Magnetic resonance imaging assessment of proteolytic enzyme concentrations and biologic properties of intraluminal thrombus in abdominal aortic aneurysms.

OBJECTIVE: The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. METHODS: Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. RESULTS: A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. CONCLUSIONS: These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.

Assessment and prevention of cartilage degeneration surrounding a focal chondral defect in the porcine model.

Focal defects in articular cartilage are unable to self-repair and, if left untreated, are a leading risk factor for osteoarthritis. This study examined cartilage degeneration surrounding a defect and then assessed whether infilling the defect prevents degeneration. We created a focal chondral defect in porcine osteochondral explants and cultured them ex vivo with and without dynamic compressive loading to decouple the role of loading. When compared to a defect in a porcine knee four weeks post-injury, this model captured loss in sulfated glycosaminoglycans (sGAGs) along the defect's edge that was observed in vivo, but this loss was not load dependent. Loading, however, reduced the indentation modulus of the surrounding cartilage. After infilling with in situ polymerized hydrogels that were soft (100 kPa) or stiff (1 MPa) and which produced swelling pressures of 13 and 310 kPa, respectively, sGAG loss was reduced. This reduction correlated with increased hydrogel stiffness and swelling pressure, but was not affected by loading. This ex vivo model recapitulates sGAG loss surrounding a defect and, when infilled with a mechanically supportive hydrogel, degeneration is minimized.

Non-Destructive Spectroscopic Assessment of High and Low Weight Bearing Articular Cartilage Correlates with Mechanical Properties.

OBJECTIVE: Autologous articular cartilage (AC) harvested for repair procedures of high weight bearing (HWB) regions of the femoral condyles is typically obtained from low weight bearing (LWB) regions, in part due to the lack of non-destructive techniques for cartilage composition assessment. Here, we demonstrate that infrared fiber optic spectroscopy can be used to non-destructively evaluate variations in compositional and mechanical properties of AC across LWB and HWB regions. DESIGN: AC plugs (N = 72) were harvested from the patellofemoral groove of juvenile bovine stifle joints, a LWB region, and femoral condyles, a HWB region. Near-infrared (NIR) and mid-infrared (MIR) fiber optic spectra were collected from plugs, and indentation tests were performed to determine the short-term and equilibrium moduli, followed by gravimetric water and biochemical analysis. RESULTS: LWB tissues had a significantly greater amount of water determined by NIR and gravimetric assay. The moduli generally increased in tissues from the patellofemoral groove to the condyles, with HWB condyle cartilage having significantly higher moduli. A greater amount of proteoglycan content was also found in HWB tissues, but no differences in collagen content. In addition, NIR-determined water correlated with short-term modulus and proteoglycan content (R = -0.40 and -0.31, respectively), and a multivariate model with NIR data was able to predict short-term modulus within 15% error. CONCLUSIONS: The properties of tissues from LWB regions differ from HWB tissues and can be determined non-destructively by infrared fiber optic spectroscopy. Clinicians may be able to use this modality to assess AC prior to harvesting osteochondral grafts for focal defect repair.

A Standardized Method of Applying Toluidine Blue Metachromatic Staining for Assessment of Chondrogenesis.

OBJECTIVES: Staining with toluidine blue is a well-established procedure for the histological assessment of cartilaginous- and chondrogenic-differentiated tissues. Being a cationic dye, toluidine blue staining visualizes proteoglycans in a tissue because of its high affinity for the sulfate groups in proteoglycans. It is generally accepted that metachromatic staining with toluidine blue represents cartilaginous matrix and that the degree of positive staining corresponds with the amount of proteoglycans. DESIGN: Articular cartilage and pellets of chondrocytes or bone marrow stromal cells were analyzed with a standardized staining procedure for toluidine blue. RESULTS: In the present study, we illustrate why such an assumption is invalid unless a detailed description of the procedure and/or reference to a detailed published method are provided. This is because the staining specificity and intensity depend, as we have shown, on the pH of the staining solution, the use of dehydration, and on staining time. CONCLUSIONS: We can, therefore, suggest a well-controlled standardized protocol for toluidine blue staining, which provides an easy and simple selective staining technique for the assessment of cartilage tissue and proteoglycan development in chondrogenic differentiation. If this procedure is not used, then investigators must provide sufficient technical information concerning the staining protocol to allow an assessment of the validity of the staining results.

Value of delayed gadolinium-enhanced magnetic resonance imaging of cartilage for the pre-operative assessment of cervical intervertebral discs.

The study was performed to preoperatively assess the cartilage integrity of cervical intervertebral discs (IVDs) using Delayed Gadolinium-Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC). Therefore, 53 cervical intervertebral discs of nine preoperative patients with neck and shoulder/arm pain scheduled for discectomy (five females, four males; mean age: 47.1 ± 8.4 years; range: 36-58 years) were included for biochemical analysis in this retrospective study. The patients underwent 3T magnetic resonance imaging (MRI) including biochemical imaging with dGEMRIC and morphological, sagittal T2 weighted (T2w) imaging. Cervical IVDs were rated using an MRI based grading system for cervical IVDs on T2w images. Region-of-interest measurements were performed in the nucleus pulposus (NP) and annulus fibrosus (AF) and a dGEMRIC index was calculated. Our results demonstrated that IVDs scheduled for discectomy showed significantly lower dGEMRIC index compared to IVDs that did not require surgical intervention in NP and AF (NP: 898.4 ± 191.9 ms vs. 1,150.3 ± 320.7 ms, p = 0.008; AF: 738.7 ± 183.8 ms vs. 984.6 ± 178.9 ms, p = 0.008). For Miyazaki score 3, the dGEMRIC indices were significantly lower in IVDs scheduled for surgery compared to non-operated discs for NP (p = 0.043) and AF (p = 0.018). In conclusion we could demonstrate that biochemical imaging with dGEMRIC is feasible in cervical IVDs. Significantly lower dGEMRIC index suggested GAG depletion in degenerated cervical IVD, scheduled for discectomy. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1824-1830, 2017.

Evaluation of correlation of articular cartilage staining for DDR2 and proteoglycans with histological tissue damage and the results of radiographic assessment in patients with early stages of knee osteoarthritis.

OBJECTIVE: To determine, if staining of articular cartilage for proteoglycans (natural element of healthy and functioning cartilage) and discoidin domain receptor 2 (DDR2) (a protein associated with articular cartilage degradation) is correlated with histological tissue damage or radiographic assessment score in patients with early stages of knee osteoarthritis (OA). METHOD: 40 patients, with early stage OA were enrolled, from whom the biopsies for histological and immunohistochemical studies were obtained from edge of the femoral condyle during the arthroscopy. Semi-quantitative computer based analysis was used to evaluate the proportion of staining in histological sections. RESULTS: No correlation was shown between the proportion of tissue stained for DDR2 and histological score or the results of radiographic assessment of tibiofemoral (TF) joint. There was a negative correlation between the proportion of tissue stained for DDR2 and radiographic grade of patellofemoral (PF) OA (Spearman r=-0.34; 95% CI -0.60 to -0.02; P=0.03). No correlation was shown between the proportion of tissue stained for proteoglycans and histological score or the results of radiographic assessment of TF and PF joints. A negative correlation was found between proportion of tissue stained for DDR2 and proteoglycans. Spearman r=-0.43; 95% CI=-0.66 to -0.12; P=0.006. CONCLUSION: Production of DDR2 in articular cartilage could be related to early stages of OA, as it is significantly correlated to decrease of staining for cartilage proteoglycans. The role of production of DDR2 in cartilage may be decreased in stages, where higher grades of OA are detected on the radiographs.

Mapping proteoglycan-bound water in cartilage: Improved specificity of matrix assessment using multiexponential transverse relaxation analysis.

Association of MR parameters with cartilage matrix components remains an area of ongoing investigation. Multiexponential analysis of nonlocalized transverse relaxation data has previously been used to quantify water compartments associated with matrix macromolecules in cartilage. We extend this to mapping the proteoglycan (PG)-bound water fraction in cartilage, using mature and young bovine nasal cartilage model systems, toward the goal of matrix component-specific imaging. PG-bound water fraction from mature and young bovine nasal cartilage was 0.31 ± 0.04 and 0.22 ± 0.06, respectively, in agreement with biochemically derived PG content and PG-to-water weight ratios. Fourier transform infrared imaging spectroscopic-derived PG maps normalized by water content (IR-PG(ww) ) showed spatial correspondence with PG-bound water fraction maps. Extensive simulation analysis demonstrated that the accuracy and precision of our determination of PG-bound water fraction was within 2%, which is well-within the observed tissue differences. Our results demonstrate the feasibility of performing imaging-based multiexponential analysis of transverse relaxation data to map PG in cartilage.

Ex vivo assessment of mouse cervical remodeling through pregnancy via 23Na MRS.

Preterm birth occurs in 12.5% of births in the United States and can lead to risk of infant death or to lifelong serious health complications. A greater understanding by which the two main processes, uterine contraction and cervical remodeling are regulated is required to reduce rates of preterm birth. The cervix must undergo extensive remodeling through pregnancy in preparation for parturition, the process of labor and delivery of young. One key aspect of this dynamic process is a change in the composition and abundance of glycosaminoglycans (GAGs) and proteoglycans within the extracellular matrix, which influences the loss of tensile strength or stiffness of the cervix during labor. 23Na NMR spectroscopy has previously been validated as a method to quantify GAGs in tissues. In the current study, the Na+ concentration was measured at several time points through pregnancy in mouse cervices using 23Na NMR spectroscopy. The Na+ concentration increased progressively during pregnancy and peaked one day before birth followed by a rapid decline after birth. The same trend was seen in GAGs as measured by a biochemical assay using independent cervix samples over the course of pregnancy. We suggest that monitoring the Na+ concentration via 23Na NMR spectroscopy can serve as an informative physiological marker in evaluating the stages of cervical remodeling ex vivo and warrants further investigation to determine its utility as a diagnostic tool for the identification of women at risk for impending preterm birth.

Assessment of human disc degeneration and proteoglycan content using T1rho-weighted magnetic resonance imaging.

STUDY DESIGN: T1rho relaxation was quantified and correlated with intervertebral disc degeneration and proteoglycan content in cadaveric human lumbar spine tissue. OBJECTIVE: To show the use of T1rho-weighted magnetic resonance imaging (MRI) for the assessment of degeneration and proteoglycan content in the human intervertebral disc. SUMMARY OF BACKGROUND DATA: Loss of proteoglycan in the nucleus pulposus occurs during early degeneration. Conventional MRI techniques cannot detect these early changes in the extracellular matrix content of the disc. T1rho MRI is sensitive to changes in proteoglycan content of articular cartilage and may, therefore, be sensitive to proteoglycan content in the intervertebral disc. METHODS: Intact human cadaveric lumbar spines were imaged on a clinical MR scanner. Average T1rho in the nucleus pulposus was calculated from quantitative T1rho maps. After MRI, the spines were dissected, and proteoglycan content of the nucleus pulposus was measured. Finally, the stage of degeneration was graded using conventional T2 images. RESULTS: T1rho decreased linearly with increasing degeneration (r = -0.76, P < 0.01) and age (r = -0.76, P < 0.01). Biochemical analysis revealed a strong linear correlation between T1rho and sulfated-glycosaminoglycan content. T1rho was moderately correlated with water content. CONCLUSIONS: Results from this study suggest that T1rho may provide a tool for the diagnosis of early degenerative changes in the disc. T1rho-weighted MRI is a noninvasive technique that may provide higher dynamic range than T2 and does not require a high static field or exogenous contrast agents.

The role of proteomics in the assessment of premature rupture of fetal membranes.

The presence and integrity of amniotic fluid is fundamental for the normal development of the human fetus during pregnancy. Its production rate changes throughout pregnancy and is mainly related to the functions of the different fetal, placental and amniotic compartments. Premature rupture of the membranes (PROM) occurs in about 5% of deliveries, with complications such as infection and preterm birth. The management of patients with PROM, regardless of gestational age, remains controversial, and it is therefore important to develop new biological tests in order to achieve accurate diagnoses by identifying the presence of specific amniotic fluid markers in vaginal environment. We recently showed the usefulness of amniotic fluid proteomics in identifying a series of peptides that were absent from the corresponding maternal plasma. Several peptides corresponded to fragments of plasma proteins. Two peptides, absent from plasma samples of pregnant women, were identified in amniotic fluid. They corresponded to the COOH-terminal parts of perlecan (SwissProt: P98160) and of agrin (SwissProt: O00468) protein cores, two major heparan sulfate proteoglycans of basement membranes. In this review we will discuss modern proteomic strategies that may improve the laboratory assessment of PROM, and will focus on some of the biochemical characteristics of agrin and perlecan fragments identified in amniotic fluid.

Correlation of radiographic and MRI parameters to morphological and biochemical assessment of intervertebral disc degeneration.

UNLABELLED: Degenerative disc disease (DDD) is a common finding in MRI scans and X-rays. However, their correlation to morphological and biochemical changes is not well established. In this study, radiological and MRI parameters of DDD were assessed and compared with morphological and biochemical findings of disc degeneration. Thirty-nine human lumbar discs (L1-S1), age 19-86 years, were harvested from eight cadavers. Within 48 h postmortem, MRIs in various spin-echo sequences and biplanar radiographs of intact spines were obtained. Individual discs with endplates were then sectioned in the mid-sagittal plane and graded according to the morphological appearance. Samples from the nucleus of each disc were harvested for biochemical analysis including water and proteoglycan contents. On MRIs, T2-signal intensity, Modic changes, disc extension beyond the interspace (DEBIT), nucleus pulposus shape, annular tears, osteophytes and endplate integrity were graded. On radiographs, an independent observer classified the parameters disc height, endplate sclerosis, osteophytes, Schmorl's nodes, intradiscal calcifications and endplate shape. General linear-regression models were used for statistical analysis. Backward elimination with a 10% significance cut-off level was used to identify the most significant parameters, which then were summed to create composite scores for radiography, MRI and the combination of both methods. The grading was performed by three observers, and a reliability analysis using Cronbach's alpha model was used to control interobserver agreement. The three radiographic parameters height-loss, osteophytes and intradiscal calcifications correlated significantly with the morphological degree of degeneration (p<0.001, R2=642). Significant differences of even one morphological grade could also be differentiated in the composite radiological score (p<0.05), except at the extremes between grades 1 and 2 and grades 4 and 5. All MRI parameters correlated significantly with the morphological grade (p<0.05); however Modic changes, T2-intensity and osteophytes accounted for 83% of the variation in the data. T2-signal intensity correlated significantly with H2O and proteoglycan content (p<0.001), and was best for detecting highly degenerated discs. Regression showed that the combined score was better correlated with the morphological grade (p<0.001, R2=775) than either the composite radiographic (p<0.001, R2=642) or composite MRI (p<0.001, R2=696) alone. Based on the combined score, a backwards elimination of the regression was performed, in which the parameters Modic changes, and T2-intensity loss (MRI) as well as calcifications (X-ray) accounted for 87% of the variability. The interobserver validation showed a high correlation for all three scores (Cronbach's alpha values ranging from 0.95 to 0.97). CONCLUSION: Selective imaging parameters and a newly created scoring scheme were found to correlate with disc degeneration as determined in a morphological manner. Surprisingly, radiographic parameters were able to distinguish different stages of degeneration, whereas MRI could only detect advanced stages of disc degeneration. We conclude that X-rays may remain a cost-effective, non-invasive in vivo-grading method to detect early disc degeneration, and, combined with MRI, correlate best with morphological and biochemical assessment of disc degeneration.

Quantitative ultrasonic assessment for detecting microscopic cartilage damage in osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent chronic conditions. The histological cartilage changes in OA include surface erosion and irregularities, deep fissures, and alterations in the staining of the matrix. The reversibility of these chondral alterations is still under debate. It is expected that clinical and basic science studies will provide the clinician with new scientific information about the natural history and optimal treatment of OA at an early stage. However, a reliable method for detecting microscopic changes in early OA has not yet been established. We have developed a novel system for evaluating articular cartilage, in which the acoustic properties of the articular cartilage are measured by introducing an ultrasonic probe into the knee joint under arthroscopy. The purpose of this study was to assess microscopic cartilage damage in OA by using this cartilage evaluation system on collagenase-treated articular cartilage in vivo and in vitro. Ultrasonic echoes from articular cartilage were converted into a wavelet map by wavelet transformation. On the wavelet map, the maximum magnitude and echo duration were selected as quantitative indices. Using these indices, the articular cartilage was examined to elucidate the relationships of the ultrasonic analysis with biochemical, biomechanical and histological analyses. In the in vitro study, the maximum magnitude decreased as the duration of collagenase digestion increased. Correlations were observed between the maximum magnitude and the proteoglycan content from biochemical findings, and the maximum magnitude and the aggregate modulus from biomechanical findings. From the histological findings, matrix staining of the surface layer to a depth of 500 mum was closely related to the maximum magnitude. In the in vivo study, the maximum magnitude decreased with increasing duration of the collagenase injection. There was a significant correlation between the maximum magnitude and the aggregate modulus. The evaluation system therefore successfully detected microscopic changes in degenerated cartilage with the use of collagen-induced OA.

Mechanical loading of chondrocytes embedded in 3D constructs: in vitro methods for assessment of morphological and metabolic response to compressive strain.

Mechanical loading of chondrocytes in 3D constructs has been used to investigate mechanotransduction and its potential for stimulating tissue-engineered cartilage repair. This chapter describes the preparation of 3D agarose or alginate constructs seeded with isolated chondrocytes and specific test rigs for applying gross compressive strain to individual constructs on a confocal microscope or for longer term compression of constructs cultured within an incubator. Experimental methods are described to quantify the level of cell deformation and the elaboration of extracellular matrix. The chapter thus provides an introduction to the experimental techniques used to examine chondrocyte mechanotransduction and downstream cell function.

Biomechanical, histologic and macroscopic assessment of articular cartilage in a sheep model of osteoarthritis.

OBJECTIVES: Our primary objective was to explore the full potential of the ovine medial meniscectomy (MMx) model of early osteoarthritis (OA) for studies to validate non-destructive articular cartilage (AC) assessments and therapeutic interventions. Our secondary objective was to re-evaluate the relationships between the different types of AC assessment after MMx in sheep. METHODS: Macroscopic assessments, dynamic shear modulus (G*), phase lag and AC thickness measurements were performed at a total of 5437 reference points on all six articular surfaces in four normal joints and 16 MMx ovine stifle (knee) joints. Comparisons with histologic assessments of gross structural damage, collagen organisation (birefringence) and proteoglycan content were possible at 702 of these points. RESULTS: Histologic gross structural damage and proteoglycan loss were seen throughout the joint with greatest severity (fibrillation) in closest proximity to the MMx site. Increases in AC (30-50%) thickness, reductions in G* (30-40%) and collagen birefringence intensity (15-30%) occurred more evenly throughout the joint. Macroscopic softening was evident only when G* declined by 80%. G* correlated with AC thickness (rho=-0.47), collagen organisation rho=0.44), gross structural damage (rho=-0.44) and proteoglycan content (rho=0.42). Multivariate analysis showed that collagen organisation contributed twice as much to dynamic shear modulus (t=6.66 as proteoglycan content (t=3.21). Collagen organisation (rho=0.11) and proteoglycan content (rho=0.09) correlated only weakly to phase lag. CONCLUSIONS: Macroscopic assessments were insensitive to AC softening suggesting that arthroscopic assessments of AC status might also perform poorly. Collagen integrity was more important for the maintenance of AC stiffness (G*) than proteoglycan content. The development of major AC softening and thickening throughout the joint following MMx suggested involvement of non-mechanical (e.g., protein and biochemical) chemical and cytokine mediated processes in addition to the disturbance in biomechanical loading. The ovine MMx model provides a setting in which the spectrum of AC changes associated with the initiation and progression of OA may be evaluated.

Qualitative and quantitative in vivo assessment of articular cartilage using magnetic resonance imaging.

The molecular organization and biochemical composition that give cartilage the viscoelasticity necessary for load distribution also convey unique magnetic resonance (MR) properties. In that context, MR imaging has the potential to detect cartilage degeneration and regeneration. Magnetization transfer (MT) imaging probes the exchange of magnetization between the bulk water pool and the water pool bound to macromolecules such as collagen and hence MT may be applied for evaluation of collagen integrity. In addition, Gd(DTPA)(2-)-induced T1 changes have been proposed as a surrogate marker of proteoglycan (PG) loss based on the principle that the paramagnetic agent Gd(DTPA)2- penetrates cartilage to an equilibrium concentration inversely proportional to the negative charge density (i.e. the PG concentration). Results obtained in vivo from MT and Gd(DTPA)(2-)-enhanced MRI acquisitions on the goat knee showed early signs of biochemical changes in response to a papain injection. A dose-dependent effect of papain was observed with both approaches over a wide range of PG depletion (i.e. T1 measurement) and collagen damage (i.e. MT measurement) as confirmed with post-mortem biochemistry and histology. Development of MRI protocols for non-invasive assessment of cartilage will facilitate diagnosis and monitoring of treatment efficacy in the clinic.

Spatial assessment of articular cartilage proteoglycans with Gd-DTPA-enhanced T1 imaging.

In Gd-DTPA-enhanced T(1) imaging of articular cartilage, the MRI contrast agent with two negative charges is understood to accumulate in tissue inversely to the negative charge of cartilage glycosaminoglycans (GAGs) of proteoglycans (PGs), and this leads to a decrease in the T(1) relaxation time of tissue relative to the charge in tissue. By assuming a constant relaxivity for Gd-DTPA in cartilage, it has further been hypothesized that the contrast agent concentration in tissue could be estimated from consecutive T(1) measurements in the absence or presence of the contrast agent. The spatial sensitivity of the technique was examined at 9.4 T in normal and PG-depleted bovine patellar cartilage samples. As a reference, spatial PG concentration was assessed with digital densitometry from safranin O-stained cartilage sections. An excellent linear correlation between spatial optical density (OD) of stained GAGs and T(1) with Gd-DTPA was observed in the control and chondroitinase ABC-treated cartilage specimens, and the MR parameter accounted for approximately 80% of the variations in GAG concentration within samples. Further, the MR-resolved Gd-DTPA concentration proved to be an even better estimate for PGs, with an improved correlation. However, the linear relation between MR parameters and PG concentration did not apply in the deep tissue, where MR measurements overestimated the PG content. While the absolute [Gd-DTPA] determination may be prone to error due to uncertainty of relaxivity in cartilage, or to other contributing factors such as variations in tissue permeability, the experimental evidence highlights the sensitivity of this technique to reflect spatial changes in cartilage PG concentration in normal and degenerated tissue.

Degradation of the cartilage collagen matrix associated with changes in chondrocytes in osteoarthrosis. Assessment by loss of background fluorescence and immunodetection of matrix components.

Articular cartilage damage and eventual loss is the primary pathological change seen in osteoarthrosis (OA). In this study we have investigated the link between turnover of the collagen matrix and changes in chondrocytes. The background fluorescence of articular cartilage, as indicated by its emission spectrum and resistance to extraction was generated by the slow non-enzymic modification of the collagen matrix by advanced glycation end products (AGEs). Assessment of changes in background fluorescence in sections of articular cartilage provided a narrative of collagen degradation. Patients without OA pathology typically had a uniform strong background fluorescence throughout the depth of the cartilage. Cartilage from OA patients showed a range of changes in background fluorescence dependent on depth from the articular surface and proximity to overt lesions. Loss of background fluorescence was centered on chondrocytes, more extensive near the surface and associated with detection of the proteoglycan epitope 7D4. Expression of type X collagen was seen in articular cartilage in the region of the interface of with subchondral bone in most OA patients but was not associated with prominent, pericellular, loss of background fluorescence. These observations are consistent with progressive cartilage damage in OA, whereby collagen turnover and loss of surface integrity is associated with chondrocyte activity similar to that seen in immature articular cartilage.

A comparative study of chemonucleolysis with recombinant human cathepsin L and chymopapain. A radiologic, histologic, and immunohistochemical assessment.

STUDY DESIGN: Investigation of the effects of recombinant human cathepsin L on intervertebral discs and comparison with the effects of chymopapain. OBJECTIVE: To evaluate the effects of cathepsin L on intervertebral discs as an agent for chemonucleolysis. SUMMARY BACKGROUND DATA: Cathepsin L is a typical cysteine proteinase that belongs to the papain superfamily. It plays a major role in intracellular proteolysis and is not believed to induce anaphylactic reactions. METHODS: In vivo: Rabbit intervertebral discs were injected with recombinant human cathepsin L, its buffer solution, and chymopapain. After 1, 4, and 16 weeks the animals were killed, and radiologic and histologic examinations were performed. In vitro: The enzymatic actions of recombinant human cathepsin L and chymopapain on human intervertebral disc proteoglycans were examined immunohistochemically using antiproteoglycan antibodies. RESULTS: In rabbit models, roentgenography showed that disc spaces treated with cathepsin L and chymopapain had become narrower 1 week after injection. Histologically, loss of safranin-O staining was observed in the anulus fibrosus of discs treated with cathepsin L. After 16 weeks, nucleus pulposus had regenerated with chondrocyte-like cells, and the safranin-O staining characteristics of the matrix also had recovered. In an immunohistochemical study, all components of the proteoglycan stained weakly after chymopapain digestion. After cathepsin L digestion, unsulfated chondroitin and core protein staining was weaker, but the chondroitin 6-sulfate staining was unaffected. CONCLUSIONS: Cathepsin L seems to be an effective agent for chemonucleolysis. Its enzymatic action on proteoglycan appears to be different from that of chymopapain.

The assessment of chondrocyte proteoglycan metabolism using molecular sieve column chromatography as compared to three commonly utilized techniques.

OBJECTIVE: Various methods have been used to quantify [35S] sulfate incorporation into proteoglycans and separate this from unincorporated [35S] sulfate. The methods currently used have not been compared for accuracy or precision, ease of completion and relative economics. This experiment evaluated and compared cetylpyridinium chloride (CPC) precipitation, potassium acetate (KAc) precipitation, rapid filtration, modified dye-binding (AB) technique to molecular weight exclusion chromatography (PD-10) as techniques to quantitate chondrocyte proteoglycan metabolism. METHODS: Each of the first three techniques was compared to the PD-10 technique as a 'gold standard' based on this technique's accuracy, precision and repeatability in molecular weight exclusion utilizing sieve chromatography. Articular cartilage was harvested, cultured and labeled with [35S] sulfate in a routine manner. Seventeen samples were processed and each was analyzed by the four different methodologies. Linear regression analysis was used to predict the linear relationship of PD-10 chromatography to the other methods (CPC, KAc, and AB) in the amount of proteoglycan synthesis in a pulse chase experiment. RESULTS: The KAc and AB methods had a significant linear relationship to the PD-10 method but the CPC did not. The AB technique was much easier than the KAc method to complete although the relative economics were similar per sample, when capital equipment costs were not considered. CONCLUSIONS: Either the KAc or AB methods compared favorably to the PD-10 method, although the AB procedure was much easier, and was more precise and more accurate. The CPC method did not compare favorably to the PD-10 method in precision or accuracy although it was easier and cost effective.