The influence of the structure (surface roughness, pore size, and porosity) of 3D printed silk-fibroin-based scaffolds on the growth of the bronchial epithelial cells in vitro

doi:10.21203/rs.3.rs-1529862/v2

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The influence of the structure (surface roughness, pore size, and porosity) of 3D printed silk-fibroin-based scaffolds on the growth of the bronchial epithelial cells in vitro

https://doi.org/10.21203/rs.3.rs-1529862/v2

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The structure (surface topography, pore size, and porosity) of 3D scaffolds plays an important role in tissue regeneration. However, the parameters of 3D printed silk-fibroin (SF)-based scaffolds suitable for tracheal epithelium growth have not been studied. In this research, we investigated the effects of 3D printed silk fibroin/Hydroxypropyl methyl cellulose (SF/HPMC) scaffolds with different structures on the behaviors of adhesion and proliferation of the tracheal epithelium in vitro. Six types of 3D printed SF/HPMC scaffolds with different surface topography, pore size and porosity were fabricated by extrusion 3D printer. The porosity of 20 wt% SF/HPMC scaffolds with rough or smooth surfaces, and 30 wt% SF/HPMC scaffolds with rough or smooth surfaces were 70.5 ±2.0%、65.5 ±6.1%、63.9 ±2.1%、59.6 ±2.1%, respectively; and the 20 wt% SF/HPMC scaffolds with rough surface had 2 types of macro-pore size, 443.9±104.1μm and 681.1±115.1μm. Normal human bronchial epithelial cell lines (BEAS-2Bcells) were cultured on these scaffolds for 7 days. The cell proliferation was detected by live/dead cell staining and CCK-8 assay, and the morphology was observed by scanning electron microscopy (SEM). Results showed the scaffolds with rough surface, higher porosity (65.5 ±6.1%) and small pore size (443.9±104.1μm) showed better effect on BEAS-2B cells proliferation than those with smooth surface, lower porosity (59.6 ±2.1%) and large pore size (681.1±115.1μm). In summary, SF/HPMC scaffolds with rough surface, higher porosity, and small pore size might facilitate BEAS-2B cells growth. This provides a research basis for selecting 3D printed SF/HPMC scaffolds with suitable structure for the repairing of tracheal defects.

Silk fibroin

3D printing

airway epithelial cell

surface morphology

porosity

Clinically, tracheectomy is required for a variety of reasons, such as trauma, congenital tracheal stenosis, and tracheal tumors. Tracheal reconstruction with a substitute is often required if the length of the trachea to be resected is greater than 50% in adults or greater than 30% in children ¹. Tissue engineering, a multidisciplinary field, which has the potential to restore, maintain, and enhance tissue and organ functions holds great promise ². The scaffolds are essential and critical in tissue engineering. The minimum requirements for successful tracheal replacement are rigidity to remain trachea patent, vascularity to facilitate graft ingrowth, and epithelial lining to prevent the accumulation of secretions and resisting bacterial colonization ³. An ideal tissue engineered scaffold should have the following demands: biocompatibility, biodegradability with a controllable degradation rate, three dimensional (3D) and porous structure to promote vascular ingrowth and transportation of nutrients, oxygen, and waste products ^4,5, a suitable surface for cell attachment and proliferation, and good mechanical properties ⁶.

Silk fibroin (SF), one of natural macromolecular protein polymer, has been used for various biotechnological and biomedical applications because of its good biocompatibility, mechanical properties and controllable degradation rates ⁷. Compared with other widely used natural polymers (collagen, gelatin, chitosan, etc.) in tissue engineering, silk fibroin has more excellent mechanical properties such as remarkable tensile strength and toughness ^8–10. Through different treatment, SF can be formed the diverse structure such as fiber, film, hydrogel, and three-dimensional porous structures which could promote the growth of fibroblasts, cartilage, mesenchymal stem cells, epithelial cells, etc. ^4,11.

Previously, our research team successfully made 3D porous SF scaffold by a traditional method of freezing-defrosting process ¹² for repairing of tracheal defect, and results showed that the 3D porous SF scaffold could promote the proliferation of tracheal epithelial cells ¹³. However, it is difficult to exactly control its structure and internal geometry ¹⁴. Three-dimensional (3D) printing has been increasingly used for ‘bioprinting,’ which allows cells, biomaterials, and bioactive molecules to be printed precisely to create 3D tissue constructs with complex geometries for a variety of biomedical applications ¹⁵. Although it is found that low pH, high temperature, alcohol and shear stress could accelerate the SF gelation process, the gelation of SF is still difficult. Hydroxypropyl methyl cellulose (HPMC), a kind of cellulose ether derivatives, can form a strong link with silk fibroin. By mixing and heating SF and HPMC, a robust hydrogel could be developed for 3D printing ¹⁶. Therefore, we used SF and HPMC to fabricate the 3D printed SF/HPMC scaffolds by an extrusion 3D printer to precisely control the structure of the scaffold.

In vitro experiments showed that the BEAS-2B cells could proliferate on the 3D printed SF/HPMC scaffold ¹⁷. The parameters of scaffold can affect cell adhesion, migration and proliferation ^4,18,19. However, the effect of 3D printed SF/HPMC scaffolds with different properties (such as surface topography, pore size, and porosity) on tracheal epithelial cells was unknown. And the influence of 3D printed scaffold properties on tracheal epithelial cells hasn’t been reported.

In this study, we investigated the appropriate parameters (surface topography, pore size, and porosity) of 3D printed SF/HPMC scaffold for the growth of tracheal epithelial cells. We fabricated 3D printed SF/HPMC scaffolds with different porosity by using different concentration of SF/HPMC solution, and the surface roughness was determined by water spraying and freezing process ¹². SF/HPMC scaffolds with various macro-pore sizes were fabricated by adjusting the printing parameters. Then the attachment and proliferation of tracheal epithelial cells on different parameters of 3D printed SF/HPMC scaffolds were assessed. It is expected that this study could provide reference data for the development of artificial tracheal grafts.

Scaffold fabrication

It is well known that the 3D porous scaffolds parameters such as surface topography, pore size, and porosity could influence the cell survival and proliferation. Over the years, many methods have been devised and investigated to promote cell attachment and subsequent cell proliferation on scaffolds by changing the surface topography, pore size, and porosity ^4,19,20. With the development of 3D printing, we could fabricate the 3D porous scaffolds with better controlled structure. Besides, previous study showed that the concentration of SF solution had a crucial influence on the micro-pore size and porosity of the SF scaffolds ^12,21. Therefore, combined with the traditional freeze-drying method ^12,21, we made the 3D printed SF/HPMC scaffolds with multi-level pores including macro-pores and micro-pores. The diameter of macro-pores could be controlled by changing the distance between printed filaments through computer, and micro-pores in the printed single filament of SF/HPMC scaffolds could be altered by changing the concentration of SF. However, we found that the surface of the 3D printed SF/HPMC scaffolds became smooth after freeze-drying. The cell adhesion and proliferation could be influenced by subtle changes in microtopography, and different cell types prefer either a smooth or a rough topography ²². Changing surface microtopography may influence cellular responses in vitro, and it may not be suitable for implantation if they are negatively affected ²³. Therefore, we tried to fabricate the 3D printed SF/HPMC scaffolds with different surface topography.

As the mixture of SF and HPMC could develop a water-insoluble hydrogel with the formation of small and uniform β-sheet structure after heating at 70℃ for 1h ¹⁶, we assumed that the ice crystals formed on the surface of the scaffold could make the surface rough if the SF/HPMC hydrogel was sprayed with water after 3D printing and then frozen at -20℃ for 24h. Results showed that fabricating 3D printed SF/HPMC scaffolds using water spraying could produce scaffolds with rougher surface than that without water spraying. Water-spraying and freeze-drying described herein is a simple method for surface modification which does not require any chemicals, and the ice crystals formed during freezing can be removed simply by sublimation.

SF/HPMC scaffold characterization

Scanning electron microscopy (SEM)

SEM was used to observe the morphology and pore size of the scaffold. The 20% SF/HPMC scaffolds sprayed with water had a rougher surface than that not sprayed with water. There were some cracks on the surface of 20% SF/HPMC scaffold sprayed with water. The surface of the water-sprayed 30% SF/HPMC scaffold formed the shape of the grid which was rougher than the other three groups. And the surface of the 30% SF/HPMC scaffolds not sprayed with water was the smoothest among the four groups. Images showed that the surface topography of 20% and 30% SF/HPMC scaffolds not sprayed with water were similar (Fig. 1A-H). The cross-section images showed that four types of scaffolds had micro-scale pores in the cross section. The 20% SF/HPMC scaffolds sprayed with water had tiered holes in the section which is similar to the 30% SF/HPMC scaffolds sprayed with water. Moreover, the 20 wt% SF/HPMC scaffolds sprayed with water had a thinner hole wall structure than the 30 wt% SF/HPMC scaffolds sprayed with water, and the connectivity of the holes was higher than that of the 30 wt% SF/HPMC scaffolds (Fig. 1H, J). The cross-sectional pore size of the 20 wt% SF/HPMC scaffolds not sprayed with water was larger than that of the 30 wt% SF/HPMC scaffolds not sprayed with water, and the hole wall was thinner than the 30 wt% SF/HPMC scaffolds not sprayed with water (Fig. 1I, K).

To investigate the influence of macro-pore size on cell proliferation, the 3D printed 20% SF/HPMC scaffold sprayed with water was used. The morphology of 3D printed SF/HPMC scaffolds with two different pore sizes was shown in Fig. 1L, M. The diameter of macro-pores of two scaffolds, which were the distance between the single printed filament, were 681.1\(\pm 115.1{\mu }\text{m}\) and 443.9\(\pm 104.1\text{u}\text{m}\), respectively.

Porosity and water content of SF/HPMC scaffolds

The porosity of 20 wt% SF/HPMC scaffolds sprayed or not sprayed with water, and 30 wt% SF/HPMC scaffolds sprayed or not sprayed with water were 70.5 ± 2.0%, 65.5 ± 6.1%, 63.9 ± 2.1% and 59.6 ± 2.1%, respectively. The difference in the porosity between 20% SF/HPMC scaffolds and 30% SF/HPMC scaffolds was statistically significant. While for the same concentration of SF/HPMC scaffolds, the porosity of water-sprayed scaffolds was numerically higher than that of non-water-sprayed scaffolds, but the difference was not statistically significant.

The water content of 20 wt% SF/HPMC scaffolds sprayed or not sprayed with water, and 30 wt% SF/HPMC scaffolds sprayed or not sprayed with water were 80.5±\(\pm\)0.8%, 79.9±0.53%, 77.1±0.7% and 68.8±2.3%, respectively. There were significant differences in the water content between 20% SF/HPMC scaffolds and 30% SF/HPMC scaffolds. And the water content of scaffolds sprayed with water was higher than that not sprayed with water in the same concentration group (Fig. 2A, B).

In addition, the porosity of 20% SF/HPMC scaffolds with large macro-pore was higher than that with small macro-pore, and its water content was lower than that with small pore. There were no significant differences in the porosity and water content between two groups (Fig. 2C, D).

In vitro evaluation

BEAS-2B cells proliferation and viability on different scaffolds

Cell proliferation and viability on SF/HPMC scaffolds with different structures were investigated by CCK-8 assay. The results showed that BEAS-2B cells on 3D printed porous scaffolds with rough surface proliferated better than those on scaffolds with smooth surface, indicating that rough surface had positive impact on BEAS-2B cells adhesion, proliferation, and migration as compared with smooth surface (Fig. 3A). The behavior of cells may be influenced by different topographic sizes, ranging from macroscale to microscale and nanoscale features ²⁴. The prepared 30% SF/HPMC scaffolds sprayed with water had the morphology of microscale grid on the surface, and this structure might facilitate the BEAS-2B cells attachment compared with those grown on the smooth surfaces, which was similar with the osteoblast ²⁵. This may be because the porous surface constituted a better substrate for cell attachment than the smooth surface ^26,27. Girault E et. al. thought that cell adhesion occurs preferentially on a surface which limits cell deformation in order for the cell to fix with a minimum of deformation energy, and thus limited roughness promotes cell adhesion ²⁸.

In addition, the porosity and pore size of the scaffolds play a crucial role in cell growth and tissue formation. Porosity and interconnection within the scaffolds are necessary for tissue to distribute oxygen as well as nutrients ²⁹. Results showed that the porosity of prepared 20% SF/HPMC scaffolds was higher than that of 30% SF/HPMC scaffolds. Cell proliferation on SF/HPMC scaffolds showed some very interesting results. The cell proliferation on 20% SF/HPMC scaffolds with high porosity was better than that on 30% SF/HPMC scaffolds with low porosity and smooth surface. However, the 30% SF/HPMC scaffolds with lower porosity and rough surface showed the best cell proliferation, suggesting that the proliferation of cells could be affected by combination of different parameters of the scaffold rather than single factor.

The 3D printed SF/HPMC scaffolds with different macro-pore size were fabricated by adjusting the distance between the filaments during the printing process. 20% SF/HPMC solution was chosen to fabricate the scaffolds with the different macro-pore size. It was observed that cells on scaffolds with smaller pore size proliferated better than that on scaffolds with larger pore size (Fig. 3B). This finding was consistent with the previous studies with 3D printed scaffold ²⁴. In general, very small pore size could limit the transport of nutrients and cell proliferation, and pore sizes above 300\(\text{u}\)m could facilitate the vascularized tissue formation ³⁰. However, cell attachment will also be affected if the pore size is too large, and negatively affecting cell spreading and differentiation ³¹. Therefore, pore size should be within a proper range to maintain the balance between the optimal pore size for cell proliferation, nutrients transportation, vascularization and specific surface area for cell attachment as well as mechanical stability ⁶.

The BEAS-2B cells viability on different SF/HMPC scaffolds was further analyzed by confocal microscopy observation of live/dead staining. The images showed that BEAS-2B cells on six types of SF/HPMC scaffolds kept alive (staining green) at 1, 3, and 7 days (Fig. 3C). No dead cells (staining red) was found during the culturing period. The cells proliferated along the single printed filament.

Cell attachment and morphology

Cell morphology on different scaffolds were studied by SEM. Images showed that cells could attach on all six types of SF/HPMC scaffolds. However, lots of cells attached on the scaffolds with rough surface (20% and 30% SF/HPMC scaffolds sprayed with water) at day 1. And cells on 30% SF/HPMC scaffolds with smooth surface were scattered and isolated with spherical shape. At day 3 and 7, SEM showed that cells grew in a sheet on the rough surface and tended to grow in clusters on the smooth surface (Fig. 4A-L); At day 7, cells on scaffolds with small pore size proliferated along the surface of scaffolds and formed cell bridging between the printed filaments (Fig. 4N, P). In addition, it seemed the cells on the 3D printed SF/HPMC scaffolds with large pores preferred to aggregate on the side wall of large pores (Fig. 4M, O), and it was difficult for them to form a bridge between the filaments. This might be inappropriate for 2D tissues such as epithelium ³⁰.

In this study, we fabricated the 3D porous SF/HPMC scaffolds by 3D printing and investigated the suitable scaffolds parameters (surface roughness, porosity, and pore size) for tracheal epithelium growth. The 3D-printing combined with vacuum freeze-drying technology enabled us to formed porous SF/HPMC scaffolds with a structure of rough surface, small pore size, and suitable porosity, which was beneficial to the exchange of nutrients and metabolites and conducive to the attachment and proliferation of BEAS-2B cells. These findings indicate that the 3D printed SF/HPMC scaffolds could be potential as a suitable scaffold for tracheal epithelium regeneration.

Materials

Polyethyleneglycol (PEG) was purchased from Ourchem (PEG20000, Ourchem, China). HPMC powders was purchased from Aladdin (Aladdin, China). Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (10% FBS) and 1% penicillin and streptomycin were purchased from Hyclone (Hyclone, USA). Calcein-AM and propidium iodide (PI) were purchased from Sigma (Sigma, USA). CCK-8 kit was purchased from Mesgen (Mesgen, China). BEAS-2B cells were purchased from ATCC.

Preparation of SF and HPMC aqueous solution

The preparation of aqueous SF solution was the same as we described in our previous research ¹². Briefly, sericin in cocoon of B. mori silkworm was removed by boiling in Na₂CO₃(0.5 wt%) aqueous solution for 45 min. Then the SF was washed with distilled water and dried at 37℃. Dried silk fibroin was dissolved in 9.3 mol/L LiBr solution at 60℃ for 1h. After been dialyzed for three days, the SF solution was dialyzed against polyethyleneglycol (PEG) (PEG20000, Ourchem, China) solution and the concentration of 30–32 wt% SF solution was obtained. Then the concentrated solution was diluted to 20 wt% and 30 wt% with deionized water respectively. HPMC powders (Aladdin, China) were dissolved in deionized water to form a 10 wt% solution.

Scaffold fabricating

In this experiment, SF/HPMC gel was prepared for printing according to the method of Luo K et al ¹⁶. To obtain the different porosity of 3D printed SF/HPMC scaffolds, the different concentration of SF solution (20 wt% and 30 wt%) was used. Scaffolds with different degrees of roughness were prepared by spraying water on the surface of the scaffold during the printing process and combining the freezing process; 20 wt% SF/HPMC scaffolds of two different pore sizes were prepared by adjusting the printing parameters. The 10 wt% HPMC solution was added to 20 wt% and 30 wt% SF solutions. The volume ration of SF to HPMC was 9:1. Two mixtures (20% SF/HPMC and 30% SF/HPMC) were poured into nozzles of the 3D printer and then transferred to a gel state at 70℃ for 1 h. 3D SF/HPMC scaffolds with pre-determined shape and dimension were fabricated by using an extrusion-based 3D bioprinting machine (Regenovo 3D Bio-printer, China). The 20% SF/HPMC and 30% SF/HPMC scaffolds were printed into a square shape. Six kinds of 3D printed SF/HPMC scaffolds were fabricated (20% SF/HPMC with smooth surface, 20% SF/HPMC with rough surface, 30% SF/HPMC with smooth surface, 30% SF/HPMC with rough surface, 20% rough surface SF/HPMC with small pore size and 20% rough surface SF/HPMC with large pore size). To obtain the rough surface of scaffolds, the scaffolds were sprayed water by using sprinkling can after the print process. Then all scaffolds were frozen at -20℃ for 24h and freeze-dried for 2 days. At last, freeze-dried 3D printed scaffolds were immersed in 75% ethanol for 1 hour at room temperature for further formation of stable β-sheet structure. The macro-pore size of 3D printed SF/HPMC scaffolds was determined by the spacing between the filaments extruded from the nozzle. To fabricate the different macro-pore size of 3D printed SF/HPMC scaffolds, 20% SF/HPMC mixture was used, and the scaffolds were sprayed with water after the completion of printing. Adjusting the spacing of printing by software, scaffolds with two different macro-pore sizes were constructed.

Scaffold characterization

Morphology of the 3D printed SF/HPMC scaffolds

The surface morphology and pore size of 3D printed SF/HPMC scaffolds were investigated using a scanning electron microscopy (SEM) (Hitachi S4800 microscope, Japan). The surface and cross sections of SF/HPMC scaffolds were coated with Au and then observed by SEM (n = 3 per group). Image J software was used to measure the diameter of the pore size of 3D printed scaffolds.

Porosity measurement

The porosity of 3D printed SF/HPMC scaffolds were measured according to liquid displacement method using hexane which is a non-solvent of SF ³⁰. The porosity was calculated according to the following formula: Porosity = [(V₁-V₃)/(V₂-V₃)] × 100% (n = 5 per group), where V₁ is the volume of hexane, V₂ is the total volume of the hexane and 3D printed SF/HPMC scaffolds impregnated with the hexane, and V₃ is the volume of the remained hexane which the hexane impregnated SF/HPMC scaffolds were removed.

Water uptake

The water uptake and the water content of the 3D printed SF/HPMC scaffolds were calculated as follows (n = 5 per group):

Water uptake= (W1-W2) / W2

Water content (%) = [ (W1-W2)/ W1] ×100%

The 3D printed SF/HPMC scaffolds were immersed in distilled water for 24h. W1 represents the wet weight of the scaffold. W2 represents the dry weight of the scaffold.

The results were presented with the average of five scaffolds.

In vitro evaluation

Cell proliferation on 3D printed SF/HPMC scaffolds

BEAS-2B cells (ATCC, USA) proliferation on 3D printed SF/HPMC scaffolds were assessed by CCK-8 assay analysis. The six types (20% SF/HPMC with smooth surface, 20% SF/HPMC with rough surface, 30% SF/HPMC with smooth surface, 30% SF/HPMC with rough surface, 20% rough surface SF/HPMC with small pore size and 20% rough surface SF/HPMC with large pore size) of printed SF/HPMC scaffolds were used. The printed SF/HPMC scaffolds with the size of 5mm length ×5 mm width × 2mm height were put in 48-well plates. The 20 ul of BEAS-2B cells suspension containing about 4.5 × 10⁴ cells were seeded on the scaffolds. After half an hour, the fresh Dulbecco's Modified Eagle Medium (DMEM) (Hyclone, USA) medium containing 10% fetal bovine serum (10% FBS) and 1% penicillin and streptomycin (Hyclone, USA) was added into each well. After 1, 3, 5 and 7 days, scaffolds were transferred to a new 48-well plate. And then 200 ul of medium containing 50 ul CCK-8 solution was added and incubated for 3 h at 37 ℃ with 5% of CO2. Then, the solution (100 µL) was pipetted into a 96-well plate, and the absorbance at 450 nm was measured using a microplate reader (Bio-Tek Instruments, USA). Six scaffolds of each group were used for each culture period.

Cell morphology on 3D printed SF/HPMC scaffolds

The morphology and proliferation of BEAS-2B cells on six types of printed SF/HPMC scaffolds were observed by SEM (Hitachi SU8000 microscope, Japan). After cultured for 1, 3, and 7 days, the BEAS-2B cell-scaffold constructs of each group were washed with phosphate-buffered saline (PBS) and were fixed in 2.5% glutaraldehyde and 1% osmic acid. Then the scaffolds were dehydrated using a graded series of ethanol. After being critical-point dried in carbon dioxide and coated with gold platinum, the cell-scaffold constructs were observed with SEM at an accelerating voltage of 10 kV.

Cell viability on 3D printed SF/HPMC scaffolds

The viability of BEAS-2B cells on six types of printed SF/HPMC scaffolds was further investigated by live/dead assay (Sigma, USA). The prepared SF/HPMC scaffolds with the same size of the above were put in 48-well plates. The BEAS-2B cells were suspended in the culture medium and 20 ul cell suspension containing 10 × 10⁴ cells were seeded into scaffolds. The cell-scaffold constructs were added with 200 ul medium after being incubated for half an hour to allow cell to adhere. After 1, 3, 7 days, 20 uM calcein-AM (staining live cells) and 1ug/ml propidium iodide (PI, staining dead cells) in PBS were added and incubated for 20 min at 37°C. The cell-scaffold constructs of each group were imaged by confocal laser scanning microscopy (CLSM) (Leica, Germany) at certain wavelengths of 488 nm (Calcein-AM) and 535 nm (PI).

Statistics

All values in this paper are expressed as mean ± standard deviation. The associated calculations were performed using Graphpad prism 6. The porosity and water content of the two scaffolds with different pore sizes, and the proliferation of BEAS-2B cells on the two scaffolds were analyzed by unpaired t-test for significance testing (p < 0.05). Other data were compared using the one-way ANOVA test. Significance levels were defined as p < 0.05.

Authors Contributions

NZ and ZC performed the experiments and wrote the manuscript. NZ and ZC contributed equally to this manuscript. XZ and ZS supervised the work. TD participated in scaffold preparation and analyses. All authors contributed to the article and approved the submitted version.

Funding

The author(s) disclosed receipt of the following financial sup- port for the research, authorship, and/or publication of this article: This work was financially supported by Shanghai Science and Technology Committee (Grant Number 17441901600), Shanghai Natural Science Foundation (Grant Number 19ZR1407700) and Application cultivation plan of Jiangxi Provincial department of science and technology (Grant Number 20212BAG70013).

Availability of data and materials

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This study was approved by Ethical Board of Huashan Hospital, Fudan University.

Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Consent for Publication

Not applicable

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No competing interests reported.

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The influence of the structure (surface roughness, pore size, and porosity) of 3D printed silk-fibroin-based scaffolds on the growth of the bronchial epithelial cells in vitro

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Abstract

Figures

Background

Results And Discussion

Scaffold fabrication

SF/HPMC scaffold characterization

Scanning electron microscopy (SEM)

Porosity and water content of SF/HPMC scaffolds

In vitro evaluation

BEAS-2B cells proliferation and viability on different scaffolds

Cell attachment and morphology

Conclusions

Materials And Methods

Materials

Preparation of SF and HPMC aqueous solution

Scaffold fabricating

Scaffold characterization

Morphology of the 3D printed SF/HPMC scaffolds

Porosity measurement

Water uptake

In vitro evaluation

Cell proliferation on 3D printed SF/HPMC scaffolds

Cell morphology on 3D printed SF/HPMC scaffolds

Cell viability on 3D printed SF/HPMC scaffolds

Statistics

Declarations

References

Additional Declarations

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