• Restorative Dentistry and Endodontics
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• 제41권4호
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• pp.296-303
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• 2016

Objectives: The purpose of the present study was to evaluate the effects of proanthocyanidin (PAC), a crosslinking agent, on the physical properties of a collagen hydrogel and the behavior of human periodontal ligament cells (hPDLCs) cultured in the scaffold. Materials and Methods: Viability of hPDLCs treated with PAC was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The physical properties of PAC treated collagen hydrogel scaffold were evaluated by the measurement of setting time, surface roughness, and differential scanning calorimetry (DSC). The behavior of the hPDLCs in the collagen scaffold was evaluated by cell morphology observation and cell numbers counting. Results: The setting time of the collagen scaffold was shortened in the presence of PAC (p < 0.05). The surface roughness of the PAC-treated collagen was higher compared to the untreated control group (p < 0.05). The thermogram of the crosslinked collagen exhibited a higher endothermic peak compared to the uncrosslinked one. Cells in the PAC-treated collagen were observed to attach in closer proximity to one another with more cytoplasmic extensions compared to cells in the untreated control group. The number of cells cultured in the PAC-treated collagen scaffolds was significantly increased compared to the untreated control (p < 0.05). Conclusions: Our results showed that PAC enhanced the physical properties of the collagen scaffold. Furthermore, the proliferation of hPDLCs cultured in the collagen scaffold crosslinked with PAC was facilitated. Conclusively, the application of PAC to the collagen scaffold may be beneficial for engineering-based periodontal ligament regeneration in delayed replantation.

• Biomolecules & Therapeutics
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• 제15권4호
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• pp.261-265
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• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.

• 대한한의학회지
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• 제27권1호
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• pp.229-239
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• 2006

Objectives : Articular cartilage is a potential target for drugs designed to inhibit the activity of matrix metalloproteinases (MMPs) to stop or slow the destruction of the proteoglycan and collagen in the cartilage extracelluar matrix. The purpose of this study was to investigate the effects of KHBJs for cartilage-protective effect in human and rabbit articular cartilage explants. Methods : The cartilage-protective effects of KHBJ were evaluated by using glycosaminoglycan degradation assay, collagen degradation assay, colorimetric analysis of MMPs activity, and histological analysis in rabbit and human cartilage explants culture. Results : KHBJs significantly inhibited GAG and collagen release of rabbit and human cartilage explant in a concentration-dependent manner. Also, KHBJs inhibited MMP-3 and MMP-13 activities from IL-$1{\alpha}$-treated cartilage explants cultures. Histological analysis indicated that KHBJ004 reduced the degradation of the cartilage matrix compared with that of IL-$1{\alpha}$-treated cartilage explants. KHBJ004 had no harmful effect on chondrocytes viability or cartilage morphology in cartilage explants. Conclusions : These results indicate that KHBJs inhibits the degradation of proteoglycan and collagen through the downregulation of MMP-3 and MMP-13 activities without affecting the viability or morphology of IL-$1{\alpha}$-stimulated rabbit and human articular cartilage explants.

• Archives of Plastic Surgery
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• 제36권6호
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• pp.679-684
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• 2009

Purpose: Human lipoaspirate cells are relatively easy to obtain in large quantities without cell culture. The aim of this in vitro pilot study was to determine the effect of cell therapy using uncultured lipoaspirate cells on cell proliferation and collagen synthesis of diabetic fibroblasts, which are the major contributing factors in wound healing. Methods: In order to get diabetic fibroblasts, dermis tissues were obtained from foot skin of diabetic patients who underwent debridements or toe amputations(n = 4). In order to isolate lipoaspirate cells, the same diabetic patients' abdominal adipose tissues were obtained by liposuction. The diabetic fibroblasts were co - cultured with or without autogenous lipoaspirate cells using porous culture plate insert. Initial numbers of the lipoaspirate cells and diabetic fibroblasts seeded were 15,000 cells/well, respectively. For cell proliferation assay, two treatment groups were included. In group I, diabetic fibroblasts were cultured with the insert having no cells, which serves as a control. In group II, the lipoaspirate cells were added in the culture plate insert. For collagen synthesis assay, one additional group(group III), in which diabetic fibroblasts were not seeded in the well and only lipoaspirate cells inside the insert were incubated without diabetic fibroblasts, was included for a reference. Results: One hundred to one hundred sixty thousand lipoaspirate cells were isolated per ml of aspirated adipose tissue. After 3 - day incubation, the mean cell numbers in group I and II were 17,294/well and 22,163/well. The mean collagen level in group I, II, and III were 29, 41, and 2 ng/ml, respectively. These results imply that both cell proliferation and collagen synthesis in the lipoaspirate cell treatment group were 28 and 44 percents higher than in the control group, respectively(p < 0.05). Conclusion: Uncultured lipoaspirate cell autografts may stimulate the wound healing activity of diabetic fibroblasts.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제35권3호
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• pp.146-152
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• 2009

Traditional surgical method or injection using filler is performed for soft tissue augmentation. Surgical methods have disadvantage of surgical morbidity. Commercially available injectable materials have the disadvantages such as resorption, short-term effect. repeated application and hypersensitivity. Significant shortcoming of cell therapy using autologous fibroblasts is delay of treatment effect. Chitosan/${\beta}$-glycerol phosphate (GP) solution has thermosensitive property and allows sol-gel transition at physiologic pH and temperature. These properties may resolve the delay of treatment effect. The purposes of this study are to evaluate the viscosity and pH changes of chitosan/${\beta}$-GP solutions and to evaluate the effect of chitosan/${\beta}$-GP solution on fibroblast proliferation and production of collagen. We measured the viscosity and pH as function of temperature, of the solution containing 1:0.7, 1:0.75, 1:0.8 chitosan (1, 10, 100, 700 kDa) /${\beta}$-GP. Fibroblasts from ears of 5 rats were cultured in chitosan/${\beta}$-GP solutions for 3 weeks. Cell proliferation and collagen contents were measured every week with WST (water-soluble tetrazolium salt) assay and Collagen assay respectively. The Results are 1) Chitosan(100 kDa<)/${\beta}$-GP solution (1:0.75) showed sol-gel transition at physiologic pH and body temperature and injectable properties. It will enable to resolve the delay in treatment effect 2) Cell proliferation and total collagen contents of the control group were increased with time. However, these decreased after the 1st week in experimental group 3) Collagen contents in the experimental group are higher than that of control group. Chitosan/${\beta}$-GP solution may provide favorable conditions for cell function

• 디지털융복합연구
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• 제14권11호
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• pp.639-644
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• 2016

본 연구의 목적은 발광 다이오드 (LED)을 인간 피부 각질 세포에 조사 시 콜라겐, 프로 콜라겐의 증식 발현을 조사하기 위해 실시되었었다. LED 조사 시 안전하게 인간의 피부에 적용할 수 있는지 여부를 결정하기 위해, LED 조사의 증식 효과는 인간 표피 각질세포에서 MTS 분석으로 결정하였다. 470nm의 파장 조사는 세포 독성 없이 mRNA의 콜라겐의 발현, 프로 콜라겐을 증가시켰으며, 이 결과는 470nm LED 조사가 피부각질세포 증식 효과와 콜라겐 합성에 영향을 미칠 수 있음을 시사한다. 또한 LED 조사시 독성 효과는 인간 피부 섬유 아세포 (HDF)에서 MTS 분석으로 결정 한 결과 세포 증식에 독성을 나타내지 않았다. 470nm LED 조사시 시험 관내 콜라겐 합성 활동을 증가시킴으로 피부미용 및 융복합적인 부분에 활용할 수 있는 기초 자료로 활용이 가능하다고 사료된다.

• Animal cells and systems
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• 제15권1호
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• pp.19-27
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• 2011

Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.

• 혜화의학회지
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• 제21권2호
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• pp.63-72
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• 2013

To develop a new anti-skin aging cosmetics or functional foods by using antioxidative activity and collagenase inhibitor, a potent collagenase or elastase inhibitor was screened from various extracts of medicinal plants and its optimal extraction condition was investigated. And antioxidative activity, antimicrobial activity and inhibitory of effect against collagenase activity were investigated. In the these results, we selected the Sanguisorba (Sanguisorba officinalis L.) that presents a potential biological activities. Sanguisorba which is very rich in triterpenoid saponin and tannins was recently reported its anti-oxidant activities and phytoestogenic activities in vivo test and many clinical studies. The experiments were carried out in vitro to determine anti-oxidant activities of Sanguisorba extracts on DPPH radical scavenging activity assay, Superoxidase scavenging activity assay, Elastase and collagenase activity assay. It show that the Sanguisorba extracts have the most significant anti-oxidant on free radical scavenging activity assay, and also inhibited significantly activities of elastase, collagenase. Further, Sanguisorba extracts are activated Type I collagen protein expression in CCD-986sk cells. These result suggest that the Sanguisorba extracts on DPPH radical scavenging activity assay, Superoxidase scavenging activity assay, elastase and collagenase activity assay, Type I collagen protein expression in CCD-986sk cells effected could be developed cosmetic ingredients for anti-aging.

• 한국응용과학기술학회지
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• 제37권4호
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• pp.820-829
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• 2020

본 연구에서는 기능성 화장품의 소재로서의 금전초(Lysimachia christinae Hance)의 항산화와 항주름 효과를 조사하였다. 최근 천연물의 주름 개선 개발의 연구가 지속적인 관심을 받고 있어 본 연구를 통해 활성산소종(reactive oxygen species, ROS) 생성과 pro-collagen 합성 및 MMPs의 연관성에 대해 알아보았다. 금전초는 70% 에탄올(LcHE)과 열수(LcHW)로 각각 추출하여 실험을 진행하였다. HaCaT cells에서 LcHE가 LcHW보다 ROS 저해효능이 더 우수하고 세포독성 결과 250 ㎍/mL 농도까지 독성을 보이지 않아 LcHE를 선택하여 주름 개선 소재연구를 진행하였다. pro-collagen 합성실험을 통하여 UVB에 의해 감소된 type-1 pro-collagen의 합성 활성을 유의미하게 확인하였다. Western blot 실험을 통하여 피부세포에서 UVB에 의해 유도된 MMPs 중 MMP-1 -3 -9의 증가를 억제함을 확인하였으며, Real time PCR을 통하여 상위단계인 mRNA levels에서도 MMP-1, MMP-2, MMP-3, MMP-9의 mRNA levels가 농도 의존적으로 유의미한 감소를 보여 추출물의 효능을 확인하였다. 위의 실험결과에 따라 UVB에 의한 주름생성과 피부 광노화를 효과적으로 예방할 수 있는 화장품의 천연소재로서의 이용이 기대된다.

• Preventive Nutrition and Food Science
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• 제16권4호
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• pp.291-298
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• 2011

Yam extracts (Dioscorea batatas) have been reported to possess a variety of functions. However, studies on its osteogenic properties are limited. In this study, we investigated the effect of ethanol and water extracts on osteoblast proliferation and bone matrix protein synthesis, type I collagen and alkaline phosphatase (ALP), using osteoblastic MC3T3-E1 cell model. MC3T3-E1 cells were cultured with yam ethanol and water extracts (0~30 mg/L) within 39 days of osteoblast differentiation period. Cell proliferation was measured by MTT assay. Bone matrix proteins were assessed by the accumulation of type I collagen and ALP activity by staining the cell layers for matrix staining. Also, the secreted (media) matrix protein concentration (type I collagen) and enzyme activity (ALP) were measured colorimetrically. Yam ethanol and water extracts stimulated cell proliferation within the range of 15~30 mg/L at 15 day treatment. The accumulation of type I collagen in the extracellular matrix, as well as secreted collagen in the media, increased with increasing doses of yam ethanol (3~15 mg/L) and water (3~30 mg/L) extracts. ALP activity was not affected by yam ethanol extracts. Our results demonstrated that yam extracts stimulated osteoblast proliferation and enhanced the accumulation of the collagenous bone matrix protein type I collagen in the extracellular matrix. These results suggest that yam extracts may be a potential activator for bone formation by increasing osteoblast proliferation and increasing bone matrix protein type I collagen. Before confirming the osteogenic action of yam, further studies for clarifying how and whereby yam extracts can stimulate this ostegenesis action are required.