• Biomolecules & Therapeutics
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• v.6 no.1
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• pp.89-94
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• 1998

CJ50001 is a recombinant human granulocyte colony-stimulating facto, (rHuG-CSF) that stimulates the formation of neutrophils from bone marrow stem cells. It was produced in E. colt and purified through refolding and several processes. We produced CS970125(300) using purified C150001 and additives in order to test the stability of CJ50001. When CS970125(300) was stored at 50'S for more than 1 week, high molecular weight proteins were formed and those proteins were detected by non-reducing SDS-PAGE, gel filtration HPLC, and Western blot. Those proteins showed single band at the same position of CJ50001 in reducing SDS-PAGE. These data indicated that those high molecular weight proteins were the multimers of C150001. In biological assays, iu viro and in viro, the multimers did not have biological activity and inhibitory action to that of CJ 50001. The mutimers did not induce toxicity in mice and rats in acute toxicity test. These results suggest that if Cs970125(300) containing CJ50001 is stored at 5$0^{\circ}C$, CJ50001 will be the multimers that do not have biological activity and inhibitory effect to CJ50001 and do not induce acute toxicity.

• Journal of Microbiology
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• v.40 no.1
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• pp.77-81
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• 2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic hematspoietic growth factor involved in the development of myeloid cells from bone marrow, and an activator of mature myeloid cells functioning in a variety of antimicrobial and inflammatory responses. Recently, recombinant GM-CSF is increasingly under clinical study for treatment of various diseases including cancer, infectious diseases and hematopoietic diseases as well as for an immune response modulator, In this study, we constructed a recombinant human GM-CSF (rhGM-CSF) expression plasmid with a pelB leader sequence and His. Tag under T7 promoter control. The expression construct was shown to produce a recombinant protein of 20 kDa in the 8M urea preparation, indicating the rhGM-CSF may be expressed as an insoluble inclusion form. The 20 kDa recombinant protein in 8M urea was transformed into the water-so1ub1e form by dialysis against PBS buffer (phosphate buffered saline). The soluble rhGM-CSF protein was shown to stimulate colony formation and cell proliferation in vitro, indicating that the rhGM-CSF could be refolded into its native form to show colony stimulating activity.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.135-141
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• 2004

Human granulocyte colony-stimulating factor (hG-CSF) is a hematopoiesis agent that principally affects the differentiation of neutrophils in the bone marrow. At present, recombinant hG-CSF is used successfully in the treatment of chemotheraphy-induced neutropenia and its indication has been expanded to bone marrow transplantation and aplastic anemia. In this study, we have constructed rhG-CSF secretion plasmid pYRC1 in which OmpA signal sequence/hG-CSF gene was expressed under the control of the T7 promoter. rhG-CSF produced in E. coli BL21 (pYRC1) grown at $37{\circ}C$ was found in aggregates. However, 15% of the periplasmic protein was soluble rhG-CSF when the E. coli BL21 (pYRC1) was cultured at $25^{\circ}C$ for 7 h in the modified MBL medium containing 10 g/$\ell$ glucose with 10 $\mu$M IPTG induction. The production of soluble rhG-CSF in E. coli BL21 (pYRC1) using fed batch culture was also studied. In the fed batch culture system, the final yield of rhG-CSF produced from E. coli BL21 (pYRC1) was increased from 4.4 mg/$\ell$to 24 mg/$\ell$by controlling the specific growth rate from $0.43 h^{-1}$ to $0.14 h^{-1}$, and optimizing the time of induction.

• Polymer(Korea)
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• v.27 no.3
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• pp.226-234
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• 2003

Ipriflavone (IP), a non-hormonal isoflavone derivative, has been shown to interfere with bone remodeling by inhibiting bone resorption and stimulating bone formation. IP consistently increased the amount of Ca incorporated into the cell layer by mesenchymal stem cells (MSCs). In this study, we developed the novel IP loaded poly(L-lactide-co-glycolide) (PLGA) scaffolds for the possibility of the application of the tissue engineered bone. IP/PLGA scaffo1ds were prepared by solvent casting/salt leaching method and were characterized by porosimeter, scanning electron microscopy, determination of residual salt amount, differential scanning calorimetry, and X-ray diffractometer, respectively. IP/PLGA scaffolds were implanted into the back of athymic nude mouse to observe the effect of IP on the osteoinduction compared with control PLGA scaffo1ds. Thin sections were cut from paraffin embedded tissues and histological sections were stained H&E, von Kossa, and immunohistochemical staining for Type I collagen and osteocalcin. It can be observed that the porosity was above 91.7% and the pore size was above 101 $\mu\textrm{m}$. Control scaffo1d and IP/PLGA scaffo1ds of 50% IP were implanted on the back of athymic nude mouse to observe the effect of IP on the induction of cells proliferation for 9 weeks. The evidence of calcification, osteoblast, and osteoid from the undifferentiated stem cells in the subcutaneous sites and other soft connective tissue sites having a preponderance of stem cells has been observed. From these results, it seems that IP plays an important role for bone induction in IP/PLCA scaffolds.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.2
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• pp.123-131
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• 2007

Introduction: Distraction osteogenesis is widely used as for bone lengthening in patients with maxillofacial deformity and alveolar bone atrophy. One of the major problems in distraction osteogenesis is long consolidation period for 2-3 months, in which the devices have to be fixed on the bone to prevent relapse. It results in scar formation on the face, disturbance of mastication and speech. This study was performed to evaluate the stimulating effect of pulsed electromagnetic field on the early bone consolidation in distraction osteogenesis. Materials and methods: Total 10 rabbit were used (5 for control group, 5 for experimental group). A vertical osteotomy in the mandibular body was performed and the distraction device was fixed. After 5 days distraction was done 1mm per a day for 7 days. A pulsed electromagnetic field (38 Gauss, 60 Hz) was applied for 8 hours per day and it continued for 5 days immediately after distraction in the experimental group. Both groups were sacrificed after 2 weeks. Histological specimens with H&E and Masson Trichrome staining were made and histomorphometrically analysed with image analyser. Results: The device for distraction osteogenesis was displaced in one animal for each group, therefore, only four animals in both groups were evaluated. In both groups, a new bone formation was observed in the distracted area after 2 weeks. The bone formation was enhanced in the experimental groups ($31.76{\pm}8.68%$) compared with control group ($9.94{\pm}3.23%$), its difference was statistically significant (p<0.001). Conclusion: This study suggests that electrical stimulation with electromagnectic field may be effective in the early bone formation after distraction osteogenesis. Further studies with large number of animals are needed before clinical application.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.27 no.6
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• pp.498-510
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• 2001

Distraction osteogenesis refers to the biological process responsible for new bone formation between bone segments by gradual distraction after osteotomy. For the past several years, various inconveniences including a protracted consolidation period that requires patients to wear a distractor frame longer, as well as higher medical costs, have not been remedied by improvements in osteotomy, distraction rate and monitoring system. Furthermore, side effects such as pin tract infections and soft tissue swelling may arise due to the long treatment period. These drawbacks form the rationale of this study which purports to seek a method by which the consolidation period can be reduced. This paper examines how platelet-rich plasma(PRP), known to facilitate osteogenesis, influences bone formation when applied in distracted area. Ten mongrel dogs, which were made to wear external distractor frames after osteotomy in both sides of the mandible, were used as subjects. After a 7day period of latency, distraction was carried out at a rate of 1mm/day for 14 consecutive days. After the onset of distraction, 2ml of PRP and a mixture of calcium gluconate and thrombine were injected into the center of the distracted callus on the left side of the mandible. The left was injected with PRP while the right side was set as the control site without PRP treatment. Execution at the onset of distraction and in 2 weeks, 4 weeks and 8 weeks after the consolidation period, clinical and radiographic tests, bone mineral density examination, histological examination and histomorphometric analysis were conducted to compare both sides. The results are summarized as follows: 1. Based on the clinical examination at two weeks, more remarkable cortical bone formation was found on the buccal and lingual side of the distracted area in the PRP treatment site than in the control site. No visual difference was found between the PRP treatment site and the control site at four eight weeks. 2. Based on the radiological examination, a distinct increase in the radiopaque appearance of the PRP treatment site was revealed at two weeks, but this increase appeared to slow down at four and eight weeks. 3. Examination of bone mineral density revealed a significant difference at two weeks with the PRP treatment site yielding density two times higher than the control site. This difference lessened after four weeks, and disappeared at eight weeks. 4. The histomorphometric examination revealed that about 20% more bony trabeculae area(20%, higher) was formed in the PRP treatment site than in the control site. In conclusion, it can be said that PRPs effect on stimulating bone formation in the PRP treatment site manifest as early as two weeks. Trabeculae formation likewise increased throughout the whole period. If this result can be applied to humans, the consolidation period can be reduced by injecting PRP into the distracted area.

• Preventive Nutrition and Food Science
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• v.8 no.1
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• pp.46-53
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• 2003

The current study investigated the effect of Korean safflower (Carthamus tinctorius L.) seed powder and its water and ethanol extracts on bone metabolism during recovery from rib-fracture induced by surgical operation in rats. 10-week-old male Sprague-Dawley rats weighing about 320 g were divided into 9 groups after arrival: 10d control (AIN 76 semi-purified diet), 10d safflower seed powder (10d SS-powder), 10d safflower seed ethanol extract (10d SS-EtOH), 10d safflower seed water extract (10d SS-$H_2O$), 20d control (AIN-76 semi-purified diet), 20d safflower heed powder (20d SS-powder), 20d safflower seed ethanol extract (20d SS-EtOH), 20d safflower seed water extract (20d SS-$H_2O$), and 20d sham-operation (20d sham), The dietary level for all the supplements was 5% based on the raw material weight. The rats were fed the experimental diets for 10 days before the rib fracture operation and for a further 10 or 20 days after the operation. A number 9 rib was fractured surgically and a sham-operation also performed. The rats were then sacrificed on the l0th or 20th day after the operation. The body weight initially decreased after the operation in all the rib-fractured groups, then gradually recovered. The concentrations of plasma osteocalcin were higher in the control group than in all the safflower-supplemented groups 10 and 20 days after the rib-fracture (p < 0.05). The bone-specific ALP (alkaline phosphatase) activity was significantly higher in the SS-EtOH group than in the other groups 20 days after the rib-fracture (p < 0.05). The level of urinary DPD (deoxypridinoline) was significantly higher in the SS-EtOH and SS-$H_2O$ groups than in the other groups 10 days after the rib-fracture. When comparing the PTH (parathyroid hormone) and calcitonin levels, the SS-$H_2O$ group exhibited the highest PTH level among the groups 10 and 20 days after the rib-fracture. Thus, it was concluded that the bone turnover during the fracture-healing period was more rapid in the rats supplemented with safflower seed powder or its fractions than in the control rats. Furthermore, the SS-$H_2O$ fraction was identified as the most effective in stimulating bone remodeling, as bone resorption and bone formation were both significantly increased during fracture healing when compared to the control group.

• Molecules and Cells
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• v.39 no.10
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• pp.734-741
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• 2016

Granulocyte-macrophage colony stimulating factor (GM-CSF) has a role in inducing emergency hematopoiesis upon exposure to inflammatory stimuli. Although GM-CSF generated murine bone marrow derived cells have been widely used as macrophages or dendritic cells in research, the exact characteristics of each cell population have not yet been defined. Here we discriminated GM-CSF grown bone marrow derived macrophages (GM-BMMs) from dendritic cells (GM-BMDCs) in several criteria. After C57BL/6J mice bone marrow cell culture for 7 days with GM-CSF supplementation, two main populations were observed in the attached cells based on MHCII and F4/80 marker expressions. GM-BMMs had $MHCII^{low}F4/80^{high}$ as well as $CD11c^+CD11b^{high}CD80^-CD64^+MerTK^+$ phenotypes. In contrast, GM-BMDCs had $MHCII^{high}F4/80^{low}$ and $CD11c^{high}CD8{\alpha}^-CD11b^+CD80^+CD64^-MerTK^{low}$ phenotypes. Interestingly, the GM-BMM population increased but GM-BMDCs decreased in a GM-CSF dose-dependent manner. Functionally, GM-BMMs showed extremely high phagocytic abilities and produced higher IL-10 upon LPS stimulation. GM-BMDCs, however, could not phagocytose as well, but were efficient at producing $TNF{\alpha}$, $IL-1{\beta}$, IL-12p70 and IL-6 as well as inducing T cell proliferation. Finally, whole transcriptome analysis revealed that GM-BMMs and GM-BMDCs are overlap with in vivo resident macrophages and dendritic cells, respectively. Taken together, our study shows the heterogeneicity of GM-CSF derived cell populations, and specifically characterizes GM-CSF derived macrophages compared to dendritic cells.

• Journal of Periodontal and Implant Science
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• v.30 no.4
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• pp.885-894
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• 2000

Deer antler has been widely prescribed in Chinese and Korean pharmacology. Although there have been several reports concerning the effects of deer antler, such as anti-aging action, anti-inflammatory activity, antifungal action and regulatory activity of the level of glucose, the effect on bone has not determined yet. The purpose of this study was to examine the effect of deer antler on osteoblast differentiation. Hexane extract(CN-H) and chloroform extract(CN-C) were acquired from deer antler(Cervus nippon) and MC3T3-E1 pre-osteoblasts were cultured in the presence or absence of each extract. Osteoblast differentiation was estimated with the formation of mineralized nodules and the mRNA expression of alkaline phosphatase(ALP), osteocalcin(OC) and bone sialoprotein(BSP) which are markers of osteoblast differentiation. Non-treated group did not show mineralized nodule. CN-C or CN-H-treated group showed minerlaized nodules in 16 days. In northern blot analysis, CN-C or CN-H-treated group showed the elevated expression of ALP, BSP and OC in 16 days. These results suggest the possibility to develop deer antler as a bone regenerative agent in periodontal therapy by showing the stimulating activity of deer antler on differentiation of osteoblast.

• Biomolecules & Therapeutics
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• v.32 no.2
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• pp.205-213
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• 2024

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.