title: Development and characterization of an acellular porcine cartilage bone matrix for use in tissue engineering.
abstract: The aim of this study was to develop a technique to decellularize a porcine cartilage bone construct with view to using this as a biological scaffold for cartilage substitution. The decellularization protocol applied freeze/thaw cycles; this was followed by cyclic incubation in hypotonic tris buffer and 0.1% (w/v) sodium dodecyl sulfate in hypotonic buffer plus protease inhibitors. Nucleases (RNase and DNase) were used to digest nucleic acids followed by disinfection using 0.1% (v/v) peracetic acid. Histological analysis confirmed the absence of visible cells within the decellularized tissue. DNA analysis revealed the near-complete removal of genomic DNA from the decellularized tissues. The decellularization process had minimal effect on the collagen content of the cartilage. However, there was a significant reduction in the glycosaminoglycan content in the decellularized tissues. There was no evidence of the expression of the major xenogeneic epitope, galactose-α-1,3-galactose. Biomechanical indentation testing of decellularized tissues showed a significant change in comparison to the fresh cartilage. This was presumed to be caused by the reduction in the glycosaminoglycan content. Biocompatibility of the acellular scaffold was determined using contact cytotoxicity assays and a galactosyltransferase knockout mouse model. Decellularized porcine cartilage tissue was found to exhibit favorable compatibility in both in vitro and in vivo tests. In conclusion, this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair. To our knowledge, this is the first study that has successfully removed whole cells and α-gal from xenogeneic cartilage and bone tissue.
authors.affiliation: Institude of Medical and Biological Engineering, The University of Leeds, Leeds, UK. ekheir@doctors.org.uk
authors.forename: Ehab
authors.initials: E
authors.lastname: Kheir
authors.forename: Thomas
authors.initials: T
authors.lastname: Stapleton
authors.forename: David
authors.initials: D
authors.lastname: Shaw
authors.forename: Zhongmin
authors.initials: Z
authors.lastname: Jin
authors.forename: John
authors.initials: J
authors.lastname: Fisher
authors.forename: Eileen
authors.initials: E
authors.lastname: Ingham
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D001857:Bone Matrix / Q000737:chemistry* / Q000166:cytology; D002356:Cartilage / Q000737:chemistry* / Q000166:cytology*; D003094:Collagen / Q000737:chemistry; D019245:Compressive Strength; D005260:Female; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D006025:Glycosaminoglycans / Q000737:chemistry; D006801:Humans; D006909:Hydroxyproline / Q000737:chemistry; D008422:Materials Testing; D051379:Mice; D018345:Mice, Knockout; D034421:Sus scrofa; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods*; D054457:Tissue Scaffolds
chemical_list: D001672:Biocompatible Materials; D006025:Glycosaminoglycans; D003094:Collagen; D005700:Galactosyltransferases; D006909:Hydroxyproline