Effect of cell-seeded hydroxyapatite scaffolds on rabbit radius bone regeneration.

Highly porous hydroxyapatite (HA) scaffolds were developed as bone graft substitutes using a template coating process, characterized, and seeded with bone marrow-derived mesenchymal stem cells (BMSCs). To test the hypothesis that cell-seeded HA scaffolds improve bone regeneration, HA scaffolds without cell seeding (HA-empty), HA scaffolds with 1.5 × 10(4) BMSCs (HA-low), and HA scaffolds with 1.5 × 10(6) BMSCs (HA-high) were implanted in a 10-mm rabbit radius segmental defect model for 4 and 8 weeks. Three different fluorochromes were administered at 2, 4, and 6 weeks after implantation to identify differences in temporal bone growth patterns. It was observed from fluorescence histomorphometry analyses that an increased rate of bone infiltration occurred from 0 to 2 weeks (p < 0.05) of implantation for the HA-high group (2.9 ± 0.5 mm) as compared with HA-empty (1.8 ± 0.8 mm) and HA-low (1.3 ± 0.2 mm) groups. No significant differences in bone formation within the scaffold or callus formation was observed between all groups after 4 weeks, with a significant increase in bone regenerated for all groups from 4 to 8 weeks (28.4% across groups). Although there was no difference in bone formation within scaffolds, callus formation was significantly higher in HA-empty scaffolds (100.9 ± 14.1 mm(3) ) when compared with HA-low (57.8 ± 7.3 mm(3) ; p ≤ 0.003) and HA-high (69.2 ± 10.4 mm(3) ; p ≤ 0.02) after 8 weeks. These data highlight the need for a better understanding of the parameters critical to the success of cell-seeded HA scaffolds for bone regeneration.

The gene expression profile of cardia intestinal metaplasia is similar to that of Barrett's esophagus, not gastric intestinal metaplasia.

The etiology and significance of cardia intestinal metaplasia (CIM) is disputed. CIM may represent a form of Barrett's esophagus due to reflux or could reflect generalized gastric intestinal metaplasia due to Helicobacter pylori. The aim of this study was to utilize gene expression data to compare CIM to Barrett's and gastric intestinal metaplasia. Endoscopic biopsies were classified by endoscopic and histologic criteria as CIM (n= 33), Barrett's (n= 25), or gastric intestinal metaplasia of the antrum or body (n= 18). The squamocolumnar and gastroesophageal junctions were aligned in CIM patients and patients with diffuse gastric intestinal metaplasia were excluded. H. pylori was tested for in the biopsies of all patients. After laser-capture microdissection, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of a panel of nine genes that has been shown to differentiate Barrett's from other foregut mucosa. Cluster analysis with linear discriminant analysis of the expression data was used to classify each sample into groups based solely on similarity of gene expression. Cluster analysis was performed for three groups (CIM vs. Barrett's vs. gastric intestinal metaplasia) and two groups (CIM + Barrett's vs. gastric intestinal metaplasia). There was no difference in H. pylori infection among groups (P= 0.66). Clustering into three groups resulted in frequent misclassification between CIM and Barrett's while misclassification of gastric intestinal metaplasia was uncommon. The CIM and Barrett's groups were then combined for two group clustering and linear discriminant analysis correctly predicted 95% of CIM and Barrett's samples and 83% of gastric intestinal metaplasia samples based on gene expression alone. In conclusion, the gene expression profiles of CIM and Barrett's esophagus were similar in 95% of biopsies and differed significantly from that of gastric intestinal metaplasia. The indistinguishable gene expression profile of CIM and BE suggests that they may share a common etiology in the majority of patients with a similar biology, and calls into question the perception that CIM is an innocuous process.

A phase II study of sequential neoadjuvant gemcitabine plus doxorubicin followed by gemcitabine plus cisplatin in patients with operable breast cancer: prediction of response using molecular profiling.

This study examined the pathological complete response (pCR) rate and safety of sequential gemcitabine-based combinations in breast cancer. We also examined gene expression profiles from tumour biopsies to identify biomarkers predictive of response. Indian women with large or locally advanced breast cancer received 4 cycles of gemcitabine 1200 mg m(-2) plus doxorubicin 60 mg m(-2) (Gem+Dox), then 4 cycles of gemcitabine 1000 mg m(-2) plus cisplatin 70 mg m(-2) (Gem+Cis), and surgery. Three alternate dosing sequences were used during cycle 1 to examine dynamic changes in molecular profiles. Of 65 women treated, 13 (24.5% of 53 patients with surgery) had a pCR and 22 (33.8%) had a complete clinical response. Patients administered Gem d1, 8 and Dox d2 in cycle 1 (20 of 65) reported more toxicities, with G3/4 neutropenic infection/febrile neutropenia (7 of 20) as the most common cycle-1 event. Four drug-related deaths occurred. In 46 of 65 patients, 10-fold cross validated supervised analyses identified gene expression patterns that predicted with >or=73% accuracy (1) clinical complete response after eight cycles, (2) overall clinical complete response, and (3) pCR. This regimen shows strong activity. Patients receiving Gem d1, 8 and Dox d2 experienced unacceptable toxicity, whereas patients on other sequences had manageable safety profiles. Gene expression patterns may predict benefit from gemcitabine-containing neoadjuvant therapy.

Cdx-2 expression in squamous and metaplastic columnar epithelia of the esophagus.

The molecular pathogenesis of Barrett's esophagus is poorly understood. Evidence suggests that at a phenotypic level, the metaplastic process begins with the transformation of squamous epithelium in the distal esophagus to cardiac mucosa, which subsequently becomes intestinalized. The homeobox gene Cdx-2 has been shown to be an important transcriptional regulator of embryonic differentiation and maintenance of adult intestinal type epithelium. We hypothesized that Cdx-2 gene expression levels increase with the phenotypic transformation of normal squamous mucosa to the intestinalized columnar mucosa of Barrett's esophagus. Endoscopic biopsies were obtained at the gastroesophageal junction in patients with symptoms of gastroesophageal reflux disease and classified according to histology: normal squamous mucosa (n = 62), cardiac mucosa (n = 19), oxynto-cardiac mucosa (n = 14), and intestinal metaplasia (n = 15). Duodenal biopsies (n = 26) served as the columnar control. After laser capture microdissection and RNA isolation, gene expression levels of Cdx-2 were measured in each tissue type by quantitative reverse transcription polymerase chain reaction. Consistent with its known function, Cdx-2 gene expression levels were highest in duodenal mucosa and nearly absent in squamous epithelium. There was a stepwise increase in Cdx-2 gene expression from cardiac to Barrett's epithelium (P < 0.001). Expression levels of Cdx-2 in cardiac and oxynto-cardiac mucosa were 40-70 times higher and Barrett's mucosa 400 times higher than that found in squamous epithelium. Relative expression of the homeobox gene Cdx-2, known to induce differentiation of intestinal type epithelium, increases in a stepwise fashion during the phenotypic transformation of distal esophageal squamous mucosa to cardiac columnar mucosa and to the intestinalized columnar mucosa of Barrett's esophagus. Therefore, Cdx-2 may be a potential biomarker to detect the early transition to Barrett's esophagus.

Improved venous return by elliptical, sequential and seamless air-cell compression.

AIM: The risk of deep vein thrombosis (DVT) in the peri-operative period is significant, but can be reduced with the use of mechanical intermittent pneumatic compression (IPC). These devices have reached widespread use in hospitals and have been found to be effective prophylactic measures against DVT. This study evaluates the latest design features of one particular IPC device in comparison to current models. METHODS: Duplex ultrasound scanning was performed on 40 lower extremities of 20 healthy volunteers before and during the application of the IPC device (VenaFlow System, Aircas, NJ, USA. Two hemodynamic parameters were measured, acceleration time from spontaneous baseline venous flow and peak vein velocity. All measurements were obtained by scanning proximal to the saphenofemoral junction in the common femoral vein in both extremities for each subject. Data were obtained from 3 compression cycles and averaged for each extremity. Results were compared with a recent prospective study form our center using a slow-filling and a rapid-filling sequential IPC devices. RESULTS: The medians for spontaneous average peak velocities at rest of the right and left lower extremities were 26 cm/s and 24.1 cm/s. The median augmented peak velocities during the compression cycle of the device in the right and left side were 79.6 cm/s and 79.0 cm/s. This represented a 306.2% increase in average peak velocity on the right side and a 327.8% increase on the left side. The median acceleration time was 305 ms +/- 40 in the left and 310 ms +/- 50 in the right limb. There was no statistically significant difference in the spontaneous and augmented velocities between the right and left lower extremities in each subject. In comparison to existing slow- and rapid-filling IPC devices the VenaFlow System had superior peak velocities and shorter acceleration times. CONCLUSION: The use of elliptical, sequential and rapid-filling compression of the leg with overlapping air-cells produces significant hemodynamic changes in the common femoral vein, which are superior to other sequential slow- or rapid-filling IPC devices. Randomized studies should be performed to determine the efficacy of this new device in DVT prevention.

Current concepts in chronic venous ulceration.

OBJECTIVES: despite numerous reports on the distribution of reflux in patients with venous ulceration, there is no consensus on the contribution of each venous system. This study was performed to evaluate the distribution of reflux in this group of patients. METHODS: a literature search from 1980 to 1998 was performed. Because duplex scanning is the best method for detecting venous reflux, we only included reports that used this diagnostic modality. All studies with less than 30 ulcerated limbs were excluded. Since most reports did not give detailed data on perforator veins, reflux in these veins was combined with the superficial and deep veins. Documented episodes of superficial or deep vein thrombosis were noted. RESULTS: thirteen studies that included 1249 ulcerated limbs fulfilled the inclusion criteria. The mean age of patients was 59 years (95% CI: 54-63, range: 14-93). Reflux was detected in 1153 (92%) of limbs. Reflux confined to the superficial veins alone was seen in 45% of limbs, in the deep veins alone in 12% and in both the superficial and deep veins in 43% of limbs. The overall involvement of the superficial veins was 88% and of the deep veins 56% (p <0. 0001). A documented episode of deep vein thrombosis was reported in only six of the 13 studies and the incidence was found to be 32%. CONCLUSIONS: reflux in the superficial veins is seen in 88% of limbs with venous ulcers (CEAP classes 5 and 6). Isolated superficial vein incompetence is detected in 45%, while reflux in the deep venous system alone is seen in only 12%. These data have significant clinical implications, since reflux in the superficial system can be easily eliminated by excision of the affected veins.

Viscoelastic properties of normal and atherosclerotic carotid arteries.

BACKGROUND: remodelling of the arterial wall occurs with ageing, even in the absence of atherosclerotic risk factors. With increasing age, arteries dilate, thicken, and get stiffer. The aim of this study was to correlate carotid artery stiffness with wall thickness and plaque presence between healthy individuals and patients with early and advanced atherosclerosis. METHODS: twenty healthy volunteers, 40 carotid segments and 90 patients, 174 carotid segments, with vascular disease were included in the study. The carotid artery was imaged longitudinally and measurements of the intimal-medial thickness (IMT) and plaque were obtained. Systolic and diastolic blood pressures were taken from each arm. The carotid artery stiffness (pressure-strain elastic modulus, Ep) was calculated in all sites from the changes in pressure and diameter. M-mode was used to detect the diameter change (systolic to diastolic) over five cardiac cycles. RESULTS: in the healthy volunteers there was no evidence of plaque or increased IMT. The mean IMT was significantly higher in the patients compared to control (0.83+/-0.27 mm vs. 0.54+/-0.08 mm, p <0.0001). The IMT had a poor correlation with Ep at lower thickness (r=0.24, p=0.08) but this association became stronger with increasing thickness (r=0.62, p<0.001). Arterial segments with an IMT 5 0.88 mm became significantly stiffer compared to the controls (p<0.001) and to patients with an IMT<0.88 mm (p <0.01). Carotid Ep was markedly greater in arterial segments with plaques than in those with increased IMT (p <0.001) and the controls (p<0.0001). CONCLUSIONS: carotid wall areas with small increase in IMT have a poor correlation with carotid artery stiffness. The carotid stiffness increases in areas with marked wall thickening and particularly in segments with plaque. The simultaneous study of vessel-wall elastic behaviour with IMT and plaque changes may increase our understanding of atherosclerotic progression and wall remodelling.

Fatal cyst formation after fetal mesencephalic allograft transplant for Parkinson's disease.

OBJECT: In recent years, fetal mesencephalic tissue transplant for the treatment of Parkinson's disease (PD) has been demonstrated to hold promise, but potential complications related to growth of allograft tissue have not been well described. This report explores the development and possible causation of a fatal cyst arising from a fetal transplant in the brain. METHODS: The authors report the case of a 52-year-old woman who underwent bilateral putamenal fetal mesencephalic allograft transplant for PD at another hospital. Twenty-three months later she presented to the authors' institution in a coma. Admission computerized tomography and magnetic resonance (MR) studies revealed a contrast-enhancing mural nodule and associated large cyst arising from the left putamen and causing brainstem compression. Despite surgical decompression of the cyst, the patient did not regain consciousness. Biopsy and autopsy specimens were obtained, along with an analysis of the cyst fluid. Genotyping of the nodule and the patient's peripheral lymphocytes by using polymerase chain reaction-based microsatellite analysis was also performed. Biopsy samples and autopsy histopathological studies showed inflammatory cells, hemosiderin-laden macrophages, and astrocytosis. Scattered neurons and multiple rests of choroid plexus were also noted. The cyst had a thin wall and contained liquid that was identical in composition to cerebrospinal fluid (CSF). Genotyping demonstrated the presence of alleles in the nodule DNA that were not present in lymphocytic DNA, indicating that the nodule contained allograft tissue. CONCLUSIONS: The authors hypothesize that the choroid plexus tissue contained in the allograft resulted in CSF production and cyst formation at the transplant site, ultimately leading to the patient's herniation syndrome. The clinical history and large size of the mural nodule indicate slow growth of this allograft site and cyst over time. This case demonstrates that unusual patterns of tissue growth can occur in the brain after fetal tissue transplant and emphasizes the need for long-term monitoring of posttransplant patients by means of MR imaging. Cell sorting should be considered to ensure transplant of pure neuronal and astroglial populations.

Insulin-like growth factor-I promotes cell proliferation in the absence of modulation of collagen phenotypes and utilizes IRS-1, not PLC-gamma 1, in corneal endothelial cells.

Corneal endothelial cells are differentiated cells and are thus incapable of physiologic regeneration. In a search for a growth factor that would promote optimal proliferation of corneal endothelial cells in the absence of other modulating activities, the effect of insulin-like growth factor-I (IGF-I) on rabbit corneal endothelial cells was studied. In addition, cellular effector molecules responsible for the signal pathway for IGF-I were studied. IGF-I at 50 ng/ml stimulated corneal endothelial cell proliferation after at least 8 h of treatment. IGF-I did not change cell shape of corneal endothelial cells: the cells treated with IGF-I at 50 ng/ml maintained polygonal morphology regardless of the duration of exposure. IGF-I did not alter collagen phenotypes either qualitatively or quantitatively: the treated cells continued to synthesize types IV and VIII collagen, as did the control cells. The steady-state levels of alpha 2(I) collagen RNA and alpha 2(IV) RNA were not altered by IGF-I treatment. Immunohistochemical analysis showed that IGF-I is present in corneal endothelium in vivo, while the underlying Descemet's membrane demonstrated no staining. Corneal endothelial cells also produce IGF binding protein-2 (IGFBP-2), which appears to bind IGF-I that has been introduced exogenously in the medium. Further investigation as to how the signals of IGF-I were transmitted for the biological activities demonstrated that the expression of insulin receptor substrate-1 (IRS-1) is up-regulated by IGF-I treatment, while PLC-gamma 1 expression is not altered by this growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)