In this study, methacrylamide chitosan modified with perfluorocarbon chains (MACF) is used as the base material to construct hydrogel dressings for treating dermal wounds. MACF hydrogels saturated with oxygen (+ O2) are examined for their ability to deliver and sustain oxygen, degrade in a biological environment, and promote wound healing in an animal model. The emerging technique of metabolomics is used to understand how MACF + O2 hydrogel dressings improve wound healing. Results indicate that MACF treatment facilitates oxygen transport rate that is two orders of magnitude greater than base MAC hydrogels. MACF hydrogel dressings are next tested in an in vivo splinted rat excisional wound healing model. Histological analysis reveals that MACF + O2 dressings improve re-epithelialization (p < 0.0001) and synthesis of collagen over controls (p < 0.01). Analysis of endogenous metabolites in the wounds using global metabolomics demonstrates that MACF + O2 dressings promotes a regenerative metabolic process directed toward hydroxyproline and collagen synthesis, with confirmation of metabolite levels within this pathway. The results of this study confirm that increased oxygen delivery through the application of MACF + O2 hydrogels enhances wound healing and metabolomics analyses provides a powerful tool to assess wound healing physiology.

Oxygen is a vital source of energy necessary to sustain and complete embryonic development. Not only is oxygen the driving force for many cellular functions and metabolism, but it is also involved in regulating stem cell fate, morphogenesis, and organogenesis. Low oxygen levels are the naturally preferred microenvironment for most processes during early development and mainly drive proliferation. Later on, more oxygen and also nutrients are needed for organogenesis and morphogenesis. Therefore, it is critical to maintain oxygen levels within a narrow range as required during development. Modulating oxygen tensions is performed via oxygen homeostasis mainly through the function of hypoxia-inducible factors. Through the function of these factors, oxygen levels are sensed and regulated in different tissues, starting from their embryonic state to adult development. To be able to mimic this process in a tissue engineering setting, it is important to understand the role and levels of oxygen in each developmental stage, from embryonic stem cell differentiation to organogenesis and morphogenesis. Taking lessons from native tissue microenvironments, researchers have explored approaches to control oxygen tensions such as hemoglobin-based, perfluorocarbon-based, and oxygen-generating biomaterials, within synthetic tissue engineering scaffolds and organoids, with the aim of overcoming insufficient or nonuniform oxygen levels and nutrient supply.

Long-term (multiple-week or -month) release of small, water-soluble molecules from hydrogels remains a significant pharmaceutical challenge, which is typically overcome at the expense of more-complicated drug carrier designs. Such approaches are payload-specific and include covalent conjugation of drugs to base materials or incorporation of micro- and nanoparticles. As a simpler alternative, here we report a mild and simple method for achieving multiple-month release of small molecules from gel-like polymer networks. Densely cross-linked matrices were prepared through ionotropic gelation of poly(allylamine hydrochloride) (PAH) with either pyrophosphate (PPi) or tripolyphosphate (TPP), all of which are commonly available commercial molecules. The loading of model small molecules (Fast Green FCF and Rhodamine B dyes) within these polymer networks increases with the payload/network binding strength and with the PAH and payload concentrations used during encapsulation. Once loaded into the PAH/PPi and PAH/TPP ionic networks, only a few percent of the payload is released over multiple months. This extended release is achieved regardless of the payload/network binding strength and likely reflects the small hydrodynamic mesh size within the gel-like matrices. Furthermore, the PAH/TPP networks show promising in vitro cytocompatibility with model cells (human dermal fibroblasts), though slight cytotoxic effects were exhibited by the PAH/PPi networks. Taken together, the above findings suggest that PAH/PPi and (especially) PAH/TPP networks might be attractive materials for the multiple-month delivery of drugs and other active molecules (e.g., fragrances or disinfectants).

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Wound healing involves multiple interrelated processes required to lead to successful healing outcomes. Phagocytosis, inflammation, cell proliferation, angiogenesis, energy production, and collagen synthesis are all directly or indirectly dependent on oxygen. Along with other critical factors, such as nutrition and comorbidities, availability of oxygen is a key determinant of healing success. Previously, we have presented a novel oxygenated hydrogel material that can be made into dressings for continuous localized oxygen delivery to wounds. In this study, an acute porcine wound model was used to test the healing benefits of these oxygenated MACF (MACF + O2) hydrogel dressings compared to controls, which included commercial Derma-GelTM hydrogel dressings. Wound closure and histological analyses were performed to assess re-epithelialization, collagen synthesis, angiogenesis, and keratinocyte maturation. Results from these assays revealed that wounds treated with MACF + O2 hydrogel dressings closed faster as compared to Derma-Gel (p<0.05). Targeted metabolomics via liquid chromatography separation and mass spectrometric detection (LC-MS/MS) and a biochemical assay determined the concentration of hydroxyproline in wound samples at days 14 and 21, showing that MACF + O2 hydrogel dressings improved wound healing via an upregulated collagen synthesis pathway as compared to Derma-Gel (p<0.05). Histological evidence showed that MACF + O2 hydrogel dressings improve new blood vessel formation and keratinocyte maturation over all other treatments.

Thread size and polymer composition are critical properties to consider for achieving a positive healing outcome with a wound dressing. Three-dimensional (3D) printed scaffolds and electrospun mats both offer distinct advantages as replaceable wound dressings. This research aims to determine if the thread size and polymer compositions of the scaffolds affect skin wound healing outcomes, an aspect that has not been adequately explored. Using a modular polymer platform, four polyester direct-write 3D printed scaffolds and electrospun mats were fabricated into wound dressings. The dressings were applied to splinted, full thickness skin wounds in an excisional wound rat model and evaluated against control wounds to which no dressing was applied. Wound closure rates and reduction of the wound bed width were not affected by the thread size or polymer composition. However, epidermal thickness was larger in wounds treated with electrospun dressings and was slightly affected by the polymer composition. Two of the four tested polymer compositions lead to delayed reorganization of granulation tissues. Moreover, enhanced angiogenesis was seen in wounds treated with 3D printed dressings compared to those treated with electrospun dressings. The results from this study can be used to inform the choice of dressing architecture and polymer compositions to achieve positive wound healing outcomes.

Antioxidants play an important role in regulating overabundant reactive oxygen species (ROS) in wound healing to reduce oxidative stress and inflammation. In this work we demonstrate for the first time that functionalization of methacrylamide chitosan (MAC) with aliphatic pentadecafluoro chains, to synthesize pentadecafluoro-octanoyl methacrylamide chitosan (MACF), enhances the antioxidant capacity of the MAC base hydrogel material, while being able to deliver oxygen for enhanced wound healing. As such MACF was shown to sequester more nitric oxide (p < 0.01) and hydroxyl (p < 0.0001) radicals as compared to MAC and controls even when delivering additional oxygen. MACF’s beneficial antioxidant capacity was further confirmed in in vitro cell culture experiments using human dermal fibroblasts stressed with 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH).

candidates, and in the past preclinical animal models have been employed that do not necessarily provide results that translate into humans. [2] It has been shown that beginning drug discovery in human cell-based platforms can decrease failure rates. [3] This is only one illustrative example highlighting the usefulness of humanized cell-based platforms in the study of human health and disease. Emerging technologies such as microphysical systems, organs-on-a-chip, 3D bioprinting, and cell spheroid/organoids offer tantalizing utility for medicine. [4] Human cell-based organoids specifically present more realistic and physiologically relevant models, [5] and provide significant utility for personalized disease modelling, drug screening, and even regenerative medicine research. [3] Organoids are miniaturized and simplified versions of organs typically derived from stem cells. They possess threedimensional microanatomy with several organ-specific differentiated and progenitor cell types. Multi-cellular spheroids present a simplified version of organoids using one organ-specific cell-type to reduce the overall complexity and are often the preferred starting point for organoid research. [6] A persistent and significant challenge for organoids and spheroids alike is maintaining sufficient oxygen perfusion to create large anatomically relevant functional tissues in vitro. [7] Past attempts to artificially improve oxygen transport includes artificial microvessels, direct fluid perfusion, and oxygen vesicles for oxygen transport. [8] Each of these techniques faces their own challenges, and despite the efforts invested in developing them, insufficient oxygenation challenges persist ultimately limiting the size of organoids/ spheroids that can be produced, the ability to maintain them long-term, and consequently their physiologic relevance to medical applications. In this study we report a novel strategy to enhance oxygen transport in 3D in vitro human cell cultures using chitosan-PFC oxygenating microgels added to cell aggregate spheroids, enabling the ability to tune local spheroid oxygenation to enhance spheroid utility. One important advantage of this approach is that it combines several mechanisms to boost oxygen transport from the surrounding environment. First, Poor oxygen transport is a major obstacle currently for 3D microtissue culture platforms, which at this time cannot be grown large enough to be truly physio logically relevant and replicate adult human organ functions. To overcome internal oxygen transport deficiencies, oxygenating microgels are formed utilizing perfluorocarbon (PFC) modified chitosan and a highly scalable water-in-oil miniemulsion method. Microgels that are on the order of a cell diameter (≈10 µm) are formed allowing them to directly associate with cells when included in 3D spheroid culture, while not being internalized. The presence of immobilized PFCs in these microgels allows for enhancement and tuning of oxygen transport when incorporated into cultured microtissues. As such, it is demonstra...