The effect of in vitro differentiated rat and dog bone marrow stromal cells on the healing of bone defect, interactions between stromal cells and implant materials and influencing factors

ABSTRACTTHE EFFECT OF IN VITRO DIFFERENTIATED RAT AND DOG BONE MARROW STROMAL CELLSON THE HEALING OF BONE DEFECT, INTERACTIONS BETWEEN STROMAL CELLS ANDIMPLANT MATERIALS AND INFLUENCING FACTORSResearch Assistant Feyzan KURTStem cells and their applications are recently discovered as an important new subject for the benefitof their role in treatment. Bone tissue plays a role in human and animal health because of its function inskeletal system and especially in pathological conditions where it takes place during wound repair andhealing process. In this study, the mechanism of differentiation of bone marrow stromal cells (BMSC) tobone cells in culture and the effecting factors in this process on the cell behavior will be investigated. In themean time, the interaction between different implant materials be used in the bone fracture therapy anddifferentiated bone cells will also be studied. It is being thought that the results of this project will support thefuture treatment applications on the human and veterinary medicine. Bone marrow is a complex tissuecomposed of hematopoietic and stromal stem cells with the potential to differentiate into adipogenic,fibroblastic, reticular, osteogenic and chondrogenic lineages. Identification of differentiation markers duringtransformation of stromal cells into osteoblasts in a time-dependent manner may be informative for cell-based tissue engineering. BMSCs promise to provide successful cell scaffolds for tissue-engineered repairof bone tissue. Although bone defects are important problem for pets, there are very few studies done withpet BMSCs in literature. Therefore, we investigated the effects of osteogenic medium (OM) on theproliferation and differentiation of rat and dog BMSCs and the biological behavior of BMSCs cultured on thedifferent materials for their attachment and differentiation to osteoblasts and also examined their role inbone repair. BMSCs from adult male rat tibia and femur were collected and cultured in alpha-MEMmedium with 15% fetal bovine serum, penicillin-- streptomycin and gentamycin. After three days of culture,the medium covering the adherent cells in culture was changed to OM containing dexamethasone, Na-bglycerophosphate and ascorbic acid. As a control, cell culture was also continued in the original mediumfor the same time period. Their proliferation capacity and viability were spectrophotometrically measuredwith MTT assay. Differentiated osteoblast like cells were fixed with 4% paraformaldehyde at 7 and 21 daysof culture, and their immunolabelling for osteoblast markers osteonectin (ON) and osteocalcin (OC) wereassessed using an indirect immunoperoxidase technique. BMSCs obtained from rat tibia and dog femurwere cultured in OM. Different pore size of Hydroxyapatite (HA-gel, HA-1), calcium phosphate basedscaffolds (CPS), titanium mesh (TiM) and matrix molecules collagen type I (COLI), COLIV and Laminin(LMN) were used as substrate and biomaterial for the differentiation of BMSCs. The BMSCs? attachmentratio and the morphology on the scaffolds were observed with scanning electron microscope (SEM).Materials with or without cells implanted to the experimental tibial fracture and examined for effect onhealing. Osteocalcin (ON)-osteonectin (ON) expression and alkaline phosphatase (ALP) activity -mineralization capacity were determined by immunohistochemistry and histochemistry respectively.Vonkossa (VK) staining technique was used to demonstrate the bone formation. Immunolabelling of ONand OC was detectable from day 7 of culture and increased steadily through to day 21. Labelling was moreintense in cells cultured with OM. BMSCs may be useful in autologous bone implant for the treatment ofbone wound healing. Similar morphological appearances were seen for all materials with the effect of OMwhere the cells change their phenotype from fibroblastic to osteoblastic. Cells with long thin processes andadhesive behavior including pattern of interconnecting cells were observed with all substrate by SEM. Theadhesive properties and osteoblastic capacity of cells determined by markers were different in eachmaterial. It seems that LMN and CPS provided more attached and adhesive cells with osteoblasticmorphology. Observation of these effects was similar for both species rat and canine. Different sizes of HAparticles did not effect the adhesive properties of these cells except HA gel. The spreading and attachmentratio of the cells on HA gel were more than the others. We found that there was heterogeneity in BMSCwith respect to differentiation capacity to the osteoblast, which was a sign of a subpopulation.Osteoinductive potential of these materials may synergistically enhance the osteodifferentiation of BMSCswith profound implications on bone-tissue-engineering applications. This combination for ideal bone couldbe used clinically in the treatment of bone defects. The main importance of this project is to reduce thetreatment expenses on the poor economical conditions of our country due to bone repair in human health,which is still expensive and has a long lasting process. It should also be recognized that stem cellapplications in human and veterinary medicine become a very important subject as an alternative forchemotherapies and operations in recent years. This subject is still being studied in different ways by bothinternational and native investigators. In this respect, the results of this project will contribute to theinternational literature.Keywords: Stem cell, bone marrow stromal cell, bone, wound healing, histology, rat, dog.