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Tissue-engineered skin equivalents based on primary isolated fibroblasts and keratinocytes have been shown to be useful tools for functional in vitro tests, including toxicological screenings and drug development. In this study, a commercially available squamous cell carcinoma (SCC) cell line SCC-25 was introduced into epidermal and full-thickness skin equivalents to generate human-based disease-in-a-dish model systems. Interestingly, when cultured either in the epidermis or dermis of full-thickness skin equivalents, SCC-25 cells formed hyper-keratinized tumor cell nests, a phenomenon that is frequently seen in the skin of patients afflicted with SCC. Raman spectroscopy was employed for the label-free cell phenotype characterization within the engineered skin equivalents and revealed the presence of differential protein patterns in keratinocytes and SCC-25 cells. To conclude, the here presented SSC disease-in-a-dish approaches offer the unique opportunity to model SSC in human skin in vitro, which will allow further insight into SSC disease progression, and the development of therapeutic strategies.

Tissue-engineered organs and implants hold promise for the replacement of damaged and diseased organs. However, the foreign body response (FBR) is a major obstacle that compromises the function of tissue-engineered constructs, typically causing them to fail. Two components of FBR are an inflammatory response and a lack of vascularization. To overcome these limitations, a collagen system was developed to release interleukin-6 (IL-6) siRNA and endothelial nitric oxide synthase (eNOS) pDNA in a staggered manner. Hollow collagen microspheres were assembled into a collagen sphere-in-hydrogel system that displayed a staggered release profile in vitro. This system was assessed in vivo in a subcutaneous rat model. The doses of IL-6 siRNA and eNOS pDNA were first individually optimized for their ability to reduce the volume fraction of inflammatory cells (7 days) and increase the length density of blood vessels (14 days), respectively. The identified optimal doses were combined, and the ability of the system to decrease the volume fraction of inflammatory cells and increase the length density of blood vessels was confirmed at both 7 and 14 days. Analysis of the tissue using Raman microspectroscopy revealed that in addition to changes in inflammation and angiogenesis, there were also changes in the extracellular matrix (ECM) at seven days. While changes in sulfated glycosaminoglycan (sGAG) content of the ECM were not detected, changes in the binding of sGAG of the ECM to growth factors were observed. Two growth factors tested, VEGF165 and bFGF showed increased binding to sGAG extracted from eNOS pDNA-treated samples at seven days, increasing the angiogenic potential of the ECM. Thus, we observe that changes in the tissue in terms of the balance of inflammation and angiogenesis as well changes in the activity of sGAG of the ECM occurs following dual delivery of nucleic acids from the collagen sphere-in-hydrogel system.