• Tissue Engineering and Regenerative Medicine
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• 제15권6호
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• pp.711-719
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• 2018

BACKGROUND: Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. METHODS: Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and $MPG^{{\Delta}NLS}$) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8 mm biopsy punch wound at 1 day to 8 wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. CONCLUSION: CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p<0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF $siRNA/MPG^{{\Delta}NLS}$ complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of $128{\pm}22nm$ (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean $90{\pm}20nm$, n = 563). CONCLUSION: Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.

• BMB Reports
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• 제46권4호
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• pp.195-200
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• 2013

To understand the corneal regeneration induced by bevacizumab, we investigated the structure changes of stroma and basement membrane regeneration. A Stick soaked in 0.5 N NaOH onto the mouse cornea and 2.5 mg/ml of bevacizumab was delivered into an alkali-burned cornea (2 ${\mu}l$) by subconjunctival injections at 1 hour and 4 days after injury. At 7 days after injury, basement membrane regeneration was observed by transmission electron microscope. Uneven and thin epithelial basement membrane, light density of hemidesmosomes, and edematous collagen fibril bundles are shown in the alkali-burned cornea. Injured epithelial basement membrane and hemidesmosomes and edematous collagen fibril bundles resulting from alkali-burned mouse cornea was repaired by bevacizumab treatment. This study demonstrates that bevacizumab can play an important role in wound healing in the cornea by accelerating the reestablishment of basement membrane integrity that leads to barriers for scar formation.

• 대한본초학회지
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• 제31권5호
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• pp.41-46
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• 2016

Objectives : The Skin is composed of multiple layers, including the epidermis, dermis, and hypodermis. It provides a vital barrier structure that protects vertebrates from external environmental antigens, solvents, ultraviolet light, microorganisms, toxins, and weather conditions. Although several biological effects of Mori Follium have been reported, beneficial effects of Mori Follium in skin health remain unclear. In this study, we prepared water extract of Mori Follium (MLE) and evaluated the effects on melanin accumulation and expression levels of skin fibril-related proteins.Methods : The cytotoxicities of MLE in B16F10 melanoma and human skin fibroblasts (HSF) were examined by MTT assay. Inhibitory effect of MLE on the α-MSH- and IBMX-induced melanosis in B16F10 melanoma was examined. The expression levels of fibronectin, collagen 1α2, and CCN2 in MLE-treated HSF were analyzed by reverse transcription-polymer chain reaction (RT-PCR) and western blotting.Results : The MLE treatment for 24 h did not affect to the B16F10 and HSF at concentrations of 1, 10, 50, 100, 200, 400 and 800 ㎍/ml. The MLE treatment for 72 h significantly and dose dependently suppressed melanin accumulation in B16F10 melanoma. In addition, the MLE treatment up-regulated expression levels of skin fibril-related genes such as fibronectin, collagen 1α2, and CCN2 in HSF. Our western blot analysis revealed MLE-induced up-regulation of skin fibril-related genes required the activation of CCN2 protein.Conclusions : In conclusion, these findings suggest that the MLE could be used in development of cosmetic natural material of maintaining healthy skin.

• 대한본초학회지
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• 제34권2호
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• pp.75-82
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• 2019

Objectives : In this study, various biological effects of Curcuma longa L. have been studied, however, beneficial effect of Curcuma longa L. in skin health remain still unclear. In this study, Curcuma longa L. water extract (CLE) was prepared. Inhibitory effect of CLE on melanin accumulation of B16F10 cells and expression levels of skin fibril-related proteins of human skin fibroblasts (HSF) were evaluated. Methods : The cytotoxic effect of CLE in B16F10 cells and HSF were examined by MTT assay. Inhibitory effect of CLE on the ${\alpha}-MSH-$ and IBMX-induced melanin accumulation and tyrosinase activity were evaluated in B16F10 cells. The expression levels of connective tissue growth factor (CCN2), Smad2, procollagen $1{\alpha}2$, collagen $1{\alpha}2$, and fibronectin in CLE-treated HSF were analyzed by western blotting. Results : The CLE treatment (concentrations 10 to $400{\mu}g/ml$) for 72 h did not affect to the B16F10 viability. However, 200 and $400{\mu}g/ml$ of CLE treatment for 24 h showed cytotoxic effect in HSF. Therefore, the concentrations 10, 50, and $100{\mu}g/ml$ of CLE were chosen in this study. The CLE treatment for 72 h dose dependently and significantly suppressed melanin accumulation and tyrosinase activity of B16F10 cells. In addition, the CLE treatment up-regulated expression levels of skin fibril-related proteins such as CCN2, Smad2, procollagen $1{\alpha}2$, collagen $1{\alpha}2$, and fibronectin. Conclusions : In conclusion, these results suggest that the CLE could be used as a natural material for skin health.

• Current Optics and Photonics
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• 제6권2호
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• pp.196-201
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• 2022

Collagen fibers tens of nanometers in size, which constitute most of the corneal volume of the human eye, are layered in a uniform direction, and adjacent fiber layers are arranged at an angle of 90° to each other. According to the results of this study, the transmittance at 45° of interlayer rotation angle is highest, and higher than that of the 90° body structure. The transmittance is examined, concerning the polarization state of the incident light; circularly polarized light case shows higher transmittance than linearly polarized. Through this, a simulation to confirm the deformed structure of collagen fibers, which show higher transmittance than the anatomical structure of the cornea, is attempted.

• Asian-Australasian Journal of Animal Sciences
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• 제15권8호
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• pp.1204-1209
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• 2002

Polymerization of heated or unheated pig skin collagen using microbial transglutaminase (MTGase) was investigated. Pig skin collagen samples were heated or left unheated, then enzymatically polymerized with MTGase. SDS-PAGE was conducted to confirm the intermolecular polymer and the results showed similar bands between samples without MTGase and unheated samples with MTGase. The polymerized product of pig skin collagen was not formed in unheated samples, even when MTGase was added during incubation. Different results were obtained from samples heated at $80^{\circ}C$ and $100^{\circ}C$ for 2 min, whereas the SDS-PAGE pattern indicated that a polymer band was generated in both cases. The heat treatment successfully modified the native structure of collagen and also made collagen more reactable in the MTGase polymerization system. Scanning Electron Microscope (SEM) investigation of pig skin collagen showed a biopolymer structure through intermolecular collagen crosslinking, while there were no intermolecular crosslinks in samples not treated with MTGase. There were no significant differences in fibril diameter between treated samples and controls. These results suggest that heat treatment of native pig skin collagen enhanced the polymerization capability of MTGase.

• Applied Microscopy
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• 제26권2호
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• pp.157-175
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• 1996

The development of flexor digital tendon of the hand was studied by electron microscopy in human fetuses ranging from 9 mm to 260 mm crown rump length. The primordium of tendons was first identified as discrete collection of mesenchymal cells at 25 mm fetus. Synovial sheath formation had commenced by 40 mm fetus and was complete by 70 mm fetus. Cell junction or adhesion sites at all ages were noted between the tendon cells. When dilatation of the synovial cavity occurred, two types of synovial cells were observed. A-type cells had numerous vesicles and large vacuoles. In contrast, B-type cells were characterized by abundant rough endoplasmic reticulum and well-developed Golgi complex. By $150mm{\sim}260mm$ fetuses, a mojority of the synovial cells were type B. The most remarkable difference between the synovial cells of full-term fetus and adult was the larger amount of collagen fibers in the latter. The vascular buds were first observed between the individual fibril bundles in the interfascicular space at 150 mm fetus. At 25 mm fetus, collagen fibrils were first noted within narrow cytoplasmic recesses which were continued with the extracellular space. Collagen fibrils were filled in almost entire extracellular space at 150 mm fetus. Besides collagen fibrils in the extracellular space small elastic fibers were also identified and followed in their development.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제20권4호
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• pp.305-315
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• 1998

흰쥐에 있어서 가령에 따라 하악과두에 가해지는 기계적 부하의 변화에 따라 하악과두 섬유층의 미세구조적 변화를 관찰, 분석한 결과를 요약하면 다음과 같다. 하악과두 섬유층의 세포는 생후 초기에는 미분화간엽세포 및 섬유모세포로 구성되어 있었다. 가령에 따라 생후 14일, 27일군 등에서는 생후 초기에 비해 교원원섬유를 활발하게 합성, 분비하는 것으로 생각되는 섬유모세포가 발달하였는데 이들 세포는 과립형 물질로 채워져 있는 내강이 대단히 확장되어 있으며, 리보솜이 치밀하게 존재하는 과립세포질세망과 골지장치가 발달되어 있었다. 또한 이들 세포내에는 세포소기관 사이에 세사가 점차로 발달하는 양상을 보였다. 생후 55일군 이후에서는, 가늘고 긴 세포돌기를 가지며, 과립세포질세망 및 골지장치가 발달되어 있어 교원섬유의 합성이 왕성한 것으로 추측되는 세포, 약간 둥글거나 다각형의 모양을 보이며, 세포질내에 사립체, 용해소체 및 골지장치가 발달하였으며, 손상된 교원섬유 혹은 변성된 세포의 흡수에 관여하는 것으로 추측되는 세포, 세포질의 상당부분이 세사로 채워져 있으며, 사립체, 용해소체 및 과립세포질내세망과 같은 세포질내 소기관은 잘 발달되어 있지 않아 가해진 기계적 자극에 대해 적응하였거나 활동이 정지된 세포 등 3종류의 세포가 관찰되었다. 세포간질의 교원원섬유는 생후 초기부터 가령에 따라 점차로 치밀하게 배열되는 양상을 보였다. 교원원섬유의 직경은 생후 1일, 7일군에서는 $38.48{\pm}3.81nm$, $38.06{\pm}3.86nm$로서 유사하였으나 생후 14일군에서는 $50.21{\pm}3.93nm$로 가장 굵은 양상을 보였으며(p<0.05), 그후 생후 27일, 55일군에서는 각기 $40.05{\pm}2.52nm$, $43.63{\pm}1.20nm$로서 점차로 가늘어져 생후 1년군에서는 $37.38{\pm}2.17nm$로서 가장 가늘어 졌다(p<0.05). 그 분포에 있어서는 생후 초기에는 직경이 30-60인 교원원섬유가 대다수를 차지하는 unimordal한 분포를 보였으나, 생후 27일 이후에는 직경이 작은 교원원섬유가 점차로 증가하는 양상을 보였다. 이상의 가령에 따른 결과로 미루어보아 하악과두 섬유층의 미세구조, 교원원섬유의 배열 및 직경의 분포에서의 변화는 가해지는 기계적 부하에 대한 적응한 결과라고 생각된다.

• Journal of Periodontal and Implant Science
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• 제30권1호
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• pp.121-134
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• 2000

Root conditioning has introduced to dissolve the smear layer and to produce surface demineralization, resulting to exposure of dentin collagen fibril and opening of dentinal tubules. The purpose of the present study was to examine the effect of different concentration and application time of tetracycline-HCL on root conditioning. Total 40 root specimen were prepared from 20 periodontitis-prone human single rooted tooth. The specimen were treated with tetracycline-HCL solution(20mg/ml, 50mg/ml, 100mg/ml)for 20 sec, 3 min, 5 min., and saline for 30 sec. The application method was rubbing method with cotton pellet. Under the scanning electron microscopy(20KV), the extent of smear removal and opening of the dentinal tubules were examined at x 3000. The following results were obtained. 1. Treatment of root specimen with saline did not remove the smear layer and open the dentinal tubules. 2. Treatment of root specimen with different concentration of tetracycline-HCL for 20 sec also did not remove the smear layer completely. 3. Treatment of root specimen with different concentration of tetracycline-HCL for 3 min opened the dentinal tubules and removed smear layer. 4. Treatment of root specimen with 50mg/ml of tetracycline-HCL for 3 min showed collagen fibril within the opened dentinal tubules. In conclusion, the effect of root conditioning with tetracycline-HCL is more dependent on the application time than the application concentration. Root conditioning with 50mg/ml tetracycline-HCL for 3 min is enough for obtaining the periodontal regeneration.

• Preventive Nutrition and Food Science
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• 제18권2호
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• pp.124-132
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• 2013

In this study, novel peptides (NIPP-1, NIPP-2) derived from Navicula incerta (microalgae) protein hydrolysate were explored for their inhibitory effects on collagen release in hepatic fibrosis with the investigation of its underlying mechanism of action. TGF-${\beta}1$ activated fibrosis in LX-2 cells was examined in the presence or absence of purified peptides NIPP-1 and NIPP-2. Besides the mechanisms of liver cell injury, protective effects of NIPP-1 and NIPP-2 were studied to show the protective mechanism against TGF-${\beta}1$ stimulated fibrogenesis. Our results showed that the core protein of NIPP-1 peptide prevented fibril formation of type I collagen, elevated the MMP level and inhibited TIMP production in a dose-dependent manner. The treatment of NIPP-1 and NIPP-2 on TGF-${\beta}1$ induced LX-2 cells alleviated hepatic fibrosis. Moreover, ${\alpha}$-SMA, TIMPs, collagen and PDGF in the NIPP-1 treated groups were significantly decreased. Therefore, it could be suggested that NIPP-1 has potential to be used in anti-fibrosis treatment.