Tgfβ signaling is required for tenocyte recruitment and functional neonatal tendon regeneration

Abstract

Tendon injuries are common with poor healing potential. The paucity of therapies for tendon injuries is due to our limited understanding of the cells and molecular pathways that drive tendon regeneration. Using a mouse model of neonatal tendon regeneration, we identified TGFβ signaling as a major molecular pathway that drives neonatal tendon regeneration. Through targeted gene deletion, small molecule inhibition, and lineage tracing, we elucidated TGFβ-dependent and TGFβ-independent mechanisms underlying tendon regeneration. Importantly, functional recovery depended on canonical TGFβ signaling and loss of function is due to impaired tenogenic cell recruitment from both Scleraxis-lineage and non-Scleraxis-lineage sources. We show that TGFβ signaling is directly required in neonatal tenocytes for recruitment and that TGFβ ligand is positively regulated in tendons. Collectively, these results show a functional role for canonical TGFβ signaling in tendon regeneration and offer new insights toward the divergent cellular activities that distinguish regenerative vs fibrotic healing.

Macrophage depletion impairs neonatal tendon regeneration

“…To better define the tenogenic phenotype of these cells, we carried out gene expression analysis at D3 and D14 by qPCR. Expression of known markers Scx , Tnmd , and Mkx was reduced in carrier treated injured limbs relative to contralateral controls at D3 ( Figure 7A ), consistent with our prior studies (8). In AP20187-treated limbs, injury induced a less predictable response.…”

“…The close proximity of macrophages to tendon cells at D3 suggests that secreted factors from macrophages may stimulate tendon cell proliferation. While TGFβ is an attractive candidate based on our gene expression array screen and has been implicated in other macrophage depletion models of tendon healing (21, 23) as well as fetal tendon healing (35, 38), our previous studies inhibiting TGFβ signaling showed no effect on tendon cell proliferation at D3 (8). The molecular regulators for early cell proliferation is therefore still unknown.…”

“…To determine functional healing when macrophages were ablated, we carried out gait analysis and tensile testing. In previous studies, we identified swing, brake, and propel parameters (normalized to stride length) were affected by injury (7, 8). Analysis of gait at 28 showed no differences with AP20187-treatment, either compared to contralateral uninjured control or compared to carrier treatment for any of the three parameters ( Figure 4 ).…”

“…We found that neonatal tendon regeneration was characterized by early proliferation of tendon cells, followed by their recruitment into the injury site to form a tenogenic neo-tendon. In the absence of tendon cell recruitment, functional properties were not restored (8). Using a similar injury model in adult mice, we also found that adult tendon healing was characterized by fibrotic scar formation, minimal tendon cell proliferation, and persistently reduced functional properties (7), consistent with studies from other groups (9–11).…”

Akt signaling is activated by TGFβ2 and impacts tenogenic induction of mesenchymal stem cells

“…Other aspects of tendon development, such as production of matrix components like fibronectin and proteoglycans, as well as collagen production and organization, may require Smad4-dependent [ 20 ] and independent [ 50 ] TGFβ1 signaling, which long-term Smad3 inhibition prohibits. Furthermore, injury in a neonatal tendon is associated with Smad2/3 activation, and Smad2/3 activation meditated by TGFβ signaling through the TGFβ type I receptor ALK4/5/7 impacts regenerative healing [ 51 ]. Another Smad protein, Smad8, has also been shown to enhance regeneration of tendon injuries when MSCs are genetically engineered to overexpress both Smad8 and its downstream target, BMP2 [ 52 , 53 ], suggesting that Smad signaling may be involved in tendon regeneration.…”