Custom-tailored tissue engineered polycaprolactone scaffolds for total disc replacement.

Degeneration of the intervertebral disc (IVD) represents a significant musculoskeletal disease burden. Tissue engineering has proposed several strategies comprising the use of biodegradable materials to prepare scaffolds that can present mechanical properties similar to those of native IVD tissues. However, this might be insufficient, since the patient's intervertebral space geometry must be replicated to allow for appropriate implant fixation and integration. Herein, we propose the use of reverse engineering and rapid prototyping techniques with the goal of preparing custom-tailored annulus fibrosus scaffolds; these techniques have previously been applied to rabbit models. The IVD reverse-engineered architecture was obtained by means of microcomputed tomography acquisition and three-dimensional modelling, resulting in a computer-aided design (CAD) that replicates the original rabbit IVD. Later, a fused deposition-modelling three-dimensional printer was used to produce the scaffolds with different geometries provided by the CAD, using polycaprolactone (PCL) with 100% infill density. The microstructure of the PCL scaffolds was investigated by scanning electron microscopy (SEM), which allowed us to observe an adequate fusion adhesion between the layers. The SEM images revealed that, up to the point of moderate resolution, the porosities manually designed in the CAD model were successfully replicated. The PCL scaffolds' three-dimensional architecture was also assessed by means of microcomputed tomography analysis. Compressive stiffness was determined using a mechanical testing system. Results showed higher values than those of human IVDs (5.9-6.7 kN mm(-1) versus 1.2 kN mm(-1), respectively). In vitro studies were performed to investigate the possible cytotoxicity of the polycaprolactone scaffolds' leachables. The results showed that the custom-tailored PCL scaffolds do not have any deleterious cytotoxic effect over annulus fibrosus cells or the mouse lung fibroblast's cell line. This study proposed a simple, rapid, and low-cost strategy to fabricate custom-tailored annulus fibrosus scaffolds. In the future, this strategy might be used in association with nucleus pulposus regeneration strategies to facilitate the development of tissue-engineered total disc replacement implants specific to each patient, with a goal of full IVD regeneration.

Heightened free radical activity in pancreatitis.

Three markers of free radical oxidation of lipids--9 cis, 11 trans isomer of linoleic acid, conjugated dienes and ultraviolet fluorescence products--were measured in the phospholipid fraction of duodenal juice collected in the first 10 min after an intravenous injection of secretin. The volume of aspirate was similar in 11 controls and in 25 patients who had sustained an attack of pancreatitis 6 weeks earlier--acute pancreatitis (AP) 10, chronic pancreatitis (CP) 15. The concentration of each marker was very significantly higher in the patients; the output of the isomer gave the best discrimination from controls; and ultraviolet fluorescence products were substantially higher in the subgroup with CP than with AP. The serum % molar ratio of the isomer to linoleic acid was measured in 25 controls, 14 AP and 17 CP patients: the highest levels were found in the CP group. Heightened hepatic free radical activity involving lipid isomerization as well as lipid peroxidation pathways is a feature of pancreatitis--probably antedating the attack and persisting well after clinical recovery--the difference between CP and AP being in the degree of abnormality. We argue that these hepatic changes mirror changes in pancreatic-acinar cells and that increased free radical activity in both organs is due to a shortfall of antioxidants in the face of cytochromes P450 induction by xenobiotics. Therefore, a combination of preventive and chain-breaking antioxidants may be useful in preventing further attacks of pancreatitis and controlling background pain in chronic disease.

Antioxidant therapy for recurrent pancreatitis: placebo-controlled trial.

Oxidant stress has been proposed as the initiating pathogenetic mechanism in pancreatitis, hence micronutrient antioxidant therapy has been assessed in patients with recurrent attacks and/or constant pancreatic pain. In a 20-week double-blind double-dummy crossover trial active treatment was given as two types of tablets providing daily doses of 600 micrograms organic selenium, 9000 IU beta carotene, 0.54 g vitamin C, 270 IU vitamin E and 2 g methionine. Of 28 patients enrolled, 20 adhered to the full protocol (idiopathic chronic 8, alcoholic chronic 7, idiopathic acute 5). Six patients had an attack whilst on placebo but none whilst on active treatment (P = 0.032). Analysis of visual analogue scoresheets to compare background pain in the 10-week period before entry and during each phase of the trial, using a 10-cm scale for each of 11 best descriptors, endorsed the beneficial effect of active treatment (placebo v baseline, P = 0.073; active v baseline, P less than 0.001; active v placebo, P = 0.049). The same trend emerged from analysis of pain-score diaries by conventional and time series methods. Micronutrient antioxidant therapy thus offers a new approach to the treatment of recurrent (non-gallstone) pancreatitis and/or pancreatic pain.

Antioxidant therapy for recurrent pancreatitis: biochemical profiles in a placebo-controlled trial.

The usefulness of micronutrient antioxidant therapy for recurrent (non-gallstone) pancreatitis has recently been endorsed by a 20-week double-blind double-dummy cross-over trial in 20 patients. Treatment was delivered as two types of tablets, providing daily doses of 600 micrograms organic selenium, 9000 i.u. beta-carotene, 0.54 g vitamin C, 270 i.u. vitamin E and 2 g methionine. We report antioxidant profiles in blood samples collected before entry, at the cross-over stage and upon completion of trial. Baseline serum concentrations of selenium, beta-carotene and vitamin E in the patients were significantly lower than in healthy controls, were unaltered by placebo and normalized by active treatment, but reverted to basal values in the subgroup that received placebo subsequently. The baseline serum concentration of a free radical marker--the 9-cis, 11-trans isomer of linoleic acid--was significantly higher in the patients than in controls, fell inexplicably in the placebo phase and fell further upon active treatment. Discriminant analysis eliminated the overlap in free radical marker and selenium concentrations between control sera on the one hand and baseline or post-placebo samples from the patients on the other: antioxidant treatment normalized the relationship between these biochemical parameters. Subnormal baseline serum levels of S-adenosylmethionine drifted downwards upon active treatment whereas a sharp rise was noted when a relapse of pancreatitis occurred during the placebo phase. The results confirm that adequate exposure to antioxidants in the active treatment phase was associated with amelioration of oxidative stress, and that there was no residual effect 10 weeks after switching over to placebo treatment. Furthermore, the paradoxical behaviour of S-adenosylmethionine may imply that the beneficial effect of micronutrient antioxidants in recurrent pancreatitis is linked with preservation of the methionine trans-sulfuration pathway in pancreatic acinar cells.

Antioxidants, enzyme induction, and chronic pancreatitis: a reappraisal following studies in patients on anticonvulsants.

Our published dietary and pharmakokinetic studies in 15 patients with idiopathic chronic pancreatitis and 15 age- and sex-matched controls suggested that a combination of subnormal antioxidant intakes and chronic induction of the cytochromes P450 facilitates the pancreatic problem. We have now attempted to determine the relative importance of these two factors by studying a group of 15 institutionalized patients with epilepsy (EP), but without abdominal pain, who were on long-term treatment with anticonvulsant inducers of cytochromes P450 so that their clearance of theophylline (which reflects cytochromes P450 activities, and thereby provides an index of antioxidant demand) was as high as in the patients with chronic pancreatitis (CP) (mean +/- s.d., 123 +/- 59 ml/kg/h versus 120 +/- 62 respectively), and significantly higher than in controls (74 +/- 16 ml/kg/h, P less than 0.02). Canonical variate analysis of the drug kinetic and dietary data provided two functions with which to separate the three groups. The first function, heavily weighted on selenium, separated the controls from the other two groups whose values were lower; the second function, equally weighted on methionine and vitamin C, separated the EP group from the CP group whose values were generally lower. The results suggest that enzyme induction per se is not the critical factor in the development of CP. Instead, suboptimal availability of antioxidants in the face of increased demand--in particular of those substances that protect cells against non-biological free radicals--may be the key consideration, a deduction reinforced by observations in patients with epilepsy who went on to develop chronic pancreatitis.

Rationale for antioxidant therapy in pancreatitis and cystic fibrosis.

The overlapping features of the acquired diseases acute pancreatitis and chronic pancreatitis on the one hand, and of chronic pancreatitis and pancreatic involvement in the congenital condition cystic fibrosis on the other, suggest that the basic mechanism of pancreatic injury may be the same in each illness. We propose that pancreatic oxidant stress is the common denominator and, furthermore, that this is facilitated by a shortfall of micronutrient antioxidants in the face of heightened free radical activity through different sources. If so antioxidant supplements should alleviate symptoms. This deduction was supported by an exploratory dose-seeking study that spanned five years in 20 patients with recurrent (non-gall stone) acute or chronic pancreatitis and confirmed by a 20-week double-blind placebo-controlled crossover trial of the successful combination (daily doses of 600 micrograms organic selenium, 0.54 g vitamin C, 9000 IU B-carotene, 270 IU vitamin E and 2 g methionine) in a further 20 cases. A randomised trial of glutathione precursors, given intravenously for 24 hours after admission in patients with a first attack of acute pancreatitis, is in progress. Long-term trials of oral antioxidant formulas are planned in patients with cystic fibrosis.

Modipafant, a new PAF antagonist: pharmacokinetics and disposition in rat, dog and man.

1. The pharmacokinetics and disposition of modipafant, a dihydropyridine PAF antagonist, were studied in rat and dog following intravenous and oral administration of the drug or its radiolabelled analogue. In addition, the pharmacokinetics were studied in man following single administration of escalating oral doses of the drug. Modipafant is a lipophilic weak base with log D(octanol) 7.4 and pKa of 4.3 and 5.3 respectively. 2. Following intravenous administration of [14C]-modipafant to rat, radioactivity is rapidly distributed throughout the body, except for the brain. A significant amount of radioactivity (probably modipafant) is rapidly distributed to the alimentary tract, particularly in the stomach. This is believed to be due to 'ion trapping' of modipafant in the acidic environment of the upper GI tract. The re-circulated modipafant may be subject to reabsorption and/or faecal excretion. 3. Following intravenous administration to rats, systemic clearance is five times greater in the male than female. The magnitude of this difference is in keeping with the clearance of other dihydropyridines such as nilvadipine. In dog, the clearance values are similar for both sexes, as expected. In this latter species, the systemic clearance decreases 6-fold with increasing dose size, indicative of saturation of a pathway of metabolism. 4. Following oral administration over a dose range of 1-12 mg/kg, modipafant is incompletely (27-67%) bioavailable in rat and dog. In the male dog, systemic exposure to drug (AUC/infinity) increased non-linearly with dose. Following oral administration to man, absorption was rapid with a mean value for Tmax of 1 h, and Cmax's ranging non-linearly from 90 to 2100 ng/ml following dosing at 12.5 to 150 mg respectively. 5. The elimination of modipafant is characterized by short half-life (mean values for t1/2 range from 1 to 3 h). However, the nature of the receptor kinetics of modipafant (slow offset) means that the drug shows a long duration of action in spite of short pharmacokinetics at pharmacologically relevant doses. 6. Following oral and intravenous administration of 14C-modipafant to rat and dog, the majority of radioactivity (mean 92%) is recovered in the faeces. The excretion of modipafant in rat and dog is characterized by metabolism, mostly to pyridine metabolites, accounting for between 38 and 75% of total clearance, the rest being cleared as unchanged drug.

Theophylline and antipyrine disposition in smoking and non-smoking epileptic subjects.

Theophylline and antipyrine disposition has been compared in smoking epileptic patients, non-smoking epileptic patients and non-smoking healthy volunteers. Although clear differences in drug clearance and half-life were evident as a result of anticonvulsant drug therapy, no effect of smoking was discernible. Thus, additive effects from induction of the hepatic microsomal monooxygenase system in man by anticonvulsant drugs and polycyclic aromatic hydrocarbons (in cigarette smoke) were not evident.

Effect of the platelet-activating factor antagonist UK-74,505 on the early and late response to allergen.

The effect of UK-74,505, a specific platelet-activating factor (PAF) antagonist, on the early (EAR) and late asthmatic response (LAR) to inhaled allergen was studied in a randomized, double-blind, placebo-controlled crossover study. A total of eight adult male atopic asthmatic subjects completed the protocol (one withdrew after screening), all having demonstrated a dual response to inhaled allergen (EAR, > 20% fall in FEV1 between 0 and 1 h; LAR, > 15% fall in FEV1 between 4 and 8 h after challenge). Subjects were studied on 2 days at least 10 days apart. After measurement of baseline FEV1, subjects ingested a single oral dose of 100 mg UK-74,505 or matched placebo (P). Allergen challenge was performed 3 h later and the FEV1 was then measured for 8 h. There was no difference between UK-74,505 and placebo in the maximum percentage change from baseline (+/- SEM) for either EAR or LAR (EAR, UK-74,505 -25.6 +/- 4.8%, P -24.0 +/- 3.3%; LAR, UK-74,505 -20.8 +/- 4.4%, P -25.7 +/- 3.8%). There was no significant difference in the area under the percentage change from baseline FEV1-time curve. Ex vivo platelet aggregation to PAF was measured at 0, 2, 6, 8, and 10 h after the dose. There was marked inhibition of platelet aggregation to PAF for 10 h following UK-74,505 but not placebo (% maximum aggregation to PAF, UK-74,505, -69.9%; P, 0.13%; p = 0.0001). Histamine challenge was performed in five patients the day before and after each study day. There was no significant difference between UK-74,505 and placebo in PD20 to histamine (mean PD20 before and after UK-74,505, 1.31 and 0.96 mumol; P, 1.32 and 1.17 mumol). UK-74,505 did not affect either the EAR or the LAR to inhaled allergen or bronchial responsiveness, despite its potency and long duration of action. This suggests that PAF does not have a major role in the acute response to inhaled allergen.

Inhibitory effect of UK,74505, a potent and specific oral platelet activating factor (PAF) receptor antagonist, on airway and systemic responses to inhaled PAF in humans.

Inhaled PAF provokes bronchoconstriction, causes peripheral blood neutropenia with rebound neutrophilia, and generates urinary production of the bronchoconstrictor eicosanoids, thromboxane (TX)A2, and the cysteinyl leukotrienes. We examined the effects of an oral PAF antagonist UK,74505 on each of these responses to a single 36 micrograms dose of inhaled PAF. In a double-blind randomized placebo-controlled crossover study, 12 normal male subjects inhaled PAF on two consecutive days, 3 and 24 h after intake of two doses of UK,74505 25 mg and 100 mg, or matched placebo (P). After P, inhalation of PAF provoked bronchoconstriction, measured at regular time points for 60 min as a change in sGaw from baseline and computed as area under the curve (AUC), induced a neutropenia at 5 min and rebound neutrophilia at 2 h, and stimulated production of urinary eicosanoids. Bronchoconstriction was maximal at 5 min but had receded at 1 h; (AUC mean [95% Cl]; 20.0 [13.2, 26.8] at 3 h; 11.0 [5.3, 16.6] at 24 h) and was completely abolished by both doses of UK,74505 at 3 h and by the higher 100 mg dose at 24 h. PAF-induced neutropenia and rebound neutrophilia were abolished by both doses of drug; neutropenia at 5 min (expressed as mean [95% Cl] change from baseline; -2.5 x 10(9)/L [-2.9, -2.1] after P; -0.3 [-0.7, 0.1] after 25 mg; 0.1 [-0.3, 0.4] after 100 mg), neutrophilia at 2 h (2.0 [-1.3, 2.6] after P; -0.2 [-0.8, 0.5] after 25 mg; -0.1 [-0.8, 0.5] after 100 mg).(ABSTRACT TRUNCATED AT 250 WORDS)