Page 504 - IJB-9-5

International Journal of Bioprinting Implantation of composites for cartilage repair

histology through fixation (10% buffered formalin), was evaluated in lieu of the medial distal defects in all the
paraffin embedding, and sectioning (5 µm). Alcian performed analyses. To fix composites within defects using
blue staining (1%, pH 1.0, Newcomer Supply) and fibrin glue (Tisseel), the fibrin glue was first applied to the
immunohistochemistry for type I collagen (COL I, mouse underlying subchondral bone, the composites were press-
monoclonal anti-collagen type I antibody, Millipore fit into the defects and manually held in place for 3 min via
Sigma) and type II collagen (COL II, mouse monoclonal application of force with a spatula and a surgical curette,
anti-collagen type II antibody, Developmental Studies and additional fibrin glue was then applied along the top of
Hybridoma Bank) were then performed as previously the composite surface.
described . The mean staining intensities of sGAG, COL After composite fixation, the patella was relocated, all
[21]
I, and COL II in composites were quantified using ImageJ instruments and retractors were removed, and the knee
software . was then ranged to ensure that the patella was stable. The
[25]
2.4. Animal procedures and stifle joint surgery joint capsule was then closed with 0 Vicryl interrupted
Skeletally mature (12–14-month-old at time of surgery) sutures, the subcutaneous tissue layer was closed with 2-0
castrated male Yucatan minipigs were acquired (Sinclair Vicryl simple interrupted sutures, and the skin layer was
Bioresources, Auxvasse, MO) and used to evaluate closed with a 3-0 monocryl running suture (all sutures
surgical fixation methods for the implantation of MEW- were procured from Ethicon, Raritan, NJ). All animals
NorHA composites in cartilage defects in vivo. All animal received post-operative analgesia, antibiotics, and anti-
procedures were approved by the Institutional Animal inflammatories, with unrestricted cage activity permitted
Care and Use Committee (IACUC) at the University 2 to 3 h after recovery from anesthesia.
of Pennsylvania. Unilateral stifle joint surgeries were
performed on the right hind limb of each animal as 2.5. Arthroscopy, micro-CT, and histological
previously described [16,26,27] . Briefly, four full-thickness evaluation of cartilage defects
chondral defects were created in the trochlear groove (two Twelve weeks after cartilage defect creation, animals were
proximal and distal medial defects and two proximal and euthanized, and the stifle joints were retrieved for post-
distal lateral defects) using a 4 mm biopsy punch and a mortem analyses. Dry arthroscopy was first performed
curette to excise cartilage within the bounds of the scored to visualize the cartilage defects in situ using an adapted
defect while ensuring the underlying subchondral bone was protocol . A 1 cm vertical incision was made to establish
[16]
not damaged. Nine total animals were used (some defects a medial subpatellar arthroscopic portal, which allowed for
were part of an alternate study). In two animals, post- the placement of a trocar and arthroscopic probe within
operative lateral patellar luxation was observed 3 weeks the medial aspect of the stifle joint. Images of each defect
after surgery, and these were excluded from the study. were then taken to qualitatively evaluate the retention of
Luxation was likely due to recovery-related complications pinned or glued composites, the smoothness of formed
and/or patellar luxation accompanied by urticaria and repair cartilage, and the integration of repair cartilage with
incisional dehiscence consistent with a previously reported the surrounding tissue . The stifle joint was dissected,
[29]
case of suture hypersensitivity in a Yucatan minipig . and cartilage defects along the trochlear groove were
[28]
macroscopically assessed to qualitatively determine the
Four groups (n = 4–6 implants/group) were assessed,
including acellular composites or composites containing retention of implants and the quality of repair cartilage
formed in defects .
[30]
pMSCs that were precultured for 28 days and implanted
with either poly(L-lactide-co-D,L-lactide) (PLDLLA) To visualize any subchondral bone remodeling or
pins (Aesculap FR736, Center Valley, PA) or fibrin glue bone resorption that occurred during the 12-week course,
(Tisseel, Baxter), as previously reported . Composites explanted cartilage defects (and healthy tissue controls)
[16]
were pinned by press-fitting into the defects, creating a were imaged via micro-CT as previously described .
[31]
pilot hole through the implant and into the subchondral Osteochondral samples were incubated in Lugol’s
bone, placing a 3-pronged fixation guide (Aesculap FR720, solution overnight at room temperature and then imaged
Center Valley, PA) on top of the composites, and then using a Scanco MicroCT 45 system (Scanco Medical,
inserting the pins into the pilot holes. In two animals, an Southeastern, PA; exposure: 600 ms, voltage: 55 kVp,
additional fifth defect was introduced on the lateral side isotropic voxel size: 10 µm), with cross-sectional and top-
of the femoral trochlea to replace medial distal defects down images of samples acquired via DragonFly software
in which insufficient fixation of implants with pins was (Object Research Systems, Montreal, Canada). After
initially achieved (i.e., poor seating of composites within micro-CT imaging, samples were fixed (10% formalin,
the created defect and misaligned pinning at the time of 24–48 h incubation overnight at 4°C) and decalcified via
fixation). Each of these additional, lateral distal defects incubation in Formical-2000 for 4 weeks (solution changed

Volume 9 Issue 5 (2023) 496 https://doi.org/10.18063/ijb.775