• Journal of the korean academy of Pediatric Dentistry
• /
• v.26 no.4
• /
• pp.652-663
• /
• 1999

Craniosynostosis, the premature fusion of cranial sutures, presumably involves disturbance of the interactions between different tissues within the cranial sutures. Interestingly, point mutaions in the genes encoding for the fibroblast growth factor receptors(FGFRs), especially FGFR2, cause various types of human craniosynostosis syndromes. To elucidate the function of these genes in the early morphogenesis of mouse cranial sutures, we first analyzed by in situ hybridization the expression of FGFR2(BEK) and osteopontin, an early marker of osteogenic differentiation, in the sagittal suture of calvaria during embryonic(E15-E18) and postnatal stage(P1-P3). FGFR2(BEK) was intensely expressed in the osteogenic fronts, whose cells undergo differentiation into osteoprogenitor cells that ultimately lay down the bone matrix. Osteopontin was expressed throughout the parietal bones excluding the osteogenic fronts, the periphery of the parietal bones. To further examine the role of FGF-mediated FGFR signaling in cranial suture, we did in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of FGF2 soaked beads onto both the osteogenic fronts and mid-mesenchyme of sagittal suture after 36 hours organ culture resulted in the increase of the tissue thickness and cell number around FGF2 beads, moreover FGF4-soaked beads implanted onto the osteogenic fronts stimulated suture closure due to an accelerated bone growth, compared to FGF4 beads placed onto mid-mesenchyme of sagittal suture and BSA control beads. In addition FGF2 induced the ectopic expression of osteopontin and Msx1 genes. Taken together, these data indicate that FGF-mediated FGFR signaling has a important role in regulating the cranial bone growth and maintenance of cranial suture, and suggest that FGF-mediated FGFR signaling is involved in regulating the balance between the cell proliferation and differentiation through inducing the expression of osteopontin and Msx1 genes.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.27 no.5
• /
• pp.446-456
• /
• 2005

Nerve growth factor(NGF) has a critical role in peripheral nerve regeneration. The aim of this study is to construct a well-functioning hNGF-$\beta$ recombinat adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with adenovirus mediated hNGF-$\beta$ gene transfection into Schwann cells. First PCR associated cloning of GFP-tagged hNGF-$\beta$ which was ligated into E1/E3 deleted adenoviral vector was performed and tranfected into E. coli to construct hNGF-$\beta$ recombinant adenovirus. After production of recombinat adenovirus in a large scale, its transfection efficiency, expression, and function were evaluated using cell lines or primarily cultured cells of HEK293 cells, Schwann cells, fibroblast(NIH3T3) and myocyte(CRH cells). GFP expression was observed in 90% of infected cells compared to uninfected cells. Total mRNA isolated from hNGF-$\beta$ recombinat adenoviru infected cells showed strong RT-PCR band, however, LacZ recombinant adenovirus infected or uninfected cells did not. NGF quantification by ELISA showed a maximal release of 18.865 +/- 0.31ng/mL at 4th day. PC-12 cells exposed to media with hNGF-$\beta$ recombinant adenovirus infected Schwann cell demonstrated higher levels of differentiation compared with controls. We generated hNGF-$\beta$ recombinant adenovirus and induced over expression of NGF successfully in nonneuronal and neuronal cells. Following these result, it is expected to develop an improved treatment strategy peripheral nerve regeneration using the hNGF-$\beta$ gene transfected cells.

• Journal of Periodontal and Implant Science
• /
• v.35 no.1
• /
• pp.153-161
• /
• 2005

파이브로넥틴은 세포외기질에 존재하는 당단백질로 세포의 부착, 이동, 성장 및 분화에 관여하며, 섬유아세포 성장인자는 세포의 증식 이동 및 분화에 영향을 주는 중요한 성장인자로 알려져 있다. 최근 연구에 의하면, 파이브로넥틴은 조골세포의 타이태늄 임플란트 표면으로 이주와 증식 및 골생성을 촉진하며, 섬유아세포 성장 인자는 파이브로넥틴에 상승작용을 한다고 보고된 바 있다. 이 실험의 목적은 파이브로넥틴 및 섬유아세포 성장인자의 복합 단백질을 이용하여 타이태늄 임플란트의 골 반응을 알아보는 것이다. 체중 2.5 kg 내외의 건강한 18 마리의 웅성가토를 준비하여 무균 사육하였고, 순수 타이태늄을 절삭가공하여 직경 3.5mm, 길이 6mm 의 machined surface를 지니는 screw type 의 임플란트를 준비하였다. 사람의 유전자를 기초로, 유전자 재조합법을 통해, 적절한 primer를 이용하여 얻은 섬유아세포 성장인자를 파이브로넥틴 III 형 분절의 9-10 번 도메인에 결합시켜 얻은 복합 단백질을 준비된 임플란트에 표면처리하여 실험군으로 하였고, 표면처리하지 않은 임플란트를 대조군으로 하여, 가토의 좌우 경골에 각각 2 개씩의 임플란트를 식립하였다. 4주 후, 가토를 희생시켜 각 경골 당 한 개의 임플란트에서 뒤틀림 제거력을 측정하였고 나머지 임플란트 식립 부위 에서는 경골을 포함하는 조직표본을 제작하였다. 조직표본상에서 골접촉이 가장 좋은 3 개의 나사산의 길이를 측정하고, 나사와 접촉하는 골의 길이를 측정하여 골-임플란트 접촉도를 구하고, 같은 부위에서 나사산 사이의 면적과 골이 차지하는 면적을 비교하여 골생성률을 얻었다. 실험군과 대조군의 결과는 Student t-test 를 이용하여 신뢰도 95% 수준에서 통계학적 유의성을 검정하였다. 파이브로넥틴과 섬유아세포 성장인자의 복합 단백질로 표면처리된 임플란트와 표면처리를 하지 않은 임플란트는 뒤틀림 제거력에서는 통계적 유의성이 나타나지 않았으나, 골-임플란트 접촉도와 골생성률에서 복합 단백질로 처리된 임플란트가 통계적으로 유의하게 높은 결과를 보였다. 이상의 연구결과로, 섬유아세포 성장인자와 파이브로넥틴 복합 단백질로 처리한 타이태늄 임플란트가 주변 골 형성을 촉진시켜, 골유합을 증진시킴을 알 수 있었다. 따라서, 복합 단백질이 타이태늄 임플란트의 성공률을 높이기 위한 표면개질 물질로 이용될 가능성을 확인할 수 있었다.

• Development and Reproduction
• /
• v.11 no.3
• /
• pp.167-177
• /
• 2007

In the present study, we isolated three human adult stem cells including adipose tissue-derived stem cells(HAD), umbilical cord-derived stem cells(HUC), and amnion-derived stem cells(HAM) and analysed their characteristics. And we examined whether HAD could be used as therapeutical cells for the heart diseases. Both HAM and HUC appeared very similar morphology but HAD was different. Doubling time of HUC was most fast, but total doubling numbers of HUC was same with HAM. Total doubling numbers of HAD was much more than others. Expression patterns of genes and proteins of three human adult stem cells were very similar. Also they were differentiated into adipocytes, osteocytes, and chondrocytes. In addition, they expressed many cardiomyocyte-related genes. But expression pattern of genes is a little different. When HAD were cultivated in the presence or absence of various combinations of BMP and FGF after 5-azacytidine expose for 24 h, expression of Cmlc-1, and ${\alpha}1c$ genes was significantly increased. However, expression of troponin T, troponin I and Kv4.3 genes was not changed. Based on these observations, it is suggested that HAD, HUC, and HAM might be used as potentially therapeutical cells for clinical application.

• The korean journal of orthodontics
• /
• v.36 no.4
• /
• pp.284-294
• /
• 2006

Objective: Activating mutations in the fibroblast growth factor receptor-2 (FGFR2) have been shown to cause syndromic craniosynostosis such as Apert and Crouzon syndromes. The purpose of this pilot study was to investigate the resultant phenotypes induced by the two distinctive bone-targeted gene constructs of FGFR2, Pro253Arg and Cys278Phe, corresponding to human Apert and Crouzon syndromes respectively. Methods: Wild type and a transgenic mouse model with normal FGFR2 were used as controls to examine the validity of the microinjection. Micro-CT and morphometric analysis on the skull revealed the following results. Results: Both Apert and Crouzon mutants of FGFR2 induced fusion of calvarial sutures and anteroposteriorly constricted facial dimension, with anterior crossbite present only in Apert mice. Apert mice differed from Crouzon mice and transgenic mice with normal FGFR2 in the anterior cranial base flexure and calvarial flexure angle which implies a possible difference in the pathogenesis of the two mutations. In contrast, the transgenic mice with normal FGFR2 displayed normal craniofacial phenotype. Conclusion: Apert and Crouzon mutations appear to lead to genotype-specific phenotypes, possibly causing the distinctive sites and sequence of synostosis in the calvaria and cranial base. The exact function of the altered FGFR2 at each suture needs further investigation.