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Journal of Orthopaedic Surgery and... Jun 2020Joints withstand huge forces, but little is known about subchondral pressures and perfusion during loading. We developed an in vitro calf foot model to explore...
BACKGROUND
Joints withstand huge forces, but little is known about subchondral pressures and perfusion during loading. We developed an in vitro calf foot model to explore intraosseous pressure (IOP) and subchondral perfusion during weight bearing.
METHODS
Freshly culled calf forefeet were perfused with serum. IOP was measured at three sites in the foot using intraosseous needles, pressure transducers, and digital recorders. IOP was measured during perfusion, with and without a tourniquet and with differing weights, including static loading and dynamic loading to resemble walking.
RESULTS
IOP varied with perfusion pressure. Static loading increased subchondral IOP whether the bone was non-perfused, perfused, or perfused with a proximal venous tourniquet (p < 0.0001). Under all perfusion states, IOP was proportional to the load (R = 0.984). Subchondral IOP often exceeded perfusion pressure. On removal of a load, IOP fell to below the pre-load value. Repetitive loading led to a falling IOP whether the foot was perfused or not.
CONCLUSION
Superimposed on a variable background IOP, increased perfusion and physiological loading caused a significant increase in subchondral IOP. Force was thereby transmitted through subchondral bone partly by hydraulic pressure. A falling IOP with repeat loading suggests that there is an intraosseous one-way valve. This offers a new understanding of subchondral perfusion physiology.
Topics: Animals; Ankle Joint; Biomechanical Phenomena; Cattle; Foot Joints; In Vitro Techniques; Male; Perfusion; Pressure; Serum; Transducers, Pressure; Weight-Bearing
PubMed: 32600340
DOI: 10.1186/s13018-020-01754-y -
Journal of Visualized Experiments : JoVE Jun 2023Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are...
Porcine models of liver ex situ normothermic machine perfusion (NMP) are increasingly being used in transplant research. Contrary to rodents, porcine livers are anatomically and physiologically close to humans, with similar organ size and bile composition. NMP preserves the liver graft at near-to-physiological conditions by recirculating a warm, oxygenated, and nutrient-enriched red blood cell-based perfusate through the liver vasculature. NMP can be used to study ischemia-reperfusion injury, preserve a liver ex situ before transplantation, assess the liver's function prior to implantation, and provide a platform for organ repair and regeneration. Alternatively, NMP with a whole blood-based perfusate can be used to mimic transplantation. Nevertheless, this model is labor-intensive, technically challenging, and carries a high financial cost. In this porcine NMP model, we use warm ischemic damaged livers (corresponding to donation after circulatory death). First, general anesthesia with mechanical ventilation is initiated, followed by the induction of warm ischemia by clamping the thoracic aorta for 60 min. Cannulas inserted in the abdominal aorta and portal vein allow flush-out of the liver with cold preservation solution. The flushed-out blood is washed with a cell saver to obtain concentrated red blood cells. Following hepatectomy, cannulas are inserted in the portal vein, hepatic artery, and infra-hepatic vena cava and connected to a closed perfusion circuit primed with a plasma expander and red blood cells. A hollow fiber oxygenator is included in the circuit and coupled to a heat exchanger to maintain a pO2 of 70-100 mmHg at 38 °C. NMP is achieved by a continuous flow directly through the artery and via a venous reservoir through the portal vein. Flows, pressures, and blood gas values are continuously monitored. To evaluate the liver injury, perfusate and tissue are sampled at predefined time points; bile is collected via a cannula in the common bile duct.
Topics: Humans; Swine; Animals; Organ Preservation; Liver; Liver Transplantation; Perfusion; Warm Ischemia
PubMed: 37358290
DOI: 10.3791/65336 -
Nature Protocols Jun 2021As engineered tissues progress toward therapeutically relevant length scales and cell densities, it is critical to deliver oxygen and nutrients throughout the tissue...
As engineered tissues progress toward therapeutically relevant length scales and cell densities, it is critical to deliver oxygen and nutrients throughout the tissue volume via perfusion through vascular networks. Furthermore, seeding of endothelial cells within these networks can recapitulate the barrier function and vascular physiology of native blood vessels. In this protocol, we describe how to fabricate and assemble customizable open-source tissue perfusion chambers and catheterize tissue constructs inside them. Human endothelial cells are seeded along the lumenal surfaces of the tissue constructs, which are subsequently connected to fluid pumping equipment. The protocol is agnostic with respect to biofabrication methodology as well as cell and material composition, and thus can enable a wide variety of experimental designs. It takes ~14 h over the course of 3 d to prepare perfusion chambers and begin a perfusion experiment. We envision that this protocol will facilitate the adoption and standardization of perfusion tissue culture methods across the fields of biomaterials and tissue engineering.
Topics: Endothelial Cells; Humans; Perfusion; Tissue Engineering
PubMed: 34031610
DOI: 10.1038/s41596-021-00533-1 -
Perfuse and Reuse: A Low-Cost Three-Dimensional-Printed Perfusion Bioreactor for Tissue Engineering.Tissue Engineering. Part C, Methods Nov 2022This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow...
This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow centimeter-scale vascularized or otherwise canalized tissue constructs to be maintained in weeks long static and/or perfusion culture at an exceptionally low cost, with intermittent live imaging and media sampling capabilities. The perfusion system includes a reusable polydimethylsiloxane (PDMS) lid generated from a three-dimensional (3D)-printed poly-lactic acid (PLA) mold and several lengths of perfusion tubing. The coverslip tissue culture chamber includes PDMS components built with 3D-printed PLA molds, as well as 3D-printed PLA frames and glass coverslips that house perfusable hydrogel constructs. As proof of concept, we fabricated a vascularized hydrogel construct, which was subjected to static and perfusion tissue culture, as well as flow studies using fluorescent beads and widefield fluorescent microscopy. This system can be readily reproduced, promoting the advancement of tissue engineering and regenerative medicine research.
Topics: Tissue Engineering; Perfusion; Bioreactors; Hydrogels; Polyesters; Printing, Three-Dimensional; Tissue Scaffolds
PubMed: 36094108
DOI: 10.1089/ten.TEC.2022.0139 -
EBioMedicine Dec 2023Normothermic regional perfusion (NRP) and hypothermic-oxygenated-perfusion (HOPE), were both shown to improve outcomes after liver transplantation from donors after...
BACKGROUND
Normothermic regional perfusion (NRP) and hypothermic-oxygenated-perfusion (HOPE), were both shown to improve outcomes after liver transplantation from donors after circulatory death (DCD). Comparative clinical and mechanistical studies are however lacking.
METHODS
A rodent model of NRP and HOPE, both in the donor, was developed. Following asystolic donor warm ischemia time (DWIT), the abdominal compartment was perfused either with a donor-blood-based-perfusate at 37 °C (NRP) or with oxygenated Belzer-MPS at 10 °C (donor-HOPE) for 2 h. Livers were then procured and underwent 5 h static cold storage (CS), followed by transplantation. Un-perfused and HOPE-treated DCD-livers (after CS) and healthy livers (DBD) with direct implantation after NRP served as controls. Endpoints included the entire spectrum of ischemia-reperfusion-injury.
FINDINGS
Healthy control livers (DBD) showed minimal signs of inflammation during 2 h NRP and achieved 100% posttransplant recipient survival. In contrast, DCD livers with 30 and 60 min DWIT suffered from greater mitochondrial injury and inflammation as measured by increased perfusate Lactate, FMN- and HMGB-1-levels with subsequent Toll-like-receptor activation during NRP. In contrast, donor-HOPE (instead of NRP) led to significantly less mitochondrial-complex-I-injury and inflammation. Results after donor-HOPE were comparable to ex-situ HOPE after CS. Most DCD-liver recipients survived when treated with one HOPE-technique (86%), compared to only 40% after NRP (p = 0.0053). Following a reduction of DWIT (15 min), DCD liver recipients achieved comparable survivals with NRP (80%).
INTERPRETATION
High-risk DCD livers benefit more from HOPE-treatment, either immediately in the donor or after cold storage. Comparative prospective clinical studies are required to translate the results.
FUNDING
Funding was provided by the Swiss National Science Foundation (grant no: 32003B-140776/1, 3200B-153012/1, 320030-189055/1, and 31IC30-166909) and supported by University Careggi (grant no 32003B-140776/1) and the OTT (grant No.: DRGT641/2019, cod.prog. 19CT03) and the Max Planck Society. Work in the A.G. laboratory was partially supported by the NIH R01NS112381 and R21NS125466 grants.
Topics: Animals; Humans; Liver Transplantation; Rodentia; Prospective Studies; Perfusion; Graft Survival; Organ Preservation; Liver; Tissue Donors; Inflammation
PubMed: 37924707
DOI: 10.1016/j.ebiom.2023.104861 -
Annals of Transplantation Aug 2021A shortage of available organs for liver transplantation has led transplant surgeons and researchers to seek for innovative approaches in hepatoprotection and... (Review)
Review
A shortage of available organs for liver transplantation has led transplant surgeons and researchers to seek for innovative approaches in hepatoprotection and improvement of marginal allografts. The most exciting development in the past decade has been continuous mechanical perfusion of livers with blood or preservation solution to mitigate ischemia-reperfusion injury in contrast to the current standard of static cold storage. Two variations of machine perfusion have emerged in clinical practice. During hypothermic oxygenated perfusion the liver is perfused using a red blood cell-free perfusate at 2-10°C. In contrast, normothermic machine perfusion mimics physiologic liver perfusion using a red blood cell-based solution at 35.5-037.5°C, offering a multitude of potential advantages. Putative effects of normothermic perfusion include abrogation of hyperfibrinolysis after reperfusion and inflammation, glycogen repletion, and regeneration of adenosine triphosphate. Research in normothermic machine perfusion focuses on development of biomarkers predicting allograft quality and susceptibility to ischemia-reperfusion injury. Moreover, normothermic perfusion of marginal allografts allows for application of a variety of therapeutic interventions potentially enhancing organ quality. Both methods need to be subjected to translational investigation and evaluation in clinical trials. A clear advantage is transformation of an emergency procedure at night into a planned daytime surgery. Current clinical trials suggest that normothermic perfusion not only increases the use of hepatic allografts but is also associated with milder ischemia-reperfusion injury, resulting in a reduced risk of early allograft dysfunction and less biliary complications, including ischemic cholangiopathy, compared to static cold storage. The aim of this review is to give a concise overview of normothermic machine perfusion and its current applications, benefits, and possible advances in the future.
Topics: Aged; Biomarkers; Humans; Liver; Liver Transplantation; Organ Preservation; Perfusion; Reperfusion Injury
PubMed: 34426566
DOI: 10.12659/AOT.931664 -
Journal of Neuroscience Methods Apr 2022Histology on fixed brain tissue is a key technique to investigate the pathophysiology of neurological disorders. Best results are obtained by perfusion fixation,...
INTRODUCTION
Histology on fixed brain tissue is a key technique to investigate the pathophysiology of neurological disorders. Best results are obtained by perfusion fixation, however, multiple protocols are available and so far the optimal perfusion pressure (PP) for the preservation of brain tissue while also maintaining vascular integrity is not defined. Therefore, the aim of our study was to investigate the effect of different PPs on the cerebral vasculature and to define the PP optimal for the preservation of both vascular integrity and tissue fixation.
MATERIAL AND METHODS
Male C57Bl6 mice, 8 weeks old, were perfused with PPs of 50/125/300 mmHg (series I) or 50/100/150/300 mmHg (series II). In series I, vascular integrity, e.g. BBB permeability, vessel diameter, and occurrence of vasospasms were investigated by spectrophotometry, light-sheet and 2-photon microscopy, respectively. In series II, we investigated vascular and neuronal artifacts and the occurrence of hemorrhage or microthrombi by light microscopy.
RESULTS
While a PP below the physiological systolic blood pressure results in the collapse of parenchymal vessels and formation of microvasospasms and microclots, a PP above the physiological systolic blood pressure dilates cerebral vessels, induces microvasospasms and disrupts the BBB. In terms of tissue integrity, our results confirm that higher PPs lead to fewer artifacts such as dark neurons or perivascular courts.
CONCLUSION
Our study demonstrates that the PP critically affects both vascular and tissue integrity in brain tissue preserved by perfusion fixation. A PP between 125 and 150 mmHg is optimal for the preservation of the cerebral vasculature and neuronal structures.
Topics: Animals; Blood-Brain Barrier; Brain; Male; Mice; Mice, Inbred C57BL; Neurons; Perfusion; Tissue Fixation
PubMed: 35151669
DOI: 10.1016/j.jneumeth.2022.109493 -
Veterinary Journal (London, England :... Jul 2022The endothelial glycocalyx (eGlx) lines the luminal surface of endothelial cells. It is critical in maintaining vascular health and when damaged contributes to many...
The endothelial glycocalyx (eGlx) lines the luminal surface of endothelial cells. It is critical in maintaining vascular health and when damaged contributes to many diseases. Its fragility makes studying the eGlx technically challenging. The current reference standard for eGlx visualisation, by electron microscopy using glutaraldehyde/Alcian blue perfusion fixation, has not been previously reported in dogs. Established techniques were applied to achieve visualisation of the eGlx in the microvasculature of reproductive tissue in five healthy dogs undergoing elective neutering. Uterine and testicular artery samples underwent perfusion fixation, in the presence of Alcian blue, prior to transmission electron microscopy imaging. Image processing software was used to determine eGlx depth. EGlx was visualised in the arteries of two dogs, one testicular and one uterine, with median (range) eGlx depths of 68.2 nm (32.1-122.9 nm) and 47.6 nm (26.1-129.4 nm) respectively. Study of the eGlx is technically challenging, particularly its direct visualisation in clinical samples. Further research is needed to develop more clinically applicable techniques to measure eGlx health.
Topics: Alcian Blue; Animals; Dogs; Endothelial Cells; Glycocalyx; Perfusion
PubMed: 35640795
DOI: 10.1016/j.tvjl.2022.105844 -
The Journal of Thoracic and... Sep 2022
Topics: Cardiopulmonary Bypass; Goals; Humans; Perfusion
PubMed: 33454092
DOI: 10.1016/j.jtcvs.2020.12.075 -
ACS Nano Nov 2021Targeted delivery of therapeutics through the use of nanoparticles (NPs) has emerged as a promising method that increases their efficacy and reduces their side effects.... (Review)
Review
Targeted delivery of therapeutics through the use of nanoparticles (NPs) has emerged as a promising method that increases their efficacy and reduces their side effects. NPs can be tailored to localize to selective tissues through conjugation to ligands that bind cell-specific receptors. Although the vast majority of nanodelivery platforms have focused on cancer therapy, efforts have begun to introduce nanotherapeutics to the fields of immunology as well as transplantation. In this review, we provide an overview from a clinician's perspective of current nanotherapeutic strategies to treat solid organ transplants with NPs during the time interval between organ harvest from the donor and placement into the recipient, an innovative technology that can provide major benefits to transplant patients. The use of normothermic machine perfusion (NMP), which is associated with preserving the function of the organ following transplantation, also provides an ideal opportunity for a localized, sustained, and controlled delivery of nanotherapeutics to the organ during this critical time period. Here, we summarize previous endeavors to improve transplantation outcomes by treating the organ with NPs prior to placement in the recipient. Investigations in this burgeoning field of research are promising, but more extensive studies are needed to overcome the physiological challenges to achieving effective nanotherapeutic delivery to transplanted organs discussed in this review.
Topics: Humans; Organ Preservation; Perfusion; Transplants; Organ Transplantation
PubMed: 34714050
DOI: 10.1021/acsnano.1c04707