FIGURE 6.

Potential outlook for the use of patient-derived cells from the cranio-/orofacial region. (A) CLP patient-derived keratinocytes (orange) and fibroblasts (green) were isolated from CLP patients and characterized by staining for E-Cad (keratinocytes) and FN (fibroblasts). Scale bars: 50 μm (Live Imaging), 25 μm (IF). These cells were used to obtain 3D skin differentiation models (top) that can be stained for H&E, the proliferation marker PCNA (red) and Loricrin (LOR, green). Note that PCNA is only detectable in basal keratinocytes, whereas LOR is present in more superficial keratinocyte layers confirming the in vivo situation. Dotted line indicates the membrane. Scale bars: 50 μm (Live Imaging), 25 μm (IF). Alternatively, the isolation of both keratinocytes and fibroblasts from the same tissue donor allows the establishment of full-thickness 3D-skin models, in which CLP keratinocytes are plated on a dermis created by the corresponding fibroblasts that also can be stained (H&E, bottom). *p < 0.05 EtOH vs. oral rinse or KSFM or DMEM. (B) Healthy gingival keratinocytes (orange) and fibroblasts (green) were used to determine cytotoxicity of a commercially available oral rinse (OR) at different dilutions after a 2 min exposure by MTT assay. Metabolic active cells are plotted as percentage of the initial cell population. (C) Cranio-/orofacial derived fibroblasts share similar characteristics with MSCs and may have a potential in personalized regenerative therapeutic approaches. Like MSCs, fibroblasts have a spindle-like morphology and are plastic-adherent, as revealed by live imaging and vinculin staining (green). Furthermore, qPCR analyses revealed that our patient-derived fibroblasts also express the typical MSC markers CD73, CD90, and CD105. *p < 0.05 MSC vs. Fbs. Alizarin Red S (AR) staining indicates the potential of PDL fibroblasts to be differentiated into bone forming cells. Scale bar: 20 μm. Data are expressed as mean ± SD. n = 3.