Chung-Chueh Chang

Differentiation of Dental Pulp Stem Cells on Gutta-Percha Scaffolds

Advances in treatment of tooth injury have shown that tooth regeneration from the pulp was a viable alternative of root canal therapy. In this study, we demonstrated that Gutta-percha, nanocomposites primarily used for obturation of the canal, are not cytotoxic and can induce differentiation of dental pulp stem cells (DPSC) in the absence of soluble mediators. Flat scaffolds were obtained by spin coating Si wafers with three Gutta-percha compounds: GuttaCore™, ProTaper™, and Lexicon™. The images of annealed surfaces showed that the nanoparticles were encapsulated, forming surfaces with root mean square (RMS) roughness of 136–211 nm. Then, by culturing DPSC on these substrates we found that after some initial difficulty in adhesion, confluent tissues were formed after 21 days. Imaging of the polyisoprene (PI) surfaces showed that biomineral deposition only occurred when dexamethasone was present in the media. Spectra obtained from the minerals was consistent with that of hydroxyapatite (HA). In contrast, HA deposition was observed on all Gutta-percha scaffolds regardless of the presence or absence of dexamethasone, implying that surface roughness may be an enabling factor in the differentiation process. These results indicate that Gutta-percha nanocomposites may be good candidates for pulp regeneration therapy.

Manipulation of cell adhesion and dynamics using RGD functionalized polymers

We have successfully synthesized an ABA tri-block co-polymer of poly(methacrylic acid)-block-poly(2-hydroxyethyl methacrylate)-block-poly(methacrylic acid), having Mw = 100k and 272k where we were able to insert RDG or RGD peptide sequences using thiol-acrylate Michael addition. A soft silicone stamp was then used to imprint a 0.4-micron wide grating of the copolymer with a period of 10 microns. The samples were then examined with atomic force microscopy after application of an external electric field and the pattern was observed to stretch by a factor of five. Cells plated onto these substrates showed clear preference for the striped patterns formed under the influence of the external field, and no preferential attachment to the patterns formed in the absence of the field. Cell migration experiments, using the agarose droplet method, performed on spun cast copolymer films showed minimal migration and adhesion on the substrates without peptides or those with only with the RDG peptide, while good adhesion and significant outward migration was observed for cells plated on the copolymers with the RGD sequence. Taken together these results confirmed our hypothesis that a smart biomimetic polymer substrate could be constructed where functional domains could be revealed selectively allowing us to mimic the natural design of engineered tissue constructs.

Using New 3D CLEM Imaging Technique to Investigate the Effects of Substrate Mechanics on Cellular Uptake of Nanoparticle

In the past years, there have been many new developments in biological imaging, ranging from synchrotron X-ray crystallography for protein structure to 3D maps of entire human body [1]. Confocal microscopy, one of the most rapidly growing areas, is commonly used to construct the 3D images of biological samples where the area of interest is stained with fluorescent dyes conjugated to specific targeting antibodies. However, the physical resolution limit of light microscopy (~0.2μm) does not permit visualization of features that are on the nanoscale. Transmission electron microscopy, TEM, is another popular technique where serial sectioning has permitted 3D reconstruction of biological elements. However, this technique is very difficult and requires extremely accurate positioning of the sections, which are each floated from the air/water interface. Lately, focused ion beam (FIB), has been introduced as a viable alternative, which can produce rapid and precise milling (sectioning) of the sample. In combination with scanning electron microscopy (SEM), it becomes a straightforward and powerful tool for 3D imaging of biological samples. Yet, despite its accuracy and high resolution, the EM techniques are not able to uniquely identify the proteins, which comprise the observed structures, or functional domains and receptors which determine the structure of the adhered cells. Hence immunohistochemical staining with fluorescently labeled antibodies has recently been used in combination with electron microsopy, otherwise known as, correlative light and electron microscopy (CLEM). It is a convenient technique which can be used to obtain comprehensive 3D images of tissues elucidating structure, composition, and cell lineage.

The Prospective Application of Graphene Loaded Poly(4-vinphylpridine) Fibrous Scaffolds on the Dental Pulp Stem Cells Proliferation and Differentiation

Dental Pulp Stem Cells (DPSCs) are a promising new approach for bone regeneration, since they are relatively easy to harvest without significant surgical access to the collection sites [1]. It is well established in the literature that DPSCs have the ability to differentiate along osteogenic, odontogenic or neural pathways. Studies have shown that the phenotype expressed is regulated by the microenvironment of the cells, or the stem cell niche where different biochemical cues, cell-cell interactions, cell-scaffold interactions are present [2]. The mechanical response of the substrate has been an important focus of many studies. However, most of these studies were performed on two dimensional structured substrates. In vivo the cellular environment is regulated by the extracellular matrix, which is fibrillar. Hence the essential questions arise as to the influence of the dimensions of the fibers on the cell differentiation. In order to study this aspect we developed a model based on polymer fibers, which serve to template the ECM proteins, and provide a structured scaffold where we can directly study the influence of topography on stem cell differentiation.