• Macromolecular Research
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• 제14권5호
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• pp.565-572
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• 2006

Recombinant human bone morphogenic protein-2 (rhBMP-2), which is known as one of the major local stimuli for osteogenic differentiation, was immobilized on the surface of hyaluronic acid (HA)-modified poly$(\varepsilon-caprolactone)$ (PCL) (HA-PCL) scaffolds to improve the attachment, proliferation, and differentiation of human bone marrow stem cells (hBMSCs) for bone tissue engineering. The rhBMP-2 proteins were directly immobilized onto the HA-modified PCL scaffolds by the chemical grafting the amine groups of proteins to carboxylic acid groups of HA. The amount of covalently bounded rhBMP-2 was measured to 1.6 pg/mg (rhBMP/HA-PCL scaffold) by using a sandwich enzyme-linked immunosorbant assay. The rhBMP-2 immobilized HA-modified-PCL scaffold exhibited the good colonization, by the newly differentiated osteoblasts, with a statistically significant increase of the rhBMP-2 release and alkaline phosphatase activity as compared with the control groups both PCL and HA-PCL scaffolds. We also found enhanced mineralization and elevated osteocalcin detection for the rhBMP-2 immobilized HA-PCL scaffolds, in vitro.

• Restorative Dentistry and Endodontics
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• 제37권1호
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• pp.34-40
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• 2012

Objectives: The aim was to confirm the stem cell-like properties of the dental pulp stromal cells and to evaluate the morphologic changes during in vitro chondrogenesis. Materials and Methods: Stromal cells were outgrown from the dental pulp tissue of the premolars. Surface markers were investigated and cell proliferation rate was compared to other mesenchymal stem cells. Multipotency of the pulp cells was confirmed by inducing osteogenesis, adipogenesis and chondrogenesis. The morphologic changes in the chondrogenic pellet during the 21 day of induction were evaluated under light microscope and transmission electron microscope. TUNEL assay was used to evaluate apoptosis within the chondrogenic pellets. Results: Pulp cells were CD90, 105 positive and CD31, 34 negative. They showed similar proliferation rate to other stem cells. Pulp cells differentiated to osteogenic, adipogenic and chondrogenic tissues. During chondrogenesis, 3-dimensional pellet was created with multi-layers, hypertrophic chondrocyte-like cells and cartilage-like extracellular matrix. However, cell morphology became irregular and apoptotic cells were increased after 7 day of chondrogenic induction. Conclusions: Pulp cells indicated mesenchymal stem cell-like characteristics. During the in vitro chondrogenesis, cellular activity was superior during the earlier phase (within 7 day) of differentiation.

• International Journal of Oral Biology
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• 제38권3호
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• pp.121-126
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• 2013

Tumor necrosis factor alpha ($TNF{\alpha}$) is a multifunctional inflammatory cytokine that regulates various cellular and biological processes. Increased levels of $TNF{\alpha}$ have been implicated in a number of human diseases including diabetes and arthritis. Sympathetic nervous system stimulation via the beta2-adrenergic receptor (${\beta}2AR$) in osteoblasts suppresses osteogenic activity. We previously reported that $TNF{\alpha}$ upregulates ${\beta}2AR$ expression in murine osteoblastic cells and that this modulation is associated with $TNF{\alpha}$ inhibition of osteoblast differentiation. In our present study, we explored whether $TNF{\alpha}$ induces ${\beta}2AR$ expression in human osteoblasts and then identified the downstream signaling pathway. Our results indicated that ${\beta}2AR$ expression was increased in Saos-2 and C2C12 cells by $TNF{\alpha}$ treatment, and that this increase was blocked by the inhibition of NF-${\kappa}B$ activation. Chromatin immunoprecipitation and luciferase reporter assay results indicated that NF-${\kappa}B$ directly binds to its cognate elements on the ${\beta}2AR$ promoter and thereby stimulates ${\beta}2AR$ expression. These findings suggest that the activation of $TNF{\alpha}$ signaling in osteoblastic cells leads to an upregulation of ${\beta}2AR$ and also that $TNF{\alpha}$ induces ${\beta}2AR$ expression in an NF-${\kappa}B$-dependent manner.

• 대한소아치과학회지
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• 제25권3호
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• pp.635-648
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• 1998

The clinical use of fluoride with a well known osteogenic action in osteoporotic patients is rational, because this condition is characterized by impaired bone formation. However, its anabolic effect has not been demonstrated well in vitro. The purpose of this study was to investigate the effects of sodium fluoride on the physiological role of osteoblastic cell. Osteoblastic cells were isolated from fetal rat calvaria. The results were as follows : 1. Mineralized nodules were shown in osteoblastic cell cultures, which had been maintained in the presence of ascorbic acid and ${\beta}-glycerophosphate$ up to 21 days. When cultures were treated with pulses of 48 hr duration before apparent mineralization was occurring, 2-fold increased in their number was detected. 2. Alkaline phosphatase activity of osteoblastic cells was inhibited by sodium fluoride in dose dependent manner. 3. The effect of sodium fluoride on the osteoblastic cell proliferation was measured by the incorporation of $[^3H]$-thymidine into DNA. As a result, sodium fluoride at $1{\sim}100{\mu}M$ increased the $[^3H]$-thymidine incorporation into DNA in a dose dependent manner. 4. The signaling mechanism activated by sodium fluoride dose-dependently enhanced the tyrosine phosphorylation of the adaptor molecule $Shc^{p66}$ and their association with Grb2, one of earlier events in a MAP kinase activation pathway cascade used by a significant subset of G protein-coupled receptors. 5. The phosphorylation of CREB(cAMP response element binding protein)was inhibited by the sodium fluoride in MC3T3E1 cells. In conclusion, the results of this study suggested that the mitogenic effect of the sodium fluoride in MC3T3E1 cell was stimulated in a dose-dependent manner and suggested "an important role for the interaction between She and Grb2" in controlling the proliferation of osteoblasts.

• Tissue Engineering and Regenerative Medicine
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• 제15권6호
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• pp.761-769
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• 2018

BACKGROUND: Bioprinting has recently appeared as a powerful tool for building complex tissue and organ structures. However, the application of bioprinting to regenerative medicine has limitations, due to the restricted choices of bio-ink for cytocompatible cell encapsulation and the integrity of the fabricated structures. METHODS: In this study, we developed hybrid bio-inks based on acrylated hyaluronic acid (HA) for immobilizing bio-active peptides and tyramine-conjugated hyaluronic acids for fast gelation. RESULTS: Conventional acrylated HA-based hydrogels have a gelation time of more than 30 min, whereas hybrid bio-ink has been rapidly gelated within 200 s. Fibroblast cells cultured in this hybrid bio-ink up to 7 days showed >90% viability. As a guidance cue for stem cell differentiation, we immobilized four different bio-active peptides: BMP-7-derived peptides (BMP-7D) and osteopontin for osteogenesis, and substance-P (SP) and Ac-SDKP (SDKP) for angiogenesis. Mesenchymal stem cells cultured in these hybrid bio-inks showed the highest angiogenic and osteogenic activity cultured in bio-ink immobilized with a SP or BMP-7D peptide. This bio-ink was loaded in a three-dimensional (3D) bioprinting device showing reproducible printing features. CONCLUSION: We have developed bio-inks that combine biochemical and mechanical cues. Biochemical cues were able to regulate differentiation of cells, and mechanical cues enabled printing structuring. This multi-functional bio-ink can be used for complex tissue engineering and regenerative medicine.

• Ocean and Polar Research
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• 제44권1호
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• pp.29-38
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• 2022

Halophytes are plants that live in harsh environments in coastal regions and are known for their diverse chemical compositions. Limonium tetragonum, a halophyte endemic to Korean shores, is known for its bioactive compounds and is utilized in folk medicine. In this study L. tetragonum extract (LHE) was used to determine and evaluate its anti-osteoporotic properties. Pre-adipocyte and pre-osteoblasts were induced to differentiate along with LHE treatment, and their differentiation was evaluated using differentiation markers. LHE treatment decreased lipid accumulation in 3T3-L1 preadipocytes during adipogenesis. Results indicated that the LHE treatment also decreased the levels of key adipogenic transcription factors: PPARγ, SREBP1c, and C/EBPα. Enhancing osteoblastogenesis by LHE treatment was confirmed in osteoblastogenesis-induced MC3T3-E1 pre-osteoblasts. Cells treated with LHE resulted in increased calcification and alkaline phosphatase (ALP) activity compared with osteoblasts without LHE treatment. Pro-osteogenic and anti-adipogenic effects were also confirmed in D1 murine mesenchymal stromal cells which are capable of differentiation into both adipocytes and osteoblasts. LHE hindered adipogenesis and enhanced osteoblastogenesis in D1 MSCs in a similar fashion. In conclusion, L. tetragonum is believed to possess the potential to be utilized as a nutraceutical ingredient against osteoporotic conditions.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제15권1호
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• pp.63-79
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• 1993

To repair bony defects with tansplanted bone in the body, fresh autogenous bone is undoubtly, the most effective bone graft for clinical applications. But the demineralized bone has the matrix-induced bone formation which was suggested by Urist in 1965. Many authors assisted that demineralized bone powder induces phenotypic conversion of mesenchymal cells into osteoblasts, with high-density bone formation. The process of inducing differentiated cells becomes osteogenic properties. The purpose of this study was to evaluate the osteoinductive capacity of allogenic freeze-dried demineralized bone block (FDD, $7{\times}7mm$) and to compare FDD with the same sue of deep-frozen allogenic bone(DF), fresh autogenous bone (A) after implantation. The histological and ultrastructural features of tissue responses were examined after 1, 2, 4, 6, 8 weeks implantation of each experimental groups in the operative site of the New Zealand white rabbits. The results were as follows : 1. Inflammatory cell infiltration generally has appeared at 1 week, but reduced at 4 weeks in each group, but most severe in DF group. 2. Osteoblastic activity has increased for 4 weeks, but decreased at 6 weeks in each group and there was no significant difference among experimental groups. 3. New bone formation has begun at 1week, least activations in A groups, and showed the revesal line of bone formation among each group at 6 to 8 weeks. 4. Bone resorption has appeared at 1 week, but disappeared at 4 weeks in both A and DF groups, but more severe in DF than A groups. 5. In ultrastructural changs, the DF group have showed the most remarkable osteoclastic activities among experimental groups. 6. Osteoid or tangled collagen fibrils near the implanted sites were replaced by more mature, lamellated bony trabeculae during bone remodeling. There was little difference among each experimental groups. 7. During the convertion osteoblasts to osteocytes which embedded within the bone matrix, there was organ-less-poor cytoplasm, increased nuclear chromatin, abundant rough endothelial reticulum (RER) in each groups. From the above the findings, the DF group shored more bone resorption and foreign body reaction than FDD and A groups, and FDD group showed more new bone formation or osteoblastic activity than DF and A groups in early stage. There was no significant difference of cellular activities among the FDD DF, and A groups according to the time.

• Nutrition Research and Practice
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• 제11권3호
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• pp.190-197
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• 2017

BACKGROUND/OBJECTIVES: Gallus gallus domesticus (GD) is a natural mutant breed of chicken in Korea with an atypical characterization of melanin in its tissue. This study investigated the effects of melanin extracts of GD on osteoblast differentiation and inhibition of osteoclast formation. MATERIALS/METHODS: The effects of the melanin extract of GD on human osteoblast MG-63 cell differentiation were examined by evaluating cell viability, osteoblast differentiation, and expression of osteoblast-specific transcription factors such as bone morphogenetic protein 2 (BMP-2), small mothers against decapentaplegic homologs 5 (SMAD5), runt-related transcription factor 2 (RUNX2), osteocalcin and type 1 collagen (COL-1) by reverse transcription-polymerase chain reaction and western blotting analysis. We investigated the inhibitory effect of melanin on the osteoclasts formation through tartrate-resistant acid phosphatase (TRAP) activity and TRAP stains in Raw 264.7 cell. RESULTS: The melanin extract of GD was not cytotoxic to MG-63 cells at concentrations of $50-250{\mu}g/mL$. Alkaline phosphatase (ALP) activity and bone mineralization of melanin extract-treated cells increased in a dose-dependent manner from 50 to $250{\mu}g/mL$ and were 149% and 129% at $250{\mu}g/mL$ concentration, respectively (P < 0.05). The levels of BMP-2, osteocalcin, and COL-1 gene expression were significantly upregulated by 1.72-, 4.44-, and 2.12-fold in melanin-treated cells than in the control cells (P < 0.05). The levels of RUNX2 and SMAD5 proteins were higher in melanin-treated cells than in control vehicle-treated cells. The melanin extract attenuated the formation of receptor activator of nuclear factor kappa-B ligand-induced TRAP-positive multinucleated RAW 264.7 cells by 22%, and was 77% cytotoxic to RAW 264.7 macrophages at a concentration of $500{\mu}g/mL$. CONCLUSIONS: This study provides evidence that the melanin extract promoted osteoblast differentiation by activating BMP/SMADs/RUNX2 signaling and regulating transcription of osteogenic genes such as ALP, type I collagen, and osteocalcin. These results suggest that the effective osteoblastic differentiation induced by melanin extract from GD makes it potentially useful in maintaining bone health.

• Journal of Oral Medicine and Pain
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• 제26권1호
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• pp.39-50
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• 2001

In order to investigate the effect of Transforming growth factor ${\beta}1$(below TGF-${\beta}1$) and osteogenic protein-1(below Op-1) onto the myogenic differentiation, C2C12 satellite myoblastic cell line was cultured and treated with both growth factors. At first morphological changes with microscopical examination were examined, and isolated total RNA to analyse mRNA expression of bone marker proteins, muscle regulatory proteins, TGF-${\beta}$ receptor and their ligands by Northern blot analysis. And cellular proliferative inducibility of both growth factors was also tested to C2C12 cells. Incubating the cell with $5ng/m{\ell}$ of TGF-${\beta}1$ until 4 days almost inhibited multinucleated myotube formation expressing muscular regulatory proteins, and induced decreasing Id proteins. However, no osteoblastic phenotypes was induced by TGF-${\beta}1$ in C2C12 cells. The mRNA expression of TGF-${\beta}$ receptors with TGF-${\beta}1$ was conversed after 48 hours cultured. Type I TGF-${\beta}$ receptor was seemed to play a role in negative signalling for inhibition of myogenic differentiation. OP-1 dose dependently induced ALP activity, osteopontine production and bone sialoprotein production at concentrations above $100ng/m{\ell}$ and osteocalcin production at concentrations above $300ng/m{\ell}$. The concentration of OP-1 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubation with above $100ng/m{\ell}$ OP-1 suppressed the expression of mRNA for muscular egulatory proteins from 2 days after incubation. Expression of Id-1, 2, 3 mRNA were stimulated by OP-1 at concentration above $300ng/m{\ell}$. When C2C12 cells were treated with both growth factors, TGF-${\beta}1$ potentiated the inhibitory effect of OP-1 on myotube formation and expression of mRNA for myogenin at 12 days. And TGF-${\beta}1$ reduced osteocalcin and bone sialoprotein production induced by OP-1 at 12 days in C2C12 cells. Both growth factor had no mitogenic effect. These results indicate that OP-1 converts the differentiation pathway of C2C12 myoblasts into that of osteoblastic lineage cells and it's not heritable, but TGF-${\beta}1$ does not and has reversible inhibitory activity on the myogenic differentiation. TGF-${\beta}1$ and OP-1 play a role in myogenic differentiation via different mechanism between them.

• 폴리머
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• 제33권1호
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• pp.26-32
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• 2009

골 결손 치료를 위해 생분해성 고분자인 PLGA에 골다공증 치료제인 이프리플라본(IP)을 함유한 미립구를 O/W 유화 용매 증발법으로 제조하였으며, 생체외 방출실험에서 IP 방출량은 HPLC로 분석하였다. SEM을 이용하여 미립구에 부착된 세포의 거동을 확인하였으며, IP가 세포에 미치는 독성평가는 CCK-8 분석방법을 이용하여 측정하였다. 골 형성 지표인 ALP 활성도를 측정하였으며, 배양된 BMSCS의 골세포로의 표현형을 확인하기 위하여 RT-PCR을 수행하였다. 방출결과에서 IP 방출은 거의 40일 이상으로 지속적이었으며, IP를 함유한 미립구에서의 세포의 부착, 성장 등이 잘 이루어짐을 확인하였고, ALP 활성도 및 RT-PCR 분석결과에서도 단독의 PLGA 미립구보다 IP를 함유한 미립구에서의 값이 더 높은 것을 확인할 수 있었다. 본 실험결과를 바탕으로 향후 서방성 제제화에 있어서 IP/PLGA 미립구를 응용하게 되면 국소지향성 골분화 주사용 지지체로서 중요한 역할을 할 것으로 보인다.