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3D Printing Osteochondral Scaffold
“living” engineered tissue or organ using bioink reservoir for growth factors which have a natural affinity
containing living cells, and thus has great potential in to ECM [40,41] . Transforming growth factor-beta (TGF-β)
regeneration medicine [17-20] . is regarded as a highly efficient chondrogenic factor .
[42]
3D bioprinting techniques can be classified into Bone morphogenetic protein-2 (BMP-2) plays a key
three distinct process categories: (i) Material extrusion , role in driving osteogenesis of BMSCs . Hence, the
[43]
[21]
(ii) material jetting , and (iii) vat polymerization combination of such a bioink with TGF-β and BMP-2 is
[22]
bioprinting . Of these, extrusion-based bioprinting effective to enhance osteochondral regeneration.
[23]
is the most prevalent employed research approach to Therefore, on the basis of our previous study, we
fabricate 3D cell-laden scaffolds due to its accessibility, employed dECM-SF bioink to fabricate 3D-printed
cost-effectiveness, and capacity to replicate tissue bilayered constructs. First, polycaprolactone (PCL) was
complexity . For extrusion-based bioprinting, various first extruded to print frame of bone layer, and the DBM
[21]
biomaterials, such as gelatin, HA, or alginate, have been bioink was printed to fill the space. The DCM bioink was
extensively used as bioink sources [24-28] . However, some used to print the cartilage layer on the bone layer. Next,
problems remain, such as the cell toxicity of the chemical we evaluated the mechanical strength and degradation
cross-link process, poor cell-material interactions, and rate of the two layers to confirm the properties of
inferior tissue formation [29,30] . In addition, only a small constructs. Furthermore, the delivery capacity of growth
percentage of cells in these materials could drive cell factors and the potential of constructs for chondrogenesis
differentiation towards target cell lineage [30,31] . Moreover, or osteogenesis were measured in vitro. Finally, we
these materials cannot represent the complexity of implanted bilayered constructs containing TGF-β1 and
extracellular matrices of repaired natural tissue. BMP-2 into the osteochondral defect and determined the
Therefore, there is an urgent need to develop a bioink that osteochondral regeneration efficacy in vivo.
is sufficient to create a tissue-specific microenvironment
with 3D cellular organization and cell-to-cell/cell-to- 2. Materials and methods
matrix communication that are typical of natural tissues. All experimental procedures involving animals have been
Decellularized extracellular matrix (dECM) has been
developed as bioink to fabricate 3D bioprinted tissues and approved and implemented in accordance with the animal
use guidelines outlined by the Medical College of Nanjing
organs [32,33] . Both biologically and functionally, dECM is Medical University (IACUC-2005033). All animal
more representative of the natural extracellular matrix subjects were treated in accordance with the National
(nECM) than other kinds of biomaterials. dECM provides Laboratory guidelines for Laboratory Animal Nursing.
a native-mimicking microenvironment for the migration,
proliferation, and differentiation of bone marrow-derived 2.1. Preparation of decellularized cartilage/
mesenchymal stem cells (BMSCs) [34,35] . Furthermore, bone ECM
BMSCs encapsulated in cartilage dECM (DCM) or
bone dECM (DBM) hydrogel can recognize and interact Decellularized DCM and DBM were prepared based
[37]
with surrounding matrix that specifically enhanced on our previously reported method . Briefly, articular
chondrogenic/osteogenic differentiation and tissue cartilage and cancellous bone segments were harvested
maturation . However, the mechanical strength of DCM from female goats (n = 12) within 6 h after sacrifice.
[36]
and DBM is insufficient because of the loss of cartilage/ These cartilage and bone segments were washed,
bone native tissue structure during the homogenization freeze-dried, and immersed in liquid nitrogen and cut
3
and solubilization process [36,37]. Silk fibroin (SF) is a into small pieces (1~2 mm ). The cartilage pieces were
natural biopolymer that is widely investigated for various rinsed with phosphate-buffered saline (PBS), while the
3D bioprinting and tissue engineering applications due to bone pieces were demineralized using an adaptation of
its remarkable mechanical properties, biocompatibility previously reported methods by submerging in 0.5 M
and biodegradation nature [38,39] . In our previous study, we hydrochloric (HCL) under agitation for 24 h, and then
reported the use of a cross-linker-free DCM-SF bioink degreased with 1:1 mixture of chloroform and methanol
in printing 3D construct which had similar mechanical for 2–3 h . Cartilage and demineralized bone pieces
[44]
properties compared with native cartilage tissue . were washed thoroughly with PBS and lyophilized
[37]
Since dECM-based bioink most likely retain before decellularization. The cartilage and bone pieces
endogenous growth factors than other kinds of bioink, it were homogenized, milled, and soaked in PBS containing
will lead to enhancement of osteochondral regeneration 0.1% w/v ethylenediaminetetraacetic acid (EDTA;
that incorporates additional exogenous growth factors Sigma-Aldrich, St. Louis, MO, USA) and 3.5% w/v
in dECM bioink. The previous studies have shown that phenylmethyl sulfonylfluoride (PMSF; Beyotime,
dECM acts as an excellent growth factor delivery system Shanghai, China) for 24 h to inhibit protease activity.
since the extracellular matrix (ECM) itself is a natural These cartilage and bone granules were treated with a 1%

86 International Journal of Bioprinting (2021)–Volume 7, Issue 4