Surface functionalization of 3D printed poly-ε-caprolactone by ultrashort laser mirostructuring and ZnO nanolayer deposition

Due to its mechanical properties and good biocompatibility, polycaprolactone (PCL) is a promising material for bone tissue regeneration. However, a major limitation to its use remains the lack of inherent antimicrobial properties and its susceptibility to bacterial colonisation and biofilm formation. A potent strategy for overcoming such issues is surface functionalisation at micro and nano level, which can have a great impact on cell-surface interaction without affecting the integrity of the material. This study presents a novel methodology for surface modification of polymers combining ultrashort laser microstructuring and atomic layer deposition of ZnO. For this purpose, the surface of 3D printed PCL scaffolds was treated with a femtosecond laser (λ=800 nm; τ=75 fs) in order to develop parallel microchannels onto which ZnO nanolayers were further deposited. The presence of ZnO on the laser structured and unstructured scaffolds was detected by X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDX). Both methods confirmed the successful layering as EDX further highlighted a trend that ZnO built up substantially more at the bottom of the microchannels rather than at the top of them. The obtained results would allow proceeding to the next step of the study – investigating the antimicrobial effect of the developed interfaces.


Introduction
Poly-ε-caprolactone is a semicrystalline aliphatic polyester with a melting point of 58 -63 °C [1].The material is composed of hydrophobic methylene and polar ester groups.This combination yields a hydrophobic product which can undergo hydrolysis in physiological conditions that can take up to several years [2].The products of the hydrolytic degradation could be further metabolized or excreted by the organism, thus no collateral inflammation is caused.Overall, PCL exhibits mechanical properties that are similar to the ones of human trabecular bone, however, these are dependent on the molecular weight and the level of crystallinity of the fabricated scaffold [3].By adjusting these two parameters, the compressive strength and modulus could either increase or decrease.These features of a potential scaffold for bone tissue engineering are of crucial importance for the effective regeneration of a damaged tissue.Despite its strong advantages, the use of PCL as an implant still holds certain limitations, the major one of which is its inertness.Because of this, PCL-based scaffolds lack good cell interaction/adhesion after implantation [4].This limitation could also lead to another post-implantation issue that is common for orthopedic implants, namely, bacterial adherence to the surface of the implant and the formation of a biofilm [5].The biofilm is characterized by bacterial aggregation and embedding in an extracellular polymeric substance matrix.Such layer provides shelter to bacteria and might often become impenetrable for antibiotics and immune cells [5].Thus, there is a high risk of developing a chronic infection that can require sequential surgeries or can lead to implant failure and rejection.There are various strategies for tackle this issue and reduce bacterial adhesion.One such approach is surface functionalization with antimicrobial agents such as peptides, antibiotics or ion releasing substrates [6].Atomic layer deposition (ALD) is a potent method for surface modification as it allows the precise development of nanolayers.During this process, one or more precursors are used to grow uniform atomic layers in a controlled manner [7].
The present study employs two strategies to overcome the limitations of PCL as a scaffold for bone tissue engineering.During the first stage, the surface of 3D printed PCL scaffolds is modified by femtosecond (fs) laser ablation in order to create ordered micropatterns that change the topography of the material in a way that could stimulate human cell adhesion and proliferation.The second stage of the experimental methodology involves the use of ALD for development of ZnO nanolayers that have been widely investigated for its antimicrobial activity.The aim of the proposed approach is to create an antimicrobial interface that could enhance bone tissue regeneration while preventing bacterial adhesion.

Fabrication of 3D printed porous polycaprolactone scaffolds
The scaffolds were fabricated similarly to the already described approach by Daskalova et al [8].Briefly, PCL pellets (average Mn: 45,000; Sigma-Aldrich, St. Louis, MO, USA) were melted at 110 °C directly in a metal cartridge of a custom-built 3D printer using a pneumatic extrusion system.The final products were extruded through a stainless steel luer-lock nozzle tip (280 μm of inner diameter) at a temperature of 110 °C.The printing parameters included: speed of printing was 100 mm/min; 1.4 bard printing pressure; 3 layers each with height of 0.2 mm; 50% of infill.

Surface processing of 3D printed PCL by ultrashort laser irradiation
In order to modify the surface topography of the 3D printed scaffolds, a Ti:sapphire mode-locked (Solstice Ace, MKS Spectra-Physics, USA) fs laser (λ=800 nm, τ=70 fs) was used.The material was irradiated by a raster scan on a vertically positioned XY translation stage (Thorlabs, NJ, USA) with applied laser fluence (F) of 4.1 J/cm 2 , scanning velocity (v) of 3.44 mm/s and repetition rate (ν) of 1 kHz.

Determination of 3D printed PCL melting point by differential scanning calorimetry (DSC)
The measurement was performed with a differential scanning calorimetry apparatus NETZSCH DSC 200 PC using the standard NETZSCH Proteus Thermal Analysis software version 4.3.1.Sample preparation included the placing of a single 3D printed scaffold in a crucible with a lid which was closed by a preparation press.The enclosed sample was inserted in the furnace of the DSC apparatus alongside an empty crucible serving as a reference to account for the influence of the heat capacity of the crucibles.During the DSC, nitrogen was used as a protective and purging gas.The gas flow rate was 20 ml/min.The temperature range for heating and cooling was set between 10 °C and 200 °C with heating and cooling rates of 3 °C/min.The thermal characteristics of the solid and liquid phases in the phase transition zone were approximated by tangential approximation.The enthalpy values were calculated according to the same method.

Atomic layer deposition of ZnO on laser-structured and unstructured PCL scaffolds
The ZnO nanolayers were grown on laser-treated 3D-printed PCL substrates using an ALD BENEQ TFS-200 system.Pulses of diethylzinc (DEZ) and deionized water (DI H2O) precursors with a duration of 300 ms were used separated by 5-s purging with nitrogen.During deposition, besides the laser treated PCL substrate, an untreated PCL and an n-Si(111) substrate were also placed in the reactor as references.Due to the low melting temperature of PCL (58 ℃) (see 3.2), the deposition was carried out at lower temperatures outside the ALD window for the system as also described by Lim et al [9].A series of depositions at low temperature with varying number of cycles were performed in order to obtain layers with rising thickness over laser-structured PCL scaffolds.The layers' thickness was estimated by ellipsometry using a Woollam M2000D ellipsometer applied to the referent n-Si(111) substrate.Table 1 provides summarized details on the ALD procedure as well as the measured thickness of the different layers.

Characterisation of functionalized PCL scaffolds by an X-ray diffraction analysis (XRD), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX)
Several methods for detection of the formed ZnO nanolayer were used.An XRD analysis was performed by using a Philips PW1050 X-ray diffractometer system (Philips, The Netherlands) utilizing a copper anode with a secondary monochromator of the diffraction beam.The range chosen for the measurement was 22° -60° 2θ with a step size of 0.05° 2θ.Obtained results were analysed with QualX2 and Crystallography Open Database.The morphology of the laser-induced micropatterns was analysed via SEM ("Lyra", Tescan Orsay Holding, Brno-Kohoutovice, Czech Republic) equipped with an EDX module (Quantax 200, Bruker, Karlsruhe, Germany) which allowed further investigation of ZnO presence.Before proceeding with the analysis, all samples were sputter coated with a thin layer of carbon.

Development of surface micropatterns for enhanced cell attachment
Ultrashort laser treatment of 3D PCL with working laser parameters of F=4.1 J/cm 2 and v=3.44 mm/s led to the formation of parallel microchannels with an average width and depth of 21 μm and 25 μm, respectively (figure 1).SEM imaging revealed defined clear patterning with small amount of material partially expulsed and re-solidified again over the microchannels.No thermally-induced collateral damage could be observed.In previous research of our group, Filipov et al developed microstructures of similar morphology and dimensions on 3D printed PCL and investigated their effects on the adhesion of osteoblastic-like cells [10].The authors found that the cells adhered preferably to the structured surfaces and not on untreated smooth scaffolds.Furthermore, the cells also aligned along the microchannels.These findings demonstrated how micropatterning could improve drastically the cellmaterial interaction and would support the results of the present study for further in vitro studies.

Differential scanning calorimetry of 3D printed PCL
The results of the analysis showed that the material exhibited a single first-order phase transition.During the heating process, a single endothermic reaction was observed and, accordingly, during the cooling process, a single exothermic peak was detected.The endothermic peak appeared to be complex, however, this was due to the inhomogeneity of the material in the capsule.During the endothermic process, PCL began melting at 58.1 °C with enthalpy values of 68.36 J/g and became liquid at 65.2 °C.
The maximum of the endothermic peak was at 62.6 °C.During the exothermic process, phase transition began at 40.2 °C with enthalpy value of -66.99 J/g, and finished at 34.1 °C.The maximum of the exothermic peak was at temperature 37.2 °C.Similar results were reported by Sánchez-González et al who designed PCL-based membranes and studied their thermal properties [11].

Characterisation of ZnO nanolayer on laser structured and unstructured PCL scaffolds
The process of ALD allowed the formation of ZnO nanolayers over surface modified and unmodified polymeric scaffolds.The nanolayers were identified via XRD and EDX analyses.The first one clearly identified characteristic planes for both PCL and ZnO on both laser treated and untreated samples (figure 2).Regarding PCL, the peaks corresponding to (110) and (200) in the range of 20° -25° could be distinguished in all samples regardless the surface laser treatment.This would mean that the laser treatment did not affect the degree of crystallinity in the polymer.The XRD analysis detected the (100) and (101) planes of ZnO in the range of 30° -40°, confirming not only the successful nanolayer growth, but also the presence of crystalline ZnO phase despite the lower temperature of deposition.Similar results were achieved by Dominguez et al who ALD of ZnO at temperature of 120°C and detected the presence of the two planes (100) and (101) [12].This could lead to the conclusion that despite working outside the optimal ALD window for ZnO, the proposed experimental methodology still allowed the development of crystalline ZnO nanolayers on the laser structured polymer [13].In order to further investigate quantitatively the differences in ZnO nanolayer formation between untreated and laser treated samples, EDX analysis was used (figure 3).There was a clear trend that followed the ellipsometry measurements on the referent n-Si(111) substrate (table 1).Following the increase in number of ALD cycles, the atomic % of ZnO on both modified and unmodified samples rose (figure 3).However, a distinct difference was noted in that the nanolayer was present in larger quantities on the laser-induced micropatterns when compared to the control scaffolds.Due to the presence of alternating structures resembled by the bottoms of the microchannels and the ridges between two adjacent microchannels, EDX allowed further comparison of ZnO presence on both.An interesting pattern was noted that for the three groups of cycles, there was a larger adsorption of ZnO at the bottom of the microchannels compared to the quantities detected on the ridges.These results could be explained with the formation of potential reactive groups or with a shift in surface energy at the regions where the fs laser interacted with the material.Thus, ZnO could be adsorbed more strongly.Research has shown that functionalisation of PCL by ZnO has been achieved by using two main approaches -by surface attachment of ZnO with a prior PCL treatment or by electrospinning.For example, Permyakova et al performed initial plasma treatment of PCL in order to create PCL-COOH layers which could further react with positively charged ZnO nanoparticles [14].Comparing literature results with the ones in the present study, it could be stated that the fs laser processing could represent a suitable one-step procedure for initial surface treatment allowing strong adsorption of ZnO without the use of additional treatments.LSB -bottom of microchannels; LST -ridge between microchannels.

Conclusion
The present paper demonstrates the preliminary results of an innovative methodology that aims at developing antimicrobial interfaces on biodegradable polymers for bone tissue engineering.The obtained initial data provided strong evidence that fs laser-induced micropatterns suitable for cell attachment could successfully improve the adsorption of ZnO.Further investigation in the laser-matter interaction would provide an insight into the mechanisms of ZnO adsorption, while future in vitro studies could confirm the antibacterial potential of the developed interface.

Figure 3 .
Figure 3. EDX analysis of ZnO at.% on laser modified and unmodified samples.LSB -bottom of microchannels; LST -ridge between microchannels.

Table 1 .
Working parameters for ZnO nanolayer growth by ALD.