Adding exogenous biglycan or decorin improves tendon formation for equine peritenon and tendon proper cells in vitro

Abstract

Background: Tendon injuries amount to one of the leading causes of career-ending injuries in horses due to the inability for tendon to completely repair and the high reinjury potential. As a result, novel therapeutics are necessary to improve repair with the goal of decreasing leg lameness and potential reinjury. Small leucine-rich repeat proteoglycans (SLRPs), a class of regulatory molecules responsible for collagen organization and maturation, may be one such therapeutic to improve tendon repair. Before SLRP supplementation can occur in vivo, proper evaluation of the effect of these molecules in vitro needs to be assessed. The objective of this study was to evaluate the effectiveness of purified bovine biglycan or decorin on tendon proper and peritenon cell populations in three-dimensional tendon constructs.

Methods: Equine tendon proper or peritenon cell seeded fibrin three-dimensional constructs were supplemented with biglycan or decorin at two concentrations (5 nM or 25 nM). The functionality and ultrastructural morphology of the constructs were assessed using biomechanics, collagen content analysis, transmission electron microscopy (TEM), and gene expression by real time - quantitative polymerase chain reaction (RT-qPCR).

Results: SLRP supplementation affected both tendon proper and peritenon cells-seeded constructs. With additional SLRPs, material and tensile properties of constructs strengthened, though ultrastructural analyses indicated production of similar-sized or smaller fibrils. Overall expression of tendon markers was bolstered more in peritenon cells supplemented with either SLRP, while supplementation of SLRPs to TP cell-derived constructs demonstrated fewer changes in tendon and extracellular matrix markers. Moreover, relative to non-supplemented tendon proper cell-seeded constructs, SLRP supplementation of the peritenon cells showed increases in mechanical strength, material properties, and collagen content.

Conclusions: The SLRP-supplemented peritenon cells produced constructs with greater mechanical and material properties than tendon proper seeded constructs, as well as increased expression of matrix assembly molecules. These findings provide evidence that SLRPs should be further investigated for their potential to improve tendon formation in engineered grafts or post-injury.

Keywords: Biglycan; Decorin; Equine; Peritenon; Tendon; Three-dimensional construct.

Fig. 1

Biomechanical properties for 5 and 25 nM purified bBGN or bDCN supplementation. a Ultimate tensile strength (UTS), b Young’s modulus, and c Maximum tensile load (MTL) were measured across five biological replicates and plotted as mean + SEM. TP: tendon proper cells; PERI: peritenon cells; CTRL: no bBGN or bDCN supplementation. Significance is based on one-sided nonparametric Wilcoxon signed-rank tests predicting improvement: *, significant as p ≤ 0.05, relative to the respective TP or PERI control; n = 5. Outliers detected by the Grubbs’ test in technical replicates (UTS, 4; Young’s modulus, 3; MTL, 2; CSA, 1; p < 0.05) were removed

Fig. 2

Collagen content for 5 and 25 nM purified bBGN or bDCN supplementation. Collagen analysis for (a) collagen content and (b) collagen fraction by dry mass in the tissues; plotted as mean + SEM. TP: tendon proper cells; PERI: peritenon cells; CTRL: no bBGN or bDCN supplementation. Significance is based on one-sided nonparametric Wilcoxon signed-rank tests predicting improvement: *, significant as p ≤ 0.05, relative to the respective TP or PERI control; n = 5

Fig. 3

Fibril diameter analysis for samples supplemented with 5 or 25 nM bovine BGN or bovine DCN. Fibril diameter distributions are given as violin plots for constructs seeded with (a) peritenon (PERI) and (b) tendon proper (TP) cells, n = 5

Fig. 4

Fibril quantity analysis by mean diameter, density, and fibrils per area of extracellular matrix. a Mean fibril diameter, b Fibril number per image, and c fibrils per area of ECM per image were counted for all treatments; plotted as mean + SEM. TP: tendon proper cells; PERI: peritenon cells; CTRL: no bBGN or bDCN supplementation; ECM: extracellular matrix. Significance is based on one-sided nonparametric Wilcoxon signed-rank tests predicting improvement: *, significant as p ≤ 0.05, relative to the respective TP or PERI control; n = 5

Fig. 5

RT-qPCR analysis of perivascular and tenogenic markers. For each gene, the gene expression was plotted against the housekeeping gene POLR2A. TP: tendon proper cells; PERI: peritenon cells; CTRL: no bBGN or bDCN supplementation. Expression is plotted in a box and whisker plot with “+” representing the mean, box representing first-third quartile, line representing median, and whisker representing range. Significance is based on one-sided nonparametric Wilcoxon signed-rank tests predicting improvement: *, significant as p ≤ 0.05, relative to the respective TP or PERI control; n = 5