Alginate hydrogels have been investigated for a broad variety of medical applications. The ability to assemble hydrogels at neutral pH and mild temperatures makes alginate a popular choice for the encapsulation and delivery of cells and proteins. Alginate has been studied extensively for the delivery of islets as a treatment for type 1 diabetes. However, poor stability of the encapsulation systems after implantation remains a challenge. In this paper, alginate was modified with 2-aminoethyl methacrylate hydrochloride (AEMA) to introduce groups that can be photoactivated to generate covalent bonds. This enabled formation of dual crosslinked structure upon exposure to ultraviolet light following initial ionic crosslinking into bead structures. The degree of methacrylation was varied and in vitro stability, long term swelling, and cell viability examined. At low levels of the methacrylation, the beads could be formed by first ionic crosslinks followed by exposure to ultraviolet light to generate covalent bonds. The methacrylated alginate resulted in more stable beads and cells were viable following encapsulation. Alginate microbeads, ionic (unmodified) and dual crosslinked, were implanted into a rat omentum pouch model. Implantation was performed with a local injection of 100 μl of 50 μg/ml of Lipopolysaccharide (LPS) to stimulate a robust inflammatory challenge in vivo. Implants were retrieved at 1 and 3 weeks for analysis. The unmodified alginate microbeads had all failed by week 1, whereas the dual-crosslinked alginate microbeads remained stable up through 3 weeks. The modified alginate microbeads may provide a more stable alternative to current alginate-based systems for cell encapsulation.

Islet transplantation is currently in clinical use as a treatment for type I diabetes, but donor shortages and long-term immunosuppression limit broad application. Alginate microcapsules coated with poly-L-ornithine can be used to encapsulate islets in an environment that allows diffusion of glucose, insulin, nutrients, and waste products while inhibiting cells and antibodies. While clinical trials are ongoing using islets encapsulated in alginate microbeads, there are concerns in regards to long-term stability. Evaluation of the local tissue response following implantation provides insight into the underlying mechanisms contributing to biomaterial failure, which can be used to the design of new material strategies. Macrophages play an important role in driving the response. In this study, the stability of alginate microbeads coated with PLO containing islets transplanted in the omentum pouch model was investigated. Biomaterial structure and the inflammatory response were characterized by X-ray phase contrast (XPC) μCT imaging, histology, and immunostaining. XPC allowed evaluation of microbead 3D structure and identification of failed and stable microbeads. A robust inflammatory response characterized by high cell density and the presence of pro-inflammatory macrophages was found around the failed grafts. The results obtained provide insight into the local tissue response and possible failure mechanisms for alginate microbeads.

Transplantation of functional islets encapsulated in stable biomaterials has the potential to cure Type I diabetes. However, the success of these materials requires the ability to quantitatively evaluate their stability. Imaging techniques that enable monitoring of biomaterial performance are critical to further development in the field. Xray phase-contrast (XPC) imaging is an emerging class of X-ray techniques that have shown significant promise for imaging biomaterial and soft tissue structures. In this study, XPC imaging techniques are shown to enable three dimensional (3D) imaging and evaluation of islet volume, alginate hydrogel structure, and local soft tissue features ex vivo. Rat islets were encapsulated in sterile ultrapurified alginate systems produced using a highthroughput microfluidic system. The encapsulated islets were implanted in omentum pouches created in a rodent model of type 1 diabetes. Microbeads were imaged with XPC imaging before implantation and as whole tissue samples after explantation from the animals. XPC microcomputed tomography (mCT) was performed with systems using tube-based and synchrotron X-ray sources. Islets could be identified within alginate beads and the islet volume was quantified in the synchrotron-based mCT volumes. Omental adipose tissue could be distinguished from inflammatory regions resulting from implanted beads in harvested samples with both XPC imaging techniques. Individual beads and the local encapsulation response were observed and quantified using quantitative measurements, which showed good agreement with histology. The 3D structure of the microbeads could be characterized with XPC imaging and failed beads could also be identified. These results point to the substantial potential of XPC imaging as a tool for imaging biomaterials in small animal models and deliver a critical step toward in vivo imaging.