The relationships among spatiotemporal collagen gene expression, histology, and biomechanics following full-length injury in the murine patellar tendon

Abstract

Tendon injuries are major orthopedic problems that worsen as the population ages. Type-I (Col1) and type-II (Col2) collagens play important roles in tendon midsubstance and tendon-to-bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology, and biomechanics up to 8 weeks following a full-length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development.

Fig. 1

The full-length, central PT defect just following surgery (a) becomes discolored during healing (b). During mechanical testing, the sample was mounted in the tibial grip (c) aided with bone cement and a staple. It was then lowered into the patella grip (d) in the testing system.

Fig. 2

H&E (A–F) and Col1-GFPtpz/Col2-ECFP (a–f) micrographs from serial sagittal sections from Normal PT (A,a) and the defect region at one (B,b), two (C,c), three (D,d), four (E,e) and five (F,f) weeks of healing.

Fig. 3

Serial H&E and Col1-GFPtpz/Col2-ECFP micrographs from the midsubstance (A–F, a–f) and tibial insertion (G–I, g–i) from normal PT (A,a,G,g) and the defect region at one (B,b,H,h), two (C,c) three (D,d,I,i), four (E,e) and five (F,f) weeks of healing. (J,j) Col2 expression can be seen within chondrocytes in articular cartilage beneath the patella.

Fig. 4

Serial H&E and Col1-GFPtpz/Col2-ECFP micrographs of contralateral sham limbs at one (A,a), two (B,b), and three (C,c) weeks depicting an increase in Col1 expression at 1-week that reduces to normal PT levels by 3-weeks post-surgery. Four and five weeks data not shown but were consistent with the 3 week time point.

Fig. 5

Average load-displacement curves. (a) The healing tissue showed significantly decreased structural properties at 2-, 5-, and 8-weeks compared to normal PT (p < 0.05). There were no significant differences among the healing tissues at any time point. The corresponding displacements at force levels recorded in the rabbit PT (21% of normal) and the goat PT (40% of normal) showed that the healing tissues were too compliant and not functional to 40% levels even after 8 weeks of healing. (b) The contralateral shams had no effect on structural properties at any time point when compared to normal PT, except the stiffness for the 2-week sham which was greater than normal (p = 0.02). Error bars indicate SEM.

Fig. 6

Comparisons of ultimate load, stiffness, ultimate stress, and modulus vs. time post-surgery normalized to the age at surgery showed improved healing in the mouse model vs previous work done in the rabbit central-third defect model. Error bars indicate SD.