Regulating temporospatial dynamics of morphogen for structure formation of the lacrimal gland by chitosan biomaterials

Accepted Manuscript Regulating temporospatial dynamics of morphogen for structure formation of the lacrimal gland by chitosan biomaterials Ya-Chuan Hsiao, Tsung-Lin Yang PII:

S0142-9612(16)30555-5

DOI:

10.1016/j.biomaterials.2016.10.012

Reference:

JBMT 17749

To appear in:

Biomaterials

Received Date: 5 August 2016 Revised Date:

9 October 2016

Accepted Date: 11 October 2016

Please cite this article as: Hsiao Y-C, Yang T-L, Regulating temporospatial dynamics of morphogen for structure formation of the lacrimal gland by chitosan biomaterials, Biomaterials (2016), doi: 10.1016/ j.biomaterials.2016.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Regulating temporospatial dynamics of morphogen

chitosan biomaterials

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for structure formation of the lacrimal gland by

Department of Otolaryngology, National Taiwan University Hospital and College of

Medicine, Taipei, Taiwan 2

Department of Ophthalmology, Zhongxing Branch, Taipei City Hospital, Taipei,

Taiwan 3

Department of Ophthalmology, College of Medicine, National Yang-Ming University,

Taipei, Taiwan

Research Center for Developmental Biology and Regenerative Medicine, National

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Ya-Chuan Hsiao1,2,3 Tsung-Lin Yang1,4,5*

Taiwan University, Taipei, Taiwan 5

Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan

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University, Taipei, 10002, Taiwan

Author for correspondence

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Tsung-Lin Yang MD. PhD. E-mail: [email protected]

#1, Sec. 1 Jen-Ai Road, Taipei 100, Taiwan Tel: +886-2-23123456 ext. 63526 Fax: +886-2-23940049

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Abstract The lacrimal gland is an important organ responsible for regulating tear synthesis and secretion. The major work of lacrimal gland (LG) is to lubricate the ocular surface and maintain the health of eyes. Functional deterioration of the

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lacrimal gland happens because of aging, diseases, or therapeutic complications, but without effective treatments till now. The LG originates from the epithelium of

ocular surface and develops by branching morphogenesis. To regenerate functional LGs, it is required to explore the way of recapitulating and facilitating the organ to establish the intricate and ramified structure. In this study, we proposed an

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approach using chitosan biomaterials to create a biomimetic environment beneficial to the branching structure formation of developing LG. The morphogenetic effect of chitosan was specific and optimized to promote LG

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branching. With chitosan, increase in temporal expression and local concentration of endogenous HGF-related molecules creates an environment around the emerging tip of LG epithelia. By efficiently enhancing downstream signaling of HGF pathways, the cellular activities and behaviors were activated to contribute to LG branching morphogenesis. The morphogenetic effect of chitosan was abolished by either ligand or receptor deprivation, or inhibition of downstream signaling

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transduction. Our results elucidated the underlying mechanism accounting for chitosan morphogenetic effects on LG, and also proposed promising approaches with chitosan to assist tissue structure formation of the LG.

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Keywords: lacrimal gland, branching morphogenesis, hepatocyte growth factor,

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regeneration, tissue engineering

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1. Introduction The lacrimal gland (LG) is responsible for tear production and secretion, providing lubrication and surface protection of eyes. Dysfunctional LG leads to ocular surface ulceration and decreases the immune responses, which may result in blindness

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without appropriate control.[1] For treating xerophthalmia, the current therapies remain palliative. The methods of tear substitution, tear retention, and inflammation control including artificial tears, punctal plugs, moisture-chamber spectacle, cyclosporine B administration are the most common therapies.[2] The causative

treatments such as salivary gland auto-transplantation are used to reduce symptoms in

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severe cases.[3] However, the different components of saliva and tears still raise

problems on the ocular surface. Until now, no effective therapeutic modalities of dysfunctional LG are developed and become clinically available.

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The LG is an excretory gland composed of numerous alveolar units. Between thousands of alveoli, an intricate and complex ramified network exists to efficiently support tear accumulation and secretion.[4] LG comes from the surface ectoderm, and also originates from differentiated epithelium of ocular surfaces.[5] During LG development, the single bud-like invagination of conjunctiva appears, and extends subsequently as a tubular sinus into periorbital mesenchyme.[6] Extensive branching

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repeatedly happens to establish the ramified structures, and followed by cell differentiation to become a functional organ. Formation of branching tissue structure is always the first step of LG organogenesis.[7] The tissue structure of LG develops by branching morphogenesis. It is an essential

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developmental process to generate ramified tissue structures frequently found in many glandular organs of excretory, vascular, and aerodigestive systems.[8] These organs benefit from branching development by creating a large surface area for biological

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functions with limited cell numbers in a confined space. The efficacy and capacity of metabolic circulation and exchanges efficiently increase with complex and branching tissue networks, and the physiological demand of multicellular organisms can be fulfilled to maintain biological activities.[9, 10] Without structure formation by branching processes, the numbers of cells and the space for regenerating a functional organ largely increase. For regenerative purpose, it is therefore necessary to recapitulate the process of structure formation of the excretory organ like LG. Although the intrinsic regulating mechanisms of LG branching have been well explored,[11] the approaches of tissue engineering that aim to reproduce the branching process for tissue regeneration are still under investigation.[12] For LG

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regeneration, explore the potential biomaterials that are competent to create a biocompatible environment to enhance the interaction between LG epithelia and mesenchyme may be beneficial for expediating tissue structure formation. Many studies have demonstrated the feasibility of using biomaterial to assist tissue structure

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formation.[13-16] It was shown that the complex tissue structure could be recapitulated assisted by biomaterials. For this part, the current progress of LG lags behind that of other organs. The biomaterial approaches principally focusing on engineering LG structure formation have not been well explored yet.

Since there are no curative therapies for xerophthalmia, there is a need to develop

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the treatment based on regenerative medicine. When no functional LG cells remain, reconstruction of the lacrimal gland for physiological function recovery is imperative.

Because LG is constituted by a complex organ structure, reconstruction of LG tissue

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is challenging. To this end, the approaching method is required to constitute three-dimensional tissue architecture to permit accommodation of sufficient cell numbers. The regenerated organ needs to encompass enough communicating network to restore total organ function. In addition, the system should be free from bio-incompatible substrata and reagents for potential requirement of future clinical translation. Therefore, in the current study, we explored the possibility of developing

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a biocompatible system capable of regulating temporal and spatial dynamics of

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endogenous morphogen to facilitate tissue structure formation of the lacrimal gland.

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2. Materials and Methods 2.1. Ex vivo explant culture of the lacrimal glands The LG explants were harvested from E16.5 ICR mice. The animal protocol was

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approved by the animal care and use committee of the National Taiwan University, and was in accordance with the guidelines. The LG explants were cultivated as the

whole explant culture, in which the culture methods had been described previously.[17] Briefly, the whole LG explants were dissected and laid flat on a membrane filter in a floating manner of culture media (supplementary information,

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Fig. S1).[18] LG explants were cultured for 48h in a 37°C humidified incubator with 5% CO2 under indicated experimental conditions.[13, 14] The LG explants were photographed and recorded at the indicated time-points during culture periods.

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Quantitation of LG branches was analyzed by the ratio obtained by dividing the branch number of experimental group over the control group. All counting processes were performed independently by different individuals who were blind to the experimental conditions. The values were determined after at least three-time repeats, and at least five explants were used for each experiment.

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2.2. Preparation of chitosan-containing culture medium

The chitosan-containing culture medium was prepared from a 2 wt.% (w/v) chitosan solution by dissolving chitosan (448869, Mn:612 kiloDaltons (kD), Sigma-Aldrich Chemical Co. St. Louis, MO, USA) in 1 M acetic acid. It was used to

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prepare chitosan-containing culture medium as described.[18-20] The concentration of chitosan used in this study was 0.3 mg/ml, determined by the tests of serial dilution of concentration.[18] Mock medium was prepared in the similar fashion as that of

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chitosan-containing medium, mixing with the same amount of acetic acid and sodium hydroxide but without chitosan. The morphogenetic effect of mock and control medium had been demonstrated not to have different morphogenetic effects.[16] It is therefore the representative control medium was used for comparison in the following assays.

2.3. Preparation of chitosan biomaterials, analogues, and related polymeric substrates. To prepare the biomaterials bearing the similar structures with chitosan, type I collagen and laminin were obtained (BD Biosciences, San Jose, CA). Poly-lysine,

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agarose, and the oligomeric forms of glycosaminoglycans including chondroitin sulfate (CS, C9819) were purchased from Sigma (St. Louis, MO). Chitosan monomers

and

analogues,

including

Nacetyl-D-glucosamine

(GluNAc),

N-acetyl-D-mannosamine (Man-NAc), N-acetyl-D-galactosamine (GalNAc), and

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D-glucosamine (GluN) were also obtained from Sigma (St. Louis, MO). Chitosan with different molecular weights (C-3646, Mn:810 kD, degree of deacetylation: 75– 85%; 448869, Mn:612 kD, degree of deacetylation: 75–85%) were also purchased from Sigma. They were all prepared in the same way with the same concentration of

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chitosan for LG explant culture. 2.4. Enzyme digestion assay

For enzyme digestion assay, chitosan was incubated with indicated enzyme prior to

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be prepared for LG explant culture. For each enzyme reaction, the procedure followed the manufacturer’s protocol by incubating chitosan with lysozyme (Sigma, L6876, 1 mg/ml), chitinase (Sigma, C6137, 1 mg/ml), or chitosanase (Sigma, C9830,1 mg/ml) respectively to digest chitosan. Then the enzyme-digested chitosan was retrieved using the membrane with specific molecular weight pore sizes (Scientific, Cellu Sep T4 and T2). Digested chitosan was then used to prepare the chitosan-containing

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medium similarly as those described above. For comparison, the same procedures were also performed in the control.

2.5. RT-PCR and quantitative PCR

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The RNA of LG explants was extracted from each conditions at indicated time-points using RNeasy mini kit (Qiagen) according to manufacturer’s protocol. Synthesis of complementary DNA was done by ReverseAid kit (Thermo Fisher).

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Semi-quantitative PCR was performed using the specific primers for HGF related genes.[18] The band intensity was quantified by a digital imager (AlphaImager, Alpha Innotech, San Leandro, CA, USA), based on the equal loading amount of cDNA adjusted by the value of GAPDH in each group. The optimal number of PCR cycles for each primer pair was applied to prevent the products from saturated.[13] Quantitative PCR (BioRad iCycler MyiQ Real Time thermocycler) was performed by SYBR green supermix (BioRad). The normalized data by GAPDH were analyzed by iQ5 software (BioRad) and shown as mRNA fold change levels.[21] All results were presented from at least triplicate reactions and three independent experiments.

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2.6. Microarray analysis Total RNA extraction was performed from the LG explants harvested from all groups (Qiagen, Valencia, CA, USA). Sufficient yield and quality of mRNA checked by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), which

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was subsequently submitted to microarray analysis. Mouse Whole Genome OneArray® v2.1 (Phalanx Biotech, HsinChu, Taiwan) was applied to determine

mRNA expression. The results were obtained from three replicates from three

independent experiments. The fluorescent target was prepared by 1µg total RNA samples using OneArray® Amino Allyl aRNA Amplification Kit (Phalanx Biotech

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Group, Taiwan) and Cy5 dyes (Amersham Pharmacia, Piscataway, NJ, USA). After hybridization at 50°C for 16 hours, the slides were scanned by an Axon 4000B

scanner (Molecular Devices, Sunnyvale, CA, USA). The Cy5 fluorescent intensities Systematic

and

random

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of every spot were analyzed by GenePix 4.1 software (Molecular Devices). errors

were

removed

using

Rosetta

Resolver

System®(Rosetta Biosoftware). The spots with the flag greater than 0 were normalized by the method of 50% media scaling normalization. The technical reproducibility was confirmed by Pearson correlation coefficient (R value > 0.975). The results of gene expression were based on the normalized spot intensities with

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significant P-value.

2.7. Immunohistochemical and immunofluorescent staining The assays of immunohistochemical and immunofluorescent staining were carried

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out as previously described.[21-23] Briefly, the LG explants were fixed with 4% paraformaldehyde in PBS and washed. The fixed LG explants were pretreated with Triton-X-100 and bovine serum albumin, and then subsequently incubated with

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primary antibodies. The following antibodies used in this study include anti-mouse HGF, anti-mouse c-Met (Abcam, Cambridge, MA); anti-mouse heparan sulfate proteoglycan (HSPG, Chemicon, Temecula, CA, USA); anti-mouse E-cadherin (Cell Signaling Technology, Danvers, MA); anti-mouse fibronectin (BD Pharmingen, San Diego, CA, USA). For immunohistochemical staining, the explants were then incubated with biotinylated secondary antibodies followed by incubation with an avidin-biotin complex (ABC Elite Kit, Vector Laboratories, Burlingame, CA, USA) for 40 min at room temperature. Finally, the stained results were developed with the enzyme substrate 3,30-diaminobenzidine and hydrogen peroxide for 3 minutes (DAB Substrate Kit, Vector Laboratories, Burlingame, CA, USA). For fluorescent staining,

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mouse Dylight 488, and rabbit Dylight 550 conjugated secondary antibody (Thermo Fisher) were used where appropriately to treat explants. They were later mounted by Fluoroshield with DAPI mounting media (Sigma-aldrich). Fluorescent images were photographed and merged by confocal microscopy (Leica SP-5). The Zen software

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(Carl Zeiss, Jena, Germany) was used for processing of confocal image stacks for three-dimensional reconstruction and analyses. 2.8. Surface plasmon resonance (SPR)

Binding affinity between growth factors and receptors were examined by SPR

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imager instrumentation (GWC Technologies, Madison, WI). Real-time monitoring of SPR intensity started when the molecules were applied on to the sensor chip.

Functionalization of the surfaces of gold-coated sensor ships was performed for

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bio-affinity immobilization assays.[24] The chemically immobilized surface was activated for covalent bond formation. The recombinant protein of growth factors and receptors were purchased from R&D (R&D Systems, Minneapolis, MN). They were prepared with the concentrations of 50 nM/mL, immobilized onto the surface, and blocked with 1% BSA. Measurements were performed using a prism coupled SPR. The Au/Cr chips were used to measure the intensities of reflected light at the angles of

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50° at 790 nm wavelength. A high flow rate (40 µl/min) was used to minimize mass transport effects. The growth factors were prepared both in chitosan-containing and control medium with concentrations of 50 nM. PBS was first applied onto the gold surface for baseline establishment. Then, the growth factors prepared in chitosan and

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control groups were injected onto immobilized surfaces. The immobilized surface was subsequently washed to remove unbound molecules. The values recorded in responses are expressed in responsive units (RU). All results were obtained form the

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experiments repeated in at least triplicate. 2.9. Ligand and carbohydrate engagement (LACE) assay A modified LACE assay was performed as previously described.[25] To test the

binding affinity of HGF in vivo, the recombinant proteins used as probes were prepared (R&D Systems, MN, USA). The LG explants harvested from culture of chitosan and control groups were washed, permeabilized, and then blocked overnight at 4°C with 10% BSA, and incubated for 3 hours with indicated recombinant protein probes (50nM). To prepare HGF-c-Met complexes, HGF was incubated with c-Met for 5 minutes prior to immerse explants. Localization of indicated probes was

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detected with antihuman Fc antibody (R&D Systems, MN, USA) after fixation. For counterstaining, anti-HSPG (Chemicon, CA, USA) was used as the primary antibodies, followed by Cy dye-conjugated secondary antibody for visualization. The

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results were photographed by confocal microscopy (SP5; Leica). 2.10. Western-blotting analyses

The LG explants were harvested after culture, and immediately frozen and solubilized in RIPA buffer for sonication. The protein extract was quantified by Bradford reagent (Bioshop), Proteins were then separated by SDS-PAG and

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transferred (BioRad). Separated-blots were first blocked, and then applied with primary antibodies overnight at 4°C, and secondary antibodies for 1 h at RT. The

primary antibodies used included ERK (4695; Cell Signaling, Beverly, MA); MA);

phospho-Akt (Ser473),

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phospho-ERK (4370; Cell Signaling); Akt, 1:2500 (9272; Cell Signaling, Beverly, 1:2500

(9271S;

Cell Signaling);

FAK and

phosphor-FAK (Abcam, Cambridge, USA); β-actin, 1:10,000 (clone AC-74; Sigma), and the secondary antibodies used were HRP-conjugated. Blots were developed by a chemiluminescent development solution (Pierce BCA protein assay kit, Thermo Scientific). The bolts were analyzed with a chemiluminescent imager (UVP

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BioSpectrum 600) with appropriate exposure time to keep intensities unsaturated. The chemiluminescence of each sample was detected by SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific), and quantitated by AlphaEaseFC 4.0 based on integration of pixel density within each band after background

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subtraction. The relative level was calculated and compared. 2.11. Neutralizing antibody assay

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The culture of LG explants were added with neutralizing antibodies against HGF

and c-Met during the culture periods. Preservative-free antibodies including anti-HGF (3µM; R&D Systems, Minneapolis, MN) and anti-c-Met (5µM; R&D Systems,

Minneapolis, MN) were applied. The LG explants were cultured in the same manner as mentioned above, and the number of branches was quantitated and compared.

2.12. Protein inhibitors Signaling perturbation of branching LG explants was performed based on the methods previously described.[23] Protein inhibitors were purchased and prepared

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with concentrations suggested by manufacturers and published subtoxic levels (Calbiochem, San Diego, CA, or Sigma-Aldrich, St. Louis, MO). Likewise, vehicle of protein inhibitors with similar concentrations were used in control groups. PD98059 (75µM; Santa Cruz Biotechnology) and LY294002 (50µM; Cayman Chemicals, Ann

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Arbor, MI) were used.[26] The LG explants were cultured with inhibitor containing media for 30 minutes, and then replaced with culture medium for subsequent 48h

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culture.

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3.Results 3.1. Enhanced branching characters of the lacrimal gland explants cultured with chitosan

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To explore the effect of chitosan-containing culture system on the lacrimal glands (LG), the LG explants were harvested at indicated stages. With increased culture

periods, the branching numbers of LG explants increased.(Fig. 1a) When chitosan was applied, LG explants survived and grew by showing similar phenotypes with

control, and increased branches with time.(Fig. 1a) When both groups were compared,

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the branching number of chitosan group was more that that of the control, either in the

first 24 and subsequent 48 hours of culture.(Fig. 1b) The results indicated that chitosan-containing system promoted the morphogenetic effect on the cultured LG

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explants.

3.2. Characterization of material properties of the morphogenetic effect of chitosan.

Since chitosan is derived from chitin and has a polymeric structure with intricate crosslinking networks, natural and synthetic materials with similar polymeric

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structures, including type I collagen, chondroitin sulfate (CS), agarose, laminin, and poly-lysine were prepared for LG explant culture.(Fig 2a) When compared with control, enhanced morphogenetic effect on LG explants were not observed among all tested materials.(Fig. 2b) The LG explants could not grow well in laminin and

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poly-lysine, showing aberrant phenotypes with reduced branching morphogenesis. Meanwhile, in light of the structural resemblance of chitosan to glycosaminoglycan (GAG),[27] CS, a member of GAGs, was tested. When CS was added, the cultured

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LG explants showed similar phenotypes to those of control, but with reduced branching numbers quantitatively.(Fig. 2a and 2b) Chitosan consists of linked moieties of GluNAc and GluN. Monomers and analogues of chitosan were prepared to determine whether the effects of chitosan were derived from these constituents. Among these analogues and monomers, cultured explants showed similar phenotypes compared to those of control, without branching enhancement.(Fig. 2c and 2d) The data suggested that the morphogenetic effect of LG was chitosan-specific, rather than a general phenomenon caused by substrata with similar chemical structures. In addition, because molecular weight of chitosan is critical in determining its biological reactions,[19] the LG explants were cultured with chitosan of different molecular

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weights. It was found that the morphogenetic effect of chitosan existed with chitosan of 612kD and 810kD molecular weight, but not for chitosan with less than 14kD molecular weight.(Fig. 2e, 2f) This suggested that the morphogenetic effects only originated from chitosan with a high molecular weight. The linkages between chitosan

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monomers are required for determining the molecular weight of chitosan. To evaluate its impact on LG morphogenetic effect, chitosan was pretreated with digested enzyme

before culture. No phenotypic alterations were found when these digested chitosan biomaterials were added to the control group, showing the preparation was not detrimental to LG branching morphogenesis. Lysozyme is capable of hydrolyzing the

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linkage between GluNAc residues in chitodextrin. When the lysozyme-treated chitosan was applied, the effect of chitosan disappeared, suggesting that GluNAc linkage was necessary for morphogenetic effects.(Fig. 2g, 2h) Chitinase cleaves the

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linkage between GluNAc monomer subunits. As such, chitinase-treated chitosan had no morphogenetic effects on cultured LG explants.(Fig. 2g, 2h) The results confirmed that GluNAc linkage was also required for chitosan effects. Chitosanase hydrolyzes the linked moieties between GluNAc and GluN. When chitosanase-treated chitosan was used, the morphogenetic effect of chitosan was absent. The results indicated that 2g, 2h)

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the linkage between GluNAc and GluN was also essential for the chitosan effects.(Fig.

3.3. Temporal expression of endogenous HGF related molecules in the LG explants cultured with chitosan.

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Since HGF has the potential to regulate ocular epithelial morphogenesis and is regarded as a promising morphogen to treat xerophthalmia,[28-30] whether its effects are regulated by chitosan is worthy of investigation. During the active branching stage

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of LG, HGF is predominantly present in the mesenchymal cells adjacent to epithelium, inferring the potential to serve as the target morphogen affected by chitosan. Given that HGF might be implicated in chitosan mediated LG morphogenesis, the expression of HGF, and related molecules were examined in both control and chitosan groups. It was found that when LG explants were cultured for 24 hours, expression of HGF and HGFa mRNA could be identified in the chitosan group. For the control, HGF and HGFa were only seen after 48 hours.(Fig. 3a, b) In the chitosan group, HGF mRNA decreased rapidly after 48 hours culture,(Fig. 3a, b, c, f) suggesting a transient

synthesis of HGF induced by chitosan. Conversely, increased c-Met mRNA expression was found in chitosan-cultured LG explants only after 48 hours,(Fig. 3a, b,

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d, f) which might respond to HGF correspondingly. In addition, expression of HAI-1 rather than HAI-2 was enhanced in chitosan group.(Fig. 3a, b, e) Increased HAI-1 was persistent in the chitosan group through the entire culture period.(Fig. 3a, b, e). It was interesting to find that the expression HGF and HGFa promptly increased when

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LG explants were cultured with chitosan. However, the effect did not persist in the following periods, a result confirmed by both qPCR and microarray analysis. These

data suggested that LG branching promoted by chitosan was based on the

immediately temporal regulation of HGF-related molecules. The stimuli of HGF appeared exactly on the time required for LG branching, and did not continue in the

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following culture period.

cultured with chitosan.

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3.4. Spatial expression of endogenous HGF related molecules in the LG explants Based on the gene profiling of HGF-related molecules in chitosan-promoted LG branching, we further examined their expression alteration by immunohistochemical and immunofluorescence staining. The staining results not only provided the change of HGF-related molecules in protein levels, but also the information of spatial distribution. HGF was mainly identified in the epithelial-mesenchymal junction of

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emerging epithelial buds by immunohistochemistry.(Fig. 4a) Because HGF was derived from stromal cells, a closed view showed HGF was present in the mesenchyme adjacent to the developing epithelial buds.(Fig. 4b) Comparing two groups, although HGF mRNA returned to the same level as that of control in the

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chitosan group after 48 hours, its protein expression was still greater in the chitosan group than control.(Fig. 4a, b) To further confirm the differences caused by chitosan, immunofluorescence was applied to demonstrate the higher intensity of HGF staining

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in chitosan group than control.(Fig. 4c, d) There was no colocalization of HGF and E-cadherin, indicating the HGF origin is from mesenchyme.(Fig. 4c) Similarly, no colocalization was found in the staining of c-Met and fibronectin, confirming that c-Met was located in epithelia and the interaction between HGF and c-Met was paracrine.(Fig. 4d) To clearly clarify the change of spatial distribution of HGF and c-Met mediated by chitosan, the immunofluorescent results were reconstructed in a three-dimensional manner. From the steric view, HGF was clearly identified along the epithelial-mesenchymal junction in LG explants. In the chitosan group, the fluorescence intensity was more obvious than in the controls.(Fig. 4e) c-Met was also specifically localized along the epithelial-mesenchymal junction.(Fig. 4f) Its

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colocalized staining with E-cadherin confirmed the epithelial localization of c-Met.(Fig. 4f) When two groups were compared, the immunofluorescent staining of c-Met was more obvious in the chitosan group. (Fig. 4f) The results provided evidence that HGF and c-Met protein expression was promoted by chitosan, and also

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confirmed that increased HGF and c-Met induced by chitosan were localized around the branching epithelia instead of spreading everywhere. The spatial distribution

regulated by chitosan created an enhanced local concentration effect of HGF-related molecules on adjacent epithelia in LG branching.

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3.5. In vitro and in vivo binding affinity of HGF-related molecules with chitosan.

In addition to temporal and spatial expression of HGF-related molecules regulated by chitosan, further tests were performed to confirm chitosan effect on the binding of

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ligand, receptor, and associated extracellular matrices. SPR analyses could provide biochemical evidence to support chitosan’s influence. Binding of HGF and c-Met was compared in the experimental settings with or without chitosan respectively. Although with same concentrations in both groups, HGF and c-Met showed greater binding affinity in the chitosan group than the control group with a higher SPR intensities.(Fig. 5a) Nonetheless, there was no difference in the time-points of initiation and

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termination of ligand-receptor interaction detection between the chitosan and the control groups.(Fig. 5a) This data showed that the in vitro binding of HGF to c-Met could be affected by chitosan. The SPR assays demonstrated the ability of chitosan to promote binding affinity of HGF ligands toward c-Met receptors.

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In addition to showing in vitro biochemical evidence of chitosan effect on ligand-receptor binding of HGF, whether similar effect also occurred in in vivo environment was tested. We used a modified LACE assay for whole-mount

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experiments of cultured LG explants to confirm endogenous binding affinity of HGF and c-Met when chitosan was present. Cultured LG explants from chitosan and control groups were probed with c-Met alone first.(Fig. 5b, 5c) HSPG was also co-stained in LACE assay. LG branching morphogenesis is dependent on the interaction between morphogens and HSPG.[31] Biochemical evidences support a strong and biological relevant interaction between HSPG and HGF-related molecules, and activation of downstream signaling follows the interaction between c-Met and HSPG in response to HGF stimuli.[32] Therefore, the LACE assay was conducted to confirm the in vivo affinity change in LG explants when affected by chitosan. By confocal microscopy, it was found that c-Met colocalized with HSGP staining.

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Similar results were also found when HGF-c-Met complexes were used as the probes. Increased staining intensity was found in the chitosan group rather than that of the control, including both probes of c-Met and HGF-c-Met complexes. With magnified views, c-Met probes were prone to show in the area of emerging epithelial tips, a

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phenomenon more obvious in chitosan than control groups. When HGF-c-Met complexes were applied for probing, staining was exclusively shown along the

epithelial-mesenchymal junction, where the emerging epithelial tips started to form branching structures.(Fig. 5c)[18] The results showed that c-Met probes were

preferentially bound by the components of endogenous basement membrane along

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adjacent epithelial borders when chitosan was present. The LACE assay of c-Met and

HGF-c-Met probes provided in vivo spatial evidence showing preferential binding of epithelial morphogenesis.

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morphogen-related molecules toward in vivo epithelial borders to facilitate ensuing

3.6. Activation of downstream signaling transduction in the LG explants cultured with chitosan

To study the molecular sequence of LG when chitosan was present for branching morphogenesis, the HGF-related signaling pathways were investigated. HGF had

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been well studied to stimulate downstream signaling including MAPK, Akt/PKB, and FAK pathways when it bound to c-Met receptor.[33] As shown in figure 6, these pathways were all activated during the branching morphogenesis of LG. With chitosan, increased phosphorylation of MAPK and Akt/PKB pathways were

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demonstrated.(Fig. 6a-d) In addition to c-Met autophosphorylation, HGF-induced activation of downstream effector molecules of MAPK and Akt/PKB pathways supported the cellular activities including cell migration, cytoskeleton reorganization,

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cell dissociation and proliferation that were altogether beneficial to LG branching morphogenesis.[33] Nonetheless, when FAK pathway was tested, no significant differences were noted between chitosan and control groups, indicating that FAK pathways were not majorly involved in the chitosan morphogenetic effect mediated by HGF. (Fig. 6e, 6f)

3.7. Effect of HGF blocking on chitosan-enhanced morphogenetic effects of cultured LG explants To further confirm the chitosan morphogenetic effect on LG mediated by HGF, functional blocking of HGF related molecules were preformed in the cultured LG

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explants. When the neutralizing HGF antibody was applied with indicated concentration, LG explants still survived to initiate branching morphogenesis in culture.(Fig. 7a) HGF blocking reduced LG branching during the same culture periods, both in the control and chitosan groups. When quantitative comparison of the

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effect of HGF blocking, it was found that the morphogenetic effects of chitosan was suppressed.(Fig. 7b) However, the branching promoting effect of chitosan still appeared, inferring the possibility that some other potential morphogens implicated in the LG branching were also activated and enhanced by chitosan. When the

downstream signaling of HGF was tested to verify the effect of HGF blocking, it was

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found that down-regulation of both MAPK and Akt/PKB phosphorylation was

shown.(Fig. 7c, d, e, f) The results confirmed that the role of HGF in LG branching

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enhanced by chitosan.

3.8. Effect of c-Met blocking on chitosan-enhanced morphogenetic effects of cultured LG explants

In addition to HGF blocking, c-Met, the corresponding receptor for HGF biological effects, was tested in LG branching when its function was blocked. With the neutralizing c-Met antibody, reduced branching numbers were found in chitosan and

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control groups.(Fig. 8a) The LG explants blocked by c-Met had fewer number of branches when compared with those treated by the control antibody. When the chitosan morphogenetic effects were evaluated, the decreasing ratio was found to be greater in chitosan group than control group, suggesting c-Met is crucial for mediating

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chitosan morphogenetic effects on LG branching.(Fig. 8b) The blocking effect was greater in c-Met blocking than HGF functional blocking by showing higher statistical significance.(Fig. 7b, 8b) In the group of c-Met blocking antibodies, the downstream

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signaling of HGF pathway including MPAK and Akt/PKB showed decreased phosphorylation in the treated explants. (Fig. 8c, d, e, f) The results further confirmed

that c-Met was essential in the LG structure formation enhanced by chitosan. 3.9. Downstream signaling of chitosan morphogenetic effects on LG branching Since chitosan could enhance HGF signaling pathways to promote LG branching

morphogenesis, the blocking effects of downstream signaling of HGF signaling pathways were examined in the chitosan-cultured LG explants to verify the functional effects. When PD98059, an inhibitors of MAPK pathway, was applied with previously-reported sub-lethal concentrations,[23] LG explants hardly survive and

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initiated branching in both groups.(Fig. 9a) Chitosan morphogenetic effect on LG explants could not sustain in the treated groups.(Fig. 9b) Western blotting confirmed the suppressive results of PD98059, and showed that MAPK/ERK phosphorylation decreased in the treated groups.(Fig. 9c, 9d) When the Akt/PKB inhibitor, LY294002,

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was applied, the LG explants still survived during the culture period.(Fig. 9e) However, no branching-promoting effects appeared. (Fig. 9f) Suppression of Akt/PKB phosphorylation was confirmed by blotting. (Fig. 9g, 9h) These results were

compatible with previous data confirming the requirement of HGF signaling pathway in chitosan enhanced LG branching. On the other hand, when the results of both

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chitosan and control groups treated by inhibitors were compared, the ratio of MAPK and Akt/PKB phosphorylation was greater in the chitosan than the control groups.[18] The results inferred a possibility that chitosan not only regulated HGF to facilitate LG

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structure formation. Taken together, these results of signaling inhibitors demonstrated that the morphogenetic effect of chitosan diminished when HGF signaling pathways were interrupted.

3.10. A proposed moled of the chitosan effect on promoting lacrimal gland structure formation by regulating HGF signaling

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According to the results gathered, we propose a model accounting for mechanism that chitosan-mediated morphogenetic effect on LG. By regulating spatial and temporal dynamics of endogenous HGF-related molecules, chitosan promotes LG structure formation.(Fig. 10). When the LG explants are exposed to chitosan,

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alteration is found in both expression levels and local distributions of HGF-related molecules. By enrichment and recruitment of endogenous HGF-related molecules, the downstream signaling was activated to increase morphogenetic activities of the cells

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located in the emerging tip of LG, which contributed to tissue structure formation of LG.

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4.Discussion Creation of a biocompatible environment to facilitate tissue morphogenesis is important for regenerative medicine because physiological function is mainly based on organized tissue structures. However, compared with the approaches exploited to

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engineer individual cells, those used for tissue structure formation are still underdeveloped. Culture of LG cells with different levels of complexity have been explored.[12] However, few can successfully rebuild the intricate and complex tissue architecture as shown in the native LG. Applying biomaterials to assist tissue

structure formation had been proven to be feasible.[13-16]. Our previous work had

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shown the morphogenetic capacity of chitosan biomaterials in promoting branching of glandular organs by providing an interactive environment.[16, 19, 20, 22, 34, 35] In

the current work, we demonstrated that LG structure formation could be recapitulated

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and promoted by chitosan-containing system. With specific properties of chitosan biomaterials fabricated in the current system, LG morphogenesis could be promoted. This is an appealing approach to regenerate tissue structure because it facilitates tissue morphogenesis and structural formation without addition of exogenous supplement of serum or bioincompatible extracts, complying with the regulation required for clinical translation.

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Branching morphogenesis is essential in the development of a variety of organs, including the lung, mammary glands, lung, kidney, vascular, and nervous systems.[8]. This process involves cell reorganization to reconstruct ramified tubular networks. The LG is composed of numerous branches and also developed by branching

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morphogenesis.[29] It usually starts from epithelial outgrowth and mesenchyme invagination assisted by dynamic and reciprocal process of epithelial-mesenchymal interactions.[36] During the process, cells need to initiate migration, shape change,

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asymmetric polarization, and tubule elongation by cell–cell and cell-extracellular matrix communication. These cellular behaviors are strictly regulated and coordinated in an appropriate manner over space and time.[37] HGF is one of the pleiotropic morphogens that can regulate various types of the

behaviors

of

epithelial

cell

including

cell

motility,

proliferation,

anchorage-independent growth, and morphogenesis. Gifted with these important biological functions, HGF accordingly contributes to branching morphogenesis of a variety of organs.[38] HGF synergizes with fibroblast growth factors in lung epithelial branching.[33] Similar phenomenon was also observed in the placenta development.[33] For the salivary gland, formation of branching morphogenesis is

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regulated by the interaction between epithelia and mesenchyme-derived HGF. HGF synthesized by the surrounding stromal tissue during the stage of robust epithelial branching can efficiently contribute to structure establishment of the salivary glands.[39]

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During LG development, HGF has been shown to express in the periocular mesenchyme near LG placodes.[40] Meanwhile, c-Met also presents in the LG.[41] HGF can regulate ocular surface epithelia from where LG organogenesis initiates.[28,

29] For epithelial cells derived from ocular surfaces, HGF increases cell motility,[42,

43] and also induces differentiation of these cells for morphogenesis.[44] In the LG

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branching promoted by chitosan, synthesis and release of HGF were shown in a

well-regulated temporal fashion. The timely enhancement of HGF and related molecules promptly trigger downstream signaling required in the emerging tip of

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epithelia, which promotes LG branching structure formation (Figure 10). HGF is synthesized by mesenchymal cells and predominantly localized in the stromal cells adjacent to epithelium; whereas the corresponding receptor, c-Met, is mainly localized on the surface of epithelial cells, showing a paracrine signaling.[33] HGF has high affinity for HSPG, a type of GAG existing in the extracellular matrix, particularly the basement membrane that is important for LG morphogenesis.[45, 46]

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The spatial distribution can sequester the ligands and locally concentrate the action of HGF exclusively to the target cells with c-Met expression. Since HSPG of cell surface mediates HGF binding and promotes c-Met signaling,[47] it is thus critical in determining the selection of cellular responses.[45] The binding capacity of HGF to

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HSPG provides the choice to tether these molecules together in specific cells for triggering downstream signaling for morphogenetic development. Chitosan is a linear polysaccharide with a variable number of randomly located

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N-acetyl-glucosamine groups. It thus shares some structural similarities with

GAG.[27] Functionally, GAG serves as a reservoir for bioactive molecules and prevents them from degradation.[48, 49] It therefore regulates spatial dynamics of bound molecules by immobilization close to activating sites, and increases local concentration for effective reactions.[50, 51] The effects of chitosan on stimulating LG branching are likely to partly originate from its resemblance with GAG. Chitosan can easily form a complex with GAG ionically or covalently.[52, 53] As shown in our results, chitosan increased binding affinity of HGF to c-Met and efficiently activated associated signaling, which was beneficial to LG morphogenesis.[54-57] Chitosan creates an environment with enhanced morphogenetic responses by retaining and

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concentrating the essential morphogens.[58] When chitosan was applied for LG branching, it was demonstrated that the interaction between endogenous HGF-related molecules and morphogenetic ECM was promoted, which work together for efficient tissue structure formation of the LG.

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There are several related molecules involved in the activating and signaling cascade of HGF. HGFA is produced originally in an inactive form, circulating in the blood,

and then activated by thrombin in target tissues. In the regenerating tissue, HAI-1 is significantly up-regulated.[59] Active HGFA selectively binds to the membranous form of HAI-1, but not to HAI-2, of epithelial cells. The membranous form of HAI-1

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not only serves as an inhibitor, but also a reservoir for HGF on the cell surface.[60]

When the complex of membranous HAI-1 and HGFA is cleaved, it can efficiently generate considerable amounts of HGFA in the pericellular area for desired cellular

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response. It is therefore HAI-1 can paradoxically localize the HGFA activity in a pericellular fashion for specific cellular condition by shedding HGFA-HAI-1 complex from the cell surface followed by the recovery of HGFA activity.[59] In the cultured LG explants whose branching was promoted by chitosan, it was found that HGFA and HAI-1 increased immediately with HGF. Presence of HGFA and HAI-1 increase the effect of HGF, and also locally concentrate these molecules around the emerging tip

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of the branching epithelia. Collectively, all effects work together to increase the morphogenetic effect of LG explants mediated by chitosan. When the HGF signaling cascade including ligand, receptors, or downstream molecules were blocked, the morphogenetic effect of chitosan was abolished in the

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cultured LG explants. It further confirmed the requirement of HGF pathways in chitosan-mediated LG branching. HGF and related molecules are essential in the branching morphogenesis during organogenesis. When either HGF or c-Met was

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blocked in the salivary gland, branching was suppressed.[39] Inhibition of HGFA activity reduced ureteric bud branching and inhibited glomerulogenesis and nephrogenesis.[61] When HAI-1 was knocked out, lethality was noted in the mutant mice during the embryonic stage because of growth retardation resulting from failed placental development and function.[62] The results altogether confirm the need of the whole regulating network of HGF morphogens and signaling in organ branching morphogenesis. When the developmental repertoire is regulated by chitosan, deprivation of these molecules of any levels might abolish the morphogenetic effect of chitosan biomaterials. When c-Met is activated by HGF, several intracellular signaling pathways are

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triggered for corresponding cell responses. Akt/PKB and MAPK are two major pathways well recognized. In the LG explants cultured with chitosan, it was found that signaling transduction of MAPK and Akt/PKB is enhanced. Transduction of MAPK pathway is coupled by the adaptor protein that links Met to Ras, playing an

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important role in disassembly of epithelial adhesion junction. When PD98059, an inhibitor for MAPK pathway, was used, cells lost the ability of moving and branching.[63] The Akt/PKB pathway is bridged to c-Met in response to HGF stimuli.

Treated by the inhibitor, LY294002, cell scattering induced by HGF disappeared.[64] Compatible with the suppressive effects of MAPK and Akt/PKB inhibitors on

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HGF-induced cell responses, the enhanced morphogenetic effects on branching LG

explants by chitosan were also abolished by these inhibitors. In addition to MAPK and Akt/PKB signaling, HGF also phosphorylates and activates FAK signaling.[65]

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Nonetheless, in the LG branching morphogenesis promoted by chitosan, no different responses were observed. These results confirmed that both Akt/PKB and MAPK pathways were principally required for HGF-induced branching morphogenesis

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promoted by chitosan.[33]

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5. Conclusion Chitosan facilitates LG tissue structure formation by regulating temporospatial dynamics of HGF-related molecules. With chitosan, increase in temporal expression and local concentration of endogenous HGF-related molecules created an

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environment along the emerging tip of LG epithelia that was beneficial for branching morphogenesis. By efficiently enhancing downstream signaling of HGF pathways, the cellular activities and behaviors were activated to assist LG branching morphogenesis.

Our results elucidated the underlying mechanism accounting for chitosan morphogenetic effects on LG, and also proposed promising approaches using chitosan

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Acknowledgements

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to promote LG structure formation.

The authors thank the Ministry of Science and Technology, the National Taiwan University Hospital, and the Taipei City Hospital, Taiwan, for their financial support. The authors also thank En-Jung Tseng, Hao-Wei Lee, Wei-zhen Lai, Tommy Chun, Ya-Shuan Chou, and the staff of the Eighth Core Lab, Department of Medical

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Research, the National Taiwan University for technical support.

Conflict of interest

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No declaration

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Figure legends Fig. 1 Effect of chitosan biomaterials on branching morphogenesis of the lacrimal glands

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(a) The LG explants were cultured in the control (Cont) and chitosan (Chi) groups for 48h. Scale bar = 100µm. (b) Quantitative analyses of branching number at 24h and 48h, and were presented as the ratio change of buds. (Student's test; ***p < 0.0001).

Fig. 2 Characterization of material properties of the morphogenetic effect of

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chitosan

(a) The effects of polymeric molecules on the branching structure formation of cultured LG explants. (Cont, control; Col I, type I collagen; CS: chondroitin sulfate;

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Aga: agarose; LN, laminin; pLys, poly-lysine) (b) Quantitative comparison with the control. (c) The effects of chitosan monomers and analogues on the cultured LG explants, with (d) quantitative results. (e) The effect of the molecular weight of the chitosan biomaterials with (f) quantitative comparison. (g) Effects of lysozyme, chitinase, and chitosanase in chitosan-mediated LG branching structure formation. (Cont, control; Cont + Lys, Cont + Chiti, Cont + Chito: control treated with lysozyme,

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chitinase, and chitosanase, respectively; Chi, chitosan; Chi + Lys, Chi + Chiti, Chi + Chito: chitosan treated with lysozyme, chitinase, and chitosanase, respectively) (h) The quantitative results for comparison. All quantification was performed at 48 h and was presented as the ratio change of buds. (Scale bar = 100µm, in a,c,e,g; Student's

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test for b, d, f, h; *p < 0.01, **p < 0.001, and ***p < 0.0001.) Fig. 3. Temporal expression of endogenous HGF related molecules in the LG

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explants cultured with chitosan (a) RT-PCR showed the expression of HGF related molecules in the LG explants of the control and chitosan groups. (b) Quantitative analyses showed the temporal alteration of LG gene profiles of both groups. The qPCR results showed mRNA change of (c) HGF and (d) c-Met during culture. (e) mRNA change of HGF related molecules after 48hr culture was shown by qPCR. (f) A heatmap demonstrated alteration of gene profiles of HGF related molecules in the LG explants cultured in both groups for 48 hours. (Genes in red, up-regulated; genes in green, down-regulated; Cont: Control groups, Chi: chitosan groups; Student's test; *p < 0.01, **p < 0.001, and ***p < 0.0001.)

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Fig. 4. Spatial expression of endogenous HGF related molecules in the LG explants cultured with chitosan. (a) Immunohistochemical staining showed that HGF was majorly expressed in the epithelial-mesenchyjmal junction of emerging epithelial buds. (arrow heads: HGF

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expression in the control group; arrows: HGF expression in the chitosan group). (b) A closed view showed HGF expression was in the mesenchyme adjacent to the developing epithelial buds. (arrow heads: HGF expression in the control group;

arrows: HGF expression in chitosan group). (c) Immunofluorescent staining demostrated the expression of HGF (red) relative to E-Cad (green) in cultured LG

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explants of control and chitosan groups. (d) Immunofluorescent staining demostrated

the expression of c-Met (red) relative to FN (green) in cultured LG explants of control and chitosan groups. (e) 3D reconstruction of immunofluorescent images demostrated

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spatial expression of colocalization of HGF and FN in cultured LG explants of control and chitosan groups. (f) 3D reconstruction of immunofluorescent images demostrated spatial expression of c-Met and E-cad colocalization in cultured LG explants of control

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(Cont:

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E-cad:E-cadherin; FN:fibronectin; Scale bar = 100µm in a, b; 50µm in c,d)

chitosan.

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Fig. 5. In vitro and in vivo binding affinity of HGF-realted molecules with (a) SPR analysis of the binding between HGF and c-Met in both groups. (b) The results of LACE assay with probes of c-Met, or HGF-c-Met complex were

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demonstrated. HSPG was used as a maker to delineate the basement membrane for confirming expresssion location of theses probes. (c) The close views of LACE results showed the binding of c-Met, or HGF-c-Met complex probes in both groups.

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(Cont: Control group; Chi:chitosan groups; HSPG: heparan sulfate proteoglycan; Scale bar = 100µm in b; 50µm in c) Fig. 6 Activation of downstream signaling transduction in the LG explants cultured with chitosan. (a) Western blot analyses showing MAPK pathway phosphorylation when LG explants were cultured with or without chitosan. (b) Quantitative analyses demonstrate a higher ratio of ERK1/2 phosphorylation in the chitosan group. (c) Phosphorylation of Akt/PKB Pathway was demonstrated in Western blotting in the LG explants of both groups. (d) Quantitative analyses demonstrate a higher ratio of

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Akt/PKB phosphorylation in the LG explants cultured with chitosan than control. (e) Phosphorylation of FAK Pathway was shown by Western blotting in both groups. (f) Quantitative analyses of the ratio of FAK phosphorylation in both groups. (Control group: Cont; Chitosan group: Chi; Student's test; *p < 0.01, **p < 0.001, and ***p <

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0.0001.) Fig. 7. Effect of HGF blocking on chitosan-enhanced morphogenetic effects of cultured LG explants.

(a) Inhibition of LG explant branching by the HGF neutralizing and control antibodies

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in the control and chitosan groups. (b) Quantitation was performed by counting branch numbers of buds in each gland at 48 h of culture. The data are expressed as the ratio between the control and chitosan groups. (c) Reduced ERK1/2 in the LG

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explants cultured with HGF neutralizing antibody was shown in the Western blotting (d) The results were confirmed quantitatively. (e) Reduced Akt phosphorylation was demonstrated in the LG treated by HGF neutralizing antibody in blotting. (f) The quantitative results were shown. (Control group: Cont; Chitosan group: Chi; Student's test; *p < 0.01, **p < 0.001, and ***p < 0.0001; Scale bar = 100µm).

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Fig. 8. Effect of c-Met blocking on chitosan-enhanced morphogenetic effects of cultured LG explants.

(a) Inhibition of LG explant branching by the c-Met neutralizing andibody was shown in the LG explants of the control and chitosan groups. (Scale bar = 100µm). (b)

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Quantitation was performed by counting branch numbers of buds in each gland at 48 h of culture. The data are expressed as the ratio between the control and chitosan groups. (c) Reduced ERK1/2 was shown in the LG explants cultured with c-Met

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neutralizing antibody by Western blotting. (d) The results were confirmed quantitatively. (e) Reduced Akt phosphorylation was demonstrated in the LG treated by c-Met neutralizing antibody. (f) The results of Western blot were quantitated. (Control group: Cont; Chitosan group: Chi; Student's test; *p < 0.01, **p < 0.001, and ***p < 0.0001.)

Fig. 9. Downstream signaling of chitosan morphogenetic effects on LG branching (a) LG explants were treated with PD98059 followed by 48 h culture in the control and chitosan groups. (b) The branching numbers of all groups were expressed as the fold-change of control average. (c) Western blot analyses confirmed reduced ERK1/2

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phosphorylation, and (d) the results were quantitated. (e) LG explants were treated with LY294002 followed by 48h culture in both grouops. (f) The branching numbers of both groups were compared. (g) Western blot analyses confirmed reduced Akt phosphorylation, and (h) the results were confirmed quantitately. (Control group: Scale bar = 100µm in a, e)

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Cont; Chitosan group: Chi; Student's test; *p < 0.01, **p < 0.001, and ***p < 0.0001;

Fig. 10. A proposed moled of the chitosan effect on promoting lacrimal gland structure formation by regulating HGF signaling.

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Chitosan timely increases local concentrations of HGF-related molecules along the

emerging tip of epithelia to activate downstream signaling for branching structure

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formation of the lacrimal glands.

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