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1.  Alfacalcidol enhances collagen quality in ovariectomized rat bones 
The aim of this study was to investigate the effects of alfacalcidol (1α(OH)D3: ALF) on bone collagen employing an ovariectomized rat model. Thirty-five 16-week-old female Sprague-Dawley rats were divided into five groups: SHAM (sham-operated + vehicle), OVX (ovariectomy + vehicle), and three ALF-treated groups, i.e. ovariectomy + 0.022μg/kg/day ALF, ovariectomy + 0.067μg/kg/day ALF and ovariectomy + 0.2 μg/kg/day ALF. After 12 weeks of treatment, tibiae were subjected to histological, biochemical and immunohistochemical analyses. Collagen matrices in OVX bone appeared as immature and poorly organized, however with the ALF treatment, it was improved in a dose-dependent manner. Contents of collagen and pyridinoline cross-link were decreased in OVX compared with SHAM, but they increased to the level comparable to SHAM in ALF-treated groups. The total aldehyde, i.e. a sum of free and those involved cross-links, in the highest dose of ALF was significantly higher than the rest of the groups (p< 0.05). In addition, the expression of lysyl oxidase was increased in the all ALF-treated groups compared with OVX (p< 0.05). In conclusion, ALF increases not only the amount of collagen but also enhances the maturation of collagen in ovariectomy-induced osteoporotic bones, which likely contributes to the improvement of bone quality.
PMCID: PMC4071133  PMID: 24809324
Vitamin D; Alfacalcidol; Collagen; Collagen cross-link; Ovariectomized rats
2.  MicroRNA-1(miR-1) inhibits chordoma cell migration and invasion by targeting Slug 
Recent studies have revealed that expression of miRNA-1(miR-1) is frequently downregulated in several cancer types including chordoma. Identifying and validating novel targets of miR-1 is useful for understanding the roles of miR-1 in chordoma. We aimed to further investigate the functions of miR-1 in chordoma. Specifically, we assessed whether restoration of miR-1 affects cell migration and invasion in chordoma, and focused on the miR-1 potential target Slug gene. Migratory and invasive activities were assessed by wound healing and Matrigel invasion assays, respectively. Cell proliferation was determined by MTT assay. Slug expression was evaluated by Western blot, immunofluorescence, and immunohistochemistry. Restoration of miR-1 expression suppressed the migratory and invasive activities of chordoma cells. Transfection of miR-1 inhibited cell proliferation both time- and dose-dependently in chordoma. miR-1 transfected cells showed inhibited Slug expression. Slug was overexpressed in chordoma cell lines and advanced chordoma tissues. In conclusion, we have shown that miR-1 directly targets the Slug gene in chordoma. Restoration of miR-1 suppressed not only proliferation, but also migratory and invasive activities, and reduced the Slug expression in chordoma cells. These results collectively indicate that miR-1/Slug pathway is a potential therapeutic target because of its crucial roles in chordoma cell growth and migration.
PMCID: PMC4123853  PMID: 24760686
miRNA-1(miR-1); Chordoma; Invasion; Migration; Slug
3.  Foot and Ankle Joint Movements Inside Orthoses for Children with Spastic CP 
We compared the ankle joint and foot segment kinematics of pediatric cerebral palsy (CP) participants walking with and without orthoses. A six segment foot model (6SF) was used to track foot motion. Holes were cut in the study orthoses so that electromagnetic markers could be directly placed on the skin. The Hinged Ankle Foot Orthoses (HAFO) allowed a significant increase in ankle dorsiflexion as compared to the barefoot condition during gait, but significantly constrained sagittal forefoot motion and forefoot sagittal range of motion (ROM) (p < 0.01), which may be detrimental. The Solid Ankle Foot Orthoses (SAFO) constrained forefoot ROM as compared to barefoot gait (p < 0.01). The 6SF model did not confirm that the SAFO can control excessive plantarflexion for those with severe plantarflexor spasticity. The supramalleolar orthosis (SMO) significantly (p < 0.01) constrained forefoot ROM as compared to barefoot gait at the beginning and end of the stance phase, which could be detrimental. The SMO had no effects observed in the coronal plane.
PMCID: PMC4502580  PMID: 24375587
cerebral palsy; orthoses; gait; foot segment model
4.  Changes in Macrophage Phenotype and Induction of Epithelial-to-Mesenchymal Transition Genes Following Acute Achilles Tenotomy and Repair 
Tendon injuries occur frequently in physically active individuals, but the clinical outcomes for these injuries can be poor. In many injured tissues the repair process is orchestrated by two types of cells, macrophages and fibroblasts. Macrophages, which have both proinflammatory (M1) and antiinflammatory (M2) phenotypes, can directly participate in tissue remodeling and direct the response of other cells through the secretion of cytokines and growth factors. In many organ systems, epithelial cells can transdifferentiate into fibroblasts, which can then regenerate damaged ECM. This process is triggered via activation of epithelial-to-mesenchymal transition (EMT) signaling programs. Most tendons are surrounded by sheets of epithelial cells, and these tissue layers could provide a source of fibroblasts to repair injured tendons. To gain greater insight into the biology of tendon repair, we performed a tenotomy and repair in Achilles tendons of adult rats and determined changes in macrophage phenotype, and ECM- and EMT-related genes over a four week time course. The results from this study suggest that changes in macrophage phenotype and activation of EMT-related programs likely contribute to the degradation and subsequent repair of injured tendon tissue.
PMCID: PMC4086481  PMID: 24700411
tendon; macrophage; epithelial-to-mesenchymal transition; repair; fibrosis
5.  High Resolution Muscle Measurements Provide Insights into Equinus Contractures in Patients with Cerebral Palsy 
Muscle contractures that occur after upper motor neuron lesion are often surgically released or lengthened. However, surgical manipulation of muscle length changes a muscle’s sarcomere length (Ls), which can affect force production. To predict effects of surgery, both macro- (fascicle length (Lf)) and micro- (Ls) level structural measurements are needed. Therefore, the purpose of this study was to quantify both Ls and Lf in patients with cerebral palsy (CP) as well as typically developing (TD) children. Soleus ultrasound images were obtained from children with CP and TD children. Lf was determined and, with the joint in the same position, CP biopsies were obtained, formalin fixed and Ls measured by laser diffraction. Since soleus Ls values were not measurable in TD children, TD Ls values were obtained using three independent methods. While average Lf did not differ between groups (CP=3.6±1.2 cm, TD=3.5±0.9 cm; p>0.6), Ls was dramatically longer in children with CP (4.07±0.45 μm vs. TD=2.17±0.24 μm; p<0.0001). While Lf values were similar between children with CP and TD children, this was due to highly stretched sarcomeres within the soleus muscle. Surgical manipulation of muscle-tendon unit length will thus alter muscle sarcomere length and change force generating capacity of the muscle.
PMCID: PMC4343320  PMID: 25242618
cerebral palsy; skeletal muscle; sarcomere; fascicle length; ultrasound
Endplate deflection frequently occurs with vertebral failure, but the relationship between the two remains poorly defined. This study examined associations between endplate deflection under compressive loading and characteristics of the neighboring subchondral bone and intervertebral disc (IVD). Ten L1 vertebrae with adjacent IVDs were dissected, compressed axially in a stepwise manner to failure, and imaged with micro-computed tomography before each loading step. From the images, deflection was measured across the surface of each endplate at each step. Trabecular microstructure and endplate volume fraction were evaluated in 5mm regions just under the superior endplate. IVDs were assessed using computed tomography and histology. A marked increase in superior endplate deflection coincided with a drop in the load-displacement curve. Endplate deflection was higher in regions with less robust bone microstructure (p<0.009), though these associations tended to weaken as loading progressed. Immediately following the ultimate point, endplate deflection was higher in regions underlying the nucleus pulposus vs. annulus fibrosus (p=0.035), irrespective of disc grade (p=0.346). These results indicate that a sudden increase in endplate deflection signals that the mechanical competence of the vertebra has been compromised. The mechanisms of endplate failure likely relate to anatomical features of the endplate, neighboring trabecular bone, and IVD.
PMCID: PMC4450106  PMID: 24700382
vertebral fracture; vertebral endplate; intervertebral disc; cancellous bone; spine biomechanics
7.  Remodeling of Murine Intrasynovial Tendon Adhesions following Injury: MMP and Neotendon Gene Expression 
Tendon injury frequently results in the formation of adhesions that reduce joint range of motion. To study the cellular, molecular, and biomechanical events involved in intrasynovial tendon healing and adhesion formation, we developed a murine flexor tendon healing model in which the flexor digitorum longus (FDL) tendon of C57BL/6 mice was transected and repaired using suture. This model was used to test the hypothesis that murine flexor tendons heal with differential expression of matrix metalloproteases (MMPs), resulting in the formation of scar tissue as well as the subsequent remodeling of scar and adhesions. Healing tendons were evaluated by histology, gene expression via real-time RT-PCR, and in situ hybridization, as well as biomechanical testing to assess the metatarsophalangeal (MTP) joint flexion range of motion (ROM) and the tensile failure properties. Tendons healed with a highly disorganized fibroblastic tissue response that was progressively remodeled through day 35 resulting in a more organized pattern of collagen fibers. Initial repair involved elevated levels of Mmp-9 at day 7, which is associated with catabolism of damaged collagen fibers. High levels of Col3 are consistent with scar tissue, and gradually transition to the expression of Col1. Scleraxis expression peaked at day 7, but the expression was limited to the original tendon adjacent to the injury site, and no expression was present in granulation tissue involved in the repair response. The MTP joint ROM with standardized force on the tendon was decreased on days 14 and 21 compared to day 0, indicating the presence of adhesions. Peak expressions of Mmp-2 and Mmp-14 were observed at day 21, associated with tendon remodeling. At day 28, two genes associated with neotendon formation, Smad8 and Gdf-5, were elevated and an improvement in MTP ROM occurred. Tensile strength of the tendon progressively increased, but by 63 days the repaired tendons had not reached the tensile strength of normal tendon. The murine model of primary tendon repair, described here, provides a novel mechanism to study the tendon healing process, and further enhances the understanding of this process at the molecular, cellular, and biomechanical level.
PMCID: PMC4472455  PMID: 19051246
flexor tendon healing; matrix metalloproteases; adhesions; mouse model; biomechanics
8.  Joint instability and cartilage compression in a mouse model of posttraumatic osteoarthritis 
Joint instability and cartilage trauma have been previously studied and identified as key mediators in the development of posttraumatic osteoarthritis (PTOA). The purpose of this study was to use an in vivo model to compare the effect of joint instability, caused by the rupture of the anterior cruciate ligament (ACL), versus cartilage compression. In this study, mice were subjected to cyclical axial loads of twelve Newtons (N) for 240 cycles or until the ACL ruptured. One and eight weeks after this procedure, knees were sectioned coronally and evaluated for osteoarthritis by histology. Using a scoring scale established by Pritzker et al. 2006, the articular cartilage across each surface was scored and combined to produce a total degeneration score. The ACL-ruptured group had a significantly greater total degeneration score than either control or compression treated joints at one and eight weeks. Additionally, only sections from ACL-ruptured knees consistently showed synovitis after one week and osteophyte formation after eight weeks. Thus, it appears using that ACL rupture consistently creates a severe osteoarthritis phenotype, while axial cartilage compression alone does not appear to be an appropriate method of inducing PTOA in vivo.
PMCID: PMC4462343  PMID: 24167068
Post-traumatic osteoarthritis (PTOA) is characterized by progressive cartilage degeneration in injured joints. Since fibronectin fragments (Fn-fs) degrade cartilage mainly through up-regulating matrix metalloproteinases (MMPs) and pro-inflammatory cytokines, we hypothesized that Fn-fs play a key role in PTOA by promoting chondrolysis in and around injured cartilage. To test this hypothesis, we profiled the catabolic events focusing on fibronectin fragmentation and proteinase expression in bovine osteochondral explants following a single blunt impact on cartilage with a drop tower device which created partial-thickness tissue damage. Injured and control explants were cultured for up to 14 days. The presence of Fn-fs, MMPs (-1, -3, -13), ADAMTS-5 in culture media and in cartilage was determined with immunoblotting. The daily proteoglycan (PG) depletion of cartilage matrix was assessed with DMMB assay. The effect of explant-conditioned media on chondrocytes was also examined with immunoblotting. Impacted cartilage released significantly higher amount of native Fn, three chondrolytic Fn-fs and PG than non-impacted controls did. Those increases coincided with up-regulation of MMP-3 both in conditioned media and in impacted cartilage. These findings support our hypothesis that PTOA may be propelled by Fn-fs which act as catabolic mediators through up-regulating cartilage-damaging proteinases.
PMCID: PMC4034576  PMID: 24610678
cartilage; fibronectin fragments; impact; matrix metalloproteinase-3; proteoglycan
10.  Changes in Tibiofemoral Forces due to Variations in Muscle Activity during Walking 
Muscles induce large forces in the tibiofemoral joint during walking and thereby influence the health of tissues like articular cartilage and menisci. It is possible to walk with a wide variety of muscle coordination patterns, but the effect of varied muscle coordination on tibiofemoral contact forces remains unclear. The goal of this study was to determine the effect of varied muscle coordination on tibiofemoral contact forces. We developed a musculoskeletal model of a subject walking with an instrumented knee implant. Using an optimization framework, we calculated the tibiofemoral forces resulting from muscle coordination that reproduced the subject’s walking dynamics. We performed a large set of optimizations in which we systematically varied the coordination of muscles to determine the influence on tibiofemoral force. Model-predicted tibiofemoral forces arising with minimum muscle activation matched in vivo forces measured during early stance, but were greater than in vivo forces during late stance. Peak tibiofemoral forces during late stance could be reduced by increasing the activation of the gluteus medius, uniarticular hip flexors, and soleus, and by decreasing the activation of the gastrocnemius and rectus femoris. These results suggest that retraining of muscle coordination could substantially reduce tibiofemoral forces during late stance.
PMCID: PMC4409006  PMID: 24615885
tibiofemoral force; walking; coordination; muscle activity; knee
11.  Development of Antisense Oligonucleotide (ASO) Technology Against Tgf-β Signaling to Prevent Scarring During Flexor Tendon Repair 
Flexor tendons (FT) in the hand provide near frictionless gliding to facilitate hand function. Upon injury and surgical repair, satisfactory healing is hampered by fibrous adhesions between the tendon and synovial sheath. In the present study we used antisense oligonucleotides (ASOs), specifically targeted to components of Tgf-β signaling, including Tgf-β1, Smad3 and Ctgf, to test the hypothesis that local delivery of ASOs and suppression of Tgf-β1 signaling would enhance murine FT healing by suppressing adhesion formation while maintaining strength. ASOs were injected in to the FT repair site at 2, 6 and 12 days post-surgery. ASO treatment suppressed target gene expression through 21 days. Treatment with Tgf-β1, Smad3 or Ctgf ASOs resulted in significant improvement in tendon gliding function at 14 and 21 days, relative to control. Consistent with a decrease in adhesions, Col3a1 expression was significantly decreased in Tgf-β1, Smad3 and Ctgf ASO treated tendons relative to control. Smad3 ASO treatment enhanced the max load at failure of healing tendons at 14 days, relative to control. Taken together, these data support the use of ASO treatment to improve FT repair, and suggest that modulation of the Tgf-β1 signaling pathway can reduce adhesions while maintaining the strength of the repair.
PMCID: PMC4416995  PMID: 25761254
Flexor tendon healing; Antisense oligonucleotides; Smad3; Ctgf; Tgf-β1
12.  Delayed Exercise Promotes Remodeling in Sub-Rupture Fatigue Damaged Tendons 
Tendinopathy is a common musculoskeletal injury whose treatment is limited by ineffective therapeutic interventions. Previously we have shown that tendons ineffectively repair early sub-rupture fatigue damage. In contrast, physiological exercise has been shown to promote remodeling of healthy tendons but its utility as a therapeutic to promote repair of fatigue damaged tendons remains unknown. Therefore, the objective of this study was to assess the utility of exercise initiated 1 and 14 days after onset of fatigue damage to promote structural repair in fatigue damaged tendons. We hypothesized that exercise initiated 14 days after fatigue loading would promote remodeling as indicated by a decrease in area of collagen matrix damage, increased procollagen I and decorin, while decreasing proteins indicative of tendinopathy. Rats engaged in 6-week exercise for 30 min/day or 60 min/day starting 1 or 14 days after fatigue loading. Initiating exercise 1-day after onset of fatigue injury led to exacerbation of matrix damage, particularly at the tendon insertion. Initiating exercise 14 days after onset of fatigue injury led to remodeling of damaged regions in the midsubstance and collagen synthesis at the insertion. Physiological exercise applied after the initial biological response to injury has dampened can potentially promote remodeling of damaged tendons.
PMCID: PMC4417076  PMID: 25732052
tendon damage; fatigue; ECM; exercise; tendinopathy
13.  Biology and mechano-response of tendon cells: progress overview and perspectives† 
In this review, we summarize the group discussions on Cell Biology & Mechanics from the 2014 ORS/ISMMS New Frontiers in Tendon Research Conference. The major discussion topics included: 1) the biology of tendon stem/progenitor cells (TSPCs) and the potential of stem cell-based tendon therapy using TSPCs and other types of stem cells, namely, embryonic and/or induced pluripotent stem cells (iPSCs), 2) the biological concept and potential impact of cellular senescence on tendon aging, tendon injury repair and the development of degenerative disease, and 3) the effects of tendon cells’ mechano-response on tendon cell fate and metabolism. For each topic, a brief overview is presented which summarizes the major points discussed by the group participants. The focus of the discussions ranged from current research progress, challenges and opportunities, to future directions on these topics. In the preparation of this manuscript, authors consulted relevant references as a part of their efforts to present an accurate view on the topics discussed.
PMCID: PMC4422159  PMID: 25728946
TSPCs; senescence; iPSCs; mechanical loading; tendon degeneration
14.  Tendon Basic Science: Development, Repair, Regeneration, and Healing 
Tendinopathy and tendon rupture are common and disabling musculoskeletal conditions. Despite the prevalence of these injuries, a limited number of investigators are conducting fundamental, basic science studies focused on understanding processes governing tendinopathies and tendon healing. Development of effective therapeutics is hindered by the lack of fundamental guiding data on the biology of tendon development, signal transduction, mechanotransduction, and basic mechanisms underlying tendon pathogenesis and healing. To propel much needed progress, the New Frontiers in Tendon Research Conference, co-sponsored by NIAMS/NIH, the Orthopaedic Research Society, and the Icahn School of Medicine at Mount Sinai, was held to promote exchange of ideas between tendon researchers and basic science experts from outside the tendon field. Discussed research areas that are underdeveloped and represent major hurdles to the progress of the field will be presented in this review. To address some of these outstanding questions, conference discussions and breakout sessions focused on six topic areas (Cell Biology and Mechanics, Functional Extracellular Matrix, Development, Mechano-biology, Scarless Healing, and Mechanisms of Injury and Repair), which are reviewed in this special issue and briefly presented in this review. Review articles in this special issue summarize the progress in the field and identify essential new research directions.
PMCID: PMC4427041  PMID: 25764524
New Frontiers; tendon conference; tendinopathy; tendon injury
15.  Validation of GAITRite and PROMIS as High-Throughput Physical Function Outcome Measures Following ACL Reconstruction 
New healthcare demands for quality measures of elective procedures, such as anterior cruciate ligament (ACL) reconstructive surgery, warrant the establishment of high through-put outcomes for high volume clinics. To this end we evaluated the PROMIS and GAITRite as physical function outcome measures to quantify early healing and post-operative complications in 106 patients at pre-op and 3, 10, 20 and 52 weeks post-ACL reconstruction with bone-tendon-bone autograft, and compared the results to the current IKDC validated outcome measure. The results showed that both PROMIS and GAITRite were significantly quicker to administer versus IKDC (p < 0.0001). Additional advantages were that PROMIS and GAITRite detected a significant decrease in physical function at 3 weeks post-op, and a significant improvement at 10 weeks post-op, versus pre-op (p<0.001), which were not detected with IKDC. GAITRite was limited by a low ceiling that could not detect improvement of physical function beyond 20 weeks, while both PROMIS and IKDC detected significant improvement out to 52 weeks postop (p<0.001). Linear regressions demonstrated a significant relationship between IKDC and PROMIS, with a combined correlation value of 0.8954 (p<.001) for all time points. Finally, ROC curve analysis demonstrated that PROMIS is a diagnostic test for poor outcomes.
PMCID: PMC3976703  PMID: 24532421
ACL reconstruction; PROMIS; Physical Function; Gait
16.  Predicting Three-dimensional Patellofemoral Kinematics from Static Imaging-Based Alignment Measures 
Patellofemoral pain syndrome causes significant discomfort and disability among much of the general population. Despite recent breakthroughs in dynamic three-dimensional imaging technologies to assess pathological patellofemoral motion, such tools remain costly for clinical diagnostics applications. Thus, this study investigated whether three-dimensional patellofemoral kinematics could be predicted from routine two-dimensional static measures of patellofemoral joint alignment quantified from magnetic resonance imaging (MRI) data acquired in full knee extension. Twenty-six volunteers clinically diagnosed with patellofemoral pain (19F/7M, 25.9±11.1 years) and twenty-six control subjects (19F/7M, 25.3±7.7 years) were included in this IRB-approved study. Static three-dimensional sagittal T1-weighted gradient recall echo and dynamic MRI scans were acquired. For the dynamic image acquisition, subjects cyclically flexed and extended their knee (at 30 cycles/minute) while a full cine-phase contrast MRI set (24 times frames of anatomic images and x, y, and z-velocity images) was acquired. From these data, static measures of patellofemoral alignment and three-dimensional patellofemoral kinematics were derived. Single and multiple regressions between static and kinematic variables were evaluated. Although shown reliable, the static MRI measures could only partially predict patellofemoral kinematics, with r2-values ranging from 16%-77%. This makes it imperitave that the current precise, accurate, 3D, dynamic imaging techniques be translated into clinical tools.
PMCID: PMC4447147  PMID: 23097251
17.  Longitudinal culture-independent pilot study of microbiota colonizing open fractures and association with severity, mechanism, location, and complication from presentation to early outpatient follow up 
Precise identification of bacteria associated with post-injury infection, co-morbidities, and outcomes could have a tremendous impact in the management and treatment of open fractures. We characterized microbiota colonizing open fractures using culture-independent, high-throughput DNA sequencing of bacterial 16S ribosomal RNA genes, and analyzed those communities with respect to injury mechanism, severity, anatomical site, and infectious complications. Thirty subjects presenting to the Hospital of the University of Pennsylvania for acute care of open fractures were enrolled in a prospective cohort study. Microbiota was collected from wound center and adjacent skin upon presentation to the emergency department, intraoperatively, and at two outpatient follow-up visits at approximately 25 and 50 days following initial presentation. Bacterial community composition and diversity colonizing open fracture wounds became increasingly similar to adjacent skin microbiota with healing. Mechanism of injury, severity, complication, and location were all associated with various aspects of microbiota diversity and composition. The results of this pilot study demonstrate the diversity and dynamism of the open fracture microbiota, and their relationship to clinical variables. Validation of these preliminary findings in larger cohorts may lead to the identification of microbiome-based biomarkers of complication risk and/or to aid in management and treatment of open fractures.
PMCID: PMC4418442  PMID: 24395335
open fracture; microbiome; 16S rRNA; bacteria; infection
18.  Prognostic Significance of miRNA-1 (miR-1) Expression in Patients with Chordoma 
Reliable prognostic biomarkers for chordoma have not yet been established. Recent studies revealed that expression of miRNA-1(miR-1) is frequently downregulated in several cancer types including chordoma. The goal of this follow-up study is to investigate the expression of miR-1 as a prognostic biomarker and further confirm the functional role of miR-1 in chordoma cell growth and proliferation. We determined the relative expression levels of miR-1 and Met in chordoma tissue samples and correlated those to clinical variables. The results showed that miR-1 was downregulated in 93.7% of chordoma tissues and expression was inversely correlated with Met expression. miR-1 expression levels also correlated with clinical prognosis. To characterize and confirm the functional role of miR-1 in the growth and proliferation of chordoma cells, miR-1 precursors were stably transfected into chordoma cell lines UCH-1 and CH-22. Cell Proliferation Assay and MTT were used to evaluate cell growth and proliferation. Restoring expression of miR-1 precursor decreased cell growth and proliferation in UCH-1 and CH-22 cells. These results indicate that suppressed miR-1 expression in chordoma may in part be a driver for tumor growth, and that miR-1 has potential to serve as prognostic biomarker and therapeutic target for chordoma patients.
PMCID: PMC4049352  PMID: 24501096
Chordoma; miR-1; Met; Prognostic Biomarker
19.  The early inflammatory response after flexor tendon healing: A gene expression and histological analysis 
Despite advances in surgical techniques over the past three decades, tendon repairs remain prone to poor clinical outcomes. Previous attempts to improve tendon healing have focused on the later stages of healing (i.e., proliferation and matrix synthesis). The early inflammatory phase of tendon healing, however, is not fully understood and its modulation during healing has not yet been studied. Therefore, the purpose of this work was to characterize the early inflammatory phase of flexor tendon healing with the goal of identifying inflammation-related targets for future treatments. Canine flexor tendons were transected and repaired using techniques identical to those used clinically. The inflammatory response was monitored for 9 days. Temporal changes in immune cell populations and gene expression of inflammation-, matrix degradation-, and extracellular matrix-related factors were examined. Gene expression patterns paralleled changes in repair-site cell populations. Of the observed changes, the most dramatic effect was a greater than 4000-fold up-regulation in the expression of the pro-inflammatory factor IL-1β. While an inflammatory response is likely necessary for healing to occur, high levels of pro-inflammatory cytokines may result in collateral tissue damage and impaired tendon healing. These findings suggest that future tendon treatment approaches consider modulation of the inflammatory phase of healing.
PMCID: PMC4076155  PMID: 24464937
inflammation; intrasynovial flexor tendon; matrix metalloproteinase; extracellular matrix; collagen
20.  Alterations in intervertebral disc composition, matrix homeostasis and biomechanical behavior in the UCD-T2DM rat model of type 2 diabetes 
Type 2 diabetes (T2D) adversely affects many tissues, and the greater incidence of discogenic low back pain among diabetic patients suggests that the intervertebral disc is affected too. Using a rat model of polygenic obese T2D, we demonstrate that diabetes compromises several aspects of disc composition, matrix homeostasis and biomechanical behavior. Coccygeal motion segments were harvested from 6-month-old lean Sprague-Dawley rats, obese Sprague-Dawley rats, and diabetic obese UCD-T2DM rats (diabetic for 69 ± 7 days). Findings indicated that diabetes but not obesity reduced disc glycosaminoglycan and water contents, and these degenerative changes correlated with increased vertebral endplate thickness and decreased endplate porosity, and with higher levels of the advanced glycation end-product (AGE) pentosidine. Consistent with their diminished glycosaminoglycan and water contents and their higher AGE levels, discs from diabetic rats were stiffer and exhibited less creep when compressed. At the matrix level, elevated expression of hypoxia-inducible genes and catabolic markers in the discs from diabetic rats coincided with increased oxidative stress and greater interactions between AGEs and one of their receptors (RAGE). Taken together, these findings indicate that endplate sclerosis, increased oxidative stress and AGE/RAGE-mediated interactions could be important factors for explaining the greater incidence of disc pathology in T2D.
PMCID: PMC4408867  PMID: 25641259
intervertebral disc degeneration; type 2 diabetes; vertebral endplate; advanced glycation end-products; pentosidine
21.  Scapular Dyskinesis is Detrimental to Shoulder Tendon Properties and Joint Mechanics in a Rat Model 
Shoulder tendon injuries are frequently seen in the presence of abnormal scapular motion, termed scapular dyskinesis. The cause and effect relationship between scapular dyskinesis and shoulder injury has not been directly defined. The objective of this study was to develop and use an animal model to examine the initiation and progression of pathological changes in the rotator cuff and biceps tendon. 60 male Sprague-Dawley rats were randomized into two groups: nerve transection (to induce scapular dyskinesis, SD) or sham nerve transection (control). The animals were sacrificed 4 and 8 weeks after surgery. Shoulder function and passive joint mechanics were evaluated over time. Tendon mechanical, histological, organizational, and compositional properties were evaluated at 4 and 8 weeks. Gross observation demonstrated alterations in scapular motion, consistent with scapular “winging”. Shoulder function, passive internal range of motion, and tendon mechanical properties were significantly altered. Histology results, consistent with tendon pathology (rounded cell shape and increased cell density), were observed and protein expression of collagen III and decorin was altered. This study presents a new model of scapular dyskinesis that can rigorously evaluate cause and effect relationships in a controlled manner. These results identify scapular dyskinesis as a causative mechanical mechanism for shoulder tendon pathology.
PMCID: PMC4410845  PMID: 25070580
scapular dyskinesis; rotator cuff; animal model
22.  Cycle-Dependent Matrix Remodeling Gene Expression Response in Fatigue-Loaded Rat Patellar Tendons 
Expression profiling of selected matrix remodeling genes was conducted to evaluate differences in molecular response to low-cycle (100) and high-cycle (7,200) sub-failure-fatigue loading of patellar tendons. Using our previously developed in vivo patellar tendon model, tendons were loaded for 100 or 7,200 cycles and expression of selected metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and collagens were quantified by real-time RT-PCR at 1- and 7-day post-loading. Expression profiles were also obtained from lacerated tendons as an acute injury model. The high-cycle group showed upregulation of TIMP-1, -2, Col3a1, and Col5a1, and downregulation TIMP-4 at both time points, upregulation of MMP-2 at 7-day post-loading and downregulation of MMP-13 and -14 at 1-day post-loading, suggesting overall repair/remodeling. In contrast, the low-cycle loaded group showed upregulation of MMP-2, -3, -13, and Col12a1 at both time points, upregulation of TIMP-1, -2, -3, Col3a1, and integrin β1 and downregulation of integrin α11 at 1-day post-loading and upregulation of Col1a1 at 7-day post-loading, consistent with a hypertrophic (adaptive) pattern. Lacerated tendons showed a typical acute wound response with upregulation of all examined remodeling genes. Differences found in tendon response to high- and low-cycle loading are suggestive of the underlying mechanisms associated with a healthy or damaging response.
PMCID: PMC4408775  PMID: 20839322
patellar tendon; fatigue loading; TIMP; MMP; collagen
23.  Intra-Articular Tibiofemoral Injection of a Nonsteroidal Anti-Inflammatory Drug has no Detrimental Effects on Joint Mechanics in a Rat Model 
Administration of intra-articular medications, including corticosteroids and analgesics, is common clinical practice for knee pathology and dysfunction. Non-steroidal anti-inflammatory drugs (NSAIDs) are another category of medication commonly prescribed for their analgesic and anti-inflammatory properties. Recent studies have demonstrated the efficacy of injectable NSAIDs in the treatment of intra-articular pathology and postoperative analgesia.1–3 However, little data exist regarding the safety of intra-articular injection, despite the increase in its application.4 Therefore, the objective of this study was to investigate the effects of intra-articular NSAID injection on articular cartilage, the anterior cruciate ligament (ACL), and joint function in the rat. Sixty-four Sprague-Dawley rats were divided into either saline (SAL) or ketorolac (NSAID) tibiofemoral single injection treatment groups. Animals were sacrificed at 2, 7, 28, and 84 days post-injection for histological and mechanical analyses. Additionally, a subset of animals underwent longitudinal ambulatory evaluation to determine joint functional properties. We hypothesized that intra-articular ketorolac injection would result in no detrimental mechanical, histological, or functional changes. No differences were reported between the NSAID and SAL groups in any of the parameters measured at any time point, demonstrating the potential safety of intra-articular NSAID administration. Therefore, NSAID injection could be further considered for clinical application in humans.
PMCID: PMC4404033  PMID: 24981310
NSAID; intra-articular; injection; knee; mechanics
24.  Defining the Phenotype of Young Healthy Nucleus Pulposus Cells: Recommendations of the Spine Research Interest Group at the 2014 Annual ORS Meeting 
Low back pain is a major physical and socioeconomic problem. Degeneration of the intervertebral disc and especially that of nucleus pulposus (NP) has been linked to low back pain. In spite of much research focusing on the NP, consensus among the research community is lacking in defining the NP cell phenotype. A consensus agreement will allow easier distinguishing of NP cells from annulus fibrosus (AF) cells and endplate chondrocytes, a better gauge of therapeutic success, and a better guidance of tissue-engineering-based regenerative strategies that attempt to replace lost NP tissue. Most importantly, a clear definition will further the understanding of physiology and function of NP cells, ultimately driving development of novel cell-based therapeutic modalities. The Spine Research Interest Group at the 2014 Annual ORS Meeting in New Orleans convened with the task of compiling a working definition of the NP cell phenotype with hope that a consensus statement will propel disc research forward into the future. Based on evaluation of recent studies describing characteristic NP markers and their physiologic relevance, we make the recommendation of the following healthy NP phenotypic markers: stabilized expression of HIF-1α, GLUT-1, aggrecan/collagen II ratio >20, Shh, Brachyury, KRT18/19, CA12, and CD24.
PMCID: PMC4399824  PMID: 25411088
nucleus pulposus; intervertebral disc; cell phenotype
25.  mTOR Regulates Fatty Infiltration through SREBP-1 and PPARg after a Combined Massive Rotator Cuff Tear and Suprascapular Nerve Injury in Rats 
Rotator cuff tears (RCTs) are among the most common injuries seen in orthopedic patients. Chronic tears can result in the development of muscular atrophy and fatty infiltration. Despite the prevalence of RCTs, little is known about the underlying molecular pathways that produce these changes. Recently, we have shown that mammalian target of rapamycin (mTOR) signaling plays an important role in muscle atrophy that results from massive RCTs in a rat model. The purpose of this study was therefore to extend our understanding of mTOR signaling and evaluate its role in fatty infiltration after a combined tendon transection and suprascapular nerve denervation surgery. Akt/mTOR signaling was significantly increased and resulted in the up-regulation of two transcription factors: SREBP-1 and PPARγ. We also saw an increase in expression of adipogenic markers: C/EBP-α and FASN. Upon treatment with rapamycin, an inhibitor of mTOR, we observed a decrease in mTOR signaling, activity of transcription factors, and reduction in fatty infiltration. Therefore, our study suggests that mTOR signaling mediates rotator cuff fatty infiltration via SREBP-1 and PPARγ. Clinically, our finding may alter current treatment methods to address rotator cuff fatty infiltration. © 2012 Orthopaedic Research Society.
PMCID: PMC4393845  PMID: 23239524
rotator cuff tear; fatty infiltration; Akt/mTOR signaling; SREBP-1; PPARγ

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