CARTILAGE 3D PRINTING - 3D Bioprinting and Nanotechnology in Tissue Engineering and Regenerative Medicine - page 264

The Bioplotter, a device previously described by Moroni et al. ( Moroni et al., 2006 ) was used to
extrude Poly[(ethylene oxide) terephthalate-co-poly(butylene) terephthalate] (PEOT/PBT) 3D fibers
for cartilage regeneration ( Moroni et al., 2008 ). The Bioplotter extruded the highly viscous polymeric
fibers layer by layer to construct a rectangular porous block. Each fiber was approximately 300 m m
thick, fiber spacing was 800 m m, and the layer height was 225 m m. Bovine chondrocytes were seeded
into 4 mm high scaffolds and a cartilaginous matrix with a Safranin O positive stain was deposited over
21 days. Scanning electron micrograph studies also revealed an uneven distribution of cells throughout
the scaffold, which is one of the major issues that confront prefabricated scaffold systems ( Carletti
et al., 2011; Wendt et al., 2006 ).
Simultaneous printing of cells and scaffold, achieved by bioprinting, has several advantages. 3D
bioprinting of cartilage and bone tissue engineering was described by Fedorovich et al. ( Fedorovich
et al., 2012 ), who used a pneumatic dispensing system (the BioScaffolder) to deposit human chondro-
cytes and bone marrow-derived mesenchymal stem cells (MSCs) in alginate into various patterns up to
10 layers thick (0.8 mm high). The alginate encapsulating MSCs were also supplemented with osteoin-
ductive materials to promote osteogenic differentiation. In vitro culture generated evidence of cartilage
formation (deposition of collagens type II and VI) and a bone-like phenotype (collagen type I, alkaline
phosphatase, osteonectin, and Alizarin red positive). In vivo implantation over 6 weeks in nude mice
resulted in limited bone formation perhaps due to the confining nature of alginate.
12.1.5 DIRECT IN SITU PRINTING
The vast majority of 3D printing techniques are directed at engineering tissue in vitro with the objec-
tive of subsequent implantation into the joint. One attractive alternative is printing engineered tissue
directly into the human body. Cohen et al. demonstrated proof of concept by delivering alginate and
demineralized bone matrix via syringe extrusion into surgically created articular defects ( Cohen
et al., 2010 ). CT scans of a cadaver bovine femur were obtained to extract the geometry of the defect.
For repair of a chondral defect, precrosslinked alginate hydrogel was extruded through a syringe. For
repair of an osteochondral defect, a paste made of demineralized bone matrix mixed in gelatin was
first extruded into the bony defect followed by alginate extrusion layered over the demineralized bone
matrix. Although no cells were involved and the femur had been devitalized, this report demonstrated
proof of concept of the potential for additive manufacturing for direct in situ printing.
Proof of concept for in situ 3D printing of live human articular chondrocytes for cartilage repair
was demonstrated by Cui et al. ( Cui et al., 2012b ). A modified thermal ink-jet printer deposited chon-
drocytes suspended in photocrosslinkable poly(ethylene) glycol dimethacrylate (PEGDMA) into a car-
tilage defect created within a bovine osteochondral (cartilage and bone) tissue explant for cartilage
repair. This study demonstrated the utility of direct cartilage repair and bioprinting by successfully
controlling the placement of individual cells with high viability, by providing a supportive environment
for secretion of cartilage ECM protein, by printing material with adequate mechanical properties, and
by generating tissue that integrated directly with the host tissue.
Poly(ethylene glycol) (PEG) macromers are biocompatible and water soluble with low viscos-
ity making them ideal for bioprinting. Cross-linking PEG generates a compressive modulus that
matches that of human cartilage ( Bryant et al., 2004 ). Most importantly, PEG has also been shown
to maintain chondrocyte viability and support the generation of cartilaginous ECMs including
the essential collagen type II and GAGs ( Bryant and Anseth, 2002; Elisseeff et al., 2000 ). In the