Final Report Summary - PEPTIDE OSTEOGEL (Determining novel peptide sequence and matrix mechanical properties to increase osteogenesis in embryonic stem cells using designer alginate hydrogels for bone The human body has an amazing ability to heal itself after injury or disease. However, these regenerative processes have their limits, and bone defects above a critical size will not heal on their own. To aid in these injuries, novel biomaterials are needed which actively encourage bone regeneration and then biodegrade once the healing process has finished. Ideally, these materials could be delivered in a minimally invasive manner, such as injection, and support the growth and differentiation of mesenchymal stem cells, which form bone in the body. The project PEPTIDE OSTEOGEL was designed to provide a novel cell-free therapy which could be injected and provide the physical and biological support needed to regenerate critical size defects. The first step in designing a hydrogel is to make the base hydrogel which can then be functionalized to improve cell adhesion. For this project we synthesized a peptide sequence that was designed to self-assemble into long nanofibers that are less than ten nanometers wide, but can reach micrometers in length. This peptide sequence features alternating hydrophilic and hydrophobic amino acids that all have a high propensity to form beta-sheets, a structure that peptides fold into common in proteins. The presence of nanofibers was confirmed using electron microscopy, and using other spectroscopic techniques the presence of beta-sheets within the hydrogels was confirmed. We then needed to ensure that the gels had the mechanical properties necessary for use in a clinical setting, as well as optimizing the gel so that stem cells will differentiate into bone forming cells. By altering the concentration of the peptide the stiffness of the gel could be controlled, and it is gelled through the addition of calcium ions. Human stem cells were cultured in these gels for several weeks to ensure that they could survive and were viable in the gels. The next step was incorporating biological activity, which required novel molecular architecture. In the body cells are surrounded by a complex arrangement of proteins and biomolecules known as the extracellular matrix (ECM). A significant amount of research has gone into incorporating proteins, short protein fragments, or other molecules that cells bind to in ECM into biomaterials. However, one aspect that has not been studied in as much detail is the importance of the spacings of these binding sequences, which can be just a few nanometers. In our system we have a well defined peptide sequence, and by selectively modifying amino acids in this sequence we can control the distance between these modifications, which allows us to space two or more peptide sequences at desired distances. To test this we found two short peptide sequences, RGDS and PHSRN, which are found in two different domains of a protein called fibronectin, and synergistically bind a single receptor on a cell. Using our system, we are able to put these two epitopes at spaces of 3.3 nm, which they are found in nature, or smaller distances (1.5 nm) or larger distances (5.5 nm) One problem limiting the use of self-assembled peptide hydrogels is that they are often brittle and fail under low strains. To address this we decided to see if it was possible to increase the strength of beta-sheet forming hydrogels by attaching the peptides to natural biopolymers to improve their strength. These 'hybrid hydrogels', containing both biopolymers and peptides feature long polymer chains that are 'cross-linked' by self-assembling peptide sequences, these bonds are not permanent and can reform after rupture.The resulting hydrogels had impressive mechanical properties, maintaining their gel state after strains of up to 20%, and recovered most of their mechanical properties after being strained to failure. Functionalizing the biopolymer with a peptide requires less than 5% of the functional groups, which leaves 95% of them available for further modification with things such as cell adhesion sequences, or sequences that bind growth factors or other biomolecules. The mechanical properties, such as stiffness and ability to maintain mechanical properties after deformation, can be tuned by changing the peptide cross-linker or the peptide density. The work carried out during this fellowship in peptide functionalized biopolymers is being continued, trying to gain a better fundamental understanding of the nature of covalent and non-covalent cross links in a hydrogel. This work has met the objectives of the grant and has been a success that has had a lasting impact on the Stevens group and European research.