Jacob F. Pollock

Mechanical and swelling characterization of poly(N-isopropyl acrylamide -co- methoxy poly(ethylene glycol) methacrylate) sol–gels

The dimensional stability and rheological properties of a series of comb-like copolymers of N-isopropyl acrylamide (NIPAAm) and methoxy poly(ethylene glycol) methacrylate (mPEGMA), poly(NIPAAm-co-mPEGMA), with varying poly(ethylene glycol) (PEG) graft densities and molecular weights were studied. The thermoresponsive character of the copolymer solutions was investigated by kinetic and equilibrium swelling, as well as by static and dynamic mechanical analysis. Surface response mapping was employed to target particular compositions and concentrations with excellent dimensional stability and a relatively large change in dynamic mechanical properties upon thermoreversible gelation. The mechanical characteristics of the gels depended strongly upon concentration of total polymer and less so upon copolymer ratio. Increased PEG graft density was shown to slow the deswelling rate and increase the equilibrium water content of the gels. Upon gelation at sol concentrations of 1-20 wt.% the materials underwent no deswelling or syneresis and maintained stable gels with a large elastic regime and high yield strain (i.e. elastic and soft but tough), even within the Pascal range of complex shear moduli. These materials are unique in that they maintained a physiologically useful lower critical solution temperature (approximately 33 degrees C), despite having a high PEG content. Copolymers with a high PEG content and low polymer fraction were conveniently transparent in the gel phase, allowing visualization of cellular activity without disrupting the microenvironment. Mesenchymal stem cells showed good viability and proliferation in three-dimensional culture within the gels, despite the lack of ligand incorporation to promote cellular interaction. Multi-component matrices can be created through simple mixing of copolymer solutions and peptide-conjugated linear polymers and proteins to produce combinatorial microenvironments with the potential for use in cell biology, tissue engineering and medical applications.

Temporal Gene Expression Profiling during Rat Femoral Marrow Ablation-Induced Intramembranous Bone Regeneration

Enhanced understanding of differential gene expression and biological pathways associated with distinct phases of intramembranous bone regeneration following femoral marrow ablation surgery will improve future advancements regarding osseointegration of joint replacement implants, biomaterials design, and bone tissue engineering. A rat femoral marrow ablation model was performed and genome-wide microarray data were obtained from samples at 1, 3, 5, 7, 10, 14, 28, and 56 days post-ablation, with intact bones serving as controls at Day 0. Bayesian model-based clustering produced eight distinct groups amongst 9,062 significant gene probe sets based on similar temporal expression profiles, which were further categorized into three major temporal classes of increased, variable, and decreased expression. Osteoblastic- and osteoclastic-associated genes were found to be significantly expressed within the increased expression groups. Chondrogenesis was not detected histologically. Adipogenic marker genes were found within variable/decreased expression groups, emphasizing that adipogenesis was inhibited during osteogenesis. Differential biological processes and pathways associated with each major temporal group were identified, and significantly expressed genes involved were visually represented by heat maps. It was determined that the increased expression group exclusively contains genes involved in pathways for matrix metalloproteinases (MMPs), Wnt signaling, TGF-β signaling, and inflammatory pathways. Only the variable expression group contains genes associated with glycolysis and gluconeogenesis, the notch signaling pathway, natural killer cell mediated cytotoxicity, and the B cell receptor signaling pathway. The decreased group exclusively consists of genes involved in heme biosynthesis, the p53 signaling pathway, and the hematopoietic cell lineage. Significant biological pathways and transcription factors expressed at each time point post-ablation were also identified. These data present the first temporal gene expression profiling analysis of the rat genome during intramembranous bone regeneration induced by femoral marrow ablation.

Designing Tunable Artificial Matrices for Stem Cell Culture

This chapter focuses on progress in the design and characterization of artificial matrices that attempt to recapitulate microenvironmental cues for in vitro stem cell culture and differentiation. The physical structure, or microarchitecture, of an artificial matrix must provide appropriate physical signals, present or allow access to biochemical cues, and permit nutrient and waste exchange. Synthetic matrices should mimic some aspects of the natural properties of the collagen scaffold and adjacent proteins of the extracellular matrix (ECM), which constitutes a highly hydrated and fibrous network that supports cell attachment, migration, and other functions. One approach to mimicking the physical structure of the ECM is the creation of matrices of nanofibers prepared with electrospun polymers. Another approach to the design of artificial matrices for stem cells is to employ a hydrogel to mimic the physical properties of the natural ECM. Hydrogels emulate the high water content and porous nature of most natural soft tissues. Mechanical design parameters for artificial matrices include elasticity, compressibility, viscoelastic behavior, and tensile strength. Controlling the mechanical properties of a material at the cellular level can help elicit a desired cell response, and, in addition, the bulk mechanical properties of the matrix must be controlled such that the matrix is able to withstand loads that may be involved in downstream applications. The mechanical properties of synthetic and natural matrices are typically characterized by either atomic force microscopy or rheology.