In Vitro Generation of Novel Functionalized Biomaterials for Use in Oral and Dental Regenerative Medicine Applications

Abstract

Recent advances in tissue engineering offer innovative clinical alternatives in dentistry and regenerative medicine. Tissue engineering combines human cells with compatible biomaterials to induce tissue regeneration. Shortening the fabrication time of biomaterials used in tissue engineering will contribute to treatment improvement, and biomaterial functionalization can be exploited to enhance scaffold properties. In this work, we have tested an alternative biofabrication method by directly including human oral mucosa tissue explants within the biomaterial for the generation of human bioengineered mouth and dental tissues for use in tissue engineering. To achieve this, acellular fibrin–agarose scaffolds (AFAS), non-functionalized fibrin-agarose oral mucosa stroma substitutes (n-FAOM), and novel functionalized fibrin-agarose oral mucosa stroma substitutes (F-FAOM) were developed and analyzed after 1, 2, and 3 weeks of in vitro development to determine extracellular matrix components as compared to native oral mucosa controls by using histochemistry and immunohistochemistry. Results demonstrate that functionalization speeds up the biofabrication method and contributes to improve the biomimetic characteristics of the scaffold in terms of extracellular matrix components and reduce the time required for in vitro tissue development.

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

“…In brief, human oral mucosa (HOM) fibroblasts were obtained by enzymatic digestion of the HOM stroma using 2 mg/ml solution of Clostridium histolyticum type I collagenase (Gibco BRL) at 37ºC for 6 hours with agitation. Isolated HOM fibroblasts were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma‐Aldrich/Merck) supplemented with 10% fetal bovine serum (Sigma‐Aldrich/Merck) and 1% antibiotics and antimycotics (100 U/ml penicillin G, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B) (Sigma‐Aldrich/Merck) using 75 cm 2 culture flasks with filter caps (Sarstedt, Nümbrecht, Germany), as previously described by the research group 15 , 16 …”

“…To determine the vascularization potential of each cell type in a three‐dimensional tissue substitute generated by tissue engineering, we generated novel models of human oral mucosa stroma (HOM) based on the previously described fibrin‐agarose biomaterial. In brief, hydrogels were developed with a mixture of human plasma combined with type VII agarose and cultured cells, as previously described 22 , 15 . To prevent gel fibrinolysis, the mixture was supplemented with tranexamic acid Amchafibrin™ (Fides Ecopharma), and 1% calcium chloride was added at the final step to induce polymerization.…”

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells

“…Evaluation of the main fibrillar and nonfibrillar components of the extracellular matrix (ECM) of the stromal substitute was carried out by histochemistry as previously described [ 25 , 26 ]. Identification of collagen fibers was performed by picrosirius red [ 27 ], whereas reticular fibers were stained with the Gomori’s reticulin histochemical method.…”

Stem Cells Regenerating the Craniofacial Skeleton: Current State-Of-The-Art and Future Directions

“…By contrast, Conejero et al found substantial bone formation when poly-L-lactic acid scaffolds seeded with osteogenically differentiated ADSCs, but not their multipotent progenitors, were implanted into rats [ 23 ]. Blanco Elices et al generated bilaminar fibrin–agarose hydrogels immersed in tranexamic acid-supplemented human plasma containing cultured ADSCs and palate-derived oral mucosa fibroblasts in an attempt to reproduce palatal hard and soft tissues in rabbits [ 24 ]. In vivo cell and tissue differentiation could regenerate the palatal tissues—however, not to control levels—following 4 weeks of observation.…”