Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering.

Rotator cuff (RC) tears represent a large proportion of musculoskeletal injuries attended to at the clinic and thereby make RC repair surgeries one of the most widely performed musculoskeletal procedures. Despite the high incidence rate of RC tears, operative treatments have provided minimal functional gains and suffer from high re-tear rates. The hypocellular nature of tendon tissue poses a limited capacity for regeneration. In recent years, great strides have been made in the area of tendonogenesis and differentiation towards tendon cells due to a greater understanding of the tendon stem cell niche, development of advanced materials, improved scaffold fabrication techniques, and delineation of the phenotype development process. Though in vitro models for tendonogenesis have shown promising results, in vivo models have been less successful. The present work investigates structured matrices mimicking the tendon microenvironment as cell delivery vehicles in a rat RC tear model. RC injuries augmented with a matrix delivering rat mesenchymal stem cells (rMSCs) showed enhanced regeneration over suture repair alone or repair with augmentation, at 6 and 12-weeks post-surgery. The local delivery of rMSCs led to increased mechanical properties and improved tissue morphology. We hypothesize that the mesenchymal stem cells function to modulate the local immune and bioactivity environment through autocrine/paracrine and/or cell homing mechanisms. This study provides evidence for improved tendon healing with biomimetic matrices and delivered MSCs with the potential for translation to larger, clinical animal models. The enhanced regenerative healing response with stem cell delivering biomimetic matrices may represent a new treatment paradigm for massive RC tendon tears

Insertion morphology of repair, matrix augmentation and matrix/rMSC repair of rat supraspinatus tendons.

<p>Native (<b>CON</b>) tendons (<b><i>A</i></b>) demonstrate a gradual transition from parallel oriented collagenous tendon tissue to bone with a cartilage intermediate. Repair (<b>R</b>) (<b><i>B</i></b>) and matrix augmented (<b>R+S</b>) (<b><i>C</i></b>) insertions have an abrupt transition. Matrix/rMSC repair (<b>R+S+C</b>) (<b><i>D</i></b>) 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.</p

Imaging and quantification of collagen organization during supraspinatus repair and augmentation.

<p>Slides from native tendon (<b>CON</b>) (<b><i>A</i></b>) and tendons harvested 12-weeks after repair (<b>R</b>) (<b><i>B</i></b>), matrix augmentation (<b>R+S</b>) (<b><i>C</i></b>) and matrix/rMSC (<b>R+S+C</b>) (<b><i>D</i></b>) 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 (<b><i>E</i></b>) from native supraspinatus tendon (<b>CON</b>) and tendons harvested 12-weeks after repair (<b>R</b>), matrix augmented repair (<b>R+S</b>) and cell seeded augmented repair (<b>R+S+C</b>). 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.</p

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.

<p>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)</p

Outline of Animal Studies.

<p>Outline of Animal Studies.</p

Tracing of donor rMSCs during supraspinatus augmentation.

<p>Rats underwent repair with matrices seeded with rMSC stained with PKH26 plasma membrane dye (half-life = 100days). The matrix (<b>White Asterisks</b>) was observed on unstained slides under differential interference contrast microscopy (<b><i>A</i></b>). Only one of three rats at 6-weeks post-surgery demonstrated a faint red fluorescent signal from donor cells (<b><i>B</i></b>). Scale bar 500 μm.</p

Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering - Fig 1

<p>Non-augmented and augmented rat supraspinatus repair model (<b><i>A</i></b>) Modified MasonAllen stitch described by Soslowsky. Purple indicates suture, * indicates areas of stress. (<b><i>B</i></b>) Integrated matrix augmentation model for supraspinatus tendon repair. Green indicates the side of cell seeding in matrix/rMSC group.</p

Representative morphology of intact, repaired, and augmented supraspinatus tendon mid-substance.

<p>Representative slide of native (<b>CON</b>) tendon demonstrates a matrix with low cellularity, with a flattened appearance (<b>blue arrow</b>) aligned to parallel collagen fibers (<b><i>A</i></b>). In contrast, repaired (<b>R</b>) tendons up to 12-weeks demonstrate a disoriented collagen matrix with a hypercellular population of larger, rounded, non-oriented cells (<b>yellow arrow</b>) (<b><i>B</i></b>). Disorganized matrix was observed in the tendon body underlying matrix augmentation (<b>R+S</b>) at 6-weeks (<b><i>C</i></b>) and 12-weeks (<b><i>E</i></b>) while matrix/rMSC (<b>R+S+C</b>) group results in fiber orientation at both time points (6-weeks (<b><i>D</i>)</b>, 12-weeks (<b><i>F</i>)</b>). Black arrows indicate anatomical stress axis. Scale Bars 100 μm.</p

Col I and Col III expression during supraspinatus repair and augmentation.

<p>Col I (<b><i>A</i>, <i>B</i>, <i>C</i>, <i>D</i></b>) and Col III (<b><i>E</i>, <i>F</i>, <i>G</i>, <i>H</i></b>) signal was observed from immunohistology staining of native supraspinatus tendon (<b>CON</b>) and tendon tissue harvested at 12-weeks after repair (<b>R</b>), matrix augmentation (<b>R+S</b>) and matrix/rMSC (<b>R+S+C</b>). 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 (<b><i>I</i></b>). 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.</p