Scleraxis is required for the development of a functional tendon enthesis - PubMed (original) (raw)

Scleraxis is required for the development of a functional tendon enthesis

Megan L Killian et al. FASEB J. 2016 Jan.

Abstract

The attachment of dissimilar materials is a major engineering challenge, yet this challenge is seemingly overcome in biology. This study aimed to determine how the transcription factor Scleraxis (Scx) influences the development and maturation of the tendon-to-bone attachment (enthesis). Mice with conditional knockout (cKO) for Scx (Scx(flx/-), Prx1Cre(+)) and wild-type [(WT) Scx(flx/+) or Scx(flx/flx)] littermates were killed at postnatal days 7-56 (P7-P56). Enthesis morphometry, histology, and collagen alignment were investigated throughout postnatal growth. Enthesis tensile mechanical properties were also assessed. Laser microdissection of distinct musculoskeletal tissues was performed at P7 for WT, cKO, and muscle-unloaded (botulinum toxin A treated) attachments for quantitative PCR. cKO mice were smaller, with altered bone shape and impaired enthesis morphology, morphometry, and organization. Structural alterations led to altered mechanical properties; cKO entheses demonstrated reduced strength and stiffness. In P7 attachments, cKO mice had reduced expression of transforming growth factor (TGF) superfamily genes in fibrocartilage compared with WT mice. In conclusion, deletion of Scx led to impairments in enthesis structure, which translated into impaired functional (i.e., mechanical) outcomes. These changes may be driven by transient signaling cues from mechanical loading and growth factors.

Keywords: attachment; musculoskeletal; postnatal; supraspinatus.

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Figures

Figure 1.

Figure 1.

A) Supraspinatus tendon-to-bone attachment of WT (left column) and cKO (right column) mice at P14, P28, and P56. Black arrowhead indicates enthesis and dotted line in WT P56 panel indicates the tidemark. Scale bar, 200 μm. B) Quantification of cell circularity (height/width), indicative of cell shape, from tendon, FC, and bone of WT (gray bars) and cKO (black bars) mice. C) P7 supraspinatus tendon-to-bone attachment after laser capture microdissection of the FC region. Similar cuts were made to isolate tendon (dotted circle) cells and supraspinatus muscle from 20–25 μm thick frozen, unfixed sections. The secondary ossification center of the bone appeared black in unstained sections and the enthesis and tendon are morphologically distinct from each other and from the bone. D_–_G) Gene expression (2−ΔCT) of bmp-4 (D), tgf-β3 (E), smo (F), and ptch1 (G), normalized to ipo8, are shown for muscle, tendon, and FC of WT (gray bars) and cKO (black bars) groups. B, bone template; E, enthesis; T, tendon. Gene expression data are log base 2-transformed. All data are presented as the mean ± 95% confidence interval. Solid line indicates significant difference between regions within a group or between groups for a specific region (P < 0.05); dashed line indicates significant difference between regions or groups (P = 0.0525).

Figure 2.

Figure 2.

A, B) Two-dimensional cut-plane of micro-CT reconstruction of proximal humerus for WT (A) and cKO (B) mice at P28 and P56, with black pixels highlighting higher density bone. Black arrow highlights supraspinatus FC. C, D) Normalized FC volume (FC volume/HH volume) (C) and FC BMD (D) were measured at P28 and P56. E, F) HH volume (E) and HH BMD (F) were measured at P14, P28, and P56. WT (gray bars) and cKO (black bars) data are represented for each time point; P14 FC volume and BMD were not measured, as the mineralized FC is not established at that time point. An over-bar indicates a significant difference (P < 0.05).

Figure 3.

Figure 3.

A, B) Polarized light micrographs of WT (A) and cKO (B) mice at P28. The regions of interest used for analysis of collagen fibril orientation are identified in dashed boxes, with tendon, enthesis, and bone and cross-polarizer orientation (45°/135°) annotated. C, D) Average distributions of extinction angles for combined WT (C) and cKO (D) analyses, represented from 45 to 135°, graphically illustrate the angular deviation of collagen fibrils in WT and cKO entheses. B, bone; E, enthesis; T, tendon. Scale bars, 200 μm.

Figure 4.

Figure 4.

Biomechanical outcomes of the supraspinatus tendon-to-bone uniaxial tensile tests. A) Representative stress-strain curves of P56 WT and cKO uniaxial tensile tests; dotted circle indicates highest measured stress for calculating ultimate stress (σmax), and the slope of the linear curve was used to calculate Young’s modulus. B, C) Ultimate strength (B) and Young's modulus (C) for WT (gray bars) and cKO (black bars) at P28 and P56. Data are presented as means ± 95% confidence interval. An overbar indicates a significant difference (P < 0.03). E, Young’s modulus.

Figure 5.

Figure 5.

A) Activity of WT and cKO mice at P42 measure for 24–120 h. a) Total activity counts in both horizontal (X and Y) directions (XT + YT; counts) for the first 24 h following a 24 h period of acclimation. b) Rearing at 0–24 and 24–48 h of activity monitor for WT and cKO mice. c) Respiratory exchange ratio for WT and cKO mice in 24 h increments for 5 d of monitoring. Significant differences between groups are indicated by solid black lines (P < 0.05). B) Relative expression of scx (a), bmp-4 (b), tgf-β3 (c), smo (d), and ptch1 (e) for WT and BTX-unloaded shoulders from LCM-isolated regions (muscle, tendon, and FC). Data are log base 2-transformed and presented as means ± 95% confidence interval. A solid overbar indicates a significant difference between regions (P < 0.05); a dashed overbar indicates a significant difference between WT and BTX groups (P = 0.0715).

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