In Vitro Evaluation of a Bioartificial Pancreas Under Various Hemodynamic Conditions

A bioartificial pancreas in which isolated islets of Langerhans are placed between two polyacrylonitrile membranes, blood circulating successively above the upper and below the lower membranes following a U-shaped circuit, has been developed. The two parts are connected by an outer loop consisting of a thin tubing. The length of this tubing determines the magnitude of the flow rate of blood through the device. The aim of this work was to determine experimentally the optimal configuration of the system containing isolated rat islets and a Krebs buffer circulating through the device. The amount of insulin released by the bioartificial pancreas was determined during a 20-mM square-wave glucose stimulation. First, the inlet pressure was set at 100 mm Hg, and the effect of the length of the tubing was investigated with two devices perfused simultaneously. For a short tubing (flow rate, 20 ml/min), a sharp increase in insulin release in response to glucose was observed; it increased within 4 min from 217 +/- 50 to 761 +/- 237 microU/500 islets/min (p less than 0.05), the peak value being reached at 11 +/- 2 min following the beginning of the stimulation. For a long tubing (flow rate, 3 ml/min), the increase in insulin release was more sluggish. It increased from 133 +/- 53 to 222 +/- 43 microU/500 islets/min at 4 min, the peak value being reached only at 20 +/- 3 min. These data are consistent with a more efficient diffusional transfer of insulin in the case of the high circulating flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vivo and In Vitro Analysis of Rat Lumbar Spine Mechanics

Background  

Rodent lumbar and caudal (tail) spine segments provide useful in vivo and in vitro models for human disc research. In vivo caudal models allow characterization of the effect of static and dynamic loads on disc mechanics of individual animals with time, but the lumbar models have required sacrifice of the animals for in vitro mechanical testing.     

In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft‐versus‐host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3‐dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4⁺ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)‐2 in vitro. Although in vivo IL‐2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL‐2–supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.     

In Vitro Effects of Prolactin and Dexamethasone on Rat Leydig Cell Aromatase Activity

In Percoll purified adult rat Leydig cells, the estradiol secretion, in presence of exogenous testosterone (200 ng/ml) is stimulated 2-fold by either LH (100 ng/ml) or dbcAMP (1 mM). The addition of prolactin (1 microgram/ml) or dexamethasone (10(-7) M) to the Leydig cell incubation medium induces a 20% increase of the basal estradiol production, whereas, under LH or dbcAMP stimulations, 46 and 41% decreases are noted; moreover, a synergistic effect between prolactin and dexamethasone was observed in presence of dbcAMP leading to a 53% diminution of the estradiol synthesis. There results suggest that either hyperprolactinemia or high doses of glucocorticoids might inhibit the Leydig cell aromatase activity in presence of LH.     

Influence of Rifampin on Capsule Formation Around Silicone Implants in a Rat Model

This study investigated the effect of rifampin on the thickness of capsules around silicone implants by bactericidal activity against Stapylococcus epidermidis. Silicone blocks (1 × 1 cm) were placed into pockets created for each of the 40 rats included in the study. In group 1, the operation was performed under aseptic conditions. In group 2, standard S. epidermidis was inoculated into the pocket, whereas rifampin and S. epidermidis were applied in group 3. In group 4, only rifampin was applied topically on implants. After 12 weeks, the peri-implant capsules were removed and examined under a photomicroscope and a scanning electron microscope. The mean thickness of the capsules was 63.307 μm in group 1, 111.538 μm in group 2, 43.076 μm in group 3, and 30.384 μm in group 4. The differences between groups 2 and 3 and groups 2 and 4 were found to be statistically significant (p < 0.001). Rifampin appears to be an agent for preventing peri-implant capsule formation.     

Tetracyclines Inhibit Rat Osteoclast Formation and Activity In Vitro and Affect Bone Turnover in Young Rats In Vivo

An experiment was designed to investigate whether systemic administration of tetracyclines (TCs) as bone fluorochrome labels could interfere with bone modeling in vivo and inhibit osteoclast formation and activity in vitro. Cell cultures of rat bone marrow macrophages revealed that TC and oxytetracycline inhibited osteoclastogenesis and bone resorption and stimulated apoptosis. Forty rats in five groups were treated with saline, calcein green, alizarin red S, TC, or oxytetracycline. Their tibias were used for histomorphometric analysis, including bone static, dynamic, and resorption parameters in the tibial proximal metaphysis. No significant differences in bone volume per tissue volume, trabecular number, trabecular thickness, trabecular separation, bone formation rate per bone surface, mineralizing surface, or mineral apposition rate were observed. TC or oxytetracycline decreased eroded surface, number of osteoclasts per bone perimeter, and osteoclast surface per bone surface by about 50%. The results demonstrated that TC and oxytetracycline inhibit rat osteoclast formation and activity in vitro, and histomorphometric parameters involved in bone turnover may be affected by the use of oxytetracycline and TC as fluorescent bone labels in vivo.     

Relationship Between Arterial Calcification and Bone Loss in a New Combined Model Rat by Ovariectomy and Vitamin D3 Plus Nicotine

Epidemiological studies have reported an association between arterial calcification and bone loss after menopause. However, the underlying mechanism of the association remains unclear. Therefore, to explore the possible mechanisms of the association, we tried to develop a new combined model rat of ovariectomy (OVX, an animal model of osteoporosis) and vitamin D₃ plus nicotine (VDN rat, an animal model of arterial calcification). We tested them by using sham-operated control rats (SC), OVX control rats (OC), and OVX plus VDN-treated rats (OVN). Dissections were performed twice at 4 (4SC, 4OC, and 4OVN) and 8 (8SC, 8OC, and 8OVN) weeks after treatment. 8OVN showed bone loss and arterial calcification, although 8OC showed only bone loss. Moreover, arterial calcium content was associated with indexes of bone loss at 8 weeks. Thus, the OVN rat is considered a good model to examine the relationship of the two disorders after menopause. Additionally, the arterial endothelin-1 (ET-1, a potent regulator of arterial calcification) levels increased in both 4OVN and 8OVN, and the level was associated with arterial calcium content at 8 weeks. Furthermore, the arterial endothelial nitric oxide synthase (eNOS) protein, which is an enzyme that produces nitric oxide (an antiatherosclerotic substance), was significantly reduced in only 8OVN. Estrogens affect the alterations of the eNOS and ET-1 proteins. Therefore, we suggest that impairment of the ET-1- and NO-producing system in arterial tissue during periods of rapid bone loss by estrogen deficiency might be a mechanism of the relationship between the two disorders seen in postmenopausal women.     

三氯生和硫酸庆大霉素两种抗菌活性骨水泥生物力学性能的实验研究

目的 通过对三氯生和硫酸庆大霉素两种抗菌活性骨水泥的进行压缩强度、弯曲强度和弹性模量测试分析,比较两种抗菌活性骨水泥的生物力学性能.方法 实验分为三氯生组、硫酸庆大霉素组及空白对照组,将三氯生和硫酸庆大霉素分别以0.5∶40、1.0∶40、1.5∶40、2.0∶40的剂量比例与骨水泥混合,制成符合实验标准的模件,每组6根,然后放在生物力学机上进行测试.结果 压缩强度检测表明,将2.0 g三氯生混入40 g骨水泥中,压缩强度为(72.4±4.14)Mpa,大于ISO 5833标准中规定的抗压强度70 Mpa,和对照组(无抗生素的骨水泥)相似.而根据ISO 5833对四点弯曲测试提出的标准,三氯生骨水泥产品弯曲模量为(1854±57) Mpa＞1 800 Mpa,弯曲强度(为51.8±2.07)Mpa＞50 Mpa.结论 生物力学实验表明,本实验剂量下的三氯生骨水泥的生物力学效果优于硫酸庆大霉素骨水泥,与无抗生素的骨水泥相似且压缩强度、弯曲模量和弯曲强度均达标.     

An In Vitro Model to Compare the Antimicrobial Activity of Silver-Coated Versus Rifampicin-Soaked Vascular Grafts

In situ replacement of infected vascular grafts is an accepted alternative to total graft excision and extraanatomic replacement. Its success relies upon the ability of the newly inserted graft to resist recurrent infection. This study compares the efficacy of two methods used to reduce the risk of graft reinfection: rifampicin soaking versus silver bonding of grafts. The grafts resistance to infection was tested in vitro in two protocols, each using a panel of seven common bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The length of time the grafts remained free of organisms was compared between the groups. Both the silver graft and the rifampicin-soaked graft were significantly better than control graft at preventing bacterial growth on the graft surface. The rifampicin inhibited the growth of the gram-positive organisms, including MRSA, significantly better than the silver graft on days 2 and 3 (p < 0.001). Conversely, the silver graft was significantly more effective against the gram-negative organisms until day 4 (p < 0.0001). Both types of graft inhibit the in vitro growth of bacteria more effectively than controls, with rifampicin being most effective against gram-positive organisms and silver being best against the gram-negative organisms.     

Assessment of Teratogenic Effects of Lidocaine in Rat Embryos Cultured In Vitro

Background: Lidocaine has been reported to cause neural tube closure defects in vitro in mice at clinically relevant concentrations. However, no studies have been conducted to further investigate this potentially hazardous effect of lidocaine. This study was aimed to reassess teratogenic effects of lidocaine in vitro in rats.

Methods: Sprague-Dawley rat embryos were explanted at 8:00 AM on gestational day 9 and were cultured in medium containing various concentrations of lidocaine. (Embryos in the control group were cultured without lidocaine). After 50 h of culture, they were evaluated for growth size and morphology, including the neural tube closure.

Results: In the presence of 250 [micro sign]m of lidocaine, embryos showed a increased incidence of situs inversus compared with control group but were otherwise normal. At 375 [micro sign]m, embryos showed slight growth retardation but no significant morphologic abnormalities. At 500 [micro sign]m, all viable embryos showed severe morphologic abnormalities. However, morphologic abnormalities were so-called nonspecific types and neural tube closure defects were not observed.     

In Vivo and In Vitro Effects of a Pulsed Electromagnetic Field on Net Calcium Flux in Rat Calvarial Bone

Although PEMF's have been found to promote fracture healing and to modulate the activity of bone cells in vitro, effects on bone metabolism are largely unexplored. A bioassay using neonatal rat calvarial bone was used to determine the early effects of a pulsing electromagnetic field (PEMF) exposure in vivo and in vitro on bone metabolic calcium exchange. Bone discs taken from whole body exposed animals (0-4 hours) show a log exposure time-dependent average increase in net Ca uptake in the 0-50% range (r2 = 0.83). This increase could be detected immediately after exposure and also after 24 hours, but not 48 hours later. Animals given whole body PEMF exposure also showed a decrease in serum calcium and did not elevate serum Ca after administration of exogenous parathyroid hormone (PTH). Bone discs from untreated rats, exposed to PEMF for 15 minutes in vitro and then assayed, showed net Ca uptake increases of a similar magnitude and also were refractory to the Ca-releasing effect of PTH. Unexposed discs responded normally to PTH by decreasing net Ca uptake. Treatment of calvarial discs with calcitonin or acetazolamide, both of which inactivate osteoclasts, made the bone refractory to further increases in Ca uptake by PEMF. These results suggest that PEMF exposure produces PTH-refractory osteoclastics and has a relatively rapid effect on increasing net bone Ca uptake, putatively due to a decrease in PTH/paracrine-mediated bone resorption.     

Contribution of the Proximal Nerve Stump in End-to-side Nerve Repair: In a Rat Model

Background

The aim of this study was to evaluate the contribution of the proximal nerve stump, in end-to-side nerve repair, to functional recovery, by modifying the classic end-to-side neurorrhaphy and suturing the proximal nerve stump to a donor nerve in a rat model of a severed median nerve.

Methods

Three experimental groups were studied: a modified end-to-side neurorrhaphy with suturing of the proximal nerve stump (double end-to-side neurorrhaphy, Group I), a classic end-to-side neurorrhaphy (Group II) and a control group without neurorrhaphy (Group III). Twenty weeks after surgery, grasping testing, muscle contractility testing, and histological studies were performed.

Results

The grasping strength, muscle contraction force and nerve fiber count were significantly higher in group I than in group II, and there was no evidence of nerve recovery in group III.

Conclusions

The contribution from the proximal nerve stump in double end-to-side nerve repair might improve axonal sprouting from the donor nerve and help achieve a better functional recovery in an end-to-side coaptation model.     

In Vitro Laser Fenestration of Aortic Stent‐Grafts: A Qualitative Analysis Under Scanning Electron Microscope

In situ fenestration of stent‐grafts allows patients with life threatening aortic pathologies to be amenable to emergent “off the shelf indications for use” percutaneous treatments as a bail out technique. Three types of aortic stent‐grafts were subjected to laser fenestration in a physiological saline solution followed by balloon angioplasty using 8, 10 or 12 mm in diameter noncompliant balloons. The morphology and the size of fenestrations were observed under optical and scanning electron microscopy. The damage to the fabrics was analyzed and quantified. The creation of fenestrations was feasible in all devices, with varying degrees of fraying and/or tearing. The monofilament twill weave (Medtronic Valiant) tore in two directions (warp and weft) while the multifilament weave fenestrations showed more fraying (Anaconda Vascutek and Zenith TX2 Cook). The size and directions of tearing were more predictable with the 8 mm diameter balloon whereas the results obtained with the 10 and 12 mm diameter balloons were more unpredictable. The fenestrations were free of melting of the yarns and blackening of the filaments. The in situ fenestration is feasible but the observed damage to the fabric constructions must be carefully considered. This procedure must currently be limited to urgent and emergent life threatening cases because it is off indications for use for approved devices.     

Behavior of Endothelial Cells Cultured on Silastic and Dacron Velour Under Flow Conditions In Vitro: Implications for Prelining Vascular Grafts with Cells

Tissue-cultured bovine aortic endothelial cells were subjected to flow in an in vitro circulatory loop designed to simulate the flow and pressure conditions in the aorta. The cells were cultured under stationary conditions on tubes of fibronectin-coated Silastic or of Dacron velour. The tubes were then added to the flow loop, which circulated complete tissue culture medium in a pulsatile mode. Light microscopy and cell counts showed that the cells not only remained adherent for up to 2 weeks under flow conditions, but also underwent hypertrophy and proliferation in response to the flow regimen.