The Relationships Among Spatiotemporal Gene Expression, Histology, and Biomechanics Following Full-Length Injury in the Murine Patellar Tendon

University of Cincinnati, Cincinnati, OH, USA.
Journal of Orthopaedic Research (Impact Factor: 2.99). 06/2011; 30(1):28-36. DOI: 10.1002/jor.21484
Source: PubMed


Tendon injuries are major orthopedic problems that worsen as the population ages. Type-I (Col1) and type-II (Col2) collagens play important roles in tendon midsubstance and tendon-to-bone insertion healing, respectively. Using double transgenic mice, this study aims to spatiotemporally monitor Col1 and Col2 gene expression, histology, and biomechanics up to 8 weeks following a full-length patellar tendon injury. Gene expression and histology were analyzed weekly for up to 5 weeks while mechanical properties were measured at 1, 2, 5, and 8 weeks. At week 1, the healing region displayed loose granulation tissue with little Col1 expression. Col1 expression peaked at 2 weeks, but the ECM was highly disorganized and hypercellular. By 3 weeks, Col1 expression had reduced and by 5 weeks, the ECM was generally aligned along the tendon axis. Col2 expression was not seen in the healing midsubstance or insertion at any time point. The biomechanics of the healing tissue was inadequate at all time points, achieving ultimate loads and stiffnesses of 48% and 63% of normal values by 8 weeks. Future studies will further characterize the cells within the healing midsubstance and insertion using tenogenic markers and compare these results to those of tendon cells during normal development.


Available from: Jason Shearn
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    • "Since SMA9 progenitor cells were found to contribute to cell turnover during tendon growth, we next determined whether they contribute to tendon healing in the adult. We utilized a full-length, central-third patellar tendon defect injury used previously [20], [25] to examine the reparative potential of SMA9+ cells in the paratenon and tendon midsubstance. One week following injury, there is notable expansion of SMA9+ cells in the thickened paratenon, which forms in response to injury (Fig. 5A–G and S5A,E,I). "
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    ABSTRACT: Unlike during embryogenesis, the identity of tissue resident progenitor cells that contribute to postnatal tendon growth and natural healing is poorly characterized. Therefore, we utilized 1) an inducible Cre driven by alpha smooth muscle actin (SMACreERT2), that identifies mesenchymal progenitors, 2) a constitutively active Cre driven by growth and differentiation factor 5 (GDF5Cre), a critical regulator of joint condensation, in combination with 3) an Ai9 Cre reporter to permanently label SMA9 and GDF5-9 populations and their progeny. In growing mice, SMA9+ cells were found in peritendinous structures and scleraxis-positive (ScxGFP+) cells within the tendon midsubstance and myotendinous junction. The progenitors within the tendon midsubstance were transiently labeled as they displayed a 4-fold expansion from day 2 to day 21 but reduced to baseline levels by day 70. SMA9+ cells were not found within tendon entheses or ligaments in the knee, suggesting a different origin. In contrast to the SMA9 population, GDF5-9+ cells extended from the bone through the enthesis and into a portion of the tendon midsubstance. GDF5-9+ cells were also found throughout the length of the ligaments, indicating a significant variation in the progenitors that contribute to tendons and ligaments. Following tendon injury, SMA9+ paratenon cells were the main contributors to the healing response. SMA9+ cells extended over the defect space at 1 week and differentiated into ScxGFP+ cells at 2 weeks, which coincided with increased collagen signal in the paratenon bridge. Thus, SMA9-labeled cells represent a unique progenitor source that contributes to the tendon midsubstance, paratenon, and myotendinous junction during growth and natural healing, while GDF5 progenitors contribute to tendon enthesis and ligament development. Understanding the mechanisms that regulate the expansion and differentiation of these progenitors may prove crucial to improving future repair strategies.
    PLoS ONE 04/2014; 9(4):e96113. DOI:10.1371/journal.pone.0096113 · 3.23 Impact Factor
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    • "These cells migrate to the wound site then express Scx as the tenascin-rich provisional matrix transitions to a collagenous matrix. While direct correlations with biomechanical properties have yet to be made, increased Scx expression during remodeling appears to be commensurate with increased biomechanical properties (Dyment et al., 2011; Scott et al., 2011). Scx expression is necessary, but certainly not sufficient, for the formation of mechanically functional tendon. "
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    ABSTRACT: Improving tendon repair using Functional Tissue Engineering (FTE) principles has been the focus of our laboratory over the last decade. Although our primary goals were initially focused only on mechanical outcomes, we are now carefully assessing the biological properties of our tissue-engineered tendon repairs so as to link biological influences with mechanics. However, given the complexities of tendon development and healing, it remains challenging to determine which aspects of tendon biology are the most important to focus on in the context of tissue engineering. To address this problem, we have formalized a strategy to identify, prioritize, and evaluate potential biological success criteria for tendon repair. We have defined numerous biological properties of normal tendon relative to cellular phenotype, extracellular matrix and tissue ultra-structure that we would like to reproduce in our tissue-engineered repairs and prioritized these biological criteria by examining their relative importance during both normal development and natural tendon healing. Here, we propose three specific biological criteria which we believe are essential for normal tendon function: (1) scleraxis-expressing cells; (2) well-organized and axially-aligned collagen fibrils having bimodal diameter distribution; and (3) a specialized tendon-to-bone insertion site. Moving forward, these biological success criteria will be used in conjunction with our already established mechanical success criteria to evaluate the effectiveness of our tissue-engineered tendon repairs.
    Journal of Biomechanics 10/2013; 47(9). DOI:10.1016/j.jbiomech.2013.10.023 · 2.75 Impact Factor
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    • "The curves represent the average of 10 specimens; error bars indicate SEM. Dyment et al., 2012). This finding, coupled with the fact that crosssectional area increased significantly in the injured tendons, indicates that failed healing is not the result of a lack of new tissue formation, but instead is due to the inability of the healing soft tissue to reintegrate with bone. "
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    ABSTRACT: Tendon-to-bone healing following acute injury is generally poor and often fails to restore normal tendon biomechanical properties. In recent years, the murine patellar tendon (PT) has become an important model system for studying tendon healing and repair due to its genetic tractability and accessible location within the knee. However, the mechanical properties of native murine PT, specifically the regional differences in tissue strains during loading, and the biomechanical outcomes of natural PT-to-bone healing have not been well characterized. Thus, in this study, we analyzed the global biomechanical properties and regional strain patterns of both normal and naturally healing murine PT at three time points (2, 5, and 8weeks) following acute surgical rupture of the tibial enthesis. Normal murine PT exhibited distinct regional variations in tissue strain, with the insertion region experiencing approximately 2.5 times greater strain than the midsubstance at failure (10.80±2.52% vs. 4.11±1.40%; mean±SEM). Injured tendons showed reduced structural (ultimate load and linear stiffness) and material (ultimate stress and linear modulus) properties compared to both normal and contralateral sham-operated tendons at all healing time points. Injured tendons also displayed increased local strain in the insertion region compared to contralateral shams at both physiologic and failure load levels. 93.3% of injured tendons failed at the tibial insertion, compared to only 60% and 66.7% of normal and sham tendons, respectively. These results indicate that 8weeks of natural tendon-to-bone healing does not restore normal biomechanical function to the murine PT following injury.
    Journal of Biomechanics 10/2013; 47(9). DOI:10.1016/j.jbiomech.2013.10.029 · 2.75 Impact Factor
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