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.

Human bone progenitor cells for clinical application: what kind of immune reaction does fetal xenograft tissue trigger in immuno-competent rats?

Tanja Cloé Hausherr, Lee Ann Laurent-Applegate, Dominique Pioletti

The potential of human fetal bone cells for successful bone regeneration has been shown in vivo. In particular, it has been demonstrated that the seeding of these cells in porous poly-(L-lactic acid)/ ß-tricalcium phosphate scaffolds improved the bone formation compared to cell-free scaffolds in skulls of rats. However, even if the outcome is an improvement of bone formation, a thorough analysis concerning any immune responses, due to the implantation of a xenograft tissue, is not known. As the immune response and skeletal system relationship may either contribute to success or failure of an implant, we were interested in evaluating the presence of any immune cells and specific reactions of human fetal cells (also called human bone progenitor cells) once implanted in femoral condyles of rats. For this purpose (1) cell-free scaffolds, (2) human bone progenitor cells or (3) osteogenic human bone progenitor cells within scaffolds were implanted over 3, 7, 14 days and 12 weeks. The key finding is that human bone progenitor cells and osteogenic human bone progenitor cells do not trigger any particular specific immune reactions in immuno-competent rats, but are noted to delay some bone formation.

Cognizant Communication Corp2017

In vivo mechanical loading combined with cell therapy in a bone tissue engineering scaffold

Tanja Cloé Hausherr

Bone defects generate a worldwide high demand for bone repair and reconstruction. The incidence of bone defect repair in our aging society is expected to increase in the next decades. As bone auto- and allografting solutions often suffer from limited supplies, there is a clinical need for alternative bone substitutes, challenging researchers in bone tissue engineering to find novel approaches. To address this challenge, several biomaterials have been combined with different approaches, such as various biomolecules delivery, cell therapy or the application of mechanical loading. In the present PhD thesis, we assessed for the first time the effect of temporal onsets of mechanical loading combined with cell therapy in a tissue engineering scaffold implanted in a bone site. For this purpose, we used a poly(L-lactic acid)/ß-tricalcium phosphate scaffold, human bone progenitor cells and two mechanical loading conditions. The choice of the scaffold was motivated by its reinforced porous structure and enhanced bioactivity, whereas, for the choice of cells, we were driven by their low immunogenicity and high capacity for osteogenic differentiation. This capacity allowed us to evaluate two different bone cell fates originating from the same cell type. We then used the same mechanical loading parameters as in previous studies on scaffolds without cells. When loading was applied a few days post-implantation (early loading) and a few weeks post-implantation (delayed loading), moderate and advantageous effects for bone repair could respectively be observed. We focused our work on (1) the investigation of a potential immune reaction triggered by xenograft cells at an early stage of implantation in immuno-competent rats and (2) the evaluation of early and delayed mechanical loadings on bone formation when combined with cell therapy. Three scaffold conditions were used: human bone progenitor cell-seeded, osteogenic human bone progenitor cell-seeded and cell-free scaffolds. Each scaffold condition was implanted bilaterally in both femoral condyles of rats. In the first part of this thesis, early immune reaction was evaluated on implanted scaffolds based on histology. The results indicated that, unlike cell-free scaffolds, both cell-seeded types of scaffolds affected the surrounding tissue by forming fibrous tissue but without triggering particular specific immune reaction. In the second part, we followed static and dynamic bone parameters inside scaffolds based on microCT scans. We showed that, independently of mechanical loading conditions, a significantly higher amount of bone was formed within cell-free scaffolds than within the two cell-seeded ones. Depending on the cell-seeded scaffold conditions, early mechanical loading had different effects on bone formation. We concluded that the timing of mechanical loading and the cell types, as well as specific combinations thereof, are crucial for a beneficial bone repair. Overall, however, cell-free scaffolds used in this study could be considered as the best solution when combined with delayed mechanical loading, as it is simple and safe from a clinical point of view without increasing regulatory restriction due to the use of cells.

EPFL2016

A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila

Jean Philippe Boquete, Bruno Lemaitre, Zongzhao Zhai

The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, we analyze at the cellular and molecular levels the differentiation program required for robust regeneration. We observe that the intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. We further demonstrate that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, our study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment.

2017

In vivo mechanical loading combined with cell therapy in a bone tissue engineering scaffold

Tanja Cloé Hausherr

EPFL2016