Search

Background Type 1 diabetes mellitus is a devastating disease for which there is currently no cure, but only lifetime management. Islet xenotransplantation is a promising technique for the restoration of blood glucose control in patients with diabetes mellitus. The purpose of this study was to explore the potential use of caprine (goat) islet cells as xenogeneic grafts in the treatment for diabetes in a mouse model. Methods Caprine pancreases were harvested and transported to the laboratory under conditions optimized to prevent ischemia. Islets were isolated, purified, and tested for functionality. Caprine islets (2000 islet equivalent) were transplanted beneath the kidney capsules of diabetic BALB/c mice under thalidomide-induced immunosuppression. Blood glucose and insulin levels of grafted mice were evaluated by glucometer and enzyme-linked immunosorbent assay kit, respectively. The functionality and quality of caprine pancreatic islet grafts were assessed by intraperitoneal glucose tolerance tests. Results The viability of purified islet cells exceeded 90%. Recipient mice exhibited normoglycemia (

In this study, isolated spermatogonial stem cells (SSCs) and Sertoli cells using enzymatic digestion from patients with maturation arrest of spermatogenesis were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum in three different groups: (1) SSCs cultured without Sertoli cells (2) SSCs co-cultured with Sertoli cells (as control group), (3) SSCs co-cultured with Sertoli cells and adding different concentrations of basic fibroblast growth factor (0.1, 1, 10 ng ml(-1)) and human leukaemia inhibitory factor (1000, 1200, 1500 unit ml(-1)) as experimental groups. The assessment of colonies every 10 days during 5-week cultures showed that in the first group, the average number and diameter of the colonies were significantly lower than in the other groups (P < 0.05). The largest number of colonies was observed in control condition (32.29 ± 9.15) in day 30. The largest diameter of colonies was formed in combination dosages of 1 ng ml(-1) basic fibroblast growth factor (bFGF) + 1500 unit ml(-1) leukaemia inhibitory factor (LIF) (302.93 ± 37.68) and 10 ng ml(-1) bFGF and 1200 unit ml(-1) LIF (262.87 ± 35.54) in day 30 respectively. Isolated SSCs were positive for spermatogonial cell markers such as Oct4, Stra8, Piwil2 and Vasa but negative for Nanog. Transplantation technique indicated that hSSCs have good efficiency for colonisation of mouse seminiferous tubules after proliferation in culture system.

The study was conducted to investigate the effect of bone tissue engineering comprised of combination of bone marrow-derived osteoblasts and corticocancellous bone graft in repairing 15 mm compact bone critical sized defect. Implant of corticocancellous bone graft alone served as group I. For the second group, bone marrow-derived osteoblasts seeded into scaffold of corticocancellous bone graft and the third group with no treatment served as the control group. Rabbits were euthanized on the fifth (only in group II) and the eighth week (all groups) postoperative. Radiographs were taken on the weekly interval. After euthanasia at the fifth and eighth weeks, the implants were harvested for, gross, histology and scanning electron microscope observations. In group I, new bone formation was only observed at the margin of defect and in the defect centre, hypertrophied chondrocytes was detected and major bone formation pattern was creeping substitution. New bone formation at margin of defect and osteogenesis at th...

The main aim of the present study was to investigate the effect of combination of bone marrow as the primary origin of osteoblast and at the same time as the seed cell and corticocancellous bone graft as the natural scaffold in repair of compact bone full thickness segmental critical sized defect in rabbits. Twelve rabbits had been divided into two groups; In Group one, fresh autogenous bone marrow aspirate has been seeded into the scaffold of autogenous corticocancellous bone graft which was utilized to repair critical size compact bone defect in mid shaft of radius. Corticocancellous bone graft alone was used as the Group 2 or control group. Up to 8 weeks, radiographs were taken to evaluate the level of osteogenicity in both groups. Rabbits were euthanized on week eight postoperative and the implants were harvested for gross, histological and scanning electron microscope observations. New bone formation and osteogenesis was observed at the margins and centre of the Group 1. Combination of mature and immature trabecullae covered the defect and bone formation pattern included osteogenesis, osteoinduction and osteocunduction. In the implant of corticocancellous bone graft alone or group 2, the major new bone formation was at the margins of the defect and osteogenesis was not observed at the centre of the defect and the major bone formation pattern was creeping substitution. As the conclusion, combination of bone marrow and corticocancellous bone graft had better results than corticocancellous bone graft alone in osteogenesis potential. Bone formation capability and critical sized defect repair was faster and more efficient and successful in Group 2 defect.

Exogenesis (cross-species) germ cell transplantation provides an opportunity to investigate fundamental aspects of spermatogenesis. In this study, testis biopsies of patients with maturation arrest of spermatogenesis during a one year ago were first minced mechanically into small pieces and then Spermatogonial Stem Cells (SSCs) and Sertoli cells isolated by the two- step enzymatic digestion, were plated and grown on DSA-Lectin coated dishes in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum. Transplantation of human spermatogonial cells into mouse recipient testis was performed on day 7 (before colony formation) and 2 weeks after culturing (colony formation). The effects of different concentrations of spermatogonial cell on quantity of transplantation and percent of colonized seminiferous tubules were assayed during 8 weeks after transplantation. The result showed that SSCs can be observed on the basement membrane of the seminiferous tubules in place of spermatogonial stem cells and proliferation occurs about 4 weeks after transplantation. The difference in donor cells concentration had more effect on colonization of mouse recipient testis (p

Hani H, TengkuAzmi TI, Abas MO, Mohd-Azmi ML, Zeenathul NA. Isolation, density purification, and in vitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study. Xenotransplantation 2010; 17: 469–480. © 2010 John Wiley & Sons A/S. Abstract: Background: Insufficient availability of human donors makes the search for alternative source of islet cells mandatory for future developments in pancreatic transplantation. The present study investigates the potential of caprine as an alternative source of pancreatic islets. The objectives of the study were to optimize techniques for caprine islet isolation and purification for culture establishment, and to subsequently assess their viable and functional potential. Methods: Caprine pancreatic tissues were collected from a local slaughterhouse and prior transported to the laboratory by maintaining the cold chain. Islets were obtained by a collagenase-based digestion and optimized isolation technique. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by staining methods and demonstrated 90% viability in the culture system. Following static incubation, an in vitro insulin secretion assay was carried out and analyzed by ELISA. Results: The islets remained satisfactorily viable for 5 days in the culture system following regular media changes. The current study has successfully optimized the isolation, purification and culture maintenance of caprine islets. Conclusion: The successful yield, viability and functionality of islets isolated from the optimized protocol provide promising potential as an alternative source of islets for diabetes and transplantation researches.

Zerumbone (ZER), a monosesquiterpene found in the subtropical ginger (Zingiber zerumbet Smith), possesses antiproliferative properties to several cancer cells lines, including the cervical, skin and colon cancers. In this study, the antitumourigenic effects of ZER were assessed in rats induced to develop liver cancer with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and dietary 2-acetylaminofluorene (AAF) (0.02%). The rats also received intraperitoneal ZER injections at 15, 30 or 60 mg/kg body wt. twice a week for 11 weeks, beginning week four post-DEN injection. The hepatocytes of positive control (DEN/AAF) rats were smaller with larger hyperchromatic nuclei than normal, showing cytoplasmic granulation and intracytoplasmic violaceous material, which were characteristics of hepatocarcinogenesis. Histopathological evaluations showed that ZER protects the rat liver from the carcinogenic effects of DEN and AAF. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AP) and alpha-fetoprotein (AFP) were significantly lower (P < 0.05) in ZER-treated than untreated rats with liver cancer. The liver malondialdehyde (MDA) concentrations significantly (P < 0.05) increased in the untreated DEN/AAF rats indicating hepatic lipid peroxidation. There was also significant (P < 0.05) reduction in the hepatic tissue glutathione (GSH) concentrations. The liver sections of untreated DEN/AAF rats also showed abundant proliferating cell nuclear antigen (PCNA), while in ZER-treated rats the expression of this antigen was significantly (P < 0.05) lowered. By the TUNEL assay, there were significantly (P < 0.05) higher numbers of apoptotic cells in DEN/AAF rats treated with ZER than those untreated. Zerumbone treatment had also increased Bax and decreased Bcl-2 protein expression in the livers of DEN/AAF rats, which suggested increased apoptosis. Even after 11 weeks of ZER treatment, there was no evidence of abnormality in the liver of normal rats. This study suggests that ZER reduces oxidative stress, inhibits proliferation, induces mitochondria-regulated apoptosis, thus minimising DEN/AAF-induced carcinogenesis in rat liver. Therefore, ZER has great potential in the treatment of liver cancers.

Restoring skeletal integrity and bone tissue regeneration is still a significant challenging issue. In this regard, bone grafting have been used to augment orthopedic repairs in human and veterinary surgery for several decades and still being under many investigation to hunt for new approaches to improve bone healing following incidences of bone complications. Bone graft is bone transplant and is categorized into autogenous and allogenic grafts as well as synthetic bone graft which are bone graft substitutes. Each of these classified grafts have some advantageous as well as a range of drawbacks, which researchers are still looking to remove those disadvantageous. Finding new instruments and new sites for graft harvests are the major concerns of researchers to diminish the morbidities of donor site in autografts. Looking for agents boosting inductivity of the allografts is the main worries of these kinds of grafting materials and finally new fabrication techniques by new pore sizes are the significant bothering for synthetic bone graft substitutes. This review would consider all grafting methods and materials that would open new windows to the bone grafting techniques.

Osteoporosis is a metabolic bone disorder, which is characterized by a decreased in the overall amount of bone mass. This is major public problem with 30% mortality rate within the first year following osteoporotic hip fracture. The altered microarchitectural structure of bone would lead to increased susceptibility to fractures. By improving the public health and aging of the population, the incidence of this disorder is also increasing. Different treatments costs of patients is showing an increasing trend, which implies that there should be a public education regarding this threatening abnormality. Prevention rather than treatment has to be publicized by correct instructing the population and especially in the women society. This review has a brief simple but concise outlook at osteoporosis its manifestation, treatment and prevention.

Background Pancreatic islets are composed of different hormone-secreting cell types. A finely balanced combination of endocrine cells in the islets regulates intraportal vein secretions and plasma nutrient levels. Every islet cell type is distinguished by its specific secretory granule pattern and hormone content, endocrine and cell signaling mechanisms, and neuronal interactions. The scarcity of pancreatic islet donors for patients with diabetes has caused a considerable interest in the field of islet xenotransplantation. Previous studies have shown that cell arrangement in the pancreatic islets of ruminants differs from that of other mammals; however, caprine islet cytoarchitecture has not yet been comprehensively described. This investigation aimed to characterize caprine islets in regard to better understanding of caprine islet structure and compare with previously reported species, by conducting a detailed analysis of islet architecture and composition using confocal microscopy and immunofluorescence staining for pancreatic islet hormones. Methodology After collection and purification of caprine islets with Euro-Ficoll density gradients, islets were considered for viability and functionality procedures with DTZ (dithizone) staining and GSIST (glucose-stimulated insulin secretion test) subsequently. Batches of islet were selected for immunostaining and study through confocal microscopy and flow cytometry. Results Histological sections of caprine pancreatic islets showed that α-cells were segregated at the periphery of β-cells. In caprine islets, α- and δ-cells remarkably were intermingled with β-cells in the mantle. Such cytoarchitecture was observed in all examined caprine pancreatic islets and was also reported for the islets of other ruminants. In both small and large caprine islets ( 150 μm in diameter, respectively), the majority of β-cells were positioned at the core and α-cells were arranged at the mantle, while some single α-cells were also observed in the islet center. We evaluated the content of β-, α-, and δ-cells by confocal microscopy (n = 35, mean ± SD; 38.01 ± 9.50%, 30.33 ± 10.11%, 2.25 ± 1.10%, respectively) and flow cytometry (n = 9, mean ± SD; 37.52 ± 9.74%, 31.72 ± 4.92%, 2.70 ± 2.81%, respectively). Our findings indicate that the caprine islets are heterogeneous in cell composition. The difference could be attributed to species-specific interaction between endocrine cells and blood. Conclusions Comparative studies of islet architecture may lead to better understanding of islet structure and cell type population arrangement. These results suggest the use of caprine islets as an addition to the supply of islets for diabetes research.

Characteristics of Human Amniotic Epithelial Cells and Bone Marrow Mesenchymal Stem Cells in a Parallel Study: Hanging Drop Colony Formation and Doubling Time Amniotic epithelial cells exhibit pluripotent capacity with some unique properties. They are considered as a good candidate in regenerative medicine. Human bone marrow mesenchymal stromal cells (hBM-MSC) are the gold standard in cell therapy procedures, but whether these cells could be replaced with hAEC remains a debate? We compared the stem cell characteristics of hAEC with hBM-MSC considering isolation, morphology, population doubling time, colony formation on substratum and in suspension, alkaline phosphatase activity and expression of some CD Markers. hAEC were round and cubical but hBM-MSC appeared spindle-shaped in culture. Both groups of cells expressed mesenchymal stem cells markers (CD44, CD90) and they did not express CD34. OCT4 was highly expressed in hAEC but not in hBM-MSC. Their proliferation rate was slower than hBM-MSC. Either of the cell groups could form colonies on substratum as well as suspension with a positive alkaline phosphatase activity. Our results showed that biological properties of human amniotic epithelial cells are to some extent similar to human bone marrow mesenchymal cells.

Background Dead islets replaced with viable islets are a promising offer to restore normal insulin production to a person with diabetes. The main reason for establishing a new islet source for transplantation is the insufficiency of human donor pancreas while using xenogeneic islets perhaps assists this problem. The expression of PDX1 is essential for the pancreas expansion. In mature β-cells, PDX1 has several critical roles such as glucose sensing, insulin synthesis, and insulin secretion. In this study, we aimed to evaluate the expression of pancreatic duodenal homeobox-1 (PDX1) in treated caprine islets in culture and to assess the protective effects of antioxidant factors on the PDX1 gene in cultured caprine islets. Materials and methods Purified islets were treated with serum-free, serum, IBMX, tocopherol, or IBMX and tocopherol media. Quantitative polymerase chain reaction and Western blotting were carried out to compare the expression levels of PDX1 in treated purified islets cultured with different media. Results Islets treated with IBMX/tocopherol exhibited the highest fold change in the relative expression of PDX1 on day 5 post-treatment (relative expression: 6.80±2.08), whereas serum-treated islets showed the lowest fold changes in PDX1 expression on day 5 post-treatment (0.67±0.36), as compared with the expression on day 1 post-treatment. Insulin production and viability tests of purified islets showed superiority of islet at supplemented serum-free media with IBMX/tocopherol compared to other cultures (53.875%±1.59%). Conclusions Our results indicated that supplemented serum-free medium with tocopherol and IBMX enhances viability and PDX1 gene expression compared to serum-added and serum-free media.

Pancreatic islet transplantation is commonly used to treat diabetes. Cell isolation and purification methods can affect the structure and function of the isolated islet cells. Thus, the development of cell isolation techniques that preserve the structure and function of pancreatic islet cells is essential for enabling successful transplantation procedures. The impact of purification procedures on cell function can be assessed by performing ultrastructure and in vivo studies. Thus, the aim of this study was to evaluate the effect of caprine islets purification procedure on islet cell ultrastructure and functional integrity prior to and post-isolation/purification. The islets were isolated from caprine pancreas by using an optimized collagenase XI-S concentration, and the cells were subsequently purified using Euro-Ficoll density gradient. In vitro viability of islets was determined by fluorescein diacetate and propidium iodide staining. Static incubation was used to assess functionality and insulin production by islet cells in culture media when exposed to various levels of glucose. Pancreatic tissues were examined by using light microscopy, fluorescence microscopy, scanning, and transmission electron microscopy. In vivo viability and functionality of caprine islets were assessed by evaluating the transplanted islets in diabetic mice. Insulin assay of glucose-stimulated insulin secretion test showed that the insulin levels increased with increasing concentration of glucose. Thus, purified islets stimulated with high glucose concentration (25 mM) secreted higher levels of insulin (0.542 ± 0.346 μg/L) than the insulin levels (0.361 ± 0.219, 0.303 ± 0.234 μg/L) secreted by exposure to low glucose concentrations (1.67 mM). Furthermore, insulin levels of recipient mice were significantly higher (p

Liver metastasis is the main cause of mortality related to colorectal cancer. CXCR4 is necessary for the outgrowth of colon cancer micrometastases. In oncology, it has been demonstrated that several human tumors release lactate dehydrogenase (LDH) into the circulation. CXCR4 gene expression and serum LDH levels are often increased in patients with colorectal cancer. Despite technological advances in cancer therapy, five-year overall survival is still around 50%. Therefore, better treatment needs to be developed. RNA interference (RNAi) is a modern and powerful tool for inhibition of gene expression. However, the rate-limiting step in this technology is effective delivery of RNAi agents. We have investigated a novel strategy of CXCR4 siRNA therapy and its effect on serum LDH levels in a BALB/C mouse model of colorectal cancer metastasis to the liver. Hepatic metastasis was established by injecting a CT26.WT mouse colon carcinoma cell line via the tail vein. Our results demonstrated that CXCR4 siRNA/ dextran-spermine nanoparticles achieved high silencing efficiency with low toxicity. Favorable localization of the nanoparticles was confirmed with CXCR4 gene expression in the liver, that was correlated with serum LDH levels. More research will be needed to determine the effect of CXCR4 silencing on serum LDH levels, which may be a useful marker for predicting liver metastasis in colorectal cancer.