Glycerol-Mediated Nanostructure Modification Leading to Improved

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Glycerol-mediated Nanostructure Modification Leading to Improved Transparency of Porous Polymeric Scaffolds for High Performance 3D Cell Imaging Shan Zhao, Zhiyuan Shen, Jingyu Wang, Xiaokang Li, Yang Zeng, Bingjie Wang, Yonghong He, and Yanan Du Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm500388m • Publication Date (Web): 02 Jun 2014 Downloaded from http://pubs.acs.org on June 9, 2014

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Glycerol-mediated Nanostructure Modification Leading to Improved Transparency of Porous Polymeric Scaffolds for High Performance 3D Cell Imaging Shan Zhaoa, Zhiyuan Shenb,c, Jingyu Wanga, Xiaokang Lia, Yang Zenga, Bingjie Wanga, Yonghong Heb and Yanan Dua* a

Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084,

China b

Laboratory of Optical Imaging and Sensing, Graduate School at Shenzhen, Tsinghua University,

Shenzhen 518055, China c

Department of Physics, Tsinghua University, Beijing 100084, China

KEYWORDS: Transparent porous scaffolds Nanostructure modification 3D cell imaging Refractive index (RI) matching ABSTRACT Glycerol is among the most commonly-used optical clearing agents for tissues clearance largely due to refractive index (RI) matching between glycerol and the submerged tissues. Here we applied glycerol as structure modifier at both macroscopic (as porogen) and nanoscopic (as nanostructure ameliorant) scales to fabricate transparent porous scaffolds made from poly (ethylene glycol) (PEG) as well as other widely-used biomaterials (e.g. PLGA, PA or gelatin), whose

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nanostructures, in the scale of light wave-length, dominantly improved the optical transmittance of the scaffolds even immersing in RI mismatched medium (e.g. culture medium or water). We further exploited the clearing mechanisms based on Mie Scattering theory illustrating that conformational changes of polymer chains induced by solvent effects of glycerol enhanced the anisotropy (i.e. directional alignment) of the nanostructures, leading to reduced crystallinity and scattering of the resulted PEG scaffolds. Our findings represent the first and systematic demonstration with both experimental and theoretical evidences in effectively clearing porous polymeric scaffolds by mechanisms other than RI matching, which could tackle the limitations of current optical imaging of cells cultured within 3D opaque porous scaffolds, such as poor visibility, low spatial resolution and small penetration depth.

Tissue optical clearance techniques enable imaging of structural and functional features of various tissues or organs1-5 (e.g. cornea, skin, brain or whole embryo) with exceptional resolution and contrasts which tremendously advance our understanding of tissue physiology and pathology. Tissue optical clearance is realized by immersion in optical clearing agents (OCA, e.g. glycerol or FocusClear®) that exhibits a refractive index (nm≈1.44-1.45) matched with the corresponding tissues thus reduces the light scattering. Here, we attempted to take advantage of OCA to improve the transparency of porous polymeric scaffolds by reducing light scattering. Porous scaffolds for three-dimensional (3D) cell culture have been increasingly adopted in cell biology and tissue engineering to provide more physiologically-relevant extracellular microenvironment than conventional two-dimensional (2D) culture on planar surface6, 7.


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Unprecedented progresses have been facilitated by 3D cell culture such as in vitro differentiation of stem cells into organotypic structures8 (e.g. optical cup9, inner ear sensory10, liver bud11), biomimetic tumor (malignant or benign) formation for deciphering cancer mechanisms and anti-cancer drug screening12, 13 as well as making transplantable cellular constructs with augmented functionalities for regenerative therapy14. However, porous polymeric scaffolds are typically isotropic and opaque, which greatly limit their optical accessibility for standard imaging tools (e.g. light microscopy). For example, naturally-derived (e.g. BD® collagen, chitosan or cellulose) or synthetic (e.g. poly (lactic-co-glycolic acid) [PLGA], poly (ethylene glycerol) [PEG)]) porous scaffolds are intrinsically opaque when immersing in saline or culture medium and impose a barrier for visualization of the cellular dynamics within the scaffolds

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. In this regard, optical imaging of 3D cells cultured in opaque scaffolds suffers

from low spatial resolution, poor contrast and limited penetration depth due to strong light scattering in the 3D milieu2, 16. Therefore, improving the optical transparency of porous scaffolds and understanding the physical basis of transparency are vital to realize high quality optical imaging of cellular behaviors in 3D porous scaffolds and will expand their applications as potential substitute for the current goldstandard 2D culture. In the present study, a commonly-used OCA for tissues, glycerol, was demonstrated to be able to effectively realize clearance of porous PEG scaffolds as well as other polymeric scaffolds (e.g. PLGA, PA and gelatin). The clearing effect of glycerol was mainly attributed to structural modification of the nano-scale scatterers within the porous polymeric scaffolds, which followed different mechanism other than refractive index matching. PEG is a widely-used biomaterial to fabricate hydrogel or porous scaffold for 3D cell culture due to its excellent biocompatibility, mechanical stability and non-fouling properties against nonspecific interactions17. To our knowledge, our work represents the first demonstration with both experimental and theoretical evidences in effectively improving the


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transparency of porous polymeric scaffolds even immersing in RI-mismatched medium (e.g. culture medium or water), which enable facile, real-time and deep-penetration optical imaging of cellular dynamics in the 3D porous scaffolds with high quality.

Experimental Section Fabrication of transparent PEG scaffolds Transparent PEG scaffolds could be obtained by including glycerol in the fabrication methods used for conventional nontransparent scaffolds as shown in Figure S2. Briefly, PEGDA(4000) basal prepolymer solution was firstly prepared by dissolving 10% w/v PEGDA, 0.5% w/v of ammonium persulfate (APS) and 0.05% w/v of N,N,N’N’-tetramethylethylenediamine (TEMED) in deionized water (DIW). 60%v/v glycerol was then mixed with the basal precursor solutions. Afterwards, the precursor mixtures were pipetted into the PMMA stencils and gelled at room temperature for 10min, which were then immersed into DIW to remove the unreacted precursors, glycerol or other impurities for 24h with 8 times DIW exchange. Finally, the stencils were frozen at -20℃ for 24h and lyophilized to obtain porous transparent scaffolds. For salt leaching (SL) methods, the precursor solution was dissolved in 40% saturated NaCl/60% glycerol, and 300mg/ml NaCl crystals (with diameter of 30-50µm) were additionally added as porogen in the precursor mixtures, which were dissolved and removed during washing. SEM and ESEM Microstructures of the scaffolds were visualized using a scanning electron microscope (FEI Quanta 200). The samples were coated with gold with a sputter coater for 90s. The surface morphologies of rehydrated scaffolds were directly observed by ESEM. Pore diameter of scaffolds were evaluated and


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statistically analyzed from scanning electron micrographs of six different areas using Image J software (National Institutes of Health). Transmittance measurement Transmittance spectra in the wavelength range of 300-900nm were recorded using microplate reader (SpectraMax M5, USA). OCT Test The self-built spectral domain OCT system used in our experiment was presented in detail18. The OCT system was based on a super luminescent diode (λ=835nm, ∆λ=40nm, Inphenix, IPSDD0804) and a single-mode fiber-optic Michelson interferometer. The interference signal of the light beams reflected from the reference and sample arms was detected by a self-made spectrometer consisted of an achromatic collimating lens (CM: f=75mm), a 1,200-line/mm transmission grating, an achromatic focusing lens (FL: f=200mm), and a line-scan charge-coupled device (CCD) camera (2048 pixels, each 14×14µm2 in size, e2v, AVIIVA SM2). The output power of the sample arm was about 2mW. The integration time of the CCD camera was set to be 200µs and 1000µs separately, equivalent to two different illumination conditions. Data acquisition and image reconstruction were implemented in Labview software (National Instrument, Austin, TX). The refractive index (RI) of the polymer was determined with the OCT system by a classic technique19. In brief, the sample was placed on top of a planar mirror for imaging acquisition. As illustrated in Figure S2, the thickness L and the optical path length S of the sample can be measured directly. Then the RI, n, may be computed:

n=

(Equation 1)


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In our experiment, 100% cross-linked PEGDA polymers were made and imaged with the OCT system. Twenty measurements were performed for each sample. OCT measures the backscattered light intensity from the sample versus axial (in depth) ranging distance. Dimension of all the OCT images is 2mm ×1mm. Lorentz-Lorenz Equation20 was used to calculate the refractive index of 100% cross-linked PEGDA polymer. n2 -1×M

RLL = n2 +2×ρ

(Equation2)

Where, n is the refractive index; M is the repeating unit molecular weight; ρ is the density; RLL is the molar refraction. For PEGDA (M=4108g/mol; ρ=1.12g/cm3), RLL =2(-CH-)+2(-COO-)+89.5×(-O-)+183×(-C -) XPS test The surface chemical compositions of scaffolds were investigated using XPS (EscaLab-250Xi, England). The spectra were collected using a Al Kα (1486 eV) X-ray source with a pass energy of 20eV. The X-rat source power was around 300W. The pressure within the XPS chamber was between 10-9Pa. The original data were subjected to deconvolution analysis using the XPSPEAK software. XRD test X-ray diffraction patterns were recorded at room temperature on a diffractometer (Bruker, Germen). The X-ray source power was around 40KV with angle of 2θ=10°-80°. Raman scattering


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A Renishaw (RM2000, England) Microscopic Confocal Raman spectrometer was used to collect the Raman spectra. A polarized fiber-optic probe head (Renishaw) backscattering geometry was used, with the polarization of the scattered photons in 0° and 90°. The excitation source was a diode-pump solidstate Nd:YAG laser (514.5 nm, 4.7 W) with a detected spot diameter of 3µm. The spectra were normalized. FT-IR The scaffold samples was homogenized in spectrophotometric grade KBr (1:20) in an agate mortar and was pressed with 3mm pellets using a hand press. The infrared spectrum was acquired using GX FTIR spectrophotometer（PerkinElmer，Inc.） with DTGS detection, at a resolution of 4cm-1 . The spectra was taken in transmission mode in the region 4000～400cm-1. The room temperature was 25℃ during the experiments. AFM AFM images were recorded using a MultiMode scanning probe microscope (MM8+Picoforce, Germany). The samples were scanned using SNL-10 Silicon nitride probes with a tip radius of 2 nm, a spring constant of 0.32 N/m or 0.06 N/m, and a resonance frequency in aqueous media of 9K Hz at an amplitude set point in the range from 0.6 to 2.5 V and a trapping force of approximately 98% of the set point at 1-2 Hz at room temperature. Poly-PEGDA dissolved in water or water/glycerol mixture solutions were injected directly into the fluid cell with the mica substrate. We started scanning the samples in about 20 min after system optimization. TEM


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For TEM imaging, a drop of Poly-PEGDA suspended in water or water/glycerol mixture solutions was deposited on a holey carbon copper grid and stained with RuO4 solution. Micrographs were recorded with a CCD camera on a H7650 microscope (Hitachi, Japan) operated at 80KV. Cell culture and staining HT1080 cell line were cultured in Dulbecco’s Modified Eagle Medium (DMEM, MUTICELL, Canada) supplemented with 10% Bovine calf serum, and 1% penicillin-steptomycin. HUVEC cells were maintained in endothelial growth media 2 kits (Sciencell, USA). hMSC cells were expanded in growth medium (BIOWIT, Beijing). All cell types were maintained at 37℃ in an incubator with 5% CO2. Cells were harvested and counted based on the general protocol and directly seeded into the scaffolds or encapsulated in 2% alginate solution. Growth mediums were refreshed every other day. Chondrogenesis was induced in a serum free, chemically defined chondrogenic medium (CM) containing 90% DMEM,100U/mL penicillin, 1000U/mL streptomycin, 0.2% fungizone antimycotic, ITS+1(10µg/mL insulin, 5.5µg/mL transferrin, 5ng/mL selenium, 0.5mg/mL bovine serum albumin,4.7µg/mL linoleic acid; Sigma), 0.1mM ascorbic acid 2-phosphate, 1.25mg/mL human serum albumin, 10-7M dexamethasone and 10ng/mL TGF- β3 (D-T). In general, negative controls were cultured in the same medium without D-T. Medium was replaced every other day. All cultures were harvested at day 21. Cell viability was tested using a live/dead assay. Scaffolds loaded with cells were first washed with DPBS and incubated with 0.5µl/ml of Calcein-AM (for live cells, Wako, Japan) and 2µl/ml of PI (for dead cells, Wako, Japan) in DPBS. For nuclei staining, cell samples were fixed in 4% paraformaldehyde in PBS for 15min followed by permeabilization with 0.1% Triton X-100 in PBS for 5min at room temperature. The samples were exposed to 100nM DAPI in PBS for 10min at 37℃. For histological analysis, scaffolds seeded with cells were rinsed in tap water for 5minutes and rinsed quickly with 1%


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acetic acid solution for no more than 10-15s, followed by staining in 0.1% safranine O solution for 5min. RNA extraction and RT-PCR analysis Total RNA was extracted from cells using an RNeasy MiniKit (Takara, Japan) following the supplier’s instructions. Briefly, the cell pellets or scaffolds seeded with cells were washed with PBS, cut into small pieces, re-suspend in 1ml TRIZOL. The final elute was stored at -80 ℃ . Total RNA concentration was determined by optical density at 260nm (OD260). Fifty nanograms of total RNA were used to synthesize cDNA using a High-Capacity cDNA Archive kit (Takara, Japan) following the supplier’s instructions. The cDNA product was stored at -80℃. The real-time RT-PCR reactions were conducted and monitored with a CFX96TM Real-Time System (Bio-RAD, USA). Taq Mans Gene Expression Assay kits (Takara, Japan) were used for transcript levels of chondrogenisis-related ECM genes including collagen type-II (Col-II), Sox9, VACN. Taq Manshuman GAPDH (Takara, Japan) was used as a housekeeping/reference transcript. All cDNA samples were analyzed for the transcript of interest and the housekeeping gene in independent reactions. Data were analyzed by CFX96 manager software. The Ct value for each sample was defined as the cycle number at which the fluorescence intensity reached a certain threshold where amplification of each target gene was within the linear region of the reaction amplification curves. Relative expression level for each gene of interest was normalized by the Ct value of Taq Manshuman housekeeping gene GAPDH using an identical procedure. Each sample was analyzed intriplicate. Imaging Time-lapse recordings were performed by means of Nikon Eclipse Ti-S microscope. This microscope is suitable for long term time-lapse in a CO2 incubator at 37℃. Microphotographs of a defined region of


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the device were taken at 5min interval for 48h. All Audio Video Interleave (AVI) files were generated by using entire time-lapse image sequence with ImageJ software. A Fluar 10× objective was used to acquire 3D fluorescence images of HUVEC cells distributed in 3D scaffold after 3days’ culture. Monte Carlo Simulation of light scattering The simulation of light backscattering was applied according to Monte Carlo method, which was developed by our previous work

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.This simulation program was applied to the propagation of photons

in an anisotropic scattering model consisting of poly-dispersed spherical and infinite long cylindrical scatterers. The cylinders are aligned following a Gaussian distribution. Densities and sizes of the spherical and cylindrical scatterers, as well as the orientation of the cylinders are variables for the simulation of different anisotropic media.

Results and Discussion As the first step to modulate the optical transmittance of the porous PEG scaffolds, the refractive index (RI) of pure poly (ethylene glycol) diacrylate (PEGDA) polymer was evaluated both by theoretical calculation according to Lorentz-Lorenz Equation20 and experimental characterization by optical coherence tomography (OCT)19, 22 (See supplementary, Figure S1). The calculated and OCT-measured RIs of cured PEGDA were 1.464 and 1.472 respectively, both of which were within the RI range of cells but mismatching the RI of aqueous medium for cell culture (RI=1.33-1.37). As expected, the porous scaffolds derived from PEG were non-transparent when rehydrated in RI-mismatched aqueous medium, regardless of the technologies for scaffold fabrication (i.e. salt leaching (SL), gas forming (GF) and cryogelation (CG)). Unexpectedly, when glycerol was supplemented in the PEGDA prepolymer solution and involved in the subsequent polymerization, the resulting porous scaffolds turned transparent after rehydration in water with all the three methods for scaffold fabrication (i.e. SL, GF, CG). The enhanced


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optical transparency for glycerol-mediated PEG scaffolds (SL+, GF+, CG+) compared with the nontransparent counterparts (SL-, GF-, CG-) were visually illustrated by clear observation of texts through the scaffolds (Figure 1A a-b, S2) and quantitatively measured with increased optical transmittances (Figure 1B, S3). OCT was used to label-free image and reveal internal pore structures of the rehydrated scaffolds. By exploiting the low coherent characteristic of a broadband light source in OCT, the light back-scattered from different layers of the porous PEG scaffold can be discriminated to provide axial resolution of the internal structures with micrometer-scale resolution (Figure 1A g-h, S3). Fibrous polymeric skeletons were clearly observed, when submerged in water, for non-transparent PEG scaffolds (SL-, GF-, CG-) exhibiting strong light backscattering. In contrast, the transparent PEG scaffolds (SL+, GF+, CG+) showed little light backscattering without visible polymeric skeletons. In addition, when the non-transparent PEG scaffolds (SL-, GF-, CG-) were submerged in glycerol instead of water, increased transmittances were observed with reduced light backscattering due to RI matching (Figure S3, S4) (the RIs of PEG and glycerol are both ~1.46). The above analysis suggested that the light backscattering reduction and optical transmittance enhancement of glycerol-treated porous PEG scaffolds (SL+, GF+, CG+) in water were realized by certain mechanism other than the RI matching.


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Figure 1. Experimental and theoretical demonstration of the transparent porous PEG scaffolds. A: Optical and structural properties of transparent (T) and nontransparent (NT) porous PEG scaffolds fabricated by salt leaching (SL) technology involved with (SL+) or without (SL-) glycerol. a) photograph of dried porous scaffolds with different heights (diameter: 4mm; heights: 0.5mm, 1mm, 2mm, 3mm); b) photograph of water-rehydrated scaffolds with blurry texts observable through SL- NTscaffold whereas clear observation was realized through SL+ T-scaffold; c, d, e, f) SEM images of porous scaffolds containing varied internal nanostructures with random spherical aggregations for SL-


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NT-scaffold and anisotropic fibrous structures for SL+ T-scaffold; g, h) OCT images of waterrehydrated SL+ T-scaffold showing much weaker interconnected porous structures due to reduced light scattering compared with SL- NT-scaffold (height: 2mm) B: Transmittance measurement of waterrehydrated SL±scaffolds. C: Transmittance measurement of water-rehydrated SL- NT-scaffolds with a wide range of pore sizes, indicating insignificant improvement of transparency by increasing pore size of the scaffolds. D: Monte Carlo Simulation of light backscattering showing large light scattering factors resulted from scatterers with dimensions comparable with the wavelength of light (i.e. from several hundred nanometer to several micron), whereas, for scatterers with radius > 10 µm (e.g. porous structures), the scattering efficiency factors sharply decreased to reach a low level. E: Spherical nanostructures with much larger scattering coefficient (µ) resulted in much stronger light scattering than cylindrical nanostructures both in theoretical effective scatterers range (the radius of scatterers d=0.110µm, the volume density of sphere scatterers n=0.01-100µm-2 or cylinder scatterers n=0.1-10µm-1) and experimental deduced scatterers range (the parameters of sphere scatterers d=0.3-3µm, n=0.11-12µm-2, the parameters of cylinder scatterers d=0.5-1.5µm, n=0.66-2µm-1). Optical physics such as Mie Scattering theory23 provides us clues for alternative mechanism of improving optical transparency of objects (e.g. biological cells, matrices or tissues), in which the total scattering cross-section equals to: C =

$ ! #%

&

, ()

(Equation 3)

where C is the total scattering cross-section, and are the radius and volume density of scattering particles, *+ is the light wavelength across the scattering medium, and ( is the relative refractive index quantified by (≡ /+ . is the refractive index of scattering particles (e.g. cells,


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=1.39-1.47), + is the refractive index of ground matter (e.g. water, saline or culture medium, + =1.33-1.37). According to Equation 3, two mechanisms, namely refractive index (RI) matching of the scattering particles (scatterers) and ground matter (to make (≈1) and structural modification of the scatterers (by changing and ) could both potentially lead to light scattering reduction. The first mechanism relies on the fact that the refractive index variation between the scatterers and ground matter determines the light scattering efficiency. Therefore, RI matching offers effective approach for improving optical transparency, which is manifested in the cases of tissue clearance as well as hydrogel-based 3D cell culture systems. In the latter case, the RI of hydrogel containing 80% (w/v) water is about 1.36 which matches the RI of the medium (Figure S5), thus facilitates clear visualization of the 3D cultured cells. We therefore tested the possibility of structural modification of scatterers as the potential mechanism for improved optical properties of the novel transparent porous scaffolds. As shown in SEM images (Figure 1A c-d, S3), transparent (T) PEG scaffolds uniformly exhibited larger pores than nontransparent (NT) counterparts regardless of the fabrication methods (i.e. SL, GF, CG). The differences in the macroscale porous structures were ruled out as determining factor for the increased light transmittance, since no significant changes in light transmittance or light backscattering （as revealed by OCT images） were observed for SL- scaffolds fabricated with a series of pore sizes ranging from 30µm to 150µm (Figure 1C, S6). According to the definition of Mie scattering, scatterers with dimensions comparable with or slightly larger than the wavelength of the incident radiation (i.e. light) gave rise to non-uniform light scattering24. Since the transmittance of porous scaffolds is hardly affected by the macro-scale structures within the porous scaffolds, we zoomed in to investigate the nano-scale structural features with similar length-scale of light (several hundred nanometers), which were determined by (radius)


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and (volume density) of the scatterers according to Equation 3. As shown in SEM images (Figure 1A e-f), coarse nano-surface with irregular aggregations comprising of spherical nanostructures was observed in SL- NT scaffolds (with radius in the range of 0.3-3µm and volume density in the range of 0.11-12µm-2), whereas, more glossy nano-surface with anisotropically cylindrical nanostructures was observed in SL+ T scaffolds (with radius in the range of 0.15-1.5µm and volume density in the range of 0.66-2µm-1). The drastic topological variances of nanostructures between the two types of scaffolds were further confirmed by ESEM, which allowed imaging of rehydrated samples in water (Figure S7). The influence of scatterers’ nanostructures on backscattering behavior of the incident light was theoretically examined by Monte Carlo simulations21, which was an effective method for investigation of light propagation in turbid media, and the turbid media was dispersed with scatterers of different nanostructures (determined by and ) (Table S3). The simulations testified experimental observations, showing the non-uniform light scattering was mainly generated by scatterers with dimensions comparable with the wavelength of light (i.e. from several hundred nanometer to several micron) (Figure 1D). For scatterers with larger radius (e.g. > 10µm), the scattering efficiency factors sharply decreased to reach a low level, further verifying the independence of light transmittance on pore sizes in the normal range of the porous scaffolds. We further simulated the effects of nano-topologies on the light scattering with two types of geometries simplified according to the SEM images, namely dispersed spheres for NT scaffolds and cylinders for T scaffolds (Figure 1E). The scattering coefficients (µ) of the spheres were larger by at least one order of magnitude than that of the cylinders both in theoretical effective scattering ranges (=0.1-10µm; =0.01-100µm-2 for spherical scatterers or =0.1-10µm-1 for cylindrical scatterers) and zoomed effective scattering ranges correlated to the geometrical features according to SEM images (=0.3-3µm, =0.11-12µm-2 for spherical scatterers within the NT scaffolds;


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=0.5-1.5µm, =0.66-2µm-1 for cylindrical scatterers within the T scaffolds). The simulations provided theoretical evidences on the feasibility of nano-structural modification on reduction of light scattering and improvement of transparency for the porous scaffolds. Since pure PEG powder was insoluble in glycerol, which also could be functional as porogen, we simplify the fabrication processes of the PEG scaffolds without using salt leaching, gas forming or cryogelation technologies. This simplified fabrication method took glycerol as structure modifier at both macroscopic (as porogen) and nanoscopic (as nanostructure ameliorant) scales and eliminated extra interference (e.g. PH, temperature) to better understand the mechanism by which glycerol affected the nanostructures within the porous scaffolds. The resulted porous PEG scaffold only involved with glycerol (Gly+) showed improved optical properties when rehydrating in water (i.e. reduced OCT light backscattering, increased light transmittance) and anisotropic nanostructural features similar to the above transparent porous PEG scaffolds (SL+, GF+, CG+) (Figure 2A, 2B).


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Figure 2. Glycerol as both porogen and nanostructural ameliorant for fabrication of transparent porous PEG scaffolds. A: Optical and structural properties of transparent PEG scaffold involved with only glycerol as porogen (Gly+). a) OCT image of water-rehydrated Gly+ scaffold with reduced light scattering; (height: 2mm) b, c) SEM images of porous Gly+ scaffold with anisotropic cylindrical


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nanostructures. B: Transmission curves of water rehydrated Gly+ scaffolds fabricated by supplemented with different volume ratio of glycerol. C: X-ray diffractograms of Gly± scaffolds. D: Polarized Raman scattering curves of Gly± scaffolds. E: XPS C1s and O core level spectra of Gly± scaffolds. F: FT-IR spectra of Gly± scaffolds. Since the scattering properties of a polymer, was profoundly dependent on the degree of crystallinity (relative content of amorphous and crystalline contributions), we further characterized the crystallinity of the PEG scaffolds by X-ray diffractograms (XRD) and polarized Raman scattering spectroscopy (Figure 2C, 2D, S8). Gly+ scaffold exhibited decreased intensity of peaks compared to the control scaffold without Gly (Gly-), indicating decreased crystallinity of PEG nanostructures. For polarized Raman spectroscopy, Gly- scaffold exhibited identical peak intensities between the 0° and 90° polarization, proving the isotropy of the crystalline nanostructures. In contrast, the peak intensities of the Gly+ scaffold were greater in the 0° polarization than that in the 90° polarization, showing anisotropic nanostructures that were more amorphous. The X-ray diffractograms showed two crystalline peaks at 19.7° and 23.9° in both of Gly+ and Glyscaffolds, which were identical to reported crystalline peaks of poly(ethylene oxide) (PEO) 25-27 (Figure 2C, S8). Since PEO shared the same [-CH2-CH2-O-] (-CCO-) structural unit with PEG polymeric network, the crystalline contribution of PEG scaffolds was mainly caused by the repeated -CCO- unit and the amorphous contribution was resulted from the remaining residues (Figure S9). The polarized Raman spectrum of Gly+ scaffold was also consistent with the reports of PEO28, both of which contained strong C-H stretching bands between 2800cm-1 and 3000cm-1 and typical spectrum of fingerprint region (500-1500cm-1) (Figure 2D, S8, Table S1). Previous studies identified that PEO in the crystalline state arranged in a 7/2 helical conformation containing 7 structural -CCO- units with 2


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helical turns, which was constructed with a succession of trans-(CCOC)-trans-(COCC)-gauche-(OCCO) bonds, namely ttg, along the chain28-30. Typical Raman peaks (843, 1073, 1141, 1281, 1482cm-1) of the 7/2 helical conformation were also observed in both Gly+ and Gly- scaffolds, indicating that the inner 7/2 helical conformation of repeated -CCO- unit did not change in both cases28, 31. Since glycerol was supplemented in the prepolymer solution, then involved in the polymerization, we investigated whether glycerol was chemically or physically incorporated into the cross-linked PEG network. Theoretically, glycerol should be inert to the reaction of covalently crosslinking among acrylates. Experimentally, both XPS and FT-IR analysis showed no significant changes in spectrum or characteristic peaks between the Gly+ and Gly- scaffolds (Figure 2E, 2F, S10, Table S2), which indicated that no residual glycerol was chemically or physically incorporated in the transparent porous scaffold. Therefore, glycerol was probably present as structural ameliorant (structural ameliorant is defined to describe the functions of glycerol to improve the nano- and macro-structures of scaffold, then optimized optical properties of scaffolds.) during the polymerization and washed off after the scaffold formation. From the above analysis of crystallinity and chemical composition, we could conclude that the primary configuration of the cross-linked PEG network did not change in the presence of glycerol (Figure 3A), thus the major effects of the supplemented glycerol were likely exerted by changing the secondary and tertiary structures of the PEG network.


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Figure 3. Solvent effect of glycerol on multi-level structural modification of PEG network deciphering potential mechanisms for transparent porous PEG scaffolds. A: Primary configuration


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of cross-linked PEG polymeric network containing repeated -CCO- (as PEO) structure. B: Secondary conformation of cross-linked PEG network modified by polar solvents. AFM (a, c) and TEM (b, d) revealed dense and clustering conformation of PEG network in water (a, b), whereas looser and more stretched conformation in glycerol (c, d). The conformational variance is proposed to be resulted from active interaction of polar glycerol molecule with -CCO- to form stronger hydrogen bonding in competition with H2O-OCC or internal hydrogen bonding. C: Tertiary structure of transparent PEG scaffold (BT+) modified by 1,2,4-butanetriol exhibited reduced crystallinity and more anisotropic nanostructure (b) to reduce light scattering as compared to the nontransparent scaffold (BT-) with typical irregular crystallographic spherulites (a). The secondary conformations of polymers are determined by the balances of inner- and inter-actions among polymer chains and their surrounding solvents32. It has been proved that glycerol contributed to improve secondary chain conformation and entanglement of polymers, which greatly affected the polymer functions33, 34. Glycerol, as a strong polar solvent with three hydroxyl groups per molecule, can actively interact with cross-linked PEG chains by forming stronger hydrogen bonding between -CCOglycerol, which might disrupt a large number of relatively weaker hydrogen bonding between -CCOH2O and internal hydrogen bonding among PEG chains. Considering the stereo-hindrance effect of glycerol molecule compared to smaller H2O molecule, the apparent flexibility of glycerol-interacted PEG chains might decreased, leading to reduced conglomerations of the polymer chain (Figure 3B). To test this hypothesis, the morphologies of lowly cross-linked PEG network dissolved in water or glycerol/water mixture were observed by AFM and TEM (Figure 3B a-d). As expected, the cross-linked PEG network tended to form dense and compact clusters in water with greater thickness (~4.4nm) as measured by AFM, while, relatively sparse and stretched clusters with lower thickness (＜2.0nm) were observed in glycerol/water system. TEM images further revealed similar morphological differences of


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polymeric PEG network in terms of density and compactness in the presence and absence of glycerol, indicating the structural modification of glycerol at the secondary conformation of the PEG network. The tertiary structures of the cross-linked PEG network mainly developed in the freeze-drying step during the processes of porous scaffold fabrication, which drove phase separation and crystallization of the polymer to produce macroscopic pores with internal nanostructures. Previous studies showed PEO polymer usually crystallized as spherulites, which were assembled from abundant lamellar crystals containing -CCO- repeating unit folded in the 7/2 helical conformation29, 32. Accordingly, the -CCOrepeating unit of the cross-linked PEG network dissolved in water (Gly-), forming dense and compact clusters in the secondary structure, was susceptible to fold into packed lamellar, then crystallize as spherulites with dimensions comparable to light wavelength thus causing strong light scattering (Figure 3C a). In the presence of glycerol (Gly+), the -CCO- repeating unit preferably interacted with glycerol by inter-hydrogen bonding leading to more rigid and stretched polymer chains as the TEM figures shown, which reduced the degree of crystallinity. The resulted cylindrical nanostructures were more amorphous which could reduce the light scattering thus improve the light transmittance (Figure 3C b). To investigate whether the glycerol-mediated clearance of porous polymeric scaffolds can be generalized and applied to other widely-used biomaterials, porous scaffolds of PLGA, gelatin and Polyacrylamide (PA) were fabricated in the presence or absence of glycerol respectively. Similar to PEG, increased light transmittances mediated by glycerol were accomplished for all three types of scaffolds compared with the corresponding counterparts fabricated by traditional salt leaching method without glycerol (Figure S11). Furthermore, the proposed solvent effects of glycerol on the multi-level structural modification of polymeric PEG network were also validated on other strong polar solvent with chemical structures similar to glycerol such as 1,2,4-butanetriol (BT). Similarly, BT also functioned as both porogen and nanostructure-ameliorant, leading to transparent porous PEG scaffold (BT+) with


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internal cylindrical nanostructures compared to more spherical nanostructures in the absence of BT (BT) (Figure 3C, S12). Finally, we demonstrated the optimal 3D imaging performances of the transparent porous PEG scaffolds (T), which could realize in-situ and deep-penetration 3D cellular imaging unachievable by conventional nontransparent porous PEG scaffolds (NT). As the first demonstration, chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was monitored and examined directly in T scaffolds, which was not feasible in NT scaffolds (Figure 4A). Upon 21 days’ differentiation, the chondrogenic cells exhibited bigger and darker morphologies compared to the undifferentiated hMSCs on day 0, which could be straightly revealed by safranine O staining of cells within the T scaffolds but not visible in the NT scaffolds (usually required sectioning into thin slices). Successful chondrogenesis in both T and NT scaffolds were further confirmed by the expression of chondrogenesis-related ECM genes (Col-Ⅱ, Sox9, VCAN) to similar levels. As the second demonstration, long-term and label-free tracking of cellular morphogenesis was achieved in-situ within the T scaffolds. As recorded by timelapse microscopy, dispersed HT1080 cells (Human Fibrosarcoma Cell Line) seeded in the 3D scaffolds gradually aggregated to form tumor spheres within 48h, which recapitulated the morphological features of in vivo tumor tissues (Figure 4B, Video 1). Lastly, due to the improved light transmittance, the imaging depth for T scaffolds can be dramatically increased which was especially important for cellular visualization within 3D constructs. As shown by 3D reconstructed fluorescence imaging of HUVEC cells stained with DAPI, a much thicker section of T scaffolds (up to 920µm) could be clearly imaged by normal fluorescence microscopy compared with the NT scaffolds (with penetration depth of only 360µm) (Figure 4C).


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Figure4. In-situ and deep-penetration 3D cellular imaging with transparent porous scaffolds. A: Insitu visualization of chondrogenic differentiation of hMSCs within transparent (T) scaffold. a) hMSCs were completely invisible in nontransparent (NT) scaffold, while, transparent (T) scaffold clearly revealed the morphologies of differentiated cells as characterized by safranine O staining (b) and expression of chondrogenesis-related genes (Col-Ⅱ, Sox9, VCAN) (c). B: Label-free tracking of tumor sphere formation based on HT1080 cells within T scaffold for 48h. C: 3D reconstructed fluorescence images of HUVEC cells within T scaffold stained with DAPI showing dramatic increase of imaging depth compared to NT scaffold. The improved imaging performances and penetration-depth of the transparent polymeric scaffolds as shown above represented key steps towards optimizing and widening the applicability of 3D porous scaffolds as well-accepted cell culture standard. Currently, optical imaging of opaque 3D porous scaffold-based culture systems typically required retrieve of the cellular samples from the culture platform by fixation and histological sectioning, followed by staining, which would affect the intactness of samples and could only provide “snapshots” of cellular dynamics at a given time point. This inefficiency severely limited our understandings of the biological processes associated with dynamic cellular behaviors and was resource and time consuming. An alternative method to track cells in 3D porous scaffolds relied on fluorescence labeling technologies (e.g. by loading with cell tracker or transfection with genes encoding fluorescence proteins), which introduced additional labeling procedures and might cause unwanted side effects to cell functions35, including cell apoptosis36, abnormal gene expression37, impaired protein interactions38. In addition, the penetration depth of microscopic imaging of cells in 3D porous scaffolds were typically unsatisfactory, partially attributed to the light scattering through the bulk scaffolds16. Imaging of deep and internal features of the 3D bulk scaffolds was still difficult, which could usually be realized by additional setups such as insertion of


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optic fibers into the scaffolds for local image acquisition39. Benefit from this novel transparent scaffold with better imaging performance and deeper imaging depth, 3D imaging process would be more simple and harmless. Integrated with microfabrication technologies (e.g. micromolding), the size (e.g. µm to cm) and architecture of transparent porous scaffolds could be further tailored to possess well-defined patterns such as hexagonal liver lobule and bifurcated branches of blood vessels mimicking in vivo tissue structures, or scaffold array chips as high throughput platform (Figure S2). In addition, the involvement of glycerol or BT tends to slightly alter the mechanical properties of the transparent porous scaffolds, which will be investigated in future studies. The mechanical property of transparent scaffolds can be further tailored to other specific applications by tuning the concentration of PEG as well as glycerol or BT. Taken together, the novel transparent porous scaffolds could potentially realize automatic cell loading (due to the highly porous structure)12, optimal cellular 3D imaging (due to transparency) and subculture for cell expansion/harvest as straightforward and easy as conventional 2D culture based on the planar substrate. As a demonstration, HeLa cells were auto-seeded, imaged and sub-cultured with good viability within the transparent PEG porous scaffold following similar operations to 2D culture. All these proved advantageous features of the transparent scaffolds marked their great potential to turn 3D culture as easy and effective as 2D culture and ultimately lead to promising in vitro culture substitute in the future (Figure S13). The clearing effect of glycerol has been validated in improving the transparence of polymeric porous scaffolds made from PEG, PLGA, gelatin and PA, but not for alginate (data not shown). Furthermore, polar solvents including glycerol and BT but not 1,4-butanediol exhibit the clearing effects. The detailed mechanisms resulted in these differences need to be further investigated to generalize and broaden the applications of this promising approach for porous scaffold clearance. It should be noted that faint


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fibrous skeletons of the current transparent porous PEG scaffolds were still slightly observable under the light microscope, which did not affect the 3D cell imaging in our applications. It would be possible to make the fibrous skeletons more transparent by further optimization of the solvent-polymer interactions (e.g. reducing the PEGDA prepolymer concentration) and could even turn the skeletons completely invisible by choosing biomaterials with RI matched with water or culture medium.

Conclusions In summary, the present work demonstrated the solvent effects of glycerol as both porogen and nanostructural ameliorant to fabricate optically transparent porous polymeric scaffolds, whose lightwavelength-scale nanostructures dominantly improved the optical properties of the scaffolds with deducted light scattering and improved light transmittance in case of refractive index mismatching. We also exploited mechanisms based on Mie Scattering theory that conformational changes of polymer chains induced by solvent effects (the balance of inner- and inter-action between the polymer chains and surrounding solvent) affected their nanostructures, then leading to changes of physical (e.g. crystallinity) and optical properties of the resulting polymeric scaffolds. Our findings represented the first and systematic study of potential solutions and mechanisms (mainly structural modification) that could effectively tackle the limitations of current 3D optical imaging in opaque 3D porous scaffolds, such as visibility, spatial resolution and penetration depth. These conceptual findings and experimental strategies are promising to be broadly applicable to optimize the optical properties of a wide range of biomaterials and improve the performances of other optical imaging techniques such as optical coherence tomography (OCT), optical coherence elastography (OCE), second-harmonic generation (SHG), bioluminescent imaging (BLI). From an application standpoint, transparent porous PEG scaffolds could be extensively useful as 3D cell culture platform and promising substitute of 2D culture


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systems for multidisciplinary applications, including cell/developmental biology, cell-based assays for drug screening, as well as cell/tissue engineering.

Supporting Information Assignment of Raman bands of scaffolds in the frequency range 500-1500cm-1 are summarized in Table S1. XPS element analysis of scaffolds are summarized in Table S2. Parameters used in Monet Carlo model are summarized in Table S3. Supporting figures are listed in the supplement. Detailed experimental protocol are included in the supporting information. “This material is available free of charge via the Internet at http://pubs.acs.org.”

Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Author Contributions S.Z. and Y.N.D. conceived the research, designed the experiments and write the manuscript. S.Z. fabricated all scaffolds with the help of B.J.W. and contributed to all other experiments. Z.Y.S. and S.Z. performed the OCT. Z.Y.S. performed the simulation. J.Y.W. transfected fluorescence HT1080 cells. X.K.L performed the time-lapse microscope. Y.Z. and S.Z. performed experiments involved with hMSC cells. S.Z. analyzed and interpreted the results, in discussion with Y.N.D and Z.Y.S. All authors have given approval to the final version of the manuscript.

Acknowledgment We thank Dr. Dengli Qiu from Bruker Co. Ltd for providing AFM apparatus and analysis. We thank all du-lab members for general assistance. This work is financially supported by the Natural Science Foundation of China (81171474, 51273106).


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For Table of Contents Use Only Title: Glycerol-mediated Nanostructure Modification Leading to Improved Transparency of Porous Polymeric Scaffolds for High Performance 3D Cell Imaging Authors: Shan Zhao, Zhiyuan Shen, Jingyu Wang, Xiaokang Li, Yang Zeng, Bingjie Wang, Yonghong He and Yanan Du


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