배양한 연골세포와 이식편에서 Calcitonin-gene-related Peptide와 Type II Collagen 면역반응 및 형태학적인 변화

정호중(Ho-Joong Jeong),배기원(Kee-Won Bae),양영철(Young-Cheoul Yang) 대한해부학회 2000 Anatomy & Cell Biology Vol.33 No.5

성숙한 동물의 관절연골과 갈비연골로부터 연골세포를 분리하는 방법의 확립 및 배양한 두 연골세포의 표현형이 이식 전까지 유지되고 있는지를 type II collagen 면역반응으로 확인하고 이식편의 조직적응성을 calcitonin-gene-related peptide (CGRP)와 type II collagen의 면역반응으로 관찰하고자 하였다. 생후 6개월 된 1.5~2 kg 내외의 성숙한 토끼의 관절연골과 갈비연골로부터 분리된 연골세포를 배양하여 전형적인 표현 형이 유지되고 있는 관절연골세포를 갈비연골막 위에 배양하고 이식하여, 이식 연골에서 CGRP와 type II collagen의 면역반응 및 연골세포의 형태학적인 변화를 조사하였다. 관절연골세포는 일차와 이차 계대배양에서 외형적 표현형을 유지하였으나, 갈비연골세포는 섬유모세포 모양의 세포로 표현형이 변화되었다. 관절연골세포를 갈비연골막에 접종했을 때 활착력이 좋았고 부착된 세포들은 납짝해지면서 가장자 리에 연골을 형성하고 있었다. 관절연골세포와 갈비연골세포의 배양에서 모든 연골세포들의 type II collagen 반응이 소실하지는 않았다. 성장이 빠르고 표현형을 잘 유지한 관절연골세포를 이식하여 형성된 이식편의 CGRP 면역반응은 전체적으로 증가하였으며, type II collagen 면역반응은 감소하였다. 전자현미경 소견에서 배양된 관절연골세포는 매우 크고 밝은 핵과 세포질내 소수의 과립들, 소포들이 풍부하였으나, 배 양시간이 경과하면서 핵이 위축되고 세포질내 많은 큰 공포들이 관찰되었으며, 갈비연골막위에 배양하면 세포막아래에 분절상으로 배열하고 있는 과립세포질세망들과 가늘고 짧은 융모들이 관찰되었다. 배양한 관절연골세포는 전형적인 연골세포의 형태를 유지하며 type II collagen 반응은 소실되지 않았다. 관절연골세포는 거의 완전한 세포소기관과 뚜렷한 핵소체와 활성화된 염색질을 가지고 있었고, 갈비연골막 접종에서는 분절상 과립세포질세망들로 보아 증식하는 미성숙한 연골세포의 형태를 나타내었다. 이식한 부위의 관절연골세포는 시간경과에 따라 세포소기관들이 소실되었으며, CGRP에 의해서 변성이 일어나 type II collagen 반응이 점차 감소된다는 것을 알 수 있었다. The purpose of this study was to investigate the immunohistochemical reaction of calcitonin-gene-related peptide (CGRP) and type II collagen and also morphological changes of cartilage implants and cultured chondrocytes isolated from the articular and costal cartilages. The chondrocytes were isolated from the head of the femur and the 11th costal cartilage of the 6 months old rabbits. De novo implants were prepared from the chondrocytes cultured on the perichondrium by culturing isolated articular chondrocytes. Cultured chondrocytes and implants were evaluated by immunohistochemical staining of CGRP and type II collagen and electron microscopy. Articular chondrocytes maintained the typical phenotype in the 1st and 2nd subcultures, but the costal chondrocytes were transformed into fibroblast-like cells. The articular chondrocytes cultured on the perichondrium were more flattened and formed the cartilage. Most chondrocytes were no loss of type II collagen immunostaining by culturing. Implants replaced by the cultured articular chondrocytes were generally increased CGRP and decreased type II collagen immunoreaction. Electron microscopically the cultured articular chondrocytes had a large euchromatic nucleus, a few granules, and abundant vesicles. During culture, the nucleus became atropy and the cytoplasm contained many large vacuoles. The chondrocytes cultured on the perichondrium showed a lot of segmented rough endoplasmic reticulum and fine short microvilli. During culture, articular chondrocytes maintained typical phenotype and type II collagen reaction. The cultured articular chondrocytes had some organelles and euchromatic nucleus with prominent nucleolus. The chondrocytes cultured on the perichondrium showed active secretion of the matrix with small vesicles and well developed endoplasmic reticulum. The implanted articular chondrocytes showed the decrease of their organelles after secretion of the marix and became increased CGRP and decreased type II collagen immunoreaction.

Potential Predictive Markers for Proliferative Capacity of Cultured Human Articular Chondrocytes: PCNA and p21

Kim, Hyeon Joo,Park, So Ra,Park, Heon Joo,Choi, Byung Hyune,Min, Byoung-Hyun Blackwell Science Inc 2005 Artificial Organs Vol.29 No.5

<P>Abstract: </P><P>The  purpose  of  this  study  was  to  investigate  age-related changes in the proliferative ability of human articular chondrocytes in culture. In addition, the possible markers for the proliferative capacity of chondrocytes were examined. Chondrocytes obtained from human articular cartilages of young (under 40 years) or old (over 60 years) individuals were expanded until their growth was arrested. The number of cells and the type II collagen phenotype were determined together with the expression levels of proliferating cell nuclear antigen (PCNA) and p21<SUP>WAF1/CIP</SUP> along with the passages of cultured chondrocytes. The results showed that young chondrocytes had higher proliferative capacity and viability than old chondrocytes. The growth arrest and the cessation in the expression of type II collagen were accompanied by down-regulation of PCNA and up-regulation of p21<SUP>WAF1/CIP</SUP> levels in both young and old chondrocytes. Notably, the expression levels of PCNA and p21<SUP>WAF1/CIP</SUP> along with the passages were correlated inversely to each other and showed distinct patterns between young and old chondrocytes. These results suggest that senescence of human articular chondrocytes leads to the decrease in the proliferative capacity and phenotypic stability. In addition, PCNA and p21 could be molecular markers that represent the status of these age-related properties of human articular chondrocytes in vitro.</P>

Fully Dedifferentiated Chondrocytes Expanded in Specific Mesenchymal Stem Cell Growth Medium with FGF2 Obtains Mesenchymal Stem Cell Phenotype In Vitro but Retains Chondrocyte Phenotype In Vivo

Lee, Jungsun,Lee, Jin-Yeon,Chae, Byung-Chul,Jang, Jeongho,Lee, EunAh,Son, Youngsook SAGE Publications 2017 Cell transplantation Vol.26 No.10

<P>Given recent progress in regenerative medicine, we need a means to expand chondrocytes in quantity without losing their regenerative capability. Although many reports have shown that growth factor supplementation can have beneficial effects, the use of growth factor–supplemented basal media has widespread effect on the characteristics of chondrocytes. Chondrocytes were in vitro cultured in the 2 most widely used chondrocyte growth media, conventional chondrocyte culture medium and mesenchymal stem cell (MSC) culture medium, both with and without fibroblast growth factor-2 (FGF2) supplementation. Their expansion rates, expressions of extracellular matrix–related factors, senescence, and differentiation potentials were examined in vitro and in vivo. Our results revealed that chondrocytes quickly dedifferentiated during expansion in all tested media, as assessed by the loss of type II collagen expression. The 2 basal media (chondrocyte culture medium vs. MSC culture medium) were associated with distinct differences in cell senescence. Consistent with the literature, FGF2 was associated with accelerated dedifferentiation during expansion culture and superior redifferentiation upon induction. However, chondrocytes expanded in FGF2-containing conventional chondrocyte culture medium showed MSC-like features, as indicated by their ability to direct ectopic bone formation and cartilage formation. In contrast, chondrocytes cultured in FGF2-supplemented MSC culture medium showed potent chondrogenesis and almost no bone formation. The present findings show that the chosen basal medium can exert profound effects on the characteristics and activity of in vitro–expanded chondrocytes and indicate that right growth factor/medium combination can help chondrocytes retain a high-level chondrogenic potential without undergoing hypertrophic transition.</P>

Inhibition of microRNA-449a prevents IL-1β-induced cartilage destruction via SIRT1

Park, K.W.,Lee, K.M.,Yoon, D.S.,Park, K.H.,Choi, W.J.,Lee, J.W.,Kim, S.H. Published for the Society by Baillère Tinda 2016 Osteoarthritis and cartilage Vol.24 No.12

<P>Objective: SIRT1 has anti-inflammatory as well as protective effects in chondrocytes. The object of this study was to investigate whether microRNA-449a regulates expression of SIRT1, which inhibits expression of catabolic genes in IL-1 beta-induced cartilage destruction. Materials and methods: MicroRNA-449a expression was determined in OA chondrocytes and IL-1 beta induced chondrocytes by real-time PCR. MicroRNA-449a binding sites on the 3'-UTR of SIRT1 mRNA and binding site conservation were examined using microRNA target prediction tools. SIRT1-overexpressing or knockdown chondrocytes were transfected with microRNA-449a or anti-microRNA-449a mimic and stimulated by IL-1 beta. Expression of catabolic and anabolic genes was examined by real-time PCR and western blotting. Finally, positive effects of anti-microRNA-449a on expression of these genes were confirmed by western analysis of OA chondrocytes. Results: Expression of microRNA-449a was increased in OA chondrocytes and IL-1 beta-induced chondrocytes. MMP-13 expression was enhanced, whereas type II collagen and SIRT1 expression were decreased in IL-1 beta-induced chondrocytes. SIRT1 overexpression resulted in decreased expression of catabolic genes such as MMPs and ADAMTSs in response to IL-1 beta, but these effects were moderated by microRNA-449a. Suppression of microRNA-449a by anti-microRNA-449a inhibited expression of catabolic genes despite IL-1 beta stimulation, but these effects were abolished in SIRT1 knockdown chondrocytes. Furthermore, expression of catabolic genes was decreased and expression of type II collagen as well as SIRT1 was restored by anti-microRNA-449a in OA chondrocytes as well as in IL-1 beta-induced chondrocytes. Conclusion: Silencing of microRNA-449a had a protective effect, inhibiting catabolic gene expression and restoring anabolic gene expression, by targeting SIRT1 in IL-1 beta-induced cartilage destruction. (C) 2016 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.</P>

Matrix Production in Chondrocytes Transfected with Sex Determining Region Y-Box 9 and Telomerase Reverse Transcriptase Genes: An In Vitro Evaluation from Monolayer Culture to Three-Dimensional Culture

Noorhidayah Md Nazir,Ahmad Hafiz Zulkifly,Kamarul Ariffin Khalid,Ismail Zainol,Zaitunnatakhin Zamli,Munirah Sha’ban 한국조직공학과 재생의학회 2019 조직공학과 재생의학 Vol.16 No.3

BACKGROUND: This study aimed to observe the cartilaginous matrix production in SRY (sex determining region Y)-box 9 (SOX9)- and/or telomerase reverse transcriptase (TERT)-transfected chondrocytes from monolayer to three-dimensional (3D) culture. METHODS: The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D ‘‘cells-scaffolds’’ constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction. RESULTS: All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D ‘‘cells-scaffolds’’ constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA. CONCLUSION: These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.

Effect of growth hormone releasing hormone on chondrocytes of osteoarthritis

( Zhuoran Li ),( Yao Li ) 대한내과학회 2022 The Korean Journal of Internal Medicine Vol.37 No.1

Background/Aims: To evaluate the effect and possible mechanism of growth hormone releasing hormone (GHRH) on chondrocytes of osteoarthritis (OA). Methods: Articular chondrocytes were cultured and the expression of GHRH receptor in chondrocytes was detected. Then recombinant adenovirus GHRH (Ad-GHRH) was transfected to one group of chondrocytes. The expression of collagen type II, matrix metalloproteinase 13 (MMP-13) and signal transducer and activator of transcription 3 (STAT3) in each experimental group was determined by Western blot. Results: The GHRH receptor was expressed in chondrocytes, and this provided a basis for further study of the role of GHRH in chondrocytes. Cell proliferation of the Ad-GHRH group was significantly higher than that of the OA group by CCK-8 assay. Compared with the OA-group, the protein expression of MMP13 was decreased in the Ad-GHRH group. Compared with the OA-group, the protein expression of collagen type II, phosphorylated STAT3 (P-STAT3) were increased in the Ad-GHRH group. Conclusions: Our results show that the GHRH receptor is expressed in chondrocytes. GHRH can promote the proliferation of chondrocytes and the synthesis of type II collagen, and increase the extracellular matrix, which is achieved by phosphorylated STAT3 protein.

Anti-osteoarthritis effects of Pomegranate, Eucommiae cortex and Achyranthis radix extracts on the primary cultured rat articular chondrocytes

Choi, Beom-Rak,Ku, Sae-Kwang,Kang, Su-Jin,Park, Hye-Rim,Sung, Mi-Sun,Lee, Young-Joon,Park, Ki-Moon Society of Preventive Korean Medicine 2017 대한예방한의학회지 Vol.21 No.3

Objectives : The objective of present study is to evaluate anti-arthritic effects of dried pomegranate concentrate powders (PCP), Eucommiae Cortex aqueous (EC) and ethanolic (ECe) extracts, Achyranthis Radix aqueous (AR) and ethanolic (ARe) extracts on the primary cultured rat articular chondrocytes. Methods : MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide) assay was performed cytotoxic effect of test substances. In addition, anti-inflammatory effects were also observed on the lipopolysaccaride (LPS) treated chondrocytes through prostaglandin $E_2\;(PGE_2)$ production and 5-lipoxygenase (LPO) activities, and inhibitory effects on metalloproteinase (MMP)-2 and MMP-9 activities were observed on the recombinant human interleukin $(rhIL)-1{\alpha}$ treated chondrocytes with their extracellular matrix (ECM) related mRNA expressions - collagen type II, SOX9 and aggrecan. Results : As results, ECe and ARe showed obvious cytotoxicity against primary cultured rat articular chondrocytes at a dose level of 10 mg/ml, respectively. However, no obvious cytotoxic effects of PCP, EC and AR were demonstrated at a dose level of 10 mg/ml, on the primary cultured rat articular chondrocytes. In addition, treatment of LPS $50{\mu}g/ml$ induced significant increases of $PGE_2$ contents and 5-LPO activities indicating inflammatory responses of the primary cultured rat articular chondrocytes, and also decreases of cell viabilities, increases of MMP-2 and MMP-9 activities with decreases of extracellular matrix (ECM) related collagen type II, SOX9 and aggrecan mRNA expressions were observed by treatment of $rhIL-1{\alpha}$ 50 ng/ml, suggesting damages on the primary cultured rat articular chondrocytes and related ECM degradations. However, these inflammatory responses and related ECM degradations were inhibited by pretreatment of all test substances, in order of PCP > ECe > ARe > EC > AR, and $rhIL-1{\alpha}$ induced chondrocytes deaths are inhibited by treatment in order of PCP > EC > AR > ECe > ARe. Conclusions : Taken together, it is expected that mixed formulation of PCP as main components with appropriate proportion of EC and AR as additional components will be achieved a potent alternative medicinal food for osteoarthritis.

Anti-osteoarthritis effects of Pomegranate, Eucommiae cortex and Achyranthis radix extracts on the primary cultured rat articular chondrocytes

최범락,구세광,강수진,박혜림,성미선,이영준,박기문 대한예방한의학회 2017 대한예방한의학회지 Vol.21 No.3

Objectives : The objective of present study is to evaluate anti-arthritic effects of dried pomegranate concentrate powders (PCP), Eucommiae Cortex aqueous (EC) and ethanolic (ECe) extracts, Achyranthis Radix aqueous (AR) and ethanolic (ARe) extracts on the primary cultured rat articular chondrocytes. Methods : MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide) assay was performed cytotoxic effect of test substances. In addition, anti-inflammatory effects were also observed on the lipopolysaccaride (LPS) treated chondrocytes through prostaglandin E2 (PGE2) production and 5-lipoxygenase (LPO) activities, and inhibitory effects on metalloproteinase (MMP)-2 and MMP-9 activities were observed on the recombinant human interleukin (rhIL)-1α treated chondrocytes with their extracellular matrix (ECM) related mRNA expressions - collagen type II, SOX9 and aggrecan. Results : As results, ECe and ARe showed obvious cytotoxicity against primary cultured rat articular chondrocytes at a dose level of 10 mg/ml, respectively. However, no obvious cytotoxic effects of PCP, EC and AR were demonstrated at a dose level of 10 mg/ml, on the primary cultured rat articular chondrocytes. In addition, treatment of LPS 50 μg/ml induced significant increases of PGE2 contents and 5-LPO activities indicating inflammatory responses of the primary cultured rat articular chondrocytes, and also decreases of cell viabilities, increases of MMP-2 and MMP-9 activities with decreases of extracellular matrix (ECM) related collagen type II, SOX9 and aggrecan mRNA expressions were observed by treatment of rhIL-1α 50 ng/ml, suggesting damages on the primary cultured rat articular chondrocytes and related ECM degradations. However, these inflammatory responses and related ECM degradations were inhibited by pretreatment of all test substances, in order of PCP › ECe › ARe › EC › AR, and rhIL-1α induced chondrocytes deaths are inhibited by treatment in order of PCP › EC › AR › ECe › ARe. Conclusions : Taken together, it is expected that mixed formulation of PCP as main components with appropriate proportion of EC and AR as additional components will be achieved a potent alternative medicinal food for osteoarthritis.

Down-Regulation of Transglutaminase 2 Stimulates Redifferentiation of Dedifferentiated Chondrocytes through Enhancing Glucose Metabolism

Ko, Kyoung-Won,Choi, Bogyu,Park, Sunghyun,Arai, Yoshie,Choi, Won Chul,Lee, Joong-Myung,Bae, Hojae,Han, In-Bo,Lee, Soo-Hong MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.11

<P>Expansion of chondrocytes for repair of articular cartilage can lead to dedifferentiation, making it difficult to obtain a sufficient quantity of chondrocytes. Although previous studies have suggested that culture in a three-dimensional environment induces redifferentiation of dedifferentiated chondrocytes, its underlying mechanisms are still poorly understood in terms of metabolism compared with a two-dimensional environment. In this study, we demonstrate that attenuation of transglutaminase 2 (TG2), a multifunctional enzyme, stimulates redifferentiation of dedifferentiated chondrocytes. Fibroblast-like morphological changes increased as TG2 expression increased in passage-dependent manner. When dedifferentiated chondrocytes were cultured in a pellet culture system, TG2 expression was reduced and glycolytic enzyme expression up-regulated. Previous studies demonstrated that TG2 influences energy metabolism, and impaired glycolytic metabolism causes chondrocyte dedifferentiation. Interestingly, TG2 knockdown improved chondrogenic gene expression, glycolytic enzyme expression, and lactate production in a monolayer culture system. Taken together, down-regulation of TG2 is involved in redifferentiaton of dedifferentiated chondrocytes through enhancing glucose metabolism.</P>

Original Article : Effect of low intensity pulsed ultrasound in activating the mitogen-activated protein kinase signaling pathway and inhibition inflammation cytokine synthesis in chondrocytes

( Eun Jung Kim ),( Gye Yeop Kim ) 물리치료재활과학회 2014 Physical therapy rehabilitation science Vol.3 No.1

Objective: Low intensity pulsed ultrasound (LIPUS) has been shown to accelerate cell proliferation and tissue healing in both animal models and clinical trials. However, details of the clinical effects of LIPUS have not been well characterized. The aim of this study was to investigate the effect of LIPUS on mitogen-activated protein kinase (MAPK) activation in rat articular chondrocytes. Design: Cross-sectional study. Methods: Chondrocyte were cultured in six well cell culture plates for 72 hours at 37oC with 5% CO2, and then exposed to LIPUS at 1.5 MHz frequency and 30-mW/cm2 power. Changes in chondrocyte activities were evaluated in response to oxydative stress in dose-dependent (0 and 300 uM) and time-dependent (0-24 hr) manner. The cell viability were analyzed using MTT [3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide]. The expression of p38 MAPK was measured using western blotting. Results: Oxidative stress was induced in rat chondrocytes using hydrogen peroxide (H2O2). The cell viability was decreased in chondrocytes after the H2O2 dose and time-dependent treatment. The p38 MAPK phosphorylation occurred at a significantly increased rate after H2O2 treated (p<0.05). Expression of p38 MAPK was decreased in the p38 inhibitor groups compared with the oxidative stress-induced chondrocyte damage via the p38 MAPK signaling pathways (p<0.05). Conclusions: It could be concluded that LIPUS can inhibit oxidative stress-induced chondrocyte damage via the p38 MAPK signaling pathways.