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Journal of Orthopaedic Research

Wiley Online Library : Journal of Orthopaedic Research

Published: 2017-11-01T00:00:00-05:00


Early Mechanical Stimulation Only Permits Timely Bone Healing in Sheep


Bone fracture healing is sensitive to the fixation stability. However, it is unclear which phases of healing are mechano-sensitive and if mechanical stimulation is required throughout repair. In this study, a novel bone defect model, which isolates an experimental fracture from functional loading, was applied in sheep to investigate if stimulation limited to the early proliferative phase is sufficient for bone healing. An active fixator controlled motion in the fracture. Animals of the control group were unstimulated. In the physiological-like group, 1 mm axial compressive movements were applied between day 5 and 21, thereafter the movements were decreased in weekly increments and stopped after 6 weeks. In the early stimulatory group, the movements were stopped after 3 weeks. The experimental fractures were evaluated with mechanical and micro-computed tomography methods after 9 weeks healing. The callus strength of the stimulated fractures (physiological-like and early stimulatory) was greater than the unstimulated control group. The control group was characterized by minimal external callus formation and a lack of bone bridging at 9 weeks. In contrast, the stimulated groups exhibited advanced healing with solid bone formation across the defect. This was confirmed quantitatively by a lower bone volume in the control group compared to the stimulated groups. The novel experimental model permits the application of a well-defined load history to an experimental bone fracture. The poor healing observed in the control group is consistent with under-stimulation. This study has shown early mechanical stimulation only is sufficient for a timely healing outcome. This article is protected by copyright. All rights reserved

New Algorithm for Simulation of Proteoglycan Loss and Collagen Degeneration in the Knee Joint: Data from the Osteoarthritis Initiative


Osteoarthritis is a harmful joint disease but prediction of disease progression is problematic. Currently, there is only one modeling framework which can be applied to predict the progression of knee osteoarthritis but it only considers degenerative changes in the collagen fibril network. Here, we have developed the framework further by considering all of the major tissue changes (proteoglycan content, fluid flow and collagen fibril network) occurring in osteoarthritis. While excessive levels of tissue stresses controlled degeneration of the collagen fibril network, excessive levels of tissue strains controlled the decrease in proteoglycan content and the increase in permeability. We created four knee joint models with increasing degrees of complexity based on the depth-wise composition. Models were tested for normal and abnormal, physiologically relevant, loading conditions in the knee. Finally, the predicted depth-wise compositional changes from each model were compared against experimentally observed compositional changes in vitro. The model incorporating the typical depth-wise composition of cartilage produced the best match with experimental observations. Consistent with earlier in vitro experiments, this model simulated the greatest proteoglycan depletion in the superficial and middle zones, while the collagen fibril degeneration was located mostly in the superficial zone. The presented algorithm can be used for predicting simultaneous collagen degeneration and proteoglycan loss during the development of knee osteoarthritis. This article is protected by copyright. All rights reserved

Integrating Soft and Hard Tissues via Interface Tissue Engineering


The enthesis, or interface between bone and soft tissues such as ligament and tendon, is prone to injury and often does not heal post-surgical intervention. Interface tissue engineering represents an integrative strategy for regenerating the native enthesis by functionally connecting soft and hard tissues and thereby improving clinical outcome. This review focuses on integrative and cell-instructive scaffold designs that target the healing of the two most commonly injured soft tissue-bone junctions: the tendon-bone interface (e.g. rotator cuff) and ligament-bone interface (e.g. anterior cruciate ligament). The inherent connectivity between soft and hard tissues is instrumental for musculoskeletal motion and is therefore a key design criterion for soft tissue regeneration. To this end, scaffold design for soft tissue regeneration have progressed from single tissue systems to the emerging focus on pre-integrated composite tissue units. Specifically, a multifaceted, bioinspired approach has been pursued wherein scaffolds are tailored to stimulate relevant cell responses using spatially patterned structural and chemical cues, growth factors, and/or mechanical stimulation. Moreover, current efforts to elucidate the essential scaffold design criteria via strategic biomimicry are emphasized as these will reduce complexity in composite tissue regeneration and ease the related burden for clinical translation. These innovative studies underscore the clinical relevance of engineering connective tissue integration and have broader impact in the formation of complex tissues for functional tissue engineering and total joint regeneration. This article is protected by copyright. All rights reserved

Distribution of Segmental Foot Kinematics in Patients with Degenerative Joint Disease of the Ankle


Degenerative joint disease (DJD) of the ankle is a debilitating chronic disease associated with severe pain and dysfunction resulting in antalgic gait alteration. Little information is available about segmental foot and ankle motion distribution during gait in ankle osteoarthritis. The aim of the current study was to dynamically characterize segmental foot and ankle kinematics of patients with severe ankle arthrosis requiring total ankle replacement. This was a prospective study involving 36 (19 M, 17 F) adult patients with a clinical diagnosis of ankle arthrosis ("DJD" group) and 36 (23 M, 13 F) healthy subjects ("Control" group). Motion data were collected at 120 Hz using a 3-D motion camera system at self-selected speed along a 6-meter walkway and processed using the Milwaukee Foot Model (MFM). The SF-36 Health Survey and Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot scale were administered to evaluate functional levels. Findings include decreases in walking speed, cadence, stride length and swing phase and reduced outcomes scores (SF-36 and AOFAS). Multisegemental motion in patients with ankle DJD demonstrates significant changes in foot mechanics characterized by altered segment kinematics and significant reduction in dynamic ROM at the tibia, hindfoot, forefoot, and hallux when compared to controls. The results demonstrate decreased temporal-spatial parameters and low outcomes scores indicative of functional limitations. Statement of Clinical Significance: Altered segment kinematics and reduced overall range of motion demonstrate how a single joint pathology affects kinematic distribution in the other segments of the foot and ankle and alters patients' overall gait. This article is protected by copyright. All rights reserved

Effects of lateral-offset sole shoes on knee adduction moment in women with medial compartment knee osteoarthritis


This study aimed to determine the impact of wearing a lateral-offset sole shoe (LOSS) on knee adduction moment (KAM) in patients with medial knee osteoarthritis (OA). From December 2012 to November 2016, patients with medial knee OA were recruited. Ninety-three knees (50 left, 43 right) of 93 female patients were analyzed. The first peak KAMs were measured with patients 1) walking barefoot, 2) walking in conventional shoes, and 3) walking in LOSSs. The patients had grade 1 (n=19), grade 2 (n=49), grade 3 (n=20), and grade 4 (n=5) knee OA. First peak KAMs differed significantly in all three conditions (p=0.031). In the post hoc analysis, first peak KAMs were significantly lower during LOSS walking than during conventional shoe walking (p=0.001), but there were no differences in peak KAMs between barefoot and LOSS walking (p=0.784). In the subgroup analysis, patients with grade 2 and 3 OA showed significantly lower first peak KAMs during LOSS walking than during conventional shoe walking (p=0.029 and p=0.011, respectively). Both the peak eversion ankle angle and moment of barefoot walking showed a significant increase compared with LOSS and conventional shoe walking, while there was no significant difference between LOSS and conventional shoe walking (p=0.612 and p=0.197, respectively). Our results suggest that LOSS wearing caused significant KAM reductions compared with conventional shoe wearing. Since LOSS wearing does not cause changes in the peak eversion ankle angle and moment during the load response, it may be an effective method to reduce the KAM in women with knee OA. This article is protected by copyright. All rights reserved

Human prosthetic joint infections are associated with myeloid-derived suppressor cells (MDSCs): implications for infection persistence


Prosthetic joint infection (PJI) is a devastating complication of joint arthroplasty surgery typified by biofilm formation. Currently, mechanisms whereby biofilms persist and evade immune-mediated clearance in immune competent patients remain largely ill-defined. Therefore, the current study characterized leukocyte infiltrates and inflammatory mediator expression in tissues from patients with PJI compared to aseptic loosening. CD33+HLA-DR-CD66b+CD14-/low granulocytic myeloid-derived suppressor cells (G-MDSCs) were the predominant leukocyte population at sites of human PJI compared to aseptic tissues. MDSCs inhibit T cell proliferation, which coincided with reduced T cells in PJIs compared to aseptic tissues. IL-10, IL-6, and CXCL1 were significantly elevated in PJI tissues and have been implicated in MDSC inhibitory activity, expansion, and recruitment, respectively, which may account for their preferential increase in PJIs. This bias towards G-MDSC accumulation during human PJI could account for the chronicity of these infections by preventing the pro-inflammatory, antimicrobial actions of immune effector cells. Clinical Significance: Animal models of PJI have revealed a critical role for MDSCs and IL-10 in promoting infection persistence; however, whether this population is prevalent during human PJI and across distinct bacterial pathogens remains unknown. This study has identified that granulocytic-MDSC infiltrates are unique to human PJIs caused by distinct bacteria, which are not associated with aseptic loosening of prosthetic joints. Better defining the immune status of human PJIs could lead to novel immune-mediated approaches to facilitate PJI clearance in combination with conventional antibiotics. This article is protected by copyright. All rights reserved

Topical Adjuvants Incompletely Remove Adherent Staphylococcus aureus from Implant Materials


Adjuvant treatments including Betadine, Dakin's solution (sodium hypochlorite), or hydrogen peroxide (H2O2) have been attempted to eradicate prosthetic joint infection caused by biofilm or intracellular bacteria. The purpose of this study was to evaluate the in vitro abilities of chemical adjuvants to decrease Staphylococcus aureus (S. aureus) biofilm presence on orthopaedic implant grade materials, including titanium, stainless steel, and cobalt chrome. S. aureus biofilms were grown for 48 hours and evaluated for baseline colony forming units/centimeter squared (CFU/cm2) and compared to treatments with Betadine, Dakin's solution, H2O2, or 1% chlorine dioxide (ClO2). Control discs (n=18) across all metals had an average of 4.2 x107 CFU/cm2. All treatments had statistically significant reductions in CFU/cm2 when compared to respective control discs (p < 0.05). For all metals combined, the most efficacious treatments were Betadine and H2O2, with an average 98% and 97% CFU/cm2 reduction, respectively. There were no significant differences between reductions seen with Betadine and H2O2, but both groups had statistically greater reductions than Dakin's solution and ClO2. There was no change in antibiotic resistance patterns after treatment. Analysis of S. aureus biofilms demonstrated a statistically significant reduction in biofilm after a five-minute treatment with the modalities, with an average two log reduction in CFU/cm2. Statement of Clinical Significance: While statistically significant reductions in CFU/cm2 were accomplished with chemical adjuvant treatments, the overall concentration of bacteria never fell below 105 CFU/cm2, leading to questionable clinical significance. Further techniques to eradicate biofilm should be investigated. This article is protected by copyright. All rights reserved

Gait patterns, symptoms and function in patients with isolated tibiofemoral osteoarthritis and combined tibiofemoral and patellofemoral osteoarthritis


The purpose of this study was to compare hip and knee biomechanics during walking in individuals with isolated TFOA, combined TFOA and PFOA, and those without knee OA, and to compare patient-reported symptoms and function in individuals with isolated TFOA and those with combined TFOA and PFOA. Participants with and without knee OA were assessed and categorized into (i) no OA, (ii) isolated TFOA, and (iii) combined TFOA and PFOA, based on Kellgren and Lawrence diagnostic criteria. Quantitative motion analyses were conducted during walking, and hip and knee kinematics and external moments were calculated. Peak values in the sagittal and frontal planes during stance phase were computed. Patient-reported symptoms and function data were obtained using the Western Ontario McMaster Universities Arthritis Index. Multivariate analyses of variance were conducted to compare between-group differences in gait and patient-reported symptoms and function data. The results showed no statistically significant differences observed in hip and knee kinematics and external moments between the three groups. Relative to those with isolated TFOA, individuals with combined TFOA and PFOA had greater pain (mean difference [95% CI]: 1.5 [0.05 to 3.1]), stiffness (0.8 [0.02 to 1.5]), and poorer function (5.4 [0.2 to 10.7]). In conclusion, the combined TFOA and PFOA radiographic disease pattern is associated with worse pain and function compared to the isolated TFOA disease pattern. The results of the present study provide no indications that treatments designed to change walking biomechanics should differ between individuals with isolated TFOA and those with combined TFOA and PFOA. This article is protected by copyright. All rights reserved

Transcriptomic Analysis of Synovial Extracellular RNA Following Knee Trauma: A Pilot Study


Traumatic knee injuries often result in damage to articular cartilage and other joint structures. Such trauma is a strong risk factor for the future development and progression of osteoarthritis (OA). The molecular mechanisms and signaling pathways modulating response to knee joint trauma remain unclear. Moreover, investigations of biomarkers influencing responses have been targeted rather than broad, unbiased discovery studies. Herein we characterize the complete complement of extracellular RNA (exRNA) in the synovial fluid of 14 subjects following knee injury. Fluid was collected during surgery from the injured knees, and from the contralateral knee in a subset, undergoing surgical repair of the ACL and/or meniscal repair/debridement. Arthroscopic grading of chondral damage in four knee compartments was performed using the Outerbridge classification. exRNA was extracted and subjected to massively parallel total RNA sequencing. Differential abundance of RNA was calculated between the subject cohorts of injured and non-injured knee, average Outerbridge score ≥ 0.5 and less, and chronic and acute injury duration defined as ≤ 4 months till surgery or longer. Overall, expression of several thousand genes was identified in the synovial fluid. Furthermore, differential expression analysis suggests a role of exRNA fragments of matrix metalloproteinases and skeletal muscle fiber genes in the response to traumatic injury. Together, these data suggest that high-throughput approaches can indicate exRNA molecular signatures following knee trauma. Future studies are required to more fully characterize the biological roles of these exRNA and the cadence of their respective release that may lead to translational treatment options for post-traumatic OA. This article is protected by copyright. All rights reserved

Biomechanical investigation of four different fixation techniques in sacrum Denis type II fracture with low bone mineral density


With increasing life expectancy, fragility fractures of the pelvic ring are seen more frequently. Although their osteosynthesis can be very challenging, specific biomechanical studies for investigation of the fixation stability are still lacking. The aim of this study was to biomechanically evaluate four different fixation methods for sacrum Denis type II fractures in osteoporotic bone. Unstable Denis type II vertical sacrum fractures were created in 16 human pelves. Their osteosynthesis was performed with one sacro-iliac screw, posterior sacral plating, triangular fixation, or spino-pelvic fixation. For that purpose, each pelvis was randomly assigned to 2 paired groups for treatment with either SI-screw/posterior sacral plating or triangular fixation/spino-pelvic fixation. Each hemi-pelvis was cyclically tested under progressively increasing axial compression. Relative interfragmentary movements were investigated via optical motion tracking analysis. Axial stiffness of triangular fixation was significantly higher versus posterior sacral plating and spino-pelvic fixation (p ≤ 0.022), but not significantly different in comparison to SI-screw fixation (p = 0.337). Cycles to 2, 3, 5 and 8 mm fracture displacement, as well as to 3, 5 and 8° gap angle at the fracture site were significantly higher for triangular fixation compared to all other groups (p≤0.041). Main failure mode for all osteosynthesis techniques was screw cutting through the bone, leading loss of fixation stability. From a biomechanical point of view, triangular fixation in sacrum Denis type II fractures demonstrated less interfragmentary movements and should be considered in unstable fragility fractures of the sacrum. This article is protected by copyright. All rights reserved

Quantifying Meniscal Kinematics in Dogs


The dog has been used extensively as an experimental model to study meniscal treatments such as meniscectomy, meniscal repair, transplantation and regeneration. However, there is very little information on meniscal kinematics in the dog. This study used MR imaging to quantify in vitro meniscal kinematics in loaded dog knees in four distinct poses: extension, flexion, internal and external rotation. A new method was used to track the meniscal poses along the convex and posteriorly tilted tibial plateau. Meniscal displacements were large, displacing 13.5 mm and 13.7 mm posteriorly on average for the lateral and medial menisci during flexion (p = 0.90). The medial anterior horn and lateral posterior horns were the most mobile structures, showing average translations of 15.9 mm and 15.1 mm, respectively. Canine menisci are highly mobile and exhibit movements that correlate closely with the relative tibiofemoral positions. This article is protected by copyright. All rights reserved

Low-dose lithium regimen enhances endochondral fracture healing in osteoporotic rodent bone


Osteoporotic bone fractures are highly prevalent and involve lengthy recovery. Lithium, commonly used in psychiatric medicine, inhibits glycogen synthase kinase-3β in the Wnt/β-catenin pathway, leading to up-regulation of osteogenesis. Our recent preclinical work demonstrated that a 20mg/kg lithium dose administered beginning 7 days post-fracture for 14 days optimally improved femoral fracture healing in healthy rats at 4 weeks post fracture (46% higher torsional strength). In this study, lithium treatment was evaluated for healing of osteoporotic bone fractures. Six-month old ovariectomized rats were subjected to closed, load-drop induced femoral diaphyseal fracture. Two regimens involving treatment initiation on day 7 and day 10, respectively, 20mg/kg/day oral dose and 14 days duration were evaluated. Femurs of lithium- vs. saline- treated rats were analyzed at 4 weeks (for day 7 onset regimen) or 6 weeks (for day 10 onset regimen) post-fracture by stereology and torsional mechanical testing. Initiation on day 10 led to a significant 50% higher maximum yield torque (primary outcome measure) at 6 weeks (309 vs. 206 N-mm, p=0.005; n=7, 7). Initiation on day 7 suggested a trend toward a more modest improvement in maximum yield torque (13%) evaluated at 4 weeks post-fracture (234 vs. 206 N-mm, p=0.10; n=10, 13). Qualitatively, lithium-treated femurs demonstrated better periosteal and mineralized callus bridging in the day 10 initiation group. Lithium is a widely-available, orally administered, low-cost drug, which represents a feasible pharmacological intervention for both healthy and osteoporotic fracture healing. This study provides important guidelines for future clinical evaluation of lithium in osteoporotic fracture patients. This article is protected by copyright. All rights reserved



During aging, skeletal muscle tissue progressively declines in mass, strength, and regenerative capacity. Decreased muscle stem cell (MuSC) number and impaired function might underlie the aging-related muscle wasting and impaired regenerative capacity. As yet, the search for factors that regulate MuSC fate and function has revealed several biochemical factors within the MuSC niche that may be responsible for the decline in MuSC regenerative capacity. This decline cannot be explained by environmental factors solely, as the MuSC potential to regenerate muscle tissue is not reversed by changing the biochemical MuSC niche composition. Here we discuss the likeliness that during physical exercise, MuSCs within their niche are subjected to mechanical loads, in particular pressure and shear stress, as well as associated deformations. We postulate that these physical cues are involved in the activation and differentiation of MuSCs as these cells contain several transmembrane sensor proteins that have been shown to be mechanosensitive in other cell types, i.e. endothelial cells and osteoprogenitors. We will specifically address age-related changes in mechanosensing in MuSCs and their niche. Insight in the physical cues applied to the MuSCs in vivo, and how these cues affect MuSC fate and function, helps to develop new therapeutic interventions to counterbalance age-related muscle loss. This requires an approach combining two- and three-dimensional live cell imaging of MuSCs within contracting muscle tissue, mathematical finite element modeling, and cell biology. This article is protected by copyright. All rights reserved

Low intensity vibration increases cartilage thickness in obese mice


Obesity is associated with an elevated risk of osteoarthritis (OA). We examined here whether high fat diet administered in young mice, compromised the attainment of articular cartilage thickness. Further, we sought to determine if low intensity vibration (LIV) could protect the retention of articular cartilage in a mouse model of diet induced obesity. Five-week-old, male, C57BL/6 mice were separated into 3 groups (n = 10): Regular diet (RD), High fat diet (HF), and HF + LIV (HFv; 90Hz, 0.2g, 30 min/d, 5 d/w) administered for 6 weeks. Additionally, an extended HF diet study was run for 6 months (LIV at 15m/d). Articular cartilage and subchondral bone morphology, and sulfated GAG content were quantified using contrast agent enhanced µCT and histology. Gene expression within femoral condyles was quantified using real-time polymerase chain reaction. Contrary to our hypothesis, HF cartilage thickness was not statistically different from RD. However, LIV increased cartilage thickness compared to HF, and the elevated thickness was maintained when diet and LIV were extended into adulthood. RT-PCR analysis showed a reduction of aggrecan expression with high fat diet, while application of LIV reduced the expression of degradative MMP-13. Further, long term HF diet resulted in subchondral bone thickening, compared to RD, providing early evidence of OA pathology—LIV suppressed the thickening, such that levels were not significantly different from RD. These data suggest that dynamic loading, via LIV, protected the retention of cartilage thickness, potentially resulting in joint surfaces better suited to endure the risks of elevated loading that parallel obesity. This article is protected by copyright. All rights reserved

Polydeoxyribonucleotide improves tendon healing following achilles tendon injury in rats


Tendon injuries are major musculoskeletal disorders. Polydeoxyribonucleotide activates the adenosine receptor subtype A2A, resulting in tissue growth and neogenesis. This experimental study confirms that polydeoxyribonucleotide can improve secretion of various growth factors, promote collagen synthesis, and restore tensile strength of the Achilles tendon in a rat model with Achilles tendon injury. Thirty-six male Sprague-Dawley rats, aged 7 weeks, were divided into two groups, and the Achilles tendon was transected and repaired using the modified Kessler's method. In the experimental group (n = 18), the rats received daily intraperitoneal administration of polydeoxyribonucleotide (8 mg/kg/day for 1, 2, or 4 weeks). The control groups received the same amount of normal saline. The rats were euthanized at 1, 2, and 4 weeks, and tissues from the repair site were harvested. The cross-sectional area of the tendon was significantly increased at 2 and 4 weeks in polydeoxyribonucleotide group (p = 0.008 and p = 0.017, respectively). Moreover, tendons in the polydeoxyribonucleotide group were more resistant to mechanical stress at 2 and 4 weeks (p = 0.041 and p = 0.041, respectively). The staining levels of collagen type I in the experimental group were significantly stronger at 2 and 4 weeks (p = 0.026 and p = 0.009, respectively). Furthermore, higher expression levels of fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor β1 were detected in the experimental group at 4 weeks (p = 0.041, p = 0.026, and p = 0.041, respectively). This study confirms that polydeoxyribonucleotide can improve the tensile strength of the rats' Achilles tendon following injury and repair. This article is protected by copyright. All rights reserved

Suppression of hyaluronan synthesis attenuates the tumorigenicity of low grade chondrosarcoma


Hyaluronan (HA) has been shown to play crucial roles in the tumorigenicity of malignant tumors. Chondrosarcoma, particularly when low-grade, is characterized by the formation of an extracellular matrix (ECM) containing abundant HA, and its drug/radiation resistance has become a clinically relevant problem. This study aimed to evaluate the effects of an HA synthesis inhibitor, 4-methylumbelliferone (MU), on ECM formation as well as antitumor effects in chondrosarcoma. We investigated the effects of MU on rat chondrosarcoma (RCS) cells with a grade I histological malignancy in vitro and in vivo grafted model. HA binding protein (HABP) stainability on and around the RCS cells was effectively reduced with treatment of MU. ECM formation was markedly suppressed by MU at a dose of 1.0mM. Cell proliferation was significantly reduced by MU at 24 hrs. Cell motility and invasion were suppressed in a dose-dependent manner by MU. No significant changes in mRNA expression of Has1-3 were observed. Furthermore, MU inhibited the growth of grafted tumors in vivo. Histologically, chondrosarcoma cells of control tumors showed a cell-clustering structure. HABP stainability was markedly decreased in the MU-treated group. These results suggest that MU exhibits antitumor effects on low-grade chondrosarcoma, via inhibition of HA accumulation and ECM formation. MU, which is an approved drug in bile therapy, could be a new off-label medication for chondrosarcomas. This article is protected by copyright. All rights reserved

Towards Engineering Distinct Multi-lamellated Outer and Inner Annulus Fibrosus Tissues


The annulus fibrosus (AF) of the intervertebral disc (IVD) has a zonal distribution of phenotypically distinct cells. The outer AF (OAF) cells produce an extracellular matrix (ECM) rich in type I collagen with little proteoglycans, whereas the ECM of the inner AF (IAF) has abundant type II collagen and proteoglycans. The inhomogeneous distribution of the ECM in the AF may reflect the complex mechanical forces that the IVD experiences. A bioengineered AF tissue should recapitulate both the inner and outer zones in order to have proper functionality. The aim of this study is to generate multi-lamellated OAF and IAF tissues with ECM compositions that resemble their zonal origin using polycarbonate urethane (PU) scaffolds. It was observed that supplementation of the media with insulin-transferrin-selenium (ITS) and proline yielded tissues with good cellularity. However, IAF cells accumulated only type I collagen, similar to OAF cells. Addition of dexamethasone and sodium pyruvate induced the accumulation of IAF tissues rich in type II collagen and aggrecan, without altering the accumulation of type I collagen in OAF tissues. Dexamethasone stimulated mitochondrial membrane potential in both tissues in the presence of sodium pyruvate, suggesting a relationship between the mitochondrial aerobic respiratory state and dexamethasone signalling during the in vitro-tissue formation by OAF and IAF cells. Inhibition of the glucocorticoid receptor blocked the stimulation of mitochondrial membrane potentials and type II collagen accumulation. In summary, biologically distinct multi-lamellated OAF and IAF tissues can be generated, which will facilitate advancement towards the goal of engineering a biological IVD replacement. This article is protected by copyright. All rights reserved

3-Dimensional Metrics of Proximal Femoral Shape Deformities in Legg-Calvé-Perthes Disease and Slipped Capital Femoral Epiphysis


Legg-Calvé-Perthes disease (LCPD) and slipped capital femoral epiphysis (SCFE) are two common pediatric hip disorders that affect the 3-dimensional shape and function of the proximal femur. This study applied the principles of continuum mechanics to statistical shape modeling (SSM) and determined 3-D metrics for the evaluation of shape deformations in normal growth, LCPD, and SCFE. CT scans were obtained from 32 patients with asymptomatic, LCPD, and SCFE hips ((0.5–0.9mm)2 in-plane resolution, 0.63mm slice thickness). SSM was performed on segmented proximal femoral surfaces, and shape deformations were described by surface displacement, strain, and growth plate angle metrics. Asymptomatic normal femurs underwent coordinated, growth-associated surface displacements and anisotropic strains that were site-specific and highest at the greater trochanter. After size- and age-based shape adjustment, LCPD femurs exhibited large displacements and surface strains in the femoral head and neck, with associated changes in femoral head growth plate angles. Mild SCFE femurs had contracted femoral neck surfaces, and surface displacements in all regions tended to increase with severity of slip. The results of this paper provide new 3-D metrics for characterizing the shape and biomechanics of the proximal femur. Statement of Clinical Significance: Quantitative 3-D metrics of shape may be useful for understanding and monitoring disease progression, identifying target regions for shape modulation therapies, and objectively evaluating the success of such therapies. This article is protected by copyright. All rights reserved

Potential Role for a Specialized β3 Integrin-Based Structure On Osteocyte Processes in Bone Mechanosensation


Osteocyte processes are an order of magnitude more sensitive to mechanical loading than their cell bodies. The mechanisms underlying this remarkable mechanosensitivity are not clear, but may be related to the infrequent αVβ3 integrin sites where the osteocyte cell processes attach to canalicular walls. These sites develop dramatically elevated strains during load-induced fluid flow in the lacunar-canalicular system and were recently shown to be primary sites for osteocyte-like MLO-Y4 cell mechanotransduction. These αVβ3 integrin sites lack typical integrin transduction mechanisms. Rather, stimulation at these sites alters Ca2+ signaling, ATP release and membrane potential. In the current studies, we tested the hypothesis that in authentic osteocytes in situ, key membrane proteins implicated in osteocyte mechanotransduction are preferentially localized at or near to β3 integrin-foci. We analyzed these spatial relationships in mouse bone osteocytes using immunohistochemistry combined with Structured Illumination Super Resolution Microscopy, a method that permits structural resolution at near electron microscopy levels in tissue sections. We discovered that the purinergic channel pannexin1, the ATP-gated purinergic receptor P2X7R and the low voltage transiently opened T-type calcium channel CaV3.2−1 all reside in close proximity to β3 integrin attachment foci on osteocyte processes, suggesting a specialized mechanotransduction complex at these sites. We further confirmed this observation on isolated osteocytes in culture using STochasitc Optical Resonance Microscopy. These findings identify a possible structural basis for the unique mechanosensation and transduction capabilities of the osteocyte process. This article is protected by copyright. All rights reserved

“Patient reported outcomes” following experimental surgery - using telemetry to assess movement in experimental ovine models


Many potential treatments for orthopaedic disease fail at the animal to human translational hurdle. One reason for this failure is that the majority of pre clinical outcome measurements emphasise structural changes, such as gross morphology and histology, and do not address pain or its alleviation, which is a key component of treatment success in man. With increasing emphasis on 'patient reported outcome measurements (PROM)' in clinical practice, in this study we have used two different telemetric methods (geolocation and Fitbark activity trackers) to measure movement behaviour, i.e. an indirect PROM, in an ovine osteoarthritis induction and an osteochondral defect model performed in adult female Welsh Mountain sheep. This study demonstrates that both systems can be used to track movement and activity of experimental sheep before and after surgery and that the Geolocator system recorded a decrease in distance moved and activity at the end of the experimental period in both models. The Fitbark activity tracker also recorded significant alterations in movement behaviour at the end of these studies and this method of recording showed a correlation between Fitbark data and radiography, macroscopic and histological scoring (well recognised outcome measurements), particularly in animals with large (10mm) defects i.e. more severe pathology. These results suggest that telemetry is able to track movement behaviour in experimental sheep and that the methodology should be considered for inclusion in outcome measures in preclinical orthopaedic research. This article is protected by copyright. All rights reserved



Osteoarthritis is a high-incidence painful and debilitating disease characterized by progressive degeneration of articular joints, which indicates a breakdown in joint homeostasis favoring catabolic processes. Biomechanical loading, associated with inflammatory and metabolic imbalances of joint, strongly contributes to the initiation and progression of the disease. Obesity is a primary risk factor for disease onset, and mechanical factors increased the risk for disease progression. Moreover, inflammatory mediators, in particular, adipose tissue-derived cytokines (better known as adipokines) play a critical role linking obesity and osteoarthritis. The present article summarizes the knowledge about the role of adipokines in cartilage and bone function, highlighting their contribution to the imbalance of joint homeostasis and, consequently, pathogenesis of osteoarthritis. This article is protected by copyright. All rights reserved

The Unfolded Protein Response Mediated by PERK is casually related to the Pathogenesis of Intervertebral Disc Degeneration


Although the number of patients with intervertebral disc (IVD) degeneration is increasing in aging societies, its etiology and pathogenesis remain elusive and there is currently no effective treatment to prevent this undesirable condition. The unfolded protein response (UPR) is a cellular machinery that plays critical roles in handling endoplasmic reticulum (ER) stress, a condition caused by the accumulation of unfolded proteins in the ER lumen. This study aimed to elucidate the potential role of the UPR mediated by pancreatic endoplasmic reticulum kinase (PERK), one of the major ER stress sensors in mammalian cells, in the development of IVD degeneration. IVD degeneration was artificially induced in Wister rats by percutaneously puncturing the coccyx IVDs and human IVDs were collected from patients who underwent spinal surgery. Expression of the UPR target genes was elevated in degenerative IVDs in both humans and rats. The induction of ER stress in annulus fibrosus cells significantly increased the transcripts for tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in a nuclear factor (NF)-κB pathway-dependent manner. The expression of TNF-α and IL-6 was significantly reduced by treatment with a selective PERK inhibitor, GSK2606414 and by gene silencing against PERK and activating transcription factor 4 (ATF4) transcripts. Our findings indicate that the UPR mediated by the PERK pathway is causally related to the development of IVD degeneration, suggesting that PERK may be a potential molecular target for suppressing the degenerative changes in IVDs. This article is protected by copyright. All rights reserved

No Difference Between Mechanical Perturbation Training with Compliant Surface and Manual Perturbation Training on Knee Functional Performance After ACL Rupture


Manual perturbation training improves dynamic knee stability and functional performance after anterior cruciate ligament rupture (ACL-rupture). However, it is limited to static standing position and does not allow time-specific perturbations at different phase of functional activities. The purpose of this study was to investigate whether administering mechanical perturbation training including compliant surface provides effects similar to manual perturbation training on knee functional measures after an acute ACL-rupture. Sixteen level I/II athletes with ACL-ruptures participated in this preliminary study. Eight patients received mechanical (Mechanical) and 8 subjects received manual perturbation training (Manual). All patients completed a functional testing (isometric quadriceps strength, single-legged hop tests) and patient-reported measures (Knee Outcome Survey- Activities of Daily Living Scale (KOS-ADLS), Global Rating Score (GRS), International Knee Documentation Committee 2000 (IKDC 2000) at pre- and post-training. 2X2 ANOVA was used for data analysis. No significant group-by-time interactions were found for all measures (p>0.18). Main effects of time were found for single hop (Pre-testing: 85.14%+21.07; Post-testing: 92.49%+17.55), triple hop (Pre-testing: 84.64%+14.17; Post-testing: 96.64%+11.14), KOS-ADLS (Pre-testing: 81.13%+11.12; Post-testing: 88.63%+12.63), GRS (Pre-testing: 68.63%+15.73; Post-testing: 78.81%+13.85), and IKDC 2000 (Pre-testing: 66.66%+9.85; Post-testing: 76.05%+14.62) (p<0.032). Administering mechanical perturbation training using compliant surfaces induce effects similar to manual perturbation training on knee functional performance after acute ACL-rupture. The clinical significance is both modes of training improve patients' functional-performance and limb-to-limb movement symmetry, and enhancing the patients' self-reported of knee functional measures after ACL rupture. Mechanical perturbation that provides a compliant surface might be utilized as part of the ACL rehabilitation training. This article is protected by copyright. All rights reserved

Hip abductor strength and fatigue are associated with activity levels more than one year after total hip replacement


Despite improvements in pain and function, people who undergo total hip arthroplasty (THR) may not always return to desired levels of physical activity (PA). The factors associated with low activity levels are not fully understood. Abductor weakness and fatigue have both been proposed as factors that limit activity in older adults or people with hip osteoarthritis, but have not been investigated after THR. We hypothesized that abductor weakness and fatigue are associated with lower activity levels in people who have undergone a THR and that fatigue mediates the association between abductor strength and activity. We evaluated 16 subjects (24±10 months post-THR; age 56.8±8.4 yrs; BMI 31±7 kg/m2). Fatigue was assessed using the PROMIS Fatigue Short-form 7a. Peak isometric hip abductor torque was assessed using a dynamometer with subjects in a sidelying position. We assessed activity level using the UCLA activity score. We used Pearson correlations to explore the associations among the variables. Next we used a three-step linear regression procedure to test whether or not fatigue acted as a mediator between abductor torque and UCLA activity scores. Higher abductor torque was associated with less fatigue (R2=0.275; p=0.037) and with higher UCLA scores (R2=0.488, p=0.003). Higher fatigue was associated with lower UCLA scores (R2 = 0. 307, p = 0.017), however there was no evidence of mediation. This suggests that addressing both abductor strength and fatigue may increase physical activity. Statement of Clinical Significance: Fatigue and abductor weakness should be evaluated in sedentary THR patients presenting for long-term follow up. This article is protected by copyright. All rights reserved

Femoral Version: Comparison Among Advanced Imaging Methods


Accurate assessment of femoral version is essential to the surgical treatment of lower extremity deformities, yet the ideal modality and technique to measure femoral version is controversial. This study explored two hypotheses: First, there is no difference in the accuracy of femoral version measurement from 2D CT, 2D MRI and 3D biplanar radiography reconstructions compared to a 3D model created from CT. Second, there is a difference between the measured version from traditional axial sections of the proximal femur compared to femoral neck oblique sections for CT and MRI. Eight adult cadaver lower extremities underwent CT, MRI and biplanar radiography. Femoral version measurements from the CT and MRI axial and oblique sections, as well as biplanar radiography reconstructions, were compared to 3D reconstructed models from CT. All five techniques underestimated femoral version compared to the 3D model, but none were statistically significantly different. Regarding the first hypothesis, all five techniques had excellent correlation (r > 0.81, p≤0.01) with the 3D model. Concerning the second hypothesis, the CT and MRI version measurements in femoral neck oblique sections were greater by 5.4° and 1.4°compared to traditional axial sections, respectively. All five techniques across three modalities provided accurate assessment of femoral version, suggesting that the treating physician's choice of modality can be determined per individual patient, not on measurement accuracy. Clinical Significance: In choosing a modality to determine femoral version, consider the advantages and disadvantages of each modality for the individual patient, using femoral neck oblique slices for CT and MRI when available. This article is protected by copyright. All rights reserved

Degradation Alters the Lubrication of Articular Cartilage by High Viscosity, Hyaluronic Acid-Based Lubricants


Hyaluronic Acid (HA) is widely injected as a viscosupplement in the treatment of osteoarthritis. Despite its extensive use, it is not currently known if cartilage degradation alters how HA-based solutions lubricate the articular surface. In this study we utilized a model of cartilage degradation by IL-1β along with a recently developed framework to study role of cartilage degradation on lubrication by clinically-approved HA-based lubricants with high viscosities. Cartilage explants were cultured up to 8 days with 10 ng/mL IL-1β. After culture, samples were examined histologically, immunohistochemically, biochemically, mechanically, topographically, and tribologically. The tribological testing analyzed both boundary and mixed lubrication modes to assess individual effects of viscosity and boundary lubricating ability. Friction testing was carried out using PBS and two clinically approved HA-based viscosupplements in a cartilage-glass configuration. After culture with IL-1β, boundary mode friction was elevated after both 4 and 8 days. Additionally, friction in mixed mode lubrication, where HA is most effective as a lubricant, was significantly elevated after 8 days of culture. As cartilage became rougher, softer, and more permeable after culture, the boundary mode plateau was extended, and as a result, significantly increased lubricant viscosities or sliding speeds were necessary to achieve effective mixed lubrication. Overall, this study revealed that lubrication of cartilage by HA is degradation-dependent and coincides with changes in mechanics and roughness. This article is protected by copyright. All rights reserved

Phlpp inhibitors block pain and cartilage degradation associated with osteoarthritis


Phlpp protein phosphatases are abnormally abundant within human osteoarthritic articular chondrocytes and may contribute to the development of osteoarthritis. Mice lacking Phlpp1 were previously shown to be resistant to post-traumatic osteoarthritis. Here a small molecule with therapeutic properties that inhibits Phlpp1 and Phlpp2 was tested for its ability to slow post-traumatic OA in mice and to stimulate anabolic pathways in human articular cartilage from OA joints. PTOA was induced in male C57Bl/6 mice by surgically destabilizing the meniscus. Seven weeks after surgery, mice received a single intra-articular injection of the Phlpp inhibitor NSC117079 or saline. Mechanical allodynia was measured with von Frey assays, mobility was tracked in an open field system, and cartilage damage was assessed histologically. A single intra-articular injection of the Phlpp inhibitor NSC117079 attenuated mechanical allodynia and slowed articular cartilage degradation in joints with a destabilized meniscus. Animals treated with the Phlpp inhibitor seven weeks after injury maintained normal activity levels, while those in the control group traveled shorter distances and were less active three months after the joint injury. NSC117079 also increased production of cartilage extracellular matrix components (glycosaminoglycans and aggrecan) in over 90% of human articular cartilage explants from OA patients and increased phosphorylation of Phlpp1 substrates (AKT2, ERK1/2 and PKC) in human articular chondrocytes. Our results indicate that Phlpp inhibitor NSC117079 is a novel osteoarthritis disease modifying drug candidate that may have palliative affects. This article is protected by copyright. All rights reserved

Expansion of Mesenchymal Stem Cells on Electrospun Scaffolds Maintains Stemness, Mechano-Responsivity, and Differentiation Potential


Mesenchymal stem cells (MSCs) hold great promise for regenerative therapies and tissue engineering applications given their multipotential differentiation capacity. However, MSC isolation and expansion are typically performed on super-physiologically stiff tissue culture plastic (TCP), which may alter their behavior and lead to unintended consequences upon implantation. In contrast, electrospun nanofibrous scaffolds possess physical and mechanical properties that are similar to that of native tissue. In this study, we investigated whether isolation and expansion of juvenile bovine MSCs directly onto electrospun nanofibrous scaffolds better preserves MSC phenotype and stemness compared to TCP. Our data show that culture of MSCs on electrospun scaffolds reduces proliferation, decreases cellular senescence, and better maintains stemness compared to cells isolated and expanded on TCP, likely due to a reduction in cell contractiility Furthermore, in contrast to electrospun scaffolds, TCP biased MSCs towards a fibrotic phenotype that persisted even after the cells were reseeded onto a different substrate. Cells pre-cultured on electrospun scaffolds exhibited a heightened response to mechanical stimuli and greater chondrogenesis in methacrylated hyaluronic acid hydrogels. These data suggest that alternative substrates that better approximate the native cell environment should be used to preserve endogenous MSC behavior and may improve their success in tissue engineering applications. This article is protected by copyright. All rights reserved

Healing in peri-implant gap with BMP-2 and systemic bisphosphonate is dependent on BMP-2 dose - a canine study


The bone-implant interface of cementless orthopedic implants can be described as a series of uneven sized gaps with discontinuous areas of direct bone-implant contact. Bridging these voids and crevices by addition of an anabolic stimulus to increase new bone formation can potentially improve osseointegration of implants. Bone Morphogenetic Protein 2 (BMP-2) stimulates osteoblast formation to increase new bone formation but indirectly stimulates osteoclast activity. In this experiment we investigate the hypothesis that osseointegration, defined as mechanical push-out and histomorphometry, depends on the dose of BMP-2 when delivered as an anabolic agent with systemic administration of the anti-resorptive agent zoledronate to curb an increase in osteoclast activity. Four porous coated titanium implants (one with each of three doses of surface-applied BMP-2 (15 µg; 60 µg; 240 µg) and control) surrounded by a 0.75 mm empty gap, were inserted into the distal femurs of each of twelve canines. Zoledronate IV (0.1 mg/kg) was administered ten days into the observation period of four weeks. Bone-implant specimens were evaluated by mechanical push-out test and histomorphometry. 15 µg implants had the best fixation on all mechanical parameters and largest surface area covered with new bone compared to control, 60 µg and 240 µg implants, as well as the highest volume of new bone in the implant gap compared to 60 µg and 240 µg implants. Results in a canine implant model demonstrated that a narrow range of BMP-2 doses have opposite effects in bridging an empty peri-implant gap with bone, when combined with systemic zoledronate. This article is protected by copyright. All rights reserved

In Vivo Correlates Between Daily Physical Activity and Intervertebral Disc Health


Physical activity impacts health and disease in multiple body tissues including the intervertebral discs. Fluid flow within the disc is an indicator of disc health that can be observed using diffusion weighted magnetic resonance imaging. We monitored activity levels of 26 participants, age 35-55yrs, using Actigraph accelerometers for four days to evaluate vigorous-intensity activity, moderate to vigorous intensity activity, and sedentary time. Participants underwent structural and diffusion weighted magnetic resonance imaging to evaluate intervertebral disc health and fluid flow. They also underwent bone density scans, carotid artery ultrasounds, a treadmill test, and a physical exam for pain, range of motion, and instability. These measures were used to correlate MRI indicators of intervertebral disc health with participant activity levels. Participants with any vigorous-intensity physical activity compared with no vigorous-intensity activity had significantly greater L5/S1 apparent diffusion coefficient values (p = 0.002), corresponding to higher freedom of diffusive movement for cellular nutrients and metabolic waste. Sagittal T2 values in the L5/S1 were also higher (p = 0.004), corresponding to a higher water content in the discs. Higher apparent diffusion coefficients were also found in participants with more than 30 minutes compared with less than 30 minutes of daily moderate to vigorous physical activity (p = 0.03), and in participants with less than 67% awake time as sedentary time compared with more than 67% sedentary time (p = 0.03). Increased dynamic loading through physical activity and decreased static loading from sedentary time benefit intervertebral disc health. Physical activity, particularly vigorous activity, is beneficial in helping maintain intervertebral disc health. This article is protected by copyright. All rights reserved

Analysis of skeletal muscle microcirculation in a porcine polytrauma model with haemorrhagic shock


Polytraumatised patients with haemorrhagic shock are prone to develop systemic complications, such as SIRS (systemic inflammatory response syndrome), ARDS (acute respiratory distress syndrome) and MOF (multiple organ failure). The pathomechanism of severe complications following trauma is multifactorial, and it is believed that microcirculatory dysfunction plays an important role. The aim of this study was to determine the changes in the microcirculation in musculature over time during shock and subsequent resuscitation in a porcine model of haemorrhagic shock and polytrauma. Twelve pigs (German Landrace) underwent femur fracture, liver laceration, blunt chest trauma and haemorrhagic shock under standard anaesthesia and intensive care monitoring. Microcirculation data were measured from the vastus lateralis muscle using a combined white light spectrometry and laser spectroscopy system every 15 minutes during the shock and resuscitation period, and at 24, 48 and 72 hours. Oxygen delivery and oxygen consumption were calculated and compared to baseline. The relative haemoglobin, local oxygen consumption and saturation values in the microcirculation were observed significantly lower during shock, however, no changes in the microcirculatory blood flow and microcirculatory oxygen delivery were observed. After resuscitation, the microcirculatory blood flow and relative haemoglobin increased and remained elevated during the whole observation period (72 hours). In this study, we observed changes in microcirculation during the trauma and shock phases. Furthermore, we also measured persistent dysfunction of the microcirculation over the observation period of three days after resuscitation and haemorrhagic shock. This article is protected by copyright. All rights reserved

Hydrogen Peroxide Induces Programmed Necrosis in Rat Nucleus Pulposus Cells through the RIP1/ RIP3-PARP-AIF Pathway


This study aimed to systematically investigate whether programmed necrosis contributes to H2O2-induced nucleus pulposus (NP) cells death and to further explore the underlying mechanism involved. Rat NP cells were subjected to different concentrations of H2O2 for various time periods. The cell viability was measured using a cell counting kit-8, and the death rate was detected by Hoechst 33258/propidium iodide staining. The programmed necrosis-related molecules receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), poly (ADP-ribose) polymerase (PARP) and apoptosis inducing factor (AIF) were determined by real-time polymerase chain reaction and western blotting, respectively. The morphologic and ultrastructural changes were examined by phasecontrast microscopy and transmission electron microscopy. In addition, the necroptosis inhibitor Necrostatin-1 (Nec-1), the PARP inhibitor diphenyl-benzoquinone (DPQ) and small interfering RNA technology were used to indirectly evaluate programmed necrosis. Our results indicated that H2O2 induced necrotic morphologic and ultrastructural changes and an elevated propidium iodide positive rate in NP cells; these effects were mediated by the upregulation of RIP1 and RIP3, hyperactivation of PARP, and translocation of AIF from mitochondria to nucleus. Additionally, NP cells necrosis was significantly attenuated by Nec-1, DPQ pretreatment and knockdown of RIP3 and AIF, while knockdown of RIP1 produced the opposite effects. In conclusion, these results suggested that under oxidative stress, RIP1/RIP3-mediated programmed necrosis, executed through the PARP-AIF pathway, played an important role in NP cell death. Protective strategies aiming to regulate programmed necrosis may exert a beneficial effect for NP cells survival, and ultimately retard intervertebral disc (IVD) degeneration. This article is protected by copyright. All rights reserved

Animal models for studying the etiology and treatment of low back pain


Chronic low back pain is a major cause of disability and health care costs. Effective treatments are inadequate for many patients. Animal models are essential to further understanding of the pain mechanism and testing potential therapies. Currently, a number of preclinical models have been developed attempting to mimic aspects of clinical conditions that contribute to low back pain. This review focused on describing these animal models and the main behavioral tests for assessing pain in each model. Animal models of low back pain can be divided into the following five categories: discogenic low back pain, radicular back pain, facet joint osteoarthritis back pain, muscle-induced low back pain, and spontaneous occurring low back pain models. These models are important not only for enhancing our knowledge of how low back pain is generated, but also for the development of novel therapeutic regimens to treat low back pain in patients. This article is protected by copyright. All rights reserved

Biomarkers for equine joint injury and osteoarthritis


We report the results of a symposium aimed at identifying validated biomarkers that can be used to complement clinical observations for diagnosis and prognosis of joint injury leading to equine osteoarthritis (OA). Biomarkers might also predict pre-fracture change that could lead to catastrophic bone failure in equine athletes. The workshop was attended by leading scientists in the fields of equine and human musculoskeletal biomarkers to enable cross-disciplinary exchange and improve knowledge in both. Detailed proceedings with strategic planning was written, added to, edited and referenced to develop this manuscript. The most recent information from work in equine and human osteoarthritic biomarkers was accumulated, including the use of personalized healthcare to stratify OA phenotypes, transcriptome analysis of anterior cruciate ligament (ACL) and meniscal injuries in the human knee. The spectrum of "wet" biomarker assays that are antibody based that have achieved usefulness in both humans and horses, imaging biomarkers and the role they can play in equine and human OA was discussed. Prediction of musculoskeletal injury in the horse remains a challenge, and the potential usefulness of spectroscopy, metabolomics, proteomics, and development of biobanks to classify biomarkers in different stages of equine and human OA were reviewed. The participants concluded that new information and studies in equine musculoskeletal biomarkers have potential translational value for humans and vice versa. OA is equally important in humans and horses, and the welfare issues associated with catastrophic musculoskeletal injury in horses add further emphasis to the need for good validated biomarkers in the horse. This article is protected by copyright. All rights reserved

The seating mechanics of head-neck modular tapers in vitro: Load-displacement measurements, moisture, and rate effects


The mechanically-assisted crevice corrosion performance of head-neck modular tapers is a significant concern in orthopedic biomaterials. Fretting crevice corrosion processes in modular tapers are thought to be influenced by a wide array of factors including seating mechanics of the junction, hence there is a need for in vitro test methods that can assess their performance. This study presented a test method to directly measure the load-displacement seating mechanics of modular tapers and used this method to compare the seating mechanics for different tapers, moisture, seating loads and seating rates. Seating mechanics were explored whereby the instantaneous load-displacement behavior of the head seating onto the neck is captured and used to define the mechanics of seating. Two distinct taper design/material combinations were assembled wet or dry using axially applied loads (500, 1000, 2000 and 4000 N) at two loading rates of 100 and 104 N/s (n = 5 for each condition) using a servohydraulic test frame. The results showed that pull-off strength scaled with seating load and ranged between 43-68% of seating load depending on sample and wetness. Tapers seated wet had higher pull-off strengths (2200 N +/− 300 N) than those seated dry (1800 N +/− 200 N, P < 0.05). Seating mechanics (load-displacement plots) varied due to sample type and due to wetness with differences in seating energy, seating stiffness and seating displacement. These results show the detailed mechanics of seating during assembly and provide significant insight into the complex interplay of factors associated with even “ideal” seating (axial, quasistatic) loading. This article is protected by copyright. All rights reserved

Sclerostin antibody enhances bone formation in a rat model of distraction osteogenesis


Neutralizing monoclonal sclerostin antibodies are effective in promoting bone formation at a systemic level and in orthopedic scenarios including closed fracture repair. In this study we examined the effects of sclerostin antibody (Scl-Ab) treatment on regenerate volume, density and strength in a rat model of distraction osteogenesis. Surgical osteotomy was performed on 179 Sprague Dawley rats. After 1 week, rats underwent distraction for 2 weeks, followed by 6 weeks for consolidation. Two treatment groups received biweekly subcutaneous Scl-AbIII (a rodent form of Scl-Ab; 25 mg/kg), either from the start of distraction onwards or restricted to the consolidation phase. These groups were compared to controls receiving saline. Measurement modalities included longitudinal DXA, ex vivo QCT and microCT, tissue histology, and biomechanical 4-point bending tests. Bone volume was increased in both Scl-Ab treatments regimens by the end of consolidation (+26-38%, p < 0.05), as assessed by microCT. This was associated with increased mineral apposition. Importantly, Scl-Ab led to increased strength in united bones, and this reached statistical significance in animals receiving Scl-Ab during consolidation only (+177%, p < 0.01, maximum load to failure). These data demonstrate that Scl-Ab treatment increases bone formation, leading to regenerates with higher bone volume and improved strength. Our data also suggest that the optimal effects of Scl-Ab treatment are achieved in the latter stages of distraction osteogenesis. These findings support further investigation into the potential clinical application of sclerostin antibody to augment bone distraction, such as limb lengthening, particularly in the prevention of refracture. This article is protected by copyright. All rights reserved

Effects of combined teriparatide and zoledronic acid on posterior lumbar vertebral fusion in an aged ovariectomized rat model of osteopenia


There has been no study regarding the effect of a combination of teriparatide (TPTD) and zoledronic acid (ZA) on vertebral fusion. In this study, we investigate the effect of single and combined TPTD and ZA treatment on lumbar vertebral fusion in aged ovariectomized (OVX) rats. Sixty two-month-old female Sprague-Dawley rats were ovariectomized and underwent bilateral L4-5 posterolateral intertransverse fusion after ten months. The OVX rats received vehicle (control) treatment, or ZA (100µg/kg, once), or TPTD (60µg/kg/2 d for 42 d), or ZA + TPTD until they were euthanized at six weeks following lumbar vertebral fusion. The lumbar spine was harvested. Bone mineral density (BMD), bone fusion, bone volume (BV), and bone formation rate (BFR)were analyzed by dual-energy X-ray absorptiometry (DXA), radiography, micro-computed tomography, and histomorphometry. Compared with vehicle (control) treatment, ZA and TPTD monotherapy increased bone volume (BV) at fusion site, and ZA + TPTD combined therapy had an additive effect. Treatment with TPTD and ZA + TPTD increased the bone fusion rate when compared with the control group. ZA monotherapy did not alter the rate of bone fusion. The TPTD and ZA + TPTD treatment groups had increased mineral apposition rate (MAR), mineralizing surfaces/bone surface ((MS/BS) and BFR/BS compared with the OVX group. Our experiment confirm that the monotherapy with TPTD and combination therapy with ZA + TPTD in an OVX rat model of osteopenia following lumbar vertebral fusion surgery increased bone fusion mass and bone fusion rate, and ZA + TPTD combined therapy had an additive effect on bone fusion mass. This article is protected by copyright. All rights reserved

Muscle Stem Cell Activation in a Mouse Model of Rotator Cuff Injury


Rotator cuff (RC) tears are frequently complicated by muscle atrophy. Muscle stem cells (MuSCs) repair damaged myofibers following injury, but their role in the prevention or pathogenesis of atrophy following RC tears remains undefined. We hypothesized that the RC MuSC population would be affected by supraspinatus (SS) and infraspinatus (IS) tendon transection (TT) compared to uninjured muscle in a mouse model of RC tear. C57BL6/J mice underwent unilateral SS and IS TT and contralateral sham surgery. At 3, 8, or 14 weeks after injury, mice were euthanized and SS and IS were harvested for FACS sorting of CD31-/CD45-/Sca1-/ITGa7 + /VCAM+ MuSCs or histological analysis. Ki-67+ MuSCs from injured muscle increased 3.4 fold at 3 weeks (p = 0.03) and 8.1 fold at 8 weeks (p = 0.04) following TT injury, but returned to baseline by 14 weeks (p = 0.91). Myod1 remained upregulated 3.3 fold at 3 weeks (p = 0.03) and 2.0 fold at 14 weeks (p = 0.0003), respectively. Myofiber cross-sectional area was decreased at both 3 and 14 weeks after injury, but the number of MuSCs per fiber remained relatively constant at 3 (p = 0.3) and 14 (p = 0.6) weeks after TT. In this study, we characterized the longitudinal effect of RC tendon injury on the MuSC population in supraspinatus and infraspinatus muscles. MuSCs are transiently activated, and are not depleted, in spite of persistent muscle atrophy. This article is protected by copyright. All rights reserved

Supraphysiological loading induces osteocyte-mediated osteoclastogenesis in a novel in vitro model for bone implant loosening


We aimed to develop an in vitro model for bone implant loosening, allowing analysis of biophysical and biological parameters contributing to mechanical instability-induced osteoclast differentiation and peri-implant bone loss. MLO-Y4-osteocytes were mechanically stimulated for 1 h by fluid shear stress using regimes simulating: (i) supraphysiological loading in the peri-prosthetic interface (2.9 ± 2.9 Pa, 1 Hz, square wave); (ii) physiologic loading in the cortical bone (0.7 ± 0.7 Pa, 5 Hz, sinusoidal wave); and (iii) stress shielding. Cellular morphological parameters, membrane-bound RANKL expression, gene expression influencing osteoclast differentiation, nitric oxide release and caspase 3/7-activity were determined. Either Mouse bone marrow cells were cultured on top of loaded osteocytes or osteocyte-conditioned medium was added to bone marrow cells. Osteoclast differentiation was assessed after 6 days. We found that osteocytes subjected to supraphysiological loading showed similar morphology and caspase 3/7-activity compared to simulated physiological loading or stress shielding. Supraphysiological stimulation of osteocytes enhanced osteoclast differentiation by 1.9-fold compared to physiological loading when cell-to-cell contact was permitted. In addition, it enhanced the number of osteoclasts using conditioned medium by 1.7-fold, membrane-bound RANKL by 3.3-fold, and nitric oxide production by 3.2-fold. The stimulatory effect of supraphysiological loading on membrane-bound RANKL and nitric oxide production was higher than that achieved by stress shielding. In conclusion, the in vitro model developed recapitulated the catabolic biological situation in the peri-prosthetic interface during instability that is associated with osteoclast differentiation and enhanced RANKL expression. The model thus provides a platform for pre-clinical testing of pharmacological interventions with potential to stop instability-induced bone implant loosening. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res In our novel in vitro model for bone implant loosening we were able to simulate supraphysiological loading that appears in the peri-prosthetic interface, leading to changes in the micro-environment around a bone implant. Stimulated osteocytes reacting to these fluid flow changes by increasing the expression of membrane-bound RANKL, thereby enhancing osteoclast formation of bone marrow cells via cell-to-cell contact (co-culture). Furthermore, supraphysiological loading of osteocytes stimulated secretion of thusfar unidentified soluble factors able to induce ostelcastogenesis on bone marrow cells.

Osteophytes and fracture calluses share developmental milestones and are diminished by unloading


Osteophytes are a typical radiographic finding during osteoarthritis (OA), but the mechanisms leading to their formation are not well known. Comparatively, fracture calluses have been studied extensively; therefore, drawing comparisons between osteophytes and fracture calluses may lead to a deeper understanding of osteophyte formation. In this study, we compared the time courses of osteophyte and fracture callus formation, and investigated mechanisms contributing to development of these structure. Additionally, we investigated the effect of mechanical unloading on the formation of both fracture calluses and osteophytes. Mice underwent either transverse femoral fracture or non-invasive anterior cruciate ligament rupture. Fracture callus and osteophyte size and ossification were evaluated after 3, 5, 7, 14, 21, or 28 days. Additional mice were subjected to hindlimb unloading after injury for 3, 7, or 14 days. Protease activity and gene expression profiles after injury were evaluated after 3 or 7 days of normal ambulation or hindlimb unloading using in vivo fluorescence reflectance imaging (FRI) and quantitative PCR. We found that fracture callus and osteophyte growth achieved similar developmental milestones, but fracture calluses formed and ossified at earlier time points. Hindlimb unloading ultimately led to a threefold decrease in chondro/osteophyte area, and a twofold decrease in fracture callus area. Unloading was also associated with decreased inflammation and protease activity in injured limbs detected with FRI, particularly following ACL rupture. qPCR analysis revealed disparate cellular responses in fractured femurs and injured joints, suggesting that fracture calluses and osteophytes may form via different inflammatory, anabolic, and catabolic pathways. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res This study compared the time courses and underlying mechanisms contributing to fracture callus and osteophyte formation in mice after femoral fracture or ACL rupture, respectively. Formation of these structures shared developmental milestones, but progressed on slightly different timelines. Both structures depended on mechanical stimuli; hindlimb unloading resulted in reduced callus and osteophyte size, inflammation, and protease activity. However, the cellular response of these structures differed at early time points, suggesting disparate formation pathways.

Differentiation of MSC and annulus fibrosus cells on genetically engineered silk fleece-membrane-composites enriched for GDF-6 or TGF-β3


Intervertebral disc (IVD) repair is a high-priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically engineered (GE)-silk from Bombyx mori containing specific growth factors to precondition human bone-marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE-silk fleece can thrive human hMSC towards an IVD-like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)-compliant GE-silk scaffolds in IVD repair and regeneration. GE-silk with growth and differentiation factor 6 (GDF-6-silk) or transforming growth factor β3 (TGF-β3, TGF-β3-silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, that is, hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation toward an NP-like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE-silk scaffold treatment approach can thrive hMSC towards a more IVD-like phenotype or can maintain the phenotype of native hAFC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Genetically enriched silk fleece scaffolds were produced by transduction of Bombyx mori larvae with a baculovirus construct containing GDF6 or TGFb3. Silk fleeces were produced under GMP-compliant conditions for the purpose of intervertebral disc repair. We found that these sericine-free GMP silk scaffolds were highly cytocompatible and that primary human bone-marrow derived stem cells can be differentiated towards intervertebral-like cells as obtained by qPCR and monitoring aggrecan and collagen type 2 ratio.

The Combination of icariin and constrained dynamic loading stimulation attenuates bone loss in ovariectomy-induced osteoporotic mice


Osteoporosis is a disease characterized by low bone mass and progressive destruction of bone microstructure, resulting in increasing the risk of fracture. Icariin (ICA) as a phytoestrogen shows osteogenic effects, and the mechanical stimulation has been demonstrated the improving effect on osteoporosis. The objective of this study was to investigate the effect of ICA in combination with constrained dynamic loading (CDL) stimulation on osteoporosis in ovariectomized (OVX) mice. The serum hormone levels, bone turnover markers, trabecular architecture, ulnar biomechanical properties, and the expression of osteoblast-related gene (alkaline phosphatase, ALP; osteocalcin, OCN; bone morphogenetic protein-2, BMP-2; Collagen I (α1), COL1; osteoprotegerin, OPG) and osteoclast-related genes (receptor activators of NF-κB ligand, RANKL; tartrate-resistant acid phosphatase, TRAP) were analyzed. The results showed that ICA + CDL treatment could increase the osteocalcin (20.85%), estradiol levels (20.61%) and decrease the TRAP activity (26.27%) significantly than CDL treatment. The combined treatment attenuated bone loss and biomechanical decrease more than single use of CDL treatment. ICA + CDL treatment significantly up-regulated the level of osteoblast-related gene expression and down-regulated the osteoclast-related genes expression; moreover, the combined treatment increased the ratio of OPG/RANKL significantly compared to ICA (72.83%) or CDL (65.63%) treatment alone. The present study demonstrates that icariin in combination with constrained dynamic loading treatment may have a therapeutic advantage over constrained dynamic loading treatment alone for the treatment of osteoporosis, which would provide new evidence for the clinical treatment of osteoporosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res The present study showed that icariin (ICA) + constrained dynamic loading (CDL) treatment could increase the osteocalcin, estradiol levels, decrease the TRAP activity, and attenuated bone loss and biomechanical decrease significantly than CDL treatment. ICA + CDL treatment significantly up-regulated the osteoblast-related gene expression and down-regulated the osteoclast-related genes expression, moreover, the combined treatment increased the ratio of OPG/RANKL significantly compared to single treatment. The present study would provide new evidence with combined stimulation for the clinical treatment of osteoporosis.

Influence of the pericellular and extracellular matrix structural properties on chondrocyte mechanics


Understanding the mechanical factors that drive the biological responses of chondrocytes is central to our interpretation of the cascade of events that lead to osteoarthritic changes in articular cartilage. Chondrocyte mechanics is complicated by changes in tissue properties that can occur as osteoarthritis (OA) progresses and by the interaction between macro-scale, tissue level, properties, and micro-scale pericellular matrix (PCM) and local extracellular matrix (ECM) properties, both of which cannot be easily studied using in vitro systems. Our objective was to study the influence of macro- and micro-scale OA-associated structural changes on chondrocyte strains. We developed a multi-scale finite element model of articular cartilage subjected to unconfined loading, for the following three conditions: (i) normal articular cartilage, (ii) OA cartilage (where macro and micro-scale changes in collagen content, matrix modulus, and permeability were modeled), and (iii) early-stage OA cartilage (where only micro-scale changes in matrix modulus were modeled). In the macro-scale model, we found that a depth-dependent strain field was induced in both healthy and OA cartilage and that the middle and superficial zones of OA cartilage had increased tensile and compressive strains. At the micro-scale, chondrocyte shear strains were sensitive to PCM and local ECM properties. In the early-OA model, micro-scale spatial softening of PCM and ECM resulted in a substantial increase (30%) of chondrocyte shear strain, even with no structural changes in macro-scale tissue properties. Our study provides evidence that micromechanical changes at the cellular level may affect chondrocyte activities before macro-scale degradations at the tissue level become apparent. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Single-dose bone pharmacokinetics of vancomycin in a porcine implant-associated osteomyelitis model


The increasing incidence of orthopaedic methicillin-resistant Staphylococcus aureus (MRSA) infections represents a significant therapeutic challenge. Being effective against MRSA, the role of vancomycin may become more important in the orthopaedic setting in the years to come. Nonetheless, vancomycin bone and soft tissue penetration during infection remains unclear. In eight pigs, implant-associated osteomyelitis was induced on day 0, using a Staphylococcus aureus strain. Following administration of 1,000 mg of vancomycin on day 5, vancomycin concentrations were obtained with microdialysis for 8 h in the implant bone cavity, in cancellous bone adjacent to the implant cavity, in subcutaneous adipose tissue (SCT) adjacent to the implant cavity, and in healthy cancellous bone and healthy SCT in the contralateral leg. Venous blood samples were also obtained. The extent of infection and inflammation was evaluated by post-mortem computed tomography scans, C-reactive protein serum levels and cultures of blood and swabs. In relation to all the implant cavities, bone destruction was found. Ranging from 0.20 to 0.74, tissue penetration, expressed as the ratio of the area under the concentration–time curve from 0 to the last measured value, was incomplete for all compartments except for healthy SCT. The lowest penetration was found in the implant cavity. In conclusion, Staphylococcus aureus implant-associated osteomyelitis was found to reduce vancomycin bone penetration, especially in the implant cavity. These findings suggest that it may be unsafe to rely solely on vancomycin therapy when treating acute osteomyelitis. Particularly when metaphyseal cavities are present, surgical debridement seems necessary. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Additive manufactured push-fit implant fixation with screw-strength pull out


Additive manufacturing offers exciting new possibilities for improving long-term metallic implant fixation in bone through enabling open porous structures for bony ingrowth. The aim of this research was to investigate how the technology could also improve initial fixation, a precursor to successful long-term fixation. A new barbed fixation mechanism, relying on flexible struts was proposed and manufactured as a push-fit peg. The technology was optimized using a synthetic bone model and compared with conventional press-fit peg controls tested over a range of interference fits. Optimum designs, achieving maximum pull-out force, were subsequently tested in a cadaveric femoral condyle model. The barbed fixation surface provided more than double the pull-out force for less than a third of the insertion force compared to the best performing conventional press-fit peg (p < 0.001). Indeed, it provided screw-strength pull out from a push-fit device (1,124 ± 146 N). This step change in implant fixation potential offers new capabilities for low profile, minimally invasive implant design, while providing new options to simplify surgery, allowing for one-piece push-fit components with high levels of initial stability. © 2017 The Authors. Journal of Orthopaedic Research Published by WileyPeriodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 9999:1–11, 2017.

Microfluidics approach to investigate the role of dynamic similitude in osteocyte mechanobiology


Fluid flow is an important regulator of cell function and metabolism in many tissues. Fluid shear stresses have been used to level the mechanical stimuli applied in vitro with what occurs in vivo. However, these experiments often lack dynamic similarity, which is necessary to ensure the validity of the model. For interstitial fluid flow, the major requirement for dynamic similarity is the Reynolds number (Re), the ratio of inertial to viscous forces, is the same between the system and model. To study the necessity of dynamic similarity for cell mechanotransduction studies, we investigated the response of osteocyte-like MLO-Y4 cells to different Re flows at the same level of fluid shear stress. Osteocytes were chosen for this study as flows applied in vitro and in vivo have Re that are orders of magnitude different. We hypothesize that osteocytes’ response to fluid flow is Re dependent. We observed that cells exposed to lower and higher Re flows developed rounded and triangular morphologies, respectively. Lower Re flows also reduced apoptosis rates compared to higher Re flows. Furthermore, MLO-Y4 cells exposed to higher Re flows had stronger calcium responses compared to lower Re flows. However, by also controlling for flow rate, the lower Re flows induced a stronger calcium response; while degradation of components of the osteocyte glycocalyx reversed this effect. This work suggests that osteocytes are highly sensitive to differences in Re, independent of just shear stresses, supporting the need for improved in vitro flow platforms that better recapitulate the physiological environment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Longitudinal changes in knee gait mechanics between 2 and 8 years after anterior cruciate ligament reconstruction


The purpose of this study was to longitudinally investigate changes in knee joint kinematics and kinetics from 2 to 8 years post-ACLR. Seventeen subjects with primary unilateral transtibial ACLR performed bilateral gait analysis approximately 2 years and 8 years post-ACLR. Seventeen matched healthy control subjects were also analyzed. Kinematic and kinetic comparisons between the ACLR and contralateral limbs over time were completed using a 2 × 2 (time, limb) repeated-measures ANOVA. Unpaired Student's t-tests were used to compare the ACLR and contralateral kinematics and kinetics to the control group. The ACLR and contralateral limbs had similar gait changes over time. Kinetic changes over time included a reduction in first (p = 0.048) and second (p < 0.001) peak extension moments, internal rotation moment (p < 0.001), adduction moment (first peak: p = 0.002, second peak: p = 0.009, impulse: p = 0.004) and an increase in peak knee flexion moment (p = 0.002). Kinematic changes over time included increases in peak knee flexion angle in the first half of stance (p = 0.026), minimum knee flexion angle in the second half of stance (p < 0.001), and average external rotation angle during stance (p = 0.007), and a reduction in average anterior femoral displacement during stance (p = 0.006). Comparison to healthy controls demonstrated improvement in some gait metrics over time. The results demonstrated longitudinal changes from 2 to 8 years after ACLR in knee joint kinetics and kinematics that have been related to clinical outcome after ACLR and the progression of knee OA, and support future larger and comprehensive investigations into long-term changes in joint mechanics in the ACLR population. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Minimal mechanical load and tissue culture conditions preserve native cell phenotype and morphology in tendon—a novel ex vivo mouse explant model


Appropriate mechanical load is essential for tendon homeostasis and optimal tissue function. Due to technical challenges in achieving physiological mechanical loads in experimental tendon model systems, the research community still lacks well-characterized models of tissue homeostasis and physiological relevance. Toward this urgent goal, we present and characterize a novel ex vivo murine tail tendon explant model. Mouse tail tendon fascicles were extracted and cultured for 6 days in a load-deprived environment or in a custom-designed bioreactor applying low magnitude mechanical load (intermittent cycles to 1% strain, at 1 Hz) in serum-free tissue culture. Cells remained viable, as did collagen structure and mechanical properties in all tested conditions. Cell morphology in mechanically loaded tendon explants approximated native tendon, whereas load-deprived tendons lost their native cell morphology. These losses were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodeling genes phenotypic of native tissue. We conclude from this study that ex vivo load deprivation of murine tendon in minimal culture medium results in a degenerative-like phenotype. We further conclude that onset of tissue degeneration can be suppressed by low-magnitude mechanical loading. Thus a minimal explant culture model featuring serum-free medium with low mechanical loads seems to provide a useful foundation for further investigations. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Low-magnitude mechanical loading in absence of serum suppresses the onset of tendon degeneration (as visualized in the frames). Murine tail tendon fascicles cultured in a bioreactor system for six days (B) approximate native tendon cell morphology (A), whereas load-deprived tendons (C) loose their native phenotype (scale bar 50 µm). These changes were reflected in altered gene expression, with mechanical loading tending to maintain tendon specific and matrix remodelling genes at levels close to those of native tissue.

IGF-1 signaling mediated cell-specific skeletal mechano-transduction


Mechanical loading preserves bone mass and stimulates bone formation, whereas skeletal unloading leads to bone loss. In addition to osteocytes, which are considered the primary sensor of mechanical load, osteoblasts, and bone specific mesenchymal stem cells also are involved. The skeletal response to mechanical signals is a complex process regulated by multiple signaling pathways including that of insulin-like growth factor-1 (IGF-1). Conditional osteocyte deletion of IGF-1 ablates the osteogenic response to mechanical loading. Similarly, osteocyte IGF-1 receptor (IGF-1R) expression is necessary for reloading-induced periosteal bone formation. Transgenic overexpression of IGF-1 in osteoblasts results in enhanced responsiveness to in vivo mechanical loading in mice, a response which is eliminated by osteoblastic conditional disruption of IGF-1 in vivo. Bone marrow derived stem cells (BMSC) from unloaded bone fail to respond to IGF-1 in vitro. IGF-1R is required for the transduction of a mechanical stimulus to downstream effectors, transduction which is lost when the IGF-1R is deleted. Although the molecular mechanisms are not yet fully elucidated, the IGF signaling pathway and its interactions with potentially interlinked signaling cascades involving integrins, the estrogen receptor, and wnt/β-catenin play an important role in regulating adaptive response of cancer bone cells to mechanical stimuli. In this review, we discuss recent advances investigating how IGF-1 and other interlinked molecules and signaling pathways regulate skeletal mechano-transduction involving different bone cells, providing an overview of the IGF-1 signaling mediated cell-specific response to mechanical stimuli. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Flash autoclave settings may influence eradication but not presence of well-established biofilms on orthopaedic implant material


Flash autoclaving is one of the most frequently utilized methods of sterilizing devices, implants or other materials. For a number of decades, it has been common practice for surgeons to remove implantable devices, flash autoclave and then reimplant them in a patient. Data have not yet indicated the potential for biofilms to survive or remain on the surface of orthopaedic-relevant materials following flash autoclave. In this study, monomicrobial and polymicrobial biofilms were grown on the surface of clinically relevant titanium materials and exposed to flash autoclave settings that included varying times and temperatures. Data indicated that when the sterilization and control temperatures of an autoclave were the same, biofilms were able to survive flash autoclaving that was performed for a short duration. Higher temperature and increased duration rendered biofilms non-viable, but none of the autoclave settings had the ability to remove or disperse the presence of biofilms from the titanium surfaces. These findings may be beneficial for facilities, clinics, or hospitals to consider if biofilms are suspected to be present on materials or devices, in particular implants that have had associated infection and are considered for re-implantation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Current and novel injectable hydrogels to treat focal chondral lesions: Properties and applicability


Focal chondral lesions and early osteoarthritis (OA) are responsible for progressive joint pain and disability in millions of people worldwide, yet there is currently no surgical joint preservation treatment available to fully restore the long term functionality of cartilage. Limitations of current treatments for cartilage defects have prompted the field of cartilage tissue engineering, which seeks to integrate engineering and biological principles to promote the growth of new cartilage to replace damaged tissue. Toward improving cartilage repair, hydrogel design has advanced in recent years to improve their utility. Injectable hydrogels have emerged as a promising scaffold due to their wide range of properties, the ability to encapsulate cells within the material, and their ability to provide cues for cell differentiation. Some of these advances include the development of improved control over in situ gelation (e.g., light), new techniques to process hydrogels (e.g., multi-layers), and better incorporation of biological signals (e.g., immobilization, controlled release, and tethering). This review summarises the innovative approaches to engineer injectable hydrogels toward cartilage repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Injectable hydrogels have emerged as a promising scaffold for cartilage regeneration due to their wide range of properties, their ability to encapsulate cells and for providing cues for cell differentiation. Some of these advances include the development of improved control over in situ gelation (e.g., light), new techniques to process hydrogels (e.g., multi-layers), and better incorporation of biological signals (e.g., immobilization, controlled release, and tethering). This review summarizes the innovative approaches to engineer injectable hydrogels toward cartilage repair.

Mechanobiology of young and aging tendons: In vivo studies with treadmill running


Tendons are unique in the sense that they are constantly subjected to large mechanical loads and that they contain tendon-specific cells, including tenocytes and tendon stem/progenitor cells. The responses of these cells to mechanical loads can be anabolic or catabolic and as a result, change the biological properties of the tendon itself that may be beneficial or detrimental. On the other hand, aging also induces aberrant changes in cellular expression of various genes and production of various types of matrix proteins in the tendon, and consequently lead to tendon degeneration and impaired healing in aging tendons; both could be improved by moderate physiological mechanical loading such as treadmill running. This article gives an overview on the mechanobiology research of young and aging animal tendons using treadmill running model. The challenges in such treadmill running studies are also discussed. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Functional properties of chondrocytes and articular cartilage using optical imaging to scanning probe microscopy


Mature chondrocytes in adult articular cartilage vary in number, size, and shape, depending on their depth in the tissue, location in the joint, and source species. Chondrocytes are the primary structural, functional, and metabolic unit in articular cartilage, the loss of which will induce fatigue to the extracellular matrix (ECM), eventually leading to failure of the cartilage and impairment of the joint as a whole. This brief review focuses on the functional and biomechanical studies of chondrocytes and articular cartilage, using microscopic imaging from optical microscopies to scanning probe microscopy. Three topics are covered in this review, including the functional studies of chondrons by optical imaging (unpolarized and polarized light and infrared light, two-photon excitation microscopy), the probing of chondrocytes and cartilage directly using microscale measurement techniques, and different imaging approaches that can measure chondrocyte mechanics and chondrocyte biological signaling under in situ and in vivo environments. Technical advancement in chondrocyte research during recent years has enabled new ways to study the biomechanical and functional properties of these cells and cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Bone remodeling and mechanobiology around implants: Insights from small animal imaging


Anchorage of orthopedic implants depends on the interfacial bonding between the implant and the host bone as well as on the mass and microstructure of peri-implant bone, with all these factors being continuously regulated by the biological process of bone (re)modeling. In osteoporotic bone, implant integration may be jeopardized not only by lower peri-implant bone quality but also by reduced intrinsic regeneration ability. The first aim of this review is to provide a critical overview of the influence of osteoporosis on bone regeneration post-implantation. Mechanical stimulation can trigger bone formation and inhibit bone resorption; thus, judicious administration of mechanical loading can be used as an effective non-pharmacological treatment to enhance implant anchorage. Our second aim is to report recent achievements on the application of external mechanical stimulation to improve the quantity of peri-implant bone. The review focuses on peri-implant bone changes in osteoporotic conditions and following mechanical loading, prevalently using small animals and in vivo monitoring approaches. We intend to demonstrate the necessity to reveal new biological information on peri-implant bone mechanobiology to better target implant anchorage and fracture fixation in osteoporotic conditions. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Novel protein therapeutic joint retention strategy based on collagen-binding Avimers


Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein–protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Current review—The rise of bacteriophage as a unique therapeutic platform in treating peri-prosthetic joint infections


Peri-prosthetic joint infection (PJI) is one of the most serious and dreaded complications after total joint replacement (TJR). Due to an aging population and the constant rise in demand for TJR, the incidence of PJI is also increasing. Successful treatment of PJI is challenging and is associated with high failure rates. One of the main causes for treatment failure is bacterial biofilm formation on implant surfaces and the adherence of biofilm bacteria on tissue and bone next to the implant. Biofilms are protective shields to bacterial cells and possess many unique properties that leads to antibiotic resistance. New therapeutic platforms are currently being explored to breakdown biofilm matrix in order to enhance the efficacy of antibiotics. Bacteriophages (phages) is one of these unique therapeutic platforms that can degrade biofilms as well as target the killing of bacterial cells. Preclinical studies of biofilm-mediated infections have demonstrated the ability of phage to eradicate biofilms and clear infections by working synergistically with antibiotics. There is strong preclinical evidence that phage can reduce the concentration of antibiotics required to treat an infection. These findings support a promising role for phages as a future clinical adjunct to antibiotics. In addition, phage therapy can be personalized to target a specific bacterial strain. Clinical studies using phage therapy are limited in Western literature; but phase I studies have established good safety profile with no adverse outcomes reported. In order to translate phage therapy to treat PJI in clinics, further preclinical testing is still required to study optimal delivery methods as well as the interaction between phage and the immune system in vivo. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Serum proteomic assessment of the progression of fracture healing


A targeted proteomic analysis of murine serum over a 35-day course of fracture healing was carried out to determine if serum proteomic changes could be used to monitor the biological progression of fracture healing. Transverse, closed femoral fractures where generated and stabilized with intramedullary fixation. A single stranded DNA aptamer-based multiplexed proteomic approach was used to assay 1,310 proteins. The transcriptomic profiles for genes matching the 1,310 proteins were obtained by microarray analysis of callus mRNA. Of the 1,310 proteins analyzed, 850 proteins showed significant differences among the time points (p-value <0.05). Ontology assessment associated these proteins with osteoblasts, monocyte/macrophage lineages, mesenchymal stem cell lines, hepatic tissues, and lymphocytes. Temporal clustering of these data identified proteins associated with inflammation, cartilage formation and bone remodeling stages of healing. VEGF, Wnt, and TGF-βsignaling pathways were restricted to the period of cartilage formation. Comparison of the proteomic and transcriptomic profiles showed that 87.5% of proteins in serum had concordant expression to their mRNA expression in the callus, while 12.5% of the protein and mRNA expression patterns were discordant. The discordant proteins that were elevated in the serum but down regulated in callus mRNA expression were related to clotting functions, allograft rejection, and complement function. While proteins down regulated in the serum and elevated in callus mRNA were associated with osteoblast function, NF-ĸb, and activin signaling. These data show the serum proteome may be used to monitor the different biological stages of fracture healing and have translational potential in assessing human fracture healing. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

5′-methylschweinfurthin G reduces chondrosarcoma tumor growth 


New treatment options are urgently required in the field of chondrosarcoma, particularly of chondrosarcomas with a well-differentiated hyaline cartilage-like extracellular matrix (e.g., collagen II and proteoglycan-rich) phenotype, notoriously resistant to drug penetration, and having potential of progression towards higher grade. We investigated the feasibility of using 5′-methylschweinfurthin G (MeSG) as a tumor suppressor agent in the Swarm rat chondrosarcoma, an intermediate- to high-grade chondrosarcoma model, having a hyaline cartilage-like phenotype. Tumor cell culture studies were performed to identify their proliferative and cytotoxicity sensitivity to MeSG. Tumor burden mice were treated with MeSG and analyzed for tumor growth, morphology and regression. The chondrosarcoma tumor cells had a half maximum cytotoxicity concentration (IC50) of 35 nM MeSG; approximately 300-fold less than freshly isolated rat chondrocytes (IC50 of 11 µM). Multiple injections of MeSG (20 mg/kg, body weight) resulted in reduced/eliminated tumor growth over a 17-day period in mice, and an 83% reduction (p = 0.023) in tumor mass. Three out of ten MeSG treated mice had complete elimination of tumor. Tumors of treated mice had a decrease in chondrosarcoma cell proliferation (p = 0.012) and an increase in cell death (p = 0.030) compared with tumors of control mice. These findings in an animal model demonstrate the effectiveness of MeSG for treatment of rat chondrosarcomas, and may have the potential use as a therapeutic option for the difficult-to-treat intermediate-to high-grade hyaline cartilage-like chondrosarcoma. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res New treatment options are urgently required in the field of chondrosarcoma. We investigated the feasibility of using 5’-methylschweinfurthin G (MeSG) as a tumor suppressor agent in the Swarm rat chondrosarcoma model. Multiple injections of MeSG with tumor burden mice resulted in reduced/eliminated tumor growth and an 83% reduction in tumor mass. These findings demonstrate the effectiveness of MeSG for treatment of rat chondrosarcomas, and may have the potential use as a therapeutic option for the difficult-to-treat hyaline cartilage-like chondrosarcoma.

Comparison of the effects of once-weekly and once-daily rhPTH (1–34) injections on promoting fracture healing in rodents


To compare the efficacy of once-weekly and once-daily subcutaneous injections of teriparatide (recombinant human parathyroid hormone 1–34) on fracture healing, 50 adult male Sprague–Dawley rats were subjected to a unilateral tibia fracture and received internal fixation with a Kirschner needle. Based on the injection dose and frequency, the rats were randomly divided into five groups (n = 10 each): subcutaneous injections of saline or 10 µg/kg/w, 20 µg/kg/w, 10 µg/kg/d, and 20 µg/kg/d teriparatide. Four weeks later, the rats were euthanatized, and the fractured tibiae were assessed using X-rays, dual-energy X-ray absorptiometry, micro-computed tomography, the three-point bending biomechanics test, and histology. Compared to the saline control group, either daily or weekly subcutaneous injections of teriparatide significantly increased bone mass, improved the bone microarchitecture, and promoted fracture healing (p < 0.05). There were no significant differences in bone mineral density (BMD), bone microstructure or bone strength between the 20 µg/kg/w and 10 µg/kg/d groups (p > 0.05). Teriparatide 20 µg weekly injections promoted bone fracture healing to the same extent as teriparatide 10 µg daily injections, which can dramatically decrease the cumulative dosage of teriparatide injections. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Effect of mechanical strain on the pluripotency of murine embryonic stem cells seeded in a collagen-I scaffold


The use of embryonic stem cells (ESC) in regenerative medicine is restricted due to the possibility of tumorigenicity after inefficient or incomplete differentiation. Studies from our group, and others, suggest that mechanical stimuli may have a suppressive effect on the pluripotency/tumorigenesis of murine ESC (mESC). Furthermore, we have demonstrated that mESC seeded in a type I collagen scaffold, and transplanted into a murine bone fracture model, demonstrated repair without tumor formation. However, it remains unknown if mechanical factors were involved in blocking tumorigenicity of the mESC. Therefore, the aims of the current study were: (i) to characterize the mechanical environment within the transplanted construct (mESC-Col I) in an in vivo murine fracture model using computational analyses; and (ii) to reproduce this mechanical environment in vitro to elucidate the role of these mechanical factors on mESC pluripotent gene expression. It was predicted that the mESC-Col I construct was subjected to an average octahedral shear strain of ∼3.8% and a compressive strain of ∼3.1% within the fracture in vivo when the murine tibia was subjected to an axial compression load of 4 N (1 Hz). When a similar strain environment was replicated experimentally in vitro, the expression patterns of marker genes for pluripotency (Oct 4, Sox 2, Nanog, Rex 1, and oncogene ERas) were significantly down-regulated. This suggests that the local micro-mechanical environment within the fracture site in vivo may be involved in regulating stem cell fate after transplantation, and that these physical factors should be considered when developing regenerative medicine strategies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

CXCL10 is upregulated in synovium and cartilage following articular fracture


The objective of this study was to investigate the expression of the chemokine CXCL10 and its role in joint tissues following articular fracture. We hypothesized that CXCL10 is upregulated following articular fracture and contributes to cartilage degradation associated with post-traumatic arthritis (PTA). To evaluate CXCL10 expression following articular fracture, gene expression was quantified in synovial tissue from knee joints of C57BL/6 mice that develop PTA following articular fracture, and MRL/MpJ mice that are protected from PTA. CXCL10 protein expression was assessed in human cartilage in normal, osteoarthritic (OA), and post-traumatic tissue using immunohistochemistry. The effects of exogenous CXCL10, alone and in combination with IL-1, on porcine cartilage explants were assessed by quantifying the release of catabolic mediators. Synovial tissue gene expression of CXCL10 was upregulated by joint trauma, peaking one day in C57BL/6 mice (25-fold) versus 3 days post-fracture in MRL/MpJ mice (15-fold). CXCL10 protein in articular cartilage was most highly expressed following trauma compared with normal and OA tissue. In a dose dependent manner, exogenous CXCL10 significantly reduced total matrix metalloproteinase (MMP) and aggrecanase activity of culture media from cartilage explants. CXCL10 also trended toward a reduction in IL-1α-stimulated total MMP activity (p = 0.09) and S-GAG (p = 0.09), but not NO release. In conclusion, CXCL10 was upregulated in synovium and chondrocytes following trauma. However, exogenous CXCL10 did not induce a catabolic response in cartilage. CXCL10 may play a role in modulating the chondrocyte response to inflammatory stimuli associated with joint injury and the progression of PTA. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res The objective of this study was to investigate the chemokine CXCL10 and its role in joint tissues following articular fracture. We hypothesized that CXCL10 is upregulated following articular fracture and contributes to cartilage degradation associated with post-traumatic arthritis (PTA). CXCL10 was upregulated in synovium and chondrocytes (see legend Fig. 2) following trauma, but did not directly induce catabolism of articular cartilage. Findings from this study suggest that CXCL10 may play a role in homeostasis of chondrocytes and modulation of inflammation.

BIO allieviated compressive mechanical force-mediated mandibular cartilage pathological changes through Wnt/β-catenin signaling activation


Osteoarthritis induced by compressive mechanical force is characterized by decreased chondrocyte proliferation and degradation of the ECM. To examine underlying mechanisms of the pathological changes of mandibular cartilage induced by compressive mechanical force, an established animal model was used to examine Wnt signaling activation by glycogen synthase kinase-3 beta (GSK3β) inhibitor 6-Bromoindirubin-3′-oxime (BIO) injection in vivo. Histological changes in mandibular cartilage were assessed via hematoxylin & eosin (HE), masson, and alcian blue staining. Immunohistochemistry and real-time PCR were performed to evaluate activation of the Wnt signaling pathway and chondrocytes proliferation markers. Chondrocytes apoptosis was examined by TUNEL staining. During the compressive mechanical force loading-mediated process, Wnt signaling was largely inhibited, which showed the inhibited expression of β-catenin and the increased expression of GSK-3β. The expression of chondrocytes proliferation markers Ki67, and proliferating cell nuclear antigen (PCNA) also decreased. With BIO injection, the Wnt signaling was restored and the proliferation of mandibular chondrocytes was also increased in the late stage (7 days) of compressive mechanical force loading. Finally, the decreasing mandibular cartilage thickness, the degradation of extracellular matrix, and the erosion of bone trabecula were subsequently restored. Also, the changes of extracellular matrix markers such as collagen II and collagen X, matrix metalloproteases, and inflammatory cytokines were reversed followed by the injection of BIO. In summary, compressive mechanical force decreased endogenously Wnt signaling, leading to impaired proliferation in chondrocytes and degradation in cartilage matrix. Restoration of Wnt signaling largely recovered the proliferation defects and alleviated the pathological changes of mandibular cartilage. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Sensitivity of serum concentration of cartilage biomarkers to 21-days of bed rest


The objective of the study was to test the hypothesis that serum levels of cartilage oligomeric matrix protein (COMP) would decrease and serum levels of tumor-necrosis factor alpha (TNF-α) and selected matrix metalloproteinases (MMPs) would increase in response to bed rest (BR) and that these changes are unaffected by the intake of potassium bicarbonate or whey protein. Seven and nine healthy male subjects participated in two 21-day 6° head down tilt crossover BR-studies with nutrition interventions. Serum samples were taken before, during, and after BR and biomarker concentrations were measured using commercial enzyme-linked immunosorbent assays. MMP-3 during BR was significantly lower than at baseline (reduction greater 20%; p < 0.001). MMP-3 increased significantly from 14 to 21 days of BR (+7%; p = 0.049). COMP during BR was significantly lower than at baseline (reduction greater 20%; p < 0.001). MMP-3 and COMP returned to baseline within 1 day after BR. MMP-9 on day 3 of BR was significantly lower than at baseline (−31%; p < 0.033) and on days 3, 5, and 14 of BR significantly lower than at the end of and after BR (reduction greater 35%; p < 0.030). The nutritional countermeasures did not affect these results. The observed changes in cartilage biomarkers may be caused by altered cartilage metabolism in response to the lack of mechanical stimulus during BR and inflammatory biomarkers may play a role in changes in biomarker levels. Clinical relevance: Immobilization independently from injury can cause altered cartilage biomarker concentration. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Palovarotene inhibits connective tissue progenitor cell proliferation in a rat model of combat-related heterotopic ossification


Heterotopic ossification (HO) develops in the extremities of wounded service members and is common in the setting of high-energy penetrating injuries and blast-related amputations. No safe and effective prophylaxis modality has been identified for this patient population. Palovarotene has been shown to reduce bone formation in traumatic and genetic models of HO. The purpose of this study was to determine the effects of Palovarotene on inflammation, progenitor cell proliferation, and gene expression following a blast-related amputation in a rodent model (n = 72 animals), as well as the ability of Raman spectroscopy to detect early HO before radiographic changes are present. Treatment with Palovarotene was found to dampen the systemic inflammatory response including the cytokines IL-6 (p = 0.01), TNF-α (p = 0.001), and IFN-γ (p = 0.03) as well as the local inflammatory response via a 76% reduction in the cellular infiltration at post-operative day (POD)-7 (p = 0.03). Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p = 0.04) and in vivo (p = 0.01). Palovarotene treated animals exhibited significantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p = 0.02). Finally, Raman spectroscopy was able to detect differences between the two groups by POD-1 (p < 0.001). These results indicate that Palovarotene inhibits traumatic HO formation through multiple inter-related mechanisms including anti-inflammatory, anti-proliferative, and gene expression modulation. Further, that Raman spectroscopy is able to detect markers of early HO formation before it becomes radiographically evident, which could facilitate earlier diagnosis and treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Elevated solute transport at sites of diffuse matrix damage in cortical bone: Implications on bone repair


Diffuse matrix damage in rat cortical bone has been observed to self-repair efficiently in 2 weeks without activating bone remodeling, and unlike the case with linear cracks, the local osteocytes at the sites of diffuse damage remain healthy. However, the reason(s) for such high efficiency of matrix repair remains unclear. We hypothesized that transport of minerals and other compounds essential for damage repair is enhanced at the damaged sites and further increased by the application of tensile loading. To test our hypothesis, diffuse damage was introduced in notched bovine wafers under cyclic tensile loading and unloading. Using the Fluorescence Recovery After Photobleaching (FRAP) approach, we measured the transport of a small fluorescent tracer (sodium fluorescein, 376 Da) in damaged versus undamaged regions and under varying tensile load magnitudes (0.2 N, 10 N, 20 N, and 30 N), which corresponded to nominal strains of 12.5, 625, 1,250, and 1,875 microstrains, respectively. We found a 37% increase in transport of fluorescein in damaged regions relative to undamaged regions and a further ∼18% increase in transport under 20 N and 30 N tension compared to the non-loaded condition, possibly due to the opening of the cracking surfaces. The elevated transport of minerals and other adhesive proteins may, at least partially, account for the highly effective repair of diffuse damage observed in vivo. Clinical Significance: Diffuse damage adversely affects bone's fracture resistance and this study provided quantitative data on elevated transport, which may be involved in repairing diffuse damage in vivo. 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Development and initial validation of a novel smoothed-particle hydrodynamics-based simulation model of trabecular bone penetration by metallic implants


A novel computational model of implant migration in trabecular bone was developed using smoothed-particle hydrodynamics (SPH), and an initial validation was performed via correlation with experimental data. Six fresh-frozen human cadaveric specimens measuring 10 × 10 × 20 mm were extracted from the proximal femurs of female donors (mean age of 82 years, range 75–90, BV/TV ratios between 17.88% and 30.49%). These specimens were then penetrated under axial loading to a depth of 10 mm with 5 mm diameter cylindrical indenters bearing either flat or sharp/conical tip designs similar to blunt and self-tapping cancellous screws, assigned in a random manner. SPH models were constructed based on microCT scans (17.33 µm) of the cadaveric specimens. Two initial specimens were used for calibration of material model parameters. The remaining four specimens were then simulated in silico using identical material model parameters. Peak forces varied between 92.0 and 365.0 N in the experiments, and 115.5–352.2 N in the SPH simulations. The concordance correlation coefficient between experimental and simulated pairs was 0.888, with a 95%CI of 0.8832–0.8926, a Pearson ρ (precision) value of 0.9396, and a bias correction factor Cb (accuracy) value of 0.945. Patterns of bone compaction were qualitatively similar; both experimental and simulated flat-tipped indenters produced dense regions of compacted material adjacent to the advancing face of the indenter, while sharp-tipped indenters deposited compacted material along their peripheries. Simulations based on SPH can produce accurate predictions of trabecular bone penetration that are useful for characterizing implant performance under high-strain loading conditions. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Wear testing of a canine hip resurfacing implant that uses highly cross-linked polyethylene


Hip resurfacing offers advantages for young, active patients afflicted with hip osteoarthritis and may also be a beneficial treatment for adult canines. Conventional hip resurfacing uses metal-on-metal bearings to preserve bone stock, but it may be feasible to use metal-on-polyethylene bearings to reduce metal wear debris while still preserving bone. This study characterized the short-term wear behavior of a novel hip resurfacing implant for canines that uses a 1.5 mm thick liner of highly cross-linked polyethylene in the acetabular component. This implant was tested in an orbital bearing machine that simulated canine gait for 1.1 million cycles. Wear of the liner was evaluated using gravimetric analysis and by measuring wear depth with an optical scanner. The liners had a steady-state mass wear rate of 0.99 ± 0.17 mg per million cycles and an average wear depth in the central liner region of 0.028 mm. No liners, shells, or femoral heads had any catastrophic failure due to yielding or fracture. These results suggest that the thin liners will not prematurely crack after implantation in canines. This is the first hip resurfacing device developed for canines, and this study is the first to characterize the in vitro wear of highly cross-linked polyethylene liners in a hip resurfacing implant. The canine implant developed in this study may be an attractive treatment option for canines afflicted with hip osteoarthritis, and since canines are the preferred animal model for human hip replacement, this implant can support the development of metal-on-polyethylene hip resurfacing technology for human patients. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res This study characterized the in vitro wear behavior of a novel hip resurfacing implant for canines that uses a 1.5 mm thick liner of highly cross-linked polyethylene. The liners had average wear rates of 0.99 mg per million cycles, and average wear depths of 0.028 mm after 1.1 million cycles. No liners, shells, or femoral heads had any catastrophic failure due to yielding or fracture. These results suggest that the thin liners will not prematurely crack after implantation in canines.

Calcium signaling of in situ chondrocytes in articular cartilage under compressive loading: Roles of calcium sources and cell membrane ion channels


Mechanical loading on articular cartilage can induce many physical and chemical stimuli on chondrocytes residing in the extracellular matrix (ECM). Intracellular calcium ([Ca2+]i) signaling is among the earliest responses of chondrocytes to physical stimuli, but the [Ca2+]i signaling of in situ chondrocytes in loaded cartilage is not fully understood due to the technical challenges in [Ca2+]i imaging of chondrocytes in a deforming ECM. This study developed a novel bi-directional microscopy loading device that enables the record of transient [Ca2+]i responses of in situ chondrocytes in loaded cartilage. It was found that compressive loading significantly promoted [Ca2+]i signaling in chondrocytes with faster [Ca2+]i oscillations in comparison to the non-loaded cartilage. Seven [Ca2+]i signaling pathways were further investigated by treating the cartilage with antagonists prior to and/or during the loading. Removal of extracellular Ca2+ ions completely abolished the [Ca2+]i responses of in situ chondrocytes, suggesting the indispensable role of extracellular Ca2+ sources in initiating the [Ca2+]i signaling in chondrocytes. Depletion of intracellular Ca2+ stores, inhibition of PLC-IP3 pathway, and block of purinergic receptors on plasma membrane led to significant reduction in the responsive rate of cells. Three types of ion channels that are regulated by different physical signals, TRPV4 (osmotic and mechanical stress), T-type VGCCs (electrical potential), and mechanical sensitive ion channels (mechanical loading) all demonstrated critical roles in controlling the [Ca2+]i responses of in situ chondrocyte in the loaded cartilage. This study provided new knowledge about the [Ca2+]i signaling and mechanobiology of chondrocytes in its natural residing environment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Sagittal plane walking patterns are related to MRI changes over 18-months in people with and without mild-moderate hip osteoarthritis


The purpose was to evaluate the association of sagittal plane gait mechanics with MRI changes in the hip joint over 18-months. Subjects with and without radiographic hip OA (n = 57) underwent MRI at baseline and 18 months for grading of cartilage lesions, bone marrow lesions (BML), cysts, and labral tears. 3D gait analyses at baseline were used for sagittal plane hip kinematics and kinetics during the stance phase. Subjects were classified as progressors or non-progressors based on increase in any MRI OA parameter. Multivariate ANOVA were used for differences in sagittal gait parameters between progressors and non-progressors at baseline while adjusting for age. Logistic regression was used to estimate the probability of being classified as a progressor or non-progressor with increasing hip flexion while adjusting for age, BMI, sex, and presence of radiographic hip OA. Of the 57, 35 were classified as non-progressors and 22 were classified as progressors. At baseline, the progressors walked with 4.5° greater hip flexion during early stance (p = 0.021) and 3.5° lesser hip extension in late stance that was nearly significant (p = 0.059). Walking with greater hip flexion at baseline was associated with a greater risk of increase in MRI defined structural changes in the hip joint (Odds Ratio = 1.1, p = 0.038). Greater hip flexion during walking was associated with a risk of structural progression of hip OA. The results may guide future interventions to alter the walking patterns and slow structural hip OA progression.© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Biomechanical investigation of two long bone growth modulation techniques by finite element simulations


Implants used to correct pathological varus–valgus deformities (VVD) and leg length discrepancies (LLD) may not be optimized for the specific treatment, as suggested by their off-label use. Detailed analysis of this issue has been limited by the poorly understood mechanical behavior of the growing physis and ignorance of the loads acting on the implants. The aim of this study was to predict and compare the loading conditions of a growth modulation implant in VVD and LLD treatments. Idealized finite element (FE) models of the juvenile distal femur treated with the Eight-Plate implant were developed for VVD and LLD. Bone growth was simulated using thermal strains. The axial force in the plate was compared between the two treatments. Case-specific plate forces were predicted by virtually reproducing the screw deformation visible on radiographs of LLD (N = 4) and VVD (N = 4) clinical cases. The simple FE models reproduced the clinical implant deformations well. The resulting forces ranged from 129 to 580 N for the VVD patients. For LLD, this range was from 295 to 1002 N per plate, that is, 590–2004 N for the entire physis. The higher forces in LLD could be explained by restricted screw divergence in the double-sided implant application. For the first time, the loading conditions of a growth modulation implant were investigated and compared between two treatments by FE analyses, and the range of case-specific loads was predicted. These simulation tools may be utilized for guiding appropriate usage and for efficient development of implants. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

In vitro loading models for tendon mechanobiology


Tendons are the connective tissue responsible for transferring force from muscles to bones. A key factor in tendon development, maturation, repair, and degradation is its biomechanical environment. Understanding tendon mechanobiology is essential for the development of injury prevention strategies, rehabilitation protocols and potentially novel treatments in tendon injury and degeneration. Despite the simple overall loading on tendon tissue, cells within the tissue in vivo experience a much more complex mechanical environment including tension, compression and shear forces. This creates a substantial challenge in the establishment of in vitro loading models of the tendon. This article reviews multiple loading models used for the study of tendon mechanobiology and summarizes the main findings. Although impressive progress has been achieved in the functionality and mimicry of in vitro loading models, an ideal platform is yet to be developed. Multidisciplinary approaches and collaborations will be the key to unveiling the tendon mechanobiology. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Influence of collars on the primary stability of cementless femoral stems: A finite element study using a diverse patient cohort


For cementless femoral stems, there is debate as to whether a collar enhances primary stability and load transfer compared to collarless designs. Finite Element (FE) analysis has the potential to compare stem designs within the same cohort, allowing for subtle performance differences to be identified, if present. Subject-specific FE models of intact and implanted femora were run for a diverse cohort (21 males, 20 females; BMI 16.4–41.2 kg/m2, age 50–80 yrs). Collared and collarless versions of Corail® (DePuy Synthes, Warsaw, IN) were sized and positioned using an automated algorithm that aligns the femoral/stem axes, preserves the head-center location, and maximizes metaphyseal fit. Joint contact and muscle forces simulating peak forces in level gait and stair climbing and were scaled to the body mass and applied to each subject. Three failure scenarios were assessed: Potential for peri-prosthetic fibrous tissue formation (stem micromotion), potential for peri-prosthetic bone damage (equivalent strains), and calcar bone remodeling (changes in strain-energy density). Comparisons were performed using paired t-tests. Only subtle differences were found (mean 90th percentile micromotion: Collared = 86 µm, collarless = 92.5 µm, mean 90th percentile interface strains: Collared = 733 µϵ, collarless = 767 µϵ, and similar remodeling stimuli were predicted). The slight differences observed were small in comparison with the inter-patient variability. Statement of clinical significance: Our results suggest that the presence/absence of a collar is unlikely to substantially alter the bone-implant biomechanics nor the initial mechanical environment. Hence, a collar is likely to have minimal clinical impact. Analysis using different femoral stem designs is recommended before generalising these findings. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Collagen XI mutation lowers susceptibility to load-induced cartilage damage in mice


Interactions among risk factors for osteoarthritis (OA) are not well understood. We investigated the combined impact of two prevalent risk factors: mechanical loading and genetically abnormal cartilage tissue properties. We used cyclic tibial compression to simulate mechanical loading in the cho/+ (Col11a1 haploinsufficient) mouse, which has abnormal collagen fibrils in cartilage due to a point mutation in the Col11a1 gene. We hypothesized that the mutant collagen would not alter phenotypic bone properties and that cho/+ mice, which develop early onset OA, would develop enhanced load-induced cartilage damage compared to their littermates. To test our hypotheses, we applied cyclic compression to the left tibiae of 6-month-old cho/+ male mice and wild-type (WT) littermates for 1, 2, and 6 weeks at moderate (4.5 N) and high (9.0 N) peak load magnitudes. We then characterized load-induced cartilage and bone changes by histology, microcomputed tomography, and immunohistochemistry. Prior to loading, cho/+ mice had less dense, thinner cortical bone compared to WT littermates. In addition, in loaded and non-loaded limbs, cho/+ mice had thicker cartilage. With high loads, cho/+ mice experienced less load-induced cartilage damage at all time points and displayed decreased matrix metalloproteinase (MMP)-13 levels compared to WT littermates. The thinner, less dense cortical bone and thicker cartilage were unexpected and may have contributed to the reduced severity of load-induced cartilage damage in cho/+ mice. Furthermore, the spontaneous proteoglycan loss resulting from the mutant collagen XI was not additive to cartilage damage from mechanical loading, suggesting that these risk factors act through independent pathways. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Etiology of developmental spinal stenosis: A genome-wide association study


Our study aimed to identify possible single nucleotide polymorphisms (SNPs) via a genome-wide association study (GWAS) approach and a candidate gene platform that were associated with lumbar developmental spinal stenosis (DSS). Southern Chinese population-based study volunteers were assessed (age range: 18–55 years). DSS was defined as the anteroposterior bony spinal canal diameter on T1-weighted axial MRI of L1 to S1. Genotyping was performed using the Illumina HumanOmniZhongHua-8 BeadChip. Using the canal diameter as the quantitative trait, genomic statistical analyses was performed. A total of 469 subjects were recruited. The mean axial AP measurements noted were: L1: 21.8 mm, L2: 21.9 mm, L3: 22.4 mm, L4: 20.2 mm, L5: 19.6 mm, and S1: 17.3 mm. Q–Q plots of genome-wide associations found significant differences in L4 and L5 measurements. More significant SNPs were found on chromosomes 8, 11, and 18. Low-density lipoprotein receptor-related protein 5 on chromosome 11 was found to be an important functional gene in canal bony development via candidate gene approach. We found two clusters in the findings with one including the upper levels (L1–L4) and the other the lower levels (L5 and S1). This is the first GWAS addressing DSS. The presence of multiple SNPs suggests a multi-factorial origin of DSS. Further analyses noted region-specific genetic predisposition, delineating distinct upper to lower lumbar regions of DSS. With better understanding of the DSS phenotype and genetic markers, the at-risk population can be identified early, preventative measures can be initiated, lifestyle/activity modification can be implemented, and more novel and precision-based therapeutics can be developed. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Effect of biomechanical stress on endogenous antioxidant networks in bovine articular cartilage


Mechanosensitve pathways in chondrocytes are essential for maintaining articular cartilage homeostasis. Traumatic loading increases cartilage oxidation and causes cell death and osteoarthritis. However, sub-lethal doses of the pro-oxidant molecule tert-Butyl hydroperoxide (tBHP) protects against loading-induced chondrocyte death. We hypothesized that compressive cyclic loading at moderate strains (<20%) causes sub-lethal cartilage oxidation that induces an adaptive increase in the endogenous antioxidant defense network. We tested this hypothesis by subjecting healthy bovine articular cartilage explants to in vitro static or cyclic (1 Hz) compressive loading at 50 kPa (15% strain, “physiologic”) versus 300 kPa (40% strain, “hyper-physiologic”) for 12 h per day for 2 days. We also treated unloaded explants with 100 μM tBHP for 12 h per day for 2 days to differentiate between biomechanical and chemical pro-oxidant stimulation. All loading conditions induced glutathione oxidation relative to unloaded controls, but only the 50 kPa cyclic loading condition increased total glutathione content (twofold). This increase was associated with a greater expression of glutamate-cysteine ligase, the rate-limiting step in glutathione synthesis, compared to 300 kPa cyclic loading. 50 kPa cyclic loading also increased the expression of superoxide dismutase-1 and peroxiredoxin-3. Like 50 kPa loading, tBHP treatment also increased total glutathione content. However, tBHP treatment and 50 kPa cyclic loading differed in their effect on the expression of genes regulating antioxidant defense and cartilage matrix synthesis and degradation. These findings suggest that glutathione metabolism is a mechanosensitive antioxidant defense pathway in chondrocytes and that intermittent pro-oxidant treatment alone is insufficient to account for all changes in mediators of cartilage homeostasis associated with cyclic loading. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Dynamic imaging demonstrates that pulsed electromagnetic fields (PEMF) suppress IL-6 transcription in bovine nucleus pulposus cells


Inflammatory cytokines play a dominant role in the pathogenesis of disc degeneration. Pulsed electromagnetic fields (PEMF) are noninvasive biophysical stimulus that has been used extensively in the orthopaedic field for many years. However, the specific cellular responses and mechanisms involved are still unclear. The objective of this study was to assess the time-dependent PEMF effects on pro-inflammatory factor IL-6 expression in disc nucleus pulposus cells using a novel green fluorescence protein (GFP) reporter system. An MS2-tagged GFP reporter system driven by IL-6 promoter was constructed to visualize PEMF treatment effect on IL-6 transcription in single living cells. IL-6-MS2 reporter-labeled cells were treated with IL-1α to mimic the in situ inflammatory environment of degenerative disc while simultaneously exposed to PEMF continuously for 4 h. Time-lapse imaging was recorded using a confocal microscope to track dynamic IL-6 transcription activity that was demonstrated by GFP. Finally, real-time RT-PCR was performed to confirm the imaging data. Live cell imaging demonstrated that pro-inflammatory factor IL-1α significantly promoted IL-6 transcription over time as compared with DMEM basal medium condition. Imaging and PCR data demonstrated that the inductive effect of IL-1α on IL-6 expression could be significantly inhibited by PEMF treatment in a time-dependent manner (early as 2 h of stimulus initiation). Our data suggest that PEMF may have a role in the clinical management of patients with chronic low back pain. Furthermore, this study shows that the MS2-tagged GFP reporter system is a useful tool for visualizing the dynamic events of mechanobiology in musculoskeletal research. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:XX–XX, 2017. Visualization of dynamic mRNA expression in individual live cells. (A1) Illustration of IL-6 expression ON and OFF in MS2-GFP system. A2) Single GFP particl[...]

Drug therapy targeting pyrophosphate slows the ossification of spinal ligaments in twy mice


The lack of an effective drug therapy against ossification of spinal ligament (OSL) warrants investigation into the therapeutic target of this disease. An endogenous inhibitor of biomineralization, pyrophosphate (PPi) is a potential therapy for ectopic ossification; however, exogenous PPi is rapidly hydrolyzed by tissue non-specific alkaline phosphatase (TNAP) present in body fluids. In this study, we examined whether a drug therapy targeting PPi is efficacious for the treatment of OSL using the Enpp1ttw/ttw (twy) mouse model. Twenty male twy mice were randomized into four groups: (i) vehicle (Control); (ii) alkaline phosphatase inhibitor levamisole (5 mg/kg/day sc continuously); (iii) levamisole + exogenous PPi (160 µmol/kg/day sc continuously); and (iv) nuclear retinoic acid receptor-γ (RARγ) agonist (6 µg/kg sc daily). The RARγ agonist, which is a proven inhibitor of ectopic endochondral ossification, was used as a positive control. Treatments commenced when the mice were 5 weeks of age and continued for 4 weeks. Longitudinal micro-computed tomography and postmortem histological analysis were performed. Administration of levamisole alone and in combination with PPi increased serum PPi concentration by 17% and 52%, respectively, compared to that in vehicle-treated mice. The development of OSL in twy mice was suppressed by levamisole + PPi and RARγ agonist treatments, but not by levamisole alone. The levamisole + PPi therapy did not cause osteoporosis, whereas RARγ agonist-treated mice developed osteoporosis. Treatment of twy mice with levamisole in combination with exogenous PPi increased serum PPi level, which slowed the progression of OSL without producing adverse effect on bone. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res

Plasticizer di(2-ethylhexyl)phthalate interferes with osteoblastogenesis and adipogenesis in a mouse model


Plasticizer di(2-ethylhexyl)phthalate (DEHP) can leach from medical devices such as blood storage bags and the tubing. Recently, epidemiological studies showed that phthalate metabolites levels in the urine are associated with low bone mineral density (BMD) in older women. The detailed effect and mechanism of DEHP on osteoblastogenesis and adipogenesis, and bone loss remain to be clarified. Here, we investigated the effect and mechanism of DEHP and its active metabolite mono(2-ethylhexyl)phthalate (MEHP) on osteoblastogenesis and adipogenesis. The in vitro study showed that osteoblast differentiation of bone marrow stromal cells (BMSCs) was significantly and dose-dependently decreased by DEHP and MEHP (10–100 µM) without cytotoxicity to BMSCs. The mRNA expressions of alkaline phosphatase, Runx2, osteocalcin (OCN), Wnt1, and β-catenin were significantly decreased in DEHP- and MEHP-treated BMSCs during differentiation. MEHP, but not DEHP, significantly increased the adipocyte differentiation of BMSCs and PPARγ mRNA expression. Both DEHP and MEHP significantly increased the ratios of phosphorylated β-catenin/β-catenin and inhibited osteoblastogenesis, which could be reversed by Wnt activator lithium chloride and PPARγ inhibitor T0070907. Moreover, exposure of mice to DEHP (1, 10, and 100 mg/kg) for 8 weeks altered BMD and microstructure. In BMSCs isolated from DEHP-treated mice, osteoblastogenesis and Runx2, Wnt1, and β-catenin expression were decreased, but adipogenesis and PPARγ expression were increased. These findings suggest that DEHP and its metabolite MEHP exposure may inhibit osteoblastogenesis and promote adipogenesis of BMSCs through the Wnt/β-catenin-regulated and thus triggering bone loss. PPARγ signaling may play an important role in MEHP- and DEHP-induced suppression of osteogenesis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Exposure of mice to plasticizer di(2-ethylhexyl)phthalate ([...]

Ischemic femoral head osteonecrosis in a piglet model causes three dimensional decrease in acetabular coverage


Legg-Calve-Perthes disease (LCPD) is a childhood form of ischemic osteonecrosis marked by development of severe femoral head deformity and premature osteoarthritis. The pathogenesis of femoral head deformity has been studied extensively using a piglet model of ischemic osteonecrosis, however, accompanying acetabular changes have not been investigated. The purpose of this study was to determine if acetabular changes accompany femoral head deformity in a well-established piglet model of LCPD and to define the acetabular changes using three dimensional computed tomography (3-D CT) and modeling. Twenty-four piglets were surgically induced with ischemic osteonecrosis on the right side. The contralateral hip was used as control. At 8 weeks postoperative, pelvi were retrieved and imaged with CT. Custom software was used to measure acetabular morphologic parameters on 3-D CT images. Moderate to severe femoral head deformities were present in all animals. Acetabula with accompanying femoral head deformity had a significant decrease in acetabular version and tilt (p [...]

Evaluating instrument responsiveness in joint function: The HOOS JR, the KOOS JR, and the PROMIS PF CAT


12345Responsiveness is the ability to detect change over time and is an important aspect of measures used to detect treatment effects. The purpose of this study was to assess the responsiveness of the HOOS JR, the KOOS JR, and the PROMIS Physical Function (PF) computerized adaptive test (CAT) in a joint reconstruction practice. 983 patients were evaluated for joint conditions between 2014 and 2017 in an orthopaedic clinic and completed the three instruments at baseline and 3 and/or 6-month follow-up visits. Average age was 61.03 years (SD = 12.33, Range = 18–90 years) and the majority of the patients were White (n = 875, 89.0%). Three-month responsiveness was calculated two ways, as between 80 and 100 days and as 90 days and beyond. Six-month responsiveness was calculated as 170–190 days and as 180 days and beyond. All changes from baseline scores were significant at the 3-, >3-, and >6-month follow-up (p < 0.05). All three measures showed large effect sizes, ranging from 0.80–1.20 at each time-point. The standardized response mean was large for each measure and at each time-point (Range = 1.06–1.53). This study demonstrated the responsiveness of the HOOS JR, KOOS JR, and the PROMIS PF in adult reconstruction patients. The PROMIS PF was consistently the most responsive instrument in this analysis. Clinical significance: The HOOS JR, KOOS JR, and PROMIS PF are useful clinical instruments for assessing treatment change and may be selected as relevant to the specific needs and conditions of the adult joint reconstruction patient population. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Labral calcification in end-stage osteoarthritis of the hip correlates with pain and clinical function


The acetabular labrum of the hip (ALH) is recognized as a clinically important structure, but knowledge about the pathophysiology of this fibrocartilage is scarce. In this prospective study we determined the prevalence of ALH calcification in patients with end-stage osteoarthritis (OA) and analyzed the relationship of cartilage calcification (CC) with hip pain and clinical function. Cohort of 80 patients (70.2 ± 7.6years) with primary OA scheduled for total hip replacement. Harris Hip Score (HHS) was recorded preoperatively. Total ALH and femoral head (FH) were sampled intraoperatively. CC of the ALH and FH was analyzed by high-resolution digital contact radiography. Histological degeneration of the ALH (Krenn-Score) and FH (OARSI-Score) was determined. Multivariate linear regression model and partial correlation analyses were performed. The prevalence of cartilage calcification both in the ALH and FH was 100%, while the amount of CC in the ALH was 1.55 times higher than in the FH (p [...]

The primary cilium as a signaling nexus for growth plate function and subsequent skeletal development


The primary cilium is a solitary, antenna-like sensory organelle with many important roles in cartilage and bone development, maintenance, and function. The primary cilium's potential role as a signaling nexus in the growth plate makes it an attractive therapeutic target for diseases and disorders associated with bone development and maintenance. Many signaling pathways that are mediated by the cilium—such as Hh, Wnt, Ihh/PTHrP, TGFβ, BMP, FGF, and Notch—are also known to influence endochondral ossification, primarily by directing growth plate formation and chondrocyte behavior. Although a few studies have demonstrated that these signaling pathways can be directly tied to the primary cilium, many pathways have yet to be evaluated in context of the cilium. This review serves to bridge this knowledge gap in the literature, as well as discuss the cilium's importance in the growth plate's ability to sense and respond to chemical and mechanical stimuli. Furthermore, we explore the importance of using the appropriate mechanism to study the cilium in vivo and suggest IFT88 deletion is the best available technique. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Possibility of quantitative T2-mapping MRI of cartilage near metal in high tibial osteotomy: A human cadaver study


T2-mapping is a widely used quantitative MRI technique in osteoarthritis research. An important challenge for its application in the context of high tibial osteotomy (HTO) is the presence of metallic fixation devices. In this study, we evaluated the possibility of performing T2-mapping after a HTO, by assessing the extent of magnetic susceptibility artifacts and the influence on T2 relaxation times caused by two commonly used fixation devices. T2-mapping with a 3D fast spin-echo sequence at three Tesla was performed on 11 human cadaveric knee joints before and after implantation of a titanium plate and screws (n = 5) or cobalt chrome staples (n = 6). Mean T2 relaxation times were calculated in six cartilage regions, located in the distal and posterior cartilage of femoral condyles and the cartilage of tibial plateaus, both medially and laterally. T2 relaxation times before and after the implantation were compared with paired t-tests and Wilcoxon rank tests. Due to the extent of the magnetic susceptibility artifact, it was not possible to segment the knee cartilage and thus calculate T2 relaxation times in the lateral weight-bearing femoral and tibial cartilage regions only in the cobalt chrome group. In all cartilage regions of the titanium implanted knees and those unaffected by artifacts due to cobalt chrome implants, T2 relaxation times did not significantly differ between the two scans. Our results suggest that accurate T2-mapping after a HTO procedure is possible in all areas after implantation of a titanium fixation device and in most areas after implantation of a cobalt chrome fixation device. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Neutral glenoid alignment in reverse shoulder arthroplasty does not guarantee decreased risk of impingement


Reverse Shoulder Arthroplasty (RSA) has gained popularity over the recent years, but impingement concerns are still present. Surgeons aim to correct pre-operative glenoid deformities to reduce impingement but it can be challenging without assistance like patient specific guides. However, it is unclear how accurate glenoid correction affects the impingement. The main objective of this study was to determine whether accurate glenoid correction to neutral version and tilt can reduce the risk of impingement. Two types of virtual surgeries were performed on 22 pre-operative arthritic shoulders: (i) “Interactive,” the glenoid baseplate could be placed with accuracy, and (ii) “Blind,” surgeons placed the RSA baseplate while they could only visualize the glenoid. The virtual models were then used in an RSA biomechanical model which recorded impingement for (i) four Range of Motion (ROM) tasks, (ii) ten Activities of Daily Living (ADL). The “Blind” method resulted in more variable glenoid placement (version and tilt) than the “Interactive” method (p = 0.001). However, both methods showed similar ROM and impingement occurrence in ADLs. The results suggest it is challenging for surgeons to accurately correct version and tilt on arthritic glenoids when only referencing off of the face of the glenoid. However, the variable glenosphere placement observed in the “Blind” method did not result in worse impingement compared to the accurate “Interactive” method. This was because both methods had similar inferior baseplate positioning which is more important than correcting version or tilt. Implantation accuracy remains important in RSA, but pre-operative planning should not just target at correcting version and tilt. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Evaluation of loading parameters for murine axial tibial loading: Stimulating cortical bone formation while reducing loading duration


Classic studies in bone mechanobiology have established the importance of loading parameters on the anabolic response. Most of these early studies were done using loading methods not currently in favor, and using non-murine species. Our objective was to re-examine the effects of several loading parameters on the response of cortical bone using the contemporary murine axial tibial compression model. We subjected tibias of 5-month old, female C57Bl/6 mice to cyclic (4 Hz) mechanical loading and examined bone formation responses using dynamic and static histomorphometry. First, using a reference protocol of 1,200 cycles/day, 5 days/week for 2 weeks, we confirmed the significant influence of peak strain magnitude on periosteal mineralizing surface (Ps.MS/BS) and bone formation rate (Ps.BFR/BS) (p  0.05). On the other hand, reducing the daily frequency of loading from 5 consecutive days/week to 3 alternate days/week significantly diminished the periosteal response, from a loading-induced increase in Ps.MS/BS of 38% (loaded vs. control) for 5 days/week to only 15% for 3 days/week (p [...]

Targeting intracellular Staphylococcus aureus to lower recurrence of orthopaedic infection


Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP), and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10× MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Biofilm formation and osteoblast invasion by Staphylococci ar[...]

Bone healing in an aged murine fracture model is characterized by sustained callus inflammation and decreased cell proliferation


Geriatric fractures take longer to heal and heal with more complications than those of younger patients; however, the mechanistic basis for this difference in healing is not well understood. To improve this understanding, we investigated cell and molecular differences in fracture healing between 5-month-old (young adult) and 25-month-old (geriatric) mice healing utilizing high-throughput analysis of gene expression. Mice underwent bilateral tibial fractures and fracture calluses were harvested at 5, 10, and 20 days post-fracture (DPF) for analysis. Global gene expression analysis was performed using Affymetrix MoGene 1.0 ST microarrays. After normalization, data were compared using ANOVA and evaluated using Principal Component Analysis (PCA), CTen, heatmap, and Incromaps analysis. PCA and cross-sectional heatmap analysis demonstrated that DPF followed by age had pronounced effects on changes in gene expression. Both un-fractured and 20 DPF aged mice showed increased expression of immune-associated genes (CXCL8, CCL8, and CCL5) and at 10 DPF, aged mice showed increased expression of matrix-associated genes, (Matn1, Ucma, Scube1, Col9a1, and Col9a3). Cten analysis suggested an enrichment of CD8+ cells and macrophages in old mice relative to young adult mice and, conversely, a greater prevalence of mast cells in young adult mice relative to old. Finally, consistent with the PCA data, the classic bone healing pathways of BMP, Indian Hedgehog, Notch and Wnt clustered according to the time post-fracture first and age second. Clinical Significance: Greater understanding of age-dependent molecular changes with healing will help form a mechanistic basis for therapies to improve patient outcomes. © 2017 Orthopaedic Research[...]

Achieving interfragmentary compression without special drilling technique or screw design


Traditional fracture fixation teaching suggests that fully threaded screws do not provide interfragmentary compression unless placed through a glide hole. Based on this assumption, pelvic surgeons typically use fully threaded screws in the treatment of comminuted transforaminal sacral fractures to limit iatrogenic neuroforaminal stenosis. Clinical experience with fully threaded screws suggests that interfragmentary compression actually does occur. We hypothesized that the use of a fully threaded screw does not produce any interfragmentary compression and that there is no difference in insertional torque between partially threaded and fully threaded screws. To test this hypothesis, fully and partially threaded 7.0 millimeter (mm) cannulated screws were placed across two synthetic bone blocks fabricated to simulate normal and osteoporotic bone. We compared two groups of normal and osteoporotic blocks for compression achieved and maximal insertional torque generated with fully threaded and partially threaded screw insertion. A micro computed tomography (CT) scan of the composite blocks was obtained to investigate for structural changes created during screw insertion. For both groups, compression was achieved with fully threaded screws and the maximal insertional torque was higher using fully threaded screws. Micro CT analysis demonstrated local bone damage with structural disruption in the near segment of the fully threaded screw path in comparison to the partially threaded. Clinical significance: this study demonstrates that compression is generated using fully threaded screws without using a predrilled glide hole. The insertional torque required to generate compression with fully threaded [...]

Optimizing a micro-computed tomography-based surrogate measurement of bone-implant contact


Histology and backscatter scanning electron microscopy (bSEM) are the current gold standard methods for quantifying bone-implant contact (BIC), but are inherently destructive. Microcomputed tomography (μCT) is a non-destructive alternative, but attempts to validate μCT-based assessment of BIC in animal models have produced conflicting results. We previously showed in a rat model using a 1.5 mm diameter titanium implant that the extent of the metal-induced artefact precluded accurate measurement of bone sufficiently close to the interface to assess BIC. Recently introduced commercial laboratory μCT scanners have smaller voxels and improved imaging capabilities, possibly overcoming this limitation. The goals of the present study were to establish an approach for optimizing μCT imaging parameters and to validate μCT-based assessment of BIC. In an empirical parametric study using a 1.5 mm diameter titanium implant, we determined 90 kVp, 88 µA, 1.5 μm isotropic voxel size, 1600 projections/180°, and 750 ms integration time to be optimal. Using specimens from an in vivo rat experiment, we found significant correlations between bSEM and μCT for BIC with the manufacturer's automated analysis routine (r = 0.716, p = 0.003) or a line-intercept method (r = 0.797, p = 0.010). Thus, this newer generation scanner's improved imaging capability reduced the extent of the metal-induced artefact zone enough to permit assessment of BIC. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9 999:XX–XX, 2017.

Vein wrapping facilitates basic fibroblast growth factor-induced heme oxygenase-1 expression following chronic nerve constriction injury


The clinical efficacy of autologous vein wrapping for recurrent compressive neuropathy has been demonstrated; however, the underlying mechanisms of this technique remain unclear. Rats were divided into chronic constriction injury (CCI) and CCI + vein wrapping (CCI + VW) groups. Mechanical allodynia was evaluated using von Frey filaments. To identify the neuroprotective factors released from veins, basic fibroblast growth factor (bFGF) mRNA expression in veins was compared to that in the sciatic nerve. The response of heme oxygenase-1 (HO-1) expression to vein wrapping was evaluated by RT-PCR and enzyme-linked immunosorbent assays. The effects of exogenous bFGF on HO-1 expression were evaluated using a sciatic nerve cell culture. Vein wrapping significantly increased the withdraw threshold levels compared to the untreated CCI group. bFGF mRNA expression in veins was higher than that in untreated sciatic nerves. HO-1 mRNA expression was induced at higher levels in sciatic nerve cells in the presence of exogenous bFGF compared to untreated control cells. HO-1 mRNA and protein expression in the sciatic nerve were also higher in the CCI + VW group compared with the CCI group. Our results suggest that vein-derived bFGF contributes to the therapeutic benefit of vein wrapping through the induction of HO-1 in the sciatic nerve. Vein wrapping is a useful technique for reducing neuropathic pain. Further understanding of the neurotrophic factors released from veins may help to optimize current procedures for treating recurrent compressive neuropathy and traumatic peripheral nerve injury, and lead to the development of new therapeutic [...]

Improved union and bone strength in a mouse model of NF1 pseudarthrosis treated with recombinant human bone morphogenetic protein-2 and zoledronic acid


Tibial pseudarthrosis associated with Neurofibromatosis type 1 (NF1) is an orthopedic condition with consistently poor clinical outcomes. Using a murine model that features localized double inactivation of the Nf1 gene in an experimental tibial fracture, we tested the effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) and/or the bisphosphonate zoledronic acid (ZA). Tibiae were harvested at 3 weeks for analysis, at which time there was negligible healing in un-treated control fractures (7% union). In contrast, rhBMP-2 and rhBMP-2/ZA groups showed significantly greater union (87% and 93%, p < 0.01 for both). Treatment with rhBMP-2 led to a 12-fold increase in callus bone volume and this was further increased in the rhBMP-2/ZA group. Mechanical testing of the healed rhBMP-2 and rhBMP-2/ZA fractures showed that the latter group had significantly higher mechanical strength and was restored to that of the un-fractured contralateral leg. Co-treatment with rhBMP-2/ZA also reduced fibrous tissue infiltration at the fracture site compared to rhBMP alone (p = 0.068). These data support the future clinical investigation of this combination of anabolic and anti-resorptive agents for the treatment of NF1 pseudarthrosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

High early post-operative complication rate after elective aseptic orthopedic implant removal of upper and lower limb


The necessity of orthopedic implant removal is under intense discussion and even if it is performed as an elective procedure, the risk of complications is present. Aim of the study was to identify parameters responsible for an increased risk of early post-operative complications after elective aseptic orthopedic implant removal. We reviewed 1,545 cases of aseptic and elective orthopedic implant removal between 2009 and 2011. The patient́s demographic data, time and duration of operation, patient́s comorbidities, and presence of complications in the first 4 weeks after implant removal were evaluated. Patients with signs of infection at the time of the surgical procedure were excluded from this study. 579 women and 966 men who underwent elective aseptic orthopedic implant removal were identified. Mean age at implant removal was 42 years and mean duration of the surgical procedure was 37 min. In this cohort, 70 patients (4.5%) underwent elective aseptic implant removal after 6pm. 52 patients (3.37%) operated on during daytime suffered from complications post-operatively and five patients (0.3%) who were operated on during the night experienced complications. The parameters age, sex, BMI, and surgeon showed no statistically significant differences for the risk of post-operative complications. Patients’ comorbidities such as diabetes seem to have influence but were not statistically significant either. Patients with revision surgery since their first operation, nocturnal surgery and longer duration of the procedure showed a statistically significant higher risk for complications, [...]

Mechanically stimulated biomarkers signal cartilage changes over 5 years consistent with disease progression in medial knee osteoarthritis patients


Using serum biomarkers to assess osteoarthritis (OA) disease state and risks of progression remain challenging. This study tested the hypothesis that changes to serum biomarkers in response to a mechanical stimulus in patients with medial knee OA signal cartilage thickness changes 5 years later. Specifically, serum concentrations of a collagen degradation marker (C1,2C) and a chondroitin sulfate synthesis marker (CS846) were measured 0.5 and 5.5 hours after a 30-min walk in 16 patients. Regional cartilage thickness changes measured from magnetic resonance images obtained at study entry and at 5-year follow-up were tested for correlations with baseline biomarker changes after mechanical stimulus, and for differences between groups stratified based on whether biomarker levels increased or decreased. Results showed that an increase in the degradation biomarker C1,2C correlated with cartilage thinning of the lateral tibia (R = −0.63, p = 0.009), whereas an increase in the synthesis marker CS846 correlated with cartilage thickening of the lateral femur (R = 0.76, p = 0.001). Changes in C1,2C and CS846 were correlated (R2 = 0.28, p = 0.037). Subjects with increased C1,2C had greater (p = 0.05) medial tibial cartilage thinning than those with decreased C1,2C. In conclusion, the mechanical stimulus appeared to metabolically link the biomarker responses where biomarker increases signaled more active OA disease states. The findings of medial cartilage thinning for patients with increases in the degradation marker and correlation of [...]

Trends in hip replacements between 1999 and 2012 in Sweden


National Registers document changes in the circumstance, practice, and outcome of surgery with the passage of time. In the context of total hip replacement (THR), registers can help elucidate the relevant factors that affect the clinical outcome. We evaluated the evolution of factors related to patient, surgical procedure, socio-economy, and various outcome parameters after merging databases of the Swedish Hip Arthroplasty Register, Statistics Sweden and the National Board of Health and Welfare. Data on 193,253 THRs (164,113 patients) operated between 1999 and 2012 were merged. We studied the evolution of surgical volume, patient demographics, socio-economic factors, surgical factors, length-of-stay, mortality rate, adverse events, re-operation and revision rates, and Patient Reported Outcome Measures (PROMs). Throughout this time period the majority of patients were operated on with a diagnosis of primary osteoarthritis. Comorbidity indices increased each year observed. The share of all-cemented implants has dropped from 92% to 68%. More than 88% of the bearings were metal-on-polyethylene. Length-of-stay decreased by 50%. There was a reduction in 30- and 90-day mortality. Re-operation and revision rates at 2 years are decreasing. The post-operative PROMs improved despite the observation of worse pre-operative pain scores getting over time. The demographics of patients receiving a THR, their comorbidities, and their primary diagnosis are changing. Notwithstanding these changes, outcomes like mortality, re-operations, revisions,[...]

Antibiotic elution from acrylic bone cement loaded with high doses of tobramycin and vancomycin


Two-stage revision treatment of prosthetic joint infection (PJI) frequently employs the use of a temporary bone cement spacer loaded with multiple antibiotic types. Tobramycin and vancomycin are commonly used antibiotics in cement spacers, however, there is no consensus on the relative concentrations and combinations that should be used. Therefore, the purpose of this study was to investigate the influence of dual antibiotic loading on the total antibiotic elution and compressive mechanical properties of acrylic bone cement. Varying concentrations of tobramycin (0–3 g) and vancomycin (0–3 g) were added either alone or in combination to acrylic cement (Palacos R), resulting in 12 experimental groups. Samples were submerged in 37°C saline for 28 d and sampled at specific time points. The collected eluent was analyzed to determine the cumulative antibiotic release. In addition, the cement's compressive mechanical properties and porosity were characterized. Interestingly, the cement with the highest concentration of antibiotics did not possess the best elution properties. Cement samples containing both 3 g of tobramycin and 2 g vancomycin demonstrated the highest cumulative antibiotic release after 28 d, which was coupled with a significant decrease in the mechanical properties and an increased porosity. The collected data also suggests that tobramycin elutes more effectively than vancomycin from cement. In conclusion, this study demonstrates that high antibiotic l[...]

Accumulation of advanced-glycation end products (AGEs) accelerates arthrogenic joint contracture in immobilized rat knee


Joint mobility decreases in the elderly and in diabetics, this process is thought to be caused by accumulation of advanced-glycation end products (AGEs). Here, we aimed to elucidate the role of AGEs in joint contracture formation in rat knees. Rats were injected with ribose or saline into the knees twice weekly for 8 weeks. Pentosidine (AGE) levels were measured in the knee-joint tissues. After serial injections, rats were subjected to unilateral knee-joint immobilization in a flexion position for various periods. At day 21, the passive knee ranges of motions (ROMs) were measured. Knee joint histopathology were assessed, and the expression of fibrotic genes in the posterior joint capsules was examined using real-time PCR. Ribose injection induced a 7.0-fold increase in pentosidine levels relative to saline injection. Joint immobilization resulted in equal myogenic ROM restriction in both groups. Arthrogenic ROM restriction was greater with ribose injection in the immobilized joints (p [...]

Patient-specific in silico models can quantify primary implant stability in elderly human bone


Secure implant fixation is challenging in osteoporotic bone. Due to the high variability in inter- and intra-patient bone quality, ex vivo mechanical testing of implants in bone is very material- and time-consuming. Alternatively, in silico models could substantially reduce costs and speed up the design of novel implants if they had the capability to capture the intricate bone microstructure. Therefore, the aim of this study was to validate a micro-finite element model of a multi-screw fracture fixation system. Eight human cadaveric humerii were scanned using micro-CT and mechanically tested to quantify bone stiffness. Osteotomy and fracture fixation were performed, followed by mechanical testing to quantify displacements at 12 different locations on the instrumented bone. For each experimental case, a micro-finite element model was created. From the micro-finite element analyses of the intact model, the patient-specific bone tissue modulus was determined such that the simulated apparent stiffness matched the measured stiffness of the intact bone. Similarly, the tissue modulus of a small damage region around each screw was determined for the instrumented bone. For validation, all in silico models were rerun using averaged material properties, resulting in an average coefficient of determination of 0.89 ± 0.04 with a slope of 0.93 ± 0.19 and a mean absolute error of 43 ± 10 μm when correlating in silico [...]

Issue Information - Cover


Issue Information - Editorial Board and TOC