Rediscovering Chemical Gardens: Self-Assembling Cytocompatible

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Rediscovering Chemical Gardens: Self-assembling Cytocompatible Protein-Intercalated Silicate-Phosphate Sponge-Mimetic Tubules Kamia Punia, Michael Anthony Bucaro, Andrew Mancuso, Christina Cuttitta, Alexandra Marsillo, Alexey Bykov, William L’Amoreaux, and Krishnaswami S Raja Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01721 • Publication Date (Web): 22 Jul 2016 Downloaded from http://pubs.acs.org on July 24, 2016

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Rediscovering Chemical Gardens: Self-assembling Cytocompatible Protein-Intercalated SilicatePhosphate Sponge-Mimetic Tubules Kamia Punia a, b, d‡, Michael Bucaro c‡, Andrew Mancuso a, b, d, Christina Cuttitta c, Alexandra Marsillo, c, d, Alexey Bykov d, e, William L’Amoreauxb, c, d, Krishnaswami S Raja* a, b, d Department of Chemistry a, Institute for Macromolecular Assemblies b, Department of Biology c of the College of Staten Island, 2800 Victory Blvd., Staten Island, NY, 10314. Graduate Center City University of New York d, 365 Fifth Avenue, New York, New York 10016. The Department of Physics, City College of New York e, 160 Convent Avenue, New York, NY 10031. KEYWORDS Biomaterial, Chemical Garden, Scaffold, Biomimetic, Assemblies

ABSTRACT The classic “chemical garden” experiment is reconstructed to produce proteinintercalated silicate-phosphate tubules that resemble tubular sponges. The constructs were synthesized by seeding calcium chloride into a solution of sodium silicate-potassium phosphate and gelatin. Sponge-mimetic tubules were fabricated with varying percentages of gelatin (0-15% w/v), in diameters ranging from 200 microns to 2 mm, characterized morphologically and compositionally, functionalized with biomolecules for cell adhesion and evaluated for

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cytocompatibility. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy analysis (EDS) experiments showed that the external surface of the tubules were relatively more amorphous in texture and carbon/protein rich in comparison to the interior surface. Transmission Electron Microscopy (TEM) images indicate a network composed of gelatin incorporated into the inorganic scaffold. The presence of gelatin in the constructs was confirmed by infrared spectroscopy. Powder X-ray diffraction (XRD) was used to identify inorganic crystalline phases in the scaffolds that are mainly composed of Ca(OH)2, NaCl and Ca2SiO4 along with a band corresponding to amorphous gelatin. Bioconjugation and coating protocols were developed to program the scaffolds with cues for cell adhesion and the resulting constructs were employed for 3D cell culture of marine (Pyrocystis lunula) and mammalian (HeLa and H9C2) cell lines. The cytocompatiblity of the constructs was demonstrated by live cell assays. We have successfully shown that these biomimetic materials can indeed support life; they serve as scaffolds which facilitate the attachment and assembly of individual cells to form multicellular entities thereby revisiting the three hundred and fifty year old effort to link chemical gardens with origins of life. Hybrid chemical garden biomaterials are programmable and readily fabricated and could be employed in tissue engineering, biomolecular materials development, 3D mammalian cell culture and by researchers investigating the origins of multicellular life.

INTRODUCTION The chemical garden experiment is a classic example of a non-equilibrium process; it is initiated by seeding or injecting water soluble salts of many multivalent cations into a highly concentrated solution of sodium silicate (water glass) or phosphate. The seeded metal salt, for example,


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calcium chloride, starts dissolving in the aqueous medium, this triggers the formation of a semipermeable membrane of calcium silicate. The ionic concentration of Ca2+ is higher inside the membrane, the resulting osmotic pressure causes the top of the membrane to rupture. The membrane formation and rupture process repeats, resulting in the formation of tubular plant-like assemblies. This experiment was first reported by Johann Glauber in 1646, he referred to the constructs as “metallic vegetation”.1 Studies describing the preparation of chemical gardens including variations in the seeding salt or the growth solutions and their characterization via advanced microscopy, Energy Dispersive X-ray Spectroscopy (EDS) and X-ray diffraction have been reported in recent literature.2,3 The facts that their formation involves a semipermeable membrane and that the final constructs closely resemble life forms (plants or fungi) led some of the greatest scientific minds including Robert Boyle, Isaac Newton and Moritz Traube to believe that chemical gardens are the key to unlocking the origins of life.4,5 Early researchers such as Wilhelm Pfeffer and Moritz Traube created semipermeable membranes based on chemical garden chemistry using copper sulfate and potassium hexacyanoferrate (II). They believed that they had created the mimics of the first artificial cells (globular semipermeable membranes across which osmosis could occur); the role that the nucleus played in cells was not yet known by Science.5 After the discovery of DNA the idea that chemical gardens are connected to the origins of life was abandoned for several decades. There has however been recent renewed interest in chemical garden chemistry after giant chemical garden assemblies were discovered at hydrothermal vent systems in the interface with ocean waters.6 These mineral assemblies are composed of membranes that maintain gradients of various chemicals and ions; the chemical composition of the constructs include compounds that


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can serve as catalysts.7 Chemical gardens have also been used to generate electrical currents; electrons produced by chemical gardens found in nature are considered as one of the sources of energy for chemolithotropic bacteria found associated with these mineral assemblies.8 These constructs are currently considered as the catalytic reactors/engines which may have produced the molecular building blocks of life: it has been shown that minerals can catalyze the formation of the small biomolecules of life and their polymerization to produce DNA and proteins.9,10 Brinicles are another class of chemical gardens observed in the ocean.11 They are composed of frozen brine and have been found associated with lipids. Scientists speculate that these lipids may have self-assembled to form the ancestors of the first living cells (protocells). The research reported in literature thus far is therefore built on the hypothesis that chemical gardens may have served as the catalytic reactors that produced the molecular building blocks of life and possibly their self-assembly to produce the ancestors of the first unicellular entity.3 The current work was inspired by the observation that the scaffolds of tubular sponges (which live attached to the ocean floor) resemble natural chemical gardens found in the hydrothermal vent systems in construction. Sponges are considered as the first animal life form; their intrinsic structure is a tubular scaffold composed of an intercalating network of the protein spongin with calcium silicate/carbonate embedded at specific locations with cue/signal molecules that promote cell adhesion and other functions. The multicellular organism (sponge) consists of choanocytes (sponge cells) attached to this scaffold.12 The choanocytes of sponges closely resemble choanoflagellates, a sister-group, which can exist in both unicellular and colonial forms and thus is often used to investigate the transition from unicellularity to the multicellularity of the sponge.13, 14 It should be noted that the protein component is not present in the case of chemical gardens prepared to date.


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In this sponge bio-inspired publication we have redefined the chemical garden experiment by seeding a concentrated sodium silicate-potassium phosphate solution containing solubilized gelatin with calcium chloride to produce protein intercalated silicate-phosphate tubules (Figure 1B and C). The morphology and composition of the resulting biomaterials was determined by a battery of techniques including light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), Energy Dispersive X-ray (EDS) analysis, IR spectroscopy and powder X-ray diffraction (XRD). Tubular constructs consisting of a network of gelatin intercalated with inorganics were observed. SEM and EDS experiments suggest that external surface of the tubules is rough in texture and carbon/protein rich whereas the interior surface has well defined crystals. Powder X- ray diffraction was used to identify inorganic crystalline phases in the scaffolds, they are mainly composed of Ca(OH)2, NaCl and Ca2SiO4 along with a band corresponding to amorphous gelatin. The three hundred and fifty year old problem of linking chemical gardens with life is revisited in a new light with this generation of chemical garden biomaterials: the focus being on whether these constructs can serve as scaffolds which facilitate the attachment and assembly of individual cells to form multicellular entities; that these materials can support life is successfully established by culturing test mammalian and marine cell lines on these constructs. To perform cell culture experiments on the constructs bioconjugation and coating protocols were first developed to systematically program the surface chemistry of the scaffolds with cue molecules which promote cell adhesion. Test marine cell line Pyrocystis lunula and mammalian cell lines HeLa and HC92 were successfully cultured on the scaffolds. Cell viability on the constructs was confirmed by chloroplast autofluorescence and fluorescein diacetate live cell assays.


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These scaffolds could be employed by a widespread scientific community including researchers probing the origins of multicellularity13,14 or as frameworks to create lab models to understand the factors that are causing the disappearance of coral reefs such as the disruptors of coral/sponge-algal symbiosis.15 The preliminary 3D mammalian cell culture experiments support the potential of further developing these biomaterials for applications such as in vitro assessment of the bioactivity of drug candidates and tissue engineering.16,17 EXPERIMENTAL General Information: Sodium silicate was obtained from Ward’s science, gelatin was procured from Kraft Foods Inc.; dipotassium hydrogen phosphate, and calcium chloride pellets (anhydrous, 4-20 mesh) from Fisher Scientific. D(+)-biotin was obtained from Acros Organics, 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC.HCl) from Advanced Chemtech Inc., Phosphate buffer saline (PBS), pH 7.4 was prepared according to standard protocol,18 N-Hydroxysuccinimide from Chem Impex Int’l Inc., avidin from EMD Millipore Corp, amino dextran, Alexa Fluor 555 phalloidin and TO-PRO-3 from Molecular Probes, fluorescein diacetate (FDA) and chitosan from Alfa Aesar and dextran coated streptavidin beads from GE Healthcare. Nuclear magnetic resonance (NMR) spectra were recorded on a Varian NMR 600 (600 MHz) spectrometer. An Agilent Technologies 845x UV-Vis spectrophotometer equipped with ChemStation Rev. A.10.01 software was used to record the UV/Vis spectra. Transmission electron microscopy (TEM) images were recorded using a FEI Tecnai G2 Twin microscope. An Amray 1910 Field Emission Scanning Electron Microscope was employed. Xray microanalysis and element mapping was obtained on an Energy-dispersive x-ray spectroscopy (EDS) detector (ThermoNoran, Madison, WI) on the above SEM. Infrared spectra were recorded using a Thermo Scientific, Nicolet 6700 Fourier Transform spectrometer. H9C2


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cells were purchased from ATCC. A Zeiss Axio Observer Z1 with incubation stage and a Leica SP2 scanning confocal laser scanning microscope were used for brightfield and fluorescence imaging. Synthesis of Sponge-Mimetic Scaffolds: Sponge-mimetic scaffolds were prepared by solubilizing gelatin (to make up 0-15% (w/v) of final solution concentration) in 3 M sodium silicate and 0.5 M potassium phosphate (K2HPO4) at 60oC followed by seeding with solid CaCl2 pellets Dissolution of the calcium chloride in the aqueous medium triggers the formation of a semipermeable membrane of calcium silicate-phosphate with intercalated gelatin. Repeated rupturing of the top of the membrane, caused by increasing osmotic pressure from Ca2+ ions in the lumen, results in cyclic depositions of protein-intercalated silicate-phosphate that gradually grow to form tubules. The sponge-mimetic tubules were allowed to develop over a period of 24 hours at 60oC. The solution was removed and exchanged with water at room temperature and stored in 70% isopropanol at 4oC. 3D Marine Cell Culture and Imaging: The dinoflagellate cell line Pyrocystis lunula was cultured on the surface of 15% gelatin incorporated silicate-phosphate tubules modified with dextran. Pyrocystis lunula cells were cultured in glass Buchner flasks, with hose barbs plugged with cotton (to provide adequate gas exchange) in L1 algal growth medium. The algal cultures were maintained at 16°C in a 12h dark-light cycle by fluorescent bulbs of 5000 Kelvin color temperature and 2500 lux. Immediately prior to use, the scaffolds were disinfected by submerging them in 70% ethyl alcohol under UV light for 10 minutes, rinsed in distilled water and further rinsed with fresh cell culture media. Dinoflagellate cells were pelleted down at 2000 x g for five minutes, resuspended in the cell culture medium and added to the constructs in sterile 48-well plates at a density of 2 x 104 cells/ml. The cells were cultured with the tubules for 2


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hours, non-adherent cells were removed by rinsing, and the cell-seeded tubules were cultured for 72 h at 16°C under the conditions described above. Autofluorescence of chloroplasts in the NIR region was utilized to image viable dinoflagellates attached to the tubules by live-cell fluorescence microscopy and confocal laser scanning microscopy (Figure 6). 3D Mammalian Cell Culture on Tubules: Capability of the tubules to support 3D cell growth was demonstrated on Poly-L-Lysine (PLL) coated gelatin-incorporated (15% w/v gelatin solution) tubules using HeLa cells, chosen for their well-established substrate adherence and availability. HeLa cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium), Nutrient mixture F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 1% ITS (human insulin, human transferrin, sodium selenite), 0.1% penicillin at 37oC, 5% CO2. The pH of the constructs in water was adjusted to 7.4 using 1% ascorbic acid, the constructs were further washed with deionized water and then sterilized in 70% ethyl alcohol under UV light for 10 minutes. This was followed by washing the constructs with fresh cell culture media. The constructs were then treated with 10 µg/mL of PLL to promote cell adhesion and rinsed with sterile distilled H2O. Cells were seeded onto constructs in complete medium in sterile 24-well polystyrene petri dishes at a density of 106 cells/cm2; after 2 hours the constructs were rinsed to remove non-adherent cells and cultured for 72 h at 37oC, 5% CO2. The HeLa cells were labeled with the cytoplasmic live cell indicator FDA and imaged on a Zeiss Axio Observer microscope and via confocal laser scanning microscopy (Figure 7b,c). The cells on the constructs were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and Alexa Fluor 555 Phalloidin and TO-PRO-3 were used to stain actin and DNA, respectively. Images were acquired on a Leica SP2 scanning confocal laser scanning microscope and


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produced on Imaris software (Bitplane). The actin component of the cells fluoresces red and is clearly visible and the cell nuclei are stained blue (Figure 7d). RESULTS AND DISCUSSION Synthesis and characterization of Chemical Garden Biomaterials: A series of spongemimetic scaffolds were prepared by solubilizing varying concentration of gelatin (0,1,3,5, 10 and 15% (w/v) of gelatin) in 3 M sodium silicate and 0.5 M potassium phosphate (K2HPO4) at 60oC followed by seeding with solid CaCl2 pellets. The calcium chloride dissolves in the aqueous medium, this produces a semipermeable membrane of calcium silicate/phosphate with intercalated gelatin, since the ionic concentration of Ca2+ is higher inside the membrane, osmotic pressure develops causing the top of the membrane to rupture, the membrane formation and rupture process repeats resulting in the formation of protein intercalated silicate-phosphate tubules. The sponge-mimetic tubules were maintained at 60oC for a period of 24 hours (we observed that constructs which were synthesized for a shorter period of time were less robust and tend to disintegrate during handling). We observed that a temperature of 60°C is required to prepare homogeneous solutions of gelatin in 3 M sodium silicate and 0.5 M potassium phosphate (K2HPO4). We have also tried growing the constructs at temperatures higher than 60°C, but this resulted in hair like structures which were not optimal for physical handling because of their fragile nature. At lower temperatures (room temperature) we did not observe any construct formation. The solution was removed and exchanged with water at room temperature and stored in 70% isopropanol at 4oC. The final constructs are robust and maintain integrity during repeated handling and over an extended period (Figure 1 B and C); Gelatin incorporated tubules of diameters ranging from approximately 200 microns (Figure 1C) to 2 mm were prepared and


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isolated. The constructs were prepared using a solution containing both silicate and phosphate ions because these are key components in hydrothermal vent systems.19

Figure 1. A) Tubular Sponge. B) Chemical garden based sponge-mimetic: protein intercalated tubular scaffolds constructed by seeding CaCl2 in a solution composed of 3M Sodium silicate, 0.5M K2HPO4 and 15% solubilized gelatin at 60oC. C) Reflected light image of gelatinintercalated silicate-phosphate scaffold tubules, Scale bar 200µm. The gelatin incorporated constructs and non-gelatin constructs were imaged using TEM; from the TEM images (Figure 2A), it is evident that the gelatin containing sample has a network possibly arising from gelatin incorporated into the inorganic scaffold. Furthermore, we observe fibers protruding out of the surface of the scaffold only in the gelatin samples. In contrast, the control sample (Figure 2B) has sharp edges and no network of any kind was observed.


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Figure 2. (A): Transmission Electron Microscopy (TEM) images of sponge-mimetic scaffolds (CaCl2 seeded in 3M sodium silicate and 0.5M K2HPO4) with 3% gelatin and (B) Control chemical gardens (CaCl2 seeded in 3M sodium silicate and 0.5M K2HPO4) without gelatin. TEM scale bars 100nm. The samples were also characterized by SEM. The 15% gelatin incorporated samples are tubular; the tubule walls are rich in texture (Figure 3A and B). The exterior of the tubule has a rough surface and is carbon / protein rich and the interior has some well-defined crystals which can be observed in the higher magnification SEM (Figure 3B). Previous studies of chemical garden materials (without gelatin) via EDS analysis have shown that the interior of the tubules are mainly composed of NaCl crystals.19,20 Energy Dispersive X-ray Spectroscopy (EDS) of the exterior of 15% gelatin incorporated tubule (Figure 3C) indicates the presence of carbon, oxygen, sodium, phosphorous, chlorine, potassium and calcium.


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Figure 3. (A) & (B) Scanning Electron Microscopy (SEM) images of tubule grown with 15% gelatin, 3M sodium silicate, 0.5M K2HPO4 seeded with CaCl2, representative EDS spectrums of (C) the outer surface of the tubule and (D) the inner surface of the tubule Scale bars (A) 1 mm, (B) 100 µm. The EDS of the inside surface of the tubule has carbon, oxygen, calcium, chlorine and traces of sodium (Figure 3D). The average element atom percent for the inner surface is 24% carbon (s.d.= 2.3%) and 64% oxygen (s.d.= 3.9%), versus 42% carbon (s.d.= 4.6%) and 43% of oxygen (s.d.=3.7%) for the outer surface. The carbon content in the outer surface is higher than the inner surface. In contrast to the gelatin incorporated sample, the control 0% gelatin sample has only trace amounts of carbon (Supplementary Information Figure S1); this indicates that gelatin has indeed been incorporated in the structural framework of the gelatin-CaCl2-seeded chemical gardens. The SEM data for the 15% gelatin incorporated sample is presented because this sample was employed for the surface modification and cell culture experiments carried out in this study. The incorporation of gelatin in the constructs was confirmed by infrared spectroscopy: the 15%


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gelatin incorporated construct has amide I peak (C = O stretch) at 1650 cm-1, amide II peak (N-H bend and C-N stretch) at 1600 cm-1, and amide A peak (N-H stretch) at 3300 cm-1 which overlaps with the OH peak. (Supplementary Information Figure S3).21 These peaks are absent in the 0% gelatin incorporated control sample (Supplementary Information Figure S2). A peak at 1050cm-1 arising from Si-O bond stretching originating from the silicate ions in the sample is also observed. A Panalytical XPert PRO powder X-ray diffractometer was used to identify the inorganic crystalline phases in the chemical garden materials generated by seeding solutions of 3M sodium silicate, 0.5M dipotassium phosphate containing 0%, 10% and 15% solubilized gelatin with CaCl2 (Figure 4). Ca(OH)2 was identified in all the samples with peaks at 18.225, 28.844, 34.237, 47.216, 54.467, 59.550, 62.703, 64.376, 71.859, 81.922, 84.733, and 86.122 (2θ values). Peaks at 23.556, 29.598, 32.748, 40.378, 43.278, 50.850, and 56.225 (2θ values) which belong to Ca2SiO4 were also observed in all the samples along with peaks from NaCl at 31.874, 45.552, and 75.348 (2θ values). An amorphous band of gelatin at 21.294(2θ value) was observed in the control gelatin sample and in the 10% to 15% gelatin, as anticipated, the gelatin band is more intense in the case of the 15% gelatin sample in comparison to the 10% gelatin sample . The gelatin band is absent in the control sample without gelatin.


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Figure 4. Powder X-ray diffraction (XRD) analyses of control gelatin sample and chemical garden materials generated by seeding solutions of 3M sodium silicate, 0.5M dipotassium phosphate containing 0%, 10% and 15% solubilized gelatin with CaCl2. XRD analysis confirms the presence of CaSiO4, Ca(OH)2 and NaCl in all the samples. Amorphous gelatin is present in the 10 and 15% gelatin incorporated samples; the intensity of the gelatin band is higher in the latter sample. Programming Surface Chemistry: Scaffolds of natural sponges and the extracellular matrix of most organisms have cue/signal molecules attached to them which promote cell attachment and other functions. A modular technology to program the surface chemistry of the scaffolds was developed. The 15% gelatin based construct (which was found to be most appropriate for cell culture) was employed for chemical modification. The lysine residues on the gelatin component of the scaffolds were biotinylated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)


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and N-hydroxysuccinimide to produce the biotinylated construct (Scheme 1b). The streptavidin/avidin-biotin complex is the strongest known non-covalent interaction (KD = 1015

M).22 Streptavidin/avidin is a protein which has four binding sites for biotin. Incubating the

biotinylated construct (Scheme 1b) with avidin would result in a construct coated with avidin which possesses a high surface density of free biotin binding sites (Scheme 1c). The resulting avidin coated constructs could then be further treated with various biotinylated cue molecules to make constructs with tailored surface properties (Scheme 1d).

Scheme 1. Modular technology for programming the sponge-mimetic scaffolds with cue molecules: a) Schematic representation of a gelatin and protein intercalated silicate-phosphate tubule. b) Biotinylated sponge-mimetic scaffold tubule. c) Avidin modified Tubule b d) Biotinylated Cue/Signal molecule decorated construct for further cell attachment studies.

Biotinylation of the gelatin intercalated scaffolds was confirmed by employing commercially available streptavidin coated dextran beads. Streptavidin coated beads were incubated with


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control constructs without biotin (Figure 5a) and with biotinylated constructs (Figure 5b) followed by washing and then imaged via SEM. The control sample without biotin has no beads attached to it (Figure 5c). It is evident from Figure 5d, that a high density of the beads are attached to the biotinylated constructs via the biotin-streptavidin interaction.

Figure 5. (a) Schematic representation of control silicate-phosphate intercalated sponge-mimetic scaffold tubule. (b) Schematic representation of Biotinylated gelatin and silicate-phosphate intercalated sponge-mimetic scaffold tubule complexed with streptavidin coated dextran beads. (c) SEM of control sample (which has not been biotinylated): the streptavidin coated beads do not attach to the surface and are removed with repeated washing of the constructs. (d) SEM of Biotinylated scaffold displaying streptavidin coated dextran beads: the beads are strongly complexed to the surface and remain attached even after thorough washing of the surface. Scale bars c and d 10µm.


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Cue Molecule Attachment and 3D Marine Cell Culture: The feasibility of using the scaffolds for 3D marine cell culture was evaluated using the crescent shaped unicellular dinoflagellate, Pyrocystis lunula. The advantages of employing this dinoflagellate strain are: (a) lack of motility (b) ease of testing cell viability (live cells fluoresce in the far red region). Pyrocystis lunula has a polysaccharide cell wall.23 To facilitate adhesion of the cells, the surface chemistry of the gelatin and silicate-phosphate intercalated sponge-mimetic scaffold tubules was programmed in multiple steps to display the polysaccharide cue molecule dextran (Scheme 1). The 15% gelatin constructs (Scheme 1a) was first biotinylated (Scheme 1b) and then incubated with avidin to produce an avidin coated construct with vacant biotin binding sites (Scheme 1c). This was followed by incubation with biotinylated dextran (see supplementary information section 7 and Figure S4, for detailed synthesis of biotinylated dextran) to produce the final construct displaying polysaccharide cues (Scheme 1d). Modification of the scaffolds with dextran was confirmed via the standard UV spectroscopy based phenol-sulfuric acid assay; color development at 480nm was observed in the dextran modified sample, no color development was observed in the control samples without dextran (see Supplementary Information Figure S5). Pyrocystis lunula was cultured on the surface of dextran modified 15% gelatin calcium chloride seeded scaffolds (Figure 6). Cell culture on the constructs was performed in accordance with protocol described in the experimental section. The dinoflagellate cells adhered very efficiently to dextran cue modified constructs; cell viability and attachment was confirmed via confocal laser scanning microscopy. It is evident from Figure 6b that the chloroplasts of the live dinoflagellates attached to the scaffolds are fluorescent. The dinoflagellate attachment and viability was also confirmed by live cell fluorescence microscopy; live cells embedded on the constructs fluoresce in the farred region (Figure 6c). The cells on the scaffolds are fluorescent over several days confirming


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the long term viability of cells on the scaffolds. The dinoflagellates did not adhere to control 15% gelatin incorporated samples which were not modified with the polysaccharide cues. These experiments clearly demonstrate the feasibility of using the non-cytotoxic chemical garden biomaterials (programmed with adhesion promoting cue molecules) for 3D marine cell culture.

Figure 6. (a) Schematic representation of Marine Dinoflagellate strain Pyrocystis lunula cultured on the surface of 15% gelatin scaffolds modified prior to cell culture by biotinylation of the gelatin component followed by incubation with avidin and further with biotinylated dextran cue molecules and then, imaged via (b) confocal laser scanning microscopy, the live dinoflagellate chloroplasts fluoresce red. (c) Live cell imaging, Zeiss cell imager; filter sets for Cy-5(red) were employed to image the live dinoflagellate chloroplasts. Scale bars (b) 200 µm and (c).


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3D Mammalian Cell Culture Experiments: The feasibility of performing 3D mammalian cell culture on the surface of Poly-lysine cue coated 15% gelatin incorporated scaffolds was evaluated using HeLa cells. Cell culture was carried out via the protocol described in the experimental methods section. HeLa cells on the constructs were labeled with cytoplasmic cell viability indicator FDA and imaged by confocal laser scanning microscopy (Figure 7b) and using a Zeiss live cell imager (Figure 7c); from the figures it is evident that the constructs are completely covered with viable cells which fluoresce green. The cells which completely cover the scaffolds are fluorescent over several days confirming the long term viability of cells on the scaffolds. The actin component of the cells on the scaffolds was stained with Alexa Fluor 555 Phalloidin and the DNA was stained with TO-PRO-3 and imaged using a confocal laser scanning microscope (Figure 7d). The cells did not show significant attachment to control 15% gelatin samples without the PLL cue. Preliminary experiments to optimize the attachment and culture of the cardiomyocytes cell line H9C2 on the constructs have also been performed; PLL and chitosan cues were found to be suitable for this cell line. Chitosan coating on 15% gelatin incorporated scaffolds was carried out in accordance with the procedure outlined in the supplementary information section 9. The coating of chitosan on the scaffolds was confirmed by UV spectroscopy based the phenol-sulfuric acid assay: color development at 480nm was observed. Cell culture was carried out in accordance with protocol described in supplementary information section 10. The H9C2 cells on the constructs were labeled with cytoplasmic live cell indicator FDA and viewed using a Zeiss live cell imager (supplementary information Figure S6.); from the figures it is evident that the construct is covered with viable cells which fluoresce green. The cell culture experiments clearly demonstrate the adherence and viability of mammalian cells on the scaffolds and their potential use for 3D mammalian cell culture.


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Figure 7. (a) Schematic of 3D cell culture of HeLa cells on calcium chloride seeded 15% gelatin-silicate-phosphate tubular scaffold coated with cue molecule Poly-L-Lysine (PLL). (b) Extended-focus image created from confocal z-stack of FDA stained HeLa cells coating a tubule. c) Extended focus image from widefield microscope of FDA fluorescence in HeLa cells on a tubule (d) Extended focus image from confocal z-stack of HeLa cells on the tubule stained for actin (Alexa Fluor 555 Phalloidin) (red) and DNA (TO-PRO-3) (blue). Scale bars (b) 200 µm, (c) 100µm (d) 50 µm. The classic chemical garden experiment has been transformed into a powerful modular technology for producing programmable protein intercalated silicate-phosphate tubules. In the current study, light microscopy (Figure 1), TEM (Figure 2) and SEM experiments along with EDS (Figure 3) demonstrate that the chemical garden biomaterials incorporating gelatin are tubular in architecture, the external surface of the tubules is protein rich and the interior surface is less carbon rich and has some well-defined crystals; the gelatin network in the construct is clearly visible in the TEM image. The presence of gelatin is further confirmed by IR


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spectroscopy (Supplementary Information Figure S3). XRD analysis indicates the presence of Ca(OH)2, NaCl, and CaSiO4 as main crystalline components of the scaffolds, along with a broad band of amorphous gelatin. (Figure 4). Among the various available proteins gelatin was deliberately employed in the construction of the scaffolds because it is soluble at elevated temperatures but forms a stable gel at room temperature thereby stabilizing and providing structural integrity to the final scaffolds. Scaffolds incorporating other proteins which do not form a gel at room temperature such as Bovine Serum Albumin (BSA) were also prepared but not employed for further studies because they were fragile in comparison to the gelatin based constructs and tended to disintegrate while handling. It is possible to use several cations other than Ca2+ to produce the constructs. Calcium was chosen because it has consistently been associated with scaffolds of sponges and several other life forms throughout the evolutionary biosphere. A series of chemical garden materials with increasing concentration of gelatin were prepared. The highest concentration of gelatin that dissolves in sodium silicate- dipotassium phosphate that we could achieve was 15 % at 600C. The resulting construct was the most cytocompatible among the samples prepared and was employed for the surface modification and cell culture experiments. The growth of the chemical gardens without the protein component has been studied in literature, the growth is buoyancy driven and bubble assisted, by varying parameters such as external pressure, concentration, reaction time one can potentially vary tubule diameter and by manipulating the bubble movement one could generate complex architectures.3 Preliminary experiments indicate that synthesis of constructs under partial vacuum produces straight tubules of narrow diameter. The growth and production of these constructs subjected to an external rotatory force results in helical architectures. Experiments to utilize these parameters to


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synthesize silicate-phosphate tubules incorporating protein of programmable diameter, length, wall thickness, and organization are currently in progress and will be reported in future. Programming the surface chemistry of the gelatin incorporated constructs via biotinylation followed by coating with avidin (as outlined in Scheme 1) results in a construct with several biotin binding sites. This opens up exciting avenues: a wide range of biotinylated signal/cue molecules which modulate properties such as cell adhesion, proliferation etc. and biotinylated nanoparticles (for a range of applications) can be easily synthesized/are commercially available.24 Biotinylated cue/signal molecules and nano/microparticles can be displayed on the surface of the scaffolds by incubating them with avidin coated scaffolds (Scheme 1d). This technology is modular and affords a general route to program the surface properties of the scaffolds for a range of possible applications. The mechanism by which multicellular animal life evolved is an unanswered question that continues to receive significant attention. The emergence of the first multicellular animal forms such as sponges occurred ~600 million years ago (the earliest fossil of a sponge discovered is 600 million years old).25 This corresponds to the end of the Precambrian ice age (melting of snowball earth). The global melting of ice was triggered by pronounced increase of volcanic activity globally which lead to the release of green-house gases and widespread flooding of the oceans with nutrient rich water. This resulted in a massive cyanobacterial (oxygen producing) bloom in the oceans and the associated global oxygenation event which lead to emergence of the unicellular organisms which could produce collagen and subsequently multicellular life.26 The cell walls of cyanobacteria are composed of peptidoglycans.27 It is likely that the increased volcanic activity (that triggered the melting of snowball earth) also produced a large number of


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natural chemical gardens because hydrothermal vents systems are associated with volcanic activity under the ocean floor. Abundant cyanobacteria and other unicellular organisms and their dead debris present in the ocean during this period in evolutionary history could have served as a rich source of cell adhesion aggregation molecules (peptidoglycans / proteins/other molecules). There has also been a recent exciting finding which shows that the bacterial strain Algoriphagus machipongonensis releases sulfanolipids which promotes the choanoflagellate strain Salpingoeca rosetta to form aggregates.28 The protein, polysaccharides/peptidoglycans/sulfanolipids ( originating from unicellular organisms) in the oceans could have attached to the surface and may have incorporated into the fabric of the natural chemical gardens formed at the volcanic hydrothermal vents at this point in evolutionary history. The resulting chemical gardens modified by the cell adhesion molecules, could then serve as the scaffolds and primordial hatcheries for choanoflagellates to attach, feed and cooperatively evolve to form the ancestors of the first sponges. In this work we are not attempting to recreate the chemistry that unicellular organisms used to produce collagen/peptidoglycans/other cell adhesion molecules or regarding how the first unicellular organism was formed. The current work focuses on a biomimetic approach to produce a chemical garden biomaterial model system and to develop well-defined modular chemistries which afford a systematic method for programming the surface of the assemblies with various cell adhesion molecules. Choanocytes (cells that line the cavity of natural sponges) feed by beating their flagella thus actively pumping water laden with nutrients into the central cavity of the sponge, the nutrients are absorbed and water passes out through the porous sponge body. The surface programmed gelatin incorporated porous scaffolds which we have developed are conveniently designed to


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mimic the structural organization present in a natural sponge scaffold. The polysaccharide (Dextran) cue modified scaffolds which we synthesized promotes the strong adhesion and viability of the test marine cell line Pyrocystis lunula. The 3D marine cell culture experiments presented here lay the groundwork and supports the feasibility of culturing choanoflagellates strains such as Salpingoeca rosetta or Stephanoeca diplocostata

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optimized scaffolds in future. The resulting supracellular entities would simulate the cellular organization in natural sponges. These studies can have a transformative impact on understanding the evolution of animal multicellularity with specific emphasis on the role that scaffolds with cues (cell adhesion molecules associated) may have played in promoting unicellular life forms to assemble and cooperatively evolve into a collective self. There has been an exponential growth of research interest in area of 3D mammalian cell culture. This is related to several advantages associated with 3D cell culture in comparison to 2D methods: (a) in vitro assessment of the bioactivity of drug candidates using 3D systems reflect in vivo systems more accurately (b) Cells in a 3D environment exhibit parameters such as gene expression and adhesion which reflect in vivo conditions very closely (c) 3D cell culture can be exploited for tissue engineering applications.16,

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here can be potentially used for 3D mammalian cell culture applications viz., they are easy to fabricate using green methods in aqueous media; the surface chemistry of the scaffolds can be varied with exquisite control via coating/bioconjugation to introduce cue/signal molecules which promote cell adhesion or other properties. Furthermore, Powder X-ray diffraction was used to identify inorganic crystalline phases in the scaffolds as mainly composed of Ca(OH)2, NaCl and Ca2SiO4 along with a band corresponding to amorphous gelatin; these materials are biocompatible or resorbable. The scaffolds are robust and can be easily manipulated. The


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preliminary feasibility of using the sponge mimetic scaffolds for mammalian cell culture was established using HeLa cells: cell attachment and viability on the tubular scaffolds was demonstrated via live cell imaging and confocal laser scanning microscopy (Figure 7). The attachment and viability of cardiomyocyte cell line H9C2 on the scaffolds was also established (Supplementary Information Figure S6). The construction of narrow diameter vascular grafts is very challenging.30 The technology developed here affords a convenient, inexpensive, green and innovative route to narrow diameter protein tubules and programmable hybrid materials that can potentially serve as scaffolds for vascular grafts. Protein intercalated calcium phosphate stigmergy scaffolds displaying cue/signal molecules that assist in the adherence and proliferation of osteoblasts could serve as templates for producing bone implants. The detailed characterization of the mechanical properties of these scaffolds and the further optimization of these constructs for 3D mammalian cell culture applications are beyond the scope of the current work and will be reported in future CONCLUSIONS Sponge mimetic scaffolds were prepared by solubilizing gelatin in 3 M sodium silicate and 0.5 M potassium phosphate (K2HPO4) at 60o C followed by seeding with solid CaCl2 pellets to produce novel chemical garden biomaterials. Light microscopy (Figure1), TEM (Figure 2) and SEM experiments along with EDS (Figure 3) indicate that the chemical garden biomaterials incorporating gelatin are tubular in architecture, the external surface of the tubules is carbon and protein rich, the interior surface has some well-defined crystals and is more inorganic in nature. The presence of gelatin is confirmed by IR spectroscopy (Supplementary Information Figure S3). Powder X-ray diffraction was used to identify inorganic crystalline phases in the scaffolds as mainly composed of Ca(OH)2, NaCl and Ca2SiO4 along with a band corresponding to


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amorphous gelatin (Figure 4). We have demonstrated the ability to modulate the surface chemistry of the tubular scaffolds with exquisite control via bioconjugation or coating to decorate the scaffolds with various cue molecules. Furthermore we have expanded the scope of this technology by synthesizing nano/micro particle decorated constructs by surface attachment (streptavidin coated dextran beads were attached to biotinylated scaffolds). The three hundred and fifty year old problem of linking chemical gardens with life has been revisited with a new generation of chemical gardens biomaterials: we have successfully shown that protein incorporated chemical garden materials can indeed support life by serving as scaffolds which facilitate the attachment and assembly of individual cells to form multicellular entities: Scaffolds with tailor-made surface properties were employed for the successful 3D cell culture of test marine ( Pyrocystis lanula and mammalian (HeLa and H9C2) cell lines. The attachment and viability of cells on the constructs was established by live cell-imaging and confocal laser scanning microscopy. 3D cell culture on surface optimized scaffolds with several other marine cell lines such as choanoflagellates and other mammalian cell lines such as osteoblasts will be explored in future; this technology will be also employed in future for biomolecular materials development. ASSOCIATED CONTENT Supporting Information. Supporting information outlining the detailed procedure to prepare the constructs and their characterization is included. The details of cell culture and imaging are also incorporated. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION


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Corresponding Author: Krishnaswami Raja [email protected], Department of Chemistry, Institute for Macromolecular Assemblies, College of Staten Island, 2800 Victory Blvd., Staten Island, NY, 10314 and Graduate Center , City University of New York, 365 Fifth Avenue, New York, New York 10016. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources We would like to acknowledge the National Science Foundation for two grants: “Acquisition of an X-ray microanalysis system with WDS spectrometer for elemental analyses” (DMR-0215745) and “Acquisition of a Confocal Microscope for Interdisciplinary Research” (DBI 0421046); these instruments were used extensively to generate the results presented. We would also like to acknowledge a Provost Research Grant. Ms. Kamia Punia would like to acknowledge support from the Rosemary O’Halloran Scholarship. Notes This paper is dedicated to Dr. Fred Naider. ACKNOWLEDGMENT We would like to thank Dr. Arenas-Mena, Cesar for access to a constant temperature room with photo-period control and Dr. Jimmie Fata for HeLa cells. We would also like to thank Dr. Alfred Levine and Dr. Michal Kruk for their valuable suggestions. Natural Sponge creative commons

image

courtesy

of

Patrick

Briggs

on

Flickr


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https://www.flickr.com/photos/87241965@N00/371542695/in/album-72157594503734186/ https://creativecommons.org/licenses/by/2.0/ REFERENCES (1) Glauber, J.R.; Philosophici, F. N.; and Jansson, J. Amsterdam 1646. (2) Cartwright, J. H. E.; Escribano, B.; and Sainz-Díaz, C. I. Chemical-Garden Formation, Morphology, and Composition. I. Effect of the Nature of the Cations. Langmuir 2011, 27, 32863293. (3) Barge, L.M.; Cardoso, S.S.S.; Cartwright, J.H.E.; Cooper, G.J.T.; Cronin, L. De Wit, A.; Doloboff, I.J.; Escribano, B.; Goldstein, R.E.; Haudin, F.; Jones, D.E.H.; Mackay, A.L.; Maselko, J.; Pagano,J.J.; Pantaleone, J.; Russell, M.J.; Sainz-Díaz, C.I.; Steinbock, O.; Stone, D.A.; Tanimoto, Y.; and Thomas, N.L. From Chemical Gardens to Chemobrionics. Chem. Rev. 2015, 115, 8652−870. (4) Newton, I. Of natures obvious laws & processes in vegetation; c. 1675. (5) Traube, M. Experimente zur Theorie der Zellenbildung und Endosmose. Arch. Anat. Physiol. 1867, 87−165. (6) Kelley, D.S.; Karson, J.A.; Früh-Green, G.L.; Yoerger, D.R.; Shank, T.M.; Butterfield, D.A.; Hayes, J.M.; Schrenk, M.O.; Olson, E.J.; Proskurowski, G.; Jakuba, M.; Bradley, A.; Larson, B.; Ludwig, K.; Glickson, D.; Buckman, K.; Bradley, A.S.; Brazelton, W.J.; Roe, K.; Elend, M.J.; Delacour, A.; Bernasconi, S.M.; Lilley, M.D.; Baross, J.A.; Summons, R.E.; and Sylva, S.P. Serpentinite-Hosted Ecosystem: The Lost City Hydrothermal Field. Science 2005, 307, 1428– 1434. (7) Russell, M. J.; Hall, A. J.; and Turner, D. In Vitro Growth Of Iron Sulphide Chimneys: Possible Culture Chambers For Origin-Of-Life Experiments. Terra Nova 1989, 1, 238–241. (8) Barge, L. M.; Abedian, Y.; Russell, M. J.; Doloboff, I. J.; Cartwright, J. H. E.; Kidd, R. D.; and Kanik, I. From Chemical Gardens to Fuel Cells: Generation of Electrical Potential and Current Across Self-Assembling Iron Mineral Membranes. Angew. Chem. Int. Ed. 2015, 54: 8184–8187. (9) Hazen, R. M.; and Sverjensky, D. A. Mineral Surfaces, Geochemical Complexities, and the Origins of Life. Cold Spring Harb Perspect Biol. 2010, v.2(5). (10) Lambert, J.F. Adsorption and Polymerization of Amino Acids on Mineral Surfaces: A Review. Orig Life Evol Biosph. 2008, 38, 211–242. (11) Cartwright, J. H. E.; Escribano, B.; González, D. L.; Sainz-Díaz, C. I.; Tuval, I. Brinicles as a Case of Inverse Chemical Gardens. Langmuir 2013, 29, 7655-7660.


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(12) Gazave, E.; Lapébie, P.; Renard, E.; Vacelet, J.; Rocher, C.; Ereskovsky, A. V.; Lavrov, D.V.; and Borchiellini, C. Molecular Phylogeny Restores The Supra-Generic Subdivision Of Homoscleromorph Sponges (Porifera, Homoscleromorpha). PLoS One 2010, 5(12): e14290. (13) Fairclough, S.R.; Dayel, M. J.; and King, N. Multicellular development in a choanoflagellate. Curr Biol. 2010, 20, R875-R876. (14) Dayel, M.J.; Alegado, R.A.; Fairclough, S.R.; Levin, T.C.; Nichols, S.A.; McDonald, K.; and King, N. Cell differentiation and morphogenesis in the colony forming choanoflagellate Salpingoeca rosetta. Dev Biol. 2011, 357(1): 73–82. (15) Parkinson J.E.; and Baums I.B. The extended phenotypes of marine symbioses: ecological and evolutionary consequences of intraspecific genetic diversity in coral-algal associations. Front Microbiol. 2014, 5:445. (16) Dhaliwal, A. 3D cell culture: A review. Mater. Methods 2012, 2:162. (17) Roth A.; and Singer, T. The application of 3D cell models to support drug safety assessment: opportunities & challenges. Adv Drug Deliv Rev. 2014, 69-70, 179-89. (18) Sambrook, J.; Fritsch, E.F.; and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second edition (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press) 1989, 3. (19) Barge, L. M.; Doloboff, I.J.; White, L.M.; Stucky, G.D.; Russell, M.J.; and Kanik I. Characterization of Iron-Phosphate-Silicate Chemical Garden Structures. Langmuir 2012, 28, 3714–3721. (20) Makki, R., Soszol, L., Pagano J. J.; and Steinbock, O. Tubular Precipitation Structures: Materials Synthesis Under Non-Equilibrium Conditions. Phil. Trans. R. Soc. A 2012, 370, 2848– 2865. (21) Pretsch, E.; Bühlmann, P.; Badertscher, M. Structure determination of organic compounds. Springer: Berlin, 2009. (22) Shi, W.; Dolai, S.; Averick, S.; Fernando, S. S.; Saltos, J. A.; L’Amoreaux, W.; Banerjee, P.; and Raja, K. A General Methodology Toward Drug/Dye Incorporated Living Copolymer−Protein Hybrids: (NIRF Dye-Glucose) Copolymer−Avidin/BSA Conjugates as Prototypes. Bioconjugate Chem. 2009, 20, 1595-1601. (23) Swift, E.; and Remsen, C. C. The cell wall of pyrocystis Spp. (Dinococcales). J. Phycol. 1970, 6, 79-86. (24) Narain, R.; Gonzales, M.; Hoffman, A. S.; Stayton, P. S.; and Krishnan, K. M. Synthesis of Monodisperse Biotinylated p(NIPAAm)-Coated Iron Oxide Magnetic Nanoparticles and their Bioconjugation to Streptavidin. Langmuir 2007, 23, 6299−6304. (25) Yin, Z.; Zhu, M.; Davidson, E.H.; Bottjer, D. J.; Zhao, F.; and Tafforeau, P. Sponge grade body fossil with cellular resolution dating 60 Myr before the Cambrian. Proc. Natl. Acad. Sci. USA 2015, 112, E1453-E1460.


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(26) Canfield, D.E; Poulton, S.W.; and Narbonne, G.M. Late-Neoproterozoic Deep-Ocean Oxygenation and the Rise of Animal Life. Science 2007, 315, 92-95. (27) Hoiczyk, E.; and Hansel, A.Cyanobacterial Cell Walls: News from an Unusual Prokaryotic Envelope. J. Bacteriol., 2000, 182, 1191–1199 (28) Dayel, M.J.; Alegado, R.A.; Fairclough, S.R.; Levin, T.C.; Nichols, S.A.; McDonald, K.; and King, N. Cell differentiation and morphogenesis in the colony-forming choanoflagellate Salpingoeca rosetta. Dev. Bio. 2011, 357, 73-82. (29) Leadbeater, B.S.C.; Yu, Q.B.; Kent, J.; and Stekel D.J. Three-dimensional images of choanoflagellate loricae. Proc R Soc Lond [Biol] 2009, 276, 3-11. (30) Smith, M.J.; McClure, M.J.; Sell, S.A.; Barnes, C.P.; Walpoth, B.H.; Simpson, D.G.; and Bowlin, G.L. Suture-reinforced electrospun polydioxanone–elastin small-diameter tubes for use in vascular tissue engineering: A feasibility study. Acta Biomater. 2008, 4, 58-66.


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Rediscovering Chemical Gardens: Self-Assembling Cytocompatible

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