Circulating Osteogenic Progenitor Cells in Mild, Moderate, and Severe Aortic Valve Stenosis.

The aim of this study was to characterize endothelial progenitor cells with osteoblastic phenotype (EPC-OCNs) and their role in individuals with varying degrees of aortic stenosis (AS). Peripheral blood mononuclear cells retrieved from blood samples of individuals with mild (n=40), moderate (n=35), or severe (n=103) AS from September 16, 2008, through March 30, 2015, were analyzed by flow cytometry for the EPC surface markers CD34, CD133, and kinase insert domain receptor (KDR) and the osteoblastic cell surface marker OCN. Levels of EPC-OCNs were correlated with AS severity and calcifications. Patients with severe AS had significantly elevated numbers of total circulating EPC-OCNs, including the EPC-OCN subtypes CD133+/OCN+, CD34+/CD133+/OCN+, and CD133+/KDR+/OCN+, compared with those with mild AS. Individuals with moderate AS also had significantly increased numbers of the circulating progenitor cell CD133+/OCN+ compared with patients with mild AS. There was a significant association between total circulating EPC-OCN levels and aortic valve (AV) calcification, AV mean gradient, and AV area measured by echocardiography. In summary, this study found the presence of circulating EPC-OCNs in patients with progressive AV stenosis. These findings might support the potential role for EPC-OCNs in the progression of AV stenosis and calcification.

In Vivo Response of Acellular Porcine Pericardial for Tissue Engineered Transcatheter Aortic Valves.

Current heart valve prostheses have limitations that include durability, inability to grow in pediatric patients, and lifelong anticoagulation. Transcatheter aortic valve replacements are minimally invasive procedures, and therefore have emerged as an alternative to traditional valve prostheses. In this experiment, the regenerative capacity of potential tissue engineered transcatheter valve scaffolds (1) acellular porcine pericardium and (2) mesenchymal stem cell-seeded acellular porcine pericardium were compared to native porcine aortic valve cusps in a rat subcutaneous model for up to 8 weeks. Immunohistochemistry, extracellular matrix evaluation, and tissue biomechanics were evaluated on the explanted tissue. Acellular valve scaffolds expressed CD163, CD31, alpha smooth muscle actin, and vimentin at each time point indicating host cell recellularization; however, MSC-seeded tissue showed greater recellularization. Inflammatory cells were observed with CD3 biomarker in native porcine pericardial tissue throughout the study. No inflammation was observed in either acellular or MSC-seeded scaffolds. There was no mechanical advantage observed in MSC-seeded tissue; however after the first week post-explant, there was a decrease in mechanical properties in all groups (p < 0.05). MSC-seeded and acellular porcine pericardium expressed decreased inflammatory response and better host-cell recellularization compared to the native porcine aortic valve cusps.

Experimental Metabolic Syndrome Model Associated with Mechanical and Structural Degenerative Changes of the Aortic Valve.

The purpose of this study was to test the hypothesis that an experimental high fat (HF) animal with metabolic syndrome results in structural degeneration of the aortic valve. Domestic pigs were divided (n = 12) and administered either a normal or HF diet. After 16-weeks, the HF diet group had increased weight (p ≤ 0.05), total cholesterol (p ≤ 0.05), and systolic and diastolic pressure (p ≤ 0.05). The aortic valve extracellular matrix showed loss of elastin fibers and increased collagen deposition in the HF diet group. Collagen was quantified with ELISA, which showed an increased concentration of collagen types 1 and 3 (p ≤ 0.05). In the HF diet group, the initial stages of microcalcification were observed. Uniaxial mechanical testing of aortic cusps revealed that the HF diet group expressed a decrease in ultimate tensile strength and elastic modulus compared to the control diet group (p ≤ 0.05). Western blot and immunohistochemistry indicated the presence of proteins: lipoprotein-associated phospholipase A2, osteopontin, and osteocalcin with an increased expression in the HF diet group. The current study demonstrates that experimental metabolic syndrome induced by a 16-week HF diet was associated with a statistically significant alteration to the physical architecture of the aortic valve.

Biomaterial characterization of off-the-shelf decellularized porcine pericardial tissue for use in prosthetic valvular applications.

Fixed pericardial tissue is commonly used for commercially available xenograft valve implants, and has proven durability, but lacks the capability to remodel and grow. Decellularized porcine pericardial tissue has the promise to outperform fixed tissue and remodel, but the decellularization process has been shown to damage the collagen structure and reduce mechanical integrity of the tissue. Therefore, a comparison of uniaxial tensile properties was performed on decellularized, decellularized-sterilized, fixed, and native porcine pericardial tissue versus native valve leaflet cusps. The results of non-parametric analysis showed statistically significant differences (p < .05) between the stiffness of decellularized versus native pericardium and native cusps as well as fixed tissue, respectively; however, decellularized tissue showed large increases in elastic properties. Porosity testing of the tissues showed no statistical difference between decellularized and decell-sterilized tissue compared with native cusps (p > .05). Scanning electron microscopy confirmed that valvular endothelial and interstitial cells colonized the decellularized pericardial surface when seeded and grown for 30 days in static culture. Collagen assays and transmission electron microscopy analysis showed limited reductions in collagen with processing; yet glycosaminoglycan assays showed great reductions in the processed pericardium relative to native cusps. Decellularized pericardium had comparatively low mechanical properties among the groups studied; yet the stiffness was comparatively similar to the native cusps and demonstrated a lack of cytotoxicity. Suture retention, accelerated wear, and hydrodynamic testing of prototype decellularized and decell-sterilized valves showed positive functionality. Sterilized tissue could mimic valvular mechanical environment in vitro, therefore making it a viable potential candidate for off-the-shelf tissue-engineered valvular applications.

A novel surgical technique for a rat subcutaneous implantation of a tissue engineered scaffold.

OBJECTIVES: Subcutaneous implantations in small animal models are currently required for preclinical studies of acellular tissue to evaluate biocompatibility, including host recellularization and immunogenic reactivity. METHODS: Three rat subcutaneous implantation methods were evaluated in six Sprague Dawley rats. An acellular xenograft made from porcine pericardium was used as the tissue-scaffold. Three implantation methods were performed; 1) Suture method is where a tissue-scaffold was implanted by suturing its border to the external oblique muscle, 2) Control method is where a tissue-scaffold was implanted without any suturing or support, 3) Frame method is where a tissue-scaffold was attached to a circular frame composed of polycaprolactone (PCL) biomaterial and placed subcutaneously. After 1 and 4 weeks, tissue-scaffolds were explanted and evaluated by hematoxylin and eosin (H&E), Masson's trichrome,Picrosirius Red, transmission electron microscopy (TEM), immunohistochemistry, and mechanical testing. RESULTS: Macroscopically, tissue-scaffold degradation with the suture method and tissue-scaffold folding with the control method were observed after 4 weeks. In comparison, the frame method demonstrated intact tissue scaffolds after 4 weeks. H&E staining showed progressive cell repopulation over the course of the experiment in all groups with acute and chronic inflammation observed in suture and control methods throughout the duration of the study. Immunohistochemistry quantification of CD3, CD 31, CD 34, CD 163, and αSMA showed a statistically significant differences between the suture, control and frame methods (P < 0.05) at both time points. The average tensile strength was 4.03 ±â€¯0.49, 7.45 ±â€¯0.49 and 5.72 ±â€¯1.34 (MPa) after 1 week and 0.55 ±â€¯0.26, 0.12 ±â€¯0.03 and 0.41 ±â€¯0.32 (MPa) after 4 weeks in the suture, control, and frame methods; respectively. TEM analysis showed an increase in inflammatory cells in both suture and control methods following implantation. CONCLUSION: Rat subcutaneous implantation with the frame method was performed with success and ease. The surgical approach used for the frame technique was found to be the best methodology for in vivo evaluation of tissue engineered acellular scaffolds, where the frame method did not compromise mechanical strength, but it reduced inflammation significantly.

Recellularization of a novel off-the-shelf valve following xenogenic implantation into the right ventricular outflow tract.

Current research on valvular heart repair has focused on tissue-engineered heart valves (TEHV) because of its potential to grow similarly to native heart valves. Decellularized xenografts are a promising solution; however, host recellularization remains challenging. In this study, decellularized porcine aortic valves were implanted into the right ventricular outflow tract (RVOT) of sheep to investigate recellularization potential. Porcine aortic valves, decellularized with sodium dodecyl sulfate (SDS), were sterilized by supercritical carbon dioxide (scCO2) and implanted into the RVOT of five juvenile polypay sheep for 5 months (n = 5). During implantation, functionality of the valves was assessed by serial echocardiography, blood tests, and right heart pulmonary artery catheterization measurements. The explanted valves were characterized through gross examination, mechanical characterization, and immunohistochemical analysis including cell viability, phenotype, proliferation, and extracellular matrix generation. Gross examination of the valve cusps demonstrated the absence of thrombosis. Bacterial and fungal stains were negative for pathogenic microbes. Immunohistochemical analysis showed the presence of myofibroblast-like cell infiltration with formation of new collagen fibrils and the existence of an endothelial layer at the surface of the explant. Analysis of cell phenotype and morphology showed no lymphoplasmacytic infiltration. Tensile mechanical testing of valve cusps revealed an increase in stiffness while strength was maintained during implantation. The increased tensile stiffness confirms the recellularization of the cusps by collagen synthesizing cells. The current study demonstrated the feasibility of the trans-species implantation of a non-fixed decellularized porcine aortic valve into the RVOT of sheep. The implantation resulted in recellularization of the valve with sufficient hemodynamic function for the 5-month study. Thus, the study supports a potential role for use of a TEHV for the treatment of valve disease in humans.

Impact of renal dysfunction on the prognostic value of the TIMI risk score in patients with non-ST elevation acute coronary syndrome.

BACKGROUND: The thrombolysis-in-myocardial-infarction risk score (TRS) is a validated risk-assessment tool based on randomized clinical trials. Its applicability to an unselected group of patients seen in general clinical practice may be limited as renal dysfunction was an exclusion criteria in the original trials upon which the TRS was determined. MATERIALS AND METHODS: Consecutive patients with non-ST elevation acute coronary syndrome were stratified based on renal function. Normal renal function was defined as a creatinine clearance (CrCl) of more than 60 ml/min, moderate renal dysfunction was defined as a CrCl of at least 30 ml/min but 60 ml/min or less, and severe renal dysfunction was defined as a CrCl of less than 30 ml/min. A TRS was calculated using the original seven criteria (TRS-7) which did not consider renal function. A second TRS was calculated using the original seven criteria plus the addition of renal dysfunction if the CrCl was 60 ml/min or less (TRS-8 ≤ 60). A third TRS was calculated using the original seven criteria plus renal dysfunction if the CrCl was less than 30 ml/min (TRS-8<30). In the calculation of both of the TRS-8, the presence of renal dysfunction was given weight equal to each of the original seven criteria. Comparisons between groups stratified by renal function were made using Pearson's χ² test or Fisher's exact test for categorical variables (presented as counts and percentages) and the unpaired t-test for continuous variables (presented as the mean ± standard deviation). The χ² test was used to compare the statistical differences between each of the three TRS and the percentage of patients achieving the primary composite outcome during the index hospitalization. The primary outcome was the composite of cardiovascular death, nonfatal myocardial infarction, or urgent coronary revascularization for documented myocardial ischemia. RESULTS: Of the 798 patients included in the analysis, 281 (35%) patients had renal dysfunction (26% had moderate dysfunction and 9% had severe dysfunction). When considered categorically, patients with moderate or severe renal dysfunction had significantly higher rates of the primary composite outcome. The three TRS (TRS-7, TRS-8 ≤ 60, and TRS-8<30) were significantly correlated with the primary composite outcome. With a calculated TRS of 5 or less, the TRS-8 ≤ 60 and the TRS<30 were not associated with a significantly higher prevalence of the composite outcome (all comparisons P>0.05). At a calculated TRS of 6 or 7, the TRS-8<30 was associated with a significantly greater prevalence of the composite outcome compared with the TRS-7 (P=0.02) and the TRS-8 ≤ 60 (P=0.02). There was no significant difference in the frequency of the composite outcome with a calculated TRS of 6 or 7 using the TRS-7 compared with the TRS-8 ≤ 60 (P=0.79). At a calculated TRS of 8, both the TRS-8<30 and TRS-8 ≤ 60 had a significantly higher prevalence of the composite outcome compared with a calculated TRS of 7 using the TRS-7 (P=0.002 for the TRS-8<30 and P=0.045 for the TRS-8 ≤ 60). At a TRS of 8, the TRS-8<30 was associated with a significantly higher composite outcome compared with the TRS-8 ≤ 60 (P=0.035). CONCLUSION: The addition of renal dysfunction to the TRS-7 as an eighth clinical criterion was associated with a higher prevalence of the primary composite outcome primarily at scores of 6 or more. When considered in the context of clinical practice, the use of the TRS-8 ≤ 60 and TRS-8<30 rather than the TRS-7 would not be expected to substantially change the management strategy for patients presenting with non-ST elevation acute coronary syndrome.