M. Garon

Detection and analysis of cartilage degeneration by spatially resolved streaming potentials

Cartilage molecular changes in osteoarthritis are most commonly related to the degradation and loss of proteoglycan and collagen fibrils of the extracellular matrix, which directly influence tissue stiffness and compression‐generated streaming potentials. In this study, we evaluated the potential of a new technique, spatially resolved mapping of streaming potentials, to non‐destructively indicate cartilage health or degeneration. Matched pairs of bovine cartilage/bone explant disks were cultured for 11 days in a serum free medium with and without interleukin‐1α (IL‐1α). The electromechanical properties (static stiffness, dynamic stiffness and streaming potentials) of cartilage disks were measured during unconfined compression using a mechanical tester coupled with a linear array of eight 50 μm diameter platinum‐iridium microelectrodes. After 11 days of culture, the proteoglycan content of IL‐1α treated disks was significantly reduced and the denatured and cleaved collagen content was increased compared to control disks. These biochemical alterations were concomitant with the reductions in the amplitudes of the static stiffness, the dynamic stiffness and the streaming potential profile as well as changes in the shape of the streaming potential profile. We found that spatial mapping of streaming potentials presents several advantages for the development of a clinical instrument to evaluate the degeneration of articular cartilage.

Streaming Potential-Based Arthroscopic Device is Sensitive to Cartilage Changes Immediately Post-Impact in an Equine Cartilage Injury Model

Models of post-traumatic osteoarthritis where early degenerative changes can be monitored are valuable for assessing potential therapeutic strategies. Current methods for evaluating cartilage mechanical properties may not capture the low-grade cartilage changes expected at these earlier time points following injury. In this study, an explant model of cartilage injury was used to determine whether streaming potential measurements by manual indentation could detect cartilage changes immediately following mechanical impact and to compare their sensitivity to biomechanical tests. Impacts were delivered ex vivo, at one of three stress levels, to specific positions on isolated adult equine trochlea. Cartilage properties were assessed by streaming potential measurements, made pre- and post-impact using a commercially available arthroscopic device, and by stress relaxation tests in unconfined compression geometry of isolated cartilage disks, providing the streaming potential integral (SPI), fibril modulus (Ef), matrix modulus (Em), and permeability (k). Histological sections were stained with Safranin-O and adjacent unstained sections examined in polarized light microscopy. Impacts were low, 17.3 ± 2.7 MPa (n = 15), medium, 27.8 ± 8.5 MPa (n = 13), or high, 48.7 ± 12.1 MPa (n = 16), and delivered using a custom-built spring-loaded device with a rise time of approximately 1 ms. SPI was significantly reduced after medium (p = 0.006) and high (p<0.001) impacts. Ef, representing collagen network stiffness, was significantly reduced in high impact samples only (p < 0.001 lateral trochlea, p = 0.042 medial trochlea), where permeability also increased (p = 0.003 lateral trochlea, p = 0.007 medial trochlea). Significant (p < 0.05, n = 68) moderate to strong correlations between SPI and Ef (r = 0.857), Em (r = 0.493), log(k) (r = -0.484), and cartilage thickness (r = -0.804) were detected. Effect sizes were higher for SPI than Ef, Em, and k, indicating greater sensitivity of electromechanical measurements to impact injury compared to purely biomechanical parameters. Histological changes due to impact were limited to the presence of superficial zone damage which increased with impact stress. Non-destructive streaming potential measurements were more sensitive to impact-related articular cartilage changes than biomechanical assessment of isolated samples using stress relaxation tests in unconfined compression geometry. Correlations between electromechanical and biomechanical methods further support the relationship between non-destructive electromechanical measurements and intrinsic cartilage properties.

Tetrapolar Measurement of Electrical Conductivity and Thickness of Articular Cartilage

A tetrapolar method to measure electrical conductivity of cartilage and bone, and to estimate the thickness of articular cartilage attached to bone, was developed. We determined the electrical conductivity of humeral head bovine articular cartilage and subchondral bone from a 1- to 2-year-old steer to be 1.14+/-0.11 S/m (mean+/-sd, n =11) and 0.306+/-0.034 S/m, (mean+/-sd, n =3), respectively. For a 4-year-old cow, articular cartilage and subchondral bone electrical conductivity were 0.88+/-0.08 S/m (mean+/-sd, n =9) and 0.179+/-0.046 S/m (mean+/-sd, n =3), respectively. Measurements on slices of cartilage taken from different distances from the articular surface of the steer did not reveal significant depth-dependence of electrical conductivity. We were able to estimate the thickness of articular cartilage with reasonable precision (<20% error) by injecting current from multiple electrode pairs with different inter-electrode distances. Requirements for the precision of this method to measure cartilage thickness include the presence of a distinct layer of calcified cartilage or bone with a much lower electrical conductivity than that of uncalcified articular cartilage, and the use of inter-electrode distances of the current injecting electrodes that are on the order of the cartilage thickness. These or similar methods present an attractive approach to the non-destructive determination of cartilage thickness, a parameter that is required in order to estimate functional properties of cartilage attached to bone, and evaluate the need for therapeutic interventions in arthritis.

Fabrication and characterization of nonplanar microelectrode array circuits for use in arthroscopic diagnosis of cartilage diseases

A process to fabricate nonplanar microelectrode array circuits was developed and the microelectrodes were characterized. These platinum microelectrode arrays are for recording streaming potential signals generated during indentation of articular cartilage. The nonplanar substrate was produced by permanent deformation of a 7-in-diameter circular stainless-steel wafer to form 32 semi-spherical caps (radius of curvature=4.65 mm and height=250 /spl mu/m) at the periphery. The wafer was covered with a 2.5-/spl mu/m-thick layer of insulating polyimide. Standard microelectronic processes were applied to produce 32 circuits (60 mm long /spl times/4 mm wide) with 37 exposed circular microelectrodes (diameter=100 /spl mu/m) centered over each semi-spherical cap. A 2.5-/spl mu/m-thick photodefinable polyimide layer encapsulated the conducting lines. Capacitances between one microelectrode and either another microelectrode or the metallic substrate were 14.6/spl plusmn/2.0 and 34.4/spl plusmn/3.3 pF, respectively, at 100 Hz. The impedance of the microelectrodes in a 0.15 M saline bath (PBS) was 0.25/spl plusmn/0.08 M/spl Omega/ while the crosstalk (V/sub induced//V/sub applied/) between two microelectrodes was 0.20/spl plusmn/0.11%, at 100 Hz. Indentation measurements were performed on articular cartilage in vitro showing streaming potentials that indicate electrode-tissue contact times and generation of streaming potentials.

Electromechanical probe and automated indentation maps are sensitive techniques in assessing early degenerated human articular cartilage.

Recent advances in the development of new drugs to halt or even reverse the progression of Osteoarthritis at an early‐stage requires new tools to detect early degeneration of articular cartilage. We investigated the ability of an electromechanical probe and an automated indentation technique to characterize entire human articular surfaces for rapid non‐destructive discrimination between early degenerated and healthy articular cartilage. Human cadaveric asymptomatic articular surfaces (four pairs of distal femurs and four pairs of tibial plateaus) were used. They were assessed ex vivo: macroscopically, electromechanically, (maps of the electromechanical quantitative parameter, QP, reflecting streaming potentials), mechanically (maps of the instantaneous modulus, IM), and through cartilage thickness. Osteochondral cores were also harvested from healthy and degenerated regions for histological assessment, biochemical analyses, and unconfined compression tests. The macroscopic visual assessment delimited three distinct regions on each articular surface: Region I was macroscopically degenerated, region II was macroscopically normal but adjacent to regions I and III was the remaining normal articular surface. Thus, each extracted core was assigned to one of the three regions. A mixed effect model revealed that only the QP (p < 0.0001) and IM (p < 0.0001) were able to statistically discriminate the three regions. Effect size was higher for QP and IM than other assessments, indicating greater sensitivity to distinguish early degeneration of cartilage. When considering the mapping feature of the QP and IM techniques, it also revealed bilateral symmetry in a moderately similar distribution pattern between bilateral joints.

Bilayer Implants Electromechanical Assessment of Regenerated Articular Cartilage in a Sheep Model

Objective To compare the regenerative capacity of 2 distinct bilayer implants for the restoration of osteochondral defects in a preliminary sheep model. Methods Critical sized osteochondral defects were treated with a novel biomimetic poly-ε-caprolactone (PCL) implant (Treatment No. 2; n = 6) or a combination of Chondro-Gide and Orthoss (Treatment No. 1; n = 6). At 19 months postoperation, repair tissue (n = 5 each) was analyzed for histology and biochemistry. Electromechanical mappings (Arthro-BST) were performed ex vivo. Results Histological scores, electromechanical quantitative parameter values, dsDNA and sGAG contents measured at the repair sites were statistically lower than those obtained from the contralateral surfaces. Electromechanical mappings and higher dsDNA and sGAG/weight levels indicated better regeneration for Treatment No. 1. However, these differences were not significant. For both treatments, Arthro-BST revealed early signs of degeneration of the cartilage surrounding the repair site. The International Cartilage Repair Society II histological scores of the repair tissue were significantly higher for Treatment No. 1 (10.3 ± 0.38 SE) compared to Treatment No. 2 (8.7 ± 0.45 SE). The parameters cell morphology and vascularization scored highest whereas tidemark formation scored the lowest. Conclusion There was cell infiltration and regeneration of bone and cartilage. However, repair was incomplete and fibrocartilaginous. There were no significant differences in the quality of regeneration between the treatments except in some histological scoring categories. The results from Arthro-BST measurements were comparable to traditional invasive/destructive methods of measuring quality of cartilage repair.

082 STREAMING POTENTIAL-BASED ARTHROSCOPIC DEVICE CAN DETECT CHANGES IMMEDIATELY FOLLOWING LOCALIZED IMPACT IN AN EQUINE IMPACT MODEL OF OSTEOARTHRITIS

Purpose: Frizzled related protein (FRZB/sFRP3) is a secreted WNT antagonist isolated from articular cartilage and expressed in developing skeletal elements. Polymorphisms in the human FRZB gene are associated with susceptibility for osteoarthritis. Induction of experimental osteoarthritis in Frzb–/– mice results in enhanced cartilage degradation associated with increased Wnt signalling, Mmp3 expression, Mmp activity and cortical bone thickness. We further studied the role of FRZB in osteoarthritis by FRZB adenoviral overexpression in the knee joint in the methylated bovine serum albumin (mBSA)-induced arthritis model. Methods: Adenovirus expressing FRZB or GFP (107 pfu/10 μl in PBS) was injected intra-articularly in the right knee joint of C57Bl/6 mice at day 0. At day 1 mBSA-induced arthritis was induced by intra-articular injection of 10 μl mBSA (20 mg/ml in PBS) followed by subcutaneous injections of IL-1β (250 ng) in the right footpad for 3 consecutive days. The left knee was used as a negative control and injected each time with 10 μl of PBS. At day 7, mice were sacrificed and the knee, synovium or articular cartilage were isolated for histology or mRNA analysis. Knee sections were stained with haematoxylin-eosin and safranin O and severity of arthritis was scored. Expression of inflammatory cytokines Il-1β, TNF-α and Il-6 and matrix-degrading enzymes Mmp3, Mmp9, Mmp13, Adamts4 and Adamts5 in synovium and articular cartilage was determined using quantitative RT-PCR. Results: Histomorphological analysis of the knee showed a significant increase in the score of inflammatory parameters such as infiltration, exsudate formation and pannus formation when FRZB was overexpressed. Cartilage damage was not different compared to GFP controls. Increased synovitis was confirmed by geneexpression analysis of inflammatory cytokines. Il-1β, TNF-α and Il-6 expression were increased in the synovium with FRZB overexpression. We also detected a decreased expression of Mmp3 in cartilage when FRZB was overexpressed and an increase of Adamts5 in synovium. Mmp9, Mmp13 and Adamts4 expression did not change in synovium and articular cartilage. The altered balance between Adamts5 and Mmp3 expression may explain the absence of changes in cartilage damage. Conclusions: In this study we demonstrate that FRZB overexpression leads to increased inflammation in mBSA-induced arthritis in mice. The increased inflammatory response appears to counteract potential chondroprotective effects of FRZB overexpression. These data further highlight the complex biology of FRZB.

Electrical potentials measured on the surface of the knee reflect the changes of the contact force in the knee joint produced by postural sway

Electroarthrography (EAG) is a novel technique for recording potentials on the knee surface that are generated by the compression of articular cartilage and that reflect both compression force and cartilage quality. The mechanical loading of the knee is achieved by transferring the subjects body weight from a bipedal stance to a unipedal stance. We hypothesized that EAG potentials change with postural sway. The study was performed on 20 normal subjects (10 male, 10 female; age 29±10.5 yrs.; mass 68.8±14.2kg; height 172.6±11.4cm). Data was recorded during 10 successive loading cycles repeated on two different days. During loading, EAG potentials were recorded with 4 electrodes placed on both sides of the knee and the ground reaction force (GRF) and the antero-posterior and medial-lateral displacements of the center of pressure (COP) were measured with a force plate. Two electromechanical models predicting the EAG signal from the GRF alone or from the GRF plus the COP displacements were computed by linear regression. The mean relative error between the four EAG signals and the predicted signals ranged from 24% to 49% for the GRF model, and from 15% to 35% for the GRF+COP model, this reduction was statistically significant at 3 electrode sites (p<0.05). The GRF+COP model also improved the repeatability of the parameters estimated on the first and second days when compared to the GRF model. In conclusion, EAG signals can be predicted by GRF and COP displacements and may reflect changes in the knee contact force due to postural sway.

Decrease of the electrical potentials measured on the surface of the knee and produced by cartilage compression during successive loading cycles

Electroarthrography (EAG) is a new technique for measuring electrical potentials appearing on the knee surface during loading that reflects cartilage quality and joint contact force. Our objective was to investigate the evolution of EAG signals during successive loading cycles. The study was conducted on 20 standing subjects who shifted their body weight to achieve knee loading. Their EAG signals were recorded during 10 successive loading cycles, and during a subsequent sequence of 10 cycles recorded after a 15min exercise period. Multiple linear regression models estimated the electro-mechanical ratio (EMR) interpreting the ability of cartilage to generate a certain potential for a given ground reaction force by taking into account this force and the center of pressure displacements during unipedal stance. The results showed that the EMR values slowly decreased with successive cycles: during the initial sequence, the correlation coefficients between EMR values and sequence numbers were significant at 3 of the 4 electrode sites (p<0.05); for the post-exercise sequence, the EMR values still decreased and were significantly lower than during the initial sequence (p<0.001). The reduction of EMR values could arise from muscle activity and habituation of the stretch reflex, and also from the time dependent electromechanical properties of cartilage. In conclusion, refraining from physical activity before the EAG measurements is important to improve measurement repeatability because of the EMR decrease. The electromechanical model confirmed the role of EAG as a natural sensor of the changes in the knee contact force and also improved EAG measurement accuracy.