Identification of pro-regenerative approaches to improve tendon healing is critically important as the fibrotic healing response impairs physical function. In the present study we tested the hypothesis that S100a4 haploinsufficiency or inhibition of S100a4 signaling improves tendon function following acute injury and surgical repair in a murine model. We demonstrate that S100a4 drives fibrotic tendon healing primarily through a cell non-autonomous process, with S100a4 haploinsufficiency promoting regenerative tendon healing. Moreover, inhibition of S100a4 signaling via antagonism of its putative receptor, RAGE, also decreases scar formation. Mechanistically, S100a4 haploinsufficiency decreases myofibroblast and macrophage content at the site of injury, with both cell populations being key drivers of fibrotic progression. Moreover, S100a4-lineage cells become α-SMA+ myofibroblasts, via loss of S100a4 expression. Using a combination of genetic mouse models, small molecule inhibitors and in vitro studies we have defined S100a4 as a novel, promising therapeutic candidate to improve tendon function after acute injury.

Aging is an important factor in disrupted homeostasis of many tissues. While an increased incidence of tendinopathy and tendon rupture are observed with aging, it is unclear whether this is due to progressive changes in tendon cell function and mechanics over time, or an impaired repair reaction from aged tendons in response to insult or injury. In the present study we examined changes in the mechanical properties of Flexor Digitorum Longus (FDL), Flexor Carpi Ulnaris (FCU), and tail fascicles in both male and female C57Bl/6 mice between 3-27 months of age to better understand the effects of sex and age on tendon homeostasis. No change in max load at failure was observed in any group over the course of aging, although there were significant decreases in toe and linear stiffness in female mice from 3-months to 15, and to 22-27-months. No changes in cell proliferation were observed with aging, although an observable decrease in cellularity occurred in 31-month old tendons. Given that aging did not dramatically alter tendon mechanical homeostasis we hypothesized that a disruption in tendon homeostasis, via acute injury would result in an impaired healing response. Significant decreases in max load, stiffness, and yield load were observed in repairs of 22-month old mice, relative to 4-month old mice. No changes in cell proliferation were observed between young and aged, however a dramatic loss of bridging collagen extracellular matrix was observed in aged repairs suggest that matrix production, but not cell proliferation leads to impaired tendon healing with aging.

29Identification of pro-regenerative approaches to improve tendon healing is of critical importance given 30 the diminished quality of life and physical function that accompanies the typical fibrotic response to 31 tendon injury. S100a4 modulates fibrosis through tissue-type dependent mechanisms, and the role of 32 S100a4 in fibrotic, scar-mediated tendon healing has not been established. In the present study we 33 tested the hypothesis that inhibition of S100a4 improves tendon function following acute injury and 34 surgical repair. We demonstrate cell non-autonomous functions of S100a4 as S100a4 35 haploinsufficiency promotes regenerative tendon healing, including decreased scar formation and 36 improved mechanical properties. Moreover, inhibition of S100a4 via antagonism of its putative 37 receptor, the Receptor for Advanced Glycation Endproducts (RAGE), also decreases scar formation. 38Mechanistically, knock-down of S100a4 decreases myofibroblast and macrophage content at the site of 39 injury, with both cell populations being key drivers of fibrotic progression. In contrast, S100a4 + cell 40 depletion displays time-dependent effects on scar formation, and consistent impairments in restoration 41 of mechanical properties, indicating a critical role for these cells in re-establishing tendon strength after 42 injury. Finally, we demonstrate, that S100a4-lineage cells become α-SMA + myofibroblasts, via loss of 43

Tendon connects muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons enable flexion of the fingers and general hand function. Injuries to the flexor tendons are common, and satisfactory healing is impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, very little is known about the molecular and cellular components of flexor tendon repair. To that end, we have previously described a murine model of flexor tendon repair that recapitulates many aspects of healing in humans including impaired range of motion and decreased mechanical properties. Here we provide an in-depth description and demonstration of this surgical procedure to completely transect and repair the flexor digitorum longus (FDL) tendon in the hind paw of the mouse. This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath the scar formation process. ii) To protect the integrity of the repair site, the tendon is release at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. In addition, we demonstrate the use of the cytospin method to identify and quantify different cell populations during healing. Isolation of sufficient cells during the healing process for flow cytometric analysis has proved quite challenging; cytospin requires far fewer cells but allows for simple immunofluorescent labeling and quantification of cells or proteins of interest.

Tendon injuries heal via scar tissue rather than regeneration. This healing response forms adhesions between the flexor tendons in the hand and surrounding tissues, resulting in impaired range of motion and hand function. Mechanistically, inflammation has been strongly linked to adhesion formation, and Prostaglandin E2 (PGE2) is associated with both adhesion formation and tendinopathy. In the present study we tested the hypothesis that deletion of the PGE2 receptor EP4 in S100a4-lineage cells would decrease adhesion formation. S100a4-Cre; EP4 flox/flox (EP4cKOS100a4) repairs healed with improved gliding function at day 14, followed by impaired gliding at day 28, relative to wild type. Interestingly, EP4cKOS100a4 resulted in only transient deletion of EP4, suggesting up-regulation of EP4 in an alternative cell population in these mice. Loss of EP4 in Scleraxis-lineage cells did not alter gliding function, suggesting that Scx-lineage cells are not the predominant EP4 expressing population. In contrast, a dramatic increase in α-SMA+, EP4+ double-positive cells were observed in EP4cKOS100a4 suggesting that EP4cKOS100a4 repairs heal with increased infiltration of EP4 expressing α-SMA myofibroblasts, identifying a potential mechanism of late up-regulation of EP4 and impaired gliding function in EP4cKOS100a4 tendon repairs.

Type II Diabetes (T2DM) dramatically impairs the tendon healing response, resulting in decreased collagen organization and mechanics relative to non-diabetic tendons. Despite this burden, there remains a paucity of information regarding the mechanisms that govern impaired healing of diabetic tendons. Mice were placed on either a high fat diet (T2DM) or low fat diet (lean) and underwent flexor tendon transection and repair surgery. Healing was assessed via mechanical testing, histology and changes in gene expression associated with collagen synthesis, matrix remodeling, and macrophage polarization. Obese/diabetic tendons healed with increased scar formation and impaired mechanical properties. Consistent with this, prolonged and excess expression of extracellular matrix (ECM) components were observed in obese/T2DM tendons. Macrophages are involved in both inflammatory and matrix deposition processes during healing. Obese/T2DM tendons healed with increased expression of markers of pro-inflammatory M1 macrophages, and elevated and prolonged expression of M2 macrophages markers that are involved in ECM deposition. Here we demonstrate that tendons from obese/diabetic mice heal with increased scar formation and increased M2 polarization, identifying excess M2 macrophage activity and matrix synthesis as a potential mechanism of the fibrotic healing phenotype observed in T2DM tendons, and as such a potential target to improve tendon healing in T2DM.

No abstract

Type II Diabetes (T2DM) dramatically impairs the tendon healing response, resulting in decreased collagen organization and mechanics relative to non-diabetic tendons. Despite this burden, there remains a paucity of information regarding the mechanisms that govern impaired healing of diabetic tendons. Mice were placed on either a high fat diet (T2DM) or low fat diet (lean) and underwent flexor tendon transection and repair surgery. Healing was assessed via mechanical testing, histology and changes in gene expression associated with collagen synthesis, matrix remodeling, and macrophage polarization. Obese/diabetic tendons healed with increased scar formation and impaired mechanical properties. Consistent with this, prolonged and excess expression of extracellular matrix (ECM) components were observed in obese/T2DM tendons. Macrophages are involved in both inflammatory and matrix deposition processes during healing. Obese/T2DM tendons healed with increased expression of markers of pro-inflammatory M1 macrophages, and elevated and prolonged expression of M2 macrophages markers that are involved in ECM deposition. Here we demonstrate that tendons from obese/diabetic mice heal with increased scar formation and increased M2 polarization, identifying excess M2 macrophage activity and matrix synthesis as a potential mechanism of the fibrotic healing phenotype observed in T2DM tendons, and as such a potential target to improve tendon healing in T2DM.