In vitro resistance of articular chondrocytes to 5-Aminolevulinic acid based photodynamic therapy

OBJECTIVE: 5-Aminolevulinic acid based photodynamic therapy (5-ALA-PDT) has revealed promising results in the treatment of inflammatory joint diseases due to the sensitivity of inflamed synovial tissue. For 5-ALA-PDT to be safe and beneficial for intra-articular applications, resistance of chondrocytes is essential to prevent cartilage damage. As no data yet exist, the aim of the present study was to assess in vitro the response of the chondrocytes to 5-ALA-PDT and to compare with osteoblasts and synovial tissue derived cells. METHODS: Bovine articular chondrocytes, osteoblasts, and synovial cells were subjected to 5-ALA-PDT in cell culture. The PpIX accumulation and the function of the cells were assessed for up to 12 days. RESULTS: Bovine chondrocytes showed lower PpIX fluorescence upon incubation with 5-ALA (0.0-2.0 mM) for 4 hours as compared to osteoblasts and synovial cells suggesting a low PpIX accumulation. After incubation with 0.5 mM 5-ALA and application of light at a dose of 20 J/cm2, chondrocytes were functionally not affected (collagen type II and aggrecan mRNA, glycosaminoglycan synthesis) whereas a decrease in the proportion of viable cells was observed in osteoblasts and synovial cells (2+/-2% and 14+/-8%, respectively; chondrocytes 91+/-13%). Chondrocytes showed a 58% reduction of 5-ALA uptake using [3H]5-ALA as compared to osteoblasts and a lower mitochondrial content as assessed by the activity of the mitochondrial marker enzyme citrate synthase (9.2+/- 3.6 mU/mg protein) than osteoblasts (32.6+/-10.5 mU/mg) and synovial cells (60.0+/-10.8 mU/mg). The reduced uptake of 5-ALA and/or the low mitochondrial content, an adaptation to their in vivo environment and the site of PpIX synthesis, presumably explains the lower PpIX content in chondrocytes and their resistance against 5-ALA-PDT. CONCLUSION: 5-ALA-PDT might represent a treatment strategy in inflammatory joint diseases without endangering the cartilage function. However, further in vitro and in vivo experiments are required to confirm this data in the authentic environment of chondrocytes, the articular cartilage.

Advanced morphological and biochemical magnetic resonance imaging of cartilage repair procedures in the knee joint at 3 Tesla

Morphological and biochemical magnetic resonance imaging (MRI) is due to high field MR systems, advanced coil technology, and sophisticated sequence protocols capable of visualizing articular cartilage in vivo with high resolution in clinical applicable scan time. Several conventional two-dimensional (2D) and three-dimensional (3D) approaches show changes in cartilage structure. Furthermore newer isotropic 3D sequences show great promise in improving cartilage imaging and additionally in diagnosing surrounding pathologies within the knee joint. Functional MR approaches are additionally able to provide a specific measure of the composition of cartilage. Cartilage physiology and ultra-structure can be determined, changes in cartilage macromolecules can be detected, and cartilage repair tissue can thus be assessed and potentially differentiated. In cartilage defects and following nonsurgical and surgical cartilage repair, morphological MRI provides the basis for diagnosis and follow-up evaluation, whereas biochemical MRI provides a deeper insight into the composition of cartilage and cartilage repair tissue. A combination of both, together with clinical evaluation, may represent a desirable multimodal approach in the future, also available in routine clinical use.

Magnetization transfer contrast and T2 mapping in the evaluation of cartilage repair tissue with 3T MRI

PURPOSE: To use magnetization transfer (MT) imaging in the visualization of healthy articular cartilage and cartilage repair tissue after different cartilage repair procedures, and to assess global as well as zonal values and compare the results to T2-relaxation. MATERIALS AND METHODS: Thirty-four patients (17 after microfracture [MFX] and 17 after matrix-associated autologous cartilage transplantation [MACT]) were examined with 3T MRI. The MT ratio (MTR) was calculated from measurements with and without MT contrast. T2-values were evaluated using a multiecho, spin-echo approach. Global (full thickness of cartilage) and zonal (deep and superficial aspect) region-of-interest assessment of cartilage repair tissue and normal-appearing cartilage was performed. RESULTS: In patients after MFX and MACT, the global MTR of cartilage repair tissue was significantly lower compared to healthy cartilage. In contrast, using T2, cartilage repair tissue showed significantly lower T2 values only after MFX, whereas after MACT, global T2 values were comparable to healthy cartilage. For zonal evaluation, MTR and T2 showed a significant stratification within healthy cartilage, and T2 additionally within cartilage repair tissue after MACT. CONCLUSION: MT imaging is capable and sensitive in the detection of differences between healthy cartilage and areas of cartilage repair and might be an additional tool in biochemical cartilage imaging. For both MTR and T2 mapping, zonal assessment is desirable.

Magnetic resonance imaging for diagnosis and assessment of cartilage defect repairs

Clinical magnetic resonance imaging (MRI) is the method of choice for the non-invasive evaluation of articular cartilage defects and the follow-up of cartilage repair procedures. The use of cartilage-sensitive sequences and a high spatial-resolution technique enables the evaluation of cartilage morphology even in the early stages of disease, as well as assessment of cartilage repair. Sequences that offer high contrast between articular cartilage and adjacent structures, such as the fat-suppressed, 3-dimensional, spoiled gradient-echo sequence and the fast spin-echo sequence, are accurate and reliable for evaluating intrachondral lesions and surface defects of articular cartilage. These sequences can also be performed together in reasonable examination times. In addition to morphology, new MRI techniques provide insight into the biochemical composition of articular cartilage and cartilage repair tissue. These techniques enable the diagnosis of early cartilage degeneration and help to monitor the effect and outcome of various surgical and non-surgical cartilage repair therapies.

Cartilage T2 assessment at 3-T MR imaging: in vivo differentiation of normal hyaline cartilage from reparative tissue after two cartilage repair procedures--initial experience

PURPOSE: To prospectively compare cartilage T2 values after microfracture therapy (MFX) and matrix-associated autologous chondrocyte transplantation (MACT) repair procedures. MATERIALS AND METHODS: The study had institutional review board approval by the ethics committee of the Medical University of Vienna; informed consent was obtained. Twenty patients who underwent MFX or MACT (10 in each group) were enrolled. For comparability, patients of each group were matched by mean age (MFX, 40.0 years +/- 15.4 [standard deviation]; MACT, 41.0 years +/- 8.9) and postoperative interval (MFX, 28.6 months +/- 5.2; MACT, 27.4 months +/- 13.1). Magnetic resonance (MR) imaging was performed with a 3-T MR imager, and T2 maps were calculated from a multiecho spin-echo measurement. Global, as well as zonal, quantitative T2 values were calculated within the cartilage repair area and within cartilage sites determined to be morphologically normal articular cartilage. Additionally, with consideration of the zonal organization, global regions of interest were subdivided into deep and superficial areas. Differences between cartilage sites and groups were calculated by using a three-way analysis of variance. RESULTS: Quantitative T2 assessment of normal native hyaline cartilage showed similar results for all patients and a significant trend of increasing T2 values from deep to superficial zones (P < .05). In cartilage repair areas after MFX, global mean T2 was significantly reduced (P < .05), whereas after MACT, mean T2 was not reduced (P > or = .05). For zonal variation, repair tissue after MFX showed no significant trend between different depths (P > or = .05), in contrast to repair tissue after MACT, in which a significant increase from deep to superficial zones (P < .05) could be observed. CONCLUSION: Quantitative T2 mapping seems to reflect differences in repair tissues formed after two surgical cartilage repair procedures. (c) RSNA, 2008.

Subcellular distribution of the insulin-like growth factor (IGF) binding proteins (IGFBPs) 2 and 3 in articular chondrocytes

The insulin-like growth factor (IGF) is a major anabolic regulator in articular cartilage. The IGF-binding proteins (IGFBPs) are increased during osteoarthritis (OA), but the function of the later proteins remains unknown. In general, the IGFBPs are pluripotential effectors capable of IGF regulation and of acting on their own to control key cell functions, including survival and proliferation. The independent functions are often associated with their cell location, and therefore this study explores the distribution of IGFBP-2 and IGFBP-3 in articular chondrocytes. Immunohistochemistry was used to localize IGFBP-2 in normal human articular cartilage. Bovine chondrocytes were used for subcellular fractionation (hypotonic cell lysis) under nonreducing conditions and nuclear purification (centrifugation on sucrose cushions). Cell fraction markers and IGFBPs were assayed in the subcellular fractions by Western immunoblot. The IHC results showed association of IGFBP-2 with chondrocytes, but not with the nuclei. Subcellular fractionation of isolated chondrocytes yielded intact nuclei as assessed at the light microscopic level; the nuclear marker histone H1 was exclusively associated with this fraction. More than 90% of the cytoplasmic marker GAPDH and all the detectable IGFBP-2 were in the cytoplasmic fraction. Immunoreactive IGFBP-3 was found in the cytoplasmic and peri-nuclear/nuclear fractions. Chondrocytes contain intracellular IGFBP-2 and IGFBP-3 but only IGFBP-3 is associated with nuclei. This suggests the hypothesis that the actions of these IGFBPs in articular cartilage extend beyond the classic modulation of IGF receptor action.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

An educational review of cartilage repair: precepts & practice - myths & misconceptions - progress & prospects.

OBJECTIVE The repair of cartilaginous lesions within synovial joints is still an unresolved and weighty clinical problem. Although research activity in this area has been indefatigably sustained, no significant progress has been made during the past decade. The aim of this educational review is to heighten the awareness amongst students and scientists of the basic issues that must be tackled and resolved before we can hope to escape from the whirlpool of stagnation into which we have fallen: cartilage repair redivivus! DESIGN Articular-cartilage lesions may be induced traumatically (e.g., by sports injuries and occupational accidents) or pathologically during the course of a degenerative disease (e.g., osteoarthritis). This review addresses the biological basis of cartilage repair and surveys current trends in treatment strategies, focussing on those that are most widely adopted by orthopaedic surgeons [viz., abrasive chondroplasty, microfracturing/microdrilling, osteochondral grafting and autologous-chondrocyte implantation (ACI)]. Also described are current research activities in the field of cartilage-tissue engineering, which, as a therapeutic principle, holds more promise for success than any other experimental approach. RESULTS AND CONCLUSIONS Tissue engineering aims to reconstitute a tissue both structurally and functionally. This process can be conducted entirely in vitro, initially in vitro and then in vivo (in situ), or entirely in vivo. Three key constituents usually form the building blocks of such an approach: a matrix scaffold, cells, and signalling molecules. Of the proposed approaches, none have yet advanced beyond the phase of experimental development to the level of clinical induction. The hurdles that need to be surmounted for ultimate success are discussed.

Accuracy of cartilage-specific 3-Tesla 3D-DESS magnetic resonance imaging in the diagnosis of chondral lesions: comparison with knee arthroscopy

BACKGROUND Arthroscopy is considered as "the gold standard" for the diagnosis of traumatic intraarticular knee lesions. However, recent developments in magnetic resonance imaging (MRI) now offer good opportunities for the indirect assessment of the integrity and structural changes of the knee articular cartilage. The study was to investigate whether cartilage-specific sequences on a 3-Tesla MRI provide accurate assessment for the detection of cartilage defects. METHODS A 3-Tesla (3-T) MRI combined with three-dimensional double-echo steady-state (3D-DESS) cartilage specific sequences was performed on 210 patients with knee pain prior to knee arthroscopy. Sensitivity, specificity, and positive and negative predictive values of magnetic resonance imaging were calculated and correlated to the arthroscopic findings of cartilaginous lesions. Lesions were classified using the modified Outerbridge classification. RESULTS For the 210 patients (1260 cartilage surfaces: patella, trochlea, medial femoral condyle, medial tibia, lateral femoral condyle, lateral tibia) evaluated, the sensitivities, specificities, positive predictive values, and negative predictive values of 3-T MRI were 83.3, 99.8, 84.4, and 99.8 %, respectively, for the detection of grade IV lesions; 74.1, 99.6, 85.2, and 99.3 %, respectively, for grade III lesions; 67.9, 99.2, 76.6, and 98.2 %, respectively, for grade II lesions; and 8.8, 99.5, 80, and 92 %, respectively, for grade I lesions. CONCLUSIONS For grade III and IV lesions, 3-T MRI combined with 3D-DESS cartilage-specific sequences represents an accurate diagnostic tool. For grade II lesions, the technique demonstrates moderate sensitivity, while for grade I lesions, the sensitivity is limited to provide reliable diagnosis compared to knee arthroscopy.

Age-Independent Cartilage Generation for Synovium-Based Autologous Chondrocyte Implantation.

The articular cartilage layer of synovial joints is commonly lesioned by trauma or by a degenerative joint disease. Attempts to repair the damage frequently involve the performance of autologous chondrocyte implantation (ACI). Healthy cartilage must be first removed from the joint, and then, on a separate occasion, following the isolation of the chondrocytes and their expansion in vitro, implanted within the lesion. The disadvantages of this therapeutic approach include the destruction of healthy cartilage-which may predispose the joint to osteoarthritic degeneration-the necessarily restricted availability of healthy tissue, the limited proliferative capacity of the donor cells-which declines with age-and the need for two surgical interventions. We postulated that it should be possible to induce synovial stem cells, which are characterized by high, age-independent, proliferative and chondrogenic differentiation capacities, to lay down cartilage within the outer juxtasynovial space after the transcutaneous implantation of a carrier bearing BMP-2 in a slow-release system. The chondrocytes could be isolated on-site and immediately used for ACI. To test this hypothesis, Chinchilla rabbits were used as an experimental model. A collagenous patch bearing BMP-2 in a slow-delivery vehicle was sutured to the inner face of the synovial membrane. The neoformed tissue was excised 5, 8, 11 and 14 days postimplantation for histological and histomorphometric analyses. Neoformed tissue was observed within the outer juxtasynovial space already on the 5th postimplantation day. It contained connective and adipose tissues, and a central nugget of growing cartilage. Between days 5 and 14, the absolute volume of cartilage increased, attaining a value of 12 mm(3) at the latter juncture. Bone was deposited in measurable quantities from the 11th day onwards, but owing to resorption, the net volume did not exceed 1.5 mm(3) (14th day). The findings confirm our hypothesis. The quantity of neoformed cartilage that is deposited after only 1 week within the outer juxtasynovial space would yield sufficient cells for ACI. Since the BMP-2-bearing patches would be implanted transcutaneously in humans, only one surgical or arthroscopic intervention would be called for. Moreover, most importantly, sufficient numbers of cells could be generated in patients of all ages.

Surgical dislocation of the hip for the fixation of acetabular fractures

Surgical dislocation of the hip in the treatment of acetabular fractures allows the femoral head to be safely displaced from the acetabulum. This permits full intra-articular acetabular and femoral inspection for the evaluation and potential treatment of cartilage lesions of the labrum and femoral head, reduction of the fracture under direct vision and avoidance of intra-articular penetration with hardware. We report 60 patients with selected types of acetabular fracture who were treated using this approach. Six were lost to follow-up and the remaining 54 were available for clinical and radiological review at a mean follow-up of 4.4 years (2 to 9). Substantial damage to the intra-articular cartilage was found in the anteromedial portion of the femoral head and the posterosuperior aspect of the acetabulum. Labral lesions were predominantly seen in the posterior acetabular area. Anatomical reduction was achieved in 50 hips (93%) which was considerably higher than that seen in previous reports. There were no cases of avascular necrosis. Four patients subsequently required total hip replacement. Good or excellent results were achieved in 44 hips (81.5%). The cumulative eight-year survivorship was 89.0% (95% confidence interval 84.5 to 94.1). Significant predictors of poor outcome were involvement of the acetabular dome and lesions of the femoral cartilage greater than grade 2. The functional mid-term results were better than those of previous reports. Surgical dislocation of the hip allows accurate reduction and a predictable mid-term outcome in the management of these difficult injuries without the risk of the development of avascular necrosis.

Vascular supply to the acetabular labrum

The acetabular labrum plays an important role in hip joint stability and protection of the articular cartilage of the hip. Despite this, few investigators have evaluated its microscopic vasculature and, to our knowledge, none has assessed its macroscopic blood supply. The purposes of this study were to identify the origin and course of the vascular supply to the acetabular labrum to determine if this blood supply is affected by a labral tear.

Numerical assessment on the effective mechanical stimuli for matrix-associated metabolism in chondrocyte-seeded constructs

The self-regeneration capacity of articular cartilage is limited, due to its avascular and aneural nature. Loaded explants and cell cultures demonstrated that chondrocyte metabolism can be regulated via physiologic loading. However, the explicit ranges of mechanical stimuli that correspond to favourable metabolic response associated with extracellular matrix (ECM) synthesis are elusive. Unsystematic protocols lacking this knowledge produce inconsistent results. This study aims to determine the intrinsic ranges of physical stimuli that increase ECM synthesis and simultaneously inhibit nitric oxide (NO) production in chondrocyte-agarose constructs, by numerically re-evaluating the experiments performed by Tsuang et al. (2008). Twelve loading patterns were simulated with poro-elastic finite element models in ABAQUS. Pressure on solid matrix, von Mises stress, maximum principle stress and pore pressure were selected as intrinsic mechanical stimuli. Their development rates and magnitudes at the steady state of cyclic loading were calculated with MATLAB at the construct level. Concurrent increase in glycosaminoglycan and collagen was observed at 2300 Pa pressure and 40 Pa/s pressure rate. Between 0-1500 Pa and 0-40 Pa/s, NO production was consistently positive with respect to controls, whereas ECM synthesis was negative in the same range. A linear correlation was found between pressure rate and NO production (R = 0.77). Stress states identified in this study are generic and could be used to develop predictive algorithms for matrix production in agarose-chondrocyte constructs of arbitrary shape, size and agarose concentration. They could also be helpful to increase the efficacy of loading protocols for avascular tissue engineering. Copyright (c) 2010 John Wiley \& Sons, Ltd.

Association between cam-type deformities and magnetic resonance imaging-detected structural hip damage: a cross-sectional study in young men

Objective Femoroacetabular impingement may be a risk factor for hip osteoarthritis in men. An underlying hip deformity of the cam type is common in asymptomatic men with nondysplastic hips. This study was undertaken to examine whether hip deformities of the cam type are associated with signs of hip abnormality, including labral lesions and articular cartilage damage, detectable on magnetic resonance imaging (MRI). Methods In this cross-sectional, population-based study in asymptomatic young men, 1,080 subjects underwent clinical examination and completed a self-report questionnaire. Of these subjects, 244 asymptomatic men with a mean age of 19.9 years underwent MRI. All MRIs were read for cam-type deformities, labral lesions, cartilage thickness, and impingement pits. The relationship between cam-type deformities and signs of joint damage were examined using logistic regression models adjusted for age and body mass index. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were determined. Results Sixty-seven definite cam-type deformities were detected. These deformities were associated with labral lesions (adjusted OR 2.77 [95% CI 1.31, 5.87]), impingement pits (adjusted OR 2.9 [95% CI 1.43, 5.93]), and labral deformities (adjusted OR 2.45 [95% CI 1.06, 5.66]). The adjusted mean difference in combined anterosuperior femoral and acetabular cartilage thickness was −0.19 mm (95% CI −0.41, 0.02) lower in those with cam-type deformities compared to those without. Conclusion Our findings indicate that the presence of a cam-type deformity is associated with MRI-detected hip damage in asymptomatic young men.

Hip arthroscopy after surgical hip dislocation: is predictive imaging possible?

Our purpose was to study the sensitivity, specificity, and predictive values for hip adhesions, labral tears, and articular cartilage lesions in patients who had open treatment for femoroacetabular impingement, had persistent symptoms, and had both magnetic resonance arthrography (MRA) with radial slices and hip arthroscopy.