completed 06/2024
The objective of this research project was to establish a 3D tissue equivalent suitable for studying implant infections. The project also aimed to address whether staphylococci can penetrate intact skin to reach an implant. This was to be achieved through four specific goals:
I. Establishment of a 3D tissue model.
II. Establishment of an infected 3D tissue model.
III. Establishment of a 3D tissue model with an intradermal implant.
IV. Establishment of a 3D tissue model with an infected implant.
I. To develop the 3D skin model, human fibroblasts were co-cultured with keratinocytes seeded on top within a collagen matrix using a transwell system. The keratinocytes were exposed to air on their apical side using the airlift technique, while nutrients were supplied basally through a permeable membrane.
II. This 3D tissue model was inoculated with iso-concentrated suspensions of biofilm-forming Staphylococcus (S.) aureus, S. epidermidis, as well as a mixture of both bacterial species, to simulate a skin infection. The control treatment was conducted with NaCl.
III. Before the maturation of the 3D tissue, a sample of a silicone implant was inserted.
IV. The 3D tissue model with the im plant from III was treated with bacteria as in II.
Histology and immunohistochemistry were used to examine the structure of the 3D tissue, including cell-cell contacts and biofilm formation. Lactate dehydrogenase (LDH) and AIF expression were measured as indicators of cell death. The cytokine profile was assessed using ELISA, and Fluorescence in situ Hybridisation (FISH) were performed to localise the bacteria in the tissue and visualise biofilm formation and activity of the cells.
I. Under airlift conditions, a mature 3D tissue model with skin-like architecture, differentiated layers, and regular cell-cell contacts developed within 30 days.
II. Mature biofilms formed. The 3D tissue cells responded with the release of proinflammatory cytokines. Despite the same bacterial concentration, the polymicrobial biofilm was more aggressive. Both bacterial species influenced each other's biofilm formation, leading to different cytokine responses. Infection led to the dissolution of cell-cell contacts.
III. A mature 3D tissue model also developed with the inclusion of an implant. Silicone was not cytotoxic in the model but induced a proinflammatory response.
IV. In the implant model, a mature biofilm developed. Despite the same bacterial concentration, the cytotoxicity was higher, and the proinflammatory cytokine response was more intense in the presence of an implant. The implant facilitated the infection process.
In summary, we successfully established a 3D tissue equivalent that provides a reproducible platform for in vitro research on biofilms, biocompatibility of foreign materials and implant infections. Due to its tissue-equivalent architecture, it is significantly superior to the previously dominant 2D cultures, enabling the testing of antimicrobial substances, implant surface treatments, and bacterial interactions within biofilms while eliminating the need for animal testing.
-cross sectoral-
Type of hazard:-various
Catchwords:rehabilitation
Description, key words:3D organoid, 3D model, skin model, implant infection, Staphylococcus