Cost effective optimised synthetic surface modification strategies for enhanced control of neuronal cell differentiation and supporting neuronal and Schwann cell viability

Enriching a biomaterial surface with specific chemical groups has previously been considered for producing surfaces that influence cell response. Silane layer deposition has previously been shown to control mesenchymal stem cell adhesion and differentiation. However, it has not been used to investigate neuronal or Schwann cell responses in vitro to date. We report on the deposition of aminosilane groups for peripheral neurons and Schwann cells studying two chain lengths: (a) 3-aminopropyl triethoxysilane (short chain-SC) and (b) 11-aminoundecyltriethoxysilane (long chain-LC) by coating glass substrates. Surfaces were characterised by water contact angle, AFM and XPS. LC – NH 2 was produced reproducibly as a homogenous surface with controlled nanotopography. Primary neuron and NG108-15 neuronal cell differentiation and primary Schwann cell responses were investigated in vitro by S100 β , p75, and GFAP antigen expression. Both amine silane surface supported neuronal and Schwann cell growth; however, neuronal differentiation was greater on LC aminosilanes versus SC. Thus, we report that silane surfaces with an optimal chain length may have potential in peripheral nerve repair for the modification and improvement of nerve guidance devices.

guidance cues for regenerating axons present in autografts. 3 Approaches used to improve hollow NGCs include the addition of intraluminal guidance scaffolds, incorporating channels and pores, coatings, and changing surface chemistry and topography.
The use of coatings has been shown to improve initial cellular adhesion with synthetic biomaterial surfaces for example, extra cellular martrix (ECM) proteins. 1 ECM coatings mimic native extracellular matrix cues and to an extent surface topography, providing the correct biochemical interactions for adhesion and potentially improving regenerative outcomes of a conduit. 3 Proteins, such as, laminin, collagen, and fibronectin improve neuronal and Schwann cell adhesion and differentiation when incorporated into bioengineered NGCs. 1 For device translation, the use of naturally derived proteins must consider cost, potential batch variation, and unwanted immune response. Alternatively, synthetic coatings are scalable, reproducible, and costeffective. 4 Synthetic coatings can control the type, level and conformation of serum proteins that adsorb. 1 Alteration of the surface chemistry can influence protein surface conformation, and influence initial adhesion, proliferation and cell differentiation. 5 Techniques such as plasma polymerization have been used to alter biomaterial surface chemistry, without altering bulk material properties. 1 Plasma deposition studies report on acrylic acid and allyamine surface modification improving SH-SY5Y neuronal cell adhesion and differentiation 6 and air plasma techniques also increasing primary Schwann cell adhesion onto modified surfaces, increasing nerve regenerative properties of conduits when grafted with peptide or growth factors. 7 However, plasma polymerization requires a high vacuum, limiting scale-up, 8 supporting a need for simpler surface modification.
Silane modification is a simple, cost effective, controllable, and scalable technique for biomaterial modification. Previous work has demonstrated enrichment of surfaces with chemical reactive groups including amines, hydroxyl, and carboxyl. 5 Silane modification can mimic the ECM by changing surface nano-topography, and substrate chemistry, providing a biological surface to enhance initial cell attachment and maintain proliferation. 9 Silane chain length has been shown to influence deposition of the chemical reactive group at submicron scale, and influence cell adhesion and differentiation due to topographic profile. 9 We have previously reported that 11-aminoundecyltriethoxysilane surfaces supported osteogenic differentiation of mesenchymal stem cells, in contrast to 3-aminopropyl triethoxysilane. 10 Aminosilanes, of varying chain lengths, were fully characterised, using water contact angle, X-ray photoelectron spectroscopy (XPS) and atomc force microscopy, to determine the effect of silane chain length on surface chemistry and topography at sub-micron level. 11-aminoundecyltriethoxysilane modified surfaces presented an ordered nanoroughness, resulting in favorable 'surface chemistry and topography' for osteoinduction, but has not been investigated for other tissue engineering applications. 10 In the context of nerve repair, one study has reported modifying chitosan using 3-aminopropyl triethoxysilane, which promoted increased Schwann cell adhesion and proliferation. 11 However, silane chain length has not been investigated thoroughly for nerve implant biomaterials. The aim of the present study was to investigate the influence of two different silane chain lengths, with known surface chemistry and topography, characterised from our previous study, 10 on neuronal and Schwann cell differentiation and phenotype.

| Preparation and modification of borosilicate glass coverslips
Glass coverslips (13mm 2 ) were modified with 3% aminosilanes (SC: 3-aminopropyl triethoxysilane; LC: 11-aminoundecyltriethoxysilane) as previously described. 10 Briefly, glass coverslips were cleaned using a 0.5 M solution of sodium hydroxide and then 1 M Nitric acid for 30 min in an ultrasonic bath, followed by washing in three changes of distilled water, and dried in a 50 C oven. Clean coverslips were then modified using the 0.1 M silanes, 3-Aminopropyl triethoxysilane (Sigma), and 11-Aminoundecyltriethoxysilane (Fluorochem), in pure isopropanol for 30 min. Samples were then rinsed with isopropanol and distilled water. All glass coverslips were sterilized with 70% ethanol for 30 min, and washed overnight with PBS before cell culture studies. Prior to cell seeding, 2 ug/ml of fibronectin (Merck), in sterile PBS, was added to clean plain glass coverslips, as the control, and incubated at 37 C in 5% CO 2 for 30 min.

| Water contact angle measurement
Dynamic contact angles of the samples in deionised purified water were measured using a Dynamic Contact Angle Tensiometer (CDCA 100, Camtel Ltd., Royston, Herts, UK) at 22 ± 0.5 C. Briefly, two samples were tightly stuck together on the unmodified side. Each sample was immersed into the wetting solution (deionised pure water) at a rate of 0.060 mm/s. The wetting force at the solid/liquid/vapour interface was recorded by an electro balance as a function of time and immersion depth and was converted into an advancing contact angle.
The values reported for dynamic advancing angles of modified glass coverslips are mean and SD of n = 4.

| X-ray photoelectron spectroscopy
Elemental surface chemistry, of aminosilane-modified substrates, was confirmed using XPS as previously described. 10

| Atomic force microscopy
Atomic force microscopy (AFM) was used to confirm surface topography, previously reported, of the NH 2 modified substrates. AFM was performed as previously described. 10 Mechanical properties of silane glass surfaces were investigated by fitting the AFM data obtained with the Derjaguin-Muller-Toporov (DMT) model to extract the elastic modulus of modified glass substrates by fitting the contact region of the retract curve close to the contact point.

| NG108-15 cell culture
An NG108-15 (ECACC 88112303) cell line was used for study. They were originally created from Sendai virus mediated fusion of a mouse neuroblastoma and rat glioma cells. They are a valuable experimental in vitro tool for quantifying neurite development as a proxy for primary neuronal cell differentiation. Herein they will be referred to as NG108-15 neuronal cells in this context, 12 with experimental culture and conditions used for experiments as previously described. 12 Cultures were maintained for 6 days, changing the culture medium to serum free Dulbecco's Modified Eagle Medium (DMEM) after 2 days in culture.

| Isolation and culture of primary Schwann cells
Rat primary Schwann cells were isolated and cultured as previously described. 13 Schwann cells were cultured up to passage 7 for experiments. 60,000 Schwann cells, per sample, were seeded onto surfaces for 6 days and medium replaced once at day 3.

| Isolation and dissociation of dorsal root ganglion bodies
Dissociation of DRGs into co-cultures of primary neurons and Schwann cells was performed using the methods as previously described. 14 Cocultures were maintained in F12 medium (containing 2 mM glutamine, 1% penicillin/streptomycin, and 0.25% amphotericin B) supplemented with the addition of 100 ug/ml bovine serum albumin (BSA), 10 μl/ml of N 2 , and 77 ng/ml of nerve growth factor (NGF). Cells were left in culture for 7 days, and medium changed at day 4.

| Live/dead analysis of NG108-15 cells and primary Schwann cells
Cell viability was confirmed by live/dead analysis, and samples imaged using confocal microscopy as previously described. 12 Cells were counted using a ITCN cell counter plugin on Image J NIH software. 15,16 2.9 | Immunolabelling of NG108-15 cells, Schwann cells and dissociated dorsal root ganglia Modified coverslips containing NG108-15 cells only, Schwann cells only, and those containing dissociated DRG cultures, were labelled for cell type specific antigens, and imaged using confocal microscopy as previously described. 12 NG108-15 cells and primary neurons were labelled for βIII-tubulin (neurite marker) and Schwann cells labelled for S100β in co-culture with primary neurons. Mono-culture of primary Schwann cells on modified substrates were labelled for S100β, Glial fibrillary acidic protein (GFAP) low-affinity nerve growth factor receptor (p75NGFR) as previously described. 13 2.10 | Neurite outgrowth and primary Schwann cell morphology assessment Four different parameters were analysed for neurite outgrowth as previously described. 12 Image analysis was conducted using a Zeiss LSM Image browser software and Image J (NIH). 15 The algorithm identified the blue (DAPI/nuclei label) and green (S100β label) channels to reveal a single red channel image identifying βIII tubulin. After conversion to greyscale, images were transferred into Image J (NIH) software and interfaced with a plugin (Neuron J). 15,17 NeuronJ (open source) was used to trace and measure neurites extending from a cell body. Regions of interested were magnified to accurately count the number of neurites extending from the cell body, and for areas where neurites crossed over. The average Schwann cell length was calculated as previously described 18 using the ruler tool, and aspect ratio calculated using the Analyze Particles tool, on NIH Image J, once images had been manually thresholded. 15,19 2.11 | Statistical analysis GraphPad Instat (GraphPad Software) was used to perform statistical analysis. One-way analysis of variance (ANOVA; p < 0.05) was conducted to analyse the differences between data sets incorporating Tukey's multiple comparisons test if p < 0.05. Two-way analysis of variance (p < 0.05) was conducted to analyse the differences between data sets when assessing live/dead cell numbers incorporating a Sidak's multiple comparisons test if p < 0.05. Data were reported as mean ± SD, p < 0.05. Each experiment was performed three independent times with each sample repeated three times as n = 3.

| Silane modification increases water contact angle
Contact angle results confirmed aminosilane deposition to glass substrates, increasing surface hydrophobicity ( Figure 1). The water contact angle of plain glass (60 ± 4 ) significantly increased to 86 ± 1 and 81 ± 2 when grafting SC and LC aminosilanes, respectively.
Values recorded were consistent with previously published data, confirming addition of NH 2 silanes to glass. 10

| Elemental confirmation of silane modification
Elemental analysis by XPS confirmed that aminosilane modified surfaces were enriched with carbon and nitrogen and conversely exhibited decreased silicon and oxygen surface functional content.
This demonstrated that aminosilanes were chemically grafted on to glass substrates successfully and consistent with previously published data confirming the presence of NH 2 modification (Table 1). 10

| Silane modification increases surface roughness and elastic modulus
AFM micrographs of plain glass substrates and SC aminosilane surfaces (Figure 2a, b) showed a patchy pattern in roughness and amine deposition. In contrast, LC surfaces had a more homogenous roughness and amine deposition. This observation was in line with our previous findings. 10 The elastic modulus of plain glass (10,820 ± 1,492 MPa) was significantly decreased when modified with SC aminosilane (8,906 ± 51 MPa) and LC aminosilanes (6,697 ± 50 MPa) (Figure 2d). In addition, the LC aminosilane modulus was significantly lower compared to modification using SC aminosilanes.

| Aminosilanes support primary Schwann cell viability
All surfaces supported primary Schwann cell attachment, with few dead cells observed (Figure 5a-e). The number of live Schwann cells F I G U R E 1 Dynamic water contact angle of glass, short chain, and long chain aminosilane modified surfaces. Water contact angle significantly increased with addition of the aminosilanes compared to the plain glass control (mean ± SD, n = 4 independent experiments; ***p < 0.001 and ****p < 0.0001 compared to plain glass) T A B L E 1 X-ray photoelectron spectroscopy (XPS) characterization of glass surfaces after aminosilane modification. XPS elemental composition is shown as percentage of carbon, silicon, oxygen, and nitrogen for glass control, short chain, and long chain aminosilane-modified surfaces (mean ± SD, n = 4)

| Long chain aminosilane supports primary Schwann cell phenotype
Primary Schwann cells were cultured on surfaces for 7 days and stained for S100β, p75NGFR, and GFAP (Figure 6a

| Long chain aminosilane supports primary neuron and Schwann cell adhesion, and supports primary neuron differentiation
Higher numbers of Schwann cells were observed on SC and LC modified glass and fibronectin coated glass, compared to plain glass, which was patchy (Figure 7a Previous studies have demonstrated that changing the chain length of silane changes the surface topography of a substrate, via deposition of amine groups, therefore controlling initial cell adhesion and influencing cellular differentiation. 9 Silane chain length has been previously shown to control osteo-induced differentiation of mesenchymal stem cells, and a similar effect was hypothesized for neuronal cell differentiation. 10 The present study investigated two different NH 2 presenting silane chain lengths: 3-aminopropyl triethoxysilane (SC) and 11-aminoundecyltriethoxysilane (LC) as potential coatings in peripheral nerve repair.
Silane modification significantly changed the surface properties of glass, increasing water contact angles compared to the unmodified glass control (Figure 1). The addition of amine groups to a surface, has been well reported to increase hydrophilicity of a surface, decreasing water contact angle. 21 10 This could be due to higher hydrophobic interaction among the long CH 2 chain, which made the amine groups, in LC modified surfaces, to be oriented outwards, which was observed in our previous study. 10 The orientation of amine groups in SC modified surfaces was random, due to no/lower hydrophobic interaction among the molecules.
Changes in nanotopography were confirmed by AFM analysis by varying silane chain length ( Figure 2). Our previous study 10 illustrated that grafting LC aminosilanes onto substrates increased surface roughness, and was consistent over the entire surface, depositing amine groups as a homogenous layer. Although surface roughness of substrates increased when grafting the SC aminosilane, roughness was patchy when compared to LC aminosilane. Rougher surface topographies have been shown to increase protein adsorption due to increased surface area, which in turn is reported to influence initial cell adhesion, and differentiation. 24 Aminosilane addition to glass substrates significantly decreased the elastic modulus of clean glass substrates when modifying the surface with LC aminosilane. However, literature suggests the addition of 3-aminopropyl triethoxysilane to substrates, and increasing the concentration, increases elastic modulus. 25  However, it is difficult to compare our results with those of previously reported studies due to the magnitude in size of the DMT moduli reported for the clean glass, SC and LC modified surfaces, as well as differences in substrates modified. Future work will investigate these findings further.
All surfaces supported NG108-15 neuronal cell adhesion and viability. However, significantly higher numbers of live cells were observed adhering to LC modified surfaces compared to SC surfaces, and plain glass, suggesting the LC aminosilane preferentially supports NG108-15 neuronal cell viability. This agrees with the study by F I G U R E 6 Confocal micrographs of primary rat Schwann cells immunolabeled for S100β (green), GFAP (red) and P75NGFR (yellow) after 6 days of culture on fibronectin controls, plain glass, SC and LC aminosilanes, and tissue culture plastic. Schwann cells were immunolabeled to confirm Schwann cell phenotype. P) Average Schwann cell length; Q) aspect ratio (length/width) to identify Schwann cell phenotype (mean ± SD, n = 3; *p < 0.05) Buttiglione et al whereby the addition of amine groups to PET surfaces, using plasma polymerization, promoted SY5Y cell adhesion, increasing the surface charge of the substrate and increasing cellular adhesion via electrostatic attraction. 29 Albumin, present in cell culture medium, has a higher affinity to both hydrophobic and rough surfaces, which would explain the increased number of live cells attached to the LC modified surfaces. 30 This study has shown that the LC aminosilane preferentially supports NG108-15 neuronal cell differentiation consistently across an exposed surface area, compared to the SC modified and plain glass coverslips. NG108-15 neuronal cells were chosen for this study as Surface modification of glass, using LC aminosilanization, has also been shown to preferentially support primary neuronal cell and Schwann cell attachment, and primary neuronal cell differentiation. cells compared to SAMs with methyl and carboxyl groups. 35 Ren et al also reported that NH 2 modified glass surfaces induced differentiation of neural stem cells, compared to control groups. 34 Both LC and SC aminosilanes supported primary neuronal cell differentiation, identified by a significant increase in the average number of neurites sprouting from each neuron. This effect was not observed using NG108-15 cells. Although it is reported that NG108-15 cells can be indicative of primary neuron responses, data gathered are not always comparable to the in vivo response. 36 This study opted to use dissociated DRGs as a primary cell model, to reduce the amount of animal usage (in line with the 3Rs) and obtain results indicative of the in vivo response. 12 It was observed that primary Schwann cells were always in close proximity to neurite outgrowth, which is important for nerve regeneration studies to observe neuronal cell-glial cell connections due to the role that Schwann cells have in nerve repair in vivo. 18 This study supports the value of more relevant cell sources, such as the dissociated DRGs for neuronal and Schwann cell studies, highlighting differences between primary cell versus immortal cell lines for investigation of novel biomaterials in peripheral nerve repair.
In summary, we report on a surface deposition method with a LC aminosilane that increases hydrophobicity, surface roughness.
This has the effect of supporting cell differentiation as determined by NG108-15 neuronal cells, and primary neurons cultured from dissociated DRGs. Both the SC and LC aminosilanes have been extensively characterised in our previous work and modification of glass substrates for neuronal cell differentiation was comparable to previous studies. 10 This is the first study to show that neuronal cell differentiation varies according to the length of the silane chain used. It also highlights the potential of silane modification as a scalable, cost effective approach, compared to using expensive ECM proteins, to functionalize existing biomaterials, used for neurosurgical scaffolds, with amine group surface modification for enhancing neuronal cell differentiation and supporting neuronal and Schwann cell viability.