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Extrusion bioprinted constructs for osteochondral tissue engineering were fabricated to study the effect of multi-material architecture on encapsulated human mesenchymal stem cells' tissue-specific matrix deposition and integration into an ex vivo porcine osteochondral explant model. Two extrusion fiber architecture groups with differing transition regions and degrees of bone- and cartilage-like bioink mixing were employed. The gradient fiber (G-Fib) architecture group showed an increase in chondral integration over time, 18.5 ± 0.7 kPa on Day 21 compared to 9.6 ± 1.6 kPa on Day 1 for the required peak push-out force, and the segmented fiber (S-Fib) architecture group did not, which corresponded to the increase in sulfated glycosaminoglycan deposition noted only in the G-Fib group and the staining for cellularity and tissue-specific matrix deposition at the fiber-defect boundary. Conversely, the S-Fib architecture was associated with significant mineralization over time, but the G-Fib architecture was not. Notably, both fiber groups also had similar chondral integration as a re-inserted osteochondral tissue control. While architecture did dictate differences in the cells' responses to their environment, architecture was not shown to distinguish a statistically significant difference in tissue integration via fiber push-out testing within a given time point or explant region. Use of this three-week osteochondral model demonstrates that these bioink formulations support the fabrication of cell-laden constructs that integrate into explanted tissue as capably as natural tissue and encapsulate osteochondral matrix-producing cells, and it also highlights the important role that spatial architecture plays in the engineering of multi-phasic tissue environments. STATEMENT OF SIGNIFICANCE: Here, an ex vivo model was used to interrogate fundamental questions about the effect of multi-material scaffold architectural choices on osteochondral tissue integration. Cell-encapsulating constructs resembling stratified osteochondral tissue were 3D printed with architecture consisting of either gradient transitions or segmented transitions between the bone-like and cartilage-like bioink regions. The printed constructs were assessed alongside re-inserted natural tissue plugs via mechanical tissue integration push-out testing, biochemical assays, and histology. Differences in osteochondral matrix deposition were observed based on architecture, and both printed groups demonstrated cartilage integration similar to the native tissue plug group. As 3D printing becomes commonplace within biomaterials and tissue engineering, this work illustrates critical 3D co-culture interactions and demonstrates the importance of considering architecture when interpreting the results of studies utilizing spatially complex, multi-material scaffolds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The osteoconductive and osteoinductive properties of materials intended for bone regeneration have been extensively tested, but the resorbability of these materials is often overlooked. Osteoclasts are responsible for bone resorption and play a crucial role in bone remodeling, which is essential for complete regeneration of bone tissue following injury. In this study we compare, for the first time, the ability of unmodified and fibrinogen (Fg)-modified chitosan (Ch) substrates to support the formation of multinucleated osteoclasts, and the potential of these cells to resorb the two substrates in vitro. Osteoclasts were differentiated from primary human peripheral blood monocytes directly on the substrates being investigated. Our results showed similar cell adhesion to unmodified and Fg-modified Ch substrates. Although the number of multinucleated osteoclasts on both Ch substrates increased throughout the culture period, by 21 days of culture significantly more highly multinucleated osteoclasts (>10 nuclei per cell) were observed on Fg-modified Ch, when compared to Ch alone. In addition, cells were tartrate-resistant acid phosphatase positive and secreted significantly more enzyme on Ch-based substrates than in control conditions. Unmodified and Fg-modified Ch resorption was investigated by fluorescence microscopy and confirmed by electron microscopy. Quantification of results obtained by fluorescence microscopy shows that Fg modification led to significantly higher substrate resorption by 17 days of culture. Our results show that osteoclasts, beyond resorbing mineralized substrates, successfully resorb a polymeric substrate (Ch), with Fg accelerating this process. Thus, in bone tissue regeneration strategies employing polymeric biomaterials, resorption may depend not only on macrophages, but also on osteoclasts., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)