2.12. Cartilage deposition in vitro in the engineered osteochondral plug

In this part, osteochondral scaffolds consisted of a cell-free bone and an ACPC-laden cartilage compartment. The bone-mimetic region was composed of a porous C-PCaP structure, (designed strand-to-strand distance = 0.7mm, diameter = 6.3mm, height of C-PCaP = 3mm), capped with a non-macroporous layer of C-PCaP struts, with an anchored microfibre mesh, prepared as described previously via combined printing. For the cartilage region, a 10% w/v GelMA hydrogel precursor solution in PBS was loaded with 2 · 10⁷ ACPCs ml⁻¹, and infused in the reinforcing microfibres linked to the C-PCaP structure. Cells were encapsulated at passage 4. To permit rapid cross-linking, the precursor solution was supplemented with a previously described visible-light responsive photoinitiator [51, 52], composed of 0.5mM tris (2,2′-bipyridyl) dichloro-ruthenium (II) hexahydrate (Sigma—Aldrich) and 5mM sodium persulfate (Sigma Aldrich), and exposed to a 1300 lumen white light lamp for 8 min. Samples were cultured in a chondrogenic medium, consisting of DMEM (Gibco, Life Technologies), supplemented with 1% v/v ITS + premix (BD biosciences), 0.2mM ASAP (Sigma Aldrich), 0.1 μM dexamethasone (Sigma Aldrich), 1% v/v HEPES, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin (Gibco, Life Technologies) and 10 ng ml⁻¹ of recombinant human transforming growth factor-β1 (TGF-β1, Peprotech). Samples were cultured for 6 weeks and harvested at two time points (day 1 and day 42) for subsequent analysis. Medium was refreshed every two days. Neo-cartilage formation in the cartilage-region of the engineered plugs, compared to the constructs composed of cell-laden reinforced GelMA only, was evaluated via immuno-histochemistry and biochemical analysis. The effect of the neo-synthesized matrix over the culture time on the mechanical strength of the interface between the bone and cartilage compartment was also assessed.

For biochemical evaluation, samples at week 1 (n = 3–6) and 6 (n = 5–14) of culture were harvested, and the chondral compartment was removed and with a razor blade and digested in papain (Papain from papaya latex, Sigma Aldrich) at 60 °C overnight. Sulphated glycosaminoglycan and DNA contents of the constructs were quantified performing a dimethylmethylene blue (DMMB, Sigma-Aldrich, The Netherlands) colorimetric assay and with a Quan-iT-Picogreen-dsDNA-kit assay (Molecular Probes, Invitrogen, Carlsbad, USA). For histological analysis, samples at day 42 (n = 3) were fixated in 4% buffered formalin. For paraffin embedding, samples were decalcified with 0.5M EDTA disodium salt for 1 day. Dehydration was performed through a graded ethanol series, followed by clearing in xylene, embedding in paraffin, and slicing into 5 μm thin sections with a microtome. Sections were stained with safranin-O and Fast Green to visualize GAGs and collagens. Immuno-histochemistry was performed to visualize collagen type I (primary antibody EPR7785, 0.0022 mg./ml., Abcam) and Collagen type II (primary antibody Col2AI II-II6313, 0.6 mg./ml., DSHB). Endogenous peroxidases were blocked via incubation with 0.3% v/v hydrogen peroxide. Antigen retrieval was performed with pronase and hyaluronidase for collagen type II and collagen type I, respectively, at 37 °C. Subsequently, sections were blocked with bovine serum albumin (BSA, 5% w/v in PBS) for 1 h at room temperature, and the primary antibody was incubated overnight at 4 °C. IgGs were used as negative controls. Horseradish peroxidase-labelled secondary antibodies were added for 1 h at room temperature, and the staining was developed using 3,3-diaminobenzidine. Nuclei were counterstained with haematoxylin and sections were mounted in DPX (Millipore).

For the osteochondral constructs, in order to visualize structure without removing the PCaP scaffold due to de-calcification steps, one formalin-fixed sample was dehydrated through a graded ethanol series and embedded in a methyl methacrylate (MMA) resin. Sections (300 μm thick) were obtained with a saw microtome (Leica SP 1600). Thereafter, all sections were stained with basic fuchsin to assess scaffold morphology. Histological slides were imaged using a light microscope (Olympus BX51, Olympus Nederland B.V.) equipped with a digital camera (Olympus DP73, Olympus Nederland B.V.).

At day 1 (n = 3) and 42 (n = 9), osteochondral structures were harvested and kept in medium to ensure hydration. To determine the strength of the connection at the interface between the cartilaginous compartment and the PCaP-based bone compartment, the same settings that were performed for cellfree structures was applied.