Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts

Biotechnol J. 2019 Mar;14(3):e1700763. doi: 10.1002/biot.201700763. Epub 2018 Aug 16.


Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.

Keywords: bone engineering; collagen crosslinks; mesenchymal stem cells; perfusion cultures; pyridinoline.

MeSH terms

  • Amino Acid Oxidoreductases / metabolism*
  • Amino Acids / metabolism
  • Cell Culture Techniques / methods
  • Cell Differentiation / physiology
  • Cells, Cultured
  • Collagen / metabolism*
  • Extracellular Matrix / metabolism
  • Extracellular Matrix / physiology
  • Humans
  • Mesenchymal Stem Cells / metabolism
  • Mesenchymal Stem Cells / physiology
  • Osteogenesis / physiology*
  • Tissue Engineering / methods
  • Tissue Scaffolds


  • Amino Acids
  • pyridinoline
  • Collagen
  • Amino Acid Oxidoreductases
  • LOXL2 protein, human