Recombinant spider silk functionalized with a motif from fibronectin

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Biological and Medical Applications of Materials and Interfaces

Recombinant spider silk functionalized with a motif from fibronectin mediates cell adhesion and growth on polymeric substrates by entrapping cells during self-assembly Christos Panagiotis Tasiopoulos, Mona Widhe, and My Hedhammar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02647 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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ACS Applied Materials & Interfaces

Recombinant spider silk functionalized with a motif from fibronectin mediates cell adhesion and growth on polymeric substrates by entrapping cells during self-assembly Christos Panagiotis Tasiopoulos, Mona Widhe, My Hedhammar* AlbaNova University Center, KTH – Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Division of Protein Science, 114 21 Stockholm, Sweden

KEYWORDS: cell seeding, recombinant spider silk, RGD binding motif, surface functionalization, revascularization applications

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ABSTRACT

In vitro endothelialization of synthetic grafts or engineered vascular constructs is considered a promising alternative to overcome shortcomings in availability of autologous vessels and in-graft complications with synthetics. A number of cell seeding techniques have been implemented to render vascular grafts accessible for cells to attach, proliferate, and spread over the surface area. Nonetheless, seeding efficiency and time needed for cells to adhere varies dramatically. Herein, we investigated a novel cell seeding approach (denoted co-seeding) that enables cells to bind to a motif from fibronectin included in a recombinant spider silk protein. Entrapment of cells occurs at the same time as the silk assembles into a nano-fibrillar coating on various substrates. Cell adhesion analysis showed that the technique can markedly improve cell seeding efficiency to non-functionalized polystyrene surfaces, as well as establish cell attachment and growth of human dermal microvascular endothelial cells on bare polyethylene terephthalate and polytetrafluoroethylene (PTFE) substrates. Scanning electron microscopy images revealed a uniform endothelial cell layer and cell-substratum compliance with the functionalized silk protein to PTFE surfaces. The co-seeding technique holds great promise as a method to reliably and quickly cellularize engineered vascular constructs as well as to in vitro endothelialize commercially available cardiovascular grafts.


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Introduction The vasculature comprises an elaborately extensive network with primary scope to facilitate gas exchange and nourish tissues. When an essential blood vessel is no longer fully functional, it has to be either replaced or bypassed. To date, conventional treatment involves either the use of autologous arteries or veins, or synthetic vascular grafts to restore normal blood flow. Despite being the most favorable grafting choice, autologous vessels are not always available, may be of poor quality, and can result in donor site morbidities after extraction1. On the contrary, synthetic grafts have been associated with chronic inflammation due to insertion site infections and poor long-term patency rates for small diameter vessels (Ø < 6mm)1-2. Reduced patency is mainly related to intimal hyperplasia in which the lumen thickens and no longer sustains an unimpeded blood flow3. In addition, synthetic grafts often fail to generate a complete endothelial cell lining and thus, thrombi may develop as a response to activation of the coagulation cascade4. In vitro endothelialization by seeding autologous endothelial cells onto lumens of inert vascular prostheses has been suggested to result in endothelium formation post-operationally5-6. The strategy has been shown to effectively provide a confluent endothelium within a short amount of time and improve long-term patency rates comparable to when vein substitutes are used7-8. Nonetheless, not yet resolved challenges in the cell seeding procedure limit the clinical applicability of in vitro endothelialization of polymeric vascular grafts. A mature morphological cell state is hard to achieve within short incubation times (< 2 hours) by gravitational or hydrostatic incorporation of endothelial cells onto synthetic surfaces. Without a mature state, cellular loss appears upon exposure to shear stresses9. Electrostatic cell seeding, on the contrary, succeeds in reducing cell adhesion time and cell loss after implantation, but lack of a standardized apparatus to be used for non-conducting synthetic materials, such as expanded 3 ACS Paragon Plus Environment


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polytetrafluoroethylene, limits its potential use10. In addition, there is not yet any solid clinical outcome supporting improved patency rates of electrostatically endothelialized polymeric vascular implants11. Alternatively, engineered vascular grafts have been proposed to overcome both deficiencies with synthetics and shortage in available autologous vessels12-15. However, biomaterial hybridization or surface modification is often necessary to promote luminal growth of endothelial cells16-18. So far, a diverse set of seeding techniques has been implemented to facilitate cell adherence, but there is not yet any method shown to be superior to the others19. To date, passive and dynamic cell seeding has been thoroughly investigated in a number of different experimental setups1, 19. Passively seeding cells is simple and the most followed procedure, but it is highly associated with low seeding efficiencies and usually fails to provide endothelial cell uniformity20. In addition, it has been reported that porosity of the engineered scaffold may be critical for the ability of cells to penetrate when statically seeded13,

21

. Dynamic approaches

instead employ either rotational forces or pressure differentials to make vascular grafts more penetrable to cells and improve uniformity by a more even cell distribution19. However, rotational systems often require extensive seeding time at low speeds, or there is a risk of altered cell morphology when higher centrifugal forces are used22-23. Vacuum systems, on the contrary, may pose a risk for contamination as cell suspension passes through the various interconnected compartments24. It is hence of great demand for a reliable and quick seeding technique to be established, preferably by utilizing a strong yet pliable material well tolerated by cells. Recombinant spider silk is a genetically engineered biopolymer with excellent mechanical properties which has been shown competent to be used as substrate for mammalian cell cultures25-26. Recently, it has been


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reported that an arginine-glycine-aspartic acid (RGD)-containing cell binding motif found in the sequence of fibronectin27 can be genetically coupled to recombinant spider silk protein 4RepCT, promoting efficient cell adhesion28. Further, this functionalized silk variant, denoted FN4RepCT, can self-assemble into stable nano-fibrillar coatings similar in morphology to structures in the extracellular matrix (ECM), which mediate cell migration and proliferation28-29. The FN4RepCT silk protein can thus be regarded a suitable candidate material to efficiently endothelialize polymeric vascular grafts. Herein, we investigated a novel cell seeding approach that enables cells to bind to the motif from fibronectin in the soluble form of FN-4RepCT silk and then be entrapped during assembly into a nano-fibrillar coating. In this way, surface functionalization of a material with cells and silk together can quickly be established without the need to pre-coat, thereby minimizing the overall seeding time dramatically. As a proof of concept, surfaces made of polymers widely used to produce cardiovascular grafts were seeded with and without FN-4RepCT silk and evaluated in terms of cell attachment, viability and uniformity.

Materials and Methods Cell cultures Human dermal microvascular endothelial cells (hDMECs) isolated from juvenile foreskin (PromoCell, Heidelberg, Germany) were cultured and expanded in complete endothelial cell growth medium MV2 (PromoCell, Heidelberg, Germany) in flasks pre-coated with 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, USA). Primary human smooth muscle cells (SMCs) isolated from coronary artery (ThermoFisher Scientific, Waltham, MA, USA) were cultured and



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expanded in complete smooth muscle cell growth medium (Gibco, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS). Human mesenchymal stem cells (hMSCs) from bone marrow (ScienCell, Carlsbad, CA, USA) were cultured and expanded in ready-to-use mesenchymal stem cell growth medium DXF (PromoCell, Heidelberg, Germany) in flasks precoated with CELLstart (Gibco, Waltham, MA, USA). HDMECs and SMCs were used in passages 6 to 8, and hMSCs at passage 3. Medium in all cell cultures was changed every second day.

Preparation of silk coatings Polystyrene 96-well plates with hydrophobic surface (Sarstedt, Nümbrecht, Germany) were coated with the wild-type 4RepCT silk protein (from now on denoted WT-silk) and 4RepCT functionalized with the RGD-containing cell binding motif from fibronectin28 (FNcc, from now on denoted FN-silk) (provided by Spiber Technologies AB, Stockholm, Sweden). Briefly, stock concentration 3.0 mg/mL of both silk variants was thawed at room temperature and spun down for a minute using a bench top centrifuge. The protein was then diluted in phosphate buffered saline (PBS) to a final concentration of 0.1 mg/mL, spun down for another minute, and finally added to respective wells (n = 8). After one hour of incubation, the protein solution, without allowed to dry, was removed and the coated surfaces were washed twice with PBS prior to be seeded. Polytetrafluoroethylene (PTFE, Wuxi Xiangjian, Wuxi City, China) and polyethylene terephthalate (PET, Sigma-Aldrich, St. Louis, MO, USA) surfaces were manually cut to fit in wells of 96-well plates and in similar fashion coated with the silk variants.


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Cell seeding Cells were harvested when reaching about 80% confluency according to commonly followed protocols. Briefly, cells were washed once with pre-warmed PBS and enzymatically detached with TrypLE Express (Life Technologies, Waltham, MA, USA) in order to be counted and prepared in 106 cells/mL solutions. In the meantime, WT- and FN-silk protein solutions were thawed at room temperature and spun down for a minute to remove any aggregates. Cell suspensions with or without the silk in equal parts (to a final concentration of 1.5 mg/mL) were further prepared. Cell densities were 3300 and 6600 cells per 10 μL for polystyrene and PET, PTFE surfaces, respectively. Uncoated and silk coated surfaces were washed twice with PBS, allowed to dry, and then seeded in quadruplicates in a drop-manner (Figure 1) with 10 μL of the prepared cell solutions. Cell drops were allowed to incubate for 30 minutes at 37 °C with 5% CO2 and 95% humidity, before removal of non-adherent cells. Cells that adhered were further analyzed or cultured according to below.

Quick adhesion assays To evaluate the degree of adhesion after 30 minutes incubation, adherent cells were washed twice with pre-warmed PBS and fixed with 96% Ethanol. Fixed cells were then washed three times with de-ionized water before stained with 0.3% Crystal Violet in Milli-Q H2O for 45 minutes. Four wells without added cells were stained as well and used as blanks. Cell attachment and morphology on polystyrene and PET surfaces were documented at 2x and 10x magnification in an inverted bright field microscope (Nikon Eclipse Ti, Tokyo, Japan) and 5x magnification for PTFE surfaces using a stereomicroscope (Nikon SMZ 745T, Tokyo, Japan). The stain bound to



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fixed cells was subsequently dissolved in 40 μL of 20% acetic acid for 30 minutes on a horizontal shaker. Once incubation was completed, 35 μL of the dissolved solution was transferred to a 384-well plate (ThermoFisher Scientific, Waltham, MA, USA) for absorbance at 595 nm to be read out by the plate reader (CLARIOstar, BMG Labtech, Ortenberg, Germany).

Cell proliferation and viability assays The viability and proliferative capacity of the drop-seeded cells were evaluated with Alamar Blue viability assay (Invitrogen, Waltham, MA, USA). After the initial 30 minutes incubation, wells of 96-well plates were gently washed before addition of complete medium and further cell culture. Cell growth was monitored between days 1 and 7 after seeding by adding Alamar blue diluted 1:10 in cell growth medium followed by 2 hours incubation. Wells (n = 4) without cells were used as blank. Fluorescence intensity of 100 μL supernatants was measured with excitation at 544 nm and emission at 595 nm using a fluorescence plate reader (CLARIOstar, BMG Labtech, Ortenberg, Germany). Live/dead viability assay (Molecular Probes, Waltham, MA, USA) was performed at the last day of culture (day 7) to visualize cells on the various substrates at 2x and 10x magnification. Cells were washed twice with pre-warmed PBS and allowed to incubate at room temperature for 30 minutes with prepared live/dead solution in respective growth medium before viewing under the fluorescence microscope (Nikon Eclipse Ti, Tokyo, Japan), and imaging with NIS elements BR.

Cell fixation and immunostaining


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At the last day of culture (day 7), cells were washed twice with pre-warmed PBS, fixed with 4% paraformaldehyde, permeabilized with 3% Triton X-100 in PBS and blocked with 5% goat serum in PBS 0.5% tween for 20 minutes, before staining with Phalloidin AlexaFlour 488 (ThermoFisher Scientific, Waltham, MA, USA) 1:40 in PBS 0.5% tween for 45 minutes. After washed twice in PBS 0.5% tween, nuclei were stained with DAPI for 10 minutes and washed twice before analyzed under the fluorescence microscope (Nikon Eclipse Ti, Tokyo, Japan). Images at 10x magnification were captured using the NIS elements BR software.

Scanning electron microscopy samples preparation Cells on PTFE surfaces were fixed overnight with 2% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M HEPES buffer. Fixation solution was then removed and fixed cells were washed three times with 0.1 M HEPES buffer for 5 minutes each, before being serially dehydrated with 50% Ethanol (2 times, for 10 minutes each), 70% Ethanol (2 times, for 10 minutes each), 95% Ethanol (2 times, for 10 minutes each), and 99.5% Ethanol (3 times, for 15 minutes each) on an agitation shaker. Chemical drying with hexamethyldisilazane (HMDS, Sigma-Aldrich, St. Louis, MO, USA) was then selected to serially dry fixed samples for 15 minutes with 2 parts 99.5% Ethanol and 1 part HMDS, 15 minutes with 1 part 99.5% Ethanol and 1 part HMDS, 15 minutes with 1 part 99.5% Ethanol and 2 parts HMDS and finally, 15 minutes with HMDS alone for 3 times. The last HMDS was let to evaporate overnight under a fume hood and samples were then mounted on specimen stubs, sputter coated with 10 nm Au/Pd and images were taken using a scanning electron microscope (Zeiss, Oberkochen, Germany).



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Statistics Multiple t tests assuming unequal variances was performed using GraphPad Prism version 6.05 for Windows (GraphPad Software, La Jolla, CA, USA). Statistical significance was considered as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 1 Illustration describing the difference between normal cell seeding and co-seeding. A drop of a prepared cell suspension was placed onto a surface, either alone or blended with FN-silk protein, and allowed to incubate for 30 minutes at 37 °C with 5% CO2 and 95% humidity.

Results Rationale of the study A silk protein functionalized with a motif from fibronectin, FN-4RepCT, has recently been shown able to spontaneously self-assemble into stable coatings with the adhesion motifs well exposed to direct cell attachment. Silk self-assembles into stable coatings within 0.5 to 1 hour and early cell adherence onto FN-silk coatings typically established within 1 hour incubation


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time28-29. In this study, cells are seeded at the same time as the FN-4RepCT protein is added, and are thus present as the silk assembles into a coating. This procedure, denoted as co-seeding, has the aim to minimize the cell incubation time. More importantly, the necessity to pre-coat substrates can then be disregarded. Since surface endothelialization has been pointed out as hard to establish in revascularization processes, endothelial cells (hDMECs) were chosen as first target to be co-seeded with the silk. Smooth muscle cells (SMCs) constitute the predominant cell type met on vascular tissues and therefore chosen as next target for co-seeding. Stromal cells derived from the mesenchyme is a favorable cell source due to multipotent capacity to differentiate into several cell types including endothelial-like cells30. Hence, human mesenchymal stem cells (hMSCs) were also evaluated herein.

Co-seeding cells together with the FN-silk protein establishes quick adhesion Early adherence of the different cell types studied was evaluated using crystal violet staining. A drop of cell suspension, with or without FN-silk, was allowed to incubate for 30 minutes and subsequently the attached cells were fixed. Micrographs (Figure 2a) show substantial cell attachment to uncoated surfaces onto which cells were co-seeded with FN-silk, as compared to same surfaces seeded with cells without any silk support. Cells co-seeded with FN-silk attained similar attachment levels as cells seeded directly onto surfaces pre-coated with the silk (Figure 2b). A similar experiment was performed with cells blended with WT-silk instead of FN-silk to confirm that cell adhesion is obtained mainly due to the RGD-containing motif and not the silk itself (Figure S1). Cell morphologies of hDMECs, SMCs, and hMSCs (representative indicated with red arrows in Figure 2a) with strongly developed anchoring points to the underlying surfaces could be noticed, whether the cells had been seeded together with FN-silk or onto a silk 11 ACS Paragon Plus Environment


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coating. On the contrary, cells on uncoated surfaces either retained an immature morphology or were utterly unable to establish adherence (Figure 2a, left panel). To further validate the notion that cells can adhere well once blended with FN-silk and then seeded, dissolving the staining solution bound to the cells and reading the respective absorbance was performed. A marked increase in absorbance was achieved from cells seeded together with FN-silk on uncoated surfaces, as compared to without (Figure 2b). From acquired absorbance values, it can be stressed that 30 minutes of incubation time was not sufficient for cells to adhere to non-functionalized substrates without the silk support. Further, no statistical difference was found between cells seeded to surfaces pre-coated with FN-silk and cells co-seeded with FN-silk to uncoated ones. Moreover, the seeding efficiency was significantly improved also when cells were co-seeded with FN-silk to surfaces pre-coated with WT-silk (Figure S2).

Figure 2 Human dermal microvascular endothelial cells (hDMECs), smooth muscle cells (SMCs) and human mesenchymal stem cells (hMSCs) seeded with or without the FN-silk protein and allowed to adhere to uncoated or


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surfaces pre-coated with FN-silk for 30 minutes. (a) Micrographs (10x magnification) of cells stained with 0.3% crystal violet. Red arrows point to adhered cells with firmly developed anchoring points. Scale bars = 100 μm. (b) Spectrophotometric absorbance of crystal violet dissolved from stained hDMECs (upper panel), SMCs (middle panel), and hMSCs (lower panel) adhered to the differently treated substrates. Grey and black bars (mean+SD, n = 4) represent adhered cells seeded with and without FN-silk, respectively. Seeding density 3300 cells / 10 μL. Statistics: ** p < 0.01 and *** p < 0.001 (multiple t tests).

Co-seeding cells together with the FN-silk protein promotes cell growth and supports viability To confirm that cells co-seeded with the silk exhibit accustomed growth profiles, Alamar Blue cell viability assay was performed to monitor cell activity at specific time points. Fluorescence intensity values (Figure 3a) show a linearly increasing growth during a culture period of 7 days for all cell types co-seeded with FN-silk to the different substrates. In fact, cell proliferation was significantly improved when cells were co-seeded with the FN-silk to uncoated surfaces, suggesting that the motif from fibronectin can guide cells to binding in the soluble form and establish growth once cells are seeded. Similar results were obtained when cells were co-seeded with FN-silk onto surfaces pre-coated with WT-silk (Figure S3d). Fluorescence images captured from live/dead stained cells after one week in culture (Figure 3b) confirmed high viability with very few dead cells encountered. Further, characteristic morphologies indicative for each cell type could be seen with no apparent cell clustering, suggesting that binding cells to the motif prior to seeding does not hamper a sound cell distribution over the seeded area. In fact, captured images (Figures S3a-c and S4) show that the cells have uniformly spread also outside of the initial seeding area.



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Figure 3 Growth rate and viability of human dermal microvascular endothelial cells (hDMECs), smooth muscle cells (SMCs) and human mesenchymal stem cells (hMSCs). (a) Alamar Blue cell viability assay (mean±SD, n = 4) of hDMECs (upper panel), SMCs (middle panel), and hMSCs (lower panel) seeded with the silk (red lines) or without (blue lines) and monitored for 7 days in culture. Seeding density 3300 cells / 10 μL. Statistics: * p < 0.05, ** p < 0.01, and *** p < 0.001 (multiple t tests). (b) Micrographs (10x magnification) of hDMECs (upper panel), SMCs (middle panel), and hMSCs (lower panel) stained for live (green)/dead (red) after 7 days in culture. Scale bars = 100 μm.


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Co-seeding endothelial cells together with the FN-silk protein enables efficient cell attachment on PET surfaces Taken into consideration the promising results with co-seeding to polystyrene surfaces, substrates more close to a clinical application were also evaluated. Hence, PET substrates, often used as a bulk material to fabricate cardiovascular grafts (Dacron®-made), but with poor cell adhesive properties, were co-seeded with hDMECs. Micrographs of hDMECs allowed to adhere for 30 minutes and subsequently stained with crystal violet (Figures 4a, 4c, and S5) showed that the addition of FN-silk protein to the cell solution prior to seeding or utilized as a coating dramatically improves cell attachment to the PET surface. Thus, the silk can be equally well used to either pre-coat PET surfaces, or blended with cells and then directly used to endothelialize bare PET substrates. In addition, the morphological appearance of adherent cells was equally good. HDMECs expanded for seven days and stained for live/dead at the last day in culture (Figures 4b and 4d) were highly viable with very few dead cells noted. Fluorescence images captured on live cells suggest that hDMECs can well tolerate PET substrates in presence of the FN-silk protein, with no obvious difference between the two different ways of seeding.

Figure 4 Human dermal microvascular endothelial cells (hDMECs) seeded and cultured onto polyethylene terephthalate (PET) surfaces. (a) Micrograph (10x magnification) of hDMECs co-seeded with FN-silk to an



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uncoated PET surface, incubated for 30 minutes and finally stained with 0.3% crystal violet. (b) Micrograph (10x magnification) of hDMECs co-seeded with FN-silk to an uncoated PET surface and stained for live (green)/dead (red) at day 7 in culture. (c) Micrograph (10x magnification) of hDMECs seeded without FN-silk to a PET surface pre-coated with FN-silk, incubated for 30 minutes and finally stained with 0.3% crystal violet. (d) Micrograph (10x magnification) of hDMECs seeded without FN-silk to a PET surface pre-coated with FN-silk and stained for live (green)/dead (red) at day 7 in culture. Seeding density 6600 cells / 10 μL. Scale bars = 100 μm.

Co-seeding endothelial cells together with the FN-silk protein promotes cell adherence and supports growth on PTFE surfaces At last, PTFE substrates, also used as bulk material to fabricate a broad range of cardiovascular grafts, were evaluated as well. The notion was to investigate whether a complete endothelium can be formed using the co-seeding approach. As expected, the nude hydrophobic PTFE substrate constituted an unfavorable milieu for hDMECs to adhere to, but the seeding process was significantly improved when cells were coseeded with FN-silk (Figure 5a). Absorbance values after early adhesion indicated no statistical difference between cells co-seeded with FN-silk onto uncoated PTFE surfaces and cells seeded onto same substrates pre-coated with FN-silk (Figure 5b). The co-seeding process could support customary cell proliferation on PTFE substrates, as illustrated by growth profiles of hDMECs monitored for seven days in expansion (Figure 5c). Similar results were obtained when hDMECs were co-seeded onto PTFE surfaces pre-coated with WT-silk (Figure S6). Growth could not be established on uncoated PTFE substrates when hDMECs were seeded without any silk support. The appearance of the endothelial layer was evaluated by F-actin staining, demonstrating a coherent monolayer of endothelial cells with spread-out morphology (Figure 5d), implying a


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sturdy anchoring and compliance with the underlying PTFE material. Provided the solid attachment, the cells could uniformly endothelialize also the surface around the initially seeded area. Moreover, equally well developed cytoskeleton arrangement could be seen when cells were co-seeded with FN-silk on an uncoated PTFE surface compared to when cells were seeded onto a PTFE surface pre-coated with FN-silk.

Figure 5 Human dermal microvascular endothelial cells (hDMECs) seeded and cultured on polytetrafluoroethylene (PTFE) surfaces. (a) Micrographs (5x magnification) of hDMECs seeded with or without FN-silk to uncoated PTFE surfaces, incubated for 30 minutes and finally stained with 0.3% crystal violet. Scale bars = 100 μm. (b)



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Spectrophotometric absorbance of crystal violet dissolved from stained hDMECs adhered to uncoated PTFE, or PTFE surfaces pre-coated with FN-silk. Grey and black bars (mean+SD, n = 4) represent adhered cells seeded with and without FN-silk, respectively. Seeding density 6600 cells / 10 μL. Statistics: * p < 0.05 and ** p < 0.01 (multiple t tests). (c) Alamar Blue cell viability assay (mean±SD, n = 4) of hDMECs seeded with the FN-silk (red lines) or without (blue lines) to uncoated PTFE, or PTFE surfaces pre-coated with FN-silk, and monitored for 7 days in culture. Statistics: * p < 0.05, ** p < 0.01, and *** p < 0.001 (multiple t tests). (d) Micrographs (10x magnification) from one week cultures showing F-actin (green) of hDMECs seeded with FN-silk onto an uncoated PTFE surface (left panel) or without the silk onto a PTFE surface pre-coated with FN-silk (right panel). Cell nuclei were stained with DAPI (blue). Scale bars = 100 μm.

SEM images reveal good compliance at the interface between PTFE material and FN-silk Fluorescence images of hDMECs stained for F-actin confirmed sound cell morphology, but the interface between the underlying material and hDMECs together with FN-silk could not be visualized by fluorescence. SEM images (Figure 6) revealed that FN-silk can well comply with the underlying PTFE substrate when hDMECs were co-seeded and expanded onto it. Further, co-seeded cells seem to have been embedded to the silk protein, thereby creating a uniform superficial layer with apparent even cell distribution. This particular observation suggests that the technique may improve the frequently occurring inability of endothelial cell lining onto PTFE-made graft surfaces.


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Figure 6 Scanning electron microscopy images of human dermal microvascular endothelial cells co-seeded with FN-silk to uncoated polytetrafluoroethylene (PTFE) surfaces. Red arrows indicate the interface between underlying PTFE material and FN-silk. Scale bars = 100 μm, except for image on lower, middle panel = 10 μm.

Discussion The 4RepCT protein functionalized with a motif from fibronectin was previously shown competent to spontaneously self-assemble into stable coatings enhancing cell adherence and proliferation28-29. Herein, the possibility to assemble silk into coatings and simultaneously entrapping cells has been investigated, and it was shown that this approach can equally well enhance cell attachment and support cell growth. The results (Figures 2b and 5b) reveal that the FN-silk mediates early cell adherence whether utilized as a pre-coating or by binding cells during assembly. Strong cell adhesion further dictates subsequent cellular fate, and especially cell migration and proliferation, with the adhesive strength depending mainly on the underlying substrate, the material which it is made of,



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and the topography31-34. Micrographs of cells co-seeded onto polystyrene, PET, and PTFE surfaces (Figures 2a, 4a, 4c, 5a, and S2a) demonstrated a solid adherence to the various substrates, with cells displaying characteristic cytoplasmic outgrowths. The pivotal role of development of cell motility protrusions for cell proliferation and cell spreading is generally regarded as indication of a sound cell-matrix crosstalk35. All cell types herein co-seeded with the FN-silk were shown to be highly proliferative (Figure 3a) as well as viable (Figure 3b), suggesting that the co-seeding technique can establish a customary cell growth based on the initial tight cell anchorage. Cells seeded to uncoated, or surfaces pre-coated with WT-silk, require the presence of the FN-silk to remain highly viable (Figures S3a-c) and proliferate (Figure S3d). Fluorescence images of co-seeded cells unveiled a uniform coverage of dense cell populations with apparent cell-cell contacts and formation of a confluent monolayer. Cell-to-cell interactions are well recognized as important for regulating endothelial cell homeostasis36, controlling stemness in mesenchymal stem cells37, and defining SMCs phenotype38. The coseeding approach was herein found to not disturb the formation of essential cell-cell contacts when all these cell types were co-seeded with FN-silk. The several cell seeding techniques that have been attempted to endothelialize vascular grafts or cellularize engineered constructs seek to define an efficient way and a minimal incubation time needed for the cells to adhere. Passive seeding of cells require an extensive seeding time (up to days), often accompanied with poor efficiencies and low uniformity. On the contrary, rotational cell seeding has succeeded in improving seeding efficiencies and reducing incubation times (12 to 72h), but it is still considered too time-consuming and thereby limiting its widespread use24. When short incubation times (< 2h) have been carried out, endothelial cell attachment was immensely impaired. With such rotational seeding technique, cells retained an


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immature, rounded morphology when seeded to polymeric vascular grafts9. The co-seeding approach examined in this study allowed cells to incubate for a short while (30 minutes) with results indicating that cell attachment with mature morphologies can be well established despite the short time. An explanation to the observed efficient cell adhesion may be given by the ability of cells to bind to the RGD-containing motif prior to substrate deposition and therefore, accelerate the surface recognition process. Functionalization of surfaces with the RGD peptide has been extensively used to facilitate cell attachment and improve cell-substratum interaction to a plethora of biomaterials39. Recombinant spider silk and silkworm silk have been also successfully modified with the RGD motif in order to enhance adherence and generate more favorable constructs compared to the nonfunctionalized equivalents28, 40-41. Nonetheless, in all previous cases the modified constructs have been utilized to functionalize surfaces or materials before cell seeding. Additionally, the time needed for those silk variants to assemble into more stable formats was lengthy and cells required a considerable amount of time to become adherent to the end-engineered matrices. Herein, the co-seeding method was found equally reliable and fast in docking cells to substrates without being pre-coated, thereby reducing the overall seeding process by one step. To date, surface functionalization of cardiovascular grafts has been proposed to reduce the risks of clot formations and intimal hyperplasia, which constitute the two main reasons in graft failure42-43. While anticoagulants, e.g. heparin, bonded to PTFE material have been successfully used to reduce in-graft thrombogenicity44, recruitment of endothelial cells is even more desired in order to populate the surface of PTFE-made grafts and generate a complete endothelium. Several strategies have been implemented to endothelialize vascular graft surfaces, spanning from incorporation of growth factors, e.g. vascular endothelial growth factor to attract



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endothelial progenitor cells, up to surface topography modifications in order to mechanically influence the adherence and proliferation of endothelial cells5. The co-seeding approach investigated herein to endothelialize PTFE surfaces was shown competent to promote efficient cell attachment and also support normal cell growth (Figures 5a and 5c). In fact, the method markedly improved cell adherence and proliferation compared to bare PTFE substrates (Figures 5b and 5c), as well as PTFE substrates pre-coated with WT-silk (Figure S6). Co-seeding with FN-silk was shown able to mediate binding of hDMECs to the RGD-containing motif during silk assembly and then evenly distributing the cells over the surface area. In addition, the resulting coherent endothelial monolayer after seven days culture (Figure 5d) revealed essential cell-cell contacts. The endothelial cell barrier is crucial in controlling blood pressure, and initiating immune responses and angiogenesis45. Further, the forming endothelium immobilizes circulating endothelial progenitor cells or acts as attractant to nearby endothelial cells to populate and aid completing it5. Scanning electron microscopy images of cells co-seeded with FN-silk onto PTFE surfaces (Figure 6) confirmed that a uniform endothelial cell layer can be achieved, where the hDMECs are adjacent enough to establish cell-to-cell communication. HDMECs were shown to be firmly embedded within the silk, indicating that co-seeding can be effectively utilized to cover exposed PTFE areas, which otherwise may compromise the formation of a confluent endothelium. Cardiovascular grafts made of Dacron® (PET) have previously been pre-coated with other proteins, e.g. collagen or fibronectin, in order to increase cell attachment and improve overall graft patency46. The co-seeding approach examined herein was found capable of establishing cell


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attachment to PET surfaces allowing hDMECs to spread over the PET surface and form a substantial cell monolayer (Figures 4b and 4d). The formation of a complete and healthy endothelium remains a complex process with a lot of factors taking part. Imperfections in the endothelial cell lining may initiate platelet activation and thrombi formation, trigger excess SMCs growth, and consequently, compromise overall graft patency. A mechanically stable medial layer of SMCs has been proposed to prevent neointimal formation, while synthesis of ECM components and remodeling has been discussed as complementary to support endothelial cell proliferation47-48. Hence, future studies that will subject co-seeded cells to mechanical stress in order to evaluate the overall cell-silk integrity are required, as well as detection of the essential ECM compounds secreted by cells which guarantee a healthy endothelium.

Conclusion In vitro endothelialization of synthetic grafts as well as cell seeding of engineered vascular constructs still remains an unsolved issue. Herein, a novel approach has been studied that enables cells to bind to a fibronectin motif of recombinant spider silk during assembly into a coating, thereby speeding up the overall seeding process. Further, the technique has been successfully used to coat and seed endothelial cells to the most relevant materials used as bulk to produce the majority of commercially available cardiovascular grafts. The investigated co-seeding method has also been found to promote cell proliferation with customary growth profiles and result in a coherent endothelial monolayer. Thus, a reliable, efficient as well as less time-consuming cell



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seeding process has been established, which in the future may be considered for quick endothelialization of engineered vascular constructs or synthetic grafts.

ASSOCIATED CONTENT Supporting Information Human dermal microvascular endothelial cells, smooth muscle cells, and human mesenchymal stem cells seeded with or without WT- and FN-silk onto uncoated, or polystyrene, polyethylene terephthalate, and polytetrafluoroethylene substrates pre-coated with WT- and FN-silk and allowed to incubate for 30 minutes before cell adhesion analysis or monitoring growth for 7 days and viability at the last day in culture (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (M.H.). ORCID Christos Panagiotis Tasiopoulos: 0000-0003-1051-9909 Mona Widhe: 0000-0001-7153-8527 24 ACS Paragon Plus Environment

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My Hedhammar: 0000-0003-0140-419X

Notes The authors declare the following competing financial interest(s): M.H. has shares in Spiber Technologies AB, a company that aims to commercialize recombinant spider silk.

ACKNOWLEDGEMENT We thank Spiber Technologies AB for generously providing the silk proteins used in all experiments of this study and Linnea Gustafsson for her kind aid in performing SEM imaging. The research was funded by the Knut and Alice Wallenberg Foundation, The Swedish Research Council and Vinnova.

ABBREVIATIONS

RGD, arginine-glycine-aspartic acid ECM, extracellular matrix FN, fibronectin motif WT, wild type hDMECs, human dermal microvascular endothelial cells SMCs, smooth muscle cells hMSCs, human mesenchymal stem cells PBS, phosphate buffered saline PET, polyethylene terephthalate PTFE, polytetrafluoroethylene 25 ACS Paragon Plus Environment


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SEM, scanning electron microscopy HMDS, hexamethyldisilazane

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TABLE OF CONTENTS


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Methods for cell seeding with functionalized spider silk. Upper panel: Classical 2-step method to seed cells onto surfaces pre-coated with a spider silk protein functionalized with a motif from fibronectin. Lower panel: New 1-step method to co-seed cells with the functionalized spider silk protein onto non-coated surfaces.



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Recombinant spider silk functionalized with a motif from fibronectin

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