Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution.

OBJECTIVES: Interactions of cells with their neighbors and influences by the surrounding extracellular matrix (ECM) is reflected in a cells transcriptome and proteome. In tissues comprised of heterogeneous cell populations or cells depending on ECM signalling cues such as those of the intervertebral disc (IVD), this information is obscured or lost when cells are pooled for the commonly used transcript analysis by quantitative PCR or RNA sequencing. Instead, these cells require means to analyse RNA transcript and protein distribution at a single cell or subcellular level to identify different cell types and functions, without removing them from their surrounding signalling cues. RESULTS: We developed a simple, sequential protocol combining RNA is situ hybridisation (RISH) and immunohistochemistry (IHC) for the simultaneous analysis of multiple transcripts alongside proteins. This allows one to characterize heterogeneous cell populations at the single cell level in the natural cell environment and signalling context, both in vivo and in vitro. This protocol is demonstrated on cells of the bovine IVD, for transcripts and proteins involved in mechanotransduction, stemness and cell proliferation. CONCLUSIONS: A simple, sequential protocol combining RISH and IHC is presented that allows for simultaneous information on RNA transcripts and proteins to characterize cells within a heterogeneous cell population and complex signalling environments such as those of the IVD.

Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc.

Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col Iα 1, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

Age estimation based on different molecular clocks in several tissues and a multivariate approach: an explorative study.

Several molecular modifications accumulate in the human organism with increasing age. Some of these "molecular clocks" in DNA and in proteins open up promising approaches for the development of methods for forensic age estimation. A natural limitation of these methods arises from the fact that the chronological age is determined only indirectly by analyzing defined molecular changes that occur during aging. These changes are not linked exclusively to the expired life span but may be influenced significantly by intrinsic and extrinsic factors in the complex process of individual aging. We tested the hypothesis that a combined use of different molecular clocks in different tissues results in more precise age estimates because this approach addresses the complex aging processes in a more comprehensive way. Two molecular clocks (accumulation of D-aspartic acid (D-Asp), accumulation of pentosidine (PEN)) in two different tissues (annulus fibrosus of intervertebral discs and elastic cartilage of the epiglottis) were analyzed in 95 cases, and uni- and multivariate models for age estimation were generated. The more parameters were included in the models for age estimation, the smaller the mean absolute errors (MAE) became. While the MAEs were 7.5-11.0 years in univariate models, a multivariate model based on the two protein clocks in the two tissues resulted in a MAE of 4.0 years. These results support our hypothesis. The tested approach of a combined analysis of different molecular clocks analyzed in different tissues opens up new possibilities in postmortem age estimation. In a next step, we will add the epigenetic clock (DNA methylation) to our protein clocks (PEN, D-Asp) and expand our set of tissues.

Temporomandibular joint imaging: current clinical applications, biochemical comparison with the intervertebral disc and knee meniscus, and opportunities for advancement.

Temporomandibular disorders encompass multiple pathologies of the temporomandibular joint that manifest as middle/inner ear symptoms, headache, and/or localized TMJ symptoms. There is an important although somewhat limited role of imaging in the diagnostic evaluation of temporomandibular disorders. In this manuscript, we provide a comprehensive review of TMJ anatomy, outline potentially important features of TMJ disc ultrastructure and biochemistry in comparison with the intervertebral disc and knee meniscus, and provide imaging examples of the TMJ abnormalities currently evaluable with MRI and CT. In addition, we provide an overview of emerging and investigational TMJ imaging techniques in order to encourage further imaging research based on the biomechanical alterations of the TMJ disc.

Ingrowth of Nociceptive Receptors into Diseased Cervical Intervertebral Disc Is Associated with Discogenic Neck Pain.

OBJECTIVE: To investigate the distribution of nociceptive nerve fibers in the cervical intervertebral discs of patients with chronic neck pain and determine whether these nociceptive nerve fibers are related to discogenic neck pain. METHODS: We collected 43 samples of cervical intervertebral discs from 34 patients with severe chronic neck pain (visual analog scale [VAS] ≥ 70 mm), 42 samples from 36 patients who suffered cervical spondylotic radiculopathy or myelopathy without neck pain or with mild neck pain (VAS ≤ 30 mm) and 32 samples from eight donators to investigate their innervation immunohistochemically using an antibody against neuropeptide substance P. RESULTS: The immunohistochemical investigation revealed that substance P-positive nerve fibers were obviously increased in number and deeply ingrown into the inner anulus fibrosus and even into the nucleus pulposus in the degenerative cervical discs of patients with severe neck pain in comparison with the discs of patients with cervical spondylotic radiculopathy or myelopathy and normal control discs (P<0.01). CONCLUSIONS: The current study may indicate a key role of nociceptive nerve fibers in the pathogenesis of neck pain of cervical disc origin.

Crystallinity dependency of the time-dependent mechanical response of polyethylene: application in total disc replacement.

Degeneration of the intervertebral disc (IVD) is a leading source of chronic low back pain or neck pain, and represents the main cause of long-term disability worldwide. In the aim to relieve pain, total disc replacement (TDR) is a valuable surgical treatment option, but the expected benefit strongly depends on the prosthesis itself. The present contribution is focused on the synthetic mimic of the native IVD in the aim to optimally restore its functional anatomy and biomechanics, and especially its time-dependency. Semi-crystalline polyethylene (PE) materials covering a wide spectrum of the crystallinity are used to propose new designs of TDR. The influence of the crystallinity on various features of the time-dependent mechanical response of the PE materials is reported over a large strain range by means of dynamic mechanical thermo-analysis and video-controlled tensile mechanical tests. The connection of the stiffness and the yield strength with the microstructure is reported in the aim to propose a model predicting the crystallinity dependency of the response variation with the frequency. New designs of TDR are proposed and implemented into an accurate computational model of a cervical spine segment in order to simulate the biomechanical response under physiological conditions. Predicted in-silico motions are found in excellent agreement with experimental data extracted from published in-vitro studies under compression and different neck movements, namely, rotation, flexion/extension and lateral bending. The simulation results are also criticized by analyzing the local stresses and the predicted biomechanical responses provided by the different prosthetic solutions in terms of time-dependency manifested by the hysteretic behavior under a cyclic movement and the frequency effect.

Comparison of B0 versus B0 and B1 field inhomogeneity correction for glycosaminoglycan chemical exchange saturation transfer imaging.

PURPOSE: The study compares glycosaminoglycan chemical exchange saturation transfer (gagCEST) imaging of intervertebral discs corrected for solely B0 inhomogeneities or both B0 and B1 inhomogeneities. METHODS: Lumbar intervertebral discs of 20 volunteers were examined with T2-weighted and gagCEST imaging. Field inhomogeneity correction was performed with B0 correction only and with correction of both B0 and B1. GagCEST effects measured by the asymmetric magnetization transfer ratio (MTRasym) and signal-to-noise ratio (SNR) were compared between both methods. RESULTS: Significant higher MTRasym and SNR values were obtained in the nucleus pulposus using B0 and B1 correction compared with B0-corrected gagCEST. The GagCEST effect was significantly different in the nucleus pulposus compared with the annulus fibrosus for both methods. CONCLUSION: The B0 and B1 field inhomogeneity correction method leads to an improved quality of gagCEST imaging in IVDs compared with only B0 correction.

The adolescent idiopathic scoliotic IVD displays advanced aggrecanolysis and a glycosaminoglycan composition similar to that of aged human and ovine IVDs.

PURPOSE: The present study was designed to ascertain how altered biomechanics in adolescent idiopathic scoliotic (AIS) intervertebral discs (IVDs) affected tissue compositions and aggrecan processing compared to age matched and aged human IVDs. Newborn, 2- and 10-year-old ovine IVDs were also examined. METHODS: Aggrecan populations were separated by Sepharose CL2B chromatography, composite agarose polyacrylamide gel electrophoresis (CAPAGE) and identified by immunoblotting. The KS and CS content of IVD tissue extracts from AIS IVDs were compared with age-matched normal adolescent IVDs and with old human IVDs. Extracts from newborn, 2- and 10-year-old ovine IVDs were also examined in a similar manner. RESULTS: Adolescent idiopathic scoliotic IVD Aggrecan populations shared similar levels of polydispersity and aggregatability with hyaluronan as old IVD proteoglycans. CAPAGE demonstrated three aggrecan populations in AIS, aged human and ovine IVDs increased polydispersity and mobility in CAPAGE. AIS IVDs had GAG compositions similar to aged human and ovine IVDs. Sulphated KS (5-D-4) and chondroitin-6-sulphate, 3-B-3(+) were markers of tissue maturation, and chondroitin-4-sulphate, 2-B-6(+) was prominent in immature IVDs but its levels were lower in mature IVDs. DISCUSSION: Sulphated KS and 3-B-3(+) CS were prominently associated with IVD maturation and AIS IVDs, while the 2-B-6(+) CS isomer was associated with immature IVD tissues. The polydispersity of aggrecan in AIS IVDs, which was similar to in old human and ovine IVDs, reflected altered processing in the AIS IVDs in response to the biomechanical microenvironments the disc cells were exposed to in AIS IVDs. These slides can be retrieved under Electronic Supplementary Material.

Vancomycin concentrations in the cervical spine after intravenous administration: results from an experimental pig study.

Background and purpose - Vancomycin may be an important drug for intravenous perioperative antimicrobial prophylaxis in spine surgery. We assessed single-dose vancomycin intervertebral disc, vertebral cancellous bone, and subcutaneous adipose tissue concentrations using microdialysis in a pig model. Material and methods - 8 female pigs received 1,000 mg of vancomycin intravenously as a single dose over 100 minutes. Microdialysis probes were placed in the C3-C4 intervertebral disc, C3 vertebral cancellous bone, and subcutaneous adipose tissue, and vancomycin concentrations were obtained over 8 hours. Venous blood samples were obtained as reference. Results - Ranging from 0.24 to 0.60, vancomycin tissue penetration, expressed as the ratio of tissue to plasma area under the concentration-time curve from 0 to the last measured value, was incomplete for all compartments. The lowest penetration was found in the intervertebral disc. The time to a mean clinically relevant minimal inhibitory concentration (MIC) of 4 µg/mL was 3, 17, 25, and 156 min for plasma, subcutaneous adipose tissue, vertebral cancellous bone, and the intervertebral disc, respectively. In contrast to the other compartments, a mean MIC of 8 µg/mL was not reached in the intervertebral disc. An approximately 3-times longer elimination rate was observed in the intervertebral disc in comparison with all the other compartments (p < 0.001), and the time to peak drug concentration was higher for all tissues compared with plasma Interpretation - Preoperative administration of 1,000 mg of vancomycin may provide adequate vancomycin tissue concentrations with a considerable delay, though tissue penetration was incomplete. However, in order also to achieve adequate intervertebral disc concentrations in all individuals and accommodating a potentially higher MIC target, supplemental application of vancomycin may be necessary.

The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration.

OBJECTIVES: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. METHODS: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. RESULTS: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1ß but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. CONCLUSIONS: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Self-Healing Dynamic Hydrogel as Injectable Shock-Absorbing Artificial Nucleus Pulposus.

The intervertebral discs (IVDs) provide unique flexibility to the spine and exceptional shock absorbing properties under impact. The inner core of the IVD, the nucleus pulposus (NP) is responsible for this adaptive behavior. Herein, we evaluate an injectable, self-healing dynamic hydrogel (DH) based on gold(I)-thiolate/disulfide (Au-S/SS) exchange as NP replacement in a spine motion segment model. For the first time, we report the application of dynamic covalent hydrogels inside biological tissues. The dynamic exchange between Au-S species and disulfide bonds (SS) resulted in self-healing ability and frequency-dependent stiffness of the hydrogel, which was also confirmed in spine motion segments. Injection of preformed DH into nucleotomized IVDs restored the full biomechanical properties of intact IVDs, including the stiffening effect observed at increasing frequencies, which cannot be achieved with conventional covalent hydrogel. DH has the potential to counteract IVD degeneration associated with high frequency vibrations. Self-healing properties, confirmed by rheology studies and macroscopic observation after injection, were required to inject preformed DH, which recovered its mechanical integrity and microstructure to act as an artificial NP. On the other hand, covalent hydrogel did not show any restoration of NP properties as this conventional material suffered irreversible damages after injection, which demonstrates that the dynamic properties are crucial for this application. The persistence of DH in the IVD space following cyclic high-frequency loading, confirmed by tomography after mechanical testing, suggests that this material would have long life span as an injectable NP replacement material.

The expression of cytokine and its significance for the intervertebral disks of Kazakhs.

BACKGROUND: Proinflammatory cytokine IL-6 and anti-inflammatory cytokine IL-10 are expressed in herniated intervertebral disks of Kazakhs, but their significance is not yet understood. METHODS: Specimens of herniated lumbar disk were collected from 30 patients with single disk herniation during lumbar discectomy. As a control, 10 specimens were collected postmortem from the cadavers of individuals who did not die from spinal disease. The clinical symptoms in all cases were evaluated preoperation by the JOA index. The expressions of IL-6 and IL-10 in the degenerative disk cells of each specimen were detected using immunohistochemical staining. The amounts of IL-6 and IL-10 in each intervertebral disk were assessed by enzyme-linked immunosorbent assay. RESULTS: The expression levels of IL-6 and IL-10 were significantly higher in the patient group than in the control group. Statistical analysis showed differences between ethnic Kazakh and Han patients in both the expression of IL-6 and JOA scores. Immunohistochemical staining showed that in the patient group, IL-6 was found in 28 patients and IL-10 was found in 27 patients. CONCLUSION: The high expression of cytokines, such as IL-6 and IL-10, has an important relationship with disk degeneration. Of course, age is another factor in cell apoptosis of degenerative disks. More research is necessary to discover how ethnicity and heredity impact the levels of expressed cytokines.

Mucin 1, a signal transduction membrane-bound mucin, is present in human disc tissue and is downregulated in vitro by exposure to IL-1ß or TNF-α.

BACKGROUND: Back pain and disc degeneration have a growing socioeconomic healthcare impact. Mucin 1 (MUC1) is a transmembrane glycoprotein whose extracellular and intracellular domains participate in cellular signaling. Little is currently known about the presence or role of MUC1 in human disc degeneration. METHODS: In this IRB-approved research study, 29 human disc specimens were analyzed for MUC1 immunohistochemical localization and gene expression, and annulus fibrosus (annulus) cells were also isolated and cultured in 3D. Microarray analysis assessed expression levels of MUC1 in healthy and degenerated disc tissue and in cells exposed to proinflammatory cytokines (IL-1ß or TNF-α). RESULTS: MUC1 was shown to be present in annulus cells at the protein level using immunochemistry, and its expression was significantly upregulated in annulus tissue from more degenerated grade V discs compared to healthier grade I-II discs (p = 0.02). A significant positive correlation was present between the percentage of MUC1-positive cells and disc grade (p = 0.009). MUC1 expression in annulus cells cultured in 3D was also analyzed following exposure to IL-1ß or TNF-α; exposure produced significant MUC1 downregulation (p = 0.0006). CONCLUSIONS: Here we present the first data for the constitutive presence of MUC1 in the human disc, and its altered expression during disc degeneration. MUC1 may have an important role in disc aging and degeneration by acting as a regulator in the hypoxic environment, helping disc cells to survive under hypoxic conditions by stabilization and by activation of HIF-1α as previously recognized in pancreatic cancer cells.

Quantitative chemical exchange saturation transfer MRI of intervertebral disc in a porcine model.

PURPOSE: Previous studies have associated low pH in intervertebral discs (IVDs) with discogenic back pain. The purpose of this study was to determine whether quantitative CEST (qCEST) MRI can be used to detect pH changes in IVDs in vivo. METHODS: The exchange rate ksw between glycosaminoglycan (GAG) protons and water protons was determined from qCEST analysis. Its dependence on pH value was investigated in GAG phantoms with varying pH and concentrations. The relationship between ksw and pH was studied further in vivo in a porcine model on a 3T MR scanner and validated using a pH meter. Sodium lactate was injected into the IVDs to induce various pH values within the discs ranging from 5 to 7. RESULTS: Phantom and animal results revealed that ksw measured using qCEST MRI is highly correlated with pH level. In the animal studies, the relationship can be described as ksw =9.2 × 106 × 10-pH + 196.9, R2 = 0.7883. CONCLUSION: The exchange rate between GAG and water protons determined from qCEST MRI is closely correlated with pH value. This technique has the potential to noninvasively measure pH in the IVDs of patients with discogenic pain. Magn Reson Med 76:1677-1683, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Evaluation of Glycosaminoglycan in the Lumbar Disc Using Chemical Exchange Saturation Transfer MR at 3.0 Tesla: Reproducibility and Correlation with Disc Degeneration.

OBJECTIVE: This study aims to explore the clinical applicability and relevance of glycosaminoglycan Chemical Exchange Saturation Transfer (gagCEST) for intervertebral disc. METHODS: 25 subjects ranging in age from 24 yrs to 74 yrs were enrolled. gagCEST was acquired using a single-slice TSE sequence on a 3T. Saturation used a continuous rectangular RF pulse with B1=0.8 µT and a fixed duration time=1100 ms. Sagittal image was obtained firstly without saturation pulse, and then saturated images were acquired at 52 offsets ranging from ±0.125 to ±7 parts per million (ppm). MR T2 relaxivity map was acquired at the identical location. Six subjects were scanned twice to assess scan-rescan reproducibility. RESULTS: GagCEST intraclass correlation coefficient (ICC) of six subjects was 0.759 for nucleus pulposus (NP) and 0.508 for annulus fibrosus (AF). Bland-Altman plots showed NP had a mean difference of 0.10% (95% limits of agreement: -3.02% to 3.22%); while that of AF was 0.34% (95% limits of agreement: -2.28% to 2.95%). For the 25 subjects, gag CEST in NP decreased as disc degeneration increased, with a similar trend to T2 relaxivity. Gag CEST of AF showed a better correlation with disc degeneration than T2 relaxivity. CONCLUSION: GagCEST in NP and AF decreased as disc degeneration increased, while gagCEST in AF showed a better correlation than T2 relaxivity.

Cartilaginous end plates: Quantitative MR imaging with very short echo times-orientation dependence and correlation with biochemical composition.

PURPOSE: To measure the T2* of the human cartilaginous end plate by using magnetic resonance (MR) imaging with very short echo times and to determine the effect of the orientation of the end plate on T2* and on relationships between T2* and biochemical composition. MATERIALS AND METHODS: This study was exempt from institutional review board approval, and informed consent was not required. Thirty-four samples of three cadaveric lumbar spines (from subjects who died at ages 51, 57, and 66 years) containing cartilaginous end plates and subchondral bone were prepared. Samples were imaged with a 3-T imager for T2* quantification by using a three-dimensional very short echo time sequence (repetition time msec/echo times msec, 30/0.075, 2, 5, 12, 18). Samples were imaged with the end plate at three orientations with respect to the constant magnetic induction field: 0°, 54.7°, and 90°. After imaging, the cartilage was assayed for its water, glycosaminoglycan, and collagen content. Pearson correlations were used to investigate the effect of orientation on the relationships between T2* and biochemical composition. RESULTS: T2* was significantly longer when measured at an orientation of 54.7° (21.8 msec ± 2.8 [± standard error of the mean]) than at 0° (10.0 msec ± 0.7, P < .001) or 90° (9.9 msec ± 0.4, P < .001). At 54.7°, T2* was highly correlated with glycosaminoglycan content (r = 0.85, P < .001), the collagen-to-glycosaminoglycan ratio (r = -0.79, P < .001), and water content (r = 0.62, P = .02); at 0° and 90°, there were no significant differences in these relationships, with a minimum P value of .19. CONCLUSION: T2* evaluation can allow noninvasive estimation of the degeneration of the cartilaginous end plate; however, the accuracy of T2*-based estimates of biochemical composition depends on the orientation of the end plate.

Detection of low back pain using pH level-dependent imaging of the intervertebral disc using the ratio of R1ρ dispersion and -OH chemical exchange saturation transfer (RROC).

PURPOSE: Low pH is associated with intervertebral disc (IVD)-generated low back pain (LBP). The purpose of this work was to develop an in vivo pH level-dependent magnetic resonance imaging (MRI) method for detecting discogenic LBP, without using exogenous contrast agents. METHODS: The ratio of R1ρ dispersion and chemical exchange saturation transfer (CEST) (RROC) was used for pH-level dependent imaging of the IVD while eliminating the effect of labile proton concentration. The technique was validated by numerical simulations and studies on phantoms and ex vivo porcine spines. Four male (ages 42.8 ± 18.3) and two female patients (ages 55.5 ± 2.1) with LBP and scheduled for discography were examined with the method on a 3.0 Tesla MR scanner. RROC measurements were compared with discography outcomes using paired t-test. RESULTS: Simulation and phantom results indicated RROC is a concentration independent and pH level-dependent technique. Porcine spine study results found higher RROC value was related to lower pH level. Painful discs based on discography had significant higher RROC values than those with negative diagnosis (P < 0.05). CONCLUSION: RROC imaging is a promising pH level dependent MRI technique that has the potential to be a noninvasive imaging tool to detect painful IVDs in vivo.

Axial T1ρ MRI as a diagnostic imaging modality to quantify proteoglycan concentration in degenerative disc disease.

PURPOSE: The aim of the study was to investigate if axial T1ρ MR images had similar accuracy as established sagittal T1ρ MRI for the assessment of proteoglycan concentration and content in intervertebral degenerated discs (IDDs). METHODS: T1ρ and T2-weighted MR images of 12 intervertebral discs (IVDs) from 3 harvested human lumbar spines (levels L1-L2 to L5-S1) were grouped across their degenerative grade (Pfirrmann scores) and analyzed using a 3T MRI scanner in the axial and sagittal views. Post-processing of axial T1ρ-weighted images was performed using a Wiener filter. Median axial T1ρ values for traced regions of interest (ROIs) on color maps were compared against ROIs in the corresponding location in the sagittal plane of each disc. Assessment of sulfated glycosaminoglycans (GAGs) content was also performed. RESULTS: Comparison of post Wiener filtered mid-axial T1ρ values in the NP with corresponding mid-sagittal values revealed no statistical difference (P > 0.05). Higher axial T1ρ and biochemically measured GAGs content corresponded to a lower Pfirrmann grading of the IVDs. A strong association between the T1ρ values and the GAG contents was observed (r = 0.85, P = 0.0002). CONCLUSIONS: The axial T1ρ methodology was validated against sagittal T1ρ providing an augmented spatial representation of IVD and can facilitate localization of focal degeneration within IVDs. T1ρ values provided a better granularity assessment of degenerative disc disease as it correlated with proteoglycan concentration. Thus, Wiener filtering is an effective tool for removing noise from T1ρ-weighted axial MR images.

Ex vivo porcine model to measure pH dependence of chemical exchange saturation transfer effect of glycosaminoglycan in the intervertebral disc.

PURPOSE: Studies have linked low pH and loss of glycosaminoglycan (GAG) in the intervertebral discs (IVDs) of patients with discogenic back pain. The purpose of this study is to determine whether the chemical exchange saturation transfer (CEST) effect of GAG (gagCEST) is pH dependent and whether it can be used to detect pH changes in IVD specimens. Iopromide, a Food and Drug Administration approved agent for CT/X-Ray, was also evaluated as a pH-sensitive CEST probe to explore the agents' potential to measure IVD pH. METHODS: The pH dependency of the CEST effect of chondroitin sulfate (containing GAG) and Iopromide phantoms was investigated at 7 T. Z-spectra from porcine IVD specimens were acquired before and after manipulating the pH with sodium lactate. Iopromide was injected into the specimens and the calibration curve was used to determine the pH status. RESULTS: Chondroitin sulfate showed a non-linear dependence of gagCEST effect with pH and gagCEST signal differences were detected in the specimens. The CEST effect of Iopromide resulted in a sigmoidal relation with pH and was used to measure pH. CONCLUSION: gagCEST is sensitive to pH and enables investigation of the IVD pH status. Iopromide CEST is independent of the local GAG concentration and has the potential for measuring pH in the IVD.

Identification of transglutaminase substrates from porcine nucleus pulposus as potential amplifiers in cross-linking cell scaffolds.

Nucleus pulposus from the porcine intervertebral disc was separated chromatographically to discover substrates of microbial transglutaminase. Highly purified proteins were prepared, among them type II collagen, the major protein of the nucleus pulposus. Determination of substrates was performed by transglutaminase-mediated incorporation of biotinylated probes displaying several glutamine and lysine donor proteins. Type II collagen was only labeled if smaller nucleus pulposus proteins were present. One of the modulating proteins was serotransferrin, a lysine donor substrate of bacterial transglutaminase. An additional substrate was the carboxy-terminal propeptide of type II collagen, chondrocalcin. Chondrocalcin, a regulator of type II collagen fibrillogenesis, occurs abundantly in juvenile cartilage and nucleus pulposus. Accordingly, the protein may be regarded as an excellent additive for the preparation of injectable stem cells in nucleus pulposus-like matrices cross-linked by microbial transglutaminase.