Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct <i>in vitro</i>

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury

Aligned Schwann cells derived from human dental pulp stem cells direct neurite growth in a tissue engineered collagen construct

A variety of traumas and diseases can cause peripheral nerve injury (PNI). In the process of spontaneous healing, Schwann cells (SC) are very important players. They not only reconstitute myelin, essential for proper nerve function, but also organize themselves into columns that lie within the endoneurial tubes to guide and support axonal regeneration from the proximal to the distal end of the injury. By contrast, large gap PNI requires artificial bridging strategies such as highly aligned scaffolds to promote directed neurite outgrowth. In the field of neural tissue engineering, hydrogels are very attractive biomaterials to encapsulate cells. Their high water content facilitates transport of oxygen, nutrients and waste and their viscoelastic properties can be relatively easy modulated. Recently, a new technique has been developed to efficiently and reproducibly align cells in a collagen type I hydrogel, thereby mimicking the environment required for nerve regeneration. This Engineered Neural Tissue (ENT) has given promising preliminary results with immortalized and primary rat SC both in vitro and in vivo. However, the use of human cells would be a significant advance in bringing this ENT from bench to bedside. Since autologous SC must be harvested from another peripheral nerve and are slowly growing in vitro, there is a high need for alternative cell types such as human dental pulp stem cells (hDPSC). hDPSC can be easily isolated from discarded teeth with very little ethical issues and their neural crest origin suggests a predisposition to differentiate into neural or glial cells. Preliminary experiments in our laboratory indicate the two-dimensional (2D) in vitro transdifferentiation capacity of human DPSC (hPDSC) into Schwann-like cells (SC-hDPSC). Here, we evaluate the potential of SC-hDPSC to align within a collagen type I hydrogel and determine the effects of this ENT on dorsal root ganglion neuron neurite outgrowth in vitro

Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM) and second harmonic generation (SHG) could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin

3D full-field quantification of cell-induced large deformations in fibrillar biomaterials by combining non-rigid image registration with label-free second harmonic generation

To advance our current understanding of cell-matrix mechanics and its importance for biomaterials development, advanced three-dimensional (3D) measurement techniques are necessary. Cell-induced deformations of the surrounding matrix are commonly derived from the displacement of embedded fiducial markers, as part of traction force microscopy (TFM) procedures. However, these fluorescent markers may alter the mechanical properties of the matrix or can be taken up by the embedded cells, and therefore influence cellular behavior and fate. In addition, the currently developed methods for calculating cell-induced deformations are generally limited to relatively small deformations, with displacement magnitudes and strains typically of the order of a few microns and less than 10% respectively. Yet, large, complex deformation fields can be expected from cells exerting tractions in fibrillar biomaterials, like collagen. To circumvent these hurdles, we present a technique for the 3D full-field quantification of large cell-generated deformations in collagen, without the need of fiducial markers. We applied non-rigid, Free Form Deformation (FFD)-based image registration to compute full-field displacements induced by MRC-5 human lung fibroblasts in a collagen type I hydrogel by solely relying on second harmonic generation (SHG) from the collagen fibrils. By executing comparative experiments, we show that comparable displacement fields can be derived from both fibrils and fluorescent beads. SHG-based fibril imaging can circumvent all described disadvantages of using fiducial markers. This approach allows measuring 3D full-field deformations under large displacement (of the order of 10 μm) and strain regimes (up to 40%). As such, it holds great promise for the study of large cell-induced deformations as an inherent component of cell-biomaterial interactions and cell-mediated biomaterial remodeling.publisher: Elsevier articletitle: 3D full-field quantification of cell-induced large deformations in fibrillar biomaterials by combining non-rigid image registration with label-free second harmonic generation journaltitle: Biomaterials articlelink: http://dx.doi.org/10.1016/j.biomaterials.2017.05.015 content_type: article copyright: © 2017 Elsevier Ltd. All rights reserved.status: publishe

The Role of Prostaglandins and COX-Enzymes in Chondrogenic Differentiation of ATDC5 Progenitor Cells

NSAIDs are used to relieve pain and decrease inflammation by inhibition of cyclooxygenase (COX)-catalyzed prostaglandin (PG) synthesis. PGs are fatty acid mediators involved in cartilage homeostasis, however the action of their synthesizing COX-enzymes in cartilage differentiation is not well understood. In this study we hypothesized that COX-1 and COX-2 have differential roles in chondrogenic differentiation.ATDC5 cells were differentiated in the presence of COX-1 (SC-560, Mofezolac) or COX-2 (NS398, Celecoxib) specific inhibitors. Specificity of the NSAIDs and inhibition of specific prostaglandin levels were determined by EIA. Prostaglandins were added during the differentiation process. Chondrogenic outcome was determined by gene- and protein expression analyses.Inhibition of COX-1 prevented Col2a1 and Col10a1 expression. Inhibition of COX-2 resulted in decreased Col10a1 expression, while Col2a1 remained unaffected. To explain this difference expression patterns of both COX-enzymes as well as specific prostaglandin concentrations were determined. Both COX-enzymes are upregulated during late chondrogenic differentiation, whereas only COX-2 is briefly expressed also early in differentiation. PGD2 and PGE2 followed the COX-2 expression pattern, whereas PGF2α and TXA2 levels remained low. Furthermore, COX inhibition resulted in decreased levels of all tested PGs, except for PGD2 and PGF2α in the COX-1 inhibited condition. Addition of PGE2 and PGF2α resulted in increased expression of chondrogenic markers, whereas TXA2 increased expression of hypertrophic markers.Our findings point towards a differential role for COX-enzymes and PG-production in chondrogenic differentiation of ATDC5 cells. Ongoing research is focusing on further elucidating the functional partition of cyclooxygenases and specific prostaglandin production

COX-1 and COX-2 specific inhibitors differentially influence prostaglandin levels during ATDC5 chondrogenic differentiation.

<p>Successful COX-1 or COX-2 inhibition by the inhibitors and possible differences in specific prostaglandin production was determined at day 14 in ATDC5 differentiation. NS398, Celecoxib, SC-50 or Mofezolac were added from the start of differentiation or from day 10 onwards. <b>A:</b> PGE₂, PGD₂, PGF_2α and TXA₂ medium concentrations were determined at day 14 in differentiation by prostaglandin specific ELISA. <b>B:</b> COX-1 and COX-2 mRNA expression at day 14 in differentiation was determined by RT-qPCR (relative to t = 0 and corrected for β-actin). In graphs, error bars represent mean ± s.e.m. (n = 3). * indicates <i>p</i> < 0.05.</p

Expression of cyclooxygenases during chondrogenic differentiation from progenitor cells.

<p><b>A:</b> Expression of COX-1 and COX-2 in growth plates was determined by IHC. RZ = resting zone, PZ = proliferative zone, HZ = hypertrophic zone. <b>B:</b> Col2a1, Col10a1, Sox9 and Runx2 mRNA expression during chondrogenic differentiation of ATDC5 cells. <b>C:</b> COX-1 and COX-2 mRNA expression. In graphs, error bars represent mean ± s.e.m.(standard error of the mean; n = 3). * indicates <i>p</i> < 0.05. <b>D:</b> Protein expression of COX-1 and COX-2 in differentiation. Molecular weight markers (kDa) are depicted on the left of immunoblots and relative quantifications are depicted on top of immunoblots. <b>E:</b> Medium concentrations of PGD₂, PGE₂, PGF_2α and TXA₂ during chondrogenic differentiation.</p

Effects of COX-1 and COX-2 specific inhibitors on chondrogenic differentiation of ATDC5 cells.

<p><b>A:</b>. Col2a1, Col10a1, Sox9 and Runx2 mRNA expression was determined at day 14 in ATDC5 differentiation. <b>B:</b> Protein expression of Col2a1 and Col10a1 at day 14 in differentiation. Molecular weight markers (kDa) are depicted on the left of immunoblots and relative quantifications are depicted on top of immunoblots. <b>C:</b> Glycosaminoglycans (GAGs) were stained and fold change of t = 14 samples was calculated as compared to t = 0 samples. Fold change (DNA) from samples from t = 14 was calculated relatively to day 0. In graphs, error bars represent mean ±s.e.m. (n = 3). * indicates <i>p</i> < 0.05.</p

Primer sequences for RT-qPCR.

<p>Primer sequences for RT-qPCR.</p