The Cellular and Matrix Dynamics of Enthesis Growth
Nathaniel A. Dyment1, Andrew P. Breidenbach2, Andrea G. Schwartz3, Ryan P. Russell1, Lindsey
Aschbacher-Smith4, Han Liu4, Yusuke Hagiwara1, Rulang Jiang4, Stavros Thomopoulos3, David L. Butler2,
David W. Rowe1.
1
University of Connecticut Health Center, Farmington, CT, USA, 2University of Cincinnati, Cincinnati, OH,
USA, 3Washington University, St Louis, MO, USA, 4Cincinnati Children's Hospital Medical Center,
Cincinnati, OH, USA.
Disclosures: N.A. Dyment: None. A.P. Breidenbach: None. A.G. Schwartz: None. R.P. Russell: None. L.
Aschbacher-Smith: None. H. Liu: None. Y. Hagiwara: None. R. Jiang: None. S. Thomopoulos: None. D.L.
Butler: None. D.W. Rowe: None.
Introduction: Compliant tendons and ligaments connect to rigid bone across a transitional tissue called
the enthesis. Several acute and chronic pathologies can affect tendon and ligament entheses. Such
injuries are particularly problematic to repair, with high re-tear rates after surgery [1]. The most
common entheses insert directly into bone across zones of unmineralized and mineralized fibrocartilage.
Hedgehog (Hh) signaling is necessary for development of this fibrocartilage [2]; however, little is known
about the mineralization process in this critical tissue. Therefore, the focus of this study is to understand
the dynamics of collagen synthesis and cellular maturation from unmineralized to mineralized
fibrocartilage within the enthesis during growth. We sought to determine: 1) how the enthesis grows by
monitoring spatiotemporal collagen expression and mineralized fibrocartilage apposition and 2)
whether Hh signaling regulates mineralized fibrocartilage production.
Methods: All protocols were IACUC approved. Different transgenic mice were used in two sets of
experiments. 1) Collagen expression and mineralized fibrocartilage apposition. 1a) Triple transgenic
Col1a1-YFP:Col2a1-CFP:Col10a1-RFP mice were assessed at postnatal days P1, P14, and P28 to monitor
temporal expression of collagen synthesis during growth. 1b) Dynamic matrix changes were measured in
Col1a1-CFP:ColX-RFP mice (4.5 and 10.5 weeks old) via mineralization labels given at 2.5
(demeclocycline), 4.5 (calcein), 6.5 (alizarin complexone), and 8.5 (demeclocycline) weeks of age. 2) Hh
signaling regulation of mineralized fibrocartilage. 2a) Inducible Gli1-CreERT2 crossed with Ai14tdTomato Cre reporter mice were injected with tamoxifen and calcein at 4.5 weeks of age and
demeclocycline at 8.5 weeks of age to trace the maturation of Hh responsive cells during growth. 2b)
Constitutive scleraxis (ScxCre) crossed with floxed smoothened (Smof/f) mice were injected with
mineralization labels similar to experiment 1b and assessed at 4 and 10 weeks of age. Repetitive imaging
cryohistology. Frozen mineralized sections were prepared using cryotape from knees, ankles, and
shoulders and each section went through up to 4 rounds of imaging. These rounds included imaging i)
fluorescent GFP reporters and/or mineralization labels, ii) collagen SHG signal via two photon imaging,
iii) alkaline phosphatase (AP) fluorescent staining, and iv) toluidine blue stain. Layered composite images
of all 4 rounds were made in Photoshop for the patellar (PT), Achilles (AT), and supraspinatus (ST)
tendons.
Results: Synthesis and maturation of collagen template during growth. Prior to mineralization, the
enthesis consisted of Col1-YFP+ cells that abut Col2-CFP chondrocytes of the primary cartilage (Fig. 1A).
At the onset of mineralization, cells at the base of the enthesis adjacent to the primary cartilage
expressed ColX-RFP and high levels of AP (Fig. 1B). These cells then transitioned to Col2-CFP
fibrochondrocytes and finally Col1-YFP cells through the transition from mineralizing to unmineralized
fibrocartilage (Fig. 1B). Following mineralization, Col1 and Col2 expression diminished while ColX
remained elevated in the mineralized fibrocartilage (Fig. 1C). Appositional growth of the enthesis and
the subchondral bone occurred in opposite directions. Appositional mineralization occured towards the
tendon body in the fibrocartilage while the subchondral bone mineralized towards the marrow space
(Fig. 2A-B, arrows). Fibrocartilage also mineralized perpendicular to while bone mineralized parallel to
the collagen orientation (Fig. 2B2). Col1-CFP expression was not seen in the rounded cells of the
enthesis fibrocartilage but was seen on the anterior surface, suggesting that appositional growth
occurred on this surface (data not shown). This was supported by the direction of mineral apposition as
well. Mineralized fibrocartilage apposition rate differed across the patellar, supraspinatus, and Achilles
tendons. The cumulative mineralized cartilage apposition between 2.5 and 10.5 weeks was significantly
different among the AT (61um), ST (85um), and PT (172um) (Fig. 2C). Hh-responsive Gli1+ cells matured
from unmineralized to mineralized fibrochondrocytes during growth. At 3 days following
tamoxifen/calcein injections, all Gli1-labeled cells were in unmineralized regions of the enthesis.
Following the 4-week chase, a portion of cells closest to the calcein label (tidemark at t=0) had matured
from unmineralized to mineralized fibrochondrocytes as they were between the 2 mineralization labels
(Fig. 3B, white arrow). ScxCre:Smof/- mice exhibited severely reduced mineralized fibrocartilage
apposition during growth compared to littermate controls. Targeted deletion of Smo in the enthesis
resulted in severely impaired mineralized fibrocartilage apposition (Fig. 3C vs D) with reduced mineral
apposition rate in all tendons investigated (50% and 60% of controls for the PT and ST, respectively). In
fact, all 3 tendons were missing at least 1 of the 4 mineralization labels and showed appreciable
disruption in AP activity at both 4 and 10 weeks of age.
Discussion: Progenitor cells that contribute to the enthesis have a common origin that traces back to a
Scx+/Sox9+ progenitor pool during embryogenesis [3-4]. As illustrated in the current study, progeny of
these cells progress through several stages of differentiation during enthesis maturation: 1) Enthesis
cells amplify during growth and synthesize a collagen template that anchors to the underlying primary
cartilage. 2) The cells then produce proteoglycans (increased toluidine blue signal) and type II collagen
as they mature into unmineralized fibrocartilage cells. 3) At the onset of mineralization, cells at the base
of the enthesis adjacent to the primary cartilage express AP and ColX and begin the production of
mineralized fibrocartilage. The mineralization continues in an appositional manner from the base of the
enthesis towards the midsubstance. Hh signaling drives the mineralization process, as ablation of Smo
leads to severe reductions in mineralized fibrocartilage production. These 3 processes lead to the
development of a mature zonal enthesis with a cell maturation gradient from the midsubstance (Col1+)
to unmineralized fibrocartilage (Col1+, Col2+, Gli1+) to mineralizing fibrocartilage (AP+, ColX+) and
finally mineralized fibrocartilage (ColX+).
Significance: An improved understanding of cellular and matrix dynamics within the enthesis during
normal growth and natural healing provides insight into how to improve repair and prevent progression
of certain chronic pathologies. These data indicate that hedgehog signaling is required for cells within
the enthesis to mature from unmineralized to mineralized fibrocartilage. These findings may direct
future insertion repair strategies to create a mechanically functional attachment by anchoring collagen
to bone, producing fibrocartilage, and maturing the fibrocartilage through coordinated Hh signaling.
ORS 2015 Annual Meeting
Paper No: 0086