Cytoskeletal regulation on polycaprolactone/graphene porous scaffolds for bone tissue engineering

Fabrication of scaffolds

The fabrication of PCL (Mn: 80,000, Sigma, USA) and PCL/G scaffolds was achieved using a solvent casting and particle leaching technique³⁷. The PCL was dissolved in chloroform at a temperature of 20 to 25°C for a duration of 12 h. The mixture was subsequently combined with different concentrations of graphene and sodium chloride (< 1μm in size) for a duration of 2 h.

Graphene was produced by transferring a graphite intercalation compound into a crucible that had been preheated to a temperature of 700 °C. This process took place in a regular furnace situated in front of a fume cupboard to ensure safety against inhaling nanoparticles. The compound was then kept in the crucible for 60 s. The layers were expanded through the use of ultrasonication, resulting in the dispersion of graphene within the solvent. After the fabrication process, the mixture was poured and then allowed to cure at ambient temperature for the duration of the night.

The chloroform underwent a 24-hour evaporation process at a temperature of 37 °C in a vacuum oven for drying purposes and was evaporated for 24 h at 37 °C in a drying vacuum oven (Deng Yng, Taiwan). To remove the porogen from scaffolds, deionized water and a water bath (BH-130D, Taiwan) were used. Furthermore, the water that was deionized was changed every 2 h and then dried in an oven at a temperature of 50 °C for a period of 12 h³⁸.

Cell culture

Scaffolds used for cell culture had dimensions of 10 × 10 × 2 mm³ and were composed of different graphene weight ratios. The specimens underwent sterilization in a 95% ethanol solution for 24 h and were subsequently washed 3 times in a 1× PBS solution to eliminate any traces of ethanol that could be left behind. Before placing the cells, scaffolds were immersed in Dulbecco’s Modified Eagle Medium (DMEM; Gibco-Invitrogen) for a duration of 3 h.

Osteoblast-like (MG-63) and mouse fibroblast (L-929) cells were obtained from 3D Global Biotech Inc, Taipei, Taiwan. The cells were grown in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin, and were incubated at 37 °C in a 5% CO₂ environment. However, the culture medium was replaced at intervals of 2 to 3 days. After reaching 80% confluency, the cells were detached and transferred to a new culture using a solution of 0.25% trypsin-EDTA (Gibco, USA)⁸.

Extract of scaffolds

Scaffolds surface area was determined with the following equation:³⁹

In the given formula, π was used to represent the value of 3.14, while r stood for the radius, and h represented the height. Furthermore, the DMEM solution was enhanced by including 10% FBS and 1% penicillin/streptomycin according to the following formula:

The samples were transferred in a conical centrifuge tube with a capacity of 50 mL and then agitated in a shaking water bath set at a temperature of 37 °C and a speed of 100 rpm for a duration of 24 h. The extracts underwent filtration using a Millipore filter unit (Sartorius, France) equipped with a 0.22 μm pore size polyethersulfone membrane.

Preparation of scaffolds and cell seeding

The scaffold used for cell culture were 10 × 10 × 2 mm³ in dimension and contained varying G weight ratios. They were sterilized by immersion in a 95% ethanol solution for 24 h, then rinsed three times with 1x PBS to remove any ethanol residue. Prior to cell seeding, the scaffolds were incubated in Dulbecco’s modified Eagle medium (DMEM; Gibco- Invitrogen) for 3 h³⁸.

For cell culture, osteoblast-like (MG-63) and L-929 cells at passage 5 (provided by 3D Global Biotech Inc, Taipei, Taiwan) were grown in culture plates with DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin. Incubation was carried out at 37°C with 5% CO₂, and the medium was refreshed every 2 ~ 3 days. When cells reached 80% confluency, they were detached using 0.25% of trypsin-EDTA (Gibco, USA), and each sample was seeded with 0.5 mL at a concentration of 10⁴ cells/mL in 24-well plates, then incubated for 14 days. The medium was refreshed every 2–3 days. Cell samples were collected from the incubator for evaluation on days 7 and 14^36,40.

To assess cell adhesion on the scaffold surface, scanning electron microscopy (SEM) was employed. After the culture medium was removed, samples were washes with 1 x PBS, then fixed in 0.6 mL of 2.5% glutaraldehyde in PBS for 30 min at 4 °C. Following two 1 x PBS washes, scaffolds were dehydrated in a graded ethanol series (30, 50, 70, 90, and 100%). The scaffolds were then placed in a critical point drying (CPD) chamber. Ethanol was replaced with liquid CO₂ by pressurizing the chamber to dissolve the ethanol in the CO₂ liquid phase, a process taking around 30 min. The chamber was gradually heated to the CO₂ critical temperature (31.1 °C ) while maintaining pressure above the critical threshold (approximately 73.8 bar). After the transition, the chamber was carefully depressurized, allowing CO₂ to convert from liquid to gas without passing through an intermediate liquid phase, thus preventing ice crystal formation. Once the pressure stabilized, the scaffolds were retrieved and stored in a desiccator until SEM analysis. For SEM visualization, the samples were gold-coated via sputter coating and observed at an accelerating voltage of 5 kV⁴¹.

Quantification of lamellipodia area (lamellipodia morphometry)

Lamellipodia region of MG-63 and L-929 cells was assessed at 7 and 14 days of incubation using Fiji software (NIH, USA). A total of samples was analyzed for each group, and after background subtraction, individual cells were selected using the wand tool, and their area and perimeter were calculated through the application of a threshold function. Lamellipodia were quantified by manually delineating regions of interest (ROIs) using a freehand tool as shown previously⁴².

Filopodia analysis using FiloQuant

A total of 5 scanning electron microscopy (SEM) images were obtained to assess cell morphology with a total of 5 samples for each group. The density and length of filopodia were analyzed using the FiloQuant plugin (version 1.52i) in ImageJ. Filopodia in the maximum intensity projections were analyzed through manual tracking and the single image tool. The filoQuant was used for filopodia length measurement. Additionally, filoQuant analysis defined the density of filopodia as the ratio of the value of identified filopodia to the length of the cell edge^22,42.

Scratch wound assay (migration)

A volume of 1 mL of mouse fibroblasts (L-929) and osteoblast-like cells (MG-63) at a concentration of 3 × 10⁴ was cultured in 12-well plates and incubated until these cells reached 90% confluency within 24 h. After the medium was discarded, the surface was scraped with a 200-µL tip, followed by rinsing with phosphate-buffered saline (PBS) to eliminate dislodged cells and debris. The extract from scaffolds was then added to each well as a chemoattractant reagent. The assay was performed using an Olympus IX73 inverted microscope at 100× magnification and processed with Gen 5.0 software. The wound edges’ distance was measured at 24 and 48 h and analyzed using ImageJ software. The calculation for wound closure was performed using the previously showed equation:⁴³.

In this equation, A_t=0h represented the initial measurement of the wound area immediately after scratching, and (:{A}_{text{t}={Delta:}text{h}}:)represented the measurement of the wound area h hours after the scratch. The migration rate could be determined using the following equation:⁴³

In this equation (:{R}_{m}) denoted the migration rate of cells, (:{W}_{i}) referred to the initial width of the wound, (:{W}_{t}) represented the final width of the wound, and t signified the duration of migration. The study used 7 samples for each group.

Tensile test

The Shimadzu universal testing machine from Japan, equipped with a 250 N load cell was used to conduct the tensile test. The experiments were conducted at ambient temperature using a crosshead, and 5 samples were created by slicing scaffolds with dimensions of 40 × 20 × 10 mm³. The stress-strain curves were used to calculate the toughness, yield strength, and yield strain of each material. The toughness was determined using an equation:¹⁸

where T represented the toughness, σ was stress, and ε was strain. The yield strength was determined using the equation:⁴⁴

where (:{sigma:}_{y}) represented the yield strength, (:{F}_{y}) was the force, and (:{A}_{y}) was the cross-sectional area, and the yield strain was calculated using the following equation:⁴⁴

where (:{epsilon:}_{gamma:})was the yield strain, (:{varDelta:L}_{gamma:}) was the total elongation, and (:{Lo}_{gamma:})was the original length.

Pore size analysis

Scaffolds’s pore diameters and morphology were analyzed through SEM (Hitachi, Japan) at an acceleration voltage of 15 kV. ImageJ was used to analyze the pores in the SEM images. Pore diameters were quantified on the SEM image using scale bars that represented a standard distance. The micrometer measurement of pore size was conducted using scaffolds of varying cross-sections as showed previously⁴⁵.

Statistical analysis

Statistical analysis was frequently used to present experimental data for individual sample groups, using the mean ± standard error (SE) format. Each experiment consisted of a minimum of 3 replicates. The data underwent analysis using SAS software, and to identify any statistically significant differences in the data, ANOVA and Tukey’s test were used. For non-normal and non-homogenous distributions, the Kruskal-Wallis test and Mann-Whitney post hoc test were used to evaluate intergroup differences. The significance levels were set at a threshold of p < 0.05⁴⁶.

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• Source: https://www.nature.com/articles/s41598-024-80467-2