Histological-histochemical Grading System Research Articles

Comparison of Different Methods of Semiquantitative Assessment and Subjective Scores for Retropatellar Articular Cartilage Evaluation in Advancing Osteoarthritis.

cartilage layer thickness, composition of the extracellular matrix (ECM), geometry and configuration of retropatellar cartilage partially differ significantly from those found at other locations and are essential for patellofemoral biomechanics. 119 serial medial and lateral patella facet samples of patients undergoing implantation of a total knee endoprosthesis of areas showing mild (Group A, macroscopically ICRS Grade 1b) respectively advanced (Group B, macroscopically ICRS Grade 3a/3b) (38 each) osteoarthritis according to the histological-histochemical grading system (HHGS) were compared with 22 healthy biopsies by means of immunohistochemistry and histology investigations. We quantified our results on the gene expression of collagen type I and II and aggrecan with real-time (RT)-PCR rsp using colourimetry for proteoglycan content. The digitized images of histology and immunohistochemistry staining with Safranin O, Alcian blue, PAS, Masson Goldner, immunostaining, e.g. for collagen I and II were also analyzed with Photoshop software. The most significant decrease in staining intensity was revealed for Safranin-O staining in Group B at the lateral patellar facet, and the most relevant increase was for Col I staining at the same site. The lateral patella site in Group B also showed the highest increase in the ratio of expression indices for the genes Col1A1 and the reference gene following the equation 2-ΔCt with a quotient of 29.6. 1. Comparisons of our retropatellar cartilage analysis with femoral and tibial studies utilizing similar techniques show significant differences. 2. Cartilage layer thickness, ECM composition, geometry and configuration are essential for patellofemoral biomechanics. 3. Consequently, there is a need for diversified approaches towards retropatellar surface during TKA as well as for advanced cartilage restoration techniques.

Comparative Assessment of Primary Osteoarthritis Progression Using Conventional Histopathology, Polarized Light Microscopy, and Immunohistochemistry.

Evaluation of collagen orientation and arrangement in articular cartilage can improve our understanding of primary osteoarthritis (OA) progression and targeted therapies. Our goal was to determine if polarized light microscopy (PLM) for collagen organization is useful in identifying early primary OA features in comparison to current standard histopathological methods. Osteochondral specimens from 90 total knee arthroplasty patients with relatively preserved lateral femoral condyle were scored using (1) histological-histochemical grading system (HHGS); (2) Osteoarthritis Research Society International (OARSI); (3) PLM-Changoor system for repair cartilage, scores ranging between 0 (totally disorganized cartilage) and 5 (healthy adult cartilage); and (4) new PLM system for primary OA cartilage with superficial zone PLM (PLM-SZ) and deep zone PLM (PLM-DZ) scores, each ranging between 0 (healthy adult SZ and DZ collagen organization) and 4 (total loss of collagen organization). Serial sections were stained for collagen I and II antibodies. Spearman correlation coefficients (rs) were determined. The associations between: (1) PLM-Changoor and HHGS or OARSI were weak (rs = -0.36) or moderate (rs = -0.56); (2) PLM-SZ and HHGS or OARSI were moderate (rs = 0.46 or rs = 0.53); and (3) PLM-DZ and HHGS or OARSI were poor (rs = 0.31 or rs = 0.21), respectively. Specimens exhibiting early and mild OA (HHGS < 5 and OARSI < 8.6) had PLM-SZ and PLM-DZ scores between 0 and 4 and between 0 and 3, respectively, and indicated new histopathological features not currently considered by HHGS/OARSI. PLM was effective at identifying early SZ and DZ collagen alterations that were not evident in the traditional scoring systems. Incorporating PLM scores and/or additional HHGS/OARSI features can help improve characterization of early primary OA cartilage.

Laser photobiomodulation for cartilage defect in animal models of knee osteoarthritis: a systematic review and meta-analysis.

To review and assess the efficacy of laser photobiomodulation for cartilage defect in animal models of knee osteoarthritis (KOA). Medline, Web of Science, and EMBASE were searched. Studies were considered if the global quality score of cartilage were parallelly reported between laser and untreated control groups. The methodological quality of each study was assessed using a modified 10-item checklist. The effect size was estimated by standardized mean difference (SMD) and pooled based on the random-effects model. Stratified analysis and regression analysis were conducted to partition potential heterogeneity. An adjusted significant level of 0.01 was acceptable. Five hundred eight initial search recordings were identified, of which 14 studies (including 274 animals) were included for quantitative analysis. The global quality scores mostly weighted by the structural integrity and chondrocyte distribution were measured by different four scales including Histologic Histochemical Grading System (HHGS), Osteoarthritis Research Society International (OARSI), Pineda, and Huang. There were considerable variances on laser parameters and irradiation time among those included studies. Overall, a moderate level of methodological qualities was determined. The synthesis results indicated that the SMD effect size was significantly larger in HHGS (z = 2.61, P = 0.01) and Huang (z = 4.90, P < 0.01) groups. Stratified by irradiance, SMD of low (< 1W/cm2) but not high (≥ 1W/cm2) level estimated significant difference (z = 5.62, P < 0.01). Meta-regression identified a significant association for SMDs and irradiation time (P < 0.01). Yet, Egger's test detected small study effect (P < 0.01). No individual study with significant variance was found in homogeneity tests. The results demonstrated the positive effect of laser photobiomodulation for cartilage defect in animal models of KOA under proper irradiance and adequate irradiation time.

Does a high-fat diet affect the development and progression of osteoarthritis in mice? A systematic review

AimsThe aim of this study was to systematically review the literature for evidence of the effect of a high-fat diet (HFD) on the onset or progression of osteoarthritis (OA) in mice.MethodsA literature search was performed in PubMed, Embase, Web of Science, and Scopus to find all studies on mice investigating the effects of HFD or Western-type diet on OA when compared with a control diet (CD). The primary outcome was the determination of cartilage loss and alteration. Secondary outcomes regarding local and systemic levels of proteins involved in inflammatory processes or cartilage metabolism were also examined when reported.ResultsIn total, 14 publications met our inclusion criteria and were included in our review. Our meta-analysis showed that, when measured by the modified Mankin Histological-Histochemical Grading System, there was a significantly higher rate of OA in mice fed a HFD than in mice on a CD (standardized mean difference (SMD) 1.27, 95% confidence interval (CI) 0.63 to 1.91). Using the Osteoarthritis Research Society International (OARSI) score, there was a trend towards HFD causing OA (SMD 0.78, 95% CI -0.04 to 1.61). In terms of OA progression, a HFD consistently worsened the progression of surgically induced OA when compared with a CD. Finally, numerous inflammatory cytokines such as tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, and leptin, among others, were found to be altered by a HFD.ConclusionA HFD seems to induce or exacerbate the progression of OA in mice. The metabolic changes and systemic inflammation brought about by a HFD appear to be key players in the onset and progression of OA.Cite this article: Bone Joint Res 2019;8:582–592.

Regional differences of tibial and femoral cartilage in the chondrocyte gene expression, immunhistochemistry and composite in different stages of osteoarthritis.

The function of articular cartilage as an avascular tissue is mainly served by collagen type II and proteoglycan molecules. Within this matrix homeostasis between production and breakdown of the matrix is exceptionally sensitive. The current study was conducted to identify regional differences in specific alterations in cartilage composition during the osteoarthritic process of the human knee joint. Therefor the changes in the expression of the key molecules of the extracellular matrix were measured in dependence of the anatomical side (femoral vs tibial) and associated with immunohistochemistry and quantitative measurement. 60 serial osteochondral femoral condyle and the tibial plateau samples of patients undergoing implantation of total knee endoprosthesis of areas showing mild (Group A, macroscopically ICRS grade 1b) respectively advanced (Group B, macroscopically ICRS grade 3a/3b) (30 each) osteoarthritis according to the histological-histochemical grading system (HHGS) were compared with 20 healthy biopsies with immunohistochemistry and histology. We quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorometrically. In group A slightly increased colour intensity was found for collagen II in deeper layers, suggesting a persisting but initially still intact repair process. But especially on the medial tibia plateau the initial Col II increase in gene expression is followed by a decrease leading to the lowest over all Col II expression on the medial plateau, here especially in the central part. There in late stage diseases the collagen type I expression was also more pronounced. Markedly decreased safranin O staining intensity was observed in the radial zone and less reduced intensity in the transitional zone with loss of zonal anatomy in 40% of the specimens in group A and all specimens in group B. Correlation between colorometrically analysed proteoglycan GAG content and aggrecan Real Time PCR is mainly weak. Tibial and femoral cartilage in contrast to patellar cartilage both are preferential exposed to compressive stresses, but presence of menisci affects the load distribution at the tibial side, which creates varying conditions for the different cartilage surfaces in the knee. As directly measured Poissońs ratio in tibial cartilage is higher but Younǵs modulus is lower than in femoral cartilage, different resulting feedback amplification loops interact with proceeding cartilage damage. The initial loss of aggrecan may support Matrix metalloproteinases (Mmps) in the access to the collagen network and the considerably differing mechanical properties at both joint surfaces result in varying increased synthesis and release of matrix degrading enzymes. The present study has identified a selection of events which reflect the response of cartilage structure and composite, chondrocytes itself and their productivity to changes in mechanical stress depending on the anatomical site.

Histopathology of naturally occurring and surgically induced osteoarthritis in mice

The morphology of lesions in mouse models of osteoarthritis (OA) has not been comprehensively characterized, in part because current histological assessments of OA focus primarily on articular cartilage (AC). In the present study, sections of murine stifle joints with naturally occurring (aged animals) and surgically induced (destabilized medial meniscus, DMM) OA were examined using a newly developed histological grading scheme that includes quantitative measurements and semiquantitative grades to evaluate multiple joint tissues. The data collected was analyzed using Principal Components Analysis (PCA); factor scores for each joint were generated. Individual parameters and factor scores were compared between surgical groups and among age groups. For comparison, the original Mankin Histological-Histochemical Grading System (HHGS) also was applied. Overall, lesions were most severe in the medial tibial plateaus. Significant changes in AC and neighboring bone were identified in surgically induced models and in naturally occurring disease. Mean factor scores provided a comprehensive evaluation of joint changes. An important new finding was that chondrocyte cell death within the AC was a commonly identified lesion and its extent significantly increased with age. While the Mankin HHGS detected significant overall differences in OA severity between surgical groups, it was not sensitive in detecting age-related differences, nor did it provide information regarding changes in individual tissues. These results demonstrate the utility of this newly developed murine OA grading scheme in identifying lesions in AC and in other joint tissues. Surgically induced changes were similar to those occurring naturally with aging.

Histopathology grading systems for characterisation of human knee osteoarthritis – reproducibility, variability, reliability, correlation, and validity

To determine the reliability, reproducibility, variability and validity of the Osteoarthritis Cartilage Histopathology (OACH) assessment system and Mankin Histological-Histochemical Grading System (HHGS) when applied to the characterisation of the osteoarthritic human knee. Osteoarthritic knees of 10 patients undergoing unilateral knee arthroplasty were assessed, and assigned Kellgren-Lawrence and Line Drawing Atlas (LDA) radiology scores. The tibial plateaux were scored using the Modified Collins (MC) and Société Française d'Arthroscopie (SFA). Three observers twice scored both the OACH and HHGS across a single complete medial and lateral tibial plateau transect taken to include the region with the most severe osteoarthritis (OA) lesion. Intra- and inter-observer reliability, reproducibility, variability and validity were assessed, and the correlation between the two histopathology scoring systems was calculated. Both histopathology scoring systems were determined to be reliable and reproducible exhibiting similar variability, when applied to characterise OA specimens sampled from a well defined patient group with knee OA. A strong correlation between the mean OACH and mean HHGS scores was identified (Spearman's ρ 0.980, P<0.0001). Both scoring systems implemented provide useful measures in the characterisation of knee osteoarthritis. It is of note that an additional parameter within the OACH score over the HHGS defines the extent of the disease, where the HHGS is a grade attributed to the most representative level of the biological aggression within the OA lesions. This study has confirmed the OACH system's utility in human knee OA and is supported by a significant correlation with the established HHGS.

Correlation between arthroscopic and histopathological grading systems of articular cartilage lesions in knee osteoarthritis

Arthroscopic and particularly histopathological assessments have been used to evaluate alterations of knee cartilage in osteoarthritis (OA). The aim of this study was to examine the correlation between an arthroscopic method to grade the severity of chondropathies and the histological/histochemical grading system (HHGS) applied to the corresponding articular cartilage areas in knee OA. The articular cartilage surface was examined by chondroscopy using the Beguin and Locker severity criteria, analysing the lesions in 72 chondroscopic areas. Afterwards, samples were obtained by dividing the cartilage surface of the medial tibiofemoral compartment of three OA knee joints into equal squares and they were evaluated histologically using the HHGS. The correlation between both grading methods was assessed using the weighted Kappa coefficient (K(w)). The results obtained with both scores showed good agreement (K(w): mean+/-standard deviation, 0.619+/-0.071). While the average HHGS scores of the chondral samples showed a better agreement with arthroscopic grades 0, I and II, the arthroscopic evaluation has a tendency to overestimate chondral lesions for histological grades III and IV. The intra- and inter-observer reliability of the HHGS evaluation of chondral lesions was excellent (Intraclass Correlation Coefficient: 0.909 and 0.941, respectively). In this study, we found a good quantitative correlation between established arthroscopic severity and histopathological scoring systems, particularly in less advanced lesions. Our results suggest that the arthroscopic method is a valuable tool in clinical research to score chondropathies in the medial femorotibial compartment of the OA knee, although some limitations should not be overlooked.

Reliability, reproducibility and variability of the traditional Histologic/Histochemical Grading System vs the new OARSI Osteoarthritis Cartilage Histopathology Assessment System

For many years, the Histologic/Histochemical Grading System (HHGS) for osteoarthritis monitoring has been used as a histological scoring system for the quality of cartilage. There are, however, some limitations using this grading system. The goal of the investigation presented in this paper was to examine the hypothesized advantage of the recently introduced Osteoarthritis Research Society International (OARSI) Cartilage Histopathology Assessment System (OOCHAS) as compared to the most frequently used HHGS by means of reliability, reproducibility, and variability evaluation as well as the correlation analysis between the two systems in goat knee articular cartilage. Nine hundred and thirty-six sections of Dutch Milk goat articular knee cartilage were scored using light microscopy. Three observers applied the HHGS for all sections and subsequently, the OOCHAS. The same scoring procedure was repeated after a minimum interval of 1 week. For each system the reliability, reproducibility and variability as well as the correlation between both systems were determined. The reliability of the OOCHAS was higher as compared to the HHGS. Both the HHGS as well the OOCHAS have an excellent intra- and inter-observer reproducibility and variability and a good positive correlation between the scores. Although the HHGS has proven to be an excellent tool for histological scoring of cartilage quality, we recommend the OOCHAS as the premium choice while stressing the importance of further research investigating the correlation of the histological results to macroscopic and biochemical parameters.

Articular cartilage degeneration after acute subchondral bone damage An experimental study in dogs with histopathological grading

Subchondral fracture patterns and bone bruises have been described and some clinical studies have shown alterations in the initially healthy cartilage after such lesions. After having performed cadaver studies, we created an animal model to produce pure subchondral damage without affecting the articular cartilage, under MRI control. We used 12 beagle dogs. For quantification of different degrees of staining, we used a grading of the sections by means of the HHGS (Histological-Histochemical Grading System) or Mankin score. In all cases, FLASH 3D sequences revealed intact cartilage in MRI after impact. The best detection of subchondral fractures was achieved in fat-suppressed TIRM sequences. Image analysis based on the HHGS showed changes in 10 of 12 samples, with a high degree of significance 6 months after the initial trauma. Correlation analysis showed loss of the physiological distribution of proteoglycans and glycoproteins in the different zones of articular cartilage. Subcategories "Structure", "Cells" and "Safranin-O Staining" also showed high significance, and the category "Tidemark Integrity" showed a tendency. Our findings indicate that acute subchondral fractures are a predictor of degenerative changes within 6 months. Modifications and supplements to rehabilitation might be needed in cases with accompanying subchondral lesions, e.g. in ACL tears.

Effects of diclofenac, aceclofenac and meloxicam on the metabolism of proteoglycans and hyaluronan in osteoarthritic human cartilage.

1. Since nonsteroidal anti-inflammatory drugs (NSAIDs) may impair the ability of the chondrocyte to repair its damaged extracellular matrix, we explored the changes in the metabolism of newly synthesized proteoglycan and hyaluronan (HA) molecules produced by aceclofenac, diclofenac and meloxicam in human osteoarthritic (OA) cartilage. 2. Explants were sampled from the medial femoral condyle and were classified by use of the Mankin's histological-histochemical grading system. Cartilage specimens exhibited moderate (M) OA in 20 subjects and had severe (S) OA in 20. 3. Cartilage explants were pulsed with [-3H]-glucosamine and chased in the absence or in the presence of 0.3 - 3 microg ml(-1) of either aceclofenac, diclofenac or meloxicam. After papain digestion, the labelled chondroitin sulphate ([-3H]-proteoglycans) and [-3H]-HA molecules present in the tissue and media were purified by anion-exchange chromatography. 4. In cartilage with MOA and SOA, the metabolic balance of proteoglycan and HA was unaffected by diclofenac. In contrast, and in a dose-dependent manner, aceclofenac and meloxicam both increased the synthesis of proteoglycans and HA in explants with MOA and SOA; these two NSAIDs also reduced significantly the net loss of [-3H]-proteoglycans and [-3H]-HA molecules from cartilage explants. 5. The data obtained in short-term in vitro cultures indicate that, at the concentrations found in synovial fluid, aceclofenac and meloxicam may exert a favourable effect on the overall metabolism of proteoglycans and HA in cartilage with MOA and SOA.

Histologic/histochemical grading system for osteoarthritic articular cartilage: reproducibility and validity.

To evaluate the reproducibility and validity of the histologic/histochemical grading system (HHGS) for osteoarthritis (OA) developed by Mankin and coworkers. Sections of human articular cartilage from macroscopically normal and osteoarthritic femoral heads were graded by the HHGS (0-14 scale) twice by 3 observers, and intra- and interobserver reproducibility was determined. The exact intra- and interobserver reproducibilities ranged from 22% to 33% and from 13% to 25%, respectively. Intra- and interobserver variation ranged from -7 to 5. Although scores were generally higher in cartilage from osteoarthritic joints as compared with cartilage from normal joints, the difference reached statistical significance for only 2 observers. Additionally, the average scores from all observers were within the definitive range for OA (>5) in 30% of sections from normal joints, and in only 47% of sections from OA joints. The results indicate that the reproducibility and the validity of the HHGS are inadequate, and an improved histopathologic grading system for OA is needed.

Effects of tenoxicam and aspirin on the metabolism of proteoglycans and hyaluronan in normal and osteoarthritic human articular cartilage

1. As nonsteroidal anti-inflammatory drugs may impair the ability of the chondrocyte to repair its damaged extracellular matrix, we explored the changes in the metabolism of newly synthesized proteoglycan (PG) and hyaluronan (HA) molecules produced by tenoxicam and aspirin in human normal cartilage explants and in osteoarthritic (OA) cartilage from age-matched donors. 2. Explants were sampled from the medial femoral condyle and were classified by use of Mankin's histological-histochemical grading system. Cartilage specimens were normal in 10 subjects, exhibited moderate OA (MOA) in 10 and had severe OA (SOA) in 10. 3. Cartilage explants were pulsed with [3H]-glucosamine and chased in the absence and in the presence of either aspirin (190 micrograms ml-1) or tenoxicam (4-16 micrograms ml-1). After papain digestion, the labelled chondroitin sulphate ([3H]-PGs) and HA([3H]-HA) molecules present in the tissue and media were purified by anion-exchange chromatography. 4. In normal cartilage as well as in explants with MOA and SOA aspirin reduced more strongly PG and HA synthesis than the loss of [3H]-HA and [3H]-PGs. 5. In normal cartilage, tenoxicam did not affect PG metabolism whereas it reduced HA synthesis in a dose-dependent manner and did not change or even increased the net loss of [3H]-HA. In contrast, in OA cartilage, tenoxicam produced a stronger reduction in the loss of [3H]-PGs than in PG synthesis and this decrease occurred at lower concentrations in cartilage with SOA (4-8 micrograms ml-1) than in cartilage with MOA (8-16 micrograms ml-1). In cartilage with MOA, the metabolic balance of HA was unaffected by tenoxicam whereas in cartilage with SOA, the drug decreased the loss of [3H]-HA and concomitantly did not change or even increased HA synthesis.6. The data obtained in short-term in vitro cultures indicate that aspirin may produce OA-like changes in normal cartilage and is likely to worsen the disease process in OA tissue. On the other hand, although tenoxicam may reduce the HA content of normal cartilage, and, in so doing, may produce OA-like lesions, this drug should not per se accelerate joint failure in OA.

Drug‐induced inhibition of proteoglycanase activity in the hulth–telhag model

Evidence has accumulated that there are increased degradative enzymes in osteoarthritis responsible for joint destruction. These enzymes are metal-dependent, and inhibited by EDTA. EDTA was administered intra-articularly to rabbits in an experimental model of osteoarthritis. There was a 25% reduction in neutral proteoglycanase activity and 75% of the animals had a reduction in the severity of the arthritis as measured on a histologic-histochemical grading system. It is suggested that future chemotherapeutic studies on arthritis might focus on enzyme inhibition.

Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. III. Distribution and metabolism of amino sugar-containing macromolecules.

Since 1960, numerous studies have supported the thesis that the synthetic activity of articular chondrocytes is increased in osteoarthritis, but several recent reports have challenged this concept. To clarify this problem fully and also to define further the products of this increased synthesis, three experiments were performed in which the distribution and rates of synthesis of amino sugar-containing macromolecules in normal and osteoarthritic cartilage from the human femoral head were assessed by biochemical analysis and studies of the incorporation of 3H-glucosamine and 35SO4. The biochemical data obtained clearly demonstrated the previously noted significant decrease in hexosamine content in osteoarthritic tissue. This decrease was principally due to a diminution in glucosamine concentration and correlated inversely with the severity of the disease process (as measured by a previously described histological-histochemical grading system). Metabolic studies showed a marked increment in the rates of incorporation of 3H-glucosamine into both the glucosamine and the galactosamine fractions of the cartilage. The increased synthesis correlated directly in a non-linear fashion with the severity of the disease. The ratio of the rate of incorporation of 3H-glucosamine into the glucosamine fraction to the rate of its incorporation into the galactosamine fraction was the same in normal and osteoarthritic samples, suggesting that the decline in glucosamine concentration was not related to a qualitative alteration of synthetic activity.