• IMMUNE NETWORK
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• v.5 no.3
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• pp.150-156
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• 2005

Background: Dendritic cells (DCs) playa critical role not only in the initiation of immune responses, but also in the induction of immune tolerance. In an effort to regulate immune responses through the modulation of antigen presenting cell (APC) function of DCs, we searched for and characterized APC function modulators from natural products. Methods: DCs were cultured in the presence of propolis components, WP and CP, and then examined for their ability to present exogenous antigen in association with major histocompatibility complexes (MHC). Results: WP and CP inhibited class I MHC-restricted presentation of exogenous antigen (cross-presentation) in a DC cell line, DC2.4 cells, and DCs generated from bone marrow cells with GM-CSF and IL-4. The inhibitory activity of WP and CP appeared to be due not only to inhibition of phagocytic activity of DCs, but also to suppression of expression of MHC molecules on DCs. We also examined the effects of WP and CP on T cells. Interestingly, WP and CP increased IL-2 production from T cells. Conclusion: These results demonstrate that WP and CP inhibit MHC-restricted presentation of exogenous antigen through down-regulation of phagocytic activity and suppression of expression of MHC molecules on DCs.

• Journal of Dental Rehabilitation and Applied Science
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• v.19 no.4
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• pp.281-290
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• 2003

Platelet-rich plasma which is made with the newly developed technique concentrating platelets 3-folds or more is also proven to be very effective method to stimulate and accelerate the healing of bone and soft tissue. This study is aimed to investigate the effect of platelet-rich plasma on the attachment of osteoblast. To evaluate the effect on human, human osteoblast cell line was cultured. Platelet-rich plasma was extracted from the blood of a healthy volunteer. The effect on the attachment was evaluated by MTT assay. To evaluate autocrine and paracrine effect on osteoblast, conditioned medium was made and compared with platelet-rich plasma. By western blot analysis, the expression of fibronectin and vitronectin in experimental groups was examined. The results were as following: The cellular attachment of osteoblast cell line increased depending on the concentration of platelet-rich plasma and conditioned medium. The amount of increasing was similar between two groups. The expression of fibronectin and vitronectin in platelet-rich plasma and conditioned medium is more than control group in western blot analysis. These findings imply that platelet-rich plasma enhance the cellular attachment by inducing fibronectin, vitronectin from osteoblast and maximize the cellular attachment by using the autocrine and paracrine effect of platelet-rich plasma.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.34 no.6
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• pp.675-681
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• 2010

It is well known that mesenchymal stem cell(MSCs) can be differentiated into fibroblasts, chondrocytes, and osteoblasts and that they develop into fibrous tissue, cartilage, or bone, as a result of mechanical stimulation. In this study, we developed a bioreactor system, which is composed of a reactor vessel that provides the required cell culture environment, an environment controlling chamber to control the media, a gas mixer, and a reactor motion control subsystem to apply mechanical stimuli to the cells. For the MSC culture, We used a poly-urethane (PU) scaffold, with a collagen coating to ensure improved cohesion ratio. Then, we transferred the cultivated MSCs in the PU scaffold, cultured the cells in the bioreactor system, and confirmed the proliferation, differentiation, and ossification processes, resulting from mechanical stimuli.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.2
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• pp.87-93
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• 2010

Introduction: In our previous studies, we isolated porcine skin-derived mesenchymal stem cells (pSDMSCs) from the ears of adult miniature pigs and evaluated the pluripotency of these pSDMSCs based on expressions of transcription factors, such as Oct-4, Sox-2, and Nanog. Moreover, the characteristic of mesenchymal stem cells was revealed by the expression of various mesenchymal stem cell markers, including CD29, CD44, CD90, and vimentin. The aim of this study was to evaluate in vivo osteogenesis after maxillary sinus lift procedures with autogenous pSDMSCs and scaffold. Materials and Methods: The autogenous pSDMSCs were isolated from the 4 miniature pigs, and cultured to 3rd passage with same methods of our previous studies. After cell membranes were labeled using a PKH26, $1{\times}10^{7}$ cells/$100{\mu}L$ of autogenous pSDMSCs were grafted into the maxillary sinus with a demineralized bone matrix (DBM) and fibrin glue scaffold. In the contralateral control side, only a scaffold was grafted, without SDMSCs. After two animals each were euthanized at 2 and 4 weeks after grafting, the in vivo osteogenesis was evaluated with histolomorphometric and osteocalcin immunohistochemical studies. Results: In vivo PKH26 expression was detected in all specimens at 2 and 4 weeks after grafting. Trabecular bone formation and osteocalcin expression were more pronounced around the grafted materials in the autogenous pSDMSCs-grafted group compared to the control group. Newly generated bone was observed growing from the periphery to the center of the grafted material. Conclusion: The results of the present study suggest that autogenous skin-derived mesenchymal stem cells grafting with a DBM and fibrin glue scaffold can be a predictable method in the maxillary sinus floor elevation technique for implant surgery.

• Journal of Periodontal and Implant Science
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• v.28 no.4
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• pp.769-786
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• 1998

The purpose of the present study is to examine the effect of cell proliferation and alkaline phosphatase activity in osteoblastic cells and to compare the bone healing ability of rat calvarial defects between the control group and the safflower seed extract treated group. Osteoblastic cells were obtained from calvariae of a fetal rat. Cells were cultured containing DMEM and safflower seed extract ($10^{-6}g/ml$, $10^{-3}g/ml$) at $37^{\circ}$ with 5% $CO_2$ in 100% humidity for 3 days. MTT was performed to examine the viability of the cells, and alkaline phosphatase activity was analyzed to examine the mineralization in vitro. Rat calvarial defects($5{\times}5mm$) in 250g Sprague-Dawly were made using round bur. Rats were administrated with safflower seed extract(0.35g/kg/day) for experimental periods. Calvarial defects were studied histopathologically and immunohistochemically at 1,4, and 8 weeks. High concentration group($10^{-3}g/ml$) of safflower seed extract significantly increased in the cell proliferation and alkaline phos phatase synthesis in osteoblastic cells. The infiltration of inflammatory cells and osteoclastic activities were decreased in the safflower seed extract treated group as compared with control group. Bone maturation was accelerated in the safflower seed extract treated group as compared to control group. No difference in osteoinductive process was observed between the control and the safflower seed extract treated group. Immunohistochemical observation revealed that protein expression of TGF-$\beta$and osteonectin during early healing phase in the safflower seed extract treated group was slightly increased as compared to control group. These results indicate that safflower seed extract promotes the healing process in bony defect of rat calvariae, and retains a potential applicability as an adjuvant therapeutic modality for regeneration of periodontal bony defect.

• Journal of Periodontal and Implant Science
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• v.32 no.1
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• pp.1-12
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• 2002

The periodontal ligament(PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. PDL-specific protein;PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the expression pattern and tissue localization of PDLs22 protein in embryonic and various postnatal stages of developing mouse using immunohistochemical staining. Embryos (E18) and postnatal (P1, P4, P5, P15, P18) were decapitated and the heads were fixed overnight in a freshly prepared solution of 4% paraformaldehyde. Some specimens were decalcified for $2{\sim}4$ weeks in a solution containing 10% of the disodium salt of ethylenediamine-tetraacetic acid (EDTA). Next, tissues were dehydrated, embedded in paraffin and sectioned serially at $6{\mu}m$ in thickness. Polyclonal antiserum raised against PDLs22 peptides, ISNKYLVKRQSRD, were made. The localization of PDLs22 in tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows; 1. Expression of PDLs22 protein was not detected in the tooth germ of bud and cap stage. 2. At the late bell stage and root formation stage, strong expression of PDLs22 protein was observed in developing tooth follicle, osteoblast-like cells, and subodontoblastic cells in the tooth pulp, but not in gingival fibroblasts, ameloblasts and odontoblasts of tooth germ 3. In erupted tooth, PDLs22 protein was intensely expressed in PDL and osteoblast-like cells of alveolar bone, but not in gingival fibroblasts, mature osteocytes and adjacent salivary glands. 4. In the developing alveolar bone and mid-palatal suture, expression of PDLs22 protein was seen in undifferentiated mesenchymal cells and osteoblast-like cells of developing mid-palatal suture, but not in mature osteocytes and chondrocytes. These results suggest that PDLs22 protein may play an important role in the differentiation of undifferentiated mesenchymal cells in the bone marrow and PDL cells, which can differentiate into multiple cell types including osteoblasts, cementoblasts, and PDL fibroblasts. However, more researches should be performed to gain a better understanding of the exact function of PDLs22 protein which related to the PDL cell differentiation.

• Food Science and Preservation
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• v.16 no.5
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• pp.782-788
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• 2009

The genotoxicological safety of gamma-irradiated egg white and yolk was examined to ensure that required safety parameters were met, and in an effort to further apply gamma-irradiation for improvement of the hygienic qualities of eggs. Egg white and yolk were irradiated at 20 kGy, much higher than the legally approved dose (less than 5 kGy), and possible genotoxicity was evaluated using in vitro and in vivo tests. The SOS chromotest employing Escherichia coli PQ37, and a chromosomal aberration test in cultured Chinese hamster lung (CHL) cells, were performed in vitro with or without metabolic activation (S9). An in vivo micronucleus development test was conducted using mouse bone marrow cells. Negative results were obtained in the SOS chromotest. The incidence of chromosomal aberration in CHL cells and the frequency of micronuclear developmentin mouse bone marrow cells treated with irradiated samples were not significantly different from those of non-irradiated controls. Thus, it may be concluded that up to 20 kGy of gamma irradiation applied to egg white and yolk did not show any genotoxic effects under our experimental conditions.

• IMMUNE NETWORK
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• v.5 no.3
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• pp.163-171
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• 2005

Background: To perform the successful dendritic cell-based cancer immunotherapy one of the main issues to be solved is the source of antigen for DC pulsing. Limitations occur by using auto-tumor lysate due to the difficulties obtaining enough tumor tissue(s) quantitatively as well as qualitatively. In this study the possibility of allogeneic tumor cell lysate as a DC pulsing antigen has been tested in mouse melanoma pulmonary me tastasis model. Methods: B16F10 melanoma cells $(1{\timeS}10^5/mouse)$ were inoculated intra venously into the C57BL/6 mouse. Therapeutic DCs were cultured from the bone marrow myeloid lineage cells with GM-CSF and IL-4 (1,000 U/ml each) for 7 days and pulsed with lysate of either autologous B16F10 (B-DC), allogeneic K1735 (C3H/He origin; K-DC) or CloneM3 (DBA2 origin; C-DC) melanoma cells for 18 hrs. Pulsed-DCs $(1{\times}10^6/mouse)_{[CGP1]}$ were injected i.p. twice with one week interval starting from the day 1 after tumor cell inoculation. Results: Without observable toxicity, allogeneic tumor cell lysate pulsed-DC induced the significantly better anti-tumor response (tumor scale: $2.7{\pm}0.3,\;0.7{\pm}0.3\;and\;0.3{\pm}0.2$ for saline, B-DC and C-DC treated group, respectively). Along with increased tumor specific lymphocyte proliferations, induction of IFN-${\gamma}$ secretion against both auto- and allo-tumor cell lysates was observed from the DC treated mice. (w/B16F10-lysate: $44.97{\pm}10.31,\;1787.94{\pm}131.18,\;1257.15{\pm}48.27$, w/CloneM3 lysate: 0, $1591.13{\pm}1.83,\;1460.47{\pm}86.05pg/ml$ for saline, B-DC and C-DC treated group, respectively) Natural killer cell activity was also increased in the mice treated with tumor cell lysate pulsed-DC ($8.9{\pm}_{[CGP2]}0.1,\;11.6{\pm}0.8\;and\;12.6{\pm}0.7%$ specific NK activity for saline, B-DC and C-DC treated group, respectively). Conclusion: Conclusively, promising data were obtained that allogeneic-tumor cell lysate can be used as a tumor antigen for DC-based cancer immunotherapy.

• Journal of Korean Biological Nursing Science
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• v.1 no.1
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• pp.25-41
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• 1999

Physiological activity of osteoblast including bone formation is known to be closely related to the increase of intracellular $Ca^{2+}$ activity($[Ca^{2+}]_i$) in osteoblast. $Ca^{2+}$ is an important intracellular messenger in diverse cellular functions, and regulation of its level is mediated by the transmembrane $Ca^{2+}$ movement via $Ca^{2+}$ channels, $Na^+-Ca^{2+}$ exchange, and by intracellular $Ca^{2+}$ movement through the intracellular stores. The purpose of this study is to investigate how the intracellular $Ca^{2+}$ is regulated in osteoblast-like cells(OLCs) by measuring $Ca^{2+}$ activity with cell imaging technique. OLCs were isolated from femur and tibia of neonatal rats, and cultured for 7 days. Cultured OLCs were loaded with a $Ca^{2+}$-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored with a cooled CCD camera. The images were processed and analyzed with an image analyzing software. The results were as follows. (1) $[Ca^{2+}]_i$ of OLC decreased as the $Ca^{2+}$ concentration in the superfusing Tyrode solution was lowered. When $Na^+$ concentration in the superfusing solution was decreased, $[Ca^{2+}]_i$ increased.. These suggest that $Ca^{2+}$ flux occurs via the $Na^+-Ca^{2+}$ exchange mechanism. (2) When $Na^+$ in the superfusing solution was removed. a transient $Ca^{2+}$, increase($Ca^{2+}$ spike) was occasionally observed. However, $Ca^{2+}$ spike was not observed after adding 1 ${\mu}M$ thapsigargin. This implies that the generation of $Ca^{2+}$ spike is mediated by the release of $Ca^{2+}$ from endoplasmic reticulum(ER). (3) As the $Ca^{2+}$ concentration in the superfusing solution was raised, the frequency of 0mM $Na^+$-induced $Ca^{2+}$ spike increased, suggesting that $Ca^{2+}$-induced $Ca^{2+}$ release(CICR) mechanism exists. (4) After $[Ca^{2+}]_i$ was decreased with the superfusion of $Ca^{2+}$-free solution containing thapsigargin, the recovery of $[Ca^{2+}]_i$ with reperfusion of 2.5mM $Ca^{2+}$ solution transiently exceeded the control level, suggesting that the depletion of $Ca^{2+}$ in ER induces $Ca^{2+}$ influx from extracellular medium via store-operated $Ca^{2+}$ influx(SOCI) mechanism. (5) $[Ca^{2+}]_i$ was not affected by the superfusion of 25mM $K^+$ Tyrode solution. These results suggest that intracellular $Ca^{2+}$ activity in osteoblast is regulated by transmembrane $Ca^{2+}$ flux via $Na^+-Ca^{2+}$ exchange, $Ca^{2+}$ release from the internal store (ER) via $Ca^{2+}$-induced $Ca^{2+}$ release, and store-operated $Ca^{2+}$ influx across the cell membrane.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.11-14
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• 2010

Human tumors, including those of the hepatobiliary system, express a number of specific antigens that can be recognized by T cells, and may provide potential targets for cancer immunotherapy. Dendritic cells (DCs) are rare leucocytes that are uniquely potent in their ability to capture, process and present antigens to T cells. The ability to culture sufficient numbers of DCs from human bone marrow or blood progenitors has attracted a great deal of interest in their potential utilization in human tumor vaccination. $CD34^+$ peripheral blood stem cells (PBSCs) were obtained from a patient with a hepatocellular carcinoma. The PBSCs were cultured in the X-VIVO 20 medium supplemented with the Flt-3 Ligand (FL), GM-CSF, IL-4 and TNF-$\alpha$ for 12 days. The morphology and functions of the cells were examined. The generated cells had the typical morphology of DCs. When the DCs were reinjected into the same patient, an augmentation of the cytotoxic T lymphocyte (CTL) activity was observed. Concomitantly, an increase in the natural killer (NK) cell activity was also detected in the patient. These results suggest that DCs-based cancer immunotherapy may become an important treatment option for cancer patients in the future.