Spatial and Temporal Expression of Molecular Markers and Cell Signals During Normal Development of the Mouse Patellar Tendon

Division of Developmental Biology, Cincinnati Children's Hospital Research Foundation, Cincinnati, OH 45229, USA.
Tissue Engineering Part A (Impact Factor: 4.64). 09/2011; 18(5-6):598-608. DOI: 10.1089/ten.TEA.2011.0338
Source: PubMed


Tendon injuries are common clinical problems and are difficult to treat. In particular, the tendon-to-bone insertion site, once damaged, does not regenerate its complex zonal arrangement. A potential treatment for tendon injuries is to replace injured tendons with bioengineered tendons. However, the bioengineering of tendon will require a detailed understanding of the normal development of tendon, which is currently lacking. Here, we use the mouse patellar tendon as a model to describe the spatial and temporal pattern of expression of molecular markers for tendon differentiation from late fetal life to 2 weeks after birth. We found that collagen I, fibromodulin, and tenomodulin were expressed throughout the tendon, whereas tenascin-C, biglycan, and cartilage oligomeric protein were concentrated in the insertion site during this period. We also identified signaling pathways that are activated both throughout the developing tendon, for example, transforming growth factor beta and bone morphogenetic protein, and specifically in the insertion site, for example, hedgehog pathway. Using a mouse line expressing green fluorescent protein in all tenocytes, we also found that tenocyte cell proliferation occurs at highest levels during late fetal life, and declines to very low levels by 2 weeks after birth. These data will allow both the functional analysis of specific signaling pathways in tenocyte development and their application to tissue-engineering studies in vitro.

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Available from: David Butler, Oct 16, 2014
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    • "At later embryonic stages, enthesis cells express Gli1 and cells within unmineralized regions of the enthesis maintain this expression during later stages of postnatal growth (Schwartz et al., 2015). At maturity, a cell phenotype gradient forms between tendon and bone with an extracellular matrix that shares characteristics from tendon and cartilage (e.g., type I and type II collagens, aggrecan, and tenascin-C) (Galatz et al., 2007; C. Liu et al., 2012; Wang et al., 2012). To better delineate the processes of enthesis formation, particularly in comparison to the adjacent tendon and primary cartilage, requires novel reporter mice (e.g., collagen I/II/X fluorescent reporter mice), multiple imaging modalities (e.g., two photon collagen imaging), and histomorphometry (e.g., quantification of mineral apposition via fluorescent labeling). "
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    ABSTRACT: The sequence of events that leads to the formation of a functionally graded enthesis is not clearly defined. The current study demonstrates that clonal expansion of Gdf5 progenitors contributes to linear growth of the enthesis. Prior to mineralization, Col1+ cells in the enthesis appose Col2+ cells of the underlying primary cartilage. At the onset of enthesis mineralization, cells at the base of the enthesis express alkaline phosphatase, Indian hedgehog, and ColX as they mineralize. The mineralization front then extends towards the tendon midsubstance as cells above the front become encapsulated in mineralized fibrocartilage over time. The hedgehog (Hh) pathway regulates this process, as Hh-responsive Gli1+ cells within the developing enthesis mature from unmineralized to mineralized fibrochondrocytes in response to activated signaling. Hh signaling is required for mineralization, as tissue-specific deletion of its obligate transducer Smoothened in the developing tendon and enthesis cells leads to significant reductions in the apposition of mineralized fibrocartilage. Together, these findings provide a spatiotemporal map of events-from expansion of the embryonic progenitor pool to synthesis of the collagen template and finally mineralization of this template-that leads to the formation of the mature zonal enthesis. These results can inform future tendon-to-bone repair strategies to create a mechanically functional enthesis in which tendon collagen fibers are anchored to bone through mineralized fibrocartilage. Copyright © 2015. Published by Elsevier Inc.
    Developmental Biology 06/2015; 405(1). DOI:10.1016/j.ydbio.2015.06.020 · 3.55 Impact Factor
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    • "Cells from these mice displayed membrane-associated red fluorescence (mTomato), except for Hhresponsive (Gli1-expressing) cells, where the red fluorescence was replaced by green fluorescence (mGFP). Hh-responsive cells were detected in the postnatal supraspinatus enthesis, consistent with previous reports for other entheses (Blitz et al., 2009; Liu et al., 2012; Wang et al., 2006). The Gli1-expressing cells were present at E16.5 when TAM was injected at E14.5, and at the immature tendon-tobone interface at P0 with E16.5 TAM injection (Fig. 1A,B). "
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    ABSTRACT: Tendon attaches to bone across a specialized tissue called the enthesis. This tissue modulates the transfer of muscle forces between two materials, i.e. tendon and bone, with vastly different mechanical properties. The enthesis for many tendons consists of a mineralized graded fibrocartilage that develops postnatally, concurrent with epiphyseal mineralization. Although it is well described that the mineralization and development of functional maturity requires muscle loading, the biological factors that modulate enthesis development are poorly understood. By genetically demarcating cells expressing Gli1 in response to Hedgehog (Hh) signaling, we discovered a unique population of Hh-responsive cells in the developing murine enthesis that were distinct from tendon fibroblasts and epiphyseal chondrocytes. Lineage-tracing experiments revealed that the Gli1 lineage cells that originate in utero eventually populate the entire mature enthesis. Muscle paralysis increased the number of Hh-responsive cells in the enthesis, demonstrating that responsiveness to Hh is modulated in part by muscle loading. Ablation of the Hh-responsive cells during the first week of postnatal development resulted in a loss of mineralized fibrocartilage, with very little tissue remodeling 5 weeks after cell ablation. Conditional deletion of smoothened, a molecule necessary for responsiveness to Ihh, from the developing tendon and enthesis altered the differentiation of enthesis progenitor cells, resulting in significantly reduced fibrocartilage mineralization and decreased biomechanical function. Taken together, these results demonstrate that Hh signaling within developing enthesis fibrocartilage cells is required for enthesis formation. © 2015. Published by The Company of Biologists Ltd.
    Development 01/2015; 142(1):196-206. DOI:10.1242/dev.112714 · 6.46 Impact Factor
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    • "During mouse patellar tendon development, all cells in the tendon were found to respond to TGFβ and BMP signaling at all stages examined, including embryonic and postnatal periods (Liu et al., 2012). In vitro micromass culture of chick mesodermal cells with TGFβ demonstrated significant upregulation of tendon markers Scx and Tnmd, with concurrent reduction in cartilage markers. "
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    ABSTRACT: As dense connective tissues connecting bone to muscle and bone to bone, respectively, tendon and ligament (T/L) arise from the somitic mesoderm, originating in a recently discovered somitic compartment, the syndetome. Inductive signals from the adjacent sclerotome and myotome upregulate expression of Scleraxis, a key transcription factor for tenogenic and ligamentogenic differentiation. Understanding T/L development is critical to establishing a knowledge base for improving the healing and repair of T/L injuries, a high-burden disease due to the intrinsically poor natural healing response. Current treatment of the three most common tendon injuries-tearing of the rotator cuff of the shoulder, flexor tendon of the hand, and Achilles tendon-include mostly surgical repair and/or conservative approaches, including biophysical modalities such as rehabilitation and cryotherapy. Unfortunately, the fibrovascular scar formed during healing possesses inferior mechanical and biochemical properties, resulting in compromised tissue functionality. Regenerative approaches have sought to augment the injured tissue with cells, scaffolds, bioactive agents, and mechanical stimulation to improve the natural healing response. The key challenges in restoring full T/L function following injury include optimal combination of these biological agents as well as their delivery to the injury site. A greater understanding of the molecular mechanisms involved in T/L development and natural healing, coupled with the capability of producing complex biomaterials to deliver multiple biofactors with high spatiotemporal resolution and specificity, should lead to regenerative procedures that more closely recapitulate T/L morphogenesis, thereby offering future patients the prospect of T/L regeneration, as opposed to simple tissue repair. Text. Birth Defects Research (Part C) 99:203-222, 2013. © 2013 Wiley Periodicals, Inc.
    Birth Defects Research Part C Embryo Today Reviews 09/2013; 99(3):203-22. DOI:10.1002/bdrc.21041 · 2.63 Impact Factor
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