Actuation of Soft Thermoresponsive Hydrogels Mechanically Stimulates Osteogenesis in Human Mesenchymal Stem Cells without Biochemical Factors.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multiple lineages and can be harvested relatively easily from adults, making them a promising cell source for regenerative therapies. While it is well-known how to consistently differentiate MSCs into adipose, chondrogenic, and osteogenic lineages by treatment with biochemical factors, the number of studies exploring how to achieve this with mechanical signals is limited. A relatively unexplored area is the effect of cyclic forces on the MSC differentiation. Recently, our group developed a thermoresponsive N-ethyl acrylamide/N-isopropylacrylamide (NIPAM/NEAM) hydrogel supplemented with gold nanorods that are able to convert near-infrared light into heat. Using light pulses allows for local hydrogel collapse and swelling with physiologically relevant force and frequency. In this study, MSCs are cultured on this hydrogel system with a patterned surface and exposed to intermittent or continuous actuation of the hydrogel for 3 days to study the effect of actuation on MSC differentiation. First, cells are harvested from the bone marrow of three donors and tested for their MSC phenotype, meeting the following criteria: the harvested cells are adherent and demonstrate a fibroblast-like bipolar morphology. They lack the expression of CD34 and CD45 but do express CD73, CD90, and CD105. Additionally, their differentiation potential into adipogenic, chondrogenic, and osteogenic lineages is validated by the addition of standardized differentiation media. Next, MSCs are exposed to intermittent or continuous actuation, which leads to a significantly enhanced cell spreading compared to nonactuated cells. Moreover, actuation results in nuclear translocation of Runt-related transcription factor 2 and the Yes-associated protein. Together, these results indicate that cyclic mechanical stimulation on a soft, ridged substrate modulates the MSC fate commitment in the direction of osteogenesis.

Bicyclic RGD peptides enhance nerve growth in synthetic PEG-based Anisogels.

Nerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and 'random screened' tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvß3 or α5ß1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvß3 or α5ß1, compared to monocyclic or linear RGD peptides, with a slight preference to αvß3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration.

Cellular responses to beating hydrogels to investigate mechanotransduction.

Cells feel the forces exerted on them by the surrounding extracellular matrix (ECM) environment and respond to them. While many cell fate processes are dictated by these forces, which are highly synchronized in space and time, abnormal force transduction is implicated in the progression of many diseases (muscular dystrophy, cancer). However, material platforms that enable transient, cyclic forces in vitro to recreate an in vivo-like scenario remain a challenge. Here, we report a hydrogel system that rapidly beats (actuates) with spatio-temporal control using a near infra-red light trigger. Small, user-defined mechanical forces (~nN) are exerted on cells growing on the hydrogel surface at frequencies up to 10 Hz, revealing insights into the effect of actuation on cell migration and the kinetics of reversible nuclear translocation of the mechanosensor protein myocardin related transcription factor A, depending on the actuation amplitude, duration and frequency.

Development of novel radiochemotherapy approaches targeting prostate tumor progenitor cells using nanohybrids.

Many tumors including prostate cancer are maintained by cancer stem cells (CSCs), which might cause tumor relapse if not eradicated during the course of treatment. Specific targeting or radiosensitization of CSCs bear promise to improve tumor curability by synergistic effects in combination with radiotherapy. Carbon nanotubes (CNTs) can be used as promising drug delivery systems for anticancer drugs such as the flavonoid catechin. Catechin is an extensively studied active ingredient of the different plants, including green tea, and it is widely recognized as co-adjuvant in cancer therapy. Here we describe the synthesis of biocompatible, catechin-loaded and gelatin-conjugated CNTs (Gel_CT_CNTs) with anticancer properties and demonstrate their potential for the eradication of prostate CSCs in combination with X-ray irradiation. Gel_CT_CNTs showed a significant enhancement of in vitro anticancer activity as compared to catechin alone. Moreover, treatment of prostate cancer cells with Gel_CT_CNT nanohybrids inhibited the tumorigenic cell population defined by a high aldehyde dehydrogenase (ALDH) activity. A combination of X-ray irradiation and treatment with Gel_CT_CNTs caused a decrease in the protein level of stem cell-related transcription factors and regulators including Nanog, Oct4 and ß-catenin and led to an increase of cancer cell radiosensitivity as demonstrated by clonogenic and spherogenic cell survival assays. Taken together, our results suggest that a combination of irradiation and Gel_CT_CNTs can be potentially used for the radiosensitization and eradication of prostate CSC populations.