Evaluación del potencial de diferenciación osteogénico de células madre mesenquimales in vitro en medio suplementado con plasma pobre en plaquetas como posible terapia en aplicaciones óseas

Abstract

The bone healing process can be limited due to conditions and pathologies such as age, diabetes, or severe anemia. This results in ineffective healing, causing an unconsolidated bone fracture. Traditionally, surgical and non-surgical methods have been used, which may not be entirely effective. This has led several investigations to focus on the potential use of Mesenchymal Stem Cells (MSCs) together with supplements for cell culture, which have presented difficulties at the time of being used in the proliferation and differentiation of these. This is why different treatments have been applied in regenerative medicine using blood-derived supplements such as Platelet Rich Plasma (PRP), since, due to its high content of growth factors, it stimulates osteogenic differentiation. However, the use of Platelet Poor Plasma (PPP) was not taken into account due to its low content of factors compared to PRP. Despite the fact that the volume obtained from PPP is greater than that of PRP. That is why in this project PPP was used as a cell culture supplement to evaluate and analyze the osteogenic differentiation potential of MSCs in vitro. Therefore, MSCs obtained from adipose tissue were cultured and differentiated in medium supplemented with three different concentrations of PPP: 5%, 10% and 15%, together with their respective control. Briefly, 4000 nucleated cells/cm^2 were cultured in generation 7 for approximately 44 days, Neubauer chamber counting was performed and cell doubling (NCPD), and doubling time (CPDT) were calculated. While for the osteogenic differentiation assay, 8000 nucleated cells/cm^2 of Generation 7 and Final Generation (resulting from proliferation) were seeded in triplicate in the aforementioned concentrations of PPP. Calcium phosphate deposition and morphology were evaluated by Alizarin Red, Rhodamine and DAPI staining at 7, 14 and 21 days of differentiation. It was obtained as a result that after the study time (week 3) the proliferation rate similar to the control was 0.9158 with a confidence interval of 0.0041 for 5% PPP, while for the other concentrations the cells reduced their proliferative capacity. Additionally, calcium deposits were evidenced, induced by the different concentrations of plasma used. There was a higher differentiation potential for the 10% and 15% concentrations, and cell death was present in both generations evaluated. Finally, with regard to cell morphology, it was evident that the cell phenotype changed after the test time, taking a wider and more elongated shape in its cytoplasm. The 15% PPP produced greater cell differentiation in Generation 7 as opposed to the final generation. Based on the results obtained and the recommendations presented, it is necessary to carry out further studies, giving rise to future research for the healing of bone tissue.

Date of Award	27 Jul 2023
Original language	Spanish (Colombia)
Awarding Institution	Universidad Autónoma de Bucaramanga Ingeniería Biomédica
Supervisor	Silvia Milena Becerra Bayona (Supervisor) & Víctor Alfonso Solarte David (Supervisor)