Noninvasive Optical Assessment of Implanted Tissue-Engineered Construct Success In Situ.

Quantitative diffuse reflectance spectroscopy (DRS) was developed for label-free, noninvasive, and real-time assessment of implanted tissue-engineered devices manufactured from primary human oral keratinocytes (six batches in two 5-patient cohorts). Constructs were implanted in a murine model for 1 and 3 weeks. DRS evaluated construct success in situ using optical absorption (hemoglobin concentration and oxygenation, attributed to revascularization) and optical scattering (attributed to cellular density and layer thickness). Destructive pre- and postimplantation histology distinguished experimental control from stressed constructs, whereas noninvasive preimplantation measures of keratinocyte glucose consumption and residual glucose in spent culture media did not. In constructs implanted for 1 week, DRS distinguished control due to stressed and compromised from healthy constructs. In constructs implanted for 3 weeks, DRS identified constructs with higher postimplantation success. These results suggest that quantitative DRS is a promising, clinically compatible technology for rapid, noninvasive, and localized tissue assessment to characterize tissue-engineered construct success in vivo. Impact statement Despite the recent advance in tissue engineering and regenerative medicine, there is still a lack of nondestructive tools to longitudinally monitor the implanted tissue-engineered devices. In this study, we demonstrate the potential of quantitative diffuse reflectance spectroscopy as a clinically viable technique for noninvasive, label-free, and rapid characterization of graft success in situ.

Photon-tissue interaction model enables quantitative optical analysis of human pancreatic tissues.

A photon-tissue interaction (PTI) model was developed and employed to analyze 96 pairs of reflectance and fluorescence spectra from freshly excised human pancreatic tissues. For each pair of spectra, the PTI model extracted a cellular nuclear size parameter from the measured reflectance, and the relative contributions of extracellular and intracellular fluorophores to the intrinsic fluorescence. The results suggest that reflectance and fluorescence spectroscopies have the potential to quantitatively distinguish among pancreatic tissue types, including normal pancreatic tissue, pancreatitis, and pancreatic adenocarcinoma.

Optical Metric Assessed Engineered Tissues Over a Range of Viability States.

Many conventional methods to assess engineered tissue morphology and viability are destructive techniques with limited utility for tissue constructs intended for implantation in patients. Sterile label-free optical molecular imaging methods analyzed tissue endogenous fluorophores without staining, noninvasively and quantitatively assessing engineered tissue, in lieu of destructive assessment methods. The objective of this study is to further investigate label-free optical metrics and their correlation with destructive methods. Tissue-engineered constructs (n = 33 constructs) fabricated with primary human oral keratinocytes (n = 10 patients) under control, thermal stress, and rapamycin treatment manufacturing conditions exhibited a range of tissue viability states, as evaluated by quantitative histology scoring, WST-1 assay, Ki-67 immunostaining imaging, and label-free optical molecular imaging methods. Both histology sections of fixed tissues and cross-sectioned label-free optical images of living tissues provided quantitative spatially selective information on local tissue morphology, but optical methods noninvasively characterized both local tissue morphology and cellular viability at the same living tissue site. Furthermore, optical metrics noninvasively assessed living tissue viability with a statistical significance consistent with the destructive tissue assays WST-1 and histology. Over the range of cell viability states created experimentally, optical metrics noninvasively and quantitatively characterized living tissue viability and correlated with the destructive WST-1 tissue assay. By providing, under sterile conditions, noninvasive metrics that were comparable with conventional destructive tissue assays, label-free optical molecular imaging has the potential to monitor and assess engineered tissue construct viability before surgical implantation.

Noninvasive Optical Assessment of Implanted Engineered Tissues Correlates with Cytokine Secretion.

Fluorescence lifetime sensing has been shown to noninvasively characterize the preimplantation health and viability of engineered tissue constructs. However, current practices to monitor postimplantation construct integration are either qualitative (visual assessment) or destructive (tissue histology). We employed label-free fluorescence lifetime spectroscopy for quantitative, noninvasive optical assessment of engineered tissue constructs that were implanted into a murine model. The portable system was designed to be suitable for intravital measurements and included a handheld probe to precisely and rapidly acquire data at multiple sites per construct. Our model tissue constructs were manufactured from primary human cells to simulate patient variability based on a standard protocol, and half of the manufactured constructs were stressed to create a range of health states. Secreted amounts of three cytokines that relate to cellular viability were measured in vitro to assess preimplantation construct health: interleukin-8 (IL-8), human ß-defensin 1 (hBD-1), and vascular endothelial growth factor (VEGF). Preimplantation cytokine secretion ranged from 1.5 to 33.5 pg/mL for IL-8, from 3.4 to 195.0 pg/mL for hBD-1, and from 0.1 to 154.3 pg/mL for VEGF. In vivo optical sensing assessed constructs at 1 and 3 weeks postimplantation. We found that at 1 week postimplantation, in vivo optical parameters correlated with in vitro preimplantation secretion levels of all three cytokines (p < 0.05). This correlation was not observed in optical measurements at 3 weeks postimplantation when histology showed that the constructs had re-epithelialized, independent of preimplantation health state, supporting the lack of a correlation. These results suggest that clinical optical diagnostic tools based on label-free fluorescence lifetime sensing of endogenous tissue fluorophores could noninvasively monitor postimplantation integration of engineered tissues.

The potential of label-free nonlinear optical molecular microscopy to non-invasively characterize the viability of engineered human tissue constructs.

Nonlinear optical molecular imaging and quantitative analytic methods were developed to non-invasively assess the viability of tissue-engineered constructs manufactured from primary human cells. Label-free optical measures of local tissue structure and biochemistry characterized morphologic and functional differences between controls and stressed constructs. Rigorous statistical analysis accounted for variability between human patients. Fluorescence intensity-based spatial assessment and metabolic sensing differentiated controls from thermally-stressed and from metabolically-stressed constructs. Fluorescence lifetime-based sensing differentiated controls from thermally-stressed constructs. Unlike traditional histological (found to be generally reliable, but destructive) and biochemical (non-invasive, but found to be unreliable) tissue analyses, label-free optical assessments had the advantages of being both non-invasive and reliable. Thus, such optical measures could serve as reliable manufacturing release criteria for cell-based tissue-engineered constructs prior to human implantation, thereby addressing a critical regulatory need in regenerative medicine.

Fluorescence lifetime imaging microscopy for quantitative biological imaging.

Fluorescence lifetime imaging microscopy (FLIM) is a method for measuring fluorophore lifetimes with microscopic spatial resolution, providing a useful tool for cell biologists to detect, visualize, and investigate structure and function of biological systems. In this chapter, we begin by introducing the basic theory of fluorescence lifetime, including the characteristics of fluorophore decay, followed by a discussion of factors affecting fluorescence lifetimes and the potential advantages of fluorescence lifetime as a source of image contrast. Experimental methods for creating lifetime maps, including both time- and frequency-domain experimental approaches, are then introduced. Then, FLIM data analysis methods are discussed, including rapid lifetime determination, multiexponential fitting, Laguerre polynomial fitting, and phasor plot analysis. After, data analysis methods are introduced that improve lifetime precision of FLIM maps based upon optimal virtual gating and total variation denoising. The chapter concludes by highlighting several recent FLIM applications for quantitative biological imaging, including Förster resonance energy transfer-FLIM, fluorescence correlation spectroscopy-FLIM, multispectral-FLIM, and multiphoton-FLIM.

Characterization of squamous cell carcinoma in an organotypic culture via subsurface non-linear optical molecular imaging.

Tristetraprolin (TTP) is an RNA-binding protein which downregulates multiple cytokines that mediate progression of head and neck squamous cell carcinoma (HNSCC). We previously showed that HNSCC cells with shRNA-mediated knockdown of TTP are more invasive than controls. In this study, we use control and TTP-deficient cells to present a novel subsurface non-linear optical molecular imaging method using a three-dimensional (3D) organotypic construct, and compare the live cell imaging data to histology of fixed tissue specimens. This manuscript describes how to prepare and image the novel organotypic system that closely mimics HNSCC in a clinical setting. The oral cancer equivalent (OCE) system allows HNSCC cells to stratify and invade beyond the basement membrane into underlying connective tissue prepared from decellularized human dermal tissue. The OCE model was inspired by tissue engineering strategies to prepare autologous transplants from human keratinocytes. Advantages of this method over previously used in vitro cancer models include the simulation of the basement membrane and complex connective tissue in the construct, in addition to the ability to track the 3D movement of live invading cells and quantify matrix destruction over time. The OCE model and novel live cell imaging strategy may be applied to study other types of 3D tissue constructs.

Enhanced photoacoustic stability of gold nanorods by silica matrix confinement.

Photoacoustic tomography (PAT) has garnered much attention for its high contrast and excellent spatial resolution of perfused tissues. Gold nanorods (GNRs) have been employed to further enhance the imaging contrast of PAT. However, the photon fluences typically needed for PA wave induction often also result in GNR shape changes that significantly reduce the efficiency of acoustic wave generation. In this work, we propose, synthesize, and evaluate amorphous silica-coated gold nanorods (GNR-Si) in an effort to improve contrast agent stability and ameliorate efficiency loss during photoacoustic (PA) wave induction. TEM and optical absorption spectra measurements of GNR and GNR-Si show that encasing GNRs within amorphous silica provides substantial protection of nanorod conformation from thermal deformation. PA signals generated by GNR-Si demonstrate considerably greater resistance to degradation of signal intensity with repetitive pulsing than do uncoated GNRs, thereby enabling much longer, high-contrast imaging sessions than previously possible. The prolongation of high-contrast imaging, and biocompatibility and easy surface functionalization for targeting ligands afforded by amorphous silica, suggest GNR-Si to be potentially significant for the clinical translation of PAT.