Simo Saarakkala

Depth-wise progression of osteoarthritis in human articular cartilage: investigation of composition, structure and biomechanics

OBJECTIVE Osteoarthritis (OA) is characterized by the changes in structure and composition of articular cartilage. However, it is not fully known, what is the depth-wise change in two major components of the cartilage solid matrix, i.e., collagen and proteoglycans (PGs), during OA progression. Further, it is unknown how the depth-wise changes affect local tissue strains during compression. Our aim was to address these issues. METHODS Data from the previous microscopic and biochemical measurements of the collagen content, distribution and orientation, PG content and distribution, water content and histological grade of normal and degenerated human patellar articular cartilage (n=73) were reanalyzed in a depth-wise manner. Using this information, a composition-based finite element (FE) model was used to estimate tissue function solely based on its composition and structure. RESULTS The orientation angle of collagen fibrils in the superficial zone of cartilage was significantly less parallel to the surface (P<0.05) in samples with early degeneration than in healthy samples. Similarly, PG content was reduced in the superficial zone in early OA (P<0.05). However, collagen content decreased significantly only at the advanced stage of OA (P<0.05). The composition-based FE model showed that under a constant stress, local tissue strains increased as OA progressed. CONCLUSION For the first time, depth-wise point-by-point statistical comparisons of structure and composition of human articular cartilage were conducted. The present results indicated that early OA is primarily characterized by the changes in collagen orientation and PG content in the superficial zone, while collagen content does not change until OA has progressed to its late stage. Our simulation results suggest that impact loads in OA joint could create a risk for tissue failure and cell death.

Novel mechano-acoustic technique and instrument for diagnosis of cartilage degeneration

Fibrillation of articular surface and depletion of proteoglycans are the structural changes related to early osteoarthrosis. These changes make cartilage softer and prone to further degeneration. The aim of the present study was to combine mechanical and acoustic measurements towards quantitative arthroscopic evaluation of cartilage quality. The performance of the novel ultrasound indentation instrument was tested with elastomers and bovine articular cartilage in vitro. The instrument was capable of measuring elastomer thickness (r = 1.000, p < 0.01, n = 8) and dynamic modulus (r = 0.994, p < 0.01, n = 13) reliably. Osteochondral plugs were tested before and after enzymatic degradation of cartilage proteoglycans by trypsin or chondroitinase ABC, and of cartilage collagens by collagenase. Trypsin and collagenase induced a mean decrease of -31.2 +/- 12.3% (+/- SD, p < 0.05) and -22.9 +/- 20.8% (p = 0.08) in dynamic modulus, respectively. Rate of cartilage deformation, i.e. creep rate, increased by +117.8 +/- 71.4% (p < 0.05) and +24.7 +/- 35.1% (p = 0.17) in trypsin and chondroitinase ABC treatments, respectively. Collagenase induced a greater decrease in the ultrasound reflection from the cartilage surface (-54.2 +/- 29.6%, p < 0.05) than trypsin (-17.1 +/- 13.5%, p = 0.08). In conclusion, combined quantitation of tissue modulus, viscoelasticity and ultrasound reflection from the cartilage surface provides a sensitive method to distinguish between normal and degenerated cartilage, and even to discern proteoglycan loss and collagen degradation from each other.

Power Doppler ultrasonography and synovitis: correlating ultrasound imaging with histopathological findings and evaluating the performance of ultrasound equipments

Objectives: To examine the validity of power Doppler ultrasound imaging to identify synovitis, using histopathology as gold standard, and to assess the performance of ultrasound equipments. Methods: 44 synovial sites in small and large joints, bursae and tendon sheaths were depicted with ultrasound. A synovial biopsy was performed on the site depicted and a synovial sample was taken for histopathological evaluation. The performance of three ultrasound devices was tested using flow phantoms. Results: A positive Doppler signal was detected in 29 of 35 (83%) of the patients with active histological inflammation. In eight additional samples, histological examination showed other pathological synovial findings and a Doppler signal was detected in five of them. No significant correlation was found between the amount of Doppler signal and histological synovitis score (r = 0.239, p = NS). The amount of subsynovial infiltration of polymorphonuclear leucocytes and surface fibrin correlated significantly with the amount of power Doppler signal: r = 0.397 (p<0.01) and 0.328 (p<0.05), respectively. The ultrasound devices differed in showing the smallest detectable flow. Conclusions: A negative Doppler signal does not exclude the possibility of synovitis. A positive Doppler signal in the synovium is an indicator of an active synovial inflammation in patients. A Doppler signal does not correlate with the extent of the inflammation and it can also be seen in other synovial reactions. It is important that the quality measurements of ultrasound devices are reported, because the results should be evaluated against the quality of the device used.

Speed of sound in normal and degenerated bovine articular cartilage

The unknown and variable speed of sound may impair accuracy of the acoustic measurement of cartilage properties. In this study, relationships between the speed of sound and cartilage composition, mechanical properties and degenerative state were studied in bovine knee and ankle cartilage (n = 62). Further, the effect of speed variation on the determination of cartilage thickness and stiffness with ultrasound (US) indentation was numerically simulated. The speed of sound was significantly (n = 32, p < 0.05) dependent on the cartilage water content (r = -0.800), uronic acid content (per wet weight, r = 0.886) and hydroxyproline content (per wet weight, r = 0.887, n = 28), Youngs modulus at equilibrium (r = 0.740), dynamic modulus (r = 0.905), and degenerative state (i.e., Mankin score) (r = -0.727). In addition to cartilage composition, mechanical and acoustic properties varied significantly between different anatomical locations. In US indentation, cartilage is indented with a US transducer. Deformation and thickness of tissue are calculated using a predefined speed of sound and used in determination of dynamic modulus. Based on the simulations, use of the mean speed of sound of 1627 m/s (whole material) induced a maximum error of 7.8% on cartilage thickness and of 6.2% on cartilage dynamic modulus, as determined with the US indentation technique (indenter diameter 3 mm). We believe that these errors are acceptable in clinical US indentation measurements.

Quantitative ultrasound imaging detects degenerative changes in articular cartilage surface and subchondral bone

Previous studies have suggested that quantitative ultrasound imaging could sensitively diagnose degeneration of the articular surface and changes in the subchondral bone during the development of osteoarthrosis (OA). We have recently introduced a new parameter, ultrasound roughness index (URI), for the quantification of cartilage surface roughness, and successfully tested it with normal and experimentally degraded articular surfaces. In this in vitro study, the applicability of URI was tested in bovine cartilage samples with spontaneously developed tissue degeneration. Simultaneously, we studied the sensitivity of quantitative ultrasound imaging to detect degenerative changes in the cartilage-bone interface. For reference, histological degenerative grade of the cartilage samples was determined. Mechanical reference measurements were also conducted. Cartilage surface roughness (URI) was significantly (p<0.05) higher in histologically degenerated samples with inferior mechanical properties. Ultrasound reflection at the cartilage-bone interface was also significantly (p<0.05) increased in degenerated samples. Furthermore, it was quantitatively confirmed that ultrasound attenuation in the overlying cartilage significantly affects the measured ultrasound reflection values from the cartilage-bone interface. To conclude, the combined ultrasound measurement of the cartilage surface roughness and ultrasound reflection at the cartilage-bone interface complement each other, and may together enable more sensitive and quantitative diagnosis of early OA or follow up after surgical cartilage repair.

Ultrasound indentation of normal and spontaneously degenerated bovine articular cartilage

OBJECTIVE We have previously developed a handheld ultrasound indentation instrument for the diagnosis of cartilage degeneration. The instrument has been demonstrated to be capable of quantifying mechanical and acoustic properties of enzymatically degraded and normal bovine articular cartilage in vitro and in situ. The aim of this study was to investigate the sensitivity of the instrument to distinguish between normal and spontaneously degenerated (e.g., in osteoarthrosis) articular cartilage in vitro. DESIGN Thirty articular cartilage samples were prepared from the bovine lateral patellae: 19 patellae with different degenerative stages and 11 patellae with visually normal appearance. Cartilage thickness, stiffness (dynamic modulus) and ultrasound reflection from the cartilage surface were measured with the handheld instrument. Subsequently, biomechanical, histological and biochemical reference measurements were conducted. RESULTS Reproducibility of the measurements with the ultrasound indentation instrument was good. Standardized coefficient of variation was < or =6.1% for thickness, dynamic modulus and reflection coefficient. Linear correlation between the dynamic modulus, measured with the ultrasound indentation instrument, and the reference dynamic modulus was high (r=0.993, n=30, P<0.05). Ultrasound reflection coefficient, as determined from the cartilage surface, showed high linear correlations (typically r(2)>0.64, n=30, P<0.05) with the cartilage composition and histological or mechanical properties. The instrument was superior compared to visual evaluation in detecting tissue degeneration. CONCLUSION This study indicates that the ultrasound indentation technique and instrument may significantly improve the early diagnosis of cartilage degeneration. The results revealed that visual evaluation is insensitive for estimating the structural and mechanical properties of articular cartilage at the initial stages of degeneration.

Application of second derivative spectroscopy for increasing molecular specificity of fourier transform infrared spectroscopic imaging of articular cartilage

OBJECTIVE Fourier transform infrared (FT-IR) spectroscopic imaging is a promising method that enables the analysis of spatial distribution of biochemical components within histological sections. However, analysis of FT-IR spectroscopic data is complicated since absorption peaks often overlap with each other. Second derivative spectroscopy is a technique which enhances the separation of overlapping peaks. The objective of this study was to evaluate the specificity of the second derivative peaks for the main tissue components of articular cartilage (AC), i.e., collagen and proteoglycans (PGs). MATERIALS AND METHODS Histological bovine AC sections were measured before and after enzymatic removal of PGs. Both formalin-fixed sections (n = 10) and cryosections (n = 6) were investigated. Relative changes in the second derivative peak heights caused by the removal of PGs were calculated for both sample groups. RESULTS The results showed that numerous peaks, e.g., peaks located at 1202 cm(-1) and 1336 cm(-1), altered less than 5% in the experiment. These peaks were assumed to be specific for collagen. In contrast, two peaks located at 1064 cm(-1) and 1376 cm(-1) were seen to alter notably, approximately 50% or more. These peaks were regarded to be specific for PGs. The changes were greater in cryosections than formalin-fixed sections. CONCLUSIONS The results of this study suggest that the second derivative spectroscopy offers a practical and more specific method than routinely used absorption spectrum analysis methods to obtain compositional information on AC with FT-IR spectroscopic imaging.

Infrared spectroscopy indicates altered bone turnover and remodeling activity in renal osteodystrophy

Renal osteodystrophy alters metabolic activity and remodeling rate of bone and also may lead to different bone composition. The objective of this study was to characterize the composition of bone in high‐turnover renal osteodystrophy patients by means of Fourier transform infrared spectroscopic imaging (FTIRI). Iliac crest biopsies from healthy bone (n = 11) and patients with renal osteodystrophy (ROD, n = 11) were used in this study. The ROD samples were from patients with hyperparathyroid disease. By using FTIRI, phosphate‐to‐amide I ratio (mineral‐to‐matrix ratio), carbonate‐to‐phosphate ratio, and carbonate‐to‐amide I ratio (turnover rate/remodeling activity), as well as the collagen cross‐link ratio (collagen maturity), were quantified. Histomorphometric analyses were conducted for comparison. The ROD samples showed significantly lower carbonate‐to‐phosphate (p < .01) and carbonate‐to‐amide I (p < .001) ratios. The spatial variation across the trabeculae highlighted a significantly lower degree of mineralization (p < .05) at the edges of the trabeculae in the ROD samples than in normal bone. Statistically significant linear correlations were found between histomorphometric parameters related to bone‐remodeling activity and number of bone cells and FTIRI‐calculated parameters based on carbonate‐to‐phosphate and carbonate‐to‐amide I ratios. Hence the results suggested that FTIRI parameters related to carbonate may be indicative of turnover and remodeling rate of bone.

Ultrasound indentation of bovine knee articular cartilage in situ

We have earlier developed a handheld ultrasound indentation instrument for the diagnosis of articular cartilage degeneration. In ultrasound indentation, cartilage is compressed with the ultrasound transducer. Tissue thickness and deformation are calculated from the A-mode ultrasound signal and the stress applied is registered with the strain gauges. In this study, the applicability of the ultrasound indentation instrument to quantify site-dependent variation in the mechano-acoustic properties of bovine knee cartilage was investigated. Osteochondral blocks (n=6 per site) were prepared from the femoral medial condyle (FMC), the lateral facet of the patello-femoral groove (LPG) and the medial tibial plateau (MTP). Cartilage stiffness (dynamic modulus, E(dyn)), as obtained with the ultrasound indentation instrument in situ, correlated highly linearly (r=0.913, p<0.01) with the values obtained using the reference material-testing device in vitro. Reproducibility (standardized coefficient of variation) of the ultrasound indentation measurements was 5.2%, 1.7% and 3.1% for E(dyn), ultrasound reflection coefficient of articular surface (R) and thickness, respectively. E(dyn) and R were site dependent (p<0.05, Kruskall-Wallis H test). E(dyn) was significantly higher (p<0.05, Kruskall-Wallis Post Hoc test) in LPG (mean+/-SD: 10.1+/-3.1MPa) than in MTP (2.9+/-1.4MPa). In FMC, E(dyn) was 4.6+/-1.3MPa. R was significantly (p<0.05) lower at MTP (2.0+/-0.7%) than at other sites (FMC: 4.2+/-0.9%; LPG: 4.4+/-0.8%). Cartilage glycosaminoglycan concentration, as quantified with the digital densitometry, correlated positively with E(dyn) (r=0.678, p<0.01) and especially with the equilibrium Youngs modulus (reference device, r=0.874, p<0.01) but it was not associated with R (r=0.294, p=0.24). We conclude that manual measurements are reproducible and the instrument may be used for detection of cartilage quality in situ. Especially, combined measurement of thickness, E(dyn) and R provides valuable diagnostic information on cartilage status.

MINIMALLY INVASIVE ULTRASOUND METHOD FOR INTRA-ARTICULAR DIAGNOSTICS OF CARTILAGE DEGENERATION

Quantitative ultrasound imaging (QUI) can be used to evaluate the integrity of articular cartilage and for diagnosing the early signs of osteoarthritis (OA). In this study, we applied a minimally invasive ultrasound imaging technique and investigated its ability to detect superficial degeneration of bovine knee articular cartilage. Intact (n=13), collagenase-digested (n=6) and mechanically degraded (n=7) osteochondral samples (dia.=25 mm) and custom-made phantoms with different degrees of surface roughness (n=8) were imaged using a high-frequency (40 MHz) QUI system. For each sample and phantom, the ultrasound reflection coefficient (R), integrated reflection coefficient (IRC) and ultrasound roughness index (URI) were determined. Furthermore, to evaluate the clinical applicability of intra-articular ultrasound (IAUS) in diagnostics, one intact bovine knee joint was investigated ex vivo using a simulated arthroscopic approach. Differences in the surface characteristics of the phantoms were detected by monitoring changes in the reflection and surface roughness parameters. Both mechanically- and enzymatically-induced degradation were sensitively diagnosed by decreased (p<0.05) reflection (R and IRC) at the cartilage surface. Furthermore, mechanical degradation was detected in the increased (p<0.05) surface roughness (URI). The intra-articular investigation of a bovine knee joint suggested that the IAUS technique may enable minimally invasive, straightforward diagnostics of the degenerative status of the articular surfaces. We conclude that quantitative IAUS imaging can be used for detecting collagen disruption and increased roughness of the articular surface. This quantitative in vivo ultrasound technique could have great clinical value in the diagnostics of joint diseases.