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Background Chondrocytes in the growth plate (GP) undergo increases in volume during different cascades of cell differentiation during longitudinal bone growth. The volume increase is reported to be the most significant variable in understanding the mechanism of long bone growth. Methods Forty‐five postnatal Sprague‐Dawley rat pups, 7‐15 days old were divided into nine age groups (P7‐P15). Five pups were allocated to each group. The rats were sacrificed and tibia and metatarsal bones were harvested. Bone lengths were measured after 0, 24, 48, and 72 hours of ex vivo incubation. Histology of bones was carried out, and GP lengths and chondrocyte densities were determined. Results There were significant differences in bone length among the age groups after 0 and 72 hours of incubation. Histological sectioning was possible in metatarsal bone from all age groups, and in tibia from 7‐ to 13‐day‐old rats. No significant differences in tibia and metatarsal GP lengths were seen among different age groups at 0 and 72 hours of incubation. Significant differences in chondrocyte densities along the epiphyseal GP of the bones between 0 and 72 hours of incubation were observed in most of the age groups. Conclusion Ex vivo growth of tibia and metatarsal bones of rats aged 7‐15 days old is possible, with percentage growth rates of 23.87 ± 0.80% and 40.38 ± 0.95% measured in tibia and metatarsal bone, respectively. Histological sectioning of bones was carried out without the need for decalcification in P7‐P13 tibia and P7‐P15 metatarsal bone. Increases in chondrocyte density along the GP influence overall bone elongation.

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 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.

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.

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.

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.

The edible red seaweed Kappaphycus alvarezii or Eucheuma cottonii is commercially cultivated in the pristine tropical seas for carrageenan production. The systemic, cellular, and molecular effects of E. cottonii 50% alcohol extract [seaweed E. cottonii ethanol extract (SECE)] on breast cancer were investigated in a rat model. Mammary tumor was induced by subcutaneously injecting LA7 cells in female rat mammary pads. After 2 weeks of cancer growth, the rats received oral administration of either SECE [150mg/kg body weight (BW) and 300mg/kg BW] or tamoxifen. Electron microscopy imaging results confirmed macrophage activity and hematoxylin and eosin staining indicated that tumor histopathological alterations were restored toward normal structures by the seaweed extract. The extract suppressed tumor development and modulated the immune responses. This was evidenced by the microscopic observations, the increased spleen weight, size, spleen CD19 B cells, and blood immunoglobulin G (IgG) levels. The extract also increased the circulating total white blood cells, lymphocytes, segmented neutrophils count, T cells (CD3), T-helper cells (CD4), cytotoxic T cell (CD8), and nuclear factor-kappa beta expressions. The extract enhanced cancer cell death, by upregulating the Birc5, Chk1, and p53 levels and downregulating the tumor growth cellular Mdm2 (transformed mouse 3T3 cell double minute 2) messenger RNA (mRNA) expression. The extract showed no toxicity at 150mg/kg BW in rats. The lectin-rich SECE showed tumor suppression by enhancing immune responses and upregulating the cancer cell apoptosis mRNA expressions.

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

Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system.

Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n=8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen-thawed SSCs were co-cultured on fresh Sertoli cells (experimental group 1), and frozen-thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co-cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen-thawed testicular cells after 2 weeks of culture had a significantly (P

Background: Currently, there has been extensive research interest for inorganic nanocrystals such as calcium phosphate, iron oxide, silicone, carbon nanotube and layered double hydroxide as a drug delivery system especially in cancer therapy. However, toxicological screening of such particles is paramount importance before use as delivery carrier. In this study we examine the biocompatibility of CaCO3 nanocrystal on NIH 3T3 cell line. Material and Methods: Transmission and field emission scanning electron microscopy (TEM and FESEM) were used for the characterisation of CaCO3 nanocrystals. Cytotoxicity and genotoxic effect of calcium carbonate nanocrystals in cultured mouse embryonic fibroblast NIH 3T3 cell line using various bioassays including MTT, and Neutral red/Trypan blue double-staining assays. LDH, BrdU and reactive oxygen species were used for toxicity analysis. Cellular morphology was examined by scanning electron microscopy (SEM) and confocal fluorescence microscope. Results: The outcome of the analyses revealed a clear rod-shaped aragonite polymorph of calcium carbonate nanocrystal. The analysed cytotoxic and genotoxicity of CaCO3 nanocrystal on NIH 3T3 cells using different bioassays revealed no significance differences as compared to control. A slight decrease in cell viability was noticed when the cells were exposed to higher concentrations of 200 to 400 µg/ml, while increase in ROS generation and LDH released at 200 and 400 µg/ml was observed. Conclusions: The study has shown that CaCO3 nanocrystal is biocompatible and non toxic to NIH 3T3 fibroblast cells. The analysed results offer a promising potential of CaCO3 nanocrystal for the development of intracellular drugs, genes and other macromolecule delivery systems.