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Title: Characterisation of the immuno-inflammatory response following operative adolescent idiopathic scoliosis and biological properties of osteoprogenitor cells harvested from vertebral bodies compared to iliac crest
Author: Fragkakis, Evangelos Minas
ISNI:       0000 0004 6494 5580
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
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In patients undergoing adolescent idiopathic scoliosis (AIS) surgery the yield and differentiation capacities of mesenchymal stem cells (MSCs) from vertebral body (VB) and iliac crest (IC) bone marrow (BM) were investigated comparing the effectiveness of two MSC-harvesting procedures [density gradient centrifugation (Lymphoprep, LMP) and red cell lysis (Ammonium Chloride, AC)]. The burden of surgery of 3 surgical approaches (posterior, posterior-costoplasty, antero-posterior), for AIS correction was also quantified. In 18 patients 10ml of BM were aspirated intraoperatively from IC and VB and MSCs were isolated using AC and LMP harvesting procedures. MSCs were enumerated and phenotypically characterised using colony-forming-unit-fibroblast (CFU-F), flow cytometry and trilineage differentiation assays. For the immuno-inflammatory study 23 patients were recruited. ELISA-assays were performed on serum from 4ml of peripheral blood, at 6 peri-operative time-points (T), measuring 4 cytokines [(Interleukin) IL-6, IL-10, IL-18 and Procalcitonin (PCT)]. Surgical parameters and gender variability were also analysed. Both IC and VB exhibited similar concentrations of MSCs, however AC yielded significantly more MSC/ml by 1.8-fold (IC, p=0.012) and 1.5-fold (VB, p=0.043), compared to LMP. Calcium and glycosaminoglycan (GAG) concentrations were both significantly higher from VB MSCs, compared to IC [Calcium: AC 8.7-fold (p=0.001), LMP 1.2-fold, GAG: AC 3.8-fold (p=0.0005), LMP 1.9-fold (p=0.003)]. Adipogenesis did not show significant differences. VB AC-harvested MSCs presented a significantly smaller colony area by 1.9-fold (p=0.039), compared to IC AC. The post-operative concentrations of IL-6 and IL-10 were always significatintly elevated at T2 and T3 points, in all 3 approaches (p < 0.01) with antero-posterior approach presenting the highest levels followed by posterior-costoplasty and posterior approaches. Although prolonged procedures and gender type affected cytokine concentrations, their levels were not statistically significant. In summary, AC isolated more effectively MSCs, without affecting their function. VBBM MSCs demonstrated higher osteo- and chondro-genic differentiation capacities, representing a valid source of MSCs in spinal surgery. The surgical stress reaction quantified with immune-inflammatory markers, was higher in more aggressive and extensile procedures, suggesting greater vigilance in identifying and preventing complications.
Supervisor: Giannoudis, Peter V. ; Jones, Jones Elena ; Pountos, Ippokratis Sponsor: Italian Society Of Trauma and Orthopaedics (SIOT) ; Yorkshire Children's Spine Foundation
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available