الفهرس 
IntroductionOnly 14 pages are availabe for public view
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Abstract
The concern of bone regeneration is at the forefront of current TE applications using a source of multipotent mesenchymal stem, scaffold, and biological mediator provide a promising alternative to the current conventional; approach to overcome their limitation and improve clinical outcome. The aim of the current study was the evaluation of bone regeneration using mesenchymal stem cells derived from human gingival versus human periodontal granulation tissues loaded on β-TCP and Ha gel in surgically created standardized CSD in rabbit’s femurs. In this study the source of gingival stem cells were taken from discarded tissues during gingivectomy procedure from a case diagnosed with inflammatory gingival enlargement. The discarded granulation tissues were harvested during periodontal surgery from a subject diagnosed with stage III grade C periodontitis and obtained from deepest part of infra-bony defect. Then, tissues were digested using collagenase enzymes to obtain suspended cells. Cells were cultured and expanded in vitro. hGMSCs and hGTMSCs at a concentration of (2-4 x 106) cells/ml were seeded on β-TCP combined with Ha gel. This study utilized adult New Zealand White rabbits for histological, immune-histochemical, and histomorphometrical evaluation of bone regeneration for CSD reconstruction using β-TCP scaffold alone and when loaded with gingival mesenchymal stem cells (hGMSCs) and periodontal granulation tissue derived undifferentiated mesenchymal stem cells (GTMSCS). The experimental part of this study was conducted from the period of March 2021 to June 2021 at Tissue Culture Laboratory, Histology Department, Faculty of Medicine, Tanta University. For isolation, cultivation, and expansion of stem cells till reaching the confluence suitable for seeding on the scaffold of β-TCP. The average period from beginning of isolation till seeding on the scaffold was 2 weeks. New Zealand white strain rabbits were selected as an animal model to perform the surgical part of this experimental study under general anesthesia and strict sterile surgical environment. A critical sized circular defect of a diameter 6 mm was created using a standardized trephine bur with depth involving the cortex. The surgical intervention was done in one day for each group in sequence of four days. The rabbits were euthanized at 2-, 4-, and 6-weeks interval to get the bone samples for further examination. During authenia, two rabbits from groups A, B, C and one rabbit from group D were sacrificed at each time interval of 2, 4, and 6 weeks, and the explanted femoral samples were evaluated macroscopically, histologically and immune-histochemically. • Study grouping: The rabbits were divided randomly into four groups; • group A (study group):- six defects belong to 6 rabbits were treated with human gingival MSCs loaded on β-TCP scaffold combined with Ha gel. • group B (study group):- six defects belong to 6 rabbits were treated with MSCs derived from human periodontal granulation tissue loaded on β-TCP scaffold combined with Ha gel. • group C (positive control group):- six defects belong to 6 rabbits were treated with β-TCP combined with Ha gel. • group D (negative control group):- three defects belong to three rabbits were left without intervention. Evaluation: A. Histological evaluation. Femur specimens were fixed and decalcified in 10 % ethylene diamine tetra acetic acid (EDTA). After decalcification, the specimens were dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax. Paraffin blocks were sectioned serially at 5μm thickness and stained for histological analysis with Hematoxylin and Eosin (H&E). B. Immuno-histochemical evaluation. Immunohistochemistry was done using markers; KI-67 (marker of cell proliferation) and Osteopontin (marker for bone regeneration) C. Histomorphometrical evaluation. Morphometrical analysis was done to evaluate cellular differentiation and bone regeneration. In general, histological findings showed, gradual healing of the CSD; neither foreign body reaction nor inflammation was seen. Bone trabeculae became thicker with time and enclosed fibro-vascular marrow spaces of varying sizes. Numerous bone ships occupy the bone marrow cavities with multiple radiating bony tongues. Almost at the end of the evaluation period, bone remodeling was observed in all examined sections. The newly formed bone was blended with the native bone surrounding the defect outlines. Bone regeneration in both study groups occurred and increased overtime; there were evident differences in the rate of bone regeneration, its distribution, mineralization and maturation during the three selected evaluation periods between the study groups (A, B) and control positive Abcam, Egypt distributer; GeneTech company. group (C) while control negative group (D) retained empty except for thin rim of newly formed bone. Immune-histochemical findings using osteopontin staining and KI-67 were complemented to each other and suggested accelerated osteogenesis and remodeling within the study group (A, B) as compared to control positive group (C). The regenerated bone tissue in the study groups was more dense, mature and homogeneously distributed in all areas of the experimental defects, including its center and peripheral border, indicating that the bone regeneration process is independent on the osteoconductive effect of the scaffold material, on the other hand, the regenerated bone tissue in the control group was developed later than the study group via the normal healing process assisted by the osteoconductive effect of the scaffold material which has been confirmed by histological and statistical analysis. Histomorphometrical evaluation using five serial slices of 5 μmthicknesses were taken from the middle of bone specimen for each rabbit at each evaluation period. The results were supplementary to the histological and immunological finding which revealed increased bone deposition and mineralization throughout the whole experimental period. In this study, the hypothesis that the hGMSCs and hGTMSCs could enhance the bone regenerating potential of β-TCP scaffold granules combined with Ha when combined together in a bioengineered constructs has been approved. Therefore, the hGMSCs or hGTMSCs / β-TCP/Ha scaffold constructs can be applied to promote bone regeneration in the repair of CSD.