Dynamic Measurement of Biological Processes

Biological processes such as tissue growth, inflammation, and angiogenesis involve a symphony of complex and dynamic interactions among multiple cell types, signaling molecules, and tissue microenvironments. Current normal tissue or tumor models typically consist of two dimensional cell culture systems or in vivo rodent animal models. The “gold standard” for measuring dynamic biological processes requires highly invasive techniques which involve sacrifice of the tissue at specific time points followed by sectioning of the tissue and histological/biochemical analysis. Therefore, we can only obtain a few isolated “static snapshots” of the complex nature of biological processes and disease progression at discrete time points and spatial positions. As a result there often exists a fundamental discrepancy between our understanding of biological processes and the actual events since key tissue response information is lost due to limited temporal and spatial information. The inability to monitor biological processes in real-time non-invasively has limited understanding of processes such as angiogenesis, wound healing, tissue regeneration, and progression of countless diseases. This, in turn, has thwarted development of effective treatments for restoring quality and length of life to millions of patients.

To address this challenge, we have developed a first-of-its-kind multifunctional system capable of simultaneous spatiotemporal molecular imaging of dynamic biologic processes and delivery of biological agents in minimally invasive and non-destructive manner within a tissue. This novel system is referred to as the “holey scaffold”. As illustrated in Fig. 1(a), a holey scaffold is built from a network of microchannels embedded in a tissue scaffold. Material for scaffolds can be selected to reflect tissue architecture and is typically composed of biodegradable synthetic (e.g. poly(glycolic acid)) or biological materials (e.g. collagen, laminin, matrigel) to promote tissue growth. Hollow microchannels can be used to perform controlled delivery of biological agents (e.g. nutrients, growth factors, cells, nanoparticles, drugs). Other microchannels can be utilized to introduce µm-scale optical fibers for non-destructive, in situ, and real-time molecular imaging of biological processes. Holey scaffolds can be viewed conceptually as a miniature microscope incorporated within a biological system, capable of fluorescence imaging.

This system will permit tissues of clinically relevant size to be grown in vitro by integrating the holey scaffold in a bioreactor. Within this framework the tissue microenvironment and signaling cues can be tightly controlled to isolate important responses without the presence of the cacophony of signals that can confound interpretation within an in vivo model. The holey scaffold can also be incorporated into in vivo animal models permitting dynamic measurement of biological phenomena within a living animal with minimal perturbation (Fig. 1b). We currently have funding to utilize the holey scaffold to improve bone and blood vessel regeneration and nanoparticle-mediated laser therapies as described below:

Project 1: Characterization of the Tumor Response to Nanoparticle-Mediated Laser Therapy

The goal of this project is to effectively characterize the spatiotemporal nanoparticle transport and dynamic photothermal/photochemical response to nanoparticle-mediated therapy for varying nanoparticle properties and laser parameters within both in vitro and in vivo tumor systems. This objective will be accomplished by creating and utilizing a novel sensing system known as the “holey scaffold” which is capable of minimally invasive and non-destructive measurement and control of dynamic biological and transport processes. The holey scaffold will measure dynamic nanoparticle mass transport and photothermal (temperature, cell viability, and HSP expression), and photochemical (reactive oxygen species production) response in real-time to a variety of nanoparticle types including carbon nanotubes and novel embodiments of carbon nanotubes and fullerenes, targeting approaches, and delivery methods (see Fig. 2). Measured tumor response will be utilized to create a multi-component treatment planning computational model for nanoparticle-mediated laser therapy. The model will consist of unique sub-models for nanoparticle transport, photothermal response, and photochemical effects based on corresponding experimentally measured data (see Fig. 1). Integration of sub-models into an overall treatment planning model will permit prediction of the tumor response and enable determination of ideal nanoparticle properties and laser parameters for maximum therapeutic effectiveness. The model will also provide additional insight into the spatiotemporal tumor response to nanoparticle-mediated laser therapies and extend our physical understanding of these processes for a wider range of experimental conditions than measured. Objectives with associated experimental measurements and computational models are shown in Fig. 1.

Funding
CBET 0955072 (M.N. Rylander) 5/10/10-5/9/15 National Science Foundation CAREER: Holey Scaffold Sensing System for Characterization of the Spatiotemporal Tumor Response to Nanoparticle-Mediated Photothermal and Photochemical Therapy

Project 2: Velocity and Wall Shear Stress Characterization in an Arterial Flow Bioreactor and Tumor Vascular System Graduate Student
Researcher: Elizabeth Voigt

Endothelial cells, the cells lining the inner surface of blood vessels, respond both mechanically and chemically to applied flow conditions. Quantification of this response will assist in the design and development of drug delivery systems. Our objective is to examine how cells grow and react to the presence of different flow environments by directly quantifying the flow conditions to which the cells are exposed. This will be done by directly measuring the flow parameters within an artificial vessel lined with living endothelial cells. We are designing and testing a system that supports the growth of human microvascular endothelial cells in an artificial artery under physiological pulsatile conditions. Particle Image Velocimetry (PIV) is being used to quantify the flow profile within the artery and assist with the design of drug delivery systems.

Initial results using our system indicate that assumptions made in previous work about the flow profiles within such artificial physiological flow systems are incorrect. Direct measurement of flow is necessary for quantitative conclusions about endothelial response to shear conditions to be made.

Funding
This work is supported in part by a Clare Boothe Luce Graduate Fellowship, AEThER, and the Nanotherapeutics and Bioheat Transfer Lab. We would like to thank Wake Forest University for providing a sample bioreactor for our redesign.

Project 3: Non-destructive real-time imaging techniques to asses cellular events and cell/biomaterials interactions
Graduate Student Researcher: Bryce Whited

The use of tissue engineered scaffolds in combination with progenitor cells has emerged as a promising strategy to restore or replace tissues damaged by disease or trauma. In addition to being biocompatible and exhibiting appropriate mechanical properties, scaffolds must be designed to sustain cell attachment, proliferation, and differentiation to ultimately produce the desired tissue once implanted in the patient. Conventional techniques used to assess successful scaffold design include cell viability stains, DNA assays, and histological sectioning/staining. While significant information can be gained from using these methodologies, they are destructive to the sample and only provide snapshots of scaffold and cell development at a limited number of time points. Consequently, key temporal and spatial information relating to tissue regeneration in the scaffold is lost utilizing these techniques. Thus, the ability to non-destructively monitor cell viability, proliferation, and differentiation in real-time is of great importance for scaffold design and tissue engineering.

To meet this challenge, we have developed a novel imaging-bioreactor system capable of monitoring the development of tissue engineered scaffolds at the cellular level non-destructively and in real time. Our system is based on the incorporation of miniature hollow-core silica fiber (HCFs) imaging microchannels into the scaffold. We have demonstrated the capability of cellular-level imaging of cells labeled with fluorescent proteins through relatively thick scaffolds (~1 mm) (Figure 5). After some modifications, our imaging system can be extended to monitor scaffold development after in-vivo implantation.

Funding:
Institute for Critical Technologies and Applied Sciences Grant 1R01HL098912-01 BRP (G. Wang) 3/01/2010-3/30/2014 NIH/NHLBI Optical Molecular Tomography for Regenerative Medicine

Relevant Papers
Whited BM, Whitney JR, Hofmann MC, Xu Y, Rylander MN. Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds. Biomaterials. 2011 Mar; 32(9):2294-304.