Engineering multi-tissue units for regenerative Medicine: Bone-tendon-muscle units of the rotator cuff.

Our body systems are comprised of numerous multi-tissue units. For the musculoskeletal system, one of the predominant functional units is comprised of bone, tendon/ligament, and muscle tissues working in tandem to facilitate locomotion. To successfully treat musculoskeletal injuries and diseases, critical consideration and thoughtful integration of clinical, biological, and engineering aspects are necessary to achieve translational bench-to-bedside research. In particular, identifying ideal biomaterial design specifications, understanding prior and recent tissue engineering advances, and judicious application of biomaterial and fabrication technologies will be crucial for addressing current clinical challenges in engineering multi-tissue units. Using rotator cuff tears as an example, insights relevant for engineering a bone-tendon-muscle multi-tissue unit are presented. This review highlights the tissue engineering strategies for musculoskeletal repair and regeneration with implications for other bone-tendon-muscle units, their derivatives, and analogous non-musculoskeletal tissue structures.

Hippo kinases MST1 and MST2 control the differentiation of the epididymal initial segment via the MEK-ERK pathway.

Although the roles of the Hippo pathway in organogenesis and tumorigenesis have been well studied in multiple organs, its role in sperm maturation and male fertility has not been investigated. The initial segment (IS) of the epididymis plays a critical role in sperm maturation. IS differentiation is governed by ERK1/2, but the mechanisms of ERK1/2 activation in IS are not fully understood. Here we show that double knockout (dKO) of mammalian sterile 20-like kinases 1 and 2 (Mst1 and Mst2), homologs of Hippo in Drosophila, in the epididymal epithelium led to male infertility in mice. Sperm in the cauda epididymides of mutant mice were immotile with flagellar angulation and severely disorganized structures. Loss of Mst1/2 activated YAP and increased proliferation and cell death in all the segments of epididymis. The mutant mice showed substantially suppressed MEK/ERK signaling in the IS and failed IS differentiation. Deletion of Yap restored the reduced MEK/ERK signaling, and partially rescued the defective IS differentiation and fertility in Mst1/2 dKO mice. Our results demonstrate that YAP inhibits the MEK/ERK pathway in IS epithelial cells, and MST1/2 control IS differentiation and fertility at least partially by repressing YAP. Taken together, the Hippo pathway is essential for sperm maturation and male fertility.

Tubule-Specific Mst1/2 Deficiency Induces CKD via YAP and Non-YAP Mechanisms.

BACKGROUND: The serine/threonine kinases MST1 and MST2 are core components of the Hippo pathway, which has been found to be critically involved in embryonic kidney development. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the pathway's main effectors. However, the biologic functions of the Hippo/YAP pathway in adult kidneys are not well understood, and the functional role of MST1 and MST2 in the kidney has not been studied. METHODS: We used immunohistochemistry to examine expression in mouse kidneys of MST1 and MST2, homologs of Hippo in Drosophila. We generated mice with tubule-specific double knockout of Mst1 and Mst2 or triple knockout of Mst1, Mst2, and Yap. PCR array and mouse inner medullary collecting duct cells were used to identify the primary target of Mst1/Mst2 deficiency. RESULTS: MST1 and MST2 were predominantly expressed in the tubular epithelial cells of adult kidneys. Deletion of Mst1/Mst2 in renal tubules increased activity of YAP but not TAZ. The kidneys of mutant mice showed progressive inflammation, tubular and glomerular damage, fibrosis, and functional impairment; these phenotypes were largely rescued by deletion of Yap in renal tubules. TNF-α expression was induced via both YAP-dependent and YAP-independent mechanisms, and TNF-α and YAP amplified the signaling activities of each other in the tubules of kidneys with double knockout of Mst1/Mst2. CONCLUSIONS: Our findings show that tubular Mst1/Mst2 deficiency leads to CKD through both the YAP and non-YAP pathways and that tubular YAP activation induces renal fibrosis. The pathogenesis seems to involve the reciprocal stimulation of TNF-α and YAP signaling activities.

Wnt16 attenuates osteoarthritis progression through a PCP/JNK-mTORC1-PTHrP cascade.

OBJECTIVES: Wnt16 is implicated in bone fracture and bone mass accrual both in animals and humans. However, its functional roles and molecular mechanism in chondrocyte differentiation and osteoarthritis (OA) pathophysiology remain largely undefined. In this study, we analysed its mechanistic association and functional relationship in OA progression in chondrocyte lineage. METHODS: The role of Wnt16 during skeletal development was examined by Col2a1-Wnt16 transgenic mice and Wnt16fl/fl;Col2a1-Cre (Wnt16-cKO) mice. OA progression was assessed by micro-CT analysis and Osteoarthritis Research Society International score after anterior cruciate ligament transection (ACLT) surgery with Wnt16 manipulation by adenovirus intra-articular injection. The molecular mechanism was investigated in vitro using 3D chondrocyte pellet culture and biochemical analyses. Histological analysis was performed in mouse joints and human cartilage specimens. RESULTS: Wnt16 overexpression in chondrocytes in mice significantly inhibited chondrocyte hypertrophy during skeletal development. Wnt16 deficiency exaggerated OA progression, whereas intra-articular injection of Ad-Wnt16 markedly attenuated ACLT-induced OA. Cellular and molecular analyses showed that, instead of ß-catenin and calcium pathways, Wnt16 activated the planar cell polarity (PCP) and JNK pathway by interacting mainly with AP2b1, and to a lesser extend Ror2 and CD146, and subsequently induced PTHrP expression through phosphor-Raptor mTORC1 pathway. CONCLUSIONS: Our findings indicate that Wnt16 activates PCP/JNK and crosstalks with mTORC1-PTHrP pathway to inhibit chondrocyte hypertrophy. Our preclinical study suggests that Wnt16 may be a potential therapeutic target for OA treatment.

Mst1/2 Kinases Modulate Glucose Uptake for Osteoblast Differentiation and Bone Formation.

Bone formation and bone homeostasis are energy-expensive processes. How they are being regulated by energy needs is not completely understood. This is of high clinical importance because diabetic-induced bone loss is common whereas the underlying mechanisms are unclear. Here, we show that Mst1/2 are important regulators for glucose uptake during osteoblast differentiation. Genetically removal of both Mst1/2 kinases simultaneously in mice in early and mature osteoblasts inhibits bone formation and bone remodeling, respectively. We found that the activity of Mst1/2 kinases is sensitive to glucose levels, and in turn, regulates glucose uptake by stabilizing key glucose transporter Glut1. In the absence of Mst1/2 kinases, Glut1 expression is loss and results in AMP-dependent protein kinase (AMPK) activation and subsequent proteasomal degradation of Runx2. The streptozotocin (STZ)-induced diabetic mouse model also recapitulates similar changes in the bone tissues. In addition, Glut1 expression regulated by Mst1/2 kinases is independent of Yap/Taz expression. Our results unravel new mechanistic insights into the orchestration of glucose level and bone homeostasis. © 2018 American Society for Bone and Mineral Research.

Osteocalcin expressing cells from tendon sheaths in mice contribute to tendon repair by activating Hedgehog signaling.

Both extrinsic and intrinsic tissues contribute to tendon repair, but the origin and molecular functions of extrinsic tissues in tendon repair are not fully understood. Here we show that tendon sheath cells harbor stem/progenitor cell properties and contribute to tendon repair by activating Hedgehog signaling. We found that Osteocalcin (Bglap) can be used as an adult tendon-sheath-specific marker in mice. Lineage tracing experiments show that Bglap-expressing cells in adult sheath tissues possess clonogenic and multipotent properties comparable to those of stem/progenitor cells isolated from tendon fibers. Transplantation of sheath tissues improves tendon repair. Mechanistically, Hh signaling in sheath tissues is necessary and sufficient to promote the proliferation of Mkx-expressing cells in sheath tissues, and its action is mediated through TGFß/Smad3 signaling. Furthermore, co-localization of GLI1+ and MKX+ cells is also found in human tendinopathy specimens. Our work reveals the molecular function of Hh signaling in extrinsic sheath tissues for tendon repair.