Effect of quercetin on chondrocyte phenotype and extracellular matrix expression / 中国天然药物

Due to the poor repair ability of cartilage tissue, regenerative medicine still faces great challenges in the repair of large articular cartilage defects. Quercetin is widely applied as a traditional Chinese medicine in tissue regeneration including liver, bone and skin tissues. However, the evidence for its effects and internal mechanisms for cartilage regeneration are limited. In the present study, the effects of quercetin on chondrocyte function were systematically evaluated by CCK8 assay, PCR assay, cartilaginous matrix staining assays, immunofluorescence assay, and western blotting. The results showed that quercetin significantly up-regulated the expression of chondrogenesis genes and stimulated the secretion of GAG (glycosaminoglycan) through activating the ERK, P38 and AKT signalling pathways in a dose-dependent manner. Furthermore, in vivo experiments revealed that quercetin-loaded silk protein scaffolds dramatically stimulated the formation of new cartilage-like tissue with higher histological scores in rat femoral cartilage defects. These data suggest that quercetin can effectively stimulate chondrogenesis in vitro and in vivo, demonstrating the potential application of quercetin in the regeneration of cartilage defects.

Nestin-positive stem cells participate in chondrocyte renewal in healthy adult lung cartilage / Células troncales nestin-positivas participan en la renovación de condrocitos en el cartílago pulmonar del adulto sano

It is considered that healthy adult cartilage has little or no capacity for renewal, and that chondrocytes maintain a stable resting phenotype and resist proliferation and differentiation throughout life. Recently we found that cell turnover in lung cartilage is possible and that nestin-positive cells may have a role in it. In this paper, we report additional findings about chondrocyte renewal in lung cartilage. Lung specimens from CD1 mice at the age of 2, 6, 12, 18 or 24 months were fixed in 10% neutral-buffered formalin and paraffin-embedded. Nestin expression was examined by an immunohistochemical peroxidase-based method. We found nestin-positive cells inside of cartilage islets and cells in division very close from them. Our findings indicate that there exist nestin-positive mesenchymal stem cells in the adult that are able to differentiate into lung chondrocytes, perhaps to maintain homeostasis or repair damaged tissue. These findings may improve our knowledge about the cartilage biology and could provide new cell candidates for cartilage tissue engineering.

Se considera que el cartílago adulto sano tiene poca o ninguna capacidad para renovarse, y que sus condrocitos permanecen en un estado de reposo estable, careciendo de las propiedades de proliferación y diferenciación. Recientemente encontramos que el recambio celular en el cartílago pulmonar es posible y que células troncales positivas para nestin pudieran tener algún papel en el mismo. En este artículo, reportamos nuevos hallazgos acerca de la renovación de condrocitos en el cartílago pulmonar. Pulmones de ratones CD1 de 2, 6, 12, 18 o 24 meses de edad se fijaron en formalina amortiguada al 10% y se incluyeron en parafina. Se analizó la expresión de nestin utilizando un método inmunohistoquímico basado en un sistema de detección con peroxidasa. Encontramos células positivas para nestin en el interior de los islotes de cartílago y células en división muy cercanas a ellas. Estos hallazgos indican que existen células madre mesenquimales positivas para nestin en el adulto con capacidad para diferenciarse en condrocitos pulmonares, probablemente para mantener la homeostasis tisular o reparar daños en el tejido. Asimismo, estos hallazgos pueden aumentar nuestra comprensión acerca de las propiedades biológicas del cartílago y podrían proporcionar nuevos candidatos para la ingeniería celular en la terapia regenerativa en enfermedades de las articulaciones.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP)

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin alpha5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.

Immortalization of human precartilaginous stem cells by transfecting SV40Tag / 华中科技大学学报（医学）（英德文版）

Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs. Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection. Colonies were isolated by puromycin selection and expanded by multiple passages. Immunohistochemistry, RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines. The positive colonies were isolated and subcultured, designated immortalized precartilaginous stem cells (IPSCs), which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR. SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting, and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR. These findings suggested that IPSCs strain with SV40Tag was constructed successfully.

Construction of Sox9 gene eukaryotic expression vector and its inductive effects on directed differentiation of bone marrow stromal cells into precartilaginous stem cells in rats / 华中科技大学学报（医学）（英德文版）

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.

Imobilização articular prolongada e remobilização / Prolonged articular immobilization and remobilization

Foram avaliados os aspectos clínicos e morfofisiológicos da articulação do joelho de ratos após imobilização prolongada e remobilização com atividade livre e natação. Trinta e quatro ratos adultos machos (Wistar) foram alocados aleatoriamente em um dos quatro grupos: sem imobilização (G1, n = 4, grupo controle), com imobilização (G2, n = 10), imobilização e remobilização com atividade livre (G3, n = 10), imobilização e remobilização com atividade livre e natação (G4, n = 10). A imobilização degenerou as células sinoviais, em relação ao controle, indicando diminuição da produção de fluido sinovial e redução do suprimento nutricional à cartilagem e, tanto a atividade livre na gaiola quanto sua associação com a natação influenciaram positivamente o retorno das condições morfológicas da cápsula, anteriores à imobilização.

The aim of this study was to evaluate the clinical and morphophysiological aspects of the rat knee articulation after immobilization and remobilization with free activity and swimming. Thirty four adult male rats (Wistar) were randomly allocated into one of four groups: without immobilization, (G1, n = 4, control group), with immobilization (G2, n = 10), immobilization and remobilization with free activity (G3, n = 10), and immobilization and remobilization with free activity and swimming (G4, n = 10). Synovial cells were degenerated by immobilization indicating decreased synovial fluid production and reduced nutritional supplying to the cartilage and both free cage activity and its association with swimming influenced positively the return of the capsule morphologic conditions to those before immobilization.

Effect of Stathmin decoy-oligodeoxynucleotides on the proliferation and differentiation of precartilainous stem cells / 华中科技大学学报（医学）（英德文版）

By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.

TGF-beta and new bone formation / TGF- ß y Neoformación Ósea

Este trabajo tiene como objetivo presentar, por medio de una revisión de la literatura, las principales características de la TGF-beta en la regulación de la neoformación ósea.

The aim of this work is to present, by a literature review, the principal characteristics of TGF-beta, in the regulation and new bone formation.

Spatio-temporal expression patterns of Runx2 isoforms in early skeletogenesis

Skeletogenesis occurs through either intramembranous or endochondral ossification. In addition, some parts of the skeletal components maintain their cartilaginous characteristics throughout life without mineralization. Runx2 is known to be a pivotal transcription factor for all skeletogenic processes. In this study, we examined the expression patterns of two major isoforms of Runx2 in early skeletogenesis. During intramembranous bone formation, Runx2-type I (Runx2-I) was widely expressed in osteoprogenitor cells and active osteoblasts, while Runx2-type II (Runx2-II) expression was stringently restricted to cells lining mineralized bones. Cells in permanent cartilage expressed collagen type II (Col-II) but never expressed Runx2 or Col-X. These permanent cartilages were well circumscribed by Runx2-I positive cells, in which Runx2-II was negative. In endochondral bone formation, Runx2 expression temporarily disappeared in Col-II-positive proliferating chondrocytes, but a secondary surge of Runx2-I expression occurred in the prehypertrophic zone before the mineralization of cartilage. Collectively, both Runx2 isoforms showed very similar expression patterns in active bone forming areas; however, Runx2-I has an exclusive role in the early commitment stage of intramembranous or endochondral bone forming processes or in cells surrounding permanent cartilage.

Chondrocyte allograft transplantation for damaged growth plate reconstruction

The growth plate is responsible for longitudinal bone growth. The problem of repair of damaged growth plate in children has never been adequately solved. The purpose of this study is to investigate the ability of the cultured chondrocyte for the prevention of bony bridge and repairment of damaged growth plate. Chondrocytes were obtained from the new born canine epiphyseal plate and was cultured in high density. Fourteen days later they formed micromass easily removable from the culture flask. Twenty dogs were divided into two groups; in group I, the medial proximal tibial growth plate was destroyed and then cultured chondrocytes were transplanted into the defect, and in group II, the medial proximal tibial growth plate was left in destroyed state. Each left leg was remained as a control. The growth pattern was observed radiographically and histologically until 16 weeks after graft. 4 weeks after the operation, the angular deformity had been observed, and 31 degrees of angulation was noted at the 16th week in group II, while there was less than 8 degrees of angulation and nearly normal growth in most of dogs of group I (8 of 10 dogs). The other 2 dogs had shown 20 degrees angulation. In group II, there was definite bony bridge on the medical proximal growth plate. In group I, initially, the cultured chondrocyte remained as a amorphous cartilagenous mass, but as time progressed, amorphous cartilagenous mass had formed cartilagenous matrix which was proved by Safranin-O staining. Although this study showed the role of cultured chondrocyte as a method of preventation of bony bridge formation and possibility to repair of growth plate, further studies should be done to prove the reconstruction of the growth plate.