Jadon, Narendra S.Bhatt, Jyotsana2021-08-042021-08-042021-03https://krishikosh.egranth.ac.in/handle/1/5810171394The present study was conducted to evaluate regenerative potential of bone marrow derived differentiated osteoblast along with β-TCP scaffold in augmenting fracture healing in rabbits and to delineate expression profile of various genes involved in fracture repair as molecular markers of healing via Real time-PCR. During first phase of study isolation and propagation of bone marrow derived MSCs, its differentiation into osteoblasts and their characterization utilizing specific techniques was done followed by second phase of clinical evaluation of therapeutic potential of undifferentiated MSCs and differentiated osteoblasts in fracture repair in rabbit model. Thirty six rabbits used in this study were randomly divided into four equal groups (A, B, C and D) having 9 animals each. Animals were anaesthetized with xylazine @ 6mg/kg followed by ketamine @ 60mg/kg, intramuscularly and a 7mm segmental defect was created in mid diaphysis of left radius bone. Animals of different groups were subjected to different treatment protocols. Animals of group I kept as negative control and only external splint was applied. In animals of group II, only β- TCP implant was used to bridge the segmental defect. Animals of group III were treated with undifferentiated MSCs along with β- TCP implant and animals of group IV were treated with differentiated osteoblasts along with β- TCP implant. Quality of fracture repair was assessed by clinical parameters, radiography, estimation of biochemical parameters, gross evaluation of test bone, histopathology, histochemistry using Van Gieson’s staining, flourochrome labelling, scanning electron microscopy and molecular assessment of bone turnover markers via Real time-PCR. Radiographic findings, gross evaluation of test bone and histopathology revealed that the key criteria for evaluating bone healing like level of callus formation, bony union between cut ends and bone remodelling were significantly (P<0.05) prominent in animals of group IV treated with differentiated osteoblasts along with β-TCP implant closely followed by animals of group III treated with undifferentiated MSCs along with β-TCP implant. Biochemical parameters showed significant (P<0.05) increase in its level on day 30 postoperatively in all the groups, however these values returned to normal level at day 45 in group III and IV signifying faster pace of healing in stem cell treated groups. Tetracycline labelling and scanning electron microscopic study of regenerated tissue from different groups of animals revealed significantly (P<0.05) superior quality of healing in animals of group IV. Gene expression profiling showed upregulation of osteopontin and osteocalcin genes, associated with remodelling, in animals of group IV whereas genes like alkaline phosphatise and collagen type-1, associated with proliferative stage of fracture healing, were upregulated in animals of group I. Results of this study reveals that animals treated with osteogenically predifferentiated MSCs (osteoblasts) showed rapid and superior quality healing of critical sized segmental bone defects as compared to animals treated with undifferentiated MSCs, β-TCP implant alone and control animals.EnglishThesis