ABSTRACT
Biodegradable scaffolds could revolutionize tissue engineering and regenerative medicine; however, in vivo matrix degradation and tissue ingrowth processes are not fully understood. Currently a large number of samples and animals are required to track biodegradation of implanted scaffolds, and such nonconsecutive single-time-point information from various batches result in inaccurate conclusions. To overcome this limitation, we developed functional biodegradable scaffolds by employing invisible near-infrared fluorescence and followed their degradation behaviors in vitro and in vivo. Using optical fluorescence imaging, the degradation could be quantified in real-time, while tissue ingrowth was tracked by measuring vascularization using magnetic resonance imaging in the same animal over a month. Moreover, we optimized the in vitro process of enzyme-based biodegradation to predict implanted scaffold behaviors in vivo, which was closely related to the site of inoculation. This combined multimodal imaging will benefit tissue engineers by saving time, reducing animal numbers, and offering more accurate conclusions.
Subject(s)
Spectroscopy, Near-Infrared , Tissue Engineering , Tissue Scaffolds , Animals , Biocompatible Materials/metabolism , Collagen/chemistry , Collagen/metabolism , Collagenases/metabolism , Infrared Rays , Magnetic Resonance Imaging , Mice , Mice, Nude , Microscopy, Fluorescence , Quaternary Ammonium Compounds/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sulfonic Acids/chemistry , SwineABSTRACT
PURPOSE: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types. DESIGN: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro. RESULTS: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing. CONCLUSIONS: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development.
Subject(s)
Immunotherapy , Oncogene Proteins, Fusion , Peptides/immunology , Sarcoma, Ewing , T-Lymphocytes, Cytotoxic , Adult , Animals , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , HLA-A2 Antigen/genetics , HLA-A2 Antigen/metabolism , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Mice , Mice, Transgenic , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/immunology , Oncogene Proteins, Fusion/metabolism , Peptides/genetics , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/immunology , Sarcoma, Ewing/pathology , Signal Transduction , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/physiology , Xenograft Model Antitumor AssaysABSTRACT
Stem cells show promise for repair of damaged cardiac tissue. Little is known with certainty, however, about the distribution of these cells once introduced in vivo. Previous attempts at tracking delivered stem cells have been hampered by the autofluorescence of host tissue and limitations of existing labeling techniques. We have developed a novel loading approach to stably label human mesenchymal stem cells with quantum dot (QD) nanoparticles. We report the optimization and validation of this long-term tracking technique and highlight several important biological applications by delivering labeled cells to the mammalian heart. The bright QD crystals illuminate exogenous stem cells in histologic sections for at least 8 weeks following delivery and permit, for the first time, the complete three-dimensional reconstruction of the locations of all stem cells following injection into the heart. Disclosure of potential conflicts of interest is found at the end of this article.
