Design optimization of the biomaterials that are used as scaffolds onto which new or regenerated tissues are expected to grow is one of the major challenges facing tissue engineers. Numerous aspects of the biomaterials design, from the molecular structure and organization to the overall macro-architecture of these systems are known to impact significantly the development of new tissue in vitro
and in vivo
]. Scaffolds provide structural support and important environmental cues to cells that populate them, thus controlling to a large extent cellular proliferation[1
], deposition of new structural proteins[9
] and ultimately the regeneration of functional tissue[4
]. Traditionally, methods such as NMR, FTIR and X-Ray spectroscopy have been used to assess biomaterials at the molecular level [13
], while SEM and TEM have been invaluable tools for characterizing the three dimensional morphology of biomaterial scaffolds [5
]. Histology and immunostaining as well as assays for determining the expression levels of specific proteins are often used to assess how scaffolds interact with cells as tissues develop[5
]. While all of these approaches provide sensitive and specific data, they are invasive. As a result, they reveal information about a single time-point along the development of a dynamically changing specimen. This limitation hinders full characterization and understanding of the relationships that exist between the structural, mechanical, architectural and biochemical properties of the scaffold and the corresponding properties of the developing tissue.
A number of optical methods have been developed to monitor non-invasively different tissue components in the context of disease diagnosis and monitoring[25
]. Such methods have only recently started to be exploited as tools for assessing different properties of the cell and matrix components of engineered tissues[30
]. The goal of this study was to determine the type of morphological and structural information that could be acquired about silk-based biomaterial scaffolds using spectral two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging. This information will be essential in developing an optical biomarker toolkit that will allow us to monitor dynamically how such scaffolds interact with and are modified by cells as engineered tissues grow either in vitro
or in vivo
. Furthermore, these optical toolkits can also be extended to other biomaterial matrices as the systems are developed and optimized.
Silk is a natural protein polymer valued for its biocompatibility, light weight, and strength [34
] Processing methods for this polymer are well established for control of morphology, mechanical properties and environmental stability [35
]. Due to these properties, silk is an excellent candidate for generating biomaterial scaffolds for engineered tissues. The Bombyx mori
silkworm silk protein, fibroin, can be described by two structural models: Silk I, consisting of type II β turn, random coil domains, and mixed structures including alpha helices, and Silk II, consisting mostly of antiparallel β pleated sheets. [14
]. The β sheet content and the alignment of these β sheet crystals, along with the non-crystalline domains of the protein, are important determinants of the bulk mechanical properties and degradation kinetics of biomaterials generated from silk[14
]. Most of theβ sheet content and orientation of these crystalline domains is lost during the processing of silk fibroin into aqueous solutions, a step required for the regeneration of new biomaterial scaffolds for tissue culture[42
]. The β sheet content and orientation can be reconstituted to different extents depending on the mode of material preparation[14
]. Thus, the non-invasive, optical assessment of β sheet content and orientation of silk fibroin during biomaterial scaffold formation was one of the specific goals of this study.
Linear optical approaches, such as fluorescence and Raman spectroscopy have been used previously to characterize materials made from silk fibroin[39
]. However, there are no studies to our knowledge on the nonlinear optical properties of silk. Nonlinear optical methods such as TPEF and SHG offer additional advantages for non-invasive imaging including excitation in the near infrared region of the spectrum, where scattering is typically lower than in the visible region, and reduced photobleaching[45
]. In TPEF and SHG two photons, typically of the same energy, interact simultaneously with a molecule and yield either fluorescence emission (TPEF) or scattering (SHG) of a single photon. In the case of SHG, the scattered photon has the same energy as the collective energy of the two incident photons (i.e. there is no net energy loss and the wavelength of the scattered photon is at exactly half the wavelength of each one of the incident photons). In the case of TPEF, the wavelength of the incident photons is approximately twice as long as the wavelength of a photon required for linear excitation, while the fluorescently emitted photons have nearly identical spectral features as those resulting from single photon excitation. Because the probability of simultaneous interaction with two photons is orders of magnitude lower than single photon interactions, TPEF and SHG processes require the presence of high photon densities. As a result, these events are confined within a small volume in the apex of a focused cone of light and automatically yield optically sectioned, depth-resolved images. The confinement of the optical effect, and the use of low energy (longer wavelength) photons results in reduced thermal and photo damage within and outside the plane of focus [45
]. Exploiting such processes in microscopic imaging platforms allows frequent sample assessment over long periods of time, without damage or contamination from elements outside the tissue culture environment.