J.-Y. Ko

In vitro chondrogenesis and in vivo repair of osteochondral defect with human induced pluripotent stem cells.

The purpose of this study was to investigate the chondrogenic features of human induced pluripotent stem cells (hiPSCs) and examine the differences in the chondrogenesis between hiPSCs and human bone marrow-derived MSCs (hBMMSCs). Embryoid bodies (EBs) were formed from undifferentiated hiPSCs. After EBs were dissociated into single cells, chondrogenic culture was performed in pellets and alginate hydrogel. Chondro-induced hiPSCs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats and evaluated after 12 weeks. The ESC markers NANOG, SSEA4 and OCT3/4 disappeared while the mesodermal marker BMP-4 appeared in chondro-induced hiPSCs. After 21 days of culture, greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation were observed from chondro-induced hiPSCs compared to chondro-induced hBMMSCs. The expression of chondrogenic markers including SOX-9, type II collagen, and aggrecan in chondro-induced hiPSCs was comparable to or greater than chondro-induced hBMMSCs. A remarkably low level of hypertrophic and osteogenic markers including type X collagen, type I collagen and Runx-2 was noted in chondro-induced hiPSCs compared to chondro-induced hBMMSCs. hiPSCs had significantly greater methylation of several CpG sites in COL10A1 promoter than hBMMSCs in either undifferentiated or chondro-induced state, suggesting an epigenetic cause of the difference in hypertrophy. The defects implanted with chondro-induced hiPSCs showed a significantly better quality of cartilage repair than the control defects, and the majority of cells in the regenerated cartilage consisted of implanted hiPSCs.

Intra-articular delivery of kartogenin-conjugated chitosan nano/microparticles for cartilage regeneration.

We developed an intra-articular (IA) drug delivery system to treat osteoarthritis (OA) that consisted of kartogenin conjugated chitosan (CHI-KGN). Kartogenin, which promotes the selective differentiation of mesenchymal stem cells (MSCs) into chondrocytes, was conjugated with low-molecular-weight chitosan (LMWCS) and medium-molecular-weight chitosan (MMWCS) by covalent coupling of kartogenin to each chitosan using an ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) catalyst. Nanoparticles (NPs, 150 ± 39 nm) or microparticles (MPs, 1.8 ± 0.54 μm) were fabricated from kartogenin conjugated-LMWCS and -MMWCS, respectively, by an ionic gelation using tripolyphosphate (TPP). The in vitro release profiles of kartogenin from the particles showed sustained release for 7 weeks. When the effects of the CHI-KGN NPs or CHI-KGN MPs were evaluated on the in vitro chondrogenic differentiation of human bone marrow MSCs (hBMMSCs), the CHI-KGN NPs and CHI-KGN MPs induced higher expression of chondrogenic markers from cultured hBMMSCs than unconjugated kartogenin. In particular, hBMMSCs treated with CHI-KGN NPs exhibited more distinct chondrogenic properties in the long-term pellet cultures than those treated with CHI-KGN MPs. The in vivo therapeutic effects of CHI-KGN NPs or CHI-KGN MPs were investigated using a surgically-induced OA model in rats. The CHI-KGN MPs showed longer retention time in the knee joint than the CHI-KGN NPs after IA injection in OA rats. The rats treated with CHI-KGN NPs or CHI-KGN MPs by IA injection showed much less degenerative changes than untreated control or rats treated with unconjugated kartogenin. In conclusion, CHI-KGN NPs or CHI-KGN MPs can be useful polymer-drug conjugates as an IA drug delivery system to treat OA.

Sulforaphane–PLGA microspheres for the intra-articular treatment of osteoarthritis

Sulforaphane (SFN) is a member of the isothiocyanate family that has anti-inflammatory action as well as anti-carcinogenic properties. The authors have devised an intra-articular injectable SFN-PLGA microsphere system that can be used for treating osteoarthritis (OA). The purpose of this study was to evaluate the in vitro and in vivo efficacy of the SFN-PLGA microsphere system. Articular chondrocytes were obtained from knee OA patients and were cultured in monolayers. The optimal concentration of SFN was obtained and the dose of SFN-PLGA microspheres was determined based on the concentration. The in vitro anti-inflammatory effect on markers such as cyclooxygenase (COX)-2, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, and matrix metalloproteinase (MMP)-2 was assessed by real-time PCR and Western blotting. The in vivo therapeutic effect of SFN-PLGA microspheres was investigated using surgically-induced rat OA model. Treatment with SFN-PLGA microspheres inhibited the mRNA and protein expression of COX-2, ADAMTS-5 and MMP-2 induced by LPS in articular chondrocytes. Intraarticular SFN-PLGA microspheres delayed the progression of surgically-induced osteoarthritis in rats. In conclusion, SFN-PLGA microspheres can be a useful injectable delivery system for treating osteoarthritis.

Intra-articular Xenotransplantation of Adipose-Derived Stromal Cells to Treat Osteoarthritis in a Goat Model

Adipose-derived stromal cells (ASCs) have been investigated as a cell source for tissue regeneration. The purpose of this study was first to confirm if medial meniscectomy induces osteoarthritis (OA) in goats within a relative short period of time, and more importantly, to investigate if systemic treatment with immunosuppressive drugs is necessary in intra-articular ASC xenotransplantation for successful regeneration of articular cartilage and prevention of joint inflammation. Eight Korean native black goats 1–2 years of age underwent medial meniscectomy. To evaluate the gross and histological appearance of articular cartilage, knee joints were re-exposed by a medial parapatellar incision at 8 weeks. After macroscopic scoring of gross appearance, cartilage biopsy specimens 6 mm in diameter were obtained from the femoral condyle in four goats. The goats were injected with single intra-articular dose of 7×106 human ASCs (hASCs) 7 days after the second arthrotomy. Four animals were treated with daily injections of cyclosporin A 10 mg/kg for 7 days, followed by a reduced dose of 5 mg/kg for another 7 days, while other 4 animals did not receive immunosuppressive therapy. All animals were sacrificed for analysis 8 weeks after injection of hASCs. OA was successfully induced 8 weeks after medial meniscectomy. Eight weeks after injection of hASCs, various signs of articular cartilage regeneration were observed. There were no significant macroscopic and histological differences between goats treated with cyclosporine and untreated goats. Interleukin-1ß and tumor necrosis factor-α level from synovial fluid did not differ between cyclosporine-treated and untreated goats. The results indicate that immunosuppressive therapy did not influence the result of ASC xenotransplantation to treat OA.

Chondrogenic and Osteogenic Induction from iPS Cells.

Articular cartilage (AC) does not heal spontaneously when injured in adults. This incapacity for self-repair after damage ultimately leads to the development of osteoarthritis. In contrast, bone repairs itself without scarring. However, complete bone healing fails to occur in large defects coming from major trauma or malignant tumor resection. Cell therapy has been investigated for these musculoskeletal conditions. Induced pluripotent stem cells (iPSCs) possess the characteristics of embryonic stem cells (ESCs) in potentially unlimited proliferation while avoiding the ethical controversies. However, several issues need to be resolved before iPSCs can be considered as a potential therapeutic measure for cartilage and bone regeneration. The authors developed protocol to examine the in vitro chondrogenesis and osteogenesis from hiPSCs and in vivo cartilage and bone regeneration using animal models.

Concave microwell plate facilitates chondrogenesis from mesenchymal stem cells

ObjectivesTo compare in vitro chondrogenesis from bone marrow-derived mesenchymal stem cells using concave microwell plates with those obtained using culture tubes.ResultsPellets cultured in concave microwell plates had a significantly higher level of GAG per DNA content and greater proteoglycan content than those cultured in tubes at day 7 and 14. Three chondrogenic markers, SOX-9, COL2A1 and aggrecan, showed significantly higher expression in pellets cultured in concave microwell plates than those cultured in tubes at day 7 and 14. At day 21, there was not a significant difference in the expression of these markers. COL10A1, the typical hypertrophy marker, was significantly lower in concave microwell plates during the whole culture period. Runx-2, a marker of hypertrophy and osteogenesis, was significantly lower at day 7 in pellets cultured in concave microwell plates than those cultured in tubes.ConclusionConcave microwell plates provide a convenient and effective tool for the study of in vitro chondrogenesis and may replace the use of propylene culture tube.

THU0462 Intra-Articular Delivery of Kartogenin-Conjugated Chitosan Nano/Microparticles for Cartilage Regeneration

Background Osteoarthritis (OA), also known as degenerative arthritis or degenerative joint disease, affects millions of people around the world. Intra-articular (IA) drug delivery can be a useful modality in OA treatment, delivering a drug directly to the main focus of the disease. The therapeutic effect of IA drug depends mostly on the efficacy of the drug delivery system, due to the short retention time and rapid clearance of soluble drugs from the joint. Kartogenin is a recently characterized material that promotes the selective differentiation of mesenchymal stem cells (MSCs) into chondrocytes, thus stimulating cartilage regeneration [1]. Objectives We developed an intra-articular (IA) drug delivery system to treat osteoarthritis (OA) that consisted of kartogenin conjugated chitosan (CHI-KGN). The aim of this study was to (1) characterize the CHI-KGN particles for sustained release and chondrogenic activity in vitro, (2) evaluate the CHI-KGN particles as novel IA drug delivery systems for IA retention and regeneration of OA joint in vivo. Methods Kartogenin was conjugated with low-molecular-weight chitosan (LMWCS) and medium-molecular-weight chitosan (MMWCS) by covalent coupling of kartogenin to each chitosan using an ethyl (dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) catalyst. The particular drug delivery systems were prepared by an ionic gelation of the CHI-KGN conjugate with tripolyphosphate (TPP) anion that can interact with cationic chitosan by electrostatic forces. Results Nanoparticles (NPs, 150±39 nm) or microparticles (MPs, 1.8±0.54 μm) were successfully fabricated from kartogenin conjugated-LMWCS and –MMWCS, respectively, by an ionic gelation using TPP. The in vitro release profiles of kartogenin from the particles showed sustained release for 7 weeks. When the effects of the CHI-KGN NPs or CHI-KGN MPs were evaluated on the in vitro chondrogenic differentiation of human bone marrow MSCs (hBMMSCs), the CHI-KGN NPs and CHI-KGN MPs induced higher expression of chondrogenic markers from cultured hBMMSCs than unconjugated kartogenin. In particular, hBMMSCs treated with CHI-KGN NPs exhibited more distinct chondrogenic properties in the long-term pellet cultures than those treated with CHI-KGN MPs. The in vivo therapeutic effects of CHI-KGN NPs or CHI-KGN MPs were investigated using a surgically-induced OA model in rats. The CHI-KGN MPs showed longer retention time in the knee joint than the CHI-KGN NPs after IA injection in OA rats. The rats treated with CHI-KGN NPs or CHI-KGN MPs by IA injection showed much less degenerative changes than untreated control or rats treated with unconjugated kartogenin. Conclusions In conclusion, CHI-KGN NPs or CHI-KGN MPs can be useful polymer-drug conjugates for an IA drug delivery system to treat OA. References Johnson K et al. A stem cell-based approach to cartilage repair. Science 2012;336:717-21. Acknowledgements This work was supported by a grant from the National Research Foundation of Korea (NRF-2013R1A1A2062978). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure of Interest None declared

THU0463 Polymeric Nanoparticles with Thermally Responsive Dual Release Profiles for Combined Therapy of Osteoarthritis

Background Osteoarthritis (OA) is a primarily noninflammatory and degenerative joint disease. However, there is growing evidence suggesting that synovial inflammation causes many of the signs and symptoms of OA [1]. It is therefore anticipated that inhibition of the inflammatory component of OA may offer an effective treatment for the disease. Regeneration of damaged cartilage is essential in long-term good result of OA therapy. However, current treatment options for OA are largely limited to either pain medication or joint replacement surgery. Kartogenin (KGN) is a recently characterized compound that promotes the selective differentiation of mesenchymal stem cells (MSCs) into chondrocytes and induces the regeneration of cartilage in OA [2]. KGN can be combined with anti-inflammatory small molecules such as diclofenac (DCF; MW =296.15 Da) in a delivery system to enhance the therapeutic effects for OA treatment. The combination can cause a rapid subsidence of inflammation and pain reduction from rapid release of DCF followed by regeneration of articular cartilage with the sustained release of KGN when used for intra-articular injection. Objectives In this study, we report the synthesis and characterization of the core-shell nanoparticles (F127/COS/KGNDCF) consisting of inner core DCF, and an outer shell of cross-linked carboxyl group-terminated pluronic F127 (F127–COOH)/chitosan oligosaccharide (COS)/KGN. The aims of this study were to (1) characterize the F127/COS/KGNDCF nanoparticles for independent dual release by thermal responsiveness, and (2) evaluate the F127/COS/KGNDCF nanoparticles as a dual drug delivery system for combined therapy. Methods KGN was conjugated covalently with COS before the nanoparticle synthesis by carbodiimide chemistry. The nanoparticles were synthesized by covalent cross-linking between COS and F127–COOH using EDC catalysis during emulsification/solvent evaporation method. Results The nanoparticles (F127/COS/KGNDCF) were ∼125 nm in size at 37°C and expanded to ∼442 nm when cooled to 4°C in aqueous solutions. Swelling and shrinking of the nanoparticles by thermal responsiveness was also controllable by the composition ratio of F127 or KGN to COS. The F127/COS/KGNDCF nanoparticles showed immediate and sustained release of DCF and KGN respectively, which was controlled independently by temperature change. The toxicity of the F127/COS/KGNDCF nanoparticles was found to be negligible. Inflammation in U937 macrophage-like cells and chondrocytes was more effectively suppressed by the F127/COS/KGNDCF nanoparticles treated with cold shock than those without cold shock treatment. Chondrogenic differentiation of bone marrow-derived mesenchymal stem cells was also enhanced by cold shock treatment of the nanoparticles. Conclusions These results suggested that thermally responsive F127/COS/KGNDCF nanoparticles could provide useful dual-function therapeutics to quench the inflammation and regenerate damaged tissue when combined with cryotherapy. References F. Berenbaum, Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!), Osteoarthr. Cartilage 21 (2013) 16-21. K. Johnson et al. A stem cell-based approach to cartilage repair, Science 336 (2012) 717-721. Acknowledgements This work was supported by a grant from the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (NRF-2013R1A1A2062978). Disclosure of Interest None declared

SAT0511 Osteogenic Differentiation of Human Induced Pluripotent Stem Cells

Background Mesenchymal stem cell derived from bone marrow have already been used for bone regeneration. A number of studies have confirmed that MSCs cultured in scaffolds can induce osteogenesis in vivo and improve healing of critical-size defects. Objectives In the present study, we generated mesodermal cell lineages from hiPSCs following treatment of embryoid bodies (EBs) with retinoic acid. Subsequently, we induced osteogenic differentiation from EBs using osteogenic medium and also test the in vivo repair of osseous defect using these osteo-induced cells. To avoid the potential safety issues associated with using viruses, we used the hiPSC line by direct delivery of four proteins fused to a cell penetrating peptide in present study. Methods Cell culture and differentiation of EB – We used the hiPSC line (SBI, cat# SC802A-1) generated by direct delivery of four proteins fused to a cell penetrating peptide. Osteogenic differentiation was induced by trypsinizing EB to a single cell suspension, diluting cells to a final concentration of 5x105 cells/ml and being cultured on gelatin-coated plates for 7 and 14 days. Analysis for osteogenic differentiation – After 7 and 14 days of in vitro culture, osteogenic differentiation was demonstated by Alizarin red S staining, alkaline phosphatase (ALP) staining, and the expression of osteogenic markers. Transplantation – The fibrin-hiPSCs and HA+β-TCP-hiPSCs constructs were implanted in the calvarial defect (f 4mm) in immunosuppressed rat. Also, these hiPSCs in fibrin were implanted in the segmental long bone defects created in the ulna of immunosuppressed of rat. Results After 7 and 14 days of osteogenic culture, alizarin red S staining and ALP staining identified calcium in these cells and osteogenic differentiation. The osteogenic marker gene and protein expression including COL1A1, Runx2, and bone sialoprotein (BSP) increased after 7 days and expression level of gene and protein after 14 days was higher than 7 days. When hiPS cells in fibrin and HA+β-TCP were implanted in calvarial defects created in the parietal bone of immunosuppressed rats, the defects implanted with osteo-induced hiPS cells showed a significantly better quality of bone repair than the control defects. Goldners trichrome staining confirmed that bone regeneration in those treated with hiPSCs-fibrin and HA+β-TCP. Immunofluorescent imaging showed that human nucleus antigens were observed in the cytoplasm of implanted hiPSC-Osteoblasts. In addition, hiPSC-Osteoblasts or hBMMSC-Osteoblasts in fibrin were implanted in the 4mm segmental defects created in the ulna of immunosuppressed rats. The defects with osteo-induced hiPS cell implantation achieved rapid bone healing compared with the control defects. Conclusions The cell culture protocol presented herein affords high yield of pure populations of hiPSC-EBs with high osteogenic capability. The osteoblasts derived from these hiPSC-EBs were able to form new mineralized and vascularized bone matrix in vivo. Once the functionality of these derived cells will be thoroughly examined in a bone repair model, the potential of the described method as a source of osteoblasts for bone tissue regenerating can be established. References Kawaguchi J, Mee PJ, Smith AG. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone. 2005 May;36(5):758-69. Acknowledgements This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (2012M3A9B4028566 & 2013R1A1A2062961). Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3442

OP0127 In Vitro Chondrogenesis and in VIVO Repair of Osteochondral Defect with Human Induced Pluripotent Stem Cells

Background Human embryonic stem cells (hESCs) have high proliferation potential and can generate differentiated progeny of all three embryonic germ layers. However, the derivation of hESCs from early embryos raises technical and ethical limitations for their use in research and the clinic. Engineered stem cells, known as iPSCs, generated from somatic cells by transduction of defined reprogramming transcription factors, typically OCT4, SOX2, KLF4, and c-MYC, open a new avenue to avoid the controversy of using hESCs. Objectives The purpose of this study was to test the in vitro chondrogenic potential and in vivo capacity for cartilage regeneration of human iPS (hiPS) cells. Methods Cell culture and differentiation of EB We used the hiPSC line (SBI, cat# SC802A-1) generated by direct delivery of four proteins fused to a cell penetrating peptide. Analysis for condrogenic differentiation After 21 days of in vitro culture, pellets were analyzed for DNA contents, GAG amount, and the expression of chondrogenic markers. Transplantation The hiPS-pellets or alginate-hiPSCs constructs were implanted in the osteochondral defect model rat. The rats received daily injections of cyclosporin A to suppress immune responses in rats. After 6 weeks, the rats were sacrificed. Results Mesodermal markers increased in EB while undifferentiated ES markers disappeared. After 21 days of chondrogenic culture in micromass pellets, GAG analysis showed that proteoglycan production was significantly greater in chondrogenic pellets than in undifferentiated hiPSCs and EBs. Safranin-O staining demonstrated that the cells in chondrogenic pellets took on the appearance of immature chondrocytes and secreted extracellular matrix. The chondrogenic marker gene and protein expression increased after 21days of pellet culture. The chondrogenic pellets derived from hiPS cells have very low expression of hypertrophic or osteogenic markers. Also, hiPS cells underwent good chondrogenic differentiation in PLGA scaffold or alginate gel as well. When hiPS cells in either pellet state or in alginate hydrogel were implanted in the osteochondral defects created on the patellar groove of immunosuppressed rats, the defects implanted with chondro-induced hiPS cells showed a significantly better quality of cartilage repair than the control defects. Conclusions In conclusion, this study provides a proof-of-principle strategy for using hiPSCs as a cell source for cartilage tissue engineering. While successful in vitro induction of chondrogenesis with improved biochemical characteristics were obtained from hiPS cells, the working mechanisms in the implantation of hiPS cells and strategies for further improvement of in vivo cartilage repair with hiPS cells should be investigated in future studies. References Wei Y, Zeng W, Wan R, Wang J, Zhou Q, Qiu S, Singh SR. Chondrogenic differentiation of induced pluripotent stem cells from osteoarthritic chondrocytes in alginate matrix. Eur Cell Mater. 2012 Jan 12;23:1-12. Kawaguchi J, Mee PJ, Smith AG. Osteogenic and chondrogenic differentiation of embryonic stem cells in response to specific growth factors. Bone. 2005 May;36(5):758-69. Disclosure of Interest None Declared