Cross-Linked Pectin Nanofibers with Enhanced Cell Adhesion

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Crosslinked pectin nanofibers with enhanced cell adhesion Sainan Chen, Sisi Cui, Hui Zhang, Xuejing Pei, Junli Hu, Yifa Zhou Zhou, and Yichun Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01605 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Crosslinked pectin nanofibers with enhanced cell adhesion Sainan Chen, †‡ Sisi Cui,§‡ Hui Zhang, † Xuejing Pei, § Junli Hu,*† Yifa Zhou,*§ Yichun Liu*† †

Key Laboratory of UV-Emitting Materials and Technology (Northeast Normal University),

Ministry of Education, Changchun, Jilin 130024, P. R. China. §

School of Life Sciences, Northeast Normal University, Changchun, Jilin 130024, P. R. China.

Keywords: pectin, nanofibers, periodate oxidation, adipic acid dihydrazide, dihydrazone crosslinking, enhanced cell adhesion.

ACS Paragon Plus Environment

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ABSTRACT: Polysaccharides display poor cell adhesion due to the lack of cell binding domains. This severely limits their applications in regenerative medicine. This study reports novel crosslinked pectin nanofibers with dramatically enhanced cell adhesion. The nanofibers are prepared by at first oxidizing pectin with periodate to generate aldehyde groups and then crosslinking the nanofibers with adipic acid dihydrazide to covalently connect pectin macromolecular chains with adipic acid dihydrazone linkers. The linkers may act as cell binding domains. Compared with traditional

Ca2+-crosslinked

pectin

nanofibers,

the

pectin

nanofibers

with

high

oxidation/crosslinking degree exhibit much enhanced cell adhesion capability. Moreover, the crosslinked pectin nanofibers exhibit excellent mechanical strength (with Young’s modulus ~ 10 MPa) and much enhanced body degradability (degrade completely in 3 weeks or longer time). The combination of excellent cell adhesion capability, mechanical strength and body degradability suggests that the crosslinked pectin nanofibers are promising candidates for in vivo applications such as tissue engineering and wound healing. This crosslinking strategy may also be used to improve cell adhesion capability of other polysaccharide materials.


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1. INTRODUCTION Since adhesion is a preliminary step for most mammal cells to grow and function, cell adhesion capability is essential for many applications of biomaterials, e.g. tissue engineering, wound healing, cell delivery and so on.1-3 Natural extracellular matrix proteins such as collagen and fibronectin are cell adherent because their molecular chains contain cell binding domains.4-6 Polysaccharides which include pectin, alginate, starch, and etc. are an important class of natural polymers for biomedical applications with the advantages of biocompatibility, biodegradation and bioactivity.7-9 However, most polysaccharides display poor cell adhesion capability due to the lack of cell binding domains.10-13 Moreover, they are hydrophilic, so they prevent cell adhesion by forming a hydration layer on surface.14 At physiological conditions, some anionic polysaccharides, such as pectin and alginate, are negatively charged. They repel cells by electrostatic interactions because most cells are also negatively charged on surface.7, 12 The poor cell adhesion capability severely limits the biomedical applications of polysaccharides. To circumvent this problem, polysaccharide materials have been modified with cell recognizing motif peptides, e.g. Arg-Gly-Asp (RGD), to improve cell adhesion.10, 12, 13, 15, 16 However, this strategy suffers from high cost and complicated synthesis. Therefore, for the biomedical applications of polysaccharides, it is highly important and challenging to develop a facile strategy to improve their cell adhesion capability. We are focusing on pectin nanofibers for biomedical applications.17-20 In this manuscript, using a periodate oxidation – adipic acid dihydrazide (ADH) crosslinking approach, we develop novel crosslinked pectin nanofibers with dramatically enhanced cell adhesion. As shown in Figure 1, the crosslinking is readily performed by at first oxidizing pectin with periodate to generate aldehyde groups and then crosslinking the nanofibers with ADH to covalently connect


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pectin macromolecular chains with adipic acid dihydrazone linkers. The linkers may act as cell binding domains and endow the crosslinked pectin nanofibers with superior cell adhesion capability. Moreover, the crosslinked pectin nanofibers exhibit excellent mechanical strength and much enhanced body degradability. The combination of excellent cell adhesion capability, mechanical strength and body degradability suggests that the crosslinked pectin nanofibers are promising candidates for in vivo applications such as tissue engineering and wound healing.

Figure 1. Schematic illustration of the preparation process of crosslinked pectin nanofibers and the possible cell binding domain.


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2. EXPERIMENTAL SECTION Materials. Pectin from apple (degree of esterification = 70.2%) was supplied by SigmaAldrich (Shanghai, China). Adipic acid dihydrazide (ADH), hydroxylamine hydrochloride, methyl orange, and polyethylene oxide (PEO, Mw = 5,000 kDa) were purchased from Aladdin Reagents (Shanghai, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and Triton X-100 were supplied by Dingguo Biotech. (Beijing, China). Sodium periodate (NaIO4), calcium chloride (CaCl2), glutaraldehyde, and all other chemicals were obtained from Xilong Chemicals (Guangzhou, China). Calcein AM/Propidium iodide double stain kits were purchased from Keygen Biotech. (Nanjing, China). Deionized water was prepared in our laboratory and used at all times.

Synthesis of dialdehyde pectins (APs). Pectin solution (5% w/w) was firstly prepared by dissolving pectin with water overnight and then mixed with NaIO4 solution in water. The final concentrations of pectin in the reaction solution were 2% w/w. The molar ratios between NaIO4 and galacturonic acid (GalA) units of pectin were set between 10:100 and 50:100. After being stirred in dark at ambient temperature (20–25 oC) for 16 h, the solutions were transferred to dialysis tubes with a cutoff molecular weight of 3500 Da and dialyzed against a large amount of water. The purified solutions were then lyophilized to obtain dialdehyde pectins (APs).

Determination of the content of aldehyde groups. The contents of aldehyde groups in APs were determined with a hydroxylamine hydrochloride titration method.21 APs (50 mg) were mixed with an aqueous solution (25 mL, pH = 4) containing hydroxylamine hydrochloride (0.25 M) and methyl orange (0.0003%). The mixtures were stirred until APs were fully dissolved. A blank hydroxylamine hydrochloride/methyl orange solution was used as a control. Sodium hydroxide solutions (0.01 M) were then added to the mixtures until the color of the


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solutions changed from red to yellow. The contents of aldehyde groups were then calculated by dividing the mole numbers of the added sodium hydroxide with the weights of the samples. The oxidation degrees were calculated by dividing the number of the oxidized saccharide units with the total number of the saccharide units.

Determination of molecular weight. The molecular weights of pristine pectin and APs were measured by high performance gel permeation chromatography using TSK-Gel columns (G4000PW, TOSOH) connected with a Shimadzu HPLC system. The column was pre-calibrated by a series of dextran standards using linear regression. Samples were dissolved at 5 mg mL-1 in sodium chloride solution (0.2 M). The solutions (20 µL) were injected into the column and eluted with sodium chloride solution (0.2 M) at a flow rate of 0.6 mL min-1. Sample peaks were monitored by a refractive index RID-10A detector (Shimadzu, Tokyo).

Electrospinnings. The electrospinnings were carried out as reported previously.17,

20

Solutions of pristine pectin (5% w/w), AP (10–15% w/w), and PEO (5% w/w) were prepared separately by dissolving the polymers with water. The pectin solutions were then mixed with the PEO solution at a certain ratio to achieve a pectin/PEO mass ratio of 80/20. A certain amount of Triton X-100, dimethyl sulfoxide and water were then added to the mixtures to reach a final concentration of Triton X-100 and dimethyl sulfoxide of 1% w/w and 5% w/w, respectively, and a polymer (pectin plus PEO) concentration of 3%, 7.5%, 9%, 10%, 10% and 10.5% w/w for pristine pectin and AP with oxidation degrees of 9%, 18%, 25%, 37% and 46%, respectively. The mixed solutions were stirred for one day and stored overnight. The solutions were then fed into a 5-mL disposable syringe capped with an 8-gaze stainless steel needle. A DC voltage of 8 kV (High DC power supply, Dalian Dingtong Technology Corp., China) was applied between the syringe tip and a grounded flat collector covered with an aluminum foil. The typical distance


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between the syringe tip and the grounded flat collector was 15 cm for pristine pectin and AP with oxidation degrees of 37% and 46%, and 20 cm for AP with oxidation degrees of 9%, 18% and 25%, respectively. All electrospinnings were carried out at temperature between 25–35 oC and humidity between 20–30%. The nanofiber mats were then lyophilized and carefully removed from the aluminum foil with a tweezer.

ADH crosslinking of electrospun AP nanofibers. Firstly, ADH solutions (5 mM) were prepared by dissolving ADH in water followed by mixing with ethanol to reach a volume content of ethanol of 50–90%. Then, AP nanofibers (5 mg) were placed into the ADH solutions (5 mL). The mixtures were then shaken at 100 rpm for 3–24 h at ambient temperature. The crosslinked nanofibers were washed with a large amount of water to remove the excess ADH, rinsed with ethanol and air dried overnight.

Ca2+ crosslinking of pristine pectin nanofibers. The pristine pectin nanofibers were crosslinked with CaCl2 as reported previously.17 Briefly, the pectin nanofibers were firstly immersed in ethanol for 5 min and pre-crosslinked in CaCl2 solution in ethanol/water mixture solvent (2% w/w, containing 95% ethanol) for 20 min. The nanofibers were then crosslinked in aqueous CaCl2 solution (40% w/w) for 2 h and rinsed with a large amount of water to remove unreacted CaCl2. The crosslinked nanofibers were then rinsed with ethanol and air dried overnight. The Ca2+-crosslinked pristine pectin nanofibers are named as Ca-pectin in the following text.

Composition characterization. The Fourier transform infrared (FTIR) spectra were collected with a FTIR spectrometer (iS10, Thermo Scientific Nicolet) equipped with a photomultiplier detector. The samples were homogeneously mixed with KBr at a weight ratio of


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1:80 and pressed into pellets. Spectra were obtained by recording 48 scans between 2000 and 800 cm−1 with a resolution of 4 cm-1.

Morphology observations. The morphologies of the nanofibers were observed through a scanning electron microscope (SEM, XL-30 ESEM FEG, Micro FEI Philips) at an acceleration voltage of 20 kV. The samples were placed on sample stages with conductive tapes and sputtercoated with a thin layer of gold before measurements. The sizes of nanofibers were determined by measuring and averaging the diameters of 50 nanofibers in SEM images with Image J (https://imagej.nih.gov/ij/).

Tensile mechanical tests. The mechanical properties of the nanofiber mats were measured with the mechanical testing machine (1121, Instron). The nanofiber mats were cut into a dimension of 6 mm × 20 mm × 0.05 mm and fixed with two clamps with a distance of 10 mm. The mats were then wetted with simulated body fluid (142 mol/L Na+, 5.0 mol/L K+, 1.5 mol/L Mg2+, 2.5 mol/L Ca2+, 147.8 mol/L Cl-, 4.2 mol/L HCO3-, 1.0 mol/L HPO42-, 0.5 mol/L SO42-, pH 7.4, 10 µL) and subjected to static tensile tests at a tensile rate of 1 mm min-1. The Young's moduli of the nanofiber mats were calculated from their stress – strain curves. Three samples were tested in parallel.

Degradation tests. The nanofibers (10 mg) were incubated in simulated body fluid (10 mL) at 37 °C. At certain time points, the nanofibers were washed with water and lyophilized. The morphologies of the samples were observed by SEM and the mass was weighed with an analytical balance with a readability of 0.01 mg (Mettler Toledo XSE). Three samples were tested in parallel.

Cell culture. Murine L929 fibroblast cells and MC3T3-E1 preosteoblast cells purchased from American Type Culture Collection were used for cell experiments and cultured in


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Dulbecco's modified Eagle’s medium supplemented with fetal calf serum (10% v/v), penicillin (100 U mL-1) and streptomycin (100 µg mL-1) at 37 °C in a humidified atmosphere containing 5% CO2. The nanofiber mats were fixed in 24-well cell culture plates using plastic rings, disinfected with 75% ethanol and dried overnight. Cells were then seeded onto the nanofiber mats at a density of 10000 cells per well and cultured at 37 °C. After being cultured for 1, 3 and 7 d, respectively, cells on nanofiber mats were observed by SEM and fluorescent imaging. For SEM imaging, the medium in the wells was removed and the nanofiber mats with cells were rinsed with water. The bound cells on the nanofiber mats were fixed with glutaraldehyde solution (2.5%) at 4 °C for 1 h. The samples were then rinsed three times with water, dehydrated in a graded series of ethanol, air dried and then subjected to SEM observation. Fluorescent imaging of live/dead cells on the nanofiber mats were performed after staining cells with Calcein AM/Propidium iodide double stain kits according to the protocol in the manual.

3. RESULTS AND DISCUSSION 3.1. Preparation of the crosslinked pectin nanofibers. As an anionic polysaccharide, pectin is mainly composed of partially methoxylated (1→4) linked-Dgalacturonic acids (GalA). As shown in Figure 1, the crosslinked pectin nanofibers were prepared with three steps: (1) partially oxidizing pectin with periodate to convert diol groups in GalA residues into two aldehyde groups; (2) electrospinning the oxidized pectin into nanofibers; (3) crosslinking the oxidized pectin nanofibers with adipic acid dihydrazide to form adipic acid dihydrazone linkers between pectin macromolecular chains.

3.1.1. Oxidation of pectin with periodate. Periodate oxidation is a general chemical reaction to functionalize polysaccharides with dialdehyde groups.22-25 Sodium periodate (NaIO4)


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cleaves the C2–C3 bonds with diols of part of the GalA residues of pectin and generates two aldehyde groups (see Figure 1). As shown in Table 1, as the NaIO4/GalA molar ratio increases from 10:100 to 50:100, the content of aldehyde groups increases linearly from 1.0 mmol g-1 to 5.1 mmol g-1, indicating that the oxidation degree increases from 9% to 46%. These results suggest that the periodate oxidation of pectin is highly efficient. The dialdehyde pectins are named as AP indexed with their corresponding oxidation degrees in the following text (Table 1). Table 1. Chemical characteristics of the dialdehyde pectins (APs). Sample name

NaIO4/GalA molar ratio (:100)

Content of aldehyde groups (mmol g-1)

Oxidation degree (%)

Molecular weight (kDa)

Pectin

0

0

0

2407

AP9

10

0.98

9.0

220

AP18

20

1.94

17.7

102

AP25

30

2.60

24.7

97

AP37

40

4.08

37.3

78

AP46

50

5.12

45.9

74

The oxidation of diols after treatment with periodate to give aldehydes in pectin is confirmed by FTIR spectroscopy. As shown in Figure 2 and S1, the peak at 1740 cm-1 in the spectrum of pristine pectin is assigned to the C=O stretching of methyl ester groups. The intensity of the peak evidently increases in the spectra of the oxidized pectins, indicating the formation of aldehyde groups which show absorption at the same wavenumber.


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Figure 2. FTIR spectra of pristine pectin, AP25, ADH, and AP25 nanofibers crosslinked with ADH for 8 h. The periodate oxidation of pectin also leads to decreased molecular weight, indicating the accompanying depolymerization of pectin. As listed in Table 1, at the low oxidation degree of 9%, the molecular weight decreases dramatically from 2406 kDa of pristine pectin to 220 kDa of AP9. As the oxidation degree increases from 9% to 41%, the molecular weight decreases gradually to 74 kDa. Depolymerization of polysaccharides during periodate oxidation has been reported previously.26-28 The depolymerization was ascribed to the scission of infrequent and specific components of the polymer chains and the hydroxyl radicals caused degradation.28 As will be shown later, pectin is resistant to biodegradation, the decreased molecular weight of oxidized pectins may benefit the hydrolysis-mediated degradation and facilitate their clearance from body.

3.1.2. Electrospinning of APs and ADH crosslinking. The APs of various oxidation degrees all can be readily electrospun into nanofibers following our previously reported method.17, 20 As shown in Figure 3, these nanofibers all exhibit smooth surface and uniform size.


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Figure 3. SEM images (a) and sizes (b) of AP nanofibers before and after ADH crosslinking. Crosslinking of the partially oxidized pectin nanofibers was carried out by immersing them in ADH solutions in ethanol/water mixture solvent for hours. Ethanol as a poor solvent of pectin is used to prevent the dissolution of AP nanofibers. Water as a good solvent of pectin is used to ensure that the AP macromolecular chains in nanofibers are sufficiently swollen to react with ADH. As can be seen from Figure 3 and S2–5, with the volume content of ethanol of 60–80%, the crosslinking with ADH does not affect the nanofibrous structure. The crosslinking time shows no obvious effects on the morphology of nanofibers. The crosslinking is due to the reaction of hydrazide groups in ADH with the aldehyde groups of the oxidized pectin chains to give dihydrazone linkers between pectin chains (Figure 1). This is confirmed by the FTIR spectra of the ADH-crosslinked AP nanofibers. As shown by Figure 2 and S1, in comparison with the original oxidized pectin, the ADH-crosslinked pectin nanofibers show evidently decreased absorption at 1740 cm-1, indicating that ADH crosslinking consume aldehyde groups. In addition, new absorption peaks at 1670 cm-1, 1550 cm-1, and 1400 cm-1, which are attributed to the C=O stretching, N-H bending, and C-H bending of adipic acid dihydrazone groups, respectively, can be clearly distinguished in the spectra of the ADHcrosslinked pectin nanofibers, although these peaks more or less overlap with the original pectin absorptions. These results demonstrate that ADH reacts with aldehyde groups in APs, forming adipic acid dihydrazone linkers.


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3.2. Mechanical and degradation properties. Although the oxidized pectins have much lower molecular weight than that of pristine pectin, the crosslinked pectin nanofibers exhibit excellent mechanical properties. Figure 4a shows the Young’s moduli and ultimate tensile strains of the ADH-crosslinked AP nanofiber mats at hydration state. For comparison, we have also prepared a control pectin nanofiber mats by crosslinking with Ca2+ to ensure water resistance. The Ca2+-crosslinked pectin nanofiber mat (Ca-pectin) has a Young’s modulus of 6.6 MPa and an ultimate tensile strain of 27.4%. For the ADH-crosslinked AP nanofiber mats, with the increase of the oxidation degree, the Young’s modulus at first decreases and then increases. The ultimate tensile strain displays opposite trend. The increased mechanical strength at high oxidation degree may be attributed to the high density of ADH crosslinking because one oxidized saccharide unit can theoretically form two crosslinkers with other oxidized saccharide units (Figure 1). The Young’s moduli of AP37 and AP46 nanofiber mats are 9.6 and 10.9 MPa, respectively, which are higher than that of the Ca-pectin nanofiber mat. This mechanical strength is comparable with that of human skin or cartilage, and may satisfy many needs in biomedical fields.29, 30


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Figure 4. Mechanical (a) and degradation (b, c) properties of ADH-crosslinked AP nanofiber mats and Ca2+-crosslinked pristine pectin nanofiber mat. The crosslinked pectin nanofibers exhibit much faster degradation rate than conventional Capectin nanofibers. Figure 4b and 4c shows the residual mass and morphologies of the crosslinked AP nanofibers after incubation in SBF for 7 to 28 d. After incubation of 28 d, the mass and morphology of the Ca2+-crosslinked pectin nanofibers are unchanged. In contrast, ADHcrosslinked pectin nanofibers of lower oxidation degrees (AP9, AP18, AP25) change


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morphology at day 14, and degrade completely in 21 d. AP37 nanofibers maintain nanofibrous morphology in 14 d but degrade completely in 28 d. The crosslinked AP46 nanofibers lose their nanofibrous morphology and more than 50% of their initial mass at 28 d. The much higher degradation rate of ADH-crosslinked AP naonfibers than Ca-pectin nanofibers can be attributed to three reasons: (1) the molecular weights of APs are far lower than that of the pristine pectin (Table 1), and thus APs are more susceptible to dissolution; (2) the β-glycosidic bonds of APs have larger rotational freedom than pristine pectin, so they are more susceptible to hydrolysis;31 (3) the hydrazone bonds between ADH and APs are hydrolysable. The enhanced degradation of the crosslinked AP nanofibers could beneficial for in vivo applications. To be used in vivo, the biomaterials are desired to degrade and clear from body after they finish their function. Pectin is resistant to biodegradation because there are not corresponding enzymes in human body.12 The conventional Ca-pectin nanofibers degrade slowly, by dissolutions after ion exchanges of Ca2+ with monovalent ions (e.g. Na+) in physiological fluids. They may occupy the space for a long time and impede the regeneration/ repair of the tissue. In addition, the degraded pectin macromolecules are of big size (high molecular weight) which exceeds the limit of kidney ultrafiltration. They may undergo bioaccumulation and cause macromolecular syndrome.32 In comparison, the ADH-crosslinked AP nanofibers degrade fast by hydrolysis of the β-glycosidic or hydrazone bonds and the degraded pectin molecules are of much smaller size (much lower molecular weight). They therefore may better support tissue regeneration/wound healing and be possible to be eliminated by kidney.

3.3. Cell adhesion. We seeded L929 fibroblast cells to study the cell adhesion capability of the crosslinked nanofiber mats. The results are shown in Figure 5 and 6. Cell adhesion includes a cascade of four steps: initial cell attachment, cell spreading, organization of actin


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cytoskeleton, and formation of focal adhesion.33-36 Pectin is not adhesive to cells because its molecular chains have no cell binding domains. It is hydrophilic, so it can prevent cell adhesion by forming a hydration layer on surface. Moreover, pectin is negatively charged at physiological conditions, so it repels negatively charged cells by electrostatic interactions. For the Ca-pectin nanofiber mat, cells keep spherical morphology at day 1 and day 3, and only start to flatten at day 7 (Figure 5). The cells initially proliferate well (cell number per mm2 increases from 94 at day 1 to 613 at day 3) but latterly proliferate slowly (to 1081 at day 7) (Figure 6a). These results indicate that cells can attach to the Ca-pectin nanofiber mat but cannot spread and proliferate well.


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Figure 5. SEM and fluorescent images of L929 fibroblast cells cultured on ADH-crosslinked AP nanofiber mats and Ca2+-crosslinked pectin nanofiber mat.


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Figure 6. Densities of L929 fibroblast cells of various morphologies on various nanofiber mats and at different time points. The spindle and spherical cells are distinguished and manually counted from the SEM images in Figure 5. As shown in Figure 5 and 6b–f, AP37 and AP46 nanofiber mats exhibit outstanding capability to support cell adhesion. At day 1, most cells have already flattened to typical spindle fibroblast


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morphology to some extent. From day 3, all cells form tight focal adhesions on these two AP nanofiber mats. The cell number per mm2 on the AP37 nanofiber mats increases from 69 at day 1 to 1805 at day 7. For AP46 nanofiber mats, the cell number per mm2 increases from 108 at day 1 to 2705 at day 7, reaching confluence. The cell adhesion on AP37 and AP46 nanofiber mats is much better than that on Ca-pectin nanofibers. The lower cell adhesion of AP9 and AP18 nanofiber mats compared with that of the pristine pectin nanofiber mat is probably attributed to their low stability in cell culture medium. As shown by the high magnification images in Figure 5, these two AP nanofiber mats have already lost their nanofibrous morphology and fused into films in cell culture medium at day 1. For AP9 and AP18 nanofiber mats, the oxidation/crosslinking may have increased cell adhesion to some extent, but the loss of nanofibrous morphology has greatly inhibited cell adhesion, leading to decreased cell adhesion. We selected another anchorage-dependent cell line, MC3T3-E1 preosteoblast cells to compare the cell adhesion capability of the pectin nanofiber mats. The results are shown in Figure 7. MC3T3-E1 preosteoblast cells cannot attach, spread nor proliferate well on nanofiber mats of Ca2+-crosslinked pectin, and APs of low oxidation degrees (AP9 and AP18). The cells can attach, spread and proliferate well on APs of high oxidation degrees (AP25, AP37 and AP46). These results confirm the excellent cell adhesion performance of the ADH-crosslinked pectin nanofibers with high oxidation degrees.


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Figure 7. Fluorescent images of MC3T3-E1 preosteoblast cells cultured on ADH-crosslinked AP nanofiber mats and Ca2+-crosslinked pectin nanofiber mat. In the above cell adhesion experiments, we observe improved cell adhesion with the increased oxidation degree (Figure 5, 6b–f, and 7). Therefore, we believe that the cell adhesion capability of the crosslinked pectin nanofiber mats may originate from the oxidation/crosslinking domains of the nanofibers. The exact mechanism is unknown yet. Possibly, their unique chemical structure enhances their interaction with proteins, that is, the adhesion mediating proteins in serum (e.g. fibronectin and vitronectin) or protein receptors on cell membrane, which facilitates cell adhesion. Biomaterials interact with proteins through hydrophobic-hydrophobic, electrostatic or covalent interactions.37, 38 As shown in Figure 1, the oxidation/crosslinking domains of pectin chains in nanofibers compose of two hydrazone groups and one alkyl spacer. The hydrazone groups may interact with proteins by hydrogen bonding and the alkyl spacer may interact with proteins by hydrophobic – hydrophobic forces. Both the two interactions may facilitate the interactions between proteins and the pectin nanofibers, and thus enhance cell adhesion.


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To be applied in vivo, pectin nanofibers must be crosslinked to achieve water resistance. Conventionally, pectin nanofibers are crosslinked with Ca2+ and suffer from poor cell adhesion and poor biodegradability. In comparison, the pectin nanofibers crosslinked with periodate oxidation - ADH crosslinking strategy exhibit comparable or even higher mechanical strength, much faster degradation rate, and much better cell adhesion. The combination of these advantages indicates that the crosslinked pectin nanofibers are superior to the conventional Ca2+crosslinked pectin nanofibers for in vivo applications. Considering that mechanical strength and the degradation rate of the biomaterials are better to match the mechanical strength and regeneration rate of the targeted tissue, the crosslinked pectin nanofibers with Young’s modulus ~ 10 MPa and degradation time of 3 weeks or longer time may specifically be suitable for cartilage tissue engineering or cutaneous wound healing. Future studies are to be undertaken in this regard. Both the periodate oxidation and ADH crosslinking reactions can be carried out with cheap chemical reagents, in aqueous conditions and at ambient temperature. The periodate oxidation ADH crosslinking strategy is thus of low cost and ease synthesis. Moreover, as periodate oxidation is versatile to many types of polysaccharide,22-25 the strategy may also be used to improve cell adhesion capacity of other polysaccharide materials.

4. CONCLUSIONS We have developed crosslinked pectin nanofibers by periodate oxidation - ADH crosslinking approach to covalently connect pectin macromolecular chains with adipic acid dihydrazone linkers. Compared with traditional Ca2+-crosslinked pectin nanofibers, the pectin nanofibers with high oxidation/crosslinking degree exhibit much enhanced cell adhesion capability. This is


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attributed to the adipic acid dihydrazone linkers, which may act as cell binding domains. Moreover, the crosslinked pectin nanofibers exhibit excellent mechanical strength and much enhanced body degradability. The combination of excellent cell adhesion capability, mechanical strength and body degradability suggest that the crosslinked pectin nanofibers are promising candidates for in vivo applications such as tissue engineering and wound healing. This crosslinking strategy may also be used to improve cell adhesion capacity of other polysaccharide materials.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge from ACS Publications website. FTIR spectra of AP9, AP18, AP37, AP46 and their nanofibers crosslinked with ADH for 8 h, and morphologies and remaining mass of AP nanofibers after being crosslinked in ADH solution in ethanol/water mixture solvent for various time.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J. Hu), [email protected] (Y. Zhou), [email protected] (Y. Liu).

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.


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Funding Sources This work was supported by the National Natural Science Foundation of China (No. 51503027, 81401516), and the 111 project (No. B13013).

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Abstract Graphic

For Table of Contents Only


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Figure 1. Schemetic illustration of the preparation process of crosslinked pectin nanofibers and the possible cell binding domain. 160x134mm (300 x 300 DPI)


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Figure 2. FTIR spectra of pristine pectin, AP25, ADH, and AP25 nanofibers crosslinked with ADH for 8 h. 80x67mm (300 x 300 DPI)



Figure 3. SEM images (a) and sizes (b) of AP nanofibers before and after ADH crosslinking. 160x42mm (300 x 300 DPI)


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Figure 4. Mechanical (a) and degradation (b, c) properties of ADH-crosslinked AP nanofiber mats and Ca2+crosslinked pristine pectin nanofiber mat. 160x153mm (300 x 300 DPI)



Figure 5. SEM and fluorescent images of L929 fibroblast cells cultured on ADH-crosslinked AP nanofiber mats and Ca2+-crosslinked pectin nanofiber mat. 160x143mm (300 x 300 DPI)


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Figure 6. Densities of L929 fibroblast cells of various morphologies on various nanofiber mats and at different time points. The spindle and spherical cells are distinguished and manually counted from the SEM images in Figure 5. 160x175mm (300 x 300 DPI)



Figure 7. Fluorescent images of MC3T3-E1 preosteoblast cells cultured on ADH-crosslinked AP nanofiber mats and Ca2+-crosslinked pectin nanofiber mat. 160x78mm (300 x 300 DPI)


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Abstract graphic 51x35mm (300 x 300 DPI)


Cross-Linked Pectin Nanofibers with Enhanced Cell Adhesion

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