Novel bisphosphonate-based cathepsin K-triggered compound targets the enthesis without impairing soft tissue-to-bone healing

Abstract

Background: Osteoadsorptive fluorogenic sentinel 3 (OFS-3) is a recently described compound that contains a bone-targeting bisphosphonate (BP) and cathepsin K (Ctsk)-triggered fluorescence signal. A prior study in a murine Achilles repair model demonstrated its effectiveness at targeting the site of tendon-to-bone repair, but the intrinsic effect of this novel bisphosphonate chaperone on tendon-to-bone healing has not been previously explored. We hypothesized that application of this bisphosphonate-fluorophore cargo conjugate would not affect the biomechanical properties or histologic appearance of tendon-bone repairs. Materials and Methods: Right hindlimb Achilles tendon-to-bone repair was performed on 12-week old male mice. Animals were divided into 2 groups of 18 each: 1) Achilles repair with OFS-3 applied directly to the repair site prior to closure, and 2) Achilles repair with saline applied prior to closure. Repaired hindlimbs from 12 animals per group were harvested at 6 weeks for biomechanical analysis with a custom 3D-printed jig. At 4 and 6 weeks, repaired hindlimbs from the remaining animals were assessed histologically using H&E, immunohistochemistry (IHC) staining for the presence of Ctsk, and second harmonic generation (SHG) imaging to evaluate collagen fibers. Results: At 6 weeks, there was no significant difference in failure load, stiffness, toughness, or displacement to failure between repaired hindlimbs that received OFS-3 versus saline. There was no difference in tissue healing on H&E or Ctsk staining on immunohistochemistry between animals that received OFS-3 versus saline. Finally, second harmonic generation imaging demonstrated no difference in collagen fiber parameters between the two groups. Conclusion: OFS-3 did not significantly affect the biomechanical properties or histologic appearance of murine Achilles tendon-to-bone repairs. This study demonstrates that OFS-3 can target the site of tendon-to-bone repair without causing intrinsic negative effects on healing. Further development of this drug delivery platform to target growth factors to the site of tendon-bone repair is warranted.

Keywords: biomechanics; enthesis; growth factor delivery; rotator cuff repair; targeted delivery.

FIGURE 1

Biomechanical testing apparatus. The apparatus consists of a hydraulic testing machine and custom 3D-printed jig.

FIGURE 2

Representative specimen after load to failure. All specimens failed at the junction of the tendon/reparative tissue and bone.

FIGURE 3

H + E results: Mouse hindfoot sagittal sections taken 4 and 6 weeks after Achilles tendon-to-bone repair followed by saline (AR) and Achilles tendon-to-bone repair followed by application of OFS-3 (AR + OFS-3), stained with H&E.

FIGURE 4

IHC results: Mouse hindfoot sagittal sections taken 4 and 6 weeks after Achilles tendon-to-bone repair followed by saline (AR) or OFS-3 (AR + OFS-3) treatment, stained with immunohistochemistry for Ctsk. Anti-Ctsk antibodies are stained brown while blue stain reflects residual hematoxylin.

FIGURE 5

OC/total bone surface area ratio between AR and AR + OFS-3 groups.

FIGURE 6

Second harmonic imaging of uninjured native Achilles. Collagen is displayed as cyan.

FIGURE 7

Second harmonic imaging of repaired specimens 6 weeks after Achilles tendon-to-bone repair followed by saline (AR) or OFS-3 (AR + OFS-3). Collagen is displayed as cyan.

FIGURE 8

Collagen fiber dimensions and alignment between groups. Values for length and width are given in µm and angles in degrees. Straightness is unitless. For alignment, a value of 1 indicates all fibers are aligned in one direction and a value of 0 indicates fibers are not aligned.