The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.
ABSTRACT Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.
- [Show abstract] [Hide abstract]
ABSTRACT: Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) superfamily, plays a key role in the induction of the differentiation of mesenchymal cells into chondrocytes to form cartilage tissue. However, it is not clear whether BMP2 regulates the proliferation of chondrocytes. In the present study, the effect of BMP2 on the proliferation of chondrocytes and its underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without BMP2 for 48 h. Cell viability was analyzed using the MTT assay. mRNA and protein expression levels of β-catenin, GSK-3β, Dvl1 and Cyclin D1 were detected using real-time RT-PCR and Western blotting, respectively. The cell cycle distribution of the chondrocytes was analyzed by flow cytometry. BMP2 stimulation was found to significantly increase cell viability. In addition, following BMP2 treatment, β-catenin, Cyclin D1 and Dvl1 expression was significantly increased, whereas GSK-3β expression was significantly decreased. Moreover, the percentage proportion of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the S phase was significantly increased. The results indicate that BMP2 promotes chondrocyte proliferation via the Wnt/β-catenin signaling pathway.Molecular Medicine Reports 07/2011; 4(4):621-6. DOI:10.3892/mmr.2011.474 · 1.48 Impact Factor
Article: Meniskus Tissue Engineering[Show abstract] [Hide abstract]
ABSTRACT: In den letzten Jahrzehnten wurde die wichtige Bedeutung des Meniskus zur Prävention der Gonarthrose erkannt. Trotzdem ist die Meniskusteilresektion nach wie vor eine der häufigsten Knieoperationen. Methoden, zerstörtes Meniskusgewebe zu ersetzen, sind bislang noch verbesserungswürdig. Hier wird dem Tissue Engineering zukünftig eine bedeutendere Rolle zukommen. Auf der Basis künstlicher oder azellularisierter natürlicher Scaffolds, die mit autologen Zellen besiedelt und durch Wachstumsfaktoren stimuliert werden, soll ein dauerhafter Meniskusersatz zukünftig möglich sein. Noch sind allerdings umfangreiche Forschungsarbeiten zur Verbesserung der gegenwärtigen Methoden notwendig. Ziel dieses Artikels ist es, einen aktuellen Überblick über das Thema zu geben. During the last centuries the important role of meniscus tissue in the prevention of osteoarthritis was recognized. Nonetheless, surgical removal of torn meniscus tissue is still one of the most often performed orthopedic operations. Methods for replacing damaged meniscus tissue must still be optimized and in this respect, tissue engineering will become more important in the future. Based on synthetic or acellular extracellular matrix scaffolds, seeded with autologous cells and stimulated by growth factors, durable meniscus replacement should be possible in the future. Currently, research is aimed at reaching these goals and this article will present the current state of the art in this field. SchlüsselwörterMeniskus–Tissue Engineering–Wachstumsfaktoren–Stammzellen–Synthetische Scaffolds KeywordsMeniscus–Tissue engineering–Growth factors–Stem cells–Synthetic scaffoldsArthroskopie 01/2011; 24(1):57-61. DOI:10.1007/s00142-010-0598-8
- [Show abstract] [Hide abstract]
ABSTRACT: Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)-regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete. Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On)-regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system. Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system- and 2-vector system-infected cells (mean +/- SD 15.5 +/- 1.1 ng/ml and 14.6 +/- 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions. The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.Arthritis & Rheumatology 03/2010; 62(7):2037-46. DOI:10.1002/art.27461 · 7.87 Impact Factor