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Embryonic stem cell-derived in vitro model of tissue inflammation following confrontation with implant materials (INFLAPLANT)
Maria Wartenberg 1 and Heinrich Sauer 2
1 Clinic of Internal Medicine I, Friedrich Schiller University Jena, 07740 Jena, Germany
2 Department of Physiology, Justus Liebig University Giessen, 35390 Giessen, Germany;
Keywords: rodents; stem cells; angiogenesis; angiogenesis; inflammation; cell cultures: 3d cultures; cell cultures: co-cultures; reduction; biocompatibility
Duration: 2 years Project Completion: 2012
Background and Aim
Artificial implants are more and more common in clinical daily routine and are used in wide diversity. This includes, for instance, dental prosthetics, bone reconstruction materials, artificial joints, vascular prosthetics, and artificial heart valves.
In recent years biocompatible polymers and biodegradable polymers have been developed for a variety of clinical applications. They have enormous potential in the processing of artificial heart valves, artificial blood vessels, occluders etc. Biodegradable polymers support the healing process, i.e. they are able to degrade in a biological surrounding.
The immuno-compatibility of implant materials is currently tested in animal experiments by analysis of immuno-rejection. In these animal experiments typical signs of inflammation are analysed, e.g. diarrhea, loss of hair, hooked posture, lethargy, and increase of leukocyte numbers in the peripheral blood.
Alternatively conventional biocompatibility testing is performed in vitro with cell cultures of mostly one cell type (fibroblasts). The cells are plated on polymer materials and cell adhesion, proliferation, cyto-toxicity is assessed. The disadvantage of this conventional in-vitro testing is the absence of an inflammatory response induced by the interaction of tissue with the material which normally occurs in vivo.
In the current project we use embryonic stem cells to generate an immuno-competent vascularised tissue (INFLAPLANT-tissue) which displays cellular inflammatory response to specific materials, and therefore allows us to draw conclusions about the biocompatibility of the respective material. The aim of this project is to replace animal experiments which are presently performed to analyse the biocompatibility of newly developed implant materials.
Our INFLAPLANT-tissue allows the study of parameters associated with inflammation, e.g. neo-angiogenesis, the differentiation of inflammatory cells and their migration next to the polymer material, the secretion of inflammatory cytokines (TNF alpha, IL-1 beta, IL-6, CRP), the generation of reactive oxygen species as well as changes in intracellular pH and intracellular Ca2+.
Method and Results
in progress (present status)
The interaction between tissues derived from embryonic stem cells (embryoid bodies) and polymer material particles is achieved in “hanging drops“ culture (Wartenberg et al., 2001). The polymer materials are developed and provided by Innovent Inc. (Jena). Following an adhesion time of 48 h, the adherent cells and the polymer particles are transferred to cell culture dishes. The following measurements are performed according to a detailed schedule.
1) Angiogenesis and inflammatory cell assay. Assessment of vascular areas and blood vessel sprouting. Determination of number and migration of leukocytes; fluorometric cytotoxicity testing: confocal laser scanning microscopy, FACS-analysis.
2) Determination of macrophage-specific mediators: ELISA-analysis.
3) Degradation of polymer materials in the co-culture with the INFLAPLANT tissue: spectroscopic determination of lactate.
4) Quantification of inflammatory markers in the supernatant: ELISA-analysis.
5) Microfluorometric determination of intracellular reactive oxygen species, pH and calcium using confocal laser scanning microscopy.
6) Type of polymers: we will use degradable polymers which are composed from two components: CLA 58 (L-lactid, caprolactone) and EP 37 (L-lactid, caprolacton methacrylate). A variation of material characteristics will be achieved by changing the mixing ratio or the sealing temperature (cooperation with INNOVENT e.V).
Conclusions and Relevance for 3R
The development of a 3-dimensional and multicellular in-vitro model for testing tissue inflammation and cellular retraction during exposition of the tissue to biodegradable polymers will reduce the number of animal experiments.
1. Control of leukocyte differentiation from embryonic stem cells upon vasculogenesis and confrontation with tumour tissue. Hannig M, Figulla HR, Sauer H, Wartenberg M.; J Cell Mol Med. 2008 Jul 4. [Epub ahead of print]
2. Polyphenols prevent cell shedding from mouse mammary cancer spheroids and inhibit cancer cell invasion in confrontation cultures derived from embryonic stem cells. Günther S, Ruhe C, Derikito MG, Böse G, Sauer H, Wartenberg M.; Cancer Lett. 2007;250:25-35.
3. Confrontation cultures of embryonic stem cells with multicellular tumor spheroids to study tumor-induced angiogenesis. Wartenberg M, Finkensieper A, Hescheler J, Sauer H.; Methods Mol Biol. 2006;331:313-28.
4. Embryonic stem cells: a novel tool for the study of antiangiogenesis and tumor-induced angiogenesis.
Wartenberg M, Dönmez F, Budde P, Sauer H. Handb Exp Pharmacol. 2006;174:53-71. Review.
5. Inhibition of tumor-induced angiogenesis and matrix-metalloproteinase expression in confrontation cultures of embryoid bodies and tumor spheroids by plant ingredients used in traditional chinese medicine. Wartenberg M, Budde P, De Mareés M, Grünheck F, Tsang SY, Huang Y, Chen ZY, Hescheler J, Sauer H.; Lab Invest. 2003;83:87-98.
6. Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells. Wartenberg M, Dönmez F, Ling FC, Acker H, Hescheler J, Sauer H.; FASEB J. 2001;15:995-1005.
Figure 1: Generation of INFLAPLANT Confrontation cultures derived from murine embryonic stem cells and polymer material particles. The embryonic stem cells were dissociated and seeded into a spinner flask culture. In this cell culture they build embryoid bodies (EBs). At day 10 the embryoid bodies were taken from the spinner flask and were inoculated in the “hanging drop” with a polymer particle of similar size. Following 24 h of adhesion the co-cultures were transferred to tissue culture dishes. They can stay there for 20 days, in this time the interaction of the INFLAPLANT tissue with the polymer will be studied.
Figure 2: Ethidiumhomodimer dead cell staining at day 16. left: staining of an embryoid body without co-culture, middle: the biocompatible polymer material (blue) is well embedded into the tissue, no increase of the number of dead cells (red) is observed. right: a badly compatible material (blue) induces a high number of dead cells (red).
Figure 3: CD68 expression (monocytes/macrophages) in control samples (embryoid bodies) and in INFLAPLANT co-culture together with a polymer particle of CLA58 (blue) (provided by INNOVENT Inc.). Note that inflammatory cells are migrating towards the biomaterial.