Page 505 - IJB-9-5

International Journal of Bioprinting Implantation of composites for cartilage repair

weekly). Thereafter, samples were embedded in paraffin, across cultured composites, and the resultant mechanical
sectioned (8 µm), and stained with either picrosirius red properties for the experimental groups investigated in the
to visualize collagen content or safranin O/fast green to porcine model.
visualize proteoglycan and collagen content, respectively,
within the repair cartilage and the underlying subchondral 3. Results and discussion
bone. The ICRS II Histological Assessment Scale (n ≥ 12 3.1. Composite fabrication and pMSC donor screen
replicates) was employed to assess safranin O/fast green- for chondrogenic potential
stained samples, with scores ranging from 0% to 100% MEW-NorHA composites were fabricated by first
(worst–best) averaged across three blinded reviewers . heating PCL into a polymer melt that is amenable to
[32]
These blinded reviewers also qualitatively selected the best melt electrowriting using a custom-built device . As the
[22]
and worst micro-CT and picrosirius red images obtained PCL polymer melt is fed through the spinneret, a high-
for each investigated experimental group.
voltage source is applied, leading to the formation of an
electrically charged polymer melt fiber. The deposition of
2.6. Indentation testing of composites and repair these fibers is then controlled using a translating collector,
cartilage allowing for the layer-by-layer fabrication of mesh
To evaluate the mechanical properties of repair cartilage structures (Figure 1A). More specifically, MEW scaffolds
12 weeks after defect creation, creep indentation testing was were formed by depositing polymer melt fibers in a 90°
performed as previously described using an Instron 5948 lay-down pattern, with 400 µm interfiber spacing between
Universal Testing System (Instron Inc., Norwood, MA) parallel fibers (Figure 1B). PCL MEW scaffolds could
with an affixed 1 mm diameter spherical indenter . Since then be combined with acellular or pMSC-laden NorHA
[33]
large deformations during physiologic creep testing may hydrogels to form MEW-NorHA composites (Figure 1C).
significantly alter the local compositional characteristics of
immature tissue constructs (i.e., acellular composites), lower To evaluate the retention of MEW-NorHA hydrogel
loads were employed during all indentation testing to ensure composites within full-thickness cartilage defects, it was
the accurate quantification of mechanical properties . first necessary to validate that the composites support
[34]
Generally, a load of 0.1 N was applied to all samples at a the chondrogenesis of adult porcine MSCs toward the
loading rate of 0.1 N/s and then held for 900 s (after the load formation of neocartilage. Adult pMSCs were selected
setpoint was reached) while the creep displacement was as an allogenic cell source to mitigate any potential
measured. Prior to testing, osteochondral samples were first immune responses upon implantation in minipigs .
[36]
fixed into place within a low-melting temperature bismuth Isolated pMSCs were age-matched to the host animals
alloy to secure samples while maintaining the cartilage (12–14 months) to ensure that they best represented the
defect surface upright. Samples were then submerged in clinically-relevant scenario in which autologous cells are
PBS and positioned under the indenter setup using a custom sourced and used within implants. Moreover, skeletally
XY positioning stage and a goniometer to ensure that the mature minipigs were selected as host animals to mitigate
cartilage surface was perpendicular to the indenter. Repair subchondral bone remodeling, which has been previously
cartilage within the center of defect samples (or directly reported in juvenile minipigs , and to recapitulate the
[27]
adjacent to pins in instances where pins were still visible higher loading environment that is normally experienced
and exposed on the cartilage surface) was then indented. in adults with cartilage defects. NorHA macromer along
After osteochondral sample fixation and decalcification, with dithiol crosslinker (DTT) and photoinitiator (LAP)
defects were cut along their midplane to determine the were mixed with pMSCs to form a suspension composed
thickness of cartilage samples. The compressive modulus, of hydrogel precursors and cells that could be readily filled
tensile modulus, and permeability of all indented samples into the interstitial spaces of the fabricated MEW scaffolds.
were then quantified by fitting the collected creep data to a Thereafter, exposure to collimated blue light initiated the
Hertzian biphasic model . thiol-ene crosslinking of pMSC-laden hydrogels within
[35]
MEW scaffolds (Figure 2A). The process and materials
2.7. Statistical analysis employed to form these composites were cytocompatible,
All statistical analyses were performed using GraphPad as evidenced by Live/Dead staining after culturing
Prism 9 software, with data reported as mean ± standard composites for 7 days (Figure 2B). Importantly, relatively
deviation and significance for all performed analyses high cell viabilities (~80%) of the encapsulated pMSCs
determined at p < 0.05. One-way analyses of variance were observed across three different porcine donors
(ANOVAs) were performed with Tukey’s honestly (Donors 1, 2, and 3), suggesting that long-term culture
significant difference (HSD) post-hoc testing to compare of pMSCs within MEW-NorHA composites is feasible,
functional outcomes between porcine donors, cell viability irrespective of donor source (Figure 2C). In addition, the

Volume 9 Issue 5 (2023) 497 https://doi.org/10.18063/ijb.775