Table 1.

Overview of Publications on the (Bio)fabrication of (Articular) Cartilage Regenerative Constructs.

		Bio-Ink		(Bio)printed Constructs
Reference	Printing Technique	Material(s)	Cells and/or Biological Cues	Mechanical Properties	Tissue Formation	Remarks
Cohen et al. (2006)18	Extrusion bioprinting	Alginate	• Full thickness bovine articular chondrocytes	• Compressive modulus (unconfined) ~1.8 ± 0.1 kPa	In vitro • Viability, ~94% • Cartilage-like tissue formation similar in printed constructs as in casted alginate or alginate beads	• Printed meniscus and intervertebral disk shapes* • Demonstrated that viability and differentiation behavior of chondrocytes were not influenced by the printing procedure
Schuurman et al. (2012)61	Extrusion printing	Alginate reinforced with PCL	• C20A4 cell-line*	• Compressive modulus (unconfined) ~6000 kPa	In vitro • Viability, ~70-90%	• First demonstration of simultaneous deposition of thermoplastic polymer and hydrogel for the fabrication of reinforced constructs
Cui et al. (2012)19	Inkjet bioprinting	PEGDMA	• Full thickness human articular chondrocytes• TGF-β1 and FGF-2 in medium during culture	• Compressive modulus (unconfined) ~30-37 kPa	In vitro • More cell proliferation and more total cartilage-like tissue formation in constructs cultured with both TGF-β1 and FGF-2 after 4 weeks of differentiation	• Demonstrated that culturing printed constructs with both TGF-β1 and FGF-2 increases proliferation and on longer term (21 days) cartilage-like tissue formation
Cui et al. (2012)70	Inkjet bioprinting	PEGDMA	• Full thickness human articular chondrocytes	• Compressive modulus (unconfined) ~38-320 kPa	Ex vivo • Viability, ~89% • Cartilage-like tissue formation increased during the first 4 weeks of culture and remained stable after (6 weeks total) • Stable construct integration in the defect after 6 weeks	• Printing directly into defects in porcine osteochondral plugs • Demonstrated the importance of rapid gelation for maintaining initial cell distribution
Schuurman et al. (2013)17	Extrusion bioprinting	GelMA with hyaluronic acidPCL reinforcement*	• Full thickness equine articular chondrocytes• Hyaluronic acid	• Compressive modulus (unconfined) for gelMA constructs: ~10-175 kPa (dependent on concentration)	In vitro • Viability, ~73-83% • Similar cartilage-like tissue formation in constructs with and without hyaluronic acid after 4 weeks differentiation	• Addition of hyaluronic acid increased the printability
Boere et al. (2014)64	Extrusion printing	GelMA (cast) reinforced with pHMGCL/PCL (printed)	• Full thickness human articular chondrocytes	• Construct failure (unconfined compression) ~2.7 N (~7.7 N when covalent bonds between gelMA and pHMGCL/PCL)	In vitro • Covalent bonds between hydrogel and reinforcement to increase construct stability and mechanical strength In vivo • Rats, subcutaneous; more collagen type II but less GAGs after 8 weeks compared to in vitro study	• Interconnected cartilage-like tissue network after 6 weeks of differentiation
Kundu et al. (2015)83	Extrusion bioprinting	Alginate reinforced with PCL	• Nasal human chondrocytes• TGF-β	• Not reported	In vitro • Viability, ~85%• More cartilage-like tissue formation in constructs with TGF-β after 4 weeks of differentiation In vivo • Immunodeficient mice, subcutaneous; moderate cartilage-like tissue formation in constructs with TGF-β after 4 weeks	• Printed constructs with a cell and growth factor-laden bio-ink, reinforced with a printed thermoplastic polymer
Kesti et al. (2015)84	Extrusion bioprinting	HAMA with HA-pNIPAAM	• Full thickness bovine articular chondrocytes	• Not reported	In vitro • Viability, low when HA-pNIPAAM remains in the culture, high when HA-pNIPAAM was eluted	• HA-pNIPAAM supports printing and can be eluted from the final construct after printing and crosslinking
Kesti et al. (2015)39	Extrusion bioprinting	Gellan with alginate	• Full thickness bovine articular chondrocytes • Hydroxyapatite particles • Cartilage extracellular matrix particles	• Tensile modulus ~116-230 kPa	In vitro • Viability, ~80-96% (60% in the center of large constructs) • Cartilage extracellular matrix particles increased cartilage-like tissue formation during 8 weeks of differentiation. However, constructs cultured in TGF-β3 supplemented medium contained most cartilage matrix	• Printed ear, nose, meniscus, and vertebral disk shapes by using support structures*
Markstedt et al. (2015)85	Extrusion bioprinting	Nanofibrillated cellulose with alginate	• Nasal human chondrocytes	• Compressive modulus (unconfined) ~75-250 kPa (depending on the ratio of both components)	In vitro • Viability, ~73-86%	• Printed ear and meniscus shapes*
Visser et al. (2015)66	Melt electrospinning	Electrospun PCL infused with gelMA	• Human articular chondrocytes in gelMA• Hyaluronic acid• Mechanical stimulation (14 days without followed by 14 days with)	• Compressive modulus (unconfined) ~80-400 kPa for hybrid construct depending on mesh porosity• Similar stress/strain curve for gelMA reinforced with a 93% porous mesh as for native articular cartilage	In vitro • Viability, ~73-86% • Expression of chondrogenic genes increased in mechanical stimulated constructs during 4 weeks of culture. No differences were found at protein level	• With low content of thermoplastic polymer fibers (7%), mechanical properties could be achieved within the range of articular cartilage
Izadifar et al. (2016)86	Extrusion bioprinting	Alginate reinforced with PCL	• Full thickness embryonic chick chondrocytes (“rounded” and “fibroblastic” subpopulations)	• Not reported	In vitro • Viability, ~77-85% • More proliferation and cartilage-like tissue formation in constructs with the “fibroblastic” chondrocyte subpopulation compared to the “rounded’ subpopulation	• Printed constructs with a cell-laden bio-ink, reinforced with a printed thermoplastic polymer • Demonstrated rapid cooling of PCL strands after printing • Different chondrocyte subpopulations give differences in cartilage-like tissue formation
Abbadessa et al. (2016)87	Extrusion bioprinting	polyHPMA-lac-PEG	• Full thickness equine articular chondrocytes• HAMA or CSMA	• Compressive modulus (unconfined) ~13-16 kPa for all combinations	In vitro • Viability, ~85-95% (printed) • Similar levels of cartilage-like tissue formation in polyHPMA-lac-PEG hydrogels as in fibrin controls after 4 weeks of culture (cast)	• Incorporation of HAMA and to a lesser extent CSMA decreased the degradation rate and improved the thermosensitive profile and printability
Mouser et al. (2016)88	Extrusion bioprinting	gelMA with gellan gum	• Full thickness equine articular chondrocytes	• Compressive modulus (unconfined) ~2.7-186 kPa depending on concentration and ratio of gelMA and gellan gum	In vitro • All evaluated concentrations supported cartilage-like tissue formation during 6 weeks of culture. Relatively high gellan gum concentrations compromised chondrogenesis and high total polymer concentrations hampered matrix distribution	• Identified yield stress as dominant factor for bioprintability • Addition of gellan gum improved filament deposition, increased construct stiffness, and supported chondrogenesis

^*Proof of concept study.

PCL = polycaprolactone; PEGDMA = poly(ethylene) glycol dimethacrylate; TGF = transforming growth factor; FGF = fibroblast growth factor; GelMA = gelatin-methacryloyl; pHMGCL/PCL = poly(hydroxylmethylglycolide-co-ϵ-caprolactone)/poly(ϵ-caprolactone); HAMA = methacrylated hyaluronic acid; HA-pNIPAAM = poly(N-isopropylacrylamide) grafted hyaluronan; polyHPMA-lac-PEG = polyethylene glycol midblock flanked by two poly[N-(2-hydroxypropyl) methacrylamide mono/dilactate]; CSMA = methacrylated chondroitin sulfate; PEGT-PBT = poly(ethylene glycol)-terephthalate-poly(butylene terephthalate).