Shin Yoon Kim

Four novel RUNX2 mutations including a splice donor site result in the cleidocranial dysplasia phenotype

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder caused by haploinsufficiency of the RUNX2 gene. In this study, we analyzed by direct sequencing RUNX2 mutations from eleven CCD patients. Four of seven mutations were novel: two nonsense mutations resulted in a translational stop at codon 50 (Q50X) and 112 (E112X); a missense mutation converted arginine to glycine at codon 131 (R131G); and an exon 1 splice donor site mutation (donor splice site GT/AT, IVS1 + 1G > A) at exon 1–intron junction resulted in the deletion of QA stretch contained in exon 1 of RUNX2. We focused on the functional analysis of the IVS1 + 1G > A mutation. A full‐length cDNA of this mutation was cloned (RUNX2Δe1) and expressed in Chinese hamster ovary (CHO) and HeLa cells. Functional analysis of RUNX2Δe1 was performed with respect to protein stability, nuclear localization, DNA binding, and transactivation activity of a downstream RUNX2 target gene. Protein stability of RUNX2Δe1 is similar to wild‐type RUNX2 as determined by Western blot analysis. Subcellular localization of RUNX2Δe1, assessed by in situ immunofluorescent staining, was observed with partial retention in both the nucleus and cytoplasm. This finding is in contrast to RUNX2 wild‐type, which is detected exclusively in the nucleus. DNA binding activity was also compromised by the RUNX2Δe1 in gel shift assay. Finally, RUNX2Δe1 blocked transactivation of the osteocalcin gene determined by transient transfection assay. Our findings demonstrate for the first time that the CCD phenotype can be caused by a splice site mutation, which results in the deletion of N‐terminus amino acids containing the QA stretch in RUNX2 that contains a previously unidentified second nuclear localization signal (NLS). We postulate that the QA sequence unique to RUNX2 contributes to a competent structure of RUNX2 that is required for nuclear localization, DNA binding, and transactivation function. J. Cell. Physiol. 207: 114–122, 2006.

Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin‐embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin‐embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT‐PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin‐embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT‐PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde‐3‐phosphate‐dehydrogenase (GAPDH) and bone‐related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen (ColXα1), a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen (Col2α1) was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.

The Gene for Aromatase, a Rate-Limiting Enzyme for Local Estrogen Biosynthesis, Is a Downstream Target Gene of Runx2 in Skeletal Tissues

ABSTRACT The essential osteoblast-related transcription factor Runx2 and the female steroid hormone estrogen are known to play pivotal roles in bone homeostasis; however, the functional interaction between Runx2- and estrogen-mediated signaling in skeletal tissues is minimally understood. Here we provide evidence that aromatase (CYP19), a rate-limiting enzyme responsible for estrogen biosynthesis in mammals, is transcriptionally regulated by Runx2. Consistent with the presence of multiple Runx2 binding sites, the binding of Runx2 to the aromatase promoter was demonstrated in vitro and confirmed in vivo by chromatin immunoprecipitation assays. The bone-specific aromatase promoter is activated by Runx2, and endogenous aromatase gene expression is upregulated by Runx2 overexpression, establishing the aromatase gene as a target of Runx2. The biological significance of the Runx2 transcriptional control of the aromatase gene is reflected by the enhanced estrogen biosynthesis in response to Runx2 in cultured cells. Reduced in vivo expression of skeletal aromatase gene and low bone mineral density are evident in Runx2 mutant mice. Collectively, these findings uncover a novel link between Runx2-mediated osteoblastogenic processes and the osteoblast-mediated biosynthesis of estrogen as an osteoprotective steroid hormone.

Surface immobilization of MEPE peptide onto HA/β-TCP ceramic particles enhances bone regeneration and remodeling†

Calcium phosphate ceramics have been widely used as scaffolds for bone regeneration. Here, to improve the osteogenic potential of hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) and to apply the bioactive peptide in situ, matrix extracellular phosphoglycoprotein (MEPE) peptide, which has been shown to stimulate osteoblast differentiation, was covalently and directionally immobilized on HA/β-TCP particles. The free-hydroxyl groups on the surface of the HA/β-TCP particles were sequentially conjugated with APTES, PEG-(SS)(2), and the synthetic MEPE peptide. Using FTIR and XPS, immobilization of the MEPE peptide on the HA/β-TCP was confirmed. Implantation of the MEPE peptide-immobilized HA/β-TCP into calvarial defect and subsequent analyses using a micro CT and histology showed significant bone regeneration and increased bone area (9.89-fold) as compared to that of unmodified HA/β-TCP. Moreover, tartrate-resistant acid phosphatase-positive osteoclasts were observed in regenerated bone by the MEPE peptide-immobilized HA/β-TCP, indicating that the bones newly formed by the MEPE peptide-immobilized HA/β-TCP are actively remodeled by osteoclasts. Therefore, our data demonstrate that MEPE peptide immobilization onto the HA/β-TCP surface stimulates bone regeneration associated with physiological bone remodeling.

Evaluation of 5% Na2O-Incorporated Calcium Metaphosphate as a Scaffold for Tissue-Engineered Bone Regeneration

As a part of the effort to develop a suitable scaffold for tissue-engineered bone regeneration, we modified calcium metaphosphate (CMP) ceramic with Na20 and evaluated its efficiency as a scaffold. We incorporate 5% Na20 into pure CMP and prepare for an average pore size of 250 or 450 µm average pore sizes. The incorporation of 5% Na2O caused reduced compressive strength and there was no change in biodegradability. The in vitro cellular attachment and proliferation rate, however, were slightly improved. The 5% Na2O-incorporated macroporous CMP ceramic-cell constructs treated with Emdogain induced ectopic bone formation more effectively than those without Emdogain treatment. These results suggest that the incorporation of 5% Na2O into pure CMP is not effective for improving the physical characteristics of pure CMP but it is positive for improving the cellular reaction and osteogenic effect with the addition of Emdogain.

Genome-wide Association Study Identification of a New Genetic Locus with Susceptibility to Osteoporotic Fracture in the Korean Population

Abstract Osteoporotic fracture (OF), along with bone mineral den-sity (BMD), is an important diagnostic parameter and a clinical predictive risk factor in the assessment of osteo-porosis in the elderly population. However, a genome- wide association study (GWAS) on OF has not yet been clarified sufficiently. To identify OF-associated genetic variants and candidate genes, we conducted a GWAS in a population-based cohort (Korean Association Resource [KARE], n=1,427 [case: 288 and control: 1139]) and per-formed a de novo replication study in hospital-based in-dividuals (Asan and Catholic Medical Center [ACMC], n=1,082 [case: 272 and control: 810]). In a combined meta-analysis, a newly identified genetic locus in an in-tergenic region at 10p11.2 (near genes FZD8 and ANKRD30A) showed the most significant association (odd ratio [OR] = 2.00, 95% confidence interval [CI] = 1.47∼2.74, p=1.27×10 −5 ) in the same direction. We provide the first evidence for a common genetic variant influencing OF and genetic information for further inves-tigation in bone metabolism.Keywords: genomewide association study, meta-analy-sis, osteoporotic fracture, intergenic

Osteogenic Repair by Bovine Bone Ash Derived Porous HA Ceramic Formed by Foaming Method

To develop a suitable scaffold optimizing bone regeneration, we developed bovine bone ash derived fully connected porous HA ceramic scaffolds adopting a foaming method. They revealed excellent biocompatibility. The attached cells on the scaffolds proliferated in multi-layers with osteoblastic differentiation. The bone defects grafted with bovine bone ash derived fully interconnected porous HA ceramics having average 500 μm sized spherical pores and average 150 μm sized interconnecting interpores with average 80% porosity were favorably healed without any pathologic changes within 3 weeks. New bone ingrowth with excellent osteoconduction through the spherical pores along the inner surface was noted from 1 week after implantation. Each spherical pore was filled with hematopoietic marrow and newly formed bone which with time was well integrated with the porous HA ceramic scaffold with time. These findings suggest that the bovine bone ash-derived fully interconnected porous HA ceramic formed by foaming method can be a promising bone substitute and a scaffold for bone tissue engineering.

Stimulatory Effect of Nano-Sized Calcium Metaphosphate Particles on Proliferation and Osteoblastic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

Recently, nanomaterials have received considerable attention because of their potential applications in the biomedical field. In the present study, we investigated the effects of nano-sized calcium metaphosphate (CMP) particles (50 nm) compared with micro-sized CMP particles (200-500 nm and 10 μm) on the proliferation and osteoblastic differentiation of human bone marrow stem cells (BMSCs). BMSCs were challenged with CMP particles with different sizes for 3, 5, and 7 days. An analysis of the proliferation revealed that the nano-sized CMP particles (50 nm) stimulated the proliferation of BMSCs up to 27.79% compared to the untreated control. This stimulatory effect of the nano-sized CMP particle was dose-dependent. CMP particles appeared to adhere on the surface of BMSCs but this did not cause distinguishable morphological changes. Moreover, all CMP particles (50 nm to 10 μm) were capable of stimulating an osteoblastic differentiation of BMSCs as accessed by alkaline phosphatase (ALP) and von Kossa stainings. Further molecular analysis revealed that all the CMP particles induced an expression of osteoblast-related genes such as osteocalcin (OC) and collagen I (Col I). Taken together, our data demonstrate that nano-sized CMP particles have the potential to stimulate the proliferation and osteoblastic differentiation of BMSCs.

In Vitro Study of HA and CMP Grit-Blasted and Acid Etched of Ti Surface

Grit-blasting using bioactive HA and biodegradable CMP followed by acid etching was done. The apatite formation of prepared Ti samples was evaluated by immersion in R-SBF. And cell viability, proliferation, and differentiation were conducted using MTT assay and ALP staining. In RSBF immersion tests, non-etched HA-blasted samples showed the faster apatite-like formation than other samples. Acid etched and non-etched HA-blasted samples showed better cell viability and proliferation compared to CMP-blasted samples after 1 and 3 days. And the cell differentiation of non-etched HA-blasted samples was better compared to etched ones, and etched and non-etched CMP-blasted samples.

Effect of Acid Etching on Surface Chemistry and Microstructure of HA and CMP Grit-Blasted Ti Surface

Grit blasting using bioactive HA and biodegradable CMP followed by acid etching has been done. HNO3 and H3PO4 were used for the etching solution by controlling the concentration and etching time to know the effect on the surface chemistry and morphology of the Ti implant. Characterization of samples was done by using SEM, EDX and surface profilometer. The contents of residues on Ti surface decreased with increasing acid concentration and etching time. It was observed that the acidic etching rate of HA grits on Ti surface was faster than that of CMP grits. And HNO3 etched more rapidly the HA and CMP grits on Ti surface, compared to H3PO4. Therefore, the surface roughness of dental implants can be controlled by having appropriate combination of acid concentration and etching time.