**4.3 Hyperphosphatemia**

	- 1.Tissue necrosis: Tumor lysis syndrome, rhabdomyolysis, hemolysis, fulminant hepatitis, severe hyperthermia
	- 2.Endocrinopathy: Hypoparathyroidism, hypothyroidism/ hyperthyroidism, adrenal insufficiency, acromegaly
	- 3.Medications: Exogenous phosphate intake (phosphate containing laxatives/enemas, TPN), Vit D toxicity, bisphosphonates, fosphenytoin

• If patient has persistent renal failure, can start oral phosphate binder. Calcium acetate can be useful in patients with concomitant hypocalcemia and should be avoided in patients with hypercalcemia, Vitamin D toxicity and Ca-Phos product>66. Sevelamer is a nonabsorbable resin that is preferred for patients

• Hypophosphatemia is defined as serum phosphate <2.5 mg/dl.

mild (or even moderate) hypophosphatemia are asymptomatic.

respiratory alkalosis, hungry bone syndrome

• Causes of hypophosphatemia are [45]:

• Patients with hypophosphatemia can present with paresthesia, tremors,

seizures, impaired heart contractility, arrythmias, muscle weakness (including the diaphragm). Usually, symptoms occur at levels <1-2.5 mg/dl. Most cases of

1.Shifting phosphate into cells: Diabetic ketoacidosis, refeeding syndrome,

on dialysis [44].

*Treatment of hyperphosphatemia.*

*Electrolytes in the ICU*

*DOI: http://dx.doi.org/10.5772/intechopen.96957*

**Figure 9.**

**155**

**4.4 Hypophosphatemia**

*Calcium Phosphate Product* ¼ *Serum Calcium x Serum Phosphate* (4)

• Acute treatment of hyperphosphatemia (see **Figure 9**) includes treating inciting event, phosphate restricted diet, fluid resuscitation and forced diuresis (acetazolamide+ � furosemide) or dialysis [44].

*Electrolytes in the ICU DOI: http://dx.doi.org/10.5772/intechopen.96957*

#### **Figure 9.**

*Treatment of hyperphosphatemia.*

• If patient has persistent renal failure, can start oral phosphate binder. Calcium acetate can be useful in patients with concomitant hypocalcemia and should be avoided in patients with hypercalcemia, Vitamin D toxicity and Ca-Phos product>66. Sevelamer is a nonabsorbable resin that is preferred for patients on dialysis [44].


*4.1.2 Descensus of the apical compartment*

A descensus of the apical compartment (defect in Level I) leads to a uterine prolapse or vaginal stump descensus (after hysterectomy, **Figure 2**). Main cause is the overstretching of the ligaments holding the uterus, in particular the sacrouterine ligament. Depending on the degree of prolapse, one speaks of partial prolapse (the cervix

**Figure 1.** *Pulsation cystocele, stadium 2. With kind permission of Dr. Christian Fünfgeld, all rights reserved.*

#### **Figure 2.**

*Vaginal stump prolapse. With kind permission of Dr. Christian Fünfgeld, all rights reserved.*

protrudes up to the introitus), prolapse (the cervix protrudes to the introitus or further) or total prolapse with a inversion of the vagina (**Figure 2**). Descensus of the apical compartment is usually less problematic, as it only becomes symptomatic when the prolapse reaches to the introitus or further (**Figure 3**) [9].


**1. Introduction**

Nanofluid, characterized by a significant increase in a number of properties compared to conventional engineered fluid [1], is found to serve in many practical applications, for example, petroleum engineering [2–5], power industry [6,7], and medical science [8,9], which has drawn particular attentions for cancer treatments in recent years. Cancer covers a huge group of diseases

which can damage any portion of the body. Nowadays, cancer is a main cause of death all over the world, around 70% of cancer deaths materialize in middle- and low-income countries. There are many treatments to cure cancer, such as surgery, radiation and chemotherapy, but these procedures may harm the normal tissue. Hanahan and Weinberg [10] have explained six cancer hallmarks, helping to differentiate features between the tumor and normal tissue, and maybe come up with better alternative therapies. These hallmarks include inducing activating invasion and metastasis, resisting cell death, angiogenesis, enabling replicative immortality, sustaining proliferative signaling, and evading growth suppressors. Based on these cancer hallmarks, latest therapies for cancer treatment have been introduced. Nowadays, nanomedicine (nanomedicine is a branch of nanotechnology, or utilization of materials less than 100 nm, applied to medicine and health sciences) is the prominent procedure for treating cancer. The nanocarriers' properties, including their targeting modifications, favorable drug release profiles, high surface-to-volume ratios, and nanoscale sizes, may authorize them to reach and target the tissue of a tumor and the deliverance of drugs in a stable and controlled manner. For cancer research, nanomaterials are available in modified shape, because to treat specific tumors, size and surface features are crucial. The size of the nanoparticle is a key attribute, which travel across the bloodstream, ensuring delivery of nanocarriers to tumor tissue. The small-scale nanoparticles can stockpile comfortably in the physiological tumor vessels and also extravagate into normal tissue. In view of many nanoparticle applications in bio-fluid flows, many researchers have concentrated their work in the field of bio-nanofluids. For instance, Prakash et al. [11] have presented the study of nanofluids which is relevant to bio-inspired nanofluid smart pump designs, which may also be exploited in smart-drug delivery. Abbas et al. [12] have provided mathematical modelling to describe the peristaltic transport of blood (blood is treated as nanofluid) and analyzed the entropy analysis. They concluded that such a study can help in analyzing blood flow in small blood vessels with elastic walls. Abdelsalam and Bhatti [13] have given a theoretical model to describe the effect of sundry variables on the feature of blood flows in the presence of nanoparticles, and suggested that Brownian motion and chemical reaction exhibit dual variation of nanoparticles' volume fraction. Shah et al. [14] have presented the theoretical study, which is applicable to the drug-delivery system, as the micro-polar nanoparticles of gold are proficient drug-delivery and drug-carrying mediums. Bhatti et al. [15] studied the two-phase flow under the effects of coagulation with peristaltic pumping through the Prandtl stress model, with magnetic field and porous medium terms. They analyzed that friction forces flourish with the altitude of clot height and particle concentration, on the other hand they are minimized with other involving factors in the problem.

It is extensively known that biological liquids, such as gastric fluids and blood, generally behave as non-Newtonian fluids. Many researchers have considered Jeffrey/viscoelastic fluid as biological (synovial, blood, gastric, chyme, and saliva) fluid. To delineate the stress relaxation effects of real fluids, the viscoelastic fluid model is appropriately competent. These effects cannot describe the usual Newtonian fluid model. In addition to this, the Jeffrey fluid model can also describe the characteristic of memory-time scale. Kahshan et al. [16] have described the Creeping flow of a viscoelastic fluid in a channel with an application to flow in a flat-plate hemodialyzer. Pandey and Tripathi [17] have explored the viscoelastic fluid flow by peristalsis in a channel in order to apply the model to the swallowing of food-bolus through the esophagus. Ellahi et al. [18] used Jeffrey fluid as bio-fluid and studied the problem of the peristaltic flow of Jeffrey fluid in a non-uniform rectangular duct, which may be applicable to modern drug delivery systems with great utility. Ramesh et al. [19] have considered Jeffery's viscoelastic formulation, which is employed in the rheology of blood. Some more studies can be seen through [20–25].

In thermodynamics, entropy is a measure of the number of specific modes in which a thermodynamic system can be organized, often called a measure of impedance or a measure of progress toward thermodynamic equilibrium. Pakdemirli and Yilbas [26] have analyzed the entropy generation mechanism of non-Newtonian fluid through a pipe. According to them, the entropy Brinkman number causes an increase in entropy generation. Entropy generation in a peristaltic

pumping problem has been presented by Souidi et al. [27]. Heat and fluid flow causing entropy generation in backward-facing step flow is suggested by Abu-Nada [28]. More studies on entropy generation in peristaltic transports are reported in [29–31], but none of these established the entropy analysis of viscoelastic nanofluids in eccentric cylinders having a peristaltic outer surface.

Keeping in mind the physiological applications of a peristaltic propulsion of viscoelastic fluids, the investigators focused on the entropy generation and Bejan number during the peristaltic transport of viscoelastic nanofluid in the annulus region of two eccentric cylinders. The equations of governing the flow are considered in the cylindrical geometry. The concerned non-dimensional system of equations is solved using optimal homotropy perturbation technique under the long wavelength and low Reynolds number assumptions. The effects of involved parameters on the pressure rise, velocity, temperature, nanoparticle concentration, entropy generation, and Bejan number are shown through graphical illustrations.


**3. Synthesis of oxygen-containing heterocycles**

### **3.1 Furans**

Furan moieties are important substructures that have been found in numerous natural products, such as kailolides and combranolides. These heterocycles are

*Synthesis of Five-Membered Heterocycles Using Water as a Solvent DOI: http://dx.doi.org/10.5772/intechopen.108929*

**Figure 11.**

*Synthesis of 5-(furan-3-yl)barbiturate/thiobarbiturate derivatives.*

#### **Figure 12.**

*Synthesis of 2-(cyclohexylamino)-3-aryl-indeno [1,2-b] furan-4-ones in water.*

also found in a variety of commercial products such as pharmaceuticals, fragrances and dyes.

A simple three-component reaction between arylglyoxal monohydrates, acetylacetone and barbituric or thiobarbituric acid resulted in the synthesis of polyfunctionalized 5-(furan-3- yl)barbiturates and 5-(furan-3-yl)thiobarbiturate derivatives in good yields (**Figure 11**). The method employs readily available starting materials, neutral reaction conditions and water as an environmentally green solvent [19].

An efficient and a simple synthesis of 2-(cyclohexylamino)-3-aryl- indeno[1,2-b] furan-4-ones was attained via a one-pot three-component reaction of aldehydes, cyclohexylisocyanide and 1,3-indandione in water for 5 h in excellent yields (**Figure 12**). Water was used as a solvent to avoid the use of other highly toxic and environmentally unfavourable solvents for this synthesis [20].


**1. Introduction**

Among pharmaceutically relevant natural and synthetic compounds, heterocyclic compounds hold a unique place. They have an incredible ability to serve as biomimetics and potential pharmacophores and are standard key components of several drugs [1, 2]. There is a critical need for new small organic molecules both in lead identification and lead optimization processes. Conventional methods of organic synthesis are often too slow to satisfy the demand for generation of such compounds. The synthetic chemists worldwide have been under tremendous pressure to produce them in good numbers and that too in an environmentally benign fashion. A typical reaction involves a reaction medium apart from reactants and reagents to form products. There are several issues that influence the choice of solvent, especially in context of green chemistry. It should be relatively non-reactive, non-toxic and non-hazardous. The solvent should also be contained, that is, it should not be released into the environment [3].

Water is one of the most intriguing media among all the alternative solvents available, owing to its peculiar properties. It is the most abundant and available molecule on the planet and many biochemical processes occur in aqueous medium. Despite its accessibility, it has not been a favourite reaction medium for organic chemists because its presence causes the decomposition of organometallic reagents, which are used preferably in dry organic solvents. In fact, water has been generally

used to work-up organic reactions and, therefore, it has been associated with a waste-production step and to the consequent obvious problems of cleaning-up water from reactants' residues arises. The low solubility of most organic compounds in water (hampering their reactivity), and the instability of many intermediates and catalysts in water are perhaps the two main causes of this problem. Today, it is a different scenario altogether. This very aqueous medium has captured the interest of organic chemists and many others, so much so that many surprising discoveries have been attributed to its use. Many organic reactions such as pericyclic, condensation, oxidation and reduction reactions can be conducted efficiently in aqueous medium and, in some cases, water is necessary to enhance the reaction rate and increase selectivity. Further, water-tolerant catalysts have been prepared that allow organometallic reactions to be carried out in aqueous medium. Reactions that were earlier thought impossible in water have today become a certainty [3].


**3. Surgical approach and technique**

posterior capsule's presence of osteophytes, on the knee X-ray lateral view. Lastly, the lateral view can be further used for sizing the femoral component and for locating the entry point of

During preoperating planning, the orthopedic surgeon selects the implant. The selection should be carried out based on the clinical evaluation and the radiological measurements, but the final decision should be taken during the operation and after the knee bone cuts and soft-tissue balancing. That is why valgus knee surgeons always have plane A and plane B in the prosthesis selection (constrained component, VVC or classical), especially in severe deformed knees.

Preferably, in proper restored soft-tissue balancing, a minimally constrained component can be implanted. Nevertheless, if significant deformity necessitates posterior cruciate ligament (PCL) sacrifice for soft-tissue balancing, the majority of surgeons agree that a more constrained posteriorly stabilized (PS) component must be used [6]. PS knee components provide some degree of posterior stabilization as well as protection against posteromedial, posterolateral, straight medial, or straight lateral translation, but it will not protect against residual medial laxity, which is one of the major considerations in achieving proper balance in VD knees [9, 10, 18].

The debated issue between posterior-stabilized (PS) and cruciate-retaining (CR) implants in VD is that the PCL is often contracted and it may limit the deformity correction [10, 21]. It is true that in specific cases, the deformity correction with an intact PCL may be difficult to be obtained, as the PCL is a secondary stabilizer [22, 23]. Above and beyond, the PS design is more stable than a CR one due to the post-cam mechanism, and the PS design allows greater lateralization of the femoral and tibial components that improves the patella tracking and minimizes the necessity performing a lateral retinacula release [1, 2]. For these reasons, in VD knees, some orthopedic surgeons prefer a contracted PCL with a PS design as simplest as to

McAuley et al. also presented that CR implants could be used in a wide range of VD osteoarthritic knees and that survival is improved when the LCL and/or the popliteus tendon are preserved. Release of both the PCL and popliteus is one of the two factors that made revision resulting from wear, osteolysis, or instability more likely, whereas, release of both the LCL and popliteus increased the likelihood of revision by 19.9 times due to more mediolateral laxity [24]. A debated issue is the amount of constraint needed to balance a VD knee. Favorito et al. proposed that the surgeons should resist the temptation, if possible, of a more highly constrained prosthesis. Although a highly constrained component may be necessary in difficult revision cases, they are infrequently necessary for primary arthroplasties [6]. In severe VD knees, the problem is that the PCL may be stretched or elongated, which means nonfunctional and these

Additionally, in extremely deficient lateral femoral condyle valgus knees, the usage of component augmentation blocks may be required. That is because the lateral femoral condyle may have had little or no distal femoral bone resected or, similarly, little to no bone resected from the chamfer and posterior cuts; then component augmentation blocks may be required

knees require either an ultra-congruent (VVC or hinged) or PC component.

the femoral canal [1, 11, 17].

60 Primary Total Knee Arthroplasty

**2.4. Component selection**

stabilize it by using a CR implant [6].

In order to understand and perform the valgus knee operative procedure, the orthopedic surgeons should consider that the lateral stabilizers are of two types: (1) the lateral collateral ligament (LCL) and the popliteal tendon who insert near the flexion-extension axis and act in both knee extension and flexion and (2) the fascia lata, the posterolateral articular capsule (PLC), the biceps and the external gastrocnemius muscles who insert remotely with respect to the axis and act only in extension [17, 25].

Many and various protocols of progressive step-wise release have been proposed during the last two decades, and as a consequence, the sequence of the lateral release remains controversial. In 2003, the SOO (*Societe d'Orthopedie de l'Ouest*—Western France Orthopedics) Society presented a classification system of four types of valgus knee, with increasing surgical difficulty to be distinguished from Type I to IV. More specifically, in Type I valgus knees, the deformity can be completely reduced, without medial laxity, and with no particular problems whereas a medial approach is possible. In case of course of patellar dislocation, a lateral approach is recommended. In Type II valgus knees, the deformity is totally or partially irreducible, nonetheless without medial laxity, and is the most frequent; and lateral release is required. In Type III, the deformity is reducible, but with medial distension laxity, and then the medial laxity should be managed. Lastly, in Type IV, the deformity is irreducible, with medial distension laxity, and a combination of Types II and III problems [25].


*Article* **Deterministic Insertion of KTP Nanoparticles into Polymeric Structures for Efficient Second-Harmonic Generation**

#### **Dam Thuy Trang Nguyen and Ngoc Diep Lai \***

Laboratoire de Photonique Quantique et Moléculaire, UMR 8537, École Normale Supérieure Paris-Saclay, Centrale Supélec, CNRS, Université Paris-Saclay, 61 avenue de Président Wilson, 94235 Cachan, France **\*** Correspondence<EMAIL_ADDRESS>Tel.: +33-(01)-47-40-55-59

Received: 27 May 2019; Accepted: 11 July 2019; Published: 17 July 2019

**Abstract:** We investigate theoretically and experimentally the creation of virtually any polymer-based photonic structure containing individual nonlinear KTiOPO4 nanoparticles (KTP NPs) using low one-photon absorption (LOPA) direct laser writing (DLW) technique. The size and shape of polymeric microstructures and the position of the nonlinear KTP crystal inside the structures, were perfectly controlled at nanoscale and on demand. Furthermore, we demonstrated an enhancement of the second-harmonic generation (SHG) by a factor of 90 when a KTP NP was inserted in a polymeric pillar. The SHG enhancement is attributed to the resonance of the fundamental light in the cavity. This enhancement varied for different KTP NPs, because of the random orientation of the KTP NPs, which affects the light/matter interaction between the fundamental light and the NP as well as the collection efficiency of the SHG signal. The experimental result are further supported by a simulation model using Finite-Difference Time-Domain (FDTD) method.

**Keywords:** direct laser writing; KTP; nonlinear optics; photonic coupling

#### **1. Introduction**

Second-harmonic generation (SHG) allows the generation of new photons with a double frequency (2*ω*) [1]. This is a second-order nonlinear optical process, thus does not provoke any absorption. Therefore, the SHG can avoid a bleaching effect and allows observations over long durations with no decrease in the signal quality, which is usually observed with fluorescent and luminescent nanoparticles (NPs) [2,3]. Moreover, the SH spectrum (2*ω*) is separated from the fundamental spectrum (*ω*), allowing spectrally filtering out the fundamental beam, resulting in an excellent contrast in nonlinear microscopy. In SHG process, a large range of excitation wavelengths can be used, depending on the nonlinear material used. Thus, for many applications, the fundamental wavelength can be selected in ranges where the absorption and scattering effects are low to avoid the losses and the photodegradation. This allows SHG to be observed in thick materials, which are very useful for bio-applications [4–8].

SHG could be performed with nonlinear materials at macro and micro scales. For some applications, such as biomarkers and nanosensors, nonlinear NPs should be used [4,8]. Several works have been devoted to fundamental studies of nonlinear properties of NPs, such as NPs of CMONS (Cyano-MethOxy-Nitro-Stilbene) [9–11] and hybrid organic/inorganic-based MnPS3 NPs [12]. Recently, Le Xuan et al. [2] have investigated KTiOPO4 (KTP) NPs, with the size in the range of 30 and 100 nm. This nonlinear crystal is commonly used due to its important nonlinear response and its transparency in visible and infrared ranges [13]. The nonlinear analyses of these NPs show that the SH signal is very high as compared to a massive KTP crystal and perfectly stable. The SHG of nonlinear NPs in general is efficient because no phase-matching is required, and those NPs could

be easily functionalized with other materials, suggesting numerous applications particularly in the biology domain.

Direct laser writing (DLW) is an excellent method allowing fabrication of any one-, two-, and three-dimensional (1D, 2D, and 3D) structures at a sub-micrometer scale [14,15]. In this method, a femtosecond laser beam is usually used. The laser beam strongly focused into a photoresist by using a large numerical aperture (NA) objective lens (OL). Only the photoresist located inside the focusing spot is polymerized/depolymerized thanks to the multi-photon absorption (MPA) effect. An arbitrary structure then can be created by moving the focusing spot inside the photoresist. However, the MPA-based DLW is rather expensive and complicated since it requires a femtosecond laser and a complex optical system. Recently, a very simple and inexpensive technique called low one-photon absorption (LOPA) DLW is demonstrated as a robust technique [16,17], allowing realization of multidimensional structures as what realized by MPA-based DLW. In the LOPA setup, a simple and low-cost, continuous-wave (cw) and low power laser is used as an excitation laser, similar to the case of conventional OPA. However, this laser wavelength is located in an ultralow absorption range of the photoresist, thus can penetrate deeply inside the material. This LOPA effect is then compensated by the high intensity at the focusing spot, resulting in a compressed polymerized spot. By moving the focusing in 3D, any structure can be created as in the case of MPA-based DLW. Moreover, it was demonstrated that LOPA DLW technique enables the creation of desirable polymeric structures containing a single gold NP [18], that cannot be obtained by a standard MPA-based DLW.

In this work, we demonstrate the use of a modified LOPA system to embed a single KTP NP in a polymeric photonic structure in order to enhance the SHG of KTP NP as well as to manipulate the SH propagation.

#### **2. Fabrication Procedure and Characterization of KTP NPs**

#### *2.1. Lopa-Based Dlw Setup*

We added a pulsed 1064 nm laser (1 ns, 24.5 kHz) into the LOPA DLW setup. This laser allowed us to investigate the SHG signal of KTP NPs, before and after coupling to the photonic structure. This infrared laser is aligned with the 532 nm laser before being focused into the sample (see Figure 1a). The system can work with the 1064 nm laser or with the 532 nm laser by switching the mirror M1. Another half-wave plate for 1064 nm–wavelength was inserted after the PBS to adjust the polarization of the fundamental laser. To measure the SH signal (532 nm), a KG5 filter is used to cut off the fundamental beam (1064 nm). The SH signal was detected by either the APD to produce a SH image, or by a spectrometer to obtain a SH spectrum. This flexible setup allows both fabrication of desired structure containing a single KTP NP at LOPA regime and characterization of SHG enhancement. The whole process is illustrated in Figure 1b.

#### *2.2. Shg Experimental Setup*

In order to measure and to characterize the SHG signal of KTP NPs, we added a pulsed 1064 nm laser (pulse duration of 1 ns and repetition rate of 24.5 kHz) into the LOPA DLW setup. This second laser is aligned so that the laser beam coincides with that of the 532 nm laser (see Figure 1a). A flip-flop mirror allows us to switch easily from one laser to the other. A half-wave plate (not shown in the Figure) for 1064 wavelength can be placed after the polarizer to control the polarization of the fundamental laser beam. To collect SHG signal at 532 nm, an infrared filter is used to cut off the fundamental light at 1064 nm. The signal is then detected by either the APD which gives a SHG image, or a spectrometer which gives a SHG spectrum of the KTP NPs. This home-built setup is very flexible for signal detection as well as structure fabrication at LOPA regime, as illustrated in Figure 1b.

**Figure 1.** (**a**) Experimental setup of the integrated optical system for fabrication of desired polymeric structures and for characterization of SHG of a single KTP NP. PZT: piezoelectric translator, OL: oil immersion microscope objective, *λ*/4: quarter-wave plate, *λ*/2: half-wave plate, BS: unpolarizing beam splitter, PBS: polarizing beam splitter, M1,2,3,4: mirrors, S: electronic shutter, L1,2: lenses, F: infrared filter, APD: avalanche photodiode. (**b**) Fabrication process of polymeric structures containing single KTP NPs. (1) Identification of KTP NPs positions by 1064 nm laser (SHG mapping); (2) Fabrication of photonic structures containing single KTP NP by 532 nm laser; (3) Development of samples; (4) Characterization (SHG by 1064 nm laser or fluorescence by 532 nm laser) of coupled structure.

#### *2.3. Ktp Nps Preparation*

KTP NPs were obtained from KTP powders, which were extracted from the polishing process of a bulk KTP crystal. The synthesis process of KTP NPs can be described as follows [2]: The KTP powders were first mixed with a polyvinylpyrrolidone polymer (PVP) using a propanol solvent (PrOH) at a controlled concentration. The solution is then placed in an ultrasonic bath for 20 min in order separate KTP NPs assembly and to create a thin layer of PVP polymer around them, which then avoiding the particles aggregation. To obtain particles with a uniform size, the solution was first passed through a filter having a hole diameter of 0.45 μm, in order to eliminate big particles, and then centrifuged with a typical speed of 11,000 rpm for 15 min. This allowed obtaining a quasi-monodisperse solution with particles size between 30 and 100 nm. We dispersed these particles again in the 0.1% PVP/PrOH mixture, which was again placed in an ultrasonic bath to obtain the final colloidal KTP solution.

#### *2.4. Sample Preparation and Fabrication Process*

To obtain a monolayer of individual KTP NPs for SHG characterization or for fabrication, we mixed the colloidal KTP solution with ethanol with a solution/ethanol ratio of 1:5 using ultrasonication for 30 min. The solution was then spin-coated on a clean glass substrate (for SHG characterization) or on photoresist thin films (for coupling), resulting in a monolayer of well separated KTP NPs.

For fabrication of coupled structures, the photoresist sample was prepared as following. First, a thin layer (500 nm or 5000 nm) of SU8 was spin-coated on a cleaned cover glass. Then, a well dispersed KTP NPs solution was spin-coated on the surface of the first SU8 layer. Finally, a second thin layer (500 nm) of SU8 was deposited on top of the KTP NPs monolayer. To remove the solvents, the sample was placed on a hot plate at 65 ◦C for 3 min and at 95 ◦C for 5 min. We note that SU8 2000.5 photoresist was used to obtain a film of 500 nm thickness and SU8 2005 photoresist for a film of 5000 nm thickness. The choice of each thickness depends on the desired structures, 2D or 3D.

The fabrication of photonic structures containing a single KTP NP is shown in Figure 1b and consists of two main steps:

We note that the two lasers have been perfectly aligned before the OL so that their focusing spots have the same transverse position, i.e., *x* and *y* coordinates. However, these spots are slightly different along the axial axis, due to the difference in wavelength. Practically, this was overcome by adjusting the *z* position in between the mapping (by 1064 nm laser) and the fabrication (by 532 nm laser) programs. After developing the sample to wash away all un-exposed photoresist, the fabricated structures were then placed again on the LOPA system in order to characterize the SH signal of KTP NPs and to compare it with that obtained before fabrication.

#### *2.5. SHG of a Single KTP NP*

We first characterized the SHG of individual KTP NPs spin-coated on glass substrate, by using the 1064 nm laser with a typical average laser power of 0.3 mW. The SH signal is very intense with respect to background noise (signal-to-background ratio of about 100), and a SHG image of an individual KTP NP is shown in Figure 2a. The intensity profile in the *x*-direction (Figure 2b) shows a full width at half maximum (FWHM) of approximately 500 nm, which agrees with the diffraction limit of the focusing spot at the 1064 nm wavelength (numerical calculation is about 480 nm). This allowed us to identify an individual KTP NP with a great precision. Figure 2c shows the spectral analysis of the SHG of a single KTP NP, obtained by adding an infrared filter to cut off the fundamental wavelength. A clear peak at 532 nm confirms the SHG of the 1064 nm laser. Figure 2d shows the temporal tracking of the SH signal. It shows a perfect stability of the SH signal for a long duration. The small variation of the SH signal is probably due to the mechanical instability of the optical setup. This excellent SHG stability allows us to optically address the same KTP NP at different moments, separated by several months, and to perform different types of measurements.

**Figure 2.** (**a**) Second-harmonic image of a KTP NP with a size of about 100 nm, obtained by scanning with a pulsed 1064 nm laser. (**b**) The SHG intensity profile in the *x*-direction, extracted from (**a**), showing a FWHM of 500 nm. (**c**) SHG spectrum. (**d**) Time evolution of the SHG signal obtained by an average power of 0.3 mW. The signal is stable for a long duration.

#### **3. Deterministic Coupling of a Single KTP Particle into a Polymeric Structure**

In order to fabricate a desired photonic structure containing a single KTP NP, we first determined the position of a KTP NP by scanning the sample with the 1064 nm laser as explained above. Experimentally, the position of a KTP NP was determined with a precision of about 10 nm. Then, the fabrication of desired polymeric structures containing the NP with well-known position was realized.  We demonstrated that the modified LOPA-based DLW technique allowed a full control of the coupling of a single KTP NP and an arbitrary polymeric structure.

#### *3.1. Control of Shape of Structures*

We first investigated the introduction of KTP NPs to structures having different shapes. Figures 3a,d show scanning electron microscope (SEM) images (top view) of a square and a triangular structures, fabricated by a laser power of 9 mW and a writing velocity of 3 μm/s. These structures were fabricated by scanning the laser spot through the SU8 film, point by point to form square/triangular structures. The distance between two adjacent points was set at 150 nm, which is smaller than the diffraction limit, resulted in continuous photoresist lines/films. Figures 3b,e show the fluorescence images of the square and triangular shapes, respectively, obtained by 532 nm laser scanning (P532 = 50 μW). Figures 3c,f represent the SHG images obtained by scanning with the 1064 nm pulsed laser. A bright spot at the center of the structure corresponds to the SHG signal emitted by the KTP NP.

**Figure 3.** (**a**) SEM image of a square structure containing a KTP NP at the center. Fluorescence image (**b**) and SHG image (**c**) of the corresponding structure, obtained by scanning with 532 nm laser and 1064 nm laser, respectively. The SHG image shows clearly that the polymeric square structure contains a KTP NP at the center. (**d**–**f**): similar SEM, fluorescence et SHG images of a triangular structure containing a KTP NP.

The first conclusion is that the LOPA-DLW allowed us to realize any structure on demand containing a single nonlinear NP. Secondly, as compared to metallic NPs [18], the nonlinear NPs do not induce a thermal effect, which may influence the structure size and shape. This is an excellent advantage which can be further exploited to embed this KTP NP to different kinds of polymeric structure.

#### *3.2. Control of NP Position in Polymeric Structures*

The control of position of the KTP NP in photonic structures is very importance, because the light intensity is usually not uniform in a resonant photonic structure. We therefore also demonstrated that LOPA based DLW allowed precisely controlling and manipulating the NP position in any polymeric structures. Several polymeric structures (e.g., triangle and circular disk) containing KTP NPs at different positions have been fabricated. Figure 4 shows fluorescence and SHG images of a series of polymeric microdisks embedding KTP NPs at different positions, from center to edge. The KTP NP position was experimentally controlled with a precision of 10 nm, that is mainly due to the diffraction limit of the optical system.

**Figure 4. Top row**: Fluorescence images of microdisk structures, obtained by scanning with a 532 nm cw laser. **Bottom row**: SH images of corresponding structures obtained by scanning with 1064 nm laser. The bright spots correspond to the SHG of KTP NPs, which are embedded at different positions of the microdisk, from center to edge. Scale bars: 1 μm.

#### *3.3. Control of Structure Size*

It was also demonstrated that by controlling the exposure dose, the size of the structure containing single KTP NPs is adjusted on demand. We fabricated various two-dimensional (2D) structures consisting of individual pillars among them the central one contains a single NP. Each pillar was fabricated by scanning the laser spot along the *z*–axis and through the photoresist film thickness (total scanning distance of 2 μm). Figure 5 shows the SEM images of fabricated structures. All pillars were fabricated with the same exposure dose (P = 6 mW, *v* = 5 μm/s), except the central one containing the KTP NP. For that, the doses were adjusted as: (top row) P = 6 mW, *v* = 4 μm/s; 3 μm/s; 2 μm/s; *v* = 1 μm/s; (bottom row) P = 9 mW, *v*= 4 μm/s; 3 μm/s; 2 μm/s; and 1 μm/s, respectively. In this example, the diameter of the central pillars increases with a step of about 50 nm. This can be controlled with a smaller step by finely controlling the laser power or the scanning speed, thanks to the non-explosion effect of the KTP NP in LOPA regime.

Thus, the second conclusion is that the LOPA DLW is a powerful technique to embed a single nonlinear NP into any PS with controlled size and shape, without any thermal effect. The position of the NP is also adjusted on demand inside the polymeric structures, which is a great advantage for photonic coupling.

#### *3.4. Coupling of KTP NP to 3D Structures*

Furthermore, we demonstrated that LOPA DLW enables the fabrication of three-dimensional (3D) structures containing a single KTP NP. For that, we used SU8 2005 (5 μm–thickness) as the structure material. Figure 6a shows the SEM image of a hexagonal air-hole membrane containing a KTP NP at the center. To support the membrane, six polymeric legs were fabricated as shown in the inset of the Figure 6a. The membrane was fabricated (laser power of 10 mW; scanning speed of 3 μm/s) by scanning the laser spot in a *xy*-plane with some missing exposures in a hexagonal configuration. Figures 6b,c show the fluorescence and SHG images of the center part of the membrane. The bright

SHG spot confirms the presence of the KTP NP in the 3D membrane structure. It is worth mentionning that it is more challenging for fabrication of 3D structures containing NPs as compared to the case of 2D structures, because the 3D structure is thicker and therefore it is more difficult to find out the position of the NPs. For this moment, we didn't find any SHG enhancement of the coupled KTP NP. This is due to the fact that the refractive index of the SU8 material is low and the periodicity of the 3D photonic membrane is not optimized to open a photonic bandgap, which is important to have a photonic coupling. This requires a long investigation and is out of the scope of this paper. However, the success of embedding a KTP NP into a 3D photonic structure is very encourageable, which suggests further investigations for fascinating applications.

**Figure 5.** SEM images of pillars structures containing single KTP NPs. All pillars were fabricated by the same exposure dose, except the central one, which was fabricated by the following parameters. **Top row**: from left to right: P = 6 mW, *v* = 4 μm/s; 3 μm/s; 2 μm/s; and 1 μm/s, respectively. **Bottom row**: from left to right: P = 9 mW, *v* = 4 μm/s; 3 μm/s; 2 μm/s; and 1 μm/s, respectively.

**Figure 6.** 3D hexagonal membrane structure containing a KTP NP. (**a**) SEM image; Inset: full side view. (**b**) Fluorescence image obtained by scanning with a 532 nm laser with low power. (**c**) SHG image, obtained by scanning with 1064 nm laser, showing the existence of a KTP NP at at center of structure.

#### **4. SHG Signal Enhancement**

In this section, as a demonstration of the coupling, we present our experimental investigation on the enhancement of the SH signal of a single KTP NP embedded in a polymeric submicropillar. This is further confirmed by a simulation model based on the FDTD method.

#### *4.1. Experimental Measurement*

In order to demonstrate the coupling effect of the fabricated structure, we have compared the SH signal of the same KTP NP obtained before and after being inserted into a polymeric pillar. The comparison was done by using the same experimental conditions, such as excitation power and measurement time.

Figure 7a shows the SH images (by a pulsed 1064 nm laser) of a single KTP NP obtained before (with polymeric film) and after fabrication (with polymeric pillar), and a fluorescence image (by a cw 532 nm laser) of the fabricated structure. It can be seen from the images that the NP exists well inside the micropillar. We note that SU8 does not emit fluorescent or SH signal when excited by 1064 nm laser, therefore only the SH signal of the KTP NP was detected. Comparing the SH signal obtained before and after fabrication, we can clearly see a great enhancement. Figure 7b shows the comparison of the SHG intensities of the same KTP NP located in a polymeric film (red curve) and in a polymeric pillar (green curve). When embedded in a pillar (diameter = 800 nm; height = 1.3 μm), the SHG intensity is enhanced up to 90 times. We have also realized many similar structures with the same fabrication parameters but containing different KTP NPs to verify the orientation dependence of the KTP NP inside the polymeric pillar. Indeed, while a bulk KTP crystal has a unique and well-defined orientation, a KTP NP is randomly oriented in space. This first affects the nonlinear interaction between the fundamental light and the nonlinear crystal, which depends strongly on the relative angle between the electric field of the fundamental beam and the main axis of the KTP crystal. Also, the amount of SH light collected by the microscope objective strongly depends on the orientation of the SH dipole and the surrounding medium. The detected SH signal is therefore the result of the photons number emitted by the nonlinear dipole and the collection efficiency of the optical system, which is defined as the ratio of the photons collected by the microscope to the total photons emitted by the dipole. Clearly, the polymeric pillar strongly enhanced the SHG emission towards the detection system thus enhancing the detected SHG signal. But the enhancement value varied from this NP to the other due to the random orientation of the KTP NP inside the pillar cavity.

#### *4.2. Numerical Simulations*

We have then performed different simulations using FDTD method to find out the answers for different questions: How the polymeric pillar cavity enhanced the incident fundamental light (at 1064 nm); How the polymeric pillar guided out the SH signal (at 532 nm) emitted from the embedded NP, and if the coupling out is affected by the NP's orientation. For these simulations, we assumed that the SH signal is emitted from an electric dipole.

We first investigated the enhancement of the fundamental light in the polymeric pillar cavity. Figure 8a shows a model used for simulation. The polymeric pillar cavity was placed on a glass substrate and a plane-wave light source was inserted inside the glass substrate, which emits a 1064 nm–wavelength in upward direction (*z*-axis). This incident light field was monitored in the (*xz*)– or (*yz*)–plane. Similar simulations were also realized for a thin SU8 film deposited on a glass substrate to verify the field enhancement in polymeric cavity. The cavity parameters such as diameter and height are swept in a large range to find out the optimum configuration of the cavity. We found that the pillar possessing parameters similar to experimental structures induces a strong enhancement of the fundamental light. Figure 8b shows the intensity of the fundamental light inside a polymeric pillar with a diameter of 800 nm and a height of 1.3 μm. Figure 8c shows the field intensity when the 1064 nm light passed though a thin film with a thickness of 1.3 μm. It is clear that, the fundamental

field is amplified and localized inside the pillar cavity, resulting in an amplification of 7 to 13 times, as compared to that inside the thin film. Therefore, when a KTP NP is embedded inside the polymeric pillar, the SHG will be enhanced by a factor in between 49 and 169. Furthermore, it was observed that a small change of NP position inside the cavity leads to a significant change of the SHG signal due to the modulation of the fundamental light field inside the cavity, as seen in Figure 8b. This suggests that the experimental determination of the NP position in the structures is very importance to obtain an optimum SHG enhancement.

**Figure 7.** (**a**) SHG images of a KTP NP obtained before and after fabrication, and a fluorescence image of the fabricated structure. In the right: SEM of corresponding fabricated structure. (**b**) Comparison of the SHG spectra intensities of the same KTP NP showing an enhancement factor of about 90; Inset: zoom-in of the SHG spectrum before fabrication.

**Figure 8.** (**a**) Illustration of the model used to simulate the coupling of a light beam and a polymeric pillar. A light beam at 1064 nm–wavelength is emitted from the source and sent towards the cavity (*n*glass = 1.5, *n*SU8 = 1.58). (**b**) Simulation of light intensity inside a polymeric pillar (diameter = 800 nm, height = 1.3 μm). (**c**) Simulation of light intensity at 1064 nm–wavelength propagating through a polymeric film (thickness = 1.3 μm).

We then investigated how the SH light is coupled out of the cavity. For simplification, we considered the nonlinear NP as a single oscillating electric dipole. Naturally, we found that the best orientation of the emitting dipole should be parallel to the glass substrate surface. We have then compared the two particular configurations: a single KTP NP embedded in a polymeric film (thickness = 1.3 μm) and a polymeric pillar (diameter = 800 nm; height = 1.3 μm). Furthermore, the emitting dipole is assumed to be embedded in polymeric material at center of the thin film or of the pillar, i.e., at 650 nm from the glass interface. The SHG detector is placed inside the glass substrate. Figure 9a shows the SH intensity distribution in the (*xz*) and (*yz*)–planes of a single emitting dipole embedded in a polymeric film (top) and in a polymeric pillar (bottom). It is evident that, thanks to the pillar cavity, the emitted light is compressed in a small cone and mostly directed towards the detector (downward direction). In contrast, in the case of the film, the light is generated in a large angle, which cannot be totally detected by the detector. To be more precise, we have numerically evaluated the collection efficiency of the emitted light as a function of the microscope objective, a main component of the detection system. Figure 9b shows the simulation result of the collection efficiency as a function of the half angle, defined as arcsin(NA/*n*), where NA is the numerical aperture of the objective lens and *n* is the refractive index of the immersion oil. In case of an objective lens with NA = 1.3 (half angle = 58.8◦), 55% of the photons emitted by the NP embedded in a polymeric pillar can be collected while this is only 11% in the case of polymeric film. The polymeric pillar cavity thus does not only amplify the fundamental light intensity, resulting in an enhancement of the nonlinear optical process, but also guide the emitted signal into the detection angle, hence enhancing the collection efficiency. Theoretically, when the KTP NP is embedded at the middle of the pillar, the best collection efficiency is obtained with a pillar having a diameter of 800 nm and a height of 1300 nm, which is quite consistent with the experimental result.

**Figure 9.** (**a**) Simulation of radiation patterns, in (*xz*) and (*yz*)–plane, of a single dipole embedded in different configurations. Top: emission diagram of a single dipole embedded in a thin film. Bottom: emission diagram of a single dipole embedded in a polymeric pillar. For both cases, the dipole was assumed to be in *x*-axis. (**b**) Calculation of the collection efficiency of the SHG signal as a function of the half angle of the objective lens for two cases. An enhancement factor of 5 is obtained.

#### **5. Conclusions**

In this work, we have first investigated the SHG of individual KTP NP with a size of about 100 nm, under a pulsed laser excitation at 1064 nm. The KTP NP shows a high nonlinear coefficient, as compared to a bulk one, and the SHG is perfectly stable at room temperature. Its transparency for both wavelengths of the fundamental and SH wavelengths leads to a high signal to noise ratio. We then demonstrated the LOPA-based DLW as an excellent technique to realize desired polymeric photonic structures containing a single KTP NP. Thanks to its transparency at 532 nm, KTP NPs did not induce explosion or damage during fabrication. Different kinds of 2D and 3D polymeric structures containing individual KTP NPs were successfully demonstrated. The size and shape of the polymeric structures and the position of the KTP NP inside the structure were demonstrated to be controlled at nanoscale. When the KTP NP is embedded in a polymeric pillar, the SHG is enhanced by a factor of 90. The SHG enhancement was varied for different KTP NPs, because of the random orientation of the NP inside the cavity. This affects the nonlinear interaction between the fundamental beam and the KTP crystal as well as the collection efficiency of the optical system. Different simulations using FDTD method have been done and shown very good agreement with the experimental results.

**Author Contributions:** D.T.T.N. and N.D.L. conceived and designed the experiments; D.T.T.N. performed the experiments, analyzed the data, and did simulations. All authors wrote, reviewed and approved the final version of manuscript.

**Funding:** This research is funded by a public grant overseen by the French National Research Agency in the frame of GRATEOM project (ANR-17-CE09-0047-01).

**Acknowledgments:** We thank L. Le Xuan and J.-F. Roch for providing nonlinear nanoparticles, I. Ledoux-Rak for fruitful discussion about nonlinear optics, and Phoebe Marcus-Porter for English revision.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


4.1.1. What Type of Space Serves as the Context for the Faunal Remains?

The architectural remains from Area T have many features that line up with Renfrew's "indicators of ritual" (Renfrew 1985, 1994; Renfrew and Bahn 2016, pp. 413–21; cf. Gilmour 2000; Zevit 2001), and such have been applied specifically in this case (Davis 2013). Indeed, the area has all of the hallmarks of a major, regional temple complex. Construction features associated with the 9th—8th c. BCE, Strata III and II in Biran's (1994) assessment, include fine ashlar masonry with slots for cedar beams typical of palaces and temples, a massive substructure for a large, elevated building, and parallel casemate side-chambers on the western and eastern boundaries of the precinct. The decorative techniques and other features, too, emphasize monumental display resonating with garden motifs typical of temples (Ackerman 2013). In the center of the complex, the base of a large altar was discovered as well as a single horn that, when compared to proportions from other altars, allows for a reconstructed altar of 4.75 × 4.75 m and, thus, apparently the largest altar ever discovered in the region.

The artifact remains, too, support the identification of the space as a temple as evidenced in the discovery of several smaller altars, seven-spouted oil lamps, perforated "incense" cups, cultic stands, metal implements associated with animal slaughter and processing, and even what has been identified as an altar kit found in situ consisting of a bowl, pair of shovels, incense pan, possibly a meat fork, and a sunken pot with charred animal remains (Greer 2010).

Evidence from the architecture and available artifacts, especially when combined with the discovery of massive concentrations of animal bone remains both in charred deposits and in deposits from the remains of meals, leaves little doubt that the space functioned as a major temple during the Iron Age IIA-B especially during the 9th–8th c. BCE (cf. Greer 2013, 2017; Davis 2013).


*Article* **3D Printing High-Consistency Enzymatic Nanocellulose Obtained from a Soda-Ethanol-O2 Pine Sawdust Pulp**

**Heli Kangas 1,\* , Fernando E. Felissia 2 , Daniel Filgueira 3 , Nanci V. Ehman 2 , María E. Vallejos 2 , Camila M. Imlauer 2 , Panu Lahtinen 1 , María C. Area 2,\* and Gary Chinga-Carrasco 3,\***

Received: 13 June 2019; Accepted: 13 July 2019; Published: 16 July 2019

**Abstract:** Soda-ethanol pulps, prepared from a forestry residue pine sawdust, were treated according to high-consistency enzymatic fibrillation technology to manufacture nanocellulose. The obtained nanocellulose was characterized and used as ink for three-dimensional (3D) printing of various structures. It was also tested for its moisture sorption capacity and cytotoxicity, as preliminary tests for evaluating its suitability for wound dressing and similar applications. During the high-consistency enzymatic treatment it was found that only the treatment of the O2-delignified pine pulp resulted in fibrillation into nano-scale. For 3D printing trials, the material needed to be fluidized further. By 3D printing, it was possible to fabricate various structures from the high-consistency enzymatic nanocellulose. However, the water sorption capacity of the structures was lower than previously seen with porous nanocellulose structures, indicating that further optimization of the material is needed. The material was found not to be cytotoxic, thus showing potential as material, e.g., for wound dressings and for printing tissue models.

**Keywords:** pine sawdust; soda ethanol pulping; nanocellulose; 3D printing; cytotoxicity

#### **1. Introduction**

Balancing environment, biodiversity, and economic development can be performed through ecosystem services, climate change mitigation strategies, and long-term food security balance. In this context, the possibility of recycling and reusing a resource represents an issue of fundamental importance in the economy of various industrial sectors. The biorefinery of agro and industrial forestry waste implies its integral use and its valorization, being able to satisfy the needs of food, raw materials, and energy, respecting the principles of sustainability. More than 65% of the plantations in Argentina are in two provinces in the Nord-East (Misiones and Corrientes), of which approximately 60% is pine, since they have very favorable conditions for the growth of species such as *P. elliottii* and *P. taeda* (yields above 20 m3 /year). Therefore, pine sawdust is the most important waste of primary wood processing. This waste is not used properly and its accumulation contributes to the pollution of the environment [1].

Fractionation is a sequence of processes, which allow for recovering the different chemical components of a raw material. Different methods have been investigated for pine sawdust fractionation, including hot water, dilute acid, steam explosion, acid organosolv, and traditional alkaline treatments

like soda-anthraquinone and kraft [2–8]. This raw material has been proven to be very recalcitrant and acid treatments before the delignification stage produced alterations in the structure that made the fractionation even more difficult [9]. On the contrary, the use of organic solvents combined with bases seems to be promising. Ethanol reduces the surface tension of the pulping liquor favoring the alkali penetration into the material structure [10]. The organic solvent also alters lignin-carbohydrate bonds by hydrolyzing lignin which is then dissolved in the organophilic phase [11]. This processing produces a pulp enriched in cellulose and a less condensed lignin. In studies comparing acid processes for bioethanol production, the organosolv treatment presents higher energy consumption than the diluted acid treatment [12]. Nevertheless, in organosolv processes, a high-quality lignin can be recovered. On the contrary, if in addition to lignin a high-added-value product such as nanocellulose is produced, the process turns auspicious [13]. In addition to the above, the alkaline processes have a much stronger recovery system than the acid ones. Another strategy for further extraction of lignin from the lignocellulosic material after pulping is oxygen delignification, using oxygen and alkali in a pressurized system. Oxygen reactions are generated by radicals which react with lignin removing it in a fraction corresponding to 25% to 65% of the initial kappa number of the pulp [14]. Kappa refers to the lignin content in a pulp, the higher the kappa number the higher the lignin content.

Nanocelluloses are promising bio-based materials for numerous applications, either as replacement of traditional oil-based materials in existing products or in generating completely new materials and products. They can be roughly divided into three categories based on their production methods and properties: Cellulose nanofibrils (CNF), Cellulose nanocrystals (CNC), and bacterial cellulose (BC). Of these, CNF are manufactured by mechanical treatments, often combined with chemical or enzymatic pre-treatment, and different manufacturing methods result in materials with variable properties. Research around different production methods of nanocelluloses has continued actively for over a decade and resulted in pilot, pre-commercial, and even commercial plants all over the world. Despite the rapid development, some challenges still remain with the traditional production technologies. Considering the mechanically manufactured nanocelluloses, the production costs are usually still high, and the resulting material is at low consistency, typically between 1%–3%. The high-water content generates problems, such as difficulties in dewatering and drying, problems in post-treatment, and restricted applicability for certain applications, such as composites or paints and coatings. In addition, long-distance transportation is not feasible leading to limited availability of the material.

To overcome the problems related to high energy consumption of nanocellulose manufacturing and low solids content of the resulting material, a high-consistency enzymatic fibrillation (HefCel) technology has been developed [15]. The benefits of the HefCel nanocellulose are its high-consistency after processing (10%–25%) and lower energy consumption compared to other manufacturing methods [16]. However, similar to other nanocelluloses, HefCel nanocellulose is a potential raw material for many different applications, such as strength additive in the middle ply of board [16] or as a barrier film in packaging materials [17].

During the last years, nanocellulose has been in the focus as a component in bioinks for three-dimensional (3D) printing [18]. The material has shear thinning behavior and consolidates rapidly after deposition on a substrate. These characteristics make it possible to deposit nanocellulose layer-by-layer in order to construct geometrically complex 3D objects. Rees et al. demonstrated the potential of nanocelluloses to be 3D-printed for constructing porous structures with potential as wound dressing materials [19]. Although nanocellulose with appropriate rheological properties are 3D printable, nanocellulose constructs require a post-treatment to be mechanically stable. Nanocellulose inks are usually combined with additional polymers such as alginate and cross-linked with Ca2+, thus yielding a mechanically stable 3D construct [20] with potential as scaffolds for tissue engineering. The composition of inks for 3D printing can be optimized with additional components to tailor the printability and shape fidelity, and to keep the stability at room temperature [21]. Most of the work on nanocellulose for 3D printing has been based on wood nanocellulose from market chemical pulp. However, recently it has been demonstrated that agro-industrial residues such as bagasse also have potential as resources for nanocellulose production and 3D printing [22].

The purpose of this work was to demonstrate and prove the suitability of a soda-ethanol pulping to yield fibres with adequate composition for production of nanocellulose by high-consistency enzymatic fibrillation, which in turn has appropriate properties for 3D printing. In addition, the cytotoxicity of the produced nanocellulose was tested to evaluate its potential suitability for wound dressing and similar applications.

#### **2. Materials and Methods**

#### *2.1. Forestry Residues*

*Pinus elliotti* and *Pinus taeda* sawdust mix was provided by a local sawmill (Forestal Eldorado and Forestal AM, Misiones). The sawdust was air-dried, screened, and maintained in closed plastic bags. The fraction passing 5 mm2 -screen was used.

Before each experiment, sawdust samples were impregnated with water overnight to eliminate bark particles by flotation.

Acid-insoluble lignin (Klason lignin) and pulp structural carbohydrates were measured according to the "Determination of Structural Carbohydrates and Lignin in Biomass" NREL/TP-510-42618. Hydrolysate samples from the aforementioned technique were neutralized with Ba(OH)2 following the methodology proposed by Kaar et al., HPLC with a SHODEX SP810 column was used to determine the carbohydrates content (glucans, xylans, mannans, galactans, and arabinans) [23]. The operational conditions used were water as eluent, 0.6 mL/min, 85 ◦C, and a refractive index detector. The final chemical composition of the pulp was completed by using an Aminex-HPX87H column (BIO-RAD), operated under the following conditions: 4 mM of H2SO4 as eluent, 0.6 mL/min, 35 ◦C, and a diode array detector.

#### *2.2. Soda Ethanol Pulping*

Soda-ethanol pulping was performed in a 7 L pressurized reactor (M/K Systems, Inc., Peabody, MA, USA), with direct heating and liquor circulation. About 500 g of dry sawdust was cooked. The pulping conditions are shown in Table 1. After the pulping stage, the spent liquor was separated from the pulp by filtration. The pulp was subjected to a 5-cycle washing with water, screened by means of a Somerville device, and properly stored in plastic bags.

**Table 1.** Pulping conditions.

% odw: oven dried weight on mass of dry wood; *v*/*v*: percentage in volume.

Residual alkali was measured from the black liquor according to the SCAN-N 33:94 method ("Residual Alkali – Hydroxide Ion Content"), and alkali consumption (%) was calculated. The Kappa number was determined following the TAPPI T236 om-99 procedure and yield was determined.

#### *2.3. Oxygen Stage*

The soda-ethanol pulps were treated with oxygen in two stages. Oxygen stages were conducted in a multipurpose reactor, equipped with a high shear rotor which generates the required agitation conditions, an oxygen inlet valve, and a heating system. In both stages the pulp was treated at 100 ◦C for 60 min, an oxygen pressure of 600 KPa and a consistency of 10%. The alkaline load was 3% on dry pulp in the first stage and 2% on dry pulp in the second stage.

#### *2.4. Chemical Composition of the Pine Sawdust Pulps*

The content of acetone extractives was analysed according to standard SCAN-CM 49:03 and the lignin content and carbohydrate composition according to SCAN-CM 71:09. The samples were freeze-dried prior to analysis. The metal (Si, Fe, Mg, Mn, Co, and Ca) contents of the pulps were determined after wet combustion by inductively coupled plasma optical emission spectrometry (ICP-OEP).

#### *2.5. Enzymatic Fibrillation*

Two different types of pine sawdust pulps, namely soda-ethanol pulp and oxygen delignified (O2) soda-ethanol pulp, were used as raw materials for producing nanocellulose according to the HefCel technology. Prior to the treatments, the pulp samples were washed into Na+ form according to the method described by Lahtinen et al. [24]. The enzymatic treatment with cellulase mixture was carried out at a consistency of 25% dry weight (% odw) for 9 h (soda ethanol) and 5.5 h (O2 delignified soda-ethanol) at 70 ◦C and pH 5 using a two-shaft sigma mixer (Jaygo Incorporated, Randolph, NJ, USA) running at 25 rpm. The enzyme dosage in both treatments was 8 mg/g and the pulp batch size 300 g (% odw). After the treatments the enzyme was inactivated by increasing the temperature in the mixer to 90 ºC for 30 min. The fibrillated material was diluted with deionised water, filtered, and washed thoroughly with deionised water. Finally, the fibrillated material was dewatered to a consistency of 18.6% (soda ethanol HefCel) and 21% (O2 soda ethanol HefCel) by filtration. Yield of the fibrillated cellulose material was 91% for the soda ethanol HefCel and 88% for the O2 delignified soda ethanol HefCel. In order to prevent contamination prior to characterization, the HefCel materials were further autoclaved at 121 ◦C for 20 min and sealed. The materials were stored at +4 ◦C until used.

After initial testing by 3D printing, the O2 delignified soda-ethanol HefCel was further fluidized. Fluidization was done with a Microfluidics microfluidizer type M110-EH. Two passes were run through the chambers having a diameter of 400 µm and 100 µm at 1,800 bar operating pressure. The first pass was at 8% solids and the second pass was at 6% solids.

#### *2.6. Characterisation of Nanocellulose*

Preliminary characterization of the HefCel nanocellulose samples included pH, conductivity, dry matter content, optical microscopy, and rheological properties. A standard portable device Metler Toledo SG2 and Inlab 413SG electrode were used for the pH and Jenway 4510 for the conductivity measurement. Dry matter content measurement was based on oven drying.

#### 2.6.1. Apparent Viscosity and Yield Value

The shear viscosities of dilute HefCel nanocellulose samples were measured by a Brookfield rheometer model RVDV-III Ultra using vane-type spindles. The samples were diluted to 5% concentration with Milli-Q water and dispersed with an Ultra-Turrax disperser at 14,000 rpm for approximately 2 min. Viscosity measurements were performed in a 250 mL Pyrex beaker, and each sample was left to settle for a minimum of 30 min at room temperature after the dispersion. This allowed the samples to regain their initial viscosity. The temperature of the samples was adjusted to 20 ± 1 ◦C. The shear viscosity was measured at 300 measuring points at 0.5 rpm and at 180 measuring points at 10 rpm. The yield stress value was recorded at 0.5 rpm. The apparent viscosities and yield values were measured twice for each sample. Light mixing was performed between the measurements.

#### 2.6.2. Residual Fibre Analysis

Characterization of residual fibres from the HefCel nanocellulose samples was carried out with a Kajaani FibreLab analyser. Each sample was firstly soaked in 0.4 g/L consistency and dispersed using a high shear laboratory blender for 2–3 min. Then the samples were further mixed in 5 L of water at 40 mg/L consistency with an impeller for 10 min. Some 50 mL of dispersed sample or 2 mg in dry weight was pipetted into the analyser and fibre analysis was performed with two repeat measurements. The number of fibres recorded by the analyser was divided by the sample amount, which gives the value for the residual fibres in the sample.

#### 2.6.3. Optical Microscopy Imaging

The HefCel nanocellulose samples were dyed with 1% Congo red solution by mixing nanocellulose and dye in a ratio of 1:1 and further diluting the dyed mixture on the microscope slide (2:1). Optical microscopy was performed with an Olympus BX 61 microscope equipped with WH10X-H eyepieces, fluorite objectives, and a ColorView 12 camera.

#### 2.6.4. Laser Profilometry

Structures of 20 mm × 40 mm were printed directly on microscopy slides (one layer), using a Regemat3D printing unit. The structures were printed with a conical nozzle (size 0.58 mm) and a flow speed of 3.0 mm/s. The structures were allowed to dry for one day at room temperature (23 ◦C). The microscopy slides were sputtered with a layer of gold for laser profilometry analysis. Ten laser profilometry images (10 × 10 mm, 1.0 µm resolution) were acquired randomly. The laser profilometry images were assessed with the SurfCharJ plugin (v. 1q) for ImageJ (v. 1.50i). The 3D plots were created with the Interactive 3D surface plot plugin (v. 2.4).

#### *2.7. Three-Dimensional (3D) Printing*

The nanocellulose gel (fluidized HefCel, 5 wt%) was used as inks for 3D printing. The 3D printing was performed with a Regemat3D bioprinter (version 1.0), equipped with the Regemat3D Designer (version 1.8, Regemat3D, Granada, Spain). Grids having a diameter of 20 mm, a height of 2 mm, and space between the tracks of 2 mm were printed. The inks were kept at room temperature (25 ◦C) for 24 h before printing. The flow speed was 3 mm/s, using a 0.58 mm conical nozzle. Fluidized HefCel was also combined with alginate (20%) before printing following the same set-up described earlier in this section. After printing, the grids were left in a solution of CaCl2 (50 mmol) for 24 h to cross-link the structure. The grids without and with alginate were freeze-dried with a Telstar LyoQuest-83 for 24 h.

The freeze-dried samples were immersed in MQ water, and the water holding capacity of the 3D-printed grids was estimated according to Equation (1):

$$\text{Moistureorption capacity (\%)} = (\text{Wt} - \text{Wo})/\text{Wo} \times 100\tag{1}$$

where, Wt is the weight of the grid at given time points and W0 is the weight of the dry grid.

#### *2.8. Cytotoxicity*

The cytotoxicity of extracts from soda-ethanol HefCel, O2 delignified soda-ethanol HefCel, and fluidized O2 delignified soda-ethanol HefCel was evaluated according to ISO 10993-5:2009 annex C (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity test) with extraction according to ISO 10993-12:2012. The HefCel samples were tested at two occasions: first, the unbleached and O2 delignified HefCel nanocellulose samples and later, the fluidized O2 delignified HefCel nanocellulose.

The MTT test is based on the measurement of the viability of the cells via metabolic activity. Yellow water-soluble MTT is metabolically reduced in viable cells to a blue-violet insoluble formazan. The number of viable cells correlates to the colour intensity determined by photometric measurements after dissolving the formazan in alcohol. Extracts from the test item, positive and negative controls, as well as blanks (extraction vehicle not containing the test item but subjected to conditions identical to those to which the test item was subjected to during extraction) were added to a subconfluent monolayer of L929 mouse fibroblast cells and incubated for 24 h at 37 ± 1 ◦C in 5% ± 1% CO2. After incubation the extracts were removed and MTT solution was added to the cells which were incubated for an additional 2 h at 37 ± 1 ◦C in 5% ± 1% CO2. Following incubation, the MTT solution was removed, 2-propanol was added, and the plates were shaken rapidly. Finally, the absorbance was measured at 570 nm (reference wavelength 650 nm) and the viability of cells was calculated. Thermanox Plastic Coverslips, Art no 174934 (Thermo Scientific NUNC) were used as the negative control and Latex rubber, Gammex 91-325 (AccuTech Ansell) as the positive control.

#### **3. Results and Discussion**

The chemical composition of the raw material, pine sawdust, is shown in Table 2. The amount of total carbohydrates (cellulose and hemicelluloses) were 62.45%, the rest of this material was composed of acid-insoluble lignin (29.16%) and extractives (2.27%).

**Table 2.** Chemical composition of pine sawdust.

% odw (% on mass of dry wood); StD (standard deviation).

The yields and kappa numbers of the unbleached and O2 delignified pulps are shown in Table 3. The O2 delignification reduced the residual lignin to almost half the kappa number of the unbleached soda-ethanol pulp in Stage 1, and to a third in Stage 2.

**Table 3.** Yield and kappa number of soda-ethanol pulps.

The chemical compositions of the unbleached soda-ethanol and O2 delignified pulp (from Stage 2) are summarized in Table 4. The O2 delignification removed almost 70% of total lignin in the unbleached pulp, which slightly increased the composition of carbohydrates in the O2 delignified pulps. The results show that a significant amount of lignin was removed from the pulp during O2 delignification, while very little changes were seen in the carbohydrate content or composition. Some metals, such as Si, Fe, and Mn, were removed from the pulp during the delignification treatment, while for some the amount increased.

treatment, while for some the amount increased.

**Table 4.** Chemical composition of soda-ethanol pulps. Total carbohydrates (%) 96.1 97.6

*Bioengineering* **2019**, *6*, x FOR PEER REVIEW 7 of 12

Some metals, such as Si, Fe, and Mn, were removed from the pulp during the delignification

**Table 4.** Chemical composition of soda-ethanol pulps.

Total Lignin (%) 4.1 1.2 Klason lignin (%) 3.7 0.7 Acid soluble lignin (%) 0.4 0.5

**Chemical Composition Unbleached Pulp O2 Delignified Pulp (Stage 2)**

#### *3.1. Nanocellulose Characterisation 3.1. Nanocellulose Characterisation*

Optical microscopy images of the HefCel nanocellulose samples provide information of their visual appearance, such as fibrillation degree and amount of unfibrillated fibres (Figure 1). Clearly, the degree of fibrillation was better in the case of the O2 delignified pulp and hardly any intact fibres could be detected. Unbleached material contained many more longer fibres after the enzymatic treatment. The bleached sample was cut into shorter fibre fragments (see Figure 1, middle) and thus, the amount of residual fibre particles was found to be higher according to the fibre analysis (Table 5). In the HefCel process, the accessibility of the enzyme to the cellulose fibre surface is a prerequisite, therefore, the lignin content or the number of adhesives or other impurities cannot be too high in the starting materials. This is probably the reason for the differences observed between the unbleached and O2 delignified HefCel samples (Table 4, Figure 1, left and middle). Optical microscopy images of the HefCel nanocellulose samples provide information of their visual appearance, such as fibrillation degree and amount of unfibrillated fibres (Figure 1). Clearly, the degree of fibrillation was better in the case of the O2 delignified pulp and hardly any intact fibres could be detected. Unbleached material contained many more longer fibres after the enzymatic treatment. The bleached sample was cut into shorter fibre fragments (see Figure 1, middle) and thus, the amount of residual fibre particles was found to be higher according to the fibre analysis (Table 5). In the HefCel process, the accessibility of the enzyme to the cellulose fibre surface is a prerequisite, therefore, the lignin content or the number of adhesives or other impurities cannot be too high in the starting materials. This is probably the reason for the differences observed between the unbleached and O2 delignified HefCel samples (Table 4, Figure 1, left and middle).

**Figure 1.** Optical microscopy images. (**left**) Unbleached HefCel, (**middle**) O2 delignified Hefcel, and (**right**) Fluidized O2 delignified HefCel. The bars are 200 µm.

**Table 5.** Characteristics of unbleached and bleached HefCel samples.

Fluidization had a significant effect on the quality of the HefCel nanocellulose. When the O2 delignified HefCel sample was fluidized twice, the amount of residual fibre particles decreased to 5923 pieces (pcs)/mg from the original value of 49,703 pcs/mg. This change in the quality was also noticed in the microscopy image (Figure 1, right). The sample became more homogeneous and the low shear apparent viscosity increased from 487 mPa\*s to 121,580 mPa\*s when measured at the fixed 5% consistency. Fluidization had a significant effect on the quality of the HefCel nanocellulose. When the O2 delignified HefCel sample was fluidized twice, the amount of residual fibre particles decreased to 5923 pieces (pcs)/mg from the original value of 49,703 pcs/mg. This change in the quality was also noticed in the microscopy image (Figure 1, right). The sample became more homogeneous and the low shear apparent viscosity increased from 487 mPa\*s to 121,580 mPa\*s when measured at the fixed 5% consistency. Fluidization had a significant effect on the quality of the HefCel nanocellulose. When the O2 delignified HefCel sample was fluidized twice, the amount of residual fibre particles decreased to 5923 pieces (pcs)/mg from the original value of 49,703 pcs/mg. This change in the quality was also noticed in the microscopy image (Figure 1, right). The sample became more homogeneous and the low shear apparent viscosity increased from 487 mPa\*s to 121,580 mPa\*s when measured at the fixed 5% consistency. The laser profilometry analysis revealed the development of the HefCel nanocellulose samples

*Bioengineering* **2019**, *6*, x FOR PEER REVIEW 8 of 12

*Bioengineering* **2019**, *6*, x FOR PEER REVIEW 8 of 12

**Figure 1.** Optical microscopy images. (**left**) Unbleached HefCel, (**middle**) O2 delignified Hefcel, and

**Figure 1.** Optical microscopy images. (**left**) Unbleached HefCel, (**middle**) O2 delignified Hefcel, and

**Table 5.** Characteristics of unbleached and bleached HefCel samples.

**Table 5.** Characteristics of unbleached and bleached HefCel samples.

Unbleached HefCel 55 1 5853 337 31,310 34 O2 delignified HefCel 0.4 0.1 487 14 49,703 2

Unbleached HefCel 55 1 5853 337 31,310 34 O2 delignified HefCel 0.4 0.1 487 14 49,703 2

**rpm (mPa\*s) StD Residual Fibres**

**rpm (mPa\*s) StD Residual Fibres**

**(pcs/mg) StD**

**(pcs/mg) StD**

**(Pa) StD Apparent Viscosity, 10**

**(Pa) StD Apparent Viscosity, 10**

(**right**) Fluidized O2 delignified HefCel. The bars are 200 µm.

(**right**) Fluidized O2 delignified HefCel. The bars are 200 µm.

**Sample Yield Value**

**Sample Yield Value**

The laser profilometry analysis revealed the development of the HefCel nanocellulose samples before and after fluidization (Figure 2). The unfibrillated residual fibres observed in Figure 1 and detected by the residual fibre analysis (Table 5) caused a roughening of the surface structure of the films (Figure 2, left). The additional fluidization of the HefCel increased the fibrillation degree, thus reducing the amount of residual fibres. The surface roughness as a function of lateral wavelength clearly showed that the roughness decreased significantly after the fluidization (see wavelengths of 40 and 320 micrometers, Figure 3). This indicates a reduction of residual fibres and an increase of the nanofibril fraction [25]. The laser profilometry analysis revealed the development of the HefCel nanocellulose samples before and after fluidization (Figure 2). The unfibrillated residual fibres observed in Figure 1 and detected by the residual fibre analysis (Table 5) caused a roughening of the surface structure of the films (Figure 2, left). The additional fluidization of the HefCel increased the fibrillation degree, thus reducing the amount of residual fibres. The surface roughness as a function of lateral wavelength clearly showed that the roughness decreased significantly after the fluidization (see wavelengths of 40 and 320 micrometers, Figure 3). This indicates a reduction of residual fibres and an increase of the nanofibril fraction [25]. before and after fluidization (Figure 2). The unfibrillated residual fibres observed in Figure 1 and detected by the residual fibre analysis (Table 5) caused a roughening of the surface structure of the films (Figure 2, left). The additional fluidization of the HefCel increased the fibrillation degree, thus reducing the amount of residual fibres. The surface roughness as a function of lateral wavelength clearly showed that the roughness decreased significantly after the fluidization (see wavelengths of 40 and 320 micrometers, Figure 3). This indicates a reduction of residual fibres and an increase of the nanofibril fraction [25].

**Figure 2.** Images from laser profilometry. (**left**) O2 delignified Hefcel and (**right**) Fluidized O2 delignified Hefcel. The lateral size of the assessed areas was 1000 × 1000 µm. The z-direction calibration bar is between –4 and 20 µm. **Figure 2.** Images from laser profilometry. (**left**) O2 delignified Hefcel and (**right**) Fluidized O2 delignified Hefcel. The lateral size of the assessed areas was 1000 × 1000 µm. The z-direction calibration bar is between −4 and 20 µm. delignified Hefcel. The lateral size of the assessed areas was 1000 × 1000 µm. The z-direction calibration bar is between –4 and 20 µm.

**Figure 3.** Laser profilometry. Surface roughness as a function of lateral wavelength. Note the large reduction of surface roughness after fluidization.

#### *3.2. 3D Printing*

Various 3D structures were printed to verify the suitability of the fibrillated material to be deposited on a substrate layer-by-layer and form a 3D object (Figure 4). The unbleached and O2 delignified HefCel nanocelluloses were not printable, probably due to a relatively large fraction of residual fibres (Figures 1–3, Table 5) that clogged the printing nozzle. However, after an additional homogenization with a microfluidizer the O2 delignified Hefcel sample performed well and the material could be *3.2. 3D printing*

reduction of surface roughness after fluidization.

extruded through a 0.4 mm nozzle (Figure 4). This behaviour was due to the additional fibrillation of residual fibres into micro- and nano-scale objects (Figure 3). homogenization with a microfluidizer the O2 delignified Hefcel sample performed well and the material could be extruded through a 0.4 mm nozzle (Figure 4). This behaviour was due to the additional fibrillation of residual fibres into micro- and nano-scale objects (Figure 3).

residual fibres (Figures 1–3, Table 5) that clogged the printing nozzle. However, after an additional

*Bioengineering* **2019**, *6*, x FOR PEER REVIEW 9 of 12

**Figure 3.** Laser profilometry. Surface roughness as a function of lateral wavelength. Note the large

Various 3D structures were printed to verify the suitability of the fibrillated material to be deposited on a substrate layer-by-layer and form a 3D object (Figure 4). The unbleached and O2

**Figure 4.** Three-dimensional (3D) printing of fluidized O2 delignified HefCel. (**left**) Circle (radius: 10 mm) and (**right**) squares (size: 20 mm × 20 mm) exemplifying the good print resolution and print fidelity. Right) Exemplification of a 3D-printed structure, i.e., a self-standing ear. **Figure 4.** Three-dimensional (3D) printing of fluidized O2 delignified HefCel. (**left**) Circle (radius: 10 mm) and (**right**) squares (size: 20 mm × 20 mm) exemplifying the good print resolution and print fidelity. Right) Exemplification of a 3D-printed structure, i.e., a self-standing ear.

Fluidized O2 delignified HefCel was also combined with alginate and cross-linked with calcium as a post-fixing step to solidify the structures (Figure 5). The 3D-printed grids were freeze-dried and immersed in water to assess the stability of the material and their water absorption capacity. The HefCel grid without alginate dissolved rapidly in water and it was not possible to quantify the water absorption capacity. The HefCel combined with alginate (20%) and cross-linked with Ca2+ performed well and the structure was stable even after several cycles of water immersion and lifting up to quantify the water absorption. The water absorption capacity reached a 450% weight increase after 78 h in water (Figure 6). This value is considerably lower than the previously reported values for porous nanocellulose structures, which reached levels of up to 10,000–16,000% water uptake [26,27] The relatively low water absorption capacity of the cross-linked HefCel sample was most probably due to two factors: (i) the relatively low fibrillation degree of HefCel and (ii) the cross-linking of the material which limits the water absorption. It is important to note that the water absorption capacity is one of the tests that are recommended for characterizing wound dressings [28]. Fluidized O2 delignified HefCel was also combined with alginate and cross-linked with calcium as a post-fixing step to solidify the structures (Figure 5). The 3D-printed grids were freeze-dried and immersed in water to assess the stability of the material and their water absorption capacity. The HefCel grid without alginate dissolved rapidly in water and it was not possible to quantify the water absorption capacity. The HefCel combined with alginate (20%) and cross-linked with Ca2+ performed well and the structure was stable even after several cycles of water immersion and lifting up to quantify the water absorption. The water absorption capacity reached a 450% weight increase after 78 h in water (Figure 6). This value is considerably lower than the previously reported values for porous nanocellulose structures, which reached levels of up to 10,000–16,000% water uptake [26,27] The relatively low water absorption capacity of the cross-linked HefCel sample was most probably due to two factors: (i) the relatively low fibrillation degree of HefCel and (ii) the cross-linking of the material which limits the water absorption. It is important to note that the water absorption capacity is one of the tests that are recommended for characterizing wound dressings [28]. *Bioengineering* **2019**, *6*, x FOR PEER REVIEW 10 of 12

**Figure 5.** (**A**) Fluidized HefCel material. (**B**) Fluidized HefCel material and 20 wt% alginate. The radius of the circles is 10 mm. **Figure 5.** (**A**) Fluidized HefCel material. (**B**) Fluidized HefCel material and 20 wt% alginate. The radius of the circles is 10 mm.

**Figure 6.** Water uptake of 3D-printed constructs containing HefCel, 20 wt% alginate and cross-linked

The tested samples (unbleached HefCel, O2 delignified HefCel, and fluidized O2 delignified HefCel nanocellulose) were found not to have cytotoxic potential according to the criteria given in the testing standard. The calculated cell viabilities are presented in Table 6. It is worth noting that the unbleached HefCel sample is just slightly above the limit to be considered not cytotoxic (70% cell viability). This indicates that this sample has a significantly lower cell viability compared to the O2 delignified and fluidized samples. The relatively lower cell viability caused by the unbleached HefCel sample may be caused by impurities and contamination that have not been removed by the pulping and nanocellulose production processes. The O2 delignified samples are subjected to more steps of processing and washing, which may have contributed to removing the potential impurities

with CaCl2. The water uptake was measured over 78 h.

*3.3. Cytotoxicity*

encountered in the raw material.

radius of the circles is 10 mm.

**Figure 6.** Water uptake of 3D-printed constructs containing HefCel, 20 wt% alginate and cross-linked **Figure 6.** Water uptake of 3D-printed constructs containing HefCel, 20 wt% alginate and cross-linked with CaCl2 . The water uptake was measured over 78 h.

with CaCl2. The water uptake was measured over 78 h.

#### *3.3. Cytotoxicity*

*3.3. Cytotoxicity*  The tested samples (unbleached HefCel, O2 delignified HefCel, and fluidized O2 delignified HefCel nanocellulose) were found not to have cytotoxic potential according to the criteria given in the testing standard. The calculated cell viabilities are presented in Table 6. It is worth noting that the unbleached HefCel sample is just slightly above the limit to be considered not cytotoxic (70% cell viability). This indicates that this sample has a significantly lower cell viability compared to the O2 delignified and fluidized samples. The relatively lower cell viability caused by the unbleached HefCel sample may be caused by impurities and contamination that have not been removed by the pulping and nanocellulose production processes. The O2 delignified samples are subjected to more steps of processing and washing, which may have contributed to removing the potential impurities The tested samples (unbleached HefCel, O2 delignified HefCel, and fluidized O2 delignified HefCel nanocellulose) were found not to have cytotoxic potential according to the criteria given in the testing standard. The calculated cell viabilities are presented in Table 6. It is worth noting that the unbleached HefCel sample is just slightly above the limit to be considered not cytotoxic (70% cell viability). This indicates that this sample has a significantly lower cell viability compared to the O2 delignified and fluidized samples. The relatively lower cell viability caused by the unbleached HefCel sample may be caused by impurities and contamination that have not been removed by the pulping and nanocellulose production processes. The O2 delignified samples are subjected to more steps of processing and washing, which may have contributed to removing the potential impurities encountered in the raw material.

encountered in the raw material. **Table 6.** Calculated average cell viabilities and cytotoxicity grading according to ISO10993-5:2009.

#### **4. Conclusions**

We provided an extensive characterization of pine sawdust pulp and produced high-consistency nanocellulose for 3D printing. O2 delignified soda-ethanol pulp was successfully fibrillated using high-consistency enzymatic fibrillation (HefCel) technology. However, a further homogenisation step by microfluidizer was needed to meet the requirements of 3D printing. After this step, various 3D structures of HefCel nanocellulose were successfully printed, with or without alginate. The structures containing alginate and cross-linked with CaCl2 were more stable against water. However, their water absorption capacity was lower compared to similar structures prepared from other types of nanocelluloses, possibly due to their limited fibrillation degree and use of a cross-linking agent. No cytotoxicity was observed for the HefCel materials studied, which is the first indication of the material suitability for wound dressing or similar applications.

**Author Contributions:** Conceptualization, H.K., M.C.A. and G.C.-C.; methodology, F.E.F, D.F., N.V.E, M.E.V., C.M.I., P.L. G.C.-C.; formal analysis, P.L., M.C.A. G.C.-C.; investigation, F.E.F, D.F., N.V.E, M.E.V., C.M.I., P.L. G.C.-C.; resources, H.K., M.C.A. and G.C.-C.; writing—original draft preparation, H.K., M.C.A. and G.C.-C.; writing—review and editing, H.K., D.F. M.E.V. P.L., M.C.A. and G.C.-C.; supervision, H.K., M.C.A. G.C.-C.; project administration, H.K., M.C.A. G.C.-C.; funding acquisition, H.K., M.C.A. and G.C.-C.

**Funding:** This work has been funded by the ValBio-3D project (Grant ELAC2015/T03-0715 Valorization of residual biomass for advanced 3D materials). The authors acknowledge the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), the Universidad Nacional de Misiones (Argentina), the Research Council of Norway (Grant no. 271054), and Academy of Finland (Agreement No. 311973, VTT) for the financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**Digital Differential Protection of Power Transformer Using Matlab**

Adel Aktaibi and M. Azizur Rahman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48624