title: Novel bilayer bacterial nanocellulose scaffold supports neocartilage formation in vitro and in vivo.
abstract: Tissue engineering provides a promising alternative therapy to the complex surgical reconstruction of auricular cartilage by using ear-shaped autologous costal cartilage. Bacterial nanocellulose (BNC) is proposed as a promising scaffold material for auricular cartilage reconstruction, as it exhibits excellent biocompatibility and secures tissue integration. Thus, this study evaluates a novel bilayer BNC scaffold for auricular cartilage tissue engineering. Bilayer BNC scaffolds, composed of a dense nanocellulose layer joined with a macroporous composite layer of nanocellulose and alginate, were seeded with human nasoseptal chondrocytes (NC) and cultured in vitro for up to 6 weeks. To scale up for clinical translation, bilayer BNC scaffolds were seeded with a low number of freshly isolated (uncultured) human NCs combined with freshly isolated human mononuclear cells (MNC) from bone marrow in alginate and subcutaneously implanted in nude mice for 8 weeks. 3D morphometric analysis showed that bilayer BNC scaffolds have a porosity of 75% and mean pore size of 50 ± 25 μm. Furthermore, endotoxin analysis and in vitro cytotoxicity testing revealed that the produced bilayer BNC scaffolds were non-pyrogenic (0.15 ± 0.09 EU/ml) and non-cytotoxic (cell viability: 97.8 ± 4.7%). This study demonstrates that bilayer BNC scaffolds offer a good mechanical stability and maintain a structural integrity while providing a porous architecture that supports cell ingrowth. Moreover, bilayer BNC scaffolds provide a suitable environment for culture-expanded NCs as well as a combination of freshly isolated NCs and MNCs to form cartilage in vitro and in vivo as demonstrated by immunohistochemistry, biochemical and biomechanical analyses.
authors.affiliation: Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden. Electronic address: hectorm@chalmers.se.
authors.forename: Héctor
authors.initials: H
authors.lastname: Martínez Ávila
authors.affiliation: Department of Otorhinolaryngology, Ulm University Medical Center, Ulm, Germany. Electronic address: evamariafeldmann@yahoo.de.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Feldmann
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Erasmus MC, University Medical Center, Rotterdam, The Netherlands. Electronic address: m.pleumeekers@erasmusmc.nl.
authors.forename: Mieke M
authors.initials: MM
authors.lastname: Pleumeekers
authors.affiliation: Institute for Biomechanics, ETH Zurich, Zurich, Switzerland. Electronic address: nluc@ethz.ch.
authors.forename: Luc
authors.initials: L
authors.lastname: Nimeskern
authors.affiliation: Institute for Biomechanics, ETH Zurich, Zurich, Switzerland. Electronic address: willykuo@gmx.ch.
authors.forename: Willy
authors.initials: W
authors.lastname: Kuo
authors.affiliation: CellCoTec BV, Bilthoven, The Netherlands. Electronic address: wilco.de.jong@cellcotec.com.
authors.forename: Willem C
authors.initials: WC
authors.lastname: de Jong
authors.affiliation: Department of Otorhinolaryngology, Ulm University Medical Center, Ulm, Germany. Electronic address: silke.schwarz@uniklinik-ulm.de.
authors.forename: Silke
authors.initials: S
authors.lastname: Schwarz
authors.affiliation: Institute for Biomechanics, ETH Zurich, Zurich, Switzerland. Electronic address: ram@ethz.ch.
authors.forename: Ralph
authors.initials: R
authors.lastname: Müller
authors.affiliation: CellCoTec BV, Bilthoven, The Netherlands. Electronic address: Jeanine.Hendriks@cellcotec.com.
authors.forename: Jeanine
authors.initials: J
authors.lastname: Hendriks
authors.affiliation: Department of Otorhinolaryngology, Ulm University Medical Center, Ulm, Germany. Electronic address: nicole.rotter@uniklinik-ulm.de.
authors.forename: Nicole
authors.initials: N
authors.lastname: Rotter
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Erasmus MC, University Medical Center, Rotterdam, The Netherlands; Department of Orthopaedics, Erasmus MC, University Medical Center, Rotterdam, The Netherlands. Electronic address: g.vanosch@erasmusmc.nl.
authors.forename: Gerjo J V M
authors.initials: GJ
authors.lastname: van Osch
authors.affiliation: Institute for Biomechanics, ETH Zurich, Zurich, Switzerland. Electronic address: kstok@ethz.ch.
authors.forename: Kathryn S
authors.initials: KS
authors.lastname: Stok
authors.affiliation: Biopolymer Technology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden. Electronic address: paul.gatenholm@chalmers.se.
authors.forename: Paul
authors.initials: P
authors.lastname: Gatenholm
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D001696:Biomechanical Phenomena; D002356:Cartilage / Q000254:growth & development*; D002460:Cell Line; D002482:Cellulose / Q000737:chemistry*; D019902:Chondrocytes / Q000166:cytology; D020219:Chondrogenesis; D004731:Endotoxins / Q000378:metabolism; D005260:Female; D041822:Gluconacetobacter xylinus / Q000737:chemistry*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008819:Mice, Nude; D008875:Middle Aged; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D040521:Subcutaneous Tissue; D054457:Tissue Scaffolds / Q000737:chemistry; D055815:Young Adult
chemical_list: D004731:Endotoxins; D002482:Cellulose
keywords: Bacterial cellulose; Ear cartilage; Mononuclear cells; Nasoseptal chondrocytes; Neo-cartilage; Tissue engineering