Elsevier

Biomaterials

Volume 30, Issue 17, June 2009, Pages 3042-3049
Biomaterials

Nuclear factor-κB bioluminescence imaging-guided transcriptomic analysis for the assessment of host–biomaterial interaction in vivo

https://doi.org/10.1016/j.biomaterials.2009.02.016Get rights and content

Abstract

Establishment of a comprehensive platform for the assessment of host–biomaterial interaction in vivo is an important issue. Nuclear factor-κB (NF-κB) is an inducible transcription factor that is activated by numerous stimuli. Therefore, NF-κB-dependent luminescent signal in transgenic mice carrying the luciferase genes was used as the guide to monitor the biomaterials-affected organs, and transcriptomic analysis was further applied to evaluate the complex host responses in affected organs in this study. In vivo imaging showed that genipin-cross-linked gelatin conduit (GGC) implantation evoked the strong NF-κB activity at 6 h in the implanted region, and transcriptomic analysis showed that the expressions of interleukin-6 (IL-6), IL-24, and IL-1 family were up-regulated. A strong luminescent signal was observed in spleen on 14 d, suggesting that GGC implantation might elicit the biological events in spleen. Transcriptomic analysis of spleen showed that 13 Kyoto Encyclopedia of Genes and Genomes pathways belonging to cell cycles, immune responses, and metabolism were significantly altered by GGC implants. Connectivity Map analysis suggested that the gene signatures of GGC were similar to those of compounds that affect lipid or glucose metabolism. GeneSetTest analysis further showed that host responses to GGC implants might be related to diseases states, especially the metabolic and cardiovascular diseases. In conclusion, our data provided a concept of molecular imaging-guided transcriptomic platform for the evaluation and the prediction of host–biomaterial interaction in vivo.

Introduction

The establishment of a comprehensive platform for the prediction and the assessment of host–biomaterial interaction in vivo is an important issue. However, host–biomaterial interaction is a very complex process. Host responses to biomaterials control the biological performances of implanted medical devices and delivery vehicles. After the implantation of biomaterials, synthetic materials dynamically adsorb proteins and other biomolecules, which trigger an inflammatory cascade comprising blood coagulation, leukocyte recruitment and adhesion, foreign body reaction, and fibrous encapsulation [1], [2]. Traditionally, standard cell-based toxicity assays are performed in vitro and the high-risk materials are removed at this early stage. Transcriptomic or proteomic analysis has also been used for understanding better the complexicity of cell–biomaterial interaction and to provide a comprehensive study of cell responses to biomaterials [3], [4], [5]. Although these cell-based systems are excellent at defining single variable effects and responses, they are limited with respect to prediction of events that might occur in vivo [6].

Biocompatibility test for the biomaterials at both pre-clinical and clinical levels is based primarily on where and how long the materials will be used in the body. In addition to the inflammatory responses in the implanted regions, biomaterials may evoke various host responses in the body distinct from the implanted regions. However, it is difficult to know which internal organs distant from the implanted regions are altered by the biomaterial implants. Nuclear factor-κB (NF-κB) is an inducible transcript factor that consists of heterodimers of RelA (p65), c-Rel, RelB, p50, and p52. NF-κB plays a central coordinator of innate and adaptive immune responses. It also has a critical role in the development of cancer, regulation of cell apoptosis, and cell cycle regulation [7], [8], [9]. When cells are exposed to stress, NF-κB translocates into the nucleus and binds to a unique decameric nucleotide sequence present in the promoter of various genes. NF-κB is activated by a large variety of signals, which typically include cytokines, mitogens, environmental particles, toxic metals, intracellular stresses, pathogen products, and ultraviolet light [10]. This property makes NF-κB a potent sensor to sense the affected cells in response to stress, and NF-κB-driven bioluminescence imaging may be used as a guide to monitor the affected organs.

In previous study, we have constructed the transgenic mice carrying the NF-κB-driven luciferase genes and have assessed the feasibility of noninvasive real-time NF-κB bioluminescence imaging on the evaluation of inflammation status in the implanted region [11]. In this study, we monitored the host responses after the implantation of genipin-cross-linked gelatin conduit (GGC) by bioluminescence imaging, and the host–biomaterials interaction was further interpreted by transcriptomic analysis. Our findings suggested that NF-κB-driven bioluminescence imaging-guided transcriptomic analysis may be used to analyze the host–biomaterial interaction in vivo.

Section snippets

Materials

The GGC implant (1.96 mm in diameter, 1.5 mm in length) and lipopolysaccharide (LPS)-immersed GGC implant were prepared as described previously [11], [12]. LPS was purchased from Sigma (St. Louis, MO) and dissolved in water at 5 mg/ml. d-Luciferin was purchased from Xenogen (Hopkinton, MA) and dissolved in phosphate-buffered saline (137 mm NaCl, 1.4 mm KH2PO4, 4.3 mm Na2HPO4, 2.7 mm KCl, pH 7.2) at 15 mg/ml. Rabbit polyclonal antibody against interleukin-1β (IL-1β) and goat polyclonal antibodies

Assessment of the NF-κB-driven bioluminescent signal in GGC-implanted mice by in vivo and ex vivo imaging

The GGC or LPS-immersed GGC implant was implanted subcutaneously in the dorsal region and the NF-κB-driven bioluminescent signals were monitored by luminescent imaging on the indicated periods (Fig. 1). In consistent with previous study [11], luminescent signal in the implanted region was initially increased and gradually decreased in all groups. NF-κB activity reached a maximal activation at 6 h. At 6 h post-implantation, a strong and specific in vivo bioluminescence around the incision was

Discussion

In previous study, we have demonstrated the feasibility of NF-κB-driven bioluminescence imaging for the evaluation of host–biomaterials interaction in implanted region [11]. In this study, we applied the bioluminescence imaging to monitor the implanted region and internal organs that were affected by GGC implantation. The complex host responses to biomaterials were further interpreted and predicted by transcriptomic analysis. Transcriptomic analysis by DNA microarray tools has evolved rapidly

Conclusion

NF-κB is activated by a variety of stimuli. NF-κB bioluminescent imaging was therefore used as a guide to monitor the affected internal organs after GGC implantation. Transcriptomic analysis was further applied to evaluate and predict the complex host responses in GGC implant-affected organs. Our data provides a concept of molecular imaging-guided transcriptomic platform for the evaluation and the prediction of host–biomaterial interaction in vivo.

Acknowledgements

We thank Miss Hsin-Yi Lo, Mr. Chia-Cheng Li, and Mr. Wei-Shuen Shen for their technical assistances. This work was supported by grants from National Research Program for Genomic Medicine, National Science and Technology Program for Agricultural Biotechnology, National Science Council, Committee on Chinese Medicine and Pharmacy, Department of Health (CCMP 96-RD-201 and CCMP 97-RD-201), and China Medical University (CMU97-064 and CMU97-CMC-004), Taiwan.

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