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International Journal of Bioprinting Biomimetic scaffolds for tendon healing

damaged tendinous tissue is located). Despite this, their (molecular biology grade), bovine serum albumin, TRI
incorporation into printed structures is challenging since reagent, calcium chloride, alginate lyase, and triton™ X-100
the methodology to incorporate them, their stability, their molecular biology reagent. Chloroform was obtained
release profile, and their bioactivity once released must be from MP biomedicals (Santa Ana, California, USA). Fast
determined. This is why, until now, their application in gene scriptase II cDNA synthesis kit was acquired from
37
3D bioprinting for tendon regeneration has been limited. Genetics Nippon (Düren, Germany). The primers for RT-
This study introduces a pioneering approach to qPCR were acquired from Applied Biosystems (Waltham,
TM
developing hydrogel-based scaffolds and tissue constructs Massachusetts, USA). LIVE/DEAD viability/cytotoxicity
customized for tendon regeneration. Notably, we designed kit was purchased from Life Technologies (Madrid, Spain).
three distinct variants: one with embedded tenocytes, Phalloidin Alexa Fluor 488 was obtained from Molecular
another loaded with VEGF, and the last one with PDGF- Probes (Eugene, Oregon, USA). Dulbecco’s phosphate-
BB. An original combination of biological materials was buffered saline (DPBS) and phosphate-buffered saline
selected: hyaluronic acid (HA), alginate (Alg), gelatin (PBS) were obtained from Lonza (Porriño, Spain). Sodium
(Gel), and fibrinogen (Fg). After formulating the ink, the chloride (NaCl) and formaldehyde 3.7–4.0% (w/v) were
rheological properties and printability were determined. acquired from Panreac AppliChem (Monza, Italy).
These properties are crucial for achieving accurate and VEGF165 and PDGF-BB were acquired from Stemcell
reproducible 3D-printed structures. Furthermore, cells Technologies (Vancouver, Canada). Human VEGF165
(tenocytes) and growth factors (VEGF165 and PDGF- standard TMB ELISA development kit and human PDGF-
BB) were incorporated into the hydrogel matrix. The BB standard ABTS ELISA development kit were purchased
viability, activity, proliferation, and gene expression of from Peprotech (London, United Kingdom).
the tenocytes in the bioprinted hydrogel-based tissue 2.2. Cell culture conditions
construct were determined. The release rates and the half- iMAT cells were grown on T-flasks pretreated with ABM’s
life values of the growth factors were established. Finally, applied cell extracellular matrix and Prigrow III culture
the potential of the factors released from the scaffold for medium. C2C12 cells were cultured using DMEM media.
promoting vascularization and cell proliferation was Both media were supplemented with 10% (v/v) FBS and
demonstrated. By elucidating the rheological behavior 1% (v/v) P/S. HUVECs were cultivated using vascular cell
and the printability of ink formulations and evaluating the basal media supplemented with endothelial cell growth
in vitro performance of the 3D-bioprinted scaffolds and kit components and 1% (v/v) P/S. All cell types were
tissue constructs, we aim to contribute to the advancement expanded at 37°C in a humid atmosphere with 5% CO .
2
of tissue engineering strategies for partial tendon ruptures The culture medium was changed frequently, and when the
repair and regeneration. culture had reached around 80% confluence, the cells were
subcultured.
2. Materials and methods
2.3. Ink and bioink preparation
2.1. Materials The ink was progressively refined through a series
Normal primary human umbilical vein endothelial cells of adjustments (set-up included in part (A) of the
(HUVECs), vascular cell basal media, endothelial cell Supplementary File). The final ink consisted of the
growth kit components, DMEM media, and immortalized following combination of biomaterials: HA 0.36% (w/v),
mouse myoblast cells (C2C12) were acquired from ATCC® Alg 1% (w/v), Gel 4.2% (w/v), and Fg 3.6% (w/v). The HA
(Manassas, Virginia, USA). Immortalized mouse Achilles and Alg were dissolved in DMEM. After both materials
tendon (iMAT) cells, ABM’s applied cell extracellular were dissolved, the Gel was added and was left overnight at
matrix, and Prigrow III culture medium were purchased 37°C. The Fg was dissolved in DMEM with 0.9% NaCl at
from ABM (Richmond, Canada). Fetal bovine serum 37°C for 4 h. The two parts of the ink were centrifuged to
(FBS) and penicillin/streptomycin (P/S) were acquired remove bubbles and then mixed.
from Gibco (San Diego, California, USA). Ultrapure low-
viscosity high guluronic acid sodium alginate (UPLVG) Prior to the incorporation of cells (iMAT) or growth
was acquired from FMC Biopolymer (Sandvika, Norway). factors (VEGF165 or PDGF-BB), they were dissolved
The following materials were obtained from Sigma-Aldrich at the desired concentration (final concentration of 1 ×
(Merck KGaA, Munich, Germany): cell counting kit-8 10 cell mL , 2 × 10 cell mL , and 5 × 10 cell mL for the
-1
6
-1
6
-1
6
assay, lactate dehydrogenase activity assay kit, hyaluronic cells, 50 ng mL for the VEGF165, and 10 ng mL , 20 ng
-1
-1
acid sodium salt (from Streptococcus equi), gelatin tested mL , and 50 ng mL for the PDGF-BB) in media inside a
-1
-1
according to Ph. Eur., fibrinogen from bovine plasma syringe. Finally, they were mixed with the rest of the ink
type I-S, thrombin from bovine plasma, 2-propanol before being transferred to a printer cartridge.
Volume 10 Issue 3 (2024) 444 doi: 10.36922/ijb.2632