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Abstract SUMMARY Tissue engineering and regenerative medicine are emerging fields that have only recently found their way into the dental field. Stem cell research has been hailed for the potential to revolutionize the future of medicine with the ability to regenerate damaged and diseased organs. Stem cells isolated from a variety of organs are capable of ignoring their cell lineage boundaries and exhibiting more plasticity in their fate(60,67). Research proved that stem cells can be used to treat many diseases such as Alzheimer’s, Parkinson’s, chronic heart diseases and periodontal diseases as well as to grow replacement teeth and bone(67). Adipose-derived stem cells possess the ability to readily be expanded in vitro and the capacity to undergo adipogenic, osteogenic, chondrogenic and myogenic (cardiomyocyte and skeletal myocyte) differentiation (103). Neurogenic differentiation potential has also been described in vitro(4,84) as has pancreatic endocrine phenotype expressing insulin(95), hepatic(7) and endothelial differentiation(5). Diseases associated with increased bone resorption such as myeloma, Paget’s disease and osteoporosis are treated by systemic doses of bisphosphonates. Locally applied bisphosphonates are emerging to help overcome the drawbacks of systemically used bisphosphonates(2). Consequently, alendronate interferes with the stability of the ruffled border and stimulates osteoclast apoptosis, which reduces bone resorption, lowers bone turnover and promotes a positive bone balance (40). Moreover, studies indicated that bisphosphonates also influence osteoblasts and increase 105 bone formation(73). Others have reported that bisphosphonates enhance osteoblast proliferation and maturation (46,81,82) and inhibit osteoblast apoptosis(50,61). In the present work, adipose derived stem cells (ADSCs) were chosen for many reasons including their capablity of differentiating into a number of mesodermal lineages including osteoblasts, availability of these cells, accessibility, suppression of apoptosis, enhancing angiogenesis and stimulation of mitosis and differentiation of tissue intrinsic reparative cascades. Another reason for choosing ADSCs was the abundancy of stem cells obtained from adipose tissue compared to other sources (6, 27). Following the isolation and culturing procedures of ADSCs the in vitro study of the effect of Aln on osteogenic differentiation of ADSCs was performed. ADSCs cultured in Aln changed their morphology and appeared cuboid or ovoid in culture and gradually lost their previous spindle-like appearance. Also, scattered brown to black nodule-like structures within the cultured cells started to appear, an event that was not seen in ordinary culture media, reflecting calcium deposition inside and outside differentiated cells. Plates showed colorimetric changes with Alizarin Red stain indicating calcium crystals deposition and successful osteogenic differentiation of ADSCs. Readings given by the spectrophotometer indicated formation of alkaline phosphatase enzyme by the cells cultured in Aln reflecting successful osteogenic differentiation of cultured ADSCs. Osteonectin gene was detected in plates on the seventh day of culture in Aln indicating successful osteoblastic differentiation of isolated cells In vivo study of the repair of critical- sized calvarial defects in different experimental groups of defects was performed. Fluorescence microscopy showed linear double tetracycline labeling, indicative of bone formation and mineralization in all cases of Aln and osteogenic media use. Absence of tetracycline labeling in control defects indicated that no mineralized bone matrix was formed during the experimental period. Histological examination of regenerated tissue in different groups using hematoxylin and eosin (H & E) stain showed that both groups (osteogenic and alendronate groups) demonstrated partial new bone formation, observed as islands within the defects. The newly formed bone- like tissue did not completely bridge the defects. However, the level of bone maturation regarding bone lamellae appearance was not different between these two groups. The newly formed bone-like tissue in the operation area had immature characteristics with many marrow spaces and disordered osteocytes. Control group defects showed healing with minimal regeneration and no evidence of newly formed bone-like tissue was noted. Fibrovascular tissue and dense bundles of residual collagen were observed. from the present study it can be concluded that locally applied Aln may be used as an alternative line of treatment to osteogenic medium to enhance bone formation. |