Fig 1.
Non-augmented and augmented rat supraspinatus repair model (A) Modified MasonAllen stitch described by Soslowsky. Purple indicates suture, * indicates areas of stress. (B) Integrated matrix augmentation model for supraspinatus tendon repair. Green indicates the side of cell seeding in matrix/rMSC group.
Table 1.
Outline of Animal Studies.
Fig 2.
Insertion morphology of repair, matrix augmentation and matrix/rMSC repair of rat supraspinatus tendons.
Native (CON) tendons (A) demonstrate a gradual transition from parallel oriented collagenous tendon tissue to bone with a cartilage intermediate. Repair (R) (B) and matrix augmented (R+S) (C) insertions have an abrupt transition. Matrix/rMSC repair (R+S+C) (D) insertions demonstrate a transition and organization similar to the intact tendon. Representative samples at 12-weeks post-surgery stained with Masson’s trichrome. Matrix/sutures are located on the surface plane above the tendon-bone insertion and are present at both 6 (not shown) and 12-weeks. Yellow arrow indicates the bone-tendon axis. Scale bars 200 μm.
Fig 3.
Representative morphology of intact, repaired, and augmented supraspinatus tendon mid-substance.
Representative slide of native (CON) tendon demonstrates a matrix with low cellularity, with a flattened appearance (blue arrow) aligned to parallel collagen fibers (A). In contrast, repaired (R) tendons up to 12-weeks demonstrate a disoriented collagen matrix with a hypercellular population of larger, rounded, non-oriented cells (yellow arrow) (B). Disorganized matrix was observed in the tendon body underlying matrix augmentation (R+S) at 6-weeks (C) and 12-weeks (E) while matrix/rMSC (R+S+C) group results in fiber orientation at both time points (6-weeks (D), 12-weeks (F)). Black arrows indicate anatomical stress axis. Scale Bars 100 μm.
Fig 4.
Col I and Col III expression during supraspinatus repair and augmentation.
Col I (A, B, C, D) and Col III (E, F, G, H) signal was observed from immunohistology staining of native supraspinatus tendon (CON) and tendon tissue harvested at 12-weeks after repair (R), matrix augmentation (R+S) and matrix/rMSC (R+S+C). Both native tendon and tendon underlying matrix/rMSC group demonstrate a small area of Col III staining. Semi-quantification of the Col I: Col III area of expression (I). Both intact tendon and tendon underlying matrix/rMSC group demonstrate a larger ratio of Col I: Col III expression. n = 3 animals per group, * = p<0.05. Scale bars 100 μm.
Fig 5.
Imaging and quantification of collagen organization during supraspinatus repair and augmentation.
Slides from native tendon (CON) (A) and tendons harvested 12-weeks after repair (R) (B), matrix augmentation (R+S) (C) and matrix/rMSC (R+S+C) (D) were stained with picrosirius red and observed under cross-polarized light. Both intact tendon and tendon underlying matrix/rMSC repair demonstrate a high level of birefringence that highlights tissue with highly oriented collagen fiber morphology. Average birefringent signaling from cross-polarized light microscopy of picrosirius red stained slides was converted to an 8-bit grayscale to quantitatively compare the degree of collagen orientation in tendon tissue (E) from native supraspinatus tendon (CON) and tendons harvested 12-weeks after repair (R), matrix augmented repair (R+S) and cell seeded augmented repair (R+S+C). Both intact tendon and tendon underlying cell seeded augmented repair demonstrate a significantly greater collagen orientation. n = 3 animals per group, * = p<0.05. Scale bars 200 μm, matrix above tendon marked by the yellow grid.
Fig 6.
Cross-sectional area (A-6-weeks, D-12-weeks), ultimate stress (B-6-weeks, E-12-weeks) and modulus (C-6-weeks, F-12-weeks) of native (CON), repaired (R), augmented supraspinatus (R+S), and matrix/rMSC (R+S+C) tendons at 6 and 12-week post-surgery.
Native tendons possessed significantly less cross-sectional area, greater ultimate stress and greater tensile modulus than all experimental groups at both time points. At 12-weeks the cross-sectional area of matrix/rMSC (R+S+C) tendon was less than the other experimental groups. There was no significant difference in cross sectional area between 6 and 12-week specimens within each experimental group. The ultimate stress (B and E) of matrix/rMSC (R+S+C) tendons was significantly greater than suture repair (R) and augmented repair (R+S) at 6 and 12-weeks. There was no significant difference in ultimate stress between 6 and 12-week specimens within each experimental group with the exception of the repair group (R). The tensile modulus (C and F) of matrix/rMSC tendon was significantly greater than suture repair (R) at 6-weeks and showed a trend of greater modulus than augmented repair (R+S) at 12-weeks. There was no significant difference in tendon modulus between 6 and 12-week specimens within each experimental group with the exception of a trend of increased stiffness for matrix/rMSC. # = p<0.05 (vs. all other groups)
Fig 7.
Tracing of donor rMSCs during supraspinatus augmentation.
Rats underwent repair with matrices seeded with rMSC stained with PKH26 plasma membrane dye (half-life = 100days). The matrix (White Asterisks) was observed on unstained slides under differential interference contrast microscopy (A). Only one of three rats at 6-weeks post-surgery demonstrated a faint red fluorescent signal from donor cells (B). Scale bar 500 μm.