BACKGROUND: In the thymus, developing thymocytes continually interact with thymic epithelial cell components. Self MHC restriction of mature T cells are imposed in the thymus through interaction of immature double positive thymocytes and thymic cortical epithelial cells. The site of negative selection, however, is a matter of debate. Through systemic injection of anti-TCR antibody or antigenic peptides, investigators suggested that most of the negative selection occurs in the thymic cortex. But the requirements for negative selection, i.e cellular counterparts and costimulatory molecules are more available in the medulla or cortico-medullary junction rather than in the thymic cortex. METHODS: The direct and indirect pathways of thymocyte death after systemic anti-TCR antibody injection were separated through several experimental systems. B6 mice were either adrenalectomized or sham-adrenalectomized to evaluate the role of endogenous glucocorticoids from adrenal gland. Role of TNF were evaluated through using TNF receptor double knockout mice. RESULTS: We found that without indirectly acting mediators such as TNF-alpha or corticosteroid, double positive thymocyte death were minimal by systemic injection of anti-TCR antibody in TNF receptor double knockout neonatal mice. Also by analyzing neonatal wild-type mice with adoptively transferred mature T cells, only peripheral activation of mature T cells could induce extensive double positive thymocyte death. CONCLUSION: Thus, systemically injected anti-TCR antibody mediated thymocyte death are mostly induced through indirect pathway.
Adrenal Cortex Hormones* ; Adrenal Glands ; Animals ; Epithelial Cells ; Glucocorticoids ; Humans ; Mice ; Mice, Knockout ; Peptides ; Receptors, Tumor Necrosis Factor ; Research Personnel ; T-Lymphocytes ; Thymocytes* ; Thymus Gland ; Tumor Necrosis Factor-alpha

Purpose: . The purpose of this study is to analyze the biomechanical differences in rotation angle-torque according to the type of implant and to compare the effects of contamination and sandblasting on the removal torque value (RTV) of abutment screws. Materials and methods: . For this study, an automated device for tightening and loosening implant screws was developed, and each 15 implant-abutment complexes of external and internal connection type were prepared, divided into three groups according to the surface treatment of the screw:control group (no treatment), experimental group-1 (artificial saliva contamination and chlorhexidine (CHX) rinsing), and experimental group-2 (artificial saliva contamination, CHX rinsing, and subsequent 50 µm Al 2 O 3 sandblasting). FirstRTV was measured for each group, followed by different post-treatment procedures for the screws, and then Second-RTV was measured. During the procedure, the removal torque value and rotation angle vs. time were recorded at a 20 data/s.A standardized protocol was followed for all the tightening and loosening procedures: screw was tightened with 30 Ncm torque and maintained for 5 s, and then rotated in the reverse direction until the torque value reached 0 Ncm. After 10min of rest period, the screws were loosened to initial point. Results: . Statistical analysis of measured data revealed that the internal connection type showed significantly higher First-RTV compared to the external connection type, however, no significant differences in Second-RTV were found. Both experimental groups 1and 2 showed a decrease in RTVs for both implant types, while the control group showed a decrease in RTV only for the internal type. The internal connection type required a significantly larger rotation angle during the tightening and loosening process. Conclusion . Within the limitations of this study, both artificial saliva contamination and CHX cleansing, as well as sandblasting, decreased RTV in both internal and external connection types. Internal connections were more susceptible to the effects of contaminants.

Purpose: . The purpose of this study is to analyze the biomechanical differences in rotation angle-torque according to the type of implant and to compare the effects of contamination and sandblasting on the removal torque value (RTV) of abutment screws. Materials and methods: . For this study, an automated device for tightening and loosening implant screws was developed, and each 15 implant-abutment complexes of external and internal connection type were prepared, divided into three groups according to the surface treatment of the screw:control group (no treatment), experimental group-1 (artificial saliva contamination and chlorhexidine (CHX) rinsing), and experimental group-2 (artificial saliva contamination, CHX rinsing, and subsequent 50 µm Al 2 O 3 sandblasting). FirstRTV was measured for each group, followed by different post-treatment procedures for the screws, and then Second-RTV was measured. During the procedure, the removal torque value and rotation angle vs. time were recorded at a 20 data/s.A standardized protocol was followed for all the tightening and loosening procedures: screw was tightened with 30 Ncm torque and maintained for 5 s, and then rotated in the reverse direction until the torque value reached 0 Ncm. After 10min of rest period, the screws were loosened to initial point. Results: . Statistical analysis of measured data revealed that the internal connection type showed significantly higher First-RTV compared to the external connection type, however, no significant differences in Second-RTV were found. Both experimental groups 1and 2 showed a decrease in RTVs for both implant types, while the control group showed a decrease in RTV only for the internal type. The internal connection type required a significantly larger rotation angle during the tightening and loosening process. Conclusion . Within the limitations of this study, both artificial saliva contamination and CHX cleansing, as well as sandblasting, decreased RTV in both internal and external connection types. Internal connections were more susceptible to the effects of contaminants.

Purpose: . The purpose of this study is to analyze the biomechanical differences in rotation angle-torque according to the type of implant and to compare the effects of contamination and sandblasting on the removal torque value (RTV) of abutment screws. Materials and methods: . For this study, an automated device for tightening and loosening implant screws was developed, and each 15 implant-abutment complexes of external and internal connection type were prepared, divided into three groups according to the surface treatment of the screw:control group (no treatment), experimental group-1 (artificial saliva contamination and chlorhexidine (CHX) rinsing), and experimental group-2 (artificial saliva contamination, CHX rinsing, and subsequent 50 µm Al 2 O 3 sandblasting). FirstRTV was measured for each group, followed by different post-treatment procedures for the screws, and then Second-RTV was measured. During the procedure, the removal torque value and rotation angle vs. time were recorded at a 20 data/s.A standardized protocol was followed for all the tightening and loosening procedures: screw was tightened with 30 Ncm torque and maintained for 5 s, and then rotated in the reverse direction until the torque value reached 0 Ncm. After 10min of rest period, the screws were loosened to initial point. Results: . Statistical analysis of measured data revealed that the internal connection type showed significantly higher First-RTV compared to the external connection type, however, no significant differences in Second-RTV were found. Both experimental groups 1and 2 showed a decrease in RTVs for both implant types, while the control group showed a decrease in RTV only for the internal type. The internal connection type required a significantly larger rotation angle during the tightening and loosening process. Conclusion . Within the limitations of this study, both artificial saliva contamination and CHX cleansing, as well as sandblasting, decreased RTV in both internal and external connection types. Internal connections were more susceptible to the effects of contaminants.

Purpose: . The purpose of this study is to analyze the biomechanical differences in rotation angle-torque according to the type of implant and to compare the effects of contamination and sandblasting on the removal torque value (RTV) of abutment screws. Materials and methods: . For this study, an automated device for tightening and loosening implant screws was developed, and each 15 implant-abutment complexes of external and internal connection type were prepared, divided into three groups according to the surface treatment of the screw:control group (no treatment), experimental group-1 (artificial saliva contamination and chlorhexidine (CHX) rinsing), and experimental group-2 (artificial saliva contamination, CHX rinsing, and subsequent 50 µm Al 2 O 3 sandblasting). FirstRTV was measured for each group, followed by different post-treatment procedures for the screws, and then Second-RTV was measured. During the procedure, the removal torque value and rotation angle vs. time were recorded at a 20 data/s.A standardized protocol was followed for all the tightening and loosening procedures: screw was tightened with 30 Ncm torque and maintained for 5 s, and then rotated in the reverse direction until the torque value reached 0 Ncm. After 10min of rest period, the screws were loosened to initial point. Results: . Statistical analysis of measured data revealed that the internal connection type showed significantly higher First-RTV compared to the external connection type, however, no significant differences in Second-RTV were found. Both experimental groups 1and 2 showed a decrease in RTVs for both implant types, while the control group showed a decrease in RTV only for the internal type. The internal connection type required a significantly larger rotation angle during the tightening and loosening process. Conclusion . Within the limitations of this study, both artificial saliva contamination and CHX cleansing, as well as sandblasting, decreased RTV in both internal and external connection types. Internal connections were more susceptible to the effects of contaminants.

Purpose: . The purpose of this study is to analyze the biomechanical differences in rotation angle-torque according to the type of implant and to compare the effects of contamination and sandblasting on the removal torque value (RTV) of abutment screws. Materials and methods: . For this study, an automated device for tightening and loosening implant screws was developed, and each 15 implant-abutment complexes of external and internal connection type were prepared, divided into three groups according to the surface treatment of the screw:control group (no treatment), experimental group-1 (artificial saliva contamination and chlorhexidine (CHX) rinsing), and experimental group-2 (artificial saliva contamination, CHX rinsing, and subsequent 50 µm Al 2 O 3 sandblasting). FirstRTV was measured for each group, followed by different post-treatment procedures for the screws, and then Second-RTV was measured. During the procedure, the removal torque value and rotation angle vs. time were recorded at a 20 data/s.A standardized protocol was followed for all the tightening and loosening procedures: screw was tightened with 30 Ncm torque and maintained for 5 s, and then rotated in the reverse direction until the torque value reached 0 Ncm. After 10min of rest period, the screws were loosened to initial point. Results: . Statistical analysis of measured data revealed that the internal connection type showed significantly higher First-RTV compared to the external connection type, however, no significant differences in Second-RTV were found. Both experimental groups 1and 2 showed a decrease in RTVs for both implant types, while the control group showed a decrease in RTV only for the internal type. The internal connection type required a significantly larger rotation angle during the tightening and loosening process. Conclusion . Within the limitations of this study, both artificial saliva contamination and CHX cleansing, as well as sandblasting, decreased RTV in both internal and external connection types. Internal connections were more susceptible to the effects of contaminants.

The use of high throughput screening (HTS) in drug development is principally for the selection new drug candidates or screening of chemical toxicants. This system minimizes the experimental environment and allows for the screening of candidates at the same time. Umbilical cordderived stem cells have some of the characteristics of fetal stem cell and have several advantages such as the ease with which they can be obtained and lack of ethical issues. To establish a HTS system, optimized conditions that mimic typical cell culture conditions in a minimal space such as 96 well plates are needed for stem cell growth. We have thus established a novel HTS system using human umbilical cord derived-mesenchymal stem cells (hUC-MSCs). To determine the optimal cell number, hUC-MSCs were serially diluted and seeded at 750, 500, 200 and 100 cells per well on 96 well plates. The maintenance efficiencies of these dilutions were compared for 3, 7, 9, and 14 days. The fetal bovine serum (FBS) concentration (20, 10, 5 and 1%) and the cell numbers (750, 500 and 200 cells/well) were compared for 3, 5 and 7 days. In addition, we evaluated the optimal conditions for cell cycle block. These four independent optimization experiments were conducted using an MTT assay. In the results, the optimal conditions for a HTS system using hUC-MSCs were determined to be 300 cell/well cultured for 8 days with 1 or 5% FBS. In addition, we demonstrated that the optimal conditions for a cell cycle block in this culture system are 48 hours in the absence of FBS. In addition, we selected four types of novel small molecule candidates using our HTS system which demonstrates the feasibility if using hUC-MSCs for this type of screen. Moreover, the four candidate compounds can be tested for stem cell research application.
Cell Count ; Cell Culture Techniques ; Cell Cycle ; Fetal Stem Cells ; Humans ; Hydrazines ; Mass Screening ; Mesenchymal Stromal Cells ; Seeds ; Stem Cell Research ; Stem Cells ; Umbilical Cord

Endothelial cells are a vital constituent of most mammalian organs and are required to maintain the integrity of these tissues. These cells also play a major role in angiogenesis, inflammatory reactions, and in the regulation of thrombosis. Angiogenesis facilitates pulp formation and produces the vessels which are essential for the maintenance of tooth homeostasis. These vessels can also be used in bone and tissue regeneration, and in surgical procedures to place implants or to remove cancerous tissue. Furthermore, endothelial cell regeneration is the most critical component of the tooth generation process. The aim of the present study was to stimulate endothelial regeneration at a site of acute cyclophosphamide (CP)-induced endothelial injury by treatment with human umbilical cord-derived endothelial/mesenchymal stem cells (hEPCs). We randomly assigned 16 to 20-week-old female NOD/SCID mice into three separate groups, a hEPC (1 x 10(5) cells) transplanted, 300mg/kg CP treated and saline (control) group. The mice were sacrificed on days 5 and 10 and blood was collected via the abdominal aorta for analysis. The alanine transaminase (ALT), aspartate aminotransferase (AST), serum alkaline phosphatase (s-ALP), and albumin (ALB) levels were then evaluated. Tissue sections from the livers and kidneys were stained with hematoxylin and eosin (HE) for microscopic analysis and were subjected to immunohistochemistry to evaluate any changes in the endothelial layer. CP treatment caused a weight reduction after one day. The kidney/body weight ratio increased in the hEPC treated animals compared with the CP only group at 10 days. Moreover, hEPC treatment resulted in reduced s-ALP, AST, ALT levels compared with the CP only group at 10 days. The CP only animals further showed endothelial injuries at five days which were recovered by hEPC treatment at 10 days. The number of CD31-positive cells was increased by hEPC treatment at both 5 and 10 days. In conclusion, the CP-induced disruption of endothelial cells is recovered by hEPC treatment, indicating that hEPC transplantation has potential benefits in the treatment of endothelial damage.
Alanine Transaminase ; Alkaline Phosphatase ; Animals ; Aorta, Abdominal ; Aspartate Aminotransferases ; Blood Vessels ; Cyclophosphamide ; Endothelial Cells ; Eosine Yellowish-(YS) ; Female ; Glycosaminoglycans ; Hematoxylin ; Homeostasis ; Humans ; Hypogonadism ; Immunohistochemistry ; Kidney ; Liver ; Mice ; Mitochondrial Diseases ; Ophthalmoplegia ; Regeneration ; Stem Cell Transplantation ; Stem Cells ; Thrombosis ; Tooth ; Transplants ; Weight Loss

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-β3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.
Adult Stem Cells ; physiology ; Aggrecans ; analysis ; Bone Morphogenetic Protein 6 ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Separation ; Chondrogenesis ; drug effects ; physiology ; Collagen Type II ; analysis ; Flow Cytometry ; Glycosaminoglycans ; analysis ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; drug effects ; physiology ; Molar, Third ; cytology ; Periodontal Ligament ; cytology ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; analysis ; Stress, Mechanical ; Tooth, Impacted ; pathology ; Transforming Growth Factor beta3 ; pharmacology

Bone marrow mesenchymal stem cells (BM MSCs) can differentiate into multi-lineage tissues. However, obtaining BM MSCs by aspiration is difficult and can be painful; therefore peripheral blood (PB) MSCs might provide an easier alternative for clinical applications. Here, we show that circulating PB MSCs proliferate as efficiently as BM MSCs in the presence of extracellular matrix (ECM) and that differentiation potential into osteoblast in vitro and in vivo. Both BM MSCs and PB MSCs developed into new bone when subcutaneously transplanted into immune-compromised mice using hydroxyapatite/tricalcium phosphate as a carrier. Furthermore, LY294002 and Wortmannin blocked mesenchymal stem cell attachment in a dose-dependent manner, suggesting a role of phosphatidylinositol 3-kinase in MSC attachment. Our data showed that the growth of PB MSCs could be regulated by interaction with the ECM and that these cells could differentiate into osteoblasts, suggesting their potential for clinical applications.
Animals ; Bone Marrow ; Extracellular Matrix ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Mice ; Osteoblasts ; Phosphatidylinositol 3-Kinase ; Phosphatidylinositols