The effect of multi-material architecture on the ex vivo osteochondral integration of bioprinted constructs

Abstract

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.

Keywords: Bioinks; Bioprinting; Explant; Extrusion; Gradient; Multi-material; Osteochondral; Tissue integration; hMSC.

Figure 1.

Dimensions and details of the osteochondral explant ex vivo model and experimental fiber groups. (a) The experimental groups and their dimensions. (b) The tissue harvesting and osteochondral defect creation processes, resulting in a 1 mm diameter cylindrical osteochondral plug with 4 mm of length, with both explant and plug cultured in well plates with osteochondral medium until study initiation. (c) Demonstration of the bioprinted fiber insertion technique, before and after swelling occurred to fill the defect diameter.

Figure 2.

Images describing the extrusion 3D printing of multi-material fiber architectures. (a) Gradient fiber printing, with alternating materials shown coming out of the nozzle (left), and representative images (right) showing where three gradient fibers were sectioned for explant insertion based on their transition region; scale bar = 4 mm. (b) Segmented fiber printing, where each segment half was printed separately before changing the bioink cartridge for multi-material printing (left) and a representative image shows where each fiber would be sectioned for explant insertion (right); scale bar = 4 mm. Representative 4× (c) brightfield and (d) fluorescent images for gradient fibers are shown next to segmented fibers (e-f), where both the suspended ceramic material and the rhodamine-B dye present in the B-INK are visible; scale bar = 500 μm. Representative SEM images of only the magnified, central bioink transition region for (g) gradient fibers and (h) segmented fibers are shown to highlight the difference in the multi-material interface, with suspended β-TCP from the B-INK highlighted with red arrows; scale bar = 300 μm. This difference is also shown in the EDS map data of the transition regions which highlight carbon, present throughout the bioink, and phosphorus, present primarily within the β-TCP, for (i) a representative gradient fiber transition region and (j) a representative segmented fiber transition region; scale bar = 200 μm. In (i-j), C stands for carbon with its detected signals given in light blue, and P stands for phosphorus with its detected signals given in green.

Figure 3.

Push-out testing for assessing integration into the ex vivo explant model. (a) Design of the push-out testing jig, which uses a piston driven into the osteochondral defect site to assess tissue integration. (b) Images demonstrating setup and progression of a push-out test. Each extruded fiber (red arrows) was kept as a sample for the biochemical assays. (c) Peak load and (d) total integration strength results from push-out testing of each sample after being split into two tissue sections, the bone (left) and cartilage (right) regions. A † indicates significant difference compared to OC plug control group at same time point; a # indicates significant difference compared to Day 1 time point within same group (p<0.05). All data n=3-5, some exclusions (listed in Supp. Table 1) were made when the piston misaligned during push-out or scraped the wall of the explant tissue during testing due to non-vertical defect creation. The legend applies to (c) & (d).

Figure 4.

Results of the biochemical assays on implanted fibers or osteochondral groups after 7 and 21 days of culture within the ex vivo model. (a) DNA content normalized to sample mass. Calcium and sGAG content are shown (b-c) normalized to sample mass or (c-d) normalized to DNA content of each sample, with results by explant section on the left, and results displayed for each entire fiber on the right, which combines data from both the top cartilage section and the bottom bone section of fibers. # indicates significant difference compared to Day 1 time point within the same group; * indicates difference between groups (p<0.05). n=5 for all Day 1 & Day 7 assay data; n=6 for all Day 21 assay data. Legend at top-right applies to all data.

Figure 5.

Brightfield microscopy of histological staining of explants with inserted fibers or osteochondral plugs after 3 weeks of culture within the ex vivo explant model, with insets highlighting activity in the chondral region. (a) The segmented S-Fib group is shown next to (b) the mixed G-Fib group and (c) the OC-Plug group. Left column: H&E staining for nuclei (dark purple), cytoplasm (pink), and general tissue structure, with arrows indicating ECM deposition and cellularity. Middle column: staining for GAGs (blue) and nuclei (purple-red), with arrows indicating cellularity and regions of matrix deposition. Right column: von Kossa staining for mineralization and Ca²⁺ content, with arrows pointing to darker regions of staining indicating mineralization. Scale bar of full explant images = 1 mm. Scale bar of inset images = 100 μm.