Next generation organotypic human models to investigate gingiva, tooth, microbe and metal interactions
Authors:   , Zhang Yan 
Published: May 2023

Oral mucosa is exposed for many years to dental medical devices which contain metals. Overexposure to metal ions leaching from these dental devices may cause inflammation of mucosa/gingiva and even result in implant failure. Therefore, there is an urgent need to develop human physiologically relevant models which mimic the complex interactions between the oral soft tissues, the microbiome and metal ions leaching from dental medical devices. In this thesis, various human organotypic gingiva and skin models of different levels of complexity were developed to address the above concerns around dental medical devices. These include sterile ex vivo skin, reconstructed human skin (RHS) and reconstructed human gingiva (RHG) where culture methods were adapted to investigate the tissue response to metals and microbes.
In Chapter 2, a full thickness ex vivo human skin to determine whether irritation and cytotoxicity occurred after topical application of nickel, palladium and titanium salts at patch-test relevant concentrations. After 24 hours of exposure, localized cytotoxicity was detected in the epidermis and dermis with separation at the basement membrane zone, formation of vacuoles, apoptotic nuclei, decreased metabolic activity and pro-inflammatory cytokine release. This chapter shows that current metal salt concentrations used in the skin patch test, by causing irritation and cytotoxicity, may confound the readout of an allergic reaction and therefore it should be considered to lower the salt concentration in the patch test.
In Chapter 3, it was investigated whether the observed cytotoxicity to metals was enhanced by the presence of the Streptococcus mitis. After metal exposure, localized apoptosis was observed in the form of condensed apoptotic nuclei and vacuole formation within the epithelium and condensed nuclei in fibroblasts within the hydrogel of both RHG and RHS. S. mitis enhanced metal-mediated apoptosis in gingiva but not in skin. Apoptosis was mediated via the extrinsic pathway via caspase 8. Activation of the execution phase of apoptosis occurred via caspases 3 and 7, and PARP-1. These results show that metal-mediated cytotoxicity can indeed be exaggerated by commensal microbes.
In Chapter 4, new strategies were introduced to the current RHG model: a) bacteria were applied within a low-concentration agar and b) penicillin and streptococcus (PS) was omitted from medium. This advanced microbe-RHG model was topically exposed to Streptococcus gordonii (commensal) or Aggregatibacter actinomycetemcomitans (pathogen) and co-cultured for 72 hours. S. gordonii did not influence RHG cytokine secretion. However, all cytokine secretion (IL-6, IL-8, CCL5, CCL20 CXCL1) secretion increased when RHG were co-cultured with A. actinomycetemcomitans. Both bacteria resulted in increased expression of RHG antimicrobial peptides (AMPs), with S. gordonii exposure resulting in the most Elafin secretion.
In Chapter 5, a model which represented the barrier properties of the biological seal between the tooth and gingiva was next developed. This model is complementary to the existing implant-RHG model already present in our laboratory. An extracted premolar was incorporated into RHG. The gingival epithelium transitioned into a sulcus and junctional epithelium which was attached tightly to both the tooth and the lamina propria hydrogel. The sulcus depth (1.24 ± 0.233mm) and junctional epithelium length (2.45 ± 0.315mm) were comparable to that of healthy individuals. It will provide an excellent means to investigate gingivitis and peri-implantitis in the future.
Taken together, our advanced organotypic skin and gingiva models enabled complex host-microbe interactions to be investigated identifying different commensal and pathogen effects on the host. This thesis shows that metal ions, which are present in many dental medical devices, do cause irritation and cytotoxicity (apoptosis) to the soft tissues (gingiva and skin) which come into contact with them. Notably, these detrimental effects are enhanced by commensal microbes present on both skin and gingiva.