Technovit 7200 resin

The samples were fixed in 10% formalin and sequentially dehydrated in 80 to 100% ethyl alcohol, infiltrated, and embedded in Technovit 7200 resin (EXAKT, Germany). The resin was solidified with a polymerization system (EXAKT, Germany), the hardened resin blocks were sectioned by using a cutting system (EXAKT, Germany) to 200 μm thick slices, and the slices were ground to a thickness of 50 μm by using a grinding system (EXAKT, Germany). The ground slices were stained with hematoxylin and eosin and the stained bone formations in the scaffolds were observed with an optical microscope.

Chondral control and cut off neocartilage samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned along the short axis into 5 μm sections to expose the full thickness of the construct. Sections were stained with hematoxylin and eosin (H&E) to illustrate morphology, safranin O/fast green to show glycosaminoglycans (GAGs), and picrosirius red to visualize collagen. Immunohistochemistry was also performed to stain for collagen I (anti-collagen I antibody, polyclonal, rabbit, Abcam, ab34710, 1:250 dilution) and II (anti-collagen II antibody, polyclonal, rabbit, Abcam ab34712, 1:4000 dilution). Native sheep subchondral bone (rich in collagen I and void of collagen II) and articular cartilage (rich in collagen II and void of collagen I) were used as immunohistochemical control tissues. Anti-rabbit IgG secondary antibodies with an ABC-HRP peroxidase kit (Vector Laboratories) and DAB peroxidase (HRP) detection (Vector Laboratories) were used. Full thickness osteochondral samples were also fixed in 10% neutral buffered formalin, serially dehydrated in ethanol, and embedded in Technovit 7200 resin (Exakt). Sections were cut from the block and polished to 40 μm and stained with Toluidine Blue to show the neocartilage-ceramic osteochondral interface.

The samples were fixed in 10% formalin and sequentially dehydrated in 80 to 100% ethyl alcohol, infiltrated, and embedded in Technovit 7200 resin (EXAKT, Germany). The resin was solidified with a polymerization system (EXAKT, Germany), the hardened resin blocks were sectioned by using a cutting system (EXAKT, Germany) to 200 μm thick slices, and the slices were ground to a thickness of 50 μm by using a grinding system (EXAKT, Germany). The ground slices were stained with hematoxylin and eosin and the stained bone formations in the scaffolds were observed by using a microscope and a digital camera.