Coating with fibronectin and stromal cell-derived factor-1α of decellularized homografts used for right ventricular outflow tract reconstruction eliminates immune response-related degeneration.

OBJECTIVE: This study assesses the performance and cellular features of decellularized ovine aortic homografts coated with stromal cell-derived factor-1α (SDF-1α) and its natural linker, fibronectin (FN), after implantation in the right ventricular outflow tract of adolescent sheep. METHODS: Right ventricular outflow tract reconstructions using cryopreserved (n = 7), decellularized (n = 8), and decellularized FN/SDF-1α-coated aortic ovine homografts (n = 6) were performed. Echocardiographic, morphologic, radiographic, histologic, and immunohistochemical examinations were performed 5 months after implantation. RESULTS: There were no hemodynamic differences between groups, except for the decellularized homografts' tendency to develop more valve regurgitation (3 of 8 grafts had regurgitation >2/4). All decellularized, but coated, grafts had normal hemodynamics. Decellularized valve conduits were less calcified than cryopreserved conduits (P < .05), but coated valve conduits were free of calcification (P < .05). The same was found for pannus in the outflow parts. Immune response (CD45(+), CD45R(+), or CD11b(+) cells) was decreased in decellularized valves compared with cryopreserved grafts, but was virtually absent (P < .05) in coated grafts. Collagen organization and density in the leaflets and walls were decreased in cryopreserved and decellularized valves, but not in coated valves (P < .05). Coating improved re-endothelialization (P < .05). CONCLUSIONS: Coating of decellularized allografts with FN/SDF-1α prevents cryopreserved heart valve-mediated immune response, conduit calcification, and pannus formation and stimulates re-endothelialization.

Functional connectivity fMRI of the rodent brain: comparison of functional connectivity networks in rat and mouse.

At present, resting state functional MRI (rsfMRI) is increasingly used in human neuropathological research. The present study aims at implementing rsfMRI in mice, a species that holds the widest variety of neurological disease models. Moreover, by acquiring rsfMRI data with a comparable protocol for anesthesia, scanning and analysis, in both rats and mice we were able to compare findings obtained in both species. The outcome of rsfMRI is different for rats and mice and depends strongly on the applied number of components in the Independent Component Analysis (ICA). The most important difference was the appearance of unilateral cortical components for the mouse resting state data compared to bilateral rat cortical networks. Furthermore, a higher number of components was needed for the ICA analysis to separate different cortical regions in mice as compared to rats.

Labeling of Luciferase/eGFP-expressing bone marrow-derived stromal cells with fluorescent micron-sized iron oxide particles improves quantitative and qualitative multimodal imaging of cellular grafts in vivo.

PURPOSE: Development of multimodal imaging strategies is currently of utmost importance for the validation of preclinical stem cell therapy studies. PROCEDURES: We performed a combined labeling strategy for bone marrow-derived stromal cells (BMSC) based on genetic modification with the reporter genes Luciferase and eGFP (BMSC-Luc/eGFP) and physical labeling with blue fluorescent micron-sized iron oxide particles (MPIO) in order to unambiguously identify BMSC localization, survival, and differentiation following engraftment in the central nervous system of mice by in vivo bioluminescence (BLI) and magnetic resonance imaging and postmortem histological analysis. RESULTS: Using this combination, a significant increase of in vivo BLI signal was observed for MPIO-labeled BMSC-Luc/eGFP. Moreover, MPIO labeling of BMSC-Luc/eGFP allows for the improved identification of implanted cells within host tissue during histological observation. CONCLUSIONS: This study describes an optimized labeling strategy for multimodal stem cell imaging resulting in improved quantitative and qualitative detection of cellular grafts.

Calcification of allograft and stentless xenograft valves for right ventricular outflow tract reconstruction: an experimental study in adolescent sheep.

OBJECTIVE: Aortic homografts were compared with pulmonary homografts in the setting of right ventricular outflow tract reconstruction in adolescent sheep. Furthermore, clinically available stentless porcine and bovine xenografts were studied as an alternative to homografts. METHODS: In 51 adolescent sheep cryopreserved aortic and pulmonary (ovine) homografts, as well as 6 different types of clinically available stentless bioprostheses (Prima Plus, Toronto SPV, Toronto BiLinx, Freestyle, Pericarbon Stentless, and Contegra) were implanted in the pulmonary position. After 5 to 6 months, the valves were explanted and studied for structural valve degeneration by means of radiographic analysis, histology, and calcium content determination. RESULTS: Pulmonary homografts calcified significantly less than aortic homografts in the wall portion. Leaflet calcification was mild, hardly detectable on radiographic analysis, and comparable between aortic and pulmonary homografts. Stentless porcine xenografts showed severe calcification in the aortic wall portion, irrespective of the antimineralization treatment. Leaflet calcification was mild and in the range of that seen in homografts. Pannus formation was present but never induced leaflet retraction or cusp immobilization. Calcification was absent in the stentless Pericarbon valve implants, but all valves showed extensive pannus overgrowth, leaflet retraction, and cusp immobilization. The Contegra valves showed wall calcification, but the leaflets were completely free of calcification and pannus. CONCLUSIONS: For right ventricular outflow tract reconstruction, the pulmonary homograft remains the first choice. All xenografts result in either calcific degeneration or cusp immobilization.

Gene expression study of monocytes/macrophages during early foreign body reaction and identification of potential precursors of myofibroblasts.

Foreign body reaction (FBR), initiated by adherence of macrophages to biomaterials, is associated with several complications. Searching for mechanisms potentially useful to overcome these complications, we have established the signaling role of monocytes/macrophages in the development of FBR and the presence of CD34(+) cells that potentially differentiate into myofibroblasts. Therefore, CD68(+) cells were in vitro activated with fibrinogen and also purified from the FBR after 3 days of implantation in rats. Gene expression profiles showed a switch from monocytes and macrophages attracted by fibrinogen to activated macrophages and eventually wound-healing macrophages. The immature FBR also contained a subpopulation of CD34(+) cells, which could be differentiated into myofibroblasts. This study showed that macrophages are the clear driving force of FBR, dependent on milieu, and myofibroblast deposition and differentiation.

Selection of an immunohistochemical panel for cardiovascular research in sheep.

Large animal research, often required as a final phase before commencing clinical trials for devices, has generally been hampered by the lack of appropriate tools to compare it with either initial small animal tests or to later evaluation in humans. Setting out to tissue engineer heart valves, we were particularly struck by the limited availability of immunohistochemical markers for sheep tissue, despite sheep being the FDA-approved animal for heart valve testing. This paper, therefore, aims to compile the available knowledge and extend the marker list with antibodies cross-reacting with sheep tissue. Thirty-seven antibodies attributed to 1 of these classes were found to be useful: (1) endothelium, (2) mesenchymal cells, myofibroblasts, and smooth muscle cells, (3) immune response, (4) primitive cells, (5) extracellular matrix, and (6) miscellaneous. Twelve had already been used in sheep tissue, but to our knowledge, the remaining 25 have not been described for use in sheep. From this result, we can conclude that the immunohistochemical panel for sheep has been extensively expanded with respect to cardiovascular research.

The remodeling of cardiovascular bioprostheses under influence of stem cell homing signal pathways.

Optimizing current heart valve replacement strategies by creating living prostheses is a necessity to alleviate complications with current bioprosthetic devices such as calcification and degeneration. Regenerative medicine, mostly in vitro tissue engineering, is the forerunner of this optimization search, yet here we show the functionality of an in vivo alternative making use of 2 homing axes for stem cells. In rats we studied the signaling pathways of stem cells on implanted bioprosthetic tissue (photooxidized bovine pericardium (POP)), by gene and protein expression analysis. We found that SDF-1alpha/CXCR4 and FN/VLA4 homing axes play a role. When we implanted vascular grafts impregnated with SDF-1alpha and/or FN as carotid artery interpositions, primitive cells were attracted from the circulation. Next, bioprosthetic heart valves, constructed from POP impregnated with SDF-1alpha and/or FN, were implanted in pulmonary position. As shown by CD90, CD34 and CD117 immunofluorescent staining they became completely recellularized after 5 months, had a normal function and biomechanical properties and specifically the combination of SDF-1alpha and FN had an optimal valve-cell phenotype.

Use of a Codman microsensor intracranial pressure probe: effects on near infrared spectroscopy measurements and cerebral hemodynamics in rats.

Intracranial pressure (ICP) monitoring is indispensable in the assessment of neurotrauma in humans and animal models. It was shown that cerebellar ICP, leaving the cortical area intact, can replace cerebral ICP in rats. While cerebral probes may induce spreading depression, the effects of a miniature cerebellar probe on near infrared spectroscopy (NIRS) measurements and cerebral hemodynamics are not known. We therefore compared a group with an ICP probe to a control group. Our experiments revealed decreased optical path lengths at 840 nm and 960 nm (both p=0.026) and a decreased cerebral blood flow (CBF, p=0.015). Despite these changes, the data found using NIRS agree with the blood sample analysis. An increased deoxyhemoglobin concentration (p=0.041) and a decreased sagittal sinus oxygen saturation (p=0.041), were found in the ICP probe group. Because the decreased CBF was accompanied by an increased arterio-venous oxygen difference (p=0.026) and unaltered cerebral metabolic rate of oxygen (p=0.485), this suggests an uncoupling. These data suggest that a cerebellar miniature Codman ICP probe induces an uncoupling of cerebral metabolism and CBF. In addition, NIRS is found to be a robust technique: even when path lengths are altered after probe insertion, physiological alterations can still be examined.

Functional and biomechanical evaluation of a completely recellularized stentless pulmonary bioprosthesis in sheep.

OBJECTIVE: In a previous study we showed that recellularization of a stentless bioprosthetic valve is stimulated 1 month after implantation in the pulmonary position, when its matrix (acellular photo-oxidized bovine pericardium) was preseeded by intraperitoneal implantation during a 3-day period. METHODS: The present study reports on the functional and biomechanical properties of such valves (n = 19) in sheep up to 5 months after implantation. Similar valves (n = 20) that were not intraperitoneally preseeded served as controls. RESULTS: Recellularization was partial in control valves and excessive in preseeded valves: 66% versus 223% of cellularity of native valves, respectively (P < .05). The valves were endothelialized and contained interstitial cells depositing new matrix (collagens and elastin). However, phenotyping revealed an increased proportion of cells with contractile properties (30%-40% alpha smooth muscle actin+) in both groups. Intraperitoneally seeded valves had thicker and shorter leaflets that were associated with mildly increased peak gradients and regurgitation. Characterization of the matrix properties revealed a gradually degrading matrix (+/-25% loss of collagen organization at 5 months) and a concomitant alteration of its biomechanical properties, that is, decreased strength, stiffness, and maximum force. However, overall valve function remained intact, and the biomechanical properties of the whole valves were superior to that of the native valves. CONCLUSION: The ectopic in vivo seeding paradigm provides full recellularization. However, the volume fraction of the cellular phenotypes is not optimal, resulting in inadequate remodeling of the valves.

Trilogy pericardial valve: hemodynamic performance and calcification in adolescent sheep.

BACKGROUND: We assessed the hemodynamic performance and calcification potential of a new design of bovine pericardial valve, the Trilogy valve (Arbor Surgical Technologies Inc, Irvine, CA). We compared this new valve with the Perimount valve (Edwards Lifesciences, Irvine, CA) in a randomized prospective study in adolescent sheep. METHODS: Nine Trilogy valves (size 21) and six Perimount valves (size 23 or 25) were implanted in the mitral position in adolescent sheep and studied during five months. Hemodynamic measurements were performed at one week, three months, and five months using transthoracic echocardiography. Valve calcification was assessed by X-ray and calcium content was measured by atomic absorption spectrometry after five months implantation in sheep. Tissues were also evaluated histologically (Von Kossa staining). RESULTS: The nine Trilogy valves had lower peak velocity, peak gradient, and mean gradient compared with the six Perimount valves. These 21-mm Trilogy valves had similar deceleration time and effective orifice area compared with the 23- and 25-mm Perimount valves. Calcification of the Trilogy valves was significantly lower than Perimount valves (p < 0.01), particularly in the commissural (p < 0.01) and free margin regions (p < 0.03). In all parameters assessed, the Trilogy valves exhibited less variation valve-to-valve compared with Perimount valves. CONCLUSIONS: These findings demonstrate that a valve designed to reduce stress in the tissue, improve leaflet kinematics, with advanced antimineralization treatment, can exhibit superior calcification resistance in the mitral position of adolescent sheep. The trilobal geometry and independent leaflet suspension design, combined with an advanced tissue treatment, appears to be a promising breakthrough in the effort to develop a more durable and hemodynamically efficient bioprosthetic valve.

The recruitment of primitive Lin(-) Sca-1(+), CD34(+), c-kit(+) and CD271(+) cells during the early intraperitoneal foreign body reaction.

Implanted materials, such as medical devices, provoke the body to initiate an inflammatory reaction, known as the foreign body reaction (FBR), which causes several complications for example in hip prostheses, silicone implants, peritoneal dialysis catheters and left ventricular assist devices. FBR is initiated by macrophage adherence and results in granulation tissue formation. The early immunobiology and development of this tissue is not completely understood, but there are indications from related myofibroblast-forming diseases such as vascular repair and fibrosis that primitive stem cells also play a role in the formation of FBR-tissue. To investigate this, acellular photo-oxidized bovine pericardium patches were implanted intraperitoneally in rats and retrieved at time-points ranging from 6h to 7 days. A significant fraction of Sca-1(+) (6h-2 days), c-kit(+), CD34(+) and CD271(+) (2-3 days) stem/progenitor cells were detected. Colony-forming and differentiation capacity of the primitive stem cells into adipo-, osteo-, and myofibroblasts were shown. The presence of these primitive cells and their myofibroblastic differentiation potential were also confirmed at RNA level. The identification of specific primitive cells during FBR may have important implications for the inflammatory responses to inert materials and their use in tissue prostheses.

In vivo cellularization of a cross-linked matrix by intraperitoneal implantation: a new tool in heart valve tissue engineering.

AIM: To use in vivo instead of in vitro cell seeding in heart valve tissue engineering. METHODS AND RESULTS: Intraperitoneally preseeded, photo-oxidized bovine pericardial pulmonary valve constructs (group 1) were compared with non-preseeded constructs (group 2) implanted in sheep. All valves functioned normally and were macroscopically intact at explantation [1 week (n = 6) and 1 month (n = 6) in each group], except for one thrombosed leaflet in a group-2 valve at 1 month. Almost 10-fold higher neomatrix deposition and doubling of the leaflet thickness were found in group 1 vs. 2 (P < 0.05). A concomitant significant decrease in leaflet length (15%) was found at 1 month in group 1. The total cross-sectional surface and total amount of collagen of the original matrix remained unchanged in both groups at all times. Immunohistochemistry showed a low immune response, stem/progenitor cell infiltration, appropriated differentiation, and spontaneous endothelialization of the valves. Significantly, increased re-cellularization was found after IP preseeding compared with spontaneous seeding: cell coverage of the leaflet was 71-100 vs. 8-26% (P < 0.05), respectively. CONCLUSION: Complete re-cellularization can be obtained by IP preseeding of an acellularized cross-linked matrix. Well-functioning valve constructs show cellularization and differentiation into myofibroblast phenotype and concomitant neomatrix deposition.

Factors influencing calcification of cardiac bioprostheses in adolescent sheep.

OBJECTIVE: We determined the possible effects of age, antimineralization treatments, circulatory implant conditions, prosthesis design, and valve-related structural aspects on valve calcification in adolescent sheep. METHODS: Calcium content was measured by means of atomic absorption spectrometry in bioprostheses implanted in 120 sheep (age <1 year) for a period of 3 or 6 months. RESULTS: Bioprostheses calcified significantly in adolescent sheep, but the extent of calcification was multifactorial. Multivariate analysis of the calcium content reveals that age, mitral or pulmonary implant position, prosthesis design (stented or stentless), structure (porcine or pericardial, wall portion or cusp), and antimineralization treatment are independent factors influencing calcification; implant duration beyond 3 months was not. In juvenile sheep (age 5 months) the wall portion, as well as the cusps of the prosthesis, calcified significantly more than in adolescent sheep (age 11 months). Irrespective of age, the cusps of valves implanted in the mitral position calcified more than those in the pulmonary position. The wall portion of stentless valves calcified more than that of stented valves, and pericardial valves calcified less than porcine valves. The surfactant (Tween 80, No-React, and alpha-amino-oleic acid) and alcohol (ethanol and octanediol) treatment significantly reduced cusp calcification; sodium dodecylsulfate did not. None of the anticalcification treatments was able to prevent wall calcification in stentless porcine valves. CONCLUSION: These findings suggest that tissue valve calcification is determined by many independent factors, which can be identified by using adolescent sheep as a preclinical in vivo model.

Pentobarbital fails to reduce cerebral oxygen consumption early after non-hemorrhagic closed head injury in rats.

It is unknown whether barbiturates suppress cerebral oxygen metabolism after cerebral trauma as they do in normal individuals. We evaluated the influence of pentobarbital on cerebral oxygen handling of normal rats and rats subjected to non-hemorrhagic closed head injury (CHI). Oxygen delivery was assessed by measuring cerebral perfusion and oxygen extraction, enabling the calculation of cerebral metabolic rate of oxygen (CMRO2). Mitochondrial function was assessed by studying changes in the oxidized cytochrome oxidase concentration. CHI caused changes in both systemic and cerebral hemodynamics. Cerebral blood flow was reduced to 66% of its control value, but the cerebral metabolic rate of oxygen remained unchanged. Pentobarbital administration induced a significant lowering of the cerebral oxygen consumption in normal rats associated with a secondary decrease in cerebral perfusion. In rats subjected to CHI, pentobarbital was unable to lower the cerebral metabolic demand and did not cause a further decrease in perfusion. Pentobarbital was unable to significantly modulate mitochondrial function in traumatized rats, whereas it exerted this effect in all control animals. We therefore conclude that, in rats subjected to CHI, pentobarbital is unable to perform its beneficial effects on the cerebral metabolism.

Beta-actin cannot be used as a control for gene expression in ovine interstitial cells derived from heart valves.

BACKGROUND AND AIM OF THE STUDY: In gene expression studies, endogenous controls that are constitutively expressed (housekeeping genes) are commonly used to normalize for variations in cDNA synthesis efficiency. In the present study, a frequently used control gene, beta-actin, was examined in ovine heart valves to evaluate its applicability as a housekeeping gene for this tissue. METHODS: Interstitial cells (IC) of the four heart valves were isolated using the outgrowth explant method. Cells were cultured under different serum conditions (10% or 20% fetal bovine serum or 20% sheep serum) up to passage (P) 5. mRNA from fresh tissue and from cells at P0 and P5 was isolated, and expression of beta-actin determined using reverse transcription-polymerase chain reaction (RT-PCR). An identical control sample was used for each PCR and each gel electrophoresis. Data were expressed as a relative value of this control sample. RESULTS: beta-Actin expression in the aortic valve was significantly lower than in other valves. The mRNA level of beta-actin was four-fold lower in freshly isolated IC than in cultured IC. Once up-regulated by in-vitro culturing conditions, beta-actin expression did not change from P0 to P5. An important increase in the variation of beta-actin expression was observed in cultured cells as compared to fresh cells. Different serum conditions did not lead to different beta-actin levels. CONCLUSION: Due to the variation in expression, beta-actin cannot be used as a reference for gene expression of ovine-derived heart valve IC in culture.

Comparison of intracranial pressure measured in the cerebral cortex and the cerebellum of the rat.

In this study, we evaluated the accuracy of intracranial pressure (ICP) measurement in rats by insertion of a miniature ICP probe in the parenchyma of the cerebellum. A comparison was made between the ICP values measured simultaneously in the parenchyma of the cerebral cortex and the cerebellum. In order to obtain a wide range of ICP, animals were subjected to a severe closed head injury (CHI), a moderate CHI or to a sham operation. ICP values ranged from 0.8 to 43.9 mmHg. After 15 min stabilisation the first measurement was taken and followed by a second measurement 25 min after onset to allow comparison of ICP changes at the two implantation sites. Linear regression analysis showed a highly significant correlation at 15 min: Y = 0.919X + 0.655 (R(2) = 0.977), and at 25 min: Y = 0.931X + 0.698 (R(2) = 0.976). The differences in ICP measurement between cerebellar and cerebral site were not significantly different from zero at both time points. Altman-Bland plots showed that the difference in ICP readings between the two locations could differ maximally by 5.3 mmHg. The largest differences were detected when high ICP values were recorded. We conclude that in rats the ICP measurement in the cerebellum is comparable to the ICP measurement in the cerebral cortex. The cerebellar ICP can be used as a valuable alternative during experimental procedures.

Cerebral blood flow assessment with indocyanine green bolus transit detection by near-infrared spectroscopy in the rat.

In this study we evaluated the feasibility of measuring cerebral blood flow in rats by monitoring the transit of an indocyanine green bolus through the brain with multiwavelength near-infrared spectroscopy. Different volumes of a 1 mg/ml indocyanine green solution (5, 15, 25, 50 microl) were injected intravenously in the search for an optimal dose. Clear transit curves were obtained with all doses and a blood flow index could easily be determined. The indocyanine green signal obtained with the bolus of 5 microl rapidly returned to baseline and interfered minimally with the haemoglobin and cytochrome oxidase signals. This dose was used in a second study to evaluate the reproducibility of the signal and the effect of hypercapnia. Two groups of rats received 7 repetitive boli of indocyanine green. In one group, 7% CO(2) was added to the gas mixture before the second, fourth and sixth indocyanine green injection. Hypercapnia consistently caused a significant increase in blood flow index, cerebral haemoglobin concentration and O(2)-saturation. In the control group these variables remained stable in time. We conclude that monitoring of the transit of an indocyanine green bolus with multiwavelength near-infrared spectroscopy can be used to assess cerebral blood flow qualitatively in rats in combination with continuous monitoring of brain oxygenation.

Nitric oxide does not inhibit cerebral cytochrome oxidase in vivo or in the reactive hyperemic phase after brief anoxia in the adult rat.

In this study, near-infrared spectroscopy was applied to examine whether cytochrome oxidase in the rat brain is inhibited by nitric oxide in vivo. During normoxia, intravenous N(G)-nitro-L-arginine methyl ester (L-NAME) administration significantly decreased the cerebral saturation of hemoglobin with oxygen but did not alter the cytochrome oxidase redox state. Anoxia significantly reduced the cytochrome oxidase. The time course of the recovery of the redox state during reoxygenation was not altered by L-NAME. The results suggest that in adult rats, cytochrome oxidase is not inhibited by nitric oxide, either in physiologic conditions or during reoxygenation after a brief anoxic period.