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

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

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Free PMC article

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

Debika Mitra et al. Biotechnol J. .
Free PMC article

Abstract

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.

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

The authors declare no commercial or financial conflict of interest.

Figures

Figure 1.
Figure 1.. Full-factorial study to determine concentrations of HL and CuSO4 required for LOX-mediated crosslinking.
Effect of three levels of HL and CuSO4 on collagen deposition in the absence of exogenous LOX at 14 (A) and 21 days (B). LOXL activity at 14 (C) and 21 days (D). For (A)-(D), one-way ANOVA and Tukey’s post hoc tests were performed to determine statistical significance, which is denoted by alphabetical letterings; groups with no significance are linked by the same letter, while groups with significance do not share a letter (n=4).
Figure 2.
Figure 2.. Effect of exogenous LOXL2 application on morphology and DNA content of constructs cultured for 21 days.
(A) Schematic representing the LOXL2 supplementation regime. (B) Gross morphology of constructs at 21 days. (C) DNA content of constructs at 21 days. (D) Representative H&E images taken at 10× magnification (scale bar represents 200 μm) to visualize cellularity of constructs with insets taken at 4× magnification (scale bar represents 500 μm). Black is indicative of scaffold and some cells are denoted by black arrows. For (C), a one-way ANOVA and Tukey’s post hoc test was performed to determine statistical significance, which is denoted by alphabetical letterings; groups with no significance are linked by the same letter, while groups with significance do not share a letter (n=4).
Figure 3.
Figure 3.. Effect of exogenous LOXL2 application on osteogenic differentiation and functional outputs for constructs in perfusion culture for 21 days.
(A) IBSP and (B) COL1A1 gene expression. (C) Calcium quantification and (D) compressive moduli of constructs. (E) Representative immunohistochemistry of constructs stained for osteocalcin (OCN). Black is indicative of scaffold (S); brown is indicative of OCN (scale bar represents 500 μm). For (A)-(D), one-way ANOVA and Tukey’s post-hoc tests were performed to determine statistical significance, which is denoted by alphabetical letterings; groups with no significance are linked by the same letter, while groups with significance do not share a letter (n=5 for gene expression; n=3-4 for calcium and compressive modulus).
Figure 4.
Figure 4.. Effect of extended culture time on morphology and DNA content.
(A) Schematic representing the LOXL2 supplementation regime for the 28-day study. (B) Gross morphology of constructs. (C) DNA content of constructs at 28 days (n=4). (D) Representative H&E images taken at 10× magnification (scale bar represents 200 μm) to visualize cellularity of constructs and insets taken at 4× magnification (scale bar represents 500 μm). Black is indicative of scaffold and some cells are denoted by black arrows. For (C), a lack of statistical significance was determined using an unpaired t-test.
Figure 5.
Figure 5.. Effect of extended culture time on osteogenic differentiation and functional outputs.
(A) IBSP and (B) COL1A1 expression at 28 days. (C) Calcium quantification and (D) compressive moduli of constructs at 28 days. (E) Total PYD content at 28 days. (F) PYD content normalized to wet weight at 28 days. (G) PYD content normalized to collagen content at 28 days. For (A)-(G), unpaired t-tests were performed to determine statistical significance, which is denoted by an asterisk; groups with significance are indicated with an asterisk symbol *p<0.05 and **p<0.01 (n=4 for gene expression; n=3 for calcium; n=5 for compressive modulus, n=5 for PYD content).

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