S.
Nedjari
a,
A.
Hébraud
a,
S.
Eap
b,
S.
Siegwald
a,
C.
Mélart
a,
N.
Benkirane-Jessel
b and
G.
Schlatter
*a
aICPEES Institut de Chimie et Procédés pour l'Energie, l'Environnement et la Santé, UMR 7515, CNRS, Université de Strasbourg, 25 Rue Becquerel, 67089 Strasbourg Cedex, France. E-mail: guy.schlatter@unistra.fr
bINSERM Unité 1109, Université de Strasbourg, 11 Rue Humann, F-67085 Strasbourg Cedex, France
First published on 28th September 2015
Electrostatic Template-Assisted Deposition (ETAD) of microparticles is described as a new process to control the deposition of microparticles by electrospraying onto a substrate. It relies on the construction of an electrostatic template by electrospinning a thin layer of fibers onto a micropatterned collector. Because the fibers cannot release their charges when they are suspended over cavities of the micropatterned collector, an electrostatic template is formed with repulsive and attractive domains. This electrostatic template is then used to guide precisely the particle deposition during the electrospraying step. Microstructured bi-layer composites with a great variety of micropatterns can thus be elaborated with any kind of materials allowing the use of the ETAD process for a wide range of applications. As a proof of concept, the ETAD process was applied for the production of composite scaffolds with poly(ε-caprolactone) nanofibers covered by a micropatterned layer of hydroxyapatite. This scaffold was then embedded in a biochip containing 21 wells and used for MG-63 cell proliferation and mineralization studies, showing their possible application in the screening of the scaffold structure for tissue engineering.
A simple fabrication technique for the elaboration of submicronic fibrous scaffolds for tissue engineering is electrospinning. Indeed, electrospun scaffolds have proven to be excellent candidates as substrates for in vivo and in vitro cell growth.8 Moreover, using this technique, fibers can be easily organized in the form of 2D9–11 or 3D12–15 microstructured scaffolds, with tailored pore sizes to allow cell colonization and infiltration.16,17 Finally, it has been demonstrated that the topography of the electrospun scaffold has got an important influence on the cell behavior.18–20
Electrospun membranes have been incorporated in biochips allowing biological experimentations in individual micro-wells having a bottom with non-structured randomly deposited nanofibers.21–23 However, to the best of our knowledge, nobody reported the elaboration of biochips allowing cell culture for a wide range of micropatterned nanofibrous structures. In this work, we developed a new process, the Electrostatic Template-Assisted Deposition (ETAD), which allows the precise spatial deposition of microparticles onto electrospun nanofibers. From this process, we elaborated microstructured biochips to screen the topography of nanofibrous scaffolds for bone tissue engineering. For such application, several groups have shown that the addition of hydroxyapatite, the major mineral component of bone, to the nanofibrous scaffolds enhance the osteophilic environment for the growth and mineralization of osteoblasts.24–26 We thus elaborated a biochip for which the bottom of each well has its own composite fibrous microstructure (bars, hexagons, blocks or mazes) made of a layer of electrospun poly(ε-caprolactone) (PCL) fibers and a layer of electrosprayed hydroxyapatite (HA) microparticles. ETAD process allowed the precise location of the HA particles onto the PCL nanofibrous layer. MG-63 osteoblast-like cells were then cultured followed by immunochemistry and alizarin red staining to evaluate the influence of the micropatterned structures on the mineralization and to demonstrate the potential of application of such biochips.
PCL polymer solutions were prepared 24 h before by dissolving poly(ε-caprolactone) (PCL) (Mw = 80 kg mol−1, PDI = 1.1, Perstrop, commercial name: CAPA 6806) into dichloromethane (DCM, Sigma-Aldrich)/N,N-dimethylformamide (DMF, ReagentPlus® ≥99%, Sigma-Aldrich) (60/40 v/v) at a concentration of 15 wt%. A 6 wt% hydroxyapatite (HA, Sigma-Aldrich, nanopowder with particles size ≤200 nm (BET), ≥97% synthetic) suspension was prepared in ethanol (Sigma-Aldrich) 48 h prior to electrospraying and ultrasonicated for 30 min (Branson Sonifier) just before processing.
In a first step, the PCL solution is electrospun during a time te (needle-to-collector distance = 13.5 cm, flow rate = 1 mL h−1, Vneedle = 25 kV). Then, in a second step, the HA suspension is electrosprayed over the layer of PCL nanofibers during 15 min (distance = 13.5 cm, flow rate = 0.62 mL h−1, Vneedle = 18 kV).
On each biochip collector, 21 wells were fabricated with 21 different structures. Each well had a diameter of 5 mm and they were spaced from each other by 5 mm. 5 different kinds of structures were studied: hexagon, bar, block, maze and random structure. The characteristic sizes Ltop and Lbot are defined and summarized for each structure (see Fig. 2 and Table 1).
Structure | Name | Controlled electrospraying for te = 90 s | Controlled electrospraying for te = 13 min | Width LHA of HA patterns for te = 13 min (μm) | % of surface area covered by HA for te = 90 s | S HA = % of surface area covered by HA for te = 13 min |
---|---|---|---|---|---|---|
Bar | Bar20,20 | Yes | No | — | 50 ± 3 | — |
Bar80,20 | Yes | No | — | 80 ± 4 | — | |
Bar20,60 | Yes | Yes | 18 ± 4 | 25 ± 2 | 23 ± 7 | |
Bar80,60 | Yes | Yes | 18 ± 5 | 57 ± 3 | 23 ± 7 | |
Bar20,80 | Yes | Yes | 15 ± 3 | 20 ± 2 | 16 ± 5 | |
Bar80,80 | Yes | No | — | 50 ± 3 | — | |
Block | Block60,80 | Yes | Yes | 17 ± 3 | 14 ± 2 | 23 ± 7 |
Block120,80 | Yes | Yes | 17 ± 4 | 28 ± 2 | 16 ± 5 | |
Block60,40 | Yes | No | — | 28 ± 2 | — | |
Block120,40 | Yes | Yes | 16 ± 2 | 44 ± 3 | 19 ± 6 | |
Hexagon | Hex20,240 | Yes | Yes | 19 ± 3 | 17 ± 2 | 16 ± 5 |
Hex20,120 | Yes | Yes | 18 ± 3 | 30 ± 2 | 27 ± 9 | |
Hex20,60 | Yes | Yes | 17 ± 3 | 48 ± 3 | 42 ± 12 | |
Hex60,240 | Yes | Yes | 16 ± 3 | 40 ± 3 | 12 ± 3 | |
Hex60,120 | Yes | Yes | 17 ± 2 | 60 ± 3 | 20 ± 6 | |
Hex60,60 | Yes | No | — | 78 ± 4 | — | |
Hex100,240 | Yes | Yes | 18 ± 2 | 54 ± 3 | 11 ± 3 | |
Hex100,120 | Yes | Yes | 17 ± 3 | 74 ± 4 | 16 ± 5 | |
Hex100,60 | Yes | No | — | 88 ± 4 | — | |
Maze | Maze20,80 | Yes | Yes | 15 ± 3 | 20 ± 2 | 14 ± 5 |
Flat | Random | Yes, random deposition | Yes | HA covers all the surface | 95 ± 5 | 85 ± 10 |
- A 50 × 50 mm PMMA plate drilled with 21 holes to form the wells, corresponding to the 21 different electrospun structures.
- A 50 × 50 mm PDMS seal with a height of 4 mm with 21 holes corresponding to the ones of the PMMA plate.
- A PCL/HA membrane obtained with the biochip collector.
- A 50 × 50 mm PMMA plate with a height of 5 mm.
Before assembling, each element of the biochip was sterilized in ethanol (except the PCL/HA membrane). The four elements were assembled together with four screws (see Fig. 4b). The assembled biochip was then exposed to UV light during 30 minutes to insure good sterilization. The PDMS seal was homemade. A 10/1 (wt/wt) mixture of PDMS-Sylgard Silicone Elastomer and Sylgard Curing Agent 184 (Dow Corning Corp., Seneffe, Belgique) was poured into a Petri dish.
Then, the mixture of PDMS-elastomer and curing agent was cured at 65 °C during 1 hour until the polymer became rigid. After cooling at room temperature, the PDMS seal was carefully peeled from the Petri dish and perforated with a punch to make 21 holes with a diameter of 5 mm.
Fig. 1 Principle of the electrostatic template-assisted deposition of electrosprayed microparticles on electrospun nanofibers. (a) Step 1 of the process: electrospinning on a micropatterned collector. (b) SEM picture showing electrospun PCL fibers deposited on a honeycomb pattern, the suspended fiber portions are positively charged. (c) Cross-section A–A of a pattern wall showing the repulsive suspended electrospun fibers and the attractive area located above the wall. (d) Step 2 of the process: electrospraying on the electrostatic template formed by the thin layer of fibers. (e) HA electrosprayed particles on a thin layer of PCL fibers obtained after only te = 45 s of electrospinning, a highly selective deposition is observed over honeycomb patterns with a wall width of Ltop = 20 μm an internal diameter ranging from Lbot = 40 to 360 μm (see Fig. 2c for the definition of Ltop and Lbot). (f) Detail of the previous picture for honeycomb patterns of internal diameter of 160 μm. (g) HA electrosprayed particles on a patterned collector without the presence of the layer of electrospun fibers: no selective deposition is observed. |
Using the ETAD process, it was possible to produce microstructured composite membranes of a large variety of pattern shapes such as bars, hexagons, blocks or maze (see Fig. 2a–j and Table 1). HA microparticles were precisely deposited on the top of the 21 different patterns of the collector in the case of the presence of a previously deposited thin layer of electrospun PCL fibers obtained for an electrospinning time te = 90 s (Fig. 2f–j). When the time te is increased to 13 min (corresponding to an average thickness of the PCL fibrous layer of roughly 80–150 μm), the controlled deposition is still observed but with some differences compared to shorter time te (see Fig. 2f′–j′). Indeed, by increasing te, the shape of the electrostatic template may change due to fiber aggregation or partial loss of the electric contact between the fibers and the top of the patterns. Thus, a first consequence of the increase of te was that whatever the pattern size Ltop, ranging from 20 to 120 μm (see Fig. 2b–e and Table 1), the corresponding width LHA of the patterns formed by the deposited HA microparticles was almost constant and ranged between 15 and 20 μm (see Table 1). Therefore, the percentage of surface area covered by the HA microparticles (SHA) was reduced when electrospraying after longer electrospinning times. While it was highly dependent on the structures of the patterns at short electrospinning times, ranging from 15 to 90% of the total surface, it became more uniform after longer times and around 15 to 30% for most of the patterns (Table 1). Regarding the bar and hexagonal patterns, HA particles were located exactly on the top of the patterns for the thinnest ones having an Ltop of 20 μm, with a low HA surface coverage of 16–20%, not really depending on te (see Fig. 2f′, 2g′ and 3). However, for patterns with larger width Ltop, the electric contact couldn't be insured over all the top surface of the patterns leading to bad electric charge release and thus the formation of repulsive areas. For such large Ltop, the contact was generally insured at the edges of the patterns leading to HA deposition only on these edges. As an example, in the case of Hex100,120, after te = 13 min of electrospinning, HA was only deposited on the edges of the patterns with SHA = 16 ± 5% (Fig. 2h′) whereas after a shorter electrospinning time, HA covered all the width Ltop of the patterns leading to SHA values as high as 74 ± 4% (see Fig. 2h). In the case of block patterns, whereas well-defined HA islands were formed at short te (SHA = 28 ± 2%, Fig. 2i), HA grids with a pitch corresponding to the distance between the centers of the blocks were observed after the longest electrospinning time te = 13 min (SHA = 16 ± 5%, Fig. 2i′). This phenomenon could be explained by the propensity of the PCL fibers to self-assemble after long times of electrospinning12 leading to the formation of bundles of fibers crossing the block gap. Indeed, such bundles, containing small amount of remaining solvent, allow the electric charges dissipation along their axes favoring thus their coverage by HA electrosprayed microparticles resulting in the formation of HA grids.
Fig. 4 (a) A microstructured composite membrane with the 21 microstructured areas. (b) The membrane integrated in the biochip and its design. |
The biochips were used to screen in parallel the effect of the different fibrous architectures on the proliferation and activity of osteoblast-like MG-63 cells. The main role of osteoblasts is to produce and secrete organic and inorganic bone extracellular matrix (ECM), called ostoid.28 The organic bone matrix is constituted of various proteins, such as collagen, osteonectin, osteocalcin, and sialoproteins. The inorganic bone matrix is constituted of calcium–phosphate salts. Osteoconductivity of the various scaffold architectures was investigated by monitoring the proliferation and ECM forming activity of osteoblasts cultured in the biochip wells.
The morphology and spatial organization of the cells were evaluated by SEM observation after 3 days of cell culture. For the largest patterns such as Hex20,240, it is observed that cells preferentially adhered on the pattern walls where HA is located (Fig. 5a). Furthermore, MG63 cells show elongated morphology suggesting a good adhesion with the fibrous substrate (Fig. 5b and c). For the smallest patterns (i.e. when the distance between the HA areas is lower than 100 μm) the cells gathered, covering all the surface of the scaffold and began forming microtissues even after only 3 days of culture (Fig. S4 in ESI†). The proliferation and localization of MG63 osteoblasts on the micropatterned scaffolds was also observed by staining the cells nuclei with DAPI after 21 days of culture (see Fig. 6a–e and S1 in ESI†), showing the good proliferation of the cells into the structured wells of the biochip. As previously shown by SEM, a higher density of cells (i.e. stronger blue intensity) is observed where HA is deposited. For hexagonal patterns (Fig. 6a), this result seems to be in contradiction with our previous study20 for which a preferential location of MG-63 cells inside the nests of the honeycomb was observed. This difference could be explained by the presence of HA microparticles insuring a better affinity with the cells as compared with the hydrophobic surface of the PCL fibers. Indeed HA particles have shown to promote adhesion and growth of MG-63 cells.29
The production of ECM by MG-63 cells was estimated by immunofluorescence assays. In contrast with DAPI, important differences are observed in the expression of osteocalcin by the cells as a function of the scaffold microstructure (Fig. 6a′–e′ and S2 in ESI†). It is obvious that some patterns show much higher fluorescence intensity than other types of patterns.
Finally, in order to evaluate the mineralization of the scaffolds by the MG-63 osteoblast-like cells, Alizarin red coloration of the membrane after 21 days of cell culture was performed. In this assay, calcium salts are stained in red and a stronger red intensity indicates the presence of higher amount of calcium salts. Because our membrane already contains a certain amount of hydroxyapatite, a negative control test has been carried out by Alizarin red coloration of a biochip membrane containing hydroxyapatite without cells (Fig. S3 in ESI†).
Fig. 6f shows a membrane after removing from the biochip and coloration by Alizarin red. A significant difference in Alizarin red intensity is observed, between this membrane, after cell culture, and an as-produced membrane, thus showing a good mineralization of the scaffolds. However, no significant differences in Alizarin red intensity could be distinguished between the different kinds of the micropatterned structures.
These results show that our biochip can be used for cell culture and are compatible with different kinds of biological tests. The membrane can be removed from the device after the cell culture and can be very easily manipulated for further observations such as microscopy.
We used this process to elaborate a new kind of biochip, dedicated to the screening of fibrous composite structures for bone regeneration applications. The biochips contain 21 wells with different micropatterned structures such as hexagon, bar, block, and maze in their bottom. Each well structure was made of electrospun PCL fibers covered by a microstructured layer of hydroxyapatite particles. MG-63 osteoblasts-like cells were then successfully cultured into the different wells of the biochip and immunochemistry as well as biochemistry assays were performed as a proof of concept. The biochip can be very easily unbuilt at the end of the cell culture and the scaffold with various microstructures separated from the setup and manipulated for microscope observation or further biological, chemical or mechanical tests.
Footnote |
† Electronic supplementary information (ESI) available: ESI includes fluorescent microscopy of MG-63 osteoblasts cell nuclei and osteocalcin expression after 21 days of culture. See DOI: 10.1039/c5ra15931h |
This journal is © The Royal Society of Chemistry 2015 |