- Email: [email protected]
Identifying synovial fluid microenvironment phenotypes in patients presenting with knee osteoarthritis
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
and translation. If we can determine which in-vivo markers select for the cells with the greatest chondrogenic capacity and exclude cells with inferior potential, this may increase the success of articular cartilage regeneration strategies; however, for this to be of use, it is critical to compare the marker profile not only between individuals but within individuals to identify the reproducibility and/or heterogeneity of the behaviour of these sub-types of MSCs.
S461
After chondrogenic differentiation the pellet size of each condition was not significantly different than the DMSO only control. However, the chondrogenic marker SOX9 was significantly upregulated in drug 102 and 107 conditions (Figure 2).
787 IDENTIFICATION OF P21 INHIBITORS TO ENHANCE CHONDROGENESIS IN OSTEOARTHRITIC SYNOVIAL MESENCHYMAL PROGENITOR CELLS K.L. Bertram, T. Irvine, P. Tailor, A. Narendran, R. Krawetz. Univ. of Calgary, Calgary, AB, Canada Purpose: Mesenchymal progenitor cells (MPCs) within the synovium have been implicated in cartilage repair both in vitro and in vivo and while MPCs are more abundant in OA joints, they demonstrate a decreased ability to differentiate into chondrocytes compared to MPCs derived from normal synovial tissue. Interestingly, mice lacking p21, have an increased repair response in many adult tissues, including cartilage; and stem cells with reduced p21 expression demonstrate an increased chondrogenic potential. However, sustained suppression of p21 leads to an increased rate of DNA damage and development of autoimmunity and cancer. It is unknown if p21 inhibition in humans can lead to similar increased tissue repair capacity, but we have demonstrated that p21 expression is increased in normal MPCs compared to OA MPCs. Therefore, temporal and local reversible inhibition of p21 in MPCs may increase their chondrogenic potential without the induction of the negative effects of sustained suppression of p21, potentially leading to novel pharmacological intervention for OA. Methods: OA MSCs were derived and purified from human knee synovium from consented patients undergoing joint replacement surgery. Normal MPCs were derived and purified from normal human knee synovium acquired from the Southern Alberta Tissue Donation Program. Compound Screening: The Xman NanoLuc (Horizon Discovery) cell line was genetically modified human colon cancer cells that produce luciferase when p21 is expressed. Luciferase was detected using a Nano-Glo Luciferase Assay (Horizon Discovery) and V3 (Perkin Elmer) plate reader. Compound library: Pharmaceutical pipeline targeted kinase inhibitors panel; 200þ small molecule kinases implicated in cancer cell growth and survival. RT-PCR: RNA was extracted using a RNA Easy Mini Kit (Qiagen) and converted to cDNA (Applied Biosystems). RT-PCR was run using chondrogenic markers (Applied Biosystems) with samples run in triplicate and 18S as a housekeeping control. Toxicity Assay: Alamar blue (Invitrogen) was used to determine metabolic toxicity. All samples were normalized to a carrier control, DMSO. Chondrogenesis: Cells were pelleted by centrifugation and cultured in 15 ml conicals in media containing growth factors to promote chondrogenesis and each respective compound at 1M. Pellet size was assessed using ImageJ software and mRNA expression was determined using RT-qPCR. Results: From the library screening, 5 compounds showed decreased luminescence as a function of drug concentration, and therefore were identified as putative upstream p21 inhibitors (Figure 1).
Figure 1. Luminescence relative to DMSO control after 24 hr exposure to candidate p21 inhibitors.There were no toxic effects on normal or OA MPCs when exposed to each drug at concentrations up to 10 M for 48 hr. p21 mRNA was then analyzed for inhibition. After 4 days of exposure each drug reduces p21 mRNA in MPCs.
Figure 2. Pellet size and SOX9 mRNA expression of MPCs following 21 days of chondrogenesis with inhibitor treatment. Conclusions: Five candidate upstream inhibitors of p21 were identified using a p21-luminescence modified cell line. There were no toxic effects of these compounds on human MPCs at concentrations up to 10M for 48hrs. p21 inhibition was confirmed in human MPCs and exposure to some of these compounds during chondrogenesis did increase the chondrogenic marker SOX9. p21 inhibition in human MPCs could lead to increased chondrogenic potential as exhibited by the p21 knockout mouse strain, leading to a novel pharmacological intervention for cartilage pathologies such as Osteoarthritis. 788 IDENTIFYING SYNOVIAL FLUID MICROENVIRONMENT PHENOTYPES IN PATIENTS PRESENTING WITH KNEE OSTEOARTHRITIS N.J. Steinmetz y, N.F. Lyons y, C.J. Centeno z. y Regenerative Sci., Broomfield, CO, USA; z CSC & Regenerative Sci., Broomfield, CO, USA Purpose: Osteoarthritis (OA) is the most common form of arthritis, and an estimated 9 million Americans suffer from symptomatic knee OA. These patients often have widely varying symptomology. Typically, these patients are initially treated with noninvasive treatments such as exercise, physical therapy, and/or a knee brace. Often, these treatments only temporarily delay the onset of more severe OA, and surgery is frequently required to best restore joint function. However, such surgeries, including total knee replacement (TKR), are sporadically effective in improving joint function and/or alleviating joint pain. Alternatively, minimally invasive procedures, such as needle based procedures, are a promising treatment option. The knee synovial fluid (SF) microenvironment (ME) contains many markers, including pro-inflammatory cytokines, anti-inflammatory cytokines, and matrix metalloproteinases (MMPs) that can be analyzed and used to characterize the status of a patient’s OA joint ME. Patients receiving minimally invasive, needle based procedures to treat OA present clinically with different subsets of symptoms such as varying levels of pain, varying ranges of activity levels, and wide ranging levels of joint swelling and inflammation. It stands to reason that there may be subsets, or phenotypes, within the SF ME of this broad OA population. The objective of this study is to determine whether the broad population of patients presenting with knee OA can be categorized into phenotypes based on the levels of several markers in the ME of the knee joint SF. Methods: This clinical research study was approved for n ¼ 200 patients by the International Cellular Medicine Society’s Institutional Review Board on August 31, 2015. Patients presenting with knee OA based on MRI imaging or KellgreneLawrence Grade 2 scores were approached to participate in the study. Prior to receiving a patented Regenexx® SD bone marrow aspirate (BMA) treatment, approximately 0.5 cc of knee SF was aspirated and analyzed for various ME markers. The knee SF was analyzed for proinflammatory cytokines, anti-inflammatory cytokines, anabolic growth factors, anti-catabolic factors, and MMP products (Fig 1). The markers were analyzed using a Quansys multiplex ELISA platform. Upon complete enrollment of the study, cluster analysis will be utilized to determine if distinct phenotypes exist within the more broad OA disease profile. Results: Synovial fluid was analyzed for a small investigatory subset (n ¼ 16) of the total approved study patients (n ¼ 200). Several anabolic growth factors, anti-inflammatory cytokines, and anti-catabolic factors
S462
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
were present at measureable levels in the SF: Ang-2, bFGF, HGF, PDGF, VEGF, TGF, TIMP-1, TIMP-2, and IL-10. Additionally, there were measureable levels of several catabolic factors and pro-inflammatory cytokines: MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, IL-1, IL-2, IL-6, IL-8, IL12p70, IL-15, IL-23,
IFNg, and TNFa. Of most notable interest are the varying levels of MMPs. With this small, investigatory pilot study, it does appear that there is SF ME phenotypes present within the greater OA patient population (Fig. 2). Conclusions: Patients who present with knee OA have different clinical presentation phenotypes, i.e., severe swelling after the Regenexx® SD procedure v. minimal swelling that resolves quickly after treatment (<1 week). It stands to reason that the anabolic and catabolic knee ME markers could be used to characterize these phenotypes on a molecular level. This could provide valuable a priori information for predicting the likelihood of success for a minimally invasive, regenerative medicine treatment regime and/or for affecting the OA ME towards a phenotype that more positively correlates with successful clinical outcomes for patients receiving regenerative medicine treatments. 789 MULTIFUNCTIONAL NANOPARTICLES FOR IMPROVING FUNCTION AND MULTIMODAL TRACKING OF MESENCHYMAL STEM CELLS FOR OSTEOARTHRITIS TREATMENT I.M. Adjei, H. Yang, L. Maldonado-Camargo, J. Dobson, C. Rinaldi, H. Jiang, B. Sharma. Univ. of Florida, Gainesville, FL, USA Purpose: Stem cell-based therapies have the potential to improve outcomes of osteoarthritis (OA) treatment by regenerating lost cartilage and/or modulating the tissue microenvironment to decrease cartilage loss. Unfortunately, the success of cell-based therapies is hindered by a decline in survival/function of implanted cells and limitations in clinically relevant technologies for monitoring cell fate in vivo. The overall goal of this research is to integrate cell imaging characteristics into a therapeutic delivery system to address critical barriers in translating stem cell therapies. We have developed a multifunctional nanoparticle system capable of 1) delivering nucleic acids and small molecule drugs to MSCs to direct their behavior, 2) enriching the population of engineered stem cells for in vivo implantation, and 3) providing multimodal imaging of cells during and post-implantation, using conventional MRI and emerging photoacoustic imaging (PAI), in order to better understand their role in the regenerative process. PAI has great potential for cell imaging, as it has greater temporal and spatial resolution compared to MRI, with the cost and convenience of a hand-held scanner. The objective of this study was to engineer the multifunctional NP system and demonstrate proof-of-principle for therapeutic delivery and imaging of MSCs. Methods: Multifunctional NPs were formulated to take advantage of the controlled release properties of poly(lactic-co-glycolic acid) (PLGA) and the magnetic and imaging contrast properties (by MRI and PAI) of iron oxide. PLGA NPs encapsulating iron oxide NPs (IONP) and plasmid DNA (magPLGA-NPs) were formulated by a modified double emulsion method. NPs were characterized for size and morphology, as well as DNA loading and integrity. Cellular uptake of NPs and transfection with reporter gene GFP were evaluated by fluorescence microscopy and flow cytometry, while cytotoxicity was determined by MTS assay. Effect of NPs on multipotency of MSCs was determined by using standard protocols to differentiate the cells into adipocytes, osteocytes and chondrocytes. Magnetic selection of cells that have taken up NPs was performed with magnetic-activated cell sorting (MACS) separation column coupled to a custom external magnet array. PA characterization of NPs and NP-loaded MSCs were performed with input wavelengths (l) of 532 or 720 nm and 1 MHz motorized immersion acoustic transducer. Results: The magPLGA NPs were 350 nm in size, with IONPs uniformly distributed within the PLGA polymer matrix. The formulation technique resulted in 94% DNA loading efficiency into the NPs, which was retained in supercoiled conformation, a characteristic necessary for gene expression. The magPLGA-NPs had linear correlation between PA signal and NP concentration for both 532 nm and 720 nm input l, with 532 nm producing stronger PA signal. MagPLGA-NPs were taken up by MSCs without any cytotoxicity, and provided sustained expression of the encapsulated plasmid DNA. Significantly, the NPs were taken up by MSCs at levels sufficient for magnetic selection of the cells at 86% efficiency, which will enable the enrichment and use of only the engineered MSCs for downstream applications. Loading NPs into MSCs did not alter their differentiation potential as the NP-loaded MSCs exhibited similar adipogenic, osteogenic and chondrogenic potential as unloaded MSC (Fig.1). MSCs loaded with magPLGA-NP were detectable at input wavelengths of 532 and 720 nm, with sensitivity of 3000 and 7000 cells/mL respectively (Fig.2). Conclusions: MSCs are heterogeneous cell population, which may contribute to the variable findings observed in clinical results. This
and translation. If we can determine which in-vivo markers select for the cells with the greatest chondrogenic capacity and exclude cells with inferior potential, this may increase the success of articular cartilage regeneration strategies; however, for this to be of use, it is critical to compare the marker profile not only between individuals but within individuals to identify the reproducibility and/or heterogeneity of the behaviour of these sub-types of MSCs.
S461
After chondrogenic differentiation the pellet size of each condition was not significantly different than the DMSO only control. However, the chondrogenic marker SOX9 was significantly upregulated in drug 102 and 107 conditions (Figure 2).
787 IDENTIFICATION OF P21 INHIBITORS TO ENHANCE CHONDROGENESIS IN OSTEOARTHRITIC SYNOVIAL MESENCHYMAL PROGENITOR CELLS K.L. Bertram, T. Irvine, P. Tailor, A. Narendran, R. Krawetz. Univ. of Calgary, Calgary, AB, Canada Purpose: Mesenchymal progenitor cells (MPCs) within the synovium have been implicated in cartilage repair both in vitro and in vivo and while MPCs are more abundant in OA joints, they demonstrate a decreased ability to differentiate into chondrocytes compared to MPCs derived from normal synovial tissue. Interestingly, mice lacking p21, have an increased repair response in many adult tissues, including cartilage; and stem cells with reduced p21 expression demonstrate an increased chondrogenic potential. However, sustained suppression of p21 leads to an increased rate of DNA damage and development of autoimmunity and cancer. It is unknown if p21 inhibition in humans can lead to similar increased tissue repair capacity, but we have demonstrated that p21 expression is increased in normal MPCs compared to OA MPCs. Therefore, temporal and local reversible inhibition of p21 in MPCs may increase their chondrogenic potential without the induction of the negative effects of sustained suppression of p21, potentially leading to novel pharmacological intervention for OA. Methods: OA MSCs were derived and purified from human knee synovium from consented patients undergoing joint replacement surgery. Normal MPCs were derived and purified from normal human knee synovium acquired from the Southern Alberta Tissue Donation Program. Compound Screening: The Xman NanoLuc (Horizon Discovery) cell line was genetically modified human colon cancer cells that produce luciferase when p21 is expressed. Luciferase was detected using a Nano-Glo Luciferase Assay (Horizon Discovery) and V3 (Perkin Elmer) plate reader. Compound library: Pharmaceutical pipeline targeted kinase inhibitors panel; 200þ small molecule kinases implicated in cancer cell growth and survival. RT-PCR: RNA was extracted using a RNA Easy Mini Kit (Qiagen) and converted to cDNA (Applied Biosystems). RT-PCR was run using chondrogenic markers (Applied Biosystems) with samples run in triplicate and 18S as a housekeeping control. Toxicity Assay: Alamar blue (Invitrogen) was used to determine metabolic toxicity. All samples were normalized to a carrier control, DMSO. Chondrogenesis: Cells were pelleted by centrifugation and cultured in 15 ml conicals in media containing growth factors to promote chondrogenesis and each respective compound at 1M. Pellet size was assessed using ImageJ software and mRNA expression was determined using RT-qPCR. Results: From the library screening, 5 compounds showed decreased luminescence as a function of drug concentration, and therefore were identified as putative upstream p21 inhibitors (Figure 1).
Figure 1. Luminescence relative to DMSO control after 24 hr exposure to candidate p21 inhibitors.There were no toxic effects on normal or OA MPCs when exposed to each drug at concentrations up to 10 M for 48 hr. p21 mRNA was then analyzed for inhibition. After 4 days of exposure each drug reduces p21 mRNA in MPCs.
Figure 2. Pellet size and SOX9 mRNA expression of MPCs following 21 days of chondrogenesis with inhibitor treatment. Conclusions: Five candidate upstream inhibitors of p21 were identified using a p21-luminescence modified cell line. There were no toxic effects of these compounds on human MPCs at concentrations up to 10M for 48hrs. p21 inhibition was confirmed in human MPCs and exposure to some of these compounds during chondrogenesis did increase the chondrogenic marker SOX9. p21 inhibition in human MPCs could lead to increased chondrogenic potential as exhibited by the p21 knockout mouse strain, leading to a novel pharmacological intervention for cartilage pathologies such as Osteoarthritis. 788 IDENTIFYING SYNOVIAL FLUID MICROENVIRONMENT PHENOTYPES IN PATIENTS PRESENTING WITH KNEE OSTEOARTHRITIS N.J. Steinmetz y, N.F. Lyons y, C.J. Centeno z. y Regenerative Sci., Broomfield, CO, USA; z CSC & Regenerative Sci., Broomfield, CO, USA Purpose: Osteoarthritis (OA) is the most common form of arthritis, and an estimated 9 million Americans suffer from symptomatic knee OA. These patients often have widely varying symptomology. Typically, these patients are initially treated with noninvasive treatments such as exercise, physical therapy, and/or a knee brace. Often, these treatments only temporarily delay the onset of more severe OA, and surgery is frequently required to best restore joint function. However, such surgeries, including total knee replacement (TKR), are sporadically effective in improving joint function and/or alleviating joint pain. Alternatively, minimally invasive procedures, such as needle based procedures, are a promising treatment option. The knee synovial fluid (SF) microenvironment (ME) contains many markers, including pro-inflammatory cytokines, anti-inflammatory cytokines, and matrix metalloproteinases (MMPs) that can be analyzed and used to characterize the status of a patient’s OA joint ME. Patients receiving minimally invasive, needle based procedures to treat OA present clinically with different subsets of symptoms such as varying levels of pain, varying ranges of activity levels, and wide ranging levels of joint swelling and inflammation. It stands to reason that there may be subsets, or phenotypes, within the SF ME of this broad OA population. The objective of this study is to determine whether the broad population of patients presenting with knee OA can be categorized into phenotypes based on the levels of several markers in the ME of the knee joint SF. Methods: This clinical research study was approved for n ¼ 200 patients by the International Cellular Medicine Society’s Institutional Review Board on August 31, 2015. Patients presenting with knee OA based on MRI imaging or KellgreneLawrence Grade 2 scores were approached to participate in the study. Prior to receiving a patented Regenexx® SD bone marrow aspirate (BMA) treatment, approximately 0.5 cc of knee SF was aspirated and analyzed for various ME markers. The knee SF was analyzed for proinflammatory cytokines, anti-inflammatory cytokines, anabolic growth factors, anti-catabolic factors, and MMP products (Fig 1). The markers were analyzed using a Quansys multiplex ELISA platform. Upon complete enrollment of the study, cluster analysis will be utilized to determine if distinct phenotypes exist within the more broad OA disease profile. Results: Synovial fluid was analyzed for a small investigatory subset (n ¼ 16) of the total approved study patients (n ¼ 200). Several anabolic growth factors, anti-inflammatory cytokines, and anti-catabolic factors
S462
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
were present at measureable levels in the SF: Ang-2, bFGF, HGF, PDGF, VEGF, TGF, TIMP-1, TIMP-2, and IL-10. Additionally, there were measureable levels of several catabolic factors and pro-inflammatory cytokines: MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, IL-1, IL-2, IL-6, IL-8, IL12p70, IL-15, IL-23,
IFNg, and TNFa. Of most notable interest are the varying levels of MMPs. With this small, investigatory pilot study, it does appear that there is SF ME phenotypes present within the greater OA patient population (Fig. 2). Conclusions: Patients who present with knee OA have different clinical presentation phenotypes, i.e., severe swelling after the Regenexx® SD procedure v. minimal swelling that resolves quickly after treatment (<1 week). It stands to reason that the anabolic and catabolic knee ME markers could be used to characterize these phenotypes on a molecular level. This could provide valuable a priori information for predicting the likelihood of success for a minimally invasive, regenerative medicine treatment regime and/or for affecting the OA ME towards a phenotype that more positively correlates with successful clinical outcomes for patients receiving regenerative medicine treatments. 789 MULTIFUNCTIONAL NANOPARTICLES FOR IMPROVING FUNCTION AND MULTIMODAL TRACKING OF MESENCHYMAL STEM CELLS FOR OSTEOARTHRITIS TREATMENT I.M. Adjei, H. Yang, L. Maldonado-Camargo, J. Dobson, C. Rinaldi, H. Jiang, B. Sharma. Univ. of Florida, Gainesville, FL, USA Purpose: Stem cell-based therapies have the potential to improve outcomes of osteoarthritis (OA) treatment by regenerating lost cartilage and/or modulating the tissue microenvironment to decrease cartilage loss. Unfortunately, the success of cell-based therapies is hindered by a decline in survival/function of implanted cells and limitations in clinically relevant technologies for monitoring cell fate in vivo. The overall goal of this research is to integrate cell imaging characteristics into a therapeutic delivery system to address critical barriers in translating stem cell therapies. We have developed a multifunctional nanoparticle system capable of 1) delivering nucleic acids and small molecule drugs to MSCs to direct their behavior, 2) enriching the population of engineered stem cells for in vivo implantation, and 3) providing multimodal imaging of cells during and post-implantation, using conventional MRI and emerging photoacoustic imaging (PAI), in order to better understand their role in the regenerative process. PAI has great potential for cell imaging, as it has greater temporal and spatial resolution compared to MRI, with the cost and convenience of a hand-held scanner. The objective of this study was to engineer the multifunctional NP system and demonstrate proof-of-principle for therapeutic delivery and imaging of MSCs. Methods: Multifunctional NPs were formulated to take advantage of the controlled release properties of poly(lactic-co-glycolic acid) (PLGA) and the magnetic and imaging contrast properties (by MRI and PAI) of iron oxide. PLGA NPs encapsulating iron oxide NPs (IONP) and plasmid DNA (magPLGA-NPs) were formulated by a modified double emulsion method. NPs were characterized for size and morphology, as well as DNA loading and integrity. Cellular uptake of NPs and transfection with reporter gene GFP were evaluated by fluorescence microscopy and flow cytometry, while cytotoxicity was determined by MTS assay. Effect of NPs on multipotency of MSCs was determined by using standard protocols to differentiate the cells into adipocytes, osteocytes and chondrocytes. Magnetic selection of cells that have taken up NPs was performed with magnetic-activated cell sorting (MACS) separation column coupled to a custom external magnet array. PA characterization of NPs and NP-loaded MSCs were performed with input wavelengths (l) of 532 or 720 nm and 1 MHz motorized immersion acoustic transducer. Results: The magPLGA NPs were 350 nm in size, with IONPs uniformly distributed within the PLGA polymer matrix. The formulation technique resulted in 94% DNA loading efficiency into the NPs, which was retained in supercoiled conformation, a characteristic necessary for gene expression. The magPLGA-NPs had linear correlation between PA signal and NP concentration for both 532 nm and 720 nm input l, with 532 nm producing stronger PA signal. MagPLGA-NPs were taken up by MSCs without any cytotoxicity, and provided sustained expression of the encapsulated plasmid DNA. Significantly, the NPs were taken up by MSCs at levels sufficient for magnetic selection of the cells at 86% efficiency, which will enable the enrichment and use of only the engineered MSCs for downstream applications. Loading NPs into MSCs did not alter their differentiation potential as the NP-loaded MSCs exhibited similar adipogenic, osteogenic and chondrogenic potential as unloaded MSC (Fig.1). MSCs loaded with magPLGA-NP were detectable at input wavelengths of 532 and 720 nm, with sensitivity of 3000 and 7000 cells/mL respectively (Fig.2). Conclusions: MSCs are heterogeneous cell population, which may contribute to the variable findings observed in clinical results. This