Enhanced functional maturation of fetal porcine hepatocytes in three-dimensional poly-L-lactic acid scaffolds: a culture condition suitable for engineered liver tissues in large-scale animal studies

To engineer liver tissues with a clinically significant size, in vivo evaluation of performance using large-scale animal studies are necessary before proceeding to human clinical trials. As pigs are the most suitable candidates, the development of culture conditions suitable for porcine hepatocyte progenitors is very important to engineer pig liver tissue equivalents. We therefore investigated the efficacy of poly-L-lactic acid (PLLA) three-dimensional (3D) scaffolds on the functional maturation of fetal porcine hepatocytes in the presence of various combinations of biofactors. Cells were isolated from pig fetuses obtained from a local slaughterhouse, and cultured for 15 days both in monolayer and PLLA scaffolds. Although 15 days of culture resulted in almost the same ratio of proliferation (about fivefold) in both monolayer and 3D PLLA culture, the PLLA culture with hepatocyte growth factor (HGF, 10 ng/ml) and sodium butylate (Sb, 1 mM) remarkably enhanced various liver-specific functions of fetal porcine hepatocytes. The final attained functions based on the numbers of immobilized cells on day 1 compared with those of day 1 monolayers; 191-fold increase in albumin secretion, 70.5-fold increase in cytochrome P450 IA1/2 capacity, 20.9-fold increase in ammonia removal, and 18.0-fold increase in urea synthesis were obtained. These functions were 2.0-3.3-fold higher than those obtained by the same period of monolayer culture. In addition, final attained unit cell-based functions on day 15 were almost comparable to the levels reported for cultures of adult porcine hepatocytes in both monolayer and 3D spheroid cultures. These results demonstrate that the use of a biodegradable polymer-based 3D culture with an appropriate combination of biofactors is a promising approach to maximize functional maturation of hepatocyte progenitors from large animals. In addition, the established culture conditions are worth using to engineer large liver tissue equivalents for pigs in large-animal-based preclinical studies.     

Non-vascularized bone grafting in scaphoid nonunion: principles and type of fixation

In scaphoid fractures, delayed diagnosis and nonunion are fairly common as a result of several factors, including the difficulty of radiographic diagnosis of non-displaced fractures and underestimation of the injury by the patient. Main factors to consider when deciding treatment are the type of fracture and fracture stability. In the stable nonunion (Type D1 according to the Filan and Herbert classification, or linear type of Ikeda’s classification), percutaneous screw fixation without bone graft is recommended. The indications of non-vascularized bone grafting are as follows: (1) arthroscopic cancellous bone graft in type D1 and cystic type of Ikeda’s classification. (2) tricortical bone graft from the iliac crest in type D2 or D3 if the possibility of avascular necrosis of the proximal fragment is excluded. In this paper, our non-vascularized bone grafting for scaphoid nonunion would like to be described mainly about principles and type of fixation.     

Low contrast dose protocol involving a 100 kVp tube voltage for hypervascular hepatocellular carcinoma in patients with renal dysfunction

Purpose

To evaluate the feasibility of a 20 % reduced contrast dose hepatic arterial phase (HAP) CT for hypervascular hepatocellular carcinoma (HCC) with 100 kVp.

Materials and methods

The study included 97 patients with hypervascular HCC who underwent dynamic CT, including HAP scanning. The 54 patients had an estimated glomerular filtration rate (eGFR) of ≥60 were scanned with our conventional 120 kVp protocol. The other 43 patients (eGFR < 60) underwent scans using a tube voltage of 100 kVp and a 20 % reduced contrast dose. We compared the estimated effective dose, image noise, tumor-liver contrast (TLC), and contrast-to-noise ratio (CNR) in the hepatic arterial phase between the two groups using the Student’s t test.

Results

Estimated effective dose and image noise were not significantly different between these groups (p = 0.67 and p = 0.20, respectively). The TLC and CNR were significantly higher for the 100 kVp protocol than for the 120 kVp protocol (52.2 HU ± 17.4 vs 40.8 HU ± 18.6, p < 0.01 and 6.8 ± 2.6 vs 5.5 ± 2.4, p = 0.01, respectively).

Conclusion

For hepatic arterial phase CT of hypervascular HCC, 100 kVp scan allows a 20 % reduction in the contrast dose without reduction in image quality compared with a standard 120 kVp CT protocol.

     

Effects of cholinesterase inhibition in supraspinal and spinal neural pathways on the micturition reflex in rats

OBJECTIVE

To investigate whether activation of brain and spinal cholinergic pathways affects the micturition reflex in rats.

MATERIALS AND METHODS

The effects of intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration of neostigmine as a cholinesterase inhibitor and oxotremorine‐M (OXO‐M) as a muscarinic acetylcholine receptor (mAChRs) agonist, on the micturition reflex were evaluated by infusion cystometrography (CMG) in urethane‐anaesthetized untreated rats or rats pretreated with capsaicin.

RESULTS

Neostigmine injected i.c.v. increased bladder capacity (BC) and pressure threshold (PT) dose‐dependently, with an increase in maximum voiding pressure (MVP) and a decrease in voiding efficiency (VE) at higher doses. Also, neostigmine injected i.t. increased the BC and PT dose‐dependently without changing MVP or VE, and these effects were not apparent in capsaicin‐pretreated rats. In both routes, atropine as an antagonist of mAChRs, but not mecamylamine as a nicotinic‐AChR antagonist, almost completely antagonized the effects of neostigmine. The rank order of potencies of the antagonists for increasing effects of BC induced by 1 nmol of neostigmine was: pirenzepine (an M₁ mAChR antagonist) = atropine > 4‐DAMP (an M₃ mAChR antagonist) >> methoctramine (an M₂ mAChR antagonist) and tropicamide (an M₄ mAChR antagonist) via the i.c.v. route; and atropine > methoctramine > pirenzepine > tropicamide and 4‐DAMP via the i.t. route, respectively. OXO‐M injected via i.c.v. and i.t. had the same effects on BC, PT, MVP and VE as neostigmine by i.c.v. and i.t., respectively.

CONCLUSIONS

These results indicate that activation of muscarinic cholinergic mechanisms by the cholinesterase inhibitor in the brain and spinal cord can inhibit the micturition reflex, mainly by affecting afferent pathways. These mAChR‐induced inhibitory effects seem to be mediated through M₁/M₃ receptor subtypes in the brain, while in the spinal cord, the M₁/M₂ receptor subtypes might be involved in inhibitory effects, which are mediated via inhibition of mechanoceptive C‐fibre afferent pathways.     

Simultaneous resection for colorectal cancer and synchronous liver metastases

Purposes

The correct timing of hepatectomy in patients with synchronous colorectal liver metastases is unclear. The aim of this study was to assess the clinical value of simultaneous resection (SR) for patients with colorectal cancer and synchronous liver metastases.

Methods

Between January 2006 and December 2013, 158 patients underwent resection of primary colorectal cancer and liver metastases. Sixty-three patients possessed synchronous colorectal liver metastases. Of those with synchronous colorectal liver metastases, 41 patients (65 %) underwent SR, and 22 (35 %) underwent delayed resection (DR). The clinicopathologic and operative data and the surgical outcomes of the patients in the SR and DR groups were retrospectively analyzed.

Results

The type of primary/liver resection, liver resection time, total blood loss volume, R0 resection rate, and morbidity rate were similar between the two groups. The SR group was associated with a shorter total postoperative hospital stay (21 vs 32 days, p < 0.001). However, the overall survival rate was similar between the two groups (3-year survival, 65.6 % in the SR group versus 66.8 % in the DR group, p = 0.054).

Conclusion

Simultaneous resection of colorectal cancer and synchronous liver metastases is associated with a comparable morbidity rate and shorter hospital stay, even when following rectal resection and major hepatectomy.

     

Detailed Analysis of Mucosal Restoration of the Small Intestine After the Cavitary Two-Layer Cold Storage Method

Small bowel transplantation (SBT) is associated with a high incidence of infectious complications because of ischemia/reperfusion (I/R) mucosal injury concomitant with potent immunosuppression. In this study, we evaluated whether the cavitary two-layer method (cTLM) could reduce I/R injury and allow early mucosal restoration, particularly after prolonged preservation and transplantation. Canine heterotopic segmental SBT was performed immediately without preservation (group 1), after 24-h preservation in UW solution (group 2) or by the cTLM (group 3). The graft samples were taken 1 h after reperfusion and on days 1, 4 and 7. We assessed graft mucosa with detailed microscopic and electromicroscopic analyses. In Group 3, histological injury and cell apoptosis after transplantation were significantly alleviated and rapidly recovered to a similar level of group 1. The mucosal restoration was morphologically completed within 4 days. In contrast, in group 2, more pronounced mucosal injury and delayed recovery were noted. Crypt cell proliferation activity was well maintained in groups 1 and 3 throughout the experimental period. Our ultrastructural analysis suggested that mitochondrial integrity achieved by the cTLM was a basal mechanism under the prompt mucosal restoration. The cTLM could reduce I/R injury, facilitate mucosal regeneration and restore the nearly normal structure early after SBT.     

A method for producing overactive bladder in the rat and investigation of the effects of GABAergic receptor agonists and glutamatergic receptor antagonists on the cystometrogram

PURPOSE: We induced radio frequency (RF) lesions in the neuronal pathway leading from the forebrain to the pontine micturition center (PMC) to produce a rat model of bladder overactivity. We studied the effects of gamma-aminobutyric acid agonists (diazepam and baclofen) and glutamate receptor antagonists (MK-801 maleate and GYKI52466 [1-(4-aminophenyl-D-4-methyl-7,8 methylenedioxy-5H-2,3-benzodiazepine] hydrochloride) on the cystometrogram and developed a possible explanation of the neuronal mechanisms underlying RF lesion induced bladder overactivity. MATERIALS AND METHOD: Seven-week-old male Sprague-Dawley rats were anesthetized with sodium pentobarbital and RF lesions were produced in the nuclei basalis. Five days later bladder contractions were induced by infusing fluid into the bladder and cystometrograms were measured in conscious rats. RESULTS: The micturition interval (MI) in rats subjected to RF lesioning was significantly shorter than that in sham operated control rats. Diazepam (0.1 and 1 mg/kg intraperitoneally), baclofen (1 mg/kg intravenously) and MK-801 (0.1 and 1 mg/kg intravenously) did not change or shortened MI in control rats but it prolonged MI in lesioned rats. GYKI52466 (0.5 and 1 mg/kg intravenously) weakly prolonged MI in lesioned rats. CONCLUSIONS: We consider that RF lesioning causes interruption of the inhibitory GABAergic neurons that lead from the forebrain to the PMC. This results in the activation of N-methyl-D-aspartate receptors in the PMC that are involved in the facilitation of voiding.     

Effects of Fuscoporia obliqua on Postprandial Glucose Excursion and Endothelial Dysfunction in Type 2 Diabetic Patients

Postprandial hyperglycemia has been reported to elicit endothelial dysfunction and provoke future cardiovascular complications. A reduction of postprandial blood glucose levels by the glucosidase inhibitor Fuscoporia obliqua was associated with a risk reduction of cardiovascular complications, but the effects of Fuscoporia obliqua on endothelial function have never been elucidated. This study is aimed to assess the efficacy of Fuscoporia obliqua on postprandial metabolic parameters and endothelial function in type 2 diabetic patients. Postprandial peak glucose (14.47±1.27 vs. 8.50±0.53 mmol/liter), plasma glucose excursion (PPGE), and change in the area under the curve (AUC) glucose after a single loading of test meal (total 450 kcal; protein 15.3%; fat 32.3%; carbohydrate 51.4%) were significantly higher in the diet-treated type 2 diabetic patients (n=14) than the age- and sex-matched controls (n=12). The peak forearm blood flow response and total reactive hyperemic flow (flow debt repayment) during reactive hyperemia, indices of resistance artery endothelial function on strain-gauge plethysmography, were unchanged before and after meal loading in the controls. But those of the diabetics were significantly decreased 120 and 240 min after the test meal. A prior administration of Fuscoporia obliqua decreased postprandial peak glucose, PPGE, and AUC glucose. The peak forearm blood flow and flow debt repayment were inversely well correlated with peak glucose, PPGE, and AUC glucose, but not with AUC insulin or the other lipid parameters. Even a single loading of the test meal was shown to impair the endothelial function in type 2 diabetic patients, and the postprandial endothelial dysfunction was improved by a prior use of Fuscoporia obliqua. Fuscoporia obliqua might reduce macrovascular complication by avoiding endothelial injury in postprandial hyperglycemic status.