**Author details**

Hayashi et al. 2012), as well as cell-to-cell communication through exosomes, containing a plethora of bioactive molecules, urges new research approaches to unravel the intricate mechanisms ensuring bone and cartilage health before and after the onset of disease states damaging the tissues in question. We have here confirmed that chondrocytes, in fact, do affect osteoclasts directly (Xiong, Onal et al. 2011), and that cytokines obtained from Th-cells, and Th17 cells in particular, are detrimental to the osteochondral phenotypes, with an additional

The present experimental setting also show that exosomes from Th17 cells may interfere with both the chondrocytic and osteoblastic phenotypes in a negative fashion (i.e. phenotype acquisition and matrix deposition), while also speeding up bone remodelling via overactivation of osteoclasts embedded in the bone adjacent to the cartilage lining. These findings are consistent with the development of cartilage loss and the appearance of osteophytes (newly formed bone in disarray) in arthritic joints due to the progressing autoimmune process

This particular report entails the use of transient microRNA manipulations to ensure acquis‐ ition of proper osteochondral phenotypes when engineering cells to replace damaged bone and cartilage in patients with inflammatory diseases targeting articular joints (in particular rheumatoid arthritis). We have shown that cell engineering, as a research field, needs to take into consideration how osteochondral cells affect osteoclasts directly, and that osteochondral cells may lose their acquired phenotypes upon exposure to cytokines (e.g. IL-1, IL-6, IL-17, and TNFα) or micro-RNA-containing exosome-like particles derived from activated Th17 cells. These detrimental effects can be counteracted by manipulating stem cell microRNA contents

When refining the search for the minimal number of effective microRNAs, it is recommended that bioinformatics approaches are used along with micro-RNA micro-arrays and marker gene transcriptomes in engineered osteochondral cells, and that maximal compatibility score (Gordeladze 2011) between them are obtained. Assessment of phenotypes obtained should include analyses of how and to which extent these cells affect osteoclasts, and whether altered (i.e. enhanced) remodelling of bone formed within an *in vivo* model system (e.g. calcium

This project was supported by EU FP6 integrated project "Genostem cells engineering for connective tissue disorders", Norwegian Center for Stem Cell research, The National Hospital,

(the optimal minimal number and species of microRNAs are yet to be defined).

characteristic of rheumatoid arthritis (Hayashi, Xu et al. 2012).

**7. Summary and future prospects**

deposits in the tibial muscle of SCID mice) of choice.

Oslo, Norway, and The Research Council of Norway.

**Acknowledgements**

activation of osteoclasts.

496 Regenerative Medicine and Tissue Engineering

Jan O. Gordeladze1,2\*, Janne E. Reseland3 , Tommy A. Karlsen1,2, Rune B. Jakobsen2 , Astrid K. Stunes4 , Unni Syversen4 , Lars Engebretsen5,6, Ståle P. Lyngstadaas3 and Christian Jorgensen7,8

\*Address all correspondence to: j.o.gordeladze@medisin.uio.no

1 Department of Medical Biochemistry, Institute of Basic Medical Science, University of Oslo, Norway

2 Norwegian Center for Stem Cell research, The National Hospital, Oslo, Norway

3 Department of Biomaterials, Faculty of Dentistry, University of Oslo, Norway

4 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway

5 Orthopaedic Center, Ullevål University Hospital, Oslo, Norway

6 Faculty of medicine, University of Oslo, Norway

7 INSERM U844, Montpellier, France

8 Unité Immuno-Rhumatologie Thérapeutique, Centre Hospitalier Universitaire (CHU) Lapeyronie, Montpellier, France

### **References**


[21] Fritz, V, Brondello, J. M, et al. (2011). Bone-metastatic prostate carcinoma favors mes‐ enchymal stem cell differentiation toward osteoblasts and reduces their osteoclasto‐ genic potential." *J Cell Biochem* , 112(11), 3234-3245.

[6] Arai, F, Miyamoto, T, et al. (1999). Commitment and differentiation of osteoclast pre‐ cursor cells by the sequential expression of c-Fms and receptor activator of nuclear

[7] Boissier, M. C. (2011). Cell and cytokine imbalances in rheumatoid synovitis." *Joint*

[8] Bonewald, L. F. (2007). Osteocytes as dynamic multifunctional cells." *Ann N Y Acad*

[9] Burgess, T. L, Qian, Y, et al. (1999). The ligand for osteoprotegerin (OPGL) directly

[10] Carvalheiro, H, & Da, J. A. Silva, et al. ((2012). Potential roles for CD8(+) T cells in

[11] Chang, M. K, Raggatt, L. J, et al. (2008). Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function in

[12] Chen, F. H, & Tuan, R. S. (2008). Mesenchymal stem cells in arthritic diseases." *Arthri‐*

[13] Cocucci, E, Racchetti, G, et al. (2009). Shedding microvesicles: artefacts no more."

[14] Davies, J, Warwick, J, et al. (1989). The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin recep‐

[15] De Broe, M. E, Wieme, R. J, et al. (1977). Spontaneous shedding of plasma membrane fragments by human cells in vivo and in vitro." *Clin Chim Acta* , 81(3), 237-245.

[16] De Smet, E, Jaecques, S. V, et al. (2007). Effect of constant strain rate, composed of varying amplitude and frequency, of early loading on peri-implant bone (re)model‐

[17] Drake, F. H, Dodds, R. A, et al. (1996). Cathepsin K, but not cathepsins B, L, or S, is abundantly expressed in human osteoclasts." *J Biol Chem* , 271(21), 12511-12516.

[18] Everts, V, Delaisse, J. M, et al. (2002). The bone lining cell: its role in cleaning How‐ ship's lacunae and initiating bone formation." *J Bone Miner Res* , 17(1), 77-90.

[19] Fermor, B, & Skerry, T. M. (1995). PTH/PTHrP receptor expression on osteoblasts and osteocytes but not resorbing bone surfaces in growing rats." *J Bone Miner Res* ,

[20] Franceschi, R. T, Xiao, G, et al. (2003). Multiple signaling pathways converge on the Cbfa1/Runx2 transcription factor to regulate osteoblast differentiation." *Connect Tis‐*

factor kappaB (RANK) receptors." *J Exp Med* , 190(12), 1741-1754.

activates mature osteoclasts." *J Cell Biol* , 145(3), 527-538.

rheumatoid arthritis." *Autoimmun Rev*.

vitro and in vivo." *J Immunol* , 181(2), 1232-1244.

*Bone Spine* , 78(3), 230-234.

*Sci* , 1116, 281-290.

498 Regenerative Medicine and Tissue Engineering

*tis Res Ther* 10(5): 223.

10(12), 1935-1943.

*sue Res* 44 Suppl , 1, 109-116.

*Trends Cell Biol* , 19(2), 43-51.

tor." *J Cell Biol* 109(4 Pt 1): 1817-1826.

ling." *J Clin Periodontol* , 34(7), 618-624.


[46] Insogna, K. L, Sahni, M, et al. (1997). Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts." *J Clin Invest* , 100(10), 2476-2485.

[33] Hassan, M. Q, Gordon, J. A, et al. (2010). A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program." *Proc*

[34] Hattersley, G, & Chambers, T. J. (1989). Calcitonin receptors as markers for osteoclas‐ tic differentiation: correlation between generation of bone-resorptive cells and cells that express calcitonin receptors in mouse bone marrow cultures." *Endocrinology* ,

[35] Hayashi, D, Xu, L, et al. (2012). Detection of osteophytes and subchondral cysts in the

[36] Heijnen, H. F, Schiel, A. E, et al. (1999). Activated platelets release two types of mem‐ brane vesicles: microvesicles by surface shedding and exosomes derived from exocy‐

[37] Heinemann, D. E, Siggelkow, H, et al. (2000). Alkaline phosphatase expression dur‐ ing monocyte differentiation. Overlapping markers as a link between monocytic cells, dendritic cells, osteoclasts and osteoblasts." *Immunobiology* , 202(1), 68-81.

[38] Heining, E, Bhushan, R, et al. (2011). Spatial segregation of BMP/Smad signaling af‐

[39] Heino, T. J, Hentunen, T. A, et al. (2002). Osteocytes inhibit osteoclastic bone resorp‐ tion through transforming growth factor-beta: enhancement by estrogen." *J Cell Bio‐*

[40] Hirota, K, Hashimoto, M, et al. (2007). T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis." *J*

[41] Hofbauer, L. C, Khosla, S, et al. (2000). The roles of osteoprotegerin and osteoprote‐ gerin ligand in the paracrine regulation of bone resorption." *J Bone Miner Res* , 15(1),

[42] Holtrop, M. E, & King, G. J. (1977). The ultrastructure of the osteoclast and its func‐

[43] Horton, M. A, Taylor, M. L, et al. (1991). Arg-Gly-Asp (RGD) peptides and the antivitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by

[44] Hu, R, Sharma, S. M, et al. (2007). Eos, MITF, and PU.1 recruit corepressors to osteo‐ clast-specific genes in committed myeloid progenitors." *Mol Cell Biol* , 27(11),

[45] Hunter, M. P, Ismail, N, et al. (2008). Detection of microRNA expression in human

tional implications." *Clin Orthop Relat Res*(123): 177-196.

peripheral blood microvesicles." *PLoS One* 3(11): e3694.

osteoclasts." *Exp Cell Res* , 195(2), 368-375.

fects osteoblast differentiation in C2C12 cells." *PLoS One* 6(10): e25163.

tosis of multivesicular bodies and alpha-granules." *Blood* , 94(11), 3791-3799.

knee with use of tomosynthesis." *Radiology* , 263(1), 206-215.

*Natl Acad Sci U S A* , 107(46), 19879-19884.

125(3), 1606-1612.

500 Regenerative Medicine and Tissue Engineering

*chem* , 85(1), 185-197.

*Exp Med* , 204(1), 41-47.

2-12.

4018-4027.


[74] Luzi, E, Marini, F, et al. (2012). The regulatory network menin-microRNA 26a as a possible target for RNA-based therapy of bone diseases." *Nucleic Acid Ther* , 22(2), 103-108.

[60] Larsen, M, Arnaud, L, et al. (2011). Multiparameter grouping delineates heterogene‐ ous populations of human IL-17 and/or IL-22 T-cell producers that share antigen spe‐

[61] Le BechecA., E. Portales-Casamar, et al. ((2011). MIR@NT@N: a framework integrat‐ ing transcription factors, microRNAs and their targets to identify sub-network motifs

[62] Legendre, F, Dudhia, J, et al. (2003). JAK/STAT but not ERK1/ERK2 pathway medi‐ ates interleukin (IL)-6/soluble IL-6R down-regulation of Type II collagen, aggrecan core, and link protein transcription in articular chondrocytes. Association with a

[63] Li, X, Qin, L, et al. (2007). Parathyroid hormone stimulates osteoblastic expression of MCP-1 to recruit and increase the fusion of pre/osteoclasts." *J Biol Chem* , 282(45),

[64] Li, X, Zhang, Y, et al. (2005). Sclerostin binds to LRP5/6 and antagonizes canonical

[65] Li, Y, Toraldo, G, et al. (2007). B cells and T cells are critical for the preservation of bone homeostasis and attainment of peak bone mass in vivo." *Blood* , 109(9),

[66] Li, Z, Hassan, M. Q, et al. (2009). Biological functions of miR-29b contribute to posi‐

[67] Li, Z, Hassan, M. Q, et al. (2008). A microRNA signature for a BMP2-induced osteo‐ blast lineage commitment program." *Proc Natl Acad Sci U S A* , 105(37), 13906-13911.

[68] Lian, J. B, Javed, A, et al. (2004). Regulatory controls for osteoblast growth and differ‐ entiation: role of Runx/Cbfa/AML factors." *Crit Rev Eukaryot Gene Expr* 14(1-2): 1-41.

[69] Lin, G. L, & Hankenson, K. D. (2011). Integration of BMP, Wnt, and notch signaling pathways in osteoblast differentiation." *J Cell Biochem* , 112(12), 3491-3501.

[70] Lories, R. (2011). The balance of tissue repair and remodeling in chronic arthritis."

[71] Lu, X, Beck, G. R, et al. (2011). Identification of the homeobox protein Prx1 (MHox, Prrx-1) as a regulator of osterix expression and mediator of tumor necrosis factor al‐

[72] Lubberts, E, Koenders, M. I, et al. (2004). Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint in‐ flammation, cartilage destruction, and bone erosion." *Arthritis Rheum* , 50(2), 650-659.

[73] Luchin, A, Purdom, G, et al. (2000). The microphthalmia transcription factor regu‐ lates expression of the tartrate-resistant acid phosphatase gene during terminal dif‐

pha action in osteoblast differentiation." *J Bone Miner Res* , 26(1), 209-219.

ferentiation of osteoclasts." *J Bone Miner Res* , 15(3), 451-460.

cificities with other T-cell subsets." *Eur J Immunol* , 41(9), 2596-2605.

in a meta-regulation network model." *BMC Bioinformatics* 12: 67.

down-regulation of SOX9 expression." *J Biol Chem* , 278(5), 2903-2912.

Wnt signaling." *J Biol Chem* , 280(20), 19883-19887.

tive regulation of osteoblast differentiation." *J Biol Chem*.

*Nat Rev Rheumatol* , 7(12), 700-707.

33098-33106.

502 Regenerative Medicine and Tissue Engineering

3839-3848.


[101] Quinn, J. M, Elliott, J, et al. (1998). A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro." *Endocrinology* , 139(10), 4424-4427.

[87] Moretti, F, Thermann, R, et al. (2010). Mechanism of translational regulation by miR-2 from sites in the 5' untranslated region or the open reading frame." *RNA* ,

[88] Motyl, K. J, & Rosen, C. J. (2012). Understanding leptin-dependent regulation of skel‐

[89] Murshed, M, Harmey, D, et al. (2005). Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone."

[90] Muys, J. J, Alkaisi, M. M, et al. (2006). Cellular transfer and AFM imaging of cancer

[91] Nakashima, T, Hayashi, M, et al. (2012). New insights into osteoclastogenic signaling

[92] Nakashima, T, & Takayanagi, H. (2009). Osteoimmunology: crosstalk between the

[93] Neve, A, Corrado, A, et al. (2011). Osteoblast physiology in normal and pathological

[94] Newby, A. C. (2008). Metalloproteinase expression in monocytes and macrophages and its relationship to atherosclerotic plaque instability." *Arterioscler Thromb Vasc Bi‐*

[95] Oh, H. J, Kido, T, et al. (2007). PIAS1 interacts with and represses SOX9 transactiva‐

[96] Palumbo, C. ultrastructural study of osteoid-osteocytes in the tibia of chick embryos."

[97] Parsonage, G, Filer, A, et al. (2008). Prolonged, granulocyte-macrophage colony-stim‐ ulating factor-dependent, neutrophil survival following rheumatoid synovial fibro‐

[98] Partridge, N. C, Jeffrey, J. J, et al. (1987). Hormonal regulation of the production of collagenase and a collagenase inhibitor activity by rat osteogenic sarcoma cells." *En‐*

[99] Pederson, L, Ruan, M, et al. (2008). Regulation of bone formation by osteoclasts in‐ volves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate." *Proc Natl*

[100] Pene, J, Chevalier, S, et al. (2008). Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes." *J Immunol* , 180(11), 7423-7430.

blast activation by IL-17 and TNFalpha." *Arthritis Res Ther* 10(2): R47.

16(12), 2493-2502.

504 Regenerative Medicine and Tissue Engineering

etal homeostasis." *Biochimie*.

*Genes Dev* , 19(9), 1093-1104.

*ol* , 28(12), 2108-2114.

*Cell Tissue Res* , 246(1), 125-131.

*docrinology* , 120(5), 1956-1962.

*Acad Sci U S A* , 105(52), 20764-20769.

cells using Bioimprint." *J Nanobiotechnology* 4: 1.

immune and bone systems." *J Clin Immunol* , 29(5), 555-567.

mechanisms." *Trends Endocrinol Metab*.

conditions." *Cell Tissue Res* , 343(2), 289-302.

tion activity." *Mol Reprod Dev* , 74(11), 1446-1455.


[130] Volpe, E, Servant, N, et al. (2008). A critical function for transforming growth factorbeta, interleukin 23 and proinflammatory cytokines in driving and modulating hu‐ man T(H)-17 responses." *Nat Immunol* , 9(6), 650-657.

[116] Takahashi, F, Takahashi, K, et al. (2004). Osteopontin is strongly expressed by alveo‐ lar macrophages in the lungs of acute respiratory distress syndrome." *Lung* , 182(3),

[117] Takarada, T, & Yoneda, Y. (2008). Pharmacological topics of bone metabolism: gluta‐

[118] Takayanagi, H, Iizuka, H, et al. (2000). Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from syn‐

[119] Tan, Y. K, & Conaghan, P. G. (2011). Imaging in rheumatoid arthritis." *Best Pract Res*

[120] Tang, Y, Wu, X, et al. (2009). TGF-beta1-induced migration of bone mesenchymal stem cells couples bone resorption with formation." *Nat Med* , 15(7), 757-765.

[121] Tani, A, Tada, Y, et al. (2012). Regeneration of tracheal epithelium using a collagen vitrigel-sponge scaffold containing basic fibroblast growth factor." *Ann Otol Rhinol*

[122] Teitelbaum, S. L. (2000). Bone resorption by osteoclasts." *Science* , 289(5484),

[123] Teitelbaum, S. L, & Ross, F. P. (2003). Genetic regulation of osteoclast development

[124] Thompson, W. R, Rubin, C. T, et al. (2012). Mechanical regulation of signaling path‐

[125] Tondravi, M. M, Mckercher, S. R, et al. (1997). Osteopetrosis in mice lacking haema‐

[126] Tran, P, Vignery, A, et al. (1982). An electron-microscopic study of the bone-remodel‐

[127] Van Bezooijen, R. L, Roelen, B. A, et al. (2004). Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist." *J Exp Med* ,

[128] Van Der Kraan, P. M. and W. B. van den Berg ((2012). Chondrocyte hypertrophy and osteoarthritis: role in initiation and progression of cartilage degeneration?" *Osteoar‐*

[129] Verborgt, O, Tatton, N. A, et al. (2002). Spatial distribution of Bax and Bcl-2 in osteo‐ cytes after bone fatigue: complementary roles in bone remodeling regulation?" *J Bone*

topoietic transcription factor PU.1." *Nature* , 386(6620), 81-84.

ing sequence in the rat." *Cell Tissue Res* , 225(2), 283-292.

mate as a signal mediator in bone." *J Pharmacol Sci* , 106(4), 536-541.

oviocytes in rheumatoid arthritis." *Arthritis Rheum* , 43(2), 259-269.

173-185.

506 Regenerative Medicine and Tissue Engineering

*Clin Rheumatol* , 25(4), 569-584.

*Laryngol* , 121(4), 261-268.

and function." *Nat Rev Genet* , 4(8), 638-649.

ways in bone." *Gene* , 503(2), 179-193.

1504-1508.

199(6), 805-814.

*thritis Cartilage* , 20(3), 223-232.

*Miner Res* , 17(5), 907-914.

