Cyclic Arginine-Glycine-Aspartate Peptides Enhance Three-Dimensional Stem Cell Osteogenic Differentiation

The role of morphogens in bone regeneration has been widely studied, whereas the effect of matrix cues, particularly on stem cell differentiation, are less well understood. In this work, we investigated the effects of arginine-glycine-aspartate (RGD) ligand conformation (linear vs cyclic RGD) on primary human bone marrow stromal cell (hBMSC) and D1 stem cell osteogenic differentiation in three-dimensional (3D) culture and compared their response with that of committed MC3T3-E1 preosteoblasts to determine whether the stage of cell differentiation altered the response to the adhesion ligands. Linear RGD densities that promoted osteogenic differentiation of committed cells (MC3T3-E1 preosteoblasts) did not induce differentiation of hBMSCs or D1 stem cells, although matrices presenting the cyclic form of this adhesion ligand enhanced osteoprogenitor differentiation in 3D culture. This may be due to enhanced integrin ligand binding. These studies indicate that biomaterial design parameters optimized for differentiated cell types may not directly translate to stem cell populations, because less-committed cells may require more instruction than differentiated cells. It is likely that design of synthetic extracellular matrices tailored to promote stem cell differentiation may enhance bone regeneration by transplanted cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78148/1/ten.tea.2007.0411.pd

Regulating Myoblast Phenotype Through Controlled Gel Stiffness and Degradation

Mechanical stiffness and degradability are important material parameters in tissue engineering. The aim of this study was to address the hypothesis that these variables regulate the function of myoblasts cultured in 2-D and 3-D microenvironments. Development of cell-interactive alginate gels with tunable degradation rates and mechanical stiffness was established by a combination of partial oxidation and bimodal molecular weight distribution. Higher gel mechanical properties (13 to 45 kPa) increased myoblast adhesion, proliferation, and differentiation in a 2-D cell culture model. Primary mouse myoblasts were more highly responsive to this cue than the C2C12 myoblast cell line. Myoblasts were then encapsulated in gels varying in degradation rate to simultaneously investigate the effect of degradation and subsequent reduction of mechanical properties on cells in a 3-D environment. C2C12 cells in more rapidly degrading gels exhibited lower proliferation, as they exited the cell cycle to differentiate, compared to those in nondegradable gels. In contrast, mouse primary myoblasts illustrated significantly higher proliferation in degradable gels than in nondegradable gels, and exhibited minimal differentiation in either type of gel. Altogether, these studies suggest that a critical balance between material degradation rate and mechanical properties may be required to regulate formation of engineered skeletal muscle tissue, and that results obtained with the C2C12 cell line may not be predictive of the response of primary myoblasts to environmental cues. The principles delineated in these studies may be useful to tailor smart biomaterials that can be applied to many other polymeric systems and tissue types.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63152/1/ten.2006.0356.pd

Cellular cross-linking of peptide modified hydrogels

Peptide modification of hydrogel-forming materials is being widely explored as a mean

Regulating myoblast phenotype through biomimetically designed hydrogels.

The loss or failure of human tissues or organs is a frequent and costly problem in terms of life and economic loss. The limitations of currently available therapies have spurred the creation of a research field entitled Tissue Engineering. Development of advanced biomaterials will play a key role in the success of tissue engineering. This thesis research focuses on designing material scaffolds presenting bioactive molecules with controllable degradation, and understanding how the signaling molecules regulate cellular function. In the first series of experiments, alginate material platforms were developed that incorporated RGD peptides as adhesion ligands while varying the ligand density, ligand affinity, and nanoscale distribution. Increasing ligand density or binding affinity led to a similar enhancement in proliferation of C2C12 cells and human primary myoblasts. The nanoscale distribution of clustered RGD influenced C2C12 cells and human primary myoblasts but proliferation was effected in an opposing manner. To understand how the RGD presentations modulate cellular function, rheological measurements and a FRET technique were further utilized to quantify the extent of receptor-ligand interactions. Higher relative numbers of bonds formed when RGD density and affinity were increased, as assessed by both approaches, and this finding correlated with cell growth rates. This suggested that varying peptide density and affinity may regulate cellular function by altering the number of bonds formed. However, the influence of nanoscale distribution could not be explained by the number of bonds. Secondly, the hypothesis that alginate gel degradation could regulate the function of myoblasts encapsulated in 3-D microenvironment was tested. Development of degradable alginate gels with tunable rates was established by a combination of partial oxidation and bimodal molecular weight distribution. Myoblasts were encapsulated in gels varying in degradation rate. C2C12 cells in degradable gels exhibited lower proliferation, due to exiting the cell cycle to differentiate, as compared to those in non-degradable gels. Mouse primary myoblasts illustrated significantly higher proliferation in degradable gels, while cells in non-degradable gels showed limited proliferation. Subsequent reduction of mechanical properties also influenced myoblast adhesion, proliferation, and differentiation in a 2-D cell culture model. Cells on stiffer gels illustrated higher spreading, proliferation, and differentiation. Altogether, the material developments in this study elucidate how one can acquire specific bioactivity from biomaterials to control desirable cellular functions and to understand these processes. The degradability of scaffolding materials might also be crucial for long-term success of tissue engineering. The principles delineated in these studies may be useful to tailor smart biomaterials that can be applied to many other polymeric systems and tissue types.Ph.D.Applied SciencesBiological SciencesBiophysicsChemical engineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125617/2/3208425.pd

Designing Scaffolds to Enhance Transplanted Myoblast Survival and Migration

Myoblast transplantation is currently limited by poor survival and integration of these cells into host musculature. Transplantation systems that enhance the viability of the cells and induce their outward migration to populate injured muscle may enhance the success of this approach to muscle regeneration. In this study, enriched populations of primary myoblasts were seeded onto delivery vehicles formed from alginate, and the role of vehicle design and local growth factor delivery in cell survival and migration were examined. Only 5 ± 2.5% of cells seeded into nanoporous alginate gels survived for 24 h and only 4 ± 0.5% migrated out of the gels. Coupling cell adhesion peptides (G4RGDSP) to the alginate prior to gelling slightly increased the viability of cells within the scaffold to 16 ± 1.4% and outward migration to 6 ± 1%. However, processing peptide-modified alginate gels to yield macroporous scaffolds, in combination with sustained delivery of HGF and FGF2 from the material, dramatically increased the viability of seeded cells over a 5-day time course and increased outward migration to 110 ± 12%. This data indicate long-term survival and migration of myoblasts placed within polymeric delivery vehicles can be greatly increased by appropriate scaffold composition, architecture, and growth factor delivery. This system may be particularly useful in the regeneration of muscle tissue and be broadly useful in the regeneration of other tissues as well.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63325/1/ten.2006.12.1295.pd