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Mesenchymal stem cells (MSCs) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, β-catenin, and yes-associated protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles × 2%) and static (1 × 2% hold) strain on nuclear translocation of β-catenin and YAP1 at 3 hours after force application. Dynamic strain induced nuclear accumulation of β-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced β-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid did not increase nuclear content of β-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds β-catenin, we considered whether β-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both β-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of β-catenin via mechanisms used for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, β-catenin, and YAP1.

 

Mesenchymal stem cells (MSCs) maintain the musculoskeletal system by differentiating into multiple lineages, including osteoblasts and adipocytes. Mechanical signals, including strain and low-intensity vibration (LIV), are important regulators of MSC differentiation via control exerted through the cell structure. Lamin A/C is a protein vital to the nuclear architecture that supports chromatin organization and differentiation and contributes to the mechanical integrity of the nucleus. We investigated whether lamin A/C and mechanoresponsiveness are functionally coupled during adipogenesis in MSCs. siRNA depletion of lamin A/C increased the nuclear area, height, and volume and decreased the circularity and stiffness. Lamin A/C depletion significantly decreased markers of adipogenesis (adiponectin, cellular lipid content) as did LIV treatment despite depletion of lamin A/C. Phosphorylation of focal adhesions in response to mechanical challenge was also preserved during loss of lamin A/C. RNA-seq showed no major adipogenic transcriptome changes resulting from LIV treatment, suggesting that LIV regulation of adipogenesis may not occur at the transcriptional level. We observed that during both lamin A/C depletion and LIV, interferon signaling was downregulated, suggesting potentially shared regulatory mechanism elements that could regulate protein translation. We conclude that the mechanoregulation of adipogenesis and the mechanical activation of focal adhesions function independently from those of lamin A/C. 

Meniscus allograft transplantations (MATs) represent established surgical procedures with proven outcomes. Yet, storage as frozen specimens and limited cellular repopulation may impair graft viability. This proof‐of‐concept study tests the feasibility of injecting allogeneic mesenchymal stromal/stem cells (MSCs) in meniscus allograft tissue. We investigated the injectable cell quantity, survival rate, migration, and proliferation ability of MSCs up to 28 days of incubation. In this controlled laboratory study, seven fresh‐frozen human allografts were injected with human allogeneic MSCs. Cells were labeled and histological characteristics were microscopically imaged up to 28 days. Mock‐injected menisci were included as negative controls in each experiment. Toluidine blue staining demonstrated that a 100‐µl volume can be injected while retracting and rotating the inserted needle. Immediately after injection, labeled MSCs were distributed throughout the injection channel and eventually migrated into the surrounding tissues. Histological assessment revealed that MSCs cluster in disc‐like shapes, parallel to the intrinsic lamination of the meniscus and around the vascular network. Quantification showed that more than 60% of cells were present in horizontally injected grafts and more than 30% were observed in vertically injected samples. On Day 14, cells adopted a spindle‐shaped morphology and exhibited proliferative and migratory behaviors. On Day 28, live/dead ratio assessment revealed an approximately 80% cell survival. The study demonstrated the feasibility of injecting doses of MSCs (>0.1 million) in meniscus allograft tissue with active cell proliferation, migration, and robust cell survival.

 

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Plasminogen activator inhibitor 1 (PAI‐1/SERPINE1), a master suppressor of fibrinolysis and protease activity, is associated with adhesions. Here, we used next‐generation RNA sequencing (RNA‐Seq) to assess genome‐wide differences in messenger RNA expression due to PAI‐1 deficiency after zone II flexor tendon injury. We used the ingenuity pathway analysis to characterize molecular pathways and biological drivers associated with differentially expressed genes (DEG). Analysis of hundreds of overlapping and DEG in PAI‐1 knockout (KO) and wild‐type mice (C57Bl/6J) during tendon healing revealed common and distinct biological processes. Pathway analysis identified cell proliferation, survival, and senescence, as well as chronic inflammation as potential drivers of fibrotic healing and adhesions in injured tendons. Importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1‐associated biological processes as unique transcriptional signatures of the healing tendon in the PAI‐1/Serpine1 KO mice. Further, transcriptomic differences due to the genetic deletion of PAI‐1 were mechanistically linked to PI3K/Akt/mTOR, PKC, and MAPK signaling cascades. These transcriptional observations provide novel insights into the biological roles of PAI‐1 in tendon healing and could identify therapeutic targets to achieve scar‐free regenerative healing of tendons. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:43–58, 2020