Mi-Lan Kang

Thermoresponsive nanospheres with independent dual drug release profiles for the treatment of osteoarthritis.

UNLABELLED Dual drug delivery of drugs with different therapeutic effects in a single system is an effective way to treat a disease. One of the main challenges in dual drug delivery is to control the release behavior of each drug independently. In this study, we devised thermo-responsive polymeric nanospheres that can provide simultaneous and independent dual drug delivery in the response to temperature change. The nanospheres based on chitosan oligosaccharide conjugated pluronic F127 grafting carboxyl group were synthesized to deliver kartogenin (KGN) and diclofenac (DCF) in a single system. To achieve the dual drug release, KGN was covalently cross-linked to the outer part of the nanosphere, and DCF was loaded into the inner core of the nanosphere. The nanospheres demonstrated immediate release of DCF and sustained release of KGN, which were independently controlled by temperature change. The nanospheres treated with cold temperature effectively suppressed lipopolysaccharide-induced inflammation in chondrocytes and macrophage-like cells. The nanospheres also induced chondrogenic differentiation of mesenchymal stem cells, which was further enhanced by cold shock treatment. Bioluminescence of the fluorescence-labeled nanospheres was significantly increased after cold treatment in vivo. The nanospheres suppressed the progression of osteoarthritis in treated rats, which was further enhanced by cold treatment. The nanospheres also reduced cyclooxygenase-2 expression in the serum and synovial membrane of treated rats, which were further decreased with cold treatment. These results suggest that the thermo-responsive nanospheres provide dual-function therapeutics possessing anti-inflammatory and chondroprotective effects which can be enhanced by cold treatment. STATEMENT OF SIGNIFICANCE We developed thermo-responsive nanospheres that can provide a useful dual-function of suppressing the inflammation and promoting chondrogenesis in the treatment of osteoarthritis. For a dual delivery system to be effective, the release behavior of each drug should be independently controlled to optimize their desired therapeutic effects. We employed rapid release of diclofenac for acute anti-inflammatory effects, and sustained release of kartogenin, a newly found molecule, for chondrogenic effects in this polymeric nanospheres. This nanosphere demonstrated immediate release of diclofenac and sustained release of kartogenin, which were independently controlled by temperature change. The effectiveness of this system to subside inflammation and regenerate cartilage in osteoarthritis was successful demonstrated through in vitro and in vivo experiments in this study. We think that this study will add a new concept to current body of knowledge in the field of drug delivery and treatment of osteoarthritis.

Vascular endothelial growth factor-transfected adipose-derived stromal cells enhance bone regeneration and neovascularization from bone marrow stromal cells

Vascular endothelial growth factor (VEGF)‐transfected adipose‐derived stromal cells (ADSCsVEGF) were devised to promote bone regeneration and neovascularization of bone marrow stromal cells (BMSCs). ADSCsVEGF were added to BMSCs and cocultured in variable proportions. ADSCsVEGF alone or ADSCsVEGF with BMSCs (BMSCs:ADSCsVEGF ratio of 1:0.025–0.5) induced significantly greater tube formation in human umbilical vein endothelial cells than untransfected ADSCs. The cocultures of BMSCs and ADSCsVEGF at ratios of 1: 0.025–0.1 showed significantly greater osteogenic differentiation and mineralization than BMSCs alone in vitro. Osteogenic markers COL1A1, OCN and BSP were most effectively induced at the BMSC: ADSCVEGF ratio of 1:0.05. Of angiogenesis‐related genes, upregulation of cathepsin Z and downregulation of early growth response 1 were observed while two osteogenesis‐related genes, osteoactivin and tetranectin, were upregulated in BMSCs/ADSCsVEGF compared to BMSCs/ADSCs. When critical size calvarial defects in rats were implanted with mixture of BMSCs and ADSCsVEGF along with hydroxyapatite/β‐tricalcium phosphate granules, BMSCs and ADSCsVEGF at the ratio of 1:0.05 showed better bone regeneration that BMSCs alone. The cotransplantation of ADSCsVEGF with BMSCs significantly increased neovascularization on the regenerated bone of the repaired defect than BMSCs alone. In conclusion, ADSCsVEGF added in small proportion to BMSCs effectively promote bone regeneration and neovascularization. Copyright

Co-transplantation of adipose and bone marrow derived stromal cells for treatment of osteonecrosis of femoral head

Osteonecrosis of femoral head (ONFH) is irreversible disease which treatment is almost depending on total joint replacement. The synergistic effect by co-transplantation of adipose-derived stromal cells (ASCs) in enhancing the osteogenic differentiation of bone marrow stromal cells (BMSCs) proved in critical sized calvarial defect model of rat in our previous study. In this study, porcine ASCs and BMSCs were co-transplanted to ONFH model of minipig. In the results of X-rays and CT, defect of cells transplantation group was complete healed up with new bone tissue while control group had unfilled bone defect. Histological findings also corroborated the radiological findings. We observed very rapid and good quality osteogenesis in combined ASCs and BMSCs implantation group compared with control group.

Angiopoietin-2 Enhances Osteogenic Differentiation of Bone Marrow Stem Cells

Our previous studies revealed that co‐transplantation of bone marrow stem cells (BMSCs) and adipose‐derived stem cells (ADSCs) can enhance bone regeneration and angiogenesis. However, it is unclear which genes are involved in the regulation of osteogenesis and/or angiogenesis during the co‐culturing of BMSCs and ADSCs. The expression patterns of genes associated with osteogenesis and/or angiogenesis were analyzed in osteogenesis‐induced BMSCs and ADSCs using an oligonucleotide microarray. Significant difference in the expression patterns of several genes were identified from hierarchical clustering and analyzed on co‐cultured BMSCs and ADSCs. Angiopoietin‐2 (ANGPT2) and activin receptor‐like kinase‐1 were significantly down‐regulated in co‐culture than culture of either BMSCs or ADSCs, while fibroblast growth factor‐9 was significantly up‐regulated in co‐culture. The effect of ANGPT2 in osteogenesis‐induced BMSCs was validated using recombinant protein and siRNA of ANGPT2. Treatment of the ANGPT2 protein significantly increased the expressions of osteogenic makers and the intensity of Alizarin red‐S staining in BMSCs. Down‐regulation of ANGPT2 significantly decreased the expression of osteogenic makers. The treatment of ANGPT2 protein to BMSCs induced significantly increased tube formation in Transwell‐co‐cultured human umbilical vein endothelial cells (HUVECs) compared with untreated control. ANGPT2 siRNA transfection showed the opposite effects. These results suggest that the treatment of ANGPT2 in BMSCs increase osteogenesis and angiogenesis in vitro, and that the enhancement of osteogenesis and angiogenesis in the co‐cultured BMSCs and ADSCs seems to be mediated by a mechanism that makes the activation of ANGPT2 unnecessary. These observations provide the first evidence for positive regulation of osteogenesis by ANGPT2 in vitro. J. Cell. Biochem. 118: 2896–2908, 2017.

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