Preparation and evaluation, in vivo and in vitro, of a construct composed of mesenchymal stem cells derived from adipose tissue, bone graft biomaterials and fibrin-rich plasma

Abstract

Abstract: In recent years, new therapies studied by bone bioengineering have been developed with the purpose of promoting bone repair defects. The creation of constructs composed of bone-substitutes, growth factors and/or mesenchymal stem cells (MSCs), emerge as a promising strategy in the treatment of these defects. Our propose is evaluate in vitro and in vivo the use of a porous hydroxyapatite and tricalcium phosphate (HA/TCP) with structure (70:30), associated with blood products (PRF) and autologous MSCs obtained from adipose tissue (ADSCs). For this purpose, 24 male New Zealand rabbits, with a body mass of 3.5-4.0 kg, will be used. The ADSCs collected from the inter-scapular region will be isolated, expanded, propagated, characterized in flow cytometer (FACs) and differentiated in osteoblasts (ADSCs-OB) in osteogenic medium for 21 days (in vitro). Blood collected (10-15 mL) from the rabbit central artery will be placed in glass-lined plastic tubes and immediately centrifuged once at 1650 and/or 3000 RPM for 5-10 minutes for PRF separation. The formation of the construct will occur by the primary combination of the PRF to the biomaterial (HA/TCP), and subsequently, by OB-ADSCs cultivate. Cell viability (by MTT), alkaline phosphatase, mineralization assay, cell morphology and adhesion (SEM) and Western blotting for osteogenic factors will be evaluated in vitro construct assays. In vivo tests, 8 mm bilateral bone defects will be performed on the parietal bone (24 rabbits), which are filled randomly with the following treatments: 1) Positive control (autologous bone); 2) Negative control (blood clot); 3) HA/TCP (70:30); 4) PRF; 5) ADSCs-OB; 6) HA/TCP (70:30) + PRF; 7) HA/TCP (70:30) + ADSCs-OB; and 8) HA/TCP (70:30) + ADSCs-OB + PRF. After the 4-week period, the calvarias region will be collected. The skulls will be scanned by microtomography to obtain/quantification of the parameters total volume of the defect (TV), bone tissue volume (BV), BV/TV ratio and BMD. Subsequently, the pieces will undergo histological processing and stained by Hematoxylin and Eosin. Descriptive histological analysis and histomorphometric will be performed. (AU)