The controlled interfacial properties of materials and modulated behaviours of cells and biomolecules on their surface are the requirements in the development of a new generation of high-performance biomaterials for regenerative medicine applications. Roughness, chemistry and mechanics of biomaterials are all sensed by cells. Organization of the environment at the nano- and the microscale, as well as chemical signals, triggers specific responses with further impact on cell fate. Particularly, human mesenchymal stem cells (hMSCs) hold a great promise in both basic developmental biology studies and regenerative medicine, as progenitors of bone cells. Their fate can be affected by various key regulatory factors (e.g. soluble growth factors, intrinsic, extrinsic environmental factors) that can be delivered by a fabricated scaffold. For example, when cultured on engineered environments that reproduce the physical features of the bone, hMSCs express tissue-specific transcription factors and consequently undergo an osteogenic fate. Therefore, producing smart bio-interfaces with targeted functionalities represents the key point in effective use of hierarchically topographical and chemical bioplatforms. In this chapter, we review laser-based approaches (e.g. Matrix-Assisted Pulsed Laser Evaporation (MAPLE), Laser-Induced Forward Transfer (LIFT), laser texturing and laser direct writing) used for the design of bio-interfaces aimed at controlling stem cell behaviour in vitro.
- laser processing
- protein-based coatings
- stem cells
Currently, there is an increased interest within regenerative medicine applications in materials and methods for controlling the interface characteristics of materials in report with the modulated behaviour of cells and biomolecules on their surface. Tissue engineering approaches rely on guided tissue regeneration using materials that serve as templates for ingrowths of host cells and tissue, or on cells that have been implanted as part of an engineered device [1-3]. The factors considered within this context refer to understanding the extracellular and intracellular factors modulating cell functions, optimizing or replicating complex scaffold architecture and arrangement
Stem cells hold a great promise for a wide range of applications in regenerative medicine as well as key elements in model systems aimed at understanding fundamental processes in the field of developmental biology. Therefore, any progress in controlling their behaviour remains an important challenge [16-18]. Stem cells, from multipotent stem cells, pluripotent embryonic stem cells to induced pluripotent stem cells, have been the subject of numerous investigations related to identifying specific intrinsic and extrinsic regulatory cues, defining niches and characterizing their potential of self-renewal and differentiation [19, 20]. Consequently, a wide variety of key regulatory factors were discovered, from soluble growth factors, extracellular matrix (ECM) interactions and cell–cell interactions to micro- and nano-engineered environments or combinations thereof.
The domains of preventive and therapeutic healthcare and tissue engineering converge into an interdisciplinary field, where the interaction between materials interfaces and proteins or cells is dictated by materials bulk but more importantly by interface characteristics. Biomaterials are used widely for studying cell–substrate interactions, scaffolding or implant functionality [15-20]. The requirements for the use of natural or synthetic biomaterials are related to the degradation or stability (mechanical integrity) within fluids and fluid transport, the presence of cell-recognizable surface chemistries, the ability to deliver active biomolecules and to induce signal transduction.
The current trend is producing either ‘smart’ biomimicking synthetic or natural composites or enhancing bulk materials (e.g. metals, ceramics and polymers) by either chemical, physical and/or topographic surface modification or surface functionalization [15-17].
Natural biomaterials include collagen, fibrinogen, hyaluronic acid, glycosaminoglycans (GAGs), laminin, heparan sulphate proteoglycans, hydroxyapatite (HA) (being bioactive, biocompatible and of similar mechanical properties as native tissue), fibrinogen and fibrin cellulose, chitosan and silk fibroin. These were and are successfully used as 2D and 3D micro- and nano-engineered environments for stem cell culture. For example, Matrigel™, which contains a variety of ECM components, was used in tissue culture as such or in combination with various growth factors for improving neovasculature formation in an ischaemic mouse model , with a view to produce tube-like vascular structures . When designing a new biomaterial, there are several aspects to take into account, such as micro- and nanoscale manipulation of scaffold composition, mechanical strength, control over porosity and 3D architecture, adequate surface area and adsorption kinetics and chemistry of bulk degradation of the scaffold, material-processing abilities, logistical issues of cost, compatibility with sterilization techniques and shelf life [14-16].
An enhanced biological response (surface–cell interactions, osseointegration, bone-to-implant interface strength, resistance for long-term functional loading) requires different surface treatments to be applied, from hydroxyapatite aggregation to Mg ion implantation, sandblasting or acid-etching. However, contamination of the surface with chemical compounds occurs often, which leads to modified biological response; so more
Therefore, besides material composition importance in addressing the current challenges in tissue engineering, the micro- and nano-fabrication methods provide the input for development of precise topographical architecture for 2D or 3D substrates. Various methods have been used, from self-assembly, lithography, photolithography, soft lithography, polymer demixing, phase separation and electrospinning [23-27] to surface roughness modification, with sandblasting, anodic oxidation and acid-etching [28, 29].
Laser-based method for surface modification is a promising alternative as it can be automated and is reproducible, does not generate contamination and, moreover, it can confer a variety of nano- and microstructures with increased roughness and stable characteristics for long-term bio-interaction assays. Laser deposition (i.e. Matrix-Assisted Pulsed Laser Evaporation) can create complex coatings onto 2D and 3D substrates, while direct writing and/or texturing of polymers represent two different approaches used to create 2D and 3D topographical features as physical guidance structures. This chapter will provide information about how bio-interfaces can influence cell fate, with a focus on their effect on stem cells and on bio-interfaces engineering using laser-based methods. In addition, it will cover the controlled regulation of stem cell response
2. Surface topographical key factors for triggering stem cell response
Various factors are responsible for the behaviour of cells in response to their own niche. Considering that surface topography and chemistry represent major factors at cell–substrate interface dictating biological reactions, it becomes crucial to elucidate the complex interplay between cells, molecular signal pathways and the effect of external factors (e.g. an implant surface, functionalization and topographic features) within well-controlled cell culture systems. Nevertheless, since, in the last years, mesenchymal stem cells (MSCs) have been ideal targets in regenerative therapies, understanding of the pathways and the cues affecting stem cell differentiation to the needed lineage is critical. The ideal tissue substrate will therefore have to deliver the optimal combination of physical and biochemical signals for the spatio-temporal control of stem cell commitment and differentiation . This section of the chapter focuses on describing current state of the art in topography controlling MSC fate.
2.1. Mechanical factors that influence stem cell differentiation
MSCs are multipotent cells able to differentiate to at least three lineages: osteoblast, chondrocyte and adipocyte. With a view to recreate specific niche microenvironments to direct MSC differentiation to a required phenotype, specific control elements have to be defined. Recent high-throughput approaches have aimed at defining specific chemical [31, 32] and/or biophysical [33, 34] factors controlling stem cell fate.
The formation, evolution and breakdown of the cellular components are highly influenced by the presence of topographical cues on the surface. The stress applied through focal adhesions
Stressing the cytoskeletal filaments networks has impact on alteration of nuclear protein assembly, gene transcription, DNA replication or RNA processing, due to nuclear shape changes, which generates modifications in chromatin organization , with further impact on gene expression programmes. Physical interactions between stem cells and ECM that govern cell fate have been reviewed in Ref. . Several lessons have been more recently learned by studying polymer scaffolds that are important for understanding how the ECM proteins regulate MSC behaviour and for the future development of customized culture systems as already available for embryonic stem cells [41, 42]. Recent studies have revealed that it is the extracellular matrix tethering that regulates stem cell fate rather than substrate/ECM stiffness
2.2. Engineering biosurface topographical cues at the micro- and nanoscale to control cell function
It has been shown that texturing of biomaterials increases cell adhesion in comparison to flat, smooth surfaces. Topography of a material can be characterized by different roughness parameters (Ra, Sa, Sm – measured in nanometres) determined by AFM, confocal microscopy, optical profilometry, SEM or tactile profilometry . Knowing that the
A variety of nano- or microtopographies within hundreds of nanometres up to tens of microns range (e.g. nanoposts and nanogratings: 150, 400 and 600 nm in diameter and width; microposts and pits: 300 nm–10 μm in diameter, 3–50 μm in height) were obtained by various techniques (photolithography, electron beam lithography, reactive imprint lithography, etching, replications, hot embossing, injection moulding) [10-16] and used to stimulate hMSC-stimulated osteogenic differentiation. Although most of the studies present interesting and promising results, only a single or a small selection of parameters related to cell response were measured. Proposed platforms designed to study MSC fate regulation by both substrate mechanics and dynamic loading in 2D and 3D are reviewed in Ref. . Noteworthy, few bio-interfaces obtained by polymer-processing approaches were able to initiate osseoinduction
2.3. Micro- and nanotopography of biosurfaces engineered by laser methods
Within the above-discussed context, by providing a control over the scale and patterning (both chemical and topographic), development in specific cell-regulating cues is implied, with the application ranging from basic cell biology to tissue engineering. Specific control over patterning involves microfabrication approaches: microcontact printing, abrasion, photolithography, hot embossing, electrospinning and laser ablation [52-57]. As most of the bioapplications require sterile conditions, laser-based techniques (i.e. laser direct irradiation or texturing, Laser-Induced Forward Transfer (LIFT), Matrix-Assisted Pulsed Laser Evaporation, photopolymerization) are contact-free techniques and could be integrated with required sterile processes. Tailoring surface textures and their features on multiple scales can be controlled by directly and precisely processing by rapid scanning of focused laser beams (nanometre range up to the millimetre range on flat and curved surfaces). Moreover, by using ultrashort pulse lasers, the mechanical properties of the materials remain unchanged after the laser processing. In the cases where controlled biocoating is necessary, by using MAPLE, the thickness, porosity, architecture of mono- or multilayer can be easily tuned by controlling laser and target parameters. Among the main advantages, these are included: no limitations in the use of materials to be deposited or structured, no difficulty in controlling the thickness of the deposited layers, the ability to deposit multilayers without interlayer blending and compatibility in processing a wide range of nanoparticles, polymeric and biologic materials [52-57].
2.3.1. Engineering microstructured thin-film biosurfaces: gradients and porous surfaces by excimer laser direct texturing
The surface texturing in the context of specific surface roughness and architecture is also required for influencing cell directionality, proliferation and differentiation as the specific surface textures can be used to influence the functional properties. Using laser texturing, the surface topography of the material is altered, thus increasing the adhesion of the cells to the substrate and promoting the growth of these in a desired direction.
The most used approach was to modify surface roughness either by mechanical methods (high-pressure blasting with metal oxide particles, high-pressure liquid jet) or by chemical means (acid treatment), presenting the disadvantage of leaving the surface contaminated or undermining the structural integrity of implants. Similar to laser-based methods like direct laser metal sintering (DLMS), laser (i.e. CO2) surface treatment modification has substantially broadened laser application for treating Ti alloys, allowing implants to be produced more economically than by traditional techniques. Laser irradiation using pulses with duration in the nanosecond (ns) to the femtosecond range can be used for direct surface texturing of a wide variety of materials: metals, ceramics and polymers.
In the recent works by Dinca
Excimer laser processing can also provide other types of surface geometries (foams, bubbles) by varying wavelength, pulse number and sample positioning. Figure 1 presents such examples of chitosan–collagen surfaces generated by two different applied fluences using single pulse irradiation (248 nm KrF laser) onto horizontally placed samples. It can be observed that the laser irradiation of the polymer films caused different modifications of their morphological characteristics, and due to the local heat and pressure generated by the laser radiation, 2D and 3D polymer ‘bubble’ (Figure 1a–c), or ‘sponge-like’ (Figure 1d–f) structures on the surface were obtained.
Obtaining microstructured thin-film surfaces and porous surfaces from naturally derived polymers (such as chitosan, collagen) could be used as artificial membranes for skin engineering and/or in cell directional growth and tissue regeneration.
2.3.2. Periodic nanostructured surfaces induced by femtosecond laser texturing
Nevertheless, machining and surface patterning of polymers and biopolymers using ultrashort pulse lasers take place with reduced mechanical and thermal deformation as compared with processing performed with longer ns laser pulses, and particular structures such as self-organizing textures can be fabricated on different materials (e.g. on metals, ceramics, semiconductors and glass). Self-organizing effects are caused by the laser material interaction and can be used to create patterns with dimensions independent of the focused laser spot size. Recent work by Rusen
OLN cells cultured on the patterned surface showed that early cell growth was conditioned by specific microtopography and indicate possible uses for the structures as cellular platform applications.
A similar effect was observed in the case of ceramics. Zirconia sheet cut with the dimension of 10 × 10 × 5 mm3 from Zirkonzahn (Zirkon Translucent-ZRAB0490, Lot ZB 0070A) was used as received before the laser irradiation (1.9 Jcm⁻2 fluence), while the traverse speed was set at 0.1 mm/sec. Droplet-shaped microcavity (DµC) arrays were obtained by setting 20 pulses per cavity and translating the samples in X, respectively Y directions with a fixed step of 35 µm (Figure 3). Control planar Zirconia substrates were used as a comparison throughout our study. It was shown that the increase in surface area induced increased spreading of cell on all directions, which triggered cell morphology modification towards polygonal shape (Figure 4a) and consequently increased circularity of stem cell nuclei (Figure 4b).
Cells grown onto DµC pattern (Figure 4, DµC, left, 2000× magnification) spread efficiently over the microcavities. Cytoskeleton filaments extended closely over their tubule-granular topography (Figure 4, DµC, right, 10000× magnification, arrows).
2.3.3. Laser engineering contact guidance structures
‘Contact guidance’ mechanism was proposed by Harrison in 1911 , and since then, micro- and nanoridges were used to control not only cellular morphology and orientation but direct cell migration as well. Intriguingly, researchers proved by time-lapse microscopy that opposite to cells on the flat surface, mouse MSCs extended forward the long, thin process and left the wider edge trailing while migrating in both directions along the tracks, when seeded onto a groove pattern (10-µm pitch and 1600-nm step height) . Consequently, cells occupy the bottom of the grooves while projecting long, thin extensions along the silicon gratings.
In order to screen for conditions to control MSC orientation and morphology, we have produced different microtopographies using excimer (Exitech KrF excimer laser, PPM-601E Gen 6 Tool), laser micromachining by mask projection with half-tone masks. By scanning the mask under the beam and the polycarbonate (PC) sample, micropatterns of different shapes (dots, cones, pyramids, pits, funnels, inverted pyramids) with a depth/height of 2.5 to 10 µm and a width of 5 to 25 µm were generated on a polycarbonate (PC) substrate.
Cells grown on microlens-like topography (Figure 5a) lay their body over the upper part of the structures and project long dendrites along these tracks (Figure 5b,c). When grown onto reversed U-shaped lines separated by deep grooves (2 µm) (Figure 5d), cells elongate and align to surface geometry. When grooves are less deep (Figure 5e) and more frequent (Figure 5f), cells are able to diverge from their aligned profile and respectively spread over a number of lines recuperating their fibroblast-like shape, while maintaining a controlled direction. Steep edges of the V-shaped lines’ topography did not have the same restrictive effect (Figure 5g), as cells are able to protrude laterally
The surface morphological characteristics of chitosan films were tailored (i.e. ridges, grooves structures) by using multiple pulses from Ti: Sapphire femtosecond laser irradiation (
Substrates bearing anisotropic microscale  or sub-micron  geometries have been shown to control hMSC alignment and elongation and consequently cell differentiation . MSC elongation was correlated with osteogenic commitment  while stem cell circularity inclines the differentiation balance towards adipogenesis . Combinations of sub-microscale texture and microgroove patterns, as well as distinct direction of the texture in relation to that of the microgrooves, have been also subject to primary cell adhesion assays [68, 69].
2.4. 2D and 3D pattern formation by Laser-Induced Forward Transfer for steering cell adhesion
LIFT is a laser-assisted direct-write process in which the materials to be transferred are in the form of a rheological fluid, polymer-based composite or fine powder, placed on a transparent support and transferred by a single pulse onto a receiver. The main key feature that separates LIFT from other direct-write techniques is that it offers the advantages of controlled and localized micrometre-size pixels/clusters, similar to inkjet printing, but faster (up to 200.000 pixels/sec) and without the limitation in the nature of the material to be transferred [73-81]. The technique does not depend on the donor material properties, which therefore allows the use of non-soluble organic compounds, and the realization of complex 2D and 3D structures onto any type of surface
Cells grown on microbead arrays (Figure 7a) spread onto flat surfaces and start projecting long dendrites along the bead-patterned array area (Figure 7b), their body adhering over the upper part of the structures (Figure 7c). Interestingly, when washed gently to check the adhesion strength, the remaining cells were grouped strictly over the microbead pattern, as seen in Figure 7d.
2.5. Biofunctional coatings onto 2D and 3D surfaces obtained by laser evaporation: Matrix-Assisted Pulsed Laser Evaporation
Although Pulsed Laser Deposition (PLD) was successfully used for deposition of few polymers (polyepichlorhydrin , poly(methyl methacrylate) , polyhydroxybutyrate  polyisobutylene (PIB)), proteins (silk protein, bovine serum albumin protein ) and crystalline HA on Ti implants  onto various substrates, when biological molecules are implied, another technique, namely MAPLE, has been used for deposition of various composites or hybrid biofunctional materials [52-57, 90, 91]. Both methods are performed under vacuum, comprising a rotating target and substrate holder, with the difference in target preparation. If for PLD the solid target is made of alloys, metals, ceramics, etc., in the case of MAPLE, the material (1–5% in weight) to be deposited is suspended into a solvent and frozen. The target is irradiated and the resultant laser plasma plume transports the molecules on the substrate placed in parallel and at a short distance (3–5 cm) [53, 90].
The coating characteristics are related to wavelength, pulse duration, repetition rate and, especially in the case of MAPLE, of solvent absorption and target composition and percentage (preferable under 5–10% in mass). For example, using the same laser parameters (Nd: YAG, 266 nm, 150 kpulses), HA coating with different aspects, from highly porous (Figure 8, left) to compact (Figure 8, right) agglomeration of HA nanoparticles, was obtained when target composition was changed by different solvents.
It is known that adherent cells may sense and respond to micro/nanoscale ECM topographical cues through cell–ECM adhesive interactions. To note, the characteristic size dimension of topographical features at the cell–ECM interface as fibrillary collagens or fibronectin fibrils in the ECM of connective tissues is in the broad size range between about 10 nm and 10 μm. The MAPLE process was shown to produce protein and protein–copolymer coatings with a wide range of morphologies and without changing the functionalities of the proteins embedded in the polymeric matrix [90, 92]. MAPLE-deposited fibronectin layers were shown to induce superior osteoprogenitor cell attachment
AFM images of chitosan and collagen reveal fibrillary, porous or conglomerate types of structures. Conglomerates can be seen especially at chitosan at low and medium fluences, while higher fluence can influence the morphology in periodic ways, creating a variety of new shapes on the surface. More importantly, MAPLE can be easily used to coat 2D and 3D structures . For example, PMMA structured substrates obtained by photolithography (Figure 11a) were coated with laminin (Figure 11b) and the effect on OLN cells was studied (Figure 11c–e). SEM images of OLN-93 cell after 2-day culture are shown in Figure 11. The cells cultured onto laminin-coated structured PMMA substrates span from bipolar elongated morphology, with preferential alignment along the line arrays with line dimensions below 10 µm.
We also used laminin (Figure 11g–j) to coat structures that were previously generated by laser irradiation of polycarbonate (PC) (Figure 11f) and studied the combined effect of topography and chemistry on MSCs. We observed that cells aligned to the 1.5-μm separated lines both on PC (Figure 11f) and on laminin-coated structure (Figure 11g–j) as compared to the random orientation they have on the laminin-coated non-irradiated PC areas (Figure 11h). A more detailed analysis showed that cells that extended partially to smooth areas between the lines (delimited by white dots in Figures 11i1 and j1) developed small protrusions from their lamellipodia (Figure 11i1,j1 – white arrows), regardless of cells that encountered the flat surface laterally (Figure 11 i–i1) or along the lines (Figure 11 j–j1). However, when cells adhered with the whole body onto the laser-processed microtopography, they ‘scanned’ the environment with long, thin filopodia (Figure 11i,j – red arrows).
2.5.1. From 2D to complex 3D biosurfaces: Multi-Photon Lithography
Multi-Photon Lithography (MPL) is a 3D printing technology that allows the construction of readily assembled structures with sub-100-nm resolution [97, 98]. It is based on the phenomenon of non-linear photon absorption and polymerization; the beam of a sub-picosecond-length pulse laser is focused inside the volume of a transparent photosensitive material, causing it to absorb two or more photons and polymerize locally. Moving the beam according to a CAD model, one can fabricate a realistic micromodel of this design.
Despite it being fairly new technology, MPL very quickly found application in tissue engineering and cell growth investigations, and, to date, several biomaterials have been employed to this purpose . These are mostly negative photoresists, such as hydrogels [100-104], acrylate materials [105, 106], the epoxy-based photoresist SU-8  and organic–inorganic hybrid materials (Figure 12)[108-113]. Special mention should be made of the 3D structuring of natural polymers and proteins, pioneered by Campagnola
There is also a lot of work on modifying natural polymers for MPL, such as polycaprolactone (Figure 13) , polylactide (Figure 14) [126, 127], gelatin and hyaluronic acid . This avenue of research has provided the most promising results, as far as biodegradable materials are concerned.
3. Conclusions and future prospects
Combining topographical features for mimicking the natural architecture of the ECM environment with chemical factors, such as biologically active molecules for controlling interfacial interaction between biological entities and materials, provides a challenging approach for identifying important factors that determine stem cell fate, such as extrinsic signals from the niche that impact gene expression.
This chapter briefly reported the recent progress in our research and others over topographical and chemical cues of biosurfaces on cells with accent on the hMSCs.
Although significant progress has been achieved in this field, the materials discussed above still have obvious limitations in practical applications that need to be overcome in the future. Our results indicate that substrate micropattern features play a key role in hMSCs’ spreading response and more importantly that even smoother patterns than grooves and ridges are able to change cell shape. Whether the substrate conformations presented are able to induce stem cell commitment to a specific differentiation pathway is a topic of future investigation.
Although the majority of the previous works in this field have used simple topographical features such as grooves, future attempts should be more focused on realistic substrates with a higher degree of biomimetic relevance to impose multidirectional cues within the cellular microenvironment. Such novel substrates will enable addressing questions on how cells globally integrate biophysical signals from their surrounding microenvironment. Furthermore, topographical features could be integrated with chemical stimuli, such as soluble factors, to enhance cellular process, such as stem cell differentiation. The continuous advancements in the field of cell–substrate topography interactions will not only benefit fundamental biological studies but also have significant implications in the field of tissue engineering through fabrication of engineering synthetic and implantable substrates with controlled features. Therefore, the design of topographical and chemical features for engineering smart bio-interfaces with multiple and synergetic functionalities represents the key point in effective use of hierarchically topographical and chemical bioplatforms targeting controlled regulation of stem cell fate.Conflict of interest
The authors declare no competing interests.
The research leading to these results has received funding from the Romanian Ministry of National Education PN-II-PT-PCCA 239/2014. V.D. acknowledges the financial support from Sciex Grant project no.12.313 and from a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-RU-TE-2011-3-0289. L.E.S. is grateful for the financial support from the European Social Fund POSDRU 2007–2013 through the contract POSDRU/89/1.5/S/60746 (postdoctoral fellowship). Partial funding was obtained from the Romanian Ministry of Research and Education, through the Romanian Academy Project 1/2011 of the Institute of Biochemistry.