Micro Injection Molded Microtopographic Polymer Plates used to Mechanically Direct Stem Cell Activity

A novel micro injection molding assembly which allows for firm yet compliant housing of silicon tooling was designed and manufactured. Microchannels were etched into a silicon wafer through the use of ultraviolet lithography (UVL) combined with deep reactive ion etching (DRIE). Injection molded polystyrene plates containing microtopography were fabricated in which multiple molding parameter studies were executed to further understand the effect of mold temperature and injection velocity on replication. Micro and nano featured polymeric substrates have tremendous potential for use in stem cell culture, as cells are exposed to and controlled by microtopography in their natural environment

Design and characterization of hybrid peptide sol -gel materials for the solid state induction of neuronal differentiation

Cell-based therapeutics are a rapidly growing area of research, with considerable promise in the treatment of neurological diseases. One of the primary limitations to neuronal cell-based devices is the necessity to maintain cells in an immature or undifferentiated state in culture prior to transplantation. In many cases, the undifferentiated cell does not express the desired characteristics for implantation. Biologically functional nanomaterials provide the ability to manipulate the direct extracellular environment surrounding cells; influencing their fate and differentiation path. The ability to engineer the interface between the cells and culture materials provides a repeatable, stable means of directing cells down a specific growth path determined by endogenous signaling pathways. This materials approach to cellular engineering can limit the need for added exogenous growth factors, feeder layers, or animal sera, in addition to creating a homogenous cell population for transplantation. In this work, hybrid peptide ormosil materials were developed; designed to mimic the developing mammalian brain during corticogenesis. These materials have been developed to enhance the GABAergic phenotype of P19 embryonic carcinoma cells and immature immortalized neurons. The ability to develop a homogenous, directed cell population has implications in stem cell research, regenerative medicine, cell-based devices and biosensing technology

Controllable Surface Expression of Bioactive Peptides Incorporated into a Silica Thin Film Matrix

Mammalian cell culture platforms often require biomolecular modification to enhance cell adhesion and proliferation, Often, these modifications are performed using self-assembled monolayers or whole protein coatings, Such its collagen. These protocols are inherently useful but generally suffer from repeatability. Undesirable conditions during self-assembly can lead to complications in the surface presentation of the biological ligands. Whole proteins are often unstable and derived from animal sources, making them less attractive for tissue engineering applications. As the biological effect of the material often depends strongly on the concentration of the integrated ligand(s), any complication due to synthesis or stability can lead to unexpected biological results. In this research, we expand upon previous work in peptide-silane modifications to sol-gel derived silica matrixes, demonstrating that the surface density of the peptide can be calibrated by simply modifying the starting liquid precursor concentration. The potential for calibration of peptide surface presentation allows for well-defined cell culture platforms that have the potential to mimic natural proteins in a stable, repeatable manner

Increasing the Potential of Bioactive Glass as a Scaffold for Bone Tissue Engineering

Bioactive glass is known for its potential as a bone scaffold due to its ability to stimulate osteogenesis and differentiation of stem cells into bone cells. In an attempt to investigate if we can increase these potentials, we decorated the structure of the bioactive glass made by the sol-gel technique with 3 peptides sequences from different proteins known for their potentials to stimulate the osteogensis process (fibronectin, BMP-2 and protein kinase CKI). This material was tested with Human Mesenchymal Stem Cells (hMSCs) and MC-3T3 preosteoblasts to see the difference in the effect on uncommitted and committed cells. The bioactive glass sol with and without the peptides was dip coated onto glass cover slips, leading to a film of the material, surface decorated with the peptides of choice. The two cell types were seeded onto the materials in standard proliferation medium without additives for differentiation induction. Cells were also grown on tissue culture treated cover slips with and without differentiation induction media as positive and negative controls, respectively. The cells were grown on the materials for a total of five weeks, and were tested at four time points (weekly from week two) by immunocytochemical assays to investigate the levels of different osteogenic markers (osteopontin, osteocalcin and osteonectin) and by qRT-PCR to investigate the mRNA potential of the same proteins. On the native bioactive glass samples, the hMSCs and the MC-3T3s adhered poorly. On peptide- decorated samples, the hMSC adhered poorly, however, the MC-3T3 cells appear to differentiate at a rate that is equal to or faster than the positive control, indicating that the peptide effect is similar to that achieved by traditional BMP-2 soluble protein techniques. This supports our hypothesis that adding specific peptide sequences known for their effects in cells adhesion, proliferation and differentiation can increase the potential of the bioactive glass as a scaffold for bone tissue engineering. The data, however, leads to some questions regarding the MC-3T3 cell model for use in further studies

journal of Biomaterials and Nanobiotechnology,

Biointerface design that targets osteogenesis is a growing area of research with significant impli- cations in biomedicine. Materials known to either support or stimulate osteogenesis are com- posed of a biomimetic ceramic material, such as bioactive glass. Bioactive glass is osteoproductive, and the potential for osteoproductivity can be enhanced by the addition of proteins or other addi- tives designed to alter functionality. In addition, soluble growth factors are often added to osteo- genic culture on bioactive glasses, further intensifying the effects of the material. In this paper, synthetic peptide combinations, covalently bound to a three-dimensional bioactive glass network, are used to mimic the effects of the whole fibronectin and bone morphogenetic proteins (BMP) 2 and 9. Peptide-silanes possessing critical binding sequences from each of these proteins are syn- thesized and used to decorate the surface of three-dimensional (3D) nano-macroporous bioactive glass. MC3T3 preosteoblast cells are then assessed for differentiation on the materials in the ab- sence of soluble differentiation cues. MC3T3 preosteoblasts undergo enhanced differentiation on the peptide-silane samples over the standard nano-macroporous bioactive glass, and the differen- tiation capacity of the cells exposes only to peptide-silane surfaces approaches that of cells grown in chemical differentiation induction media

Maintenance and Neuronal Cell Differentiation of Neural Stem Cells C17.2 Correlated to Medium Availability Sets Design Criteria in Microfluidic Systems

<div><p>Background</p><p>Neural stem cells (NSCs) play an important role in developing potential cell-based therapeutics for neurodegenerative disease. Microfluidics has proven a powerful tool in mechanistic studies of NSC differentiation. However, NSCs are prone to differentiate when the nutrients are limited, which occurs unfavorable by fast medium consumption in miniaturized culture environment. For mechanistic studies of NSCs in microfluidics, it is vital that neuronal cell differentiation is triggered by controlled factors only. Thus, we studied the correlation between available cell medium and spontaneous neuronal cell differentiation of C17.2 NSCs in standard culture medium, and proposed the necessary microfluidic design criteria to prevent undesirable cell phenotype changes.</p><p>Methodology/Principal Findings</p><p>A series of microchannels with specific geometric parameters were designed to provide different amount of medium to the cells over time. A medium factor (<i>MF</i>, defined as the volume of stem cell culture medium divided by total number of cells at seeding and number of hours between medium replacement) successfully correlated the amount of medium available to each cell averaged over time to neuronal cell differentiation. <i>MF</i> smaller than 8.3×10⁴ µm³/cell⋅hour produced significant neuronal cell differentiation marked by cell morphological change and significantly more cells with positive β-tubulin-III and MAP2 staining than the control. When <i>MF</i> was equal or greater than 8.3×10⁴ µm³/cell⋅hour, minimal spontaneous neuronal cell differentiation happened relative to the control. <i>MF</i> had minimal relation with the average neurite length.</p><p>Significance</p><p><i>MF</i>s can be controlled easily to maintain the stem cell status of C17.2 NSCs or to induce spontaneous neuronal cell differentiation in standard stem cell culture medium. This finding is useful in designing microfluidic culture platforms for controllable NSC maintenance and differentiation. This study also offers insight about consumption rate of serum molecules involved in maintaining the stemness of NSCs.</p></div

Behavior of C17.2 cells cultured in 2000 µm microchannels with 24, 48 and 96 hour feeding intervals.

<p>(A) The β-tubulin-III positive cell counts per mm² over time. (B) The neuronal cell counts per mm² over time. (C) The average neurite length per mm² over time. Groups lacking neuronal cells were labeled with “O” in Figure 5B and Figure 5C. The control (with “c”) was C17.2 NSCs seeded at the same surface density in FluoroDishes but without microchannels and fed every 48 hours as in standard subculture protocols. The * above the bars indicated a statistical difference between the sample and the control by two-tailed Student’s t test (<i>p</i><0.05). The * below the bars indicated a statistical difference in the group by one way ANOVA (<i>p</i><0.05). The ** in Figure 5C indicates a significant difference due exclusively to the samples with no neurite outgrowth. N≥15.</p

Summary of cell phenotypes with microchannel heights of 50 µm to 1000 µm and feeding frequencies of 12 hours to 48 hours.

<p>Groups with small <i>MF</i>s (towards top right corner of each graph) generally had more obvious neuronal cell differentiation as demonstrated by brighter colors representing higher density of cells with positive β-tubulin-III staining and neural morphology (first and second columns). The neurite length (third column), however, did not seem to have a strong correlation with the <i>MF</i>. The samples without noticeable neurite outgrowth were marked with a cross sign.</p

Neuronal cell differentiation of C17.2 cells cultured in 50 µm microchannels with 12, 24 and 48 hour feeding intervals.

<p>(A) The cell morphological change over 3 weeks. All groups showed biomarker staining and morphological change consistent with neuronal cell differentiation. Red: Nestin. Green: β-tubulin-III. Blue: cell nuclei. Scale bar = 100 µm. (B) The β-tubulin-III positive cell counts per mm² over time. (C) The neuronal cell counts per mm² over time. (D) The average neurite length measurement over time. In Figures 3B–3D, the control (with “c”) was C17.2 NSCs seeded at the same surface density in FluoroDishes but without microchannels and fed every 48 hours as in standard subculture protocols. Data were shown as mean ± standard deviation. The * above the bars indicated a statistical difference between the sample and the control by two-tailed Student’s t test (<i>p</i><0.05). The * below the bars indicated a statistical difference in the group by one way ANOVA (<i>p</i><0.05). N≥15.</p