Magnetic Resonance Water Proton Relaxation in Protein Solutions and Tissue: T1ρ Dispersion Characterization

Background

Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data.

Principal Findings

Water proton T₁, T₂, and T_1ρ of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T₂ and T_1ρ, but did not change high-field T₁. T_1ρ dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T_1ρ(0) values that closely approximated T₂. The T_1ρ dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T_1ρ dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz.

Conclusions

Proton exchange is a minor pathway for tissue T₁ and T_1ρ relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T_1ρ to T₁.     

Measurement of Myocardial T1ρ with a Motion Corrected,Parametric Mapping Sequence in Humans

Purpose

To develop a robust T_1ρ magnetic resonance imaging (MRI) sequence for assessment of myocardial disease in humans.

Materials and Methods

We developed a breath-held T_1ρ mapping method using a single-shot, T_1ρ-prepared balanced steady-state free-precession (bSSFP) sequence. The magnetization trajectory was simulated to identify sources of T_1ρ error. To limit motion artifacts, an optical flow-based image registration method was used to align T_1ρ images. The reproducibility and accuracy of these methods was assessed in phantoms and 10 healthy subjects. Results are shown in 1 patient with pre-ventricular contractions (PVCs), 1 patient with chronic myocardial infarction (MI) and 2 patients with hypertrophic cardiomyopathy (HCM).

Results

In phantoms, the mean bias was 1.0 ± 2.7 msec (100 msec phantom) and 0.9 ± 0.9 msec (60 msec phantom) at 60 bpm and 2.2 ± 3.2 msec (100 msec) and 1.4 ± 0.9 msec (60 msec) at 80 bpm. The coefficient of variation (COV) was 2.2 (100 msec) and 1.3 (60 msec) at 60 bpm and 2.6 (100 msec) and 1.4 (60 msec) at 80 bpm. Motion correction improved the alignment of T_1ρ images in subjects, as determined by the increase in Dice Score Coefficient (DSC) from 0.76 to 0.88. T_1ρ reproducibility was high (COV < 0.05, intra-class correlation coefficient (ICC) = 0.85–0.97). Mean myocardial T_1ρ value in healthy subjects was 63.5 ± 4.6 msec. There was good correspondence between late-gadolinium enhanced (LGE) MRI and increased T_1ρ relaxation times in patients.

Conclusion

Single-shot, motion corrected, spin echo, spin lock MRI permits 2D T_1ρ mapping in a breath-hold with good accuracy and precision.     

Magnetic Resonance Imaging Is Sensitive to Pathological Amelioration in a Model for Laminin-Deficient Congenital Muscular Dystrophy (MDC1A)

Purpose

To elucidate the reliability of MRI as a non-invasive tool for assessing in vivo muscle health and pathological amelioration in response to Losartan (Angiotensin II Type 1 receptor blocker) in DyW mice (mouse model for Laminin-deficient Congenital Muscular Dystrophy Type 1A).

Methods

Multiparametric MR quantifications along with histological/biochemical analyses were utilized to measure muscle volume and composition in untreated and Losartan-treated 7-week old DyW mice.

Results

MRI shows that DyW mice have significantly less hind limb muscle volume and areas of hyperintensity that are absent in WT muscle. DyW mice also have significantly elevated muscle levels (suggestive of inflammation and edema). Muscle T₂ returned to WT levels in response to Losartan treatment. When considering only muscle pixels without T₂ elevation, DyW T₂ levels are significantly lower than WT (suggestive of fibrosis) whereas Losartan-treated animals do not demonstrate this decrease in muscle T₂. MRI measurements suggestive of elevated inflammation and fibrosis corroborate with increased Mac-1 positive cells as well as increased Picrosirius red staining/COL1a gene expression that is returned to WT levels in response to Losartan.

Conclusions

MRI is sensitive to and tightly corresponds with pathological changes in DyW mice and thus is a viable and effective non-invasive tool for assessing pathological changes.     

Tumor T1 Relaxation Time for Assessing Response to Bevacizumab Anti-Angiogenic Therapy in a Mouse Ovarian Cancer Model

Purpose

To assess whether T₁ relaxation time of tumors may be used to assess response to bevacizumab anti-angiogenic therapy. Procedures: 12 female nude mice bearing subcutaneous SKOV3ip1-LC ovarian tumors were administered bevacizumab (6.25ug/g, n=6) or PBS (control, n=6) therapy twice a week for two weeks. T₁ maps of tumors were generated before, two days, and 2 weeks after initiating therapy. Tumor weight was assessed by MR and at necropsy. Histology for microvessel density, proliferation, and apoptosis was performed.

Results

Bevacizumab treatment resulted in tumor growth inhibition (p<0.04, n=6), confirming therapeutic efficacy. Tumor T₁ relaxation times increased in bevacizumab treated mice 2 days and 2 weeks after initiating therapy (p<.05, n=6). Microvessel density decreased 59% and cell proliferation (Ki67+) decreased 50% in the bevacizumab treatment group (p<.001, n=6), but not apoptosis.

Conclusions

Findings suggest that increased tumor T₁ relaxation time is associated with response to bevacizumab therapy in ovarian cancer model and might serve as an early indicator of response.     

Demineralized Bone Matrix Combined Bone Marrow Mesenchymal Stem Cells,Bone Morphogenetic Protein-2 and Transforming Growth Factor-β3 Gene Promoted Pig Cartilage Defect Repair

Objectives

To investigate whether a combination of demineralized bone matrix (DBM) and bone marrow mesenchymal stem cells (BMSCs) infected with adenovirus-mediated- bone morphogenetic protein (Ad-BMP-2) and transforming growth factor-β3 (Ad-TGF-β3) promotes the repair of the full-thickness cartilage lesions in pig model.

Methods

BMSCs isolated from pig were cultured and infected with Ad-BMP-2(B group), Ad-TGF-β3 (T group), Ad-BMP-2 + Ad-TGF-β3(BT group), cells infected with empty Ad served as a negative group(N group), the expression of the BMP-2 and TGF-β3 were confirmed by immunofluorescence, PCR, and ELISA, the expression of SOX-9, type II collagen(COL-2A), aggrecan (ACAN) in each group were evaluated by real-time PCR at 1w, 2w, 3w, respectively. The chondrogenic differentiation of BMSCs was evaluated by type II collagen at 21d with immunohistochemical staining. The third-passage BMSCs infected with Ad-BMP-2 and Ad-TGF-β3 were suspended and cultured with DBM for 6 days to construct a new type of tissue engineering scaffold to repair full-thickness cartilage lesions in the femur condyles of pig knee, the regenerated tissue was evaluated at 1,2 and 3 months after surgery by gross appearance, H&E, safranin O staining and O''driscoll score.

Results

Ad-BMP-2 and Ad-TGF-β3 (BT group) infected cells acquired strong type II collagen staining compared with Ad-BMP-2 (B group) and Ad-TGF-β3 (T group) along. The Ad-BMP-2 and Ad-TGF-β3 infected BMSCs adhered and propagated well in DBM and the new type of tissue engineering scaffold produced hyaline cartilage morphology containing a stronger type II collagen and safranin O staining, the O''driscoll score was higher than other groups.

Conclusions

The DBM compound with Ad-BMP-2 and Ad-TGF-β3 infected BMSCs scaffold has a good biocompatibility and could well induce cartilage regeneration to repair the defects of joint cartilage. This technology may be efficiently employed for cartilage lesions repair in vivo.     

Comparison of Dixon Sequences for Estimation of Percent Breast Fibroglandular Tissue

Objectives

To evaluate sources of error in the Magnetic Resonance Imaging (MRI) measurement of percent fibroglandular tissue (%FGT) using two-point Dixon sequences for fat-water separation.

Methods

Ten female volunteers (median age: 31 yrs, range: 23–50 yrs) gave informed consent following Research Ethics Committee approval. Each volunteer was scanned twice following repositioning to enable an estimation of measurement repeatability from high-resolution gradient-echo (GRE) proton-density (PD)-weighted Dixon sequences. Differences in measures of %FGT attributable to resolution, T₁ weighting and sequence type were assessed by comparison of this Dixon sequence with low-resolution GRE PD-weighted Dixon data, and against gradient-echo (GRE) or spin-echo (SE) based T₁-weighted Dixon datasets, respectively.

Results

%FGT measurement from high-resolution PD-weighted Dixon sequences had a coefficient of repeatability of ±4.3%. There was no significant difference in %FGT between high-resolution and low-resolution PD-weighted data. Values of %FGT from GRE and SE T₁-weighted data were strongly correlated with that derived from PD-weighted data (r = 0.995 and 0.96, respectively). However, both sequences exhibited higher mean %FGT by 2.9% (p < 0.0001) and 12.6% (p < 0.0001), respectively, in comparison with PD-weighted data; the increase in %FGT from the SE T₁-weighted sequence was significantly larger at lower breast densities.

Conclusion

Although measurement of %FGT at low resolution is feasible, T₁ weighting and sequence type impact on the accuracy of Dixon-based %FGT measurements; Dixon MRI protocols for %FGT measurement should be carefully considered, particularly for longitudinal or multi-centre studies.     

High Resolution T1ρ Mapping of In Vivo Human Knee Cartilage at 7T

Purpose

Spin lattice relaxation time in rotating frame (T1ρ) mapping of human knee cartilage has shown promise in detecting biochemical changes during osteoarthritis. Due to higher field strength, MRI at 7T has advantages in term of SNR compared to clinical MR scanners and this can be used to increase in image resolution. Objective of current study was to evaluate the feasibility of high resolution T1ρ mapping of in vivo human knee cartilage at 7T MR scanner.

Materials and Methods

In this study we have used a T1ρ prepared GRE pulse sequence for obtaining high resolution (in plan resolution  = 0.2 mm²) T1ρ MRI of human knee cartilage at 7T. The effect of a global and localized reference frequency and reference voltage setting on B₀, B₁ and T1ρ maps in cartilage was evaluated. Test-retest reliability results of T1ρ values from asymptomatic subjects as well as T1ρ maps from abnormal cartilage of two human subjects are presented. These results are compared with T1ρ MRI data obtained from 3T.

Results

Our approach enabled acquisition of 3D-T1ρ data within allowed SAR limits at 7T. SNR of cartilage on T1ρ weighted images was greater than 90. Off-resonance effects present in the cartilage B₀, B₁ and T1ρ maps obtained using global shim and reference frequency and voltage setting, were reduced by the proposed localized reference frequency and voltage setting. T1ρ values of cartilage obtained with the localized approach were reproducible. Abnormal knee cartilage showed elevated T1ρ values in affected regions. T1ρ values at 7T were significantly lower (p<0.05) compared to those obtained at 3T.

Conclusion

In summary, by using proposed localized frequency and voltage setting approach, high-resolution 3D-T1ρ maps of in vivo human knee cartilage can be obtained in clinically acceptable scan times (<30 min) and SAR constraints, which provides the ability to characterize cartilage molecular integrity.     

T1 Relaxation Time in Lungs of Asymptomatic Smokers

Purpose

Interest in using T₁ as a potential MRI biomarker of chronic obstructive pulmonary disease (COPD) has recently increased. Since tobacco smoking is the major risk factor for development of COPD, the aim for this study was to examine whether tobacco smoking, pack-years (PY), influenced T₁ of the lung parenchyma in asymptomatic current smokers.

Materials and Methods

Lung T₁ measurements from 35 subjects, 23 never smokers and 12 current smokers were retrospectively analyzed from an institutional review board approved study. All 35 subjects underwent pulmonary function test (PFT) measurements and lung T_1, with similar T₁ measurement protocols. A backward linear model of T₁ as a function of FEV₁, FVC, weight, height, age and PY was tested.

Results

A significant correlation between lung T₁ and PY was found with a negative slope of -3.2 ms/year (95% confidence interval [CI] [-5.8, -0.6], p = 0.02), when adjusted for age and height. Lung T₁ shortens with ageing among all subjects, -4.0 ms/year (95%CI [-6.3, -1.7], p = 0.001), and among the never smokers, -3.7 ms/year (95%CI [-6.0, -1.3], p = 0.003).

Conclusions

A correlation between lung T₁ and PY when adjusted for both age and height was found, and T₁ of the lung shortens with ageing. Accordingly, PY and age can be significant confounding factors when T₁ is used as a biomarker in lung MRI studies that must be taken into account to detect underlying patterns of disease.     

Anti-Apoptotic Effects of 3,3’,5-Triiodo-L-Thyronine in the Liver of Brain-Dead Rats

Background

Thyroid hormone treatment in brain-dead organ donors has been extensively studied and applied in the clinical setting. However, its clinical applicability remains controversial due to a varying degree of success and a lack of mechanistic understanding about the therapeutic effects of 3,3’,5-Triiodo-L-thyronine (T₃). T₃ pre-conditioning leads to anti-apoptotic and pro-mitotic effects in liver tissue following ischemia/reperfusion injury. Therefore, we aimed to study the effects of T₃ pre-conditioning in the liver of brain-dead rats.

Methods

Brain death (BD) was induced in mechanically ventilated rats by inflation of a Fogarty catheter in the epidural space. T₃ (0.1 mg/kg) or vehicle was administered intraperitoneally 2 h prior to BD induction. After 4 h of BD, serum and liver tissue were collected. RT-qPCR, routine biochemistry, and immunohistochemistry were performed.

Results

Brain-dead animals treated with T₃ had lower plasma levels of AST and ALT, reduced Bax gene expression, and less hepatic cleaved Caspase-3 activation compared to brain-dead animals treated with vehicle. Interestingly, no differences in the expression of inflammatory genes (IL-6, MCP-1, IL-1β) or the presence of pro-mitotic markers (Cyclin-D and Ki-67) were found in brain-dead animals treated with T₃ compared to vehicle-treated animals.

Conclusion

T₃ pre-conditioning leads to beneficial effects in the liver of brain-dead rats as seen by lower cellular injury and reduced apoptosis, and supports the suggested role of T₃ hormone therapy in the management of brain-dead donors.     

Alginate Microspheres Containing Temperature Sensitive Liposomes (TSL) for MR-Guided Embolization and Triggered Release of Doxorubicin

Objective

The objective of this study was to develop and characterize alginate microspheres suitable for embolization with on-demand triggered doxorubicin (DOX) release and whereby the microspheres as well as the drug releasing process can be visualized in vivo using MRI.

Methods and Findings

For this purpose, barium crosslinked alginate microspheres were loaded with temperature sensitive liposomes (TSL/TSL-Ba-ms), which release their payload upon mild hyperthermia. These TSL contained DOX and [Gd(HPDO3A)(H₂O)], a T₁ MRI contrast agent, for real time visualization of the release. Empty alginate microspheres crosslinked with holmium ions (T₂* MRI contrast agent, Ho-ms) were mixed with TSL-Ba-ms to allow microsphere visualization. TSL-Ba-ms and Ho-ms were prepared with a homemade spray device and sized by sieving. Encapsulation of TSL in barium crosslinked microspheres changed the triggered release properties only slightly: 95% of the loaded DOX was released from free TSL vs. 86% release for TSL-Ba-ms within 30 seconds in 50% FBS at 42°C. TSL-Ba-ms (76 ± 41 μm) and Ho-ms (64 ± 29 μm) had a comparable size, which most likely will result in a similar in vivo tissue distribution after an i.v. co-injection and therefore Ho-ms can be used as tracer for the TSL-Ba-ms. MR imaging of a TSL-Ba-ms and Ho-ms mixture (ratio 95:5) before and after hyperthermia allowed in vitro and in vivo visualization of microsphere deposition (T₂*-weighted images) as well as temperature-triggered release (T₁-weighted images). The [Gd(HPDO3A)(H₂O)] release and clusters of microspheres containing holmium ions were visualized in a VX₂ tumor model in a rabbit using MRI.

Conclusions

In conclusion, these TSL-Ba-ms and Ho-ms are promising systems for real-time, MR-guided embolization and triggered release of drugs in vivo.     

Allogeneic Bone Marrow Transplant from MRL/MpJ Super-Healer Mice Does Not Improve Articular Cartilage Repair in the C57Bl/6 Strain

Background

Articular cartilage has been the focus of multiple strategies to improve its regenerative/ repair capacity. The Murphy Roths Large (MRL/MpJ) “super-healer” mouse demonstrates an unusual enhanced regenerative capacity in many tissues and provides an opportunity to further study endogenous cartilage repair. The objective of this study was to test whether the super-healer phenotype could be transferred from MRL/MpJ to non-healer C57Bl/6 mice by allogeneic bone marrow transplant.

Methodology

The healing of 2mm ear punches and full thickness cartilage defects was measured 4 and 8 weeks after injury in control C57Bl/6 and MRL/MpJ “super-healer” mice, and in radiation chimeras reconstituted with bone marrow from the other mouse strain. Healing was assessed using ear hole diameter measurement, a 14 point histological scoring scale for the cartilage defect and an adapted version of the Osteoarthritis Research Society International scale for assessment of osteoarthritis in mouse knee joints.

Principal Findings

Normal and chimeric MRL mice showed significantly better healing of articular cartilage and ear wounds along with less severe signs of osteoarthritis after cartilage injury than the control strain. Contrary to our hypothesis, however, bone marrow transplant from MRL mice did not confer improved healing on the C57Bl/6 chimeras, either in regards to ear wound healing or cartilage repair.

Conclusion and Significance

The elusive cellular basis for the MRL regenerative phenotype still requires additional study and may possibly be dependent on additional cell types external to the bone marrow.     

SERPINE2 Inhibits IL-1α-Induced MMP-13 Expression in Human Chondrocytes: Involvement of ERK/NF-κB/AP-1 Pathways

Objectives

Osteoarthritis (OA) is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs) in chondrocytes, contributing thus to the extracellular matrix (ECM) degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2), under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored.

Methods

SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis.

Results

Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1.

Conclusions

Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.     

The Effect of Protease Inhibitors on the Induction of Osteoarthritis-Related Biomarkers in Bovine Full-Depth Cartilage Explants

Objective

The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors.

Methods

Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF-α) and Oncostatin M (OSM) were cultured for 21 days with or without a number of inhibitors targeting different types of proteases. Monoclonal antibodies were raised against the active sites of ADAMTS-4, -5, MMP-9 and -13, and 4 ELISAs were developed and technically validated. In addition, the established AGNxI (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explants-conditioned media.

Results

We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E³⁷³-³⁷⁴A bond cleavage in aggrecan in this setting; and iii) The compensatory mechanism could be triggered following the blockage of the enzyme caused by inhibitors.

Conclusions

ADAMTS-4 appeared to be the major protease for the generation of ³⁷⁴ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage breakdown.     

Simultaneous Determination of Glass Transition Temperatures of Several Polymers

Aims

A simple and easy optical method is proposed for the determination of glass transition temperature (T_g) of polymers.

Methods & Results

T_g was determined using the technique of microsphere imaging to monitor the variation of the refractive index of polymer microsphere as a function of temperature. It was demonstrated that the method can eliminate most thermal lag and has sensitivity about six fold higher than the conventional method in T_g determination. So the determined T_g is more accurate and varies less with cooling/heating rate than that obtained by conventional methods. The most attractive character of the method is that it can simultaneously determine the T_g of several polymers in a single experiment, so it can greatly save experimental time and heating energy.

Conclusion

The method is not only applicable for polymer microspheres, but also for the materials with arbitrary shapes. Therefore, it is expected to be broadly applied to different fundamental researches and practical applications of polymers.     

Deletion of Dual Specificity Phosphatase 1 Does Not Predispose Mice to Increased Spontaneous Osteoarthritis

Background

Osteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse).

Results

Utilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups.

Conclusion

Loss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA.     

Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

Objective

Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.

Methods

For in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.

Results

In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.

Conclusion

Cartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.     

Transient anabolic effects accompany epidermal growth factor receptor signal activation in articular cartilage in vivo

Introduction

Signals from the epidermal growth factor receptor (EGFR) have typically been considered to provide catabolic activities in articular cartilage, and accordingly have been suggested to have a causal role in osteoarthritis progression. The aim of this study was to determine in vivo roles for endogenous EGFR signal activation in articular cartilage.

Methods

Transgenic mice with conditional, limb-targeted deletion of the endogenous intracellular EGFR inhibitor Mig-6 were generated using CreLoxP (Mig-6-flox; Prx1Cre) recombination. Histology, histochemical staining and immunohistochemistry were used to confirm activation of EGFR signaling in the articular cartilage and joints, and to analyze phenotypic consequences of Mig-6 loss on articular cartilage morphology, proliferation, expression of progenitor cell markers, presence of chondrocyte hypertrophy and degradation of articular cartilage matrix.

Results

The articular cartilage of Mig-6-conditional knockout (Mig-6-cko) mice was dramatically and significantly thicker than normal articular cartilage at 6 and 12 weeks of age. Mig-6-cko articular cartilage contained a population of chondrocytes in which EGFR signaling was activated, and which were three to four times more proliferative than normal Mig-6-flox articular chondrocytes. These cells expressed high levels of the master chondrogenic regulatory factor Sox9, as well as high levels of putative progenitor cell markers including superficial zone protein (SZP), growth and differentiation factor-5 (GDF-5) and Notch1. Expression levels were also high for activated β-catenin and the transforming growth factor beta (TGF-β) mediators phospho-Smad2/3 (pSmad2/3). Anabolic effects of EGFR activation in articular cartilage were followed by catabolic events, including matrix degradation, as determined by accumulation of aggrecan cleavage fragments, and onset of hypertrophy as determined by type × collagen expression. By 16 weeks of age, the articular cartilage of Mig-6-cko knees was no longer thickened and was degenerating.

Conclusions

These results demonstrate unexpected anabolic effects of EGFR signal activation in articular cartilage, and suggest the hypothesis that these effects may promote the expansion and/or activity of an endogenous EGFR-responsive cell population within the articular cartilage.     

Scan-Rescan Reproducibility of High Resolution Magnetic Resonance Imaging of Atherosclerotic Plaque in the Middle Cerebral Artery

Purpose

To evaluate the scan-rescan reproducibility of high-resolution magnetic resonance imaging (MRI) of middle cerebral artery (MCA) plaque, and calculate the number of subjects needed for future longitudinal clinical studies.

Material and Methods

Twenty two patients with MCA plaque were scanned twice by a T₂-weighted fast-spin-echo sequence at 3T. Areas and volumes of MCA lumen, total vessel and plaque were quantified and compared between two repeated scans. Agreement and measurement error was quantified by intraclass correlation coefficient (ICC) and coefficient of variance (CV) as defined by standard deviation (SD) of pair wise difference / mean. Sample size needed to detect 5% to 20% changes in area/volume was calculated using 80% power and 5% significance level.

Results

There was no significant different between the area and volume measurements of two repeated scans (p>0.05) with good agreement (ICC range 0.97–0.98 for area and 0.99 for volume). Relatively small measurement errors were observed with CVs range 6.1%-11.8% for area quantification and 4.9%-8.0% for volume quantification. Volume measurements tended to have 19.7% to 32.2% smaller CVs compared with area measurements. Sample size calculation showed a group of 47 patients was sufficient to detect 5% to 10% changes in MCA area/volume.

Conclusion

High resolution MRI is feasible for quantifying intracranial plaque area and volume in longitudinal clinical studies with low scan-rescan variability. Volume measurement tends to be more reproducible compared with area measurements.     

Magnetic Resonance Assessment of Hypertrophic and Pseudo-Hypertrophic Changes in Lower Leg Muscles of Boys with Duchenne Muscular Dystrophy and Their Relationship to Functional Measurements

Introduction

The primary objectives of this study were to evaluate contractile and non-contractile content of lower leg muscles of boys with Duchenne muscular dystrophy (DMD) and determine the relationships between non-contractile content and functional abilities.

Methods

Lower leg muscles of thirty-two boys with DMD and sixteen age matched unaffected controls were imaged. Non-contractile content, contractile cross sectional area and non-contractile cross sectional area of lower leg muscles (tibialis anterior, extensor digitorum longus, peroneal, medial gastrocnemius and soleus) were assessed by magnetic resonance imaging (MRI). Muscle strength, timed functional tests and the Brooke lower extremity score were also assessed.

Results

Non-contractile content of lower leg muscles (peroneal, medial gastrocnemius, and soleus) was significantly greater than control group (p<0.05). Non-contractile content of lower leg muscles correlated with Brooke score (r_s = 0.64-0.84) and 30 feet walk (r_s = 0.66-0.80). Dorsiflexor (DF) and plantarflexor (PF) specific torque was significantly different between the groups.

Discussion

Overall, non-contractile content of the lower leg muscles was greater in DMD than controls. Furthermore, there was an age dependent increase in contractile content in the medial gastrocnemius of boys with DMD. The findings of this study suggest that T₁ weighted MR images can be used to monitor disease progression and provide a quantitative estimate of contractile and non-contractile content of tissue in children with DMD.     

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism

Introduction

Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.

Methods

We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.

Results

Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻⁷ M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.

Conclusions

We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.