· web viewin terms of bone scaffolds, the porous size of scaffolds has a significant effect on...

Preparation and Characterization of the Silk Fibroin 3D Scaffolds with Porous and Interconnected Structure

Jun Song1, 2, Xiaoqin Wang1*, Jiashen Li2*,

1Soochow University, Renai Road, Suzhou, Jiangsu, 215123, China2School of Materials, University of Manchester, Manchester, M13 9PL, UK

*Corresponding author’s email: [email protected] Presenting author’s email address:

1st author’s email address: [email protected]

Abstract

Silk fibroin (SF) is wildly used in the field of biomedical science. Considering the contradiction between suitable pore size and outstanding mechanical properties, this research focused on the topic of preparing the 3D scaffolds with large pore sizes and high interconnectivity. For preparing the silk fibroin 3D scaffold samples, three kind of porogen were applied in two method which paraffin spheres were used in freeze-drying and sodium chloride (NaCl) and sodium carbonate (Na2CO3) were used in leaching respectively. Different pore size of porogen were used to control the pore size and porosities of prepared scaffold samples. Some basic properties of scaffold samples were characterized including surface morphology, chemical structure, porosity and mechanical performance. The best porosity of sample reached to 93% in the rearch.

Keywords: Silk fibroin; Paraffin spheres; Tissue engineering; Porous scaffold

1. Introductions

The silk fashions can be traced back to thousands of years ago. However, the silk did not be considered as biomaterial or other kinds of advanced materials until 1990s. One of the most emerging subjects in this century, tissue engineering, contents varieties of knowledge not only limit to molecular biology but also material engineering[1]. Tissue engineering tries to heal or replace human organs and tissues, if injured, by means of certain scaffolds. The main function of silk as biomaterial, regenerated silk fibroin scaffold, is the enhancement and support to the organs and assistance of tissue reconstruction[2].

Biomaterials for tissue engineering applications could be itemized as following classifications: 1) non-biodegradable materials, such as nylon, dacron and expanded polytetrafluoroethylene (ePTFE). This type of biomaterial is the initially developed one, and is widely applied in the clinical environment now. 2) Artificial synthetic biodegradable polymers: Diversified polymers have already been exploited as tissue engineering, including poly Glycolide (PGA), poly L-lactic acid (PLA), poly Caprolactone (PCL), poly L-lactide-co-glycolide (PLGA), polyethylene oxide (PEO), et al. The major advantages of synthetic materials are the wealth of sources, convince in processing and adjustable biochemistry or physical properties [3]. The disadvantage of polymers cannot be ignored. The hydrophobic polymers lead to the difficulty of cells adhesion and proliferation. Also, the degradation products of artificial synthetic material are acids, which bring about inflammatory reaction [4]. 3) Natural materials: Apart from silk fibroin, collagen, fibrous protein gel and chitosan are taken into consideration for tissue engineering. This kind of biomaterial outperforms the synthetic polymers in cells adhered growth [5]. Easy preparation and low toxicity are the other superiority for natural material whilst its mechanical properties usually under instability when the degradation starts [6]. 4) Combined extracellular matrix materials: It is the unity of artificial synthetic polymer and natural material. It is believed of combined extracellular matrix could hold the good characters from its both

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raw materials. However, the preparation materials and methods are still need to explore in the future studies.

Silk fibroin, derived from Bombyx mori, is a widely used natural protein polymer [7, 8]. The raw silk is consisted of two insoluble proteins, sericin and fibroin [9]. The sericin is smooth and glossy, which coats two independent filaments of fibroin. To obtain pure fibroin, the norming stage is boiling the silk to degum the sericin. The dissolve of fibroin is a huge topic for many researchers. Matsumoto (1996) was one of the first to dissolve the fibroin by using neutral salt (LiBr · H2O–EtOH–H2O) [10]. A similar study by Park reached different method, finding CaCl2–EtOH–H2O ternary solution could dissolve fibroin in room temperature [11]. The two methods stated above are the most favorable of dissolve fibroin.

In recent years, silk fibroin has been applied to 3D tissue engineering scaffolds, but there is a contradiction between its aperture size and mechanical properties [12]. In terms of bone scaffolds, the porous size of scaffolds has a significant effect on the long-term implant efficiency of bone filler and bone tissue[13], while the cellular metabolic efficiency is also related to the scaffold pore size [14].

The research of our predecessors shows that efficacious preparation method of the 3D scaffold needs to take into account the two points, intrinsic advantages of the mechanical properties and biocompatibility, for the material. At present, the focus of 3D tissue engineering scaffold research is to enable cells could grow inside scaffolds [1], a three-dimensional and porous space, to make sure these cells have the desired morphology and function of the human bodies. Large pore size three-dimensional silk fibroin scaffolds can give cells enough space to grow in, whereas the scaffolds have good connectivity pores is also the effect of this experiment hope to achieve.

Further, the chosen processing method also needs to be able to control the key parameters, pore size and porosity, in 3D scaffolds. On the one hand, higher porosity scaffolds facilitate cell growth and migration [15]. On the other hand, the larger pore size scaffolds will improve newly grown cells to adhere to the inner surface because of the higher specific surface area to satisfy the replacement and recovery of the function of the human organ [16]. In particular, the mechanical properties of the scaffolds are of particular importance for the hard tissue parts of the body, such as bone and cartilage tissue. Although the mechanical properties depend mainly on the nature of the material, processing also has some impact on it [17, 18]. In general, scaffold morphology is very important for its function. Therefore, the preparation methods of tissue engineering scaffold capable of producing irregular three-dimensional shape are better [19]. The current situation is that scientists have found a variety of techniques for the preparation of tissue engineering scaffolds, each of which has obvious advantages, but there is no single method efficacious enough to dominate the arena [20].

Freeze-drying method is also named as vacuum freeze-drying technique. Specifically, the wet polymer or scaffold sample is freeze-formed at a relatively low temperature (typically -80 °C, -20 °C, et al.), and the frozen sample is placed in a high vacuum dryer, the use of sublimation principle to remove water in the sample, the final polymer completely dry method[21]. Freeze-drying is an efficient technique for making porous structural scaffolds. The pores left by the sublimation of ice crystals formed at a lower temperature allow the scaffold to avoid cracking caused by stress changes and control over the formation of cryogenic ice crystal [22]. Pore structure changes, prepared a complex microstructure, porosity can change a wide range of high porosity, good mechanical properties of materials. The scaffold porosity obtained by the method can reach more than 90% and the pore size is about 100 μm [23]. One of the features that the method may utilize is that the resulting scaffolds will be oriented, substantially in one direction, and occluded in the other direction. Studies have shown that the pore structure is affected by solvent crystallization [24].

Particle leaching method is also called as salt leaching method [25]. The specific operation steps are: dispersing the selected salt particles in the silk protein solution into a mold such as a plate or a syringe in. Solvent are removed by freeze-drying. In this case, the silk protein has formed a stable crystal structure in the silk protein-salt composite scaffold and the connection rate is good, and the salt particles are removed by the reaction or water washing. The porosity of scaffolds in such preparation methods can be changed by adding or reducing salt particles [26]. The pore diameter of the scaffold prepared by the method is generally between 100 μm and 800μm [27].

This research seeks to address the coordination among mechanical properties, pore size and porosity.

2. Materials and methods

2.1 Materials

Raw silk of Bombyx Mori was purchased from Zhejiang Shengzhou Xiehe Silk Co. Ltd. (China). The paraffin and dialysis tube were purchased from Sigma-Aldrich Co. Ltd. (USA). Salts and organics are purchased form Sinopharm Chemical Reagent Co. Ltd. (China). All other compounds were of analytical purity.

2.2 Preparation of silk fibroin solution

The Bombyx Mori raw silk was degummed to remove sericin that is an extracted protein in the surface of fibroin. The degumming process was to boil the silk in a 0.2% (w/w) sodium carbonate solution for 30 minutes, then thoroughly rinse the fibers with distilled water, remove surface ions, and store in a ventilated plate for 12 h. The degummed silk was dissolved in a 9.3 M lithium bromide aqueous solution at a concentration of 10% by weight at room temperature. The dissolved silk fibroin was dialyzed against a semi-permeable cellulose tube (molecular weight cut-off of 12,000-14,000 Daltons) to give a pure silk fibroin solution free of any neutral salts. The concentration of the silk fibroin solution was concentrated by dialysis with polyethylene glycol (PEG; 20 kilo Dalton) and cellulose tube with molecular weight cut-off 12-16 kD (kilo Dalton) at 25 °C and 48 hours, and distilled water diluting.

30% (v/v) glycerol (Gly) was added to the silk fibroin solution to ensure a mass ratio of SF:Gly of 10:3, and the mixture was uniformly mixed under magnetic stirring (stirring for 15 minutes, stirring speed of 200) Then add 80% polyethylene glycol (PEG; 400 Daltons) to the mixture solution to ensure a mass ratio of SF: glycerol: PEG = 10:5:3 (v/v/v), mix well stirring 15 minutes with stirring speed 200 rpm.

2.3 Preparation of porogens

This study designed three porogens for the treatment of three-dimensional silk fibroin scaffolds.The particle diameters of sodium chloride (NaCl) and sodium carbonate (Na2CO3) were

respectively controlled in the range of 200 μm to 400 μm using a standard test sieve.The paraffin particle preparation process is critical to controlling the size of the finished paraffin

particles. The gelatin was dissolved in distilled water at 80 ° C at a concentration of 30 g / L. The paraffin was added to the gelatin solution to a final concentration of 200 g / L (paraffin w / v). After the paraffin is dissolved, the liquid system is more like an emulsion than a solution. The emulsion was stirred for 15 minutes and then the emulsion was poured into ice water (0 ° C). Paraffin particles in the range of 200 μm to 800 μm were obtained by filtration and thoroughly washed with ultrapure water and dried at room temperature for 3 days. It should be noted that the paraffin/gelatin emulsion is thoroughly stirred at a temperature not lower than 80 ° C to produce smaller bubbles in the emulsion, and smaller bubbles can ensure paraffin particles having a particle size of 200 to 800. Productivity during the experiment. This method of preparing paraffin microspheres is called a dispersion curing method.

In this experiment, the effect of the rotational speed on the size of the paraffin microspheres was also designed during emulsion agitation. Experiments have shown that the rotational speed has little effect on the size of the paraffin microspheres in the range of 400-800 rpm.

2.4 Preparation of silk fibroin scaffolds

A 5-ml syringe was chosen as the experimental stent mold. The plunger at the back of the syringe was pulled out and a quantity of paraffin microspheres or sodium chloride particles or sodium carbonate particles were weighed into the syringe to ensure that the porogen particles reached a height of 2 ml above the syringe marking line. Push the plunger back into the syringe and then gently press.

Insert the needle into the silk fibroin solution and slowly aspirate the solution so that the level of the solution in the syringe is slightly above the porogen height.

Considering that three porogens were set in this study, different treatment methods were applied in this case. The porogen was selected to be paraffin, stored in a refrigerator at -80 ° C for 12 hours, and then dried in a freeze dryer for 3 days. Place a selective sodium chloride or sodium bicarbonate group at room temperature while removing the plunger and waiting for the silk fibroin solution to dry.

After all of the stent samples have dried, they are replaced from the syringe. The paraffin-based porogen was selectively immersed in n-hexane for 2 days, the paraffin was dissolved and washed off, and n-hexane was replaced with cyclohexane. After the replacement, the stent sample was placed in a ventilated plate.

The porogen of the sodium chloride group is selectively immersed in distilled water to dissolve the sodium chloride in water for removal. The porogen was selected to be a sodium carbonate group, and dilute hydrochloric acid (HCl, 10% v/v) was used, and the scaffold sample was reacted under a hydrochloric acid solution to remove the porogen. It is worth noting that when processing the pure silk fibroin solution group, considering that the scaffold itself is soluble in water, it needs to be placed in 90% methanol (CH3OH) for 2 h before treatment. This processing method is considered to be cross-linking of fibroin.

Fig. 1 Schematic for preparing the scaffolds

2.5 Characterization

In this experiment, a total of 5 different porogens were designed, which were: a) paraffin microspheres, 200-300 μm; b) paraffin microspheres, 300-400 μm; c) paraffin microspheres, 400-600 μm; d 300 μm to 400 μm; e) sodium carbonate particles: 300 μm to 400 μm.

The surface morphology and pore structure of the silk fibroin 3D scaffold were observed using a Wield-emission scanning electron microscope (HITACHI S4800, Japan). Samples need to be processed prior to observation. Each set of dried samples was selected, a small piece was cut with a blade, mounted on a sample stage, and then a thin layer of gold was applied over the sample. Set the charging voltage to 3kV and the working distance (WD) = 12.4mm.

Table 1 the group details of scaffolds

Groups Porogen Solution Concentrations

Ratio(porogen:fibroin

solution)

Preparation method

1 Paraffin,200-300 μm

SF+Glycerol +PEG 10% 1.2 g : 1 ml freeze drying





4Sodium chloride,

200-400 μm

SF+Glycerol +PEG 10% 1 g : 1 ml room

temperature



6Sodium chloride,

200-400 μm


temperature

7Sodium

carbonate,200-400 μm


temperature

8 Paraffin,300-400 μm SF 10% 1.2 g : 1 ml freeze drying

9Sodium chloride,

200-400 μmSF 10% 1 g : 1 ml room

temperature

10Sodium

carbonate,200-400 μm

SF 10% 1 g : 1 ml room temperature

The porosity of the stent material refers to the percentage of the pore space in the stent to the total volume of the stent, indicated by the letter ρ. Based on the Archimedes principle, the porosity of the scaffold material is determined by cutting the sample into regular, appropriately sized sheets prior to measurement. Porosity measurement requires the use of an electronic balance. The centrifuge tube was first placed on an electronic balance, and n-hexane was added to about 20 g, which was recorded as mass m1. The stent sample was then placed in a centrifuge tube and the mass m2 was labeled. After standing for 4 hours, the sample was changed, and the mass of the centrifuge tube and the remaining n-hexane solution was m3. Porosity can be calculated by the following equation:

ρ = (m1- m3) / (m2- m3) (1)

Infrared Spectroscopy is mainly used for quantitative infrared analysis to illustrate the molecular structure, spectrum and chemical properties of a sample. The experimental instrument was a Smart Fourier Transform Infrared Spectrometer (Nicolet-5700, USA). It should be prepared to cut the dried

sample into small pieces with a razor blade, place it on a smart Omni handle, rotate the sampler and stop after hearing the sound. Some samples were required to be compressed prior to testing, and a 1 mg sample and 0.2 g of potassium bromide (KBr) powder were mixed to prepare tablets at a constant temperature of 20 ° C and a humidity of 65%.

Compression modulus is the basic mechanical property of a three-dimensional tissue engineering scaffold. The experimental instrument was a mechanical testing machine (INSTRON-5967/3365, USA). Four parallel samples were prepared for each set, all with a height of 12 mm and a diameter of 13 mm. Set the deformation to 30% and the test speed to 20 mm per minute, then place the sample above the center of the fixture during the test.

3. Results and discussion

3.1 Surface morphology

Through the observation and photographing of the fluorescence microscope, it can be seen that the paraffin microspheres prepared by the dispersion solidification method exhibit a three-dimensional spherical shape with a smooth surface and good dispersibility under dry conditions. The sodium chloride particles exhibit cubic crystals and have a certain degree of symmetry. The sodium carbonate particles behave as irregular white particles.

Fig. 2 Fluorescence Microscope of porogen samples: a) paraffin microspheres, 200-300 μm; b) paraffin microspheres, 300-400 μm; c) paraffin microspheres, 400-600 μm; d) 300 μm to 400 μm; e)

Sodium carbonate particles: 300 μm to 400 μm.

Three different particle size paraffin particles were compared (Fig. 2). The black bars in the lower right corner of the chart indicate a 100 μm scale, and the white bars in the lower right corner of b indicate a 100 μm scale. Groups a, b, and c were set to compare the effects of stent porosity, compression, and other properties under the same material porogen at different particle sizes. Comparing the 300-400 μm particles of three different materials (Fig. 2), the white bars in the lower right corner of the b and d images are 100 μm, and the black bars in the lower right corner of the e image are 100 μm. Groups b, d, and e were set to compare the effect of different material porogens on stent porosity, compression, and other properties at the same particle size.

Fig. 3 SEM of scaffold samplesGroup 1-3 (line1): SF with addition, paraffin porogens;Group 5-7 (line 2): SF with addition, three kinds of porogens of same particle sizeGroup 8-10 (line 3): SF without addition, three kinds of porogens of same particle sizeGroup 4 (left): SF with addition, Sodium chloride porogens.

The SEM photograph of Group 1 showed dense but isolated pores, while the pores in the SEM photograph of Group 2 showed a relatively loose fragment-like structure. The SEM image of the third set of stents has a circular and well-perforated pore structure, particularly a layer-to-layer height connection within the stent.

Like the other groups that selected paraffin as a porogen, Group 5 has a dense network of porous structures. The structure of the pores in Group 6 was not uniform, but highly connected, and the portions of the stent were dendritic, showing satisfactory performance. The number of holes in the 7th set of stents is very small. Although there are small holes, there is essentially no connection and the distribution is dispersed, so the experimental purpose in this group cannot be achieved.

The stent set 8 has a morphology similar to the other groups, and paraffin wax is selected as the porogen. The shape of the holes is not regular enough, but it has good thoroughness in this group. The ninth group has a large number of elliptical and circular pores, and is well connected and has a good three-dimensional shape. The space formed by the 10th set of scaffolds is very small and far from the intended experimental purpose.

The scaffold set 3 was less distinguished by SEM images, with groups 2 and 13 showing a layered structure showing a certain degree of orientation. Group 5 presents a more rounded hole.

3.2 Chemical structure

From the above figure (left), groups 5, 6 and 7, in which 10% pure SF solution scaffolds were treated with paraffin, sodium chloride and sodium carbonate respectively, it can be seen that the secondary structure of the three groups is significantly different. In Groups 5 and 7 as porogens, the characteristic peak of amide I was 1644 cm-1, which means that the group exhibited unstable random coils and α- in the scaffold due to limitations of infrared spectroscopy. The spiral structure, which cannot be further distinguished. However, the characteristic peak of the amide I in the scaffold of Group 6 was 1628 cm-1, that is, the conformation was a stable β-sheet structure. The configuration of the Group 7 amide II was also more unstable than the Groups 6 and 7. The most obvious difference is that there is a characteristic peak of about 1390 cm-1 in the 7th set of scaffolds. The amide III component was not apparent in all three stent groups.

From the above (right), groups 8, 9 and 10, select paraffin in a 15% SF solution scaffold, sodium chloride and sodium carbonate as porogens, respectively, and add glycerol (Gly) and polyethylene glycol. (PEG) has a large impact on the performance of each scaffold sample. In the secondary structure, all three groups of amide I characteristic peaks were 1627 cm-1. At this time, SF not only exhibited a β-sheet conformation, but also exhibited a transition from 1628 cm-1 to 1627 cm -1 at a concentration of 10%, indicating that the concentration of the silk fibroin solution had a certain influence on the characteristic peak. The comparison of the characteristic peaks of the three groups of amide II and amide III showed little difference in the secondary structure.

Fig. 4 FT-IR comparison of scaffold samples, upper left: different size paraffin porogens scaffolds; bottom left: different solutions scaffolds; upper right: different porogens scaffolds with addition; bottom

right: different porogens scaffolds without addition

In this study, stents that were neither glycerol nor polyethylene glycol formed a stable β-sheet structure, and stents containing glycerol and polyethylene glycol affirmed literature review in all aspects of characterization: 1) Moderate Compressive strength is an improvement in the specificity of the flexibility of the silk fibroin scaffold, indicating that the additive acts as a plasticizer; 2) by comparing the FT-IR of each group, the amide I characteristic peak of the scaffold appears at 1628 cm-1, indicating that these groups The silk fibroin in the main exhibits a stable Silk II structure.

3.3 Mechanical properties

Considering the compressibility of Fig. 5, the compressive strength of the scaffold with sodium chloride particles as the porogen is significantly higher than that of the paraffin as the porogen. In combination with porosity, the low porosity of the seventh set of compressive strength is much higher than the other groups.

The group using sodium chloride as the porogen was again superior to the paraffin wax selection in terms of compressive strength to verify that the group having a low porosity would have better mechanical properties. As the pore size becomes larger, the compressive strength of the stent becomes weaker. Considering the compression performance in Fig. 5, as the pore size becomes larger, the compressive strength of the stent becomes weak. The results of comparing mechanical properties and porosity are just the opposite. The second group has the worst compressive strength, the eighth group is the best, and the difference between the three is still small, and the compressive strength is at a poor level.

Fig. 5 Strength compression of scaffold samples

Fig. 5 illustrates Groups 1-3: added SF, paraffin porogen; Group 4 (left): added SF, sodium chloride porogen; Group 5-7: added SF, three of the same particle size Porogen; Groups 8-10 (Line 3): No SF, three porogens of the same particle size.

Comparing the numbers of compressive strength and porosity, it was found that there is a roughly inverse relationship between porosity and compressive strength. In short, the lower the porosity, the higher the compressive strength. At 15% concentration SF of the additive, the balance of mechanical properties and porosity is better, while under other conditions, the choice of paraffin particles or sodium chloride particles as porogen has little effect on porosity and mechanical properties.

3.4 Porosity

Considering the porosity in Table 2, scaffold porosity of pure SF solution is low, and the highest grouping is 11 with sodium chloride as porogen. However, the difference between Group 5 and 6 is not obvious. Significantly lower porosity is the Group 7 scaffold with sodium carbonate as porogen.

The higher concentration of silk fibroin solution decreased the porosity as a whole, while the paraffin as a porogen still had higher porosity under the same concentration. Upon the same conditions, the larger the pore size is, the higher the porosity of the scaffold will be. The porosity of Group 2 was the best, but the difference of the three groups (2, 5, 8) was not obvious, all had good porosity.

Table 2 the porosity of group samplesGroups 1 2 3 4 5 6 7 8 9 10Porosity 85% 90% 93% 58% 82% 87% 33% 76% 43% 59%

4. Conclusion

This experiment designed one of the pore formers using sodium carbonate as a contrast agent. Characterization of the experiments indicated that sodium carbonate was not a suitable material for

the porogen used as a silk fibroin tissue engineering scaffold. The scaffold prepared with the sodium carbonate porogen has the following disadvantages: 1) The number of pores in the scaffold is small by SEM, and the connectivity is poor; 2) The secondary structure of silk fibroin is partially observed in FTIR. In the observation group using sodium carbonate as a porogen, most of the amides were still in a random crimp state; 3) low porosity and high compressibility resulted in limited practical applications of the scaffolds of these subgroups.

Comparing the numbers of compressive strength and porosity, it was found that there is a roughly inverse relationship between porosity and compressive strength. In short, the lower the porosity, the higher the compressive strength. At 15% concentration SF of the additive, the balance of mechanical properties and porosity is better, while under other conditions, the choice of paraffin particles or sodium chloride particles as porogen has little effect on porosity and mechanical properties.

The experimental group of paraffin and sodium chloride porogen achieved good preparation results. In the case of other characterizing properties, the paraffin groups as porogens are predominantly layered, and the sodium chloride groups as porogens are predominantly spherical or elliptical. In terms of continuity, the choice of paraffin groups is generally superior to the choice of sodium chloride grouping. For solution concentrations, the higher concentration group had better penetration results than the lower concentration group, but formed a more irregular pore shape.

5. Acknowledgement

I wish to acknowledge all the people who supported me during the research. Dr. Jiashen Li has given me many suggestions about the academic writing and courage me to keep finishing the work. Also, Dr. Jiashen provided plenty of literatures for reading. Then, I want to say thank to Prof. Xiaoqin, who pointed out the project proposal and taught me the operational rules in the lab. This project is original from my undergraduate dissertation in Soochow University, and was finished in University of Manchester finally.

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