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Nano-indentation methodology for cartilage quality assessment
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Figure 3. Dedifferentiation increased cilia and proliferation.
S343
p¼0.248). Needle size (23G n¼33 or 21G n¼18) had no effect on IOP (t test¼0.141). Needle site (femoral head n¼6, femoral condyle n¼7, upper tibia n¼32) had no influence on IOP (analysis of variance p¼0.188). The majority of recordings showed a pulsatile wave form. At a recording speed of 12.5mm/sec the wave was seen to be synchronous with the arterial pulse. At a 1/60th slower recording speed of 12.5mm/min the arterial pulse was obliterated but an underlying wave synchronous with respiration was seen. After bolus drug administration a third or ‘circulation time wave’ was seen. This wave lasted some 90 seconds and could be followed for one or two passes. After a saline clearance bolus injection the IOP and PP fell. Recovery took up to 15 minutes. After aspiration to clear the needle recovery was within 30 seconds. There was a correlation between the IOP and the mean blood pressure (Pearson p<0.001) There was a close correlation between IOP and its associated pulse pressure (p<0.0001). Conclusions: These results show that IOP is not a static venous back pressure but is closely related to the arterial supply circulation. The variability in IOP is a manifestation of the size of vessel encountered by the needle tip or pool in the microcirculation in which the pressure is being measured. A single IOP measurement in isolation is meaningless. The physiology of the subchondral microcirculation may be important in understanding blood supply to the cartilage in health and in osteoarthritis.
Conclusions: Changes in primary cilia length affects cilia function including mechanotransduction and hedgehog signalling and may also affect other cilia signalling pathways. As hypothesized, the reduction in mechano-responsiveness and hedgehog signalling with extended 2D culture is due to the associated elongation of primary cilia. To further identify the underpinning mechanisms, ongoing studies are using super resolution microscopy to examine whether cilia elongation at P5 is associated with alterations in cilia expression of ARL13B, and putative mechanoreceptors polycystin 2 and TRPV4. The observed reductions in mechano-responsiveness and ligand-induced hedgehog signalling with passage have important implications for the success of cartilage tissue engineering. Future work will investigate whether modulation of cilia length can maintain or rescue mechano- and hedgehog-responsiveness during expansion of chondrocytes for cartilage tissue engineering. 562 FACTORS AFFECTING PHYSIOLOGY OF INTRAOSSEOUS PRESSURE MEASUREMENT M. Beverly, J. Urban, D. Murray. Oxford Univ., Oxford, United Kingdom Purpose: Intraosseous pressure (IOP) has been studied for 50 or more years and is of interest in diseases such as osteoarthritis and osteonecrosis and in bone pain. IOP in cancellous bone has usually been thought to have a static value due to venous back pressure or tissue turgor. We measured IOP in patients before surgery but the results varied. We therefore set out to assess factors which might influence the physiology of IOP at rest in healthy subchondral cancellous bone in a standardized animal model. Methods: With ethical approval IOP was studied in 21 anaesthetised rabbits. Intraosseous pressure was measured through needles inserted directly into the subchondral bone of the femoral head, femoral condyle or upper tibia. Pressure was also measured in the left femoral artery in 5 animals. A heparinized saline filled line was connected from the intraosseous needle to pressure transducers (Bell and Howell or Druck PDCR75) and to a four channel chart recorder (Lectromed MX4P- 31). The transducers were calibrated on a 0-60 mmHg scale. Needle clearance was occasionally required using a 0.5ml saline bolus. Results: Basal intraosseous pressure (IOP) was measured in 21 individuals at 51 sites and varied considerably between different subjects and also at different sites in the same subject. The average IOP (n¼51) was 23.8mmHg (SD 13.6mmHg) with a pulse pressure (PP) of 4.0mmHg (SD 4.2mmHg). Gender (female n¼14, male n¼7) had no effect on IOP (t test¼0.58). Weight (2,920g - 5,560g) had no effect on IOP (Pearson correlation
563 NANO-INDENTATION METHODOLOGY FOR CARTILAGE QUALITY ASSESSMENT C. Lavet, P. Ammann. Service of Bone Diseases, Geneva, Switzerland Purpose: To date, histological analysis is the gold-standard research tool for assessing cartilage integrity. Indentation features the benefits of assessing the main function of cartilage (shock absorption and force transmission) by its biomechanical parameters which could be altered by numerous factors. Furthermore significant cartilage metabolic disturbances can occur in cartilage in early stage of osteoarthrosis (OA) and prior to morphometric alterations. Therefore it could also be assumed that a normal macroscopic appearance of the cartilage does not ensure the absence of pathology. Despite the wide range of cartilage biomechanical testing methods in samples harvested from human or big animals’ models, rodents’ models which have an increasingly prominent role in OA research, are lacking of biomechanical assessment of their cartilage. Therefore, it is mandatory to develop a technique dedicated to the objective evaluation of the cartilage tissue quality. Methods: The original positioning of the femur allows to indent three different areas of interest of the cartilage surface corresponding to area physiologically exposed to load with various forces and orientation, various timing along the day and various frequency. Two different methods were used. Monotonic indentation was defined as a set of 5 successive indentations (controlled in force) within the region of interest. Each indentation was 200 mm distant from each other and were performed in the same sagittal plan of the medial condyle (at the center). Cyclic indentation was defined as a set of 5 successive cyclic
S344
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
indentations. All the 5 cyclic indentations were done with a quadratic load increment (from 0.05mN to 8 mN) resulting in 15 successive indentations at the same location repeated 5 times at 200 mm distant location. This allowed to investigate cartilage at different depth. In both type monotonic and cyclic indentation methods, elastic modulus (E), work of indentation (WI, area under the curve) and depth of indentation (h) were assessed. In the cyclic method they were assessed for each indentation force performed resulting in the determination of biomechanical material level properties at different depths. Distal femur were harvested from four 10 week-old female Lewis rats. Based on monotonic indentation, we first evaluated the reproducibility of the measurement by repositioning the indenter’s probe between each set of three monotonic indentations in the zone 1 of the medial condyle. Then, within each zone investigated and for each evaluated parameters, we determined in-vivo variability, defined by the average of the dispersion of the data between the 5 indentations which compose the monotonic indentation. In order to address the sensibility of the monotonic indentation, we assessed the difference between each investigated zone within the medial condyle (by t-test). Same investigations were also performed using cyclic indentation methodology within each zone of the medial condyle. Results: The mean reproducibility after probe repositioning was less than 10% for all evaluated parameters. In-vivo variability of all evaluated parameters within each investigated zone of interest was around 15% (range: 5-25%) in medial condyle as well as in lateral condyle. Despite this variability within each investigated zone we were able to determined distinct biomechanical properties relative to the zone investigated. In the medial condyle, in zone 1 as compared to zone 2 and 3, indentation depth was approximatively three times greater (p<.001) while WI and E were respectively four and two times greater (p<.05 and p<.001). Biomechanical properties were equivalent in zone 2 and 3. Based on the literature, this mapping of the cartilage biomechanical properties could correlate with in-vivo joint stress: since zone 1 is the most loaded zone during rat ambulation it could present the most elasticity. Using cyclic indentation method, at each investigated force inducing assessment of the hyaline cartilage thickness (without bone interaction), similar differences were observed as in the monotonic indentation method. This technic also succeeded to assess biomechanical deterioration of the cartilage in meniscectomized rats and in rats submitted to protein malnutrition (results presented at OARSI 2016 congress). Conclusions: These results indicate that the bioindentation of rat femoral cartilage is feasible, reproducible and enough sensible to detect biomechanical alterations between different zone of the cartilage as well as in biologic challenges.
564 DEVELOPING AN IN VITRO MODEL OF HEALTHY AND OA MEDIAL KNEE COMPARTMENTAL LOADING N. Khatib y, A. Metcalfe z, E. Blain y, D. Mason y, C. Holt y. y Cardiff Univ., Cardiff, United Kingdom; z Warwick Univ., Warwick, United Kingdom Purpose: A growing area of research aims to recreate ‘physiological’ and ‘pathological’ loading of the knee joint in an in vitro environment. Tissue loading models investigating the effect of uniaxial compression loading on chondral explants are employed to explore osteoarthritis (OA) development. There has been desire to study medial knee compartment loading as this is known to be a commonly affected area of knee tissue in OA progress. According to data acquired from instrumented knee replacement studies, external knee adduction moments (EKAMs) have been identified as a surrogate measure for medial contact force during gait. Therefore, a novel loading model has been explored, utilizing EKAM waveforms extracted from healthy subjects or late-stage OA patient dynamic knee gait analysis, to simulate ‘physiological’ and ‘pathological’ medial knee compartmental loading in an in vitro environment. The purpose of the study is to determine if there is a difference in tissue response (cell death, structural glycosaminoglycan release and peak deformation change) to loading with the two distinct waveforms. Methods: Dynamic EKAM waveforms for healthy volunteers and latestage (pre-total knee replacement) OA patients were measured using a 9-camera motion capture system (Qualisys) and force plates (Bertec Corp, USA), and extracted using Qualisys Track Manager and Visual 3D softwares (C-motion, USA). The main discrepancies were used to create two possible extremes of waveform (NL ¼ normal, OA ¼ osteoarthritic), to allow distinction of the two groups. 6mm disc cartilage explants (n¼12) were extracted from metacarpophalangeal joints of 7-10 day old bovine calves using sterile 6mm biopsy punches, stabilized in culture for 24 hours, and randomly separated into 3 groups subjected to either 1800 cycles of NL or OA waveforms at 1Hz with a peak load of 2.5MPa (maintains cartilage turnover), or an unloaded control. The application of cyclic compression load and measurement of peak explant deformation was made using the BOSE ElectroForce 3200® (Bose Corporation®, USA). Cell viability was measured using lactate dehydrogenase cytotoxicity assay on explant media 1h post loading (Invitrogen). Glycosaminoglycan (GAG) release was measured using a demethymethylene blue assay on explant media 1h post loading (DMMB, Invitrogen). Statistics were calculated and analysed using SPSS software. Results: The DMMB assays revealed there was no significant difference in GAG release into the explant media between the NL waveform loaded explants and unloaded controls, however there was a significantly higher (p<0.05) release in OA waveform loaded explants compared to controls. There was no statistical difference in GAG release directly between NL and OA waveforms. This result correlated with our LDH release assay, where similarly there was a significantly higher (p<0.05) relative cell death in OA loaded explants compared to unloaded controls, but with no significant difference compared to NL loaded. Mean relative cell death was averagely higher in the OA waveform group compared to NL waveform, but with no statistical significance to back up this result. Averagely higher peak explant deformation was determined in OA loaded explants compared to NL groups at every load cycle milestone (every 200 cycles), but with no statistical differences between the two groups for the duration of the loading. Conclusions: The results revealed by this study suggest that cell death and structural GAG release in bovine hyaline explant discs are weakly increased in response to OA EKAM waveforms in comparison to NL waveforms, and stronger in comparison to unloaded controls. The expected notion that increased peak deformation of explants as a result of OA waveform loading compared to NL may have a negative impact on tissue viability has been weakly supported by the results, however not proven. Higher explant sample sizes are required to investigate further and conclude these results.
Meniscus, Muscle, Tendon & Ligament Biology 565 PROGNOSTIC FACTORS FOR THE LONG-TERM CLINICAL OUTCOME OF ARTHROSCOPIC PARTIAL MENISCECTOMY - A SYSTEMATIC REVIEW S. Eijgenraam, D. Meuffels, S. Bierma-Zeinstra, D. van Yperen, M. Reijman. Erasmus MC, Rotterdam, Netherlands
Figure 3. Dedifferentiation increased cilia and proliferation.
S343
p¼0.248). Needle size (23G n¼33 or 21G n¼18) had no effect on IOP (t test¼0.141). Needle site (femoral head n¼6, femoral condyle n¼7, upper tibia n¼32) had no influence on IOP (analysis of variance p¼0.188). The majority of recordings showed a pulsatile wave form. At a recording speed of 12.5mm/sec the wave was seen to be synchronous with the arterial pulse. At a 1/60th slower recording speed of 12.5mm/min the arterial pulse was obliterated but an underlying wave synchronous with respiration was seen. After bolus drug administration a third or ‘circulation time wave’ was seen. This wave lasted some 90 seconds and could be followed for one or two passes. After a saline clearance bolus injection the IOP and PP fell. Recovery took up to 15 minutes. After aspiration to clear the needle recovery was within 30 seconds. There was a correlation between the IOP and the mean blood pressure (Pearson p<0.001) There was a close correlation between IOP and its associated pulse pressure (p<0.0001). Conclusions: These results show that IOP is not a static venous back pressure but is closely related to the arterial supply circulation. The variability in IOP is a manifestation of the size of vessel encountered by the needle tip or pool in the microcirculation in which the pressure is being measured. A single IOP measurement in isolation is meaningless. The physiology of the subchondral microcirculation may be important in understanding blood supply to the cartilage in health and in osteoarthritis.
Conclusions: Changes in primary cilia length affects cilia function including mechanotransduction and hedgehog signalling and may also affect other cilia signalling pathways. As hypothesized, the reduction in mechano-responsiveness and hedgehog signalling with extended 2D culture is due to the associated elongation of primary cilia. To further identify the underpinning mechanisms, ongoing studies are using super resolution microscopy to examine whether cilia elongation at P5 is associated with alterations in cilia expression of ARL13B, and putative mechanoreceptors polycystin 2 and TRPV4. The observed reductions in mechano-responsiveness and ligand-induced hedgehog signalling with passage have important implications for the success of cartilage tissue engineering. Future work will investigate whether modulation of cilia length can maintain or rescue mechano- and hedgehog-responsiveness during expansion of chondrocytes for cartilage tissue engineering. 562 FACTORS AFFECTING PHYSIOLOGY OF INTRAOSSEOUS PRESSURE MEASUREMENT M. Beverly, J. Urban, D. Murray. Oxford Univ., Oxford, United Kingdom Purpose: Intraosseous pressure (IOP) has been studied for 50 or more years and is of interest in diseases such as osteoarthritis and osteonecrosis and in bone pain. IOP in cancellous bone has usually been thought to have a static value due to venous back pressure or tissue turgor. We measured IOP in patients before surgery but the results varied. We therefore set out to assess factors which might influence the physiology of IOP at rest in healthy subchondral cancellous bone in a standardized animal model. Methods: With ethical approval IOP was studied in 21 anaesthetised rabbits. Intraosseous pressure was measured through needles inserted directly into the subchondral bone of the femoral head, femoral condyle or upper tibia. Pressure was also measured in the left femoral artery in 5 animals. A heparinized saline filled line was connected from the intraosseous needle to pressure transducers (Bell and Howell or Druck PDCR75) and to a four channel chart recorder (Lectromed MX4P- 31). The transducers were calibrated on a 0-60 mmHg scale. Needle clearance was occasionally required using a 0.5ml saline bolus. Results: Basal intraosseous pressure (IOP) was measured in 21 individuals at 51 sites and varied considerably between different subjects and also at different sites in the same subject. The average IOP (n¼51) was 23.8mmHg (SD 13.6mmHg) with a pulse pressure (PP) of 4.0mmHg (SD 4.2mmHg). Gender (female n¼14, male n¼7) had no effect on IOP (t test¼0.58). Weight (2,920g - 5,560g) had no effect on IOP (Pearson correlation
563 NANO-INDENTATION METHODOLOGY FOR CARTILAGE QUALITY ASSESSMENT C. Lavet, P. Ammann. Service of Bone Diseases, Geneva, Switzerland Purpose: To date, histological analysis is the gold-standard research tool for assessing cartilage integrity. Indentation features the benefits of assessing the main function of cartilage (shock absorption and force transmission) by its biomechanical parameters which could be altered by numerous factors. Furthermore significant cartilage metabolic disturbances can occur in cartilage in early stage of osteoarthrosis (OA) and prior to morphometric alterations. Therefore it could also be assumed that a normal macroscopic appearance of the cartilage does not ensure the absence of pathology. Despite the wide range of cartilage biomechanical testing methods in samples harvested from human or big animals’ models, rodents’ models which have an increasingly prominent role in OA research, are lacking of biomechanical assessment of their cartilage. Therefore, it is mandatory to develop a technique dedicated to the objective evaluation of the cartilage tissue quality. Methods: The original positioning of the femur allows to indent three different areas of interest of the cartilage surface corresponding to area physiologically exposed to load with various forces and orientation, various timing along the day and various frequency. Two different methods were used. Monotonic indentation was defined as a set of 5 successive indentations (controlled in force) within the region of interest. Each indentation was 200 mm distant from each other and were performed in the same sagittal plan of the medial condyle (at the center). Cyclic indentation was defined as a set of 5 successive cyclic
S344
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
indentations. All the 5 cyclic indentations were done with a quadratic load increment (from 0.05mN to 8 mN) resulting in 15 successive indentations at the same location repeated 5 times at 200 mm distant location. This allowed to investigate cartilage at different depth. In both type monotonic and cyclic indentation methods, elastic modulus (E), work of indentation (WI, area under the curve) and depth of indentation (h) were assessed. In the cyclic method they were assessed for each indentation force performed resulting in the determination of biomechanical material level properties at different depths. Distal femur were harvested from four 10 week-old female Lewis rats. Based on monotonic indentation, we first evaluated the reproducibility of the measurement by repositioning the indenter’s probe between each set of three monotonic indentations in the zone 1 of the medial condyle. Then, within each zone investigated and for each evaluated parameters, we determined in-vivo variability, defined by the average of the dispersion of the data between the 5 indentations which compose the monotonic indentation. In order to address the sensibility of the monotonic indentation, we assessed the difference between each investigated zone within the medial condyle (by t-test). Same investigations were also performed using cyclic indentation methodology within each zone of the medial condyle. Results: The mean reproducibility after probe repositioning was less than 10% for all evaluated parameters. In-vivo variability of all evaluated parameters within each investigated zone of interest was around 15% (range: 5-25%) in medial condyle as well as in lateral condyle. Despite this variability within each investigated zone we were able to determined distinct biomechanical properties relative to the zone investigated. In the medial condyle, in zone 1 as compared to zone 2 and 3, indentation depth was approximatively three times greater (p<.001) while WI and E were respectively four and two times greater (p<.05 and p<.001). Biomechanical properties were equivalent in zone 2 and 3. Based on the literature, this mapping of the cartilage biomechanical properties could correlate with in-vivo joint stress: since zone 1 is the most loaded zone during rat ambulation it could present the most elasticity. Using cyclic indentation method, at each investigated force inducing assessment of the hyaline cartilage thickness (without bone interaction), similar differences were observed as in the monotonic indentation method. This technic also succeeded to assess biomechanical deterioration of the cartilage in meniscectomized rats and in rats submitted to protein malnutrition (results presented at OARSI 2016 congress). Conclusions: These results indicate that the bioindentation of rat femoral cartilage is feasible, reproducible and enough sensible to detect biomechanical alterations between different zone of the cartilage as well as in biologic challenges.
564 DEVELOPING AN IN VITRO MODEL OF HEALTHY AND OA MEDIAL KNEE COMPARTMENTAL LOADING N. Khatib y, A. Metcalfe z, E. Blain y, D. Mason y, C. Holt y. y Cardiff Univ., Cardiff, United Kingdom; z Warwick Univ., Warwick, United Kingdom Purpose: A growing area of research aims to recreate ‘physiological’ and ‘pathological’ loading of the knee joint in an in vitro environment. Tissue loading models investigating the effect of uniaxial compression loading on chondral explants are employed to explore osteoarthritis (OA) development. There has been desire to study medial knee compartment loading as this is known to be a commonly affected area of knee tissue in OA progress. According to data acquired from instrumented knee replacement studies, external knee adduction moments (EKAMs) have been identified as a surrogate measure for medial contact force during gait. Therefore, a novel loading model has been explored, utilizing EKAM waveforms extracted from healthy subjects or late-stage OA patient dynamic knee gait analysis, to simulate ‘physiological’ and ‘pathological’ medial knee compartmental loading in an in vitro environment. The purpose of the study is to determine if there is a difference in tissue response (cell death, structural glycosaminoglycan release and peak deformation change) to loading with the two distinct waveforms. Methods: Dynamic EKAM waveforms for healthy volunteers and latestage (pre-total knee replacement) OA patients were measured using a 9-camera motion capture system (Qualisys) and force plates (Bertec Corp, USA), and extracted using Qualisys Track Manager and Visual 3D softwares (C-motion, USA). The main discrepancies were used to create two possible extremes of waveform (NL ¼ normal, OA ¼ osteoarthritic), to allow distinction of the two groups. 6mm disc cartilage explants (n¼12) were extracted from metacarpophalangeal joints of 7-10 day old bovine calves using sterile 6mm biopsy punches, stabilized in culture for 24 hours, and randomly separated into 3 groups subjected to either 1800 cycles of NL or OA waveforms at 1Hz with a peak load of 2.5MPa (maintains cartilage turnover), or an unloaded control. The application of cyclic compression load and measurement of peak explant deformation was made using the BOSE ElectroForce 3200® (Bose Corporation®, USA). Cell viability was measured using lactate dehydrogenase cytotoxicity assay on explant media 1h post loading (Invitrogen). Glycosaminoglycan (GAG) release was measured using a demethymethylene blue assay on explant media 1h post loading (DMMB, Invitrogen). Statistics were calculated and analysed using SPSS software. Results: The DMMB assays revealed there was no significant difference in GAG release into the explant media between the NL waveform loaded explants and unloaded controls, however there was a significantly higher (p<0.05) release in OA waveform loaded explants compared to controls. There was no statistical difference in GAG release directly between NL and OA waveforms. This result correlated with our LDH release assay, where similarly there was a significantly higher (p<0.05) relative cell death in OA loaded explants compared to unloaded controls, but with no significant difference compared to NL loaded. Mean relative cell death was averagely higher in the OA waveform group compared to NL waveform, but with no statistical significance to back up this result. Averagely higher peak explant deformation was determined in OA loaded explants compared to NL groups at every load cycle milestone (every 200 cycles), but with no statistical differences between the two groups for the duration of the loading. Conclusions: The results revealed by this study suggest that cell death and structural GAG release in bovine hyaline explant discs are weakly increased in response to OA EKAM waveforms in comparison to NL waveforms, and stronger in comparison to unloaded controls. The expected notion that increased peak deformation of explants as a result of OA waveform loading compared to NL may have a negative impact on tissue viability has been weakly supported by the results, however not proven. Higher explant sample sizes are required to investigate further and conclude these results.
Meniscus, Muscle, Tendon & Ligament Biology 565 PROGNOSTIC FACTORS FOR THE LONG-TERM CLINICAL OUTCOME OF ARTHROSCOPIC PARTIAL MENISCECTOMY - A SYSTEMATIC REVIEW S. Eijgenraam, D. Meuffels, S. Bierma-Zeinstra, D. van Yperen, M. Reijman. Erasmus MC, Rotterdam, Netherlands