Bioprinted constructs of differentiated primary osteocytes: A Co-Culture Model with osteoclasts for biomedical research

Osteocytes play a major role in the regulation of bone remodelling and homeostatis and should therefore be more prominently incorporated into in vitro bone models. Bioprinting is a versatile method to generate spatially defined tissue-like structures, however bioprinting of osteocytes remains challenging due to their post-mitotic nature. This study therefore investigates the transition of primary human osteoblasts (hOB) into osteocytes in bioprinted constructs, with respect to bioink composition and growth factor supplementation. Osteocytes were successfully differentiated from hOB, within bioprinted constructs via using alginate/methylcellulose/gelatin ink, alginate/methylcellulose/egg white ink and alginate/methylcellulose/human plasma ink (Alg/MC/Pl). Osteocytic morphology and marker expression was confirmed by fluorescence microscopy and gene expression analysis. Moreover, a significant upregulation of late osteocytic markers (e.g. SOST and MEPE) was observed under low serum conditions (2%) compared with cultures maintained in 10 % fetal calf serum (FCS). Human platelet lysate (hPL), evaluated as an alternative to FCS, also demonstrated the capacity to support osteocyte differentiation. Furthermore, hPL was successfully used for hOB pre-differentiation. In the presence of 2 % hPL, a higher number of multinucleated osteoclasts along with an elevated activity of osteoclast-specific enzymes (tartrate-resistant acid phosphatase, cathepsin K and carbonic anhydrase 2) in comparison to 2 % FCS was observed. This high potential of hPL to support osteoclastogenesis opens the way for physiologically-relevant in vitro bone models comprising both osteocytes and osteoclasts. Indirect co-cultures of human osteoclasts and human osteocytes, bioprinted and differentiated in Alg/MC/Pl showed expression of all relevant osteoclast and osteocyte markers. 
&#xD.