사람 귀 연골 세포주와 조직공학술을 이용한 신생 연골의 재생

정재호,김승업,박병윤,박명철,임현이,이명애,홍현석 대한성형외과학회 2003 Archives of Plastic Surgery Vol.30 No.5

The purpose of these studies was an establishment of human auricular chondrocyte cell line using retrovirus mediated v-myc transfer, characterizing the human auricular chondrocyte cell line by type II collagen mRNA expression and transplantation of human auricular cell line into immunological incompetent nude mice to establish neocartilage formation. Also, I evaluated the growth rate of chondrocyte cell line to measure the cellular proliferative potency. I have established the human auricular chondrocyte cell line integrated v-myc and confirmed by v-myc transduced Myc protein expression by immunohistochemistry and immunoblotting study. And, growth rate of established human auricular chondrocyte cell line increased 4 folds times faster than primarily cultured human auricular chondrocyte. The established human auricular chondrocyte had type II collagen mRNA upto 8 months in monolayer culture. And we observed formation of neocartilage on the back of nude mice using chondrocyte cell line/fibrin glue polymer at 12 weeks transplantation.

일산화질소에 의해 유도된 연골 세포 사멸에서의 항세포사멸유전자 Bcl-XL의 방어 효과

한창환 ( Chang Whan Han ),신윤학 ( Yun Hak Shin ),권순용 ( Soon Yong Kwon ),조윤경 ( Yun Kyoung Cho ),지보근 ( Bo Keun Jee ),김영율 ( Young Yul Kim ),김순희 ( Soon Hee Kim ),박기숙 ( Ki Sook Park ),강길선 ( Gil Son Khang ) 한국조직공학과 재생의학회 2005 조직공학과 재생의학 Vol.2 No.2

To evaluate whether transfection of human chondrocytes with an anti-apoptotic gene, Bcl-XL, can prevent nitric oxide (NO)-induced chondrocyte apoptosis. We stably transduced early passage chondrocytes with an anti- apoptotic gene Bcl-XL and retrovirus. Bcl-XL transducants were compared with chondrocytes transduced in parallel with a Lac-Z gene. After the cells were treated with 500 microM of SNP, the process of apoptosis was assessed by trypan blue exclusion, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The functional aspect of the chondrocyte was measured by proteoglycan synthesis assay. Chondrocytes were cultivated in NO, the chondrocytes that were either not transfected or transfected with Lac-Z exhibited a decrease in viable cell number in comparison to the Bcl-XL transfected chondrocytes. The NO significantly increased fragmentation of DNA in chondrocytes that were either not transfected or transfected with Lac-Z, and this increase in DNA fragmentation was significantly greater than in the chondrocytes transfected with Bcl-XL. Flow cytometric result showed that apoptotic chondrocytes were significantly higher in the transfected and Lac-Z transfected chondrocytes than Bcl-XL transfected chondrocytes. Chondrocytes transfected with Bcl-XL were protected from NO-induced impairment of proteoglycan synthesis response to NO. From the present study, we have demonstrate that NO-induced chondrocytes death involve mechanisms subjected to regulation by an anti-apoptotic protein, Bcl-XL. Therefore, Bcl-XL gene therapy has the potential to protect apoptosis in human chondrocyte.

Cordycepin inhibits chondrocyte hypertrophy of mesenchymal stem cells through PI3K/Bapx1 and Notch signaling pathway

( Zhen Cao ),( Ce Dou ),( Jianmei Li ),( Xiangyu Tang ),( Junyu Xiang ),( Chunrong Zhao ),( Lingyu Zhu ),( Yun Bai ),( Qiang Xiang ),( Shiwu Dong ) 생화학분자생물학회 2016 BMB Reports Vol.49 No.10

Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering to repair articular cartilage defects. However, hypertrophy of chondrocytes derived from MSCs might hinder the stabilization of hyaline cartilage. Thus, it is very important to find a suitable way to maintain the chondrogenic phenotype of chondrocytes. It has been reported that cordycepin has anti-inflammatory and anti-tumor functions. However, the role of cordycepin in chondrocyte hypertrophy remains unclear. Therefore, the objective of this study was to determine the effect of cordycepin on chondrogenesis and chondrocyte hypertrophy in MSCs and ATDC5 cells. Cordycepin upregulated chondrogenic markers including Sox9 and collagen type II while down-regulated hypertrophic markers including Runx2 and collagen type X. Further exploration showed that cordycepin promoted chondrogenesis through inhibiting Nrf2 while activating BMP signaling. Besides, cordycepin suppressed chondrocyte hypertrophy through PI3K/Bapx1 pathway and Notch signaling. Our results indicated cordycepin had the potential to maintain chondrocyte phenotype and reconstruct engineered cartilage. [BMB Reports 2016; 49(10): 548-553]

LGR5 Modulates Differentiated Phenotypes of Chondrocytes Through PI3K/AKT Signaling Pathway

Wu Xu,Fu Yaoyao,Ma Jing,Li Chenlong,He Aijuan,Zhang Tianyu 한국조직공학과 재생의학회 2024 조직공학과 재생의학 Vol.21 No.5

Background: Tissue engineering is increasingly viewed as a promising avenue for functional cartilage reconstruction. However, chondrocyte dedifferentiation during in vitro culture remains an obstacle for clinical translation of tissue engineered cartilage. Re-differentiated induction have been employed to induce dedifferentiated chondrocytes back to their original phenotype. Regrettably, these strategies have been proven to be only moderately effective. Methods: To explore underlying mechanism, RNA transcriptome sequencing was conducted on primary chondrocytes (P0), dedifferentiated chondrocytes (P5), and redifferentiated chondrocytes (redifferentiation-induction of P5, P5.R). Based on multiple bioinformatics analysis, LGR5 was identified as a target gene. Subsequently, stable cell lines with LGR5 knocking-down and overexpression were established using P0 chondrocytes. The phenotypic changes in P1 and P5 chondrocytes with either LGR5 knockdown or overexpression were assessed to ascertain the potential influence of LGR5 dysregulation on chondrocyte phenotypes. Regulatory mechanism was then investigated using bioinformatic analysis, protein–protein docking, immunofluorescence co-localization and immunoprecipitation. Results: The current study found that dysregulation of LGR5 can significantly impact the dedifferentiated phenotypes of chondrocytes (P5). Upregulation of LGR5 appears to activate the PI3K/AKT signal via increasing the phosphorylation levels of AKT (p-AKT1). Moreover, the increase of p-AKT1 may stabilize β-catenin and enhance the intensity of Wnt/β-catenin signal, and help to restore the dedifferentated phenotype of chondrocytes. Conclusion: LGR5 can modulate the phenotypes of chondrocytes in P5 passage through PI3K/AKT signaling pathway.

천연 알긴산을 지지체로 한 삼차원 배양에서 연골세포의 생존력

류유정 ( You Jeong Lyou ),김상경 ( Sang Gyung Kim ),최연희 ( Yeon Hee Choi ),최정윤 ( Jung Yoon Choe ),김채기 ( Chae Gi Kim ),김종기 ( Jong Ki Kim ),윤연희 ( Yeon Hee Yoon ),신임희 ( Im Hee Shin ),박상옥 ( Sang Ock Park ) 대한류마티스학회 2003 대한류마티스학회지 Vol.10 No.3

Objective: Articular cartilage has a highly limited capacity to repair because of lack of blood supply. There have been no effective modality to regenerate the articular cartilage and prevent degenerative changes. It is necessary to proliferate the cells in vitro, however the cells lose their phenotype during in vitro monolayer culture. Although it is not enough to increase the number of the cells in the three dimensional culture, it is a effective way to maintain their original phenotype expression. Alginate has been used as a good source of scaffold in chondrocyte three dimensional culture. The objective of this study was to find the most favorable scaffold for chondrocyte viability among various alginate extracted from natural source in chondrocyte three dimensional culture. Methods: The alginate extracted from brown seaweed, Undaria pinnatifida and sea tangle, Laminaria japonica inhabitating near Korean sea and commercially available alginate were used. Chondrocytes isolated from adult pig were used. Three kinds of chondrocyte-alginate bead were made and incubated for forty-four days. Cellular viability and glycosaminoglycan (GAG) content were measured and compared. SPSS Version 10.0 was used for data analysis. Results: Viability of chondrocyte and GAG content were increased as a function of time. Alginate from brown seaweed, U. pinnatifida appeared more favorable to maintain chondrocyte viability than others. The total GAG content was similar among three kinds of alginate Conclusion: Alginate extracted from natural see weed, especially brown seaweed, may be a good source to maintain chondrocyte viability in three dimensional culture.

축두하악관절판 연골세포증식에 대한 배양액의 비교

한승규,김우경,Mark E. Mason 大韓成形外科學會 1999 Archives of Plastic Surgery Vol.26 No.4

Recent advances in cell culture and tissue engineering permit the successful treatment of deep articular defects with autologous chondrocyte transplantation. While chondrocyte culturing from articular cartilage is commonplace, in vitro growth of chondrocytes from temporomandibular joint fibrocartilage has not been defined. Likewise, the concept of autologous chondrocyte transplantation has not been applied to the temporomandibular joint. Since fibrocartilage differs structurally and functionally from articular cartilage, an effective culturing method for this tissue is essential as well. The ultimate goal of this project is to enable the use of autologous temporomandibular joint chondrocyte transplantation for the regeneration of damaged or missing components of the joint. The aim of this pilot study is to develop an effective cell culturing technique for chondrocytes harvested from the temporomandibular joint disc of dogs. Cartilage of the temporomandibular joint disc of drug-free mongrel dogs was dissected, dissociated, and centrifuged for chondrocyte collection. Blood specimen was collected from the same animal to produce the autologous serum. Chondrocytes were then dispersed in 24-well plates and incubated in one of four media, DMEM (Dulbecco`s modified Eagle`s medium), Ham`s F-12, DMEM/F-12, or Iscove`s MDM(modified Dulbecco`s medium). Each medium was also mixed with either 10% fetal bovine serum or 10% autologous serum. Temporomandibular joint disc chondrocytic proliferation was tested in all the culture media with sera on the fifth day. The initial plating count was held constant throughout at 1×10⁴ cells/well and eight samples were evaluated in each culture. The results demonstrated that fetal bovine serum worked much better than autologous serum in all evaluated media and that DMEM/F-12 mixed with 10% fetal bovine serum was the most optimal culture condition for expansion of temporomandibular joint disc chondrocytes.

마우스 성장판 결손부에 이식된 배아기 간(幹)세포와 연골세포의 형태학적 비교

권오숙,박중규,허대영,배기원,양영철 인제대학교 2001 仁濟醫學 Vol.22 No.1

Objectives: We aimed to investigate the morphological changes of embryonic stem cells and chondrocytes. We transplanted into experimental defect of mouse growth plate. Methods and Materials: The embryonic stem cells of blastocysts were collected from superovulated mice on day 4 after the vaginal plug checked. The chondrocytes were obtained from the growth plate of the 2 weeks old mice. Embryonic stem cells and chondrocytes were transplanted into the experimental defect of the growth plate. The morphological changes of these transplanted cells were investigated under the light microscope and electron microscope. Results: Embryonic stem cells and chondrocytes began to proliferate at the 8th day and the 6th day of the culture respectively. The cell size was not decreased embryonic stem cell, but chondrocytes. The survival of embryonic stem cells began to be at the 4th day and the chondrocytes at the 8th day after transplantation on the defect. During healing process, the PCNA reaction of embryonic stem cells appeared at the 4th day, and chondrocytes at the 8th day after transplantation. The number of PCNA positive embryonic stem cells began to be increased at the 8th day, and maintained by the 20th day. The number of PCNA positive chondrocytes began to be increased at the 10th day and decreased by the 20th day. On the electron microscopic finding, the embryonic stem cells were a large euchromatic nucleus with distinct nucleolus and contained well-developed rough endoplasmic reticulum by the 10th day. At the 14th and 20th day, transplanted embryonic stem cell showed the markedly decreased cytoplasm, and had a few rough endoplasmic reticulum and small vesicles and long slender processes. Chondrocytes had well-developed rough endoplasmic reticulum by the 14th day after the transplantation. At 14th day, the long slender process of the transplanted chondrocytes separated their cytoplasm from the extracelluar matrix. Transplanted chondrocytes began to be changed into the typical chondrocytic shape with euchromatic nucleus and rich free ribosomes at the 20th day. Conclusions: The embryonic stem cell proliferated slower than the chondrocyte, but took root faster than the chondrocyte on the epiphyseal defect. Embryonic stem cells which proliferated long-standing were gradually differentiated into the chondrocytic cell and finally maintained undifferentiated cells. Chondrocytes took root slowly and were rapidly differentiated into well differentiated typical chondrocytes.

태반추출물이 인간 연골세포의 증식과 분화에 미치는 영향

허준,서만수,박세정,임영국,신준호,정호윤,조병채,박재우 대한성형외과학회 2006 Archives of Plastic Surgery Vol.33 No.5

Purpose: The isolated human chondrocytes for cartilage reconstruction and transplantation presents a major problem as these cells would change biologically in vitro. For more effective applications of these cells in the clinical field, it is necessary to get a large amount of cells in a short period without affecting their function and phenotype.Methods: This study reports the effects of placenta extract on chondrocytes in vitro. We initiated this study on the basis of the hypothesis that placenta extract can influence both the proliferation of chondrocytes and their biologic functions(for example, to express cell specific gene or to produce their own extracellular matrix). Chondrocytes in monolayer culture with or without placenta extract were collected and analyzed by MTT assay, ECM assay, and RT-PCR.Results: Placenta extract stimulated the proliferation of chondrocytes in monolayer culture. The phenotype of chondrocytes was well maintained during the expansion in monolayers. Chondrocytes expanded in the presence of placenta extract produced ECM, glycosaminoglycan, abundantly. Compared to chondrocyte expanded in culture medium only, chondrocytes expanded with placenta extract demonstrated higher COL2A1 expression that was biochemically comparable to primary chondrocytes. This study provides an evidence that placenta extract is helpful to expand chondrocytes during tissue cultivation, to maintain their differentiated phenotype and to promote their function. Conclusion: These results suggest that placenta extract during cultivation play an important role in controlling cell behaviors. Furthermore, these results provide a biologic basis for cartilage tissue engineering.

Modulation of Apoptosis and Differentiation by the Treatment of Sulfasalazine in Rabbit Articular Chondrocytes

Won Kil Lee,Jin Seok Kang 한국독성학회 2016 Toxicological Research Vol.32 No.2

This study was conducted to examine the cellular regulatory mechanisms of sulfasalazine (SSZ) in rabbit articular chondrocytes treated with sodium nitroprusside (SNP). Cell phenotype was determined, and the MTT assay, Western blot analysis and immunofluorescence staining of type II collagen was performed in control, SNP-treated and SNP plus SSZ (50~200 μg/mL) rabbit articular chondrocytes. Cellular proliferation was decreased significantly in the SNP-treated group compared with that in the control (p < 0.01). SSZ treatment clearly increased the SNP-reduced proliferation levels in a concentration-dependent manner (p < 0.01). SNP treatment induced significant dedifferentiation and inflammation compared with control chondrocytes (p < 0.01). Type II collagen expression levels increased in a concentration-dependent manner in response to SSZ treatment but were unaltered in SNP-treated chondrocytes (p < 0.05 and < 0.01, respectively). Cylooxygenase-2 (COX-2) expression increased in a concentration-dependent manner in response to SSZ treatment but was unaltered in SNP-treated chondrocytes (p < 0.05). Immunofluorescence staining showed that SSZ treatment increased type II collagen expression compared with that in SNP-treated chondrocytes. Furthermore, phosphorylated extracellular regulated kinase (pERK) expression levels were decreased significantly in the SNP-treated group compared with those in control chondrocytes (p < 0.01). Expression levels of pERK increased in a concentration-dependent manner by SSZ but were unaltered in SNP-treated chondrocytes. pp38 kinase expression levels increased in a concentrationdependent manner by SSZ but were unaltered in control chondrocytes (p < 0.01). In summary, SSZ significantly inhibited nitric oxide-induced cell death and dedifferentiation, and regulated extracellular regulated kinases 1 and 2 and p38 kinase in rabbit articular chondrocytes.

육미지황탕가미방(六味地黃湯加味方) 약침이 연골세포에 미치는 영향

최원주,민상연,김장현,Choi, Won-Joo,Min, Sang Yeon,Kim, Jang Hyun 대한한방소아과학회 2010 대한한방소아과학회지 Vol.24 No.1

Objectives The purpose of this study is to examine the effect of chondrocyte re-differentiation in using ukmijihwang-Tang gamibang aqua-acupuncture. Methods In this study, chondrocytes were extracted from New Zealand white rabbit's knee joint, and cultured in monolayer after collagenase treatment. In the third passage, after the mRNA transcript of the type II collagen significantly reduced, diluted Yukmijihwang-Tang gamibang were added to cultured of chondrocyte, and its effect on the mRNA expression of type II collagen was quantitatively evaluated. Results As a result of treatment with Yukmijihwang-Tang gamibang in vitro for 48 hours, the mRNA expression of type II collagen was up-regulated. In addition, the result of H&E-staining in vivo indicated that chondrocyte-like tissues were formed in repairing injured cartilages after 12 weeks of treatment with Yukmijihwang-Tang gamibang. Conclusions These results indicated that Yukmijihwang-Tang gamibang was effective on the recovery of chondrocyte phenotype, and could be used for cartilage regeneration in arthritic diseases. However, more clinical study of Oriental medical treatment for this case might be also needed.