Live reporting for hypoxia: Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O₂). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs.     

Glutamate affects dendritic morphology of neurons grown on compliant substrates

Brain stiffness changes in response to injury or disease. As a secondary consequence, glutamate is released from neurons and astroglia. Two types of glutamate receptors, N‐methyl‐d ‐aspartate (NMDA) and α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors, sense mechanotransduction, leading to downstream signaling in neurons. Recently, our group reported that these two receptors affect dendrite morphology in hippocampal neurons grown on compliant substrates. Blocking receptor activity has distinct effects on dendrites, depending on whether neurons are grown on soft or stiff gels. In the current study, we examine whether exposure to glutamate itself alters stiffness‐mediated changes to dendrites in hippocampal neurons. We find that glutamate augments changes seen when neurons are grown on soft gels of 300 or 600 Pa, but in contrast, glutamate attenuates changes seen when neurons are grown on stiff gels of 3,000 Pa. These results suggest that there is interplay between mechanosensing and glutamate receptor activation in determining dendrite morphology in neurons. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1128–1132, 2015     

Chondroitin sulfate and dynamic loading alter chondrogenesis of human MSCs in PEG hydrogels

While biochemical and biomechanical cues are known to play important roles in directing stem cell differentiation, there remains little known regarding how these inextricably linked biological cues impact the differentiation fate of human marrow stromal cells (hMSCs). This study investigates the chondrogenic differentiation potential of hMSCs when encapsulated in a three dimensional (3D) hydrogel and exposed to a biochemical cue, chondroitin sulfate (ChS), a biomechanical cue, dynamic loading, and their combination. hMSCs were encapsulated in bioinert poly(ethylene glycol) (PEG) hydrogels only, PEG hydrogels modified with covalently incorporated methacrylated ChS and cultured under free swelling conditions or subjected to delayed intermittent dynamic loading for 2 weeks. The 3D hydrogel environment led to the expression of chondrogenic genes (SOX9) and proteins (aggrecan and collagen II), but also upregulated hypertrophic genes (RUNX2 and Col X mRNA) and proteins (collagen X), while the application of loading generally led to a downregulation in chondrogenic proteins (collagen II). The presence of ChS led to elevated levels of aggrecan, but also collagen I, protein expression and when combined with dynamic loading downregulated, but did not suppress, hypertrophic genes (Col X and RUNX2) and collagen I protein expression. Taken together, this study demonstrates that while the 3D environment induces early terminal differentiation during chondrogenesis of hMSCs, the incorporation of ChS into PEG hydrogels may slow the terminal differentiation process down the hypertrophic lineage particularly when dynamic loading is applied. Biotechnol. Bioeng. 2012; 109: 2671–2682. © 2012 Wiley Periodicals, Inc.     

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e. low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.     

Thermosensitive hybrid hyaluronan/p(HPMAm-lac)-PEG hydrogels enhance cartilage regeneration in a mouse model of osteoarthritis

Osteoarthritis (OA), due to cartilage degeneration, is one of the leading causes of disability worldwide. Currently, there are not efficacious therapies to reverse cartilage degeneration. In this study we evaluated the potential of hybrid hydrogels, composed of a biodegradable and thermosensitive triblock copolymer cross-linked via Michael addition to thiolated hyaluronic acid, in contrasting inflammatory processes underlying OA. Hydrogels composed of different w/w % concentrations of hyaluronan were investigated for their degradation behavior and capacity to release the polysaccharide in a sustained fashion. It was found that hyaluronic acid was controllably released during network degradation with a zero-order release kinetics, and the release rate depended on cross-link density and degradation kinetics of the hydrogels. When locally administered in vivo in an OA mouse model, the hydrogels demonstrated the ability to restore, to some extent, bone remineralization, proteoglycan production, levels of Sox-9 and Runx-2. Furthermore, the downregulation of proinflammatory mediators, such as TNF-α, NFkB, and RANKL and proinflammatory cytokines was observed. In summary, the investigated hydrogel technology represents an ideal candidate for the potential encapsulation and release of drugs relevant in the field of OA. In this context, the hydrogel matrix could act in synergy with the drug, in reversing phenomena of inflammation, cartilage disruption, and bone demineralization associated with OA.     

Affinity‐Triggered Assemblies Based on a Designed Peptide–Peptide Affinity Pair

Affinity‐triggered assemblies rely on affinity interactions as the driving force to assemble physically crosslinked networks. WW domains are small hydrophobic proteins binding to proline‐rich peptides that are typically produced in the insoluble form. Previous works attempted the biological production of the full WW domain in tandem to generate multivalent components for affinity‐triggered hydrogels. In this work, an alternative approach is followed by engineering a 13‐mer minimal version of the WW domain that retains the ability to bind to target proline‐rich peptides. Both ligand and target peptides are produced chemically and conjugated to multivalent polyethylene glycol, yielding two components. Upon mixing together, they form soft biocompatible affinity‐triggered assemblies, stable in stem cell culture media, and display mechanical properties in the same order of magnitude as for those hydrogels formed with the full WW protein in tandem.     

Anti-calcification of bovine pericardium for bioprosthetic heart valves after surface modification with hyaluronic acid derivatives

Surface modification of glutaraldehyde fixed bovine pericardium (GFBP) was successfully carried out with hyaluronic acid (HA) derivatives. At first, HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional group into the carboxyl group of HA backbone. Then, GFBP was surface modified by grafting HA-ADH to the free aldehyde groups on the tissue and the subsequent HA-ADH hydrogel coating. HA-ADH hydrogels could be prepared through selective crosslinking at low pH between hydrazide groups of HA-ADH and crosslinkers containing succinimmidyl moieties with minimized protein denaturation. When HA-ADH hydrogels were prepared at low pH of 4.8 in the presence of erythropoietin (EPO) as a model protein, EPO release was continued up to 85% of total amount of loaded EPO for 4 days. To the contrary, only 30% of EPO was released from HA-ADH hydrogels prepared at pH=7.4, which might be due to the denaturation of EPO during the crosslinking reaction. Because the carboxyl groups on the glucuronic acid residues are recognition sites for HA degradation by hyaluronidase, the HA-ADH hydrogels degraded more slowly than HA hydrogels prepared by the crosslinking reaction of divinyl sulfone with hydroxyl groups of HA. Following a two-week subcutaneous implantation in osteopontin-null mice, clinically significant levels of calcification were observed for the positive controls without any surface modification. However, the calcification of surface modified GFBP with HA-ADH and HA-ADH hydrogels was drastically reduced by more than 85% of the positive controls. The anti-calcification effect of HA surface modification was also confirmed by microscopic analysis of explan ted tissue after staining with Alizarin Red S for calcium, which followed the trend as observed with calcium quantification.     

Hydrogels as extracellular matrix mimics for 3D cell culture

Methods for culturing mammalian cells ex vivo are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Two‐dimensional culture has been the paradigm for typical in vitro cell culture; however, it has been demonstrated that cells behave more natively when cultured in three‐dimensional environments. Permissive, synthetic hydrogels and promoting, natural hydrogels have become popular as three‐dimensional cell culture platforms; yet, both of these systems possess limitations. In this perspective, we discuss the use of both synthetic and natural hydrogels as scaffolds for three‐dimensional cell culture as well as synthetic hydrogels that incorporate sophisticated biochemical and mechanical cues as mimics of the native extracellular matrix. Ultimately, advances in synthetic–biologic hydrogel hybrids are needed to provide robust platforms for investigating cell physiology and fabricating tissue outside of the organism. Biotechnol. Bioeng. 2009;103: 655–663. © 2009 Wiley Periodicals, Inc.     

Circular dichroism studies of low molecular weight hydrogelators: The use of SRCD and addressing practical issues

Circular dichroism (CD) spectroscopy has been used extensively for the investigation of the conformation and configuration of chiral molecules, but its use for evaluating the mode of self‐assembly in soft materials has been limited. Herein, we report a protocol for the study of such materials by electronic CD spectroscopy using commercial/benchtop instruments and synchrotron radiation (SR) using the B23 beamline available at Diamond Light Source. The use of the B23 beamtime for SRCD was advantageous because of the unique enhanced spatial resolution achieved because of its highly collimated and small beamlight cross section (ca. 250 μm) and higher photon flux in the far UV region (175‐250 nm) enhancing the signal‐to‐noise ratio relative to benchtop CD instruments. A set of low molecular weight (LMW) hydrogelators, comprising two Fmoc‐protected enantiomeric monosaccharides and one Fmoc dipeptide (Fmoc‐FF), were studied. The research focused on the optimization of sample preparation and handling, which then enabled the characterization of sample conformational homogeneity and thermal stability. CD spectroscopy, in combination with other spectroscopic techniques and microscopy, will allow a better insight into the self‐assembly of chiral building blocks into higher order structural architectures.