Introduction: Biological tissues are composed of multiple components organized hierarchically to yield specific functions that cannot be achieved by homogeneous presentation of each component alone. Despite advances in biomaterial designs, engineering constructs with complexity that matches the properties and functions of native tissues remains an ongoing challenge[1]. We have developed novel strategies to create biodegradable tissue engineering scaffolds that mimic the physical architectures and biomolecule compositions in native tissues[2]-[4]. Recently, we combined advanced scaffold fabrication techniques with a peptide-polymer conjugate-based functionalization approach to generate polymer scaffolds with dynamic and spatially organized functionalities to guide bioactivity[3],[4]. Here, we designed and synthesized peptide-polymer conjugates with specific peptide sequences to initiate biomineralization or bind hyaluronic acid (HA) to produce osteochondral scaffolds that promote bone and cartilage regeneration simultaneously in a single construct.
Materials and Methods: Peptides that initiate mineralization (E3) or bind HA (HAbind) were covalently linked to a biodegradable polymer poly(ε-caprolactone) (PCL) using methods described previously[3]. The resulting peptide-PCL conjugates were electrospun alone or in opposing concentrations to spatially guide mineral formation on one side and HA-binding on the opposite. Scaffolds were incubated in simulated body fluid (SBF) to mineralize the surface before seeding cells. Human mesenchymal stem cells (hMSCs) were seeded onto unmodified PCL, mineralizing only (E3-PCL), HA-binding only (HAbind-PCL), or dual functional (HAbind/E3-PCL) scaffolds and cultured in basic growth media without growth factors for up to 42 days. Samples were evaluated for chondrogenesis, osteogenesis, and overall spatial tissue development.
Results and Discussion: Our functionalization strategy produced scaffolds with peptides presented in discrete locations within the scaffold to guide bioactivity with preferential mineralization and HA-binding in the HAbind/E3-PCL scaffold (Figure 1). HAbind-PCL encouraged early chondrogenesis shown by upregulation of Sox9 at Days 7 and 42 while the E3-PCL triggered early osteogenesis with upregulation of Runx2 and Sox9 at Day 42. Interestingly, the dual functional scaffolds demonstrated a synergistic improvement for chondrogenic biomolecules from Day 14 and osteogenic biomolecules at Day 42. Histological and immunohistochemical analysis at Day 42 revealed distinct spatial tissue development and tissue maturation in the HAbind/E3-PCL scaffolds (Figure 2) compared to controls.
Conclusions: The results demonstrated that HA-binding and mineralizing peptides on the scaffold surface spatially guided hMSC chondrogenesis and osteogenesis, respectively, without exogenous factors. The dual presentation of these peptides within a scaffold resulted in a synergistic improvement in chondrogenic and osteogenic properties and spatial regeneration of osteochondral-specific tissues. Spatial organization of bioactivity within a single construct introduces the potential to engineer translational scaffolds that guide in vivo differentiation and functional tissue regeneration.
References:
[1] Place, E. S.; Evans, N. D.; Stevens, M. M. Complexity in Biomaterials for Tissue Engineering. Nature Materials 2009, 8, 457–470.
[2] Steele, J. A. M.; McCullen, S. D.; Callanan, A.; Autefage, H.; Accardi, M. A.; Dini, D.; Stevens, M. M. Combinatorial Scaffold Morphologies for Zonal Articular Cartilage Engineering. Acta Biomaterialia 2014, 10, 2065–2075.
[3] Chow, L. W.; Armgarth, A.; St-Pierre, J.-P.; Bertazzo, S.; Gentilini, C.; Aurisicchio, C.; McCullen, S. D.; Steele, J. A. M.; Stevens, M. M. Peptide-Directed Spatial Organization of Biomolecules in Dynamic Gradient Scaffolds. Advanced Healthcare Materials 2014, 3, 1381–1386.
[4] Harrison, R. H.; Steele, J. A. M.; Chapman, R.; Gormley, A. J.; Chow, L. W.; Mahat, M. M.; Podhorska, L.; Palgrave, R. G.; Payne, D. J.; Hettiaratchy, S. P.; et al. Modular and Versatile Spatial Functionalization of Tissue Engineering Scaffolds Through Fiber‐Initiated Controlled Radical Polymerization. Adv. Funct. Mater. 2015, 25, 5748-5757.