Redo Research Articles

Effects of alendronate on adipogenic differentiation of bone marrow stromal cells derived from ovariectomized rats

Objective To explore the effects of alendronate on adipogenic differentiation of bone marrow stremal cells (BMSCs) and the role of mitogen-activated protein kinases (MAPK) signal pathway in this process. Methods BMSCs were derived from 9-month-old ovariectomized SD rats and exposed to 0.01, 0.1, 1, 10 μmol/L of alendronate for 2 weeks. The number of BMSCs was counted under light microscope after oil red O staining. The expression of peroxisome proliferators activated receptor-γ, 2 (PPAR-γ2) was measured by RT-PCR. The effect of alendronate on MAPK signal pathway was detected by Western blot. Results After two weeks of induction of BMSCs by alendronate, BMSCs with positive oil red O staining significantly decreased as the increase of the concentration of alendronate (P <0.01), so did the expression of PPAR-γ2. The expression level of PPAR 2 increased when exposing BMSCs to ERK1/2 or JNK specific inhibitors, PD98059 and SP600125 for two weeks. However, the expression level of PPAR 2 decreased when exposing BMSCs to SB203580 (an inhibitor of p38) for two weeks. Condusion Alendronate can inhibit adipogenic differentiation of BMSCs derived from ovariectomized rats in a doso-dependent manner by activating ERK1/2 and JNK rather than p38. Key words: Alendronate; Bone marrow stromal cells; Mitogen-activated protein kinases; Osteoporosis

Anterior sagittal approach for anorectal malformations in female children: early results.

To determine the technical applicability and early postoperative outcome of anterior sagittal approach for anorectal malformations in female children. Case series. Surgical Unit B of National Institute of Child Health (NICH), Karachi, from April to November 2007. Female patients with congenital anorectal malformation who underwent anorectoplasty through anterior sagittal approach were included in the study. Surgery was done either as primary or staged procedure (with initial colostomy or cut back). Operative details were recorded. Follow-up was done in OPD. Thirty patients with mean age of 11.5 months underwent anorectoplasty through anterior sagittal approach. Eighteen patients had ASARP as a primary procedure. Staged procedure with initial colostomy was done in 9 patients. Initial cut back was done in 2 cases and one redo surgery done. Vaginal tear occurred in one, while partial tear of most distal part of fistula occurred in 4 children. At follow-up, 2 patients with primary ASARP developed wound infection with superficial disruption. Bleeding with wound disruption occurred in one case. Anal mucosal prolapse, anastomotic stricture and recurrent fistula occurred in one patient each. Cosmetic appearance of perineum was good in 10, satisfactory in 5 and poor in 3. Among patients staged with colostomy, bleeding with wound disruption, anal stenosis and retraction occurred in one case each. Cosmetic results were good in 7, satisfactory and poor in one case each. Two patients with initial cut back did not have any complication and one operated for disrupted wound developed disruption again. Anorectoplasty can satisfactorily be done through anterior sagittal approach in females with anorectal malformations. Primary ASARP has almost the same results as staged procedure, which should be done in selected patients.

Adipose tissue engineering with human adipose-derived stem cells and fibrin glue injectable scaffold

To explore the possibility of building tissue-engineered adipose tissue and find a new approach for repairing soft tissue defects. Using enzymatic digestion, adipose tissue-derived stem cells (ASCs) were extracted from the lipid part of human liposuction aspirate, cultured, and underwent adipogenic induction or not. The adipogenic-induced and non-adipogenic-induced ASCs were labeled with 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate (DiI), a fluorescent marker, in vitro to be used as seed cells. Then, they were combined with injectable fibrin glue scaffold at 1 x 10(7)/ml cell density. Six athymic BALB/C mice underwent subcutaneous injection of adipogenic-induced ASCs with fibrin glue scaffold at the density of 1 x 10(7) cells/ml into the left side of the low back (induced group), subcutaneous injection of non-adipogenic-induced ASCs into the right side of the low back (non-induced group), and subcutaneous injection of injectable fibrin glue scaffold into the middle part of the neck (blank control group), with 0.2 ml per injection. Twelve weeks later the mice were killed and the implants were taken out. The wet weight was measured. HE and oil red O staining and light and fluorescence microscopy were used for morphological observation. Adipose tissue-like new-born tissues were found in the injection sites of the induced and un-induced groups. The average wet-weight of the induced group new-born tissue was (28 +/- 15) mg, significantly heavier than that of the un-induced group [(22 +/- 16) mg, P < 0.01]. HE staining and oil red O staining confirmed that the new-born tissue of the induced group was mature adipose tissue and DiI fluorescent staining approved its exogenousness. Most part of the new-born tissues of the un-induced group was fibroid tissue with only a few mature adipose tissues. ASCs extracted from the lipid part after liposuction can be used as seed cells, mixed, after adipose-induction, with injectable scaffold of fibrin glue, and injected into the body to build mature adipose tissue.

Biologic characteristics of rat bone marrow mesenchymal stem cells cultured in vitro

To investigate biological characteristics of rat bone marrow mesenchymal stem cells (MSC) cultured in vitro and to explore their potential applications. MSC were isolated from rat bone marrow by density gradient centrifugation and were induced to differentiation. Flow cytometry was used to characterize their surface antigen expression, cell cycle status and cell growth parameters. Telomerase activity was determined by TRAP-ELISA assay. Fusiform MSC became larger and flattener with increasing passages of culture. After the fourth passage, the MSC showed an immunophenotype of CD29 (94.75% +/- 3.68%), CD71 (95.43% +/- 2.23%), and CD90 (98.08% +/- 3.88%). After the seventh passage, MSC with such immunophenotype decreased with CD29: 50.00% +/- 3.35%, CD71: 50.70% +/- 2.43%, and CD90: 48.60% +/- 2.83%. Cells with such immunoprofile completely disappeared after passage 9. Overall, MSC grew faster during the first 5 passages. The number of MSC in S and G(2)/M phases were 38.36% +/- 2.01% and those in G(0)/G(1) phase were 61.64% +/- 2.13% after 3 passages. The cell growth decreased after passage 7. Percentage of MSCs in S and G(2)/M phases was 10.83% +/- 1.63% and that in G(0)/G(1) was 89.17% +/- 1.96% after passage 12, after which the cells failed to further divide. After passage 9, MSCs lost their ability to differentiate to Von Kossa and oil red O positive staining cells. In addition, telomerase activity of MSC also gradually decreased with the prolonged passages, from the original 52.7% +/- 0.78% to no telomerase activity. The biological and immunophenotypical characteristics of cultured MSC showed obvious alterations with increasing numbers of passage of culture.

Primary culture of human omental preadipocytes and study of their biological properties

To develop a primary culture method of human omental preadipocytes and to study their biological properties, such as hyperplasia, hypertrophy and endocrine secretion of human visceral adipose tissue. Using enzyme-digesting method, fibroblast-like cells from the human omental adipose tissues were cultured. The morphological changes of the cultured cells were observed and the growth curve was drawn by MTT method. The intracytoplasmic lipid of the cultured cells was determined by oil red O staining. The leptin and adiponectin levels in the culture supernatants were measured by ELISA. The cultured fibroblast-like cells were homogeneous. Proliferation of cells began at the 3 rd day and the cell numbers increased in indicial way from the 3 rd day to the 9 th day. The doubling time of cells was about 60 hours. During the process of induction by conditional medium, the cells became round and larger, and more adipose droplets were aggregated. On the 21 st day, more than 90% of the cells became adipocytes. Leptin secretion was detected at low level in the preadipocytes and continuously increased during differentiation, with a peak on day 17. It remained constant from day 17 onward. Unlike leptin, adiponectin secretion was not detected until day 7 after induction, when differentiated adipocytes had already been observed. Its secretion increased dramatically between days 7 and 17, and reached a maximum level on day 17, but had a significant reduction on day 21. Extraction of intracytoplasmic lipid stained with oil red O and detection of leptin and adiponectin both verified the isolated cells were preadipocytes functioning actively. A human omental preadipocytes model has been established and different secretion patterns of leptin and adiponectin secretion related to preadipocyte differentiation has been characterized. Adiponectin may be proposed as a specific marker for preadipocyte differentiation.

Pediatric assist with the Medos and Excor systems in small children.

Pediatric long-term ventricular support with paracorporeal assist devices is performed in only a few institutions. We report on our experience with two pediatric paracorporeal devices, which have been implanted in neonates, infants, and small children. Seven children with ages ranging from 2 weeks to 6 years and a body weight of 3 to 19 kg were provided with either a Medos or a BerlinHeart System. The underlying heart diseases included dilative cardiomyopathy (n = 3), endocardial fibroelastosis (n = 2), Ebstein anomaly, and status post redo aortic valve replacement (n = 1). All children were in New York Heart Association class IV and were inotrope dependent. Three children were provided with a Medos system and 4 children with a BerlinHeart Excor device. In 6 cases, left ventricular support, and in 1 case, right ventricular support was performed. All patients were stabilized with univentricular mechanical support. The perioperative course was uneventful, and end-organ function was well recovered. Reexploration for bleeding and evacuation of mediastinal blood clots was necessary in all three neonates but not in any of the older infants. Severe thromboembolic events were only noticed in the neonates. Successful bridge to transplantation was performed in 6 of the 7 patients (87.5%). Our late results have been quite encouraging, as they readily prove that pediatric long-term mechanical support is possible with a high quality of life and an acceptable low complication rate.

Ross procedure for pediatric aortic valve disease.

Currently, prosthetic selection for aortic valve replacement is still debatable in pediatric patients with aortic valve disease. Ross procedure is a time-consuming and demanding technique which may produce good results. This study assessed the results obtained with various methods of right ventricle outflow tract (RVOT) reconstruction in patients receiving Ross procedure. From September 1996 to December 2003, 13 pediatric patients underwent Ross procedure for aortic valve disease. Their ages ranged from 1 month to 17 years (mean, 6.29 +/- 3.62 years; median, 7 years) and 2 patients were less than 1 year of age. Previous procedures in these patients included balloon dilation of the aortic valve in 10 and surgical aortic valvuloplasty in 2. RVOT was reconstructed with heterografts in 3, homografts in 3 or without extracardiac conduits in 7. There was 1 in-hospital death (7.7%) and 1 late death from a non-cardiac cause. One patient developed infective endocarditis with periaortic abscess 2 weeks after the Ross procedure. Eleven patients were followed for a mean of 3.3 years (range, 8 months to 8 years). There was no significant pressure gradient across the neoaorta. Severe stenosis developed gradually in 3 patients who had RVOT reconstruction with heterograft conduits. All 3 had received RVOT redo operation. None of the patients who received RVOT reconstruction without extracardiac conduits had significant pulmonary stenosis but 6 had regurgitation (nil 1, mild 3, moderate 3). No redo operation was required during follow-up (range, 8 months to 4 years). All of the 11 survivors were in New York Heart Association functional class I. This study found satisfactory results of Ross procedure in pediatric aortic valve disease. RVOT reconstruction without extracardiac conduit is a feasible alternative despite the availability of homograft.

Identification and isolation of human dental pulp stem cells

To isolate and cultivate human dental pulp stem cells (DPSCs). Pulp tissue was removed from healthy young human teeth extracted for orthodontic purposes. The pulp was digested by Type I collagenase and dispase. Then single-cell suspensions were obtained by filter and cultivated. The clones were identified by expression of STRO-1. Under the conditions of inducement, clones were identified by activity of alkaline phosphatase (ALP), formation of mineralized nodule and expression of dentin sialoprotein (DSP), and by Oil Red-O dyeing and expressing of PPARr2. The clones had positive expression of STRO-1. When stimulated to differentiation, these cells took on dramatically high activity of ALP, had the ability of mineralization and expressed DSP. These cells also had ability to trans-differentiate into adipocytes. There are stem cells in human dental pulp tissues, which can be isolated and cultivated.

Limited Left Thoracotomy for Reoperative Coronary Artery Bypass Grafting without Cardiopulmonary Bypass for Circumflex Grafting in Patients with Patent Internal Thoracic Artery Graft.

Abstract Background: We describe our experience with the limited left thoracotomy strategy for reoperative coronary artery bypass graft (CABG)to the circumflex coronary artery system, emphasizing the indications, our particular operative technique, and early clinical follow-up. Methods: From January 2001 to January 2002, 8 consecutive patients underwent redo revascularization via limited left thoracotomy and without cardiopulmonary bypass. This operation was indicated for patients with recurrent myocardial ischemia confined to the lateral wall of the left ventricle, especially if a patent left internal thoracic artery (LITA)-to-left anterior descending coronary artery (LAD)graft was present. Results: All 8 patients underwent successful redo revascularization via limited left thoracotomy. Eight patients received 14 saphenous vein grafts (mean 1.7 grafts/patient). No instances of postoperative myocardial infarction or death occurred. During a follow-up period ranging from 1 to 12 months (mean, 5. 2 months), all patients were asymptomatic and without evidence of ischemia or infarction. Conclusions: For select patients who have patent LITA grafted into the LAD and who need redo CABG to the coronary artery circumflex system, the limited left thoracotomy approach without cardiopulmonary bypass is a safe operation and a less invasive alternative to repeat sternotomy and conventional CABG.

Off-Pump Coronary Artery Bypass Grafts Assessment by Multislice Computed Tomography.

Abstract Background: Selective coronary angiography is the standard but invasive procedure for postoperative assessment of coronary artery bypass graft patency. The aim of this prospective study is to evaluate the multislice computed tomography (CT) as a means of postoperative patency assessment and anastomotic site control of arterial and venous coronary bypass grafts performed with off-pump coronary artery bypass techniques. Methods: Over a 6-month period, 20 patients underwent isolated coronary artery bypass (beating heart technique) and benefited, 7 days later, from a patency and anastomotic site control by multidetector angio multislice CT with cardiac gating. Results: Whole internal thoracic artery bypasses and venous grafts were visualized perfectly on their entire length, including the anastomotic site, and 3-dimensional reconstruction was possible. The relationship between cardiac cavities and the bypasses were well visualized, allowing quantification of bypass stenosis ensured by software analysis. Conclusions: Postoperative control of coronary bypasses is possible by multislice CT with a very satisfactory resolution, thus making it possible to check the patency of coronary bypasses and the quality of anastomosis with a noninvasive method. Three-dimensional reconstructions are very useful in the event of redo surgery.

Preoperative evaluation and postoperative prediction of hemostatic function with thromboelastography in patients undergoing redo cardiac surgery.

Patients who receive cardiac procedures, in particular "redo" ones, often suffer complications from massive bleeding, largely due to bypass-induced coagulopathies. Cardiopulmonary bypass (CPB) may cause damage of the blood components, both in terms of quality and quantity. In order to investigate the qualitative changes of blood constituents with special regard to coagulation resulting from the complex insult of previous cardiac surgery, thromboelastography (TEG) was used to analyze the whole clotting process. Seventy-four patients who underwent cardiac surgery with CPB were prospectively studied. Of them, 32 patients received "redo" cardiac surgery. Blood samples for routine laboratory coagulation tests (RCT) and TEG examination were drawn before and after cardiopulmonary bypass. Clinically significant bleeding was defined if the chest tube drainage was greater than 100 ml/h for 3 consecutive h or greater than 300 ml in 1 h during the first 8 h after surgery. Prebypass and postbypass coagulation parameters were compared and the percentage of accuracy, false positive and false negative rate were deduced from calculation. In the TEG tracings, preoperative alpha angle and maximum amplitude were significantly decreased in the "redo" group when compared with primary group, indicating less competent platelet function and platelet-fibrin interaction. Lower platelet count was also found by conventional coagulation tests in "redo" patients. Postoperatively, higher percentage of excessive hemorrhage was also noted in the "redo" group (42.8% vs. 27.5% in primary group). However, a much lower predictive accuracy was found in "redo" patients in comparison with primary cardiac patients (53.5% vs. 90%). We concluded that thromboelastography failed to predict postoperative hemorrhage in "redo" cardiac patients and the graphic recordings derived could not be treated as a guide of transfusion therapy. We thought that inferior preoperative hemostatic status and severer coagulopathy might be responsible for the differences between "redo" and primary cardiac patients.

Late occlusion of aortofemoral bypass graft: surgical treatment.

Surgical treatment for graft thrombosis is often difficult and its outcome is complicated by a high incidence of rethrombosis. The records of 803 patients (1261 limbs) treated with aortofemoral bypass graft for peripheral vascular disease were reviewed. Among these, 71 patients (63 men and eight women with a mean age of 60.2 years) needed an overall number of 125 reoperations. The mean follow-up was 6.3 years. Smoking was significantly higher in these patients compared with those who were not reoperated on. Intimal hyperplasia was the most common lesion responsible for graft thrombosis. Before 'redo' surgery 20 patients with recent thrombosis received thrombolytic treatment; one was subsequently treated with percutaneous transluminal angioplasty and the others underwent reoperation. The best patency rates were achieved with reconstruction of the distal anastomosis with a graft interposition or with a patch angioplasty. Isolated thrombectomy was complicated by early rethrombosis in 53.3% of cases. The overall amputation rate was 14%, the operative mortality rate 5.6%, and the incidence of complications 16.8%. At follow-up a cumulative secondary patency rate of 81.7% at 5 years (74.5% at 10 years), a limb salvage rate of 80.5%, and a survival rate of 89.2% at 10 years were recorded. It is concluded that the only indications for redo surgery following graft thrombosis are severe limb ischaemia or disabling claudication.

Factor XIIIa expression in juvenile xanthogranuloma.

A method was developed to measure in vitro on human skin the effectiveness of barrier creams against three dyes (eosin, methyl-violet and oil red O) with different n-octanol/water partition coefficients (0.19, 29.8 and 165, respectively). Some galenic properties (water washability, water content and viscosity) of the products were also evaluated to try to understand the mechanisms of such a protection. The barrier creams were assayed by measurements of the dyes in the epidermis of protected skin samples after an application time of 30 min. Whereas some products showed some degree of protection, as claimed on the packaging, we demonstrated in several cases disagreement with the manufacturer's information. Surprisingly, petrolatum was found to provide the best protection of all tested products in our in vitro model. There was no correlation between the galenic parameters of the assayed products and the level of protections, indicating that neither the water content nor the consistence of the formulations influenced the protection effectiveness. In conclusion, regarding the possible skin effects of some irritants, our results stress that barrier creams should be used with caution, knowing the protection limits of some of the formulations marketed.

NAS to redo atomic studies found to be flawed

NAS to redo atomic studies found to be flawed

Cytoplasmic granularity of the renal medullary interstitial cells in experimental hypertension.

Abstract The renal medullary (papillae and medullae) interstitial cells have prominent features suggesting an actively secretory cell. These include the presence of numerous mitochondria, prominent Golgi bodies, smooth-surfaced endoplasmic reticula, and osmiophilic granules which are stained for neutral lipids with oil Red-O. The interstitial cells were studied as a possible production site for the antihypertensive factor-a neutral lipid. Experimental hypertension was induced in Sprague-Dawley rats by administration of excessive sodium chloride or by injections of DOCA or with a combination of both agents. The same agents were administered to separate groups of animals with and without unilateral nephrectomy. The granularity within 100 interstitial cells was evaluated quantitatively in 0.5 μ sections stained with methylene blue and azure II. Granules within interstitial cells were also counted in electron micrographs of 50 cells chosen at random. Hypertension developed in a significant number of animals after administration of DOCA and also after administration of saline with DOCA, in both uninephrectomized and non-nephrectomized animals. Animals were more susceptible to experimental hypertension following unilateral nephrectomy. Ultrastructural studies of the renal medullary interstitial cell in control animals revealed a relatively constant number of cytoplasmic granules (8.8 ± 1.2). In animals undergoing unilateral nephrectomy and given NaCl with DOCA, the osmiophilic granularity was significantly increased (p

The morphologic distribution of intravenously injected fatty chyle and artificial fat emulsion in rats and dogs.

Abstract The distribution of fat in certain organs of rats and dogs was studied primarily by frozen sections stained with oil red O, at intervals from ten minutes to twenty-four hours after rapid intravenous administration of 0.6 to 1.2 Gm. per kilogram of olive oil in emulsified form. This was compared with the distribution of fat following similarly injected fatty dog chyle as well as that of olive oil administered by stomach tube. Olive oil in the amount of 2 ml. given by stomach tube to rats could not be identified in spleen, liver, or lungs. The fat of injected artificial emulsions always appeared by ten minutes after injection in large amounts in the marginal zone of the splenic red pulp. In contrast, the fat of dog chyle was never concentrated here. Within ten minutes after injection of chyle, fat appeared in the parenchymal cells of the liver of dogs; at this interval after artificial emulsion, fat was concentrated in the Kupffer cells. The evidence from rat livers was clouded by variable fat content in the livers of untreated rats. Kupffer cells of rats contained fat only in recently anesthetized animals which had received emulsion; there was some evidence that this fat was passed on to the parenchymal cells. Chyle fat was never concentrated in the Kupffer cells. Lung capillaries of rats were packed with fat ten minutes after injections of artificial emulsion, but at subsequent intervals only scattered droplets remained in the septal macrophages. Chyle fat was never concentrated in the lung. Fat did not appear in significant quantities outside the blood vessels in cervical lymph nodes, kidney, heart, thymus, stomach, duodenum, or ileum after injections of either chyle or emulsion. The sheathed arteries of the dog spleen were sites of a striking concentration of fat in dogs receiving either emulsion or chyle. No concentration of fat could be detected in the rat spleen that would correspond to sheathed arteries. Possible causes of the considerable variation in results are discussed and the need for a labeled fat for future experiments is indicated.