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

Abstract

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.

A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton’s jelly mesenchymal stromal cells for cell therapy and tissue engineering

“…In the same sense, it would be necessary to evaluate if the effects of FIBRIAGAR-3D are related to the concentration of cells cultured within this matrix. Although we used the cell concentration that previously allowed us to generate a pre-vascularized oral mucosa substitute by tissue engineering ( Blanco-Elices et al, 2021 ), alternative cell concentrations should also be evaluated.…”

Fibrin and Marine-Derived Agaroses for the Generation of Human Bioartificial Tissues: An Ex Vivo and In Vivo Study

“…Development of an ideal biomaterial for clinical use is one of the main objectives of current research in tissue engineering. Although FA biomaterial showed promising results in tissue engineering of the cornea, skin, nerve, tendon, sclera and oral mucosa, the biocompatibility and biomechanical properties of this biomaterial still need to be improved [ 27 , 28 , 29 , 30 , 31 , 32 ]. In this regard, we recently demonstrated that different types of marine agaroses could have specific biological and biomechanical properties, suggesting that not all currently available marine agaroses polysaccharides show the same behavior both in vitro and in vivo [ 23 ].…”

“…In the present work, we generated several types of bioartificial tissues containing human fibroblasts embedded within fibrin and fibrin-agarose biomaterials, as previously described [ 23 , 28 ]. For the tissues containing fibrin biomaterials without agarose (F-0 group), we mixed 760 µL of human plasma, 75 µL of DMEM containing 5000 detached fibroblasts, 15 µL of tranexamic acid (Amchafibrin 5 mg/mL, MEDA Pharma SL, Madrid, Spain), 50 µL of a 2% CaCl 2 (Merck) and 100 µL of PBS, per mL of final volume.…”

“…Fibrin-agarose (FA) biomaterials were first described by our group for the generation of cornea substitutes with promising results [ 27 ]. Subsequently, FA scaffolds were applied to the generation of numerous tissues and organs, such as the skin, nerve, tendon, sclera and oral mucosa [ 28 , 29 , 30 , 31 , 32 ]. Previous studies showed that the combination of these two materials resulted in a significant improvement of the biomaterials’ biomechanical properties as compared to fibrin hydrogels, especially when nanostructuration is applied [ 33 , 34 ].…”

Development of secretome-based strategies to improve cell culture protocols in tissue engineering

“…To generate primary cell cultures of human MSC, we obtained samples of human adipose tissue, dental pulp, and umbilical cord according to previously described protocols 19 – 21 . Samples corresponding to three different donors were used (n = 3 for each tissue).…”