Evaluation of fibrin-gelatin hydrogel as biopaper for application in skin bioprinting: An in-vitro study

2.1.1. Fibrinogen and thrombin extraction

We employed a simple combination of physical and chemical precipitation methods to purify fibrinogen and thrombin from human blood for the purpose of preparation fibrin hydrogel (Schematic 1). Briefly, fibrinogen and thrombin were isolated from fresh whole human blood after separation of plasma under a mild centrifugation (3000 rpm, 2°C, 20 min) followed by keeping it in −8°C for 48 h. Before extraction, the plasma was slowly thawed in °C for another 48 h. The fibrinogen precipitated after adding 15 ml protamine sulfate (Exir Pharmaceutical co, Iran) to 45 ml plasma and collected using centrifugation (1600 rpm, 2°C, and 15 min). The extracted fibrinogen underwent a freeze drying procedure to have a fine powder. For thrombin extraction, the plasma thawed in the incubator (3°C) followed by adding 0.1 g of kaolin (Merck, Germany) to the plasma (30 ml). 13.5 ml of CaCl₂ solution (0.0146 g/ml) added to the plasma and the mixture transferred to the incubator (3°C, 10 min). The gel like part of solution was removed and the supernatant was dried under freeze drying. Schematic 1 shows the major steps of the fibrinogen and thrombin production procedure.

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Schematic 1.

A schematic representation of fibronectin and thrombin extraction from blood.

In order to accurately examine the properties of different compositions of fibrin and gelatin, 4 different weight compositions with fibrin: gelatin ratios of 1:0 (pure fibrin), 3:1, 1:1 and 1:3 were prepared, which will be referred to as FG1, FG2, FG3 and FG4, respectively.

Briefly, the required amounts of extracted fibrin and gelatin (Type B, Merck, Germany) were weighted so as to result in a final concentration of 10 weight percent for each sample. In order to prepare fibrinogen solution, fibrinogen powder was dissolved in double distilled water and stirred for 10 min at 300 rpm. After adding gelatin powder at 6°C, and then stirring for 10 min at 300 rpm, the resulting clear solution was poured into the mold and thrombin enzyme solution was added (equal amount of fibrinogen). The desired hybrid hydrogel was formed by reaching to the environmental temperature (Schematic 2) [21]. The prepared samples were held at °C before utilization in experiments.

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Schematic 2.

A schematic representation of gelatin/fibrin hydrogel.

2.2.1. Water uptake analysis

After preparing the films, they were cut to 1 × 1 cm² square samples. These dried samples were weighted ( W 0 ) and then immersed in water for 24 h. The samples were weighted 1, 2, 12 and 24 h after immersion, ( W w ) finally the percent of water absorption at each time point was calculated using the following formulation: (1) S r = W w − W 0 W 0 × 100

2.2.2. In vitro biodegradation analysis

Degradation rate and level of hydrogel samples were examined in phosphate buffer (PBS) environment at 3°C. After fabrication of the samples, they were cut in 5 mm thicknesses so as to be divided into 4 equal parts. Each sample was immersed in 5 ml of phosphate buffer solution for at most 7 days and the medium was changed every day. On days 1, 2, 3 and 7 after immersion, a sample was taken out and dehydrated using freeze drying method and the dry weight was determined. Degradation level was calculated based on the alterations in dry weight using the following formulation: (2) Degradation % = W 0 − W 1 W 0 × 100 Where, W 0 and W 1 represent the initial weight of hydrogel before water absorption and the dry weight of hydrogel after immersion in phosphate buffer, respectively.

2.2.3. Glucose absorption test

After preparation of samples, glucose powder was dissolved in 20 ml of water to give a 30 mM solution. Then, each sample was immersed in glucose solution and after closing the cap of bottle, kept it in 3°C water bath. Absorption of solute by the hydrogel leads to a reduction in its concentration in the solution surrounding the hydrogel. Glucose absorption levels after 5, 15, 30 and 60 min were estimated by measuring glucose concentration in the surrounding solution, using an IME-DC blood glucose meter (IME-DC, Germany), via sampling the solution at consecutive time points.

2.2.4. Mechanical compression test

Cylindrical hydrogel samples were subjected to unconfined compression with a rate of 2 mm/s. Using the initial length and cross section area of the samples and the resulting force-displacement data, stress-strain curves were obtained. Although we attempted to minimize the slippage of samples between grips, little slippage was inevitable due to the horizontality of the grips.

2.2.5. Water vapor transmission test

The resistance to transmission of water vapor is usually measured by diffusivity. A mathematical model for calculating water vapor permeability (WVP) of synthetic polymer films has been previously developed (Eq. (3)): (3) W V P = W V T R × Δ Z P ( R 2 − R 1 ) Here, Δ Z is film thickness in m, P is the vapor pressure of pure water at 25°C which equals 3169 Pa, ( R 1 − R 2 ) representing the difference in wetness between the two sides of the film and WVTR is the water vapor transmission rate which can be calculated using the following formulation: (4) W V T R ≡ Δ W A × Δ t Where Δ W represents the weight of transmitted water, A indicates film area in m² and Δ t is test time [4].

In order to perform the experiment, samples of the four different compositions were first prepared as circular films (3.5 cm in diameter and 1 mm in thickness) and then utilized as sealed caps on the mouth of glass bottles. Thereafter, they were placed in a 90% humidity incubator for 24 h and then weighted. Weight difference obtained for each sample was equal to the amount of water passed through the hydrogel film and used in Eqs (3) and (4) to calculate water vapor diffusivity.

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Fig. 1.

The petri dish containing hanging drops containing fibroblast cells.

2.3.1. Cell culture

Frozen fibroblasts (fibroblast cell line 3T3) were provided by Razi pharmaceutical research center of Iran University of medical sciences. After thawing, cells were transferred to a T25 culture flask containing 5 ml of DMEM supplemented with 10% fetal bovine serum and 1% pen-Strep and incubated at 3°C and 5% CO₂. The medium was changed every 3 day and cells with 90% confluency were passaged using trypsin/EDTA.

2.3.2. Cell pellet culture

Hanging drop method was used to prepare cell pellets [6]. Briefly, the prepared cell suspension containing 4 × 10 4 cells/ml was dropped onto the cover of a petri dish (Fig. 1). After carefully inverting the cover and placing it on the petri dish containing a little water to prevent dryness, hanging drops were incubated at 3°C and 5% CO₂ to obtain cell. Figure 3 illustrates the petri dish containing hanging drops.

2.4.1. Evaluation of cell pellet appearance

Uniformity in size and appearance is a desired feature when characterizing cell pellets, hence in the current study, prepared pellets were examined after 1, 2 and 4 days using an optical microscope and classified based on their shapes and sizes [6].

2.4.2. Cell viability within cell pellets

In order to assess cell viability, cells inside the pellet were first separated using collagenase enzyme and then stained with trypan blue. Dead cells were stained blue and viable cells remained uncolored. Cells were counted and the results were expressed in percentage.

2.4.3. Interaction between cell pellets and substrate

The success of the interaction between cellular ink and biopaper, depends on their ability in creating an integrated cellular structure as well as the rate of tissue fusion. Measuring of adjacent cell pellets contact angle was used to evaluate tissue formation ability by placing two cell pellets inside the hydrogel, after which contact angle between the two adjacent cell aggregates was measured using images taken at consecutive days [7].

First, a sample of the characterized hybrid hydrogel was prepared on a disk (with a diameter of 2 cm and a height of 0.5 cm) and placed in a 3.5 cm diameter plate which was then added to 3 ml of medium culture. After washing cell pellets with PBS, they were injected into the hydrogel disk using a syringe similar to that of the bio printer. Making use of an optical microscope, two adjacent pellets with appropriate position relative to each other were chosen and marked for future observations. Thereafter, they were incubated at 3°C and 5% CO₂ for 10 days and the medium was changed every day. The ability and rate of tissue formation was quantitatively examined by calculating contact angles recorded in images.

3.1.1. Water uptake

The similarity between the amounts of water trapped in hydrogel and water content of the target tissue is an indication of success in mimicking native ECM by the fabricated scaffold [17].The water uptake analysis of samples at different time points was summarized in Fig. 2(a). As it was expected, water absorption was increased over time in a logarithmic manner so that slope of the plot was decreased with time (Fig. 2(b)). The amount of absorbed water was essentially decreased with time and therefore, the amount of water absorbed in the first hour is not identical to that at other time points. This might be assigned to the saturation of hydrogel with water, and hence reaching the equilibrium point. Finally, slope of the plot reached zero, implying that the sample has reached its maximum absorption level.

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Fig. 2.

Water uptake behavior of different samples at different time points. (a) Water uptake (%) vs. sample fibrin content after 1, 2, 3 and 4 h. (b) The typical water uptake cure of FG2 during incubation time till 24 h. (c) Water uptake of samples as a function of fibrin content at 24 h.

Comparing different samples demonstrated that increasing gelatin percentage could enhance water absorption at all 4 time points. For instance, water uptake of samples (FG1 to FG4) after 24 h showed a linear relation as a function of the fibrin content (Fig. 2(c)). So the desirable water uptake could be adjusted based on controlling fibrin content in hybrid hydrogel.

Samples with higher gelatin contents (25 to 50% fibrin) seemed more suitable in terms of water uptake. This can be explained by the chemical and three dimensional (3D) structures of gelatin and fibrin as well as their crosslinking and gelation behavior. 3D microstructure of gelatin may provide more porosity in the network resulting in more water absorption. On the other hand, since gelatin is a temperature-sensitive hydrogel and fibrin is an enzymatic one, less compaction and organization is achieved during gelation and hence, more porosity is provided in the network to absorb more water.

Degradation analysis results were presented in Fig. 3. As it is shown, the degradation rate was not constant during the time and showed fast increasing over time. It was speculated that following an initial degradation of the samples, their integrated structure might be ruptured and the rate of degradation accelerated. Comparison of different samples demonstrated the amount of degradation elevated by adding fibrin components of the samples (Fig. 3(b)).

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Fig. 3.

Degradation behavior of fibrin-gelatin hydrogel samples at different time points (a). Percent of degradation vs. sample fibrin content at day 7. (b) The typical degradation cure of FG2 during incubation time. (c) Degradation (%) of samples as a function of fibrin content.

Considering degradation rate, it could be possible to estimate the time required for complete degradation of each sample. In this regard, FG1 (pure fibrin) seems to be completely degraded after 14 days when full rupture of the structure as well as the release of fibrin fibers was evident. At this time point, FG2 and FG3 showed almost fast degradations too. Surprisingly FG4, with 25% fibrin, significantly kept its structural integrity, and appeared to need at least 3 weeks for full degradation, which seems a long time for our desired application. This observation can be directly assigned to the chemical and 3D structures of fibrin and gelatin as well as their cross-linking and gelation performance and the strength of produced bonds in the structure.

As it was previously mentioned, degradation of the hydrogel with 75% gelatin was not high enough for our intended application. Since very fast degradation of the hydrogel with 75% fibrin, might disturb cell growth and does not allow complete production of extracellular matrix by cells. Meanwhile, Hydrogels containing 30–50% fibrin provide optimized degradation conditions.

Duo to importance of glucose as a main cell nutrient, glucose absorption was investigated. As it was shown in Fig. 4, the percent of absorbed glucose was increased over time where the initial rate of absorption was the same for all samples and the rate decreased by time. This may be due to saturation of hydrogel with glucose, and therefore, absorbing less glucose over time. FG4 showed more absorbed glucose compared to other samples at initial and final testing time. Although the trend of glucose absorption was like FG 1 > FG 2 > FG 3 , there was no significant difference among these three samples at 60 min.

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Fig. 4.

Glucose absorption profile of fibrin-gelatin hydrogel during time.

It is expected blending hydrogels and forming blend network changes pore size of pure ones [10]. Blending gelatin with fibrin at different weight ratios could provide various microstructures with different integrity and pore size where the glucose diffusion showed decreasing with increasing gelatin content.

Finally, all samples with different ratios of gelatin and fibrin showed almost similar values of glucose absorption in the range of 4–4.5 mM/L, and seemed suitable compared to the native tissues of the body, in terms of providing required nutrients for cell growth.

Force-displacement data obtained by the compression test were used to determine young’s modulus of the samples as the main parameter for comparison with mechanical properties of native skin so as to evaluate the similarity of this target tissue. Because of the low mechanical strength of FG1 and FG2 (with 100% and 75% fibrin, respectively), they could not be appropriately captured by grips of the device and therefore were reported as samples with no mechanical strength. This was already expected for samples with low gelatin contents, as fibrin is a low-strength material and gelatin was expected to mechanically support the structure. The resulting data for 25% and 50% gelatin-containing samples were utilized to obtain stress-strain curves (using initial length and cross section area data), in which the slope of the linear region demonstrates the elasticity modulus. The resulting values of young’s modulus for FG3 and FG4 compared to pure gelatin hydrogel were depicted in Fig. 5. As it is evident, fibrin contents between 45% to 50% result in young’s modulus values in the range of 37 to 40 kPa, which is an acceptable range compared to the native skin [2,3,15].

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Fig. 5.

Mechanical analysis of sample Young’s modulus under compression test compared to pure gelatin.

Water vapor transmission is usually studied in skin-related researches, because low ability of desired dressings in water vapor transmission results in water retention in the wound while excess levels of such ability will cause wound dryness [16].

Based on the obtained results, reported in Table 1, water vapor permeability is considerably increased by increasing fibrin content of the hybrid hydrogel.

Based on the all characterization performed on the fibrin-gelatin biopaper with different composition, FG3 with 1:1 fibrin:gelatin ratio was selected as an optimized hydrogel composition for further analysis in present of cell aggregates.

3.2.1. Evaluating of cell pellets

Appearance of cell pellets were examined on days 1, 2 and 4 after culture. As it is obvious from Fig. 6(a) (for typical three drops), the utilized 3D culture protocol, hanging drop, resulted in almost uniform pellets in terms of appearance and morphology. Half-sphere shapes and the suitable cell confluency could be also observed.

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Fig. 6.

Evaluation of cell pallets formed by hanging drop 3D culture. (a) Cell pellets formation during culture time (b) size distribution of prepared cell pallets.

Size is a critical cell aggregate characteristic. It strongly affects cell viability, particularly by affecting the oxygen and nutrient availability to the central portion of the aggregate [6]. The diameter distribution among 100 formed pellets is illustrated in Fig. 6(b). The majority of pellets were associated with diameters between 200 to 250 μm, which is an ideal range of bioink aggregates.

Keeping cell viability for the cells in the core of cell aggregate is necessary to continue aggregate culturing inside hydrogel. The estimated cell viability percentage was around 83%. Although this viability seems not enough in a common cell culture condition considering high local cellular density after deposition of aggregates into the hydrogel, cell damage could be negligible. In addition, using trypsin enzyme might damage cell and results decreasing cell viability.