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Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Background: Various multimodal analgesic approaches have been proposed for spine surgery. The authors evaluated the effect of using a combination of four nonopioid analgesics versus placebo on Quality of Recovery, postoperative opioid consumption, and pain scores., Methods: Adults having multilevel spine surgery who were at high risk for postoperative pain were double-blind randomized to placebos or the combination of single preoperative oral doses of acetaminophen 1,000 mg and gabapentin 600 mg, an infusion of ketamine 5 µg/kg/min throughout surgery, and an infusion of lidocaine 1.5 mg/kg/h intraoperatively and during the initial hour of recovery. Postoperative analgesia included acetaminophen, gabapentin, and opioids. The primary outcome was the Quality of Recovery 15-questionnaire (0 to 150 points, with 15% considered to be a clinically important difference) assessed on the third postoperative day. Secondary outcomes were opioid use in morphine equivalents (with 20% considered to be a clinically important change) and verbal-response pain scores (0 to 10, with a 1-point change considered important) over the initial postoperative 48 h., Results: The trial was stopped early for futility per a priori guidelines. The average duration ± SD of surgery was 5.4 ± 2.1 h. The mean ± SD Quality of Recovery score was 109 ± 25 in the pathway patients (n = 150) versus 109 ± 23 in the placebo group (n = 149); estimated difference in means was 0 (95% CI, -6 to 6, P = 0.920). Pain management within the initial 48 postoperative hours was not superior in analgesic pathway group: 48-h opioid consumption median (Q1, Q3) was 72 (48, 113) mg in the analgesic pathway group and 75 (50, 152) mg in the placebo group, with the difference in medians being -9 (97.5% CI, -23 to 5, P = 0.175) mg. Mean 48-h pain scores were 4.8 ± 1.8 in the analgesic pathway group versus 5.2 ± 1.9 in the placebo group, with the difference in means being -0.4 (97.5% CI; -0.8, 0.1, P = 0.094)., Conclusions: An analgesic pathway based on preoperative acetaminophen and gabapentin, combined with intraoperative infusions of lidocaine and ketamine, did not improve recovery in patients who had multilevel spine surgery.

Stroke is the third leading cause of death and the leading cause of disability in contemporary society. Aneurysmal subarachnoid hemorrhage (aSAH) is a hemorrhagic stroke which accounts for 7% of all stroke cases and 22 to 25% of cerebrovascular deaths. Aneurysmal subarachnoid hemorrhage is a very complex disease and many controversies on its pathophysiology and management have not yet been settled. The aim of this review is to present the most recent evidence-based advances in the pathophysiology and perioperative management of aSAH.

Purpose: To demonstrate that results similar to high-volume academic centers can be achieved in the community setting when treating abdominal aortic aneurysm (AAA) using endovascular techniques, given appropriate volume and skill sets., Methods: A retrospective review was conducted of 342 consecutive patients who underwent endovascular aneurysm repair (EVAR) by surgeons in a community hospital group from October 1999 through September 2005. In this population, 245 (71.6%) patients were treated with EVAR and 97 (28.4%) with open surgical repair. Of the 245 EVAR patients (203 men; mean age 73.4+/-9.2 years), 218 AneuRx, 19 Ancure, 6 Excluder, and 2 Zenith stent-grafts were implanted by 2 vascular surgeons to exclude AAAs with a mean diameter of 54+/-11 mm. Patients were followed postoperatively with office visits and computed tomography at 1, 6, and 12 months and annually thereafter., Results: Technical success was achieved in 99.6% (244/245) with 1 intraoperative conversion. Mean operative time was 131+/-57 minutes, with a mean contrast load of 161.6+/-65.5 mL. Thirty-five (14.3%) patients required intraoperative blood transfusion. Length of stay was 2.3+/-4.5 days. Thirty-day mortality was 0.8% (2/245). Secondary procedures were required in 15 (6.1%) patients. Kaplan-Meier estimates of freedom from secondary interventions were 98%, 98%, 95%, and 90% at 12, 24, 36, and 48 months, respectively. At the same time points, freedom from surgical conversion was 99%, 99%, 97%, and 96%, and freedom from aneurysm-related death was 97%, 96%, 96%, and 96%., Conclusion: Endovascular AAA repair provides a less invasive method of managing aortic disease with resultant low perioperative mortality. Results in our community hospital demonstrate that this technology can be applied outside an academic environment in nearly three quarters of the population with excellent short and long-term results.

Background: The recommendation for population- based cystic fibrosis (CF) carrier screening by the American College of Medical Genetics for the 25 most prevalent mutations and 6 polymorphisms in the CF transmembrane regulatory gene has greatly increased clinical laboratory test volumes. We describe the development and technical validation of a DNA chip in a 96-well format to allow for high-throughput genotype analysis., Methods: The CF Portrait chip contains an 8 x 8 array of capture probes and controls to detect all requisite alleles. Single-tube multiplex PCR with 15 biotin-labeled primer pairs was used to amplify sequences containing all single-nucleotide polymorphisms to be interrogated. Detection of a thin-film signal created by hybridization of multiplex PCR-amplified DNA to complementary capture probes was performed with an automated image analysis instrument, NucleoSight. Allele classification, data formatting, and uploading to a laboratory information system were fully automated., Results: The described platform correctly classified all mutations and polymorphisms and can screen approximately 1300 patient samples in a 10-h shift. Final validation was performed by two separate 1000-sample comparisons with Roche CF Gold line probe strips and the Applera CF OLA, Ver 3.0. The CF Portrait Biochip made no errors during this validation, whereas the Applera assay made seven miscalls of the IVS-8 5T/7T/9T polymorphism, Conclusions: The CF Portrait platform is an automated, high-throughput, DNA chip-based assay capable of accurately classifying all CF mutations in the recommended screening panel, including the IVS-8 5T/7T/9T polymorphism.

A comprehensive investigation of the incidence, distribution, progression and chemical composition of Abeta deposits in the brains of two young (5 years) and seven aged (25-30 years) rhesus monkeys was conducted to determine the similarity of this phenomenon to that in the human. The brains of the young rhesus were devoid of Abeta deposits. In contrast, Abeta deposits were observed within the cerebral cortex of all aged animals. In animals with mild Abeta burden, deposits were observed primarily in association cortical zones. In animals with moderate Abeta burden, many paralimbic cortical zones also contained Abeta deposits. Finally, in an animal with a heavy burden of Abeta, core limbic cortical zones were also involved. The primary sensory and motor cortices were relatively free of Abeta deposits. A higher proportion of plaques contained Abeta40 as compared with Abeta42. Abeta deposits contained a number of other constituents. Cholinesterases were present in nearly 50% of plaques and displayed the exact same biochemical characteristics as those in the human. Nearly 20% of Abeta deposits also contained apolipoprotein E and a smaller proportion contained heparin sulfate proteoglycans and alpha1-anti-chymotrypsin. The latter three chemicals were present in many compact plaques. These results indicate that the distribution, progression and chemical composition of plaques in the aged rhesus monkey display many similarities to those observed in the aged human and Alzheimer's disease. Therefore, despite some differences from the human, the aged rhesus may be a good model for studies of the pathological effects of Abeta in the primate brain.

Subependymal giant cell astrocytoma (SEGCA) is a benign, slow-growing glial tumor that manifests with signs and symptoms of obstructive hydrocephalus most often in adolescent patients with tuberous sclerosis complex (TSC). Neonatal highly aggressive SEGCA is very rare. We report a 5-month-old child with TSC presenting with a cystic mass lesion in the left frontal lobe as well as multiple other periventricular masses. After initial conservative treatment, the child was readmitted with intractable seizures, a massive increase in the size of the left frontal lobe tumor and obstructive hydrocephalus. Despite surgical interventions, the child succumbed to the intracranial lesions. In this report, we discuss the challenges of managing SEGCA and the importance of further studies, including genetic studies, that may lead to a better understanding of its pathophysiology., (Copyright 2002 S. Karger AG, Basel)

The purpose of this study was to determine the physiologic effects of cigarette smoke exposure and dietary cholesterol on the availability of nitric oxide in carotid vascular rings. New Zealand white rabbits were placed in an airflow chamber for 3 hr/day over an 8-week period and were exposed to smoke from 600 cigarettes/per day added to the chamber inflow by a robotic smoke generator. New Zealand white rabbits, made hypercholesterolemic, and one group fed a normal diet, were similarly placed in the chamber without exposure to cigarette smoke. In those exposed groups, serum cotinine and cholesterol levels were consistently elevated. After the 8-week period, the carotid arteries were harvested. The vessels were cut into 3-mm rings which were suspended from pressure transducers. The rings were contracted with potassium chloride (KCl) to determine vessel integrity. One ring from each carotid was maximally contracted with 1 x 10(-3) molar norepinephrine (NE) while the experimental ring was contracted to 50% of maximum. Relaxation of the rings was achieved by adding incremental doses of acetylcholine. Our results showed that endothelial dysfunction, as measured by acetylcholine-mediated vasorelaxation, occurs in the rabbit carotid artery when exposed to high dietary cholesterol. Cigarette exposure alone in this particular vessel did not result in significant alteration in acetylcholine-mediated vasorelaxation.

Pheochromocytomas are a rare cause of hypertension in pregnancy. Laparoscopic adrenalectomy has been used effectively and safely in nonpregnant patients with pheochromocytoma, with the resultant benefits to the patients of less postoperative pain, shorter hospital stay, and quicker return to normal activities than is associated with open techniques. This represents the first report of a laparoscopic adrenalectomy for pheochromocytoma in a pregnant woman. Issues that are unique to laparoscopic surgery in pregnant patients are discussed.

Background: Endothelial injury after ischemia and reperfusion is characterized by an increase in permeability, cellular edema, and loss of acetylcholine-mediated vasorelaxation. Three hours of ischemia followed by 2 hr of reperfusion in the New Zealand white rabbit hindlimb has been shown to result in loss of acetylcholine-induced superficial femoral artery vasorelaxation. The purpose of this study was to evaluate the effect of intraarterial pentoxyfylline (PTX) on this endothelial injury., Methods: New Zealand white rabbits underwent 3 hr of complete hindlimb ischemia followed by 2 hr of reperfusion. Twenty milliliters of either 100 microM PTX or normal saline was infused over 20 min via the circumflex iliac artery at initiation of reperfusion. Superficial femoral artery rings were then evaluated in vitro for endothelial cell-mediated relaxation. Rings were exposed to standardized incremental doses of acetylcholine after norepinephrine-induced contraction and percentage relaxation was measured. Sections of arteries were also sent for hematoxylin and eosin staining., Results: Similar contraction responses following NE stimulation were observed between control and PTX-treated rings. Control rings relaxed a mean of 14.97 +/- 3.64, 23.17 +/- 5.61, and 31.84 +/- 8.43% in response to acetylcholine doses of 6 x 10(-8), 1 x 10(-7), and 1.5 x 10(-7) M, respectively. In contrast, PTX-treated segments relaxed a mean of 47.52 +/- 8.88, 62.32 +/- 6.83, and 76.73 +/- 4.91% to the same doses of acetylcholine. Differences in relaxation between control and PTX-treated vessels were significantly different at each dose (P < 0.05, Student's t test). Histologic examination of the PTX-treated and control arteries revealed an intact endothelium without morphologic differences between the two groups., Conclusion: In this model of rabbit hindlimb ischemia, endothelial cell-mediated vasorelaxation was preserved with the administration of intraarterial PTX during reperfusion compared to controls. The different relaxation responses could not be attributed to altered arterial contractility in response to norepinephrine, or explained by histologic changes in the arterial wall. These studies demonstrate a potential modality for therapeutic intervention to reduce reperfusion injury after acute limb ischemia.

Recombinant alphaviruses have been used as vehicles for delivery and expression of heterologous genes in mammalian, avian and insect cell lines. We have used a Sindbis replicon virus (Sinreplac) able to express the E. coli lacZ gene to compare the efficiency of transduction in one insect, six mammalian cell lines and cultured rat dorsal neurons which apparently express beta-galactosidase over a 30-day time period. Results show that different cell lines were transduced with varying degrees of efficiency and that this efficiency could be improved in some cell lines by packaging the replicon with a helper derived from a more neurovirulent strain of Sindbis.

Acute ischemia followed by reperfusion in skeletal muscle is associated with tissue edema and necrosis. The purpose of this study was to demonstrate superficial femoral artery endothelial injury following complete ischemia with reperfusion. New Zealand white rabbits underwent total devascularization of one hindlimb for 3 hr followed by 0, 1, and 2 hr of reperfusion. Control rabbits underwent a sham operation. Superficial femoral artery rings were then studied for acetylcholine induced relaxation in vitro. The response to acetylcholine was measured as percentage relaxation at three incremental doses (1 x 10(-7) , 3 x 10(-7) and 5 x 10(-7) M). The ischemia-only (26.30 +/- 7.07, 62.63 +/- 8.64, 88.08 +/- 5.25%) and the 1-hr reperfusion group (19.35 +/- 12.99, 39.24 +/- 15.78, 62.01 +/- 14.03%) showed no significant difference (P > or = 0.05, Student's t test) in relaxation as compared to the control group (13.73 +/- 2.11, 47.88 +/- 7.23, 72.44 +/- 9.00%). The 2-hr reperfusion group (6.10 +/- 1.02, 15.33 +/- 2.56, 34.67 +/- 6.31%), however, had a significant loss of relaxation at all three doses of acetylcholine compared to that seen in the control group (P < or = 0.05, Student's t test). In this model of complete ischemia, superficial femoral artery rings lose their ability to relax in response to acetylcholine following 3 hr of ischemia and 2 hr of reperfusion, demonstrating endothelial injury. However, immediately after 3 hr of ischemia or ischemia followed by only 1 hr of reperfusion, superficial femoral artery rings did not lose their ability to relax in response to acetylcholine. This study identifies a window of opportunity for therapeutic intervention after ischemia and prior to endothelial injury from reperfusion.

Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540-mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540-mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540-sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye-binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate-induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.

Merocyanine 540 (MC540) is a photosensitizing dye that has been used in several preclinical models and in a phase I clinical trial for the extracorporeal purging of tumor cells from autologous bone marrow grafts. The mechanism of the cytotoxic activity of MC540 is not yet fully understood, and the subcellular targets of MC540-mediated photodynamic damage remain to be identified. The human neutrophil provides an attractive model with which to study the effects of photoactivated MC540 on several well-defined cellular functions. As we report in this paper, simultaneous exposure of neutrophils to MC540 and light inhibited phagocytosis, random migration, chemotaxis, hydrogen peroxide production, and oxygen consumption. By contrast, the ability of neutrophils to kill engulfed bacteria and to produce superoxide radical was not compromised. Intracellular ATP levels and the activities of the cytosolic enzymes superoxide dismutase, catalase, and myeloperoxidase were only slightly reduced. Even in HL-60 leukemia cells, which bind more dye and are more readily killed by MC540-mediated photodynamic therapy than neutrophils, superoxide dismutase, catalase, and myeloperoxidase activities remained at normal or near-normal levels. These results are compatible with the view that plasma membrane components are primary targets of MC540-mediated photodynamic damage.

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.

Merocyanine 540 (MC 540), a photosensitizing dye, has been used in preclinical studies and in a phase I clinical trial for the purging of leukemia, lymphoma, and neuroblastoma cells from bone marrow grafts. We evaluated MC 540 as an agent for the inactivation of brain tumor cell lines of medulloblastoma or glioma origin. The U373 glioma and 74SA medulloblastoma demonstrated significantly reduced survival as determined by in vitro clonogenic assay compared to normal glial cells when exposed to MC 540 and light. U87 glioma and Daoy medulloblastoma, however, were less sensitive than normal glial cells to MC 540 photoinactivation. In vivo injection of MC 540 into mice with malignant brain tumors disclosed greater dye incorporation into the malignant tissue compared with normal control mice brains or normal tissue surrounding the brain tumor. Increased uptake of MC 540 was observed in mice injected with either photosensitive (U373 and 74SA) or photoresistant (Daoy) cell lines. These data suggest that MC 540 may be an effective agent against certain brain tumors and that dye uptake in vivo does not reflect photosensitivity.

Analysis of 5000 consecutive flexible fiberoptic sigmoidoscopies form the basis of this report. It is concluded that this method of examination of the distal large bowel is not only safe and comfortable for the patient but is a more appropriate examination than the rigid proctosigmoidoscopy because of the significant increase in pathologic material found. This examination has proven practical and acceptable in a multispecialty clinic setting and has completely replaced rigid proctosigmoidoscopy. Flexible sigmoidoscopy is now the standard "routine" examination of the rectum and distal colon. The rationale for this conclusion is presented in this timely report.