Effect of age and proteoglycan deficiency on collagen fiber re-alignment and mechanical properties in mouse supraspinatus tendon

Abstract

Collagen fiber realignment is one mechanism by which tendon responds to load. Re-alignment is altered when the structure of tendon is altered, such as in the natural process of aging or with alterations of matrix proteins, such as proteoglycan expression. While changes in re-alignment and mechanical properties have been investigated recently during development, they have not been studied in (1) aged tendons, or (2) in the absence of key proteoglycans. Collagen fiber re-alignment and the corresponding mechanical properties are quantified throughout tensile mechanical testing in both the insertion site and the midsubstance of mouse supraspinatus tendons in wild type (WT), decorin-null (Dcn(-/-)), and biglycan-null (Bgn(-/-)) mice at three different ages (90 days, 300 days, and 570 days). Percent relaxation was significantly decreased with age in the WT and Dcn(-/-) tendons, but not in the Bgn(-/-) tendons. Changes with age were found in the linear modulus at the insertion site where the 300 day group was greater than the 90 day and 570 day group in the Bgn(-/-) tendons and the 90 day group was smaller than the 300 day and 570 day groups in the Dcn(-/-) tendons. However, no changes in modulus were found across age in WT tendons were found. The midsubstance fibers of the WT and Bgn(-/-) tendons were initially less aligned with increasing age. The re-alignment was significantly altered with age in the WT tendons, with older groups responding to load later in the mechanical test. This was also seen in the Dcn(-/-) midsubstance and the Bgn(-/-) insertion, but not in the other locations. Although some studies have found changes in the WT mechanical properties with age, this study did not support those findings. However, it did show fiber re-alignment changes at both locations with age, suggesting a breakdown of tendon's ability to respond to load in later ages. In the proteoglycan-null tendons however, there were changes in the mechanical properties, accompanied only by location-dependent re-alignment changes, suggesting a site-specific role for these molecules in loading. Finally, changes in the mechanical properties did not occur in concert with changes in re-alignment, suggesting that typical mechanical property measurements alone are insufficient to describe how structural alterations affect tendon's response to load.

Fig. 1

(a) Image of tendon showing stain lines for regional analysis, (b) testing setup of Instron integrated with polarized light system, and (c) mechanical testing protocol. Images were taken for alignment analysis at: (1) before preconditioning, (2) after preconditioning, (3) after the initial displacement of the stress relaxation (SR), (4) after a return to zero displacement, (5) at the transition strain, and (6) at a point in the linear-region. (Not to scale)

Fig. 2

Mechanical changes are present across age and across genotype in the (a) cross-sectional area, (b) percent relaxation, (c) transition strain at the insertion site, and (d) transition stress at the midsubstance

Fig. 3

(a) Significant changes with age were found in the linear modulus at the insertion site with the Bgn^-/- and Dcn^-/- tendons. (b) No changes in the midsubstance linear modulus were found.

Fig. 4

The initial circular variance of collagen fibers in the midsubstance of the tendon increased with age in the WT and Bgn^-/- tendons, denoting a less aligned tendon with age (larger distribution of fiber angles)

Fig. 5

Re-alignment of wild type (WT) tendons at later ages shows a progressively later response of tendons to the application of load in both the insertion site and the midsubstance. Data is presented as a representative sample for each group with population statistics noted. Each line connects the alignment at one point of the mechanical test to the alignment at the next point, i.e., the ‘B’ segment connects the ‘before preconditioning (no. 1 in Fig. 1)’ point to the ‘after preconditioning (no. 2 in Fig. 1)’ point. This segment therefore represents the change in alignment, or the re-alignment, occurring during the preconditioning segment of the mechanical test. A significant change in re-alignment is represented as a line segmented in bold with a significance star (*p <0.025).

Fig. 6

Re-alignment of decorin-null (Dcn^-/-) tendons at later ages shows a progressively later response of the tendons to the application of load at the midsubstance but not at the insertion site. Data is presented as representative samples with population statistics for nonparametric data.

Fig. 7

Re-alignment of biglycan-null (Bgn^-/-) tendons at later ages shows a progressively later response of the tendons to the application of load in the insertion but not in the midsubstance. Data is presented as representative samples with population statistics for nonparametric data.