Periodontitis is a dental disease which damages the supporting structures of teeth, such as the alveolar bone, and can lead to eventual tooth loss. Periodontitis has been shown to be present in a mild to severe form in 24.4% of adults aged between 30 and 34 years, which increases to 70.1% prevalence in adults aged 65 years and over [1
]. If periodontitis is not treated early enough, or the periodontal condition continues to decline, it may be necessary for surgical intervention. Barrier membranes can be used in conjunction with guided bone regeneration (GBR) to help repair the damage caused by periodontitis. GBR promotes and directs the growth of new bone, whilst the barrier membrane secludes the defect site from infiltration by fast-growing connective and epithelial tissues which would otherwise fill the defect space. In the field of implantology, dental barrier membranes are used to aid with the fixation of dental implants in over 40% of implantations to improve bone augmentation [2
The ideal properties for a barrier membrane are: to have a controllable degradation rate; to be biocompatible; to prevent surrounding tissues from collapsing into the defect space; and to provide cell occlusivity [2
Current commercial barrier membranes are produced out of materials which are either non-resorbable and require a secondary surgery for their extraction, or made from resorbable materials which can have poor structural integrity or degrade into acidic by-products [3
]. Silk could be considered as a possible alternative material as it already has a long history of use as a medical material [4
]. More recently, research into silk for tissue engineering applications has increased due to the development of regenerated silk fibroin (RSF) structures, such as sponges, films, hydrogels, and mats [6
]. Silk will only degrade in the presence of enzymes and degrades into non-harmful free amino acids and peptides [8
]. Many structural characteristics, such as biodegradation rate and mechanical strength, can be adapted by using regenerated silk fibroin [10
]. These adaptable properties make it ideal for use as a barrier membrane or tissue engineering scaffold material where control over all aspects of the material is required. Other properties of silk which make it a desirable material include versatility of sterilisation techniques [7
], solvent- and water-based processing, and the ability to modify chemical groups along its structure [15
Silk fibroin has several polymorphs: silk I, silk II, and silk III. Silk I has an unordered structure which is water soluble and present within the silkworm gland before spinning; silk II has a crystalline structure that is non-water soluble and produced during spinning from the silk worm spinneret; silk III is an unstable structure which forms at the water–air interface. As regards the current research, silk I and silk II are of interest.
Silk I consists of an unordered fibroin structure, mostly composed of α-helix and random coils, whilst silk II is mostly composed of a crystalline β-sheet structure. Silk I can be transformed into silk II by exposure to methanol or potassium chloride [16
], stretching [19
], as well as through heat treatments [20
]. Methanol dehydrates the unordered random coil structural component of silk I, converting it into anti-parallel β-sheets and thereby creating a water-insoluble silk II structure [16
]. The ability to change silk I to silk II make it ideal for processing and manufacture.
Current methods for producing RSF films involve either casting, spin drying, or electrospinning; however, these methods have a limited control over the final structure and require additional procedural steps to treat the films and improve their mechanical properties [6
]. Reactive inkjet printing offers complete control over film design and structure, as well as the possibility of combining film manufacture with a methanol treatment to induce β-sheet crystallinity. Previously, we have reported the reactive inkjet printing of RSF, where we have demonstrated the flexibility in film design and production when using an inkjet printer [21
]. This paper will establish the characteristics of RSF solutions for inkjet printing and printed RSF films for use in tissue engineering, and more specifically for dental barrier membranes. We have also produced films with the inclusion of nano-hydroxyapatite (nHA). The inclusion of nHA within a tissue engineering construct has been shown to improve osteogenic activity and therefore improve bone cell interaction [22
]. The ability to include bioactive components, such as nHA, could prove beneficial for improving site regeneration after periodontal surgery.
Droplet formation and stability are key to producing reliable and repeatable experiments with reactive inkjet printing. The two key factors which influence droplet formation and stability are the applied waveform and the rheology of the ink. It was therefore important to analyse the RSF and nHA/RSF inks before using them for printing. Viscosity has been linked to the stability of droplets by preventing instabilities from forming before droplet detachment [38
]; however, viscosities which are too high will dampen out acoustic waves before a droplet is formed. Surface tensions are required to hold a meniscus at the nozzle and prevent flooding of the nozzle tip. High surface tensions will cause faster separation of the droplet from the nozzle as well as larger droplet formation [38
RSF inks showed a slight drop in surface tensions with increased concentration. This could be explained by the work of Yang et al. [41
], who modelled the RSF protein at the liquid-air interface and suggested two separate models for high and low RSF concentrations. RSF molecules were modelled as multi-block amphiphilic macromolecules, which at low concentrations are arranged into helical silk III or β-sheet silk II conformations at the liquid-air interface, and produce a high surface elasticity. As the concentration of the RSF solutions increases, the air-water interface becomes more crowded with RSF molecules. A lack of space at the surface causes RSF molecules to protrude out of the surface and into a hairpin-like configuration, decreasing surface elasticity [41
]. The experiment by Yang et al. looked at RSF concentrations over a much larger range than those used in this study; however, it is suggested that the slight drop in surface tension experienced by the higher concentrated solutions could be the result of changing concentrations of RSF molecules with a hairpin-like conformation at the liquid-air interface.
Surface tensions of the nHA/RSF inks showed significant changes with each nHA concentration. The ink containing only nHA and no RSF (nHA/RSF ink 100%) had a slightly higher surface tension than water, and the addition of 25 dried wt % nHA to RSF only caused a slight increase of surface tension. However, the combination of both nHA and RSF, where the dried nHA weight concentration was 50% and 75%, created a synergistic effect which caused a significant increase in surface tension.
Over the range of dynamic viscosity measurements, the RSF inks appeared to be less stable at low concentrations. However, as the shear rate increased and the inks approached shear independence, the inks became more stable. At shear rates around 100 s−1
, the RSF inks began to form a Newtonian plateau and by a shear rate of 1000 s−1
had become shear-independent. The nHA/RSF inks were more stable at low concentrations in comparison to the RSF inks; however, they took longer to reach shear independence. Both RSF and nHA/RSF inks had reached shear independence by a shear rate of 2000 s−1
, which is a mid-range shear rate experienced during printing [42
]. As a shear rate of 2000 s−1
is comparable to inkjet printing forces, viscosity measurements for Z number calculations were taken from this position.
When Newtonian fluids are analysed for printing, the zero viscosity (the viscosity at a very low shear rate) is used to calculate the Z number. However, for non-Newtonian fluids the viscosity becomes a function of shear, whereby the zero viscosity can be vastly different to that of the infinite viscosity. According to Yoo et al., the ejection of a droplet is associated with the infinite shear viscosity and not the zero viscosity [43
]. Therefore, all Z number calculations used the infinite viscosity value and the instability of the RSF inks at low concentrations was not considered a problem.
Z numbers correctly predicted a stable droplet formation for the RSF inks. However, the highest concentration inks were susceptible to crusting-over during printing and therefore RSF ink 100 mg·mL−1 was chosen as the ideal ink to produce the RSF films. The Z numbers for the nHA/RSF inks with an aperture size of 80 μm were all well above the predicted stable range. However, all inks were tested for printing with an 80 μm printhead and each ink was shown to have a stable droplet formation. Therefore, all nHA/RSF inks were considered suitable for further printing.
The crystallinity data showed that a small volume of methanol could induce a large proportion of the RSF to become crystalline. The degree of RSF crystallinity doubled between RSF films without any methanol treatment (RSF films Cast and 100%) and the RSF film which had been exposed to the smallest volume of methanol (RSF film 25%). However, after this initial transition, RSF crystallinity did not significantly change up until RSF film 50%, which was produced with a 1:1 volume ratio of RSF to methanol. Significant changes in crystallinity were then observed between RSF films 50% and 25%. A peak crystallinity was reached with RSF film 25% which was similar to a cast RSF film, submerged in methanol for 4 days, as well as that of a native Bombyx Mori silkworm cocoon, suggesting a complete transition of silk I to silk II. The crystallinity data also showed that printing alone without methanol treatment did not induce crystallinity due to shear, as there was no difference in crystallinity between RSF films Cast and 100%. This is important, as it demonstrates that all structural changes observed by the printing of different volumes of methanol are caused by interactions with the methanol alone.
The inclusion of nHA within the composite ink was shown to affect the transition of silk I to silk II, as nHA/RSF films showed similar RSF crystallinity to that of RSF films without methanol treatment. Additional research by the authors showed that Fourier deconvolution of the nHA/RSF films had a larger volume of β-turns compared to the pure RSF films [44
]. Previously, Yamane et al. suggested that β-turns are a precursor to a β-sheet structure [45
]. This is supported by Wilson et al. who created a model amorphous fibroin peptide chain, which, when exposed to methanol, gradually transitioned into a crystalline silk II structure. During the transition, an intermediate state appeared which consisted of a high proportion of β-turns [46
]. This could suggest that the presence of nHA within the composite ink was hindering the conversion of silk I to silk II, possibly by reduced contact with the methanol, and that only a partial transition occurred.
The peaks and troughs on the RSF films visible in the light microscopy photos were caused by the spacing of the printed RSF droplets. The initial spacing of the droplets was chosen to produce a uniform layer and create a flat film. As multiple layers of RSF were printed, the RSF droplets were no longer interacting with the glass coverslip and were instead interacting with dried RSF film. As lines of droplets are visible on the surface of the films, it would indicate that the RSF films were slightly more hydrophobic than that of the glass coverslips, causing the droplets to spread over a smaller area. The only film where no droplets were visible was on RSF film 100%, which had had no methanol treatment. No visible droplets on the surface of RSF film 100% could be an indication that the methanol treatment was causing the films to become more hydrophobic, which makes sense when one considers that the addition of methanol results in the production of insoluble silk II.
Significant cracking of the films, which began to form on RSF film 66%, became larger and more frequent up until RSF film 25%. As there are no cracks visible on RSF film 100%, the cracking could be the result of rapid dehydration of the RSF caused by methanol. Larger volumes of methanol would have had longer to diffuse into the RSF before evaporating and therefore caused more prolific crack propagation. The effects of crack formation on the surface of the films will have to be monitored for further development of RSF barrier membranes. A rougher surface could aid with cellular interactions; however, the cracking may cause problems with the structural integrity of the membranes.
Crystallinity of the RSF films was shown to influence water droplet contact angles. When RSF is in an amorphous state, polar groups along the molecule have a random orientation which produces a high surface energy and a more hydrophilic surface. During crystallisation, the polar groups are used for hydrogen bonding to produce a β-sheet structure [47
]. As the polar groups are positioned within the β-sheet layers, the surface energy is reduced which increases hydrophobicity.
As observed with the light microscopy photographs, films which included larger volumes of methanol had the roughest surfaces. Increasing the volume of methanol caused larger, deeper, and more frequent cracking to occur and could be observed visually in Figure 5
, which caused the roughness values of the RSF films to increase. The cracking of the films was most likely caused by the rapid dehydration of the RSF and was proportional to the volume of methanol printed.
Degradation of the RSF films was studied and compared by immersing them in either an enzymatic solution of protease XIV or in phosphate buffered saline (PBS) over an 8 day period. The enzymatic solution facilitated the breakdown of the fibroin structure and therefore produced faster degradation rates. Degradation within the PBS solutions should show the proportion of RSF films being actively broken down by enzymatic activity and how much RSF is lost simply due to dissolution of the water-soluble structures.
The largest mass loss for RSF films degraded with protease XIV had occurred by day 1, and was proportional to film crystallinity. Crystallinity continued to affect the degradation of the films, as by the final day of the protease XIV degradation study, mass loss was shown to be related to film crystallinity. It was expected that the RSF films degraded in PBS would have experienced mass losses by the first day which related to film crystallinity, as the non-crystalline water-soluble silk structures were dissolved. However, it was only RSF film 100%, which had had no methanol treatment, which experienced similar degradation rates in both degradation media.
A potential reason for the methanol-treated RSF films having different degradation profiles could be due to the way the films are produced. During printing, each printed layer of RSF solution is very thin, and it is necessary to print multiple layers to build up the mass of the film. A layer-by-layer approach to producing the films meant that layers of methanol were printed between sequential layers of RSF solution. Printing methanol between layers of RSF could have produced a film with a non-uniform structure, whereby layers of unordered silk I were encapsulated under layers of ordered silk II. Films exposed to larger volumes of methanol had higher crystallinities, which could represent thicker layers of silk II. Larger volumes of methanol would require longer to evaporate off the substrate. The longer evaporation times would increase RSF exposure to methanol, enabling it to diffuse further into the RSF film, converting unordered silk I into silk II. Therefore, films which have had a longer exposure to methanol would have thicker layers of silk II with denser crystal packing, encapsulating the unordered silk I beneath.
Degradation of the nHA/RSF films with protease XIV showed mass losses relative to the nHA concentration. Films with higher nHA contents were shown to experience larger mass losses. nHA would have been unsusceptible to proteolytic degradation; however, as the RSF degraded, it could have released the nHA into the surrounding solution. Therefore, the more concentrated films would have released larger quantities of nHA into the surrounding solution, which resulted in larger mass losses. The nHA/RSF films had also been shown to have a lower RSF crystallinity than that of the pure RSF films. Consequently, some of the mass loss could also be attributed to the dissolution of silk I content. The RSF did not degrade at the same rate as that of the pure RSF films with a similar crystallinity. This could be because of a higher β-turns content. β-turns are associated with a water-soluble silk I structure, however it has previously been shown that RSF films with a high β-turn content are water-insoluble [48
]. nHA content was not shown to cause substantial differences between the degradation rates of the nHA/RSF films in PBS solution, except for the pure nHA film.
nHA/RSF film 100% experienced the largest mass loss in both the PBS and protease XIV solutions. This could have been caused by a lack of RSF binding the nHA crystals together, which, upon washing of the films, would be more vulnerable to becoming dislodged and washed away. The lack of structural stability of the printed nHA could explain the similarity of degradation rates of nHA/RSF film 100% in both degradation solutions.