Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure.

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-ß1 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-ß1, 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.

Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line.

BACKGROUND: Type 1 diabetes mellitus is one of the metabolic diseases that cause insulin-producing pancreatic ß cells be destroyed by immune system self-reactive T cells. Recent-ly, new treatment methods have been developed including use of the stem cells, ß islet cells transplantation and gene therapy by viral and non-viral gene constructs. OBJECTIVES: The aim of this project was preparing the non-viral vector containing the glucose inducible insulin gene and using it in the NIH3T3 cell line. MATERIALS AND METHODS: Cloning was carried out by standard methods. Total RNA was extracted from pancreatic tissue, RNA was converted to cDNA using RT-PCR reaction and preproinsulin gene was amplified using specific primers. PNMTCH plasmid was extract-ed and digested by NotI, HindIII, and MTIIA and ChoRE genes were purified and cloned into pcDNA3.1 (-) plasmid and named pcDNAMTCh. Finally, the preproinsulin genes were cloned into pcDNA3.1 (-) plasmid and pcDNAMTChIns was built. RESULTS: The cloned gene constructs were evaluated by restriction enzyme digestion and RT-PCR. The NIH3T3 cells were transfected by plasmid naked DNA containing preproinsu-lin gene and expression was confirmed by Reverse Transcriptase PCR and Western Blot-ting Techniques. CONCLUSIONS: Gel electrophoresis of PCR products confirmed that cloning was per-formed correctly. The expression of preproinsulin gene in recombinant plasmid in NI-H3T3 cell line was observed for the first time. The findings in this study can be the basis of further research on diabetes mellitus type 1 gene therapy on animals.