105 research outputs found
Test and validation of a bioreactor for the stimulation of engineered cartilage constructs with combined regimens of hydrostatic pressure and interstitial perfusion.
No full textAn integrated experimental-computational approach for the study of engineered cartilage constructs subjected to combined regimens of hydrostatic pressure and interstitial perfusion
No full textAn experimental approach for the study of engineered cartilage constructs subjected to combined regimens of hydrostatic pressure and interstitial perfusion
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Discovery and engineering of an aldehyde tolerant 2-deoxy-d-ribose 5-phosphate aldolase (Dera) from pectobacterium atrosepticum
No full textDERA (2-Deoxy-D-ribose 5-phosphate aldolase) is the only known aldolase that accepts two aldehyde substrates, which makes it an attractive catalyst for the synthesis of a chiral polyol motif that is present in several pharmaceuticals, such as atorvastatin and pravastatin. However, inactivation of the enzyme in the presence of aldehydes hinders its practical application. Whole cells of Pectobacterium atrosepticum were reported to exhibit good tolerance toward acetaldehyde and to afford 2-deoxyribose 5-phosphate with good yields. The DERA gene (PaDERA) was identified, and both the wild-type and a C49M mutant were heterologously expressed in Escherichia coli. The purification protocol was optimized and an initial biochemical characterization was conducted. Unlike other DERAs, which show a maximal activity between pH 4.0 and 7.5, PaDERA presented an optimum pH in the alkaline range between 8.0 and 9.0. This could warrant its use for specific syntheses in the future. PaDERA also displayed fourfold higher specific activity than DERA from E. coli (EcDERA) and displayed a promising acetaldehyde resistance outside the whole-cell environment. The C49M mutation, which was previously identified to increase acetaldehyde tolerance in EcDERA, also led to significant improvements in the acetaldehyde tolerance of PaDERA.ChemE/Product and Process EngineeringBT/Biocatalysi
Unseeded Elastomeric Single Leaflets Retain Function and Remodel After Implant In Ovine Pulmonary Outflow Tract
No full textCurrent materials for heart valve replacement and repair are limited by the inability to grow or remodel. Tissue engineered valves offer the potential to overcome these disadvantages by creating living structures, but is limited by the availability of biocompatible scaffold materials with desirable biomechanical properties. We assessed the in vivo performance of a novel scaffold poly(carbonate urethane) urea (PCUU), fabricated by electrospinning and implanted in the pulmonary outflow tract of sheep. PCUU was electrospun into elastomeric sheets of thickness ranging from 120-180 μm. Using cardiopulmonary bypass we replaced the native anterior pulmonary leaflet with an acellular PCUU leaflet. Valve function was evaluated by epicardial echocardiography at implant and explant at weeks 1 (n=3), 3 (n=3), 6 (n=3) and 16 (n=3). Histological, immunohistochemical, molecular imaging analyses and multi-photon imaging were performed on the explanted leaflets. Echocardiography demonstrated mobile functioning leaflets, with zero to mild pulmonary regurgitation. Molecular imaging showed increased levels of proteolytic activity and macrophage accumulation. Histology showed persistence of scaffold material up to 16 weeks with cellular infiltration throughout the leaflet. Picrosirius red revealed mature collagen deposition along the arterial surface of the construct at 6 and 16 weeks. These findings were corroborated by multi-photon analysis showing highly aligned collagen fibers across the leaflets. Both surfaces of the engineered leaflets were consistently covered with CD31 positive cells. The majority of cells expressed α-SMA and MMP2. CD45 positive cells, suggesting hematogenous origin, were found throughout the leaflet. These results suggest that: 1) PCUU can be a suitable polymer for valve bioengineering; 2) cell pre-seeding may not be required for tissue formation or remodeling for a functional engineered valve; 3) host cells seem to populate the leaflet either by migration from adjacent tissue or by attachment from circulating blood; 4) mature matrix orientation and increased proteolytic activity suggests active tissue remodeling. Longer term implants and the role of scaffold pre-seeding will require further study
Mesenchymal stromal cells improve survival during sepsis in the absence of heme oxygenase-1: the importance of neutrophils
No full textThe use of mesenchymal stromal cells (MSCs) for treatment of bacterial infections, including systemic processes like sepsis, is an evolving field of investigation. This study was designed to investigate the potential use of MSCs, harvested from compact bone, and their interactions with the innate immune system, during polymicrobial sepsis induced by cecal ligation and puncture (CLP). We also wanted to elucidate the role of endogenous heme oxygenase (HO)-1 in MSCs during a systemic bacterial infection. MSCs harvested from the bones of HO-1 deficient (-/-) and wild-type (+/+) mice improved the survival of HO-1(-/-) and HO-1(+/+) recipient mice when administered after the onset of polymicrobial sepsis induced by CLP, compared with the administration of fibroblast control cells. The MSCs, originating from compact bone in mice, enhanced the ability of neutrophils to phagocytize bacteria in vitro and in vivo and to promote bacterial clearance in the peritoneum and blood after CLP. Moreover, after depleting neutrophils in recipient mice, the beneficial effects of MSCs were entirely lost, demonstrating the importance of neutrophils for this MSC response. MSCs also decreased multiple organ injury in susceptible HO-1(-/-) mice, when administered after the onset of sepsis. Taken together, these data demonstrate that the beneficial effects of treatment with MSCs after the onset of polymicrobial sepsis is not dependent on endogenous HO-1 expression, and that neutrophils are crucial for this therapeutic response
Modified CD4 + t-cell response in recipients of old cardiac allografts
No full textWith an increasing demand, organs from elderly donors are more frequently utilized for transplantation. Herein, we analyzed the impact of donor age on CD4 + T-cell responses with regard to regulatory and effector mechanisms. Young (3 months) BM12 recipients were engrafted with young or old (18 months) B6 cardiac allografts. Systemic CD4 + T-cell responses and intragraft changes were monitored and compared to age-matched syngenic transplant controls. While elderly, nonmanipulated hearts contained significantly elevated frequencies of donor-derived leukocytes prior to transplantation, allograft survival was age-independent. T-cell activation, however, was delayed and associated with a compromised immune response in mixed lymphocyte cultures (MLR; P = 0.0002) early after transplantation (day 14). During the time course after transplantation, recipients of old grafts demonstrated an augmented immune response as shown by significantly higher frequencies of activated CD4 + T-cells and a stronger in vitro alloreactivity (MLR; ELISPOT; P < 0.01). In parallel, frequencies of regulatory T-cells had increased systemically and overall fewer CD4 + T-cells were detected intragraft. Interestingly, changes in the CD4 + T-cell response were not reflected by graft morphology. Of note, transplantation of young and old syngenic hearts did not show age-related differences of the CD4 + T-cells response suggesting that old grafts can recover from a period of short cold ischemia time. Our data suggest that donor age is associated with an augmented CD4 + T-cells response which did not affect graft survival in our model. These findings contribute to a better understanding of the immune response following the engraftment of older donor organs
TIM-3 : a novel regulatory molecule of alloimmune activation
No full textT cell Ig domain and mucin domain (TIM)-3 has previously been established as a central regulator of Th1 responses and immune tolerance. In this study, we examined its functions in allograft rejection in a murine model of vascularized cardiac transplantation. TIM-3 was constitutively expressed on dendritic cells and natural regulatory T cells (Tregs) but only detected on CD4 +FoxP3- and CD8+ T cells in acutely rejecting graft recipients. A blocking anti-TIM-3 mAb accelerated allograft rejection only in the presence of host CD4+ T cells. Accelerated rejection was accompanied by increased frequencies of alloreactive IFN-γ-, IL-6-, and IL-17-producing splenocytes, enhanced CD8+ cytotoxicity against alloantigen, increased alloantibody production, and a decline in peripheral and intragraft Treg/effector T cell ratio. Enhanced IL-6 production by CD4 + T cells after TIM-3 blockade plays a central role in acceleration of rejection. Using an established alloreactivity TCR transgenic model, blockade of TIM-3 increased allospecific effector T cells, enhanced Th1 and Th17 polarization, and resulted in a decreased frequency of overall number of allospecific Tregs. The latter is due to inhibition in induction of adaptive Tregs rather than prevention of expansion of allospecific natural Tregs. In vitro, targeting TIM-3 did not inhibit nTreg-mediated suppression of Th1 alloreactive cells but increased IL-17 production by effector T cells. In summary, TIM-3 is a key regulatory molecule of alloimmunity through its ability to broadly modulate CD4+ T cell differentiation, thus recalibrating the effector and regulatory arms of the alloimmune response
A Blood-Resistant Surgical Glue for Minimally Invasive Repair of Vessels and Heart Defects
No full textCurrently, there are no clinically approved surgical glues that are nontoxic, bind strongly to tissue, and work well
withinwet and highly dynamic environments within the body. This is especially relevant tominimally invasive surgery
that is increasingly performed to reduce postoperative complications, recovery times, and patient discomfort. We
describe the engineering of a bioinspired elastic and biocompatible hydrophobic light-activated adhesive (HLAA) that
achieves a strong level of adhesion to wet tissue and is not compromised by preexposure to blood. The HLAA
provided an on-demand hemostatic seal, within seconds of light application, when applied to high-pressure large
blood vessels and cardiac wall defects in pigs. HLAA-coated patches attached to the interventricular septum in a
beating porcine heart and resisted supraphysiologic pressures by remaining attached for 24 hours, which is relevant
to intracardiac interventions in humans. The HLAA could be used for many cardiovascular and surgical applications,
with immediate application in repair of vascular defects and surgical hemostasis
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