(194l) Osteoblast Adhesion and Proliferation on Multi-Functional Polyampholyte Hydrogels with Covalently Attached Sibling Proteins

There has been increasing interest in using polyampholyte polymers in tissue engineering due to their unique multi-functional properties. Polyampholyte polymers have been shown to be resistant to nonspecific protein adsorption, while being able to covalently attach biomolecules. The mechanical properties of these polymers are also tunable by changing the cross-linker density as well as the underlying monomer composition. Therefore polyampholyte hydrogels represent a promising platform technology for tissue engineering. In this work we are developing a polyampholyte hydrogel composed of equimolar mixtures of [2-(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and 2-carboxyethyl acrylate (CAA) as a scaffold for bone tissue engineering. This scaffold is being used as a delivery platform, to screen the osteoblast response to individual members of the SIBLING (small integrin binding N-linked glycoprotein) family of proteins. SIBLING proteins are the primary non-collagenous proteins found in mineralized tissue and they all contain a cell binding RGD amino acid sequence, a collagen binding domain, and a hydroxyapatite binding domain. This family includes osteopontin (OPN), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), dentin sialoprotein (DSP), dentin phosphoprotein (DPP) and dentin matrix protein that is naturally cleaved into N-terminal (DMP1-N) and C-terminal (DMP1-C) fractions. Following hydrogel synthesis, individual SIBLING proteins were conjugated to the hydrogel using EDC/NHS chemistry. The initial osteoblast recruitment was investigated using 2 hour cell adhesion assays and the short term response of the cells was investigated following 24 hours of culture. Hydrogels with conjugated OPN exhibited the highest cell recruitment after 2 hours, but these cells demonstrated a rounded morphology. At the same time, DMP1-N and DSP showed approximately half of the number of cells compared to OPN, but the cells were present in a spread morphology. Additional characterizations were completed following 24 hours of cell adhesion to these scaffolds and the initial stages of bone matrix production was evaluated. In the absence of SIBLING proteins, these scaffolds prevent the adhesion of osteoblast cells, confirming that the presence of the SIBLING proteins is the driving force for cell adhesion. These results support the conclusion that polyampholyte hydrogels with conjugated SIBLING proteins represent a promising platform for bone tissue engineering.