Hydraulic Joint Function and Osteoarthritis.

¼ This review of bone perfusion work introduces a new field of subchondral physiology.¼ Intraosseous pressure (IOP) measured through an intraosseous needle reflects conditions only at the needle tip rather than being a constant for the whole bone.¼ Measurements of IOP in vitro and in vivo, with and without proximal vascular occlusion, show that at rest, bone is perfused at normal physiological pressures.¼ A subtraction perfusion range or bandwidth at the needle tip offers a better measure of bone health than a single IOP.¼ With ordinary loads, very great subchondral pressures are generated.¼ Subchondral tissues are relatively delicate but are microflexible with bone fat being essentially liquid at body temperature.¼ Collectively, the subchondral tissues transmit load mainly through hydraulic pressure to the trabeculae and cortical shaft.¼ White vascular marks on normal magnetic resonance imaging scans are present but are lost in early osteoarthritis.¼ Histological studies confirm the presence of those vascular marks and also choke valves capable of supporting hydraulic pressure load transmission.¼ Osteoarthritis seems to be at least partly a vasculomechanical disease.¼ Understanding subchondral physiology will be key to better classification, control, prognosis, and treatment of osteoarthritis.

Walking on water: subchondral vascular physiology explains how joints work and why they become osteoarthritic.

This review of bone perfusion introduces a new field of joint physiology, important in understanding osteoarthritis. Intraosseous pressure (IOP) reflects conditions at the needle tip rather than being a constant for the whole bone. Measurements of IOP in vitro and in vivo, with and without proximal vascular occlusion confirm that cancellous bone is perfused at normal physiological pressures. Alternate proximal vascular occlusion may be used to give a perfusion range or bandwidth at the needle tip more useful than a single IOP measure. Bone fat is essentially liquid at body temperature. Subchondral tissues are relatively delicate but are micro-flexible. They tolerate huge pressures with loading. Collectively, the subchondral tissues transmit load mainly by hydraulic pressure to the trabeculae and cortical shaft. Normal MRI scans demonstrate subchondral vascular marks which are lost in early osteoarthritis. Histological studies confirm the presence of those marks and possible subcortical choke valves which support hydraulic pressure load transmission. Osteoarthritis appears to be at least partly a vasculo-mechanical disease. Understanding subchondral vascular physiology will be key to better MRI classification and prevention, control, prognosis and treatment of osteoarthritis and other bone diseases.

Subchondral pressures and perfusion during weight bearing.

BACKGROUND: Joints withstand huge forces, but little is known about subchondral pressures and perfusion during loading. We developed an in vitro calf foot model to explore intraosseous pressure (IOP) and subchondral perfusion during weight bearing. METHODS: Freshly culled calf forefeet were perfused with serum. IOP was measured at three sites in the foot using intraosseous needles, pressure transducers, and digital recorders. IOP was measured during perfusion, with and without a tourniquet and with differing weights, including static loading and dynamic loading to resemble walking. RESULTS: IOP varied with perfusion pressure. Static loading increased subchondral IOP whether the bone was non-perfused, perfused, or perfused with a proximal venous tourniquet (p < 0.0001). Under all perfusion states, IOP was proportional to the load (R2 = 0.984). Subchondral IOP often exceeded perfusion pressure. On removal of a load, IOP fell to below the pre-load value. Repetitive loading led to a falling IOP whether the foot was perfused or not. CONCLUSION: Superimposed on a variable background IOP, increased perfusion and physiological loading caused a significant increase in subchondral IOP. Force was thereby transmitted through subchondral bone partly by hydraulic pressure. A falling IOP with repeat loading suggests that there is an intraosseous one-way valve. This offers a new understanding of subchondral perfusion physiology.

An in vitro model to explore subchondral perfusion and intraosseous pressure.

BACKGROUND: Little is known about subchondral perfusion physiology. We developed a 3Rs (Replace, Reduce, Refine) compliant in vitro calf foot model to explore perfusion and intraosseous pressure (IOP). METHODS: Calf feet were catheterised and perfused with serum. IOP was measured at three sites, the metacarpal diaphysis (MCD), metacarpal subchondral epiphysis (MCS) and proximal phalanx diaphysis (PPD) using intraosseous needles with pressure transducers and digital recorders. Fresh (< 4 h post mortem) and old feet (> 4 h post mortem) were perfused at different pressures, with and without a proximal tourniquet. RESULTS: There was a wide range in basal IOP with a mean IOP of 30.0 mmHg, SD 14.4, range 7.6 mmHg to 52.7 mmHg (n = 40 records) in 15 subjects. There was no significant difference between the three sites tested (p = 0.54, 0.12 and 0.051). At each individual site IOP correlated with perfusion pressure (r = 0.993). With a proximal venous tourniquet, IOP increased from 15.1 mmHg (SD 11.3 mmHg) to 44.9 mmHg (SD 24 mmHg), p < 0.0001, n = 9. Filling and emptying curves during perfusion and with using a tourniquet were similar, indicating that the model behaves in an elastic hydrodynamic manner. In fresh feet IOP peaked after about 1 min irrespective of perfusion pressure, possibly due to auto regulation. Older feet showed a continuously rising IOP and became oedematous. There was no significant difference in IOP between fresh and old feet perfused with serum at 150 cms pressure for 1 min. CONCLUSION: Though basal intraosseous pressure varies, IOP behaves predictably. IOP measurements reflect the perfusion microclimate at the individual needle tip. This 3Rs compliant model will be used for further exploration of subchondral perfusion physiology with loading.

Upper tibial MRI vascular marks lost in early knee osteoarthritis.

BACKGROUND: We describe upper tibial radiating vascular marks on MRI scans. They are lost in early osteoarthritis (OA). METHODS: A literature search revealed no previous description of upper tibial MRI radial vascular marks. Fifty-six consecutive patients with anteroposterior knee X-rays and an axial PD_SPAIR MRI scan of the same knee within 1 year were studied. Their mean age was 53.1 years (range 22-85) with 27 males and 29 females. The medial and lateral compartments of each knee were scored for osteoarthritis using the Kellgren-Lawrence (K-L) classification. Marks on the MRI scans were counted by layer and quadrant position. RESULTS: Radial vascular marks were present in the first axial upper tibial subchondral slice, peaked between 6 and 10 mm depth and were absent by 16 mm depth. There was no association with age, left or right knee, BMI, or weight. There was more K-L graded OA medially and more vascular marks laterally. There was an inverse correlation between the number of marks and early grades of osteoarthritis medially (p < 0.001) and laterally (p < 0.002). CONCLUSION: We demonstrate previously undescribed subchondral vascular marks on axial MRI scans of the tibia and their inverse correlation with the presence and severity of early knee osteoarthritis. Our work offers a new insight into the possible vascular aetiology of osteoarthritis and potentially a means of earlier diagnosis and a therapeutic target.

Factors affecting intraosseous pressure measurement.

BACKGROUND: Although a raised intraosseous pressure (IOP) has been found in osteoarthritis and osteonecrosis, the normal physiology of subchondral circulation is poorly understood. We developed an animal model and explored the physiology of normal subchondral perfusion and IOP. METHODS: In 21 anaesthetised rabbits, 44 intraosseous needles were placed in the subchondral bone of the femoral head (n = 6), femoral condyle (n = 7) or proximal tibia (n = 31). Needles were connected to pressure transducers and a chart recorder. In 14 subjects, the proximal femoral artery and vein were clamped alternately. In five subjects, arterial pressure was measured simultaneously in the opposite femoral artery. RESULTS: The average IOP at all 44 sites was 24.5 mmHg with variability within SD 6.8 and between subjects SD 11.5. IOP was not significantly influenced by gender, weight, site or size of a needle. Needle clearance by flushing caused a prolonged drop in IOP whereas after clearance by aspiration, recovery was rapid. IOP recordings exhibited wave patterns synchronous with the arterial pulse, with respiration and with drug circulation time. There was a correlation between IOP and blood pressure (13 sites in 5 subjects, Pearson correlation 0.829, p < 0.0005). There was a correlation between IOP and the associated pulse pressure (PP) in 44 sites among 21 subjects (Pearson correlation 0.788, p < 0.001). In 14 subjects (31 sites), arterial occlusion caused a significant reduction in IOP and loss of PP (p < 0.0001). Venous occlusion significantly raised IOP with preservation of the PP (p < 0.012). CONCLUSION: Our study shows that subchondral cancellous bone behaves as a perfused tissue and that IOP is mainly a reflection of arterial supply. A single measure of IOP is variable and reflects only perfusion at the needle tip rather than being a measure of venous back pressure. Alternate proximal vessel clamping offers a new means of exploring the physiology of subchondral perfusion. We describe a model that will allow further study of IOP such as during loading.

Poly A tail length analysis of in vitro transcribed mRNA by LC-MS.

The 3'-polyadenosine (poly A) tail of in vitro transcribed (IVT) mRNA was studied using liquid chromatography coupled to mass spectrometry (LC-MS). Poly A tails were cleaved from the mRNA using ribonuclease T1 followed by isolation with dT magnetic beads. Extracted tails were then analyzed by LC-MS which provided tail length information at single-nucleotide resolution. A 2100-nt mRNA with plasmid-encoded poly A tail lengths of either 27, 64, 100, or 117 nucleotides was used for these studies as enzymatically added poly A tails showed significant length heterogeneity. The number of As observed in the tails closely matched Sanger sequencing results of the DNA template, and even minor plasmid populations with sequence variations were detected. When the plasmid sequence contained a discreet number of poly As in the tail, analysis revealed a distribution that included tails longer than the encoded tail lengths. These observations were consistent with transcriptional slippage of T7 RNAP taking place within a poly A sequence. The type of RNAP did not alter the observed tail distribution, and comparison of T3, T7, and SP6 showed all three RNAPs produced equivalent tail length distributions. The addition of a sequence at the 3' end of the poly A tail did, however, produce narrower tail length distributions which supports a previously described model of slippage where the 3' end can be locked in place by having a G or C after the poly nucleotide region. Graphical abstract Determination of mRNA poly A tail length using magnetic beads and LC-MS.

Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS.

A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping. Graphical abstract mRNA 5' end analysis with RNAse H cleavage and capture probe.

Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

The use of strong anion-exchange (SAX) magnetic particles for the extraction of therapeutic siRNA and their analysis by liquid chromatography/mass spectrometry.

Traditional methods for extracting oligonucleotides from serum and other biological fluids are often time-consuming and require multiple steps. Magnetic particle based separation of oligonucleotides has gained importance recently due to the advantages of simplicity and high efficiency. Here we report the development and optimization of commercially available strong anion-exchange (SAX) magnetic beads for the extraction of siRNA from human serum. The beads allowed for rapid extraction of siRNA from human serum in 100-200 µL of liquid chromatography/mass spectrometry (LC/MS)-compatible buffer in less than 1 h for a 96-well plate with no further drying steps. Due to the strong cation-binding properties of oligonucleotides, volatile ammonium salts such as triethylammonium bicarbonate (TEAB), ammonium bicarbonate, and NH(4) Cl were used to elute the siRNA from the beads. For more hydrophobic siRNA sequences, the addition of 5-10% organic solvent was required for elution. The recovery of chemically modified siRNA from human serum was around 80% for two types of beads examined; however, the recovery for highly modified sequences differed greatly between the two types of beads. In addition to extracting highly modified oligonucleotides, the SAX beads were also able to extract liposomal formulated siRNAs from serum with no interference from the lipid formulation. The extraction of siRNA from human serum was linear over the tested range of 50 ng/mL to 10 µg/mL. Using this extraction methodology, we have created a workflow to monitor siRNA serum stability by LC/MS. Initial observations confirm that RNase A type degradation with strand cleavage on the 3' side of uridine or cytosine is the dominant cleavage pattern in serum. This finding has implications for the selection and modification of therapeutic siRNAs and demonstrates the utility of magnetic beads as a simple and rapid extraction technique for siRNA.

Applications of mass spectrometry to the study of siRNA.

RNA interference (RNAi) has quickly become a well-established laboratory tool for regulating gene expression and is currently being explored for its therapeutic potential. The design and use of double-stranded RNA oligonucleotides as therapeutics to trigger the RNAi mechanism and a greater effort to understand the RNAi pathway itself is driving the development of analytical techniques that can characterize these oligonucleotides. Electrospray (ESI) and MALDI have been used routinely to analyze oligonucleotides and their ability to provide mass and sequence information has made them ideal for this application. Reviewed here is the work done to date on the use of ESI and MALDI for the study of RNAi oligonucleotides as well as the strategies and issues associated with siRNA analysis by mass spectrometry. While there is not a large body of literature on the specific application of mass spectrometry to RNAi, the work done in this area is a good demonstration of the range of experiments that can be conducted and the value that ESI and MALDI can provide to the RNAi field.

Sequence confirmation of modified oligonucleotides using chemical degradation, electrospray ionization, time-of-flight, and tandem mass spectrometry.

We report the sequencing of highly modified oligonucleotides containing a mixture of 2'-deoxy, 2'-fluoro, 2'-O-methyl, abasic, and ribonucleotides. The passenger and guide strands each containing 48% and 86% of modified nucleotides, respectively, are representative sequences of synthetic short interfering RNAs (siRNAs). We describe herein the sequence confirmation of both strands using a series of robust chemical reactions, followed by analysis via ESI-TOF and ion trap mass spectrometry (ITMS). The following method enables the rapid sequence confirmation of highly modified oligonucleotides.

Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry.

On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1 Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes.

Examination of real-time polymerase chain reaction methods for the detection and quantification of modified siRNA.

With the ongoing efforts to develop siRNA-based therapeutics, there is a need for high-throughput detection and quantification of siRNA. Here we report the application of four reverse-transcriptase RT-PCR-based assays for the detection of 2'-deoxy-2'-fluoro and 2'-O-methyl-modified therapeutic siRNA in mouse plasma and tissue. These assays take advantage of the dynamic range, sensitivity, specificity, and high-throughput potential found in PCR assays. Three of these assays require design and optimization of primers and/or probes specific to the siRNA while the fourth utilizes a "universal" TaqMan probe that is independent of the siRNA sequence, thereby reducing method development time and cost. For the universal assay the range of detection in mouse plasma was 500 to 5e(-5) pg/microl for four of five model Luciferase sequences tested. We found that the universal RT-PCR assay had comparable or better sensitivity and specificity than the other three assays. The universal design provides a rapid, sensitive, and specific assay with minimal method development time that will be well suited for high-throughput analysis of various siRNA sequences.

Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex.

The ocular metabolism of an siRNA duplex, SIRNA-027, was examined by ion-pair reversed-phase liquid chromatography (IP-RP-LC) coupled to electrospray ionization mass spectrometry (ESI-MS). The RNA duplex was injected intraocularly into the eyes of New Zealand white rabbits. Rabbits were sacrificed at different timepoints and the vitreous and retina/choroid tissue analyzed for siRNA by IP-RP-LC-MS. The method used a hexafluoroisopropanol (HFIP)/triethylamine (TEA) ion-pairing buffer with a methanol gradient. Using electrospray ionization, the duplex was preserved in the gas phase for analysis by a triple quadrupole mass spectrometer. With this methodology metabolites from rabbit ocular vitreous humor and retina/choroid tissue were identified and a pattern of siRNA degradation was established. Results showed that the duplex was metabolized predominantly from one end. This end of the siRNA duplex was calculated to have the weakest binding energy of the two ends indicating that the ability of the siRNA to split into single strands is a factor in its degradation.

Liquid chromatography/electrospray mass spectrometric analysis of metabolites from an inhibitory RNA duplex.

Liquid chromatography/mass spectrometry (LC/MS) was used as a method for analyzing the metabolites of a model short interfering RNA (siRNA) duplex. The model siRNA duplex incorporated oligonucleotide stabilizing and protecting chemistries as these have been shown to increase the half-life of oligonucleotides. Two complementary 23 nucleotide single strands were joined to form the duplex. The intact duplex was analyzed using ion-pair reversed-phase chromatography coupled to electrospray ionization mass spectrometry (ESI-MS). The method used a hexafluoroisopropanol/triethylamine ion-pairing buffer with a methanol gradient to separate single-stranded oligonucleotide components from the duplex. This buffer system with ESI also preserved the duplex in the gas phase for analysis by a triple quadrupole mass spectrometer. Using this methodology, in vitro and in vivo metabolites from urine and rabbit ocular vitreous humor were determined and a pattern of duplex siRNA degradation was established. The masses of the metabolites were determined by ESI-MS and used with the known sequence of the siRNA duplex to identify the metabolites. Over the time course of the metabolism experiments it was shown that the breakdown products of the siRNA are consistent with the nuclease protection given by chemical modifications and that the duplex structure adds additional stability compared to the single strands alone. This study demonstrates that the ability of LC/MS to analyze duplex oligonucleotides has unique benefits for the study of siRNA metabolism.

The transfibular approach for distal tibial osteochondroma: an alternative technique for excision.

Surgical excision of osteochondromata occurring at the lateral aspect of the distal tibia is hampered by the difficult access to this area. Current techniques use an anterior approach, but this makes access to the posterior aspect of the tibia difficult. The authors report on 2 cases in which removal, and subsequent replacement of distal fibula and fixation with a semitubular plate, allowed complete excision of a distal tibial osteochondroma and satisfactory outcomes. This procedure provides an alternative technique to the anterior approach previously described. The literature appears to contain no similar previous reports.

Evaluation of a micro volume pulsed ultrafiltration cell for screening ligands in non-covalent complexes.

A pulsed ultrafiltration cell with a 35 microL binding chamber was evaluated for its ability to screen ligands that formed non-covalent complexes with protein targets. The cell was tested with ligands to the targets of carbonic anhydrase and serum albumin. Non-covalent ligand binding to both of these targets was observed and bound ligands were eluted from the cell in less than five min. The cell was also demonstrated to effectively screen a methanolic fermentation broth extract spiked with a known inhibitor to carbonic anhydrase. In addition to detecting specific binding events, the pulsed ultrafiltration method was investigated for its ability to distinguish non-specific binding events. Using carbonic anhydrase with the zinc-binding site removed, it was found that non-specific complexes observed when using electrospray ionization alone were not detected when using the pulsed ultrafiltration mass spectrometry method.