Bioactive micropatterning of apatite immobilizing cell adhesion protein by laser-induced forward transfer with a shock absorber

: The additive patterning of apatite with good biocompatibility and osteoconductivity is a useful technique for the production and surface functionalization of biomaterials. We developed this technique through our laser-induced forward transfer (LIFT) process using a laser-absorbing sacriﬁcial layer in combination with a shock-absorbing polydimethylsiloxane (PDMS) receiver. With the PDMS shock-absorbing function, even the brittle apatite and that immobilizing the cell adhesion protein ﬁbronectin (Fn-apatite) were successfully transferred and micropatterned while maintaining their dense, ﬁlmy state. The laser pulse energy eﬀect was investigated, leading to the optimum energy range just above the transfer threshold. The apatite and Fn-apatite micropatterns exhibited superior cytocompatibility compared to the PDMS surface, and could potentially be used for cellular micromanipulation


Introduction
LIFT is a potential candidate for next-generation additive manufacturing because it would enable the additive micropatterning of a variety of materials, such as inks [1], metals [2][3][4][5][6][7], semiconductors [8,9], oxides [10][11][12][13][14], organic materials [15,16], biomaterials [17][18][19] silver nanopastes [20,21], and other materials [22,23], even at a micron/submicron resolution under atmospheric and room-temperature conditions.The LIFT process involves the laser irradiation of a donor material or a sacrificial layer that absorbs the laser light, leading to a laser-induced change such as heating, melting, ablation, and so forth.The laser-induced change generally induces a transient excitation field with a high temperature and/or pressure, which transfers the donor material toward a receiver substrate placed against the donor within approximately a pulse duration.
Hydroxyapatite (hereafter, apatite), Ca 10 (PO 4 ) 6 (OH) 2 , is the main inorganic component of the bones and teeth.Apatite exhibits osteoconductivity and good biocompatibility with human soft and hard tissues.Thus, apatite coatings are applied to various implant materials (e.g., artificial bones and tooth roots) to improve their functionality using various coating techniques, such as plasma spray and pulsed laser deposition (PLD) [24][25][26].These conventional apatite coating techniques can provide a high-quality apatite film.However, they are usually non-area-specific and require high temperatures and/or long processing times.For example, a biomimetic process is a nonthermal coating technique that uses a supersaturated calcium phosphate solution as a coating medium [27,28], but it generally takes several hours to days to form a micron-thick apatite coating.Accordingly, a laser-assisted biomimetic process was recently developed to overcome this drawback [29].The process still requires laser processing for as long as 30 min.A rapid and nonthermal apatite coating achieved by the LIFT process will provide an easy and versatile method for fabricating materials with biofunctional patterning and with a finely tailored surface functionality.
The present study developed a LIFT process for apatite micropatterning.Using PDMS as a shock-absorbing receiver, even the brittle apatite could be transferred and micropatterned by the LIFT while keeping its dense, filmy state.A cell adhesion protein fibronectin (Fn) was immobilized in the apatite donor film to explore herein the feasibility of protein-immobilized apatite micropatterning.We demonstrate the bioactive micropatterning of apatite and Fnimmobilized apatite (Fn-apatite) by our LIFT process, which enhanced cell adhesion and localization.

Preparation of the apatite and fibronectin-immobilized apatite donor films by the biomimetic process
Figure 1 shows a flowchart for the preparation of the apatite and Fn-apatite donor films by a precursor-assisted biomimetic process [27,28].A polyethylene terephthalate (PET) substrate was used as a transparent support, on which a carbon thin film with a thickness of approximately 50 nm was preformed as a sacrificial layer via vapor deposition.The carbon-preformed substrate (denoted as C/PET) was subjected to oxygen plasma treatment (0.1 W/cm 2 , 30 Pa, 30 s) for surface activation.
To form an apatite film, the plasma-treated C/PET substrate was thrice dipped alternately in 0.  [30]).A Fn-apatite film was formed on the C/PET substrate by the same method, except that Fn (from bovine plasma, Sigma-Aldrich) was supplemented at a final concentration of 40 µg/mL to the CaCl 2 and K 2 HPO 4 •3H 2 O solutions for the alternate dipping treatment and to the supersaturated calcium phosphate solution for the film growth.Fn is a kind of protein found in mammalian serum, and has biological activity promoting cell adhesion and spreading.We previously confirmed that Fn is immobilized within an apatite film formed by a similar precursor-assisted biomimetic process [28].After the film formation, the substrate was washed with ultrapure water and dried before use.For the Fn-apatite donor film, the substrate was freeze-dried to reduce the risk of Fn denaturation.

Film characterization
The surface morphology of the donor films was monitored by a scanning electron microscope (SEM).The film thickness was also estimated by the depth measurements of the laser-irradiated spots on the donor films after the LIFT process.The crystalline structure was evaluated by thin-film X-ray diffraction (XRD) using a Rigaku SmartLab diffractometer with a grazing angle of 0.3°.The 2θ angles ranged from 3°to 50°at 3°/min scanning speed.

LIFT system for the additive micropatterning of the apatite films
Figure 2 shows the LIFT system used in the present experiment.A high-repetition nanosecond pulsed laser (λ = 1064 nm, 10 kHz, fwhm 40 ns) was used as a light source.The Gaussian laser beam was scanned with galvanometer mirrors and the fθ lens system.Apatite has insufficient light absorption for the LIFT in the wavelength range from UV to near-IR; hence, a carbon film was inserted between a transparent support and the donor film as a laser-absorbing sacrificial layer to efficiently lead to a laser-induced change such as laser ablation and resultant film transfer.Each laser pulse induces the transfer of the donor film to a receiver substrate; therefore, the transferred apatite film has a shape that corresponds to the laser spot.Here the donor film and the receiver substrate were placed in direct contact in our LIFT system to reduce the laser pulse energy threshold for transfer and the impact upon transfer.
Fig. 2. Schematic of the LIFT system for the apatite micropattern fabrication.Each nanosecond (ns) laser pulse is absorbed at a carbon sacrificial layer between a transparent PET support and an apatite donor film, resulting in the transfer of the apatite donor to a receiver with a micropattern corresponding to the laser spot.

Cell adhesion assay
We assayed the cell adhesion properties of the apatite and Fn-apatite films before and after the LIFT process by a cell culture test.We used a widely used epithelial cell line CHO-K1 derived from the Chinese hamster ovary.CHO-K1 cells are adherent cells.They adhere to solid surfaces for growth.For the cell culture test, the plasma-treated C/PET substrate was sterilized by exposure to ethylene oxide gas, then coated with apatite or Fn-apatite donor film under aseptic conditions.The thus-prepared sterile donor sample (apatite/C/PET or Fn-apatite/C/PET) and sterilized PDMS receiver were set in a sterilized and sealed holder for the LIFT process to prevent contamination by microorganisms.
The CHO-K1 cells (RIKEN BioResource Center) were seeded (5 × 10 4 cells/0.5 mL medium/well) on each sample using a medium (RPMI-1640) with 10% fetal bovine serum and a 24-well cell culture plate.After culturing at 37°C in 5% CO 2 for 24-27 h, the cells on the sample were rinsed with phosphate-buffered saline to remove the non-adhering cells from the sample surface, fixed with paraformaldehyde, and stained by crystal violet (a dye commonly used for staining cells) for optical microscopy.The culture period was determined from the preliminary test varying the observation timing to enable the observation of the morphology of each cell and the cell density on the sample surface.

Donor film characterization
Figures 3(a) and (b) show the top-and 30°-view SEM images for the apatite/C/PET donor sample, respectively.These high-magnification SEM images showed that the film has a characteristic porous structure, which is a typical morphology of the calcium phosphate films prepared by our biomimetic method [28].Figure 3(c) depicts a top-view SEM image for the Fn-apatite/C/PET donor sample and a porous surface similar with the apatite/C/PET sample, although the pore size was smaller.The depth measurements of the laser-irradiated spots after the LIFT process indicated that the donor film thickness was approximately 500 and 300 nm for the apatite and Fn-apatite films, respectively.Based on these results, the addition of Fn made the film thinner in our method.
Figure 3(d) shows the thin-film XRD pattern for the Fn-apatite/C/PET sample.Two diffraction peaks assigned to hydroxyapatite appeared at 2θ = 26°and 32°(red circles, Fig. 3(d)).Furthermore, no other sharp peaks specific to different calcium phosphate crystalline phases like octacalcium phosphate were found.The broad diffraction peaks at 20°and 45°can be assigned to a PET substrate.These results confirmed that the Fn-apatite/C/PET donor film has apatite crystallites, regardless of thinning.

LIFT of the apatite films using the shock-absorbing PDMS receiver
The LIFT is effective in the delivery and patterning of various kinds of materials.However, challenges still remain in the transfer of brittle films like inorganic apatite films.The LIFT is generally accompanied by the collision between a transferred donor film and a receiver substrate; hence, brittle donor films easily fracture upon transfer.We hypothesized that this problem can be overcome by employing shock-absorbing materials as a receiver because they can absorb and buffer the impact shock.Polymer materials generally exhibit both elasticity and viscosity during deformation.The dynamic mechanical test, which measures the response of the material to sinusoidal or other cyclic stresses, is suitable for the rigorous evaluation of such rheological characteristics of the materials [31].The complex modulus, which is experimentally determined by applying a sinusoidal stress, is resolved into two components, namely storage modulus E and loss modulus E .While the former is the elastic component that is directly proportional to the energy storage during deformation, the latter represents the viscous one that is proportional to the energy loss as heat.The tangent of the phase angle (δ) between stress and strain, namely the loss tangent (tan δ = E /E ), is the ratio of the energy lost to the energy stored.Thus, an increase in tan δ indicates that the material is more able to absorb more energy, acting more as a shock absorber.For example, the value of tan δ for a typical PET is approximately 10 −2 (at a measured frequency of 1 Hz, room temperature) [32].In contrast, the tan δ of PDMS is approximately one order of magnitude larger than that of PET, indicating that the PDMS has a higher shock absorption ability.
To verify this hypothesis, we transferred apatite films by the LIFT toward two different receivers: PDMS (soft surface) and PET (hard surface).Figure 4 shows the confocal laser scanning microscopic images of the apatite microchips transferred onto the (a) PDMS and (b) PET receivers.The transferred apatite chips on the PDMS receiver retained their dense, filmy state, and had a circular shape corresponding to the laser beam spot.In contrast, the apatite film was observed to crush on the PET receiver.Figure 4(c) depicts the model diagrams of the impact force generated on these two receivers during the LIFT.When the receiver is PDMS (left), the impact shock upon film transfer can be dissipated as heat and effectively absorbed by the receiver, thereby reducing the destructive impact on the apatite film.In contrast, when a receiver is made of PET (right), the impact shock does not dissipate much as heat, and is stored near the surface, resulting in a film fracture.The results confirm that the PDMS receiver functioned as a shock absorber, enabling the LIFT of even brittle apatite films.

Pulse energy dependence of the LIFT for the Fn-immobilizing apatite
Next, we used the Fn-apatite donor film for the LIFT to provide apatite patterns with additional functionality.As shown in the inset in Fig. 5(b), the Fn-apatite film was successfully transferred and micropatterned on the PDMS receiver at a laser pulse energy of 110 µJ/pulse.The transferred microchip had almost no cracks and a shape corresponding to the laser beam, which confirmed a high-quality film transfer.Thus, the present LIFT process is applicable not only to the apatite films, but also to the Fn-apatite films.The laser pulse energy dependence of the morphology and the size of the transferred Fn-apatite micropatterns was explored in detail to investigate the LIFT behavior.Figure 5(a) shows the morphology change with the laser pulse energy of the laser-irradiated spots on the C/PET (left) and Fn-apatite/C/PET (center) samples of the corresponding Fn-apatite micropatterns formed on the PDMS receiver (right).The laser pulse passed through the PET support and reached the carbon film in the case of both C/PET and Fn-apatite/C/PET samples.As a result of the high laser absorption by the carbon film, laser-ablated spots formed on the former sample even at 50 µJ/pulse.By contrast, the latter sample with the top Fn-apatite film exhibited no clear change in the surface structure after the laser irradiation below 90 µJ/pulse.Roughly a factor of two was found between these two samples in terms of the laser pulse energy threshold for the laser-induced surface morphology change.A certain portion of the incident energy η abs • E p was absorbed in proportion to the absorption coefficient of the material after reflection when a laser pulse energy E p was applied on a material.In the case of general laser ablation, this energy is distributed as: where E abl denotes the ablation energy necessary for the evaporation of the materials; E v is the kinetic and thermal energies of the vapor; and E res.heat is the energy of the residual heat concentrated around the ablation area [33].In the case of the LIFT of a material coated with a donor film and a sacrificial layer, laser light absorption mainly occurs at the sacrificial layer, leading to a laser-induced change of the sacrificial layer E s,change , such as heating, melting, ablation, and so forth.Part of this then becomes an energy term used for the propulsion of the donor material to the receiver E d,transfer .Thus, η s,abs • E p is described as follows: E d,transfer mainly depends on the energy of adhesion of the donor film to the sacrificial layer and the energy to extrude the donor film; hence, it varies with the combination of the sacrificial and donor materials and the density and thickness of the donor film.The higher laser pulse energy threshold for the laser-induced surface morphology change on the Fn-apatite/C/PET sample as compared to that on the C/PET sample stems from the energy term E d,transfer in Eq. ( 2).At a laser pulse energy of 90 µJ/pulse (ablation threshold), the transfer of the Fn-apatite film to the PDMS receiver began to occur as shown in the center and right images in Fig. 5(a).Crack-free Fn-apatite micropatterns formed on the PDMS receiver in the energy range from 90 to 130 µJ/pulse.Cracks started to appear on the transferred films as the pulse energy increased above 140 µJ/pulse, eventually leading to a film fracture.These facts show an optimum energy range for high-quality film transfer just above the transfer threshold.
Figure 5(b) plots the average diameter of the laser spots formed on the C/PET (black triangles) and Fn-apatite/C/PET (open red circles) samples and of the corresponding Fn-apatite microchips transferred onto the PDMS receiver (blue circles) versus the laser pulse energy.The average diameter was determined by calculating the average value of the minor and major diameters of the elliptical shape reflecting the laser beam spot from the sample number n = 3.The average diameter of the laser-ablated spots on the C/PET sample monotonically increased with the increasing laser pulse energy and matched the spot diameter observed on the Fn-apatite donor film and the diameters of the transferred Fn-apatite microchips, which confirmed that the main driving force for Fn-apatite transfer was the laser ablation of the carbon film.The diameter of the Fn-apatite microchips greatly differed from that of the laser spot on the Fn-apatite donor film at a laser pulse energy of 250 µJ/pulse because the film severely fractured during the transfer.The laser pulse energy for the LIFT must be optimized to achieve a high-quality film transfer without causing crack formation and film fracture.) than on the Fn-free apatite film.Many of the cells on the latter film remained spherical (Fig. 6(a)).The dark purple spots, which are 10 to several tens of micrometers in size, are the stained cells.This result indicates the superior cell adhesion properties of the former film compared to the latter, which was caused by the physiological activity of Fn in the Fn-apatite donor film to promote cell adhesion and spreading as previously reported [28].These results confirm the cell adhesion activity of the Fn-apatite donor film before laser irradiation.Figures 6(c) and (d) show the optical microscopic images of the CHO-K1 cells cultured on the apatite and Fn-apatite micropatterns, respectively, prepared on the PDMS receiver by the LIFT.The pale purple patterns of the circular disks with diameters of approximately 100 µm depict the apatite (c) and Fn-apatite (d) micropatterns formed by the LIFT.The cells densely accumulated on both of these micropatterns, whereas those on the outer PDMS surface were sparse.This result suggests that the apatite and Fn-apatite micropatterns prepared by the LIFT process exhibited superior cytocompatibility for the CHO-K1 cells than PDMS, which is in agreement with previous reports on apatite-coated siloxane-based polymers [34,35].The enhanced cell adhesion on the micropatterns can be attributed to the presence of apatite, which has high adsorption affinity, with serum proteins including fibronectin and vitronectin [36].In addition, many of the cells on the Fn-apatite micropatterns extended and flattened (Fig. 6(d)), as in the case of the as-prepared donor film (Fig. 6(b)).This result might be attributed to the cell adhesion activity of Fn in the Fn-apatite micropatterns, although further studies are needed to clarify this hypothesis.Note that some of the cells were localized and aligned along the periphery of the apatite and Fn-apatite chips (Figs.6(c) and (d)).This trend shows the potential cell manipulation capability of these micropatterns (i.e., cell alignment (direction of cell elongation)).The localization and adhesion might also be controllable by the micropatterns prepared by our LIFT technique.

Summary
The additive micropatterning of both apatite and Fn-apatite was successfully demonstrated herein by our LIFT process, which uses a laser-absorbing carbon sacrificial layer and a shock-absorbing PDMS receiver.By tuning the laser pulse energy, we confirmed an optimum energy range just above the transfer threshold.The CHO-K1 cell culture experiments revealed the good cytocompatibility of the apatite and Fn-apatite micropatterns.The present LIFT process could be useful in additive manufacturing and surface functionalization of biomedical materials.

Fig. 1 .
Fig. 1.Process flowchart for the preparation of the apatite and fibronectin (Fn)-immobilized apatite (Fn-apatite) donor films on a carbon sacrificial layer-coated PET support (C/PET) by a precursor-assisted biomimetic process.The Fn proteins are immobilized within the apatite donor film by supplementing the CaCl 2 , K 2 HPO 4 •3H 2 O, and supersaturated calcium phosphate solutions with Fn.

Fig. 3 .
Fig. 3. SEM images from the (a) top-and (b) 30°-view observation for the apatite/C/PET donor sample.(c) Top-view SEM image and (d) thin-film XRD pattern for the Fnapatite/C/PET donor sample.

Fig. 4 .
Fig. 4. Confocal laser scanning microscopy images of the apatite microchips transferred by the LIFT onto the (a) PDMS and (b) PET receivers.(c) Model diagram of the impact force generated on the PDMS (left) and PET (right) receivers during the film transfer.The impact force can be dispersed and absorbed by the PDMS receiver, while the impact does not dissipate much as heat, and is stored near the PET surface, resulting in a film fracture.

Fig. 5 .
Fig. 5. Laser pulse energy dependence of the morphology and size of the Fn-apatite micropatterns.(a) Confocal laser scanning microscopy images of the laser-irradiated spots on the C/PET (left) and Fn-apatite/C/PET (center) samples and the corresponding Fn-apatite micropatterns formed on the PDMS receiver (right).(b) Variation of the average diameter with the laser pulse energy of the laser-irradiated spots on the C/PET (black triangles) and Fn-apatite/C/PET (red open circles) samples and of the corresponding transferred Fn-apatite microchips (blue circles).

3. 4 .
Cell adhesion properties of the apatite and the Fn-apatite donor films and micropatterns prepared by the LIFT First, we characterized the cell adhesion properties of the as-prepared apatite and Fn-apatite donor films (Figs.6(a) and (b)).After culturing, more cells extended and flattened on the Fn-apatite donor film (Fig. 6(b)

Fig. 6 .
Fig. 6.Optical microscopic images of the CHO-K1 cells (dark purple spots with 10 to several tens of micrometers in size) cultured on the (a) apatite/C/PET and (b) Fn-apatite/C/PET donor samples and the corresponding (c) apatite and (d) Fn-apatite micropatterns on PDMS prepared by the LIFT.The CHO-K1 cells are fixed and stained by crystal violet for 24 h (c, d) or 27 h (a, b) after seeding.In (c) and (d), cells were observed more densely on the micropatterns, suggesting a higher cytocompatibility of the transferred apatite and Fn-apatite compared to PDMS.