**4. Conclusions**

The main conclusions from this study are (1) it was possible to infect chicken embryos with *S. aureus*, one of the main types of bacteria found in the persistent infection associated with chronic wounds, and maintain the embryos' survival for up to 48 h; (2) survival of the embryos varied with the dose of bacteria inoculum and with the use and time of application after infection of an antibiotic (streptomycin in this study); (3) in infected ye<sup>t</sup> viable embryos, the blood vessels network of the CAM was maintained with minimal disruption; and (4) microbiological tests can confirm infection of the embryo, but quantification is difficult.

In summary, we report here preliminary data towards the development of a live ex vivo model of persistent infection that can be used for pre-screening biomaterials intended for treating chronic wounds for their antimicrobial and angiogenic potential. This model is relatively simple, quick, and low-cost, and mimics the in vivo situation more closely than traditionally used antimicrobial tests using agar plates and dilution assays. In addition, keeping in accordance with the principles of the National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3R's), this model does not require administrative procedures for obtaining ethics committee approval for animal experimentation. Nevertheless, we do acknowledge that further work is still needed towards further optimisation to study the interaction of biomaterials with two different strains of bacteria commonly found in chronic wounds, in order to assess their antimicrobial and angiogenic properties. By publishing these preliminary results, we hope that not only our group but others within the scientific community further this research towards the establishment of biomimetic and reproducible ex vivo models of persistent infection. Such models would not only be useful in the research of treatments for chronic wounds, but also in those applications where infection is an important factor to take into account when investigating new therapies.

**Author Contributions:** Conceptualization, V.S. and N.K.; methodology, V.S., N.K., and J.B.; investigation and data acquisition, J.B., V.S., and N.K.; data analysis E.G.-G.; writing—original draft preparation, E.G.-G.; writing—review and editing, E.G.-G., J.B., N.K., and V.S.; supervision, V.S. and E.G.-G.; funding acquisition, E.G.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Restoration of Appearance and Function Trust (RAFT, U.K., registered charity number 299811).

**Conflicts of Interest:** The authors declare no conflict of interest.
