Veterinary Research Forum

Abstract This study aimed to investigate the effects of zoledronic acid (ZA) and antibacterial CM11 peptide on the osteoinduction and antibacterial properties of bioactive glass (BG). The bioactive glass/gelatin (BG/Gel) composite was synthesized using the sol-gel method. The 2-x minimum inhibitory concentration of the peptide and 4.00 mg mL^-1 of ZA were added to the BG/Gel during fabrication. The BG/Gel composite morphological and structural characteristics and anti-bacterial activities were analyzed using Fourier transform infra-red spectroscopy, scanning electron microscopy and disk diffusion test, respectively. The release of the peptide and ZA from BG/Gel was measured using ultra-violet spectroscopy. After 14 days, the effects of the peptide/ ZA-containing BG/Gel (PZ-BG/Gel) on the growth and differentiation of mesenchymal stem cells were evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide, calcium and alkaline phosphatase assays, immunocytochemical staining for osteocalcin (OCN) and real-time polymerase chain reaction for OCN, type I collagen, bone morphogenetic protein 2 and Runt-related transcription factor-2 genes. The disk diffusion test showed the anti-microbial activity of the scaffold against multi-drug-resistant isolates of Pseudomonas aeruginosa and Staphylococcus aureus. Analyses showed a significantly higher level of stem cells differentiation into the osteo-genic cells in PZ-BG/Gel scaffold compared to BG/Gel scaffold alone. Accordingly, osteoblast markers were significantly increased in comparison with the control. In conclusion, the osteo-induction and antibacterial properties of BG-based scaffold can be improved using ZA and CM11.

Abstract Zinc as an essential trace element was reported to be involved in regulation of the growth and aging of cells. In this study, rat adipose-derived mesenchymal stem cells were exposed to extremely low frequency electromagnetic field (ELF-EMF) of 50 Hz and 20 mT to evaluate whether exposure to ELF-EMF in the presence of zinc sulfate (ZnSO₄) affects the telomerase reverse transcriptase (TERT) gene expression and aging in mesenchymal stem cells (MSCs). The cell plates were divided into four groups including group I (control without ZnSO₄ and ELF-EMF exposure); group II (ELF-EMF-exposure without ZnSO₄); group III (ZnSO₄ treatment without ELF-EMF exposure) and group ІV (ELF-EMF exposure with ZnSO₄). In the presence of different concentrations of ZnSO₄,cells viability, TERT gene expression and percentage of senescent cells were evaluated using colorimetric assay, real-time PCR and senescence-associated β-galactosidase activity assay, respectively. In this experiment, cells were exposed to ELF-EMF for 30 min per day for 21 days in the presence and absence of ZnSO₄. The results revealed that ELF-EMF leads to a decrease in the expression of TERT gene and increase in the percentage of senescent cells. However, the ZnSO₄ could significantly increase the TERT gene expression and decrease the aging of ELF-EMF-exposed MSCs. It seems that ZnSO₄ may be a beneficial agent to delay aging of ELF-EMF-exposed MSCs due to the induction of TERT gene expression. Zinc as an essential trace element was reported to be involved in regulation of the growth and aging of cells. In this study, rat adipose-derived mesenchymal stem cells were exposed to extremely low frequency electromagnetic field (ELF-EMF) of 50 Hz and 20 mT to evaluate whether exposure to ELF-EMF in the presence of zinc sulfate (ZnSO₄) affects the telomerase reverse transcriptase (TERT) gene expression and aging in mesenchymal stem cells (MSCs). The cell plates were divided into four groups including group I (control without ZnSO₄ and ELF-EMF exposure); group II (ELF-EMF-exposure without ZnSO₄); group III (ZnSO₄ treatment without ELF-EMF exposure) and group ІV (ELF-EMF exposure with ZnSO₄). In the presence of different concentrations of ZnSO₄,cells viability, TERT gene expression and percentage of senescent cells were evaluated using colorimetric assay, real-time PCR and senescence-associated β-galactosidase activity assay, respectively. In this experiment, cells were exposed to ELF-EMF for 30 min per day for 21 days in the presence and absence of ZnSO₄. The results revealed that ELF-EMF leads to a decrease in the expression of TERT gene and increase in the percentage of senescent cells. However, the ZnSO₄ could significantly increase the TERT gene expression and decrease the aging of ELF-EMF-exposed MSCs. It seems that ZnSO₄ may be a beneficial agent to delay aging of ELF-EMF-exposed MSCs due to the induction of TERT gene expression.

Abstract The aim of this study was to investigate the interactions between rat intestine decellularized scaffold and human adipose derived mesenchymal stem cells. Rat large intestine was dissected in fragments and decellularized by physicochemical methods. The scaffolds were loaded by human adipose derived mesenchymal stem cells expressing green fluorescent protein. Microscopic sections were prepared from the scaffolds after two weeks of culture with stem cells and studied by histological methods. The interactions of scaffolds with MSCs were also studied by electron microscopy. Histological and electron microscopy studies revealed human mesenchymal stem cell adhesion, migration, division and maintenance during the 14 days of culture in vitro. According to the results, scaffolds prepared from rat intestine matrix could be a suitable scaffold for studying in vitro cell behaviors such as division, migration and attachment. These various behaviors of cultured cells might be due to inductive effects of the extracellular matrix derived scaffold. However, more investigations are required to discover the exact effects of this scaffold and its interactions with mesenchymal stem cells.

Abstract In vitro expansion of mesenchymal stem cell (MSCs) into large number is necessary for their application in cell-based treatment of articular cartilage defects. On the other hand, some studies have indicated that BIO (6-Bromoindirubin-3-Oxime) possesses mitogenic effects on cell culture. The objective of the present study was to examine the effect of BIO on in vitro expansion and chondrogenic differentiation of mouse marrow-derived MSCs. The culture was established using bone marrow tissue obtained from 10 NMRI mice. MSC nature of the isolated cells was verified according to the minimal criteria proposed for MSC. Passaged-3 cells were seeded in 24-well culture plates and treated by 0.05, 0.01, 0.1, 1.0 and 1.5 µM BIO for seven days. The culture without BIO was taken as the control. At the end of cultivation period, the cultures were examined for viable cell number which was then used to calculate population doubling time (PDT). The BIO with higher proliferation-promoting effect was investigated for its chondrogenic effect on MSC culture. There was significantly more viable cells at the cultures treated by 0.1 µM BIO. At this culture the cells tended to double their population in rapid rate (each 43.07 hr) than the cells treated with the other BIO concentrations (p < 0.05). Interestingly treatment of MSC chondrogenic culture with 0.1 µM BIO led to the up-regulation of cartilage specific genes including aggrecan, collagen II and sox9. In conclusion BIO at 0.1 µM could enhance mouse MSC in vitro proliferation as well as their chondrogenic differentiation. These findings would be of great importance for the field of regenerative medicine.

Abstract Rabbits have the capacity to regenerate holes in their ears by forming a blastema, a tissue that is made up of a group of undifferentiated cells. The purpose of the present study was to isolate and characterize blastema progenitor cells and compare them with marrow mesenchymal stem cells (MSCs). Five New Zealand white male rabbits were used in the present study. A 2-mm hole was created in the animal ears. After 4 days, the blastema ring formed in the periphery of the hole was removed and cultivated. The cells were expanded through several subcultures and compared with the MSCs derived from the marrow of same animal in terms of in vitro differentiation capacity, growth kinetics and culture requirements for optimal proliferation. The primary cultures from both cells tended to be heterogeneous. Fibroblastic cells became progressively dominant with advancing passages. Similar to MSCs blastema passaged-3 cells succeeded to differentiate into bone, cartilage and adipose cell lineages. Even lineage specific genes tended to express in higher level in blastema cells compared to MSCs (p < 0.05). Moreover blastema cells appeared more proliferative; producing more colonies (p < 0.05). While blastema cells showed extensive proliferation in 15% fetal bovine serum (FBS), MSCs displayed higher expansion rate at 10% FBS. In conclusion, blastema from rabbit ear contains a population of fibroblastic cells much similar in characteristic to bone marrow mesenchymal stem cells. However, the two cells were different in the level of lineage-specific gene expression, the growth curve characteristics and the culture requirements.