X-ray CT in Phase Contrast Enhancement Geometry of Alginate Microbeads in a Whole-Animal Model.

Imaging soft biomaterials in vivo is a significant challenge, as most conventional techniques are limited by biomaterial contrast, penetration depth, or spatial resolution. Exogeneous contrast agents used to increase contrast may also alter material properties or exhibit local toxicity. The capability to observe biomaterial constructs in vivo without introducing exogenous contrast would improve preclinical testing and evaluation. Conventional X-ray Computed Tomography allows fast, high-resolution imaging at high penetration depth, but biomaterial contrast is low. Previous studies employing X-ray phase contrast (XPC) and utilizing a synchrotron source provided support for the significant potential of XPC in imaging biomaterials without contrast agents. In this study, XPC tomography was used to image alginate hydrogel microspheres within a small animal omental pouch model using a commercially available X-ray source. Multilayer microbeads could be identified in the XPC images with volumetric and structural information not possible in histological analysis. The number of microbeads present and microbead volume and diameter could be quantified from the images. The results of this study show that XPC tomography can be a useful tool for monitoring of implanted soft biomaterials in small animal models.

Imaging of Hydrogel Microsphere Structure and Foreign Body Response Based on Endogenous X-Ray Phase Contrast.

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. X-ray 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 high-throughput 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 (µCT) 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 µCT 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.

Biomaterials with persistent growth factor gradients in vivo accelerate vascularized tissue formation.

Gradients of soluble factors play an important role in many biological processes, including blood vessel assembly. Gradients can be studied in detail in vitro, but methods that enable the study of spatially distributed soluble factors and multi-cellular processes in vivo are limited. Here, we report on a method for the generation of persistent in vivo gradients of growth factors in a three-dimensional (3D) biomaterial system. Fibrin loaded porous poly (ethylene glycol) (PEG) scaffolds were generated using a particulate leaching method. Platelet derived growth factor BB (PDGF-BB) was encapsulated into poly (lactic-co-glycolic acid) (PLGA) microspheres which were placed distal to the tissue-material interface. PLGA provides sustained release of PDGF-BB and its diffusion through the porous structure results in gradient formation. Gradients within the scaffold were confirmed in vivo using near-infrared fluorescence imaging and gradients were present for more than 3 weeks. The diffusion of PDGF-BB was modeled and verified with in vivo imaging findings. The depth of tissue invasion and density of blood vessels formed in response to the biomaterial increased with magnitude of the gradient. This biomaterial system allows for generation of sustained growth factor gradients for the study of tissue response to gradients in vivo.

Agent-based modeling of porous scaffold degradation and vascularization: Optimal scaffold design based on architecture and degradation dynamics.

A multi-layer agent-based model (ABM) of biomaterial scaffold vascularization is extended to consider the effects of scaffold degradation kinetics on blood vessel formation. A degradation model describing the bulk disintegration of porous hydrogels is incorporated into the ABM. The combined degradation-angiogenesis model is used to investigate growing blood vessel networks in the presence of a degradable scaffold structure. Simulation results indicate that higher porosity, larger mean pore size, and rapid degradation allow faster vascularization when not considering the structural support of the scaffold. However, premature loss of structural support results in failure for the material. A strategy using multi-layer scaffold with different degradation rates in each layer was investigated as a way to address this issue. Vascularization was improved with the multi-layered scaffold model compared to the single-layer model. The ABM developed provides insight into the characteristics that influence the selection of optimal geometric parameters and degradation behavior of scaffolds, and enables easy refinement of the model as new knowledge about the underlying biological phenomena becomes available. STATEMENT OF SIGNIFICANCE: This paper proposes a multi-layer agent-based model (ABM) of biomaterial scaffold vascularization integrated with a structural-kinetic model describing bulk degradation of porous hydrogels to consider the effects of scaffold degradation kinetics on blood vessel formation. This enables the assessment of scaffold characteristics and in particular the disintegration characteristics of the scaffold on angiogenesis. Simulation results indicate that higher porosity, larger mean pore size, and rapid degradation allow faster vascularization when not considering the structural support of the scaffold. However, premature loss of structural support by scaffold disintegration results in failure of the material and disruption of angiogenesis. A strategy using multi-layer scaffold with different degradation rates in each layer was investigated as away to address this issue. Vascularization was improved with the multi-layered scaffold model compared to the single-layer model. The ABM developed provides insight into the characteristics that influence the selection of optimal geometric and degradation characteristics of tissue engineering scaffolds.

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.

Evaluating 3D-printed biomaterials as scaffolds for vascularized bone tissue engineering.

There is an unmet need for a consistent set of tools for the evaluation of 3D-printed constructs. A toolbox developed to design, characterize, and evaluate 3D-printed poly(propylene fumarate) scaffolds is proposed for vascularized engineered tissues. This toolbox combines modular design and non-destructive fabricated design evaluation, evaluates biocompatibility and mechanical properties, and models angiogenesis.

Agent-based modeling of osteogenic differentiation of mesenchymal stem cells in porous biomaterials.

Mesenchymal stem cells (MSC) have shown promise in tissue engineering applications due to their potential for differentiating into mesenchymal tissues such as osteocytes, chondrocytes, and adipocytes and releasing proteins to promote tissue regeneration. One application involves seeding MSCs in biomaterial scaffolds to promote osteogenesis in the repair of bone defects following implantation. However, predicting in vivo survival and differentiation of MSCs in biomaterials is challenging. Rapid and stable vascularization of scaffolds is required to supply nutrients and oxygen that MSCs need to survive as well as to go through osteogenic differentiation. The objective of this study is to develop an agent-based model and simulator that can be used to investigate the effects of using gradient growth factors on survival and differentiation of MSCs seeded in scaffolds. An agent-based model is developed to simulate the MSC behavior. The effect of vascular endothelial growth factor (VEGF) and bone morphogenic protein-2 (BMP-2) on both survival and osteogenic differentiation is studied. Results showed that the survival ratio of MSCs can be enhanced by increasing VEGF concentration. BMP-2 caused a slight increase on survival ratio. Osteogenesis strongly depends on the VEGF concentration as well because of its effect on vascularization. BMP-2 increased the osteogenic differentiation of MSCs.

Imaging of poly(α-hydroxy-ester) scaffolds with X-ray phase-contrast microcomputed tomography.

Porous scaffolds based on poly(α-hydroxy-esters) are under investigation in many tissue engineering applications. A biological response to these materials is driven, in part, by their three-dimensional (3D) structure. The ability to evaluate quantitatively the material structure in tissue-engineering applications is important for the continued development of these polymer-based approaches. X-ray imaging techniques based on phase contrast (PC) have shown a tremendous promise for a number of biomedical applications owing to their ability to provide a contrast based on alternative X-ray properties (refraction and scatter) in addition to X-ray absorption. In this research, poly(α-hydroxy-ester) scaffolds were synthesized and imaged by X-ray PC microcomputed tomography. The 3D images depicting the X-ray attenuation and phase-shifting properties were reconstructed from the measurement data. The scaffold structure could be imaged by X-ray PC in both cell culture conditions and within the tissue. The 3D images allowed for quantification of scaffold properties and automatic segmentation of scaffolds from the surrounding hard and soft tissues. These results provide evidence of the significant potential of techniques based on X-ray PC for imaging polymer scaffolds.