Alyssa A. Appel

Imaging challenges in biomaterials and tissue engineering

Biomaterials are employed in the fields of tissue engineering and regenerative medicine (TERM) in order to enhance the regeneration or replacement of tissue function and/or structure. The unique environments resulting from the presence of biomaterials, cells, and tissues result in distinct challenges in regards to monitoring and assessing the results of these interventions. Imaging technologies for three-dimensional (3D) analysis have been identified as a strategic priority in TERM research. Traditionally, histological and immunohistochemical techniques have been used to evaluate engineered tissues. However, these methods do not allow for an accurate volume assessment, are invasive, and do not provide information on functional status. Imaging techniques are needed that enable non-destructive, longitudinal, quantitative, and three-dimensional analysis of TERM strategies. This review focuses on evaluating the application of available imaging modalities for assessment of biomaterials and tissue in TERM applications. Included is a discussion of limitations of these techniques and identification of areas for further development.

Design of a composite biomaterial system for tissue engineering applications

Biomaterials that regulate vascularized tissue formation have the potential to contribute to new methods of tissue replacement and reconstruction. The goal of this study was to develop a porous, degradable tissue engineering scaffold that could deliver multiple growth factors and regulate vessel assembly within the porous structure of the material. Porous hydrogels of poly(ethylene glycol)-co-(L-lactic acid) (PEG-PLLA) were prepared via salt leaching. The degradation time of the hydrogels could be controlled between 1 and 7 weeks, based on hydrogel composition. Fibrin was incorporated into the interconnected pores of the hydrogels to promote neovascularization and as a reservoir for rapid (<5 days) growth factor delivery. Poly(lactic-co-glycolic acid) (PLGA) microspheres were incorporated into the degradable polymeric hydrogel scaffold to allow sustained (>30 days) growth factor delivery. Fibroblast growth factor-1 (FGF-1) and platelet-derived growth factor-BB (PDGF-BB) were delivered from the system owing to their roles in the promotion of angiogenesis and vascular stabilization, respectively. Hydrogels tested in vivo with a subcutaneous implantation model were selected based on the results from in vitro degradation and growth factor release kinetics. Dual growth factor delivery promoted significantly more tissue ingrowth in the scaffold compared with blank or single growth factor delivery. The sequential delivery of FGF-1 following PDGF-BB promoted more persistent and mature blood vessels. In conclusion, a biomaterials system was developed to provide structural support for tissue regeneration, as well as delivery of growth factors that stimulate neovascularization within the structure prior to complete degradation.

Fibrin-Loaded Porous Poly(Ethylene Glycol) Hydrogels as Scaffold Materials for Vascularized Tissue Formation

Vascular network formation within biomaterial scaffolds is essential for the generation of properly functioning engineered tissues. In this study, a method is described for generating composite hydrogels in which porous poly(ethylene glycol) (PEG) hydrogels serve as scaffolds for mechanical and structural support, and fibrin is loaded within the pores to induce vascularized tissue formation. Porous PEG hydrogels were generated by a salt leaching technique with 100-150-μm pore size and thrombin (Tb) preloaded within the scaffold. Fibrinogen (Fg) was loaded into pores with varying concentrations and polymerized into fibrin due to the presence of Tb, with loading efficiencies ranging from 79.9% to 82.4%. Fibrin was distributed throughout the entire porous hydrogels, lasted for greater than 20 days, and increased hydrogel mechanical stiffness. A rodent subcutaneous implant model was used to evaluate the influence of fibrin loading on in vivo response. At weeks 1, 2, and 3, all hydrogels had significant tissue invasion, but no difference in the depth of invasion was found with the Fg concentration. Hydrogels with fibrin loading induced more vascularization, with a significantly higher vascular density at 20 mg/mL (week 1) and 40 mg/mL (weeks 2 and 3) Fg concentration compared to hydrogels without fibrin. In conclusion, we have developed a composite hydrogel that supports rapid vascularized tissue ingrowth, and thus holds great potential for tissue engineering applications.

X-ray phase contrast imaging of calcified tissue and biomaterial structure in bioreactor engineered tissues

Tissues engineered in bioreactor systems have been used clinically to replace damaged tissues and organs. In addition, these systems are under continued development for many tissue engineering applications. The ability to quantitatively assess material structure and tissue formation is critical for evaluating bioreactor efficacy and for preimplantation assessment of tissue quality. Techniques that allow for the nondestructive and longitudinal monitoring of large engineered tissues within the bioreactor systems will be essential for the translation of these strategies to viable clinical therapies. X‐ray Phase Contrast (XPC) imaging techniques have shown tremendous promise for a number of biomedical applications owing to their ability to provide image contrast based on multiple X‐ray properties, including absorption, refraction, and scatter. In this research, mesenchymal stem cell‐seeded alginate hydrogels were prepared and cultured under osteogenic conditions in a perfusion bioreactor. The constructs were imaged at various time points using XPC microcomputed tomography (µCT). Imaging was performed with systems using both synchrotron‐ and tube‐based X‐ray sources. XPC µCT allowed for simultaneous three‐dimensional (3D) quantification of hydrogel size and mineralization, as well as spatial information on hydrogel structure and mineralization. Samples were processed for histological evaluation and XPC showed similar features to histology and quantitative analysis consistent with the histomorphometry. These results provide evidence of the significant potential of techniques based on XPC for noninvasive 3D imaging engineered tissues grown in bioreactors. Biotechnol. Bioeng. 2015;112: 612–620.

X-ray imaging of poly(ethylene glycol) hydrogels without contrast agents.

Hydrogels have shown promise for a number of tissue engineering applications. However, their high water content results in little or no image contrast when using conventional X-ray imaging techniques. X-ray imaging techniques based on phase-contrast have shown promise for biomedical application due to their ability to provide information about the X-ray refraction properties of samples. Nonporous and porous poly(ethylene glycol) hydrogels were synthesized and imaged using a synchrotron light source employing a silicon analyzer crystal and an X-ray energy of 40-keV. Data were acquired at 21 angular analyzer positions spanning the range of -5 to 5 μrad. Images that depict the projected X-ray absorption, refraction, and ultra-small-angle scatter (USAXS) properties of the hydrogels were reconstructed from the measurement data. The poly(ethylene glycol) hydrogels could be discerned from surrounding water and soft tissue in the refraction image but not the absorption or USAXS images. In addition, the refraction images of the porous hydrogels have a speckle pattern resulting in increased image texture in comparison to nonporous hydrogels. To our knowledge, this is the first study to show that X-ray phase-contrast imaging techniques can identify and provide detail on hydrogel structure without the addition of contrast agents.

Long-Term Function of Alginate-Encapsulated Islets.

Human trials have demonstrated the feasibility of alginate-encapsulated islet cells for the treatment of type 1 diabetes. Encapsulated islets can be protected from the hosts immune system and remain viable and functional following transplantation. However, the long-term success of these therapies requires that alginate microcapsules maintain their immunoprotective capacity and stability in vivo for sustained periods. In part, as a consequence of different encapsulation strategies, islet encapsulation studies have produced inconsistent results in regard to graft functioning time, stability, and overall metabolic benefits. Alginate composition (proportion of M- and G-blocks), alginate purity, the cross-linking ions (calcium or barium), and the presence or absence of additional polymer coating layers influence the success of cell encapsulation. This review summarizes the outcomes of long-term studies of alginate-encapsulated islet transplants in animals and humans and provides a critical discussion of the graft failure mechanisms, including issues with graft biocompatibility, transplantation site, and integrity of the encapsulated islet grafts. Strategies to improve the mechanical stability of alginate capsules and methods for monitoring graft survival and function in vivo are presented.

Pore Interconnectivity Influences Growth Factor-Mediated Vascularization in Sphere-Templated Hydrogels.

Rapid and controlled vascularization within biomaterials is essential for many applications in regenerative medicine. The extent of vascularization is influenced by a number of factors, including scaffold architecture. While properties such as pore size and total porosity have been studied extensively, the importance of controlling the interconnectivity of pores has received less attention. A sintering method was used to generate hydrogel scaffolds with controlled pore interconnectivity. Poly(methyl methacrylate) microspheres were used as a sacrificial agent to generate porous poly(ethylene glycol) diacrylate hydrogels with interconnectivity varying based on microsphere sintering conditions. Interconnectivity levels increased with sintering time and temperature with resultant hydrogel structure showing agreement with template structure. Porous hydrogels with a narrow pore size distribution (130-150 μm) and varying interconnectivity were investigated for their ability to influence vascularization in response to gradients of platelet-derived growth factor-BB (PDGF-BB). A rodent subcutaneous model was used to evaluate vascularized tissue formation in the hydrogels in vivo. Vascularized tissue invasion varied with interconnectivity. At week 3, higher interconnectivity hydrogels had completely vascularized with twice as much invasion. Interconnectivity also influenced PDGF-BB transport within the scaffolds. An agent-based model was used to explore the relative roles of steric and transport effects on the observed results. In conclusion, a technique for the preparation of hydrogels with controlled pore interconnectivity has been developed and evaluated. This method has been used to show that pore interconnectivity can independently influence vascularization of biomaterials.

Effects of inhibition of neuronal nitric oxide synthase on basal retinal blood flow regulation.

Nitric oxide (NO) has been observed to regulate blood flow under basal and stimulated conditions in the retina. Recent evidence suggests that NO produced by neuronal nitric oxide synthase (nNOS) may regulate blood flow in addition to that produced by endothelial nitric oxide synthase (eNOS). The objective of the current study was to investigate the contribution of NO produced by nNOS in the regulation of basal retinal blood flow. A non-specific NOS inhibitor N (G)-nitro-l-arginine methyl ester (l-NAME) and the specific nNOS inhibitors 1-(2-trifluoromethylphenyl) imidazole (TRIM) and (4S)-N-(4-amino-5 [aminoethyl] aminopentyl)-N-nitroguanidine (AAAN) were injected into the vitreous (intravitreal) of Long-Evans rats. Vessel diameters, velocities and volumetric blood flow rates (VBF) in the retinal circulation were determined prior to and in 30-min intervals for 4-4.5h after injection. In addition, the basal amount of nNOS in the rat retina was quantified using a specific enzyme linked immunoassay (ELISA). Treatment with l-NAME and TRIM significantly decreased diameters and VBF. Compared with saline, treatment with l-NAME and TRIM produced a significant (p<0.001) decrease of approximately 12-17% in vessel diameters. Treatment with AAAN significantly decreased vessel diameters and venous VBF. Compared with saline AAAN produced a significant decrease of approximately 7% in arterial (p<0.001) and 5% in venous (p=0.011) diameters, respectively. The amount of nNOS in the rat retina was 0.17+/- 0.0147 pmol mg(-1) of dry retina. The results suggest that though inhibition of nNOS decreases basal diameters, constant VBF is maintained in the retinal circulation.

Evaluation of the Tissue Response to Alginate Encapsulated Islets in an Omentum Pouch Model

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.

This paper is a winner in the Undergraduate category for the SFB awards: Evaluation of the tissue response to alginate encapsulated islets in an omentum pouch model.

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.