The aim of this thesis is to demonstrate the potential of VHH in tissue engineering applications, with a focus on bone and cartilage tissue regeneration. After a general introduction to this thesis in chapter 1, the selection of VHH targeting growth factors is described in chapter 2. VHH were selected to target growth factors relevant in skeletal tissue engineering and VHH were found to modulate BMP activity with high affinity. Chapter 3 describes the immobilization of VHH and its potential to reversibly immobilize BMP6 to a surface. The strategy consisted in the combination of orthogonal supramolecular interactions for the immobilization of the polyhistidine-tagged VHH at a surface and its subsequent ability to bind and release BMP6. The system was able to deliver BMP6 and improved osteogenic activity of MSC-like cells. In chapter 4, two strategies are presented for the incorporation of VHH in biomaterials. One approach consisted in the incorporation of VHH in electrospun fibers of poly(ethylene oxide). In this procedure the VHH were homogeneously distributed in the fibers. The VHH remained biologically active after the electrospinning process and were able to potentiate osteogenic differentiation. In the second approach, VHH were incorporated in an in situ gelating dextran-based hydrogel. Retention and controlled release of VHH was shown. Chapter 5 presents another approach for directional VHH immobilization on biomaterial surface. Based on genetic engineering, VHH were engineered with a unpaired cysteine at the C-terminus capable of directional grafting to poly(trimethylene carbonate). VHH were able to capture and release BMP6 whilst BMP6 remained functional for cell differentiation. In chapter 6, a VHH targeting bone mimicking ceramic hydroxyapatite (HA) was selected and tested in vivo in mouse for bone binding capacities. Fluorescently labelled VHH successfully showed specific accumulation in the skeleton. Chapter 7 presents a bivalent VHH with dual specificity towards BMP7 and HA. The bivalent VHH permits a non-covalent functionalization of biomaterials with growth factors. Chapter 8 presents a set of VHH targeting DKK1, a key molecule in degenerative and inflammatory joint disease. VHH were incorporated in injectable thermo reversible hydrogels resulting in a controlled release of VHH over time. In chapter 9, different strategies were explored to select VHH targeting osteoarthritic or healthy cartilage. One approach consisted in the use of a non-immunized phage library targeting macroscopically looking arthritic and healthy human cartilage. The second approach consisted of engineering a VHH with a peptide sequence, previously identified, targeting collagen type II. The peptide sequence was inserted in the complementary determining region 3 of the VHH. Unfortunately, neither of these approaches resulted in in the selection of VHH with the desired effect. Finally, chapter 10 discusses the results obtained in this thesis and gives an outlook on the potential applications of VHH in tissue engineering.
|Award date||4 Sept 2014|
|Place of Publication||Enschede|
|Publication status||Published - 4 Sept 2014|