Sensors for in vitro bone tissue engineering applications
- Autores: Johan Mikael Gustavsson
- Directores de la Tesis: Elisabet Engel López (dir. tes.)
- Lectura: En la Universitat Politècnica de Catalunya (UPC) ( España ) en 2011
- Idioma: inglés
- Tribunal Calificador de la Tesis: Josep Antoni Planell Estany (presid.), Montserrat Español Pons (secret.), Miguel Ángel Mateos Timoneda (voc.), Josep Samitier Martí (voc.), Pedro Granja Granja (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
This doctoral thesis explores how ion sensors can provide spatial and temporal control of specific cellular and biomaterial activity related to bone tissue engineering applications. First it was investigated the influence of different osteoblast-like cell models on the ionic extracellular environment (IEE) in vitro. Rat-derived mesenchymal stem cells (rMSCs) and SAOS-2 cells were observed to express high alkaline phosphatase (ALP) activity, and as a consequence they increased the concentration of inorganic phosphorus (Pi) in culture medium containing B-glycerophosphate. On the contrary, MG63 cells showed low ALP activity and did not influence [Pi]. Moreover, cell-induced calcium deposition in the extracellular matrix was observed both in mature SAOS-2 and rMSC layers but not in MG63 layers, and coincided with decreased [Ca2+] of the cell culture medium. Fluctuations in the IEE with respect to Pi and Ca2+ may therefore be indicative of specific osteoblast activity. Second, the ion reactivity of calcium-deficient hydroxyapatite (CDHA), a scaffold candidate material for bone TE, was systematically investigated in vitro by exposing it during different time periods to culture media of varying chemical composition. Traditional sorption models described experimental data well, revealing both significant sorption of Ca2+ onto CDHA and acidification of all culture media. Interestingly, different chemical composition of culture medium provoked opposite ion reactivity of CDHA with respect to Pi. Consequently, cellular sensitivity to dynamic IEEs may cause different cellular response using different culture media. Third, the effects of the dynamic IEE induced by CDHA on cellular behaviour were evaluated by growing SAOS-2 cells in semi-permeable inserts and in close proximity to CDHA. Cells proliferated well and their ALP-activity was modified mainly in time rather than in absolute levels. While cellular ALP-activity created conditions for Ca2+-deposition in the extracellular matrix in absence of CDHA, presence of CDHA caused competition between cells and material for Ca2+ and Pi which initially impeded cell-induced Ca2+-deposition. However, as sorption of Pi onto CDHA gradually decreased with time, conditions for bone mineralisation were created also in presence of CDHA. Obtained results indicate that sorption of Pi rather than sorption of Ca2+ was the main limiter for bone mineralisation in presence of CDHA. Fluctuations in the IEE of bone TE applications awoke interest in developing a generic setup for potentiometric ion and pH measurements online. Traditional Ca2+-selective electrodes for measurements in small volumes (easily down to 0.1 mL) were fabricated. They exhibited a Nernstian response to Ca2+, and were little influenced by other major extracellular ions. Moreover, the electrodes resisted sterilisation through UV radiation, did not induce any cytotoxic effects in contact with osteoblasts, and the electrode potential was subject only to minor drift during longer measurements in cell culture medium. The electrodes were used to successfully monitor sorption of Ca2+ onto CDHA when immersed in culture medium, as well as osteoblast-induced Ca2+-deposition in mature extracellular matrix during time frames of 24 hours. Also, all-solid-state potentiometric microelectrodes based on iridium oxide were prepared for real-time monitoring of pH in traditionally inaccessible bone TE environments. Specifically, pH was measured inside curing bone cement (a-tri calcium phosphate) as well as at its immediate interface with extracellular fluid. In both cases the developed pH microelectrodes indicated how the material initially provoked an alkaline environment, which gradually acidified with time. Absolute pH variations caused by the material were of such magnitude that they should be considered upon drug loadings and/or implantation.
