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10.1186/s11671-024-04110-9
Research
Studies on cytocompatibility of human dermal fibroblasts on carbon nanofiber nanoparticle-containing bioprinted constructs
Raja Iruthayapandi Selestin 1
Kim Chuntae 12
Kang Moon Sung 3
Joung Yoon Ki 245
Lee Jong Hun foodguy@gachon.ac.kr

6
Han Dong-Wook nanohan@pusan.ac.kr

13
1 https://ror.org/01an57a31 grid.262229.f 0000 0001 0719 8572 Institute of Nano-Bio Convergence, Pusan National University, Busan, 46241 Republic of Korea
2 https://ror.org/04qh86j58 grid.496416.8 0000 0004 5934 6655 Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul, 02792 Republic of Korea
3 https://ror.org/01an57a31 grid.262229.f 0000 0001 0719 8572 Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, 46241 Republic of Korea
4 https://ror.org/000qzf213 grid.412786.e 0000 0004 1791 8264 Division of Bio-Medical Science and Technology, KIST School, University of Science and Technology, Seoul, 02792 Republic of Korea
5 https://ror.org/01zqcg218 grid.289247.2 0000 0001 2171 7818 Department of Conversing Science and Technology, KIST Graduate School, Kyung Hee University (KHU), Seoul, 02453 Republic of Korea
6 https://ror.org/03ryywt80 grid.256155.0 0000 0004 0647 2973 Department of Food Science and Biotechnology, Gachon University, Seongnam, 13120 Republic of Korea
13 9 2024
13 9 2024
12 2024
19 1 14926 6 2024
10 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Functional nanocomposite-based printable inks impart strength, mechanical stability, and bioactivity to the printed matrix due to the presence of nanomaterials or nanostructures. Carbonaceous nanomaterials are known to improve the electrical conductivity, osteoconductivity, mechanical, and thermal properties of printed materials. In the current work, we have incorporated carbon nanofiber nanoparticles (CNF NPs) into methacrylated gelatin (GelMA) to investigate whether the resulting nanocomposite printable ink constructs (GelMA-CNF NPs) promote cell proliferation. Two kinds of printable constructs, cell-laden bioink and biomaterial ink, were prepared by incorporating various concentrations of CNF NPs (50, 100, and 150 µg/mL). The CNF NPs improved the mechanical strength and dielectric properties of the printed constructs. The in vitro cell line studies using normal human dermal fibroblasts (nHDF) demonstrated that CNF NPs are involved in cell-material interaction without affecting cellular morphology. Though the presence of NPs did not affect cellular viability on the initial days of treatment, it caused cytotoxicity to the cells on days 4 and 7 of the treatment. A significant level of cytotoxicity was observed in the highly CNF-concentrated bioink scaffolds (100 and 150 µg/mL). The unfavorable outcomes of the current work necessitate further study of employing functionalized CNF NPs to achieve enhanced cell proliferation in GelMA-CNF NPs-based bioprinted constructs and advance the application of skin tissue regeneration.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-024-04110-9.

Keywords

Nanocomposite bioinks
Carbon nanofiber nanoparticles
Normal human dermal fibroblasts
Skin tissue regeneration
http://dx.doi.org/10.13039/501100003725 National Research Foundation of Korea 2022R1A1A01064416 RS-2024-00406152 Raja Iruthayapandi Selestin Han Dong-Wook issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
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pmcIntroduction

Tissue engineering dealing with scaffolds, cells, and growth factors is a promising strategy to heal severely damaged tissues or organs [1]. Among these, scaffolds act as a structural platform, allowing for cell attachment and migration. Specifically, hydrogel-type scaffolds provide an amicable environment by maintaining abundant water and nutrients and by diffusing metabolic wastes [2]. Three-dimensional (3D) bioprinting of hydrogels makes tissue engineering capable of developing 3D functioning tissue constructs with the aid of personalized, specific computer-aided designs. Printable inks can be categorized into cell-laden bioinks and biomaterial inks, depending on the inclusion of cells [3]. The major components of a typical scaffold-based bioink are polymer hydrogels, cells, and biocompatible additives, including nanomaterials [4]. The nanomaterials incorporated into the bioink could act as fillers to improve mechanical strength, thermal stability, and shape fidelity and exhibit electroconductive, osteoconductive, or antimicrobial properties [5, 6].

Literature reports reveal that functionalized carbon-based nanomaterials have the potential to promote cell proliferation of neural stem cells, bone marrow mesenchymal stem cells, fibroblasts, pre-osteoblasts, and endothelial cells in their hybrid hydrogel scaffolds [7–10]. The reason is that carbon nanomaterials act as nanofillers and electroactive materials to provide mechanical and electrical cues to the microenvironment of their reinforced biomaterials. As the cell fate is determined by the biochemical, electrical, mechanical, and structural cues present in the microenvironments of the scaffold, designing biomaterials presenting one or several microenvironmental cues advances tissue regeneration by closely imitating the target tissues [11, 12]. Hence, carbonaceous materials-reinforced biomaterials are known as electroconductive or electroactive scaffolds [13]. For instance, NIH-3T3 fibroblasts-loaded graphene oxide (GO)-containing GelMA bioink improved cell viability with tunable mechanical stiffness and electrical conductivity [14]. Izadifar et al. [15] fabricated hybrid hydrogel constructs consisting of alginate-coated carbon nanotubes (CNTs)-incorporated, human coronary artery endothelial cells (HCAECs)-laden methacrylated collagen. They reported that HCAECs presented enhanced cellular proliferation and differentiation after 10 days of incubation with a selected CNT mass ratio. Further, the constructs were utilized as potentially prevascularized hybrid cardiac patches with upgraded biomechanical and electrical behavior. CNF is one of the less-explored 1D carbonaceous nanomaterials in tissue regeneration. It also exhibits its unique properties of electrical conductivity and mechanical strength, like CNTs and GO. Though CNF materials have been widely explored in energy applications, including supercapacitors, batteries, and solar cells [16], their uses in biomedical applications are still limited.

CNF materials can be prepared in either two ways: bottom-up approaches like chemical vapor deposition or top-down approaches like the calcination of electrospun nanofibers. The CNF produced by the electrospinning approach has several advantages over vapor-grown nanofibers in the aspects of generation of continuous nanofiber, bulk preparation, easy purification, and being economically inexpensive [17, 18]. Considering these advantages, exploring CNF-containing materials would replace expensive vapor-grown CNF and other carbonaceous materials in tissue engineering scaffolds. Extrusion bioprinting was deployed to pneumatically (via pressurized air) inject a continuous strand of the bioink [19]. There are four extensively employed bioprinting techniques, i.e., extrusion-based, inkjet-based (material-jetting), vat-photopolymerization (stereolithography), and laser-assisted methods [20–23]. Extrusion-based 3D printing has some remarkable advantages compared to other fabrication strategies. The method is capable of fabricating tissue constructs with a high cell density of about 108 cells/mL and achieving continuous deposition of filaments with better structural integrity [24]. The cell viability in the fabricated constructs is between 40 and 80%; however, it can be improved up to 97% if printing parameters such as pressure, deposition rate, and temperature are optimized. The other drawbacks of this bioprinting method are its low printing speed and medium resolution [20]. GelMA is a well-known hybrid gel system in which methacryloyl substituent groups are involved in photo-cross-linkage during UV curing in the presence of a suitable photo-crosslinker. This printable ink has been reported to be compatible with many biomolecules and nanofillers [25].

The main objective of the current work is to investigate the cell proliferation potential of various concentrated CNF nanomaterials in two kinds of bioprinted constructs: cell-laden bioink and biomaterial ink. We optimized the UV-curing time during the fabrication of GelMA gel to know its potential to hold the cells in the matrix before incorporating CNF NPs into the gel. Then, we explored the morphology, mechanical, and dielectric properties of the GelMA-CNF constructs using relevant physicochemical characterizations. In vitro cell line studies of the printed constructs using nHDF cells were analyzed to examine the cell biocompatibility and cell proliferation of the constructs. The key findings and prospective recommendations to improve the cell proliferation potential of the constructs have been discussed.

Methods

Materials

GelMA granules from type A gelatin from porcine skin (15–400 kDa, gel strength ~ 300) were kindly provided by 3D Materials Co., Ltd. (Anyang-si, Republic of Korea). 2-hydroxy-4′-(2-hydroxyethoxy)-2-methyl-propiophenone (Irgacure 2959) was obtained from TCI Co., Tokyo, Japan. Polyacrylonitrile (150 kDa) and N, N-dimethyl formamide (DMF, 99.8%) were obtained from Sigma-Aldrich. The solvents used were of analytical grade, and the chemicals obtained were utilized without further purification.

Preparation of CNF NPs

We prepared CNF NPs following the procedures mentioned in our previous work [26]. In brief, 10% (w/v) of PAN solution in DMF was taken into a 10 mL syringe to prepare electrospun nanofiber mats. The distance between the needle tip and the aluminum foil collector was 10 cm. During electrospinning, the flow rate of solution, applied voltage, and rotor speed were 0.8 mL/h, 15 kV, and 1000 rpm, respectively. The temperature and humidity maintained in the chamber were 25 °C and 35%, respectively. The as-prepared nanofiber mat was subjected to subsequent processes to produce CNF NPs: heat stabilization in open air (2 h, 280 °C), calcination treatment (1 h, 800 °C, 5 °C/min) under nitrogen gas atmosphere, and manual grinding using a pestle and mortar. The impurities of the nanoparticles were removed by washing with acetone and subsequently dried in a hot air oven at 100 °C. The air-dried nanoparticles were then stored at room temperature until further use for characterization.

Fabrication of GelMA-CNF printed constructs

0.1 g of GelMA (5%, w/v) was dissolved in 1.5 mL of DMEM, and freshly prepared 0.4% of irgacure solution (30 µL of EtOH and 70 µL of DMEM) was added to it. Irgacure was sparingly soluble in water, and hence EtOH was added to increase its dissolution in the aqueous solution. Subsequently, 0.4 mL of cell-containing DMEM was added. Aliquots of CNF NPs solution (20 mg/mL) were mixed to have a final concentration of 0, 50, 100, and 150 µg/mL in the total volume (2 mL) of bioink precursor solution. The respective samples were labeled as GelMA, GM50, GM100, and GM150, as shown in Table 1.Table 1 The composition of GelMA-CNF NPs in the printable precursor solutions and their respective labels have been listed

GelMA (w/v) (%)	Irgacure (w/v) (%)	CNF NPs (µg/mL)	Volume of ink precursors (mL)	Label	
5	0.4	0	2	GelMA	
5	0.4	50	2	GM50	
5	0.4	100	2	GM100	
5	0.4	150	2	GM150	

Film-like printed tissue constructs of GelMA-CNF NPs were fabricated in a 6-well plate using an extrusion-based 3D bioprinter (BioX, Cellink, Sweden). Before extrusion, the cell-laden printable ink was pre-cooled for 8 min at 4 °C in a refrigerator. The bioprinting parameters were as follows: print bed temperature, 10 °C; pneumatic pressure, 60 kPa; nozzle gauge, 20 G-type; printing speed, 2.6 mm/s speed; grid pattern infill density, 100%; single layer, 20 mm × 20 mm × 1 mm; gel setting time after UV-curing, 5 min. The population of nHDF cells was 5 × 105 cells/mL. As shown in Fig. 1, the cells were incubated with CNF NPs in bioink before bioprinting, whereas the cells were seeded on biomaterial ink after fabrication. The UV-curing time was optimized by printing GelMA bioink by varying UV exposure times to 2, 3, 4, and 5 min with a wavelength of 365 nm before the preparation of nanoparticle-containing ink.Fig. 1 Schematic demonstration of the fabrication of A a film-like printed biomaterial ink construct from CNF NPs-incorporated GelMA hydrogel and B cell-laden CNF NPs-incorporated GelMA hydrogel. Cell proliferation in two different constructs was investigated. Irgacure was used to crosslink GelMA monomers under UV light. CNF NPs were prepared by the subsequent processes of stabilization, calcination, and grinding

Instrumental characterizations

Field emission Scanning electron micrographs (FESEM, Zeiss Supra-40) of the lyophilized samples were captured with a 4- or 10 kV voltage. The specimen preparation for SEM involves stubbing the samples on carbon tape and subsequently sputter-coating to apply an electroconductive platinum layer. The size of the nanoparticles and pore size in the printed constructs were measured using Image J software (Version 1.41). Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of the samples were measured in the wavelength range of 4000–600 cm−1 using a FTIR spectrum GX (PerkinElmer Inc., Waltham, MA). The compressive stress–strain measurements were performed on printed constructs after submerging them in DMEM medium for 3 days at 37 °C. The ElectroForce 5500 TA electromechanical universal testing machine with a load cell of 100 N was applied at a crosshead speed of 1 mm/min for the measurement. The compressive modulus of the printed constructs was calculated from the slope of the linear region of the stress–strain curves. Measurements were carried out in triplicate, and the average of Young’s modulus has been reported. Alternative current (AC) impedance analyses of the printed ink samples (without cells) were carried out using a CH instrumental electrochemical analyzer (CHI-model 660D, U.S.A.) in PBS medium using the frequency range of 10–2 to 105 Hz. The wet GelMA-CNF NPs biomaterial constructs acted as working electrodes. Platinum wire and saturated calomel were the counter and reference electrodes, respectively. The initial electric field (E) in AC impedance measurement was obtained from the open-circuit potential value of the experimental set-up [27].

Cell maintenance

The nHDF cell line was procured from the American Type Culture Collection (ATCC, Rockville, MD), and the cultivation of cells was processed in complete Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Daegu, Korea) augmented with 10% fetal bovine serum (FBS, Welgene, Daegu, Korea) and 1% penicillin–streptomycin solution. The cells were maintained in an incubator at 37 °C with a 5% CO2 atmosphere. The media was refreshed every 48 h. The constructs were transferred to another 6-well plate after 12 h of cell incubation, as there may be some leakage of cells into the surrounding area that disturbs the cell measurement results.

Measurement of cell viability in printed constructs

The cells in the printed constructs were grown for different periods, i.e., 1, 4, and 7 days (bioink) or 2, 4, and 6 days (biomaterial ink), and the cell viability of nHDF cells was determined using the cell counting kit-8 (CCK-8, Boya Biotech, China). For the CCK assay, the reagent solution was prepared by mixing CCK-8 with DMEM in a proportion of 1:9 (v/v). The culture medium of tissue constructs was replaced by 2 mL of CCK reagent solution. After incubation in the cell maintenance chamber for 1.5 h, 100 μL of the supernatant was transferred to a 96-well plate. The absorbance was recorded at 450 nm using a microplate reader.

Phase contrast, fluorescent, and confocal laser scanning microscope images were captured to observe the number of viable cells and the 3D view of cell distribution in printed constructs, respectively. A BX51M optical microscope (Olympus Co., Japan) supplied with a fluorescence light source and filters was utilized to monitor phase contrast and fluorescent images. For visualizing fluorescent images, the samples were stained with a dye mixture of calcein-AM and ethidium homodimer-1 to identify live and dead cells, respectively. In the case of CLSM images (CLSM 800, Zeiss, Germany), the samples were washed using PBS, and the cells were fixed in buffered formalin for 15 min. Then, the cells were subjected to permeabilization using 0.5% Triton X-100 (10 min) and blocking using 2.5% bovine serum albumin (10 min). Subsequently, the samples were incubated with 2 mL of a dye mixture of DAPI and rhodamine B for 20 min to stain the nuclei and cytoplasm, respectively.

Statistical analysis

All the qualitative data were presented with a mean ± standard deviation. The number of samples was three (n = 3) in each experiment. The statistically significant difference (p < 0.05) of different groups compared with control was provided using the one-way ANOVA method with a post-hoc Tukey test.

Results and discussion

Micrographs and spectral characteristics

We prepared CNF NPs from electrospun PAN through the subsequent thermal heating processes of stabilization and carbonization to prepare GelMA-CNF printable constructs. SEM images were captured to observe the morphology of PAN nanofibers, CNF nanofibers, and CNF NPs, as shown in Fig. 2. The nanofibers were cylindrical in shape; however, the fiber diameter of CNF (320 ± 62) was less than that of PAN (550 ± 83). The reduction in fiber diameter might have occurred during the thermal transformation of PAN into CNF. The CNF NPs maintained a cylindrical shape but had a different size range of 650–4100 nm. Further, most of the nanoparticles were found aggregated.Fig. 2 SEM images showing the surface morphology of PAN nanofibers, CNF nanofibers, and CNF NPs. Scale bar, 1 µm

A typical bioink should be soft and biocompatible to support the cellular growth; however too much soft hydrogel scaffold can disintegrate or lose the potential to hold the cells inside the scaffold’s matrix [28]. Hence, it is important to optimize the UV-exposure time, in which a maximum number of cells and cell viability can be achieved in the gelMA network. As shown in Figs. S1, S2, a greater number of viable cells was observed in constructs irradiated with 2 or 3 min of UV exposure than 4 min- and 5 min- irradiated constructs. The cell leakage is gradually decreased from 2 to 4 min of UV exposure in the constructs. These observations indicate that lesser UV irradiation time causes more cell leakage with a possible reason of less cross-linked network in the matrix and higher UV exposure significantly affects cell viability. Among the samples, only 3 min-irradiated constructs maintained a larger cell viability with less leakage of cells. Thus, we preferred 3 min of UV exposure as the optimized irradiation time for the fabrication of GelMA-CNF printed constructs. Previously, Boularaoui et al. fabricated a 3D conductive tissue construct of GelMA and MXene nanosheets/gold nanoparticles and examined the spreading of C2C12 cells [29]. They carried out experiments to optimize the concentration of GelMA hydrogel (2, 4, and 6%) with different UV exposure times, either 2 or 4 min at 365 nm, using a 0.1% LAP (lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate) photoinitiator. They observed that 2% concentrated GelMA with 4 min of UV exposure demonstrated a higher cell viability and elongation on 7 days of treatment than that of other tested samples with different concentrations and times.

We have shown digital images of film-like printed constructs of GelMA-CNF NPs prepared in DMEM medium after UV-irradiation in Fig. 3A. An increase in black contrast was observed according to the increased concentration of CNF NPs in the constructs. The FESEM images of lyophilized GelMA-CNF samples (Fig. S3) demonstrated a reduction in pore size from GelMA to GM150, as follows: GelMA (78 ± 8 µm), GM50 (60 ± 5 µm), GM100 (37 ± 4 µm), and GM150 (28 ± 3 µm). The reason was attributed to the physical cross-linkages of nanoparticles within the GelMA network. ATR-FTIR spectral measurements of lyophilized GelMA-CNF NPs constructs were carried out to identify the functional groups of components (Fig. 3B). The FTIR spectrum of CNF showed a peak at 1627 cm−1, representing vibrational bands of C–N, C=N, and C=C. Another peak centered around 1113 cm−1 indicated the vibrational bands of O–H, N–H, and C–O–C [26]. The GelMA spectrum displayed typical peaks of 1241, 1526, and 1639 cm−1, related to N–H, C–N, and C=O vibrational bands, respectively, which implied the presence of amide I, II, and III bonds [30]. Both the spectra of GelMA and GelMA-CNF NPs appeared similar in their traces, as the peaks of CNF NPs overlapped with the traces of GelMA. The mechanical property of the printed constructs was determined by the compressive stress–strain curves, as shown in Fig. 3C. The GelMA construct failed at an average stress of 2.8 kPa and strain of 2.8%, while the higher CNF-concentrated construct (GM150) fractured at a higher average stress of 28.8 kPa and strain of 2.5%. The average stress values of GM50 and GM100 were within the range of 11–13.5 kPa. The average compressive moduli of the GelMA, GM50, GM100, and GM150 were 6.4, 14.5, 16.4, and 37 kPa, respectively. The results indicate that CNF NPs have strong physical hydrogen bonding with the UV-crosslinked GelMA network, and so increasing the concentration of CNF NPs in the constructs yielded a higher compressive modulus.Fig. 3 A Digital images of GelMA and GelMA-CNF NPs (GM50, GM100, and GM150) film-like printed constructs. The dimension of each construct is 2 cm × 2 cm × 1 mm. B ATR-FTIR spectra of CNF NPs, GelMA, and GM150; C compressive stress–strain relationship curves of the constructs; and D Nyquist admittance plots of the constructs

AC impedance analyses were useful to determine the dielectric behavior of the constructs in PBS medium (Fig. 3D). Dielectric materials experience orientational polarization due to the polar molecules, which entrap electric charge at lower frequencies [31]. GelMA contains polar molecules such as NH2, COOH, and OH groups and acts as a charge carrier [25]. CNF NPs produced from PAN, due to thermal oxidation and carbonization processes, exhibit a polyconjugated system with mobile π-electrons [32]. Hence, when CNF NPs are blended with GelMA, the net polarized moieties present in the constructs increase, which is inferred from the steepness of the Nyquist admittance curves. Sui et al. reported that the loading of 5 wt.% CNF to the polypropylene (PP) matrix demonstrated a larger dielectric constant than the neat PP polymer under the wide sweep frequencies of 10–2 to 106 Hz. They revealed that the addition of CNF caused extra polarization in the PP composites under the alternative electric field [33]. A literature report reveals that an increase in the dielectric behavior of a material influences cell attachment and proliferation of adherent cells like fibroblasts [34].

Cell viability in GelMA-CNF NPs bioink constructs

In this section, we investigated cell viability and proliferation of nHDF cells in GelMA-CNF NPs-based bioink printed constructs on days 1, 4, and 7 of post-incubation. As shown in Fig. 4, the bioink samples have shown concentration-dependent cytotoxicity, though they have endorsed increased cell proliferation on increasing days. Each CNF NPs-containing sample witnessed a drastic decrease in cell numbers on days 4 and 7 compared to the control (GelMA). The samples, GM100 and GM150, have shown a significant reduction (p > 0.05) in cell viability compared to the control on days 4 and 7 of cell incubation. The respective phase contrast microscope images (Fig. 5) showed that the cells are spherical in all the samples on day 1 of post-incubation. On days 4 and 7, we observed the growth of cells with extended filopodia and their proliferation. These observations indicate that the cell morphology was not affected by the presence of CNF NPs in the constructs. A homogenous cell spreading across the matrix of GelMA was noticed in the control. Further, cell-nanomaterial interaction was observed in the GelMA-CNF NPs due to CNF’s chemoattractant property. In our previous work, we reported that CNF NPs could act as chemoattractants to enable increased cells’ migration in a wound scratch assay using NIH3T3 mouse embryonic fibroblast [26]. Fluorescence microscope images of live and dead cells after staining by calcein and ethidium homodimer-1, respectively, have been shown in Fig. 6. The number of viable cells found decreased in the higher CNF NPs-concentrated bioinks. The cell distribution throughout the matrix on day 7 was visualized using 3D confocal microscope images (Fig. 7). All the samples, except GM150, have shown a good cell distribution throughout the matrix. However, the control demonstrated a higher cell density compared to GM50 and GM100 (Videos S1–S4).Fig. 4 CCK-assay demonstrating cell viability of nHDF cells on GelMA-CNF NPs-based bioink. The statistical difference (p < 0.05) has been denoted by an asterisk symbol

Fig. 5 Cell biocompatibility of nHDF cells on GelMA-CNF NPs based bioink printed constructs. Phase contrast microscope images of cells in the scaffold have been shown. The CNF NPs interacting with the cells on day 4 appeared as dark black spots, denoted by red arrow marks. The scale bar is 100 µm

Fig. 6 Cell spreading and viability of nHDF cells on GelMA-CNF NPs based bioink printed constructs. Fluorescent microscope images of cells spread inside the scaffold matrices on day 7 have been shown. Live and dead cells were identified by Calcein AM and ethidium homodimer-1 staining. The scale bar is 200 µm

Fig. 7 3D Cell distribution of nHDF cells on GelMA-CNF NPs based bioink printed constructs. Confocal microscope images of cells in the constructs on day 7 of the investigation have been displayed. The scale bar is 2400 µm

The in vitro biological measurements of cell-laden GelMA-CNF printed constructs show a significant decrease in cell population from control to GM150 on days 4 and 7 of post-treatment. According to literature reports, the cells engulf the interacting NPs through the endocytosis process, eventually causing their own cell death, which may reduce the number of viable cells [35]. Another explanation for the complete cell death could be that the presence of localized hindrances to forming cell–cell communication and heterogeneous matrix stiffness might prevent the maturation of some cells inside the hydrogel matrix. For instance, Shin et al. [36] fabricated NIH-3T3 fibroblast-laden carbon nanotubes (CNT)-GelMA hybrid bioink constructs with increasing concentrations of CNTs (0.1, 0.25, and 0.5 mg/mL) and determined cellular viability for 48 h. They observed a uniform distribution of viable cells within the 3D structure. However, they reported that there was a significant loss of cells in the 3D culture due to the shear stress produced during bioprinting or low oxygen and nutrient diffusion in the matrix microenvironment. Further, they concluded that the cell viability of NIH-3T3 was indirectly proportional to longer incubation times in the CNT-GelMA construct with an increased concentration of nanomaterials. Loh et al. investigated the cellular proliferation of human dermal fibroblasts in a bacterial nanocellulose composite hydrogel system and reported that the cells could not proliferate even after 7 days of seeding, though cell viability was maintained. The reason was attributed to the low porosity of the hydrogels, which could limit the nutrient supply and diffusion of waste [37]. Along with these literature reports, our work shows that carbonaceous nanomaterials, including CNF, support fibroblast cell viability and cell-material interaction during the early stages of treatment in a bioprinted construct. However, in the later stages of treatment, the cells die for a variety of possible reasons, including the engulfment of nanoparticles by the cells.

Cell viability in GelMA-CNF NPs biomaterial ink constructs

It is essential to study the cellular growth on the surface of the tissue construct, as it interacts immediately with the tissue cells when applied to the defect area. Hence, we investigated cell proliferation of nHDF cells in GelMA-CNF NPs-based biomaterial ink on days 2, 4, and 6 of post-incubation. Though the biomaterial ink constructs imitated the cell proliferation pattern of the bioink constructs on increasing days, the difference in cell population was not found to be significant compared to the control (Fig. 8). The phase contrast and fluorescence microscope images demonstrating the cellular growth on the constructs’ surface have been shown in Figs. 9, 10, respectively. An extended cell network with amicable cell–cell communication and a smaller number of dead cells has been observed in all the samples. Unlike bioink, the cells grown in biomaterial ink constructs do not suffer shear stress or the harmful effects of UV irradiation as the cells are seeded after the fabrication. Being localized on the surface of the scaffold, the cells could access O2 and nutrients from the culture medium without any obstacles [38]. For these reasons, the cellular growth in biomaterial ink was not significantly affected, as observed in bioink constructs. In a similar work by Serafin et al. [39], an electroconductive 3D-printed biomaterial ink scaffold was developed using a hybrid composite of alginate, gelatin, and CNFs (Alg-Gel-CNFs). The concentration of CNFs varied from 0.5 to 5% (w/v). The in vitro results demonstrated that all the Alg-Gel-CNFs scaffolds showed an increase in cell proliferation but without significant difference compared to Alg-Gel scaffolds.Fig. 8 CCK-assay demonstrating cell viability of nHDF cells in GelMA-CNF NPs-based biomaterial ink constructs

Fig. 9 Cell biocompatibility of nHDF cells on GelMA-CNF NPs based biomaterial ink printed constructs. Phase contrast microscope images of cells grown on the surface of the tissue constructs have been shown with a scale bar of 100 µm

Fig. 10 Cell spreading and viability of nHDF cells on the surface of GelMA-CNF NPs based biomaterial ink printed constructs. Fluorescent microscope images of cells on GelMA-CNF NPs biomaterial ink constructs on day 6 of the investigation. Live and dead assay (Calcein AM/Ethidium homodimer-1). The scale bar is 200 µm

Combining the in vitro cell biocompatibility profiles of both film-like printed tissue constructs of GelMA-CNF-based bioink and biomaterial ink, we understand that CNF NPs readily interact with nHDF cells without altering the cellular morphology at the investigated concentration range. Both kinds of printed constructs showed dose-dependent cytotoxicity; however, the higher CNF-concentrated bioink constructs (GM100 and GM150) demonstrated a significant decrease in cell population compared to control. In our previous work, we showed that the lower CNF (100 µg/mL)-concentrated electrospun PCL nanofiber mats demonstrated a greater cell proliferation profile than the neat PCL nanofiber mat and the higher CNF (400, 1600, and 3200 µg/mL)-concentrated PCL mats [26]. Similarly, we assumed that low-concentrated GelMA-CNF NPs, GM50 or GM100, could promote cell viability and proliferation compared to the neat GelMA construct, but the result was unexpectedly different. The reason could be that the CNF NPs in PCL-CNF nanofiber mats were found buried into the nanofiber’s core or partly exposed to the culture medium, while, in the current work, the NPs readily interacted with fibroblasts and were engulfed by them in the hydrogel-printed constructs, causing a dose-dependent toxicity. This observation suggests that preventing the direct exposure of freely available CNF NPs to the fibroblasts could be an effective strategy to improve cell viability and proliferation of fibroblasts in the constructs. According to literature research by Shin et al. [36], GelMA-coated CNT suspension (50 μg/mL) had a significantly greater viable cell population of NIH-3T3 cells than that of bare CNTs. Wang et al. [40] revealed that a chitosan-functionalized graphene oxide (cGO)-dispersed chitosan hydrogel system exhibited greater cytocompatibility of RS1 fibroblasts than chitosan hydrogel on 3 and 5 days of incubation. Therefore, we propose that employing functionalized CNF (fCNF) materials using a biocompatible polymer may promote cell proliferation in GelMA-fCNF constructs in comparison to GelMA and bare CNF NPs-based constructs; however, more research is required to confirm this.

Conclusion

CNF NPs were prepared by the thermal conversion of electrospun PAN nanofiber into carbon nanofiber through stabilization and carbonization. Two kinds of GelMA-CNF NPs-based film-like printed tissue constructs (bioink and biomaterial ink) were fabricated using an extrusion bioprinter. The concentrations of CNF NPs (50, 100, and 150 µg/mL) in GelMA-CNF NPs varied to investigate whether the NPs influence cell proliferation. Though CNF NPs improved the mechanical strength and dielectric properties of their hybrid composites, they reduced cell proliferation in a dose-dependent manner at the investigated concentration range of NPs. The bioink-printed constructs experienced a significant cell reduction on days 4 and 7 of post-treatment compared to the control; however, the difference in cell biocompatibility among biomaterial ink-printed constructs was not significant. It was suggested that functionalized CNF-containing GelMA constructs could demonstrate enhanced cell proliferation potential and progress their skin tissue regeneration ability.

Supplementary Information

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (No. RS-2024-00406152), and the NRF grant funded by the Ministry of Education (No. 2022R1A1A01064416).

Author contributions

Conceptualization, I.S.R. J.H.L. and D.-W.H.; methodology and investigation, I.S.R., C.K., M.S.K. and Y.K.J.; writing—original draft preparation, I.S.R. and D.-W.H.; supervision, J.H.L. and D.-W.H. All authors have read and agreed to the published version of the manuscript. All authors read and approved the final manuscript.

Data availability

All data supporting the findings of this study are available within the paper.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

The authors approve to publish on Discover Nano if the manuscript is accepted.

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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