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Biomedical subjects

Ung-il Chung

Publications and source records attributed to Ung-il Chung.

At least 19 recordsLinked to original sources
PubMed2006

Tailor-made tricalcium phosphate bone implant directly fabricated by a three-dimensional ink-jet printer.

Rapid prototyping (RP) is a molding technique that builds a three-dimensional (3D) model from computer-aided design (CAD) data. We fabricated new tailor-made bone implants (TIs) from alpha-tricalcium phosphate powder using an RP ink-jet printer based on computed tomography (CT) data, and evaluated their safety and efficacy. CT data of the skulls of seven beagle dogs were obtained and converted to CAD data, and bone defects were virtually made in the skull bilaterally. TIs were designed to fit the defects and were fabricated using the 3D ink-jet printer with six horizontal cylindrical holes running through the implants, designed for possible facilitation of vascular invasion and bone regeneration. As a control, hydroxyapatite implants (HIs) were cut manually from porous hydroxyapatite blocks. Then, craniectomy was performed to create real skull defects, and TIs and HIs were implanted. After implantation, CT was performed regularly, and the animals were euthanized at 24 weeks. No major side effects were observed. CT analysis showed narrowing of the cylindrical holes; bony bridging between the implants and the temporal bone was observed only for TIs. Histological analysis revealed substantial new bone formation inside the cylindrical holes in the TIs, while mainly connective tissues invaded the porous structures in HIs. Bone marrow was observed only in TIs. Osteoclasts were seen to resorb regenerated bone from inside the cylindrical holes and to invade and probably resorb the TIs. These data suggest that TIs are a safe and effective bone substitute, possessing osteoconductivity comparable with that of HIs.

Animals↗
PubMed2006

Regulation of bone formation by adiponectin through autocrine/paracrine and endocrine pathways.

Since interaction between bone and lipid metabolism has been suggested, this study investigated the regulation of bone metabolism by adiponectin, a representative adipokine, by analyzing deficient and overexpressing transgenic mice. We initially confirmed that adiponectin and its receptors were expressed in osteoblastic and osteoclastic cells, indicating that adiponectin can act on bone not only through an endocrine pathway as a hormone secreted from fat tissue, but also through an autocrine/paracrine pathway. There was no abnormality in bone mass or turnover of adiponectin-deficient (Ad-/-) mice, possibly due to an equivalent balance of the two pathways. In the culture of bone marrow cells from the Ad-/- mice, osteogenesis was decreased compared to the wild-type (WT) cell culture, indicating a positive effect of endogenous adiponectin through the autocrine/paracrine pathway. To examine the endocrine action of adiponectin, we analyzed transgenic mice overexpressing adiponectin in the liver, and found no abnormality in the bone. Addition of recombinant adiponectin in cultured osteoprogenitor cells suppressed osteogenesis, suggesting that the direct action of circulating adiponectin was negative for bone formation. In the presence of insulin, however, this suppression was blunted, and adiponectin enhanced the insulin-induced phosphorylations of the main downstream molecule insulin receptor substrate-1 and Akt. These lines of results suggest three distinct adiponectin actions on bone formation: a positive action through the autocrine/paracrine pathway by locally produced adiponectin, a negative action through the direct pathway by circulating adiponectin, and a positive action through the indirect pathway by circulating adiponectin via enhancement of the insulin signaling.

Adiponectin↗
PubMed2006

Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2.

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Alkaline Phosphatase↗
PubMed2006

Carminerin contributes to chondrocyte calcification during endochondral ossification.

Endochondral ossification is an essential process not only for physiological skeletal development and growth, but also for pathological disorders. We recently identified a novel cartilage-specific molecule, carminerin (also known as cystatin 10 and encoded by Cst10), which is upregulated in synchrony with cartilage maturation and stimulates the later differentiation of cultured chondrocytes. Although carminerin-deficient (Cst10-/-) mice developed and grew normally, they had a microscopic decrease in the calcification of hypertrophic chondrocytes at the growth plate. When we created experimental models of pathological endochondral ossification, we observed suppression of chondrocyte calcification during formation of osteoarthritic osteophytes, age-related ectopic ossification and healing of bone fractures in Cst10-/- mice. Cultured Cst10-/- chondrocytes showed a reduction in calcification with activation of an SRY site in the promoter of the gene encoding nucleotide pyrophosphatase phosphodiesterase 1 (NPP1, encoded by Enpp1). Functional NPP1 is required for carminerin deficiency to suppress the pathological endochondral ossifications listed above. Carminerin is the first cartilage-specific protein that contributes to chondrocyte calcification during endochondral ossification under physiological and pathological conditions through the transcriptional inhibition of NPP1.

Animals↗
PubMed2006

Nkx3.2/Bapx1 acts as a negative regulator of chondrocyte maturation.

Parathyroid hormone-related protein (PTHrP) is essential to maintain a pool of dividing, immature chondrocytes in the growth plate of long bones. In chick and mouse, expression of Nkx3.2/Bapx1 in the growth plate is restricted to the proliferative zone and is down regulated as chondrocyte maturation begins. Nkx3.2/Bapx1 expression is lost in the growth plates of mice engineered to lack PTHrP signaling and, conversely, is maintained by ectopic expression of PTHrP in developing bones. Artificially preventing Nkx3.2/Bapx1 downregulation, by forced expression of either retroviral-encoded PTHrP or Nkx3.2 inhibits chondrocyte maturation. Although wild-type Nkx3.2 blocks chondrocyte maturation by acting as a transcriptional repressor, a ;reverse function' mutant of Nkx3.2 that has been converted into a transcriptional activator conversely accelerates chondrocyte maturation. Nkx3.2 represses expression of the chondrocyte maturation factor Runx2, and Runx2 mis-expression can rescue the Nkx3.2-induced blockade of chondrocyte maturation. Taken together, these results suggest that PTHrP signals block chondrocyte hypertrophy by, in part, maintaining the expression of Nkx3.2/Bapx1, which in turn represses the expression of genes required for chondrocyte maturation.

Animals↗
PubMed2006

Contribution of runt-related transcription factor 2 to the pathogenesis of osteoarthritis in mice after induction of knee joint instability.

OBJECTIVE: By producing instability in mouse knee joints, we attempted to determine the involvement of runt-related transcription factor 2 (RUNX-2), which is required for chondrocyte hypertrophy, in the development of osteoarthritis (OA). METHODS: An experimental mouse OA model was created by surgical transection of the medial collateral ligament and resection of the medial meniscus of the knee joints of heterozygous RUNX-2-deficient (Runx2+/-) mice and wild-type littermates. Cartilage destruction and osteophyte formation in the medial tibial cartilage were compared by histologic and radiographic analyses. Localization of type X collagen and matrix metalloproteinase 13 (MMP-13) was examined by immunohistochemistry. Localization of RUNX-2 was determined by X-Gal staining in heterozygous RUNX-2-deficient mice with the lacZ gene insertion at the Runx2-deletion site (Runx2+/lacZ). Messenger RNA levels of type X collagen, MMP-13, and RUNX-2 were examined by real-time reverse transcriptase-polymerase chain reaction analysis. RESULTS: RUNX-2 was induced in the articular cartilage of wild-type mice at the early stage of OA, almost simultaneously with type X collagen but earlier than MMP-13. Runx2+/- and Runx2+/lacZ mice showed normal skeletal development and articular cartilage; however, after induction of knee joint instability, they exhibited decreased cartilage destruction and osteophyte formation, along with reduced type X collagen and MMP-13 expression, as compared with wild-type mice. CONCLUSION: RUNX-2 contributes to the pathogenesis of OA through chondrocyte hypertrophy and matrix breakdown after the induction of joint instability.

Animals↗
PubMed2006

A PEG-based biocompatible block catiomer with high buffering capacity for the construction of polyplex micelles showing efficient gene transfer toward primary cells.

Nonviral gene vectors from synthetic catiomers (polyplexes) are a promising alternative to viral vectors. In particular, many recent efforts have been devoted to the construction of biocompatible polyplexes for in vivo nonviral gene therapy. A promising approach in this regard is the use of poly(ethylene glycol) (PEG)-based block catiomers, which form a nanoscaled core-shell polyplex with biocompatible PEG palisades. In this study, a series of PEG-based block catiomers with different amine functionalities were newly prepared by a simple and affordable synthetic procedure based on an aminolysis reaction, and their utility as gene carriers was investigated. This study revealed that the block catiomers carrying the ethylenediamine unit at the side chain are capable of efficient and less toxic transfection even toward primary cells, highlighting critical structural factors of the cationic units in the construction of polyplex-type gene vectors. Moreover, the availability of the polyplex micelle for transfection with primary osteoblasts will facilitate its use for bone regeneration in vivo mediated by nonviral gene transfection.

Animals↗
PubMed2006

Cartilage tissue engineering using human auricular chondrocytes embedded in different hydrogel materials.

To seek a suitable scaffold for cartilage tissue engineering, we compared various hydrogel materials originating from animals, plants, or synthetic peptides. Human auricular chondrocytes were embedded in atelopeptide collagen, alginate, or PuraMatrix, all of which are or will soon be clinically available. The chondrocytes in the atelopeptide collagen proliferated well, while the others showed no proliferation. A high-cell density culture within each hydrogel enhanced the expression of collagen type II mRNA, when compared with that without hydrogel. By stimulation with insulin and BMP-2, collagen type II and glycosaminoglycan were significantly accumulated within all hydrogels. Chondrocytes in the atelopeptide collagen showed high expression of beta1 integrin, seemingly promoting cell-matrix signaling. The N-cadherin expression was inhibited in the alginate, implying that decrease in cell-to-cell contacts may maintain chondrocyte activity. The matrix synthesis in PuraMatrix was less than that in others, while its Young's modulus was the lowest, suggesting a weakness in gelling ability and storage of cells and matrices. Considering biological effects and clinical availability, atelopeptide collagen may be accessible for clinical use. However, because synthetic peptides can control the risk of disease transmission and immunoreactivities, some improvement in gelling ability would provide a more useful hydrogel for ideal cartilage regeneration.

Atrial Appendage↗
PubMed2006

[Recent advances in cartilage metabolism research].

The past few years have seen an enormous increase in the knowledge of the signaling pathways and the transcription factors that control endochondral bone development. Recent studies of rare chondrodysplasias that exhibit disproportionate dwarfism due to the impairment of endochondral ossification have identified many responsible genes and have clarified pathogenic mechanisms of these diseases by using molecular genetics and biology. The details of human diseases related to collagen type II alpha1 (COL2A1), collagen type X alpha1 (COL10A1), parathyroid hormone (PTH) /parathyroid hormone-related protein (PTHrP) receptor, guanylate cyclase B (GC-B), sry-box 9 (SOX9), runt-related transcription factor 2 (RUNX2), which play important roles in the process of endochondral ossification, will be outlined.

Animals↗
PubMed2005

Mechanism of osteogenic induction by FK506 via BMP/Smad pathways.

FK506 is an immunosuppressant that exerts effects by binding to FK506-binding protein 12 (FKBP12). Recently, FK506 has also been reported to promote osteogenic differentiation when administered locally or in vitro in combination with bone morphogenetic proteins (BMPs), although the underlying mechanism remains unclarified. The present study initially showed that FK506 alone at a higher concentration (1muM) induced osteogenic differentiation of mesenchymal cell lines, which was suppressed by adenoviral introduction of Smad6. FK506 rapidly activates the BMP-dependent Smads in the absence of BMPs, and the activation was blocked by Smad6. Overexpression of FKBP12, which was reported to block the ligand-independent activation of BMP type I receptor A (BMPRIA), suppressed Smad signaling induced by FK506, but not that induced by BMP2. BMPRIA and FKBP12 bound to each other, and this binding was suppressed by FK506. These data suggest that FK506 promotes osteogenic differentiation by activating BMP receptors through interacting with FKBP12.

Animals↗
PubMed2005

Involvement of endogenous bone morphogenetic protein (BMP) 2 and BMP6 in bone formation.

Although accumulated evidence has shown the bone anabolic effects of bone morphogenetic proteins (BMPs) that were exogenously applied in vitro and in vivo, the roles of endogenous BMPs during bone formation remain to be clarified. This study initially investigated expression patterns of BMPs in the mouse long bone and found that BMP2 and BMP6 were the main subtypes expressed in hypertrophic chondrocytes that induce endochondral bone formation. We then examined the involvement of the combination of these BMPs in bone formation in vivo by generating the compound-deficient mice (Bmp2+/-;Bmp6-/-). Under physiological conditions, these mice exhibited moderate growth retardation compared with the wild-type (WT) littermates during the observation period up to 52 weeks of age. Both the fetal and adult compound-deficient mice showed a reduction in the trabecular bone volume with suppressed bone formation, but normal bone resorption, whereas the single deficient mice (Bmp2+/- or Bmp6-/-) did not. When a fracture was created at the femoral midshaft and the bone healing was analyzed, the endochondral bone formation, but not intramembranous bone formation, was impaired by the compound deficiency. In the cultures of bone marrow cells, however, there was no difference in osteogenic differentiation between WT and compound-deficient cells in the presence or absence of the exogenous BMP2. We thus concluded that endogenous BMP2 and BMP6 cooperatively play pivotal roles in bone formation under both physiological and pathological conditions.

Animals↗
PubMed2005

Synergistic effects of FGF-2 with insulin or IGF-I on the proliferation of human auricular chondrocytes.

Chondrocyte preparation with the safety and efficiency is the first step in cartilage regenerative medicine. To prepare a chondrocyte proliferation medium that does not contain fetal bovine serum (FBS) and that provides more than a 1000-fold increase in cell numbers within approximately 1 month, we attempted to use the medium containing 5% human serum (HS), but it exerted no more than twofold increase in 2 weeks. To compensate for the limited proliferation ability in HS, we investigated the combinational effects of 12 factors [i.e., fibroblast growth factor (FGF)-2, insulin-like growth factor (IGF)-I, insulin, bone morphogenetic protein-2, parathyroid hormone, growth hormone, dexamethasone, 1alpha25-dihydroxy vitamin D3, L-3,3',5'-triodothyronine, interleukine-1 receptor antagonist, 17beta-estradiol, and testosterone] on the proliferation of human auricular chondrocytes by analysis of variance in fractional factorial design. As a result, FGF-2, dexamethasone, insulin, and IGF-I possessed promotional effects on proliferation, while the combination of FGF-2 with insulin or IGF-I synergistically enhanced the proliferation. Actually, the chondrocytes increased 7.5-fold in number in 2 weeks in a medium containing 5% HS with 10 ng/ml FGF-2, while the cell number synergistically gained a 10-12-fold increase with 5 microg/ml insulin or 100 ng/ml IGF-I in the same period. The proliferation effects were more enhanced at a concentration of 100 ng/ml for FGF-2, and especially for the combination of 100 ng/ml FGF-2 and 5 microg/ml insulin (approximately 16-fold within 2 weeks). In the long-term culture with repeated passaging, this combination provided more than 10,000-fold within 8 weeks (i.e., passage 4). Thus, we concluded that such a combination of FGF-2 with insulin or IGF-I may be useful for promotion of auricular chondrocyte proliferation in a clinical application for cartilage regeneration.

Adolescent↗
PubMed2004

Stimulatory G protein directly regulates hypertrophic differentiation of growth plate cartilage in vivo.

Stimulatory heterotrimeric G protein (Gs) transduces signals from various cell-surface receptors to adenylyl cyclases, which generate cAMP. The alpha subunit of Gs (Gsalpha) is encoded by GNAS (Gnas in mice), and heterozygous Gsalpha inactivating mutations lead to Albright hereditary osteodystrophy. The in vivo role of Gsalpha in skeletogenesis is largely unknown, because of early embryonic lethality of mice with disruption of Gnas exon 2 (Gnas(E2-/E2-)) and the absence of easily detectable phenotypes in growth plate chondrocytes of heterozygous mutant mice (Gnas(+/E2-)). We generated chimeric mice containing wild-type cells and either Gnas(E2-/E2-) or Gnas(+/E2-) cells. Gnas(E2-/E2-) chondrocytes phenocopied PTH/PTHrP receptor (PPR)(-/-) cells by prematurely undergoing hypertrophy. Introduction of a transgene expressing Gsalpha, one of several gene products that include Gnas exon 2, into Gnas(E2-/E2-) cells prevented premature hypertrophy. Gsalpha mRNA expression detected by real-time RT-PCR analysis was reduced to approximately half that of the wild-type in both paternal and maternal Gnas(+/E2-) growth plate chondrocytes, indicating biallelic expression of Gsalpha in these cells. Hypertrophy of Gnas(+/E2-) chondrocytes was modestly but significantly premature in chimeric growth plates of mice containing wild-type and Gnas(+/E2-) cells. These data suggest that Gsalpha is the primary mediator of the actions of PPR in growth plate chondrocytes and that there is haploinsufficiency of Gsalpha signaling in Gnas(+/E2-) chondrocytes.

Animals↗
PubMed2004

SRC-1 is necessary for skeletal responses to sex hormones in both males and females.

UNLABELLED: We created SRC-1(-/-) mice by mating floxed SRC-1 mice with CMV-Cre transgenic mice. The SRC-1(-/-) mice showed high turnover osteopenia under physiological conditions and hardly responded to osteoanabolic actions of exogenous androgen and estrogen in males and females, respectively, after gonadectomies, indicating that SRC-1 is essential for the maintenance of bone mass by sex hormones. INTRODUCTION: Steroid receptor coactivator-1 (SRC-1) is the first identified coactivator of nuclear receptors. This study investigated the role of SRC-1 in skeletal tissues of males and females using the deficient (SRC-1(-/-)) mice. MATERIALS AND METHODS: SRC-1(-/-) mice were generated by mating our original floxed SRC-1 mice with CMV-Cre transgenic mice. Bone metabolism between 24-week-old SRC-1(-/-) and wildtype (WT) littermates under physiological conditions was compared in males and females by radiological, histological, and biochemical analyses. Difference of skeletal responses to steroid hormones was examined by gonadectomies and exogenous administration experiments with the hormones. Statistical analysis was performed by ANOVA determined by posthoc testing using Bonferroni's method. RESULTS AND CONCLUSIONS: Although SRC-1(-/-) mice showed no abnormality in growth or major organs, both males and females showed osteopenia with high bone turnover in the trabecular bones, but not in the cortical bones, compared with WT littermates. Their serum levels of sex hormones were upregulated, suggesting a compensatory reaction for the insensitivity to these hormones. Gonadectomies caused decreases in BMDs of SRC-1(-/-) and WT mice to the same levels; however, replacement with 5 alpha-dihydrotestosterone and 17 beta-estradiol in males and females, respectively, failed to restore the bone loss in SRC-1(-/-), whereas the WT bone volume was increased to the sham-operated levels. In contrast, bone loss by administered prednisolone was similarly seen in SRC-1(-/-) and WT mice. We conclude that SRC-1 is essential for the maintenance of bone mass by sex hormones, but not for the catabolic action of glucocorticoid, under both physiological and pathological conditions.

Animals↗
PubMed2004

Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton.

Indian hedgehog (Ihh) is indispensable for development of the osteoblast lineage in the endochondral skeleton. In order to determine whether Ihh is directly required for osteoblast differentiation, we have genetically manipulated smoothened (Smo), which encodes a transmembrane protein that is essential for transducing all Hedgehog (Hh) signals. Removal of Smo from perichondrial cells by the Cre-LoxP approach prevents formation of a normal bone collar and also abolishes development of the primary spongiosa. Analysis of chimeric embryos composed of wild-type and Smo(n/n) cells indicates that Smo(n/n) cells fail to contribute to osteoblasts in either the bone collar or the primary spongiosa but generate ectopic chondrocytes. In order to assess whether Ihh is sufficient to induce bone formation in vivo, we have analyzed the bone collar in the long bones of embryos in which Ihh was artificially expressed in all chondrocytes by the UAS-GAL4 bigenic system. Although ectopic Ihh does not induce overt ossification along the entire cartilage anlage, it promotes progression of the bone collar toward the epiphysis, suggesting a synergistic effect between ectopic Ihh and endogenous factors such as the bone morphogenetic proteins (BMPs). In keeping with this model, Hh signaling is further found to be required in BMP-induced osteogenesis in cultures of a limb-bud cell line. Taken together, these results demonstrate that Ihh signaling is directly required for the osteoblast lineage in the developing long bones and that Ihh functions in conjunction with other factors such as BMPs to induce osteoblast differentiation. We suggest that Ihh acts in vivo on a potential progenitor cell to promote osteoblast and prevent chondrocyte differentiation.

Animals↗