Optimization of human skin keratinocyte culture protocols using bioactive molecules derived from olive oil.

Skin damage due to severe burns can compromise patient life. Current tissue engineering methods allow the generation of human skin substitutes for clinical use. However, this process is time-consuming, as the keratinocytes required to generate artificial skin have a low proliferation rate in culture. In this study, we evaluated the pro-proliferative effects of three natural biomolecules isolated from olive oil: phenolic extract (PE), DL-3,4-dihydroxyphenyl glycol (DHFG), and oleuropein (OLP), on cultured human skin keratinocytes. The results showed that PE and OLP increased the proliferation of immortalized human skin keratinocytes, especially at concentrations of 10 and 5 µg/mL, respectively, without altering cell viability. In contrast, DHFG did not produce a significant improvement in keratinocyte proliferation. In normal human skin keratinocytes obtained from skin biopsies, we found that PE, but not OLP, could increase the number of keratinocyte colonies and the area occupied by these cells. Furthermore, this effect was associated with increased KI-67 and Proliferating cell nuclear antigen (PCNA) gene expression. Thus, we propose that PE positively affects keratinocyte proliferation and could be used in culture protocols to improve bioartificial skin generation by tissue engineering.

Histochemical and Immunohistochemical Methods for the Identification of Proteoglycans.

Proteoglycans (PGs) are non-fibrillar extracellular matrix (ECM) molecules composed by a protein core and glycosaminoglycan (GAG) chains. These molecules are present in all tissues playing essential structural, biomechanical, and biological roles. In addition, PGs can regulate cell behavior due to their versatility and ability to interact with other ECM molecules, growth factors, and cells. The distribution of PGs can be evaluated by histochemical and immunohistochemical methods. Histochemical methods aimed to provide a useful overview of the presence and distribution pattern of certain groups of PGs. In contrast, immunohistochemical procedures aimed the identification of highly specific target molecules. In this chapter we described Alcian Blue, Safranin O, and Toluidine Blue histochemical methods for the screening of PGs in tissue sections. Finally, we describe the immunohistochemical procedures for specific identification of PGs (decorin, biglycan, and versican) in formaldehyde-fixed and paraffin-embedded tissues.

Tissue Fixation and Processing for the Histological Identification of Lipids.

Lipids are a heterogeneous group of substances characterized by their solubility in organic solvents and insolubility in water. Lipids can be found as normal components of different tissues and organs, and they can be affected by several pathological conditions. The histochemical identification of lipids plays an important role in the histopathological diagnosis and research, but successful staining depends on adequate fixation and processing of the tissue. Here we describe methods to fix, cryoprotect, and process tissue samples for the histochemical identification of lipids in frozen or paraffin-embedded tissues.

Generation of a novel model of bioengineered human oral mucosa with increased vascularization potential.

OBJECTIVE: The aim of this study was to generate novel models of bioartificial human oral mucosa with increased vascularization potential for future use as an advanced therapies medicinal product, by using different vascular and mesenchymal stem cell sources. BACKGROUND: Oral mucosa substitutes could contribute to the clinical treatment of complex diseases affecting the oral cavity. Although several models of artificial oral mucosa have been described, biointegration is a major issue that could be favored by the generation of novel substitutes with increased vascularization potential once grafted in vivo. METHODS: Three types of mesenchymal stem cells (MSCs) were obtained from adipose tissue, bone marrow, and dental pulp, and their in vitro potential was evaluated by inducing differentiation to the endothelial lineage using conditioning media. Then, 3D models of human artificial oral mucosa were generated using biocompatible fibrin-agarose biomaterials combined with human oral mucosa fibroblasts and each type of MSC before and after induction to the endothelial lineage, using human umbilical vein endothelial cells (HUVEC) as controls. The vascularization potential of each oral mucosa substitute was assessed in vitro and in vivo in nude mice. RESULTS: In vitro induction of MSCs kept in culture was able to increase the expression of VEGF, CD31, and vWF endothelial markers, especially in bone marrow and dental pulp-MSCs, and numerous proteins with a role in vasculogenesis become overexpressed. Then, in vivo grafting resulted in a significant increase in blood vessels formation at the interface area between the graft and the host tissues, with significantly positive expression of VEGF, CD31, vWF, and CD34 as compared to negative controls, especially when pre-differentiated MSCs derived from bone marrow and dental pulp were used. In addition, a significantly higher number of cells committed to the endothelial lineage expressing the same endothelial markers were found within the bioartificial tissue. CONCLUSION: Our results suggest that the use of pre-differentiated MSCs could contribute to a rapid generation of a vascular network that may favor in vivo biointegration of bioengineered human oral mucosa substitutes.

Generation of a novel human dermal substitute functionalized with antibiotic-loaded nanostructured lipid carriers (NLCs) with antimicrobial properties for tissue engineering.

BACKGROUND: Treatment of patients affected by severe burns is challenging, especially due to the high risk of Pseudomonas infection. In the present work, we have generated a novel model of bioartificial human dermis substitute by tissue engineering to treat infected wounds using fibrin-agarose biomaterials functionalized with nanostructured lipid carriers (NLCs) loaded with two anti-Pseudomonas antibiotics: sodium colistimethate (SCM) and amikacin (AMK). RESULTS: Results show that the novel tissue-like substitutes have strong antibacterial effect on Pseudomonas cultures, directly proportional to the NLC concentration. Free DNA quantification, WST-1 and Caspase 7 immunohistochemical assays in the functionalized dermis substitute demonstrated that neither cell viability nor cell proliferation were affected by functionalization in most study groups. Furthermore, immunohistochemistry for PCNA and KI67 and histochemistry for collagen and proteoglycans revealed that cells proliferated and were metabolically active in the functionalized tissue with no differences with controls. When functionalized tissues were biomechanically characterized, we found that NLCs were able to improve some of the major biomechanical properties of these artificial tissues, although this strongly depended on the type and concentration of NLCs. CONCLUSIONS: These results suggest that functionalization of fibrin-agarose human dermal substitutes with antibiotic-loaded NLCs is able to improve the antibacterial and biomechanical properties of these substitutes with no detectable side effects. This opens the door to future clinical use of functionalized tissues.