**Engineering of Inflammation-Resistant Osteochondral Cells**

Jan O. Gordeladze, Janne E. Reseland, Tommy A. Karlsen, Rune B. Jakobsen, Astrid K. Stunes, Unni Syversen, Lars Engebretsen, Ståle P. Lyngstadaas and Christian Jorgensen

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55544

**1. Introduction**

### **1.1. Clinical issue**

Osteoblasts and chondrocytes engineered from various sources of stem cells to replace dam‐ aged bone or cartilage in an inflamed joint in patients with inflammatory diseases like osteoar‐ thritis or osteoarthrosis need to be made resilient to the impact of the inflamed environment withinthe joint cavity (Neve,Corrado et al. 2011;vanderKraanandvandenBerg 2012).Clinical experience has clearly shown that the engineered osteochondral cells relatively quickly tend to lose their defined cell phenotype (Chen and Tuan 2008; Kastrinaki and Papadaki 2009), while also tending to perpetuate bone loss through activation of osteoclasts, thus yielding a negative bone remodelling volume (Lories 2011; Goldring 2012). The present concept of manipulation of stemcellmicroRNAlevelsenvisagesoneroute,bywhichonemayobtainengineeredosteochon‐ dral cells with a better potential of producing bone and cartilage *in vivo*, and to withstand the putative detrimental effect of immune cell (e.g. T-helper cell = Th-cell) influence constituted by a plethora of cytokines and exosome-conveyed microRNAs.

#### **1.2. Previous findings**

We have previously shown that osteochondral cells can be derived from human mesenchymal stem cells (hMSCs) by manipulating the levels of a group of microRNAs, i.e. mir-16, mir-24, mir-125b, mir-149, mir-328, and mir-339 (Gordeladze 2009; Gordeladze, Djouad et al. 2009). All

© 2013 Gordeladze et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

these microRNAs are low in differentiated osteoblasts, but highly expressed in stem cells and differentiated chondrocytes. Especially mir-149 seems to serve as a switch, both stimulating chondrogenesis, while suppressing osteoblastogenesis in differentiating hMSCs. Further‐ more, mir-328 and mir-339 are the better determinants of the osteochondral cell phenotypes, since manipulations of their intracellular levels enabled us to reciprocally trans-differentiate osteoblasts to chondrocytes at day 7-14 in 2D and 3D cultures *in vitro* (Gordeladze, Djouad et al. 2009; Gordeladze 2011). Hence, we propose that these six microRNAs, and mir-149, mir-328, and mir-339 in particular, might be able to influence the osteochondral phenotypes in such a way that they are able to maintain their phenotype *in vitro* and *in vivo* for an extended period of time, while under the influence of a Th-cell invaded arthritic luminal space.

#### **1.3. Experimental procedures**

The material used in a defined set of experiments consisted of hMSCs differentiated towards osteoblasts in monolayer cultures or in scaffolds (hydroxyapatite or alginate beads, respec‐ tively), hPBMCs (human peripheral blood monocytic cells) differentiated towards osteoclasts and grown on bovine dentine slices, exosomes (100,000xg pellets) from activated Th-17 cells, and SCID mice. The cells were transfected with scrambled microRNA, mir-16, mir-24, mir-125b, mir-149, mir-328 or mir-339 pre-microRNAs or antago-microRNAs, using amounts known to suppress or enhance endogenous levels approximately 5 times (Gordeladze 2009). Human MSCs, osteoblasts or chondrocytes were grown in a medium supplemented with chemokines (Interleukin-1 = IL-1, Interleukin-6 = IL-6, and Interleukin-17A = IL-17A, and TNFα), or over-layered with exosomes from Th17 cells, either in the absence or presence of dentine slices over-layered with osteoclasts differentiated from hPBMCs, using RANK-L and M-CSF (Fritz, Brondello et al. 2011). Subsequent to a certain period of time (7-21 days), osteochondral cells were either analysed for phenotypic characteristics (e.g. matrix production, microRNA levels, phenotypic gene expression) or injected (without or with mature osteoclasts) into the tibial muscle of SCID mice. After 14 days of *in vivo* incubation, the *de novo* formed osteochondral tissues were analysed for phenotypic markers of injected cells by Q-PCR, as well as matrix characteristics (e.g. matrix protein immunohistochemistry, hydroxyapatite production, or GAG = glucosaminglycan over DNA ratio).

#### **1.4. Results**

These experiments showed the following:


for osteoblasts), we observed that the osteochondral phenotypes were disturbed, along with a concomitantly enhanced potential for osteoclast activation.


#### **1.5. Conclusion**

these microRNAs are low in differentiated osteoblasts, but highly expressed in stem cells and differentiated chondrocytes. Especially mir-149 seems to serve as a switch, both stimulating chondrogenesis, while suppressing osteoblastogenesis in differentiating hMSCs. Further‐ more, mir-328 and mir-339 are the better determinants of the osteochondral cell phenotypes, since manipulations of their intracellular levels enabled us to reciprocally trans-differentiate osteoblasts to chondrocytes at day 7-14 in 2D and 3D cultures *in vitro* (Gordeladze, Djouad et al. 2009; Gordeladze 2011). Hence, we propose that these six microRNAs, and mir-149, mir-328, and mir-339 in particular, might be able to influence the osteochondral phenotypes in such a way that they are able to maintain their phenotype *in vitro* and *in vivo* for an extended period of

The material used in a defined set of experiments consisted of hMSCs differentiated towards osteoblasts in monolayer cultures or in scaffolds (hydroxyapatite or alginate beads, respec‐ tively), hPBMCs (human peripheral blood monocytic cells) differentiated towards osteoclasts and grown on bovine dentine slices, exosomes (100,000xg pellets) from activated Th-17 cells, and SCID mice. The cells were transfected with scrambled microRNA, mir-16, mir-24, mir-125b, mir-149, mir-328 or mir-339 pre-microRNAs or antago-microRNAs, using amounts known to suppress or enhance endogenous levels approximately 5 times (Gordeladze 2009). Human MSCs, osteoblasts or chondrocytes were grown in a medium supplemented with chemokines (Interleukin-1 = IL-1, Interleukin-6 = IL-6, and Interleukin-17A = IL-17A, and TNFα), or over-layered with exosomes from Th17 cells, either in the absence or presence of dentine slices over-layered with osteoclasts differentiated from hPBMCs, using RANK-L and M-CSF (Fritz, Brondello et al. 2011). Subsequent to a certain period of time (7-21 days), osteochondral cells were either analysed for phenotypic characteristics (e.g. matrix production, microRNA levels, phenotypic gene expression) or injected (without or with mature osteoclasts) into the tibial muscle of SCID mice. After 14 days of *in vivo* incubation, the *de novo* formed osteochondral tissues were analysed for phenotypic markers of injected cells by Q-PCR, as well as matrix characteristics (e.g. matrix protein immunohistochemistry, hydroxyapatite

**1.** Human MSCs, pre-chondrocytes or pre-osteoblasts lost many of their phenotypic characteristics as matrix builders upon exposure to either pre-microRNAs (osteoblasts)

**2.** These cells, when grown in co-culture with differentiated osteoclasts, appeared to enhance osteoclast multi-nucleation, gene expression, and ensuing dentine resorption surface area.

**3.** By exposing these cells to either artificial, i.e. "inflamed" synovial fluid (growth medium fortified with Th17 cell like profile of cytokines), exosome-like fractions obtained from activated Th17 cells, or certain pre-microRNAs (i.e. mir-222 for chondrocytes or mir-22

time, while under the influence of a Th-cell invaded arthritic luminal space.

production, or GAG = glucosaminglycan over DNA ratio).

These experiments showed the following:

or antago-microRNAs (chondrocytes).

**1.3. Experimental procedures**

476 Regenerative Medicine and Tissue Engineering

**1.4. Results**

Manipulating pertinent microRNAs in osteoblast or chondrocytes derived from hMSCs (or other sources, such as hADSCs = human adipose tissue derived stem cells) may facilitate differentiation to proper, functional cell phenotypes, which are also resilient to the detrimental impact from inflammatory chemokines and/or microRNAs, proteins or mRNAs transferred from Th-cells (via exosome-like vehicles) to engineered osteoblasts or chondrocytes.
