Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies.

The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEK[G3S]3WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co2+ ions. The TetrHis motif contains four 6 residues-long ß-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co2+ on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.

The effectiveness and mechanical properties of chemotherapy-impregnated cement in Ewing sarcoma.

Bone cement is often used in the surgical treatment of Ewing sarcoma (ES). Chemotherapy-impregnated cement (CIC) has never been tested in slowing ES growth. The purpose of the study is to determine if CIC can decrease cell proliferation, and to assess changes in the mechanical qualities of the cement. Chemotherapeutic agents including doxorubicin, cisplatin, etoposide, and SF2523 were mixed with bone cement. ES cells were plated and exposed to cell growth media that had contained CIC or regular bone cement (RBC) as a control, and cell proliferation assays were performed daily for 3 days. Mechanical testing on RBC and CIC was also performed. There was a significant decrease (p < 0.001) in cell proliferation among all cells treated with CIC compared to cells treated with RBC by 48 h postexposure. Additionally, there was a synergistic effectiveness of the CIC noted when multiple antineoplastic agents were combined. Three-point bending tests did not reveal substantial reductions in tolerated maximum bending load and maximal displacement at maximal bending load between CIC and RBC. Statement of Clinical Significance: CIC does appear to be effective at decreasing cell growth and does not appear to substantially alter the mechanical properties of the cement.

Spaceflight-Associated Vascular Remodeling and Gene Expression in Mouse Calvaria.

Astronauts suffer from a loss of bone mass at a rate of 1.5% per month from lower regions of the body during the course of long-duration (>30 days) spaceflight, a phenomenon that poses important risks for returning crew. Conversely, a gain in bone mass may occur in non-load bearing regions of the body as related to microgravity-induced cephalad fluid shift. Representing non-load bearing regions with mouse calvaria and leveraging the STS-131 (15-day) and BION-M1 (30-day) flights, we examined spatial and temporal calvarial vascular remodeling and gene expression related to microgravity exposure compared between spaceflight (SF) and ground control (GC) cohorts. We examined parasagittal capillary numbers and structures in calvaria from 16 to 23 week-old C57BL/6 female mice (GC, n = 4; SF, n = 5) from STS-131 and 19-20 week-old C57BL/6 male mice (GC, n = 6; SF, n = 6) from BION-M1 using a robust isolectin-IB4 vessel marker. We found that the vessel diameter reduces significantly in mice exposed to 15 days of spaceflight relative to control. Capillarization increases by 30% (SF vs. GC, p = 0.054) in SF mice compared to GC mice. The vessel numbers and diameter remain unchanged in BION-M1 mice calvarial section. We next analyzed the parietal pro-angiogenic (VEGFA) and pro-osteogenic gene (BMP-2, DMP1, RUNX2 and OCN) expression in BION-M1 mice using quantitative RT-PCR. VEGFA gene expression increased 15-fold while BMP-2 gene expression increased 11-fold in flight mice compared to GC. The linkage between vascular morphology and gene expression in the SF conditions suggests that angiogenesis may be important in the regulation of pathological bone growth in non-weight bearing regions of the body. Short-duration microgravity-mediated bone restructuring has implications in planning effective countermeasures for long-duration flights and extraterrestrial human habitation.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

An automated platform for structural analysis of membrane proteins through serial crystallography.

Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.

7TM proteins are not necessarily GPCRs.

In this review article, we summarize the current knowledge on a large and diverse superfamily of seven-pass transmembrane proteins functionally independent from the GPCR superfamily. We include the newest research findings about their physiological roles and their mechanism of action. In particular, we concentrate on the structural basis for the newly discovered amide hydrolase activity, with a focus on adiponectin receptors for which structures are available. Finally, we discuss the remaining challenges in understanding the activation and signaling of these intramembrane proteins and suggest how regulation of the amide hydrolase activity may help in development of new therapeutic agents.

Ultrashort Time to Echo Magnetic Resonance Evaluation of Calcium Pyrophosphate Crystal Deposition in Human Menisci.

OBJECTIVES: In human menisci, we aimed to investigate whether calcium pyrophosphate crystal deposition (CPPD) affects biomechanical and quantitative MR properties, and their zonal distribution. MATERIALS AND METHODS: From 9 cadaveric knees, sectioned triangular meniscus pieces were harvested. Samples were classified into "normal" or "CPPD" groups based upon visual inspection. Micro computed tomography scan verified CPPD. Using magnetic resonance imaging, ultrashort echo time (UTE) T2* and spin echo (SE) T2, quantitative values in 3 zones (red, red-white, and white) were determined. Using biomechanical test, indentation forces in the same zones were determined. Effects of CPPD and meniscal zone on indentation force and quantitative MR values were compared. RESULTS: On UTE MRI scans, CPPD-affected menisci exhibited punctate dark regions, found mostly (92%) in avascular white and red-white zones. Indentation forces were significantly higher for CPPD samples in the red-white (all P < 0.02) and white (all P < 0.004) zones but not in the vascular red zone (all P > 0.2). Similarly, UTE T2* red zone values were similar between both groups (~6.6 milliseconds, P = 0.8), whereas in the red-white and white zones, CPPD samples had significantly lower values (~5.1 milliseconds, P = 0.005 to 0.007). In contrast, SE T2 values showed no difference with CPPD (P = 0.12 to 0.16). UTE T2*, but not SE T2, correlated significantly with indentation force (R = -0.29, P = 0.009). CONCLUSIONS: Dark CPP deposits were detectable on UTE images featuring high signal intensity from surrounding meniscal tissue. Preliminary results indicate that CPP deposits were almost exclusively found in the avascular zones. Compared with normal, CPPD menisci featured higher indentation stiffness and lower UTE T2* values in the affected zones.

Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy.

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.

Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

From the international space station to the clinic: how prolonged unloading may disrupt lumbar spine stability.

BACKGROUND CONTEXT: Prolonged microgravity exposure is associated with localized low back pain and an elevated risk of post-flight disc herniation. Although the mechanisms by which microgravity impairs the spine are unclear, they should be foundational for developing in-flight countermeasures for maintaining astronaut spine health. Because human spine anatomy has adapted to upright posture on Earth, observations of how spaceflight affects the spine should also provide new and potentially important information on spine biomechanics that benefit the general population. PURPOSE: This study compares quantitative measures of lumbar spine anatomy, health, and biomechanics in astronauts before and after 6 months of microgravity exposure on board the International Space Station (ISS). STUDY DESIGN: This is a prospective longitudinal study. SAMPLE: Six astronaut crewmember volunteers from the National Aeronautics and Space Administration (NASA) with 6-month missions aboard the ISS comprised our study sample. OUTCOME MEASURES: For multifidus and erector spinae at L3-L4, measures include cross-sectional area (CSA), functional cross-sectional area (FCSA), and FCSA/CSA. Other measures include supine lumbar lordosis (L1-S1), active (standing) and passive (lying) flexion-extension range of motion (FE ROM) for each lumbar disc segment, disc water content from T2-weighted intensity, Pfirrmann grade, vertebral end plate pathology, and subject-reported incidence of chronic low back pain or disc injuries at 1-year follow-up. METHODS: 3T magnetic resonance imaging and dynamic fluoroscopy of the lumbar spine were collected for each subject at two time points: approximately 30 days before launch (pre-flight) and 1 day following 6 months spaceflight on the ISS (post-flight). Outcome measures were compared between time points using paired t tests and regression analyses. RESULTS: Supine lumbar lordosis decreased (flattened) by an average of 11% (p=.019). Active FE ROM decreased for the middle three lumbar discs (L2-L3: -22.1%, p=.049; L3-L4: -17.3%, p=.016; L4-L5: -30.3%, p=.004). By contrast, no significant passive FE ROM changes in these discs were observed (p>.05). Disc water content did not differ systematically from pre- to post-flight. Multifidus and erector spinae changed variably between subjects, with five of six subjects experiencing an average decrease 20% for FCSA and 8%-9% for CSA in both muscles. For all subjects, changes in multifidus FCSA strongly correlated with changes in lordosis (r2=0.86, p=.008) and active FE ROM at L4-L5 (r2=0.94, p=.007). Additionally, changes in multifidus FCSA/CSA correlated with changes in lordosis (r2=0.69, p=.03). Although multifidus-associated changes in lordosis and ROM were present among all subjects, only those with severe, pre-flight end plate irregularities (two of six subjects) had post-flight lumbar symptoms (including chronic low back pain or disc herniation). CONCLUSIONS: We observed that multifidus atrophy, rather than intervertebral disc swelling, associated strongly with lumbar flattening and increased stiffness. Because these changes have been previously linked with detrimental spine biomechanics and pain in terrestrial populations, when combined with evidence of pre-flight vertebral end plate insufficiency, they may elevate injury risk for astronauts upon return to gravity loading. Our results also have implications for deconditioned spines on Earth. We anticipate that our results will inform new astronaut countermeasures that target the multifidus muscles, and research on the role of muscular stability in relation to chronic low back pain and disc injury.

Operative findings and complications associated with adjunctive hip arthroscopy in 95 hips undergoing periacetabular osteotomy.

PURPOSE: to report our initial experience with adjunctive hip arthroscopy and periacetabular osteotomy (PAO). METHODS: Retrospective review of patients who underwent PAO and ipsilateral hip arthroscopy between 2003 and 2013. Indications for arthroscopy were mechanical symptoms and/or positive magnetic resonance imaging to suggest intra-articular pathology including chondrolabral lesions, ligamentum teres tears and hypertrophy, and synovitis. Preoperative Tönnis arthritis grades, age, sex, operative findings and treatment, complications, and, if available, hip survival were recorded. RESULTS: Of 78 patients (95 hips) included, 64 (82%) were female and 14 (18%) male, average age was 30.6 (14-63) years. Tönnis grades were 0 in 40 hips (42%), 1 in 45 hips (47%), and 2 in 10 hips (11%). No Tönnis 3 hips were included. 84% of hips demonstrated labral pathology. 92% had chondromalacia, which was severe enough in 4 patients to warrant cancellation of PAO; all 4 have subsequently required total hip replacement at short-term follow-up. Labral debridement was performed in 73 hips and refixation in 7. 5 postoperative complications occurred - none major, including 1 fluid extravasation, 1 case of heterotopic ossification, and 3 transient neuropraxias. CONCLUSIONS: Intraarticular pathology is highly prevalent in patients undergoing PAO with mechanical symptoms, and can be safely managed by adjunctive arthroscopy. Complications were minimal in our series and are comparable to reports of PAO without arthroscopy. Arthroscopy can treat pathology that is inaccessible during standalone PAO and permits avoidance of routine arthrotomy, surgical dislocation, rectus release, and futile PAO in cases with severe articular damage.

An improved process for the production of highly purified recombinant thaumatin tagged-variants.

The sweetest tasting molecule known is the protein thaumatin, first isolated from the katemfe fruit, Thaumatococcus daniellii. Thaumatin is used in the food and beverage industry as a low-calorie sugar substitute. Thaumatin interacts with taste receptors in the oral cavity eliciting a persistent sweet taste and a bitter, liquorice flavor. Recombinant thaumatin was expressed in Pichia pastoris and through a co-expression strategy with a molecular chaperone, yields of one engineered thaumatin variant increased by greater than two-fold. A detailed purification strategy for thaumatin is reported resulting in a homogenous sample recovered at a yield of 42%. The recombinant thaumatins were extensively characterised using size exclusion chromatography for homogeneity, reversed-phase HPLC for purity (99%), peptide digest LC-MS/MS for sequence determination, and circular dichroism and tryptophan fluorescence spectroscopies for conformational characterisation. These new thaumatin variants are amenable for bioconjugation, providing chemical biology tools for thaumatin:taste receptor interaction studies.

A bacterial chloroform reductive dehalogenase: purification and biochemical characterization.

We report herein the purification of a chloroform (CF)-reducing enzyme, TmrA, from the membrane fraction of a strict anaerobe Dehalobacter sp. strain UNSWDHB to apparent homogeneity with an approximate 23-fold increase in relative purity compared to crude lysate. The membrane fraction obtained by ultracentrifugation was solubilized in Triton X-100 in the presence of glycerol, followed by purification by anion exchange chromatography. The molecular mass of the purified TmrA was determined to be 44.5 kDa by SDS-PAGE and MALDI-TOF/TOF. The purified dehalogenase reductively dechlorinated CF to dichloromethane in vitro with reduced methyl viologen as the electron donor at a specific activity of (1.27 ± 0.04) × 103 units mg protein-1 . The optimum temperature and pH for the activity were 45°C and 7.2, respectively. The UV-visible spectrometric analysis indicated the presence of a corrinoid and two [4Fe-4S] clusters, predicted from the amino acid sequence. This is the first report of the production, purification and biochemical characterization of a CF reductive dehalogenase.

Lumbar Spine Paraspinal Muscle and Intervertebral Disc Height Changes in Astronauts After Long-Duration Spaceflight on the International Space Station.

STUDY DESIGN: Prospective case series. OBJECTIVE: Evaluate lumbar paraspinal muscle (PSM) cross-sectional area and intervertebral disc (IVD) height changes induced by a 6-month space mission on the International Space Station. The long-term objective of this project is to promote spine health and prevent spinal injury during space missions and here on Earth. SUMMARY OF BACKGROUND DATA: National Aeronautics and Space Administration (NASA) crewmembers have a 4.3 times higher risk of herniated IVDs, compared with the general and military aviator populations. The highest risk occurs during the first year after a mission. Microgravity exposure during long-duration spaceflights results in approximately 5 cm lengthening of body height, spinal pain, and skeletal deconditioning. How the PSMs and IVDs respond during spaceflight is not well described. METHODS: Six NASA crewmembers were imaged supine with a 3 Tesla magnetic resonance imaging. Imaging was conducted preflight, immediately postflight, and then 33 to 67 days after landing. Functional cross-sectional area (FCSA) measurements of the PSMs were performed at the L3-4 level. FCSA was measured by grayscale thresholding within the posterior lumbar extensors to isolate lean muscle on T2-weighted scans. IVD heights were measured at the anterior, middle, and posterior sections of all lumbar levels. Repeated measures analysis of variance was used to determine significance at P < 0.05, followed by post-hoc testing. RESULTS: Paraspinal lean muscle mass, as indicated by the FCSA, decreased from 86% of the total PSM cross-sectional area down to 72%, immediately after the mission. Recovery of 68% of the postflight loss occurred during the next 6 weeks, still leaving a significantly lower lean muscle fractional content compared with preflight values. In contrast, lumbar IVD heights were not appreciably different at any time point. CONCLUSION: The data reveal lumbar spine PSM atrophy after long-duration spaceflight. Some FCSA recovery was seen with 46 days postflight in a terrestrial environment, but it remained incomplete compared with preflight levels. LEVEL OF EVIDENCE: 4.

24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats.

Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] accumulates in articular cartilage following injection of [3H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1ß with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1ß in a dose-dependent manner, and its effect was partially mediated through the TGF-ß1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis.

Thickness of the Meniscal Lamellar Layer: Correlation with Indentation Stiffness and Comparison of Normal and Abnormally Thick Layers by Using Multiparametric Ultrashort Echo Time MR Imaging.

Purpose To determine the relationship between lamellar layer thickness on ultrashort echo time (UTE) magnetic resonance (MR) images and indentation stiffness of human menisci and to compare quantitative MR imaging values between two groups with normal and abnormally thick lamellar layers. Materials and Methods This was a HIPAA-compliant, institutional review board-approved study. Nine meniscal pieces were obtained from seven donors without gross meniscal pathologic results (mean age, 57.4 years ± 14.5 [standard deviation]). UTE MR imaging and T2, UTE T2*, and UTE T1ρ mapping were performed. The presence of abnormal lamellar layer thickening was determined and thicknesses were measured. Indentation testing was performed. Correlation between the thickness and indentation stiffness was assessed, and mean quantitative MR imaging values were compared between the groups. Results Thirteen normal lamellar layers had mean thickness of 232 µm ± 85 and indentation peak force of 1.37 g ± 0.87. Four abnormally thick lamellar layers showed mean thickness of 353.14 µm ± 98.36 and peak force 0.72 g ± 0.31. In most cases, normal thicknesses showed highly positive correlation with the indentation peak force (r = 0.493-0.912; P < .001 to .05). However, the thickness in two abnormal lamellar layers showed highly negative correlation (r = -0.90, P < .001; and r = -0.23, P = .042) and no significant correlation in the others. T2, UTE T2*, and UTE T1ρ values in abnormally thick lamellar layers were increased compared with values in normal lamellar layers, although only the UTE T2* value showed significant difference (P = .010). Conclusion Variation of lamellar layer thickness in normal human menisci was evident on two-dimensional UTE images. In normal lamellar layers, thickness is highly and positively correlated with surface indentation stiffness. UTE T2* values may be used to differentiate between normal and abnormally thickened lamellar layers. (©) RSNA, 2016.

High-resolution morphologic and ultrashort time-to-echo quantitative magnetic resonance imaging of the temporomandibular joint.

OBJECTIVE: To implement high-resolution morphologic and quantitative magnetic resonance imaging (MRI) of the temporomandibular joint (TMJ) using ultrashort time-to-echo (UTE) techniques in cadavers and volunteers. METHODS: This study was approved by the institutional review board. TMJs of cadavers and volunteers were imaged on a 3-T MR system. High-resolution morphologic and quantitative sequences using conventional and UTE techniques were performed in cadaveric TMJs. Morphologic and UTE quantitative sequences were performed in asymptomatic and symptomatic volunteers. RESULTS: Morphologic evaluation demonstrated the TMJ structures in open- and closed-mouth position. UTE techniques facilitated the visualization of the disc and fibrocartilage. Quantitative UTE MRI was successfully performed ex vivo and in vivo, reflecting the degree of degeneration. There was a difference in the mean UTE T2* values between asymptomatic and symptomatic volunteers. CONCLUSIONS: MRI evaluation of the TMJ using UTE techniques allows characterization of the internal structure and quantification of the MR properties of the disc. Quantitative UTE MRI can be performed in vivo with short scan times.

Ultrashort echo time magnetization transfer (UTE-MT) imaging of cortical bone.

Magnetization transfer (MT) imaging is one way to indirectly assess pools of protons with fast transverse relaxation. However, conventional MT imaging sequences are not applicable to short T2 tissues such as cortical bone. Ultrashort echo time (UTE) sequences with TE values as low as 8 µs can detect signals from different water components in cortical bone. In this study we aim to evaluate two-dimensional UTE-MT imaging of cortical bone and its application in assessing cortical bone porosity as measured by micro-computed tomography (µCT) and biomechanical properties. In total, 38 human cadaveric distal femur and proximal tibia bones were sectioned to produce 122 rectangular pieces of cortical bone for quantitative UTE-MT MR imaging, µCT, and biomechanical testing. Off-resonance saturation ratios (OSRs) with a series of MT pulse frequency offsets (Δf) were calculated and compared with porosity assessed with µCT, as well as elastic (modulus, yield stress, and strain) and failure (ultimate stress, failure strain, and energy) properties, using Pearson correlation and linear regression. A moderately strong negative correlation was observed between OSR and µCT porosity (R(2) = 0.46-0.51), while a moderate positive correlation was observed between OSR and yield stress (R(2) = 0.25-0.30) and failure stress (R(2) = 0.31-0.35), and a weak positive correlation (R(2) = 0.09-0.12) between OSR and Young's modulus at all off-resonance saturation frequencies. OSR determined with the UTE-MT sequence provides quantitative information on cortical bone and is sensitive to µCT porosity and biomechanical function.

Radiofrequency stimulation for potential healing of meniscal injuries in the avascular zone.

We conducted this study to evaluate the effect of radiofrequency (RF) stimulation with suture repair on the healing of tears in the meniscal white-white zone. Fifty-four New Zealand white rabbits underwent surgically induced meniscal injuries within the white-white region. RF was applied using a 0.8-mm TOPAZ MicroDebrider RF wand (ArthroCare) at level 4 for 500 milliseconds. Rabbits were sacrificed at 28 and 84 days for gross and histologic analysis by 3 blinded observers and at 9, 28, and 84 days for biochemical examination. Biochemical analyses included evaluation of cell proliferation (3H-thymidine), as well as mitogenic (IGF-1, bFGF) and angiogenic (VEGF, αV) factors. Of specimens repaired with RF combined with suture, 19 (58%) showed a degree of gross morphologic and histologic healing. No significant healing was seen in specimens with either no repair or repair with suture alone. We observed a 40% increase in cellular proliferation when RF supplementation was used (P<.05). With regards to mitogenic and angiogenic markers (IGF-1, bFGF, VEGF, and αV), there was a significant increase in groups treated with RF at 9 and 28 days (P>0.05). RF supplementation of avascular zone meniscal repairs may lead to an increased healing response.

Use of platelet-rich plasma to enhance tendon function and cellularity.

Clinical studies have shown inconsistent healing with subjective improvement after use of platelet-rich plasma (PRP) for tendinosis and partial tendon tears. We conducted a study to assess changes after injecting PRP into an intact rabbit patellar tendon (PT) model. In the study group (n = 10), an incision was made over the PT and PRP was injected into multiple sites on the PT. The control group (n = 8) was injected with saline. PTs were harvested 7 and 28 days after injection. Hematoxylin-eosin staining showed hypercellularity in the PRP group at 7 days, but the effect was not as marked at 28 days. At 7 days, polarized microscopy showed increased crimp density of collagen in the PRP group, compared with the control group, demonstrating up-regulation in collagen matrix. Cellular proliferation measured by tritiated thymidine was also significantly increased (P = .02) in the PRP group, compared with the control group, but the difference was not as significant at 28 days. At 7 and 28 days, there were no significant changes in basic fibroblast growth factor, insulin-like growth factor, vascular endothelial growth factor, or platelet-derived growth factor with 2B chains. Injection of PRP into rabbit PT enhances collagen remodeling and hypercellularity with increased metabolic activity, which could have a positive effect on healing.