Table 1.
Summaries of the Technologies and Properties of the Bioprinted Constructs for Cartilage Tissue Engineering.
| Study | Materials | Cell Source | Animal Model | Mechanical Reinforcement | Compressive Modulus | Zonal Structure | In Vitro | In Vivo | |
|---|---|---|---|---|---|---|---|---|---|
| Scaffold-based bioprinting (EBB) | Daly et al. (2016) | Agarose, alginate, GelMA, and PEGMA | MSC | — | Extruded PCL fibers | Without PCL: <40 kPa, with PCL: ~2-3 MPa | — | ● MSC-laden alginate hydrogels stained intensely for sulfated GAG and COL-II. GelMA and PEGMA stained stronger for COL-I ● High levels of cell viability were observed in all bioinks postprinting ● Alginate and agarose hydrogels best supported the development of hyaline-like cartilage, and GelMA and PEGMA supported the development of a more fibrocartilage-like tissue. |
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| Markstedt et al. (2015) | NFC and alginate | hNC | — | — | ~35 kPa | — | ● Cell viability was 73% and 86% after 1 and 7 days of printing. | — | |
| Nguyen et al. (2017) | NFC, alginate and HA | iPSCs and chondrocytes | — | — | — | — | ● Low proliferation and phenotypic changes away from pluripotency were seen in NFC/HA. ● In 3D-bioprinted NFC/A constructs, pluripotency was initially maintained, and hyaline-like cartilaginous tissue with COL-II expression and lacking tumorigenic Oct4 expression was observed after 5 weeks. ● A marked increase in cell number within the cartilaginous tissue was detected. |
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| Mouser et al. (2016) | GelMa-GG | Primary equine chondrocytes | — | — | ~3-200 kPa | — | ● The addition of GG supported chondrogenesis, evidenced by presence of GAGs. ● High GG concentrations compromised cartilage matrix production and distribution, and even higher concentrations resulted in cell encapsulation. |
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| Abbadessa et al. (2016) | M15P10 and CSMA | ATDC5 cells | — | — | ~8-60 kPa | — | ● Embedded cells remained viable and proliferating over a culture period of 6 days. | — | |
| Abbadessa et al. (2016) | M10P10, CSMA and HAMA | Primary equine chondrocytes | — | — | ~15 kPa | — | ● The cell-laden hydrogel supported expression of proteoglycans, COL-II and VI for 42 days culture. | — | |
| Mouser et al. (2017) | polyHPMA-lac-PEG, HAMA, and PCL | Primary equine chondrocytes | — | Extruded PCL fibers | Without PCL: ~15-30 kPA, withPCL: ~4-8 MPa | — | ● GAG and COL-II content increased with HAMA concentrations (0.25%–0.5%) compared with HAMA-free hydrogels ● A relatively high HAMA concentration (1%) resulted in increased fibrocartilage formation. ● The polyHPMA-lac-PEG hydrogels with 0.5% HAMA was optimal for cartilage-like tissue formation. |
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| Constantini et al. (2016) | GelMA, CS-AEMA, and HAMA | BM-MSCs | — | — | ~50-100 kPa | — | ● Cell viability was ~90% for all the hydrogels ● Hydrogel with alginate, GelMA and CS-AEMA was the best candidate in neocartilage formation with the highest COL-II/COL-I and COL-II/COL-X ratios. ● Addition of HAMA favored the differentiation toward hypertrophic cartilage. |
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| Kundu et al. (2015) | PCL and alginate | Human chondrocytes | Mouse | Extruded PCL fibers | — | — | ● PCL-alginate gels with TGFβ showed higher ECM formation (GAG and total collagen content). | ● Implants after 4 weeks revealed enhanced cartilage tissue (GAG) and COL-II fibril formation in the PCL-alginate gel with TGFβ | |
| Izadifar et al. (2016) | PCL and alginate | Embryonic chick chondrocytes and ATDC5 cell line | — | Extruded PCL fibers | — | — | ● Rounded cells had higher COL-II mRNA levels than the fibroblastic cells, while fibroblastic cells had higher COL-II mRNA levels than the rounded cells after biofabrication ● Rounded and fibroblastic cells demonstrated high ● viability in hybrid constructs ● Fibroblastic cells had higher proliferation than rounded ● cells in hybrid constructs ● Fibroblastic cells produced more Alcian blue–stained matrix than the rounded cells |
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| Olubamiji et al.(2017) | PCL and alginate | ATDC5 cell line | Mouse | Extruded PCL fibers | — | — | — | ● Cells within the cartilage constructs remained viable over 21 days postimplantation. ● Progressive secretion of cartilage matrix in implanted cartilage constructs (sulfated GAG and COLII) over 21 days ● SR-inline-PCI-CT enabled noninvasive visualization of the individual components of cartilage constructs, surrounding host tissues and their structural changes postimplantation. |
|
| Yang et al. (2018) | COL-I, agarose and alginate | Rat primary chondrocytes | — | — | ~25-70 kPa | — | ● SA/COL facilitated cell adhesion, accelerated cell proliferation and enhanced the expression of cartilage specific genes (Aggeacan, COL-Il, and Sox9). ● Lower expression of COL-I was present in SA/COL group than SA and SA/AG groups, indicating that SA/COL suppressed dedifferentiation of chondrocytes and preserved the phenotype. |
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| Scaffold-based bioprinting (DBB) | Cui et al. (2012) | PEGDMA | Human articular chondrocytes | — | — | ~40-90 kPa | — | ● The presence of TGFβ1 is essential for the induction or maintenance of the chondrocyte phenotype (COL-II and aggrecan). ● Pre-culture or co-culture with FGF-2 to stimulate cell proliferation does not interfere with the chondrogenic effect of TGF-β1. ● FGF-2/TGF-β1 treated constructs showed overall higher amount of proteoglycan content ● ECM production per chondrocyte in low cell density was much higher than that in high cell seeding density. |
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| Cui et al. (2012) | PEGDMA | Human articular chondrocytes | — | — | ~300-500 kPa | — | ● Viability of printed chondrocytes increased 26% in simultaneous polymerization than polymerized after printing. ● Printed construct attached firmly with surrounding tissue and showed greater proteoglycan deposition at the interface of implant and native cartilage. ● Printed cartilage in 3D OC plugs had elevated GAG content comparing to that without OC plugs. |
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| Gao et al. (2015) | PEGDMA and GelMA | BM-MSCs | — | — | ~40-60 kPa | — | ● The procedure showed a good biocompatibility ● Gene expression analysis showed both osteogenic and chondrogenic differentiation was improved by PEG-GelMA in comparison with PEG alone. |
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| Gao et al. (2015) | Peptide-conjugated PEG | BM-MSCs | — | — | ~30-70 kPa | — | ● Cell viability of >85% ● The bioprinted bone and cartilage tissue demonstrated excellent mineral and cartilage matrix deposition with osteogenic and chondrogenic differentiation ● Bioprinted PEG-peptide scaffold inhibited hMSC hypertrophy during chondrogenic differentiation |
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| Gao et al. (2017) | PEGDA | BM-MSCs | Mouse | — | ~75-100 kPa | — | NR2F2 overexpressed MSCs showed significantly enhanced chondrogenesis in monolayer, 3D pellet, and hypoxia cultures | ● Vascularized tissue membrane was formed surrounding the constructs ● More proteoglycan deposition was found in scaffold using NR2F2 overexpressed cells comparing with the control group. |
|
| Xu et al. (2013) | Collagen, fibrinogen and PCL | Rabbit articular chondrocytes | Mouse | Electrospun PCL fibers | ~1.8 MPa | Alternant hydrogel and electrospun layers | ● Cell viability >80% one week after printing ● Constructs formed cartilage-like tissues as evidenced by the deposition of COL-II and GAG |
● Dense and well-organized collagen formation, GAG and COL-II production were observed after 8 weeks of implantation | |
| Scaffold-based bioprinting (LBB) | Zhu et al. (2018) | GelMA, PEGDA and PLGA | BM-MSCs | — | — | ~1-18 MPa | — | ● Cells grown on 5%/10% (PEGDA/GelMA) hydrogel present the highest cell viability and proliferation rate. ● The TGF-β1 embedded in nanospheres can keep a sustained release up to 21 d and improve chondrogenic differentiation. |
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| Scaffold-based bioprinting (zonally stratified arrangement) | Levato et al. (2014) | PLA and GelMA-GG | MSC | — | PLA microcarriers | ~30-50 kPa | Cartilage region and bone region | ● Microcarrier encapsulation facilitated cell adhesion and supported osteogenic differentiation and bone matrix deposition by MSCs. ● Microcarrier-cell complexes displayed a high viability after the automated printing process |
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| Levato et al. (2017) | GelMA and pluronic F-127 | ACPCs, MSCs and chondrocytes | — | — | ~100-190 kPa | ACPC-laden and MSC-laden zones | ● ACPCs outperformed chondrocytes in terms of sulphated GAG ● MSCs produced significantly more sulphated GAG and had higher gene expression of COL-II and aggrecan than both ACPCs and chondrocytes. ● ACPCs had the lowest gene expression levels of COL-X, and the highest expression of PRG4 ● MSC/ACPC co-cultured matrices displayed the highest overall sulphated GAG concentration than MSC/chondrocytes and ACPC/chondrocytes. ● In bioprinted zonal-like constructs, different distributions of sulphated GAG, COL-I and II were observed in ACPC- and MSC-laden zones |
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| Ren et al. (2016) | COL-II | Chondrocytes from New Zealand white rabbits | — | — | — | Cell density gradient | ● Gradient cell distribution patterns were established and maintained ● Cell viability was >90% ● GAG content was positively correlated with the total cell density ● Cellular biosynthetic ability was affected by both the total cell density and the cell distribution pattern. |
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| Shim et al. (2016) | Atelocollagen, HA and PCL | Human mesenchymal stromal cells | Rabbits | Extruded PCL fibers | — | Subchondral bone layer and superficial cartilage layer | ● Cell viability was ~90% ● The cells in the atelocollagen hydrogel layer exhibited increases in ALP, COL-I, and OSX genes ● The cells in the CB[6]/DAH-HA hydrogel exhibited increases in ACAN, COL-II, and SOX9 genes ● Cells in atelocollagen showed Runx2 expression and calcium deposition, and cells cultured in CB[6]/DAH-HA showed COL-II and GAG deposition. |
● The defect treated by layered scaffold was covered with neotissue without fiber exposure and exhibited a smooth surface. ● Layered scaffold showed a remarkable capability for the osteochondral regeneration at week 8. The newly regenerated cartilage tissues were smoothly integrated with ends of the host cartilage tissue. ● GAG, COL-II and X were strongly expressed in only layered scaffold. |
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| Scaffold-free bioprinting | Yu et al. (2016) | Cell ink | Primary cattle chondrocytes | — | — | ~1-5 MPa | — | ● Cell viability in strands was > 85% ● Tissue strands showed slightly higher proteoglycan production than native cartilage, and significant amount of COL II and aggrecan ● Printed tissue also had a significant amount of proteoglycan formation and the interface of each tissue strand was well integrated. ● High sulfated GAG content and cell density, and chondrocytes with rounded morphology were observed in printed construct ● In a bovine osteochondral model, the explants tissue adhered to the defect, stayed intact and exhibited proteoglycan-rich ECM. |
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| In situ bioprinting | O’Connell et al. (2016) | GelMA/HAMA | Human infrapatellar fat pad derived adipose stem cells | — | — | — | — | ● Cell viability was >97% | — |
| Duchi et al. (2017) | GelMA/HAMA | Sheep ADSCs | — | — | ~9-380 kPa | — | ● UV light exposure at 700 mW/cm2 did not significantly affect cell viability compared with the untreated cells ● Cells printed by coaxial configuration were observed to retain viability and proliferative capability ● The mono-axial bioprinting configuration shows a viability decrease by 30% compared with coaxial printing. |
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| Di Bella et al. (2017) | GelMA/HAMA | Sheep MSCs | Sheep | — | ~0.5 MPa | — | — | ● There was better overall macroscopic appearance in the handheld printed group compared with control groups. ● Handheld printed construct showed a higher amount of newly regenerated cartilage with chondrocytes columnar alignment, and the absence of subchondral bone deformation or collapse ● Handheld printed construct showed positive Safranin O and COL-II staining. |
Note: GelMA = gelatin-metacryloyl; PEGMA = poly(ethylene glycol) methyl ether methacrylate; MSC = mesenchymal stem cell; PCL = polycaprolactone; GAG = glycosaminoglycans; COL-I, II, VI and X = collagen types; NFC = nanofibrillated cellulose; hNC = human nasoseptal chondrocytes; HA = hyaluronic acid; iPSCs = induced pluripotent stem cells; NFC/A = nanofibrillated cellulose/alginate; GG = gellan gum; CSMA = methacrylated chondroitin sulfate; HAMA = methacrylated hyaluronic acid; PEG = polyethylene glycol; CS-AEMA = chondroitin sulfate amino ethyl methacrylate; BM-MSCs = bone marrow-derived mesenchymal stem cells; TGF-β = transforming growth factor-β; ECM = extracellular matrix; SR-inline-PCI-CT = synchrotron radiation inline phase contrast imaging computed tomography; SA = sodium alginate; AG = agarose; PEGDMA = poly(ethylene glycol) dimethacrylate; FGF-2 = fibroblast growth factor-2; OC = osteochondral; PEGDA = polyethylene glycol diacrylate; NR2F2 = nuclear receptor subfamily 2 group F member 2; PLGA = poly(lactic-co-glycolic acid); PLA = polylactic acid; ACPCs = articular cartilage-resident chondroprogenitor cells; PRG4 = proteoglycan 4; ALP = alkaline phosphatase; Osx = osterix; CB[6]-HA = cucurbit[6]uril-conjugated hyaluronic acid; ACAN = aggrecan; Runx2 = runt-related transcription factor 2; ADSCs = adipose-derived stem cells; UV = ultraviolet.