Page 17 - IJB-9-1

International Journal of Bioprinting Biocompatible materials and Multi Jet Fusion

thinning behavior [51,52] . The CCNC concentration of 1.4% is cell viability within the bioprinted construct was observed
optimal for preventing tears and clogs with higher values of at all time points. However, the decrease in cell count
the storage modulus and maintaining print integrity. From indicates that chondrocytes do not proliferate inside the
the rheological analysis, the selected bioink formulation is construct, which is contrary to other research conducted
4.0% gelatin, 0.75% alginate, and 1.4% CCNC. on bioink composed of alginate and nanofibrillated
[60]
We have formulated a printable bioink with the lowest cellulose . In the future, the identification of proliferation
shear stress and the highest printing accuracy by selecting markers, like Ki-67, should be carried out to prove the
[61]
the lowest possible concentration of components. The best presence of proliferating cells . The transcriptional
printing accuracy was obtained with a 25 G nozzle for control of the avascular meniscus phenotype is regulated
pressure below 55 kPa. This pressure is applicable for 3D by transcription factors SOX-9 and SOX-8 that upregulate
[62]
bioprinting since higher pressures might increase shear COL2α1, COL11α2, and ACAN expressions . Products of
stress in the nozzle and damage the cell membrane . The these genes, namely collagen type II, type XI, and aggrecan,
[53]
shear forces exerted on cells may elicit alterations in the gene are the main structural proteins of the cartilaginous ECM.
expression profile. Excess mechanical stress downregulates Endochondral ossification is a process in which bones
collagen type I and II expressions and upregulates matrix replace the hyaline cartilage; hence, it is important to
[63]
metallopeptidase (MMP) 1 and 13 [24,54] . The MMPs observe the expression of osteogenesis marker genes .
encode collagenases that are involved in endochondral RUNX family transcription factor 2 (RUNX2) is the main
ossification or osteoarthritis through the degradation transcription factor associated with osteogenesis. The
of ECM proteins, such as collagen type II and aggrecan. change in SOX9 and RUNX2 expressions reduces COL2α1
This situation is highly undesirable for cartilage tissue expression and initiates collagen type X synthesis, followed
engineering. An attempt was made to perform a dynamic by increased collagen type I synthesis. Due to low yields
mechanical analysis (DMA 242 D, Netzsch) to compare of RNA extractions, only five genes’ expressions were
the mechanical strength of the constructs; however, the measured (Table 2). A significant change was observed
scaffolds were too soft for the analyzer’s detector (results only in the expression of COL2A1, which increased during
not shown). We intended to repeat a dynamic mechanical the culture. The high accumulation of type II collagen
analysis following the production of ECM proteins by cells. is characteristic of the inner (white-white) and middle
[64]
It is a feasible step since our bioprinted constructs were (white-red) zones of the meniscus . However, collagen
stable in culture medium for more than six months (results type I is still the most prevalent in the native meniscus.
not shown). Another possibility is to enhance mechanical There were no observable significant changes in the rest
properties by introducing other materials, like PCL, as of the analyzed genes. Perhaps, longer culture periods may
mentioned in the introduction [10,11,14] . allow for the observation of more significant changes.
The present study has several limitations. A good
Various crosslinking strategies may also be used to
control the mechanical stress and bioprinting parameters. practice in bioink research is to conduct disintegration
studies and pore size evaluation with the diffusion of
Gelatin with chemical modification can be subjected to nutrients . The absence of these tests is due to the
[23]
enzymatic crosslinking to enable 3D bioprinting . Besides, limited number of constructs, ensuing from the cells’ low
[55]
gelatin can be crosslinked with a chemical crosslinker, such proliferative capacity and the high cell count required for
as glutaraldehyde, which was used with a hydrogel composed 3D bioprinting, which is a challenge often underscored in
of alginate, gelatin, and nanocellulose and compared with the tissue engineering community [5,65,66] . Our team is also
the Ca alginate crosslinking . Based on mechanical and working on this issue (including 3D scaffold-free cultures
2+
[56]
structural differences, the divalent cation crosslinking of and mesenchymal stem cell application).
alginate was considered most suitable for 3D bioprinting.
The selection of divalent ions and their concentration also Bioink with higher component concentrations (1.25%
influences the mechanical properties of alginate hydrogel; alginate, 20% gelatin, and 0.25% of cellulose nanofiber)
for example, strontium ions create more durable constructs was also proven succesful for meniscal bioprinting . The
[7]
than calcium ions . Moreover, the proper use of cations viability of fibrochondorocytes was equally high (> 95 %).
[57]
can direct cell differentiation. Cobalt ions (Co ) mimic The most relevant differences were bioprinting with a wider
2+
hypoxic conditions by inhibiting hypoxia-inducible nozzle (22 G) in comparison to the results presented in this
factors . Research performed on human mesenchymal study (25 G). The other bioink composed of 4% alginate,
[58]
stem cells encapsulated in alginate beads crosslinked with 35% gelatine, and 2% carboxymethyl cellulose was also
Co revealed significant changes in cartilage-specific gene succesfully used for extrusion into the negative mould .
[67]
2+
expression . Live/dead assay and real-time PCR were Encapsulated MG-63 osteosarcoma cells proliferated and
[59]
performed to assess the biocompatibility of bioink. The high produced collagen inside the construct.
Volume 9 Issue 1 (2023) 9 https://doi.org/10.18063/ijb.v9i1.621