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1.  Evaluation of Proximal Joint Kinematics and Muscle Strength Following ACL Reconstruction Surgery in Female Athletes 
Background\Purpose
Despite the intense focus on outcomes following an anterior cruciate ligament (ACL) reconstruction, it is not yet known whether unresolved abnormal hip and trunk neuromuscular control exists. The purpose of this study was to compare trunk and hip kinematics during running, hip abductor and external rotator strength, and trunk control between females who had undergone an ACL reconstruction and healthy control participants.
Methods
We compared 20 ACL reconstructed females to 20 healthy individuals, measuring abduction and external rotation strength, a trunk control test, and performed an instrumented gait evaluation during running. Comparisons between groups were made for non-sagittal peak hip angles, forward trunk lean, trunk ipsilateral lean at initial contact, trunk control and hip abduction and external rotation strength.
Results
We found no significant differences in hip abduction (p = 0.25), hip external rotation strength (p = 0.63), peak hip adduction (p = 0.11) or hip internal rotation angle (p = 0.47). The ACL group did have a significantly greater ipsilateral trunk lean (p = 0.028), forward lean (p = 0.004), and had higher errors on the trunk stability test (p = 0.007).
Conclusion
We found significant differences in trunk control, suggesting further attention should be devoted to this component of rehabilitation.
doi:10.1002/jor.22678
PMCID: PMC4314615  PMID: 25044305
biomechanics; strength; trunk; knee; running
2.  Inhibition of beta-catenin signaling by Pb leads to incomplete fracture healing 
There is strong evidence in the clinical literature to suggest that elevated lead (Pb) exposure impairs fracture healing. Since Pb has been demonstrated to inhibit bone formation, and Wnt signaling is an important anabolic pathway in chondrocyte maturation and endochondral ossification, we investigated the impact of Wnt therapy on Pb-exposed mice undergoing bone repair in a mouse tibial fracture model. We established that tibial fracture calluses from Pb-treated mice were smaller and contained less mineralized tissue than vehicle controls. This resulted in the persistence of immature cartilage in the callus and decreased β-catenin levels. Reduction of β-catenin protein was concurrent with systemic elevation of LRP5/6 antagonists DKK1 and sclerostin in Pb-exposed mice throughout fracture healing. β-catenin stimulation by the GSK3 inhibitor BIO reversed these molecular changes and restored the amount of mineralized callus. Overall, Pb is identified as a potent inhibitor of endochondral ossification in vivo with correlated effects on bone healing with noted deficits in β-catenin signaling, suggesting the Wnt/β-catenin as a pivotal pathway in the influence of Pb on fracture repair.
doi:10.1002/jor.22677
PMCID: PMC4314692  PMID: 25044211
3.  [No title available] 
PMCID: PMC3945668  PMID: 24122902
4.  [No title available] 
PMCID: PMC3978382  PMID: 24108673
5.  Transforming Growth Factor-β (TGF-β) Expression is Increased in the Subsynovial Connective Tissues of Patients with Idiopathic Carpal Tunnel Syndrome 
Non-inflammatory fibrosis of the subsynovial connective tissue (SSCT) is a hallmark of carpal tunnel syndrome (CTS). The etiology of this finding and its relationship to the development of CTS remain poorly understood. Recent studies have found that transforming growth factor-β (TGF-β) plays a central role in fibrosis. The purpose of this study was to investigate the expression of TGF-β and connective tissue growth factor (CTGF), a downstream mediator of TGF-β, in the pathogenesis of CTS. We compared SSCT specimens from 26 idiopathic CTS patients with specimens from 10 human cadaver controls with no previous diagnosis of CTS. Immunohistochemistry was performed to determine levels TGF-β1, CTGF, collagen 1(Col1) and collagen 3 (Col3) expression. TGF-β1(P<0.01), CTGF (P<0.01), and Col3 (P<0.01) were increased in SSCT of CTS patients compared with control tissue. In addition, a strong positive correlation was found between TGF-β1 and CTGF, (R2=0.80, p<0.01) and a moderate positive correlation between Col3 and TGF-β1 (R2=0.49, p<0.01). These finding suggest that there is an increased expression of TGF-β and CTGF, a TGF-β regulated protein, and that this TGF-β activation may be responsible for SSCT fibrosis in CTS patients.
doi:10.1002/jor.22485
PMCID: PMC4095953  PMID: 24014274
Carpal Tunnel Syndrome; Subsynovial Connective Tissue; TGF-β; CTGF
6.  Comparison of loading rate-dependent injury modes in a murine model of post-traumatic osteoarthritis 
Post-traumatic osteoarthritis (PTOA) is a common long-term consequence of joint injuries such as anterior cruciate ligament (ACL) rupture. In this study we used a tibial compression overload mouse model to compare knee injury induced at low speed (1 mm/s), which creates an avulsion fracture, to injury induced at high speed (500 mm/s), which induces midsubstance tear of the ACL. Mice were sacrificed at 0 days, 10 days, 12 weeks, or 16 weeks post-injury, and joints were analyzed with micro-computed tomography, whole joint histology, and biomechanical laxity testing. Knee injury with both injury modes caused considerable trabecular bone loss by 10 days post-injury, with the Low Speed Injury group (avulsion) exhibiting a greater amount of bone loss than the High Speed Injury group (midsubstance tear). Immediately after injury, both injury modes resulted in greater than 2-fold increases in total AP joint laxity relative to control knees. By 12 and 16 weeks post-injury, total AP laxity was restored to uninjured control values, possibly due to knee stabilization via osteophyte formation. This model presents an opportunity to explore fundamental questions regarding the role of bone turnover in PTOA, and the findings of this study support a biomechanical mechanism of osteophyte formation following injury.
doi:10.1002/jor.22480
PMCID: PMC4140447  PMID: 24019199
Mouse model; Post-traumatic osteoarthritis; ACL injury; Joint stability; Osteophyte
7.  The Effect of Low and High Velocity Tendon Excursion on the Mechanical Properties of Human Cadaver Subsynovial Connective Tissue 
SUMMARY
Fibrosis of the subsynovial connective tissue (SSCT) in the carpal tunnel is the most common histological finding in carpal tunnel syndrome (CTS). Fibrosis may result from damaged SSCT. Previous studies found that with low-velocity (2 mm/s), tendon excursions can irreversibly damage the SSCT. We investigated the effect of tendon excursion velocity in the generation of SSCT damage. Nine human cadaver wrists were used. Three repeated cycles of ramp-stretch testing were performed simulating 40, 60, 90 and 120% of the middle finger flexor tendon superficialis physiological excursion with an excursion velocity of 60 mm/s. Energy and force were calculated and normalized by values obtained in the first cycle for each excursion level. Data were compared with low-velocity excursion data. For high-velocity excursions, a significant drop in the excursion energy ratio was first observed at an excursion level of 60% physiological excursion (P<0.024) and that for low-velocity excursions was first observed at 90% physiological excursion (P<0.038). Furthermore, the energy ratio was lower at 60% for high velocities (P≤0.039). Increasing velocity lowers the SSCT damage threshold. This finding may be relevant for understanding the pathogenesis of SSCT fibrosis, such as that accompanying CTS, and a relationship with occupational factors.
doi:10.1002/jor.22489
PMCID: PMC3845004  PMID: 24038298
Carpal Tunnel; Subsynovial Connective Tissue; Biomechanics; Human Cadaver; Velocity
8.  Differential ubiquitin-proteasome and autophagy signaling following rotator cuff tears and suprascapular nerve injury 
Previous studies have evaluated role of Akt/mTOR signaling in rotator cuff muscle atrophy and determined that there was differential in signaling following tendon transection (TT) and suprascapular nerve (SSN) denervation (DN), suggesting that atrophy following TT and DN was modulated by different protein degradation pathways. In this study, two muscle proteolytic systems that have been shown to be potent regulators of muscle atrophy in other injury models, the ubiquitin-proteasome pathway and autophagy, were evaluated following TT and DN. In addition to examining protein degradation, this study assessed protein synthesis rate following these two surgical models to understand how the balance between protein degradation and synthesis results in atrophy following rotator cuff injury. In contrast to the traditional theory that protein synthesis is decreased during muscle atrophy, this study suggests that protein synthesis is up-regulated in rotator cuff muscle atrophy following both surgical models. While the ubiquitin-proteasome pathway was a major contributor to the atrophy seen following DN, autophagy was a major contributor following TT. The findings of this study suggest that protein degradation is the primary factor contributing to atrophy following rotator cuff injury. However, different proteolytic pathways are activated if SSN injury is involved.
doi:10.1002/jor.22482
PMCID: PMC3856942  PMID: 24018537
rotator cuff tear; denervation; muscle atrophy; ubiquitin-proteasome; autophagy; protein synthesis
9.  Effects of ACL reconstruction surgery on muscle activity of the lower limb during a jump-cut maneuver in males and females 
We compared muscle activity of the quadriceps, hamstring, and gastrocnemius muscles when ACL-intact (ACLINT) and ACL-reconstructed (ACLREC) male and female subjects performed a jump-cut task. Surface electromyography sensors were used to evaluate time to peak muscle activity and muscle activity ratios. Rectus femoris (RF) and vastus medialis (VM) peak timing was 71 ms and 78 ms earlier in ACLINT than in ACLREC subjects, respectively. Biceps femoris (BF) peak timing was 90 ms earlier in ACLINT than in ACLREC subjects and 75 ms earlier in females than in males. Medial gastrocnemius (MG) muscle peak timing was 77 ms earlier in ACLINT than in ACLREC subjects. Lateral gastrocnemius (LG) and MG muscle peak times were 106 ms and 87 ms earlier in females than in males, respectively. The RF, VM, BF and MG peaked later in ACLREC than in ACLINT subjects. There was evidence suggesting that the loading phase quadriceps:hamstring (quad:ham) muscle activity ratio was greater in ACLREC than in ACLINT subjects. Finally, the injury risk phase quad:ham muscle activity ratio was found to be 4.8 times greater in females than in males. In conclusion, there are differences in muscle activity related to ACL status and sex that could potentially help explain graft failure risk and the sex bias.
doi:10.1002/jor.22470
PMCID: PMC3808517  PMID: 23966333
ACL; Injury; Reconstruction; Muscle; EMG
10.  Analysis of microRNAs expressions in chondrosarcoma 
MicroRNAs (miRNAs) are small noncoding RNAs capable of inhibiting gene expression post-transcriptionally and expression profiling can provide therapeutic targets and tools for cancer diagnosis. Chondrosarcoma is a mesenchymal tumor with unknown cause and differentiation status. Here, we profiled miRNA expression of chondrosarcoma, namely clinical samples from human conventional chondrosarcoma tissue, established chondrosarcoma cell lines, and primary non-tumorous adult articular chondrocytes, by miRNA array and quantitative real-time PCR. A wide variety of miRNAs were differently downregulated in chondrosarcoma compared to non-tumorous articular chondrocytes; 27 miRNAs: miR-10b, 23b, 24-1*, 27b, 100, 134, 136, 136*, 138, 181d, 186, 193b, 221*, 222, 335, 337-5p, 376a, 376a*, 376b, 376c, 377, 454, 495, 497, 505, 574-3p, and 660, were significantly downregulated in chondrosarcoma and only 2: miR-96 and 183, were upregulated. We further validated the expression levels of miRNAs by quantitative real-time PCR for miR-181a, let-7a, 100, 222, 136, 376a, and 335 in extended number of chondrosarcoma clinical samples. Among them, all except miR-181a were found to be significantly downregulated in chondrosarcoma derived samples. The findings provide potential diagnostic value and new molecular understanding of chondrosarcoma.
doi:10.1002/jor.22457
PMCID: PMC3835201  PMID: 23940002
microRNA; chondrosarcoma; cartilage; sarcoma; malignancy
11.  Alfacalcidol enhances collagen quality in ovariectomized rat bones 
Journal of Orthopaedic Research  2014;32(8):1030-1036.
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, that is, ovariectomy + 0.022 µg/kg/day ALF, ovariectomy + 0.067 µg/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, that is, 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. © 2014 The Authors. Journal of Orthopaedic Research. Published by Wiley Periodicals, Inc. J Orthop Res 32:1030–1036, 2014.
doi:10.1002/jor.22642
PMCID: PMC4071133  PMID: 24809324
vitamin D; alfacalcidol; collagen; collagen cross-link; ovariectomized rats
12.  Effects of Low Dose X-Ray Irradiation on Porcine Articular Cartilage Explants 
Ionizing radiation therapy is a crucial treatment for cancer, but can damage surrounding normal tissues. Damage to articular cartilage leading to arthropathy can occur at irradiated sites. It is unclear whether this response is due to damaging surrounding skeletal structures or direct effects on cartilage. In this study, we showed that irradiation with 2 Gy of X-rays causes a significant reduction in the stiffness of porcine explants 1 week post-irradiation. By using both microindentation and indentation-type atomic force microscopy, ionizing radiation reduces stiffness in both the superficial zone and throughout the entire thickness of the tissue. Young’s modulus values were 75% and 60% lower in 2 Gy irradiated samples when compared with controls using microindentation and nanoindentation, respectively. Glycosaminoglycans (GAGs) released into the culture media of irradiated samples was nearly 100% greater at 24 hours after exposure. While collagen content in the tissue is similar between groups, GAG content is 55% lower in irradiated explants compared with controls by one week. Therefore, the irradiated explants are unable to recover from the initial loss of GAGs by one week. This acute loss of GAGs is a likely contributor to the reduction in modulus seen after exposure to ionizing radiation.
doi:10.1002/jor.22406
PMCID: PMC3792489  PMID: 23913833
articular cartilage; radiation exposure; cartilage mechanics; atomic force microscopy; glycosaminoglycans
13.  Energy Metabolism of Intervertebral Disc Under Mechanical Loading 
Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra-cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression-mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism.
doi:10.1002/jor.22436
PMCID: PMC3787952  PMID: 23843186
IVD; mechanical loading; ATP; lactate; energy metabolism
14.  Tendon Gradient Mineralization for Tendon to Bone Interface Integration 
Tendon-to-bone integration is a great challenge for tendon or ligament reconstruction regardless of use of autograft or allograft tendons. We mineralized the tendon, thus transforming the tendon-to-bone into a “bone-to-bone” interface for healing. Sixty dog flexor digitorum profundus (FDP) tendons were divided randomly into 5 groups: 1) normal FDP tendon, 2) CaP (Non-extraction and mineralization without fetuin), 3) CaPEXT (Extraction by Na2HPO4 and mineralization without fetuin), 4) CaPFetuin (Non-extraction and mineralization with fetuin), and 5) CaPEXTFetuin (Extraction and mineralization with fetuin). The calcium and phosphate content significantly increased in tendons treated with combination of extraction and fetuin compared to the other treatments. Histology also revealed a dense mineral deposition throughout the tendon outer layers and penetrated into the tendon to a depth of 200 μm in a graded manner. Compressive moduli were significantly lower in the four mineralized groups compared with normal control group. No significant differences in maximum failure strength or stiffness were found in the suture pull-out test among all groups. Mineralization of tendon alters the interface from tendon to bone into mineralized tendon to bone, which may facilitate tendon-to-bone junction healing following tendon or ligament reconstruction.
doi:10.1002/jor.22412
PMCID: PMC4121959  PMID: 23939935
Tendon Mineralization; Graded Mineral; Tendon-to-Bone Healing; Tendon Allografts
15.  Mechanical Properties of the Extra-Fibrillar Matrix of Human Annulus Fibrosus are Location and Age Dependent 
The mechanical behavior of the annulus fibrosus (AF) of the intervertebral disc can be modeled as a mixture of fibers, extra-fibrillar matrix (EFM), ions, and fluid. However, the properties of the EFM have not been measured directly. We measured mechanical properties of the human EFM at several locations, determined the effect of age and degeneration, and evaluated whether changes in EFM properties correspond to AF compositional changes. EFM mechanical properties were measured using a method that combines osmotic loading and confined compression. AF samples were dissected from several locations, and mechanical properties were correlated with age, degeneration, and composition. EFM modulus was found to range between 10 and 50 kPa, increasing nonlinearly with compression magnitude and being highest in the AF outer-anterior region. EFM properties were not correlated with composition or degeneration. However, the EFM modulus, its relative contribution to tissue modulus, and model parameters were correlated with age. These measurements will result in more accurate predictions of deformations in the intervertebral disc. Additionally, parameters such as permeability and diffusivity used for biotransport analysis of glucose and other solutes depend on EFM deformation. Consequently, the accuracy of biotransport simulations will be greatly improved.
doi:10.1002/jor.22430
PMCID: PMC4164199  PMID: 23818058
16.  Temporal Effect of In Vivo Tendon Fatigue Loading on the Apoptotic Response Explained in the Context of Number of Fatigue Loading Cycles and Initial Damage Parameters 
Accumulation of damage is a leading factor in the development of tendinopathy. Apoptosis has been implicated in tendinopathy, but the biological mechanisms responsible for initiation and progression of these injuries are poorly understood. We assessed the relationship between initial induced damage and apoptotic activity 3 and 7 days after fatigue loading. We hypothesized that greater apoptotic activity (i) will be associated with greater induced damage and higher number of fatigue loading cycles, and (ii) will be higher at 7 than at 3 days after loading. Left patellar tendons were fatigue loaded for either 100 or 7,200 cycles. Diagnostic tests were applied before and after fatigue loading to determine the effect of fatigue loading on hysteresis, elongation, and loading and unloading stiffness (damage parameters). Cleaved Caspase-3 staining was used to identify and calculate the percent apoptosis in the patellar tendon. While no difference in apoptotic activity occurred between the 100 and 7,200 cycle groups, greater apoptotic activity was associated with greater induced damage. Apoptotic activity was higher at 7 than 3 days after loading. We expect that the decreasing number of healthy cells that can repair the induced damage in the tendon predispose it to further injury.
doi:10.1002/jor.22639
PMCID: PMC4209741  PMID: 24838769
damage accumulation; tendinopathy; apoptosis; cluster analysis; tendon fatigue
17.  The Biology of Anterior Cruciate Ligament Injury and Repair: Kappa Delta Ann Doner Vaughn Award Paper 2013 
Anterior cruciate ligament (ACL) injuries are currently treated by removing the injured ligament and replacing it with a tendon graft. Recent studies have examined alternative treatment methods, including repair and regeneration of the injured ligament. In order to make such an approach feasible, a basic understanding of ACL biology and its response to injury is needed. Identification of obstacles to native ACL healing can then be identified and potentially resolved using tissue engineering strategies - first, with in vitro screening assays, and then with in vivo models of efficacy and safety. This Perspectives paper outlines this path of discovery for optimizing ACL healing using a bio-enhanced repair technique. This journey has required constructing indices of functional tissue response, pioneering physiologically-based methods of biomechanical testing, developing and validating clinically relevant animal models, and creating and optimizing translationally feasible scaffolds, surgical techniques and biologic additives. Using this systematic translational approach, “bio-enhanced” ACL repair has been advanced to the point where it may become an option for future treatment of acute ACL injuries and the prevention of subsequent post-traumatic osteoarthritis associated with this injury.
doi:10.1002/jor.22420
PMCID: PMC3750083  PMID: 23818453
18.  Integrin-Mediated Interactions with Extracellular Matrix Proteins for Nucleus Pulposus Cells of the Human Intervertebral Disc 
The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3 and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival and biosynthesis in cell-based therapeutics.
doi:10.1002/jor.22395
PMCID: PMC3826265  PMID: 23737292
intervertebral disc; nucleus pulposus; integrin; extracellular matrix; human
19.  Increasing both CoCrMo-alloy Particle Size and Surface Irregularity Induces Increased Macrophage Inflammasome Activation In vitro Potentially through Lysosomal Destabilization Mechanisms 
Recent investigations indicate that innate immune “danger-signaling” pathways mediate metal implant debris induced-inflammatory responses, e.g. NALP3 inflammasome. How the physical characteristics of particles, (size, shape and chemical composition) affect this inflammatory reactivity remains controversial. We examined the role of Cobalt-Chromium-Molybdenum (CoCrMo) alloy particle shape and size on human macrophage phagocytosis, lysosomal destabilization, and inflammasome activation. Round/smooth vs. irregularly shaped/rough CoCrMo-alloy particles of ~1µm and 6 to 7µm diameter were investigated for differential lysosomal damage and inflammasome activation in human monocytes/macrophages. While spherical/smooth 1µm CoCrMo-alloy particles did not measurably affect macrophage IL-1β production, irregular 1µm CoCrMo-alloy particles induced significant IL-1β increases over controls. Both round/smooth particles and irregular CoCrMo-alloy particles that were 6 to 7µ min size induced >10-fold increases in IL-1β production compared to similarly shaped smaller particles (p<0.05). Larger irregular particles induced a greater degree of intracellular lysosomal damage and a >3-fold increase in IL-1β vs. similarly sized round/smooth particles (at an equal dose, particles/cell). CoCrMo-alloy particle-size-induced IL-1β production was dependent on the lysosomal protease Cathepsin B, further supporting lysosomal destabilization as causative in inflammation. Phagocytosable larger/irregular shaped particles (6µm) demonstrated the greatest lysosomal destabilization (observed immunofluorescently) and inflammatory reactivity when compared on an equal dose basis (particles/cell) to smaller/spherical 1µm particles in vitro.
doi:10.1002/jor.22411
PMCID: PMC4028039  PMID: 23794526
Inflammasome; Monocytes/macrophages; Lysosomal destabilization; Cathepsin B; Metal particles
20.  Involvement of ERCC1 in the Pathogenesis of Osteoarthritis Through the Modulation of Apoptosis and Cellular Senescence 
DNA damage is a cause of age related pathologies, including osteoarthritis (OA). Excision repair cross complementation group 1 (ERCC1) is an endonuclease required for DNA damage repair. In this study we investigated the function of ERCC1 in chondrocytes and its association with the pathophysiology of OA. ERCC1 expression in normal and osteoarthritic cartilage was assessed, as were changes in ERCC1 expression in chondrocytes under catabolic stress. Inhibiting ERCC1 in chondrocytes under interleukin-1β stimulation using small interfering RNA (siRNA) was also evaluated. Finally, cellular senescence and apoptosis were examined in relation to ERCC1 function. ERCC1 expression was decreased in OA cartilage and increased within 4 h of exposure to interleukin (IL)-1β, but decreased after 12 h. The inhibition of ERCC1 by siRNA increased the expression of matrix metallopeptidase 13 and decreased collagen type II. ERCC1 inhibition also increased the number of apoptotic and senescent cells. The inhibition of ERCC1 in chondrocytes increased their expression of OA related proteins, apoptosis, cellular senescence, and hypertrophic-like changes which suggest that ERCC1 is critical for protecting human chondrocytes (HCs) from catabolic stresses and provides insights into the pathophysiology of OA and a potential target for its treatment.
doi:10.1002/jor.22656
PMCID: PMC4134687  PMID: 24964749
ERCC1; osteoarthritis; apoptosis; senescence; interleukin (IL)-1β
21.  Signaling through the small G-protein Cdc42 is involved in insulin-like growth factor-I resistance in aging articular chondrocytes 
During aging, chondrocytes become unresponsive to insulin-like growth factor-I (IGF-I). This study examined the role of Cdc42 (cell-division-cycle 42) in IGF-I signaling during aging. Experiments were performed using cartilage and chondrocytes isolated from horses ages 1 day – 25 years. Northern analysis was used to examine expression of the small GTPases Cdc42, Rac, and RhoA. Western analysis was utilized to assess total Cdc42 (GTP + GDP-bound); active, GTP-Cdc42 was assessed using a pull-down assay with western analysis. GTP-Cdc42 was also measured following IGF-I treatment. Gene expression for Cdc42 and Rac were decreased in mature samples, but there was no difference in total Cdc42 (GTP+GDP-bound) protein expression due to age. GTP-Cdc42 was significantly greater in prepubescent samples compared to other age groups. IGF-I diminished the GTP-bound state of Cdc42 in prepubescent chondrocytes, however, this effect was lost during aging. No differences in results were observed due to sample type; i.e. cartilage tissues vs. isolated chondrocytes. These studies suggest that loss of IGF-I – mediated regulation of Cdc42 activation may be a mechanism for the chondrocyte unresponsive state during aging. Further, the activation state of Cdc42, measured in native and IGF-I-treated cartilage tissue for the first time, is similar to that of isolated chondrocytes, indicating that the activation state of small G-proteins is not affected by isolation of chondrocytes from the extracellular matrix. Continued studies will identify the upstream regulators of Cdc42 which will further elucidate the molecular mechanism of IGF-I resistance during aging thereby providing insight into targeted strategies for age-related osteoarthritis.
doi:10.1002/jor.20185
PMCID: PMC4154065  PMID: 16779831
22.  Stem Cell-Mediated Accelerated Bone Healing Observed with In Vivo Molecular and Small Animal Imaging Technologies in a Model of Skeletal Injury 
Adult stem cells are promising therapeutic reagents for skeletal regeneration. We hope to validate by molecular imaging technologies the in vivo life cycle of adipose-derived multipotent cells (ADMCs) in an animal model of skeletal injury. Primary ADMCs were lentivirally transfected with a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. Bioluminescence imaging (BLI), [18F]FHBG (9-(fluoro-hydroxy-methyl-butyl-guanine)-micro-PET, [18F]Fluoride ion micro-PET and micro-CT were performed to monitor stem cells and their effect. Bioluminescence microscopy and immunohistochemistry were done for histological confirmation. BLI showed ADMC’s traffic from the lungs then to the injury site. BLI microscopy and immunohistochemistry confirmed the ADMCs in the bone defect. Micro-CT measurements showed increased bone healing in the cell-injected group compared to the noninjected group at postoperative day 7 (p <0.05). Systemically administered ADMC’s traffic to the site of skeletal injury and facilitate bone healing, as demonstrated by molecular and small animal imaging. Molecular imaging technologies can validate the usage of adult adipose tissue-derived multipotent cells to promote fracture healing. Imaging can in the future help establish therapeutic strategies including dosage and administration route.
doi:10.1002/jor.20736
PMCID: PMC4154812  PMID: 18752273
stem cell; imaging; cell tracking
23.  Statistical Shape Modeling of Cam Femoroacetabular Impingement 
Statistical shape modeling (SSM) was used to quantify 3D variation and morphologic differences between femurs with and without cam femoroacetabular impingement (FAI). 3D surfaces were generated from CT scans of femurs from 41 controls and 30 cam FAI patients. SSM correspondence particles were optimally positioned on each surface using a gradient descent energy function. Mean shapes for groups were defined. Morphological differences between group mean shapes and between the control mean and individual patients were calculated. Principal component analysis described anatomical variation. Among all femurs, the first six modes (or principal components) captured significant variations, which comprised 84% of cumulative variation. The first two modes, which described trochanteric height and femoral neck width, were significantly different between groups. The mean cam femur shape protruded above the control mean by a maximum of 3.3 mm with sustained protrusions of 2.5–3.0 mm along the anterolateral head-neck junction/distal anterior neck. SSM described variations in femoral morphology that corresponded well with areas prone to damage. Shape variation described by the first two modes may facilitate objective characterization of cam FAI deformities; variation beyond may be inherent population variance. SSM could characterize disease severity and guide surgical resection of bone.
doi:10.1002/jor.22389
PMCID: PMC4137561  PMID: 23832798
hip; statistical shape modeling; femoroacetabular impingement; cam
24.  Quantitative T2* (T2 Star) Relaxation Times Predict Site Specific Proteoglycan Content and Residual Mechanics of the Intervertebral Disc Throughout Degeneration 
Degeneration alters the biochemical composition of the disc, affecting the mechanical integrity leading to spinal instability. Quantitative T2* MRI probes water mobility within the macromolecular network, a potentially more sensitive assessment of disc health. We determined the relationship between T2* relaxation time and proteoglycan content, collagen content, and compressive mechanics throughout the degenerative spectrum. Eighteen human cadaveric lumbar (L4–L5) discs were imaged using T2* MRI. The T2* relaxation time at five locations (nucleous pulposus or NP, anterior annulus fibrosis or AF, posterior AF, inner AF, and outer AF) was correlated with sulfated-glycosaminoglycan (s-GAG) content, hydroxyproline content, and residual stress and strain at each location. T2* relaxation times were significantly correlated with s-GAG contents in all test locations and were particularly strong in the NP (r = 0.944; p < 0.001) and inner AF (r = 0.782; p < 0.001). T2* relaxation times were also significantly correlated with both residual stresses and excised strains in the NP (r = 0.857; p < 0.001: r = 0.816; p < 0.001), inner AF (r = 0.535; p = 0.022: r = 0.516; p = 0.028), and outer AF (r = 0.668; p = 0.002: r = 0.458; p = 0.041). These strong correlations highlight T2* MRI’s ability to predict the biochemical and mechanical health of the disc. T2* MRI assessment of disc health is a clinically viable tool showing promise as a biomarker for distinguishing degenerative changes.
doi:10.1002/jor.22633
PMCID: PMC4136382  PMID: 24788830
disc degeneration; quantitative magnetic resonance imaging; T2* (T2 star); proteoglycan; biomechanics
25.  Transient Hypoxia Improves Matrix Properties in Tissue Engineered Cartilage 
Adult articular cartilage is a hypoxic tissue, with oxygen tension ranging from <10% at the cartilage surface to <1% in the deepest layers. In addition to spatial gradients, cartilage development is also associated with temporal changes in oxygen tension. However, a vast majority of cartilage tissue engineering protocols involves cultivation of chondrocytes or their progenitors under ambient oxygen concentration (21% O2), that is, significantly above physiological levels in either developing or adult cartilage. Our study was designed to test the hypothesis that transient hypoxia followed by normoxic conditions results in improved quality of engineered cartilaginous ECM. To this end, we systematically compared the effects of normoxia (21% O2 for 28 days), hypoxia (5% O2 for 28 days) and transient hypoxia—reoxygenation (5% O2 for 7 days and 21% O2 for 21 days) on the matrix composition and expression of the chondrogenic genes in cartilage constructs engineered in vitro. We demonstrated that reoxygenation had the most effect on the expression of cartilaginous genes including COL2A1, ACAN, and SOX9 and increased tissue concentrations of amounts of glycosaminoglycans and type II collagen. The equilibrium Young’s moduli of tissues grown under transient hypoxia (510.01 ± 28.15 kPa) and under normoxic conditions (417.60 ± 68.46 kPa) were significantly higher than those measured under hypoxic conditions (279.61 ± 20.52 kPa). These data suggest that the cultivation protocols utilizing transient hypoxia with reoxygenation have high potential for efficient cartilage tissue engineering, but need further optimization in order to achieve higher mechanical functionality of engineered constructs.
doi:10.1002/jor.22275
PMCID: PMC4136653  PMID: 23203946
tissue engineering; cartilage; hypoxia; hydrogel; extracellular matrix

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