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1.
Tissue Eng Part C Methods ; 27(5): 287-295, 2021 05.
Article in English | MEDLINE | ID: mdl-33726570

ABSTRACT

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.


Subject(s)
Tissue Engineering , Tissue Scaffolds , Animals , Cell Count , Humans , Keratinocytes , Mice
2.
Tissue Eng Part C Methods ; 24(4): 214-221, 2018 04.
Article in English | MEDLINE | ID: mdl-29448894

ABSTRACT

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.


Subject(s)
Cytokines/metabolism , Image Processing, Computer-Assisted/methods , Keratinocytes/transplantation , Microscopy, Fluorescence/methods , Mouth Mucosa/transplantation , Tissue Engineering/methods , Animals , Cell Survival , Female , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Mice, SCID , Mouth Mucosa/cytology , Mouth Mucosa/metabolism , Tissue Scaffolds , Transplantation, Heterologous
3.
Exp Biol Med (Maywood) ; 238(11): 1233-41, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-24085785

ABSTRACT

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.


Subject(s)
Carcinoma, Squamous Cell/pathology , Head and Neck Neoplasms/pathology , Tristetraprolin/genetics , Cell Culture Techniques , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Optical Imaging/methods , Tissue Engineering/methods , Tristetraprolin/metabolism , Tumor Cells, Cultured
4.
Gastrointest Endosc ; 76(6): 1197-206.e1-5, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23022051

ABSTRACT

BACKGROUND: Fluorescent-labeled peptides are being developed to improve the endoscopic detection of colonic dysplasia. OBJECTIVE: To demonstrate a near-infrared peptide multimer that functions as a phage mimic for in vivo detection of colonic adenomas. DESIGN: A peptide multimer was synthesized by using trilysine as a dendritic wedge to mimic the presentation of peptides on phage, and all peptides, including the multimer, were fluorescent-labeled with Cy5.5. SETTING: Small-animal imaging facility. ANIMAL SUBJECTS: Genetically engineered CPC;Apc mice that spontaneously develop colonic adenomas. INTERVENTION: Near-infrared-labeled AKPGYLS peptide multimer was administered topically into the distal colons of the mice, and endoscopic images of adenomas were captured. Fluorescence intensities were quantified by target-to-background (T/B) ratios, and adenoma dimensions were measured with calipers after imaging. Validation of specific peptide binding was performed on cryosectioned specimens and cells by using confocal microscopy and flow cytometry. MAIN OUTCOME MEASUREMENTS: Fluorescence T/B ratios from colonic adenomas and adjacent normal-appearing mucosa. RESULTS: AKP-multimer, monomer, trilysine core, and Cy5.5 resulted in mean (± SD) T/B ratios of 3.85 ± 0.25, 2.21 ± 0.13, 1.56 ± 0.12, and 1.19 ± 0.11, respectively, P < .01 on in vivo imaging. Peptide multimer showed higher contrast and greater specificity for dysplastic crypts as compared with other probes. Peptide multimer demonstrated significantly greater binding to HT29 cells on flow cytometry and fluorescence microscopy in comparison to monomer and trilysine core. A binding affinity of 6.4 nm/L and time constant of 0.1136 minutes(-1) (8.8 minutes) was measured for multimer. LIMITATIONS: Only distal colonic adenomas were imaged. CONCLUSION: Peptide multimers combine strengths of multiple individual peptides to enhance binding interactions and demonstrate significantly higher specificity and affinity for tumor targets.


Subject(s)
Adenoma/diagnosis , Colonic Neoplasms/diagnosis , Colonoscopy/methods , Molecular Imaging/methods , Optical Imaging/methods , Peptides , Amino Acid Sequence , Animals , Bacteriophages , Carbocyanines , Chemistry Techniques, Synthetic , Flow Cytometry , Fluorescent Dyes , HT29 Cells , Humans , Mice , Mice, Transgenic , Peptide Library , Peptides/chemical synthesis
5.
J Biophotonics ; 4(7-8): 471-81, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21751414

ABSTRACT

The future of endoscopy will be dictated by rapid technological advances in the development of light sources, optical fibers, and miniature scanners that will allow for images to be collected in multiple spectral regimes, with greater tissue penetration, and in three dimensions. These engineering breakthroughs will be integrated with novel molecular probes that are highly specific for unique proteins to target diseased tissues. Applications include early cancer detection by imaging molecular changes that occur before gross morphological abnormalities, personalized medicine by visualizing molecular targets specific to individual patients, and image guided therapy by localizing tumor margins and monitoring for recurrence.


Subject(s)
Endoscopy, Gastrointestinal/methods , Endoscopy, Gastrointestinal/trends , Animals , Cell Line, Tumor , Endoscopy, Gastrointestinal/instrumentation , Humans , Miniaturization , Molecular Imaging , Photons
6.
Biomed Opt Express ; 2(4): 981-6, 2011 Mar 28.
Article in English | MEDLINE | ID: mdl-21483619

ABSTRACT

We validate specific binding activity of a fluorescence-labeled peptide to colorectal dysplasia in living mice using a miniature, flexible, fiber microendoscope that passes through the instrument channel of an endoscope. The microendoscope delivers excitation light at 473 nm through a fiber-optic bundle with outer diameter of 680 µm to collect en face images at 10 Hz with 4 µm lateral resolution. We applied the FITC-labeled peptide QPIHPNNM topically to colonic mucosa in genetically engineered mice that spontaneously develop adenomas. More than two-fold greater fluorescence intensity was measured from adenomas compared to adjacent normal-appearing mucosa. Images of adenomas showed irregular morphology characteristic of dysplasia.

7.
Mol Imaging Biol ; 13(6): 1157-62, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21136184

ABSTRACT

PURPOSE: We engineered a flexible fiber-optic microendoscope for longitudinal optical imaging studies in a mouse model of disseminated ovarian cancer. PROCEDURES: The microendoscope delivers 470 nm excitation light from a light-emitting diode through a fiber-optic bundle with outer diameter of 680 µm. Optics were optimized to maximize power and lateral resolution. We used this instrument to repetitively monitor intraperitoneal growth of HeyA8 ovarian cancer cells stably transduced with green fluorescent protein over 4 weeks. RESULTS: The microendoscope achieves 0.7 mW power and lateral resolution of 4 µm. Initial in vivo imaging studies visualized single cells and small clusters of malignant cells with subsequent studies showing tumor masses and vasculature. We also resolved single cells within intraperitoneal tumor masses. CONCLUSIONS: These studies establish microendoscope technology with single cell resolution for minimally-invasive, longitudinal imaging in living animals. This technology will advance future molecular imaging studies of ovarian cancer and other diseases.


Subject(s)
Endoscopes , Microscopy, Confocal/instrumentation , Molecular Imaging/instrumentation , Molecular Imaging/methods , Optics and Photonics/instrumentation , Ovarian Neoplasms/pathology , Animals , Cell Line, Tumor , Female , Green Fluorescent Proteins , Mice , Models, Biological , Reproducibility of Results , Xenograft Model Antitumor Assays
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