Understanding the signals that direct biomineralization is paramount for successful synthesis of self-assembled artificial bone and dentin biomimetic materials. Bone and seashell are biominerals shown to undergo both whole cell and single protein directed biomineralization. Furthermore, mammalian osteoblasts have been shown to produce shell-like structures in the presence of shells. Bone and nacre chips with no growth factors incubated with osteoblasts demonstrated biomineralization in the intervening gap: new bone near the bone chip and new nacre near the nacre chip. Understanding the concomitant changes in the microenvironment and their impact on osteoblast function and differentiation will enable the design of biomaterials that can create or direct specific functions. The hypothesis of this project is that water soluble proteins from seashell mother of pearl (nacre) cause osteoblasts to build bone and nacre. Nacre-initiated osteogenesis occurs in vivo, but not the production of new nacre. Despite its osteogenicity, nacre is not widely used as an implant material due to its limited dimensions and in vivo results that vary with preparation procedures. Regardless, nacre's presence changes the cellular microenvironment and causes functional changes in mammalian osteoblasts that direct them to build mollusk shell material. This behavior does not occur with the geological or synthetic mineral comprising nacre (aragonite), supporting our hypothesis that soluble factors are responsible. Nacre provides a model for engineering such factors into biomaterials.

The specific aims of this proposal are: 1) to determine the differences in gene expression of osteoblasts producing bone and osteoblasts producing nacre; 2) to determine the different proteins in the microenvironment of osteoblasts producing nacre vs. those producing bone; and 3) to pattern hydrogels with DMP1 and N40 (Dentin Matrix Protein 1, a hydroxyapatite nucleation protein in bone and dentin; and Nacre 40, an aragonite nucleation protein in nacre) to direct biomineralization in desired patterns.

Tissue engineering has the potential to answer this need. Biomaterials can be formed through synthetic processes, biomimetic processes such as self-assembly directed by proteins or through tissue engineering. Biomimetically created biomaterials devoid of cells show varying results as bone graft materials, often failing to initiate key biological functions in bone formation. Ultimately only live osteoblasts generate new bone tissue, and the key to developing a graft that is osteogenic lies in designing a material that contains or attracts osteogenic cells. Although tissue engineered osteoprogenitor constructs may form bone, this bone does not have the equivalent structure or biomechanical properties as native bone, and in some cases bone is not formed. Tatebe et al. found bone marrow mesenchymal stem cells contributed more to cartilage repair than bone formation in a rabbit full-thickness osteochondral defect model. Their results illustrate the importance of microenvironmental signals and that those defining bone phenotype are necessary for bone formation. Tissue engineering aims to return structure and function to a defect by augmenting the host healing response by treatment with cells, morphogens and scaffolds17. In tissue engineering, host cells are cultured in an artificial scaffold with morphogens to promote the desired cellular function. The cultured cells are directed by the choice of scaffold and morphogens to proliferate, migrate and differentiate throughout the scaffold, which slowly degrades and resorbs, eventually leading to the formation of a desired tissue that secretes its own extracellular matrix and morphogens, a final step necessary for tissue regeneration. This project proposes using nacre to induce osteoblasts to form bone and nacre in order to uncover novel methods to direct osteoblast function, and in turn utilize these methods to develop orthopedic biomaterials for tissue engineering applications.

Three 8 week experiments were performed in which the conditions were repeated as above, except with the goal of patterning the mineralization in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. PEGDA was polymerized with PEG-RGDS and with appropriate bone or nacre chips, and these were seeded with osteoblasts. Media were collected and frozen for MALDI-TOF mass spectrometry and SDS-PAGE.

Understanding the signals that direct biomineralization is paramount for successful synthesis of self-assembled artificial bone and dentin biomimetic materials. Bone and seashell are biominerals shown to undergo both whole cell and single protein directed biomineralization. Furthermore, mammalian osteoblasts have been shown to produce shell-like structures in the presence of shells. Bone and nacre chips with no growth factors incubated with osteoblasts demonstrated biomineralization in the intervening gap: new bone near the bone chip and new nacre near the nacre chip. Understanding the concomitant changes in the microenvironment and their impact on osteoblast function and differentiation will enable the design of biomaterials that can create or direct specific functions.

The hypothesis of this project is that water soluble proteins from seashell mother of pearl (nacre) cause osteoblasts to build bone and nacre. Nacre-initiated osteogenesis occurs in vivo, but not the production of new nacre. Despite its osteogenicity, nacre is not widely used as an implant material due to its limited dimensions and in vivo results that vary with preparation procedures. Regardless, nacre's presence changes the cellular microenvironment and causes functional changes in mammalian osteoblasts that direct them to build mollusk shell material. This behavior does not occur with the geological or synthetic mineral comprising nacre (aragonite), supporting our hypothesis that soluble factors are responsible. Nacre provides a model for engineering such factors into biomaterials.

Specific Aims:

To determine the differences in gene expression of osteoblasts producing bone and osteoblasts producing nacre.

To determine the different proteins in the microenvironment of osteoblasts producing nacre vs. those producing bone.

To pattern hydrogels with Dentin Matrix Protein 1 (DMP1), a hydroxyapatite nucleation protein in bone and dentin, and Nacre 40 (N40), an aragonite nucleation protein in nacre to direct biomineralization in desired patterns.