- Email: [email protected]
Establishment of a Human Synovium and Cartilage Co-Culture
S276
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
tonic (100 mOsm/kg water) condition. Thereafter, enzymatic samples were incubated at 37 ºC and for 1 hour in collagenase type II (0.5 mM, Worthington Biochemical Corporation) in their corresponding osmolalities with a sufficient supply of Caþþ. Cross-linking samples were incubated in L-threose (50mM, Sigma Aldrich) instead for four consecutive days using the same incubation parameters as the enzymatic experiment. Post-treatment the samples were re-equilibrated (400 mOsm/kg water and 4 C) for 1 hour and micro-indentation in the same area as in the initial step was performed. The relative difference in the average effective Young's modulus was used as an indication of the chemical activities that created either collagen cleavage and lowered the stiffness (collagenase type II) or created crosslinks and increased stiffness (L-threose). Moreover, surface color of the specimens after incubation in L-threose was quantified as an indication of non-enzymatic cross-linking efficiency. Results: The loss in the average effective Young's modulus for the samples treated with collagenase type II was lower under hyper-osmolality condition (Figure 1). Within all groups, we observed an osmolality-dependent trend with respect to the cross-linking process (Figure 2). Increase of the osmolality is associated with a higher chance of collagen fibril cross-linking as suggested by the changes in the average effective Young’s modulus. Furthermore, color analysis of cartilage surface confirmed the increase in cross-linking i.e. toward dark yellow, as the osmolality increases (Figure 3). Conclusions: Chemical reactions are believed to be responsible for several types of collagen fibril alterations within articular cartilage, namely enzymatic cleavage occurring in OA and advanced glycation or non-enzymatic cross-linking occurring in ageing. One way to reduce these glycation reactions is to enhance the pre-stress in the collagen fibers, which can be accomplished by low osmolality in the external bath or by increased proteoglycan content. In the current study, we showed that collagen fibril pre-stress plays a role in controlling the degradation induced by bacterial collagenase as well, although in a reverse way compared to the glycation processes. Nevertheless, the effect of osmolality i.e. collagen fibril pre-stress on the enzymatic activity of MMPs is yet to be investigated as a complementary to this research. Therefore, mechanical forces in the macro scale could substantially affect the molecular processes within collagen fibrils in articular cartilage which could offer potential strategies to restrict them and consequently prolong the functional lifetime of articular cartilage.
Figure 1: Changes in effective Young's modulus as a result of enzymatic treatment with collagenase II.
Figure 2: Changes in effective Young's modulus as a result of non-enzymatic cross-linking treatment.
Figure 3: Analysis of cartilage surface color post-treatment with L-threose.
437 ESTABLISHMENT OF A HUMAN SYNOVIUM AND CARTILAGE COCULTURE C. Kjelgaard-Petersen y, z, A.-C. Bay-Jensen y, T. Christiansen x, €gglund z, A. Siebuhr y, C.S. Thudium y. y NordicbioSci., M.A. Karsdal y, P. Ha Herlev, Denmark; z Technical Univ. of Denmark, Kgs. Lyngby, Denmark; x Gentofte Univ. Hosp., Gentofte, Denmark Purpose: Osteoarthritis (OA) is a degenerative joint disease with a lowgrade inflammatory component that leads to an altered turnover of extracellular matrix (ECM), not only in the cartilage, but also in the synovium and bone. The diseased tissues are believed to interact with each other and initiate and drive OA. However, the mechanism behind this interaction and the effect on the ECM turnover are unknown. The aim of this study was to establish an ex vivo co-culture model of the cartilage and synovium to study the interaction between the two tissues and its effect on the ECM turnover. Methods: Human synovium and cartilage were obtained from endstage OA patients undergoing total knee replacement. The synovium were cut into explants of 30±4 mg and the cartilage were punched with a 5 mm biopsy punches. After isolation of the two tissues, they were immediately added together in one well. Additionally both tissues were also cultured alone. The tissues were cultured for 14 days with OSM [10ng/mL] and TNFa [20ng/mL] (OþT), alone (w/o), or with OþT and GM6001 10 mM. GM6001 was only included in the co-culture system. The metabolic activity was measured with alamar blue weekly. Conditioned media were removed three times a week and fresh treatment added. The conditioned media were used for biomarker measurement. Four biomarkers, C3M, AGNx1, FFGV, and C2M, were measured by ELISA in the conditioned media. Results: The human co-culture of the synovium and cartilage were cultured for 14 days. The explants were metabolic active throughout the study. However, the metabolic activity of the synovium dropped after 7 days on culture. Four biomarkers of the joint ECM turnover were measured in the conditioned medium and the accumulated biomarker over 14 days were calculated based on measurements of four time points. OþT increased the release of C3M 3.2-fold compared to w/o in the co-culture and 8.6-fold (P¼0.016) compared to w/o in the synovium alone (Fig.1a). OþT increased the release of C2M 4.9-fold compared to w/o in the co-culture (P¼0.012), 2-fold compared to non-treated synovium (P¼0.041), and 2.4-fold compared to non-treated cartilage (P<0.001) (Fig. 1b). The MMP-mediated aggrecanse degradation, FFGV, was increased in 12.9-fold in response to OþT compared to w/o in the co-culture (P¼0.003) and 7.1-fold in cartilage alone (P¼0.002) (Fig. 1c). AGNx1, aggrecanase mediated degradation of aggrecan, was similarly released in response to OþT treatment compared to w/o, 2.2-fold increase in the co-culture (P¼0.008) and 3.7-fold increase from cartilage alone (P<0.001) (Fig. 1d). Neither FFGV nor AGNx1 were released from synovium alone (Fig. 1c and d). No increased biomarker release was measured in the co-culture without external cytokine stimuli. The release of C3M, C2M and FFGV were MMP-depended as GM6001 inhibited the release (Fig. 1).
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
Conclusions: It was possible to culture the synovium and cartilage together for 14 days. The pro-inflammatory cytokines OSM and TNFa induced release of C3M, C2M, FFGV, and AGNx1 from the co-culture, where C3M originated from the synovium, C2M originated primarily from the cartilage, and FFGV and AGNx1 only originated from the cartilage. This model provides a tool to study effect of potential drugs on the synovium and cartilage together.
Mechanobiology 438 CHONDROCYTE-SPECIFIC LOSS OF THE BRANCHED ACTIN MEDIATOR ARP2/3 RESULTS IN GROWTH PLATE FUSION AND PROTEOGLYCAN LOSS IN ARTICULAR CARTILAGE
S277
Results: Mice with constitutive loss of Arp2/3 in chondrocytes showed reduced viability (5/90 pups; expected ratio 1/4). Mutant mice that did survive were smaller than littermate controls and showed disorganization of the growth plate with secondary ossification failure (Fig. 1). When Arp2/3 loss was induced at 4 months, growth plates showed partial closure at 6 months and complete fusion at 12 months (Fig. 2). The articular cartilage structure at 6 and 12 months was not affected by Arp2/3 loss (Fig. 3a). However, the proteoglycan content was significantly reduced at 12 months in mice with Arp2/3 loss as compared to other groups (Fig. 3b). Osteophytes were noted in 4 out of 8 mice with Arp2/3 loss at 12 months as compared to 1 out of 6 controls. Using an in vitro assay of cell-matrix interaction, growth plate and articular chondrocytes directly isolated from 3 week old mice with induced Arp2/3 loss showed less spread cell area on fibronectin and type I collagen coated dishes as compared to heterozygous controls (all combinations p<0.01). Conclusions: The ability of chondrocytes to sense and respond to extra-cellular matrix cues plays important roles during both development and homeostasis. Arp2/3 appears to mediate chondrocytematrix interactions, as cells with Arp2/3 loss were unable to spread normally on collagen and fibronectin surfaces. Growth plate chondrocytes with constitutive Arp2/3 loss failed to organize the highly ordered columns that result in longitudinal bone growth. When Arp2/ 3 loss was induced after skeletal maturity, growth plates progressively fused in a manner that does not typically occur in mice. This suggests that cell-matrix interactions or other Arp2/3 functions contribute to the active maintenance of the growth plate throughout adulthood. Loss of Arp2/3 in articular chondrocytes did not result in extensive erosion of cartilage matrix at 12 months, but did cause reduced staining for proteoglycans and more osteophytes. These observations suggest a role for the actin cytoskeleton in maintaining cartilage homeostasis over the long-term, although longer follow-up may be necessary to determine whether loss of Arp2/3 would result in more severe OA. This work has implications for understanding developmental phenomena such as chondrodysplasias and catch-up growth, as well as providing insight into the mechanisms that govern how cell-matrix interactions in articular cartilage may alter the risk for OA progression.
B.O. Diekman, J.D. Rotty, V. Ulici, J.E. Bear, R.F. Loeser, Jr., N.E. Sharpless. Univ. of North Carolina Chapel Hill, Chapel Hill, NC, USA Purpose: The actin cytoskeleton regulates cell shape changes that are associated with chondrogenic differentiation in the developing growth plate and with phenotypic changes to articular chondrocytes during monolayer culture. However, it has been challenging to determine the role of the actin cytoskeleton during cartilage homeostasis in vivo. The Arp2/3 complex generates branched actin and is involved in many cellular processes including cell motility in response to extra-cellular matrix cues. We hypothesized that chondrocyte-specific deletion of Arp2/3 in vivo would alter cell-matrix interactions and result in both growth plate disorganization and premature osteoarthritis (OA). Methods: C57BL/6 mice harboring flanking loxP sites around a critical exon of an Arp2/3 subunit (p34) were crossed to two chondrocytespecific Cre driver strains. Type II collagen (Col2)-Cre resulted in constitutive loss of Arp2/3, whereas Aggrecan-CreERT2 was used to induce Arp2/3 loss by intraperitoneal injection of tamoxifen. The cellular organization of the tibial growth plate was analyzed using hematoxylin and eosin stained histological sections. Mid-coronal sections stained with Safranin-O were evaluated for disrupted articular cartilage structure and proteoglycan loss using established semiquantitative scales of 0e12 for medial and lateral sides of the tibial and femoral cartilage. Statistical analysis was performed by ANOVA and Tukey's post-hoc test with greater than 6 mice per group. Live cell imaging was used to quantify cell spreading in isolated cells after induction of Arp2/3 loss in vivo. Pure populations of growth plate and articular chondrocytes were sorted based on fluorescence in cells successfully recombined by Aggrecan-CreERT2 using a lox-stop-lox zsgreen reporter allele.
Figure 1: H&E stained section of tibiae from 26 day old mice. Homozygous Arp2/3 loss was driven by a Col2-Cre (right) and littermate control retaining one copy of Arp2/3 is shown (left). Boxes indicate approximate location of inset (bottom). All scale bars are 50 mm.
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
tonic (100 mOsm/kg water) condition. Thereafter, enzymatic samples were incubated at 37 ºC and for 1 hour in collagenase type II (0.5 mM, Worthington Biochemical Corporation) in their corresponding osmolalities with a sufficient supply of Caþþ. Cross-linking samples were incubated in L-threose (50mM, Sigma Aldrich) instead for four consecutive days using the same incubation parameters as the enzymatic experiment. Post-treatment the samples were re-equilibrated (400 mOsm/kg water and 4 C) for 1 hour and micro-indentation in the same area as in the initial step was performed. The relative difference in the average effective Young's modulus was used as an indication of the chemical activities that created either collagen cleavage and lowered the stiffness (collagenase type II) or created crosslinks and increased stiffness (L-threose). Moreover, surface color of the specimens after incubation in L-threose was quantified as an indication of non-enzymatic cross-linking efficiency. Results: The loss in the average effective Young's modulus for the samples treated with collagenase type II was lower under hyper-osmolality condition (Figure 1). Within all groups, we observed an osmolality-dependent trend with respect to the cross-linking process (Figure 2). Increase of the osmolality is associated with a higher chance of collagen fibril cross-linking as suggested by the changes in the average effective Young’s modulus. Furthermore, color analysis of cartilage surface confirmed the increase in cross-linking i.e. toward dark yellow, as the osmolality increases (Figure 3). Conclusions: Chemical reactions are believed to be responsible for several types of collagen fibril alterations within articular cartilage, namely enzymatic cleavage occurring in OA and advanced glycation or non-enzymatic cross-linking occurring in ageing. One way to reduce these glycation reactions is to enhance the pre-stress in the collagen fibers, which can be accomplished by low osmolality in the external bath or by increased proteoglycan content. In the current study, we showed that collagen fibril pre-stress plays a role in controlling the degradation induced by bacterial collagenase as well, although in a reverse way compared to the glycation processes. Nevertheless, the effect of osmolality i.e. collagen fibril pre-stress on the enzymatic activity of MMPs is yet to be investigated as a complementary to this research. Therefore, mechanical forces in the macro scale could substantially affect the molecular processes within collagen fibrils in articular cartilage which could offer potential strategies to restrict them and consequently prolong the functional lifetime of articular cartilage.
Figure 1: Changes in effective Young's modulus as a result of enzymatic treatment with collagenase II.
Figure 2: Changes in effective Young's modulus as a result of non-enzymatic cross-linking treatment.
Figure 3: Analysis of cartilage surface color post-treatment with L-threose.
437 ESTABLISHMENT OF A HUMAN SYNOVIUM AND CARTILAGE COCULTURE C. Kjelgaard-Petersen y, z, A.-C. Bay-Jensen y, T. Christiansen x, €gglund z, A. Siebuhr y, C.S. Thudium y. y NordicbioSci., M.A. Karsdal y, P. Ha Herlev, Denmark; z Technical Univ. of Denmark, Kgs. Lyngby, Denmark; x Gentofte Univ. Hosp., Gentofte, Denmark Purpose: Osteoarthritis (OA) is a degenerative joint disease with a lowgrade inflammatory component that leads to an altered turnover of extracellular matrix (ECM), not only in the cartilage, but also in the synovium and bone. The diseased tissues are believed to interact with each other and initiate and drive OA. However, the mechanism behind this interaction and the effect on the ECM turnover are unknown. The aim of this study was to establish an ex vivo co-culture model of the cartilage and synovium to study the interaction between the two tissues and its effect on the ECM turnover. Methods: Human synovium and cartilage were obtained from endstage OA patients undergoing total knee replacement. The synovium were cut into explants of 30±4 mg and the cartilage were punched with a 5 mm biopsy punches. After isolation of the two tissues, they were immediately added together in one well. Additionally both tissues were also cultured alone. The tissues were cultured for 14 days with OSM [10ng/mL] and TNFa [20ng/mL] (OþT), alone (w/o), or with OþT and GM6001 10 mM. GM6001 was only included in the co-culture system. The metabolic activity was measured with alamar blue weekly. Conditioned media were removed three times a week and fresh treatment added. The conditioned media were used for biomarker measurement. Four biomarkers, C3M, AGNx1, FFGV, and C2M, were measured by ELISA in the conditioned media. Results: The human co-culture of the synovium and cartilage were cultured for 14 days. The explants were metabolic active throughout the study. However, the metabolic activity of the synovium dropped after 7 days on culture. Four biomarkers of the joint ECM turnover were measured in the conditioned medium and the accumulated biomarker over 14 days were calculated based on measurements of four time points. OþT increased the release of C3M 3.2-fold compared to w/o in the co-culture and 8.6-fold (P¼0.016) compared to w/o in the synovium alone (Fig.1a). OþT increased the release of C2M 4.9-fold compared to w/o in the co-culture (P¼0.012), 2-fold compared to non-treated synovium (P¼0.041), and 2.4-fold compared to non-treated cartilage (P<0.001) (Fig. 1b). The MMP-mediated aggrecanse degradation, FFGV, was increased in 12.9-fold in response to OþT compared to w/o in the co-culture (P¼0.003) and 7.1-fold in cartilage alone (P¼0.002) (Fig. 1c). AGNx1, aggrecanase mediated degradation of aggrecan, was similarly released in response to OþT treatment compared to w/o, 2.2-fold increase in the co-culture (P¼0.008) and 3.7-fold increase from cartilage alone (P<0.001) (Fig. 1d). Neither FFGV nor AGNx1 were released from synovium alone (Fig. 1c and d). No increased biomarker release was measured in the co-culture without external cytokine stimuli. The release of C3M, C2M and FFGV were MMP-depended as GM6001 inhibited the release (Fig. 1).
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
Conclusions: It was possible to culture the synovium and cartilage together for 14 days. The pro-inflammatory cytokines OSM and TNFa induced release of C3M, C2M, FFGV, and AGNx1 from the co-culture, where C3M originated from the synovium, C2M originated primarily from the cartilage, and FFGV and AGNx1 only originated from the cartilage. This model provides a tool to study effect of potential drugs on the synovium and cartilage together.
Mechanobiology 438 CHONDROCYTE-SPECIFIC LOSS OF THE BRANCHED ACTIN MEDIATOR ARP2/3 RESULTS IN GROWTH PLATE FUSION AND PROTEOGLYCAN LOSS IN ARTICULAR CARTILAGE
S277
Results: Mice with constitutive loss of Arp2/3 in chondrocytes showed reduced viability (5/90 pups; expected ratio 1/4). Mutant mice that did survive were smaller than littermate controls and showed disorganization of the growth plate with secondary ossification failure (Fig. 1). When Arp2/3 loss was induced at 4 months, growth plates showed partial closure at 6 months and complete fusion at 12 months (Fig. 2). The articular cartilage structure at 6 and 12 months was not affected by Arp2/3 loss (Fig. 3a). However, the proteoglycan content was significantly reduced at 12 months in mice with Arp2/3 loss as compared to other groups (Fig. 3b). Osteophytes were noted in 4 out of 8 mice with Arp2/3 loss at 12 months as compared to 1 out of 6 controls. Using an in vitro assay of cell-matrix interaction, growth plate and articular chondrocytes directly isolated from 3 week old mice with induced Arp2/3 loss showed less spread cell area on fibronectin and type I collagen coated dishes as compared to heterozygous controls (all combinations p<0.01). Conclusions: The ability of chondrocytes to sense and respond to extra-cellular matrix cues plays important roles during both development and homeostasis. Arp2/3 appears to mediate chondrocytematrix interactions, as cells with Arp2/3 loss were unable to spread normally on collagen and fibronectin surfaces. Growth plate chondrocytes with constitutive Arp2/3 loss failed to organize the highly ordered columns that result in longitudinal bone growth. When Arp2/ 3 loss was induced after skeletal maturity, growth plates progressively fused in a manner that does not typically occur in mice. This suggests that cell-matrix interactions or other Arp2/3 functions contribute to the active maintenance of the growth plate throughout adulthood. Loss of Arp2/3 in articular chondrocytes did not result in extensive erosion of cartilage matrix at 12 months, but did cause reduced staining for proteoglycans and more osteophytes. These observations suggest a role for the actin cytoskeleton in maintaining cartilage homeostasis over the long-term, although longer follow-up may be necessary to determine whether loss of Arp2/3 would result in more severe OA. This work has implications for understanding developmental phenomena such as chondrodysplasias and catch-up growth, as well as providing insight into the mechanisms that govern how cell-matrix interactions in articular cartilage may alter the risk for OA progression.
B.O. Diekman, J.D. Rotty, V. Ulici, J.E. Bear, R.F. Loeser, Jr., N.E. Sharpless. Univ. of North Carolina Chapel Hill, Chapel Hill, NC, USA Purpose: The actin cytoskeleton regulates cell shape changes that are associated with chondrogenic differentiation in the developing growth plate and with phenotypic changes to articular chondrocytes during monolayer culture. However, it has been challenging to determine the role of the actin cytoskeleton during cartilage homeostasis in vivo. The Arp2/3 complex generates branched actin and is involved in many cellular processes including cell motility in response to extra-cellular matrix cues. We hypothesized that chondrocyte-specific deletion of Arp2/3 in vivo would alter cell-matrix interactions and result in both growth plate disorganization and premature osteoarthritis (OA). Methods: C57BL/6 mice harboring flanking loxP sites around a critical exon of an Arp2/3 subunit (p34) were crossed to two chondrocytespecific Cre driver strains. Type II collagen (Col2)-Cre resulted in constitutive loss of Arp2/3, whereas Aggrecan-CreERT2 was used to induce Arp2/3 loss by intraperitoneal injection of tamoxifen. The cellular organization of the tibial growth plate was analyzed using hematoxylin and eosin stained histological sections. Mid-coronal sections stained with Safranin-O were evaluated for disrupted articular cartilage structure and proteoglycan loss using established semiquantitative scales of 0e12 for medial and lateral sides of the tibial and femoral cartilage. Statistical analysis was performed by ANOVA and Tukey's post-hoc test with greater than 6 mice per group. Live cell imaging was used to quantify cell spreading in isolated cells after induction of Arp2/3 loss in vivo. Pure populations of growth plate and articular chondrocytes were sorted based on fluorescence in cells successfully recombined by Aggrecan-CreERT2 using a lox-stop-lox zsgreen reporter allele.
Figure 1: H&E stained section of tibiae from 26 day old mice. Homozygous Arp2/3 loss was driven by a Col2-Cre (right) and littermate control retaining one copy of Arp2/3 is shown (left). Boxes indicate approximate location of inset (bottom). All scale bars are 50 mm.