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Development of Optimized Copolymers and Delivery Formulations to Scavenge Reactive Oxygen Species and Prevent Joint Damage from Post-Traumatic Osteoarthritis
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
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Figure 1: Exemplary depth-wise histograms for normal (A) and degenerated (B) AC. On the degenerated sample (B) wider histogram and two peaks (red arrows) can be observed in the zone 3. The VOIs selected for analysis are indicated with green and blue rectangles for zones 2 and 3, respectively. Results: With increasing degeneration, widening of histogram is seen in the depth-wise histogram visualizations (Fig 1). This widening is more pronounced in zone 3 occasionally characterized by a histogram with two or more peaks (Fig 1B, red arrows). No clear degenerationdependence can be observed in the peak intensity (Fig 2, left column). However, with increased degeneration FWHM shows an increasing trend (Fig 2, right column).
415 HETEROGENEITY IN EXTRACELLULAR MATRIX OF ARTICULAR CARTILAGE AS A FUNCTION OF OSTEOARTHRITIS PROGRESSION AS CHARACTERIZED BY MICRO-CT € y, x, S.S. Karhula y, k, M. Valkealahti y, ¶, T.J. Ylitalo y, z, M. Finnila €m z, K.P. Pritzker #, yy, S. Saarakkala y, ¶, P. Lehenkari y, ¶, E. Hæggstro H.J. Nieminen y, z. y Univ. of Oulu, Oulu, Finland; z Univ. of Helsinki, Helsinki, Finland; x Univ. of Eastern Finlad, Kuopio, Finland; k Infotech Oulu, Oulu, Finland; ¶ Oulu Univ. Hosp., Oulu, Finland; # Univ. of Toronto, Toronto, ON, Canada; yy Mount Sinai Hosp., Toronto, ON, Canada Purpose: Phosphotungstic acid (PTA) provides contrast in articular cartilage (AC) in microcomputed tomography (mCT). The contrast is associated with the content of collagenous proteins in the extracellular matrix (ECM). ECM goes through morphological modifications during osteoarthritis (OA) process characterized by chemical modifications and structural degeneration. It has been demonstrated in earlier literature that ECM heterogeneity is associated with OA. In this study, we aimed to quantify the heterogeneity using simple histogram derived-parameters from PTA-stained cartilage samples scanned with mCT. Methods: Human osteochondral cylinders (n ¼ 36, diameter ¼ 2.0 mm) were obtained from tibial plateu and femoral condyle of 19 patients undergoing total knee arthroplasty (ethics approval PPSHP 78/2013; consents obtained). Samples were subjected to formalin fixation (5 d), after which they were immersed for 48h (n ¼ 34) to 72 h (n ¼ 2) in 70% EtOH containing 1% w/v PTA. Stained samples were imaged with mCT (Skyscan 1272; Bruker microCT, Kontich, Belgium; scanning parameters: 45 kV, 222 mA, 3.2 mm voxel side length, 3050 ms, 2 frames/projection, 1200 projections, and with 0.25 mm Aluminium filter). Projections were reconstructed using NRecon -software (v.1.6.10.4.; Bruker microCT). From the reconstructed data AC volume was segmented (CTAnalyser, v. 1.16.4.1; Bruker microCT) and the AC surface was aligned horizontally using Dataviewer -software (v.1.5.2.4; Bruker microCT). Histogram was calculated for each horizontal (parallel to AC surface) slice (Fig. 1) to describe the depth-wise heterogeneity. Analysis volumes of interest (VOI) (150 150 30 voxels, 480 480 96 mm) were automatically selected from center of the cylinder at relative AC depths of 15 % (Zone 2) and 65 % (Zone 3) from AC surface. Three samples were excluded because segmentation and VOI selection artefacts. Intensity histograms were calculated for each VOI and peak position and full width at half maximum (FWHM) was calculated for each histogram. For reference OARSI grade was evaluated from the orthogonal center slices of reconstructed mCT data.
Figure 2: Histogram peak intensity and FWHM as function of OARSI. Variance between samples increases greatly as function of OARSI. Increasing trend of FWHM in both zone 2 and zone 3 indicates the increase in contrast heterogeneity. Conclusions: Our histogram-based approximation of heterogeneity is in line with earlier literature suggesting that heterogeneity of AC increases with OA progression. However, it appears that presented histogram approach is not a very specific marker to OA progression. A possible explanation to this might be that OARSI grade is a marker for OA progression, whereas the heterogeneity of ECM (histogram parameters) contains information on both OA severity and reaction. Our future studies will investigate if other histogram based approaches could provide further information on ECM heterogeneity and OA progression.
Inflammation and Immunity 416 DEVELOPMENT OF OPTIMIZED COPOLYMERS AND DELIVERY FORMULATIONS TO SCAVENGE REACTIVE OXYGEN SPECIES AND PREVENT JOINT DAMAGE FROM POST-TRAUMATIC OSTEOARTHRITIS T.E. Kavanaugh y, E.A. Dailing y, H. Cho z, K.A. Hasty z, C.L. Duvall y. y Vanderbilt Univ., Nashville, TN, USA; z Univ. of Tennessee Hlth.Sci. Ctr., Memphis, TN, USA Purpose: Post-traumatic osteoarthritis (PTOA) occurs after a traumatic injury to the bone or soft tissue and there is currently no cure. Reactive
S266
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
oxygen species (ROS) are elevated at sites of joint injury and can cause cell-damaging and pro-inflammatory “oxidative stress” that propagates the tissue degenerative process of PTOA. We sought to develop a sustained and active hybrid microparticle that can be delivery locally and sustainably scavenge both hydrogen peroxide and superoxide ROS. To scavenge hydrogen peroxide, we utilize poly(propylene sulfide) (PPS), which undergoes a phase change from hydrophobic to hydrophilic when oxidized by hydrogen peroxide to provide a drug release and microparticle degradation mechanism. Oil-in-water emulsion allows for fabrication of PPS microparticles loaded with hydrophobic molecules, but loading efficiency is very low for water-soluble small molecules drugs such as Tempol (4-hydroxy Tempo), a powerful superoxide dismutase mimetic. To improve loading, we synthesized a polymeric form of TEMPO (poly(Tempo)), and much of this work is focused on optimization of poly(Tempo) composition and bioactivity. We hypothesize that local, delivery of polyTempo-PPS microparticles can to prevent the onset of PTOA after a joint injury Methods: Reversible addition-fragmentation chain-transfer (RAFT) polymerization was used to synthesize a polymeric form of Tempo (polyTempo) (Figure 1A). Co-polymers of 4-amino Tempo and the small hydrophilic monomer dimethylacrylamide (DMA) (DMA-co-Tempo) were synthesized at different molar ratios to tune Tempo density along the backbone (10e100 mol% Tempo). Cell free ROS scavenging of the copolymers was measured based on ferric reducing antioxidant power (FRAP). Intracellular ROS of the copolymers was also measured in RAW 264.7 macrophages using dichlorofluorescin diacetate (DCFDA). Composite microparticles of DMA-co-Tempo (0:100) were synthesized with PPS via oil-in-water emulsion; PPS serves both as a H2O2 “sponge” and also as a bioresponsive depot that triggers release of therapeutic cargo in response to ROS. Using a non-invasive, mechanical loading murine model of PTOA, polyTempo and polyTempo-PPS microparticles were delivered via intraarticular (IA) injection. MMPSense 750 and MabCII 680, a collagen II specific antibody, were used to monitor the protease activity and cartilage damage, respectively, in vivo. Results: DMA-co-Tempo polymers at DMA:Tempo ratios of 0:100, 50:50, 60:40, 70:30, 75:25, 80:20 and 90:10 have increasing water solubility and increased Tempo bioavailability. The DMA-co-Tempo ratio of 70:30 produced the highest antioxidant power, measured by FRAP, resulting in equal antioxidant power to Tempol and increased antioxidant power 10-fold over polyTempo (DMA-co-Tempo 0:100) (Figure 1B).
Figure 1: DMA-co-Tempo synthesis and characterization. (A) DMA-coTempo is synthesized via a combination of RAFT polymerization and postpolymerization grafting of amine modified Tempo to Pentafluorophenol acrylate(PFPA) (B) Total antioxidant power of DMA-co-Tempo polymers is determined using a Ferric Reducing Antioxidant Power (FRAP) assay.
In preliminary studies, treatment of RAW 264.7s with DMA-co-Tempo 75:25 produced 84% intracellular ROS scavenging, while DMA-coTempo 75:25-PPS microparticles scavenged 97% of intracellular ROS following LPS stimulation (Figure 2A). In a murine model of PTOA, polyTempo-PPS microparticles prevented cartilage damage. Microparticles reduced MMP activity and MabCII 680 binding by 40% and 31%, respectively, following IA injections in mouse knees with mechanicallyinduced PTOA (Figure 2B,C).
Figure 2: Combined PPS and Tempol microparticles improve ROS scavenging and PTOA progression in vitro and in vivo. (A) RAW 264.7 macrophages were pre-treated with DMA-co-Tempo polymers or PPS-DMAco-Tempo microparticles, then stimulated with LPS. Intracellular ROS was measured with DCFDA. PPS-DMA-co-Tempo 75:25 microparticles significantly reduced ROS compared to polymer or PPS alone. (B) Representative images of mice subjected to bilateral mechanically induced PTOA and treated with PPS-polyTempo and injected with MMPSense 750. (C) Quantified MMPSense 750 fluorescence of saline, PPS, and PPS-polyTempo treated PTOA mice. Conclusions: The increased intracellular ROS scavenging of polyTempo-PPS microparticles relative to polyTempo alone confirmed a beneficial effect of formulation of polyTempo- into PPS hybrid microparticles. PolyTempo-PPS hybrid microspheres also effectively reduced ROS in vitro and reduced protease activity and resultant cartilage damage in an in vivo model of PTOA. Follow up studies are needed to integrate more bioavailalbe DMA-co-TEMPO polymers within hybrid PPS particles and to further optimize the hybrid PPS-TEMPO formulation for blocking progression of PTOA. 417 EXPRESSION OF INDUCIBLE PROSTAGLANDIN E SYNTHASE-1 (MPGES-1) IN CHONDROCYTES IS REGULATED BY MAP KINASE PHOSPHATASE-1 (MKP-1) €m a € la €inen, E. Nummenmaa, R. Nieminen, E. Moilanen. L. Tuure, M. Ha Univ. of Tampere, Tampere, Finland Purpose: Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the production of prostaglandin E2 (PGE2), the most abundant prostanoid related to inflammation and inflammatory pain in (osteo)arthritis. mPGES-1 is a terminal enzyme situated downstream of COX-2 in the prostaglandin synthesis pathway and it is overexpressed along COX-2 under inflammatory conditions, such as in OA joints. Inhibition of COX enzymes affects the production of all prostanoids and the known adverse effects of currently used nonsteroidal anti-inflammatory drugs (NSAIDs), such as gastrointestinal bleedings or cardiovascular problems, are considered to result from this. It is therefore proposed that a specific inhibitor of mPGES-1 expression or activity would retain similar therapeutic effects to NSAIDs through decreased inflammation-associated PGE2synthesis, whereas the formation of prostanoids other than PGE2 would stay intact. mPGES-1 is therefore considered as a potential drug target for the treatment of inflammation and pain in osteoarthritis and related diseases with a better safety profile compared to NSAIDs. Mitogen-activated protein kinase phosphatase 1 (MKP-1) is an enzyme that dephosphorylates and thus deactivates the essential inflammatory pathways, MAP kinases, and in this regard acts as a limiting factor in inflammation. MKP-1 has also been shown to mediate the effects of some anti-inflammatory drugs, including glucocorticoids. In the present study, we investigated the role of MKP-1 in the regulation of mPGES-1 expression in cartilage and tested the hypothesis that gluco-
S265
Figure 1: Exemplary depth-wise histograms for normal (A) and degenerated (B) AC. On the degenerated sample (B) wider histogram and two peaks (red arrows) can be observed in the zone 3. The VOIs selected for analysis are indicated with green and blue rectangles for zones 2 and 3, respectively. Results: With increasing degeneration, widening of histogram is seen in the depth-wise histogram visualizations (Fig 1). This widening is more pronounced in zone 3 occasionally characterized by a histogram with two or more peaks (Fig 1B, red arrows). No clear degenerationdependence can be observed in the peak intensity (Fig 2, left column). However, with increased degeneration FWHM shows an increasing trend (Fig 2, right column).
415 HETEROGENEITY IN EXTRACELLULAR MATRIX OF ARTICULAR CARTILAGE AS A FUNCTION OF OSTEOARTHRITIS PROGRESSION AS CHARACTERIZED BY MICRO-CT € y, x, S.S. Karhula y, k, M. Valkealahti y, ¶, T.J. Ylitalo y, z, M. Finnila €m z, K.P. Pritzker #, yy, S. Saarakkala y, ¶, P. Lehenkari y, ¶, E. Hæggstro H.J. Nieminen y, z. y Univ. of Oulu, Oulu, Finland; z Univ. of Helsinki, Helsinki, Finland; x Univ. of Eastern Finlad, Kuopio, Finland; k Infotech Oulu, Oulu, Finland; ¶ Oulu Univ. Hosp., Oulu, Finland; # Univ. of Toronto, Toronto, ON, Canada; yy Mount Sinai Hosp., Toronto, ON, Canada Purpose: Phosphotungstic acid (PTA) provides contrast in articular cartilage (AC) in microcomputed tomography (mCT). The contrast is associated with the content of collagenous proteins in the extracellular matrix (ECM). ECM goes through morphological modifications during osteoarthritis (OA) process characterized by chemical modifications and structural degeneration. It has been demonstrated in earlier literature that ECM heterogeneity is associated with OA. In this study, we aimed to quantify the heterogeneity using simple histogram derived-parameters from PTA-stained cartilage samples scanned with mCT. Methods: Human osteochondral cylinders (n ¼ 36, diameter ¼ 2.0 mm) were obtained from tibial plateu and femoral condyle of 19 patients undergoing total knee arthroplasty (ethics approval PPSHP 78/2013; consents obtained). Samples were subjected to formalin fixation (5 d), after which they were immersed for 48h (n ¼ 34) to 72 h (n ¼ 2) in 70% EtOH containing 1% w/v PTA. Stained samples were imaged with mCT (Skyscan 1272; Bruker microCT, Kontich, Belgium; scanning parameters: 45 kV, 222 mA, 3.2 mm voxel side length, 3050 ms, 2 frames/projection, 1200 projections, and with 0.25 mm Aluminium filter). Projections were reconstructed using NRecon -software (v.1.6.10.4.; Bruker microCT). From the reconstructed data AC volume was segmented (CTAnalyser, v. 1.16.4.1; Bruker microCT) and the AC surface was aligned horizontally using Dataviewer -software (v.1.5.2.4; Bruker microCT). Histogram was calculated for each horizontal (parallel to AC surface) slice (Fig. 1) to describe the depth-wise heterogeneity. Analysis volumes of interest (VOI) (150 150 30 voxels, 480 480 96 mm) were automatically selected from center of the cylinder at relative AC depths of 15 % (Zone 2) and 65 % (Zone 3) from AC surface. Three samples were excluded because segmentation and VOI selection artefacts. Intensity histograms were calculated for each VOI and peak position and full width at half maximum (FWHM) was calculated for each histogram. For reference OARSI grade was evaluated from the orthogonal center slices of reconstructed mCT data.
Figure 2: Histogram peak intensity and FWHM as function of OARSI. Variance between samples increases greatly as function of OARSI. Increasing trend of FWHM in both zone 2 and zone 3 indicates the increase in contrast heterogeneity. Conclusions: Our histogram-based approximation of heterogeneity is in line with earlier literature suggesting that heterogeneity of AC increases with OA progression. However, it appears that presented histogram approach is not a very specific marker to OA progression. A possible explanation to this might be that OARSI grade is a marker for OA progression, whereas the heterogeneity of ECM (histogram parameters) contains information on both OA severity and reaction. Our future studies will investigate if other histogram based approaches could provide further information on ECM heterogeneity and OA progression.
Inflammation and Immunity 416 DEVELOPMENT OF OPTIMIZED COPOLYMERS AND DELIVERY FORMULATIONS TO SCAVENGE REACTIVE OXYGEN SPECIES AND PREVENT JOINT DAMAGE FROM POST-TRAUMATIC OSTEOARTHRITIS T.E. Kavanaugh y, E.A. Dailing y, H. Cho z, K.A. Hasty z, C.L. Duvall y. y Vanderbilt Univ., Nashville, TN, USA; z Univ. of Tennessee Hlth.Sci. Ctr., Memphis, TN, USA Purpose: Post-traumatic osteoarthritis (PTOA) occurs after a traumatic injury to the bone or soft tissue and there is currently no cure. Reactive
S266
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
oxygen species (ROS) are elevated at sites of joint injury and can cause cell-damaging and pro-inflammatory “oxidative stress” that propagates the tissue degenerative process of PTOA. We sought to develop a sustained and active hybrid microparticle that can be delivery locally and sustainably scavenge both hydrogen peroxide and superoxide ROS. To scavenge hydrogen peroxide, we utilize poly(propylene sulfide) (PPS), which undergoes a phase change from hydrophobic to hydrophilic when oxidized by hydrogen peroxide to provide a drug release and microparticle degradation mechanism. Oil-in-water emulsion allows for fabrication of PPS microparticles loaded with hydrophobic molecules, but loading efficiency is very low for water-soluble small molecules drugs such as Tempol (4-hydroxy Tempo), a powerful superoxide dismutase mimetic. To improve loading, we synthesized a polymeric form of TEMPO (poly(Tempo)), and much of this work is focused on optimization of poly(Tempo) composition and bioactivity. We hypothesize that local, delivery of polyTempo-PPS microparticles can to prevent the onset of PTOA after a joint injury Methods: Reversible addition-fragmentation chain-transfer (RAFT) polymerization was used to synthesize a polymeric form of Tempo (polyTempo) (Figure 1A). Co-polymers of 4-amino Tempo and the small hydrophilic monomer dimethylacrylamide (DMA) (DMA-co-Tempo) were synthesized at different molar ratios to tune Tempo density along the backbone (10e100 mol% Tempo). Cell free ROS scavenging of the copolymers was measured based on ferric reducing antioxidant power (FRAP). Intracellular ROS of the copolymers was also measured in RAW 264.7 macrophages using dichlorofluorescin diacetate (DCFDA). Composite microparticles of DMA-co-Tempo (0:100) were synthesized with PPS via oil-in-water emulsion; PPS serves both as a H2O2 “sponge” and also as a bioresponsive depot that triggers release of therapeutic cargo in response to ROS. Using a non-invasive, mechanical loading murine model of PTOA, polyTempo and polyTempo-PPS microparticles were delivered via intraarticular (IA) injection. MMPSense 750 and MabCII 680, a collagen II specific antibody, were used to monitor the protease activity and cartilage damage, respectively, in vivo. Results: DMA-co-Tempo polymers at DMA:Tempo ratios of 0:100, 50:50, 60:40, 70:30, 75:25, 80:20 and 90:10 have increasing water solubility and increased Tempo bioavailability. The DMA-co-Tempo ratio of 70:30 produced the highest antioxidant power, measured by FRAP, resulting in equal antioxidant power to Tempol and increased antioxidant power 10-fold over polyTempo (DMA-co-Tempo 0:100) (Figure 1B).
Figure 1: DMA-co-Tempo synthesis and characterization. (A) DMA-coTempo is synthesized via a combination of RAFT polymerization and postpolymerization grafting of amine modified Tempo to Pentafluorophenol acrylate(PFPA) (B) Total antioxidant power of DMA-co-Tempo polymers is determined using a Ferric Reducing Antioxidant Power (FRAP) assay.
In preliminary studies, treatment of RAW 264.7s with DMA-co-Tempo 75:25 produced 84% intracellular ROS scavenging, while DMA-coTempo 75:25-PPS microparticles scavenged 97% of intracellular ROS following LPS stimulation (Figure 2A). In a murine model of PTOA, polyTempo-PPS microparticles prevented cartilage damage. Microparticles reduced MMP activity and MabCII 680 binding by 40% and 31%, respectively, following IA injections in mouse knees with mechanicallyinduced PTOA (Figure 2B,C).
Figure 2: Combined PPS and Tempol microparticles improve ROS scavenging and PTOA progression in vitro and in vivo. (A) RAW 264.7 macrophages were pre-treated with DMA-co-Tempo polymers or PPS-DMAco-Tempo microparticles, then stimulated with LPS. Intracellular ROS was measured with DCFDA. PPS-DMA-co-Tempo 75:25 microparticles significantly reduced ROS compared to polymer or PPS alone. (B) Representative images of mice subjected to bilateral mechanically induced PTOA and treated with PPS-polyTempo and injected with MMPSense 750. (C) Quantified MMPSense 750 fluorescence of saline, PPS, and PPS-polyTempo treated PTOA mice. Conclusions: The increased intracellular ROS scavenging of polyTempo-PPS microparticles relative to polyTempo alone confirmed a beneficial effect of formulation of polyTempo- into PPS hybrid microparticles. PolyTempo-PPS hybrid microspheres also effectively reduced ROS in vitro and reduced protease activity and resultant cartilage damage in an in vivo model of PTOA. Follow up studies are needed to integrate more bioavailalbe DMA-co-TEMPO polymers within hybrid PPS particles and to further optimize the hybrid PPS-TEMPO formulation for blocking progression of PTOA. 417 EXPRESSION OF INDUCIBLE PROSTAGLANDIN E SYNTHASE-1 (MPGES-1) IN CHONDROCYTES IS REGULATED BY MAP KINASE PHOSPHATASE-1 (MKP-1) €m a € la €inen, E. Nummenmaa, R. Nieminen, E. Moilanen. L. Tuure, M. Ha Univ. of Tampere, Tampere, Finland Purpose: Microsomal PGE synthase-1 (mPGES-1) is an inducible enzyme that catalyzes the production of prostaglandin E2 (PGE2), the most abundant prostanoid related to inflammation and inflammatory pain in (osteo)arthritis. mPGES-1 is a terminal enzyme situated downstream of COX-2 in the prostaglandin synthesis pathway and it is overexpressed along COX-2 under inflammatory conditions, such as in OA joints. Inhibition of COX enzymes affects the production of all prostanoids and the known adverse effects of currently used nonsteroidal anti-inflammatory drugs (NSAIDs), such as gastrointestinal bleedings or cardiovascular problems, are considered to result from this. It is therefore proposed that a specific inhibitor of mPGES-1 expression or activity would retain similar therapeutic effects to NSAIDs through decreased inflammation-associated PGE2synthesis, whereas the formation of prostanoids other than PGE2 would stay intact. mPGES-1 is therefore considered as a potential drug target for the treatment of inflammation and pain in osteoarthritis and related diseases with a better safety profile compared to NSAIDs. Mitogen-activated protein kinase phosphatase 1 (MKP-1) is an enzyme that dephosphorylates and thus deactivates the essential inflammatory pathways, MAP kinases, and in this regard acts as a limiting factor in inflammation. MKP-1 has also been shown to mediate the effects of some anti-inflammatory drugs, including glucocorticoids. In the present study, we investigated the role of MKP-1 in the regulation of mPGES-1 expression in cartilage and tested the hypothesis that gluco-