Page 503 - IJB-9-5

International Journal of Bioprinting Implantation of composites for cartilage repair

reinforced with melt electrowritten PCL supported juvenile and any precipitates were removed via filtration. The
bovine mesenchymal stromal cell (MSC) chondrogenesis degree of modification (~22%) was quantified via H-NMR
1
and neocartilage formation while also achieving high spectroscopy (Bruker Neo400 360 MHz) to confirm
initial construct mechanical properties . The compressive the successful conjugation of norbornene groups to the
[21]
moduli and biochemical content achieved in these MEW- HA backbone. After synthesis, NorHA was dissolved in
NorHA composites in vitro approached that of native phosphate-buffered saline (PBS) with LAP photoinitiator
tissues and, taken together with their ability to integrate (0.05%) and dithiothreitol (DTT) crosslinker (0.54 mM) to
with native articular cartilage ex vivo, motivated additional obtain a macromer precursor solution.
exploration of their therapeutic potential in a clinically- The prepared macromer solutions were pipetted into the
relevant model of cartilage damage. Thus, the aims of box-structured PCL MEW meshes with or without porcine
this study were (i) to explore adult porcine MSC behavior MSCs (pMSCs, P1, 20 × 10 cells/mL) and irradiated
6
within MEW-NorHA composites and (ii) to elucidate how with blue light (400–500 nm, Omnicure lamp with an
surgical fixation methods influence the efficacy of these affixed collimator, I = 10 mW/cm ) for 5 min to obtain
2
composites in a porcine model of cartilage damage.
composites similar to those investigated in our previous
studies (i.e., 400 mm interfiber spacing within PCL MEW
2. Materials and methods meshes and NorHA hydrogels with a compressive modulus
[21]
2.1. Materials of ~2 kPa) . Porcine MSCs were isolated from bone
Sodium hyaluronic acid was obtained from Lifecore marrow aspirates of three adult Yucatan minipigs (12–14
Biomedical (Chaska, MN), and lithium phenyl-2,4,6- months old; Sinclair Research, Auxvasse, MO) via plastic
trimethylbenzoylphosphinate (LAP) was obtained from adherence and cultured in Dulbecco’s modified eagle
Colorado Photopolymer Solutions (Boulder, CO). Unless medium (DMEM) supplemented with 10% fetal bovine
otherwise specified, all other reagents and materials were serum (FBS) and 1% penicillin/streptomycin (P/S). After
purchased from Sigma-Aldrich (St. Louis, MO). fabrication, composites were cultured in chondrogenic
media (1% insulin–transferrin–selenium solution, 2.5 µg/
2.2. MEW-NorHA composite fabrication and mL amphotericin B, 1 × 10 M sodium pyruvate, 50 µg/mL
−3
cell culture ascorbic acid 2-phosphate, 40 µg/mL L-proline, 1 × 10 M
−7
First, PCL (Purasorb PC 12, Corbion Inc., Gorinchem, dexamethasone, and 10 ng/mL TGF-β3) for up to 28 days.
Netherlands) box-structured MEW meshes (diameter
~4 mm, height ~1 mm, 400 μm fiber interspacing) were 2.3. Composite characterization
fabricated as previously reported . In short, a custom- Live/Dead staining with calcein AM and ethidium
[22]
built MEW device was used to heat PCL to 90°C, creating homodimer was first performed in accordance with the
a polymer melt (heating system from TR 400, HKEtec, manufacturer’s instructions (Invitrogen) after 7 days of
Germany) that could then be fed through an electrically culture in chondrogenic media to evaluate encapsulated
charged 23G spinneret (power supply from Heinzinger pMSC cell viability. Cell viability was quantified using
Electronic GmbH, Rosenheim, Germany). PCL polymer ImageJ software as the number of live cells per total cells
melt fibers (~20 μm) were then layered in a 90° lay-down within images acquired via epifluorescence microscopy
pattern onto a computer-controlled translating collector (n ≥ 3 hydrogels, 9 images per sample).
(acceleration voltage = 5.5 kV, spinning gap = 3.3 mm, After culture in chondrogenic media for 28 days,
−1
E = 1.3 kV mm ) to obtain MEW scaffolds. Finally, disc- constructs were evaluated for biomechanical and
shaped scaffolds were extracted from initially fabricated biochemical properties. First, the compressive moduli of
sheets of MEW scaffolds using 4 mm biopsy punches. composites were determined via unconfined, uniaxial
NorHA was synthesized, as previously described, via compressive testing with a constant loading rate of 0.2 N/
benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium min (Q800 DMA, TA Instruments). The modulus was
hexafluorophosphate (BOP) coupling . Briefly, quantified as the slope of the stress–strain curves between
[23]
sodium hyaluronic acid was first converted into its 10% and 20% strain. Samples were then minced and
tetrabutylammonium salt form (HA-TBA), which was then digested via incubation with papain and hyaluronidase
dissolved in anhydrous dimethyl sulfoxide (DMSO). HA- overnight at 60°C . Sulfated glycosaminoglycan (sGAG)
[21]
TBA was then reacted with 5-norbornene-2-methylamine content, collagen (COL) content, and DNA content were
in the presence of BOP under inert nitrogen for 2 h at room determined via the dimethylmethylene blue assay, the
temperature. The reaction solution was then quenched hydroxyproline (OHP) assay (Abcam Hydroxyproline
with the addition of cold distilled water. To purify the Assay Kit, ab222941), and the Picogreen dsDNA assay,
crude product, the solution was dialyzed (8–10 total days), respectively . Separate samples were processed for
[24]

Volume 9 Issue 5 (2023) 495 https://doi.org/10.18063/ijb.775