Résumé
BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-β1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-β1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.
Sujets)
Humains , Agrécanes , Bleu Alcian , Moelle osseuse , Trame osseuse , Cartilage , Cartilage articulaire , Collagène de type I , Collagène de type II , Éosine jaunâtre , Matrice extracellulaire , Technique d'immunofluorescence , Expression des gènes , Hématoxyline , Pression hydrostatique , Techniques in vitro , Cellules souches mésenchymateuses , Ostéocalcine , Réaction de polymérisation en chaine en temps réel , ARN messagerRésumé
Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds. In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker. The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells [ASC] in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis. These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo