Page 481 - IJB-9-6

International Journal of Bioprinting Bioprinting cell-laden protein-based hydrogel

demonstrating the bone matrix were seen 3 weeks post- integrating well with the nearby cartilage, and showed
implantation [117] . Another study in the field of cartilage high levels of glycosaminoglycans (GAGs) distribution
TE evaluated the degradation of polyethylene glycol compared to the non-porous scaffold-treated group having
diacrylate (PEGDA)/gelatin/silk methacrylate (SilMA) severe irregularity in the articular surface and defect
bioink (crosslinking approach: photo-crosslinking with lesions with centers that were not regenerated 4 weeks post-
0.2 w/v LAP at 405 nm wavelength, light-emitting diode implantation [121] . Multiple evaluations have revealed that
(LED) light intensity 1000 mW/cm, and exposure time of in vivo printed scaffolds with pore sizes of approximately
20 s) (6%, 9%, and 3% w/v) encapsulating primary porcine 300 μm promote osteogenesis because of their higher
chondrocytes (cell density: 2 × 10 cells/mL) printed permeability and capacity for vascularization; on the other
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using extrusion-based printing and reported that delay hand, smaller pore sizes around 100 μm are more favorable
in the degradation rate was seen after adding PEGDA to for chondrogenesis [122] . Moreover, a porosity gradient
the composition, which was suitable for the load-bearing in the radial direction can be found in the structure of
cartilage repair. Specifically, 90% degradation and minimal bone, in which the mean porosity is enhanced from the
degradation of the printed hydrogel were observed after 28 cortical bone toward the trabecular bone [123,124] . To mimic
days when incubating in protease enzyme and phosphate- this unique structure, a novel experiment employed DLP-
buffered saline solution, respectively [118] . In the case of based bioprinting system including a microfluidic mixer
bone regeneration, an innovative experiment on the digital chip to print hMSCs-loaded 10% wt GelMA bioinks mixed
light processing (DLP)-based bioprinting of MC3T3-E1 with 10% wt GelMA solution comprising the porogen
preosteoblasts-encapsulated SilMA bioinks (crosslinking (crosslinking approach: photo-crosslinking with 2.20 × 10
-3
approach: photo-crosslinking with 0.2% wt LAP at 405 M Tris(2,2-bipyridyl) dichlororuthenium (II) hexahydrate
nm wavelength, visible blue light, and exposure time of 13 [Ru]/sodium persulfate [SPS] at 450 nm wavelength, blue
s for each layer) (cell density: 2 × 10 cells/mL) revealed light, and exposure time of 30 or 60 s for each layer) (cell
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that between three groups of 10%, 15%, and 25% w/v density: 2 × 10 cells/mL). The final printed construct
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SilMA scaffolds with degradation percentages of 91.0 ± was three gradual zones featuring various pore sizes (12,
2.27%, 64.8 ± 3.2%, and 48.6 ± 2.15% at 21 days, the 15% 29, and 65 µm). Further tests illustrated the improved
SilMA construct was the most efficient among the others spreading of the encapsulated cells within the portions with
in supporting the proliferation and attachment of the larger pore sizes 7 days post-bioprinting. In comparison
embedded cells [119] . with the hydrogel segment that was mixed with 0.5% wt
porogen, the cell cluster sizes were promoted to 2.5-fold
3.1.3. Porosity-related parameters and 4-fold in the gel regions containing 1.5% and 3.0% wt
Porosity-related parameters associated with bioprinted porogen, respectively. Thereafter, bone morphogenetic
protein-based structures can affect in vitro cellular protein-2 (BMP-2) was integrated within the bioink in
behaviors, in addition to in vivo tissue development [120] . order to enhance the hMSCs’ osteogenesis. The growth of
Specifically, the presence of a porous structure into a MSCs, as cell clusters, filled in the pore areas and enhanced
printed scaffold is necessary for the diffusion of nutrients, cell proliferation in portions having higher porogen
cellular viability, cell migration, and proliferation, as well concentration were observed, and improved expression
as in vivo tissue regeneration [121] . Utilizing cell-laden of runt-related transcription factor 2 (RUNX2) (an
porcine tendon-derived collagen bioinks (1%, 3%, and osteoprogenitor in early stages) in the regions with larger
5% wt), porous collagen scaffolds crosslinked via genipin pores and higher GF concentrations was confirmed [125] .
were printed and compared with non-porous ones, both
fabricated via an extrusion printing system. Mostly live 3.1.4. Crosslinking process
rabbit articular chondrocytes (cell density: 1 × 10 cells/ The crosslinking process is another biophysical cue
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mL) were found in the porous constructs’ cross-sectional influencing the behaviors of encapsulated cells in the
live and dead image, whereas dead cells were mostly PBHs. For crosslinking of PBHs, physical, chemical, and
present in the non-porous scaffolds’ core after 7 days of enzymatic crosslinking approaches can be employed.
cultivation. Indeed, the death of cells could be owing to Importantly, the crosslinker itself, its concentration, and
the restricted nutrition penetrability of the collagen and the time of crosslinking affect the mechanical features of
the lack of a porous structure to compensate for this printed constructs and embedded cell properties [126-128] .
limitation. Notably, female New Zealand White rabbits Physical crosslinking involves using temperature or pH to
with osteochondral defects were used as animal models, form reversible interactions within the protein structure,
and the porous bioprinted collagen scaffold-treated group influencing cell viability due to the sensitivity of cells to
displayed significantly improved in vivo regeneration changes in temperature and pH. Furthermore, chemical
of cartilage, possessed newly-formed hyaline cartilage crosslinking utilizes chemical agents to create covalent

Volume 9 Issue 6 (2023) 473 https://doi.org/10.36922/ijb.1089