds were studied in an in vitro flow chamber using baboon blood. RESULTS The decellularized arteries showed an absence of endothelial lining, and an intact basement membrane. The seeding process produced a complete endothelial layer on the surfaces of the arteries. After perfusion with whole blood, no thrombi were formed in the control arteries and re-endothelialized vessels. Widespread platelet activation and adhesion occurred in the decellularized vessels despite a relatively intact basal membrane. CONCLUSIONS This study supports the development of re-endothelialized tissue engineered small-vessel conduits.

Reprogramming Bone Marrow Stem Cells to Functional Endothelial Cells in a Mini Pig Animal Model.

BACKGROUND The aims of this study were to compare the morphological, biochemical, and functional properties of reprogrammed bone marrow stem cell (BMSC)-derived arterial endothelial cells (AECs) and venous endothelial cells (VECs), following adenosine triphosphate (ATP)-stimulation in a mini pig animal model. MATERIAL AND METHODS Bone marrow aspiration was performed in six adult mini pigs. Harvested mononuclear cells were isolated, cultured, and treated with vascular endothelial growth factor (VEGF) (16 µg/ml). Transformed cells were characterized using immunofluorescence staining for CD31 and von Willebrandt factor (vWF) and expression of endothelial nitric oxide synthase (eNOS). Cell release of nitric oxide (cNO) was measured using spectrophotometry. Matrigel assays were used to investigate angiogenesis in transformed BMSCs. RESULTS Reprogrammed BMSCs in culture showed a typical cobblestone-like pattern of growth. Immunofluorescence staining was positive for CD31 and vWF expression. Expression of eNOS, using immunofluorescence staining and Western blot, showed no difference between the reprogrammed BMSCs and VECs. Spectrophotometric examination following stimulation with 10mmol/l ATP, showed comparable cNO release for reprogrammed BMSCs (10.87±1.76 pmol/106 cells/min) and VECs (13.23±2.16 pmol/10^6 cells/min), but reduced cNO release for AECS (3.44±0.75 pmol/10^6 cells/min). Matrigel assay for angiogenesis showed vascular tube formation of differentiated BMSC endothelial cells (grade 3.25). BMSCs cultured without VEGF did not demonstrate vascular tube formation. CONCLUSIONS The findings of this study showed that eNOS expression and release of NO could be used to show that BMSCs can be reprogrammed to functional VECs and AECs.

Cardiology-cardiothoracic subspeciality training in South Africa: a position paper of the South Africa Heart Association.

Over the past decades, South Africa has undergone rapid demographic changes, which have led to marked increases in specific cardiac disease categories, such as rheumatic heart disease (now predominantly presenting in young adults with advanced and symptomatic disease) and coronary artery disease (with rapidly increasing prevalence in middle age). The lack of screening facilities, delayed diagnosis and inadequate care at primary, secondary and tertiary levels have led to a large burden of patients with heart failure. This leads to suffering of the patients and substantial costs to society and the healthcare system. In this position paper, the South African Heart Association (SA Heart) National Council members have summarised the current state of cardiology, cardiothoracic surgery and paediatric cardiology reigning in South Africa. Our report demonstrates that there has been minimal change in the number of successfully qualified specialists over the last decade and, therefore, a de facto decline per capita. We summarise the major gaps in training and possible interventions to transform the healthcare system, dealing with the colliding epidemic of communicable disease and the rapidly expanding epidemic of non-communicable disease, including cardiac disease.

Twenty Years of Adult Congenital Heart Surgery: A Single-Center African Experience.

BACKGROUND: Lack of data exists on the surgical management of adult congenital heart disease on the African continent. This study was undertaken to describe the clinical profile and surgical outcome of adult patients with congenital heart disease undergoing surgery in a single-center African study population. METHODS: A retrospective medical chart review of consecutive adult patients (≥18 years) undergoing congenital heart surgery between October 1995 and December 2015 at our institution was undertaken. We described cardiac diagnosis, diagnostic complexity, risk profile, and surgical morbidity and mortality. RESULTS: Data were collected of 233 surgical procedures performed in 219 patients (45.6% males). The most common diagnostic category was septal defects (41.2%), followed by right heart lesions (17.2%), left heart lesions (12.4%), thoracic arteries (9.0%), and conduit failure (6.9%). Twenty-four percent of patients presented in functional class III or IV, and 46% of patients met the criteria for the simple Bethesda diagnostic class. Preoperative risk factors were identified in 19.8% of patients. Corrective surgery was performed in 71.7% of cases, reoperative surgery in 27.6%, and palliative surgery in 0.8%. Right ventricle to pulmonary artery conduit placement comprised 53.1% of reoperations. The overall hospital mortality was 1.7%. Postoperative complications occurred in 26.3% of cases. CONCLUSIONS: This study presents a detailed description of this emerging population in a developing world context. Our outcomes data suggest that adult congenital heart disease surgery is feasible in a Southern African tertiary referral center with low operative mortality and acceptable morbidity.

Does prolonged post-mortem cold ischemic harvesting time influence cryopreserved pulmonary homograft tissue integrity?

This study investigated cryopreserved pulmonary homograft (CPA) structural integrity after prolonged cold ischemic harvesting times in a juvenile sheep model. Three groups with different post-mortem cold ischemic harvesting times were studied, i.e. Group 1 (24 h, n = 10); group 2 (48 h, n = 10); group 3 (72 h, n = 10). In each group, 5 CPAs were studied in vitro after cryopreservation and thawing. The other 5 CPAs were implanted in juvenile sheep for a minimum of 180 days. Serology samples were obtained and echocardiography was performed before euthanasia. Hematoxylin and eosin (H&E), scanning electron microscopy (SEM), von Kossa, Picrosirius red, α-actin, immunohistochemistry [von Willebrand factor (vWF), CD4, CD31 and CD34] and calcium content analyses were performed on explanted CPAs. The in vitro and in vivo studies failed to demonstrate any change in tensile strength, Young's Modulus and thermal denaturation (Td) results between the groups. SEM demonstrated a reduction in endothelial cells (50 % at 24 h, 60.9 % at 48 h and 40.9 % at 72 h), but H&E could not demonstrate autolysis in any CPA in vitro. All cultures were negative. In the explanted groups, IgE, IgM and IgG results were inconclusive. Echocardiography demonstrated normal valve function in all groups. H&E and Picrosirius red staining confirmed tissue integrity. vWF, CD31 and CD34 staining confirmed a monolayer of endothelial cells in all explanted valves. Calcium content of explanted CPA leaflets was similar. This experimental study supports the concept of prolonging the cold ischemic harvesting time of cryopreserved homografts to reduce homograft shortage.