Study of Cell Adhesion and Tracking Methods for in Vivo Cell Localisation in a Tissue-Engineering Scaffold
Bone fracture healing problems, because of the loss of tissue regeneration, can affect many old or multi-pathologic patients around the world. Among the therapeutic strategies, bone substitutes, such as poly-(L-lactitve acid) with 5% wt. % ß-tricalcium phosphate (PLA/5%ß-TCP) scaffolds, are promising as a clinical solution. One strategy to tune their osteoinductive properties is the addition of bone progenitor cells. Therefore, cell- free and cell-seeded PLA/5% ß-TCP scaffolds were implanted into femoral condyles of rats to promote bone regeneration. The in vivo study showed that the bone growth inside a cell-seeded scaffold with human bone progenitor cells was delayed compared to cell-free scaffold. What happened to the cells? Did they trigger an immune response, migrate or die? Therefore, the goals of this project are to evaluate the cell adhesion in vitro to the scaffold and to propose a reliable cell labeling method to track them in future in vivo studies. In the first aim, the adhesion of the cells was qualitatively assessed by the cell morphology and the presence of fibronectin using fluorescence imaging. To quantify their adherence, seeded scaffolds were washed up to 3 times with a 0.9 % NaCl solution. The luminescence of the scaffolds and the washing solutions was measured using a cell viability assay. These experiments demonstrated that after 72h of culture, the measured adherent bone progenitor cells on the scaffolds decreased after 3 washes down to 50 % while it stayed stable after 14 days of culture. In the second aim, we focused on methods to label the cells either once implanted or prior to implantation. The first method was to translate the in situ hybridization protocol with the human specific Alu sequence from paraffin embedded samples to resin embedded ones. The obtained results were so far inconclusive. Concerning the labeling of the cells before implantation, the transmembrane label PKH26 was investigated. Its staining efficacy was high just after labeling, while it decreased drastically over time due to cell division.