Microgrooves encourage endothelial cell adhesion and organization

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Article

Microgrooves encourage endothelial cell adhesion and organization on shape memory polymer surfaces Tina Govindarajan, and Robin Shandas ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00833 • Publication Date (Web): 03 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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Microgrooves encourage endothelial cell adhesion and organization on shape memory polymer surfaces Tina Govindarajan1 and Robin Shandas2,* 1

Department of Bioengineering, University of Colorado at Denver | Anschutz Medical Campus;

[email protected] 2

Department of Bioengineering, University of Colorado at Denver | Anschutz Medical Campus;

[email protected] Keywords: Shape memory polymer, microgrooves, endothelial cells, adhesion, alignment

Abstract

Cardiovascular stents have become the mainstay for treating coronary and other vascular diseases; however, the need for long-term anti-platelet therapies continues to drive research on novel materials and strategies to promote in situ endothelialization of these devices, which should decrease local thrombotic response. Shape memory polymers (SMPs) have shown promise as polymer stents due to their self-deployment capabilities and vascular biocompatibility. We previously demonstrated isotropic endothelial cell adhesion on the unmodified surfaces of a family of SMPs previously developed by our group. Here, we evaluate whether endothelial cells align preferentially along microgrooved versus unpatterned surfaces of these SMPs. Results show that micropatterning SMP surfaces enhances natural surface


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hydrophobicity, which helps to promote endothelial cell attachment and alignment along the grooves. With the addition of microgrooves to the SMP surface, this class of SMPs may provide an improved surface and material for next-generation blood contacting devices.

Research Highlights:

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50-µm-width microgrooves were introduced to shape memory polymer (SMP) surfaces using a simple, robust and cost-effective metal-printed molding method

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Microgrooves increase surface hydrophobicity and roughness

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Endothelial cell adhesion is generally greater on microgrooved surfaces compared to unpatterned analogues

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Endothelial cells demonstrate increased alignment parallel to the microgrooves, whereas adherent cells on unpatterned surfaces are randomly oriented

Introduction

Although cardiovascular disease (CVD) is the leading cause of death globally, treatment methods continue to be limited 1. Initially, surgical intervention was the preferred method for opening occluded vessels, but the invasiveness of surgery and lack of support to arteries frequently resulted in vessel re-occlusion, limiting this approach. Balloon angioplasty and bare metal stents (BMS) provide a less invasive method for opening and supporting narrowed vessels; however, complications such as restenosis and thrombosis from reduced biocompatibility often prompt reintervention in many patients 2, 3. Drug eluting stents (DES) reduce restenosis due to the addition of an anti-proliferative agent, but delayed re-endothelialization and resulting late stage thrombosis continue to be prevalent issues 2, 4. Many of these limitations in current stent


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technologies are due to material noncompliance and reduced surface patency, which result in sub-optimal patient outcomes. These issues provide continued support for research into improving stent performance 5, 6.

Endothelialization of stents and other blood-contacting devices facilitates proper function and integration with surrounding tissue 3, 7. However, reduced compatibility between stents and their respective physiological environments promotes poor endothelial cell recruitment and adhesion. Endothelialization decreases device rejection and eliminates the need for long term anti-platelet therapies due to the generation of an anti-thrombotic and anti-proliferative environment created by healthy endothelial cells 3. Current in vitro cell seeding on devices prior to implantation has resulted in reduced thrombosis and improved integration, but these methods may not be feasible in practice due to their laborious and highly specialized nature 1, 5, 8. In situ re-endothelialization of implanted medical devices would be the preferred approach 5, 7, 8.

Mechanical, chemical, and/or topographical alterations to stent materials may promote endothelialization and are thus important considerations in the design of such devices 9-12. Chemical, physical and biofunctional surface modification methods have been shown to encourage in situ endothelialization 13, 14. In this regard, surface patterning may be particularly attractive since it not only promotes cell adhesion, but also encourages cell alignment for a variety of cell types, including endothelial cells, all with far less potential regulatory burden than that required for chemical or biological additions 15-17. Grooves and ridges are some of the most common features used to encourage cell attachment and promote cell alignment 18, 19. Cell alignment is essential for proper biological and mechanical function in most native tissues, but many biomedical device materials do not contain the structural cues necessary to encourage such


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organization 20, 21. In native blood vessel under healthy flow conditions, endothelial cells align along the direction of flow, whereas in unhealthy flow conditions, ECs demonstrate isotropic orientations, which may result in a more thrombogenic and inflammatory environment 22, 23.

Shape memory polymers (SMPs) are customizable, smart materials that can recover their original shape following deformation 24, 25.. For biomedical devices, SMPs have been used in cardiovascular, orthopedic, and dental applications 26-28. Our group has previously developed SMPs for stents, embolic coils, hernia meshes, etc. 29-35. Prior research on this family of SMPs for stent use has largely focused on the mechanical properties for stent deployment and implantation and to confirm low cytotoxicity 31, 32. We recently evaluated endothelial cell adhesion on the surface of these acrylate-based SMPs and found that certain formulations, specifically those containing a higher weight percent ratio of the acrylate monomer, encourage endothelial cells to adhere without requiring surface modification 30. However, cells on these surfaces were randomly oriented; a key next step is to evaluate whether axially oriented topographical surface modifications facilitate endothelial cell alignment.

To study this further, we investigated techniques for micropatterning the surface of these acrylate-based SMPs, and then evaluated the extent of endothelial cell attachment onto patterned and unpatterned surfaces. Since the unpatterned surfaces encourage cell adhesion, micropatterning should further optimize the surface for cell adhesion and survival. Shallow microgrooves were created using 3D metal printed molds, a technique that offers a scalable, costeffective and practical approach to topographically pattern polymerizable materials. To the best of our knowledge, surface patterning of this family of SMPs for endothelial cell adhesion and organization has not yet been studied. In this investigation, three SMP formulations with varying


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crosslinker molecular weights were analyzed. These particular formulations, which contained high weight percent ratios of acrylate, previously demonstrated endothelial cell adhesion capabilities, but did not contain the topographical cues needed to encourage alignment. Unpatterned and microgrooved substrate surface properties were assessed using scanning electron microscopy (SEM), atomic force microscopy (AFM) and contact angle measurements. Endothelial cell adhesion and alignment were evaluated for up to seven days using fluorescence microscopy.

Materials and Methods

Micropatterned Mold Fabrication

Micropatterned metal plates were fabricated using an EOSINT M270 3D metal printer (EOS, Munich, Germany). The length, width and height of the metal print were specified to match the dimensions of standard microscope slides using SolidWorks (Waltham, MA, USA). The surface of the metal printed piece consisted of approximately 50-μm-width repeating, shallow grooves. This microgroove pattern is a result of the direct metal laser-sintering method (DMLS) used by the metal printer to manufacture parts. This pattern was subsequently used for micropatterning of the SMP surface via molding.

Synthesis

The components for shape memory polymer fabrication, monomer tert-butyl acrylate (tBA) and crosslinker poly(ethylene glycol) dimethacrylate (PEGDMA) with average molecular weights (Mn) of 550 and 750, were purchased from Sigma-Aldrich (St. Louis, MO, USA).


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PEGDMA1000 was obtained from Polysciences (Warrington, PA, USA). Polymerization was facilitated by photoinitiator 2,2 – dimethoxy-2-phenylacetophenone (DMPA), also obtained from Sigma-Aldrich.

Three different monomer mixtures were used for this study (i.e., one mixture for each PEGDMA molecular weight) and all were an 80:20 wt% ratio of tBA:PEGDMA, similar to previous methods30. Monomer mixtures were injected into molds composed of a standard microscope slide (Thermo Fisher Scientific, Waltham MA, USA) and a metal plate, separated by a 1.33mm silicone spacer (Mcmaster-Carr, Elmhurst, IL, USA). The unpatterned SMP surfaces were created with a polished steel plate, while the metal printed piece was used for the microgrooved SMPs. The pre-polymers were then cured under ultraviolet (UV) radiation from a Dymax Model 200 Light Curing System (Dymax, Torrington, CT, USA) of wavelength 365nm for 10 minutes pulsed (30 seconds on, 30 seconds off) followed by 10 minutes uninterrupted curing. The samples were then removed from the molds at 90°C and post-cured in an oven at 75°C overnight, similar to previous methods 30.

Dynamic mechanical analysis (DMA) was then conducted on a Q100 DMA (TA Instruments, New Castle, DE, USA) to ensure that the thermomechanical bulk properties of the SMPs remained consistent between unpatterned and microgrooved samples. Three SMP samples per formulation and surface condition were cut into 20mm x 5mm x 1mm specimens, equilibrated to 0°C and ramped to 100°C at a rate of 3°C/min, as performed previously 34.

Scanning Electron Microscopy (SEM)


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The surfaces of the unpatterned and microgrooved SMPs were imaged using a JEOL ASM 6010LV (JEOL USA, Peabody, MA, USA). Prior to SEM imaging, the SMPs were sputter coated for 30 seconds using a Leica EM ACE200 (Leica Microsystems, IL, USA; EM Laboratory, Children’s Hospital Colorado). SEM micrographs were used to ensure that the unpatterned surfaces did not exhibit any organized surface features and to verify micropatterning and groove width of the microgrooved SMP surfaces.

Atomic Force Microscopy (AFM)

Surface topography and surface roughness were obtained using atomic force microscopy (AFM). Five different samples per formulation were cleaned with ethanol and air-dried to remove any debris prior to imaging. Topographical data and images were obtained using a JPK AFM system (JPK, Berlin, Germany). Image post-processing was completed using Gwyddion open source software (Gwyddion, Brno, Czech Republic). The root mean square roughness coefficient, Rms, which provides quantitative information of the sample surface. Rms, measured by the standard deviation of the distribution of surface heights of the sample, was also obtained from Gwyddion 36

.

Contact Angle

Contact angle measurements and wettability of SMP samples were obtained using a Kudos Precision Instruments DropMeter A60 (Manhattan, NY, USA). Wettability of each formulation was measured by applying 10 µL water droplets to each surface and measuring the angle formed between the water droplet and the surface of the sample. Measurements were taken ten seconds after the water droplet was introduced to the surface of the SMP to maintain consistency. Contact


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angles were measured using SurfaceMeter Elements computer software (NBSI, Ningbo City, China). Five different samples were analyzed per surface, per SMP formulation. Five drops were applied to each SMP sample surface and five measurements were taken per drop.

Cell Culture

Obtained from the endothelium of the umbilical vein, human umbilical vein endothelial cells (HUVECs) are a common cell model for angiogenesis and re-endothelialization studies. HUVECs were the chosen cell model for this re-endothelialization study because they are robust, which makes them a favorable cell type for use in such studies 7.

Prior to cell culture experiments, HUVECs (Lonza, Walkersville, MD, USA) were seeded in T75 flasks using complete growth medium: EBM-2 Cell Culture Bullet Kit (Lonza, Walkersville, MD, USA). HUVECs were maintained in standard cell culture conditions of 37°C and 5% CO2 in a humidified incubator. HUVECs and HUVEC-SMP samples were observed daily under a Nikon inverted light microscope (Nikon, Melville, NY, USA). Cells were washed with HEPES, 1M, buffer (Life Technologies, Carlsbad, CA, USA) prior to changing media. Media was changed every other day, and cells were passaged at 80-90% confluence. Cell passages four through seven were used for cell seeding on SMP substrates. At least three independent experiments were performed, and all experiments were conducted in triplicate.

Endothelial Cell Attachment

SMP substrates were submerged in ethanol, air-dried and subsequently steam sterilized prior to HUVEC seeding. Steam sterilization has been previously used to sterilize acrylate-based SMPs


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successfully without disrupting shape memory capabilities or other material properties31, 34. HUVECs were then plated at a seeding density 1× 105 cells/mL per well on 10mm diameter SMP substrates in coverslip bottom 24-well plates and allowed to attach. Cell-adherent SMPs were monitored daily for proper cell growth and absence of contamination using transmission microscopy.

Cell viability was quantitatively assessed at three time points: 1 day, 3 days and 7 days after cell introduction. Endothelial cell attachment and viability was assessed using the Live/Dead Cell Imaging Kit (488/570) (Life Technologies, Carlsbad, CA, USA). Live cells, which were actively attached to the substrate, emit green fluorescence, while dead cells emit red fluorescence. Complete cell medium was changed every other day during the study. Images were obtained using a Zeiss Axiovert A1 inverted microscope (Zeiss, Thornwood, New York, USA). At least five images from three replicate experiments were used for cell attachment counting using ImageJ software (NIH, Bethesda, MD, USA).

CD31, Actin and Nucleus Orientation

After 7 days of sub-culture, cell-attached SMP samples were fixed using 4% paraformaldehyde in PBS for 10 minutes. Fixed samples were then submerged in 0.1% Triton-X for permeabilization prior to staining with Anti-CD31 (Abcam, Cambridge, MA, USA), which was used per manufacturer instructions. In addition to Anti-CD31, phalloidin and DAPI were also used according to manufacturer instructions. DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), (MilliporeSigma, Burlington, MA, USA) was used to visualize nucleus alignment, which was measured as 0° if the nucleus position was perpendicular to the groove while nuclei parallel to the groove were measured at 90°. The alignment angle was measured


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using ImageJ software (NIH, Bethesda, MD, USA). Filamentous F-actin was visualized using Alexa 568 Phalloidin Actin Stain (Thermo Fisher Scientific, Waltham, MA, USA) and confirmed cell alignment. Fixed cell imaging was performed on a Zeiss AxioObserver inverted microscope (Zeiss, Thornwood, New York, USA).

Statistical Analysis

The data were expressed as mean ± standard deviation (µ ± SD). Statistical analysis was performed using MATLAB (MathWorks, Natick, MA, USA) and significance was determined using a two-tailed t-test with α-level of significance of 0.05 when comparing unpatterned vs. micropatterned groups. When comparing more than two groups, analysis was conducted using a two-way ANOVA and the Tukey’s Honest Significant Difference Test assessed the significance between individual samples if ANOVA determined significance of the sample set.

Results

Synthesis

Dynamic mechanical analysis (DMA) confirmed the consistency of bulk properties, specifically the glass transition temperature as measured by the peak of the tan delta as well as the storage modulus, between unpatterned and micropatterned SMPs, similar to prior results (Figure 1) 30, 35. Data confirmed that microgroove introduction does not significantly affect Tg, Tonset or Tg range, as demonstrated in Table 1.


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Figure 1. Storage modulus and tan delta curves for 80:20 wt% unpatterned and microgrooved SMPs. Both storage modulus and tan delta display overlap between unpatterned and microgrooved surfaces.

Table 1. Tg, Tonset and Tgrange (n = 3,) for unpatterned and microgrooved 80:20 wt% tBA:PEGDMA550, 750, 1000 SMPs.


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Scanning Electron Microscopy (SEM)

Scanning electron micrographs qualitatively depicted pattern transfer to the surface of micropatterned SMPs as well as the lack of periodic, pattern-like surface features on unpatterned surfaces. As shown in Figure 2, the unpatterned surfaces lack repetitive surface features while the patterned surfaces exhibit repeating shallow microgrooves, confirming pattern transfer from the mold to the SMP surface. The microgroove widths, which ranged from 55μm to 60μm, were measured using ImageJ (NIH, Bethesda, MD, USA).

Figure 2. SEM micrographs verified pattern transfer and surface feature presence, or lack thereof, on unpatterned and micropatterned SMP surfaces. Unpatterned surfaces exhibit topographical randomness. Micropatterned surfaces display shallow grooves with widths ranging from approximately 55 to 60 μm (n=3). Scale Bar: 100 µm.

Atomic Force Microscopy (AFM)

Atomic Force Microscopy (AFM) was used to quantify the topography of each SMP surface, by measurement of surface roughness 37. The root mean square surface coefficient (Rms), which provides a quantitative measure of surface roughness, was obtained from AFM data. Micropatterned surfaces exhibited 11-14% higher roughness compared to unpatterned SMP


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surfaces, but surface roughness did not demonstrate statistically significant differences between unpatterned SMPs and their microgrooved analogues (Figure 3). The roughness of unpatterned SMP samples, polymerized in glass molds, has been previously reported by our group and similar results for unpatterned surfaces were confirmed 30.

Figure 3. Roughness of unpatterned vs. microgrooved 80:20 wt% tBA:PEGDMA SMP surfaces. Microgrooved surfaces exhibit 11%-14% increased roughness compared to unpatterned SMP surfaces. A 2-tailed t-test was used to determine significance between unpatterned and microgrooved surfaces, n = 5. While there is an increase in roughness between unpatterned and microgrooved surfaces, the differences are not statistically significant. Topographical views of unpatterned and microgrooved tBA:PEGDMA550, tBA:PEGDMA750 and tBA:PEGDMA1000 surfaces confirm that pattern introduction further increases roughness compared to unpatterned surfaces.


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Contact Angle

Surface wettability was determined by measuring the contact angles formed between a 10μL drop of diH2O and the SMP surface. Unpatterned SMP surfaces consistently exhibited contact angles close to 90°, indicating slightly hydrophobic surfaces. Micropatterning the SMP surfaces decreased wettability by 3-6%, as seen in other studies using different materials 38. Notably, significant differences between wettability of unpatterned vs microgrooved surfaces is confirmed and are shown in Figure 4.

Figure 4. Wettability of unpatterned vs. microgrooved SMP surfaces. Hydrophobicity is 3%-6% greater for microgrooved surfaces, indicating a small increase in hydrophobicity between unpatterned and microgrooved surfaces. A 2-tailed t-test determined significance between unpatterned and microgrooved surfaces, *** corresponds to p < 0.001. n =5.

Endothelial Cell Attachment


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Endothelial cell attachment and viability was assessed using Live/Dead imaging fluorescence microscopy. Live cells are displayed in green, whereas dead cells fluoresce red. NucBlue Live Cell ReadyProbes was used to mark cell nuclei as indicated in blue (Figure 5).


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Figure 5. Endothelial cell attachment at 1 day, 3 days and 7 days after cell introduction assessed using Live/Dead Cell Imaging and NucBlue Live Cell Stain to mark nuclei. Microgrooved SMPs exhibited greater cell attachment compared to unpatterned surfaces. There is also some evidence of cell organization in the direction of the grooves. Scale Bar: 200μm. Note: some of these SMPs, specifically the formulations containing PEGDMA750 and PEGDMA1000, occasionally absorb the NucBlue stain; this is particularly evident on microgrooved surfaces.

Although all SMPs and topographies demonstrate endothelial cell adhesion and increases in living cells during the study, endothelial cell attachment is greater on micropatterned surfaces compared to their unpatterned analogues, for all SMP formulations, at all timepoints, after endothelial cell seeding, as shown in Figure 6A. The 80:20 wt% tBA:PEGDMA550 microgrooved surfaces displayed the highest endothelial cell adhesion, which was measured by counting live, surface-adherent cells, as displayed in Figure 6. It should be noted, however, that the 80:20 wt% tBA:PEGDMA1000 microgrooved surface consistently experienced the greatest increases in cell presence compared to its unpatterned analogue. Occasionally, increases in dead cells on micropatterned vs. unpatterned surfaces are present, but the percentage of dead cells on microgrooved surfaces remains equal to or lower than their unpatterned counterparts. Long-term cell cultures, up to seven days, depict similar trends as microgrooved surfaces and continue to show higher endothelial cell presence compared to unpatterned surfaces

In addition to cell adhesion, live/dead data was also used to assess cell proliferation. Cell proliferation was estimated as the increase in cell presence on each surface relative to the initial day 1 cell counts and displayed as percentage increase in adherent cells (Figure 6B). Although the unpatterned surfaces initially demonstrated less cell adhesion, these same unpatterned SMP


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surfaces typically demonstrated greater increases in cell presence on day 3 and day 7 compared to their microgrooved analogues. However, microgrooved surfaces ultimately demonstrated the greatest overall cell adhesion compared to their respective unpatterned analogues.


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Figure 6. A) Approximate cell presence, live and dead, on all unpatterned vs. microgrooved SMPs. Microgrooved surfaces for the most part demonstrate statistically significant increases in cell presence compared to unpatterned SMP surfaces. Dead endothelial cell presence is typically comparable between unpatterned and microgrooved surfaces.)B) Percentage increase in EC presence on unpatterned and microgrooved SMPs. Unpatterned surfaces demonstrate a higher percentage increase in cells, but cell presence remains highest on microgrooved surfaces compared to unpatterned ones. Significance between unpatterned and microgrooved surfaces was determined using a 2-tailed, unpaired t-test, * corresponds to p < 0.05, ** corresponds to p < 0.01, *** corresponds to p < 0.001. Significance between SMPs of varying crosslinker lengths and unpatterned and microgrooved surfaces was determined using a 2-way ANOVA, * corresponds to p < 0.05, ** corresponds to p < 0.01, *** corresponds to p < 0.001.

Endothelial Cell Alignment: Filamentous Actin and Nuclei Orientation

Endothelial cell alignment was assessed by measuring nuclear orientation and alignment angle relative to the groove, as displayed in Figure 7. For cells adhering to microgrooved surfaces, nucleus orientation angles of 90° indicate cellular position parallel to the direction of the groove. Endothelial cells are more randomly oriented on unpatterned surfaces, whereas on micropatterned surfaces, ECs orient themselves in the direction of the groove, depicted by increases in the percentage of cells for orientation angles 80°-90°. Cells on unpatterned surfaces are more isotropically distributed, demonstrating a lack of organization. Nucleus orientation, or lack thereof, was further verified by normalizing to a gaussian fit function.


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Figure 7. Endothelial cell alignment as measured by nuclei and actin organization on 80:20 wt% tBA:PEGDMA550, 80:20 wt% tBA:PEGDMA750 and 80:20 wt% tBA:PEGDMA1000. Increases in nuclei orientation at 80°-90° on microgrooved surfaces indicate that cells display higher organization on microgrooved surfaces compared to their unpatterned counterparts. Nucleus orientation was confirmed by normalizing data to a gaussian fit function.

Discussion

Despite advances in cardiovascular stent technologies, issues such as restenosis and thrombosis continue to occur, requiring reintervention to prevent further complications. Rapid reendothelialization may eliminate, or at least reduce, the need for antiproliferative drugs by promoting a local anti-inflammatory setting; several studies have shown that the presence of a well-functioning endothelium promotes an anti-thrombotic and anti-stenotic local environment 22, 39

. Thus, rapidly restoring the endothelial lining could greatly improve device success 40.

Numerous methods have been implemented to accomplish rapid endothelial restoration, but the time-cost and complexity of some methods limit their utility. We present a straightforward and customizable strategy for microgroove implementation on shape memory polymer surfaces to optimize endothelial cell adhesion by encouraging organized attachment.

This exploratory study examined the effect of introducing microgrooves to the surface of acrylate-based shape memory polymers to increase endothelial cell adhesion and organization. Previous studies from our group have examined thermomechanical behavior, the shape memory effect and recovery, biocompatibility and cytotoxicity of these tBA:PEGDMA SMPs 31, 32, 34, 35, 41

. Recently, our group demonstrated endothelial growth onto a family of unmodified acrylate-

based SMP surfaces, but adherent cells lacked spatial organization 30. In this previous study, the


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SMP formulations with weight percent ratios of 80:20 tBA:PEGDMA demonstrated improved cell adhesion and survival compared to the formulations containing greater amounts of PEGDMA; as such, these formulations were selected for surface optimization through microgroove introduction. Studies have shown that endothelial cells adhering to microgrooved surfaces demonstrate increased viability, migration and organization relative to unpatterned surfaces 4, 42.

Dynamic mechanical analysis (DMA) data confirmed that pattern introduction had little effect on the bulk properties of the SMP. SMPs are attractive materials for stent fabrication due to their low cost, ease of fabrication, customizability, drug loading potential, and compliance-matching ability, among others 32, 43. Previous studies by our group have demonstrated the thermomechanical utility of these SMPs for use in cardiovascular stents 32. Here, we aimed to limit modification of the material to the surface, to ensure that the bulk properties, such as modulus and activation temperature, were maintained and could potentially be used for safe and minimally invasive stent delivery 32, 33, 35. While the shape memory effect was not extensively investigated here, these formulations were selected, in part, for their previously validated shape memory capabilities. Ensuring that the changes introduced here did not change thermomechanical properties of the bulk material was therefore important 30, 32, 34, 35. Although the fabrication method was slightly modified compared to previous work by our group, there were no significant differences in Tg, Tonset and Tg range between unpatterned and microgrooved SMPs, confirming minimal effect on thermomechanical properties.

Successful pattern transfer from a metal printed mold to the shape memory polymer surface via cast molding was confirmed using scanning electron micrographs of the polymer surfaces.


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Molding methods allow for pattern introduction while limiting chemical variation between the groove, ridge and sides compared to etching or embossing 44, 45. The primary advantage of using the 3D metal printing approach is that it offers a rapid, reproducible, and customizable method for mold fabrication, joining the growing list of low-cost/high volume manufacturing techniques 14, 46

. Current topographical patterning methods, many of which originated in the semiconductor

industry, produce highly precise and complex surface features, which have utility in the study of cell function, but the high cost and specialization associated with these methods often reduces translational and scaling capability 47-49.

Surfaces were also quantitatively measured by assessing roughness and wettability. Surface roughness was obtained from AFM data and showed that roughness was lower on unpatterned surfaces compared to microgrooved ones, consistent with previous studies 44, 50. The unpatterned SMPs contained random surface features, which increased roughness compared to SMPs fabricated in smooth glass molds, but the addition of microgrooves resulted in the highest observed surface roughness 30. Pattern introduction has been shown to increase the contact points on a surface, which would contribute to increased surface roughness; further, rougher surfaces have been shown to be more favorable to cell adhesion 51, 52. Micropatterned surfaces also displayed larger water contact angles, indicating an increase in hydrophobicity, consistent with related studies 50, 53. Higher hydrophobicity has been shown to increase cell attachment for certain cell types, including endothelial cells 54, 55. Pareta et. al, found that endothelial cells preferentially adhere to a surface at a specific surface energy, which is influenced by wettability 37

. Increases in both roughness and hydrophobicity contribute to increased presence in cell

adhesion to the microgrooved surfaces, as confirmed by the data presented here, but individual contributions are difficult to discern 52, 56.


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Several groups have investigated endothelial cell adhesion on SMPs; for example, the EC adhesion capabilities of thermoplastic, degradable block copolymers, electrospun SMP scaffolds, etc., have been investigated and it has been shown that these materials typically promote angiogenesis 57-59. Our group previously demonstrated endothelial cell adhesion to unpatterned, acrylate-based SMPs with varying compositions 30. Here, we selected the most promising of previously evaluated materials in our lab and further optimized the surface topographically through the introduction of microgrooves using a molding process. Microgroove introduction to the surface allows for the topographical modification while still maintaining the cell adhesion capabilities of the base material. The data confirmed that microgrooved surfaces increase cell adhesion, similar to results seen on other materials, including metals as well as polymers 4, 37, 40, 60

. Microgrooved SMPs initially displayed 20-30% higher cell presence one day after cell

seeding compared to their unpatterned analogues. The continued increase in cell presence up to seven days after initial cell introduction, which was notably greater on unpatterned surfaces compared to their microgrooved counterparts, further confirmed cytocompatibility of both unpatterned and microgrooved surfaces.

The improved functionality of endothelial cells on microgrooved surfaces most likely contributed to the greater cell adhesion initially, and subsequent sustained survival. Lu et. al., demonstrated similar results on titanium surfaces, in which microgrooved titanium displayed higher initial cell presence 42. At the end of the study time period, the 80:20 wt% tBA:PEGDMA550 microgrooved SMP displayed the highest cell adhesion of the formulations and surfaces examined. The higher cell adhesion to this specific surface may be a result of the combined benefits of material stiffness and microgroove addition. Our group previously found that endothelial cells prefer stiffer SMP surfaces, such as the one found on 80:20


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tBA:PEGDMA550, and the addition of microgrooves may have further optimized the surface for cell adhesion and viability30. Notably, the 80:20 wt% tBA:PEGDMA1000 SMPs experienced the greatest increases in cell adhesion between unpatterned and microgrooved surfaces, which further supports the motivation for microgroove addition to the SMP surface. Additionally, Huang et. al., determined that endothelial cells adhering to microgrooved titanium oxide surfaces displayed reduced adhesiveness to monocytes and platelets, promoting an anti-inflammatory and anti-thrombotic phenotype, an added benefit to microgroove introduction 23.

In addition to increased cell adhesion and viability, microgrooved surfaces also encouraged cell organization. Alignment was assessed by measuring the orientation angle of the nuclei 21. Microgrooved surfaces appear to encourage endothelial cell alignment through contact guidance, complementing results from other work 61-63. The 50μm width grooves have been previously implemented on a wide range of materials and demonstrated successful endothelial cell organization compared to unpatterned surfaces 21, 40, 64. Prior studies on other SMPs, specifically those with microwrinkled surfaces, have indicated that the addition of groove-like nano- and micro-topographies also encourages cell alignment 65-67. Other studies have shown that microtextured surfaces may contain local differences in surface energy, directing the spatial arrangement and conformation of adsorbed cell adhesion proteins, which dictates cell attachment to a surface 56.

To further improve cell alignment, it may be valuable to reduce groove width to more accurately match elongated cell size or increase groove depth; these and other changes will be investigated in future work 64, 68.In addition to improving cell alignment, further decreasing groove width may also generate more surface contact points by increasing material surface area, improving cell


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adhesion as well as proliferation69. Further, implementing a combination of micro- and nanogrooves, or a combination of other topographies such as wrinkles, waves, pits, etc., may provide added benefits compared to using a single surface feature dimension alone70. Recent studies by Brasch et. al., have found that the orientation of cellular Golgi bodies may be a better indicator of cell adhesion, which may also provide insight into cell orientation preferences65. It should be noted that the dimensions of the artifact micropattern were dictated by the specific model of the printer material deposition head and thus may be unique to the specific metal printer used; use of a different printer material deposition head may generate microgrooves of varying widths and/or depths or other topographies. This study was terminated 7 days after EC introduction, but cell survival beyond this time point, up to 14 or 21 days will be of additional interest. Assessment of EC adhesion on SMPs of varied composition, but similar activation temperatures, may provide additional insight into the nuances of cell adhesion on these materials and will be investigated in future work. In-depth studies regarding the effect of programming these materials on subsequent EC adhesion would also increase the clinical relevance of this work. Finally, in vivo studies, as well as verifying the effect of steam sterilization on SMP thermomechanical properties, are required to validate the results from these in vitro studies for implementation in implanted, blood-contacting devices.

The benefits of cell organization are not limited to endothelial cells in the vascular environment. Muscle tissue, nerve tissue, smooth muscle cells in vascular tissue and periodontal tissue all require cell alignment to maintain proper cellular function 16, 62, 68. The generic, yet customizable, nature of this approach may allow for the extension of this technique to a variety of materials and cell types for tissue engineering.


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Conclusions

A metal-printed mold casting method was used to generate a microgrooved, acrylate-based shape memory polymer surface to encourage increased endothelial cell attachment and alignment. Endothelial cells appear to prefer microgrooved surfaces, which display increased roughness and hydrophobicity, compared to their unpatterned analogues, resulting in increased cell presence on microgrooved surfaces. Additionally, microgrooved surfaces increase endothelial cell alignment compared to unpatterned surfaces. Our findings suggest that the introduction of microgrooves may further optimize the surface compatibility of these SMPs for implanted, blood-contacting biomedical devices.

Author Contributions: T.G. took the lead on conducting the experiments and preparing the manuscript. R.S. provided guidance and feedback during the studies as well as suggestions and editing during manuscript preparation; R.S. also provided funding.

Funding Sources: The authors would like to acknowledge support from the NIH (T32 HL072738, K24 HL081506). Acknowledgements: A special thanks to Stephen Huddle for his assistance with metal printing, Jennifer Wagner for her assistance with the goniometer, Eric Wartchow for his assistance with SEM imaging, Dr. Brisa Pena for her assistance with AFM and Dr. Emily Beck for AxioObserver support. ABBREVIATIONS SMP: shape memory polymer tBA: tert-butyl acrylate DMPA: 2,2 – dimethoxy-2-phenylacetophenone PEGDMA: poly(ethylene glycol) dimethacrylate DMA: dynamic mechanical analysis SEM: scanning electron microscopy AFM: atomic force microscopy HUVEC: human umbilical vein endothelial cell


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Microgrooves encourage endothelial cell adhesion and organization

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