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1.  Yin-Yang 1, a new player in early heart development 
Circulation research  2013;112(6):876-877.
doi:10.1161/CIRCRESAHA.113.300947
PMCID: PMC3955736  PMID: 23493301
2.  Odontoid pannus formation in a patient with ankylosing spondylitis causing atlanto-axial instability 
BMJ Case Reports  2012;2012:bcr1120115178.
Ankylosing spondylitis is one of the commonest inflammatory diseases of the axial skeleton and can be complicated by atlanto-axial instability. This serious and likely underestimated complication can be easily overlooked. However, there are clear features which can help alert suspicion to initiate the appropriate investigations with imaging that is very effective at diagnosing and assessing this complication. The authors report an unusual case where odontoid pannus formation, akin to that seen in rheumatoid arthritis, was the underlying cause.
doi:10.1136/bcr.11.2011.5178
PMCID: PMC3291001  PMID: 22665557
3.  Fatigue is Specific to Working Muscles: No Cross-over with Single-leg Cycling in Trained Cyclists 
Fatigue induced via a maximal isometric contraction of a single-limb muscle group can evoke a “cross-over” of fatigue that reduces voluntary muscle activation and maximum isometric force in the rested contralateral homologous muscle group. We asked whether a cross-over of fatigue also occurs when fatigue is induced via high-intensity endurance exercise involving a substantial muscle mass. Specifically, we used high-intensity single-leg cycling to induce fatigue and evaluated associated effects on maximum cycling power (Pmax) in the fatigued ipsilateral leg (FATleg) as well as the rested contralateral leg (RESTleg). On separate days, 12 trained cyclists performed right leg Pmax trials before and again 30s, 3, 5, and 10min after a cycling time trial (TT, 10min) performed either with their right or left leg. Fatigue was estimated by comparing exercise-induced changes in Pmax and maximum handgrip isometric force (Fmax). Mean power produced during the right and left leg TT’s did not differ (203±8 vs. 199±8W). Compared to pre-TT, FATleg Pmax was reduced by 22±3% at 30s post-TT and remained reduced by 9±2% at 5min post-TT (both P<0.05). Despite considerable power loss in the FATleg, post-TT RESTleg Pmax (596–603W) did not differ from pre-TT values (596±35W). There were no alterations in handgrip Fmax (529–547N). Our data suggest that any potential cross-over of fatigue, if present at all, was not sufficient to measurably compromise RESTleg Pmax in trained cyclists. These results along with the lack of changes in handgrip Fmax indicate that impairments in maximal voluntary neuromuscular function were specific to working muscles.
doi:10.1007/s00421-012-2455-0
PMCID: PMC3934423  PMID: 22806085
Muscle fatigue; central fatigue; neuromuscular function; contralateral limb
4.  Mechanical Stress and ATP Synthesis are coupled by Mitochondrial Oxidants in Articular Cartilage 
Metabolic adaptation of articular cartilage under joint loading is evident and matrix synthesis seems to be critically tied to ATP. Chondrocytes utilize the glycolytic pathway for energy requirements but seem to require mitochondrial reactive oxygen species (ROS) to sustain ATP synthesis. The role of ROS in regulating ATP reserves under a mechanically active environment is not clear. It is believed that physiological strains cause deformation of the mitochondria, potentially releasing ROS for energy production. We hypothesized that mechanical loading stimulates ATP synthesis via mitochondrial release of ROS. Bovine osteochondral explants were dynamically loaded at 0.5Hz with amplitude of 0.25MPa for 1 Hour. Cartilage response to mechanical loading was assessed by imaging with dihydroethidium (ROS indicator) and a Luciferase based ATP assay. Electron transport inhibitor rotenone and mitochondrial ROS scavenger MitoQ significantly suppressed mechanically induced ROS production and ATP synthesis. Our findings indicate that mitochondrial ROS are produced as a result of physiological mechanical strains. Taken together with our previous findings of ROS involvement in blunt impact injuries, mitochondrial ROS are important contributors to cartilage metabolic adaptation and their precise role in the pathogenesis of osteoarthritis warrants further investigation.
doi:10.1002/jor.22223
PMCID: PMC3678272  PMID: 22930474
Articular Cartilage; Oxidants; Reactive Oxygen Species; Mechanical stress; Glycolysis
5.  Comparative digital cartilage histology for human and common osteoarthritis models 
Orthopedic research and reviews  2013;2013(5):13-20.
Purpose
This study addresses the species-specific and site-specific details of weight-bearing articular cartilage zone depths and chondrocyte distributions among humans and common osteoarthritis (OA) animal models using contemporary digital imaging tools. Histological analysis is the gold-standard research tool for evaluating cartilage health, OA severity, and treatment efficacy. Historically, evaluations were made by expert analysts. However, state-of-the-art tools have been developed that allow for digitization of entire histological sections for computer-aided analysis. Large volumes of common digital cartilage metrics directly complement elucidation of trends in OA inducement and concomitant potential treatments.
Materials and methods
Sixteen fresh human knees, 26 adult New Zealand rabbit stifles, and 104 bovine lateral plateaus were measured for four cartilage zones and the cell densities within each zone. Each knee was divided into four weight-bearing sites: the medial and lateral plateaus and femoral condyles.
Results
One-way analysis of variance followed by pairwise multiple comparisons (Holm–Sidak method at a significance of 0.05) clearly confirmed the variability between cartilage depths at each site, between sites in the same species, and between weight-bearing articular cartilage definitions in different species.
Conclusion
The present study clearly demonstrates multisite, multispecies differences in normal weight-bearing articular cartilage, which can be objectively quantified by a common digital histology imaging technique. The clear site-specific differences in normal cartilage must be taken into consideration when characterizing the pathoetiology of OA models. Together, these provide a path to consistently analyze the volume and variety of histologic slides necessarily generated by studies of OA progression and potential treatments in different species.
doi:10.2147/ORR.S38400
PMCID: PMC3899351  PMID: 24465137
knee; osteoarthritis; imaging; rabbit; bovine; cell density
6.  Comparison of T1ρ, dGEMRIC, and Quantitative T2 MRI in Pre-Operative ACL Rupture Patients 
Academic radiology  2012;20(1):99-107.
Rationale and Objectives
T1ρ, dGEMRIC and T2-mapping have shown sensitivity toward different osteoarthritic-associated compositional changes after joint injury, but have not been studied concomitantly in vivo. We hypothesized that these MRI sequences can be used to measure early glycosaminoglycan (GAG) losses and collagen disruption in cartilage of ACL rupture patients.
Materials and Methods
Thirteen acute ACL rupture patients were each imaged during a four hour pre-surgery work-up to acquire a fast-spin-echo-based T1ρ sequence, a multi-echo spin-echo T2 sequence, and a T1-weighted inversion recovery sequence with a gadolinium contrast agent (dGEMRIC) an average of 55.7 days post-injury. After acquisition, the three sequences’ relaxation times were analytically compared.
Results
Site-specific differences were evinced, but non-significant differences in mean relaxation time between layers of the same region and sequence were observed (ANOVA, p<0.05). Spearman’s correlation coefficients of 0.542 (T1ρ vs. T2, p<0.05), −0.026 (T1ρ vs. dGEMRIC, p=0.585) and −0.095 (T2 vs. dGEMRIC, p<0.05), were found.
Conclusion
No appreciable focal GAG loss was detected by dGEMRIC, and T2 was generally elevated in the early acute phase of blunt trauma injury. In contrast, both general and focal elevations in T1ρ relaxation times were identified, indicating an acute increase in unbound water in the matrix after blunt trauma, and show that patient-specific cartilage changes occur within otherwise healthy, young patients. Further investigation into each sequence’s long-term significance is warranted to help clinicians decide which sequence(s) will be the most useful for osteoarthritis prognosis given the challenge of concomitantly acquiring all three in a busy clinical setting.
doi:10.1016/j.acra.2012.07.009
PMCID: PMC3525789  PMID: 22981604
Quantitative MRI; Cartilage; ACL-rupture; Injury assessment
7.  A Novel Impaction Technique to Create Experimental Articular Fractures in Large Animal Joints 
Objective
A novel impaction fracture insult technique, developed for modeling post-traumatic osteoarthritis in porcine hocks in vivo, was tested to determine the extent to which it could replicate the cell-level cartilage pathology in human clinical intra-articular fractures.
Design
Eight fresh porcine hocks (whole-joint specimens with fully viable chondrocytes) were subjected to fracture insult. From the fractured distal tibial surfaces, osteoarticular fragments were immediately sampled and cultured in vitro for 48 hours. These samples were analyzed for the distribution and progression of chondrocyte death, using the Live/Dead assay. Five control joints, in which “fractures” were simulated by means of surgical osteotomy, were also similarly analyzed.
Results
In the impaction-fractured joints, chondrocyte death was concentrated in regions adjacent to fracture lines (near-fracture regions), as evidenced by fractional cell death significantly higher (p < 0.0001) than in central non-fracture (control) regions. Although nominally similar spatial distribution patterns were identified in the osteotomized joints, fractional cell death in the near-osteotomy regions was nine-fold lower (p < 0.0001) than in the near-fracture regions. Cell death in the near-fracture regions increased monotonically during 48 hours after impaction, dominantly within 1 mm from the fracture lines.
Conclusion
The impaction-fractured joints exhibited chondrocyte death characteristics reasonably consistent with those in human intra-articular fractures, but were strikingly different from those in “fractures” simulated by surgical osteotomy. These observations support promise of this new impaction fracture technique as a mechanical insult modality to replicate the pathophysiology of human intra-articular fractures in large animal joints in vivo.
doi:10.1016/j.joca.2012.10.004
PMCID: PMC3538937  PMID: 23069855
intra-articular fracture; animal modeling; mechanical insult; cartilage injury; chondrocyte death
8.  Pleiotropic and isoform-specific functions for Pitx2 in superior colliculus and hypothalamic neuronal development 
Transcriptional regulation of gene expression during development is critical for proper neuronal differentiation and migration. Alternative splicing and differential isoform expression have been demonstrated for most mammalian genes, but their specific contributions to gene function are not well understood. In mice, the transcription factor gene Pitx2 is expressed as three different isoforms (PITX2A, PITX2B, and PITX2C) which have unique amino termini and common DNA binding homeodomains and carboxyl termini. The specific roles of these isoforms in neuronal development are not known. Here we report the onset of Pitx2ab and Pitx2c isoform-specific expression by E9.5 in the developing mouse brain. Using isoform-specific Pitx2 deletion mouse strains, we show that collicular neuron migration requires PITX2AB and that collicular GABAergic differentiation and targeting of hypothalamic projections require unique Pitx2 isoform dosage. These results provide insights into Pitx2 dosage and isoform-specific requirements underlying midbrain and hypothalamic development.
doi:10.1016/j.mcn.2012.11.007
PMCID: PMC3540135  PMID: 23147109
migration; transcription factor; midbrain; isoform; differentiation; axon
9.  Prejudice-Related Events and Traumatic Stress Among Heterosexuals and Lesbians, Gay Men and Bisexuals 
This mixed-methods study examined associations between prejudice events and posttraumatic stress disorder (PTSD) among 382 lesbians, gays, and bisexuals (LGB) and 126 heterosexuals. Using the Composite International Diagnostic Interview, we assessed PTSD but relaxed Criterion A1, that is, allowed prejudice events that did not involve threat to life or physical integrity to also qualify as traumatic. First, we tested whether exposure to prejudice events differed with respect to sexual orientation and race. White LGBs were more likely than White heterosexuals to encounter a prejudice event, but Black and Latino LGBs were no more likely than White LGBs to experience a prejudice event. Second, we used qualitative analysis to examine the prejudice events that precipitated relaxed Criterion A1 PTSD among 8 participants. Two specific themes emerged: the need to make major changes and compromised sense of safety and security following exposure to the prejudice event.
PMCID: PMC3860584  PMID: 24348008
lesbian, gay and bisexual; PTSD; Criterion A1; prejudice; discrimination
10.  Correction: MicroRNA-17-92, a Direct Ap-2α Transcriptional Target, Modulates T-Box Factor Activity in Orofacial Clefting 
PLoS Genetics  2013;9(12):10.1371/annotation/90602bc3-5052-49ac-a7fb-33210d7c8b4d.
doi:10.1371/annotation/90602bc3-5052-49ac-a7fb-33210d7c8b4d
PMCID: PMC3867558
11.  Cartilage Abnormalities Associated with Defects of Chondrocytic Primary Cilia in Bardet-Biedl Syndrome Mutant Mice 
SUMMARY
Primary cilia are found on nearly every mammalian cell, including osteocytes, fibroblasts, and chondrocytes. However, the functions of primary cilia have not been extensively studied in these cells, particularly chondrocytes. Interestingly, defects in the primary cilium result in skeletal defects such as polydactyly in Bardet-Biedl Syndrome (BBS), a ciliary disorder that also results in obesity, retinopathy, and cognitive impairments (1–4). Wild-type mice and mutant mice of the ciliary proteins Bbs1, Bbs2, and Bbs6 were evaluated with respect to histological and biochemical differences in chondrocytes from articular cartilage and xiphoid processes. Using immunofluorescence microscopy, chondrocytic cilia were visualized from the load-bearing joints and non-load-bearing xiphoid processes. Significant differences in ciliary morphology were not identified between mutant and wild-type mice. However, after expanding chondrocytes in cell culture and implanting them in solid agarose matrix, it was seen that the fraction of ciliated cells in cultures from mutant mice was significantly lower than in the wild-type cultures (p<.05). In addition, in Safranin-O-stained whole joint sections, Bbs mutant mice had significantly lower articular joint thickness (p<.05) and lower proteoglycan content saturation (p<.05) than wild-type mice. Moreover, there were statistically significant differences of cell distribution between Bbs mutant and wild-type mice (p<.05), indicating that mutant articular cartilage had changes consistent with early signs of osteoarthritis. These data indicate that Bbs genes and their functions in the chondrocytic primary cilium are important for normal articular cartilage maintenance.
doi:10.1002/jor.20855
PMCID: PMC3845817  PMID: 19195025
chondrocyte; primary cilium; articular cartilage; Bardet-Biedl syndrome; osteoarthritis
12.  MicroRNAs get to the heart of development 
eLife  2013;2:e01710.
A microRNA regulates the expression of a network of genes in the heart to ensure that progenitor cells develop into strongly contractile cardiac muscle.
doi:10.7554/eLife.01710
PMCID: PMC3833033  PMID: 24252874
microRNA-1; cardiac; sarcomere; Telokin; genetics; smooth muscle gene expression; Mouse
13.  FREQUENCY CONTENT OF CARTILAGE IMPACT FORCE SIGNAL REFLECTS ACUTE HISTOLOGIC STRUCTURAL DAMAGE 
Cartilage  2012;3(4):314-322.
Objective
The objective of this study was to determine if acute cartilage impact damage could be predicted by a quantification of the frequency content of the impact force signal.
Design
Osteochondral specimens excised from bovine lateral tibial plateaus were impacted with one of six impact energies. Each impact force signal underwent frequency analysis, with the amount of higher-frequency content (percent of frequency spectrum above 1 KHz) being registered. Specimens were histologically evaluated to assess acute structural damage (articular surface cracking and cartilage crushing) resulting from the impact.
Results
Acute histologic structural damage to the cartilage had higher concordance with the high-frequency content measure than with other mechanical impact measures (delivered impact energy, impact maximum stress, and impact maximum stress rate of change).
Conclusions
This result suggests that the frequency content of an impact force signal, specifically the proportion of higher-frequency components, can be used as a quick surrogate measure for acute structural cartilage injury. Taking advantage of this relationship could reduce the time and expense of histological processing needed to morphologically assess cartilage damage, especially for purposes of initial screening when evaluating new impaction protocols.
doi:10.1177/1947603511430706
PMCID: PMC3760429  PMID: 24015324
Articular cartilage; histology; impact testing; impact injury; post-traumatic osteoarthritis
14.  MicroRNA-17-92, a Direct Ap-2α Transcriptional Target, Modulates T-Box Factor Activity in Orofacial Clefting 
PLoS Genetics  2013;9(9):e1003785.
Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.
Author Summary
CL/P are very common birth defects in humans. The genetic mechanism underlying CL/P pathogenesis is poorly understood. MiRs, small non-coding RNAs that function to post-transcriptionally regulate gene expression, have been identified as pivotal modulators of various developmental events and diseases. To date, there is no individual miR or miR cluster that has been identified as functionally essential in mammalian CL/P. Here, we have discovered that deletion of miR-17-92 cluster in mice results in craniofacial malformations including CL/P. Importantly, MIR-17-92 is located on a critical human chromosome region associated with 13q deletion syndrome, a chromosomal disorder that presents with defects including CL/P, suggesting the advantages of our animal model to study human disease. Moreover, our work demonstrated that miR-17-92 cluster directly repressed T-box factors, which have critical functions during craniofacial development. We further showed that miR-17-92 was directly activated by Bmp-signaling and transcription factor AP-2α. Together, our work identified a novel miR-mediated transcriptional network underlying CL/P, providing new insights into craniofacial developmental biology.
doi:10.1371/journal.pgen.1003785
PMCID: PMC3777996  PMID: 24068957
15.  THE POTENTIAL OF HUMAN ALLOGENEIC JUVENILE CHONDROCYTES FOR RESTORATION OF ARTICULAR CARTILAGE 
Background
Donor site morbidity, limited numbers of cells, loss of phenotype during ex vivo expansion, and age-related decline in chondrogenic activity present critical obstacles to the use of autologous chondrocyte implantation for cartilage repair. Chondrocytes from juvenile cadaveric donors may represent an alternative to autologous cells.
Hypothesis/Purpose
The authors hypothesized that juvenile chondrocyte would show stronger and more stable chondrogenic activity than adult cells in vitro and that juvenile cells pose little risk of immunologic incompatibility in adult hosts.
Study Design
Controlled laboratory study.
Methods
Cartilage samples were from juvenile (<13 years old) and adult (> 13 years old) donors. The chondrogenic activity of freshly isolated human articular chondrocytes and of expanded cells after monolayer culture was measured by proteoglycan assay, gene expression analysis, and histology. Lymphocyte proliferation assays were used to assess immunogenic activity.
Results
Proteoglycan content in neocartilage produced by juvenile chondrocytes was 100-fold higher than in neocartilage produced by adult cells. Collagen type II and type IX mRNAs in fresh juvenile chondrocytes were 100- and 700-fold higher, respectively, than in adult chondrocytes. The distributions of collagens II and IX were similar in native juvenile cartilage and in neocartilage made by juvenile cells. Juvenile cells grew significantly faster in monolayer cultures than adult cells (p = 0.002) and proteoglycan levels produced in agarose culture was significantly higher in juvenile cells than in adult cells after multiple passages (p < 0.001). Juvenile chondrocytes did not stimulate lymphocyte proliferation.
Conclusions
These results document a dramatic age related decline in human chondrocyte chondrogenic potential and show that allogeneic juvenile chondrocytes do not stimulate an immunologic response in vivo.
Clinical Relevance
Juvenile human chondrocytes have greater potential to restore articular cartilage than adult cells, and may be transplanted without the fear of rejection, suggesting a new allogeneic approach to restoring articular cartilage in older individuals.
doi:10.1177/0363546510361950
PMCID: PMC3774103  PMID: 20423988
juvenile human chondrocytes; neocartilage; agarose culture; immunogenicity; serial expansion; cartilage repair; chondrocyte transplantation; aging
16.  Differential Roles of CXCL2 and CXCL3 and Their Receptors in Regulating Normal and Asthmatic Airway Smooth Muscle Cell Migration 
Structural cell migration plays a central role in the pathophysiology of several diseases, including asthma. Previously, we established that IL-17–induced (CXCL1, CXCL2, and CXCL3) production promoted airway smooth muscle cell (ASMC) migration, and consequently we sought to investigate the molecular mechanism of CXC-induced ASMC migration. Recombinant human CXCL1, CXCL2, and CXCL3 were used to assess migration of human primary ASMCs from normal and asthmatic subjects using a modified Boyden chamber. Neutralizing Abs or small interfering RNA (siRNA) knockdown and pharmacological inhibitors of PI3K, ERK1/2, and p38 MAPK pathways were used to investigate the receptors and the signaling pathways involved in CXC-induced ASMC migration, respectively. We established the ability of CXCL2 and CXCL3, but not CXCL1, to induce ASMC migration at the tested concentrations using normal ASMCs. We found CXCL2-induced ASMC migration to be dependent on p38 MAPK and CXCR2, whereas CXCL3-induced migration was dependent on p38 and ERK1/2 MAPK pathways via CXCR1 and CXCR2. While investigating the effect of CXCL2 and CXCL3 on asthmatic ASMC migration, we found that they induced greater migration of asthmatic ASMCs compared with normal ones. Interestingly, unlike normal ASMCs, CXCL2- and CXCL3-induced asthmatic ASMC migration was mainly mediated by the PI3K pathway through CXCR1. In conclusion, our results establish a new role of CXCR1 in ASMC migration and demonstrate the diverse mechanisms by which CXCL2 and CXCL3 mediate normal and asthmatic ASMC migration, suggesting that they may play a role in the pathogenesis of airway remodeling in asthma.
doi:10.4049/jimmunol.1203421
PMCID: PMC3748335  PMID: 23904157
17.  Co-cultures of adult and juvenile chondrocytes compared with adult and juvenile chondral fragments: in vitro matrix production 
Background
The use of allogenic juvenile chondrocytes or autologous chondral fragments has shown promising laboratory results for the repair of chondral lesions.
Hypothesis/Purpose
The purpose of the study was to evaluate in vitro the extracellular matrix production of mixed adult/juvenile cultures of both chondrocytes (part 1) and minced cartilage fragments (part 2). The authors hypothesized that juvenile chondrocytes would not affect matrix production when mixed with adult chondrocytes or cartilage fragments.
Study design
Controlled laboratory study.
Methods
Cartilage sources consisted of three adult and three juvenile (human) donors. In part 1, per each donor, juvenile chondrocytes were mixed with adult chondrocytes in five different proportions: 100, 50, 25, 12.5 and 0 %. Three-dimensional cultures in low melt agarose were performed. At 6 weeks, biochemical and histological analyses were performed. In part 2, isolated adult, isolated juvenile, and mixed three-dimensional cultures (1:1) were performed with chondral fragments (<1mm), both with low melt agarose and a hyaluronic acid scaffold. At 2 and 6 weeks, cultures were evaluated with biochemical and histological analyses.
Results
Part 1: biochemical and histological analyses showed that isolated juvenile cultures performed significantly better than mixed and isolated adult cultures. No significant differences were noted between mixed cultures (1:1) and isolated adult cultures. Part 2: biochemical and histological results at 6 weeks showed that mixed cartilage fragment cultures performed better than isolated adult cultures in terms of PG/DNA ratio (p=0.014), percentage of safranin-O positive cells (p=0.012), Bern score (p=0.001), and Collagen type II. No statistical difference was noted between juvenile and mixed cultures.
Conclusion
Extracellular matrix production of juvenile chondrocytes is inhibited by adult chondrocytes. The addition of juvenile cartilage fragments to adult fragments improves matrix production, with a positive interaction between the two sources.
Clinical relevance
Even if the underlying mechanisms are still unknown, this study describes the behavior of juvenile/adult co-cultures using both chondrocytes and cartilage fragments, with potential for new research and clinical applications.
doi:10.1177/0363546511417172
PMCID: PMC3708454  PMID: 21828366
juvenile; adult; chondrocytes; cartilage fragments; co-culture; cartilage repair
18.  OXIDANT CONDITIONING PROTECTS CARTILAGE FROM MECHANICALLY-INDUCED DAMAGE 
Articular cartilage degeneration in osteoarthritis has been linked to abnormal mechanical stresses that are known to cause chondrocyte apoptosis and metabolic derangement in in vitro models. Evidence implicating oxidative damage as the immediate cause of these harmful effects suggests that the anti-oxidant defenses of chondrocytes might influence their tolerance for mechanical injury. Based on evidence that anti-oxidant defenses in many cell types are stimulated by moderate oxidant exposure, we hypothesized that oxidant pre-conditioning would reduce acute chondrocyte death and proteoglycan depletion in cartilage explants after exposure to abnormal mechanical stresses. Porcine cartilage explants were treated every 48 hours with tert-butyl hydrogen peroxide (tBHP) at non-lethal concentrations (25, 100, 250, 500 µM) for a varying number of times (1, 2 or 4) prior to a bout of unconfined axial compression (5 MPa, 1 Hz, 1800 cycles). When compared with untreated controls, tBHP had significant positive effects on post-compression viability, lactate production, and proteoglycan losses. Overall, the most effective regime was 100 µM tBHP applied 4 times. RNA analysis revealed significant effects of 100 µM tBHP on gene expression. Catalase, hypoxia-inducible factor-1alpha (HIF-1α), and glyceraldehyde 6-phosphate dehydrogenase (GAPDH) were significantly increased relative to untreated controls in explants treated 4 times with 100 µM tBHP, a regime that also resulted in a significant decrease in matrix metalloproteinase-3 (MMP-3) expression. These findings demonstrate that repeated exposure of cartilage to sub-lethal concentrations of peroxide can moderate the acute effects of mechanical stress, a conclusion supported by evidence of peroxide-induced changes in gene expression that could render chondrocytes more resistant to oxidative damage.
doi:10.1002/jor.21072
PMCID: PMC3708667  PMID: 20058262
Cartilage; mechanical stress; oxidant
19.  TOWARDS A NEW SPATIAL REPRESENTATION OF BONE REMODELING 
Irregular bone remodeling is associated with a number of bone diseases such as osteoporosis and multiple myeloma. Computational and mathematical modeling can aid in therapy and treatment as well as understanding fundamental biology. Different approaches to modeling give insight into different aspects of a phenomena so it is useful to have an arsenal of various computational and mathematical models. Here we develop a mathematical representation of bone remodeling that can effectively describe many aspects of the complicated geometries and spatial behavior observed.
There is a sharp interface between bone and marrow regions. Also the surface of bone moves in and out, i.e. in the normal direction, due to remodeling. Based on these observations we employ the use of a level-set function to represent the spatial behavior of remodeling. We elaborate on a temporal model for osteoclast and osteoblast population dynamics to determine the change in bone mass which influences how the interface between bone and marrow changes.
We exhibit simulations based on our computational model that show the motion of the interface between bone and marrow as a consequence of bone remodeling. The simulations show that it is possible to capture spatial behavior of bone remodeling in complicated geometries as they occur in vitro and in vivo.
By employing the level set approach it is possible to develop computational and mathematical representations of the spatial behavior of bone remodeling. By including in this formalism further details, such as more complex cytokine interactions and accurate parameter values, it is possible to obtain simulations of phenomena related to bone remodeling with spatial behavior much as in vitro and in vivo. This makes it possible to perform in silica experiments more closely resembling experimental observations.
PMCID: PMC3708700  PMID: 22901065
Bone remodeling; level-set equation; cytokines; osteoclast; osteoblast
20.  Organ-Level Histological and Biomechanical Responses from Localized Osteoarticular Injury in the Rabbit Knee 
The processes of whole-joint osteoarthritis development following localized joint injuries are not well understood. To demonstrate this local-to-global linkage, it was hypothesized that a localized osteoarticular injury in the rabbit knee would not only cause biomechanical and histological abnormalities in the involved compartment, but also concurrent histological changes in the non-involved compartment. Twenty rabbits had an acute osteoarticular injury that involved localized joint incongruity (a 2 mm osteochondral defect created in the weight-bearing area of the medial femoral condyle), while another twenty received control sham surgery. At the time of sacrifice at eight or sixteen weeks post-surgery, the experimental knees were subjected to sagittal-plane laxity measurement, followed by cartilage histo-morphological evaluation using the Mankin score. The immediate effects of defect creation on joint stability and contact mechanics were explored in concomitant rabbit cadaver experimentation. The injured animals had cartilage histological scores significantly higher than in the sham surgery group (p<0.01) on the medial femoral, medial tibial, and lateral femoral surfaces (predominantly on the medial surfaces), accompanied by slight (mean 20%) increase of sagittal-plane laxity. Immediate injury-associated alterations in the medial compartment contact mechanics were also demonstrated. Localized osteoarticular injury in this survival animal model resulted in global joint histological changes.
doi:10.1002/jor.21259
PMCID: PMC3700429  PMID: 20886656
joint injury; osteoarthritis; cartilage histology; rabbit knee; osteochondral defect
21.  Myocardial infarction in a young adult following the consumption of a caffeinated energy drink 
BMJ Case Reports  2011;2011:bcr0220113854.
This report summarises the case of a 19-year-old male, with a history of gastro-oesophageal reflux disease, who presented to hospital with an acute chest pain. An electrocardiographic and biochemical diagnosis of ST elevation myocardial infarction was made; however, subsequent coronary angiography and echocardiography were both normal. In the week preceding the admission, the patient had consumed large quantities of a popular energy drink and the authors believe this may have implicated the development of his coronary event. This is an association that has been suggested previously and this report briefly summarises the evidence supporting the connection.
doi:10.1136/bcr.02.2011.3854
PMCID: PMC3128328  PMID: 22693185
22.  REPEATED MEASUREMENT OF MECHANICAL PROPERTIES IN VIABLE OSTEOCHONDRAL EXPLANTS FOLLOWING A SINGLE BLUNT IMPACT INJURY 
Objective
To develop a method for repeated same-site measurement of mechanical properties suitable for the detection of degenerative changes in a biologically-active explant model after a single blunt impact injury.
Background
Focal blunt impact injuries to articular surfaces lead to local cartilage degeneration and loss of mechanical properties. We employed a repeated measurement methodology to determine variations in mechanical same-site properties before and after injury in living cartilage with the hypothesis that normalization with initial mechanical properties may provide a clearer evaluation of impact effects and improve our understanding of the biologic responses to impact injury.
Materials and Methods
Bovine osteochondral explants were cultured for up to 14 days after impact injury. Indentation tests were performed before and after impact injury to assess relative changes in mechanical properties.
Results
Creep strain increased significantly in impacted explants after 7 days and in both impacted and control explants after 14 days. Further analysis at 14 days revealed decreases in stretch factor β, creep time constant and local compressive modulus.
Conclusion
A repeated measures methodology reliably detected changes in mechanical behavior viable osteochondral explants after a single impact injury.
PMCID: PMC3684069  PMID: 22204121
23.  Automated Objective Scoring of Histologically Apparent Cartilage Degeneration Using a Custom Image Analysis Program 
Histologic assessment of cartilage degradation has traditionally involved semiquantitative techniques, the most commonly utilized being the Mankin scale. Such assessments depend on human observer subjectivity, and thus have drawn criticism on the basis of associated inter- and intraobserver variability. We report a newly developed computational image analysis procedure for fully automated and fully objective assessment of the Mankin scale. Image processing routines were developed in a widely used programming environment (Matlab®) to analyze cartilage degradation. One hundred and twenty-five histology images incorporating a wide range of degradation features were analyzed by the algorithm and by seven observers experienced in cartilage histologic assessment. Based on random effects linear statistical models, the computer program performed well, showing a correlation of 0.88 between its Mankin scores and latent (average of human observers’) image scores. Regarding the four subcomponents of the Mankin scale, computer program correlations with observer scores were best for surface defect and proteoglycan depletion, but less favorable for cellularity and tidemark invasion. While limitations exist with image processing techniques, the new algorithm provides an objective and automated method for analyzing cartilage histology sections, consistent with human observer grading.
doi:10.1002/jor.20779
PMCID: PMC3682491  PMID: 18972361
osteoarthritis; cartilage; image processing; comparative histology
24.  FRS2α-mediated FGF signals suppress premature differentiation of cardiac stem cells through regulating autophagy activity 
Circulation research  2011;110(4):e29-e39.
Rational
Although the fibroblast growth factor (FGF) signaling axis plays important roles in heart development, the molecular mechanism by which the FGF regulates cardiogenesis is not fully understood.
Objective
To investigate the mechanism by which FGF signaling regulates cardiac progenitor cell differentiation.
Methods and results
Using mice with tissue-specific ablation of FGF receptors and FGF receptor substrate 2α (Frs2α) in heart progenitor cells, we demonstrate that that disruption of FGF signaling leads to premature differentiation of cardiac progenitor cells in mice. Using embryoid body (EB) cultures of mouse embryonic stem cells (ESCs), we reveal that FGF signaling promotes mesoderm differentiation in ESCs, but inhibits cardiomyocyte differentiation of the mesoderm cells at later stages. Furthermore, we also report that inhibiting FRS2α-mediated signals increases autophagy and that activating autophagy promotes myocardial differentiation and vice versa.
Conclusions
The results indicate that the FGF/FRS2α-mediated signals prevent premature differentiation of heart progenitor cells through suppressing autophagy. The findings provide the first evidence that autophagy plays a role in heart progenitor differentiation.
doi:10.1161/CIRCRESAHA.111.255950
PMCID: PMC3677753  PMID: 22207710
FGF; autophagy; heart development; second heart field; premature differentiation; heart defect
25.  The Role of Osteocytes in Targeted Bone Remodeling: A Mathematical Model 
PLoS ONE  2013;8(5):e63884.
Until recently many studies of bone remodeling at the cellular level have focused on the behavior of mature osteoblasts and osteoclasts, and their respective precursor cells, with the role of osteocytes and bone lining cells left largely unexplored. This is particularly true with respect to the mathematical modeling of bone remodeling. However, there is increasing evidence that osteocytes play important roles in the cycle of targeted bone remodeling, in serving as a significant source of RANKL to support osteoclastogenesis, and in secreting the bone formation inhibitor sclerostin. Moreover, there is also increasing interest in sclerostin, an osteocyte-secreted bone formation inhibitor, and its role in regulating local response to changes in the bone microenvironment. Here we develop a cell population model of bone remodeling that includes the role of osteocytes, sclerostin, and allows for the possibility of RANKL expression by osteocyte cell populations. We have aimed to give a simple, yet still tractable, model that remains faithful to the underlying system based on the known literature. This model extends and complements many of the existing mathematical models for bone remodeling, but can be used to explore aspects of the process of bone remodeling that were previously beyond the scope of prior modeling work. Through numerical simulations we demonstrate that our model can be used to explore theoretically many of the qualitative features of the role of osteocytes in bone biology as presented in recent literature.
doi:10.1371/journal.pone.0063884
PMCID: PMC3661588  PMID: 23717504

Results 1-25 (130)