Use of growth factors and adhesive ligands to promote connective tissue progenitor colony formation from fresh marrow

Abstract

The current gold standard for bone graft material is autologous bone, which provides mechanical support, possesses factors that promote bone formation, and contains connective tissue progenitors (CTPs), a heterogeneous population of connective tissue stem and progenitor cells that contribute to neotissue formation. A major limitation to autologous bone grafts is the risk of surgical complications associated with graft harvesting as well as significant donor-site morbidity. Available bone graft substitutes are not as efficacious as autologous bone, resulting in a prescient need for improved bone grafting materials. A promising tissue engineering approach involves the use of bioactive biomaterials that can promote the selective retention of CTPs from pre-seeded autologous bone marrow. When presented in a tethered form, EGF has been shown to promote the survival and enhance the adhesion of culture expanded CTPs. Therefore, the hypothesis of this work was that tethered EGF could be used to enhance the retention of osteogenic CTPs from freshly aspirated bone marrow. Numerous adhesion ligands and growth factors have been investigated for use as candidates for the functionalization of bioactive materials. In this work, we showed that synergy-RGD peptides, which incorporate the putative synergy site on fibronectin, can promote cell adhesion through both a5pl and av33 integrins. We then investigated the effects of tethered EGF on CTP colony formation in the context of defined adhesion environments using a functionalizable comb copolymer. We found that tethered EGF increased the colony forming efficiency of CTPs from fresh human marrow when cell attachment was promoted by either non-specific protein adsorption, fibronectin pre adsorption, or through the synergy-RGD ligand. In contrast, soluble EGF did not increase colony formation, demonstrating the importance of the modality of ligand presentation.

(cont.) Quantitative image analysis also suggested that while tethered EGF did not promote increased osteogenesis at early times after cell seeding tethered EGF may induce the proliferation and migration of cells within osteogenic colonies. These results provide important insight into both the study of the effect of EGF on CTP behavior, as well as the use of tethered EGF as a potential ligand for use in biomaterials that promote the selective retention of CTPs.