Continuous Perfusion Research Articles

A successful case of continuous local antibiotics perfusion for osteomyelitis after osteosynthesis

A successful case of continuous local antibiotics perfusion for osteomyelitis after osteosynthesis

A Lyapunov based heuristic to speed up convergence of a feedback optimization framework with experiment batches-application to bioprocess manufacturing

In this work a heuristic to speed up the convergence of a feedback-based optimization scheme, when experiments can be run in batches, is discussed. The proposed approach allows to select the most promising experiment in the batch, as the one maximising the decrease of an associated Lyapunov function, and to define the inputs for the next batch, based on this. We suggest the application of the scheme to a biological setting, with the goal of maximizing the concentration of a product of interest in a bioreactor under a continuous perfusion framework, while at the same time minimizing the yield of a toxic byproduct. The potential of the approach is exposed by means of a simple synthetic example.

Illustrated Technique of “Branch-First” Total Aortic Arch Replacement

Technical aspects of aortic arch replacement have evolved over the years with significant focus on reducing the risk of operative morbidity and mortality. Recent developments in surgical methods relate to optimizing strategies for neuroprotection, distal organ perfusion and myocardial protection. We describe the branch-first technique for aortic arch replacement using a trifurcation graft with a side perfusion port (TAPP). It simplifies the delivery of continuous antegrade cerebral perfusion, and takes advantage of intracranial and extracranial networks to augment contralateral cerebral perfusion. Consequently, it allows for moderate levels of hypothermia and avoids distal circulatory arrest in many cases. In cases where distal circulatory arrest is required, it affords a longer safe duration of distal arrest and allows aortic pathology to be completely and meticulously corrected without time pressures.

Two pumps or one pump? A comparison of human liver normothermic machine perfusion devices for transplantation.

Normothermic machine perfusion provides continuous perfusion to ex situ hepatic grafts through the portal vein and the hepatic artery. Because the portal vein has high flow with low pressure and the hepatic artery has low flow with high pressure, different types of perfusion machines have been employed to match the two vessels' infusion hemodynamics. We compared transplanted human livers perfused through a 2-pump (n=9) versus a 1-pump perfusion system (n=6) where a C-clamp is used as a tubing constrictor to regulate hemodynamics. There was no significant difference between groups in portal vein or hepatic artery flow rate. The 1-pump group had more hemoglobin in the perfusate. However, there was no significant difference in plasma hemoglobin between the 2-pump and 1-pump groups at each time point or in the change in levels, proving no hemolysis occurred due to C-clamp tube constriction. After transplantation, the 2-pump group had two cases of early allograft dysfunction (EAD), whereas the 1-pump group had no EAD. There was no graft failure or patient death in either group during follow-up ranging from 20-52months. Our data show that the 1-pump design provided the same hemodynamic output as the 2-pump design, with no additional hemolytic risk, but with the benefits of lower costs, easier transport and faster and simpler setting.

The use of ex-vivo liver perfusion circuit in sheep model: Preliminary report.

Objectives:To describe a novel animal model for ex-vivo liver perfusion.Methods:This study was carried out at King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, between September 2016 and January 2019. We assembled a perfusion circuit operated by a continuous pressure-driven arterial pump with continuous portal and arterial pressure and volume measurements. We used normothermic oxygenated perfusate. The livers used were retrieved from the sheep.Results:Ex-vivo continuous perfusion of the liver was achieved for up to 9 hours with stable pressure and volume in both hepatic artery and portal vein. In 4 experiments the arterial pressure was kept in a range of 48-52 mmHg with a mean of 51.75±4.31 resulting in arterial volume at steady state of 223.5±48.25 ml/minute (95% confidence level). At steady state the mean portal pressure was 16.25±1.45 mmHg with a mean volume of 854±313.75 ml/minute (95% confidence level). Bile production was observed during the perfusion period. Hemodynamic parameters were similar to the physiological parameters observed in normothermic perfusion model of the porcine liver.Conclusion:A normothermic oxygenated ex-vivo perfusion circuit was successfully constructed using the sheep liver. A sustainable functional circuit with physiological hemodynamic parameters was achieved. Further study on sheep model seems to be feasible.

Human Organs‐on‐Chips: A Review of the State‐of‐the‐Art, Current Prospects, and Future Challenges

New emerging technologies, remarkably miniaturized 3D organ models and microfluidics, enable simulation of the real in vitro microenvironment ex vivo more closely. There are many fascinating features of innovative organ-on-a-chip (OOC) technology, including the possibility of integrating semipermeable and/or stretchable membranes, creating continuous perfusion of fluids into microchannels and chambers (while maintaining laminar flow regime), embedding microdevices like microsensors, microstimulators, micro heaters, or different cell lines, along with other 3D cell culture technologies. OOC systems are designed to imitate the structure and function of human organs, ranging from breathing lungs to beating hearts. This technology is expected to be able to revolutionize cell biology studies, personalized precision medicine, drug development process, and cancer diagnosis/treatment. OOC systems can significantly reduce the cost associated with tedious drug development processes and the risk of adverse drug reactions in the body, which makes drug screening more effective. The review mainly focus on presenting an overview of the several previously developed OOC systems accompanied by subjects relevant to pharmacy-, cancer-, and placenta-on-a-chip. The challenging issues and opportunities related to these systems are discussed, along with a future perspective for this technology.

Intraoperative adenosine for pheochromocytoma with myocardial infarction and SARS-CoV-2. Case report

Introduction: Pheochromocytomas are neuroendocrine tumors capable of synthetizing, storing and releasing catecholaminergic hormones that may lead to life-threatening hemodynamic instability. The COVID-19 pandemic has increased the risks and perioperative complexity of the patients undergoing pheochromocytoma-associated adrenalectomy. This article discusses the use of adenosine for the management of hypertensive crisis during this intervention, as well as the need to individualize the suitable timing for surgery after recent COVID-19 infection.
 Case presentation: This article discusses the case of a patient with a finding of right adrenal incidentaloma; further studies determined a metanephrines secreting pheochromocytoma. Following hospital admission for preoperative optimization, the eve of the procedure the patient developed an acute myocardial infarction and subsequently SARS-CoV-2 symptomatic infection. Intraoperatively, hypertensive peaks were managed with continuous adenosine perfusion. The patient was discharged after 48 hours.
 Conclusions: Preoperative optimization positively influences the intraoperative management of patients with pheochromocytoma. The intraoperative use of adenosine allows for adequate and safe control of hypertensive crises. Each situation must be individualized in patients pending surgery, with a recent COVID-19 infection.

Non-Standard Management of Target Vessels With the Inner Branch Arch Endograft: A Single-Center Retrospective Study

Purpose: The purpose of this study was to evaluate early and mid-term results of non-standard management of the supraaortic target vessels with the use of the inner branch arch endograft in a single high-volume center. Material and methods: A single-center retrospective study including all patients undergoing implantation of an inner branch arch endograft from December 2012 to March 2021, who presented a non-standard management of the supraaortic target vessels (any bypass other than a left carotid-subclavian or landing in a dissected target vessel). Technical success, mortality, reinterventions, endoleak (EL), and aortic remodeling at follow-up were analyzed. Results: Twenty-four patients were included. In 17 (71%) cases, the non-standard management was related to innominate artery (IA) compromise (12 with IA dissection, 2 with short IA, 2 with short proximal aortic landing zone that required occlusion of IA, 1 with occluded IA after open arch repair). Two (8%) cases were related to an aberrant right subclavian artery (RSA), 1 patient (4%) due to the concomitant presence of a left vertebral artery (LVA) arising from the arch and an occluded left subclavian artery (LSA), and another patient presented with an occluded LSA distal to a dominant vertebral artery. Three (13%) cases were exclusively related to management in patients with genetic aortic syndromes. Twenty (83%) patients had a previous type A aortic dissection. Ten (42%) patients presented a thoracic or thoracoabdominal aortic aneurysm and 8 (33%) patients an arch aneurysm, 6 of them associated to false lumen (FL) perfusion. There were 2 (8%) perioperative minor strokes, and 1 patient with perioperative mortality. Seven patients presented an early type I endoleak, all resolved at follow-up. Seven patients required reinterventions during follow-up (7 reinterventions related to continuous false lumen perfusion, 3 related to Type Ia endoleak, 2 related to surgical bypass). All patients who presented with FL perfusion had complete FL thrombosis at follow-up. No patient presented aneurysm growth at follow-up. Conclusions: The use of the inner branch arch endograft with a non-standard management of the supraaortic target vessels is a possible option. Despite a high reintervention rate, regression or stability of the aneurysmal diameter was achieved in all the patients with follow-up.

COMPLETE REPAIR OF TETRALOGY OF FALLOT (TOF) ON BEATING HEART SURGERY WITHOUT AORTIC CROSS CLAMPING : REPORT OF A CASE

Introduction: Reperfusion injury is a well-known phenomenon that occurred in cardioplegic techniques with cardiopulmonary bypass. Therefore great effort is made to prevent reperfusion injury. Beating Heart Continuous Coronary Perfusion (BHCCP) surgery is one of the alternative techniques to improve an ischemic reperfusion injury in open-heart surgery, either in pediatric or in adult. This technique can be done in complete repair of tetralogy of Fallot (TOF). Purpose: To report our first experience in doing complete repair of TOF using beating heart technique without aortic cross-clamping Case Report: A five-year-old boy came on to ER on January 6th, 2012, with a history of cyanotic since birth. His echocardiography and catheterization concluded a tetralogy of Fallot with a McGoon ratio of 1.95 (the diameter of RPA and LPA were 9.14 mm and 8.79 mm, respectively). A complete repair of TOF was done using beating heart surgery without aortic cross-clamping technique. One month post-surgery evaluation, there weren't any neurological or motor disorders. Echocardiography results showed no residual pulmonary stenosis, but 0.2 mm residual VSD was found. Conclusion: This heart rate technique is an alternative method of myocardial protection in cardiac surgery. In several cardiac centres globally, this technique is relatively safe and can be used during surgery, especially in the total correction of TOF.

Uterine artery pseudoaneurysm after treatment of cesarean scar pregnancy: a case report

BackgroundPseudoaneurysms are formed when a local arterial wall ruptures, leading to hemorrhage and hematoma adjacent to the artery. Continuous perfusion of the injured artery increases the pressure in the lumen of the pseudoaneurysm. It may rupture and lead to massive hemorrhage that could be life-threatening. Cesarean scar pregnancy (CSP) is an ectopic pregnancy where the gestational sac is implanted in the cesarean scar. Uterine artery pseudoaneurysm (UAP) after CSP treatment is rare.Case presentationWe report the case of a 36-year-old Chinese woman who presented with acute massive vaginal bleeding 53 days after transabdominal scar pregnancy excision. Doppler ultrasound confirmed UAP. Selective uterine artery embolization (UAE) failed because of the thin and curved blood vessels. The lesion decreased in size after transvaginal ultrasound-guided direct thrombin injection (UGTI); however, massive vaginal bleeding recurred and endangered the patient’s life. The uterus was removed thereafter.ConclusionsUAP is a rare complication after CSP treatment that can lead to fatal massive hemorrhage. Ultrasound should be reexamined regularly after treatment of CSP. In case of unexplained vaginal bleeding, we should be alert to the existence of UAP and the possibility of rupture and take effective diagnosis and treatment measures promptly.

Continuous gastric saline perfusion elicits cardiovascular responses in freshwater rainbow trout (Oncorhynchus mykiss)

When in seawater, rainbow trout (Oncorhynchus mykiss) drink to avoid dehydration and display stroke volume (SV) mediated elevations in cardiac output (CO) and an increased proportion of CO is diverted to the gastrointestinal tract as compared to when in freshwater. These cardiovascular alterations are associated with distinct reductions in systemic and gastrointestinal vascular resistance (RSys and RGI, respectively). Although increased gastrointestinal blood flow (GBF) is likely essential for osmoregulation in seawater, the sensory functions and mechanisms driving the vascular resistance changes and other associated cardiovascular changes in euryhaline fishes remain poorly understood. Here, we examined whether internal gastrointestinal mechanisms responsive to osmotic changes mediate the cardiovascular changes typically observed in seawater, by comparing the cardiovascular responses of freshwater-acclimated rainbow trout receiving continuous (for 4 days) gastric perfusion with half-strength seawater (½ SW, ~ 17 ppt) to control fish (i.e., no perfusion). We show that perfusion with ½ SW causes significantly larger increases in CO, SV and GBF, as well as reductions in RSys and RGI, compared with the control, whilst there were no significant differences in blood composition between treatments. Taken together, our data suggest that increased gastrointestinal luminal osmolality is sensed directly in the gut, and at least partly, mediates cardiovascular responses previously observed in SW acclimated rainbow trout. Even though a potential role of mechano-receptor stimulation from gastrointestinal volume loading in eliciting these cardiovascular responses cannot be excluded, our study indicates the presence of internal gastrointestinal milieu-sensing mechanisms that affect cardiovascular responses when environmental salinity changes.

Functional characterization of a bioengineered liver after heterotopic implantation in pigs

Organ bioengineering offers a promising solution to the persistent shortage of donor organs. However, the progression of this technology toward clinical use has been hindered by the challenges of reconstituting a functional vascular network, directing the engraftment of specific functional cell types, and defining appropriate culture conditions to concurrently support the health and phenotypic stability of diverse cell lineages. We previously demonstrated the ability to functionally reendothelialize the vasculature of a clinically scaled decellularized liver scaffold with human umbilical vein endothelial cells (HUVECs) and to sustain continuous perfusion in a large animal recovery model. We now report a method for seeding and engrafting primary porcine hepatocytes into a bioengineered liver (BEL) scaffold previously reendothelialized with HUVECs. The resulting BELs were competent for albumin production, ammonia detoxification and urea synthesis, indicating the presence of a functional hepatocyte compartment. BELs additionally slowed ammonia accumulation during in vivo perfusion in a porcine model of surgically induced acute liver failure. Following explant of the graft, BEL parenchyma showed maintenance of canonical endothelial and hepatocyte markers. Taken together, these results support the feasibility of engineering a clinically scaled functional BEL and establish a platform for optimizing the seeding and engraftment of additional liver specific cells.

Acute Kidney Injury After Neonatal Aortic Arch Surgery: Deep Hypothermic Circulatory Arrest Versus Moderate Hypothermia With Distal Aortic Perfusion.

Acute kidney injury (AKI) is a common complication observed after neonatal aortic arch repair. We studied its incidence after procedures carried out using deep hypothermic circulatory arrest (DHCA) versus moderate hypothermia with distal aortic perfusion (MHDP), usually through the common femoral artery. In both groups, continuous regional cerebral perfusion (RCP) was used during the time required for aortic arch repair. A total of 125 neonates underwent aortic arch repair. Between 2007 and 2012, DHCA with RCP was used in 51 neonates. From 2013 to 2019, MHDP with RCP was performed on 74 newborns. Operative complexity was similar in both periods. Acute kidney injury was defined as a significant elevation of serum creatinine and was classified according to the neonatal modified n-KDIGO (neonatal Kidney Disease: Improving Global Outcomes) stages 1 to 3 (Kidney Disease Improving: Global Outcomes). Acute kidney injury was observed in a total of 68 patients (68/125: 54.4%). In the majority (44/68: 64.7%), n-KDIGO stage 1 occurred. Stage 2 (n = 14) and stage 3 (n = 10) were observed more frequently after DHCA versus MHDP: 29.4% (15/51) versus 12.2% (9/74), P = .02. At cardiopulmonary bypass end, lactate levels were significantly higher (P = .001) after DHCA: 3.4 (2.9-4.3) mmol/L compared to 2.7 (2.3-3.7) mmol/L after MHDP. Early mortality was 12% (15/125) in the entire cohort. It was 17.6% (9/51) after DHCA versus 8.1% (6/74) after MHDP, however not statistically significant (P = .16). Mild (stage 1) AKI occurred frequently after neonatal aortic arch repair. The use of MHDP was associated with a significantly lower incidence of moderate (stage 2) and severe (stage 3) AKI forms.

Continuous local antibiotic perfusion for patients with surgical site infection after instrumented spinal surgery; a novel technique to retain the implants

ObjectivesTo describe the novel technique of continuous local antibiotic perfusion (CLAP) for a surgical site infection (SSI) after instrumented spinal surgery. MethodsCLAP was applied to 4 continuous patients at our institution who developed SSI after instrumented spinal surgery. ResultsAll 4 patients were successfully treated and the infection was controlled. The implant was retained in all patients. The duration of CLAP ranged from 2 to 3 weeks. The blood level of the antibiotic used (gentamicin) at 1 week after the initiation of CLAP did not increase in any patient. No other adverse events occurred in any patient. Dramatic improvements in laboratory parameters, including the white blood cell (WBC) counts and C-reactive protein (CRP) levels were seen in every patient at 1 week after the initiation of CLAP. ConclusionsCLAP controlled the infection without severe adverse events in all 4 patients, and the implants were retained. Despite its cost and the discomfort of patients, CLAP may become an option for the treatment of SSIs after instrumented spinal surgery. A large number of case series are needed to verify the efficacy of CLAP for patients with SSIs after instrumented spinal surgery.

The growth of xenotransplanted hearts can be reduced with growth hormone receptor knockout pig donors

ObjectiveGenetically engineered pigs are thought to be an alternative organ source for patients in end-stage heart failure unable to receive a timely allograft. However, cardiac xenografts exhibit growth and diastolic heart failure within 1 month after transplantation. Grafts function for up to 6 months, but only after administration of temsirolimus and afterload-reducing agents to reduce this growth. In this study we investigated the growth and hemodynamics of growth hormone receptor (GHR) knockout xenografts, without the use of adjuncts to prevent intrinsic graft growth after transplantation. MethodsGenetically engineered pig hearts were transplanted orthotopically into weight-matched baboons between 15 and 30 kg, using continuous perfusion preservation before implantation (n = 5). Xenografts included knockout of carbohydrate antigens and knockin of human transgenes for thromboregulation, complement regulation, and inflammation reduction (grafts with intact growth hormone, n = 2). Three grafts contained the additional knockout of GHR (GHR knockout grafts; n = 3). Transthoracic echocardiograms were obtained twice monthly and comprehensively analyzed by a blinded cardiologist. Hemodynamics were measured longitudinally after transplantation. ResultsAll xenografts demonstrated life-supporting function after transplantation. There was no difference in intrinsic growth, measured using septal and posterior wall thickness and left ventricular mass, on transthoracic echocardiogram out to 1 month in either GHR knockout or GHR intact grafts. However, hypertrophy of the septal and posterior wall was markedly elevated by 2 months post transplantation. There was minimal hypertrophy out to 6 months in GHR knockout grafts. Physiologic mismatch was present in all grafts after transplantation, which is largely independent of growth. ConclusionsXenografts with GHR knockout show reduced post-transplantation xenograft growth using echocardiography >6 months after transplantation, without the need for other adjuncts.

Intra-medullary antibiotics perfusion (iMAP) for the control of fracture-related infection early after osteosynthesis.

In our hospital, cases of bone and soft tissue infections have been treated with continuous local antibiotics perfusion that allows for continuous circulation of antibiotics throughout the infected lesion. We termed this treatment "intramedullary antibiotics perfusion (iMAP)" for bone infection such as fracture-related infection (FRI) and "intrasoft tissue antibiotics perfusion" for soft tissue infection. Many cases are treated with both modalities. To introduce iMAP, this study focused on the patients with FRI treated with iMAP and reviewed their treatment outcomes. We included 10 patients with FRI treated with iMAP between 2004 and 2017. The iMAP needles were inserted near the infected lesion, and an aminoglycoside antimicrobial was continuously administered. Patient characteristics, pathogenic bacteria, administered antibiotics, duration of administration, concentrations of antibiotics in blood and leachate fluid, fracture union rate, implant retention rate, and complications were studied. The mean age of patients was 59.9years, and the mean follow-up period was 2.5years. Affected bones were the tibia (n = 8), humerus (n = 1), and fibula (n = 1). Deep infections developed on average 29.9days after osteosynthesis. Pathogenic bacteria were methicillin-susceptible Staphylococcus aureus (n = 6), methicillin-resistant S. aureus (n = 2), and unknown (n = 2). Average iMAP duration was 17.1days. In all patients, infection was eradicated while preserving the implants, and fracture union was achieved without complications. iMAP is a novel local drug delivery system allowing high concentrations of antibiotics to be administered without complications and is useful in the treatment of FRI.

Modeling of the Human Bone Environment: Mechanical Stimuli Guide Mesenchymal Stem Cell-Extracellular Matrix Interactions.

In bone tissue engineering, the design of in vitro models able to recreate both the chemical composition, the structural architecture, and the overall mechanical environment of the native tissue is still often neglected. In this study, we apply a bioreactor system where human bone-marrow hMSCs are seeded in human femoral head-derived decellularized bone scaffolds and subjected to dynamic culture, i.e., shear stress induced by continuous cell culture medium perfusion at 1.7 mL/min flow rate and compressive stress by 10% uniaxial load at 1 Hz for 1 h per day. In silico modeling revealed that continuous medium flow generates a mean shear stress of 8.5 mPa sensed by hMSCs seeded on 3D bone scaffolds. Experimentally, both dynamic conditions improved cell repopulation within the scaffold and boosted ECM production compared with static controls. Early response of hMSCs to mechanical stimuli comprises evident cell shape changes and stronger integrin-mediated adhesion to the matrix. Stress-induced Col6 and SPP1 gene expression suggests an early hMSC commitment towards osteogenic lineage independent of Runx2 signaling. This study provides a foundation for exploring the early effects of external mechanical stimuli on hMSC behavior in a biologically meaningful in vitro environment, opening new opportunities to study bone development, remodeling, and pathologies.

Novel Human Podocyte Cell Model Carrying G2/G2 APOL1 High-Risk Genotype.

Apolipoprotein L1 (APOL1) high-risk genotypes (HRG), G1 and G2, increase the risk of various non-diabetic kidney diseases in the African population. To date, the precise mechanisms by which APOL1 risk variants induce injury on podocytes and other kidney cells remain unclear. Trying to unravel these mechanisms, most studies have used animal or cell models created by gene editing. We developed and characterised conditionally immortalised human podocyte cell lines derived from urine of a donor carrying APOL1 HRG G2/G2. Following induction of APOL1 expression by polyinosinic-polycytidylic acid (poly(I:C)), we assessed functional features of APOL1-induced podocyte dysfunction. As control, APOL1 wild type (G0/G0) podocyte cell line previously generated from a Caucasian donor was used. Upon exposure to poly(I:C), G2/G2 and G0/G0 podocytes upregulated APOL1 expression resulting in podocytes detachment, decreased cells viability and increased apoptosis rate in a genotype-independent manner. Nevertheless, G2/G2 podocyte cell lines exhibited altered features, including upregulation of CD2AP, alteration of cytoskeleton, reduction of autophagic flux and increased permeability in an in vitro model under continuous perfusion. The human APOL1 G2/G2 podocyte cell model is a useful tool for unravelling the mechanisms of APOL1-induced podocyte injury and the cellular functions of APOL1.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Pass through the Filtration Barrier and Protect Podocytes in a 3D Glomerular Model under Continuous Perfusion

Background:Dynamic cultures, characterized by continuous fluid reperfusion, elicit physiological responses from cultured cells. Mesenchymal stem cell-derived EVs (MSC-EVs) has been proposed as a novel approach in treating several renal diseases, including acute glomerular damage, by using traditional two-dimensional cell cultures and in vivo models. We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion.Methods:To this end, we set up a 3D glomerular model culturing human glomerular endothelial cells and podocytes inside a bioreactor on the opposite sides of a porous membrane coated with type IV collagen. The bioreactor was connected to a circuit that allowed fluid passage at the rate of 80 µl/min. To mimic glomerular damage, the system was subjected to doxorubicin administration in the presence of therapeutic MSC-EVs.Results:The integrity of the glomerular basal membrane in the 3D glomerulus was assessed by a permeability assay, demonstrating that the co-culture could limit the passage of albumin through the filtration barrier. In dynamic conditions, serum EVs engineered with cel-miR-39 passed through the glomerular barrier and transferred the exogenous microRNA to podocyte cell lines. Doxorubicin treatment increased podocyte apoptosis, whereas MSC-EV within the endothelial circuit protected podocytes from damage, decreasing cell death and albumin permeability.Conclusion:Using an innovative millifluidic model, able to mimic the human glomerular barrier, we were able to trace the EV passage and therapeutic effect in dynamic conditions.

QS4: Optimizing The Decellularization Of The Rodent Epigastric Free Flap: A Comparison Of Automated SDS-based Protocols

Purpose:Raising flaps to cover complex wounds with exposed critical structures are lengthy operations that result in donor site morbidity. Tissue engineering research is developing with great promise to build replacement tissues without morbidity. Decellularization removes whole cells and cell debris, and is the initial step to create a scaffold with an intact vascular network. Benchmark measurement of the overall cellular level is quantification of the DNA content, where 50 ng/mg is classically considered as a threshold. Although perfusion decellularization and recellularization approaches have shown exceptional promise in whole organ engineering, there is minimal crossover into the microsurgical field. Sodium dodecyl sulfate (SDS)-based protocols are known to have deleterious effects on the ultrastructure and capillary network of the scaffolds, but remain the predominant choice for decellularization protocols. This study aims to optimize the SDS exposure protocol for automated decellularization by comparing different SDS perfusion times to gain better understanding of the balance between decellularization and scaffold preservation.Methods:A 3D-printed closed-system bioreactor capable of continuously perfusing fluid throughout the vasculature was used for decellularization of free flaps. 2x2 cm fasciocutaneous free flaps from the epigastric region of the rat were harvested, and the vascular pedicle was isolated as a single artery and vein. Three flaps were evaluated in each group. The artery (inflow) and vein (outflow) were cannulated to monitor preservation of the vasculature. 1% SDS solution was perfused in different durations (3, 5 and 10 days) based on several protocols found in the literature. Automated SDS perfusion was followed by 1 day of 1% Triton X-100 and 1 day of 1xPhosphate-buffered saline (PBS), all of which were at the perfusion pressure of 120 mmHg.Results:For vasculature analysis, continual perfusion into the artery and out of the vein within the bioreactor was assessed throughout the decellularization process. H&E, Masson’s trichrome, and Verhoeff-Van Gieson staining were performed to assess architecture and locale of residual nuclei. Residual DNA was quantified by the fluorescent marker PicoGreen. 5 days of 1% SDS solution had the least residual DNA content (1.309±0.807 ng/mg) followed by 10 days (12.684±14.184 ng/mg) and 3 days (82.387±71.595 ng/mg), p<0.001. The DNA content ratio of skin over subcutaneous tissues was consistent across all protocols with skin having twice as much residual DNA after each protocol. The vascular network was visualized for qualitative assessment with the perfusion of hardening cast to create contrast against soft tissue to visualize with microCT imaging.Conclusion:A decellularization protocol of 5 days of 1% SDS solution followed by 1 day of 1% Triton X-100 and 1 day of 1xPBS was the most successful to keep the residual DNA content at a minimum, while preserving the structural integrity of the tissues. The bioreactor is capable of running automated and repeatable protocols using several solutions and continuously perfusing fluid throughout the vasculature of a free flap. This compact and integrated system can decrease hands-on time and be used in the future for further recellularization of scaffolds to bioengineer soft tissue replacement without donor site morbidity.