Peptoid-Cross-Linked Hydrogel Stiffness Modulates Human Mesenchymal Stromal Cell Immunoregulatory Potential in the Presence of Interferon-Gamma.

Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross-linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein-1, macrophage colony-stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN-γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN-γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN-γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN-γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN-γ.

Crosslinker structure modulates bulk mechanical properties and dictates hMSC behavior on hyaluronic acid hydrogels.

Synthetic hydrogels are attractive platforms due in part to their highly tunable mechanics, which impact cell behavior and secretory profile. These mechanics are often controlled by altering the number of crosslinks or the total polymer concentration in the gel, leading to structure-property relationships that inherently couple network connectivity to the overall modulus. In contrast, the native extracellular matrix (ECM) contains structured biopolymers that enable stiff gels even at low polymer content, facilitating 3D cell culture and permeability of soluble factors. To mimic the hierarchical order of natural ECM, this work describes a synthetic hydrogel system in which mechanics are tuned using the structure of sequence-defined peptoid crosslinkers, while fixing network connectivity. Peptoid crosslinkers with different secondary structures are investigated: 1) a helical, molecularly stiff peptoid, 2) a non-helical, less stiff peptoid, and 3) an unstructured, relatively flexible peptoid. Bulk hydrogel storage modulus increases when crosslinkers of higher chain stiffness are used. In-vitro studies assess the viability, proliferation, cell morphology, and immunomodulatory activity of human mesenchymal stem cells (hMSCs) on each hydrogel substrate. Matrix mechanics regulate the morphology of hMSCs on the developed substrates, and all of the hydrogels studied upregulate IDO production over culture on TCP. Softer substrates further this upregulation to a plateau. Overall, this system offers a biomimetic strategy for decoupling hydrogel storage modulus from network connectivity, enabling systematic study of biomaterial properties on hMSC behavior and enhancement of cellular functionality for therapeutic applications. STATEMENT OF SIGNIFICANCE: Various strategies to tune hydrogel mechanics have been developed to control human mesenchymal stem cell (hMSC) behavior and regulate their immunomodulatory potential. However, these strategies typically couple mechanics to network connectivity, which in turn changes other hydrogel properties such as permeability that may have unintended effects on hMSC behavior. This work presents a strategy to tune hydrogel mechanics using crosslinkers with different secondary structure and molecular rigidity. This strategy successfully decouples hydrogel moduli from crosslinker stoichiometry and mimics the hierarchical nature of the native extracellular matrix. The moduli of the developed hydrogels led to significant impacts on hMSC morphology and proliferation, and increased immunomodulatory potential, indicating that molecular rigidity is a promising avenue to control engineered ECM mechanics for therapeutic applications.

Biodegradable microneedle patch for delivery of meloxicam for managing pain in cattle.

Microneedle patches are a promising source for transdermal diffusion of macromolecules and are designed to painlessly penetrate the skin. In this study, a biodegradable chitosan microneedle patch to deliver meloxicam for managing pain in cattle was tested. The potential of reuse of the polymeric solution to fabricate the patches, optimization of fabrication, morphological analysis of the microneedle patch and analysis of preservation of the chemical composition after sterilization were evaluated. In-vitro analysis consisted of studying in-vitro penetration mechanical properties, compression testing analysis of microneedle patch, and in-vitro drug release analysis. In-vivo studies were performed to analyze the dissolution capability of the microneedle patch. Results regarding the physical characteristics, chemical composition, and mechanical properties confirmed that rheological properties of the chitosan solution, present significant differences over time, demonstrating that reusing the solution on the fourth day results in failure patches. Morphological characteristics and chemical composition studies revealed that the process of sterilization (ethylene oxide gas) needed for implanting the patches into the skin did not affect the properties of microneedle patches. In-vitro studies showed that approximately 33.02 ± 3.88% of the meloxicam was released over 7 days. A full penetration of the microneedles into the skin can be obtained by applying approximately 3.2 N. In-vivo studies demonstrated that microneedle patches were capable of swelling and dissolving, exhibiting a dissolution percentage of more than 50% of the original height of microneedle after 7 days. No abnormal tissue, swelling, or inflammation was observed in the implanted area. The results of this work show that chitosan biodegradable microneedle patches may be useful to deliver meloxicam to improve pain management of cattle with positive effects for commercial manufacturing.

Immunomodulatory functions of human mesenchymal stromal cells are enhanced when cultured on HEP/COL multilayers supplemented with interferon-gamma.

Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for cell therapies due to their immunosuppressive capacity that can be enhanced in the presence of interferon-gamma (IFN-γ). In this study, multilayers of heparin (HEP) and collagen (COL) (HEP/COL) were used as a bioactive surface to enhance the immunomodulatory activity of hMSCs using soluble IFN-γ. Multilayers were formed, via layer-by-layer assembly, varying the final layer between COL and HEP and supplemented with IFN-γ in the culture medium. We evaluated the viability, adhesion, real-time growth, differentiation, and immunomodulatory activity of hMSCs on (HEP/COL) multilayers. HMSCs viability, adhesion, and growth were superior when cultured on (HEP/COL) multilayers compared to tissue culture plastic. We also confirmed that hMSCs osteogenic and adipogenic differentiation remained unaffected when cultured in (HEP/COL) multilayers in the presence of IFN-γ. We measured the immunomodulatory activity of hMSCs by measuring the level of indoleamine 2,3-dioxygenase (IDO) expression. IDO expression was higher on (HEP/COL) multilayers treated with IFN-γ. Lastly, we evaluated the suppression of peripheral blood mononuclear cell (PBMC) proliferation when co-cultured with hMSCs on (HEP/COL) multilayers with IFN-γ. hMSCs cultured in (HEP/COL) multilayers in the presence of soluble IFN-γ have a greater capacity to suppress PBMC proliferation. Altogether, (HEP/COL) multilayers with IFN-γ in culture medium provides a potent means of enhancing and sustaining immunomodulatory activity to control hMSCs immunomodulation.

Heparin/collagen surface coatings modulate the growth, secretome, and morphology of human mesenchymal stromal cell response to interferon-gamma.

The therapeutic potential of human mesenchymal stromal cells (h-MSC) is dependent on the viability and secretory capacity of cells both modulated by the culture environment. Our previous studies introduced heparin and collagen I (HEP/COL) alternating stacked layers as a potential substrate to enhance the secretion of immunosuppressive factors of h-MSCs. Herein, we examined the impact of HEP/COL multilayers on the growth, morphology, and secretome of bone marrow and adipose-derived h-MSCs. The physicochemical properties and stability of the HEP/COL coatings were confirmed at 0 and 30 days. Cell growth was examined using cell culture media supplemented with 2 and 10% serum for 5 days. Results showed that HEP/COL multilayers supported h-MSC growth in 2% serum at levels equivalent to 10% serum. COL and HEP as single component coatings had limited impact on cell growth. Senescent studies performed over three sequential passages showed that HEP/COL multilayers did not impair the replicative capacity of h-MSCs. Examination of 27 cytokines showed significant enhancements in eight factors, including intracellular indoleamine 2, 3-dioxygenase, on HEP/COL multilayers when stimulated with interferon-gamma (IFN-γ). Image-based analysis of cell micrographs showed that serum influences h-MSC morphology; however, HEP-ended multilayers generated distinct morphological changes in response to IFN-γ, suggesting an optical detectable assessment of h-MSCs immunosuppressive potency. This study supports HEP/COL multilayers as a culture substrate for undifferentiated h-MSCs cultured in reduced serum conditions.

Design, characterization, and modeling of a chitosan microneedle patch for transdermal delivery of meloxicam as a pain management strategy for use in cattle.

This work describes the formulation and evaluation of a chitosan microneedle patch for the transdermal delivery of meloxicam to manage pain in cattle. Microneedle patches composed of chitosan and chitosan/meloxicam were evaluated regarding their chemical composition, uniformity of physical characteristics, capacity to penetrate the skin, and response to thermal and thermo-mechanical changes. Microneedle patches were prepared by varying the percentage of acetic acid used during solution preparation, including 90% (v/v), 50% (v/v), and 10% (v/v). In addition, drug release was assessed by modeling different percentages of penetration into the skin and the number of microneedles on the microneedle patch. Scanning electron microscopy confirmed the presence of microneedles uniformly organized on the patch surface for each percentage of acetic acid used. Fourier transform infrared spectroscopy revealed that 10% (v/v) of acetic acid in the solution was a suitable condition to preserve the characteristic bands of chitosan (amide I and amide II) and meloxicam (amine NH stretch and CO stretch) as compared to 90% (v/v) and 50% (v/v) of acetic acid used during the solution preparation. The resultant microneedle patches were successful in penetrating the skin in a cow's cadaver ear. Results demonstrated that the average depth penetration measured after complete dehydration of the penetrated skin was approximately 78 ± 1 µm. Chitosan and chitosan/meloxicam microneedle patches with higher acetic acid percentages reflected greater resistance to compressive force as temperature increased. Time-dependent simulation of the transport of diluted species by COMSOL revealed that the transdermal drug delivery increases in function to the increment of the number of microneedles on the surface patch and percentage of penetration per microneedle. One patch released a drug concentration of 3.57 × 10-5 mol/m3 in the skin per week, which represents the 26.2% of what is needed for pain management in cattle, established as 1.43 × 10-4 mol/m3. These results demonstrate that chitosan/meloxicam microneedles patches may be suitable to manage pain in cattle after routine procedures.

Methods for the Assembly and Characterization of Polyelectrolyte Multilayers as Microenvironments to Modulate Human Mesenchymal Stromal Cell Response.

Thin films are of interest in materials design because they allow for the modification of surface properties of materials while the bulk properties of the material are largely unaffected. In this work, we outline methods for the assembly of thin films using a technique known as layer-by-layer (LbL). Furthermore, their interactions with human mesenchymal stromal cells (hMSCs) are discussed. hMSCs are a subject of growing interest because of their potential to treat or cure diseases, given their immunosuppressive properties, multipotent differentiation capabilities, and tissue regeneration capabilities. Numerous improvements and modifications have been suggested for the harvesting, treatment, and culture of hMSCs prior to their administration in human subjects. Here, we discuss methods to assess the interactions of hMSCs with thin LbL-assembled films of heparin and collagen. Three different methods are discussed. The first details the preparation of heparin/collagen multilayers on different surfaces and the seeding of cells on these multilayers. The second method details the characterization of multilayers, including techniques to assess the thickness, roughness, and surface charge of the multilayers, as well as in situ deposition of multilayers. The third method details the analysis of cell interactions with the multilayers, including techniques to assess proliferation, viability, real-time monitoring of hMSC behavior, analysis of hMSC-adhesive proteins on the multilayers, immunomodulatory factor expression of hMSCs, and cytokine expression on heparin/collagen multilayers. We propose that the methods described in this work will assist in the design and characterization of LbL-assembled thin films and the analysis of hMSCs cultured on these thin films.

Real-time monitoring of human Schwann cells on heparin-collagen coatings reveals enhanced adhesion and growth factor response.

In this work, we evaluate the enhancing effect of six bilayers of heparin/collagen (HEP/COL)6 layer-by-layer coatings on human Schwann cell (hSCs) adhesion and proliferation in the presence or absence of nerve growth factor (NGF). hSCs behavior and in vitro bioactivity were studied during six days of culture using end-point viability and proliferation assays as well as an impedance-based real-time monitoring system. An end-point viability assay revealed that hSCs cultured on the (HEP/COL)6 coatings increased their growth by more than 230% compared to controls. However, an EdU proliferation assay revealed that the proliferation rate of hSCs in all conditions were similar, with 45% of cells proliferating after 18 hours of incubation. Fluorescence microscopy revealed that hSCs spreading was similar between the tissue culture plastic control and the (HEP/COL)6. The presence of NGF in solution resulted in cells with a larger spread area. Real-time monitoring of hSCs seeded on (HEP/COL)6 with and without NGF reveals that initial cell adhesion is improved by the presence of the (HEP/COL)6 coatings, and it is further improved by the presence of NGF. Our results suggest that (HEP/COL)6 coatings enhance Schwann cell behavior and response to NGF. This simple modification could be applied to current nerve regeneration strategies to improve the repair of damaged nerve.

Effects of Physical, Chemical, and Biological Stimulus on h-MSC Expansion and Their Functional Characteristics.

Human adult mesenchymal stem or stromal cells (h-MSC) therapy has gained considerable attention due to the potential to treat or cure diseases given their immunosuppressive properties and tissue regeneration capabilities. Researchers have explored diverse strategies to promote high h-MSC production without losing functional characteristics or properties. Physical stimulus including stiffness, geometry, and topography, chemical stimulus, like varying the surface chemistry, and biochemical stimuli such as cytokines, hormones, small molecules, and herbal extracts have been studied but have yet to be translated to industrial manufacturing practice. In this review, we describe the role of those stimuli on h-MSC manufacturing, and how these stimuli positively promote h-MSC properties, impacting the cell manufacturing field for cell-based therapies. In addition, we discuss other process considerations such as bioreactor design, good manufacturing practice, and the importance of the cell donor and ethics factors for manufacturing potent h-MSC.

Heparin/Collagen Coatings Improve Human Mesenchymal Stromal Cell Response to Interferon Gamma.

Mesenchymal stromal cells (MSC) are a promising source for cell-based therapies as they secrete a myriad of reparative factors in response to inflammatory stimuli. In this study, multilayers of heparin and collagen (HEP/COL) were used as a bioactive surface coating to enhance human MSC (hMSC) response to soluble interferon-gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, varying the final layer between COL and HEP and supplemented with IFN-γ in the culture medium. hMSC adhesion, proliferation, and cytokine expression were assessed. Infrared variable angle spectroscopic ellipsometry confirmed film chemistry, thickness, and roughness. COL-ending films of 12 layers of HEP/COL had an average thickness of 129 ± 5.8 nm, and 13 layers (HEP-ending) were 178 ± 28.3 nm thick. Changes in temperature between 25-37 °C did not have significant effects on film chemistry, thickness, or roughness. An EdU incorporation assay revealed that IFN-γ had an antiproliferative effect in all conditions evaluated except when hMSCs were cultured on HEP-ending films supplemented with IFN-γ. Moreover, hMSCs cultured on HEP-ending films supplemented with IFN-γ had a higher cytokine expression as compared with cells cultured on tissue culture polystyrene, COL-ending films with and without IFN-γ, and HEP-ending films without IFN-γ as measured by Luminex assay. Finally, immunostaining revealed strong integrin binding and FAK phosphorylation for each condition. This study shows that HEP/COL films can modulate hMSC response to soluble factors, which may be exploited in cell manufacturing practices.

Simultaneous characterization of physical, chemical, and thermal properties of polymeric multilayers using infrared spectroscopic ellipsometry.

In this study, multilayered films of polyethylenimine/poly (sodium-p-styrene sulfonate) (PEI)/(PSS) and type I collagen/heparin sodium (COL)/(HEP) were fabricated using the layer-by-layer technique, and fully characterized using Infrared Variable Angle Spectroscopic Ellipsometry (IRVASE) to simultaneously analyze the chemistry, thickness, and roughness of the multilayers with respect to changes in pH of the washing solution, and changes in temperature. Film topography and Young's modulus were obtained by atomic force microscopy (AFM) and nanoindentation. Our results show that with IRVASE it is possible to analyze the thickness of the multilayers prepared using a washing solution of pH 5, obtaining values of 71.7 nm and 40.3 nm for three bilayers of PEI/PSS and COL/HEP, respectively. Film roughness varies between multilayer systems, obtaining values of 37.76 nm for three bilayers of PEI/PSS and 33.58 nm for three bilayers of COL/HEP. Increasing the pH of the washing solution for PEI/PSS yielded thinner films that were less susceptible to thermal induced changes in film chemistry in the range of 25 - 150 °C. PEI/PSS films decreased in thickness with increasing temperature up to 75 °C, whereas above 75 °C film thickness increased. Through IRVASE, a transition temperature for the PEI/PSS multilayers was observed at 75 °C. Temperatures above 37 °C drastically alter the chemistry and the thickness of the COL/HEP multilayers indicating a possible degradation of the polymers. We obtained, through nanoindentation, a Young's modulus of 15000 kPa and 9000 kPa for 12 bilayers of PEI/PSS and COL/HEP, respectively. These results demonstrate that, using IRVASE, we can simultaneously evaluate the physical, chemical, and thermal properties of synthetic and natural multilayered polymeric films.

Aptamer-Based Impedimetric Assay of Arsenite in Water: Interfacial Properties and Performance.

In this work, we explore the use of electrochemical methods (i.e., impedance) along with the arsenic-specific aptamer (ArsSApt) to fabricate and study the interfacial properties of an arsenic (As(III)) sensor. The ArsSApt layer was self-assembled on a gold substrate, and upon binding of As(III), a detectable change in the impedimetric signal was recorded because of conformational changes at the interfacial layer. These interfacial changes are linearly correlated with the concentration of arsenic present in the system. This target-induced signal was utilized for the selective detection of As(III) with a linear dynamic range of 0.05-10 ppm and minimum detectable concentrations of ca. 0.8 µM. The proposed system proved to be successful mainly because of the combination of a highly sensitive electrochemical platform and the recognized specificity of the ArsSApt toward its target molecule. Also, the interaction between the ArsSApt and the target molecule (i.e., arsenic) was explored in depth. The obtained results in this work are aimed at proving the development of a simple and environmentally benign sensor for the detection of As(III) as well as in elucidating the possible interactions between the ArsSApt and arsenic molecules.