Fig 1.
Overview of optimized decellularization procedure used in study.
Harvested cartilage plugs were frozen at -20°C then subjected to two dry FTCs followed by two wet FTCs with hypotonic buffer. Plugs were then incubated in multiwell plates on an orbital shaker with high frequency agitation (220 RPM) for the remaining steps. Samples were submerged in a 0.125 U/ml chABC solution to digest GAGs. This was followed by three wash cycles of alternating hypotonic buffer and 0.1% (w/v) SDS detergent to increase cell membrane permeability and promote cell lysis. Finally, DNase and RNase were added to degrade nuclear material.
Fig 2.
Overview of recellularization procedure.
Three channels were created in the decellularized cartilage scaffolds. The cells were pipetted onto the scaffolds in two steps. For the first step, half of the cell suspension was pipetted onto the deep zone (DZ) surface and the samples were centrifuged. A syringe was used to create a vacuum in the centrifuge tubes to maximize seeding. For the second step, the remaining cell suspension was pipetted onto the superficial zone (SZ) surface. Samples were cultured in serum-free chondrogenic medium.
Fig 3.
Safranin-O and Fast Green staining of longitudinal cross sections of decellularized cartilage.
Compared to native cartilage (Ntv), both groups of decellularized cartilage had significantly less proteoglycan staining, with more proteoglycan removal in the chABC-treated scaffolds compared to the PBS controls. Scale bar = 100 μm and magnification = 100X.
Fig 4.
H&E staining of longitudinal cross sections of decellularized cartilage.
Compared to native cartilage (Ntv), both groups of decellularized cartilage contained less cellular material in the superficial, middle, and deep zones. The PBS group had slightly more residual nucleic material than the chABC group, especially in the deep zone (G-I). Scale bar = 100 μm and magnification = 100X.
Fig 5.
Biochemical content and mechanical properties of decellularized cartilage.
(A) dsDNA/WW, as quantified by the PicoGreen assay. The decellularization procedure significantly reduced dsDNA/WW from native cartilage (Ntv), with no significant difference between the PBS and chABC groups. (B) GAG/WW, as quantified by the DMMB assay. Both decellularized groups had significantly lower GAG compared to native cartilage. (C) Collagen/WW, as quantified by the hydroxyproline assay. Collagen content was maintained after both decellularization procedures. (D) Equilibrium modulus, represented as percent of pre-decellularization equilibrium modulus. Both groups showed a decrease in equilibrium modulus after decellularization. Data is plotted as arithmetic mean ± SEM; asterisks denote significant differences from native cartilage (p<0.05; n = 5–13).
Table 1.
Mean Biochemical and Mechanical Properties of Decellularized Cartilage as Percentages of Native Values.
Fig 6.
Picrosirius Red staining of longitudinal cross sections of decellularized cartilage, viewed with cross-polarized light.
Regions of high collagen fiber alignment appear with greater intensity. Collagen fiber alignment in the superficial (A-C) and deep (D-F) zones was maintained after decellularization. Scale bar = 100 μm and magnification = 100X.
Fig 7.
Summary of Recellularization Results.
(A-N) Fluorescent labeling of seeded SDSCs. Seeded SDSCs were labeled with red DiI and all cells (seeded and native) were labeled with blue DAPI; fluorescent images were superimposed onto differential interference contrast images to more easily view infiltration. (A-F) Within 6 days, SDSCs attached to all outer surfaces, with some infiltration into the deep zone. (G-L) After 28 days, the outer cell layers grew thicker and there were generally more cells in the deep zone. (M-N) SDSCs were found throughout the lengths of the channels (channel borders indicated by small white arrows). (O) dsDNA/WW after 28 days in culture, as quantified by the PicoGreen assay. The seeded groups had significantly more dsDNA/WW compared to their respective unseeded controls, with no significant difference between the seeded PBS and chABC groups. For histology, scale bar = 100 μm and magnification = 200X for superficial and middle zones and 100X for circumference and channels. For biochemistry, data is plotted as arithmetic mean ± SEM; asterisks denote significant differences between groups (p<0.05; n = 6–10).